10069801
full text
Y1173F
protein
substitution
true negative
EGF receptor mutants used in Ab P2 binding studies Number of receptors per cell ( 105) 0.025 1.41 0.56 2.49 0.70 0.77 0.25 0.45 1.85 0.25 0.61 0.50 0.39 Cell type NIH3T3 CL17 MI31 MI32 MI33 MI34 MI35 MI36 MI37 MI38 MI40 MI41 Dc63 Receptor type Untransfected Wild-type Y1173F Y1148F Y(1173-1148)F Y(1173-1148-1068)F Y(1148-1068)F Y(1173-1068)F Y1068F Y1086F Y992F Y(1173-1148-1086-1068-992)F C-terminal deletion of 63 amino acids lacking tyrosine 1173 and 1148 Biosynthetic Labeling of the EGF Receptor Twenty hours after subculturing, cell monolayers were washed with methionine- and cysteine-free DMEM containing 2% dialyzed newborn calf serum and were preincubated in the same medium at 37C for 1 h.
Because the EGF receptor mutants MI31 (Y1173F) and MI32 (Y1148F) showed no change in antibody binding, we also investigated whether a double mutant (MI33) of the EGF receptor in which the tyrosines at Vol.
Y1148F
protein
substitution
true negative
EGF receptor mutants used in Ab P2 binding studies Number of receptors per cell ( 105) 0.025 1.41 0.56 2.49 0.70 0.77 0.25 0.45 1.85 0.25 0.61 0.50 0.39 Cell type NIH3T3 CL17 MI31 MI32 MI33 MI34 MI35 MI36 MI37 MI38 MI40 MI41 Dc63 Receptor type Untransfected Wild-type Y1173F Y1148F Y(1173-1148)F Y(1173-1148-1068)F Y(1148-1068)F Y(1173-1068)F Y1068F Y1086F Y992F Y(1173-1148-1086-1068-992)F C-terminal deletion of 63 amino acids lacking tyrosine 1173 and 1148 Biosynthetic Labeling of the EGF Receptor Twenty hours after subculturing, cell monolayers were washed with methionine- and cysteine-free DMEM containing 2% dialyzed newborn calf serum and were preincubated in the same medium at 37C for 1 h.
Because the EGF receptor mutants MI31 (Y1173F) and MI32 (Y1148F) showed no change in antibody binding, we also investigated whether a double mutant (MI33) of the EGF receptor in which the tyrosines at Vol.
Y3F
protein
substitution
true negative
Our present studies using either EGF receptor C-terminal deletion mutants or point mutations (Tyr3 Phe) and our previous studies on antibody inhibition by P2-derived peptides suggest that Gln-Gln in combination with Asp-Glu-Glu forms a high-affinity complex with Ab P2 and that such complex formation is dependent on tyrosine phosphorylation.
Using Tyr3 Phe substitution mutants, we report here that phosphorylation of Tyr 992, 1068, and 1086 that are located between the tripeptides Asp-Glu-Glu and Tyr-Gln-Gln is highly critical in conformational change of the receptor as determined by Ab P2 binding; however, Tyr 1148 that is part of one of the tripeptides and Tyr 1173 play no role in the conformational change.
Because the phosphorylation patterns of the mutants used in our studies were not characterized previously, we considered the possibility that single Tyr3 Phe substitution at 992, 1068, or 1086 adversely affects the phosphorylation of the other two tyrosine 529 A.
Single Tyr3 Phe substitution at 992, 1068, or 1086 drastically reduces the binding of Ab P2 to the 32P-labeled EGF receptor.
As seen in Figure 5, b-d, when the peptides from the single Tyr3 Phe substitution were analyzed, all the peaks except the peak corresponding to the mutated tyrosine could be detected, suggesting that a single Tyr3 Phe substitution has no negative effect on the phosphorylation of the other tyrosine residues.
Thus, it will be of interest to investigate the effect of Tyr3 Phe substitution on the Vmax for autophosphorylation and for exogenous substrate phosphorylation.
Y1086F
protein
substitution
true negative
EGF receptor mutants used in Ab P2 binding studies Number of receptors per cell ( 105) 0.025 1.41 0.56 2.49 0.70 0.77 0.25 0.45 1.85 0.25 0.61 0.50 0.39 Cell type NIH3T3 CL17 MI31 MI32 MI33 MI34 MI35 MI36 MI37 MI38 MI40 MI41 Dc63 Receptor type Untransfected Wild-type Y1173F Y1148F Y(1173-1148)F Y(1173-1148-1068)F Y(1148-1068)F Y(1173-1068)F Y1068F Y1086F Y992F Y(1173-1148-1086-1068-992)F C-terminal deletion of 63 amino acids lacking tyrosine 1173 and 1148 Biosynthetic Labeling of the EGF Receptor Twenty hours after subculturing, cell monolayers were washed with methionine- and cysteine-free DMEM containing 2% dialyzed newborn calf serum and were preincubated in the same medium at 37C for 1 h.
Y1068F
protein
substitution
true negative
EGF receptor mutants used in Ab P2 binding studies Number of receptors per cell ( 105) 0.025 1.41 0.56 2.49 0.70 0.77 0.25 0.45 1.85 0.25 0.61 0.50 0.39 Cell type NIH3T3 CL17 MI31 MI32 MI33 MI34 MI35 MI36 MI37 MI38 MI40 MI41 Dc63 Receptor type Untransfected Wild-type Y1173F Y1148F Y(1173-1148)F Y(1173-1148-1068)F Y(1148-1068)F Y(1173-1068)F Y1068F Y1086F Y992F Y(1173-1148-1086-1068-992)F C-terminal deletion of 63 amino acids lacking tyrosine 1173 and 1148 Biosynthetic Labeling of the EGF Receptor Twenty hours after subculturing, cell monolayers were washed with methionine- and cysteine-free DMEM containing 2% dialyzed newborn calf serum and were preincubated in the same medium at 37C for 1 h.
Y992F
protein
substitution
true negative
EGF receptor mutants used in Ab P2 binding studies Number of receptors per cell ( 105) 0.025 1.41 0.56 2.49 0.70 0.77 0.25 0.45 1.85 0.25 0.61 0.50 0.39 Cell type NIH3T3 CL17 MI31 MI32 MI33 MI34 MI35 MI36 MI37 MI38 MI40 MI41 Dc63 Receptor type Untransfected Wild-type Y1173F Y1148F Y(1173-1148)F Y(1173-1148-1068)F Y(1148-1068)F Y(1173-1068)F Y1068F Y1086F Y992F Y(1173-1148-1086-1068-992)F C-terminal deletion of 63 amino acids lacking tyrosine 1173 and 1148 Biosynthetic Labeling of the EGF Receptor Twenty hours after subculturing, cell monolayers were washed with methionine- and cysteine-free DMEM containing 2% dialyzed newborn calf serum and were preincubated in the same medium at 37C for 1 h.
12000708
full text
V560D
protein
substitution
P10721
true positive
Mutations Detected in Incidental GISTs Case 1 2 3 4 5 6 7 8 9 10 11 12 13 Exon 11 Deletion KVVEEING 558565 R* Point mutation V559D* Deletion KPMYEVQWK 550558* Deletion D570 (homozygous)* Deletion NYVYIDPTQL 567576 KV* Point mutation V560D* Deletion QKPMYEVQWK 549558Q (homozygous)* Deletion KPMYEVQWK 550558* Deletion YIDPTQLPY 570578* Point mutation V559D* WTDHPLC WT* WT* DHPLC Exon 9 Exon 13 Exon 17 WTDHPLC WTDHPLC WTDHPLC WTDHPLC WTDHPLC Insertion AY 502503* WTDHPLC WTDHPLC WTDHPLC WTDHPLC WTDHPLC WTDHPLC WTDHPLC *Sequence confirmed.
V559D
protein
substitution
P10721
true positive
Mutations Detected in Incidental GISTs Case 1 2 3 4 5 6 7 8 9 10 11 12 13 Exon 11 Deletion KVVEEING 558565 R* Point mutation V559D* Deletion KPMYEVQWK 550558* Deletion D570 (homozygous)* Deletion NYVYIDPTQL 567576 KV* Point mutation V560D* Deletion QKPMYEVQWK 549558Q (homozygous)* Deletion KPMYEVQWK 550558* Deletion YIDPTQLPY 570578* Point mutation V559D* WTDHPLC WT* WT* DHPLC Exon 9 Exon 13 Exon 17 WTDHPLC WTDHPLC WTDHPLC WTDHPLC WTDHPLC Insertion AY 502503* WTDHPLC WTDHPLC WTDHPLC WTDHPLC WTDHPLC WTDHPLC WTDHPLC *Sequence confirmed.
10515870
full text
W282R
protein
substitution
true negative
EPO Equilibrium Binding Studies on Selected Clones of 32D Cells Containing Variant Forms of the EPO-R Cell Line kd (pmol/L) Receptor No./Cell R2 Value 32D.EPO-R:1-483 (wild-type) 32D.EPO-R:1-411 32D.EPO-R:1-373 32D.EPO-R:1-321 32D.EPO-R:1-252 32D.EPO-R:1-483(W282R) 32D.EPO-R:1-483(YF) 220 260 345 267 325 383 224 258 373 285 382 315 650 576 555 402 209 834 1280 1650 1413 1256 1554 1300 1158 1447 675 1055 1252 6550 6882 5897 1718 734 .95 .93 .95 .94 .93 .95 .96 .93 .94 .94 .95 .94 .95 .93 .94 .95 .93 To determine if the cytoplasmic domain of the EPO-R regulates EPO internalization, a series of cytoplasmic tail truncated EPO-R variants were constructed (Fig 1A).
The Initial Rate of EPO Internalization and Extent of Bound EPO Internalized for 32D Cell Lines Expressing EPO-R Variants EPO-R Variant Internalization Rate (%/min) EPO Internalized at 40 Minutes (% initially bound) 1-483 (wild-type) 1-411 1-373 1-321 1-252 1-483(W282R) 1-483(YF) 3.2 3.6 4.2 3.8 1.0 3.0 2.9 54 50 58 55 20 52 45 The initial rate of internalization was determined from the slope of the linear portion of the curve between 0 and 10 minutes.
In the case of the EGF-R, receptor kinase activation and tyrosine phosphorylation of the receptor and cellular substrates can regulate endocytosis of the receptor.13,19 The membrane proximal region of the EPO-R required for internalization of bound EPO contains the Box1 and Box2 motifs, which are the minimal region of the EPO-R required for mitogenic responses and JAK2 binding site.20,21 To determine whether EPO-induced JAK2 tyrosine kinase activation is required for internalization of bound EPO, we expressed a mitogenically inactive form of the EPO-R, EPO-R(W282R), in 32D cells (Fig 1B).
In addition, there was no change in the tyrosinephosphorylated protein profile of 32D.EPO-R(W282R) cells upon addition of EPO (Fig 6A), and neither the EPOR(W282R) (Fig 6B) nor JAK2 (Fig 6C) was tyrosine phosphorylated in response to EPO.
Finally, we were unable to detect any activation of JAK2 kinase activity in 32D.EPO-R(W282R) cells in response to EPO, confirming the results of Witthuhn et al8 (not shown).
Similarly, EPO-R(W282R), which also was not tyrosine phosphorylated in response to EPO, internalized receptor-bound EPO as well as cells containing wild-type EPO-R (Fig 7 and Table 2).
32D cells containing EPO-R(W282R) or EPO-R(YF) do not proliferate in response to EPO.
( ) MTT assay of 32D clones containing wild-type EPO-R; ( ) EPO-R(1-373); ( ) EPO-R(W282R); and ( ) EPO-R(YF).
Lanes 1 and 2, 32D.EPO-R(1-483), wild-type cells; lanes 3 and 4, 32D.EPO-R(1-373) cells; lanes 5 and 6, 32D.EPO-R(YF) cells; and lanes 7 and 8, 32D.EPO-R(W282R) cells.
The blot was then stripped and reprobed with antisera against the EPO-R (lower panel); arrowheads on the left indicate the position of wild-type (wt) EPO-R, EPO-R(W282R) and EPO-R(YF), IgG, and EPO-R(1-373), respectively.
Lanes 1 through 3, 32D.EPO-R(1-483) wt cells; lanes 4 through 6, 32D.EPO-R(1-373) cells; lanes 7 and 8, 32D.EPO-R(YF) cells; and lanes 9 and 10, 32D.EPO-R(W282R) cells.
Lanes 1 and 2, 32D.EPO-R(1-483) wt cells; lanes 3 and 4, 32D.EPO-R(1-373) cells; lanes 5 and 6, 32D.EPO-R(YF) cells; and lanes 7 and 8, 32D.EPO-R(W282R) cells.
( ) Wild-type EPO-R(1-483); ( ) EPO-R(W282R); and ( ) EPO-R(YF).
Secondly, nonfunctional EPO-Rs [eg, EPOR(YF) and EPO-R(W282R)] that should not induce expression of CIS internalize EPO as well as functional, wild-type EPO-R.
Downregulation of activated EGF-R is thought to be important in modulating cellular responses to ligand.27 Whether the intrinsic tyrosine kinase activity of the activated RTKs and subsequent tyrosine phosphorylation of sites in the cytoplasmic tail of RTKs are directly required for their internalization remains controversial.13 Recent results suggest that EGF-R kinase activity as well as tyrosine phosphorylation of the receptor cytoplasmic tail and other cytosolic substrates are indeed required for sequestration of the EGF-R into clathrin-coated pits and downmodulation of its signals.28,29 Other studies suggest that receptor tyrosine kinase activity is not required for targeting the EGF-R to lysosomes, but rather that tyrosine kinase activity amplifies the conformation changes induced by ligand binding by exposing or generating other internalization and lysosomal targeting sequences.30 We have shown that mitogenically inactive forms of the EPO-R [EPOR(W282R)] do not affect the internalization of bound EPO or of receptor downregulation.
We have shown that mitogenically inactive or attenuated EPO-Rs (eg, W282R, EPO-R:YF, and 1-321) do not exhibit any alteration in the rate or extent of receptor-bound EPO internalization as compared with cells containing functional EPO-Rs (eg, wt, 1-411, and 1-373).
Goldsmith for providing EPO-R(W282R) and EPO-R(YF) plasmids, respectively; Abbott Laboratories for purified EPO; Dr Sally York for helpful discussions; and Drs S.
11818458
full text
D1232V
protein
substitution
true negative
However, when MSP was included in cell cultures together with LPS or LPS plus IFN- , the percentages http://www.jleukbio.org Establishment of Raw264.7 cell lines expressing a constitutively active RON mutant Transfection of Raw264.7 cells with pcDNA3.1 vector containing a mutant RON cDNA with D1232V substitution was performed as described [25].
The addition of MSP also did not show a significantly protective effect on these cells, because D1232V is a constitutively active RON mutant.
To further demonstrate that RON activation protects macrophages from LPS-induced apoptosis, Raw264.7 cells transfected with a constitutively active RON mutant (D1232V) [26] were included in experiments.
However, the expression of the D1232V mutant rendered cells less sensitive to LPS-induced apoptosis, as evident by the Correlation of MSP-induced iNOS inhibition and cell survival in LPS-stimulated Raw264.7 cells Because LPS-induced NO is the principle molecule responsible for macrophage apoptosis, we sought to determine the effect of RON activation on LPS-induced iNOS expression to correlate the apoptotic effect of LPS.
10574949
full text
L321M
protein
substitution
true positive
P22607
Adding a third point mutation, L321M (H327R/S325H/L321M) significantly enhanced FGF2 binding.
H327R
protein
substitution
true positive
P22607
The H327R, S325H, or the double mutation (H327R/S325H) resulted in only a small increase in FGF2 binding (Fig.
Adding a third point mutation, L321M (H327R/S325H/L321M) significantly enhanced FGF2 binding.
S325H
protein
substitution
true positive
P22607
The H327R, S325H, or the double mutation (H327R/S325H) resulted in only a small increase in FGF2 binding (Fig.
Adding a third point mutation, L321M (H327R/S325H/L321M) significantly enhanced FGF2 binding.
10490604
full text
K273E
protein
substitution
true negative
pSX mLck Myc K273E (kinase dead) was made by subcloning the XbaI-EcoRI fragment of pGEM4Z mLck Myc K273E (generous gift from J.
pSX mLck Myc was made by replacing the SacII-EcoRI fragment of pSX mLck (67) with that from pGEM4Z mLck Myc K273E.
(C) Jurkat-TAg cells were transiently cotransfected with HA-70Z G306E or HA-70Z and either pSX SRa (vector), wt Fyn Myc, Fyn Myc K295R (kinase dead), wt Lck Myc, or Lck Myc K273E (kinase dead) and further treated as for panel A.
G306E
protein
substitution
true negative
pSX SR , pSX HA Cbl, and pSX HA 70Z (13) as well as the G306E and Y700F/Y731F/Y774F (here referred to as Y3F) mutant derivatives of pSX HA Cbl and pSX HA 70Z (63) were previously described.
pSC65 HA 70Z G306E was made by replacing the PshAI-BglII fragment from pSC65 HA Cbl with that from pSX HA 70Z G306E (63).
(A) Jurkat-TAg cells were transiently cotransfected with HA-70Z G306E or HA-70Z and either Myc epitope-tagged wt, K369R (kinase dead), or Y493F ZAP-70 constructs.
(C) Jurkat-TAg cells were transiently cotransfected with HA-70Z G306E or HA-70Z and either pSX SRa (vector), wt Fyn Myc, Fyn Myc K295R (kinase dead), wt Lck Myc, or Lck Myc K273E (kinase dead) and further treated as for panel A.
Thus, we cotransfected Jurkat-TAg cells with either 70Z Cbl or its inactive G306E mutant derivative together with empty vector, wt ZAP-70 or kinase-dead ZAP-70 (ZAP-70 K369R).
Consistent with recent findings (63, 70), overexpression of 70Z Cbl but not its G306E mutant derivative led to constitutive upregulation of NFAT in unstimulated Jurkat T cells (Fig.
We therefore used the 70Z G306E mutant construct as a negative control in subsequent experiments.
Coexpression of wt ZAP-70 but not kinase-dead ZAP-70 with the inactive 70Z Cbl G306E mutant led to a two- to threefold increase over basal NFAT activation in Jurkat T cells (Fig.
Coexpression of wt but not kinase-dead forms of Fyn and Lck with 70Z G306E constitutively upregulated NFAT activity in unstimulated Jurkat T cells (Fig.
To distinguish between these two possibilities, coexpression studies were performed with 70Z Cbl or the inactivated 70Z Cbl G306E mutant, as a control, together with various ZAP-70 constructs.
Coexpression of the 70Z G306E construct with ZAP-70 Y292F resulted in constitutive NFAT activation that was increased relative to coexpression of 70Z Cbl G306E with wt ZAP-70 or the vector control (Fig.
4A; compare columns 4 and 5) even though NFAT activation induced by wt ZAP-70 (in the presence of 70Z G306E) was less than that induced by ZAP-70 Y292F (Fig.
4A, column 6) relative to the expression of 70Z G306E with ZAP-70 Y292F (Fig.
To test this idea, Jurkat-TAg cells were cotransfected with ZAP-70 and either the inactivated 70Z G306E (as a negative control), 70Z, wt Cbl, or the Cbl G306E mutant.
1 and 4, overexpression of ZAP-70 in the presence of 70Z G306E resulted in a two- to threefold enhancement of NFAT activation relative to expression of the inactive 70Z G306E alone.
Moreover, Cbl-mediated inhibition of ZAP-70-induced NFAT activation was blocked by the Cbl G306E mutation (Fig.
Y869F
protein
substitution
true negative
Y674F/ Y700F/Y731F/Y735F/Y774F/Y869F/Y871F (here referred to as Y7F) mutant derivatives of pSX HA Cbl and pSX HA 70Z were made by replacing the 3 SacII-KpnI fragment of pSX HA Cbl Y5F or pSX HA 70Z Y5F with that from the mutagenized pSX HA Cbl Y869F/Y871F construct.
Y735F
protein
substitution
true negative
Y674F/Y700F/ Y731F/Y735F/Y774F (here referred to as Y5F) mutant derivatives of pSX HA Cbl and pSX HA 70Z were made by replacing the BglII-NotI fragment of pSX HA Cbl or pSX HA 70Z with that from pAlter Max HA Cbl Y5F constructs (gifts from A.
Y674F/ Y700F/Y731F/Y735F/Y774F/Y869F/Y871F (here referred to as Y7F) mutant derivatives of pSX HA Cbl and pSX HA 70Z were made by replacing the 3 SacII-KpnI fragment of pSX HA Cbl Y5F or pSX HA 70Z Y5F with that from the mutagenized pSX HA Cbl Y869F/Y871F construct.
Y3F
protein
substitution
true negative
pSX SR , pSX HA Cbl, and pSX HA 70Z (13) as well as the G306E and Y700F/Y731F/Y774F (here referred to as Y3F) mutant derivatives of pSX HA Cbl and pSX HA 70Z (63) were previously described.
pSC65, pSC65 HA Cbl, and pSC65 HA 70Z (43) Y3F mutant derivatives of pSC 65 HA Cbl and pSC65 HA 70Z were described previously (63).
Whether the other tyrosines (Tyr674, Tyr735, Tyr869, and Tyr871) undergo phosphorylation is not known, but we previously reported residual tyrosine phosphorylation of wt and 70Z Cbl Y3F triple mutants under both basal and OKT3-stimulated conditions (63).
We previously reported that Tyr700, Tyr731, and Tyr774 represent the major phosphorylation sites in c-Cbl but that wt and 70Z Cbl Y3F triple tyrosine mutants still display residual basal and activation-induced tyrosine phosphorylation (63).
observed in the wt and 70Z Cbl Y3F mutants was virtually eliminated in the wt and 70Z Cbl Y7F mutants (Fig.
Compared to the Y3F and Y7F constructs, the wt and 70Z Cbl Y5F constructs displayed intermediate levels of tyrosine phosphorylation (data not shown).
Importantly, and in agreement with the data obtained with the 1-655 truncation constructs, the Y7F and, to a lesser extent, Y5F and Y3F mutations not only blocked but even enhanced NFAT activation by 70Z Cbl (Fig.
Although we initially reported that the 70Z Cbl Y3F mutation showed a relatively small but not consistently observed increase in basal NFAT activation (63), additional experiments revealed that the 70Z Y3F mutant showed an approximately 50% increase in NFAT activation relative to 70Z Cbl in four of six experiments.
Not a point mutation
Y7F
protein
substitution
true negative
Y674F/ Y700F/Y731F/Y735F/Y774F/Y869F/Y871F (here referred to as Y7F) mutant derivatives of pSX HA Cbl and pSX HA 70Z were made by replacing the 3 SacII-KpnI fragment of pSX HA Cbl Y5F or pSX HA 70Z Y5F with that from the mutagenized pSX HA Cbl Y869F/Y871F construct.
The Y5F and Y7F mutant derivatives of pSC65 HA Cbl and pSC65 HA 70Z were made by replacing the 3 BglII-KpnI fragment of pSC65 HA Cbl or 70Z with that from the respective pSX HA Cbl mutant derivatives.
We therefore tested whether mutation of two additional tyrosines (Y674F and Y735F) in the Y5F constructs or mutation of all seven tyrosines (including Y869F and Y871F) in the Y7F constructs eliminated Cbl tyrosine phosphorylation.
observed in the wt and 70Z Cbl Y3F mutants was virtually eliminated in the wt and 70Z Cbl Y7F mutants (Fig.
Scanning densitometry revealed that the level of tyrosine phosphorylation in the wt and 70Z Cbl Y7F mutants was less than 2% of that observed in the unmutated constructs (data not shown).
Compared to the Y3F and Y7F constructs, the wt and 70Z Cbl Y5F constructs displayed intermediate levels of tyrosine phosphorylation (data not shown).
Importantly, and in agreement with the data obtained with the 1-655 truncation constructs, the Y7F and, to a lesser extent, Y5F and Y3F mutations not only blocked but even enhanced NFAT activation by 70Z Cbl (Fig.
Thus, inactivation of this second negative regulatory function in the 70Z Y7F or 70Z 655 truncation mutants leads to enhanced NFAT activation relative to the 70Z Cbl oncoprotein.
Not a point mutation
Y871F
protein
substitution
true negative
Y674F/ Y700F/Y731F/Y735F/Y774F/Y869F/Y871F (here referred to as Y7F) mutant derivatives of pSX HA Cbl and pSX HA 70Z were made by replacing the 3 SacII-KpnI fragment of pSX HA Cbl Y5F or pSX HA 70Z Y5F with that from the mutagenized pSX HA Cbl Y869F/Y871F construct.
We therefore tested whether mutation of two additional tyrosines (Y674F and Y735F) in the Y5F constructs or mutation of all seven tyrosines (including Y869F and Y871F) in the Y7F constructs eliminated Cbl tyrosine phosphorylation.
Y774F
protein
substitution
true negative
pSX SR , pSX HA Cbl, and pSX HA 70Z (13) as well as the G306E and Y700F/Y731F/Y774F (here referred to as Y3F) mutant derivatives of pSX HA Cbl and pSX HA 70Z (63) were previously described.
Y674F/Y700F/ Y731F/Y735F/Y774F (here referred to as Y5F) mutant derivatives of pSX HA Cbl and pSX HA 70Z were made by replacing the BglII-NotI fragment of pSX HA Cbl or pSX HA 70Z with that from pAlter Max HA Cbl Y5F constructs (gifts from A.
Y674F/ Y700F/Y731F/Y735F/Y774F/Y869F/Y871F (here referred to as Y7F) mutant derivatives of pSX HA Cbl and pSX HA 70Z were made by replacing the 3 SacII-KpnI fragment of pSX HA Cbl Y5F or pSX HA 70Z Y5F with that from the mutagenized pSX HA Cbl Y869F/Y871F construct.
Y700F
protein
substitution
true negative
pSX SR , pSX HA Cbl, and pSX HA 70Z (13) as well as the G306E and Y700F/Y731F/Y774F (here referred to as Y3F) mutant derivatives of pSX HA Cbl and pSX HA 70Z (63) were previously described.
Y674F/Y700F/ Y731F/Y735F/Y774F (here referred to as Y5F) mutant derivatives of pSX HA Cbl and pSX HA 70Z were made by replacing the BglII-NotI fragment of pSX HA Cbl or pSX HA 70Z with that from pAlter Max HA Cbl Y5F constructs (gifts from A.
Y674F/ Y700F/Y731F/Y735F/Y774F/Y869F/Y871F (here referred to as Y7F) mutant derivatives of pSX HA Cbl and pSX HA 70Z were made by replacing the 3 SacII-KpnI fragment of pSX HA Cbl Y5F or pSX HA 70Z Y5F with that from the mutagenized pSX HA Cbl Y869F/Y871F construct.
K369R
protein
substitution
true negative
pSX ZAP-70 Myc and its Y292F, K369R (kinase dead), Y492F, and Y493F mutant derivatives were described previously (67).
(A) Jurkat-TAg cells were transiently cotransfected with HA-70Z G306E or HA-70Z and either Myc epitope-tagged wt, K369R (kinase dead), or Y493F ZAP-70 constructs.
Thus, we cotransfected Jurkat-TAg cells with either 70Z Cbl or its inactive G306E mutant derivative together with empty vector, wt ZAP-70 or kinase-dead ZAP-70 (ZAP-70 K369R).
Y493F
protein
substitution
true negative
pSX ZAP-70 Myc and its Y292F, K369R (kinase dead), Y492F, and Y493F mutant derivatives were described previously (67).
(A) Jurkat-TAg cells were transiently cotransfected with HA-70Z G306E or HA-70Z and either Myc epitope-tagged wt, K369R (kinase dead), or Y493F ZAP-70 constructs.
To determine whether activation of ZAP-70 by Src family PTKs was required for 70Z Cbl-mediated NFAT activation, the effect of the ZAP-70 Y493F mutation on 70Z Cblmediated NFAT activation was also evaluated.
The ZAP-70 Y493F mutation blocked 70Z Cbl-mediated NFAT activation as efficiently as kinase-dead ZAP-70 (Fig.
The specificity of the Y493F-mediated blockade is indicated by the fact that the ZAP-70 Y492F mutation, which enhances NFAT activation in unstimulated Jurkat T cells (reference 71 and Fig.
Y5F
protein
substitution
true negative
Y674F/Y700F/ Y731F/Y735F/Y774F (here referred to as Y5F) mutant derivatives of pSX HA Cbl and pSX HA 70Z were made by replacing the BglII-NotI fragment of pSX HA Cbl or pSX HA 70Z with that from pAlter Max HA Cbl Y5F constructs (gifts from A.
Y674F/ Y700F/Y731F/Y735F/Y774F/Y869F/Y871F (here referred to as Y7F) mutant derivatives of pSX HA Cbl and pSX HA 70Z were made by replacing the 3 SacII-KpnI fragment of pSX HA Cbl Y5F or pSX HA 70Z Y5F with that from the mutagenized pSX HA Cbl Y869F/Y871F construct.
The Y5F and Y7F mutant derivatives of pSC65 HA Cbl and pSC65 HA 70Z were made by replacing the 3 BglII-KpnI fragment of pSC65 HA Cbl or 70Z with that from the respective pSX HA Cbl mutant derivatives.
We therefore tested whether mutation of two additional tyrosines (Y674F and Y735F) in the Y5F constructs or mutation of all seven tyrosines (including Y869F and Y871F) in the Y7F constructs eliminated Cbl tyrosine phosphorylation.
Compared to the Y3F and Y7F constructs, the wt and 70Z Cbl Y5F constructs displayed intermediate levels of tyrosine phosphorylation (data not shown).
Importantly, and in agreement with the data obtained with the 1-655 truncation constructs, the Y7F and, to a lesser extent, Y5F and Y3F mutations not only blocked but even enhanced NFAT activation by 70Z Cbl (Fig.
Not a point mutation
K295R
protein
substitution
true negative
pXS mFyn Myc and pXS mFyn Myc K295R (kinase dead) were described previously (16).
(C) Jurkat-TAg cells were transiently cotransfected with HA-70Z G306E or HA-70Z and either pSX SRa (vector), wt Fyn Myc, Fyn Myc K295R (kinase dead), wt Lck Myc, or Lck Myc K273E (kinase dead) and further treated as for panel A.
Y292F
protein
substitution
true negative
Further, 70Z Cbl does not cooperate with ZAP-70 Y292F to upregulate NFAT, indicating that 70Z Cbl and ZAP-70 do not activate parallel signalling pathways.
Interestingly, the ZAP-70 Y292F mutation does not affect the interaction of ZAP-70 with phosphorylated ITAMs in the receptor, nor does it affect ZAP-70 tyrosine phosphorylation, ZAP-70 kinase activity, or the ability of ZAP-70 to reconstitute B-cell receptor-mediated Ca2 mobilization in Syk-deficient DT40 cells (24, 71).
Moreover, transient overexpression of 70Z Cbl in Jurkat T cells leads to constitutive upregulation of NFAT and AP-1 transcriptional activity (29, 63), similar to what has been observed upon overexpression of ZAP-70 Y292F (24, 71).
pSX ZAP-70 Myc and its Y292F, K369R (kinase dead), Y492F, and Y493F mutant derivatives were described previously (67).
To distinguish between these models, we tested whether 70Z Cbl-mediated NFAT activation depends on (i) tyrosine phosphorylation of 70Z Cbl and (ii) synergism with the ZAP-70 Y292F mutation.
This model predicts that overexpression of activated ZAP-70 Y292F does not cooperate with 70Z Cbl overexpression to upregulate NFAT compared to the overexpression of either construct alone.
Coexpression of the 70Z G306E construct with ZAP-70 Y292F resulted in constitutive NFAT activation that was increased relative to coexpression of 70Z Cbl G306E with wt ZAP-70 or the vector control (Fig.
This is consistent with previously published data demonstrating that overexpression of ZAP-70 Y292F alone upregulates NFAT activity (reference 71 and Fig.
Importantly, coexpression of ZAP-70 Y292F with 70Z Cbl did not lead to enhanced NFAT activation relative to the expression of either construct alone (Fig.
4A; compare columns 4 and 5) even though NFAT activation induced by wt ZAP-70 (in the presence of 70Z G306E) was less than that induced by ZAP-70 Y292F (Fig.
It should be noted that the slightly decreased NFAT activation observed upon coexpression of 70Z Cbl with ZAP-70 Y292F (Fig.
4A, column 6) relative to the expression of 70Z G306E with ZAP-70 Y292F (Fig.
In contrast to cells overexpressing wt ZAP70, the great majority of TCR-associated ZAP-70 proteins in cells overexpressing the ZAP-70 Y292F mutant will carry the VOL.
70Z Cbl does not synergize with the ZAP-70 Y292F mutant to upregulate NFAT.
Y292F mutation.
This will essentially block the effect of 70Z Cbl, leading to lack of cooperativity between 70Z Cbl and ZAP-70 Y292F in the induction of NFAT.
It should be noted that the upregulation of NFAT by overexpression of ZAP-70 Y292F depends on intact SH2 and kinase domains (71), indicating that the ZAP-70 Y292F protein must be recruited to phosphorylated ITAMs and activated by Src family kinases to upregulate NFAT activity.
4 provide evidence that 70Z Cbl functionally interacts with the ZAP-70 pY292 phosphotyrosine residue in vivo and demonstrate that the 70Z Cbl oncoprotein does not synergize with the ZAP-70 Y292F mutant to upregulate NFAT.
Similarly, overexpression of ZAP-70 Y292F, which disrupts the interaction of ZAP-70 with the Cbl PTB domain in vitro and in vivo (31, 32), does not detectably affect ZAP-70 tyrosine phosphorylation, ZAP-70 kinase activity, recruitment of ZAP-70 to phosphorylated ITAMs, or the ability of ZAP-70 to reconsti- tute Ca2 mobilization in Syk-deficient DT40 cells compared to wt ZAP-70 (24, 71).
Although we cannot exclude the possibility that the effect of ZAP-70 Y292F or 70Z Cbl overexpression on the specific enzymatic activity of ZAP-70 is too subtle to be detected by biochemical methods, these observations suggest that the effect of ZAP-70 Y292F and 70Z Cbl overexpression on NFAT activation results primarily from an effect on the expression levels of the activated ZAP-70 enzyme and/or the duration of ZAP-70 activation.
Y731F
protein
substitution
true negative
pSX SR , pSX HA Cbl, and pSX HA 70Z (13) as well as the G306E and Y700F/Y731F/Y774F (here referred to as Y3F) mutant derivatives of pSX HA Cbl and pSX HA 70Z (63) were previously described.
Y674F/Y700F/ Y731F/Y735F/Y774F (here referred to as Y5F) mutant derivatives of pSX HA Cbl and pSX HA 70Z were made by replacing the BglII-NotI fragment of pSX HA Cbl or pSX HA 70Z with that from pAlter Max HA Cbl Y5F constructs (gifts from A.
Y674F/ Y700F/Y731F/Y735F/Y774F/Y869F/Y871F (here referred to as Y7F) mutant derivatives of pSX HA Cbl and pSX HA 70Z were made by replacing the 3 SacII-KpnI fragment of pSX HA Cbl Y5F or pSX HA 70Z Y5F with that from the mutagenized pSX HA Cbl Y869F/Y871F construct.
Y674F
protein
substitution
true negative
Y674F/Y700F/ Y731F/Y735F/Y774F (here referred to as Y5F) mutant derivatives of pSX HA Cbl and pSX HA 70Z were made by replacing the BglII-NotI fragment of pSX HA Cbl or pSX HA 70Z with that from pAlter Max HA Cbl Y5F constructs (gifts from A.
Y674F/ Y700F/Y731F/Y735F/Y774F/Y869F/Y871F (here referred to as Y7F) mutant derivatives of pSX HA Cbl and pSX HA 70Z were made by replacing the 3 SacII-KpnI fragment of pSX HA Cbl Y5F or pSX HA 70Z Y5F with that from the mutagenized pSX HA Cbl Y869F/Y871F construct.
G315E
protein
substitution
true negative
elegans indicate that the G315E loss-of-function allele of Sli-1 rescues vulval development induced by a reduction-offunction allele of the Let23 epidermal growth factor receptor (EGFR) homolog (69).
Y492F
protein
substitution
true negative
pSX ZAP-70 Myc and its Y292F, K369R (kinase dead), Y492F, and Y493F mutant derivatives were described previously (67).
The specificity of the Y493F-mediated blockade is indicated by the fact that the ZAP-70 Y492F mutation, which enhances NFAT activation in unstimulated Jurkat T cells (reference 71 and Fig.
In striking contrast, the Y492F mutation enhances the specific enzymatic activity of ZAP-70 (references 24 and 67 and data not shown).
10463620
full text
12754375
full text
W557R
protein
substitution
P10721
true positive
For comparison, histological sections of neoplastic and hyperplastic lesions of a human patient with familial GIST, resulting from a missense mutation, Kit-W557R (G.S., unpublished work), are shown (Fig.
8662889
full text
Y1349F
protein
substitution
true positive
P08581
Mutant Y1356F runs ahead of mutant Y1349F because Tyr1356 is more heavily phosphorylated in vitro than Tyr1349 (8).
Similarly active is a Tyr 3 Phe mutant lacking, in addition to Tyr1349 and Tyr1356, also the most carboxyl-terminal tyrosine residue (Y1349F/Y1356F/Y1365F).
DNA Foci/10 g DNA Relative transforming activity % of TPR-MET 14121 Vector TPR-MET TPR-MET TPR-MET TPR-MET TPR-MET TPR-MET TPR-MET TPR-MET 0 Y1349F Y1356F Y1365F Y1349F/Y1356F N1358H H1351N Lys 322 202 17 346 0 35 466 0 2 50 30 17 3 40 0 100 62 5 107 0 11 145 0 FIG.
Cells expressing TrkMet mutants Y1349F and Y1365F were also induced to scatter by NGF, in a manner indistinguishable from wild type (not shown).
A completely amotile phenotype was apparent only in MDCK cells expressing the Trk-Met double mutant Y1349F/Y1356F (as indicated on Fig.
14122 Ras Requirement in Met-mediated Growth and Motility the chimera Y1349F/Y1356F.
Y1365F
protein
substitution
true positive
P08581
Similarly active is a Tyr 3 Phe mutant lacking, in addition to Tyr1349 and Tyr1356, also the most carboxyl-terminal tyrosine residue (Y1349F/Y1356F/Y1365F).
DNA Foci/10 g DNA Relative transforming activity % of TPR-MET 14121 Vector TPR-MET TPR-MET TPR-MET TPR-MET TPR-MET TPR-MET TPR-MET TPR-MET 0 Y1349F Y1356F Y1365F Y1349F/Y1356F N1358H H1351N Lys 322 202 17 346 0 35 466 0 2 50 30 17 3 40 0 100 62 5 107 0 11 145 0 FIG.
Cells expressing TrkMet mutants Y1349F and Y1365F were also induced to scatter by NGF, in a manner indistinguishable from wild type (not shown).
In the particular experiment shown NGF treatment of MDCK cells expressing mutant Y1365F caused only a partial MAP kinase shift.
H1351N
protein
substitution
true positive
P08581
RESULTS Met kinase Activity Is Unaffected by Carboxyl-terminal Point Mutations--To abrogate Grb2 binding, asparagine 1358 (which confers Grb2 specificity to phosphotyrosine 1356) was converted into histidine (mutant N1358H), while to enhance Grb2 binding, histidine 1351 was converted into asparagine (mutant H1351N).
Mutant H1351N and N1358H migrate exactly like wild type Tpr-Met, indicating that the Asn 7 His mutations in 2 do not affect the level of phosphorylation of the upstream tyrosine residues.
DNA Foci/10 g DNA Relative transforming activity % of TPR-MET 14121 Vector TPR-MET TPR-MET TPR-MET TPR-MET TPR-MET TPR-MET TPR-MET TPR-MET 0 Y1349F Y1356F Y1365F Y1349F/Y1356F N1358H H1351N Lys 322 202 17 346 0 35 466 0 2 50 30 17 3 40 0 100 62 5 107 0 11 145 0 FIG.
Similar experiments carried out with the H1351N Tpr-Met mutant gave the same results as wild type Tpr-Met (not shown), indicating that the presence of the asparagine in 2 does not prevent all other effectors from binding to Tyr1349.
The increase in the number of foci brought about by the introduction of a second Grb2 binding site (mutant H1351N) shows that the Tpr-Met transforming potential can be further enhanced by recruitment of additional Grb2/SOS complex.
Trk-Met mutants N1358H and H1351N are equally capable of going beyond the "spreading" stage and are able to elicit a full scatter response, indicating that also the third signal mentioned above is passed on regardless of the residue in position 2 of either tyrosine.
N1358H
protein
substitution
true positive
P08581
GST-fusion proteins of the SH2 domains indicated (C-SH2 domain of p85, N-SH2 domain of phospholipase-C , full-size Grb2 and Shc) were immobilized (approximately 500 ng/point) on glutathione-Sepharose beads and incubated with lysates from COS-1 cells transiently expressing wild type Tpr-Met or the N1358H mutant.
RESULTS Met kinase Activity Is Unaffected by Carboxyl-terminal Point Mutations--To abrogate Grb2 binding, asparagine 1358 (which confers Grb2 specificity to phosphotyrosine 1356) was converted into histidine (mutant N1358H), while to enhance Grb2 binding, histidine 1351 was converted into asparagine (mutant H1351N).
Mutant H1351N and N1358H migrate exactly like wild type Tpr-Met, indicating that the Asn 7 His mutations in 2 do not affect the level of phosphorylation of the upstream tyrosine residues.
Grb2 Binding Is Selectively Abrogated by the Asn 3 His Mutation in Position 1358 --The ability of the N1358H Tpr-Met mutant to bind downstream effectors was tested in in vitro association experiments.
DNA Foci/10 g DNA Relative transforming activity % of TPR-MET 14121 Vector TPR-MET TPR-MET TPR-MET TPR-MET TPR-MET TPR-MET TPR-MET TPR-MET 0 Y1349F Y1356F Y1365F Y1349F/Y1356F N1358H H1351N Lys 322 202 17 346 0 35 466 0 2 50 30 17 3 40 0 100 62 5 107 0 11 145 0 FIG.
phospholipase-C , Grb2, and Shc (produced as GST fusion proteins) were used to precipitate wild type and the N1358H Tpr-Met mutant from lysates of transfected COS-1 cells.
Surprisingly, mutation N1358H (which uncouples Grb2 from the chimeric receptor and is as drastic as Y1356F on transformation), did not interfere at all with motility.
These results indicate that in mutant N1358H phosphorylation of Tyr1356 allows other effectors to pass enough signal to cause a MAP kinase shift.
5 shows that NGF induced a MAP kinase shift in all clones capable of motility, included those expressing the Trk-Met chimera N1358H.
uate their ability to activate the Ras kinase cascade, both Tpr-Met mutants N1358H and Y1356F were 50% as efficient as wild type.2 This level of residual signal is evidently not adequate for transformation.
Surprisingly (considering their similar inhibitory effect on transformation), the mutation which specifically uncouples Grb2 from Tyr1356 in the Met receptor (N1358H) was permissive for a bona fide scatter response in MDCK cells, while the mutation involving the loss of this tyrosine (Tyr1356) did interfere with motility.
Furthermore, following NGF stimulation, the N1358H Trk-Met chimera induced a MAP kinase shift equivalent to that caused by wild type Trk-Met, while the Y1356F Trk-Met chimera induced only a partial MAP kinase shift.
This suggests that when the N1358H mutation is present within the context of a receptor in MDCK cells, Tyr1356 mediates recruitment to the membrane of other effectors (i.e.
Our stable clones do not allow us to evaluate the proliferative potential of the N1358H chimeric receptor, since HGF/SF is not a mitogen for MDCK cells.
Trk-Met mutants N1358H and H1351N are equally capable of going beyond the "spreading" stage and are able to elicit a full scatter response, indicating that also the third signal mentioned above is passed on regardless of the residue in position 2 of either tyrosine.
Mutant N1358H, which is likely to be impaired in mediating growth but is competent in transducing motility, seems particularly interesting in terms of its possible application to the study of the HGF/SF-Met pair in vivo.
Y1356F
protein
substitution
true positive
P08581
Mutant Y1356F runs ahead of mutant Y1349F because Tyr1356 is more heavily phosphorylated in vitro than Tyr1349 (8).
Similarly active is a Tyr 3 Phe mutant lacking, in addition to Tyr1349 and Tyr1356, also the most carboxyl-terminal tyrosine residue (Y1349F/Y1356F/Y1365F).
DNA Foci/10 g DNA Relative transforming activity % of TPR-MET 14121 Vector TPR-MET TPR-MET TPR-MET TPR-MET TPR-MET TPR-MET TPR-MET TPR-MET 0 Y1349F Y1356F Y1365F Y1349F/Y1356F N1358H H1351N Lys 322 202 17 346 0 35 466 0 2 50 30 17 3 40 0 100 62 5 107 0 11 145 0 FIG.
Table I shows that selective uncoupling of Grb2 from Tpr-Met drastically lowers its transforming efficiency, bringing it close to the level of the Y1356F mutant.
Cells expressing mutant Y1356F (which is drastically impaired in its ability to transform cells) when treated with NGF did not fully scatter, although they underwent morphological changes.
A completely amotile phenotype was apparent only in MDCK cells expressing the Trk-Met double mutant Y1349F/Y1356F (as indicated on Fig.
Surprisingly, mutation N1358H (which uncouples Grb2 from the chimeric receptor and is as drastic as Y1356F on transformation), did not interfere at all with motility.
14122 Ras Requirement in Met-mediated Growth and Motility the chimera Y1349F/Y1356F.
MAP kinase was only partially shifted in cells expressing the chimera Y1356F and not shifted at all in the amotile clones expressing FIG.
uate their ability to activate the Ras kinase cascade, both Tpr-Met mutants N1358H and Y1356F were 50% as efficient as wild type.2 This level of residual signal is evidently not adequate for transformation.
Furthermore, following NGF stimulation, the N1358H Trk-Met chimera induced a MAP kinase shift equivalent to that caused by wild type Trk-Met, while the Y1356F Trk-Met chimera induced only a partial MAP kinase shift.
11689702
full text
V677A
protein
substitution
true negative
(p70S6K; C-18) from Santa Cruz Biotechnology; anti-SHC (06203), anti-phospho-STAT1 (Y701), anti-phospho-STAT5 (Tyr694), and antiphosphotyrosine (4G10); anti-phospho-AKT/PKB (Ser473) and anti-AKT (PhosphoPlus AKT [Ser473] antibody kit; New England Biolabs); anti-BCL2 and anti-STAT3 (Transduction Laboratories); anti-P-MAPK (V677A; Promega); anti-phosphoSTAT3 (Tyr705) and anti-phospho-p70S6K (Thr389) (Cell Signaling Technology); and anti-STAT5B (R&D systems).
V701A
protein
substitution
true negative
P16056
(p70S6K; C-18) from Santa Cruz Biotechnology; anti-SHC (06203), anti-phospho-STAT1 (Y701), anti-phospho-STAT5 (Tyr694), and antiphosphotyrosine (4G10); anti-phospho-AKT/PKB (Ser473) and anti-AKT (PhosphoPlus AKT [Ser473] antibody kit; New England Biolabs); anti-BCL2 and anti-STAT3 (Transduction Laboratories); anti-P-MAPK (V677A; Promega); anti-phosphoSTAT3 (Tyr705) and anti-phospho-p70S6K (Thr389) (Cell Signaling Technology); and anti-STAT5B (R&D systems).
10512882
full text
K454R
protein
substitution
true positive
Q05397
A catalytically inactive variant of FAK, FAKK454R, has been described (Hildebrand et al., 1993).
A double mutant, FAK397F/K454R, was also created by ligating a fragment of FAK containing the Y397F point mutation (nucleotide 1 to 1381) to a fragment of FAK containing the K454R mutation (nucleotides 1382 to 3248) using the Bsp EI site at nucleotide 1381.
The mutants analyzed included FAK397F, FAK576F/577F, a variant with phenylalanine substituted for two regulatory sites of tyrosine phosphorylation that are substrates for pp60src, and FAK454R, which is catalytically defective.
Despite its catalytic inactivity, FAK454R was tyrosine-phosphorylated when expressed in CE cells, albeit at reduced levels relative to wild-type FAK (Figure 5B, lane 7).
A double mutant, FAK454R/397F, with point mutations destroying both catalytic activity and the autophosphorylation site, contained no phosphotyrosine when expressed alone; when coexpressed with pp60c-src or p59fyn, it did not exhibit enhanced tyrosine phosphorylation (Figure 5A, lanes 11 and 12; 5B, lanes 9 and 10).
The low level of phosphotyrosine detected upon coexpression of pp60c-src and p59fyn with FAK454R/397F is likely due to the presence of wild-type endogenous FAK in the immune complex.
As anticipated, both FAK397F and FAK454R/397F failed to associate with pp60c-src and p59fyn (Figure 6, A and B).
FAK454R also induced tyrosine phosphorylation of paxillin when coexpressed with p59fyn or pp60c-src (Figure 7, B and C, lanes 8 and 7, respectively).
FAK576F/577F and FAK454R could also be coimmunoprecipitated with pp60c-src and p59fyn (Figure 6, A and B; our unpubVol.
(A) FAK was analyzed from CE cells (lane 1) or CE cells expressing RCAS B src (lane 2), RCAS A FAK (lane 3), RCAS A FAK RCAS B src (lane 4), RCAS A FAK397F (lane 5), RCAS A FAK397F RCAS B src (lane 6), RCAS A FAK576/577FF (lane 7), RCAS A FAK576/577FF RCAS B src (lane 8), RCAS A FAK454R (lane 9), RCAS A FAK454R RCAS B src (lane 10), RCAS A FAK454R/397F (lane 11), or RCAS A FAK454R/397F RCAS B src (lane 12).
(B) FAK was analyzed from CE cells (lane 1) or CE cells expressing RCAS B fyn (lane 2), RCAS A FAK (lane 3), RCAS A FAK RCAS B fyn (lane 4), RCAS A FAK397F (lane 5), RCAS A FAK397F RCAS B fyn (lane 6), RCAS A FAK454R (lane 7), RCAS A FAK454R RCAS B fyn (lane 8), RCAS A FAK454R/397F (lane 9), or RCAS A FAK454R/397F RCAS B fyn (lane 10).
(C) Twenty-five micrograms of whole-cell lysate from CE cells (lane 1) or CE cells expressing RCAS B src (lane 2), RCAS A FAK RCAS B src (lane 3), RCAS A FAK397F RCAS B src (lane 4), RCAS A FAK576/577FF RCAS B src (lane 5), RCAS A FAK454R RCAS B src (lane 6), or RCAS A FAK454R/397F RCAS B src (lane 7) was Western-blotted with a src antibody.
(D) Twenty-five micrograms of whole-cell lysate from CE cells (lane 1) or CE cells expressing RCAS B fyn (lane 2), RCAS A FAK RCAS B fyn (lane 3), RCAS A FAK397F RCAS B fyn (lane 4), RCAS A FAK454R RCAS B fyn (lane 5), or RCAS A FAK454R/397F RCAS B fyn (lane 6) was Westernblotted with an fyn antibody.
(A) Src immune complexes from lysates of CE cells (lane 1) or CE cells expressing RCAS B src (lane 2), RCAS A FAK RCAS B src (lane 3), RCAS A FAK397F RCAS B src (lane 4), RCAS A FAK576/577FF RCAS B src (lane 5), RCAS A FAK454R RCAS B src (lane 6), or RCAS A FAK454R/397F RCAS B src (lane 7) were analyzed.
(B) Fyn immune complexes from lysates of CE cells expressing RCAS B fyn (lane 1), RCAS A FAK RCAS B fyn (lane 2), RCAS A FAK397F RCAS B fyn (lane 3), RCAS A FAK454R RCAS B fyn (lane 4), or RCAS A FAK454R/397F RCAS B fyn (lane 5) were analyzed.
(B) Paxillin was immunoprecipitated from lysates of CE cells (lane 1) or CE cells expressing RCAS B fyn (lane 2), RCAS A FAK (lane 3), RCAS A FAK RCAS B fyn (lane 4), RCAS A FAK397F (lane 5), RCAS A FAK397F RCAS B fyn (lane 6), RCAS A FAK454R (lane 7), RCAS A FAK454R RCAS 454R/397F B fyn (lane 8), RCAS A FAK (lane 9), or RCAS A FAK454R/397F RCAS B fyn (lane 10).
(C) Paxillin was immunoprecipitated from lysates of CE cells (lane 1) or CE cells expressing RCAS A FAK (lane 2), RCAS B Src (lane 3), RCAS A FAK RCAS B Src (lane 4), RCAS A FAK397F RCAS B Src (lane 5), RCAS A FAK576/577FF RCAS B fyn (lane 6), or RCAS A FAK454R RCAS B fyn (lane 7).
K576F
protein
substitution
true negative
The mutants analyzed included FAK397F, FAK576F/577F, a variant with phenylalanine substituted for two regulatory sites of tyrosine phosphorylation that are substrates for pp60src, and FAK454R, which is catalytically defective.
The phosphotyrosine content of FAK576F/577F was increased in cells coexpressing pp60c-src and p59fyn (Figure 5A, lanes 7 and 8; our 3494 unpublished results).
FAK576F/577F induced tyrosine phosphorylation of paxillin when coexpressed with pp60c-src or p59fyn, indicating that phosphorylation of these regulatory sites is not essential for signaling to paxillin (Figure 7, A and C, lanes 6).
FAK576F/577F and FAK454R could also be coimmunoprecipitated with pp60c-src and p59fyn (Figure 6, A and B; our unpubVol.
K397F
protein
substitution
true negative
Coexpression of exogenous wild-type FAK, but not FAK397F, rescues the cell-spreading defect (Richardson et al., 1997).
Expression of a membrane-bound, CD2-FAK chimeric molecule in MDCK cells blocks anoikis (Frisch et al., 1996); however, a CD2FAK397F mutant cannot block anoikis in MDCK cells held in suspension (Frisch et al., 1996).
A double mutant, FAK397F/K454R, was also created by ligating a fragment of FAK containing the Y397F point mutation (nucleotide 1 to 1381) to a fragment of FAK containing the K454R mutation (nucleotides 1382 to 3248) using the Bsp EI site at nucleotide 1381.
The mutants analyzed included FAK397F, FAK576F/577F, a variant with phenylalanine substituted for two regulatory sites of tyrosine phosphorylation that are substrates for pp60src, and FAK454R, which is catalytically defective.
As previously described, FAK397F contains little if any phosphotyrosine (Figure 5, A and B, lanes 5).
Coexpression with p59fyn did not enhance phosphorylation of FAK397F (Figure 5B, lane 6).
In some experiments, coexpression with pp60c-src induced a small increase in the tyrosine phosphorylation of FAK397F (Figure 5A, lane 6), and in others it did not.
As anticipated, both FAK397F and FAK454R/397F failed to associate with pp60c-src and p59fyn (Figure 6, A and B).
FAK397F failed to induce paxillin phosphorylation when coexpressed with pp60c-src or p59fyn, demonstrating that the physical association of the two PTKs was necessary for downstream signaling (Figure 7, B and C, lanes 6 and 5, respectively).
The mechanism by which catalytically defective FAK could send a signal appeared to be via recruitment of the Src family PTK because the double mutant (FAK397F/454R) was defective for induction of paxillin phosphorylation (Figure 7B, lane 10; our unpublished results).
(A) FAK was analyzed from CE cells (lane 1) or CE cells expressing RCAS B src (lane 2), RCAS A FAK (lane 3), RCAS A FAK RCAS B src (lane 4), RCAS A FAK397F (lane 5), RCAS A FAK397F RCAS B src (lane 6), RCAS A FAK576/577FF (lane 7), RCAS A FAK576/577FF RCAS B src (lane 8), RCAS A FAK454R (lane 9), RCAS A FAK454R RCAS B src (lane 10), RCAS A FAK454R/397F (lane 11), or RCAS A FAK454R/397F RCAS B src (lane 12).
(B) FAK was analyzed from CE cells (lane 1) or CE cells expressing RCAS B fyn (lane 2), RCAS A FAK (lane 3), RCAS A FAK RCAS B fyn (lane 4), RCAS A FAK397F (lane 5), RCAS A FAK397F RCAS B fyn (lane 6), RCAS A FAK454R (lane 7), RCAS A FAK454R RCAS B fyn (lane 8), RCAS A FAK454R/397F (lane 9), or RCAS A FAK454R/397F RCAS B fyn (lane 10).
(C) Twenty-five micrograms of whole-cell lysate from CE cells (lane 1) or CE cells expressing RCAS B src (lane 2), RCAS A FAK RCAS B src (lane 3), RCAS A FAK397F RCAS B src (lane 4), RCAS A FAK576/577FF RCAS B src (lane 5), RCAS A FAK454R RCAS B src (lane 6), or RCAS A FAK454R/397F RCAS B src (lane 7) was Western-blotted with a src antibody.
(D) Twenty-five micrograms of whole-cell lysate from CE cells (lane 1) or CE cells expressing RCAS B fyn (lane 2), RCAS A FAK RCAS B fyn (lane 3), RCAS A FAK397F RCAS B fyn (lane 4), RCAS A FAK454R RCAS B fyn (lane 5), or RCAS A FAK454R/397F RCAS B fyn (lane 6) was Westernblotted with an fyn antibody.
(A) Src immune complexes from lysates of CE cells (lane 1) or CE cells expressing RCAS B src (lane 2), RCAS A FAK RCAS B src (lane 3), RCAS A FAK397F RCAS B src (lane 4), RCAS A FAK576/577FF RCAS B src (lane 5), RCAS A FAK454R RCAS B src (lane 6), or RCAS A FAK454R/397F RCAS B src (lane 7) were analyzed.
(B) Fyn immune complexes from lysates of CE cells expressing RCAS B fyn (lane 1), RCAS A FAK RCAS B fyn (lane 2), RCAS A FAK397F RCAS B fyn (lane 3), RCAS A FAK454R RCAS B fyn (lane 4), or RCAS A FAK454R/397F RCAS B fyn (lane 5) were analyzed.
To determine whether the relocalization of pp60c-src was dependent on binding FAK, a mutant with a phenylalanine for tyrosine substitution at residue 397, FAK397F, was coexpressed with pp60c-src.
FAK397 is discretely localized to focal adhesions (Figure 11, A and C); however, coexpression of FAK397F with pp60c-src did not induce the same dramatic change in the cellular localization of pp60c-src that occurred upon coexpression with wild-type FAK (Figure 11, B and D).
(B) Paxillin was immunoprecipitated from lysates of CE cells (lane 1) or CE cells expressing RCAS B fyn (lane 2), RCAS A FAK (lane 3), RCAS A FAK RCAS B fyn (lane 4), RCAS A FAK397F (lane 5), RCAS A FAK397F RCAS B fyn (lane 6), RCAS A FAK454R (lane 7), RCAS A FAK454R RCAS 454R/397F B fyn (lane 8), RCAS A FAK (lane 9), or RCAS A FAK454R/397F RCAS B fyn (lane 10).
(C) Paxillin was immunoprecipitated from lysates of CE cells (lane 1) or CE cells expressing RCAS A FAK (lane 2), RCAS B Src (lane 3), RCAS A FAK RCAS B Src (lane 4), RCAS A FAK397F RCAS B Src (lane 5), RCAS A FAK576/577FF RCAS B fyn (lane 6), or RCAS A FAK454R RCAS B fyn (lane 7).
In contrast, coexpression of pp60c-src with FAK397F did not cause a dramatic relocalization of pp60c-src, although these cells could sometimes exhibit very faint focal adhesion localization of pp60c-src.
It is possible that the small amount of pp60c-src that may be found in focal adhesions in FAK397F cells may be a result of SH3-mediated interactions.
The fact that FAK397F does not effectively alter the localization of pp60c-src suggests that the autophosphorylation/Src SH2 domain binding site plays an important role in the relocalization of pp60c-src.
CE cells expressing RCAS B c-src and RCAS A FAK397F were fixed, permeabilized, and stained with BC4 to detect FAK localization (A and C) and with EC10 to detect Src localization (B and D).
A430V
protein
substitution
true negative
SrcA430V contains a valine substitution for alanine at residue 430, a residue that is highly conserved in protein kinases, and exhibits 10% of the activity of wild-type pp60c-src (Wilson et al., 1989).
One mutant, Src PK, has a deletion from residues 260 to 513 inclusive, and the second, SrcA430V, has a single substitution of a valine for alanine at residue 430.
Expression of SrcA430V did not alter the tyrosine phosphorylation of cellular proteins 3493 M.D.
Western blotting demonstrated expression of FAK, pp60c-src, and SrcA430V.
(B) Twenty-five micrograms of whole-cell lysate from CE cells (lane 1) or CE cells expressing RCAS A FAK (lane 2), RCAS B SrcA430V (lane 3), RCAS A FAK RCAS B SrcA430V (lane 4), or RCAS A FAK RCAS B c-src (lane 5) were analyzed by Western blotting using phosphotyrosine antibodies (top panel), mAb 2A7 to detect FAK (middle panel), or mAb EC10 to detect pp60c-src (bottom panel).
Y397F
protein
substitution
Q05397
true positive
A double mutant, FAK397F/K454R, was also created by ligating a fragment of FAK containing the Y397F point mutation (nucleotide 1 to 1381) to a fragment of FAK containing the K454R mutation (nucleotides 1382 to 3248) using the Bsp EI site at nucleotide 1381.
10216104
full text
9545268
full text
F99Y
protein
substitution
true negative
2, FKBP12 variants bearing D37L or F99Y mutations expressed greatly reduced inhibitory effects.
In line with this interpretation, two site-directed FKBP12 variants, D37L and F99Y, with reduced PPIase activity exhibited reduced ability to suppress EGF receptor tyrosine phosphorylation (30).
D37L
protein
substitution
true negative
2, FKBP12 variants bearing D37L or F99Y mutations expressed greatly reduced inhibitory effects.
In line with this interpretation, two site-directed FKBP12 variants, D37L and F99Y, with reduced PPIase activity exhibited reduced ability to suppress EGF receptor tyrosine phosphorylation (30).
11581249
full text
K179M
protein
substitution
true negative
EXPERIMENTAL PROCEDURES DNA Construction--The mouse cDNAs of PIPK and - , the Nterminal truncated mutants ( 1197( ); 1238( )), and the PIPK kinase dead mutant (K179M) were subcloned into pCDNA3 or the Sindbis vector Toto1000:3 2J (13) without a tag or with a GFP, GST, or HA-tag as indicated.
10402467
full text
S2173A
protein
substitution
true negative
The expression construct encoding dominant negative MEK1 (S2173 A) (dn-MEK1) in pBABE is previously described (23).
10781592
full text
10465268
full text
Y1062F
protein
substitution
true positive
P07949
As shown in Table 1, all of the I1057S, N1059A, L1061P, and Y1062F mutations significantly impaired the transforming activity of Ret-MEN2A, although the impairment by the Y1062F mutation was more severe than other mutations.
The activity of Ret-MEN2A with the I1057S/N1059A double mutation was reduced to a degree similar to that of RetMEN2A with the Y1062F mutation (Table 1).
Transforming activity of the mutant RET genes DNAa Focus-forming activity (foci/ g DNA)b c-RET51 RET(C634R)51 RET(C634R, Y1062F)51 RET(C634R, I1057S)51 RET(C634R, N1059A)51 RET(C634R, I1057S, N1059A)51 RET(C634R, L1061P)51 0 100150 2030 5070 3050 2030 3050 a Each mutant cDNA was fused to a Moloney murine leukemia virus long terminal repeat.
3A, the amounts of Ret-MEN2A with the I1057S, N1059A, I1057S/N1059A, L1061P, or Y1062F mutation coprecipitated with Shc were markedly decreased compared with that of the original Ret-MEN2A coprecipitated with Shc.
Mutations in the I-E-N-K-L sequence, however, did not completely abolish the transforming activity of RetMEN2A or its ability to bind to Shc as observed for Ret with the Y1062F mutation.
N1059A
protein
substitution
true positive
P07949
Substitution of Ser for Ile1057 (I1057S), Ala for Asn1059 (N1059A), or Pro for Leu1061 (L1061P) in this sequence significantly decreased the transforming activity of Ret with the multiple endocrine neoplasm type 2A (MEN2A) mutation as well as the binding affinity of Shc to it in vivo and in vitro, indicating that these three amino acids play a role in Shc binding.
As shown in Table 1, all of the I1057S, N1059A, L1061P, and Y1062F mutations significantly impaired the transforming activity of Ret-MEN2A, although the impairment by the Y1062F mutation was more severe than other mutations.
The activity of Ret-MEN2A with the I1057S/N1059A double mutation was reduced to a degree similar to that of RetMEN2A with the Y1062F mutation (Table 1).
Transforming activity of the mutant RET genes DNAa Focus-forming activity (foci/ g DNA)b c-RET51 RET(C634R)51 RET(C634R, Y1062F)51 RET(C634R, I1057S)51 RET(C634R, N1059A)51 RET(C634R, I1057S, N1059A)51 RET(C634R, L1061P)51 0 100150 2030 5070 3050 2030 3050 a Each mutant cDNA was fused to a Moloney murine leukemia virus long terminal repeat.
3A, the amounts of Ret-MEN2A with the I1057S, N1059A, I1057S/N1059A, L1061P, or Y1062F mutation coprecipitated with Shc were markedly decreased compared with that of the original Ret-MEN2A coprecipitated with Shc.
In particular, in the case of Ret-MEN2A with the I1057S/N1059A mutation, coprecipitation with Shc was almost undetectable.
I1057S
protein
substitution
true positive
P07949
Substitution of Ser for Ile1057 (I1057S), Ala for Asn1059 (N1059A), or Pro for Leu1061 (L1061P) in this sequence significantly decreased the transforming activity of Ret with the multiple endocrine neoplasm type 2A (MEN2A) mutation as well as the binding affinity of Shc to it in vivo and in vitro, indicating that these three amino acids play a role in Shc binding.
As shown in Table 1, all of the I1057S, N1059A, L1061P, and Y1062F mutations significantly impaired the transforming activity of Ret-MEN2A, although the impairment by the Y1062F mutation was more severe than other mutations.
The activity of Ret-MEN2A with the I1057S/N1059A double mutation was reduced to a degree similar to that of RetMEN2A with the Y1062F mutation (Table 1).
Transforming activity of the mutant RET genes DNAa Focus-forming activity (foci/ g DNA)b c-RET51 RET(C634R)51 RET(C634R, Y1062F)51 RET(C634R, I1057S)51 RET(C634R, N1059A)51 RET(C634R, I1057S, N1059A)51 RET(C634R, L1061P)51 0 100150 2030 5070 3050 2030 3050 a Each mutant cDNA was fused to a Moloney murine leukemia virus long terminal repeat.
3A, the amounts of Ret-MEN2A with the I1057S, N1059A, I1057S/N1059A, L1061P, or Y1062F mutation coprecipitated with Shc were markedly decreased compared with that of the original Ret-MEN2A coprecipitated with Shc.
In particular, in the case of Ret-MEN2A with the I1057S/N1059A mutation, coprecipitation with Shc was almost undetectable.
The phosphorylation of the immu- noprecipitated 46- and 52-kDa Shc (66-kDa Shc was not visible in this experiment) was significantly reduced in the NIH-3T3 cells expressing each mutant Ret protein, except for the cells expressing Ret-MEN2A with the I1057S mutation (Fig.
The binding of fulllength Shc as well as its PTB domain to Ret-MEN2A was markedly decreased by the mutations examined, although the degree of the reduction by the I1057S mutation was lower than that by other mutations (Fig.
C634R
protein
substitution
true positive
P07949
These mutations were introduced into the Ret long isoform complementary DNA with a MEN2A (Cys6343 Arg, C634R) mutation.
Transforming activity of the mutant RET genes DNAa Focus-forming activity (foci/ g DNA)b c-RET51 RET(C634R)51 RET(C634R, Y1062F)51 RET(C634R, I1057S)51 RET(C634R, N1059A)51 RET(C634R, I1057S, N1059A)51 RET(C634R, L1061P)51 0 100150 2030 5070 3050 2030 3050 a Each mutant cDNA was fused to a Moloney murine leukemia virus long terminal repeat.
Each mutation was introduced into Ret 51type with a MEN2A (C634R) mutation.
Transforming activity of three RET isoforms DNAa Focus-forming activity (foci/ g DNA)b c-RET51 RET(C634R)51 RET(C634R)9 RET(C634R)43 0 100150 7090 1020 a Each mutant cDNA was fused to a Moloney murine leukemia virus long terminal repeat.
When the transforming activities of three isoforms with the C634R mutation (designated Ret-MEN2A 9type, 43type, and 51type) were investigated, the activity of Ret-MEN2A 43type was very low compared with those of Ret-MEN2A 9type and 51type (Table 2).
M1064T
protein
substitution
true positive
P07949
Several Hirschsprung mutations, including the L1061P and M1064T mutations, were also found in the carboxyl-terminal sequence of Ret.
(28) recently reported that the M1064T mutation specifically causes a reduction in Shc PTB domain binding to Tyr1062 in vitro as well as Ret-dependent Shc phos- Downloaded from endo.endojournals.org on December 5, 2005 BINDING SEQUENCE OF SHC PTB DOMAIN IN RET 3997 phorylation in vivo.
L1061P
protein
substitution
true positive
P07949
Substitution of Ser for Ile1057 (I1057S), Ala for Asn1059 (N1059A), or Pro for Leu1061 (L1061P) in this sequence significantly decreased the transforming activity of Ret with the multiple endocrine neoplasm type 2A (MEN2A) mutation as well as the binding affinity of Shc to it in vivo and in vitro, indicating that these three amino acids play a role in Shc binding.
As shown in Table 1, all of the I1057S, N1059A, L1061P, and Y1062F mutations significantly impaired the transforming activity of Ret-MEN2A, although the impairment by the Y1062F mutation was more severe than other mutations.
Transforming activity of the mutant RET genes DNAa Focus-forming activity (foci/ g DNA)b c-RET51 RET(C634R)51 RET(C634R, Y1062F)51 RET(C634R, I1057S)51 RET(C634R, N1059A)51 RET(C634R, I1057S, N1059A)51 RET(C634R, L1061P)51 0 100150 2030 5070 3050 2030 3050 a Each mutant cDNA was fused to a Moloney murine leukemia virus long terminal repeat.
3A, the amounts of Ret-MEN2A with the I1057S, N1059A, I1057S/N1059A, L1061P, or Y1062F mutation coprecipitated with Shc were markedly decreased compared with that of the original Ret-MEN2A coprecipitated with Shc.
Several Hirschsprung mutations, including the L1061P and M1064T mutations, were also found in the carboxyl-terminal sequence of Ret.
Interestingly, our present study also suggested that the L1061P mutation could impair the intracellular signaling of Ret by decreasing its binding affinity to the Shc PTB domain.
10234043
full text
12750701
full text
D835V
protein
substitution
true positive
P36888
Sequence analysis showed one each of D835 Y, D835 V, and D835 H.
In the patient with AML1-ETO, the second nucleotide A of D835 was substituted with T, resulting in an Asp to Val amino-acid change (D835 V).
D835Y
protein
substitution
true positive
P36888
Sequence analysis showed one each of D835 Y, D835 V, and D835 H.
The substitution for the first nucleotide G of D835 with T, resulting in an Asp to Tyr change (D835 Y) was found in the other APL patient with L-type PML-RARa.
D835H
protein
substitution
true positive
P36888
Sequence analysis showed one each of D835 Y, D835 V, and D835 H.
The substitution for the first nucleotide G of D835 with C, resulting in an Asp to His change (D835 H) was found in the APL patient with V-type PML-RARa.
10077632
full text
10786674
full text
Y705F
protein
substitution
true negative
The cDNA of DN-STAT3 (Y705F; Ref.
To further determine the role of IL-6-activated STAT3 in ARmediated gene activation in response to IL-6, we decided to investigate the effect of a DN form of STAT3 (Y705F-STAT3, in which a phenylalanine was substituted for tyrosine at position 705) on ARmediated gene activation in response to IL-6.
Y705F-STAT3 has been shown to be able to inhibit tyrosine phosphorylation of Tyr705 of the wild-type STAT3 and thus inhibit STAT3-mediated gene activation (19).
LNCaP cells were cotransfected with ARE3-tk-luc (B) or Fib-luc (C) along with pcDNA3 carrying DN-STAT3 (Y705F-STAT3) or vector alone and then stimulated with or without IL-6 and R1881 as indicated.
10100709
full text
10428759
full text
V80W
protein
substitution
true negative
V664E
protein
substitution
true positive
P04626
The oncogenic allele of neu contains a point mutation [Val(664) 3 Glu] in the predicted TM sequence of its product, which causes increased tyrosine kinase activity and increased aggregation of the receptor protein (27, 28).
V80A
protein
substitution
true negative
(39) have shown that a Val(80) 3 Ala mutation was not as effective in disrupting dimerization as was predicted from the detergent studies.
9444958
full text
H1351N
protein
substitution
P08581
true positive
The 110 kDa protein is specically phosphorylated by Tpr-Met on tyrosine residues as shown by phosphoaminoacid analysis Table 1 Point mutations in the Met-multifunctional docking site WT Y1349F Y1356F Y1349 1356F N1358H H1351N YVHVNATYVNV FVHVNATYVNV YVHVNATFVNV FVHVNATFVNV YVHVNATYVHV YVNVNATYVNV (Figure 1c).
As a control the same lter-blot was probed with anti-Gab1 antibody Met binding to Gab1 requires Grb2 A Bardelli et al tyrosine 1356) was substituted with a histidine (TprMetN1358H); and a mutant with an additional Grb-2 binding was obtained by replacing histidine 1351 with an asparagine (Tpr-MetH1351N).
Binding is unaected by introduction of a second Grb2 binding site (Tpr-MetH1351N).
Binding is slightly increased by introduction of a second Grb2 binding site (Tpr-MetH1351N).
Cell lysates were preincubated with peptides blocking 3105 Y1349-1356F a K1110A Y1349F Y1356F N1358H 193 kD -- 112 kD -- 86 kD -- 70 kD -- H1351N wt p110GAb1 Blot anti-Gab1 b 29 kD -- p24Grb2 20 kD -- Blot anti-Grb2 c 70 kD -- p65Tpr-Met 57 kD -- Blot anti-Met d 70 kD -- p65Tpr-Met 57 kD -- Blot anti-PTyr Figure 2 Met-coupling to Gab1 requires a functional Grb2 binding site.
Binding and phosphorylation of Gab1 correlates with the transforming potential of Tpr-Met Y1349-1356F a K1110A Y1349F Y1356F N1358H H1351N wt p65Tpr-Met Blot anti-Met b p65Tpr-Met Signaling by the Met-receptor is mediated by the multifunctional docking site (Ponzetto et al., 1994).
Replacement of Y1349 and Y1356 with phenylalanine (Tpr-Met1349 1356F mutant) completely abrogates the oncogenic potential of Tpr-Met by impairing recep- a a IP anti-Gab1 H1351N FR 112 kD -- wt p110Gab1 86 kD -- Blot anti-PTyr N1351N N1358H Y1349F Y1356F wt b 112 kD -- p110Gab1 112 kD -- p110Gab1 b 86 kD -- Blot anti-Gab1 IP anti-Gab1 H1351N Ctr IP FR p65Tpr-Met 57 kD -- Ctr antibody c wt 70 kD -- p85PI3K IP anti-Gab1 Blot anti-p85 p80SHPTP2 Blot anti-SHPTP2 p110Gab1 p140PLC Figure 6 Correlation between the transforming potential of TprMet mutants with the ability to bind and phosphorylate Gab1.
(a) Lysates of normal (FR) and Tpr-Met transformed broblasts (Tpr-Metwt and Tpr-MetH1351N) were immunoprecipitated with anti-Gab1 antibodies.
N1358H
protein
substitution
P08581
true positive
The 110 kDa protein is specically phosphorylated by Tpr-Met on tyrosine residues as shown by phosphoaminoacid analysis Table 1 Point mutations in the Met-multifunctional docking site WT Y1349F Y1356F Y1349 1356F N1358H H1351N YVHVNATYVNV FVHVNATYVNV YVHVNATFVNV FVHVNATFVNV YVHVNATYVHV YVNVNATYVNV (Figure 1c).
As a control the same lter-blot was probed with anti-Gab1 antibody Met binding to Gab1 requires Grb2 A Bardelli et al tyrosine 1356) was substituted with a histidine (TprMetN1358H); and a mutant with an additional Grb-2 binding was obtained by replacing histidine 1351 with an asparagine (Tpr-MetH1351N).
Interestingly, the N1358H mutation (Tpr-MetN1358H), which selectively impairs the Met/ Grb2 interaction, also dramatically aects the Met/ Gab1 interaction (Figure 2a and 2b).
Furthermore, the N1358H mutation, which selectively impairs the Met/Grb2 association, also reduces the Met/MBD interaction.
Cell lysates were preincubated with peptides blocking 3105 Y1349-1356F a K1110A Y1349F Y1356F N1358H 193 kD -- 112 kD -- 86 kD -- 70 kD -- H1351N wt p110GAb1 Blot anti-Gab1 b 29 kD -- p24Grb2 20 kD -- Blot anti-Grb2 c 70 kD -- p65Tpr-Met 57 kD -- Blot anti-Met d 70 kD -- p65Tpr-Met 57 kD -- Blot anti-PTyr Figure 2 Met-coupling to Gab1 requires a functional Grb2 binding site.
Binding and phosphorylation of Gab1 correlates with the transforming potential of Tpr-Met Y1349-1356F a K1110A Y1349F Y1356F N1358H H1351N wt p65Tpr-Met Blot anti-Met b p65Tpr-Met Signaling by the Met-receptor is mediated by the multifunctional docking site (Ponzetto et al., 1994).
Replacement of Y1349 and Y1356 with phenylalanine (Tpr-Met1349 1356F mutant) completely abrogates the oncogenic potential of Tpr-Met by impairing recep- a a IP anti-Gab1 H1351N FR 112 kD -- wt p110Gab1 86 kD -- Blot anti-PTyr N1351N N1358H Y1349F Y1356F wt b 112 kD -- p110Gab1 112 kD -- p110Gab1 b 86 kD -- Blot anti-Gab1 IP anti-Gab1 H1351N Ctr IP FR p65Tpr-Met 57 kD -- Ctr antibody c wt 70 kD -- p85PI3K IP anti-Gab1 Blot anti-p85 p80SHPTP2 Blot anti-SHPTP2 p110Gab1 p140PLC Figure 6 Correlation between the transforming potential of TprMet mutants with the ability to bind and phosphorylate Gab1.
Interestingly, we found that two mutations (Y1356F and N1358H) which selectively uncouple Grb2 from the receptor (Ponzetto et al., 1996), also abrogate Gab1 binding in intact cells.
Y1349F
protein
substitution
P08581
true positive
The 110 kDa protein is specically phosphorylated by Tpr-Met on tyrosine residues as shown by phosphoaminoacid analysis Table 1 Point mutations in the Met-multifunctional docking site WT Y1349F Y1356F Y1349 1356F N1358H H1351N YVHVNATYVNV FVHVNATYVNV YVHVNATFVNV FVHVNATFVNV YVHVNATYVHV YVNVNATYVNV (Figure 1c).
Cell lysates were preincubated with peptides blocking 3105 Y1349-1356F a K1110A Y1349F Y1356F N1358H 193 kD -- 112 kD -- 86 kD -- 70 kD -- H1351N wt p110GAb1 Blot anti-Gab1 b 29 kD -- p24Grb2 20 kD -- Blot anti-Grb2 c 70 kD -- p65Tpr-Met 57 kD -- Blot anti-Met d 70 kD -- p65Tpr-Met 57 kD -- Blot anti-PTyr Figure 2 Met-coupling to Gab1 requires a functional Grb2 binding site.
Binding and phosphorylation of Gab1 correlates with the transforming potential of Tpr-Met Y1349-1356F a K1110A Y1349F Y1356F N1358H H1351N wt p65Tpr-Met Blot anti-Met b p65Tpr-Met Signaling by the Met-receptor is mediated by the multifunctional docking site (Ponzetto et al., 1994).
Replacement of Y1349 and Y1356 with phenylalanine (Tpr-Met1349 1356F mutant) completely abrogates the oncogenic potential of Tpr-Met by impairing recep- a a IP anti-Gab1 H1351N FR 112 kD -- wt p110Gab1 86 kD -- Blot anti-PTyr N1351N N1358H Y1349F Y1356F wt b 112 kD -- p110Gab1 112 kD -- p110Gab1 b 86 kD -- Blot anti-Gab1 IP anti-Gab1 H1351N Ctr IP FR p65Tpr-Met 57 kD -- Ctr antibody c wt 70 kD -- p85PI3K IP anti-Gab1 Blot anti-p85 p80SHPTP2 Blot anti-SHPTP2 p110Gab1 p140PLC Figure 6 Correlation between the transforming potential of TprMet mutants with the ability to bind and phosphorylate Gab1.
Y1356F
protein
substitution
P08581
true positive
The 110 kDa protein is specically phosphorylated by Tpr-Met on tyrosine residues as shown by phosphoaminoacid analysis Table 1 Point mutations in the Met-multifunctional docking site WT Y1349F Y1356F Y1349 1356F N1358H H1351N YVHVNATYVNV FVHVNATYVNV YVHVNATFVNV FVHVNATFVNV YVHVNATYVHV YVNVNATYVNV (Figure 1c).
Cell lysates were preincubated with peptides blocking 3105 Y1349-1356F a K1110A Y1349F Y1356F N1358H 193 kD -- 112 kD -- 86 kD -- 70 kD -- H1351N wt p110GAb1 Blot anti-Gab1 b 29 kD -- p24Grb2 20 kD -- Blot anti-Grb2 c 70 kD -- p65Tpr-Met 57 kD -- Blot anti-Met d 70 kD -- p65Tpr-Met 57 kD -- Blot anti-PTyr Figure 2 Met-coupling to Gab1 requires a functional Grb2 binding site.
Binding and phosphorylation of Gab1 correlates with the transforming potential of Tpr-Met Y1349-1356F a K1110A Y1349F Y1356F N1358H H1351N wt p65Tpr-Met Blot anti-Met b p65Tpr-Met Signaling by the Met-receptor is mediated by the multifunctional docking site (Ponzetto et al., 1994).
Replacement of Y1349 and Y1356 with phenylalanine (Tpr-Met1349 1356F mutant) completely abrogates the oncogenic potential of Tpr-Met by impairing recep- a a IP anti-Gab1 H1351N FR 112 kD -- wt p110Gab1 86 kD -- Blot anti-PTyr N1351N N1358H Y1349F Y1356F wt b 112 kD -- p110Gab1 112 kD -- p110Gab1 b 86 kD -- Blot anti-Gab1 IP anti-Gab1 H1351N Ctr IP FR p65Tpr-Met 57 kD -- Ctr antibody c wt 70 kD -- p85PI3K IP anti-Gab1 Blot anti-p85 p80SHPTP2 Blot anti-SHPTP2 p110Gab1 p140PLC Figure 6 Correlation between the transforming potential of TprMet mutants with the ability to bind and phosphorylate Gab1.
Interestingly, we found that two mutations (Y1356F and N1358H) which selectively uncouple Grb2 from the receptor (Ponzetto et al., 1996), also abrogate Gab1 binding in intact cells.
K1110A
protein
substitution
P08581
true positive
In addition, a kinaseinactive mutant (Tpr-MetK1110A) was also included as a negative control.
We found that Tpr-Metwt but not the kinaseinactive mutant (Tpr-MetK1110A) interacts with Gab1 indicating that the association is phosphorylationdependent.
Cell lysates were preincubated with peptides blocking 3105 Y1349-1356F a K1110A Y1349F Y1356F N1358H 193 kD -- 112 kD -- 86 kD -- 70 kD -- H1351N wt p110GAb1 Blot anti-Gab1 b 29 kD -- p24Grb2 20 kD -- Blot anti-Grb2 c 70 kD -- p65Tpr-Met 57 kD -- Blot anti-Met d 70 kD -- p65Tpr-Met 57 kD -- Blot anti-PTyr Figure 2 Met-coupling to Gab1 requires a functional Grb2 binding site.
Tpr Met proteins are present in similar amounts and phosphorylated at comparable levels with the exception of the kinase-inactive mutant (Tpr-MetK1110A).
Binding and phosphorylation of Gab1 correlates with the transforming potential of Tpr-Met Y1349-1356F a K1110A Y1349F Y1356F N1358H H1351N wt p65Tpr-Met Blot anti-Met b p65Tpr-Met Signaling by the Met-receptor is mediated by the multifunctional docking site (Ponzetto et al., 1994).
11742534
full text
W652A
protein
substitution
true negative
Several point mutations in the membraneproximal region of gp130 (W652A, P671P672A, F676A, Y683F, where W, A, P, F and Y are tryptophan, alanine, proline, phenylalanine and tyrosine) did not affect Jak1 association.
Most importantly, signal- ling by the receptor with the box1 mutation W652A was totally abrogated.
Among several point mutations, the most striking effect was observed for mutation of the aromatic Abbreviations used : EMSA, electrophoretic-mobility-shift assay ; EPOR, erythropoietin receptor ; Jak, Janus kinase ; LIF, leukaemia inhibitory factor ; OSM (R), oncostatin M (receptor) ; sIL-6R, soluble IL-6 receptor ; STAT, signal transducer and activator of transcription ; W652A etc., Trp652 Ala mutant etc.
The PCR fragments were introduced into the pSVL-based expression vectors for gp130 [20] using the restriction enzymes EcoRIBstEII for the construct W652A, BstEIIKpn2I for the constructs box2 and P671AP672A, and EcoRIAspI for the construct F676A.
Behrmann Figure 3 STAT activation is abrogated by point mutation W652A and by deletion of box2, while P671A/P672A and F676A lead to a modulation of STAT activity COS-7 cells were transiently co-transfected with IL-5Rgp130 and IL-5Rgp130 wild-type or with two mutant expression constructs as depicted in the upper left panel.
Figure 2 Binding of Jak1 to the IL-5R-gp130 mutant receptors (A) Mutations W652A, P671/P672A, F676A, Y683F and B do not affect Jak association.
Also a single amino acid exchange, W652A, in the box1 region did not affect Jak1 association (lane 3).
Interestingly, the single amino acid substitution in the box1-region, W652A, totally prevented the activation of STAT1 (Figure 3A), although this mutein bound Jak1 (Figure 2A).
109 Point mutation of W652 to alanine in gp130 leads to an impaired Jak1 activation The mutant construct W652A did not affect Jak1 binding, but totally abolished the gp130-induced DNA-binding activity of STATs.
To determine further at which point signal transduction stalls, we were interested as to whether Jak1 still becomes activated on stimulation of the W652A receptor mutein.
Interestingly, the single amino acid substitution W652A totally prevented Jak1 phosphorylation (Figure 4, middle panel) and, not surprisingly, phosphorylation of STAT transcription factors (Figure 4, lower panel).
Figure 4 W652A Jak1 activation is impaired by the single amino acid exchange Jak association to both chains of a gp130 dimer is essential, but not sufficient, to promote Jak1 activation The use of heterodimeric receptor chimaeras based on the IL5R- and -chains enabled us to induce the dimerization of the cytoplasmic parts of a gp130 W652A mutant with a wild-type receptor construct.
Interestingly, IL-5-induced dimerization of one wild-type with a mutant W652A chain leads only to a marginal STAT1 signal.
Here we show that the role of box1 in cytokine receptor signalling cannot be only restricted to mere Jak binding : a receptor mutant with a single amino acid substitution within box1, W652A, displayed a binding behaviour comparable with that of the wild-type receptor, but was unable to mediate STAT1 activation in COS-7 cells.
Even when a W652A mutant receptor was dimerized with the cytoplasmic part of wild-type gp130, no signalling occured, indicating that the mutation acts dominantly.
P656A
protein
substitution
true negative
Overexpressed Jak1 could not be co-precipitated with a construct lacking the box1 region (Figure 2A) or with the inactive W666A construct (results not shown), but overexpression of Jak1 also resulted in co-precipitation with the otherwise nonbinding IL-5Rgp130P656AP658A receptor which is mutated # 2002 Biochemical Society gp130 contributes to Jak activation the Jaks involves structural reorganization of the Jakgp130 binding interface, and that residues such as W652 are involved in this dynamic process.
F676A
protein
substitution
true negative
Several point mutations in the membraneproximal region of gp130 (W652A, P671P672A, F676A, Y683F, where W, A, P, F and Y are tryptophan, alanine, proline, phenylalanine and tyrosine) did not affect Jak1 association.
However, stimulation of chimaeric receptors with the mutations P671P672A and F676A in the interbox1 ox2 region resulted in a reduced activation of STAT (signal transducer and activator of transcription) transcription factors.
The PCR fragments were introduced into the pSVL-based expression vectors for gp130 [20] using the restriction enzymes EcoRIBstEII for the construct W652A, BstEIIKpn2I for the constructs box2 and P671AP672A, and EcoRIAspI for the construct F676A.
Behrmann Figure 3 STAT activation is abrogated by point mutation W652A and by deletion of box2, while P671A/P672A and F676A lead to a modulation of STAT activity COS-7 cells were transiently co-transfected with IL-5Rgp130 and IL-5Rgp130 wild-type or with two mutant expression constructs as depicted in the upper left panel.
(B) A Personal Imager-FX and Quantity-One software (both from Bio-Rad) were used to quantify the signals for wild-type, P671A/P672A and F676A.
Figure 2 Binding of Jak1 to the IL-5R-gp130 mutant receptors (A) Mutations W652A, P671/P672A, F676A, Y683F and B do not affect Jak association.
The constructs with point mutations in the interbox1 ox2 region P671AP672A, F676A and Y683F bound Jak1 as effectively as the wild-type receptor (lanes 4, 6, and 7).
The muteins P671AP672A and F676A mediated a reproducibly reduced STAT1 DNA-binding activity (Figure 3B).
P672A
protein
substitution
true negative
Several point mutations in the membraneproximal region of gp130 (W652A, P671P672A, F676A, Y683F, where W, A, P, F and Y are tryptophan, alanine, proline, phenylalanine and tyrosine) did not affect Jak1 association.
However, stimulation of chimaeric receptors with the mutations P671P672A and F676A in the interbox1 ox2 region resulted in a reduced activation of STAT (signal transducer and activator of transcription) transcription factors.
The PCR fragments were introduced into the pSVL-based expression vectors for gp130 [20] using the restriction enzymes EcoRIBstEII for the construct W652A, BstEIIKpn2I for the constructs box2 and P671AP672A, and EcoRIAspI for the construct F676A.
Behrmann Figure 3 STAT activation is abrogated by point mutation W652A and by deletion of box2, while P671A/P672A and F676A lead to a modulation of STAT activity COS-7 cells were transiently co-transfected with IL-5Rgp130 and IL-5Rgp130 wild-type or with two mutant expression constructs as depicted in the upper left panel.
(B) A Personal Imager-FX and Quantity-One software (both from Bio-Rad) were used to quantify the signals for wild-type, P671A/P672A and F676A.
Figure 2 Binding of Jak1 to the IL-5R-gp130 mutant receptors (A) Mutations W652A, P671/P672A, F676A, Y683F and B do not affect Jak association.
The constructs with point mutations in the interbox1 ox2 region P671AP672A, F676A and Y683F bound Jak1 as effectively as the wild-type receptor (lanes 4, 6, and 7).
The muteins P671AP672A and F676A mediated a reproducibly reduced STAT1 DNA-binding activity (Figure 3B).
W666A
protein
substitution
true negative
Overexpressed Jak1 could not be co-precipitated with a construct lacking the box1 region (Figure 2A) or with the inactive W666A construct (results not shown), but overexpression of Jak1 also resulted in co-precipitation with the otherwise nonbinding IL-5Rgp130P656AP658A receptor which is mutated # 2002 Biochemical Society gp130 contributes to Jak activation the Jaks involves structural reorganization of the Jakgp130 binding interface, and that residues such as W652 are involved in this dynamic process.
Y683F
protein
substitution
true negative
Several point mutations in the membraneproximal region of gp130 (W652A, P671P672A, F676A, Y683F, where W, A, P, F and Y are tryptophan, alanine, proline, phenylalanine and tyrosine) did not affect Jak1 association.
Gp130-Y683F was generated by introducing the BglIISalIfragment of pSVL-EPORgp130-6F [21] into the pSVL-based expression vector for gp130 (EPOR refers to erythropoietin receptor).
Figure 2 Binding of Jak1 to the IL-5R-gp130 mutant receptors (A) Mutations W652A, P671/P672A, F676A, Y683F and B do not affect Jak association.
The constructs with point mutations in the interbox1ox2 region P671AP672A, F676A and Y683F bound Jak1 as effectively as the wild-type receptor (lanes 4, 6, and 7).
P671A
protein
substitution
true negative
The PCR fragments were introduced into the pSVL-based expression vectors for gp130 [20] using the restriction enzymes EcoRIBstEII for the construct W652A, BstEIIKpn2I for the constructs box2 and P671AP672A, and EcoRIAspI for the construct F676A.
Behrmann Figure 3 STAT activation is abrogated by point mutation W652A and by deletion of box2, while P671A/P672A and F676A lead to a modulation of STAT activity COS-7 cells were transiently co-transfected with IL-5Rgp130 and IL-5Rgp130 wild-type or with two mutant expression constructs as depicted in the upper left panel.
(B) A Personal Imager-FX and Quantity-One software (both from Bio-Rad) were used to quantify the signals for wild-type, P671A/P672A and F676A.
The constructs with point mutations in the interbox1 ox2 region P671AP672A, F676A and Y683F bound Jak1 as effectively as the wild-type receptor (lanes 4, 6, and 7).
The muteins P671AP672A and F676A mediated a reproducibly reduced STAT1 DNA-binding activity (Figure 3B).
10200283
full text
G380R
protein
substitution
true positive
P22607
Capecchi, University of Utah, Salt Lake City, UT, and approved February 12, 1999 (received for review December 24, 1998) ABSTRACT Achondroplasia, the most common form of dwarfism in man, is a dominant genetic disorder caused by a point mutation (G380R) in the transmembrane region of fibroblast growth factor receptor 3 (FGFR3).
Achondroplasia, the most common form of dwarfism, was shown to be linked to a single point mutation, G380R, in the transmembrane region of FGFR3 (5, 6).
Several experiments at the cellular level indicated that the Ach mutation (G380R) results in a constitutive activation of the receptor in a ligand-independent manner (1113).
targeting to introduce the achondroplasia mutation (G380R) into murine FGFR3.
G374R
protein
substitution
true positive
Q61851
This point mutation (FGFR3G374R) was incorporated into a targeting construct, including a floxed neo cassette in intron 4.
Lanes: 1, wild type; 2, FGFR3G374R heterozygote; 3, FGFR3G374R homozygote.
(D) Detection of neo deletion in offspring from mating FGFR3G374Rneo heterozygote with PGK-Cre m mice.
Lanes: 1 and 5, control FGFR3G374Rneo heterozygote; 2 and 6, control wild-type mouse; 3 and 7, FGFR3G374Rneo heterozygote; 4 and 8, wild-type littermates.
Homozygotes of the FGFR3G374Rneo mutation display a phenotype very similar to the targeted loss of function mutation of FGFR3.
Surprisingly, FGFR3G374Rneo homozygotes (abbreviated as neo neo ) exhibited a phenotype very similar to the targeted loss-offunction mutation of FGFR3 (9, 10).
The gross structure of FGFR3 gene in the dwarf mice (containing the FGFR3G374Rneo mutation, abbreviated here as neo ) was identical to that of the wild type, as judged by hybridization of FGFR3 cDNA to Southern blots of DNA digested with three different enzymes (data not shown).
Anatomical defects in FGFR3G374Rneo heterozygotes.
Mice from a cross between mutant FGFR3G374R heterozygotes and PGK-Crem mice were weighed daily from birth.
At 4 weeks of age, tail DNA was analyzed by Southern blotting to determine the genotype (FGFR3G374Rneo or ; see Fig.
There was complete correspondence between the FGFR3G374Rneo genotype and the weight curve of the affected mice (lower curve).
Lanes: 1 and 5, control wild-type mouse; 2 and 6, FGFR3G374Rneo heterozygote dwarf mouse; 3 and 7, FGFR3G374Rneo heterozygote, showing both FGFR3 RNA and the high molecular weight RNA that also hybridized with neo; 4 and 8, FGFR3G374Rneo homozygote RNA is devoid of normal size FGFR3 RNA.
Lanes: 1 and 4, control wild-type mouse; 2 and 5, FGFR3G374Rneo heterozygote dwarf mouse; 3 and 6, FGFR3G374Rneo heterozygote.
Histological analysis of the growth plates from the tibia of neo FGFR3 heterozygotes (FGFR3G374Rneo ) (Right) and wild-type mice (Left).
(A and B) Tibia of 30-day-old normal mouse (A) and its FGFR3G374Rneo littermate (B).
In the FGFR3G374Rneo mouse, the tibia is smaller, and the overall organization of the columns is disrupted and markedly shortened, with very few cells in the proliferative and hypertrophic zones.
The phenotype of the neo heterozygotes that carried the G374R point mutation is a model for human achondroplasia (Fig.
9294615
full text
K650E
protein
substitution
true positive
P22607
Activating mutations in the kinase domain of the receptor tyrosine kinases c-kit (D814Y; equivalent to A883F mutation in MEN 2B DP Smith et al 1214 RET codon 898) and FGFR3 (K650E; equivalent to RET codon 908) which also alter the catalytic substrate specicity have recently been reported (Webster et al., 1996; Piao et al., 1996; Su et al., 1997), and in the case of the c-kit mutation the substrate specicity change is related to that seen in M918T-RET.
Bold residues beneath the aligned sequence illustrate disease-causing activating mutations (c-kit: D816V and D820G in mast cell leukaemia (Piao et al., 1996; Pignon et al., 1997); FGFR3: K650E in thanatophoric dysplasia type II (Webster et al., 1996); c-met: V1238I, D1246N and Y1248C in familial renal carcinoma and D1246H, Y1248H and M1268T in sporadic renal carcinoma (Schmidt et al., 1997); RET: M918T and A883F in MEN 2B).
Mutations in the `activation loop' of other receptor tyrosine kinases (D816V in c-kit and K650E in FGFR3; Figure 3) have been demonstrated to cause ligand-independent activation.
The FGFR3 K650E mutation has been suggested to alter conformation of the `activation loop' and stabilise it in a non-inhibitory conformation such that the dimerisation-induced phosphorylation of the two tyrosine residues in this region is no longer required for activation (Webster et al., 1996; Mohammadi et al., 1996a,b).
D816V
protein
substitution
true positive
P10721
Bold residues beneath the aligned sequence illustrate disease-causing activating mutations (c-kit: D816V and D820G in mast cell leukaemia (Piao et al., 1996; Pignon et al., 1997); FGFR3: K650E in thanatophoric dysplasia type II (Webster et al., 1996); c-met: V1238I, D1246N and Y1248C in familial renal carcinoma and D1246H, Y1248H and M1268T in sporadic renal carcinoma (Schmidt et al., 1997); RET: M918T and A883F in MEN 2B).
Mutations in the `activation loop' of other receptor tyrosine kinases (D816V in c-kit and K650E in FGFR3; Figure 3) have been demonstrated to cause ligand-independent activation.
V804L
protein
substitution
true positive
P07949
In addition there have been seven families reported with mutations (E768D, V804L) in the intracellular kinase domain (Eng et al., 1995, 1996; Eng and Mulligan, 1997; Bolino et al., 1995).
V1238I
protein
substitution
true negative
P08581-2
Bold residues beneath the aligned sequence illustrate disease-causing activating mutations (c-kit: D816V and D820G in mast cell leukaemia (Piao et al., 1996; Pignon et al., 1997); FGFR3: K650E in thanatophoric dysplasia type II (Webster et al., 1996); c-met: V1238I, D1246N and Y1248C in familial renal carcinoma and D1246H, Y1248H and M1268T in sporadic renal carcinoma (Schmidt et al., 1997); RET: M918T and A883F in MEN 2B).
Further receptor tyrosine kinase mutations in this region (D820G in c-kit and V1238I, D1246N, D1246H, Y1248C, Y1248H, M1268T in c-met; Figure 3) are thought to be activating.
Y1248H
protein
substitution
true positive
P08581-2
Bold residues beneath the aligned sequence illustrate disease-causing activating mutations (c-kit: D816V and D820G in mast cell leukaemia (Piao et al., 1996; Pignon et al., 1997); FGFR3: K650E in thanatophoric dysplasia type II (Webster et al., 1996); c-met: V1238I, D1246N and Y1248C in familial renal carcinoma and D1246H, Y1248H and M1268T in sporadic renal carcinoma (Schmidt et al., 1997); RET: M918T and A883F in MEN 2B).
Further receptor tyrosine kinase mutations in this region (D820G in c-kit and V1238I, D1246N, D1246H, Y1248C, Y1248H, M1268T in c-met; Figure 3) are thought to be activating.
E768D
protein
substitution
true positive
P07949
In addition there have been seven families reported with mutations (E768D, V804L) in the intracellular kinase domain (Eng et al., 1995, 1996; Eng and Mulligan, 1997; Bolino et al., 1995).
M1268T
protein
substitution
true positive
P08581-2
Bold residues beneath the aligned sequence illustrate disease-causing activating mutations (c-kit: D816V and D820G in mast cell leukaemia (Piao et al., 1996; Pignon et al., 1997); FGFR3: K650E in thanatophoric dysplasia type II (Webster et al., 1996); c-met: V1238I, D1246N and Y1248C in familial renal carcinoma and D1246H, Y1248H and M1268T in sporadic renal carcinoma (Schmidt et al., 1997); RET: M918T and A883F in MEN 2B).
Further receptor tyrosine kinase mutations in this region (D820G in c-kit and V1238I, D1246N, D1246H, Y1248C, Y1248H, M1268T in c-met; Figure 3) are thought to be activating.
D820G
protein
substitution
true positive
P10721
Bold residues beneath the aligned sequence illustrate disease-causing activating mutations (c-kit: D816V and D820G in mast cell leukaemia (Piao et al., 1996; Pignon et al., 1997); FGFR3: K650E in thanatophoric dysplasia type II (Webster et al., 1996); c-met: V1238I, D1246N and Y1248C in familial renal carcinoma and D1246H, Y1248H and M1268T in sporadic renal carcinoma (Schmidt et al., 1997); RET: M918T and A883F in MEN 2B).
Further receptor tyrosine kinase mutations in this region (D820G in c-kit and V1238I, D1246N, D1246H, Y1248C, Y1248H, M1268T in c-met; Figure 3) are thought to be activating.
D1246H
protein
substitution
true positive
P08581-2
Bold residues beneath the aligned sequence illustrate disease-causing activating mutations (c-kit: D816V and D820G in mast cell leukaemia (Piao et al., 1996; Pignon et al., 1997); FGFR3: K650E in thanatophoric dysplasia type II (Webster et al., 1996); c-met: V1238I, D1246N and Y1248C in familial renal carcinoma and D1246H, Y1248H and M1268T in sporadic renal carcinoma (Schmidt et al., 1997); RET: M918T and A883F in MEN 2B).
Further receptor tyrosine kinase mutations in this region (D820G in c-kit and V1238I, D1246N, D1246H, Y1248C, Y1248H, M1268T in c-met; Figure 3) are thought to be activating.
Y1248C
protein
substitution
true positive
P08581-2
Bold residues beneath the aligned sequence illustrate disease-causing activating mutations (c-kit: D816V and D820G in mast cell leukaemia (Piao et al., 1996; Pignon et al., 1997); FGFR3: K650E in thanatophoric dysplasia type II (Webster et al., 1996); c-met: V1238I, D1246N and Y1248C in familial renal carcinoma and D1246H, Y1248H and M1268T in sporadic renal carcinoma (Schmidt et al., 1997); RET: M918T and A883F in MEN 2B).
Further receptor tyrosine kinase mutations in this region (D820G in c-kit and V1238I, D1246N, D1246H, Y1248C, Y1248H, M1268T in c-met; Figure 3) are thought to be activating.
M918T
protein
substitution
true positive
P07949
A single RET mutation, resulting in the substitution M918T, has been identied in 94% of cases of MEN 2B (which consists of MTC, pheochromocytoma and developmental abnormalities).
Ninety-four percent of MEN 2B cases (75 of 80 analysed) are caused by a single mutation (M918T) in the tyrosine kinase domain of RET (Eng et al., 1994, 1996; Hofstra et al., 1994; Carlson et al., 1994 and this paper).
The MEN 2B-like mutation M918T occurs in approximately 40% of sporadic MTC.
Activating mutations in the kinase domain of the receptor tyrosine kinases c-kit (D814Y; equivalent to A883F mutation in MEN 2B DP Smith et al 1214 RET codon 898) and FGFR3 (K650E; equivalent to RET codon 908) which also alter the catalytic substrate specicity have recently been reported (Webster et al., 1996; Piao et al., 1996; Su et al., 1997), and in the case of the c-kit mutation the substrate specicity change is related to that seen in M918T-RET.
Consequently we decided to analyse this region of RET (exon 15) in three cases of clinically clear MEN 2B (two de novo and one familial) which do not harbour the M918T mutation.
Results Three patients with clinically clear MEN 2B (see Materials and methods) were screened for the MEN 2B-associated RET mutation M918T as previously described (Eng et al., 1994), and were found to have the wild-type sequence at codon 918.
Seventy-ve of these 80 have an identical mutation, M918T.
Clinically the patients with the A883F mutation have no features to distinguish them from the 94% of MEN 2B patients in which the disease-causing mutation is M918T.
Bold residues beneath the aligned sequence illustrate disease-causing activating mutations (c-kit: D816V and D820G in mast cell leukaemia (Piao et al., 1996; Pignon et al., 1997); FGFR3: K650E in thanatophoric dysplasia type II (Webster et al., 1996); c-met: V1238I, D1246N and Y1248C in familial renal carcinoma and D1246H, Y1248H and M1268T in sporadic renal carcinoma (Schmidt et al., 1997); RET: M918T and A883F in MEN 2B).
(1988) from a two base substitution, and this may account for its rarity relative to the M918T, which results from a single base substitution.
In clinical management, mutation analysis of cases with an MEN 2B phenotype should start with a search for the M918T mutation, and if this is negative, for A883F.
The tumours are similar to those seen in MEN 2A, and are generally ascribed to consitutive activation of the RET kinase; whereas the developmental abnormalities are specic to MEN 2B and are therefore attributed to the altered substrate specicity of the tyrosine kinase which results from the M918T mutation.
The M918T mutation characteristic of MEN 2B also occurs frequently as a somatic mutation in sporadic MTC (approximately 40%), but the cysteine codon mutations characteristic of MEN 2A are rare.
The explanation for this is unclear, but is also speculatively ascribed to the altered substrate specicity of the M918T mutation.
The MEN 2B mutations M918T and A883F lie in the distal half of the split kinase domain, either side of the highly conserved `activation loop' which is seen in receptor and non-receptor tyrosine kinases (Figure 3).
In accordance with this model, introduction of the mutation Y905?F abolishes MEN2A-RET activity, but not M918T-RET activity (Iwashita et al., 1996).
The M918T mutation lies within the substrate binding pocket of the tyrosine kinase domain (Carlson et al., 1995; Hanks et al., 1988), and whereas a M residue is seen at the equivalent position in most A883F mutation in MEN 2B DP Smith et al 1216 receptor tyrosine kinases, a T is seen in most cytoplasmic tyrosine kinases.
Hence it has been predicted that the substrate binding pocket of M918T-RET will be altered so as to prefer the substrates of cytoplasmic kinases.
And indeed the M918T-RET kinase domain does phosphorylate synthetic peptide substrates of cytoplasmic kinases more eciently than the wild-type kinase domain (Songyang et al., 1995).
In addition, changes in M918T-RET autophosphorylation have been observed (Santoro et al., 1995; Liu et al., 1996).
If the developmental abnormalities seen in MEN 2B are due to substrate specicity changes, one would expect that the changes caused by the M918T and A883F mutations would be closely similar.
For example, it will be interesting to determine if the A883F mutant shares with the M918T mutant reduced phosphorylation of the Grb2 docking site Y1096 (Liu et al., 1996), and increased ability to phosphorylate the optimal synthetic substrates of src and abl (Songyang et al., 1995).
A883F
protein
substitution
true positive
P07949
Here we report the identication of a new germline RET mutation (A883F) in two de novo cases of MEN 2B.
Activating mutations in the kinase domain of the receptor tyrosine kinases c-kit (D814Y; equivalent to A883F mutation in MEN 2B DP Smith et al 1214 RET codon 898) and FGFR3 (K650E; equivalent to RET codon 908) which also alter the catalytic substrate specicity have recently been reported (Webster et al., 1996; Piao et al., 1996; Su et al., 1997), and in the case of the c-kit mutation the substrate specicity change is related to that seen in M918T-RET.
No mutation in exon 15 was found in the familial case, in agreement with a previous report (Toogood et al., 1995), but in both de novo cases the mutation A883F was identied.
We have analysed three of the remaining ve cases, and have identied the mutation A883F in two of these.
Both are de novo cases of MEN 2B, and in each case the A883F mutation is a de novo RET mutation not present in either parent.
This is very strong evidence to suggest that A883F is the disease causing mutation in these patients.
In the third case, we have previously reported nding no abnormality on direct sequencing of exons 2 to 20 including exonintron boundaries and the 3' alternative splice junction of RET (Toogood et al., 1995); and here we conrm that the A883F mutation is absent.
Clinically the patients with the A883F mutation have no features to distinguish them from the 94% of MEN 2B patients in which the disease-causing mutation is M918T.
The A883F mutation results A MOTHER CGT A FATHER CGT A MEN 2B CGT T C/T G/T Figure 1 Sequence of codon 883 in a de novo MEN 2B patient (MEN231) and the unaected parents.
Exon 15 amplicons (primers E15-f2 to E15-r2) were sequenced with primer E15-f2 (see Materials and methods) A883F mutation in MEN 2B DP Smith et al 1215 Figure 3 (a) Schematic representation of the RET receptor tyrosine kinase showing the position of disease-causing mutations.
Bold residues beneath the aligned sequence illustrate disease-causing activating mutations (c-kit: D816V and D820G in mast cell leukaemia (Piao et al., 1996; Pignon et al., 1997); FGFR3: K650E in thanatophoric dysplasia type II (Webster et al., 1996); c-met: V1238I, D1246N and Y1248C in familial renal carcinoma and D1246H, Y1248H and M1268T in sporadic renal carcinoma (Schmidt et al., 1997); RET: M918T and A883F in MEN 2B).
In clinical management, mutation analysis of cases with an MEN 2B phenotype should start with a search for the M918T mutation, and if this is negative, for A883F.
Although the A883F mutation has been reported in sporadic MTC, it is uncommon (four of 111 analysed; Eng and Mulligan, 1997; Marsh et al., 1996).
The MEN 2B mutations M918T and A883F lie in the distal half of the split kinase domain, either side of the highly conserved `activation loop' which is seen in receptor and non-receptor tyrosine kinases (Figure 3).
The M918T mutation lies within the substrate binding pocket of the tyrosine kinase domain (Carlson et al., 1995; Hanks et al., 1988), and whereas a M residue is seen at the equivalent position in most A883F mutation in MEN 2B DP Smith et al 1216 receptor tyrosine kinases, a T is seen in most cytoplasmic tyrosine kinases.
The A883F mutation also lies within a region involved in substrate recognition (Hanks et al., 1988), but the F residue is not seen in cytoplasmic kinases, and in fact a F residue at the equivalent position is very uncommon in tyrosine kinases.
If the developmental abnormalities seen in MEN 2B are due to substrate specicity changes, one would expect that the changes caused by the M918T and A883F mutations would be closely similar.
For example, it will be interesting to determine if the A883F mutant shares with the M918T mutant reduced phosphorylation of the Grb2 docking site Y1096 (Liu et al., 1996), and increased ability to phosphorylate the optimal synthetic substrates of src and abl (Songyang et al., 1995).
A883F mutation in MEN 2B DP Smith et al References Asai N, Iwashita T, Matsuyama M and Takahashi M.
D1246N
protein
substitution
true positive
P08581-2
Bold residues beneath the aligned sequence illustrate disease-causing activating mutations (c-kit: D816V and D820G in mast cell leukaemia (Piao et al., 1996; Pignon et al., 1997); FGFR3: K650E in thanatophoric dysplasia type II (Webster et al., 1996); c-met: V1238I, D1246N and Y1248C in familial renal carcinoma and D1246H, Y1248H and M1268T in sporadic renal carcinoma (Schmidt et al., 1997); RET: M918T and A883F in MEN 2B).
Further receptor tyrosine kinase mutations in this region (D820G in c-kit and V1238I, D1246N, D1246H, Y1248C, Y1248H, M1268T in c-met; Figure 3) are thought to be activating.
D814Y
protein
substitution
true negative
Activating mutations in the kinase domain of the receptor tyrosine kinases c-kit (D814Y; equivalent to A883F mutation in MEN 2B DP Smith et al 1214 RET codon 898) and FGFR3 (K650E; equivalent to RET codon 908) which also alter the catalytic substrate specicity have recently been reported (Webster et al., 1996; Piao et al., 1996; Su et al., 1997), and in the case of the c-kit mutation the substrate specicity change is related to that seen in M918T-RET.
really D816Y in P10721
10229868
full text
10454545
full text
Y19F
protein
substitution
true negative
Results of experiments using a nonphosphorylatable CDC28Y19F allele suggested that the checkpoint stimulated two inhibitory pathways, one that promoted phosphorylation at tyrosine 19 (Y19) and a poorly characterized second pathway that did not require Cdc28p Y19 phosphorylation.
Comparison of this allele with the nonphosphorylatable CDC28Y19F allele suggested that Swe1p is still able to inhibit CDC28Y19F in a phosphorylation-independent manner and that both the Y19 phosphorylation-dependent and -independent checkpoint pathways in fact reflect Swe1p inhibition of Cdc28p.
Strain Relevant genotype DLY1 ..........................................................................................................................MATa bar1 JMY1208 ....................................................................................................................MATa zds1 HIS3 zds2 URA3 GAL:MIH1:TRP1 GAP:SWE1:HIS2 JMY1222 ....................................................................................................................MAT zds1 HIS3 zds2 ura3 GAL:MIH1:TRP1 GAP:SWE1:HIS2 JMY1207 ....................................................................................................................MAT zds1 HIS3 zds2 URA3 swe1 LEU2 DLY1028 ....................................................................................................................MATa swe1 LEU2 bar1 DLY115 ......................................................................................................................MAT cdc28-4 JMY1380 ....................................................................................................................MATa CDC28E12K bar1 JMY1390 ....................................................................................................................MATa GAL:SWE1:LEU2 CDC28Y19F bar1 JMY1386 ....................................................................................................................MATa GAL:SWE1:LEU2 CDC28E12K bar1 DLY2626 ....................................................................................................................MATa GAL:SWE1:LEU2 bar1 JMY1362 ....................................................................................................................MATa CDC28E12K:URA3:CDC28 bar1 JMY1364 ....................................................................................................................MATa CDC28Y19F:URA3:CDC28 bar1 JMY1365 ....................................................................................................................MATa CDC28E12K:URA3:CDC28 swe1 LEU2 bar1 JMY1367 ....................................................................................................................MATa CDC28Y19F:URA3:CDC28 swe1 LEU2 bar1 JMY1428 ....................................................................................................................MATa swe1K473P-12Xmyc:TRP1 bar1 JMY1429 ....................................................................................................................MATa CDC28E12K:URA3:CDC28 swe1K473P-12Xmyc:TRP1 bar1 DLY101 ......................................................................................................................MATa cdc28-4 bar1 JMY1455 ....................................................................................................................MATa cdc28-4 GAL:SWE1-12Xmyc:URA3 bar1 JMY1456 ....................................................................................................................MATa cdc28-4 GAL:swe1K473P-12Xmyc:URA3 bar1 RSY16.........................................................................................................................MATa/MAT GAL:GST-CDC28:LEU2/GAL:CLB2:LEU2 RSY17.........................................................................................................................MATa/MAT GAL:GST-CDC28Y19F:LEU2/GAL:CLB2:LEU2 Furthermore, our experiments suggest that catalytically inactive Swe1p can respond to a signal from the morphogenesis checkpoint to inhibit Cdc28p.
The previously described swe1 LEU2 and GAL:SWE1:: LEU2 (7), GAP:SWE1::HIS2 (31), GAL:SWE1-myc::URA3 and GAL:MIH1:: TRP1 (23), zds1 HIS3 (4), GAL:CLB2::LEU2 (34), cdc28-4 (37), zds2 URA3, gal:SWE1K473P-myc::URA3, SWE1K473P-myc::TRP1, CDC28Y19F, and CDC28E12K alleles were introduced into the BF264-15DU background by direct transformation.
To create the CDC28Y19F:URA3:CDC28 allele (strains JMY1364 and JMY1367), the XhoI/BamHI DNA fragment from pSF38 (33) carrying CDC28Y19F as well as approximately 340 bp upstream and 790 bp downstream of the CDC28Y19F ORF was ligated into the corresponding sites of pRS306 (32), a URA3 integrating vector.
To construct and integrate the GAL:GST:CDC28 and GAL:GST:CDC28Y19F alleles (strains RSY16 and RSY17), the glutathione S-transferase (GST) sequence plus the adjacent multiple cloning site from pGEX-KG (14) was amplified by PCR with primers that placed BglII sites at the ends of the PCR product.
A 1.8-kb NdeI (start codon)-to-BamHI (3 of the stop codon) CDC28 fragment from pRD47 (7) and the similar CDC28Y19F NdeI/ BamHI fragment from pSF38 were blunt-end ligated into the XhoI site of YIpG2:GST, placing the CDC28 alleles in frame with GST, to create YIpG2: GST-CDC28 and YIpG2:GST-CDC28-YF, respectively.
4 (pJM1042 [CDC28], pJM1046 [CDC28E12K], and pAL88 [CDC28Y19F]) were made by cloning XhoI/ BamHI fragments containing CDC28 alleles into the corresponding sites of pRS316.
CDC28 was isolated from pRD47, CDC28E12K was isolated from a gap-repaired plasmid rescued from a CDC28E12K mutant (see below), and CDC28Y19F came from pSF38.
Strains DLY2626 (CDC28 GAL: SWE1), JMY1390 (CDC28Y19F GAL:SWE1), and JMY1386 (CDC28E12K GAL: SWE1) were grown in YEPS and induced to express Swe1p by growth for 4 h after addition of galactose (2%).
Yeast strains were grown to a density of 0.5 107 to 1 107 cells/ml in YEPS either at 30C for RSY16 (GAL:CDC28-GST GAL:CLB2) and RSY17 (GAL:CDC28Y19F-GST CHECKPOINT-DEFECTIVE CDC28 MUTANT 5983 GAL:CLB2) or at 24C for DLY101 (cdc28-4 control strain), JMY1455 (GAL: SWE1-myc cdc28-4), and JMY1456 (GAL:SWE1K473P-myc cdc28-4).
To purify GST-Cdc28p and GST-Cdc28pY19F, a 50% glutathione bead slurry (Pharmacia) was added to 500 g of total protein in 1 ml of Nonidet P-40 (NP-40) lysis buffer (50 mM Tris [pH 7.5], 5 mM EDTA, 1 mM sodium pyrophosphate, 150 mM NaCl, 1% NP-40, 1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 2 g each of aprotinin, pepstatin, and leupeptin per ml) with 10 mM dithiothreitol (DTT).
Immediately before use in the kinase assay, the eluted GST-Cdc28p and GST-Cdc28pY19F proteins were diluted 1:1 with reaction buffer (7.5 mM MgCl2, 20 mM Tris [pH 7.5]).
The final kinase reaction mixture contained 7.5 mM MgCl2, 20 mM Tris (pH 7.5), 2 g of histone H1, 0.1 nM ATP, 10 Ci of [ -32P]ATP (3,000 Ci/mmol), 10 l of Swe1p-Myc (or Swe1pK473P-Myc) beads, and 1 l of GSTCdc28p (or GST-Cdc28pY19F) in a total volume of 45 l.
However, Wang and Burke (36) reported that the poor growth and morphologic abnormalities of zds1 zds2 mutants could be rescued by expression of the CDC28Y19F allele that cannot be phosphorylated at Y19.
Both the morphogenesis checkpoint defect exhibited by CDC28E12K and its lack of a cell cycle defect in the unperturbed cycle are reminiscent of the CDC28Y19F mutant that cannot be phosphorylated by Swe1p.
This reagent detected wild-type Cdc28p but not Cdc28pY19F, confirming its specificity for Y19-phosphorylated Cdc28p (Fig.
Comparison of the CDC28Y19F and CDC28E12K mutants.
The repeated independent isolation of the CDC28E12K mutation and the failure to isolate a CDC28Y19F mutation (which would require a single A-to-T change) seemed surprising.
To determine whether this was a chance result or whether there might be phenotypic differences between the E12K and Y19F alleles, we transformed these alleles on a low-copy-number plasmid into the strains used for the genetic screen.
zds1 zds2 mutants containing the CDC28Y19F allele continued to display abnormal morphologies including elongated buds, although to a lesser degree than the parent strain (Fig.
In contrast, cells containing the mutant CDC28E12K allele underwent nuclear division at around 2 h, while cells containing the CDC28Y19F allele underwent nuclear division at around 2.5 h (Fig.
Thus, the CDC28E12K allele was more potent than the CDC28Y19F allele both in rescuing zds1 zds2 cells and in abolishing the morphogenesis checkpoint.
GAL:SWE1 strains DLY2626 (GAL:SWE1), and JMY1386 (CDC28 containing wild-type (DLY2626), Y19F (JMY1390), and E12K (JMY1386) alleles of Cdc28p were induced to overexpress Swe1p by growth for 4 h after addition of Cks1 beads were then used to precipitate the different Cdc28p proteins, which were immunoblotted with anti-phosphogalactose to cells growing in YEPS medium.
In previous studies, we had interpreted the residual checkpoint-induced G2 delay in cells containing the CDC28Y19F allele to indicate the existence of a second pathway capable of inhibiting Cdc28p in response to morphogenesis insults (21).
The CDC28E12K allele fully rescues the zds1 zds2 mutant, while the CDC28Y19F allele only partially rescues cell morphology.
(A) Spot assays of a zds1 zds2 GAL:MIH1 strain (JMY1222) transformed with a centromere plasmid (pRS316) carrying CDC28 or the CDC28E12K or CDC28Y19F alleles, grown on YEPG and YEPD at 30C for 3 days and 2 days, respectively.
Both the CDC28E12K and CDC28Y19F alleles rescue the growth defect in a zds1 zds2 strain.
This finding suggests that both the E12K and Y19F forms of Cdc28p are still sensitive (to a reduced extent) to inhibition by Swe1p: Cdc28pE12K is more resistant to inhibition by Swe1p than Cdc28pY19F.
Booher and colleagues have reported that Swe1p can inhibit Cdc28pY19F in vitro, in a manner that is sensitive to dilution (7).
We confirmed the observation that Swe1p could inhibit Cdc28pY19F as well as wild-type Cdc28p (Fig.
Under these conditions, Swe1p inhibited wild-type and Y19F Cdc28p to similar extents (Fig.
Morphogenesis checkpoint-induced delay of nuclear division in CDC28Y19F and CDC28E12K mutants.
Strains used were DLY1 (SWE1 CDC28), JMY1362 (SWE1 CDC28E12K), JMY1364 (SWE1 CDC28Y19F), DLY1028 (swe1 CDC28), JMY1365 (swe1 CDC28E12K), and JMY1367 (swe1 CDC28Y19F).
(A) Immunoprecipitates containing Swe1p from strains expressing (WT [wild type]) or not expressing ( ) GAL:SWE1-myc were mixed as indicated with GST-Cdc28p/Clb2p (WT) or GST-Cdc28pY19F/Clb2p (Y19F) complexes isolated from yeast and subsequently assayed for Cdc28p-dependent histone H1 kinase activity as described in Materials and Methods.
Swe1p inhibited wild-type and Y19F Cdc28p to similar extents.
Swe1p inhibits the nonphosphorylatable Cdc28pY19F.
Based on previous findings with strains containing the CDC28Y19F allele, we concluded that in addition to cell cycle arrest mediated by phosphorylation of Y19 in Cdc28p, the morphogenesis checkpoint induced a second phosphorylation-independent arrest pathway (21).
The data presented in this report suggest that this second pathway is not distinct, but rather is also mediated by Swe1p, which can still inhibit the Y19F form of Cdc28p.
In that study, the effects of deleting SWE1 were found to be more severe than those associated with the CDC28Y19F allele, leading the authors to speculate that additional targets of Swe1p might be involved.
However, given our findings we suggest that the relative ineffectiveness of the CDC28Y19F allele might simply reflect residual inhibition of the encoded mutant protein by Swe1p.
K473P
protein
substitution
true negative
Strain Relevant genotype DLY1 ..........................................................................................................................MATa bar1 JMY1208 ....................................................................................................................MATa zds1 HIS3 zds2 URA3 GAL:MIH1:TRP1 GAP:SWE1:HIS2 JMY1222 ....................................................................................................................MAT zds1 HIS3 zds2 ura3 GAL:MIH1:TRP1 GAP:SWE1:HIS2 JMY1207 ....................................................................................................................MAT zds1 HIS3 zds2 URA3 swe1 LEU2 DLY1028 ....................................................................................................................MATa swe1 LEU2 bar1 DLY115 ......................................................................................................................MAT cdc28-4 JMY1380 ....................................................................................................................MATa CDC28E12K bar1 JMY1390 ....................................................................................................................MATa GAL:SWE1:LEU2 CDC28Y19F bar1 JMY1386 ....................................................................................................................MATa GAL:SWE1:LEU2 CDC28E12K bar1 DLY2626 ....................................................................................................................MATa GAL:SWE1:LEU2 bar1 JMY1362 ....................................................................................................................MATa CDC28E12K:URA3:CDC28 bar1 JMY1364 ....................................................................................................................MATa CDC28Y19F:URA3:CDC28 bar1 JMY1365 ....................................................................................................................MATa CDC28E12K:URA3:CDC28 swe1 LEU2 bar1 JMY1367 ....................................................................................................................MATa CDC28Y19F:URA3:CDC28 swe1 LEU2 bar1 JMY1428 ....................................................................................................................MATa swe1K473P-12Xmyc:TRP1 bar1 JMY1429 ....................................................................................................................MATa CDC28E12K:URA3:CDC28 swe1K473P-12Xmyc:TRP1 bar1 DLY101 ......................................................................................................................MATa cdc28-4 bar1 JMY1455 ....................................................................................................................MATa cdc28-4 GAL:SWE1-12Xmyc:URA3 bar1 JMY1456 ....................................................................................................................MATa cdc28-4 GAL:swe1K473P-12Xmyc:URA3 bar1 RSY16.........................................................................................................................MATa/MAT GAL:GST-CDC28:LEU2/GAL:CLB2:LEU2 RSY17.........................................................................................................................MATa/MAT GAL:GST-CDC28Y19F:LEU2/GAL:CLB2:LEU2 Furthermore, our experiments suggest that catalytically inactive Swe1p can respond to a signal from the morphogenesis checkpoint to inhibit Cdc28p.
The previously described swe1 LEU2 and GAL:SWE1:: LEU2 (7), GAP:SWE1::HIS2 (31), GAL:SWE1-myc::URA3 and GAL:MIH1:: TRP1 (23), zds1 HIS3 (4), GAL:CLB2::LEU2 (34), cdc28-4 (37), zds2 URA3, gal:SWE1K473P-myc::URA3, SWE1K473P-myc::TRP1, CDC28Y19F, and CDC28E12K alleles were introduced into the BF264-15DU background by direct transformation.
Two PCR fragments (up- and downstream from the K473 position) were amplified by using primers that altered K473 to P (actually, through a transcribing error, one of the primers altered the K to P while the other altered the K to R; subsequent sequencing demonstrated that the mutant generated was K473P).
The two PCR products were then mixed in an overlap PCR with the outside primers to generate a SWE1 fragment with an internal K473P mutation.
The resulting GAL:swe1K473P plasmid (pRAS17) was sequenced to confirm the presence of the K473P mutation and the absence of any other PCR-generated mutations.
The GAL:swe1K473P-myc allele (strain JMY1456) was constructed by first transferring GAL:swe1K473P into the integrating pRS306 vector and then swapping the C terminus and 3 noncoding fragment of SWE1 (KpnI BamHI) in that plasmid with the corresponding fragment from GAL:SWE1-myc (23), generating pRAS12.
To make a SWE1K473P-myc allele transcribed from the SWE1 promoter (strains JMY1428 and JMY1429), the EcoRI/BamHI DNA fragment containing the 3 end of SWE1K473P-myc and downstream sequence was isolated from pRAS12 and ligated into the corresponding sites of pRS304, a TRP1 integrating vector (32).
This plasmid, pJM1050, was digested with BglII, which cuts 5 of the K473P mutation within the SWE1 ORF, to target integration at the SWE1 locus.
19, 1999 Integration creates a full-length SWE1K473P-myc allele under control of the endogenous SWE1 promoter with an adjacent 5 truncated allele of swe1.
Yeast strains were grown to a density of 0.5 107 to 1 107 cells/ml in YEPS either at 30C for RSY16 (GAL:CDC28-GST GAL:CLB2) and RSY17 (GAL:CDC28Y19F-GST CHECKPOINT-DEFECTIVE CDC28 MUTANT 5983 GAL:CLB2) or at 24C for DLY101 (cdc28-4 control strain), JMY1455 (GAL: SWE1-myc cdc28-4), and JMY1456 (GAL:SWE1K473P-myc cdc28-4).
To purify Swe1p-Myc and Swe1pK473P-Myc, 5 l of anti-Myc antibody 9E10 was added to 2.5 mg of total protein in 1 ml of NP-40 lysis buffer and mixed for 1 h at 4C.
The final kinase reaction mixture contained 7.5 mM MgCl2, 20 mM Tris (pH 7.5), 2 g of histone H1, 0.1 nM ATP, 10 Ci of [ -32P]ATP (3,000 Ci/mmol), 10 l of Swe1p-Myc (or Swe1pK473P-Myc) beads, and 1 l of GSTCdc28p (or GST-Cdc28pY19F) in a total volume of 45 l.
To distinguish between these possibilities, we generated a swe1 mutant containing a K473P change in subdomain II of the catalytic domain.
In mixing experiments similar to those described above, we found that Swe1pK473P was capable of inhibiting Cdc28p histone H1 kinase activity to the same extent as wildtype Swe1p (Fig.
Endogenous levels of Swe1pK473P provide a partial checkpoint response.
To test this, we generated a strain in which Myc-tagged Swe1pK473P was expressed at the SWE1 genomic locus from its own promoter (see Materials and Methods).
Cells containing wild-type Swe1p, catalytically inactive Swe1pK473P, or no Swe1p were then synchronized, and their response to actin perturbation by Lat-A was monitored (Fig.
Cells containing Swe1pK473P displayed an intermediate response, delaying but not blocking nuclear division (Fig.
Morphogenesis checkpoint-induced delay of nuclear division in a swe1K473P mutant.
Strains were DLY1 (WT [wild type]), DLY1028 (swe1 ), and JMY1428 (swe1K473P-myc).
(B) Assays were performed as for panel A but with immunoprecipitated Swe1p (WT) or Swe1pK473P (K473P).
10419537
full text
H335F
protein
substitution
true negative
Clones with low background expression of luciferase and high inducibility in the presence of doxycycline were chosen for transfection with the reporter plasmid pUHD 10 3 containing either wild-type PLC- 1 or a lipase inactive PLC- 1 (H335F/H380F), each containing a carboxylterminal HA-tag.
In contrast, when cells expressing a dominant negative H335F/H380F mutant of PLC- 1 under a tetracyclineinducible promoter were incubated with doxycycline, the PI3kinase inhibitor LY294002 effectively inhibited PDGF-BB-induced chemotaxis (Fig.
H380F
protein
substitution
true negative
Clones with low background expression of luciferase and high inducibility in the presence of doxycycline were chosen for transfection with the reporter plasmid pUHD 10 3 containing either wild-type PLC- 1 or a lipase inactive PLC- 1 (H335F/H380F), each containing a carboxylterminal HA-tag.
In contrast, when cells expressing a dominant negative H335F/H380F mutant of PLC- 1 under a tetracyclineinducible promoter were incubated with doxycycline, the PI3kinase inhibitor LY294002 effectively inhibited PDGF-BB-induced chemotaxis (Fig.
Y934F
protein
substitution
true negative
Overactivation of Phospholipase C- 1 Renders Platelet-derived Growth Factor -Receptor-expressing Cells Independent of the Phosphatidylinositol 3-Kinase Pathway for Chemotaxis* (Received for publication, January 13, 1999, and in revised form, May 6, 1999) Lars Ronnstrand, Agneta Siegbahn, Charlotte Rorsman, Matilda Johnell, Klaus Hansen, and Carl-Henrik Heldin From the Ludwig Institute for Cancer Research, Biomedical Centre, Box 595, S-751 24 Uppsala, Sweden and the Department of Clinical Chemistry, University Hospital, S-751 85 Uppsala, Sweden We have previously shown that porcine aortic endothelial cells expressing the Y934F platelet-derived growth factor (PDGF) -receptor mutant respond to PDGF-BB in a chemotaxis assay at about 100-fold lower concentration than do wild-type PDGF -receptor-expressing cells (Hansen, K., Johnell, M., Siegbahn, A., Rorsman, C., Engstrom, U., Wernstedt, C., Heldin, C.-H., and Ronnstrand, L.
To assess whether increased activation of PLC- 1 is the cause of the hyperchemotactic behavior of the Y934F mutant cell line, we constructed cell lines expressing either wildtype or a catalytically compromised version of PLC- 1 under a tetracycline-inducible promoter.
The present study was undertaken to investigate the mechanisms behind the increased chemotactic response in the Y934F mutant cell line.
We present data here that phosphorylation and activation of PLC- 1 are considerably increased in the Y934F mutant receptor cell line.
Two-dimensional tryptic phosphopeptide maps of PLC- 1 from wild-type PDGF -receptor-expressing PAE cells or cells expressing the Y934F mutant PDGF -receptor.
A, PAE/wt cells without PDGF-BB stimulation; B, PAE/wt cells with PDGF-BB stimulation; C, PAE/Y934F cells without PDGF-BB stimulation; and D, PAE/ Y934F cells with PDGF-BB stimulation.
It was also demonstrated that tyrosine phosphorylation of PLC- 1 in response to PDGF-BB was increased in cells expressing the Y934F mutant receptor.
To assess whether the increase in phosphorylation was selective for a particular tyrosine residue, and whether additional phosphorylation sites appeared in cells expressing the Y934F mutant, cells expressing the wild-type PDGF -receptor and the Y934F mutant receptor were labeled with [32P]orthophosphate.
A general increase in the intensity of peptides phosphorylated in response to PDGF-BB could be seen in the Y934F mutant cell line, compared with cells expressing the wild-type PDGF -receptor (Fig.
No selective increase in the phosphorylation of any peptide, nor any new phosphopeptides, were seen in PLC- 1 from Y934F mutant cells.
Independence of PKC and PI3-Kinase for PLC- 1 Activity-- Previously, we have shown that the increased chemotactic response seen in the Y934F mutant receptor cell line could be inhibited by bisindolylmaleimide, a protein kinase C inhibitor, while leaving the wild-type receptor-induced chemotactic response virtually unaffected.
In contrast, an inhibitor of PI3kinase, LY294002, effectively inhibited the chemotactic response seen in wild-type receptor-expressing cells, with little effect on the chemotactic response elicited by the Y934F mutant receptor (5).
The inhibitor of protein kinase C, bisindolylmaleimide, showed no effect on PLC- 1 activity, neither in wild-type receptor cells nor in the Y934F mutant cells, whereas the PI3-kinase inhibitor LY294002 slightly inhibited the PLC- 1 activity in both cell lines.
Effect of LY294002 and GF109203X on PDGF-BB stimulated inositol phosphate formation in wild-type PDGF -receptor-expressing cells and in cells expressing the Y934F mutant PDGF -receptor.
Cells were either treated with Me2SO (control), 20 nM GF109203X (GF), or 2.8 M LY294002 (LY) Establishment of Stable Cell Lines Containing Wild-type or Dominant Negative PLC- 1 under the Control of an Inducible Promoter--In order to investigate whether increased PLC- 1 activity causes increased chemotaxis and thus could explain the hyperchemotactic phenotype of the Y934F mutant cell line, wild-type PDGF -receptor-expressing cells were transfected with wild-type or catalytically compromised HA-tagged human PLC- 1 under the control of a tetracycline-inducible promoter.
Conditions were optimized so that the expression level of PLC- 1 would lead to a response in IP3 release after PDGF-BB stimulation similar to that seen in the Y934F cells (Fig.
In accordance with the Y934F mutant cells, PDGF -receptor cells overexpressing wild-type PLC- 1 responded to PDGF-BB with chemotaxis at concentrations almost 100-fold lower compared with control cells without induction of PLC- 1 expression (Fig.
was seen in cells expressing the Y934F mutant compared with cells expressing wild-type PDGF -receptor (data not shown).
The reason for the increased phosphorylation of PLC- 1 in cells expressing the Y934F mutant PDGF -receptor is not known; it is possible that the receptor kinase in the Y934F mutant has altered kinetics toward PLC- 1 as a substrate.
The magnitude of IP3 production produced in the Y934F mutant cell line was about 3-fold higher than in wild-type receptor-expressing cells.
The chemotactic response seen in the Y934F mutant cell line was, in contrast to the response seen in the wild-type PDGF FIG.
10867021
full text
H402F
protein
substitution
true negative
Inhibitory effects of Pyk2H-K457A (PKM) were less than that of Pyk2H402F.
Y881F
protein
substitution
true positive
P70600
Ltd., Hachioji, Tokyo 192-8512, and the Department of Biochemistry, Cancer Research Institute, Sapporo Medical University School of Medicine, Sapporo 060-8556, Japan We established Jurkat transfectants that overexpress Pyk2 or its mutants, K457A (lysine 457 was mutated to alanine), Pyk2-Y402F (tyrosine 402 to phenylalanine), and Pyk2-Y881F to investigate the role of Pyk2 in T cell activation.
However, no tyrosine phosphorylation of Pyk2-Y402F was detected while more than 60% of the tyrosine phosphorylation was observed in Pyk2-Y881F.
In the clones expressing a mutant Pyk2Y402F (tyrosine 402 to phenylalanine) but not expressing Pyk2-Y881F, both tyrosine phosphorylation and activation of Pyk2 that occur upon TCR stimulation were prevented.
Hematopoietic cell-specific Pyk2 (Pyk2H) and point mutants of kinase-inactive PKM (K457A, lysine 457 mutated to alanine), autophosphorylation site (Y402F, tyrosine 402 to phenylalanine), and Grb2 SH2-binding site (Y881F, tyrosine 881 to phenylalanine) mutants were constructed by PCR-based mutagenesis.
BD, JTPyk2H, JT-K457A, JT-Y402F, and JT-Y881F (107 cells) were stimulated with or without anti-CD3 mAb (OKT3) for 3 min and solubilized by Triton X-100 lysis buffer.
tants that overexpressed Pyk2 (JT-Pyk2), Pyk2H (JT-Pyk2H), and mutant Pyk2Hs including Pyk2H-K457A (lysine 457 was changed to alanine, JT-K457A), Pyk2H-Y402F (tyrosine 402 to phenylalanine, JT-Y402F), Pyk2H-Y881F (tyrosine 881 to phenylalanine, JT-Y881F), or vehicle (pME18s neo) alone (JTpME18s) (Fig.
As expected, substitution of tyrosine residues for phenylalanine abolished the phosphorylation of the corresponding sites (Pyk2Y402F (panel B) and Pyk2-Y881F (panel C)).
Pyk2H-Y402F Prevents Activation of Endogenous Pyk2 upon TCR Stimulation--To investigate further the role of tyrosine 402 and tyrosine 881 in the tyrosine phosphorylation of Pyk2 in the activated T cells, we examined the tyrosine phosphorylation of Pyk2H-Y402F and Pyk2H-Y881F in these Jurkat transfectants after stimulation with OKT3.
2A, no tyrosine phosphorylation was observed in Pyk2H-Y402F, while that of Pyk2H-Y881F was 60 70% of Pyk2H, indicating that tyrosine 402 is indispensable for the phosphorylation of Pyk2 upon T cell activation.
A, the lysates from JT-Pyk2H, JT-Y402F, or JT-Y881F (107 cells) stimulated with anti-CD3 mAb for 3 min were immunoblotted with anti-phosphotyrosine mAb (4G10).
The same numbers of JT-Y881F and JT-Y402F were similarly stimulated with anti-CD3 mAb.
association of Fyn SH2 with Pyk2H or Pyk2H-Y881F but not with Pyk2H-Y402F (Fig.
In both systems, the JNK activity after stimulation was enhanced by 30 80% over control cells in JT-Pyk2, JTPyk2H, and JT-Y881F, while reduced in JT-K457A and JTY402F by 30% and 60%, respectively (Fig.
A, upper, JT-pME18s, JT-Pyk2, JT-Pyk2H, JT-K457A, JTY881F, and JT-Y402F (2 107 cells) were stimulated with anti-CD3 mAb plus anti-CD28 mAb for 5 min.
B, JT-pME18s, JT-Pyk2, JT-Pyk2H, JT-K457A, JT-Y881F, and JT-Y402F (107 cells) were stimulated with anti-CD3 mAb plus anti-CD28 mAb for 10 min.
E (upper), JT-pME18s, JT-Pyk2, JT-Pyk2H, JT-K457A, JT-Y881F, and JT-Y402F (2 107 cells) were stimulated with anti-CD3 mAb plus anti-CD28 mAb for 5 min.
A, upper, JT-pME18s, JT-Pyk2, JT-Pyk2H, JT-K457A, JT-Y881F, and JT-Y402F (2 107 cells) were stimulated with anti-CD3 mAb for 5 min.
B, JTpME18s, JT-Pyk2, JT-Pyk2H, JT-K457A, JT-Y881F, and JT-Y402F (107 cells) were stimulated with anti-CD3 mAb for 10 min.
Some extent of the increase in the amount of IL-2 secreted by JT-Pyk2, JT-Pyk2H, or JT-Pyk2H-Y881F and a weak decrease in that by JT-K457A were reproducibly observed.
A, JT-pME18s, JT-Pyk2, JT-Pyk2H, JT-K457A, JT-Y881F, and JTY402F (2 106 cells) were cultured in the presence of anti-CD3 and anti-CD28 mAbs for 24 h, and the amount of IL-2 in the supernatant was measured by ELISA.
C, JT-pME18s, JT-Pyk2, JT-Pyk2H, JT-Y402F, and JT-Y881F (4 107 cells) were stimulated with anti-CD3 mAb for 3 min and lysed by 1% Brij 97 lysis buffer, followed by immunoprecipitation by antiFLAG mAb.
Some extent of the increase in the IL-2 production was observed in JT-Pyk2, JT-Pyk2H, or JTPyk2H-Y881F and a weak decrease in that in JT-K457A.
Y402F
protein
substitution
true positive
P70600
Ltd., Hachioji, Tokyo 192-8512, and the Department of Biochemistry, Cancer Research Institute, Sapporo Medical University School of Medicine, Sapporo 060-8556, Japan We established Jurkat transfectants that overexpress Pyk2 or its mutants, K457A (lysine 457 was mutated to alanine), Pyk2-Y402F (tyrosine 402 to phenylalanine), and Pyk2-Y881F to investigate the role of Pyk2 in T cell activation.
However, no tyrosine phosphorylation of Pyk2-Y402F was detected while more than 60% of the tyrosine phosphorylation was observed in Pyk2-Y881F.
Pyk2-Y402F inhibited the activation of endogenous Pyk2.
The degree of activation of both c-Jun NH2-terminal kinase and p38 mitogen-activated protein kinase but not extracellular signal-regulated protein kinase after concurrent ligation of T cell antigen receptor and CD28 was reduced by more than 50% in the clones expressing Pyk2-Y402F.
Consistent with this inhibition, IL-2 production was significantly diminished in the Pyk2-Y402Fexpressing clones.
In the clones expressing a mutant Pyk2Y402F (tyrosine 402 to phenylalanine) but not expressing Pyk2-Y881F, both tyrosine phosphorylation and activation of Pyk2 that occur upon TCR stimulation were prevented.
The degree of activation of both JNK and p38 MAP kinase after concurrent ligation of TCR and CD28 was also reduced by more than 50% in Pyk2H-Y402F-expressing cells.
Hematopoietic cell-specific Pyk2 (Pyk2H) and point mutants of kinase-inactive PKM (K457A, lysine 457 mutated to alanine), autophosphorylation site (Y402F, tyrosine 402 to phenylalanine), and Grb2 SH2-binding site (Y881F, tyrosine 881 to phenylalanine) mutants were constructed by PCR-based mutagenesis.
BD, JTPyk2H, JT-K457A, JT-Y402F, and JT-Y881F (107 cells) were stimulated with or without anti-CD3 mAb (OKT3) for 3 min and solubilized by Triton X-100 lysis buffer.
tants that overexpressed Pyk2 (JT-Pyk2), Pyk2H (JT-Pyk2H), and mutant Pyk2Hs including Pyk2H-K457A (lysine 457 was changed to alanine, JT-K457A), Pyk2H-Y402F (tyrosine 402 to phenylalanine, JT-Y402F), Pyk2H-Y881F (tyrosine 881 to phenylalanine, JT-Y881F), or vehicle (pME18s neo) alone (JTpME18s) (Fig.
As expected, substitution of tyrosine residues for phenylalanine abolished the phosphorylation of the corresponding sites (Pyk2Y402F (panel B) and Pyk2-Y881F (panel C)).
Pyk2H-Y402F Prevents Activation of Endogenous Pyk2 upon TCR Stimulation--To investigate further the role of tyrosine 402 and tyrosine 881 in the tyrosine phosphorylation of Pyk2 in the activated T cells, we examined the tyrosine phosphorylation of Pyk2H-Y402F and Pyk2H-Y881F in these Jurkat transfectants after stimulation with OKT3.
2A, no tyrosine phosphorylation was observed in Pyk2H-Y402F, while that of Pyk2H-Y881F was 60 70% of Pyk2H, indicating that tyrosine 402 is indispensable for the phosphorylation of Pyk2 upon T cell activation.
Pyk2H-Y402F prevents activation of endogenous Pyk2 upon TCR stimulation.
A, the lysates from JT-Pyk2H, JT-Y402F, or JT-Y881F (107 cells) stimulated with anti-CD3 mAb for 3 min were immunoblotted with anti-phosphotyrosine mAb (4G10).
The same numbers of JT-Y881F and JT-Y402F were similarly stimulated with anti-CD3 mAb.
C, the lysates from JT-pME18s and JT-Y402F (4 107 cells) stimulated or not with anti-CD3 mAb for 3 min were immunoprecipitated with anti-Pyk2 Ab (N-19) and immunoblotted with 4G10.
D, the same immunoprecipitates as in B from JT-pME18s, JT-Y402F and JT-K457A (4 107 cells) were subjected to in vitro kinase reactions using poly(Glu-Tyr) (4:1) as an exogenous substrate.
association of Fyn SH2 with Pyk2H or Pyk2H-Y881F but not with Pyk2H-Y402F (Fig.
2 (C and D), Pyk2HY402F provided a dominant inhibitory effect on both tyrosine phosphorylation and activation of endogenous Pyk2.
2D) of endogenous Pyk2 were significantly prevented in the TCR-stimulated JT-Pyk2-Y402F cells as compared with those of JT-pME18s.
In both systems, the JNK activity after stimulation was enhanced by 30 80% over control cells in JT-Pyk2, JTPyk2H, and JT-Y881F, while reduced in JT-K457A and JTY402F by 30% and 60%, respectively (Fig.
These inhibitory effects of Pyk2H-K457A and Pyk2H-Y402F were confirmed by using three to four independent clones that ex- FIG.
A, upper, JT-pME18s, JT-Pyk2, JT-Pyk2H, JT-K457A, JTY881F, and JT-Y402F (2 107 cells) were stimulated with anti-CD3 mAb plus anti-CD28 mAb for 5 min.
B, JT-pME18s, JT-Pyk2, JT-Pyk2H, JT-K457A, JT-Y881F, and JT-Y402F (107 cells) were stimulated with anti-CD3 mAb plus anti-CD28 mAb for 10 min.
C, 3 clones of JT-pME18s, 3 clones of JT-K457A, and 4 clones of JT-Y402F (2 107 cells) were stimulated with anti-CD3 mAb plus CD28 mAb for 5 min.
E (upper), JT-pME18s, JT-Pyk2, JT-Pyk2H, JT-K457A, JT-Y881F, and JT-Y402F (2 107 cells) were stimulated with anti-CD3 mAb plus anti-CD28 mAb for 5 min.
In the respective clones, about 30% (JT-K457A) and 60% (JT-Y402F) of the JNK activity were reduced compared with that of JT-pME18s clones.
A, upper, JT-pME18s, JT-Pyk2, JT-Pyk2H, JT-K457A, JT-Y881F, and JT-Y402F (2 107 cells) were stimulated with anti-CD3 mAb for 5 min.
B, JTpME18s, JT-Pyk2, JT-Pyk2H, JT-K457A, JT-Y881F, and JT-Y402F (107 cells) were stimulated with anti-CD3 mAb for 10 min.
Similar with the JNK activation described above, p38 MAP kinase activation was significantly inhibited in JT-Y402F and JT-457A.
The amount of IL-2 in the culture supernatant of JT-Y402F was significantly reduced (p 0.05), as shown in Fig.
A, JT-pME18s, JT-Pyk2, JT-Pyk2H, JT-K457A, JT-Y881F, and JTY402F (2 106 cells) were cultured in the presence of anti-CD3 and anti-CD28 mAbs for 24 h, and the amount of IL-2 in the supernatant was measured by ELISA.
B, JT-pME18s, JT-K457A, and JT-Y402F (107 cells) were cultured for 7 h in the absence (none) or presence of anti-CD3 and anti-CD28 mAbs, and their total RNA were extracted by acid guanidinium-phenolchloroform method followed by reverse transcription using hexanucleotide primers to obtain template cDNA for PCR.
consistent with the secreted amount of IL-2, the level of IL-2 mRNA was significantly low in Pyk2-Y402F.
Thus, the amount of IL-2 mRNA was reduced by 50 60% in JT-Pyk2-Y402F, while slight decrease was observed in JT-Pyk2-K457A.
C, JT-pME18s, JT-Pyk2, JT-Pyk2H, JT-Y402F, and JT-Y881F (4 107 cells) were stimulated with anti-CD3 mAb for 3 min and lysed by 1% Brij 97 lysis buffer, followed by immunoprecipitation by antiFLAG mAb.
Overexpression of Pyk2H-Y402F significantly prevented IL-2 production by Jurkat.
Pyk2H-Y402F inhibited at most 60 70% of these responses.
One is that Pyk2-dependent pathway is the major one leading to activation of JNK but exogenous Pyk2H-Y402F cannot prevent activation of Pyk2 completely, as demonstrated in Fig.
K457A
protein
substitution
true positive
P70600
Ltd., Hachioji, Tokyo 192-8512, and the Department of Biochemistry, Cancer Research Institute, Sapporo Medical University School of Medicine, Sapporo 060-8556, Japan We established Jurkat transfectants that overexpress Pyk2 or its mutants, K457A (lysine 457 was mutated to alanine), Pyk2-Y402F (tyrosine 402 to phenylalanine), and Pyk2-Y881F to investigate the role of Pyk2 in T cell activation.
Pyk2 as well as kinase-inactive Pyk2-K457A, was phosphorylated at tyrosine residues 402, 580, and 881 upon T cell antigen receptor cross-linking, indicating that these residues are phosphorylated by other tyrosine kinase(s).
Hematopoietic cell-specific Pyk2 (Pyk2H) and point mutants of kinase-inactive PKM (K457A, lysine 457 mutated to alanine), autophosphorylation site (Y402F, tyrosine 402 to phenylalanine), and Grb2 SH2-binding site (Y881F, tyrosine 881 to phenylalanine) mutants were constructed by PCR-based mutagenesis.
BD, JTPyk2H, JT-K457A, JT-Y402F, and JT-Y881F (107 cells) were stimulated with or without anti-CD3 mAb (OKT3) for 3 min and solubilized by Triton X-100 lysis buffer.
tants that overexpressed Pyk2 (JT-Pyk2), Pyk2H (JT-Pyk2H), and mutant Pyk2Hs including Pyk2H-K457A (lysine 457 was changed to alanine, JT-K457A), Pyk2H-Y402F (tyrosine 402 to phenylalanine, JT-Y402F), Pyk2H-Y881F (tyrosine 881 to phenylalanine, JT-Y881F), or vehicle (pME18s neo) alone (JTpME18s) (Fig.
It is noted that a kinase-negative mutant, Pyk2H-K457A (PKM) underwent tyrosine phosphorylation at Tyr-402, Tyr-881, and Tyr-580 to similar levels as Pyk2H (Fig.
D, the same immunoprecipitates as in B from JT-pME18s, JT-Y402F and JT-K457A (4 107 cells) were subjected to in vitro kinase reactions using poly(Glu-Tyr) (4:1) as an exogenous substrate.
PKM(K457A) also inhibited the activation of Pyk2 (Fig.
Kinase activity of Pyk2H-K457A was negligible, and that of Pyk2H-Y402 was little activated upon TCR stimulation (Fig.
In both systems, the JNK activity after stimulation was enhanced by 30 80% over control cells in JT-Pyk2, JTPyk2H, and JT-Y881F, while reduced in JT-K457A and JTY402F by 30% and 60%, respectively (Fig.
These inhibitory effects of Pyk2H-K457A and Pyk2H-Y402F were confirmed by using three to four independent clones that ex- FIG.
A, upper, JT-pME18s, JT-Pyk2, JT-Pyk2H, JT-K457A, JTY881F, and JT-Y402F (2 107 cells) were stimulated with anti-CD3 mAb plus anti-CD28 mAb for 5 min.
B, JT-pME18s, JT-Pyk2, JT-Pyk2H, JT-K457A, JT-Y881F, and JT-Y402F (107 cells) were stimulated with anti-CD3 mAb plus anti-CD28 mAb for 10 min.
C, 3 clones of JT-pME18s, 3 clones of JT-K457A, and 4 clones of JT-Y402F (2 107 cells) were stimulated with anti-CD3 mAb plus CD28 mAb for 5 min.
E (upper), JT-pME18s, JT-Pyk2, JT-Pyk2H, JT-K457A, JT-Y881F, and JT-Y402F (2 107 cells) were stimulated with anti-CD3 mAb plus anti-CD28 mAb for 5 min.
In the respective clones, about 30% (JT-K457A) and 60% (JT-Y402F) of the JNK activity were reduced compared with that of JT-pME18s clones.
A, upper, JT-pME18s, JT-Pyk2, JT-Pyk2H, JT-K457A, JT-Y881F, and JT-Y402F (2 107 cells) were stimulated with anti-CD3 mAb for 5 min.
B, JTpME18s, JT-Pyk2, JT-Pyk2H, JT-K457A, JT-Y881F, and JT-Y402F (107 cells) were stimulated with anti-CD3 mAb for 10 min.
Some extent of the increase in the amount of IL-2 secreted by JT-Pyk2, JT-Pyk2H, or JT-Pyk2H-Y881F and a weak decrease in that by JT-K457A were reproducibly observed.
A, JT-pME18s, JT-Pyk2, JT-Pyk2H, JT-K457A, JT-Y881F, and JTY402F (2 106 cells) were cultured in the presence of anti-CD3 and anti-CD28 mAbs for 24 h, and the amount of IL-2 in the supernatant was measured by ELISA.
B, JT-pME18s, JT-K457A, and JT-Y402F (107 cells) were cultured for 7 h in the absence (none) or presence of anti-CD3 and anti-CD28 mAbs, and their total RNA were extracted by acid guanidinium-phenolchloroform method followed by reverse transcription using hexanucleotide primers to obtain template cDNA for PCR.
Thus, the amount of IL-2 mRNA was reduced by 50 60% in JT-Pyk2-Y402F, while slight decrease was observed in JT-Pyk2-K457A.
Inhibitory effects of Pyk2H-K457A (PKM) were less than that of Pyk2H402F.
Some extent of the increase in the IL-2 production was observed in JT-Pyk2, JT-Pyk2H, or JTPyk2H-Y881F and a weak decrease in that in JT-K457A.
In our study, the equivalent degree of tyrosine phosphorylation occurred in the kinase inactive form of Pyk2H, PKM (Pyk2HK457A), suggesting that tyrosine kinase other than Pyk2 phosphorylates Pyk2.
10825157
full text
Y182V
protein
substitution
true negative
Mutagenesis of the three putative high affinity Crk SH2 binding motifs in the N-terminal region of paxillin (Tyr31Ser-Tyr-Pro, Tyr118-Ser-Phe-Pro, and Tyr182-Val-Ile-Pro (26)) virtually abrogated NGF-inducible paxillin phosphorylation (Fig.
Y222F
protein
substitution
true negative
Transient expression of a c-Crk II Tyr222 point mutant (c-Crk Y222F) in 293T cells induces hyperphosphorylation of paxillin on Tyr31 and enhances complex formation between c-Crk Y222F and paxillin as well as c-Crk Y222F and c-Abl, suggesting that c-Crk II Tyr222 phosphorylation induces both the dissociation of the Crk SH2 domain from paxillin and the Crk SH3 domain from c-Abl.
PC12 cells overexpressing c-Crk Y222F manifested a defect in cellular adhesion and neuritogenesis that led to detachment of cells from the extracellular matrix, thus demonstrating the biological significance of c-Crk II tyrosine phosphorylation in NGF-dependent morphogenesis.
To generate clonal PC12 cell lines stably overexpressing Crk protein, cells were transfected with 18 g of pEBG-c-Crk II or pEBG-c-Crk Y222F plasmid DNA and 2 g of pMEXneo using the LipofectAMINE reagent (Life Technologies, Inc.), and cell lines were expanded as described previously (4).
We found that a high level of c-Crk II or c-Crk Y222F expression was unstable in PC12 cells over multiple passage.2 Therefore, cell lines were routinely monitored for consistent levels of of c-Crk II or c-Crk Y222F expression, and when necessary, aliquots of early passaged lines were re-established from liquid nitrogen stocks.
Generation of and Infections with Recombinant c-Crk-containing Retroviruses--A cDNA encoding full-length c-Crk II or c-Crk Y222F was subcloned into a bicistronic pCX-bsr retroviral vector in which c-Crk II and green fluorescent protein (GFP) are expressed from the same transcript via an internal ribosome entry site.
The significance of this mechanism is highlighted by the findings that a c-Crk II Tyr222 mutant (c-Crk Y222F), while causing constitutive complex formation between c-Crk and paxillin as well as c-Crk and c-Abl, impairs NGF-dependent cellular spreading and neurite outgrowth.
TrkAoverexpressing PC12-615 cells, c-Crk II overexpressing PC12 cells, and c-Crk Y222F-expressing PC12 cells were maintained in DMEM containing 10% calf serum and 5% horse serum with 200 g/ml G418.
Briefly, avian c-crk II and c-crk Y222F were subcloned into the 2 M.
Immunofluorescence and Confocal Microscopy--Wild-type PC12 and c-Crk II- or c-Crk Y222F-expressing PC12 cells were plated on glass coverslips coated with collagen type IV and cultured in the presence of NGF for the indicated times (5).
Toward this goal, wild-type c-Crk II or the point mutant of c-Crk II at Tyr222 (c-Crk Y222F) were coexpressed with TrkA in 293T cells.
Using either an anti-phosphotyrosine (Tyr(P)) antisera or phospho-c-Crk-specific Tyr222 (pCrk) antisera, we found that wild-type c-Crk II, but not c-Crk Y222F, was phosphorylated upon NGF treatment (Fig.
D, 293T cells were co-transferred with 1.0 g of cDNA encoding TrkA and 0.2 g of either c-Crk II or c-Crk Y222F constructs as indicated.
To examine the effect of c-Crk Tyr222 phosphorylation on the cellular turnover of Crk/paxillin complexes, 293T cells co-expressing TrkA, FLAG-tagged paxillin, and wild-type c-Crk II or Y222F c-Crk were analyzed (Fig.
Expression of c-Crk Y222F resulted in an increased level of cel- c-Crk Phosphorylation and NGF Signaling 24791 FIG.
3A, lanes 2 and 3 in anti-Tyr(P) (anti-pTyr) blot), and a much greater proportion of the total pool of c-Crk Y222F existed as a complex with paxillin compared with the wild-type c-Crk II, as evident from co-immunoprecipitation analysis (Fig.
Given the hyperphosphorylation of paxillin in cells expressing Y222F c-Crk II and the fact that paxillin can serve as a substrate for Abl following NGF stimulation, we investigated the effect of Y222F c-Crk II on the cellular interaction between c-Abl and the SH3 domain of Crk (Fig.
When 293T cells expressing wild-type c-Abl and c-Crk Y222F were immunoprecipitated with anti-Crk antibodies, the activity of c-Abl associated with c-Crk Y222F was more than 4-fold greater than that associated with equivalent amounts of wild-type c-Crk II (Fig.
Expression of c-Crk Y222F leads to increased paxillin tyrosine phosphorylation and results in enhanced Crk/paxillin association.
A, 293T cells were cotransfected with plasmid vectors encoding FLAG-tagged paxillin and TrkA together with wild-type c-Crk II or c-Crk Y222F as indicated.
B, c-Crk Y222F increases complex formation between Crk and paxillin.
Expression of c-Crk Y222F enhances the association of Crk with Abl and with paxillin.
The lower two panels showed the appropriate expression of c-Abl and wild-type c-Crk II or c-Crk Y222F in the total lysate.
co-precipitating with c-Crk Y222F under these conditions (Fig.
4A were immunoprecipitated with anti-FLAG antibodies (to precipitate paxillin), there was also a much greater pool of Abl co-precipitating with paxillin in c-Crk II Y222F-expressing 293T cells, as well as a greater pool of Crk in these complexes (Fig.
D, PC12-615 cells were infected with recombinant retroviral vectors for GFP (lane 1), wild-type c-Crk II (lanes 2 and 3), or c-Crk Y222F (lanes 4 and 5).
NGF-induced Dissociation of c-Crk II-Paxillin Complex in PC12 Cells Is Also Inhibited by c-Crk Y222F--The transient transfection experiments in 293T cell system suggests a model in which c-Crk II Tyr222 phosphorylation is required for the regulated turnover of Crk-paxillin-Abl protein complexes.
To test this hypothesis, wild-type c-Crk II or c-Crk Y222F were transiently expressed in PC12-615 cells using recombinant Crk-expressing retrovirus (Fig.
In contrast, expression of c-Crk Y222F promoted Crk/paxillin association in the basal state and resulted in hy- perphosphorylation of paxillin.
Mutation of c-Crk II Tyr222 Impairs Cellular Adhesion and NGF-induced Neuritogenesis--The enhanced interaction of hyperphosphorylated paxillin with c-Crk Y222F could either positively or negatively modulate the cytoskeletal dynamics that are critical for NGF-induced neuritogenesis.
To examine the effects of c-Crk Y222F overexpression during NGF-inducible neuritogenesis, wild-type c-Crk II and c-Crk Y222F were expressed in PC12 cells by stable gene transfer.
Independent PC12 cell lines overexpressing c-Crk II or c-Crk Y222F at 23 times the endogenous c-Crk II levels were clonally expanded (Fig.
Whereas parental PC12 cells or c-Crk II-overexpressing PC12 cells demonstrated rapid c-Crk II tyrosine phosphorylation following NGF stimulation, no endogenous c-Crk II tyrosine phosphorylation was observed in NGF-treated c-Crk Y222F-expressing cells (Fig.
When NGF-stimulated neurite outgrowth was compared between the three cell types, we found that the c-Crk Y222F-expressing PC12 cells tended to form aggregates with short or no visible neuritic processes, even after 72 h of NGF treatment (Fig.
To determine if paxillin localization was perturbed upon c-Crk Y222F expression, immunolocalization of paxillin was performed.
With Y222F c-Crk-expression, 24794 c-Crk Phosphorylation and NGF Signaling FIG.
c-Crk Y222F inhibits NGFdependent neuritogenesis and causes paxillin mislocalization.
A, Wild-type and c-Crk Y222F-expressing PC12 cells were treated for 5 min in the presence or absence of 100 ng/ml NGF as indicated.
B, PC12 cells (panels i-iii), c-Crk II-expressing PC12 cells (panels iv-vi), or c-Crk Y222Fexpressing PC12 cells (panels vii-ix) were treated with 50 ng/ml NGF for 72 h (top 3 panels) or 36 h (bottom 6 panels).
The possible mislocalization of paxillin in c-Crk Y222F-expressing PC12 cells together with the established role of paxillin and c-Crk II in focal adhesion assembly and in integrinmediated cellular adhesion prompted us to evaluate the effects of Y222F c-Crk on cell attachment and adherence to extracellular matrix (Fig.
In contrast, c-Crk Y222F-expressing PC12 cells were significantly impaired in their ability to attach to collagen IV (approximately 25%) yet maintained ability to adhere to poly-D-lysine.
Since a decrease in substrate adhesion might have a direct effect on cellular flattening, an area/height ratio analysis was carried out as an independent criterion for assessing the effect of c-Crk Y222F on PC12 cytoskeleton (Fig.
Using BSA-DiI (which stains plasma membranes) followed by optical sectioning with confocal microscopy, we found that both native and c-Crk II-expressing PC12 cells exhibit similar area/height ratios, whereas c-Crk Y222F expression changes the cell shape from flat and polygonal to round, as revealed by the decrease ( 50%) in area/height ratios.
Because stable cell lines exhibiting defects in cellular adhe- sion may result in instability of c-Crk expression with passaging, transient overexpression of c-Crk II or c-Crk Y222F in the PC12-615 cells with the marker gene GFP was utilized for further analysis (Fig.
Consistent with the results using stable PC12 transfectants, GFP-positive cells overexpressing c-Crk Y222F exhibited a round morphology compared with those expressing GFP alone (not shown).
Similarly, in a quantitative cell attachment assay, the ratio of poorly adherent/ attached GFP-positive cells was approximately 3-fold higher in the Crk Y222F-expressing cells compared with the vector alone-infected cells, whereas cells overexpressing wild-type cCrk II exhibited a modest increase in detachment (a 1.6-fold increase).
Overexpression of c-Crk Y222F in cells stabilized an association between c-Crk II and paxillin and c-Abl, resulting in persistent complex formation of these proteins and hyperphosphorylation of paxillin and led to a defect in cell adhesion and neuritogenesis in PC12 cells.
c-Crk Y222F impairs integrin-mediated cell attachment and causes detachment of PC12 cells.
A, control PC12 cells, c-Crk II, or c-Crk Y222F-expressing PC12 cells were plated for 6 h on either 1.0 mg/ml BSA or 2 mg/ml collagen IV-coated surfaces as indicated and assayed for attachment by a quantitative cell attachment assay.
B, serial x-y confocal sections obtained from parental PC12 cells, c-Crk II expressing PC12 cells, or c-Crk Y222F-expressing PC12 cells (from left to right).
C, PC12 cells were infected with recombinant retroviral vectors expressing GFP alone or coexpressing GFP with either wild-type c-Crk II or c-Crk Y222F as in Fig.
Interesting, in a recent study with CrkL, Y207F CrkL, which corresponds to Y222F in c-Crk II, increases the level of paxillin tyrosine phosphorylation compared with native CrkL (18), suggesting there may be functional redundancy of Crk proteins regulating cell adhesion.
In the c-Crk Y222F-expressing PC12 cells, hyperphosphorylated paxillin is unable to interact with focal contacts and actin microfilaments in the lamellipodia of advancing growth cones.
The present findings that c-Crk Y222F overexpression manifests an apparent defect in cytoskeletal organization, despite leading to persistent complex formation between Crk, paxillin, and c-Abl implies that purely inductive signaling is insufficient for the effects of Crk on cellular adhesion and migration.
Although a definitive proof of this hypothesis may require "real time" analysis, the enhanced association of hyperphosphorylated paxillin and c-Abl with the c-Crk Y222F mutant (this study) is consistent with the dynamic molecular interactions, as suggested by this model.
Accordingly, when the pathway required for turnover is impaired upon introduction of the Y222F mutation, c-Abl-mediated phosphorylation of paxillin is not counterbalanced by the formation of phospho-c-Crk II monomer.
As a result, phosphorylated paxillin remains associated with c-Crk (Y222F) c-Crk Phosphorylation and NGF Signaling Acknowledgments--We thank Christopher Turner, Steven Hanks, David Baltimore, David Kaplan, Hisataka Sabe, Tohru Ouchi, and Tadashi Yamamoto for generous gifts of reagents and Kathrin Kirsch and Hidesaburo Hanafusa for critical comments on the manuscript.
Y31F
protein
substitution
true negative
The following sequences (5 -3 ) of the sense mutagenic oligonucleotides were used, with mismatches indicated in uppercase: for the Y31F mutation, gag gaa acg cct tTc tcc tac cca act g; for Y118F, gag gaa cac gtg tTc agc ttc cca aac; and for Y182F, g acc gga cct cac tT t gtc atc cca gag.
Paxillin mutants containing a Y31F substitution exhibited the most significant reduction ( 70%) in Trk A-dependent phosphorylation after NGF treatment (Fig.
To demonstrate that Tyr31 represents the major NGF-inducible Crk binding motif in paxillin, 293T cells co-expressing TrkA and c-Crk II with either FLAG-tagged paxillin, FLAG-tagged Y31F paxillin, or FLAG-tagged paxillin Y31F,Y118F,Y182F paxillin, were immunoprecipitated with anti-Crk antibodies (Fig.
As indicated, mutation of Tyr31 in paxillin significantly disrupted binding to c-Crk II, which was not further abrogated upon co-expression of Y31F,Y118F,Y182F paxillin with TrkA and c-Crk II (Fig.
In the inset, the relative binding of c-Crk II to wild-type paxillin, Y31F paxillin, or Y31F,Y118F,Y182F paxillin is compared.
K295M
protein
substitution
true negative
Expression plasmids for TrkA (pcDNA3-trkA), kinase-dead c-Src (pBabe K295M src), or kinase-dead FAK (pCR K454M FAK) were provided by David Kaplan (McGill University), Hisataka Sabe (Osaka Bioscience institute), and Steven Hanks (University of Virginia), respectively.
Moreover, when paxillin was co-transfected with TrkA in 293T cells in the presence of either kinase-deficient c-Abl (K290M c-Abl), kinase-deficient FAK (K454M FAK), or kinasedeficient-Src (K295M c-Src), K290M c-Abl exhibited the most significant inhibition in Trk A-induced paxillin phosphorylation (Fig.
Y118F
protein
substitution
true negative
The following sequences (5 -3 ) of the sense mutagenic oligonucleotides were used, with mismatches indicated in uppercase: for the Y31F mutation, gag gaa acg cct tTc tcc tac cca act g; for Y118F, gag gaa cac gtg tTc agc ttc cca aac; and for Y182F, g acc gga cct cac tT t gtc atc cca gag.
To demonstrate that Tyr31 represents the major NGF-inducible Crk binding motif in paxillin, 293T cells co-expressing TrkA and c-Crk II with either FLAG-tagged paxillin, FLAG-tagged Y31F paxillin, or FLAG-tagged paxillin Y31F,Y118F,Y182F paxillin, were immunoprecipitated with anti-Crk antibodies (Fig.
As indicated, mutation of Tyr31 in paxillin significantly disrupted binding to c-Crk II, which was not further abrogated upon co-expression of Y31F,Y118F,Y182F paxillin with TrkA and c-Crk II (Fig.
In the inset, the relative binding of c-Crk II to wild-type paxillin, Y31F paxillin, or Y31F,Y118F,Y182F paxillin is compared.
K416M
protein
substitution
true negative
293T cells were contransfected with vectors encoding paxillin and TrkA and one of the kinase-deficient constructs encoding c-Abl (K290M), FAK (K454M), or c-Src (K416M) as indicated.
Y207F
protein
substitution
true negative
Interesting, in a recent study with CrkL, Y207F CrkL, which corresponds to Y222F in c-Crk II, increases the level of paxillin tyrosine phosphorylation compared with native CrkL (18), suggesting there may be functional redundancy of Crk proteins regulating cell adhesion.
Y182F
protein
substitution
true negative
The following sequences (5 -3 ) of the sense mutagenic oligonucleotides were used, with mismatches indicated in uppercase: for the Y31F mutation, gag gaa acg cct tTc tcc tac cca act g; for Y118F, gag gaa cac gtg tTc agc ttc cca aac; and for Y182F, g acc gga cct cac tT t gtc atc cca gag.
1B), whereas a Y113F,Y182F paxil- lin double mutant with an intact Tyr31 was less affected.
To demonstrate that Tyr31 represents the major NGF-inducible Crk binding motif in paxillin, 293T cells co-expressing TrkA and c-Crk II with either FLAG-tagged paxillin, FLAG-tagged Y31F paxillin, or FLAG-tagged paxillin Y31F,Y118F,Y182F paxillin, were immunoprecipitated with anti-Crk antibodies (Fig.
As indicated, mutation of Tyr31 in paxillin significantly disrupted binding to c-Crk II, which was not further abrogated upon co-expression of Y31F,Y118F,Y182F paxillin with TrkA and c-Crk II (Fig.
In the inset, the relative binding of c-Crk II to wild-type paxillin, Y31F paxillin, or Y31F,Y118F,Y182F paxillin is compared.
K454M
protein
substitution
true negative
Expression plasmids for TrkA (pcDNA3-trkA), kinase-dead c-Src (pBabe K295M src), or kinase-dead FAK (pCR K454M FAK) were provided by David Kaplan (McGill University), Hisataka Sabe (Osaka Bioscience institute), and Steven Hanks (University of Virginia), respectively.
Moreover, when paxillin was co-transfected with TrkA in 293T cells in the presence of either kinase-deficient c-Abl (K290M c-Abl), kinase-deficient FAK (K454M FAK), or kinasedeficient-Src (K295M c-Src), K290M c-Abl exhibited the most significant inhibition in Trk A-induced paxillin phosphorylation (Fig.
293T cells were contransfected with vectors encoding paxillin and TrkA and one of the kinase-deficient constructs encoding c-Abl (K290M), FAK (K454M), or c-Src (K416M) as indicated.
Y113F
protein
substitution
true negative
1B), whereas a Y113F,Y182F paxil- lin double mutant with an intact Tyr31 was less affected.
K290M
protein
substitution
true positive
P00520-4
Wild-type and kinase-deficient (K290M) murine type IV c-abl (43) were provided by David Baltimore as described previously (44) and were subcloned into pcDNA3 (Invitrogen).
To determine whether NGF-inducible paxillin tyrosine phosphorylation is also dependent on c-Abl, FLAG-tagged paxillin and TrkA were transiently co-expressed with either wild-type c-Abl or the kinase-deficient mutant K290M c-Abl (Fig.
However, expression of K290M c-Abl abolished the phosphorylation of paxillin following Trk A activation (lane 4).
Indeed, both wild-type and K290M c-Abl formed stable complexes with paxillin in co-precipitation analysis (Fig.
2B, anti-Abl blot; lanes 2 4), although as expected, no paxillinassociated kinase activity was detected when K290M c-Abl was expressed (Fig.
Moreover, when paxillin was co-transfected with TrkA in 293T cells in the presence of either kinase-deficient c-Abl (K290M c-Abl), kinase-deficient FAK (K454M FAK), or kinasedeficient-Src (K295M c-Src), K290M c-Abl exhibited the most significant inhibition in Trk A-induced paxillin phosphorylation (Fig.
293T cells were contransfected with vectors encoding paxillin and TrkA and one of the kinase-deficient constructs encoding c-Abl (K290M), FAK (K454M), or c-Src (K416M) as indicated.
Y118S
protein
substitution
true negative
Mutagenesis of the three putative high affinity Crk SH2 binding motifs in the N-terminal region of paxillin (Tyr31Ser-Tyr-Pro, Tyr118-Ser-Phe-Pro, and Tyr182-Val-Ile-Pro (26)) virtually abrogated NGF-inducible paxillin phosphorylation (Fig.
Y31S
protein
substitution
true negative
Mutagenesis of the three putative high affinity Crk SH2 binding motifs in the N-terminal region of paxillin (Tyr31Ser-Tyr-Pro, Tyr118-Ser-Phe-Pro, and Tyr182-Val-Ile-Pro (26)) virtually abrogated NGF-inducible paxillin phosphorylation (Fig.
W170K
protein
substitution
true negative
No Abl activity associated with the c-Crk W170K SH3 domain mutant (not shown), confirming previous studies that the interaction between Abl and Crk is dependent on the Crk SH3 domain (17).
12444546
full text
S17N
protein
substitution
true negative
To further clarify the involvement of Ras and Rap in the PLCe-dependent accumulation of IP3, a dominant-negative mutant of Ras (HaRasS17N) or a Rap GTPase-activating protein (SPA-1) was expressed in BaF3-PDGFR(Y977F/Y989F)/PLCe Figure 3 Activation of Ras and Rap following PDGF stimulation.
Ha-RasS17N potently inhibited the PDGFtriggered IP3 production, but a slight increase in the IP3 level was observed with delayed time kinetics at 10 to 20 min.
BaF3-PDGFR(Y977F/ Y989F)/PLCe cells were mock-infected (circles) or infected with a retrovirus expressing Ha-RasS17N (triangles) or SPA-1 (squares).
(e) Effect of Ha-RasS17N and SPA-1.
Inhibition of Ras- and Rapmediated activation of PLCe by retroviral expression of Ha-RasS17N and SPA-1, respectively, almost completely abolished cell growth, suggesting that PLCe activities in distinct subcellular regions, such as the plasma membrane and the Golgi apparatus, are indispensable for cell growth (Figure 6d).
In contrast, Ras mediates the rapid and transient Oncogene activation of PLCe as demonstrated by the effect of Ha-RasS17N (Figure 5).
Notably, PDGF-dependent growth of PLCe-expressing BaF3-derived cells was totally sensitive to the inhibitory action of Ha-RasS17N or SPA-1 (Figure 6).
The HaRasS17N cDNA was subcloned into the retroviral vector pLPCX containing the puromycin-resistant gene (Clontech), generating pLPCX-Ha-RasS17N.
Retroviral gene transfer The packaging cell line Phoenix Eco (American Type Culture Collection No; SD3444) was transfected with pLPCX-HaRasS17N and pLXSN-FLAG-SPA-1 and cultured in the presence of puromycin (1 mg/ml, for pLPCX-Ha-RasS17N) or G418 (0.3 mg/ml, for pLXSN-FLAG-SPA-1) for 2 weeks.
BaF3-PDGFR(Y977F/Y989F)/PLCe cells infected with the Ha-RasS17N virus were selected by puromycin (1 mg/ml).
Expression of Ha-RasS17N and SPA-1 was confirmed by immunoblotting.
G12V
protein
substitution
true negative
Ha-RasG12V or its effector region mutant Ha-RasG12V,Y32F was expressed with or without PLCe in COS-7 cells, and inositol phosphates were quantitated.
Rap1AG12V or its effector region mutant Rap1AG12V,D38A was expressed with or without PLCe in COS-7 cells, and inositol phosphates were quantitated.
(n=3) Ras- and Rap1-dependent activation of PLCe C Song et al 8107 2001), coexpression of a constitutively active form of Ha-Ras, Ha-RasG12V, resulted in an increase in the level of inositol phosphates in PLCe-expressing COS-7 cells, reflecting the activation of PLCe (Figure 1a).
An effector region mutant of Ha-Ras, Ha-RasG12V,Y32F, which did not bind to PLCe (Song et al., 2001), exerted no effect on inositol phosphates production, supporting a notion that the observed activation of PLCe is mediated through direct binding (Figure 1a).
Similarly, an activated form of Rap1A (Rap1AG12V), but not its effector region mutant (Rap1AG12V,D38A) that did not bind to PLCe (data not shown), increased the level of inositol phosphates (Figure 1b).
Hemagglutinin (HA)-tagged small GTPases (Ha-Ras, Ha-RasG12V, Ha-RasG12V,Y32F, Rap1AG12V, Rap1AG12V,D38A, Rap2BG12V, RinG29V and MRasQ71L) were subcloned into the mammalian expression vector pEF-BOS (Mizushima and Nagata, 1990).
D38A
protein
substitution
true negative
Rap1AG12V or its effector region mutant Rap1AG12V,D38A was expressed with or without PLCe in COS-7 cells, and inositol phosphates were quantitated.
Similarly, an activated form of Rap1A (Rap1AG12V), but not its effector region mutant (Rap1AG12V,D38A) that did not bind to PLCe (data not shown), increased the level of inositol phosphates (Figure 1b).
Hemagglutinin (HA)-tagged small GTPases (Ha-Ras, Ha-RasG12V, Ha-RasG12V,Y32F, Rap1AG12V, Rap1AG12V,D38A, Rap2BG12V, RinG29V and MRasQ71L) were subcloned into the mammalian expression vector pEF-BOS (Mizushima and Nagata, 1990).
Y32F
protein
substitution
true negative
Ha-RasG12V or its effector region mutant Ha-RasG12V,Y32F was expressed with or without PLCe in COS-7 cells, and inositol phosphates were quantitated.
An effector region mutant of Ha-Ras, Ha-RasG12V,Y32F, which did not bind to PLCe (Song et al., 2001), exerted no effect on inositol phosphates production, supporting a notion that the observed activation of PLCe is mediated through direct binding (Figure 1a).
Hemagglutinin (HA)-tagged small GTPases (Ha-Ras, Ha-RasG12V, Ha-RasG12V,Y32F, Rap1AG12V, Rap1AG12V,D38A, Rap2BG12V, RinG29V and MRasQ71L) were subcloned into the mammalian expression vector pEF-BOS (Mizushima and Nagata, 1990).
Y1009F
protein
substitution
true positive
P09619
The cDNA encoding full-length PLCe or PLCe lacking the N-terminal portion including the CDC25 homology domain (PLCeDN) was introduced into a hematopoietic BaF3 cell line-derived clone carrying the PDGF receptor mutant PDGFR(Y977F/Y989F) (designated BaF3-PDGFR(Y977F/Y989F)) (Satoh et al., 1993).
Resulting stable transfectant clones (designated BaF3-PDGFR (Y977F/Y989F)/PLCe and BaF3-PDGFR(Y977F/ Y989F)/PLCeDN, respectively) were subjected to further analyses.
In BaF3-PDGFR(Y977F/Y989F)/PLCe cells, PDGF caused a rapid and transient increase in Ha-RasGTP with a peak level at 1 to 5 min (Figure 3a).
In BaF3-PDGFR(Y977F/Y989F)/ PLCeDN cells, PDGF-dependent Rap1GTP formation became transient as shown in Figure 3c, suggesting that the CDC25 homology domain is required for the sustained Rap1 activation observed in the cells expressing full-length PLCe.
The time course of Rap1 activation in BaF3-PDGFR(Y977F/Y989F) cells was similar to that of BaF3-PDGFR(Y977F/Y989F)/ PLCeDN cells (data not shown).
To further clarify the involvement of Ras and Rap in the PLCe-dependent accumulation of IP3, a dominant-negative mutant of Ras (HaRasS17N) or a Rap GTPase-activating protein (SPA-1) was expressed in BaF3-PDGFR(Y977F/Y989F)/PLCe Figure 3 Activation of Ras and Rap following PDGF stimulation.
(a) BaF3-PDGFR(Y977F/Y989F)/PLCe cells were transiently transfected with the HA-tagged Ha-Ras cDNA.
(b) The GTP-bound form of endogenous Rap1 in BaF3PDGFR(Y977F/Y989F)/PLCe cells was detected by the pulldown assay following PDGF stimulation for indicated times.
(c) The GTP-bound form of endogenous Rap1 in BaF3PDGFR(Y977F/Y989F)/PLCeDN cells was detected by the pulldown assay following PDGF stimulation for indicated times.
(a) BaF3-PDGFR(Y977F/Y989F) cells were stimulated with IL-3 (circles) or PDGF (triangles), and intracellular IP3 levels were quantitated.
(b) BaF3-PDGFR(Y977F/Y989F)/PLCe cells were stimulated with IL-3 (circles) or PDGF (triangles), and intracellular IP3 levels were quantitated.
(c) BaF3PDGFR(Y977F/Y989F)/PLCeDN cells were stimulated with IL3 (circles) or PDGF (triangles), and intracellular IP3 levels were quantitated.
Indeed, prolonged IP3 production was impaired in BaF3PDGFR(Y977F/Y989F)/PLCeDN cells, and consequently the time course resembled that observed in the SPA-1-expressing cells (Figures 4c and 5).
Cell number increase of two BaF3PDGFR(Y977F/Y989F)/PLCe cell clones (#8 and #10) in the presence or absence of PDGF is illustrated in Figure 6.
PDGF did not stimulate growth of BaF3PDGFR(Y977F/Y989F) and BaF3-PDGFR(Y977F/ Y989F)/vector cells.
Additionally, growth rates of the two clones in the presence of IL-3 were higher compared to that of BaF3PDGFR(Y977F/Y989F) cells.
Apoptosis of the two clones was also prevented when stimulated by PDGF although BaF3-PDGFR(Y977F/Y989F) cells underwent apoptosis in the presence of PDGF.
Therefore, Figure 5 Effect of dominant-negative Ras and the Rap GTPaseactivating protein SPA-1 on PDGF-induced production of IP3 in BaF3-PDGFR(Y977F/Y989F)/PLCe cells.
BaF3-PDGFR(Y977F/ Y989F)/PLCe cells were mock-infected (circles) or infected with a retrovirus expressing Ha-RasS17N (triangles) or SPA-1 (squares).
(a) Proliferation of BaF3-PDGFR(Y977F/Y989F) cells.
BaF3PDGFR(Y977F/Y989F) cells (1.06105 cells) were cultivated in the presence of FBS (circles), FBS plus PDGF (triangles) or FBS plus IL-3 (squares), and cell numbers were counted.
(b) Proliferation of BaF3-PDGFR(Y977F/ Y989F)/vector cells.
BaF3-PDGFR(Y977F/Y989F)/vector cells were cultivated as in (a).
(c) Proliferation of BaF3-PDGFR(Y977F/Y989F)/PLCe (clone #8) cells.
BaF3-PDGFR(Y977F/Y989F)/PLCe (clone #8) cells were cultivated as in (a).
(d) Proliferation of BaF3-PDGFR(Y977F/Y989F)/PLCe (clone #10) cells.
BaF3PDGFR(Y977F/Y989F)/PLCe (clone #10) cells were cultivated as in (a).
BaF3-PDGFR(Y977F/Y989F)/PLCe (clone #8) cells were infected with retroviruses expressing indicated proteins.
BaF3-PDGFR(Y977F/Y989F)/PLCe (clone #8) cells did not undergo apoptosis under growth factor-deprived conditions (Figure 6b), which may be due to basal unstimulated activity of PLCe at a high expression level.
BaF3-PDGFR(Y977F/Y989F) cells (Satoh et al., 1993) were cultured in RPMI 1640 supplemented with 10% FBS, IL-3 (approximately 1 nM) and G418 (0.3 mg/ ml).
pFLAG-CMV2-PLCe, pFLAG-CMV2-PLCeDN or pFLAG-CMV2 (vector) was introduced into BaF3PDGFR(Y977F/Y989F) cells with a hygromycin B-resistant marker plasmid by electroporation essentially as described (Satoh et al., 1993).
Stable transfectants (designated BaF3-PDGFR(Y977F/Y989F)/PLCe, BaF3-PDGFR(Y977F/ Y989F)/PLCeDN and BaF3-PDGFR(Y977F/Y989F)/vector, respectively) were selected and cultured in RPMI 1640 supplemented with 10% FBS, IL-3 (approximately 1 nM), G418 (0.3 mg/ml) and hygromycin B (0.5 mg/ml).
BaF3-PDGFR(Y977F/Y989F)/PLCe cells infected with the Ha-RasS17N virus were selected by puromycin (1 mg/ml).
In vivo GEF (pull-down) assay BaF3-PDGFR(Y977F/Y989F)/PLCe cells transfected with pEF-BOS-HA-Ha-Ras by electroporation (for Ha-Ras) or References Baron CL and Malhotra V.
BaF3-PDGFR(Y977F/Y989F)/PLCe cells (for Rap1) were serum-starved in RPMI 1640 supplemented with bovine serum albumin (1 mg/ml) for 2 h.
G29V
protein
substitution
true negative
Hemagglutinin (HA)-tagged small GTPases (Ha-Ras, Ha-RasG12V, Ha-RasG12V,Y32F, Rap1AG12V, Rap1AG12V,D38A, Rap2BG12V, RinG29V and MRasQ71L) were subcloned into the mammalian expression vector pEF-BOS (Mizushima and Nagata, 1990).
Y1021F
protein
substitution
true positive
P09619
The cDNA encoding full-length PLCe or PLCe lacking the N-terminal portion including the CDC25 homology domain (PLCeDN) was introduced into a hematopoietic BaF3 cell line-derived clone carrying the PDGF receptor mutant PDGFR(Y977F/Y989F) (designated BaF3-PDGFR(Y977F/Y989F)) (Satoh et al., 1993).
Resulting stable transfectant clones (designated BaF3-PDGFR (Y977F/Y989F)/PLCe and BaF3-PDGFR(Y977F/ Y989F)/PLCeDN, respectively) were subjected to further analyses.
In BaF3-PDGFR(Y977F/Y989F)/PLCe cells, PDGF caused a rapid and transient increase in Ha-RasGTP with a peak level at 1 to 5 min (Figure 3a).
In BaF3-PDGFR(Y977F/Y989F)/ PLCeDN cells, PDGF-dependent Rap1GTP formation became transient as shown in Figure 3c, suggesting that the CDC25 homology domain is required for the sustained Rap1 activation observed in the cells expressing full-length PLCe.
The time course of Rap1 activation in BaF3-PDGFR(Y977F/Y989F) cells was similar to that of BaF3-PDGFR(Y977F/Y989F)/ PLCeDN cells (data not shown).
To further clarify the involvement of Ras and Rap in the PLCe-dependent accumulation of IP3, a dominant-negative mutant of Ras (HaRasS17N) or a Rap GTPase-activating protein (SPA-1) was expressed in BaF3-PDGFR(Y977F/Y989F)/PLCe Figure 3 Activation of Ras and Rap following PDGF stimulation.
(a) BaF3-PDGFR(Y977F/Y989F)/PLCe cells were transiently transfected with the HA-tagged Ha-Ras cDNA.
(b) The GTP-bound form of endogenous Rap1 in BaF3PDGFR(Y977F/Y989F)/PLCe cells was detected by the pulldown assay following PDGF stimulation for indicated times.
(c) The GTP-bound form of endogenous Rap1 in BaF3PDGFR(Y977F/Y989F)/PLCeDN cells was detected by the pulldown assay following PDGF stimulation for indicated times.
(a) BaF3-PDGFR(Y977F/Y989F) cells were stimulated with IL-3 (circles) or PDGF (triangles), and intracellular IP3 levels were quantitated.
(b) BaF3-PDGFR(Y977F/Y989F)/PLCe cells were stimulated with IL-3 (circles) or PDGF (triangles), and intracellular IP3 levels were quantitated.
(c) BaF3PDGFR(Y977F/Y989F)/PLCeDN cells were stimulated with IL3 (circles) or PDGF (triangles), and intracellular IP3 levels were quantitated.
Indeed, prolonged IP3 production was impaired in BaF3PDGFR(Y977F/Y989F)/PLCeDN cells, and consequently the time course resembled that observed in the SPA-1-expressing cells (Figures 4c and 5).
Cell number increase of two BaF3PDGFR(Y977F/Y989F)/PLCe cell clones (#8 and #10) in the presence or absence of PDGF is illustrated in Figure 6.
PDGF did not stimulate growth of BaF3PDGFR(Y977F/Y989F) and BaF3-PDGFR(Y977F/ Y989F)/vector cells.
Additionally, growth rates of the two clones in the presence of IL-3 were higher compared to that of BaF3PDGFR(Y977F/Y989F) cells.
Apoptosis of the two clones was also prevented when stimulated by PDGF although BaF3-PDGFR(Y977F/Y989F) cells underwent apoptosis in the presence of PDGF.
Therefore, Figure 5 Effect of dominant-negative Ras and the Rap GTPaseactivating protein SPA-1 on PDGF-induced production of IP3 in BaF3-PDGFR(Y977F/Y989F)/PLCe cells.
BaF3-PDGFR(Y977F/ Y989F)/PLCe cells were mock-infected (circles) or infected with a retrovirus expressing Ha-RasS17N (triangles) or SPA-1 (squares).
(a) Proliferation of BaF3-PDGFR(Y977F/Y989F) cells.
BaF3PDGFR(Y977F/Y989F) cells (1.06105 cells) were cultivated in the presence of FBS (circles), FBS plus PDGF (triangles) or FBS plus IL-3 (squares), and cell numbers were counted.
(b) Proliferation of BaF3-PDGFR(Y977F/ Y989F)/vector cells.
BaF3-PDGFR(Y977F/Y989F)/vector cells were cultivated as in (a).
(c) Proliferation of BaF3-PDGFR(Y977F/Y989F)/PLCe (clone #8) cells.
BaF3-PDGFR(Y977F/Y989F)/PLCe (clone #8) cells were cultivated as in (a).
(d) Proliferation of BaF3-PDGFR(Y977F/Y989F)/PLCe (clone #10) cells.
BaF3PDGFR(Y977F/Y989F)/PLCe (clone #10) cells were cultivated as in (a).
BaF3-PDGFR(Y977F/Y989F)/PLCe (clone #8) cells were infected with retroviruses expressing indicated proteins.
BaF3-PDGFR(Y977F/Y989F)/PLCe (clone #8) cells did not undergo apoptosis under growth factor-deprived conditions (Figure 6b), which may be due to basal unstimulated activity of PLCe at a high expression level.
BaF3-PDGFR(Y977F/Y989F) cells (Satoh et al., 1993) were cultured in RPMI 1640 supplemented with 10% FBS, IL-3 (approximately 1 nM) and G418 (0.3 mg/ ml).
pFLAG-CMV2-PLCe, pFLAG-CMV2-PLCeDN or pFLAG-CMV2 (vector) was introduced into BaF3PDGFR(Y977F/Y989F) cells with a hygromycin B-resistant marker plasmid by electroporation essentially as described (Satoh et al., 1993).
Stable transfectants (designated BaF3-PDGFR(Y977F/Y989F)/PLCe, BaF3-PDGFR(Y977F/ Y989F)/PLCeDN and BaF3-PDGFR(Y977F/Y989F)/vector, respectively) were selected and cultured in RPMI 1640 supplemented with 10% FBS, IL-3 (approximately 1 nM), G418 (0.3 mg/ml) and hygromycin B (0.5 mg/ml).
BaF3-PDGFR(Y977F/Y989F)/PLCe cells infected with the Ha-RasS17N virus were selected by puromycin (1 mg/ml).
In vivo GEF (pull-down) assay BaF3-PDGFR(Y977F/Y989F)/PLCe cells transfected with pEF-BOS-HA-Ha-Ras by electroporation (for Ha-Ras) or References Baron CL and Malhotra V.
BaF3-PDGFR(Y977F/Y989F)/PLCe cells (for Rap1) were serum-starved in RPMI 1640 supplemented with bovine serum albumin (1 mg/ml) for 2 h.
Q71L
protein
substitution
true negative
Hemagglutinin (HA)-tagged small GTPases (Ha-Ras, Ha-RasG12V, Ha-RasG12V,Y32F, Rap1AG12V, Rap1AG12V,D38A, Rap2BG12V, RinG29V and MRasQ71L) were subcloned into the mammalian expression vector pEF-BOS (Mizushima and Nagata, 1990).
9565426
full text
10473622
full text
P228A
protein
substitution
true negative
The Hck SH2-kinase linker Pro to Ala substitution mutants (P225A single mutant and P225A/P228A double mutant, referred to hereafter as Hck-2PA) were generated using the GeneEditor in vitro site-directed mutagenesis system from Promega.
The Hck linker-tail combination mutant (P225A/P228A/Y501F; Hck-2PAYF) was generated by swapping a unique restriction fragment containing the Hck-YF mutation with the corresponding fragment in Hck-2PA.
mutation belongs to P08631, which is a 'Protein-tyrosine kinase' instead of 'Tyrosine-protein kinase'
Y501F
protein
substitution
true negative
Construction of kinase-defective (K269E; Hck-KE) and tail-activated (Y501F; Hck-YF) mutants of Hck as well as the Nef mutant lacking the proline residues essential for SH3 binding (Nef-PA) has been described elsewhere (23, 25).
The Hck linker-tail combination mutant (P225A/P228A/Y501F; Hck-2PAYF) was generated by swapping a unique restriction fragment containing the Hck-YF mutation with the corresponding fragment in Hck-2PA.
mutation belongs to P08631, which is a 'Protein-tyrosine kinase' instead of 'Tyrosine-protein kinase'
P225A
protein
substitution
true negative
The Hck SH2-kinase linker Pro to Ala substitution mutants (P225A single mutant and P225A/P228A double mutant, referred to hereafter as Hck-2PA) were generated using the GeneEditor in vitro site-directed mutagenesis system from Promega.
The Hck linker-tail combination mutant (P225A/P228A/Y501F; Hck-2PAYF) was generated by swapping a unique restriction fragment containing the Hck-YF mutation with the corresponding fragment in Hck-2PA.
The resulting mutant (Hck-P225A) exhibited less than 10% of the Hck-2PA transforming and tyrosine kinase activities in Rat-2 cells, suggesting that both linker proline residues contribute to negative regulation as predicted by the crystal structure (data not shown).
Unlike Hck-2PA, co-expression of Hck-P225A with Nef resulted in enhanced focus-forming activity, providing additional evidence for regulation by the linker despite loss of Pro-225.
mutation belongs to P08631, which is a 'Protein-tyrosine kinase' instead of 'Tyrosine-protein kinase'
K249E
protein
substitution
true negative
In contrast to our results, however, this c-Src linker mutant (K249E/P250E) showed much lower focus-forming activity when compared with c-Src activated by mutation of the conserved tail tyrosine residue, despite similar kinase activity.
P250E
protein
substitution
true negative
In contrast to our results, however, this c-Src linker mutant (K249E/P250E) showed much lower focus-forming activity when compared with c-Src activated by mutation of the conserved tail tyrosine residue, despite similar kinase activity.
K269E
protein
substitution
true negative
Construction of kinase-defective (K269E; Hck-KE) and tail-activated (Y501F; Hck-YF) mutants of Hck as well as the Nef mutant lacking the proline residues essential for SH3 binding (Nef-PA) has been described elsewhere (23, 25).
W260A
protein
substitution
true negative
(22) changed this Trp to Ala (Hck-W260A mutant) and observed higher specific activity than wild-type Hck.
We observed that Hck-W260A exhibits focus-forming activity in the Rat-2 transformation assay (data not shown).
This difference may result from the altered accessability of the Hck-W260A SH2 and SH3 domains to critical transformation-related signaling partners.
12231543
full text
G12D
protein
substitution
true negative
Experimental Design: To investigate the efficacy of this delivery method in normal and neoplastic lung, an adenoviral vector expressing -galactosidase was administered by jet nebulization to K-rasLA1 mice, which develop lung adenocarcinomas through activation of a latent allele carrying mutant K-ras (G12D).
We conducted studies on K-rasLA1 mice, which carry a latent K-ras allele with two copies of exon 1, one wild type and the other mutant (G12D; Ref.
HB56B cells were transiently transfected for 1 h with expression vectors (5 g) containing K-ras (G12D) or empty vector using LipofectAMINE (Life Technologies, Inc.) followed by infection for 48 h with calcium phosphate-precipitated Ad-MKK4 (KR) (24) or Ad5CMV (104 particles).
We examined the ability of Ad-MKK4 (KR) to inhibit MKK4 activation by K-ras (G12D) in HB56B immortalized HBE cells, which contain no K-ras gene mutations at codons 12, 13, or 61 (22).
HB56B cells were transiently transfected with K-ras (G12D) and infected with Ad-MKK4 (KR).
HB56B cells were transiently transfected with a plasmid containing K-ras (G12D) or empty vector ( ) and infected for 48 h with Ad-MKK4 (KR) or empty virus ( ).
K-ras (G12D) increased JNK activity, and MKK4 (KR) inhibited JNK activation by K-ras (G12D) (Fig.
3), indicating that MKK4 (KR) effectively blocked K-ras (G12D)dependent signaling in HBE cells.
10228162
full text
10228163
full text
D716A
protein
substitution
true negative
HeLa cells expressing wild-type HA-2 (WT, circles), HA-D176A 2 (D716A, squares) or HA-D176A/W421A 2 (D176A/W421A, triangles) mutants were grown in the presence ( Tet) or absence of tetracycline (Tet).
D176A
protein
substitution
true negative
Point mutations D176A and W421A disrupt the interaction of 2 with internalization signals without interfering with incorporation into AP-2 To identify residues in 2 that are essential for the interaction with YXX internalization signals, several 2 mutants were generated.
(B) COS-1 cells transfected with the wild-type or D176A mutant HA-2 were lysed, and the lysates were incubated with glutathioneagarose beads containing GSTEGFR or GST alone.
In GST pull-down experiments with in vitro translated 2, only the D176A mutation completely abolished the 2 interaction with TGN38, whereas the M209A mutation resulted in a partial inhibition (Figure 3A, upper panel).
Surprisingly, binding of 2 transiently expressed in COS-1 cells to GSTEGFR was only moderately affected by D176A mutation (Figure 3B).
That D176A and W421 are directly involved in the interaction with internalization signal peptides has now been demonstrated by the crystal structure data published during the preparation of this manuscript (Owen and Evans, 1998).
As shown in Figure 4D, the D176A/W421A HA-2 was severely impaired in its ability to bind GSTEGFR.
In contrast to truncations, the point mutations D176A and W421A did not affect the ability of 2 to coimmunoprecipitate with -adaptins (Figure 5A).
Figure 5B demonstrates that the double mutant D176A/W421A expressed in HeLa cells was also immunoprecipitated with -adaptins.
As demonstrated for wild-type HA-2 (Figure 2) when the expression of D176A/W421A 2 was induced by tetracycline withdrawal, the mutant displaced endogenous 2 from the cellular AP-2 complexes (Figure 5B).
Inducible overexpression of the D176A/W421A 2 mutant inhibits interaction of AP-2 with the EGF receptor To investigate how the displacement of endogenous 2 by the mutant 2 affects interaction between the whole AP-2 complex and YXX signals, lysates from HeLa cells expressing either wild-type or D176A/W421A 2 were incubated with GSTEGFR.
In contrast, when expression of HA-2-D176A/W421A was induced by the removal of tetracycline, binding of AP-2 to GSTEGFR was abolished completely (Figure 6B).
(B) Wild-type (WT), truncation mutants (C 409, C 420 and C 428) and point mutants D176A and W421A of HA-2 were 35S-labeled by in vitro translation in reticulocyte lysates, and the lysates were incubated with glutathioneagarose beads containing GSTTGN38 or GST alone.
(C) HEK293 cells transfected with wild-type HA-2, D176A or W421 mutants of HA-2 were lysed, and incubated with glutathioneagarose beads containing GSTEGFR or GSTEGFRY974A.
(D) HeLa cells grown in the absence of tetracycline to express wild-type HA-2 or HA-2D176A/W421A mutant were lysed, and the lysates were incubated with glutathioneagarose beads containing GSTEGFR, GST EGFRY974A or GST alone.
(A) HEK293 cells were transfected with wild-type HA-2 (WT), truncation mutants of HA-2 with the last residue being 420 (C 420) or 428 (C 428), HA-2D176A (D176A) or HA-2W412A constructs.
(B) HeLa cells expressing (TET) or not expressing ( TET) HA-2D176A/ W421A were lysed and AP-2 was precipitated using AP.6 antibody.
Figure 6B demonstrates that the EGF receptor interaction with AP-1, detected by the presence of -adaptin in GSTEGFR precipitates, was not affected by the overexpression of D176A/W421A mutant 2.
D176A/W421A 2 mutant inhibits interaction of AP-2 with the EGF receptor C-terminus.
GSTEGFR, GSTEGFRY974A or GST alone were bound to glutathioneagarose beads, and incubated with lysates from HeLa cells expressing (TET) or not expressing ( TET) either wild-type HA-2 (A) or D176A/W421A HA-2 (B).
Three cell lines, inducibly expressing wildtype, single mutant D176A 2 and double mutant D176A/ W421A 2, were examined for the rates of internalization of labeled transferrin and EGF.
HeLa cells expressing wild-type HA-2 (WT, circles), HA-D176A 2 (D716A, squares) or HA-D176A/W421A 2 (D176A/W421A, triangles) mutants were grown in the presence ( Tet) or absence of tetracycline (Tet).
Four single cell clones expressing the D176A/W421A mutant were examined.
The results are representative of several experiments with two clones of cells expressing the D176A/W421A 2 mutant.
transferrin receptor endocytosis as a result of the expression of the D176A/W421A mutant resulted in a 4-fold increase in the surface pool of these receptors (data not shown).
The single mutant D176A 2 imposed an intermediate effect on transferrin endocytosis (Figure 7).
In GST pull-down experiments, however, only mutation D176A significantly affected 2 interaction.
Interestingly, whereas the D176A mutation strongly inhibited 2 binding to the YQRL motif of TGN38, the interaction of this mutant with the YRAL sequence of the EGF receptor was only partially affected.
To suppress completely the interaction of 2 with several types of YXX consensus internalization signals, the double mutant D176A/W421A was used in expression studies.
The crystal structure demonstrates multiple interactions within the binding pocket, possibly explaining why the mutation D176A had only partial effects on the EGF receptor interaction with 2.
However, as the D176A/ W421A2 mutations completely abrogated the binding abilities of full-length 2, region 102125 does not appear to be essential for the interaction with YXX motifs.
M209A
protein
substitution
true negative
In GST pull-down experiments with in vitro translated 2, only the D176A mutation completely abolished the 2 interaction with TGN38, whereas the M209A mutation resulted in a partial inhibition (Figure 3A, upper panel).
W421A
protein
substitution
true negative
Point mutations D176A and W421A disrupt the interaction of 2 with internalization signals without interfering with incorporation into AP-2 To identify residues in 2 that are essential for the interaction with YXX internalization signals, several 2 mutants were generated.
Consistent with this hypothesis, the substitution of W421 by alanine (W421A) completely abolished the interaction of HA-2 with both GSTTGN38 (Figure 4B) and GST EGFR (Figure 4C).
As shown in Figure 4D, the D176A/W421A HA-2 was severely impaired in its ability to bind GSTEGFR.
In contrast to truncations, the point mutations D176A and W421A did not affect the ability of 2 to coimmunoprecipitate with -adaptins (Figure 5A).
Figure 5B demonstrates that the double mutant D176A/W421A expressed in HeLa cells was also immunoprecipitated with -adaptins.
As demonstrated for wild-type HA-2 (Figure 2) when the expression of D176A/W421A 2 was induced by tetracycline withdrawal, the mutant displaced endogenous 2 from the cellular AP-2 complexes (Figure 5B).
Inducible overexpression of the D176A/W421A 2 mutant inhibits interaction of AP-2 with the EGF receptor To investigate how the displacement of endogenous 2 by the mutant 2 affects interaction between the whole AP-2 complex and YXX signals, lysates from HeLa cells expressing either wild-type or D176A/W421A 2 were incubated with GSTEGFR.
In contrast, when expression of HA-2-D176A/W421A was induced by the removal of tetracycline, binding of AP-2 to GSTEGFR was abolished completely (Figure 6B).
(B) Wild-type (WT), truncation mutants (C 409, C 420 and C 428) and point mutants D176A and W421A of HA-2 were 35S-labeled by in vitro translation in reticulocyte lysates, and the lysates were incubated with glutathioneagarose beads containing GSTTGN38 or GST alone.
The W421A mutant consistently displayed high non-specific binding to glutathioneagarose beads.
(D) HeLa cells grown in the absence of tetracycline to express wild-type HA-2 or HA-2D176A/W421A mutant were lysed, and the lysates were incubated with glutathioneagarose beads containing GSTEGFR, GST EGFRY974A or GST alone.
(B) HeLa cells expressing (TET) or not expressing ( TET) HA-2D176A/ W421A were lysed and AP-2 was precipitated using AP.6 antibody.
Figure 6B demonstrates that the EGF receptor interaction with AP-1, detected by the presence of -adaptin in GSTEGFR precipitates, was not affected by the overexpression of D176A/W421A mutant 2.
D176A/W421A 2 mutant inhibits interaction of AP-2 with the EGF receptor C-terminus.
GSTEGFR, GSTEGFRY974A or GST alone were bound to glutathioneagarose beads, and incubated with lysates from HeLa cells expressing (TET) or not expressing ( TET) either wild-type HA-2 (A) or D176A/W421A HA-2 (B).
Three cell lines, inducibly expressing wildtype, single mutant D176A 2 and double mutant D176A/ W421A 2, were examined for the rates of internalization of labeled transferrin and EGF.
HeLa cells expressing wild-type HA-2 (WT, circles), HA-D176A 2 (D716A, squares) or HA-D176A/W421A 2 (D176A/W421A, triangles) mutants were grown in the presence ( Tet) or absence of tetracycline (Tet).
Four single cell clones expressing the D176A/W421A mutant were examined.
The results are representative of several experiments with two clones of cells expressing the D176A/W421A 2 mutant.
transferrin receptor endocytosis as a result of the expression of the D176A/W421A mutant resulted in a 4-fold increase in the surface pool of these receptors (data not shown).
To suppress completely the interaction of 2 with several types of YXX consensus internalization signals, the double mutant D176A/W421A was used in expression studies.
However, as the D176A/ W421A2 mutations completely abrogated the binding abilities of full-length 2, region 102125 does not appear to be essential for the interaction with YXX motifs.
W412A
protein
substitution
true negative
(A) HEK293 cells were transfected with wild-type HA-2 (WT), truncation mutants of HA-2 with the last residue being 420 (C 420) or 428 (C 428), HA-2D176A (D176A) or HA-2W412A constructs.
Y998A
protein
substitution
true positive
P00533
To control for nonspecific interactions, GST alone or a GSTEGFR mutant, whose AP-binding sequence Y974RAL (Sorkin et al., 1996) was mutated to ARAL (GSTEGFRY974A), were used.
It was found that AP-2 obtained from cells expressing wild-type HA-2 bound very efficiently to GSTEGFR and did not bind to either the GST EGFRY974A mutant or GST alone (Figure 6A).
(C) HEK293 cells transfected with wild-type HA-2, D176A or W421 mutants of HA-2 were lysed, and incubated with glutathioneagarose beads containing GSTEGFR or GSTEGFRY974A.
(D) HeLa cells grown in the absence of tetracycline to express wild-type HA-2 or HA-2D176A/W421A mutant were lysed, and the lysates were incubated with glutathioneagarose beads containing GSTEGFR, GST EGFRY974A or GST alone.
GSTEGFR, GSTEGFRY974A or GST alone were bound to glutathioneagarose beads, and incubated with lysates from HeLa cells expressing (TET) or not expressing ( TET) either wild-type HA-2 (A) or D176A/W421A HA-2 (B).
A tyrosine to alanine mutation of the residue equivalent to Tyr974 in the holo-EGF receptor was introduced into GSTEGFR using the QuickChange method to yield GSTEGFRY974A.
GSTEGFR and GSTEGFRY974A were made in E.coli B834 (DE3) pLysS, which were grown in LB (supplemented with ampicillin and chloramphenicol) to A260 ~0.6.
In binding experiments with HA-2 expressed in vivo, beads loaded with GST, GSTEGFR and GSTEGFRY974A were washed once in CMF-PBS and incubated with mammalian cell lysates for 3 h at 4C.
12393381
full text
F131del
protein
substitution
true negative
Biological and clinical data Rearrangement IgH DJ DJ No No No No No No FR3 No VG1JP1/2-VD1VG11JP1/2 Mutated S140 ins Splice site ex4/int4 No Supportive care FR1 No Germ line -- -- Yes AraC-anthracycline No Germ line -- -- Yes AraC-anthracycline Yes No No No Germ line -- -- Yes AraC-anthracycline Yes No Germ line -- -- Yes AraC-anthracycline No No Germ line -- -- Yes AraC-anthracycline No No Mutated R139 ter del No Supportive care No -- -- -- 28 10 -- -- No Mutated D171G del Yes AraC-anthracycline Yes 56 No Mutated R135G del Yes AraC-anthracycline Yes 20 No Mutated C114ins F131del Yes AraC-anthracycline Yes 6 6 20 56 2 2 1 28 11 1 1 TCR AML1 gene mutation First allele Second allele Induction regimen Intensive Treatment CR achievement CR duration, mo Survival, mo AML1 gene alteration Patient No No No Yes No No No Yes No No Sex Age, y Karyotype FLT3 duplication 1 M 45 Not done 2 M 38 46,XY [20] 3 F 64 46,XX [19] ROUMIER et al 4 M 86 Not done 5 F 45 46,XX,inv (3)(q21q26) [1]/45,XX,id.
R174Q
protein
substitution
true negative
Biological and clinical data (continued) Rearrangement IgH No No Germ line Yes AraC-anthracycline No -- TCR AML1 gene mutation First allele Second allele Induction regimen 1 Intensive Treatment CR achievement CR duration, mo AML1 gene alteration Survival, mo Patient No Sex Age, y Karyotype FLT3 duplication 18 M 61 47,XY,i(21)(q10), mar[14]/47, XY,idem,del(3)(p11), der(11)add(11)(p15)add (11)(q25), 18, min.ish der(11)(MLL )[2] /46,XY [1] Yes Yes No No Mutated R139 ter del Yes AraC-anthracycline Yes No No Germ line -- -- Yes AraC-anthracycline No -- 7.5 19 M 64 47,XY, 21, der(21)(q2?) 2 .ish der(21)(AML1 3)[16] 1 10 20 M 29 79,XXYY, 2, 3, 5, 6, 7, 9, 10, 11,15, 15, 16, 17, 18, 21 [4]/ 46,XY [25] No No No No No No Yes No No Germ line -- -- Yes No No Germ line -- -- Yes No VG9J1J2 Mutated G138 ins del No FR3 FR1 No Mutated R139 ter del No No No Germ line -- -- No Supportive care Supportive care Supportive care AraC-anthracycline AraC-anthracycline No VG1J1J2 Germ line -- -- Yes AraC-anthracycline No No Germ line -- -- No Supportive care No Yes No No No No Yes 21 M 76 45,XY, 21.ish (21)(wcp21 ) 1[12]/ 46,XY [21] -- 7 -- -- -- -- 7 1 10 1 1 1 2 8 22 M 53 48,XY, del (3)(p2?), del (3)(p2?), 19 [13]/ 46,XY[18] BLOOD, 15 FEBRUARY 2003 VOLUME 101, NUMBER 4 23 M 77 Not done 24 M 86 Not done 25 M 81 Not done 26 M 38 46,XY, ins(10;?)(q23;?)[3]/ 46, XY [32] 27 M 36 46,XY, t(6;12)(q25;q11), t(8;9)(q24;q33), t(11;16)(q13;q13) [29]/46, XY [2] Yes No No Yes No No No Germ line -- No No Germ line -- No No Germ line -- -- -- -- No No Germ line -- -- FR3 FR1 No Mutated R174Q Ins.
G138C
protein
substitution
true negative
7 [4]/ 46,XX [8] 6 F 43 45,XX,inv (3)(q21q26), 7 [16] 7 F 46 46,XX [20] 8 M 23 46,XY [20] 9 F 49 Not done 10 F 35 45-46,XX X[3],del(5)(q15q33), 7, add(7)(p22), 17, 20, add(20)(p13)[2] mar1, r[cp44]/ 44-46, XX-X[3],idem,del(3)(q21)[3], ?der(3)t(3:?)[3], del(5) (q15q33)[4], 6, 8[4], 14[6], add(16)(q22)[6], 17[2], 17[4],add(17)(p13)[3), m2[7], m3[5][cp]7 Not done Not done No No No Germ line Not done Not done Germ line -- -- No Yes Not done Not done Germ line -- -- No Supportive care Supportive care AraCanthracycline-VP 16 11 F 80 Not done No No No -- -- -- 1 1 1 12 M 80 46,XY [20] 13 M 48 45,XX, 1, add(I)(q?10), 5, 7,der(7)t(7;?)(p?;?), 12, 17, i(17)(q10), 4 mar, dmin [17]/ 46, XY [1] ishder(1) wcp1 , wcp7 , mar1der(1)del(1)(q22) (wcp1 ;wcp5 ),mar2 der(5) (wcp5 ), mar3 der(5) (?:5?;7p14 qter)(wcp5 ;wcp7 ) No Yes No No Germ line No No Mutated R49S -- del(38 bp) -- 14 M 78 47,XY,del(20)(q13.1), mar [5]/ 47,id, der(13;13)(q10;q10) [16] No Yes Supportive care AraC-anthracyclineVP16 No Yes -- 12 1 12 15 M 28 46,XY, ins(10;11)(p14;q14q23).ish der(10)(CALM ,AF10 )[15] / 46,XY [7] No No No No DJ No Mutated Mutated 16 M 78 Not done N119del R135M del G138C Yes No AraC-anthracycline Supportive care No No -- -- 1 1 BLOOD, 15 FEBRUARY 2003 VOLUME 101, NUMBER 4 17 M 78 Not done Table 1.
R49S
protein
substitution
true negative
7 [4]/ 46,XX [8] 6 F 43 45,XX,inv (3)(q21q26), 7 [16] 7 F 46 46,XX [20] 8 M 23 46,XY [20] 9 F 49 Not done 10 F 35 45-46,XX X[3],del(5)(q15q33), 7, add(7)(p22), 17, 20, add(20)(p13)[2] mar1, r[cp44]/ 44-46, XX-X[3],idem,del(3)(q21)[3], ?der(3)t(3:?)[3], del(5) (q15q33)[4], 6, 8[4], 14[6], add(16)(q22)[6], 17[2], 17[4],add(17)(p13)[3), m2[7], m3[5][cp]7 Not done Not done No No No Germ line Not done Not done Germ line -- -- No Yes Not done Not done Germ line -- -- No Supportive care Supportive care AraCanthracycline-VP 16 11 F 80 Not done No No No -- -- -- 1 1 1 12 M 80 46,XY [20] 13 M 48 45,XX, 1, add(I)(q?10), 5, 7,der(7)t(7;?)(p?;?), 12, 17, i(17)(q10), 4 mar, dmin [17]/ 46, XY [1] ishder(1) wcp1 , wcp7 , mar1der(1)del(1)(q22) (wcp1 ;wcp5 ),mar2 der(5) (wcp5 ), mar3 der(5) (?:5?;7p14 qter)(wcp5 ;wcp7 ) No Yes No No Germ line No No Mutated R49S -- del(38 bp) -- 14 M 78 47,XY,del(20)(q13.1), mar [5]/ 47,id, der(13;13)(q10;q10) [16] No Yes Supportive care AraC-anthracyclineVP16 No Yes -- 12 1 12 15 M 28 46,XY, ins(10;11)(p14;q14q23).ish der(10)(CALM ,AF10 )[15] / 46,XY [7] No No No No DJ No Mutated Mutated 16 M 78 Not done N119del R135M del G138C Yes No AraC-anthracycline Supportive care No No -- -- 1 1 BLOOD, 15 FEBRUARY 2003 VOLUME 101, NUMBER 4 17 M 78 Not done Table 1.
D171G
protein
substitution
true negative
Biological and clinical data Rearrangement IgH DJ DJ No No No No No No FR3 No VG1JP1/2-VD1VG11JP1/2 Mutated S140 ins Splice site ex4/int4 No Supportive care FR1 No Germ line -- -- Yes AraC-anthracycline No Germ line -- -- Yes AraC-anthracycline Yes No No No Germ line -- -- Yes AraC-anthracycline Yes No Germ line -- -- Yes AraC-anthracycline No No Germ line -- -- Yes AraC-anthracycline No No Mutated R139 ter del No Supportive care No -- -- -- 28 10 -- -- No Mutated D171G del Yes AraC-anthracycline Yes 56 No Mutated R135G del Yes AraC-anthracycline Yes 20 No Mutated C114ins F131del Yes AraC-anthracycline Yes 6 6 20 56 2 2 1 28 11 1 1 TCR AML1 gene mutation First allele Second allele Induction regimen Intensive Treatment CR achievement CR duration, mo Survival, mo AML1 gene alteration Patient No No No Yes No No No Yes No No Sex Age, y Karyotype FLT3 duplication 1 M 45 Not done 2 M 38 46,XY [20] 3 F 64 46,XX [19] ROUMIER et al 4 M 86 Not done 5 F 45 46,XX,inv (3)(q21q26) [1]/45,XX,id.
N119del
protein
substitution
true negative
7 [4]/ 46,XX [8] 6 F 43 45,XX,inv (3)(q21q26), 7 [16] 7 F 46 46,XX [20] 8 M 23 46,XY [20] 9 F 49 Not done 10 F 35 45-46,XX X[3],del(5)(q15q33), 7, add(7)(p22), 17, 20, add(20)(p13)[2] mar1, r[cp44]/ 44-46, XX-X[3],idem,del(3)(q21)[3], ?der(3)t(3:?)[3], del(5) (q15q33)[4], 6, 8[4], 14[6], add(16)(q22)[6], 17[2], 17[4],add(17)(p13)[3), m2[7], m3[5][cp]7 Not done Not done No No No Germ line Not done Not done Germ line -- -- No Yes Not done Not done Germ line -- -- No Supportive care Supportive care AraCanthracycline-VP 16 11 F 80 Not done No No No -- -- -- 1 1 1 12 M 80 46,XY [20] 13 M 48 45,XX, 1, add(I)(q?10), 5, 7,der(7)t(7;?)(p?;?), 12, 17, i(17)(q10), 4 mar, dmin [17]/ 46, XY [1] ishder(1) wcp1 , wcp7 , mar1der(1)del(1)(q22) (wcp1 ;wcp5 ),mar2 der(5) (wcp5 ), mar3 der(5) (?:5?;7p14 qter)(wcp5 ;wcp7 ) No Yes No No Germ line No No Mutated R49S -- del(38 bp) -- 14 M 78 47,XY,del(20)(q13.1), mar [5]/ 47,id, der(13;13)(q10;q10) [16] No Yes Supportive care AraC-anthracyclineVP16 No Yes -- 12 1 12 15 M 28 46,XY, ins(10;11)(p14;q14q23).ish der(10)(CALM ,AF10 )[15] / 46,XY [7] No No No No DJ No Mutated Mutated 16 M 78 Not done N119del R135M del G138C Yes No AraC-anthracycline Supportive care No No -- -- 1 1 BLOOD, 15 FEBRUARY 2003 VOLUME 101, NUMBER 4 17 M 78 Not done Table 1.
R174G
protein
substitution
true negative
del (5)(q15q35) [1] No No FR3 No No No 58 M 82 Not done Germ line Mutated -- R135ins -- R174G No No Supportive care Supportive care No No -- -- 1 1 BLOOD, 15 FEBRUARY 2003 VOLUME 101, NUMBER 4 59 M 79 Not done BLOOD, 15 FEBRUARY 2003 VOLUME 101, NUMBER 4 CLINICAL AND BIOLOGIC FEATURES OF M0 AML 1281 Table 2.
R135G
protein
substitution
true negative
Biological and clinical data Rearrangement IgH DJ DJ No No No No No No FR3 No VG1JP1/2-VD1VG11JP1/2 Mutated S140 ins Splice site ex4/int4 No Supportive care FR1 No Germ line -- -- Yes AraC-anthracycline No Germ line -- -- Yes AraC-anthracycline Yes No No No Germ line -- -- Yes AraC-anthracycline Yes No Germ line -- -- Yes AraC-anthracycline No No Germ line -- -- Yes AraC-anthracycline No No Mutated R139 ter del No Supportive care No -- -- -- 28 10 -- -- No Mutated D171G del Yes AraC-anthracycline Yes 56 No Mutated R135G del Yes AraC-anthracycline Yes 20 No Mutated C114ins F131del Yes AraC-anthracycline Yes 6 6 20 56 2 2 1 28 11 1 1 TCR AML1 gene mutation First allele Second allele Induction regimen Intensive Treatment CR achievement CR duration, mo Survival, mo AML1 gene alteration Patient No No No Yes No No No Yes No No Sex Age, y Karyotype FLT3 duplication 1 M 45 Not done 2 M 38 46,XY [20] 3 F 64 46,XX [19] ROUMIER et al 4 M 86 Not done 5 F 45 46,XX,inv (3)(q21q26) [1]/45,XX,id.
R135M
protein
substitution
true negative
7 [4]/ 46,XX [8] 6 F 43 45,XX,inv (3)(q21q26), 7 [16] 7 F 46 46,XX [20] 8 M 23 46,XY [20] 9 F 49 Not done 10 F 35 45-46,XX X[3],del(5)(q15q33), 7, add(7)(p22), 17, 20, add(20)(p13)[2] mar1, r[cp44]/ 44-46, XX-X[3],idem,del(3)(q21)[3], ?der(3)t(3:?)[3], del(5) (q15q33)[4], 6, 8[4], 14[6], add(16)(q22)[6], 17[2], 17[4],add(17)(p13)[3), m2[7], m3[5][cp]7 Not done Not done No No No Germ line Not done Not done Germ line -- -- No Yes Not done Not done Germ line -- -- No Supportive care Supportive care AraCanthracycline-VP 16 11 F 80 Not done No No No -- -- -- 1 1 1 12 M 80 46,XY [20] 13 M 48 45,XX, 1, add(I)(q?10), 5, 7,der(7)t(7;?)(p?;?), 12, 17, i(17)(q10), 4 mar, dmin [17]/ 46, XY [1] ishder(1) wcp1 , wcp7 , mar1der(1)del(1)(q22) (wcp1 ;wcp5 ),mar2 der(5) (wcp5 ), mar3 der(5) (?:5?;7p14 qter)(wcp5 ;wcp7 ) No Yes No No Germ line No No Mutated R49S -- del(38 bp) -- 14 M 78 47,XY,del(20)(q13.1), mar [5]/ 47,id, der(13;13)(q10;q10) [16] No Yes Supportive care AraC-anthracyclineVP16 No Yes -- 12 1 12 15 M 28 46,XY, ins(10;11)(p14;q14q23).ish der(10)(CALM ,AF10 )[15] / 46,XY [7] No No No No DJ No Mutated Mutated 16 M 78 Not done N119del R135M del G138C Yes No AraC-anthracycline Supportive care No No -- -- 1 1 BLOOD, 15 FEBRUARY 2003 VOLUME 101, NUMBER 4 17 M 78 Not done Table 1.
9845547
full text
D814V
protein
substitution
true positive
P05532
The identification of the KitD814H mutation in an erythroleukemia is also significant, because it appears that Kit codon 814 mutations are only transforming in some cell types, as KitD814V is unable to transform fibroblasts, in contrast to the wild-type receptor stimulated with SCF.43 Activating mutations have been shown to interfere with the normal cellular processing of the Kit receptor, normally present as 125-kD and 145-kD glycoproteins.
D814H
protein
substitution
true positive
P05532
cDNAs of the full-length coding sequence of the wild-type murine c-kit gene and c-kitD814H were excised from the pCR2.1 vector with Xba I and Kpn I restriction enzymes and ligated into these sites of the expression vector pcDNA3 (Invitrogen).
These showed only the wild-type sequence expressed in ELM-D cells, but both wildtype and mutant sequences at nucleotide 2468 in ELM-I-1 cells, showing that ELM-I-1 cells retain expression of the wild-type Kit receptor in addition to the KitD814H mutant.
Expression of c-kitD814H abrogates the SCF dependence of ELM-D cells.
To determine whether the mutation in c-kit is responsible for the growth factor independence of ELM-I-1 cells, we introduced the c-kitD814H mutation into ELM-D cells.
Expression constructs for c-kit and c-kitD814H were created using the vector pcDNA3 (Invitrogen).
Expression and tyrosine phosphorylation of Kit and MAPK in ELM cells expressing KitD814H and wild-type Kit.
(A) pcDNA3 vector (Invitrogen) and expression constructs for c-kit and c-kitD814H based on pcDNA3 were transfected into the Q2BN Quail fibroblastic cell line and expression of Kit protein was analyzed by Western blotting with anti-Kit antibodies.
(B) Phosphorylation of the Kit receptor in ELM-I-1 cells and ELM-D cells stably transfected with wild-type c-kit or c-kitD814H expression vectors.
In contrast, 10 of 27 clones transfected with the KitD814H mutant cDNA were growth factor independent.
However, only 8 of 18 of the latter clones tested showed detectable expression of the mutant KitD814H vector by RT-PCR (data not shown), and all of these 8 expressing clones continued to proliferate in the absence of SCF.
Thus, in all cases, expression of KitD814H resulted in SCF-independent growth.
Effects of the c-kitD814H mutation on the proliferation and clonegenicity of ELM-D cells.
To determine the effects of the c-kitD814H mutation on the proliferation and survival of ELM cells, the growth of ELM-Kit, ELM-KitDH, and ELM-I-1 cells was compared in a microtiter well proliferation assay and a semisolid medium colony assay in the presence and absence of additional growth factors.
These experiments showed that the KitD814H mutation not only allows ELM cells to continue to proliferate in the absence of SCF, but also gives ELM-KitDH cells a growth advantage compared with ELM-Kit cells maintained in the concentration of SCF we have previously shown to be optimal for ELM growth28 (10 ng/mL; Fig 4A).
Thus, the KitD814H mutation clearly confers a selective advantage over the wild-type Kit in terms of proliferation and clonegenicity.
Effects of the c-kitD814H mutation on the differentiation of ELM-D cells.
One of the obvious characteristics of the ELM-I-1 cells in which the KitD814H mutation was identified is that they do not undergo erythroid differentiation in response to Epo or upon removal from SCF or stroma, as ELM-D cells do.26,28 We therefore investigated the effects of the KitD814H Fig 4.
Expression of c-kitD814H in ELM cells causes SCFindpendence and enhanced proliferation and cloning efficiency.
The effect of KitD814H on the proliferation and differentiation of ELM cells in response to SCF and/or Epo.
Effects of the KitD814H mutation on expression of p66Shc, Fli-1, and Bcl-2.
Besides the c-kitD814H mutation, a number of other differences have been identified between ELM-I-1 and ELM-D cells: they express high levels of the ets protein Fli-126 and the regulator of apoptosis Bcl-2 (J.
We therefore tested whether any of these other changes in ELM-I-1 cell gene expression are induced by expression of the c-kitD814H mutant.
However, neither wild-type Kit nor KitD814H induced expression of Bcl-2 or Fli-1 in ELM cells, compared with the ELM-I-1 control (Fig 6B and C).
Comparison of the effects of wild-type c-kit and c-kitD814H expression in ELM-D cells on (A) the pattern of Shc isoforms expression, (B) bcl-2 mRNA, and (C) Fli-1 protein.
This might be due to the stromal expression of the membrane bound form of SCF causing a more persistent intracellular Kit phosphorylation signal than the soluble form of the ligand.38 Our data here would argue against this, because the KitD814H mutation produces a constantly elevated level of receptor signaling and yet does not inhibit Epo-induced differentiation.
Although a spontaneous D814H mutation has not been previously described, aspartic acid 814 has been identified as a hot spot for c-kit activating mutations, being mutated to a valine or a tyrosine in mast cell lines and mastocytosis.19-21 Mutation of aspartic acid 814 to tyrosine has also been shown to alter the substrate specificity of the Kit Kit RECEPTOR MUTATION IN ELM ERYTHROLEUKEMIA 4805 receptor kinase.39 Aspartic acid 814 lies within the activation loop of the cytoplasmic kinase domain.
Mutagenesis has shown that many amino acids introduced in place of aspartic acid 814 allow some level of ligand-independent kinase activity.42 In these studies, the D814H mutation was described as modestly activating and displayed accelerated ligand-independent degradation relative to the wild-type receptor when expressed in kidney 293 cells.
The identification of the KitD814H mutation in an erythroleukemia is also significant, because it appears that Kit codon 814 mutations are only transforming in some cell types, as KitD814V is unable to transform fibroblasts, in contrast to the wild-type receptor stimulated with SCF.43 Activating mutations have been shown to interfere with the normal cellular processing of the Kit receptor, normally present as 125-kD and 145-kD glycoproteins.
We show that expression of mutant KitD814H in ELM cells results in the reduced expression of p145 and its complete absence when expressed in Quail fibroblasts.
Our data show constitutively high levels of MAPK phosphorylation in unstimulated ELM cells expressing KitD814H and in the stroma-independent ELM-I-1 cells from which the mutant c-kit cDNA was cloned.
Because there is evidence that a quantitatively different level or duration of MAPK activation can result in a qualitatively different cellular response,56 the constant level of MAPK activation caused by the KitD814H mutation may be significant.
Recent data show that, unlike the p46 and p52 Shc isoforms, p66 does not transform fibroblasts and inhibits, rather than enhances, EGF-induced activation of MAPK and the c-fos promoter.32,33 This implicates loss of p66 expression as a potential mechanism that may increase the sensitivity of cells expressing the KitD814H mutant to extracellular stimuli, and this may have been a factor in the selection of growth factor-independent ELM-I-1 cells during ELM erythroleukemia progression.
15166264
full text
D816V
protein
substitution
true positive
P10721
The only major diagnostic criterion for SM is the histological demonstration of compact tissue infiltrates, consisting of at least 10 to 15 mast cells.2 Recently, immunohistochemistry on routinely processed bone marrow biopsy specimens demonstrated that CD25 is expressed exclusively on mast cells of those cases with morphologically and molecular biologically confirmed mastocytosis, but not on mast cells in states of mast cell hyperplasia, enabling an abnormal or neoplastic phenotype of mast cells to be defined.3 CD25 expression on mast cells, prominent spindling of mast cells, and the demonstration of a point mutation of C-KIT (D816V) are three of the four minor criteria that are needed to establish a diagnosis of SM.
of cases 1 1 7 C-KIT mutation (N) D816V (1) D816V (1) D816V (5) D816Y (1) wt (1) wt (1) D816V (2) No amplification D816V (2) wt (1) D816V (1) D816Y (1) D816V (2) 16 CD25+ 1/1 1/1 7/7 RESULTS Regarding the morphological evaluation of routinely processed bone marrow biopsy trephines, SM, irrespective of the subtype, comprised approximately 0.3% of all haematological diagnoses (approximately 19 500), and nearly 1.5% of all myelogenous neoplasms (approximately 4100).
The mast cells coexpressed KIT (CD117) MPS: PVR MPS: CIM AML M1 AML M2 AML M4 Plasma cell myeloma NC (probably MDS/ MPS) Total 1 2 1 2 1 2 2 20 1/1 2/2 1/1 2/2 1/1 2/2 2/2 20 AML, acute myeloid leukaemia; CIM, chronic idiopathic myelofibrosis; CMML, chronic myelomonocytic leukaemia; D816V, Asp816RVal; D816Y, Asp816RTyr; MDS/MPS, myelodysplastic/myeloproliferative syndrome; NC, not classifiable; PVR, polycythaemia vera rubra; RAEB1, refractory anaemia with excess of blasts (510%); RARS, refractory anaemia with ringed sideroblasts; wt, wild-type.
Nevertheless, because three minor criteria (``spindling'', ``expression of CD25'', and ``D816V point mutation'') could be demonstrated, a diagnosis of SM-AHNMD was established.
Because the point mutation D816V was detected in this initial trephine biopsy specimen, this case also fulfilled three minor diagnostic criteria, enabling the diagnosis of SM-AHNMD to be made.
Molecular biological findings revealed the typical point mutation within the C-KIT protooncogene (D816V) in 14 of 20 cases, and uncommon point mutations (D816Y) in a further two cases associated with CMML and AMLM2.
Sixteen of the 20 cases with SM-AHNMD showed a point mutation of the C-KIT protooncogene, mostly of the common type D816V (14 of 16).
Most (14 of 16) of these point mutations belonged to the so called common type, D816V, a finding that agrees with recently published data.17 The uncommon point mutation D816Y was detected twice.
In this regard, it is also noteworthy that imatinib is unlikely to be an effective agent in such patients because the D816V mutation of C-KIT converts KIT to an imatinib resistant kinase receptor.20 This fact is underlined by our findings in a patient with AML type M2 with t(8;21) in whom treatment resulted in long standing complete haematological and cytogenetic remission of the AML but the disclosure and persistence of SM.14 In addition, a patient with AML associated with mast cell leukaemia was reported to achieve complete molecular remission of the AML soon after induction chemotherapy, whereas the mastocytosis persisted after standard chemotherapy and allogeneic stem cell transplantation.22 To summarise, SM-AHNMD is a primarily morphological diagnosis based on a thorough investigation of bone marrow trephine specimens including tryptase and CD25 immunohistochemistry.
D816H
protein
substitution
true positive
P10721
However, the point mutation recently described by Pullarkat et al in three of five (Asp816His) SM-AHNMDs was not found in our patients.18 It also seems that the variability of C-KIT mutations in bone marrow infiltrates of mastocytosis is very much lower than in cutaneous mastocytosis.6 ``Cells coexpressing tryptase and CD25 in the setting of a haematological disease are probably abnormal mast cells and therefore can be regarded as a diagnostic clue for mastocytosis'' In a small proportion of the AHNMDs, other clonal markers were detected--for example, the 8;21 translocation in one of our patients with SM-AMLM2 and monoclonal gammopathies or paraproteinaemias (IgM/k and IgG/l, respectively) in the two patients with SM plasma cell myeloma.14 15 One of these patients presented clinically with a secondary intestinal amyloidosis, and the bone marrow biopsy was then performed to assess or exclude plasma cell myeloma.15 The detection of SM was unexpected in this patient.
D816Y
protein
substitution
true positive
P10721
of cases 1 1 7 C-KIT mutation (N) D816V (1) D816V (1) D816V (5) D816Y (1) wt (1) wt (1) D816V (2) No amplification D816V (2) wt (1) D816V (1) D816Y (1) D816V (2) 16 CD25+ 1/1 1/1 7/7 RESULTS Regarding the morphological evaluation of routinely processed bone marrow biopsy trephines, SM, irrespective of the subtype, comprised approximately 0.3% of all haematological diagnoses (approximately 19 500), and nearly 1.5% of all myelogenous neoplasms (approximately 4100).
The mast cells coexpressed KIT (CD117) MPS: PVR MPS: CIM AML M1 AML M2 AML M4 Plasma cell myeloma NC (probably MDS/ MPS) Total 1 2 1 2 1 2 2 20 1/1 2/2 1/1 2/2 1/1 2/2 2/2 20 AML, acute myeloid leukaemia; CIM, chronic idiopathic myelofibrosis; CMML, chronic myelomonocytic leukaemia; D816V, Asp816RVal; D816Y, Asp816RTyr; MDS/MPS, myelodysplastic/myeloproliferative syndrome; NC, not classifiable; PVR, polycythaemia vera rubra; RAEB1, refractory anaemia with excess of blasts (510%); RARS, refractory anaemia with ringed sideroblasts; wt, wild-type.
Molecular biological findings revealed the typical point mutation within the C-KIT protooncogene (D816V) in 14 of 20 cases, and uncommon point mutations (D816Y) in a further two cases associated with CMML and AMLM2.
Most (14 of 16) of these point mutations belonged to the so called common type, D816V, a finding that agrees with recently published data.17 The uncommon point mutation D816Y was detected twice.
10449770
full text
G306E
protein
substitution
true negative
Wild-type or G306E mutant cDNA encoding the Cbl N-terminal 25351 region was subcloned into the pGEX 4T-3 (Amersham Pharmacia Biotech) and pEBM vector (provided by B.
Moreover, the Cbl-N G306E mutation did not affect the interaction of Cbl-N with SLAP (Fig.
GST-SLAP and myc-Cbl-N (wild type or G306E) were transiently transfected into COS-7 cells.
Both wild-type Cbl-N and the G306E mutant coprecipitated GSTSLAP (Fig.
(A) GST-Cbl-N wildtype or G306E mutant proteins were overexpressed in E.
Cell lysates were incubated with glutathione-Sepharose-bound GST-Cbl-N and GST-G306E.
More interestingly, the well-defined point mutation in Cbl, G306E, disrupted the binding of Cbl-N to ZAP-70 but did not diminish the Cbl-N and SLAP interaction.
The SH2 domain, in which the G306E mutant presides, is responsible for most of the interactions with ZAP-70-specific phosphopeptide, whereas the A-B loop in the four-helix bundle helps to form the binding pocket.
Because the Cbl-N and SLAP interaction does not depend on G306E or tyrosine phosphorylation, it may represent a new form of interaction mediated by the four-helix bundle or EF hand in Cbl-N.
C20H
protein
substitution
true negative
The anti-LAT polyclonal antibody (UpState Biotechnology), anti-Syk mAb (Santa Cruz Biotechnology), antiZAP-70 mAb (Transduction Laboratories, Lexington, KY), antiCD3 mAb (Santa Cruz Biotechnology), horseradish peroxidaselinked antiphosphotyrosine mAb (RC20H, Transduction Laboratories), sheep anti-mouse IgG Dynabeads M-280 (Dynal), and polyclonal rabbit anti-mouse IgG antibody (RAM) (Southern Biotechnology Associates) were purchased from commercial sources.
The c-src kinase activity and the expression of GAL4BD-Cbl-N fusion protein were tested by Western blot analysis of the whole-cell lysates with antiphosphotyrosine antibody (anti-p-Tyr) RC20H and anti-GAL4BD antibody, respectively.
R111K
protein
substitution
true negative
A point mutation in the SH2 domain (R111K) of SLAP markedly diminished this interaction, whereas a 64-aa truncation in the SLAP tail region ( T) had no effect, indicating that the association between SLAP and ZAP-70 is mediated by the SH2 domain of SLAP.
Consistent with our observation in COS-7 cells, the R111K point mutation in the SH2 domain of SLAP abolished its associations with phospho-tyrosyl proteins in activated Jurkat T cells.
10727422
full text
E381A
protein
substitution
true negative
Briefly, 10 l of secreted ExoS ExoS(E381A) were added to HeLa cell lysate and incubated with [$#P]NAD (Dupont-NEN, Stevenage, Herts, U.K.) for 1 h at 37 mC.
To achieve this, wild-type ExoS and a set of ExoS mutants, ExoS(E381A), ExoS(98-232) and ExoS(98232, E381A) were employed (Figure 1A).
Amino acid residue 381 in the catalytic domain of ExoS [ExoS(E381A)] is essential for ADP-ribosyltransferase activity [26,32].
Two further mutants, ExoS(98-232) and ExoS(98-232, E381A), were constructed, based on the shared Cell culture, cell lysis and immunoprecipitation HeLa and NIH3T3 cells were grown in minimal essential medium and RPMI 1640 respectively, supplemented with 10 % (vv) fetal-bovine sera and 100 unitsml penicillin.
(i) Wild-type ExoS [YPIII (pIB251, pTS103)] ; (ii) ExoS mutant harbouring a E381A mutation oExoS(E381A) [YPIII (pIB251, pTS106)]q ; (iii) ExoS mutant with deletion of amino acids 98232 oExoS(98232) [YPIII (pIB251, pTS103, 98232)]q ; (iv) ExoS(E381A) mutant with deletion of amino acids 98232 oExoS(E381A,98232) [YPIII(pIB251, pTS106, 98232)]q.
Lane 1, ExoS (wild type) ; lane 2, ExoS(98232) ; lane 3, ExoS(E381A) ; lane 4, ExoS(E381A, 98232) ; lane 5 : mock HeLa cell infection [YPIII (pIB251)] ; lane 6, uninfected HeLa cells.
pseudotuberculosis expressing wild-type ExoS or ExoS(E381A ; # 2000 Biochemical Society Exoenzyme S modification and inhibition of Ras in vivo 219 A Figure 2 Ras modification in HeLa cells infected with bacteria expressing either ExoS or ExoS(E381A) (A) HeLa cells were harvested after ExoS/ExoS(E381A) infection at the indicated time points.
(B) Endogenous Ras modification in vitro by bacterially expressed and purified ExoS (wt) or ExoS(E381A).
HeLa cell lysates were incubated for 1 h at 37 mC in the presence of [32P]NAD with purified ExoS/ExoS(E381A) or buffer control [26].
Figure 3 In vivo time course showing the nucleotide bound to p21Ras HeLa and NIH3T3 (V-12, K-Ras) cells were uninfected (k) or infected for the indicated times with bacteria expressing ExoS (A), ExoS(E381A) (B) or which had been mock infected (M).
observed with a bacterial strain expressing the ExoS(E381A) mutant.
To investigate if the change in migration of Ras proteins observed (Figure 2A) was due to ExoS ADP ribosylation activity, HeLa cells were harvested and the lysate was incubated with [$#P]NAD and purified bacterially expressed wild-type ExoS protein or the ExoS(E381A) mutant protein for 60 min.
A slower migrating form of Ras was visible when the HeLa cell lysate had been exposed to wild-type ExoS, which was not observed when the mutant ExoS(E381A) protein was used.
ExoS(98-232, E381A), however, showed a similarly compromised ability to modify Ras proteins as the ExoS(E381A) (see Figure 4).
Lanes 1 and 2, uninfected HeLa cell lysates ; lane 3, mock infection ; lane 4, infection with ExoS(E381A, 98232) ; lane 5, infection with ExoS(E381A) ; lane 6, infection with ExoS(98232) ; lane 7, infection with wild type ExoS.
In contrast, mock infection or infection of HeLa cells with the ExoS(E381A) mutant protein did not inhibit RasGTP loading in response to EGF (Figure 3).
Furthermore, Ras immunoprecipitation from [$#P]Pi-labelled NIH3T3(V12, K-Ras) cells, after infection for 80 min with either ExoS, ExoS(E381A) or mock infected, resulted in no change in hydrolysis of GTP-bound Ras, compared with non-infected NIH3T3(V12,K-Ras) cells (Figure 3).
Conversely, co-precipitation of Ras proteins with GSTRafRBD was detected upon EGF receptor stimulation after infection with either ExoS(E381A) or ExoS(98-232, E381A) (compare lanes 2 and 3 with 4 and 5).
Tyrosine phosphorylation of Shc (SHC) was examined in (lane 1) unstimulated (k) and (lanes 27) EGF-stimulated (j) HeLa cells treated for 60 min as follows : lanes 1 and 2, uninfected HeLa cell lysates ; lane 3, mock infection ; lane 4, infection with ExoS(E381A,98232) ; lane 5, infection with ExoS(E381A) ; lane 6, infection with ExoS(98 232) ; lane 7, infection with wild-type ExoS.
In contrast, infection of HeLa cells with the ExoS(E381A) mutant or mock infection did not inhibit RasGTP loading in response to EGF.
Interaction between GSTRaf RBD and Ras is only observed when cells are infected with ExoS mutants with limited catalytic capacity, such as ExoS(E381A) and ExoS(98-232, E381A).
This effect was not seen with cells infected with ExoS(E381A) or ExoS(98-232, E381A), suggesting that catalytically impaired ExoS mutant proteins are unable to block PKBAkt and Erk 12 activation upon stimulation with EGF.
Cells were infected for 60 min as follows : lanes 1 and 2, uninfected cells ; lane 3, mock infection ; lane 4, infection with ExoS(E381A,98232) ; lane 5, infection with ExoS(E381A) ; lane 6, infection with ExoS(98232) ; lane 7, infection with wild-type ExoS.
This effect was not seen with cells infected with ExoS(E381A) and ExoS(98-232, E381A) for 40, 60 or 80 min (Figure 6 ; M.
15190072
full text
S252W
protein
substitution
true positive
P21802
We show that the overactive FGFR2 S252W mutation induced decreased Src family kinase tyrosine phosphorylation and activity associated with decreased Lyn and Fyn protein expression in human osteoblasts.
In human osteoblasts, we have shown that single missense point mutations (S252W and P253R) located in the linker region between the second and third extracellular immunoglobulin domains of FGFR2 activate the expression of early and late osteoblast differentiation genes, including alkaline phosphatase (ALP), type I collagen (COLIA1), and osteocalcin in vitro and in vivo (11, 12), a phenotype that is mediated in part through protein kinase C activation (13, 14).
In this work, we show that the activating FGFR2 S252W mutation enhances c-Cbldependent down-regulation of Lyn and Fyn and subsequent inhibition of their kinase activity.
The overactive FGFR2 S252W mutation decreases total Src kinase activity and Lyn and Fyn protein expression in osteoblasts.
RESULTS The Overactive FGFR2 S252W Mutation Reduces Src Kinase Activity in Osteoblasts--To assess whether activation of FGFR2 induced by the gain-of-function S252W mutation alters total Src activity in mutant osteoblasts, we assessed the phosphorylation state of Src family kinases by Western blot analysis using a substrate that is phosphorylated by all Src family members.
1A, we observed a significant decrease in phosphorylation of Src family kinases at the tyrosine corresponding to Try416 in cells bearing the activating S252W mutation compared with control cells (Fig.
These results show that the overactive FGFR2 S252W mutation reduces the amount of Lyn and Fyn, resulting in decreased Lyn and Fyn kinase activity, which contributes to the decreased total Src kinase activity in mutant osteoblasts.
These results indicate that the reduced Lyn and Fyn protein levels induced by the overactive FGFR2 S252W mutation result in part from their increased degradation by the proteasome and that this effect contributes to the ALP up-regulation induced by the overactive FGFR2 mutation in osteoblasts.
We previously reported that the FGFR2 S252W mutation results in down-regulation of FGFR2 protein levels in mutant osteoblasts (12).
This suggests that c-Cbl-dependent ubiquitination of Lyn (but not Fyn) by the activating FGFR2 S252W mutation may be dependent on interaction with the PTB domain of c-Cbl.
6 (A and B), indicate that ALP up-regulation induced by the overactive FGFR2 S252W mutation results, at least in part, from Lyn and Fyn down-regulation mediated by c-Cbl interaction via the RING domain and possibly the PTB domain.
We used these c-Cbl mutants as dominant-negative vectors to assess the role of c-Cbl-interacting domains in FGFR2 degradation induced by the S252W mutation.
We have shown here that FGFR2 activation induced by the overactive FGFR2 S252W mutation induced c-Cbl-mediated Lyn and Fyn down-regulation, resulting in decreased Lyn and Fyn protein levels and activity and increased expression of early markers of osteoblast differentiation.
The RING and PTB domains of c-Cbl are involved in FGFR2 degradation induced by the overactive FGFR2 S252W mutation.
We had previously shown that constitutive activation of FGFR2 by the FGFR2 S252W mutation induces FGFR downregulation in mutant osteoblasts in vitro and in vivo (12).
P253R
protein
substitution
true positive
P21802
In human osteoblasts, we have shown that single missense point mutations (S252W and P253R) located in the linker region between the second and third extracellular immunoglobulin domains of FGFR2 activate the expression of early and late osteoblast differentiation genes, including alkaline phosphatase (ALP), type I collagen (COLIA1), and osteocalcin in vitro and in vivo (11, 12), a phenotype that is mediated in part through protein kinase C activation (13, 14).
10969784
full text
11577078
full text
R544E
protein
substitution
true negative
Phosphorylation of Tyr-551 could lead to Btk activation by triggering an exchange of hydrogen-bonded pairs from Glu-445/Arg-544 to Glu-445/Lys-430 and subsequent rotation of helix C within the N-terminal lobe of the catalytic domain (32).
R28C
protein
substitution
true positive
P07332
Some PH domain residues mutated in XLA patients (such as R28C) are within the inositol binding region, indicating the importance of the inositol binding and the consequent membrane localization
10085136
full text
Y580F
protein
substitution
true positive
Q14289
Mutated CADTK cDNAs were amplified by Pfu DNA polymerase (Stratagene) with complementary DNA mutagenic oligonucleotides for designed mutations (K457A, D567N, Y402F, Y881F, K457A/Y402F, Y579F/Y580F CADTK).
Unlike the Y402F mutation, the Y579F/ Y580F CADTK mutant exhibited partial tyrosine autophosphorylation when overexpressed, suggesting that the residual Y579F/Y580F CADTK kinase activity autophosphorylates Tyr402.
While CADTK Y579F/Y580F does have slightly higher autophosphorylation and kinase activity than Y402F, it seems unlikely that the difference is due to endogenous Src/Fyn activation of overexpressed CADTK.
Y925F
protein
substitution
true positive
Q05397
The human FAK cDNA was used to make the mutants Y397F, Y576F/Y577F, and Y925F FAK by the same method.
We investigated the potential CADTK-dependent tyrosine phosphorylation sites on FAK by co-transfecting wtCADTK and FAK mutants, kdFAK, Y397F, Y576F/Y577F, and Y925F FAK in 293(T) cells (Fig.
D567N
protein
substitution
true positive
Q14289
Mutated CADTK cDNAs were amplified by Pfu DNA polymerase (Stratagene) with complementary DNA mutagenic oligonucleotides for designed mutations (K457A, D567N, Y402F, Y881F, K457A/Y402F, Y579F/Y580F CADTK).
1A, mutation of the CADTK Mg2 -ATP-binding site residues (K457A or D567N) abolished tyrosine kinase activity as assessed by poly(Glu4:Tyr) phosphorylation as well as CADTK tyrosine autophosphorylation (Fig.
Y527F
protein
substitution
true positive
P12931-2
EXPERIMENTAL PROCEDURES Materials--Yes and Y527F Src were kindly provided by Drs.
Y397F
protein
substitution
true positive
Q05397
The human FAK cDNA was used to make the mutants Y397F, Y576F/Y577F, and Y925F FAK by the same method.
We investigated the potential CADTK-dependent tyrosine phosphorylation sites on FAK by co-transfecting wtCADTK and FAK mutants, kdFAK, Y397F, Y576F/Y577F, and Y925F FAK in 293(T) cells (Fig.
Y576F
protein
substitution
true positive
Q05397
The human FAK cDNA was used to make the mutants Y397F, Y576F/Y577F, and Y925F FAK by the same method.
We investigated the potential CADTK-dependent tyrosine phosphorylation sites on FAK by co-transfecting wtCADTK and FAK mutants, kdFAK, Y397F, Y576F/Y577F, and Y925F FAK in 293(T) cells (Fig.
Y577F
protein
substitution
true positive
Q05397
The human FAK cDNA was used to make the mutants Y397F, Y576F/Y577F, and Y925F FAK by the same method.
We investigated the potential CADTK-dependent tyrosine phosphorylation sites on FAK by co-transfecting wtCADTK and FAK mutants, kdFAK, Y397F, Y576F/Y577F, and Y925F FAK in 293(T) cells (Fig.
Y881F
protein
substitution
true positive
Q14289
Mutated CADTK cDNAs were amplified by Pfu DNA polymerase (Stratagene) with complementary DNA mutagenic oligonucleotides for designed mutations (K457A, D567N, Y402F, Y881F, K457A/Y402F, Y579F/Y580F CADTK).
C, kdCADTK or Y402F/Y881F CADTK were co-expressed with Src or Fyn in 293(T) cells.
Finally, double mutants, K457A/Y402F and Y402F/Y881F, produced the same effect as the single mutants, K457A and Y402F, respectively, suggesting that the single mutation of K457A and Y402F produced the dominant effect.
However, Src and Fyn were also capable of substantially phosphorylating the Y402F/Y881F CADTK double mutant (Fig.
Y579F
protein
substitution
true positive
Q14289
Mutated CADTK cDNAs were amplified by Pfu DNA polymerase (Stratagene) with complementary DNA mutagenic oligonucleotides for designed mutations (K457A, D567N, Y402F, Y881F, K457A/Y402F, Y579F/Y580F CADTK).
Unlike the Y402F mutation, the Y579F/ Y580F CADTK mutant exhibited partial tyrosine autophosphorylation when overexpressed, suggesting that the residual Y579F/Y580F CADTK kinase activity autophosphorylates Tyr402.
While CADTK Y579F/Y580F does have slightly higher autophosphorylation and kinase activity than Y402F, it seems unlikely that the difference is due to endogenous Src/Fyn activation of overexpressed CADTK.
K454A
protein
substitution
true positive
Q05397
To further examine the regulation of these two kinases, the kinase-deficient CADTK (kdCADTK), K457A CADTK, and kinase-deficient FAK (kdFAK) K454A FAK were transiently coexpressed with the Src family tyrosine kinase members, Src, Fyn, Yes, and Lck.
Y402F
protein
substitution
true positive
Q14289
Mutated CADTK cDNAs were amplified by Pfu DNA polymerase (Stratagene) with complementary DNA mutagenic oligonucleotides for designed mutations (K457A, D567N, Y402F, Y881F, K457A/Y402F, Y579F/Y580F CADTK).
C, kdCADTK or Y402F/Y881F CADTK were co-expressed with Src or Fyn in 293(T) cells.
These data, taken together with the Y402F mutant data, suggest that Tyr579/Tyr580 are not major tyrosine autophosphorylation sites but that transient tyrosine phosphorylation of these sites may be required for full or continued CADTK activation.
Finally, double mutants, K457A/Y402F and Y402F/Y881F, produced the same effect as the single mutants, K457A and Y402F, respectively, suggesting that the single mutation of K457A and Y402F produced the dominant effect.
However, Src and Fyn were also capable of substantially phosphorylating the Y402F/Y881F CADTK double mutant (Fig.
We doubt that these results (the difference between wtCADTK and Y402F CADTK) are confounded by associated Src family tyrosine kinases (or other co-precipitated endogenous kinases) because these experiments have been repeated using RIPA buffer containing SDS, which reverses the association with other molecules, e.g.
As expected, the Y402D mutation was not autophosphorylated, but the reduction of CADTK immunocomplex kinase activity was similar to Cross-talk between CADTK and FAK that of the Y402F mutant (data not shown).
Unlike the Y402F mutation, the Y579F/ Y580F CADTK mutant exhibited partial tyrosine autophosphorylation when overexpressed, suggesting that the residual Y579F/Y580F CADTK kinase activity autophosphorylates Tyr402.
Surprisingly, there is no Tyr579/Tyr589 tyrosine phosphorylation in the Y402F mutant despite detectable tyrosine kinase activity (Fig.
While CADTK Y579F/Y580F does have slightly higher autophosphorylation and kinase activity than Y402F, it seems unlikely that the difference is due to endogenous Src/Fyn activation of overexpressed CADTK.
Y402D
protein
substitution
true positive
Q14289
We attempted to test the first hypothesis by introducing a permanent negative charge at this site by mutating Tyr402 to aspartic acid (Y402D).
As expected, the Y402D mutation was not autophosphorylated, but the reduction of CADTK immunocomplex kinase activity was similar to Cross-talk between CADTK and FAK that of the Y402F mutant (data not shown).
K457A
protein
substitution
true positive
Q14289
Mutated CADTK cDNAs were amplified by Pfu DNA polymerase (Stratagene) with complementary DNA mutagenic oligonucleotides for designed mutations (K457A, D567N, Y402F, Y881F, K457A/Y402F, Y579F/Y580F CADTK).
1A, mutation of the CADTK Mg2 -ATP-binding site residues (K457A or D567N) abolished tyrosine kinase activity as assessed by poly(Glu4:Tyr) phosphorylation as well as CADTK tyrosine autophosphorylation (Fig.
Finally, double mutants, K457A/Y402F and Y402F/Y881F, produced the same effect as the single mutants, K457A and Y402F, respectively, suggesting that the single mutation of K457A and Y402F produced the dominant effect.
To further examine the regulation of these two kinases, the kinase-deficient CADTK (kdCADTK), K457A CADTK, and kinase-deficient FAK (kdFAK) K454A FAK were transiently coexpressed with the Src family tyrosine kinase members, Src, Fyn, Yes, and Lck.
11381259
full text
G93A
protein
substitution
true negative
These neuropathological signs are reminiscent of those found in mice expressing a mutated SOD1G93A (superoxide dismutase) transgene, an animal model of familial ALS (ref.
10085131
full text
V658L
protein
substitution
true negative
GRK-specific mAb and ELISA Procedures--Synthetic peptides that correspond to amino acids Val-658 to Leu-689 of bovine GRK2 and Leu-658 to Leu-688 of bovine GRK3, respectively, were synthesized on an Applied Biosystems 430A peptide synthesizer using FastMoc chemistry, and the products were purified by high pressure liquid chromatography.
A480F
protein
substitution
true negative
For the quantitation of GRK2, the mAb C5/1 (18), which recognizes a conserved sequence (Ala-480 to Phe-488) present in both GRK2 and GRK3, was adsorbed into wells of microtiter plates (5 g/ml) in 50 mM carbonate, pH 10.6.
10754312
full text
R307K
protein
substitution
true positive
Q06187
Mutation R307K in the Btk SH2 domain has been shown to abrogate both the regulatory effect of Btk to the sustained increases in intracellular Ca2 and the phosphorylation of PLC isoforms (71).
Y361C
protein
substitution
true positive
Q06187
We have analyzed the structural and functional effects of six disease-related amino acid substitutions in the SH2 domain: G302E, R307G, Y334S, L358F, Y361C, and H362Q.
SH2 domain mutations were divided into three categories: 1) Functional mutations, which affect residues presumably participating directly in pY binding (R307G); 2) structural mutations that, via conformational change, not only impair pY binding, but severely derange the structure of the SH2 domain and possibly interfere with the overall conformation of the Btk molecule (G302E, Y334S, L358F, and H362Q); and 3) structural-functional mutations, which contain features from both categories above (Y361C).
In this study, we have analyzed the structural and functional properties of six Btk SH2 missense mutations viz G302E, R307G, Y334S, L358F, Y361C, and H362Q, and one nonpatient mutation C337S (Fig.
The SH2 variant Y361C was constructed using the QuickChange mutagenesis kit (Stratagene) and PCR using native SH2 (271383) as the template.
From 20 XLA-causing amino acid substitutions identified to date in the Btk SH2 domain (18), we have selected six missense mutations, i.e., G302E, R307G, Y334S, L358F, Y361C, and H362Q, and one nonpatient mutation C337S for further analysis (Fig.
Fermentation conditions of different SH2 domain variants and levels of soluble GST fusion proteins in the lysates Expression Conditions Used for SH2 GST-Proteins GST-Fusion Protein Time (h) Temperature (C) GST Activity of Soluble Protein in the Lysate ( A340/min/ml of lysate) Relative Amount of Soluble Fusion Protein in the Lysate (%) 4173 Native SH2 G302E R307G Y334S L358F Y361C H362Q 34 16 16 16 16 16 16 37 16 16 16 16 22 16 710 30 168 36 18 38 82 100 4 24 5 3 5 12 The nature of Btk SH2 polypeptide termini is critical for domain solubility The expression level of the various native and GST-SH2 fusion proteins, as estimated from both lysate and pellet, was similar, but their solubility differed considerably.
The CD spectrum of C337S and Y361C (Fig.
The minor changes observed in the conformation of Y361C might contribute to the ligand binding.
C337S and Y361C do not change the amount of -strands, while the results of the estimation of -helices using the different programs were contradictory.
The solubility and binding assays of Btk SH2 constructsa Relative Solubility of SH2 Proteins SH2-GST fusion Digested with thrombin Protein Bound to pY-Sepharose (percentage SD) SH2 Protein Native SH2 G302Eb R307Gb Y334Sb L358Fb Y361Cb H362Qb C337Sc a -- 100 13 4.2 9.0 4.8 1.0 1.5 137 1.2 2.6 0.1 2.0 0.6 0.7 1.8 0.6 ( The long-term solubility on a time-scale from several hours ( ) to weeks ) of the GST-fused or thrombin-digested SH2 domains.
Upper, The CD spectrum of the native Btk SH2 domain (black) is compared with the spectra of SH2 domains containing mutations R307G (green), C337S (blue), and Y361C (red).
The mutations in adjacent residues, Y361C and H362Q, had the lowest pY-resin-binding values, 1% and 1.5%, respectively, but differed considerably in secondary structure.
Moreover, in the G302E, L358F, and Y361C mutant forms, the loss of pY-binding ability is accompanied by changes in protein conformation.
Our results show that the missense mutation Y361C leads to only minor alterations in the CD spectrum in spite of an essentially complete inability to bind to pY-Sepharose.
Based on these results, we regard G302E, Y334S, L358F, and H362Q as structural mutations and Y361C as structural-functional mutation.
In addition, the t1/2 of Y361C was decreased 3-fold in B cells (57).
R307G
protein
substitution
true positive
Q06187
We have analyzed the structural and functional effects of six disease-related amino acid substitutions in the SH2 domain: G302E, R307G, Y334S, L358F, Y361C, and H362Q.
Based on circular dichroism analysis, the conformation of five of the XLA mutants studied differs from the native Btk SH2 domain, while mutant R307G is structurally identical.
SH2 domain mutations were divided into three categories: 1) Functional mutations, which affect residues presumably participating directly in pY binding (R307G); 2) structural mutations that, via conformational change, not only impair pY binding, but severely derange the structure of the SH2 domain and possibly interfere with the overall conformation of the Btk molecule (G302E, Y334S, L358F, and H362Q); and 3) structural-functional mutations, which contain features from both categories above (Y361C).
In this study, we have analyzed the structural and functional properties of six Btk SH2 missense mutations viz G302E, R307G, Y334S, L358F, Y361C, and H362Q, and one nonpatient mutation C337S (Fig.
For in vivo studies, human cDNA of the native Btk and constructs carrying the mutations G302E, R307G, and Y334S at their SH2 domains were cloned into pSGT (42) and used to transfect COS-7 cells using standard calcium phosphate precipitation (43, 44).
The overlap extension PCR technique (40) was used to create the mutants G302E, R307G, Y334S, C337S, L358F, and H362Q with SH2 (274 381) or SH2 (271383) as the templates.
From 20 XLA-causing amino acid substitutions identified to date in the Btk SH2 domain (18), we have selected six missense mutations, i.e., G302E, R307G, Y334S, L358F, Y361C, and H362Q, and one nonpatient mutation C337S for further analysis (Fig.
Fermentation conditions of different SH2 domain variants and levels of soluble GST fusion proteins in the lysates Expression Conditions Used for SH2 GST-Proteins GST-Fusion Protein Time (h) Temperature (C) GST Activity of Soluble Protein in the Lysate ( A340/min/ml of lysate) Relative Amount of Soluble Fusion Protein in the Lysate (%) 4173 Native SH2 G302E R307G Y334S L358F Y361C H362Q 34 16 16 16 16 16 16 37 16 16 16 16 22 16 710 30 168 36 18 38 82 100 4 24 5 3 5 12 The nature of Btk SH2 polypeptide termini is critical for domain solubility The expression level of the various native and GST-SH2 fusion proteins, as estimated from both lysate and pellet, was similar, but their solubility differed considerably.
The solubility of transiently expressed native Btk and Btk proteins containing mutations at G302E, R307G, and Y334S was studied in COS-7 cells.
The conformation of Btk SH2 mutant R307G remained unchanged, while the conformation of the other mutants was altered Proteins can be classified based on the content and organization of secondary structure elements.
The spectra of the native Btk and mutant R307G are identical (Fig.
The R307G mutation does not affect the secondary structure.
The solubility and binding assays of Btk SH2 constructsa Relative Solubility of SH2 Proteins SH2-GST fusion Digested with thrombin Protein Bound to pY-Sepharose (percentage SD) SH2 Protein Native SH2 G302Eb R307Gb Y334Sb L358Fb Y361Cb H362Qb C337Sc a -- 100 13 4.2 9.0 4.8 1.0 1.5 137 1.2 2.6 0.1 2.0 0.6 0.7 1.8 0.6 ( The long-term solubility on a time-scale from several hours ( ) to weeks ) of the GST-fused or thrombin-digested SH2 domains.
Upper, The CD spectrum of the native Btk SH2 domain (black) is compared with the spectra of SH2 domains containing mutations R307G (green), C337S (blue), and Y361C (red).
The R307G mutation reduces binding to the pY-Sepharose to 4.5% (Table II), although its secondary structure remains unaltered according to the CD analysis (Fig.
Only one mutant, R307G, has decreased pY binding and unchanged SH2 domain conformation.
Thus, R307G may be described as a functional mutation, resulting in loss of a critical pYbinding residue and destroying the important interaction between guanidinium and phosphate groups (Fig.
Therefore, the highly reduced pY-binding capacity of R307G is easily understood.
Two different mutations have been identified from XLA patients, R307G and R307T.
Northern blot of the R307G mutation showed a Btk transcript of normal size and abundance (63).
According to our results, R307G does not alter protein structure, but it almost completely abolishes the pY binding.
The substitution removes the essential pY interaction, and R307G is therefore classified as a functional mutation.
Y223A
protein
substitution
true positive
Q06187
Binding of SH2 domains to pY-Sepharose The GST-fusion proteins of wild-type Btk SH2 and SH3 (Y223A) domains and mutation-containing SH2 variants were dialyzed against buffer A.
Another GST-fusion construct, Btk SH3 (Y223A), was used as the control for unspecific adsorption.
R307T
protein
substitution
true positive
Q06187
Two different mutations have been identified from XLA patients, R307G and R307T.
R175L
protein
substitution
true negative
Moreover, mutation R175L, at the corresponding position of c-Src, impairs the regulation of c-Src kinase activity most obviously by preventing the association of the phosphorylated tail with the SH2 domain (67, 68).
H362Q
protein
substitution
true positive
Q06187
We have analyzed the structural and functional effects of six disease-related amino acid substitutions in the SH2 domain: G302E, R307G, Y334S, L358F, Y361C, and H362Q.
SH2 domain mutations were divided into three categories: 1) Functional mutations, which affect residues presumably participating directly in pY binding (R307G); 2) structural mutations that, via conformational change, not only impair pY binding, but severely derange the structure of the SH2 domain and possibly interfere with the overall conformation of the Btk molecule (G302E, Y334S, L358F, and H362Q); and 3) structural-functional mutations, which contain features from both categories above (Y361C).
In this study, we have analyzed the structural and functional properties of six Btk SH2 missense mutations viz G302E, R307G, Y334S, L358F, Y361C, and H362Q, and one nonpatient mutation C337S (Fig.
The overlap extension PCR technique (40) was used to create the mutants G302E, R307G, Y334S, C337S, L358F, and H362Q with SH2 (274 381) or SH2 (271383) as the templates.
From 20 XLA-causing amino acid substitutions identified to date in the Btk SH2 domain (18), we have selected six missense mutations, i.e., G302E, R307G, Y334S, L358F, Y361C, and H362Q, and one nonpatient mutation C337S for further analysis (Fig.
Fermentation conditions of different SH2 domain variants and levels of soluble GST fusion proteins in the lysates Expression Conditions Used for SH2 GST-Proteins GST-Fusion Protein Time (h) Temperature (C) GST Activity of Soluble Protein in the Lysate ( A340/min/ml of lysate) Relative Amount of Soluble Fusion Protein in the Lysate (%) 4173 Native SH2 G302E R307G Y334S L358F Y361C H362Q 34 16 16 16 16 16 16 37 16 16 16 16 22 16 710 30 168 36 18 38 82 100 4 24 5 3 5 12 The nature of Btk SH2 polypeptide termini is critical for domain solubility The expression level of the various native and GST-SH2 fusion proteins, as estimated from both lysate and pellet, was similar, but their solubility differed considerably.
As an exception, H362Q fused with GST showed relatively high GST activity, but it had a remarkably strong tendency to aggregate during thrombin digestion.
The spectra of G302E, Y334S, and H362Q have only one minimum at 207208 nm.
The solubility and binding assays of Btk SH2 constructsa Relative Solubility of SH2 Proteins SH2-GST fusion Digested with thrombin Protein Bound to pY-Sepharose (percentage SD) SH2 Protein Native SH2 G302Eb R307Gb Y334Sb L358Fb Y361Cb H362Qb C337Sc a -- 100 13 4.2 9.0 4.8 1.0 1.5 137 1.2 2.6 0.1 2.0 0.6 0.7 1.8 0.6 ( The long-term solubility on a time-scale from several hours ( ) to weeks ) of the GST-fused or thrombin-digested SH2 domains.
Lower, The CD spectra of the native SH2 (black), G302E (green), Y334S (magenta), L358F (blue), and H362Q (red) SH2 domains.
The Y334S and H362Q mutations affect both the and structures.
The mutations in adjacent residues, Y361C and H362Q, had the lowest pY-resin-binding values, 1% and 1.5%, respectively, but differed considerably in secondary structure.
2), changes in secondary structure elements, and diminished pY-binding abilities (Table II) indicate that mutations Y334S and H362Q have both structural and functional contributions.
The H362Q mutation leads to a conformational change in the SH2 domain structure, and it markedly disturbs the pY-peptide binding.
Based on these results, we regard G302E, Y334S, L358F, and H362Q as structural mutations and Y361C as structural-functional mutation.
A missense mutation affecting the same residue, H362Q, has been reported previously in the Btk SH2 domain, leading also to a classical form of XLA (54).
H362R
protein
substitution
true positive
Q06187
Also, we present a novel Btk SH2 missense mutation, H362R, leading to classical XLA.
In addition, we have identified a new XLAcausing missense mutation, H362R, located in the Btk SH2 domain.
Results A novel Btk SH2 domain missense mutation, H362R, leads to a classical XLA To elucidate the structure-function relationships of the Btk, and its connection to signal transduction and XLA disease, we are studying the functional and conformational effects of XLA mutations.
During preparation of the manuscript, we identified a novel SH2 missense mutation, H362R, causing a classical form of XLA.
The mutation replaces the basic and non- ionized histidine residue with basic and charged arginine residue at aa 362 in the Btk SH2 domain (BTKbase PIN H362R (1), accession number A0542).
By using SSCP analysis, we demonstrated a new, severe XLA-causing missense mutation, H362R, in the Btk SH2 domain.
C337S
protein
substitution
true positive
Q06187
In this study, we have analyzed the structural and functional properties of six Btk SH2 missense mutations viz G302E, R307G, Y334S, L358F, Y361C, and H362Q, and one nonpatient mutation C337S (Fig.
The overlap extension PCR technique (40) was used to create the mutants G302E, R307G, Y334S, C337S, L358F, and H362Q with SH2 (274 381) or SH2 (271383) as the templates.
From 20 XLA-causing amino acid substitutions identified to date in the Btk SH2 domain (18), we have selected six missense mutations, i.e., G302E, R307G, Y334S, L358F, Y361C, and H362Q, and one nonpatient mutation C337S for further analysis (Fig.
The CD spectrum of C337S and Y361C (Fig.
C337S and Y361C do not change the amount of -strands, while the results of the estimation of -helices using the different programs were contradictory.
The solubility and binding assays of Btk SH2 constructsa Relative Solubility of SH2 Proteins SH2-GST fusion Digested with thrombin Protein Bound to pY-Sepharose (percentage SD) SH2 Protein Native SH2 G302Eb R307Gb Y334Sb L358Fb Y361Cb H362Qb C337Sc a -- 100 13 4.2 9.0 4.8 1.0 1.5 137 1.2 2.6 0.1 2.0 0.6 0.7 1.8 0.6 ( The long-term solubility on a time-scale from several hours ( ) to weeks ) of the GST-fused or thrombin-digested SH2 domains.
c C337S was not made as a GST-fusion construct.
Upper, The CD spectrum of the native Btk SH2 domain (black) is compared with the spectra of SH2 domains containing mutations R307G (green), C337S (blue), and Y361C (red).
The binding of a domain with missense mutation C337S, not known to cause XLA, had an increased affinity (137%) and a solubility that was at least as high as that of the native SH2 domain.
Y334S
protein
substitution
true positive
Q06187
We have analyzed the structural and functional effects of six disease-related amino acid substitutions in the SH2 domain: G302E, R307G, Y334S, L358F, Y361C, and H362Q.
SH2 domain mutations were divided into three categories: 1) Functional mutations, which affect residues presumably participating directly in pY binding (R307G); 2) structural mutations that, via conformational change, not only impair pY binding, but severely derange the structure of the SH2 domain and possibly interfere with the overall conformation of the Btk molecule (G302E, Y334S, L358F, and H362Q); and 3) structural-functional mutations, which contain features from both categories above (Y361C).
In this study, we have analyzed the structural and functional properties of six Btk SH2 missense mutations viz G302E, R307G, Y334S, L358F, Y361C, and H362Q, and one nonpatient mutation C337S (Fig.
For in vivo studies, human cDNA of the native Btk and constructs carrying the mutations G302E, R307G, and Y334S at their SH2 domains were cloned into pSGT (42) and used to transfect COS-7 cells using standard calcium phosphate precipitation (43, 44).
The overlap extension PCR technique (40) was used to create the mutants G302E, R307G, Y334S, C337S, L358F, and H362Q with SH2 (274 381) or SH2 (271383) as the templates.
From 20 XLA-causing amino acid substitutions identified to date in the Btk SH2 domain (18), we have selected six missense mutations, i.e., G302E, R307G, Y334S, L358F, Y361C, and H362Q, and one nonpatient mutation C337S for further analysis (Fig.
Fermentation conditions of different SH2 domain variants and levels of soluble GST fusion proteins in the lysates Expression Conditions Used for SH2 GST-Proteins GST-Fusion Protein Time (h) Temperature (C) GST Activity of Soluble Protein in the Lysate ( A340/min/ml of lysate) Relative Amount of Soluble Fusion Protein in the Lysate (%) 4173 Native SH2 G302E R307G Y334S L358F Y361C H362Q 34 16 16 16 16 16 16 37 16 16 16 16 22 16 710 30 168 36 18 38 82 100 4 24 5 3 5 12 The nature of Btk SH2 polypeptide termini is critical for domain solubility The expression level of the various native and GST-SH2 fusion proteins, as estimated from both lysate and pellet, was similar, but their solubility differed considerably.
The solubility of transiently expressed native Btk and Btk proteins containing mutations at G302E, R307G, and Y334S was studied in COS-7 cells.
The spectra of G302E, Y334S, and H362Q have only one minimum at 207208 nm.
The solubility and binding assays of Btk SH2 constructsa Relative Solubility of SH2 Proteins SH2-GST fusion Digested with thrombin Protein Bound to pY-Sepharose (percentage SD) SH2 Protein Native SH2 G302Eb R307Gb Y334Sb L358Fb Y361Cb H362Qb C337Sc a -- 100 13 4.2 9.0 4.8 1.0 1.5 137 1.2 2.6 0.1 2.0 0.6 0.7 1.8 0.6 ( The long-term solubility on a time-scale from several hours ( ) to weeks ) of the GST-fused or thrombin-digested SH2 domains.
Lower, The CD spectra of the native SH2 (black), G302E (green), Y334S (magenta), L358F (blue), and H362Q (red) SH2 domains.
The Y334S and H362Q mutations affect both the and structures.
The level of pY binding of mutant L358F was about 5%, and that of the mutants G302E and Y334S was about 10% compared with the native SH2.
2), changes in secondary structure elements, and diminished pY-binding abilities (Table II) indicate that mutations Y334S and H362Q have both structural and functional contributions.
Based on these results, we regard G302E, Y334S, L358F, and H362Q as structural mutations and Y361C as structural-functional mutation.
R307A
protein
substitution
true positive
Q06187
Noteworthily, by using a Btk gene carrying a R307A mutation, it Discussion The XLA mutations are scattered all along the BTK gene, and the distribution of the mutations in the five domains is approximately according to the length of the domain, except for the TH domain (18).
L358F
protein
substitution
true positive
Q06187
We have analyzed the structural and functional effects of six disease-related amino acid substitutions in the SH2 domain: G302E, R307G, Y334S, L358F, Y361C, and H362Q.
SH2 domain mutations were divided into three categories: 1) Functional mutations, which affect residues presumably participating directly in pY binding (R307G); 2) structural mutations that, via conformational change, not only impair pY binding, but severely derange the structure of the SH2 domain and possibly interfere with the overall conformation of the Btk molecule (G302E, Y334S, L358F, and H362Q); and 3) structural-functional mutations, which contain features from both categories above (Y361C).
In this study, we have analyzed the structural and functional properties of six Btk SH2 missense mutations viz G302E, R307G, Y334S, L358F, Y361C, and H362Q, and one nonpatient mutation C337S (Fig.
The overlap extension PCR technique (40) was used to create the mutants G302E, R307G, Y334S, C337S, L358F, and H362Q with SH2 (274 381) or SH2 (271383) as the templates.
From 20 XLA-causing amino acid substitutions identified to date in the Btk SH2 domain (18), we have selected six missense mutations, i.e., G302E, R307G, Y334S, L358F, Y361C, and H362Q, and one nonpatient mutation C337S for further analysis (Fig.
Fermentation conditions of different SH2 domain variants and levels of soluble GST fusion proteins in the lysates Expression Conditions Used for SH2 GST-Proteins GST-Fusion Protein Time (h) Temperature (C) GST Activity of Soluble Protein in the Lysate ( A340/min/ml of lysate) Relative Amount of Soluble Fusion Protein in the Lysate (%) 4173 Native SH2 G302E R307G Y334S L358F Y361C H362Q 34 16 16 16 16 16 16 37 16 16 16 16 22 16 710 30 168 36 18 38 82 100 4 24 5 3 5 12 The nature of Btk SH2 polypeptide termini is critical for domain solubility The expression level of the various native and GST-SH2 fusion proteins, as estimated from both lysate and pellet, was similar, but their solubility differed considerably.
The spectrum of the L358F differs clearly from all the others by having two minima at 207 and 214 nm.
The solubility and binding assays of Btk SH2 constructsa Relative Solubility of SH2 Proteins SH2-GST fusion Digested with thrombin Protein Bound to pY-Sepharose (percentage SD) SH2 Protein Native SH2 G302Eb R307Gb Y334Sb L358Fb Y361Cb H362Qb C337Sc a -- 100 13 4.2 9.0 4.8 1.0 1.5 137 1.2 2.6 0.1 2.0 0.6 0.7 1.8 0.6 ( The long-term solubility on a time-scale from several hours ( ) to weeks ) of the GST-fused or thrombin-digested SH2 domains.
Lower, The CD spectra of the native SH2 (black), G302E (green), Y334S (magenta), L358F (blue), and H362Q (red) SH2 domains.
L358F mutation increases -helical content.
The -helix content of G302E mutation and -strand content of L358F mutation could not be interpreted because of large variation in the results.
The level of pY binding of mutant L358F was about 5%, and that of the mutants G302E and Y334S was about 10% compared with the native SH2.
Moreover, in the G302E, L358F, and Y361C mutant forms, the loss of pY-binding ability is accompanied by changes in protein conformation.
Based on these results, we regard G302E, Y334S, L358F, and H362Q as structural mutations and Y361C as structural-functional mutation.
Mutations G302E and L358F decrease the Btk level in monocytes in vivo (56).
G302E
protein
substitution
true positive
Q06187
We have analyzed the structural and functional effects of six disease-related amino acid substitutions in the SH2 domain: G302E, R307G, Y334S, L358F, Y361C, and H362Q.
SH2 domain mutations were divided into three categories: 1) Functional mutations, which affect residues presumably participating directly in pY binding (R307G); 2) structural mutations that, via conformational change, not only impair pY binding, but severely derange the structure of the SH2 domain and possibly interfere with the overall conformation of the Btk molecule (G302E, Y334S, L358F, and H362Q); and 3) structural-functional mutations, which contain features from both categories above (Y361C).
In this study, we have analyzed the structural and functional properties of six Btk SH2 missense mutations viz G302E, R307G, Y334S, L358F, Y361C, and H362Q, and one nonpatient mutation C337S (Fig.
For in vivo studies, human cDNA of the native Btk and constructs carrying the mutations G302E, R307G, and Y334S at their SH2 domains were cloned into pSGT (42) and used to transfect COS-7 cells using standard calcium phosphate precipitation (43, 44).
The overlap extension PCR technique (40) was used to create the mutants G302E, R307G, Y334S, C337S, L358F, and H362Q with SH2 (274 381) or SH2 (271383) as the templates.
From 20 XLA-causing amino acid substitutions identified to date in the Btk SH2 domain (18), we have selected six missense mutations, i.e., G302E, R307G, Y334S, L358F, Y361C, and H362Q, and one nonpatient mutation C337S for further analysis (Fig.
Fermentation conditions of different SH2 domain variants and levels of soluble GST fusion proteins in the lysates Expression Conditions Used for SH2 GST-Proteins GST-Fusion Protein Time (h) Temperature (C) GST Activity of Soluble Protein in the Lysate ( A340/min/ml of lysate) Relative Amount of Soluble Fusion Protein in the Lysate (%) 4173 Native SH2 G302E R307G Y334S L358F Y361C H362Q 34 16 16 16 16 16 16 37 16 16 16 16 22 16 710 30 168 36 18 38 82 100 4 24 5 3 5 12 The nature of Btk SH2 polypeptide termini is critical for domain solubility The expression level of the various native and GST-SH2 fusion proteins, as estimated from both lysate and pellet, was similar, but their solubility differed considerably.
The solubility of transiently expressed native Btk and Btk proteins containing mutations at G302E, R307G, and Y334S was studied in COS-7 cells.
The spectra of G302E, Y334S, and H362Q have only one minimum at 207208 nm.
The G302E mutation decreases -strand content slightly, and the Table II.
The solubility and binding assays of Btk SH2 constructsa Relative Solubility of SH2 Proteins SH2-GST fusion Digested with thrombin Protein Bound to pY-Sepharose (percentage SD) SH2 Protein Native SH2 G302Eb R307Gb Y334Sb L358Fb Y361Cb H362Qb C337Sc a -- 100 13 4.2 9.0 4.8 1.0 1.5 137 1.2 2.6 0.1 2.0 0.6 0.7 1.8 0.6 ( The long-term solubility on a time-scale from several hours ( ) to weeks ) of the GST-fused or thrombin-digested SH2 domains.
Lower, The CD spectra of the native SH2 (black), G302E (green), Y334S (magenta), L358F (blue), and H362Q (red) SH2 domains.
The -helix content of G302E mutation and -strand content of L358F mutation could not be interpreted because of large variation in the results.
The level of pY binding of mutant L358F was about 5%, and that of the mutants G302E and Y334S was about 10% compared with the native SH2.
Moreover, in the G302E, L358F, and Y361C mutant forms, the loss of pY-binding ability is accompanied by changes in protein conformation.
According to our results, the G302E mutant folds in a structurally altered manner, which leads to decreased pY-binding ability and to reduced solubility.
The substitution G302E introduces an acidic residue at a conserved site in the loop connecting the A-helix and the B-strand.
Without a conformational change in the G302E SH2 domain, the side chain of the bulky glutamate can be predicted to cause a clash with the K374 side chain.
Based on these results, we regard G302E, Y334S, L358F, and H362Q as structural mutations and Y361C as structural-functional mutation.
Mutations G302E and L358F decrease the Btk level in monocytes in vivo (56).
The mutation G302E re- 4176 has been shown that a functional SH2 domain is also important for the ability of Btk to act as a negative regulator of Fas-mediated apoptosis (77).
12446614
full text
Y807F
protein
substitution
true positive
P09581
In contrast, those expressing Y559F generate fewer OCs, whereas theY807F mutant is incapable of osteoclastogenesis.
Finally, although mature OCs expressing Y559F exhibit impaired bone resorption, those bearing Y807F do not.
In contrast, substantially fewer OCs were derived by Epo/ RANKL treatment of cells bearing either Y559F or Y807F mutation (Fig.
Epo were 51/0, 55/52, 54/5, 54/48, 55/45, 46/42, 52/17, and 55/45 in vector alone (vehicle), wild-type EpoR/c-Fms, Y549F, Y697F, Y706F, Y721F, Y807F, and Y921F mutants, respectively.
On the other hand, Epo and RANKL treatment generated numerous OCs using as targets pOCs expressing wild-type chimera (control), Y697F, Y706F, Y721, Y807F, and Y921F mutants.
Although vector alone (vehicle) generated no osteoclasts, as expected, the number of TRAP-positive multinucleated cells was slightly lower with Y807F and was almost zero in the case of Y559F.
With the exception of Y559F and Y807F, cells expressing both wild-type and mutant EpoR2/c-Fms form numerous osteoclasts in the presence of Epo and RANKL.
For example, expression of the Y807F point mutant in NIH-3T3 cells leads to almost complete loss of proliferation in response to M-CSF.
Finally, in the IL-3-dependent FDC-P1 line, activation of c-Fms Y807F again enhances proliferation (16).
Both wild-type and all mutants, with the exception of Y559F, generated numerous resorptive pits, whereas apoptotic OCs were seen in Y807F mutant.
Y721F
protein
substitution
true positive
P09581
Cells expressing chimeric receptors with Y697F, Y706F, Y721F, and Y921F single point mutations generate normal numbers of bone-resorbing OCs, with normal bone-resorbing activity when treated with RANKL and Epo.
Epo were 51/0, 55/52, 54/5, 54/48, 55/45, 46/42, 52/17, and 55/45 in vector alone (vehicle), wild-type EpoR/c-Fms, Y549F, Y697F, Y706F, Y721F, Y807F, and Y921F mutants, respectively.
Y706F
protein
substitution
true positive
P09581
Cells expressing chimeric receptors with Y697F, Y706F, Y721F, and Y921F single point mutations generate normal numbers of bone-resorbing OCs, with normal bone-resorbing activity when treated with RANKL and Epo.
Epo were 51/0, 55/52, 54/5, 54/48, 55/45, 46/42, 52/17, and 55/45 in vector alone (vehicle), wild-type EpoR/c-Fms, Y549F, Y697F, Y706F, Y721F, Y807F, and Y921F mutants, respectively.
On the other hand, Epo and RANKL treatment generated numerous OCs using as targets pOCs expressing wild-type chimera (control), Y697F, Y706F, Y721, Y807F, and Y921F mutants.
Y599F
protein
substitution
true negative
Once again, cells bearing a Y to F mutation at positions 697, 706, 721, or 921 in the cytoplasmic tail all generated numerous pits, whereas those bearing Y599F were functionally inactive (Fig.
Y697F
protein
substitution
true positive
P09581
Cells expressing chimeric receptors with Y697F, Y706F, Y721F, and Y921F single point mutations generate normal numbers of bone-resorbing OCs, with normal bone-resorbing activity when treated with RANKL and Epo.
Epo were 51/0, 55/52, 54/5, 54/48, 55/45, 46/42, 52/17, and 55/45 in vector alone (vehicle), wild-type EpoR/c-Fms, Y549F, Y697F, Y706F, Y721F, Y807F, and Y921F mutants, respectively.
On the other hand, Epo and RANKL treatment generated numerous OCs using as targets pOCs expressing wild-type chimera (control), Y697F, Y706F, Y721, Y807F, and Y921F mutants.
Y921F
protein
substitution
true positive
P09581
Cells expressing chimeric receptors with Y697F, Y706F, Y721F, and Y921F single point mutations generate normal numbers of bone-resorbing OCs, with normal bone-resorbing activity when treated with RANKL and Epo.
Epo were 51/0, 55/52, 54/5, 54/48, 55/45, 46/42, 52/17, and 55/45 in vector alone (vehicle), wild-type EpoR/c-Fms, Y549F, Y697F, Y706F, Y721F, Y807F, and Y921F mutants, respectively.
On the other hand, Epo and RANKL treatment generated numerous OCs using as targets pOCs expressing wild-type chimera (control), Y697F, Y706F, Y721, Y807F, and Y921F mutants.
Y549F
protein
substitution
true negative
Epo were 51/0, 55/52, 54/5, 54/48, 55/45, 46/42, 52/17, and 55/45 in vector alone (vehicle), wild-type EpoR/c-Fms, Y549F, Y697F, Y706F, Y721F, Y807F, and Y921F mutants, respectively.
Y559F
protein
substitution
true positive
P09581
In contrast, those expressing Y559F generate fewer OCs, whereas theY807F mutant is incapable of osteoclastogenesis.
Finally, although mature OCs expressing Y559F exhibit impaired bone resorption, those bearing Y807F do not.
In contrast, substantially fewer OCs were derived by Epo/ RANKL treatment of cells bearing either Y559F or Y807F mutation (Fig.
Although vector alone (vehicle) generated no osteoclasts, as expected, the number of TRAP-positive multinucleated cells was slightly lower with Y807F and was almost zero in the case of Y559F.
These data demonstrate that pOCs expressing all mutant receptors except Y559F can fully differentiate into mature OCs from pOCs within 24 h.
TRAP staining of cells kept in culture for 2 d revealed that, once again, all mutants except Y559F generated numerous OCs (Fig.
pOCs bearing the Y559F mutant generated equivalent numbers of OCs and pits compared with the wild-type chimera receptor when cultured in the presence of M-CSF and RANKL (data not shown).
With the exception of Y559F and Y807F, cells expressing both wild-type and mutant EpoR2/c-Fms form numerous osteoclasts in the presence of Epo and RANKL.
When cells were exposed to Epo and RANKL, no osteoclasts and only a few such cells were detected in vector alone (Vehicle) and Y559F mutant, respectively.
Both wild-type and all mutants, with the exception of Y559F, generated numerous resorptive pits, whereas apoptotic OCs were seen in Y807F mutant.
9808578
full text
K290M
protein
substitution
true positive
P00519-2
To evaluate the contributions of various ABL domains to biochemical signaling and biological effects, chimeric receptors were constructed in which the ABL SH3 domain was deleted ( SH3), the SH2 domain was deleted ( SH2), the C-terminal actin-binding domain was deleted ( ABD), or kinase activity was eliminated by a point mutation, K290M (KD).
ABD, actin-binding domain (c-terminal 167 amino acids) deletion; SH2, Src-homology domain 2 deletion; SH3, Src-homology domain 3 deletion; KD, kinase dead has a point mutation at K290M.
11468192
full text
Q576R
protein
substitution
true negative
Brief report Association of the Q576R polymorphism in the interleukin-4 receptor with indolent mastocytosis limited to the skin Trisha Daley, Dean D.
Because addition of interleukin-4 (IL-4) to mast cell cultures is reported to induce apoptosis, the hypothesis was considered that individuals carrying the gain-of-function polymorphism Q576R in the cytoplasmic domain of the -subunit of the IL-4 receptor (IL4R) might be relatively resistant to the gain-of-function mutation in c-kit.
To assess this possibility, 36 patients with either cutaneous or systemic mastocytosis were studied for association with the Q576R polymorphism.
The Q576R polymorphism was found more frequently in those with disease limited to skin and who exhibited lower levels of surrogate disease markers.
These data suggest that the Q576R IL-4R - chain polymorphism may mitigate disease expression and confer a better prognosis in patients with mastocytosis.
Among these, the gain-of- function polymorphism, Q576R (single-letter amino acid codes), appears to have clinical relevance, because it has been shown to be associated with atopy, severe asthma, and renal allograft rejection.10-12 We thus hypothesized that patients expressing the Q576R IL-4RA polymorphism might be relatively protected from severe mast cell hyperplasia resulting from activating mutations in c-kit and other yet-to-be discovered events that promote mast cell hyperplasia.
Patients carrying the Q576R polymorphism do appear to have more limited forms of mastocytosis, suggesting that this polymorphism confers relative protection from adverse events that lead to pathologic mast cell hyperplasia.
Detection of Q576R polymorphism Total RNA was extracted from peripheral blood mononuclear cells and reverse transcribed to complementary DNA (cDNA) as reported.6 This cDNA was used as a template to amplify a 307-bp product containing the region encoding the IL-4RA Q576R polymorphism by nested polymerase chain reaction (PCR) as described.10 Presence of the polymorphism and its allelic status were determined by direct PCR sequencing using the Big Dye Terminator kit (PE Biosystems, Foster City, CA) and analysis on a capillary automated sequencer (ABI Prism 310 Genetic Analyzer, PE Biosystems).
The IL-4RA Q576R polymorphism in patients with mastocytosis P All patients Cutaneous Systemic ISM AHD Total Adult onset Cutaneous Systemic ISM AHD Total Pediatric onset Cutaneous Systemic ISM AHD Total 6 1 1 0 7 3 4 3 1 7 66.7 20 25 0 50 3 1 1 0 4 1 17 13 4 18 75 5.6 7.1 0 18.2 Figure 1.
Q576R polymorphism and mastocytosis.
IL-4RA Q576R polymorphism is associated with lower levels of surrogate disease markers, tryptase (A) and soluble CD117 (sCD117) (B), in mastocytosis.
Results and discussion We first determined the frequency of the IL-4RA Q576R polymorphism in individuals with and without mastocytosis.
Thus, the frequency of the IL-4RA Q576R polymorphism in patients with mastocytosis was not different from that of the general population.
We next compared the frequency of the IL-4RA Q576R polymorphism in patients with mastocytosis assigned to different categories based on disease extent and prognosis.
In conclusion, this study shows that the presence of the IL-4RA Q576R polymorphism in a patient with mastocytosis is associated with a lower mast cell burden as determined both by extent of clinical disease and by circulating levels of surrogate disease markers.
This finding suggests a protective role for IL-4RA Q576R polymorphism in the limitation of tissue mast cell numbers in patients with mastocytosis.
10657996
full text
Y1230H
protein
substitution
true positive
P08581
Image mutation
V1188L
protein
substitution
true positive
P08581
Image mutation
D1228H
protein
substitution
P08581
true positive
Three of these mutations (D1228N, D1228H, and M1250T) are located in codons homologous to those mutated in the tyrosine kinase receptors Kit and Ret.
A luciferase construct containing a fos-responsive promoter was induced 8- and 7-fold by coexpression of the transforming mutants MET M1250T and MET D1228H, respectively (Fig.
Conversely, MET M1250T and MET D1228H mutants, which display the highest transforming potential in vitro, develop shorter, mainly unbranched tubules.
The decreased invasive and morphogenetic ability of MET M1250T and MET D1228H mutants, which also interact with Pi3Kinase, albeit at a lower level than MET L1195V and MET Y1230C, could be due to an unbalanced activation of the Ras and Pi3Kinase pathways as sustained activation of the Ras pathway causes disorganized growth resulting in reduced invasion and tubulogenesis (42).
Mutants belonging to one group, including MET M1250T and MET D1228H, display increased tyrosine kinase activity, stimulate efficiently the Ras pathway, and transform recipient cells in focus forming assays.
D1228N
protein
substitution
P08581
true positive
Three of these mutations (D1228N, D1228H, and M1250T) are located in codons homologous to those mutated in the tyrosine kinase receptors Kit and Ret.
Y1230C
protein
substitution
P08581
true positive
In the same assay, MET PRC mutants MET L1195V and MET Y1230C, which are almost devoid of transforming ability, induce the luciferase construct to a lower degree, displaying a 60% reduced ability to activate the Ras pathway.
MLP 29 cells expressing MET L1195V and MET Y1230C mutants (which are only weakly transforming in a focus forming assay) showed the best morphogenetic response, developing many long tubular structures (Fig.
3B, cells expressing MET L1195V and MET Y1230C mutants, which also induced branched morphogenesis, were effective in invading the reconstituted basal membrane.
Pull-down experiments of the MET PRC mutants with the GST protein fused to the amino-terminal SH2 domain of p85 showed that MET L1195V and MET Y1230C mutants, displaying the best invasive and tubulogenic ability, interact very efficiently with Pi3Kinase (Fig.
The decreased invasive and morphogenetic ability of MET M1250T and MET D1228H mutants, which also interact with Pi3Kinase, albeit at a lower level than MET L1195V and MET Y1230C, could be due to an unbalanced activation of the Ras and Pi3Kinase pathways as sustained activation of the Ras pathway causes disorganized growth resulting in reduced invasion and tubulogenesis (42).
4A, cells expressing MET L1195V and MET Y1230C mutants display an increased resistance to apoptosis, which is further enhanced by HGF stimulation.
Conversely, the other group of mutations, including MET L1195V and MET Y1230C, are almost devoid of in vitro transforming potential but are effective in inducing protection from apoptosis, sustaining anchorage-independent growth, and promoting invasion.
The most effective mutants were MET L1195V and METY1230C, which were also more resistant to apoptosis.
M1250T
protein
substitution
P08581
true positive
Three of these mutations (D1228N, D1228H, and M1250T) are located in codons homologous to those mutated in the tyrosine kinase receptors Kit and Ret.
FurtherBIOLOGICAL PROPERTIES OF METPRC MUTANTS more, the MET PRC mutant with the highest transforming ability (MET M1250T) also displays the highest catalytic activity.
A luciferase construct containing a fos-responsive promoter was induced 8- and 7-fold by coexpression of the transforming mutants MET M1250T and MET D1228H, respectively (Fig.
Conversely, MET M1250T and MET D1228H mutants, which display the highest transforming potential in vitro, develop shorter, mainly unbranched tubules.
The decreased invasive and morphogenetic ability of MET M1250T and MET D1228H mutants, which also interact with Pi3Kinase, albeit at a lower level than MET L1195V and MET Y1230C, could be due to an unbalanced activation of the Ras and Pi3Kinase pathways as sustained activation of the Ras pathway causes disorganized growth resulting in reduced invasion and tubulogenesis (42).
Mutants belonging to one group, including MET M1250T and MET D1228H, display increased tyrosine kinase activity, stimulate efficiently the Ras pathway, and transform recipient cells in focus forming assays.
L1195V
protein
substitution
P08581
true positive
In the same assay, MET PRC mutants MET L1195V and MET Y1230C, which are almost devoid of transforming ability, induce the luciferase construct to a lower degree, displaying a 60% reduced ability to activate the Ras pathway.
MLP 29 cells expressing MET L1195V and MET Y1230C mutants (which are only weakly transforming in a focus forming assay) showed the best morphogenetic response, developing many long tubular structures (Fig.
3B, cells expressing MET L1195V and MET Y1230C mutants, which also induced branched morphogenesis, were effective in invading the reconstituted basal membrane.
Pull-down experiments of the MET PRC mutants with the GST protein fused to the amino-terminal SH2 domain of p85 showed that MET L1195V and MET Y1230C mutants, displaying the best invasive and tubulogenic ability, interact very efficiently with Pi3Kinase (Fig.
The decreased invasive and morphogenetic ability of MET M1250T and MET D1228H mutants, which also interact with Pi3Kinase, albeit at a lower level than MET L1195V and MET Y1230C, could be due to an unbalanced activation of the Ras and Pi3Kinase pathways as sustained activation of the Ras pathway causes disorganized growth resulting in reduced invasion and tubulogenesis (42).
4A, cells expressing MET L1195V and MET Y1230C mutants display an increased resistance to apoptosis, which is further enhanced by HGF stimulation.
Conversely, the other group of mutations, including MET L1195V and MET Y1230C, are almost devoid of in vitro transforming potential but are effective in inducing protection from apoptosis, sustaining anchorage-independent growth, and promoting invasion.
The most effective mutants were MET L1195V and METY1230C, which were also more resistant to apoptosis.
V1220I
protein
substitution
true positive
P08581
Image mutation
M1131T
protein
substitution
true positive
P08581
Image mutation
10455177
full text
10500188
full text
11309396
full text
Y68H
protein
substitution
true negative
A recent report sug- 25351 25352 Isolation of Active Forms of the MAPK p38/Hog1 TABLE I Sequences of primers used in polymerase chain reaction and site-directed mutagenesis reactions Primer name Primer sequence HOG15 -Y68H HOGI-3 -Y68H HOGI-5 -D170A HOGI-5 -A314T HOGI-3 -A314T HOGI-5 -F318L HOGI-3 -F318L HOGI-5 -F318S HOGI-3 -F318S HOGI-5 -W320R HOGI-3 -W320R HOGI-5 -F322L HOGI-3 -F322L HOGI-5 -W332R HOGI-3 -W332R NP-1 (T174A; Y176F) NP-2 (T174A; Y176F) QC-D170A1 ( Y176F) QC-D170A2 ( Y176F) 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 -tcc -cca -cgg -cca -cga -gaa -gtg -gaa -gtg -cga -gcg -gcc -gat -gat -gta -caa -ccc -gtc -gcc act aat tct acg act cca cca cca cca tgc tca aag cag ctg cat gac tgt tag tgt gca gtc agc gat tgg gta atc gta atc caa tta ttc cgt cct cat cct agt caa cat gtg gac aag gaa cat gcc gag gcc gga gtt aag gat cat gtc aac caa atc gaa ttg ctg ctc aat cca gcc gat ctt gat ctt cga tgc tgg taa gat acg atg tag ttc agg gcc gac tca gta tta gcc ggc gcc ggc tcg cga cac ggt acc ccg gca tgg aag ggc aaa gtc agc acc ctg aag atc aag atc gca tcg ctt gcc cgg ggt ggc aaa ccc ttg agg a-3 ccc gat gtt ctc ggc tcc ggc ctt aac aat aat cgt atc ttt caa ctc aat aca cat ctg gaa c-3 tca gcc cat gat tac gat tac taa ttg gac cga gtt gac gtt agc aaa tct aat aag ccg tgg tgg tgg tgg tga gca gct act atg agg tcc ctg tga tgc g-3 ttc g-3 ttg g-3 cac-3 ttc-3 cac-3 ttc-3 cgc-3 tcg-3 gat c-3 tgg c-3 atg tac-3 cag atc-3 act aga tac tac agg g-3 cca ttt gag ggt ctt g-3 cag gc-3 tag ac-3 gested that these interactions create an interface for homodimerization of the kinase molecules.
of colonies harboring the mutation Y68H D170A A314Ta F318L F318S W320R F322L W332R N391Da 4 1 1 17 3 2 3 9 1 a For unknown reasons, two mutants, N391D and A314T, were not able to rescue pbs2 cells when expressed from a strong promoter and containing an HA tag.
The second mutation in the NH2 terminus is Y68H.
Namely, Hog1Y68H, Hog1D170A, Hog1W320R, Hog1F322L, and Hog1W332R mutants are also hyperactive under any growth conditions but manifest their maximal activity after salt induction.
First, mutants Y68H, D170A, A314T, and W320R are phosphorylated in the absence of salt stimulation, at a significantly higher level than that of the wild type Hog1.
Hog1 mutants Y68H, D170A, and F318L show low levels of phosphorylation in pbs2 cells.
Insertion of appropriate mutations into JNK (equivalent to the HOG1 mutations Y68H and W322R (Fig.
F318L
protein
substitution
true negative
A recent report sug- 25351 25352 Isolation of Active Forms of the MAPK p38/Hog1 TABLE I Sequences of primers used in polymerase chain reaction and site-directed mutagenesis reactions Primer name Primer sequence HOG15 -Y68H HOGI-3 -Y68H HOGI-5 -D170A HOGI-5 -A314T HOGI-3 -A314T HOGI-5 -F318L HOGI-3 -F318L HOGI-5 -F318S HOGI-3 -F318S HOGI-5 -W320R HOGI-3 -W320R HOGI-5 -F322L HOGI-3 -F322L HOGI-5 -W332R HOGI-3 -W332R NP-1 (T174A; Y176F) NP-2 (T174A; Y176F) QC-D170A1 ( Y176F) QC-D170A2 ( Y176F) 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 -tcc -cca -cgg -cca -cga -gaa -gtg -gaa -gtg -cga -gcg -gcc -gat -gat -gta -caa -ccc -gtc -gcc act aat tct acg act cca cca cca cca tgc tca aag cag ctg cat gac tgt tag tgt gca gtc agc gat tgg gta atc gta atc caa tta ttc cgt cct cat cct agt caa cat gtg gac aag gaa cat gcc gag gcc gga gtt aag gat cat gtc aac caa atc gaa ttg ctg ctc aat cca gcc gat ctt gat ctt cga tgc tgg taa gat acg atg tag ttc agg gcc gac tca gta tta gcc ggc gcc ggc tcg cga cac ggt acc ccg gca tgg aag ggc aaa gtc agc acc ctg aag atc aag atc gca tcg ctt gcc cgg ggt ggc aaa ccc ttg agg a-3 ccc gat gtt ctc ggc tcc ggc ctt aac aat aat cgt atc ttt caa ctc aat aca cat ctg gaa c-3 tca gcc cat gat tac gat tac taa ttg gac cga gtt gac gtt agc aaa tct aat aag ccg tgg tgg tgg tgg tga gca gct act atg agg tcc ctg tga tgc g-3 ttc g-3 ttg g-3 cac-3 ttc-3 cac-3 ttc-3 cgc-3 tcg-3 gat c-3 tgg c-3 atg tac-3 cag atc-3 act aga tac tac agg g-3 cca ttt gag ggt ctt g-3 cag gc-3 tag ac-3 gested that these interactions create an interface for homodimerization of the kinase molecules.
of colonies harboring the mutation Y68H D170A A314Ta F318L F318S W320R F322L W332R N391Da 4 1 1 17 3 2 3 9 1 a For unknown reasons, two mutants, N391D and A314T, were not able to rescue pbs2 cells when expressed from a strong promoter and containing an HA tag.
Particularly high basal kinase activity was measured for Hog1F318L and Hog1F318S.
Thus, Hog1F318L and Hog1F318S man- Isolation of Active Forms of the MAPK p38/Hog1 25355 FIG.
Not only Hog1F318L and Hog1F318S but also all the other mutants showed very high catalytic activity in the absence of any stimulation.
Strikingly, mutants F318L and F318S expressed in hog1 seem to be labile.
6B (upper panel, lanes 11 and 12 and lower panel, lanes 5 and 6), in lysates prepared from hog1 cells expressing Hog1F318L or Hog1F318S, a 35-kDa protein reacted with the anti-HA antibodies.
Thus, Hog1F318L and Hog1F318S are intact proteins in vivo but are susceptible to cleavage when vigorous lysis procedures are used.
Note that Hog1F318L and Hog1F318S molecules are fully functional in hog1 cells and allow growth on salt (Fig.
Hog1 mutants Y68H, D170A, and F318L show low levels of phosphorylation in pbs2 cells.
3)) and into ERK2 (equivalent to the HOG1 mutations D170A, A314T, F318S, and F318L (Fig.
Hog1F318S and Hog1F318L) are stronger than others and show very high basal activity in vivo.
F327L
protein
substitution
true negative
The p38Phe327Leu and p38Phe327Ser, as well as a wild type p38 protein, were expressed in E.
We found that p38Phe327Leu and p38Phe327Ser have acquired the intrinsic capability for autophosphorylation (Fig.
T174A
protein
substitution
true negative
A recent report sug- 25351 25352 Isolation of Active Forms of the MAPK p38/Hog1 TABLE I Sequences of primers used in polymerase chain reaction and site-directed mutagenesis reactions Primer name Primer sequence HOG15 -Y68H HOGI-3 -Y68H HOGI-5 -D170A HOGI-5 -A314T HOGI-3 -A314T HOGI-5 -F318L HOGI-3 -F318L HOGI-5 -F318S HOGI-3 -F318S HOGI-5 -W320R HOGI-3 -W320R HOGI-5 -F322L HOGI-3 -F322L HOGI-5 -W332R HOGI-3 -W332R NP-1 (T174A; Y176F) NP-2 (T174A; Y176F) QC-D170A1 ( Y176F) QC-D170A2 ( Y176F) 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 -tcc -cca -cgg -cca -cga -gaa -gtg -gaa -gtg -cga -gcg -gcc -gat -gat -gta -caa -ccc -gtc -gcc act aat tct acg act cca cca cca cca tgc tca aag cag ctg cat gac tgt tag tgt gca gtc agc gat tgg gta atc gta atc caa tta ttc cgt cct cat cct agt caa cat gtg gac aag gaa cat gcc gag gcc gga gtt aag gat cat gtc aac caa atc gaa ttg ctg ctc aat cca gcc gat ctt gat ctt cga tgc tgg taa gat acg atg tag ttc agg gcc gac tca gta tta gcc ggc gcc ggc tcg cga cac ggt acc ccg gca tgg aag ggc aaa gtc agc acc ctg aag atc aag atc gca tcg ctt gcc cgg ggt ggc aaa ccc ttg agg a-3 ccc gat gtt ctc ggc tcc ggc ctt aac aat aat cgt atc ttt caa ctc aat aca cat ctg gaa c-3 tca gcc cat gat tac gat tac taa ttg gac cga gtt gac gtt agc aaa tct aat aag ccg tgg tgg tgg tgg tga gca gct act atg agg tcc ctg tga tgc g-3 ttc g-3 ttg g-3 cac-3 ttc-3 cac-3 ttc-3 cgc-3 tcg-3 gat c-3 tgg c-3 atg tac-3 cag atc-3 act aga tac tac agg g-3 cca ttt gag ggt ctt g-3 cag gc-3 tag ac-3 gested that these interactions create an interface for homodimerization of the kinase molecules.
Active Hog1 mutants containing the mutation Y176F can rescue yeast from high salt concentrations but cannot when containing the T174A mutation.
Hog1 wild type and mutants on the left side contained the mutation T174A, and on the right side they contained the mutation Y176F.
F322L
protein
substitution
true negative
A recent report sug- 25351 25352 Isolation of Active Forms of the MAPK p38/Hog1 TABLE I Sequences of primers used in polymerase chain reaction and site-directed mutagenesis reactions Primer name Primer sequence HOG15 -Y68H HOGI-3 -Y68H HOGI-5 -D170A HOGI-5 -A314T HOGI-3 -A314T HOGI-5 -F318L HOGI-3 -F318L HOGI-5 -F318S HOGI-3 -F318S HOGI-5 -W320R HOGI-3 -W320R HOGI-5 -F322L HOGI-3 -F322L HOGI-5 -W332R HOGI-3 -W332R NP-1 (T174A; Y176F) NP-2 (T174A; Y176F) QC-D170A1 ( Y176F) QC-D170A2 ( Y176F) 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 -tcc -cca -cgg -cca -cga -gaa -gtg -gaa -gtg -cga -gcg -gcc -gat -gat -gta -caa -ccc -gtc -gcc act aat tct acg act cca cca cca cca tgc tca aag cag ctg cat gac tgt tag tgt gca gtc agc gat tgg gta atc gta atc caa tta ttc cgt cct cat cct agt caa cat gtg gac aag gaa cat gcc gag gcc gga gtt aag gat cat gtc aac caa atc gaa ttg ctg ctc aat cca gcc gat ctt gat ctt cga tgc tgg taa gat acg atg tag ttc agg gcc gac tca gta tta gcc ggc gcc ggc tcg cga cac ggt acc ccg gca tgg aag ggc aaa gtc agc acc ctg aag atc aag atc gca tcg ctt gcc cgg ggt ggc aaa ccc ttg agg a-3 ccc gat gtt ctc ggc tcc ggc ctt aac aat aat cgt atc ttt caa ctc aat aca cat ctg gaa c-3 tca gcc cat gat tac gat tac taa ttg gac cga gtt gac gtt agc aaa tct aat aag ccg tgg tgg tgg tgg tga gca gct act atg agg tcc ctg tga tgc g-3 ttc g-3 ttg g-3 cac-3 ttc-3 cac-3 ttc-3 cgc-3 tcg-3 gat c-3 tgg c-3 atg tac-3 cag atc-3 act aga tac tac agg g-3 cca ttt gag ggt ctt g-3 cag gc-3 tag ac-3 gested that these interactions create an interface for homodimerization of the kinase molecules.
of colonies harboring the mutation Y68H D170A A314Ta F318L F318S W320R F322L W332R N391Da 4 1 1 17 3 2 3 9 1 a For unknown reasons, two mutants, N391D and A314T, were not able to rescue pbs2 cells when expressed from a strong promoter and containing an HA tag.
Namely, Hog1Y68H, Hog1D170A, Hog1W320R, Hog1F322L, and Hog1W332R mutants are also hyperactive under any growth conditions but manifest their maximal activity after salt induction.
D170A
protein
substitution
true negative
A recent report sug- 25351 25352 Isolation of Active Forms of the MAPK p38/Hog1 TABLE I Sequences of primers used in polymerase chain reaction and site-directed mutagenesis reactions Primer name Primer sequence HOG15 -Y68H HOGI-3 -Y68H HOGI-5 -D170A HOGI-5 -A314T HOGI-3 -A314T HOGI-5 -F318L HOGI-3 -F318L HOGI-5 -F318S HOGI-3 -F318S HOGI-5 -W320R HOGI-3 -W320R HOGI-5 -F322L HOGI-3 -F322L HOGI-5 -W332R HOGI-3 -W332R NP-1 (T174A; Y176F) NP-2 (T174A; Y176F) QC-D170A1 ( Y176F) QC-D170A2 ( Y176F) 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 -tcc -cca -cgg -cca -cga -gaa -gtg -gaa -gtg -cga -gcg -gcc -gat -gat -gta -caa -ccc -gtc -gcc act aat tct acg act cca cca cca cca tgc tca aag cag ctg cat gac tgt tag tgt gca gtc agc gat tgg gta atc gta atc caa tta ttc cgt cct cat cct agt caa cat gtg gac aag gaa cat gcc gag gcc gga gtt aag gat cat gtc aac caa atc gaa ttg ctg ctc aat cca gcc gat ctt gat ctt cga tgc tgg taa gat acg atg tag ttc agg gcc gac tca gta tta gcc ggc gcc ggc tcg cga cac ggt acc ccg gca tgg aag ggc aaa gtc agc acc ctg aag atc aag atc gca tcg ctt gcc cgg ggt ggc aaa ccc ttg agg a-3 ccc gat gtt ctc ggc tcc ggc ctt aac aat aat cgt atc ttt caa ctc aat aca cat ctg gaa c-3 tca gcc cat gat tac gat tac taa ttg gac cga gtt gac gtt agc aaa tct aat aag ccg tgg tgg tgg tgg tga gca gct act atg agg tcc ctg tga tgc g-3 ttc g-3 ttg g-3 cac-3 ttc-3 cac-3 ttc-3 cgc-3 tcg-3 gat c-3 tgg c-3 atg tac-3 cag atc-3 act aga tac tac agg g-3 cca ttt gag ggt ctt g-3 cag gc-3 tag ac-3 gested that these interactions create an interface for homodimerization of the kinase molecules.
of colonies harboring the mutation Y68H D170A A314Ta F318L F318S W320R F322L W332R N391Da 4 1 1 17 3 2 3 9 1 a For unknown reasons, two mutants, N391D and A314T, were not able to rescue pbs2 cells when expressed from a strong promoter and containing an HA tag.
One mutation (D170A) is located just four amino acids from the phosphoacceptor Thr174.
Namely, Hog1Y68H, Hog1D170A, Hog1W320R, Hog1F322L, and Hog1W332R mutants are also hyperactive under any growth conditions but manifest their maximal activity after salt induction.
First, mutants Y68H, D170A, A314T, and W320R are phosphorylated in the absence of salt stimulation, at a significantly higher level than that of the wild type Hog1.
8 for Hog1D170AY176F).
Hog1 mutants Y68H, D170A, and F318L show low levels of phosphorylation in pbs2 cells.
Replacement of only Tyr176 with Phe does not abolish the activity of Hog1D170A (indicated with one asterisk; A, left plate, row 5).
3)) and into ERK2 (equivalent to the HOG1 mutations D170A, A314T, F318S, and F318L (Fig.
Additional support for this notion comes from the D170A mutation.
Y176F
protein
substitution
true negative
A recent report sug- 25351 25352 Isolation of Active Forms of the MAPK p38/Hog1 TABLE I Sequences of primers used in polymerase chain reaction and site-directed mutagenesis reactions Primer name Primer sequence HOG15 -Y68H HOGI-3 -Y68H HOGI-5 -D170A HOGI-5 -A314T HOGI-3 -A314T HOGI-5 -F318L HOGI-3 -F318L HOGI-5 -F318S HOGI-3 -F318S HOGI-5 -W320R HOGI-3 -W320R HOGI-5 -F322L HOGI-3 -F322L HOGI-5 -W332R HOGI-3 -W332R NP-1 (T174A; Y176F) NP-2 (T174A; Y176F) QC-D170A1 ( Y176F) QC-D170A2 ( Y176F) 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 -tcc -cca -cgg -cca -cga -gaa -gtg -gaa -gtg -cga -gcg -gcc -gat -gat -gta -caa -ccc -gtc -gcc act aat tct acg act cca cca cca cca tgc tca aag cag ctg cat gac tgt tag tgt gca gtc agc gat tgg gta atc gta atc caa tta ttc cgt cct cat cct agt caa cat gtg gac aag gaa cat gcc gag gcc gga gtt aag gat cat gtc aac caa atc gaa ttg ctg ctc aat cca gcc gat ctt gat ctt cga tgc tgg taa gat acg atg tag ttc agg gcc gac tca gta tta gcc ggc gcc ggc tcg cga cac ggt acc ccg gca tgg aag ggc aaa gtc agc acc ctg aag atc aag atc gca tcg ctt gcc cgg ggt ggc aaa ccc ttg agg a-3 ccc gat gtt ctc ggc tcc ggc ctt aac aat aat cgt atc ttt caa ctc aat aca cat ctg gaa c-3 tca gcc cat gat tac gat tac taa ttg gac cga gtt gac gtt agc aaa tct aat aag ccg tgg tgg tgg tgg tga gca gct act atg agg tcc ctg tga tgc g-3 ttc g-3 ttg g-3 cac-3 ttc-3 cac-3 ttc-3 cgc-3 tcg-3 gat c-3 tgg c-3 atg tac-3 cag atc-3 act aga tac tac agg g-3 cca ttt gag ggt ctt g-3 cag gc-3 tag ac-3 gested that these interactions create an interface for homodimerization of the kinase molecules.
8 for Hog1D170AY176F).
Only Hog1A314T and Hog1W320R were affected by the Y176F mutation.
Active Hog1 mutants containing the mutation Y176F can rescue yeast from high salt concentrations but cannot when containing the T174A mutation.
Hog1 wild type and mutants on the left side contained the mutation T174A, and on the right side they contained the mutation Y176F.
W322R
protein
substitution
true negative
Insertion of appropriate mutations into JNK (equivalent to the HOG1 mutations Y68H and W322R (Fig.
N391D
protein
substitution
true negative
of colonies harboring the mutation Y68H D170A A314Ta F318L F318S W320R F322L W332R N391Da 4 1 1 17 3 2 3 9 1 a For unknown reasons, two mutants, N391D and A314T, were not able to rescue pbs2 cells when expressed from a strong promoter and containing an HA tag.
W320R
protein
substitution
true negative
A recent report sug- 25351 25352 Isolation of Active Forms of the MAPK p38/Hog1 TABLE I Sequences of primers used in polymerase chain reaction and site-directed mutagenesis reactions Primer name Primer sequence HOG15 -Y68H HOGI-3 -Y68H HOGI-5 -D170A HOGI-5 -A314T HOGI-3 -A314T HOGI-5 -F318L HOGI-3 -F318L HOGI-5 -F318S HOGI-3 -F318S HOGI-5 -W320R HOGI-3 -W320R HOGI-5 -F322L HOGI-3 -F322L HOGI-5 -W332R HOGI-3 -W332R NP-1 (T174A; Y176F) NP-2 (T174A; Y176F) QC-D170A1 ( Y176F) QC-D170A2 ( Y176F) 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 -tcc -cca -cgg -cca -cga -gaa -gtg -gaa -gtg -cga -gcg -gcc -gat -gat -gta -caa -ccc -gtc -gcc act aat tct acg act cca cca cca cca tgc tca aag cag ctg cat gac tgt tag tgt gca gtc agc gat tgg gta atc gta atc caa tta ttc cgt cct cat cct agt caa cat gtg gac aag gaa cat gcc gag gcc gga gtt aag gat cat gtc aac caa atc gaa ttg ctg ctc aat cca gcc gat ctt gat ctt cga tgc tgg taa gat acg atg tag ttc agg gcc gac tca gta tta gcc ggc gcc ggc tcg cga cac ggt acc ccg gca tgg aag ggc aaa gtc agc acc ctg aag atc aag atc gca tcg ctt gcc cgg ggt ggc aaa ccc ttg agg a-3 ccc gat gtt ctc ggc tcc ggc ctt aac aat aat cgt atc ttt caa ctc aat aca cat ctg gaa c-3 tca gcc cat gat tac gat tac taa ttg gac cga gtt gac gtt agc aaa tct aat aag ccg tgg tgg tgg tgg tga gca gct act atg agg tcc ctg tga tgc g-3 ttc g-3 ttg g-3 cac-3 ttc-3 cac-3 ttc-3 cgc-3 tcg-3 gat c-3 tgg c-3 atg tac-3 cag atc-3 act aga tac tac agg g-3 cca ttt gag ggt ctt g-3 cag gc-3 tag ac-3 gested that these interactions create an interface for homodimerization of the kinase molecules.
of colonies harboring the mutation Y68H D170A A314Ta F318L F318S W320R F322L W332R N391Da 4 1 1 17 3 2 3 9 1 a For unknown reasons, two mutants, N391D and A314T, were not able to rescue pbs2 cells when expressed from a strong promoter and containing an HA tag.
3B) reveals that all mutations but one, W320R, occur in residues that are conserved in at least one subfamily.
Namely, Hog1Y68H, Hog1D170A, Hog1W320R, Hog1F322L, and Hog1W332R mutants are also hyperactive under any growth conditions but manifest their maximal activity after salt induction.
First, mutants Y68H, D170A, A314T, and W320R are phosphorylated in the absence of salt stimulation, at a significantly higher level than that of the wild type Hog1.
We expected that all mutants (and in particular F318S, W320R, and W332R, which are not phosphorylated at all in pbs2 cells) would tolerate replacement of Thr174 with Ala and Tyr176 with Phe and would remain active.
Only Hog1A314T and Hog1W320R were affected by the Y176F mutation.
Most significantly, when expressed in pbs2 cells, the active Hog1 mutants F318S, W320R, and W332R show no detectable phosphorylation (Fig.
A314T
protein
substitution
true negative
A recent report sug- 25351 25352 Isolation of Active Forms of the MAPK p38/Hog1 TABLE I Sequences of primers used in polymerase chain reaction and site-directed mutagenesis reactions Primer name Primer sequence HOG15 -Y68H HOGI-3 -Y68H HOGI-5 -D170A HOGI-5 -A314T HOGI-3 -A314T HOGI-5 -F318L HOGI-3 -F318L HOGI-5 -F318S HOGI-3 -F318S HOGI-5 -W320R HOGI-3 -W320R HOGI-5 -F322L HOGI-3 -F322L HOGI-5 -W332R HOGI-3 -W332R NP-1 (T174A; Y176F) NP-2 (T174A; Y176F) QC-D170A1 ( Y176F) QC-D170A2 ( Y176F) 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 -tcc -cca -cgg -cca -cga -gaa -gtg -gaa -gtg -cga -gcg -gcc -gat -gat -gta -caa -ccc -gtc -gcc act aat tct acg act cca cca cca cca tgc tca aag cag ctg cat gac tgt tag tgt gca gtc agc gat tgg gta atc gta atc caa tta ttc cgt cct cat cct agt caa cat gtg gac aag gaa cat gcc gag gcc gga gtt aag gat cat gtc aac caa atc gaa ttg ctg ctc aat cca gcc gat ctt gat ctt cga tgc tgg taa gat acg atg tag ttc agg gcc gac tca gta tta gcc ggc gcc ggc tcg cga cac ggt acc ccg gca tgg aag ggc aaa gtc agc acc ctg aag atc aag atc gca tcg ctt gcc cgg ggt ggc aaa ccc ttg agg a-3 ccc gat gtt ctc ggc tcc ggc ctt aac aat aat cgt atc ttt caa ctc aat aca cat ctg gaa c-3 tca gcc cat gat tac gat tac taa ttg gac cga gtt gac gtt agc aaa tct aat aag ccg tgg tgg tgg tgg tga gca gct act atg agg tcc ctg tga tgc g-3 ttc g-3 ttg g-3 cac-3 ttc-3 cac-3 ttc-3 cgc-3 tcg-3 gat c-3 tgg c-3 atg tac-3 cag atc-3 act aga tac tac agg g-3 cca ttt gag ggt ctt g-3 cag gc-3 tag ac-3 gested that these interactions create an interface for homodimerization of the kinase molecules.
of colonies harboring the mutation Y68H D170A A314Ta F318L F318S W320R F322L W332R N391Da 4 1 1 17 3 2 3 9 1 a For unknown reasons, two mutants, N391D and A314T, were not able to rescue pbs2 cells when expressed from a strong promoter and containing an HA tag.
First, mutants Y68H, D170A, A314T, and W320R are phosphorylated in the absence of salt stimulation, at a significantly higher level than that of the wild type Hog1.
Only Hog1A314T and Hog1W320R were affected by the Y176F mutation.
3)) and into ERK2 (equivalent to the HOG1 mutations D170A, A314T, F318S, and F318L (Fig.
W332R
protein
substitution
true negative
A recent report sug- 25351 25352 Isolation of Active Forms of the MAPK p38/Hog1 TABLE I Sequences of primers used in polymerase chain reaction and site-directed mutagenesis reactions Primer name Primer sequence HOG15 -Y68H HOGI-3 -Y68H HOGI-5 -D170A HOGI-5 -A314T HOGI-3 -A314T HOGI-5 -F318L HOGI-3 -F318L HOGI-5 -F318S HOGI-3 -F318S HOGI-5 -W320R HOGI-3 -W320R HOGI-5 -F322L HOGI-3 -F322L HOGI-5 -W332R HOGI-3 -W332R NP-1 (T174A; Y176F) NP-2 (T174A; Y176F) QC-D170A1 ( Y176F) QC-D170A2 ( Y176F) 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 -tcc -cca -cgg -cca -cga -gaa -gtg -gaa -gtg -cga -gcg -gcc -gat -gat -gta -caa -ccc -gtc -gcc act aat tct acg act cca cca cca cca tgc tca aag cag ctg cat gac tgt tag tgt gca gtc agc gat tgg gta atc gta atc caa tta ttc cgt cct cat cct agt caa cat gtg gac aag gaa cat gcc gag gcc gga gtt aag gat cat gtc aac caa atc gaa ttg ctg ctc aat cca gcc gat ctt gat ctt cga tgc tgg taa gat acg atg tag ttc agg gcc gac tca gta tta gcc ggc gcc ggc tcg cga cac ggt acc ccg gca tgg aag ggc aaa gtc agc acc ctg aag atc aag atc gca tcg ctt gcc cgg ggt ggc aaa ccc ttg agg a-3 ccc gat gtt ctc ggc tcc ggc ctt aac aat aat cgt atc ttt caa ctc aat aca cat ctg gaa c-3 tca gcc cat gat tac gat tac taa ttg gac cga gtt gac gtt agc aaa tct aat aag ccg tgg tgg tgg tgg tga gca gct act atg agg tcc ctg tga tgc g-3 ttc g-3 ttg g-3 cac-3 ttc-3 cac-3 ttc-3 cgc-3 tcg-3 gat c-3 tgg c-3 atg tac-3 cag atc-3 act aga tac tac agg g-3 cca ttt gag ggt ctt g-3 cag gc-3 tag ac-3 gested that these interactions create an interface for homodimerization of the kinase molecules.
of colonies harboring the mutation Y68H D170A A314Ta F318L F318S W320R F322L W332R N391Da 4 1 1 17 3 2 3 9 1 a For unknown reasons, two mutants, N391D and A314T, were not able to rescue pbs2 cells when expressed from a strong promoter and containing an HA tag.
Namely, Hog1Y68H, Hog1D170A, Hog1W320R, Hog1F322L, and Hog1W332R mutants are also hyperactive under any growth conditions but manifest their maximal activity after salt induction.
We expected that all mutants (and in particular F318S, W320R, and W332R, which are not phosphorylated at all in pbs2 cells) would tolerate replacement of Thr174 with Ala and Tyr176 with Phe and would remain active.
Most significantly, when expressed in pbs2 cells, the active Hog1 mutants F318S, W320R, and W332R show no detectable phosphorylation (Fig.
F327S
protein
substitution
true negative
The p38Phe327Leu and p38Phe327Ser, as well as a wild type p38 protein, were expressed in E.
We found that p38Phe327Leu and p38Phe327Ser have acquired the intrinsic capability for autophosphorylation (Fig.
F318S
protein
substitution
true positive
P08018
A recent report sug- 25351 25352 Isolation of Active Forms of the MAPK p38/Hog1 TABLE I Sequences of primers used in polymerase chain reaction and site-directed mutagenesis reactions Primer name Primer sequence HOG15 -Y68H HOGI-3 -Y68H HOGI-5 -D170A HOGI-5 -A314T HOGI-3 -A314T HOGI-5 -F318L HOGI-3 -F318L HOGI-5 -F318S HOGI-3 -F318S HOGI-5 -W320R HOGI-3 -W320R HOGI-5 -F322L HOGI-3 -F322L HOGI-5 -W332R HOGI-3 -W332R NP-1 (T174A; Y176F) NP-2 (T174A; Y176F) QC-D170A1 ( Y176F) QC-D170A2 ( Y176F) 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 -tcc -cca -cgg -cca -cga -gaa -gtg -gaa -gtg -cga -gcg -gcc -gat -gat -gta -caa -ccc -gtc -gcc act aat tct acg act cca cca cca cca tgc tca aag cag ctg cat gac tgt tag tgt gca gtc agc gat tgg gta atc gta atc caa tta ttc cgt cct cat cct agt caa cat gtg gac aag gaa cat gcc gag gcc gga gtt aag gat cat gtc aac caa atc gaa ttg ctg ctc aat cca gcc gat ctt gat ctt cga tgc tgg taa gat acg atg tag ttc agg gcc gac tca gta tta gcc ggc gcc ggc tcg cga cac ggt acc ccg gca tgg aag ggc aaa gtc agc acc ctg aag atc aag atc gca tcg ctt gcc cgg ggt ggc aaa ccc ttg agg a-3 ccc gat gtt ctc ggc tcc ggc ctt aac aat aat cgt atc ttt caa ctc aat aca cat ctg gaa c-3 tca gcc cat gat tac gat tac taa ttg gac cga gtt gac gtt agc aaa tct aat aag ccg tgg tgg tgg tgg tga gca gct act atg agg tcc ctg tga tgc g-3 ttc g-3 ttg g-3 cac-3 ttc-3 cac-3 ttc-3 cgc-3 tcg-3 gat c-3 tgg c-3 atg tac-3 cag atc-3 act aga tac tac agg g-3 cca ttt gag ggt ctt g-3 cag gc-3 tag ac-3 gested that these interactions create an interface for homodimerization of the kinase molecules.
of colonies harboring the mutation Y68H D170A A314Ta F318L F318S W320R F322L W332R N391Da 4 1 1 17 3 2 3 9 1 a For unknown reasons, two mutants, N391D and A314T, were not able to rescue pbs2 cells when expressed from a strong promoter and containing an HA tag.
Particularly high basal kinase activity was measured for Hog1F318L and Hog1F318S.
Thus, Hog1F318L and Hog1F318S man- Isolation of Active Forms of the MAPK p38/Hog1 25355 FIG.
Not only Hog1F318L and Hog1F318S but also all the other mutants showed very high catalytic activity in the absence of any stimulation.
Strikingly, mutants F318L and F318S expressed in hog1 seem to be labile.
6B (upper panel, lanes 11 and 12 and lower panel, lanes 5 and 6), in lysates prepared from hog1 cells expressing Hog1F318L or Hog1F318S, a 35-kDa protein reacted with the anti-HA antibodies.
Thus, Hog1F318L and Hog1F318S are intact proteins in vivo but are susceptible to cleavage when vigorous lysis procedures are used.
Note that Hog1F318L and Hog1F318S molecules are fully functional in hog1 cells and allow growth on salt (Fig.
We expected that all mutants (and in particular F318S, W320R, and W332R, which are not phosphorylated at all in pbs2 cells) would tolerate replacement of Thr174 with Ala and Tyr176 with Phe and would remain active.
Most significantly, when expressed in pbs2 cells, the active Hog1 mutants F318S, W320R, and W332R show no detectable phosphorylation (Fig.
The positive control was Hog1F318S expressed in pbs2 cells (first row, right side); the negative control was wild type Hog1 expressed in pbs2 cells (first row, left side).
3)) and into ERK2 (equivalent to the HOG1 mutations D170A, A314T, F318S, and F318L (Fig.
Hog1F318S and Hog1F318L) are stronger than others and show very high basal activity in vivo.
15146033
full text
11278817
full text
T266A
protein
substitution
true negative
Since the amino acids surrounding Thr266 appeared most homologous against a consensus MAP kinase site, we mutated the threonine residue by replacing it with an alanine to create Sp1-T266A.
The use of Sp1-T266A in transfection studies revealed the following results.
Although transfection of Sp1-T266A alone into stable Hep G2 cells augmented activity of pAI.474-CAT, the activity was not significantly different from wild-type Sp1.
10212216
full text
K1294L
protein
substitution
true negative
Y950, mutation to Phe at tyrosine 950; 3Y, tyrosines 1131, 1135, and 1136 mutated to Phe; Y1250 1251, tyrosines 1250 and 1251 mutated to Phe; 6 serine, serines 1272, 1278, and 1280 1283 mutated to alanine; 4 basic aa, R1289F,H1290L,H1293F,K1294L; 12931294, H1293F,K1294L; 1245, wild type IGF-IR truncated at residue 1245.
Y950F
protein
substitution
true negative
Using the same conditions, we can see that the three mutant receptors, Y950F, Y1250F/Y1251F, and d1245, have lost the ability to induce differentiation (Fig.
However, the Y950F mutant, which fails to induce differentiation, also fails to increase Shc phosphorylation and to co-precipitate Grb2.
5) Shc phosphorylation is decreased in cells expressing the two mutant receptors that fail to induce differentiation (Y950F and Y1250F/Y1251F).
Indeed, the 32D cells expressing the Y950F mutant receptor completely fail to phosphorylate Shc.
H1290L
protein
substitution
true negative
Y950, mutation to Phe at tyrosine 950; 3Y, tyrosines 1131, 1135, and 1136 mutated to Phe; Y1250 1251, tyrosines 1250 and 1251 mutated to Phe; 6 serine, serines 1272, 1278, and 1280 1283 mutated to alanine; 4 basic aa, R1289F,H1290L,H1293F,K1294L; 12931294, H1293F,K1294L; 1245, wild type IGF-IR truncated at residue 1245.
Y1251F
protein
substitution
true negative
Using the same conditions, we can see that the three mutant receptors, Y950F, Y1250F/Y1251F, and d1245, have lost the ability to induce differentiation (Fig.
5) Shc phosphorylation is decreased in cells expressing the two mutant receptors that fail to induce differentiation (Y950F and Y1250F/Y1251F).
Y1250F
protein
substitution
true negative
Using the same conditions, we can see that the three mutant receptors, Y950F, Y1250F/Y1251F, and d1245, have lost the ability to induce differentiation (Fig.
5) Shc phosphorylation is decreased in cells expressing the two mutant receptors that fail to induce differentiation (Y950F and Y1250F/Y1251F).
R1289F
protein
substitution
true negative
Y950, mutation to Phe at tyrosine 950; 3Y, tyrosines 1131, 1135, and 1136 mutated to Phe; Y1250 1251, tyrosines 1250 and 1251 mutated to Phe; 6 serine, serines 1272, 1278, and 1280 1283 mutated to alanine; 4 basic aa, R1289F,H1290L,H1293F,K1294L; 12931294, H1293F,K1294L; 1245, wild type IGF-IR truncated at residue 1245.
H1293F
protein
substitution
true negative
Y950, mutation to Phe at tyrosine 950; 3Y, tyrosines 1131, 1135, and 1136 mutated to Phe; Y1250 1251, tyrosines 1250 and 1251 mutated to Phe; 6 serine, serines 1272, 1278, and 1280 1283 mutated to alanine; 4 basic aa, R1289F,H1290L,H1293F,K1294L; 12931294, H1293F,K1294L; 1245, wild type IGF-IR truncated at residue 1245.
10617656
full text
10811866
full text
K445Q
protein
substitution
true positive
P51813
Etk(kq) harbors a point mutation (K445Q) in its ATP binding pocket, resulting in the loss of its kinase activity (22).
E42K
protein
substitution
true positive
P51813
A substitution of glutamate residue 42 to lysine (E42K) in the PH domain of Etk gives rise to the dominant-negative expression construct Etk(DN) (38).
10962552
full text
K401S
protein
substitution
true negative
The activity of the Lys401 to Ser (K401S) and Thr407 to Ser (T407S) mutants are most similar to Wt and appear to have primarily lost phosphorylation of a 31 33 kDa doublet while substitution of Thr407 to either Ala (T407A) or Met (T407M) disrupts mutually exclusive protein phosphorylations.
Off by 1 numbering for mutations in P06239
K405D
protein
substitution
true negative
The K405D, K405G, T407D, and T407R mutants all exhibited autokinase activities that were 500 1200 times less than that of Wt Lck (data not shown).
However, the four mutants in this region analysed in vitro (K405D, K405G, T407D, and T407R) were also inactive upon immunoprecipitation.
To generate the point mutations at K405, double-stranded NarI SI oligos (K405D: 5'c gcc aag ttt ccc att gac tgg acg gcc cca g3'; K405F: 5'c gcc aag ttt ccc att ttc tgg acg gcc cca g3'; K405G: 5'c gcc aag ttt ccc att ggc tgg acg gcc cca g3') were subcloned into the NarI SI digested synk-pGEM3zf+ backbone.
Off by 1 numbering for mutations in P06239
K405G
protein
substitution
true negative
The K405D, K405G, T407D, and T407R mutants all exhibited autokinase activities that were 500 1200 times less than that of Wt Lck (data not shown).
However, the four mutants in this region analysed in vitro (K405D, K405G, T407D, and T407R) were also inactive upon immunoprecipitation.
To generate the point mutations at K405, double-stranded NarI SI oligos (K405D: 5'c gcc aag ttt ccc att gac tgg acg gcc cca g3'; K405F: 5'c gcc aag ttt ccc att ttc tgg acg gcc cca g3'; K405G: 5'c gcc aag ttt ccc att ggc tgg acg gcc cca g3') were subcloned into the NarI SI digested synk-pGEM3zf+ backbone.
Off by 1 numbering for mutations in P06239
K405F
protein
substitution
true negative
To generate the point mutations at K405, double-stranded NarI SI oligos (K405D: 5'c gcc aag ttt ccc att gac tgg acg gcc cca g3'; K405F: 5'c gcc aag ttt ccc att ttc tgg acg gcc cca g3'; K405G: 5'c gcc aag ttt ccc att ggc tgg acg gcc cca g3') were subcloned into the NarI SI digested synk-pGEM3zf+ backbone.
Off by 1 numbering for mutations in P06239
G399A
protein
substitution
true negative
The Gly399 to Ala (G399A) mutant displays a unique phosphorylation pattern from the Lys401 and Thr407 mutants.
Off by 1 numbering for mutations in P06239
E390M
protein
substitution
true negative
Mutation of each site to bulkier (Met; E390M) or charged residues (Asp; L388D) measurably reduces kinase activity towards bacterial proteins (as does the double mutant L388D/E390M).
Wt Lck, Lck containing activation loop mutations below Y394 (L388D, L388N, E390M, E390T, and L388D/E390M), and corresponding Y505F double mutants were subcloned into the prokaryotic expression vector pGEX-2T.
Drug-resistant pools of BalbC 3T3 broblasts expressing either Wt Lck, Lck containing activation loop mutations N-terminal to Y394 (L388D, L388N, E390M, E390T, and L388D/E390M), and corresponding Y505F double mutants were analysed for dierences in PTyr content by Western blotting.
The levels of PTyr associated with the E390M and E390T single mutants were similar to that of Wt Lck, presumably due to Csk downregulation at Y505.
However, the L388D, L388N, and L388D/E390M mutants display increased PTyr levels compared to Wt Lck and have increased abilities to phosphorylate cellular proteins (more easily visible on longer exposures).
The subtle Oncogene Effects of activation loop mutations on Lck regulation LE Laham et al 3964 dierences in substrate specicity observed when the L388D, E390M, and L388D/E390M mutants were expressed in bacteria are not detectable upon expression in broblasts.
Similarly, most of the mutants show slightly decreased kinase activity in a Y505F background and also display a level of morphological transformation which is slightly down from the Y505F single mutant, with the L388D/E390M/Y505F triple mutant having the least transformed morphology.
We show that mutations in the conserved amino terminal region of the activation loop (L388D, L388N, and L388D/E390M) activate Lck in vivo, coincident with increased levels of PTyr on the kinase, but have little eect on substrate specicity.
To generate the activation loop mutations N-terminal to Y394, doublestranded NheI BssHII oligos (L388D: 5'cta gca cgc gat att gag gac aac gag tac aca g3'; L388N: 5'cta gca cgc aat att gag gac aac gag tac aca g3'; E390M: 5'cta gca cgc ctc att atg gac aac gag tac aca g3'; E390T: 5'cta gca cgc ctc att acc gac aac gag tac aca g3'; L388D/E390M: 5'cta gca cgc gat att atg gac aac gag tac aca g3') were subcloned into the NheI BssHII digested synk-pGEM3zf+ backbone.
Off by 1 numbering for mutations in P06239
E390T
protein
substitution
true negative
Mutations at Leu388 to Asn (L388N) and at Glu390 to Thr (E390T) have little aect on activity, both in the Wt and Y505F backgrounds.
Off by 1 numbering for mutations in P06239
Y394F
protein
substitution
true negative
To analyse the eects of activation loop mutations on autophosphorylation and subsequent activation of the kinase, Lck Y394F and Y394-downstream mutants were expressed in both bacterial and mammalian expression systems in combination with Y505F mutations.
Specically, the YCPK/Y505F and YNQQ/Y505F mutants were able to phosphorylate proteins more readily than the YGQQ/ Y505F and Y394F/Y505F mutants (Figure 3b).
Indeed Y394 phosphorylation is not necessary for in vitro kinase activity as the Y394F and the Y394F/Y505F Lck mutants isolated from either mammalian cells (Figure 3d) or insect cells (data not shown) are still active.
As shown in Figures 3b and 4a, substitution of the three amino acids downstream of Y394 (TAR) to CPK, GQQ, and NQQ does not aect the level of Lck Y505 phosphorylation when expressed in broblasts as the Wt Lck, the Y394-downstream mutants, and Y394F Lck all contain similar levels of PTyr.
Many previous in vivo labeling and phosphate mapping studies have shown that both Wt and Y394F Lck expressed in broblasts only contain tyrosine phosphorylation at the C-terminal downregulatory site Y505 (Abraham and Veillette, 1990; Amrein and Sefton, 1988; Gervais et al., 1993; Weil and Veillette, 1994).
Wt Lck, Lck containing activation loop mutations at or immediately downstream of Y394 (Y394F, YCPK, YGQQ, YNQQ), and corresponding Y505F double mutants were subcloned into the prokaryotic expression vector pGEX-2T.
Mouse broblasts expressing Wt Lck, Lck containing activation loop mutations at or immediately downstream to Y394 (Y394F, YCPK, YGQQ, YNQQ), and corresponding Y505F double mutants were analysed for dierences in PTyr and Lck as described in Figure 2.
To create the specic activation loop mutations at, or immediately C-terminal to Y394, double-stranded NheI NarI oligos (Y394F:5'cta gca cgc ctc att gag gac aac gag ttc aca gcg cgc gag gg3'; YCPK: 5'cta gca cgc ctc att gag gac aac gag tac tgc cct aag gag gg3'; YGQQ: 5'cta gca cgc ctc att gag gac aac gag tac gga cag cag gag gg3'; YNQQ: 5'cta gca cgc ctc att gag gac aac gag tac aac cag cag gag gg3') were subcloned into the NheI NarI digested synk-pGEM3zf+ backbone.
For construction of Y505F, YCPK, YGQQ, YNQQ and Y394F mutants in mammalian expression vectors, Wt Lck and mutant cDNA's were subcloned into pLNCX (Clontech, Palo Alto, CA, USA).
Kinase inactive Lck constructs were generated by creating K273R mutations in the pLNCX expression vectors by substituting the 739 bp BglII SalI fragments from K273R synk (Carrera et al., 1993) into each of the Wt, Y394F, YCPK, YNQQ, and Y505F pLNCX mutants.
Off by 1 numbering for mutations in P06239
K273R
protein
substitution
true negative
To eliminate the possibility of any background autophosphorylation at Y394, the PTyr were re-examined using constructs containing an additional Lys273 to Arg mutation (K273R), which has been shown to inactivate the kinase domain by disrupting the phosphate-transfer reaction.
Off by 1 numbering for mutations in P06239
T407M
protein
substitution
true negative
The activity of the Lys401 to Ser (K401S) and Thr407 to Ser (T407S) mutants are most similar to Wt and appear to have primarily lost phosphorylation of a 31 33 kDa doublet while substitution of Thr407 to either Ala (T407A) or Met (T407M) disrupts mutually exclusive protein phosphorylations.
Wt Lck, Lck containing activation loop mutations C-terminal to Y394 (G399A, K401S, T407A, T407M, T407S), and the corresponding Y505F double mutants were subcloned into the prokaryotic expression vector pGEX-2T.
Mouse broblasts expressing Wt Lck, Lck containing activation loop mutations C-terminal to Y394 (G399A, K401S, T407A, T407M, T407S), and the corresponding Y505F double mutants were analysed for dierences in PTyr content by Western blotting as described in Figure 2.
The upstream PCR oligo at the HincII site was 5'tgc tct aga cat ggc agc cca aat tgc3' while the downstream PCR oligos containing the point mutations are summarized as follows: G399A: 5'cgg ggt acc gta gtt aat ggc ctc tgg ggc cgt cca ctt aat ggg aaa ctt ggc ggc ctc3'; K401S: 5'cgg ggt acc gta gtt aat ggc ctc tgg ggc cgt cca ctt aat ggg aaa gct ggc tcc3'; F402I: 5'cgg ggt acc gta gtt aat ggc ctc tgg ggc cgt cca ctt aat ggg gat ctt gcc3'; F402A: 5'cgg ggt acc gta gtt aat ggc ctc tgg ggc cgt cca ctt aat ggg tgc ctt ggc3'; P403S: 5'cgg ggt acc gta gtt aat ggc ctc tgg ggc cgt cca ctt aat tga aaa ctt3'; P403T: 5'cgg ggt acc gta gtt aat ggc ctc tgg ggc cgt cca ctt aat tgt aaa ctt3'; W406Y: 5'cgg ggt acc gta gtt aat ggc ctc tgg ggc cgt gta ctt aat 3'; T407A: 5'cgg ggt acc gta gtt aat ggc ctc tgg ggc tgc cca ctt aat3'; T407D:5'cgg ggt acc gta gtt aat ggc ctc tgg ggc gtc cca ctt aat3'; T407M: 5'cgg ggt acc gta gtt ggc ctc tgg ggc cat cca ctt aat3'; T407R: 5'cgg ggt acc gta gtt aat ggc ctc tgg ggc cct cca ctt aat3'; T407S: 5'cgg ggt acc gta gtt aat aac ctc tgg ggc tga cca ctt aat3'.
Off by 1 numbering for mutations in P06239
L388N
protein
substitution
true negative
Mutations at Leu388 to Asn (L388N) and at Glu390 to Thr (E390T) have little aect on activity, both in the Wt and Y505F backgrounds.
Off by 1 numbering for mutations in P06239
T407A
protein
substitution
true negative
The activity of the Lys401 to Ser (K401S) and Thr407 to Ser (T407S) mutants are most similar to Wt and appear to have primarily lost phosphorylation of a 31 33 kDa doublet while substitution of Thr407 to either Ala (T407A) or Met (T407M) disrupts mutually exclusive protein phosphorylations.
When these mutants were expressed in a Y505F background, the order of activity levels mimicked that observed in bacteria with the K401S mutant being most similar to Y505F followed by the T407A and G399A mutants.
Wt Lck, Lck containing activation loop mutations C-terminal to Y394 (G399A, K401S, T407A, T407M, T407S), and the corresponding Y505F double mutants were subcloned into the prokaryotic expression vector pGEX-2T.
Mouse broblasts expressing Wt Lck, Lck containing activation loop mutations C-terminal to Y394 (G399A, K401S, T407A, T407M, T407S), and the corresponding Y505F double mutants were analysed for dierences in PTyr content by Western blotting as described in Figure 2.
The upstream PCR oligo at the HincII site was 5'tgc tct aga cat ggc agc cca aat tgc3' while the downstream PCR oligos containing the point mutations are summarized as follows: G399A: 5'cgg ggt acc gta gtt aat ggc ctc tgg ggc cgt cca ctt aat ggg aaa ctt ggc ggc ctc3'; K401S: 5'cgg ggt acc gta gtt aat ggc ctc tgg ggc cgt cca ctt aat ggg aaa gct ggc tcc3'; F402I: 5'cgg ggt acc gta gtt aat ggc ctc tgg ggc cgt cca ctt aat ggg gat ctt gcc3'; F402A: 5'cgg ggt acc gta gtt aat ggc ctc tgg ggc cgt cca ctt aat ggg tgc ctt ggc3'; P403S: 5'cgg ggt acc gta gtt aat ggc ctc tgg ggc cgt cca ctt aat tga aaa ctt3'; P403T: 5'cgg ggt acc gta gtt aat ggc ctc tgg ggc cgt cca ctt aat tgt aaa ctt3'; W406Y: 5'cgg ggt acc gta gtt aat ggc ctc tgg ggc cgt gta ctt aat 3'; T407A: 5'cgg ggt acc gta gtt aat ggc ctc tgg ggc tgc cca ctt aat3'; T407D:5'cgg ggt acc gta gtt aat ggc ctc tgg ggc gtc cca ctt aat3'; T407M: 5'cgg ggt acc gta gtt ggc ctc tgg ggc cat cca ctt aat3'; T407R: 5'cgg ggt acc gta gtt aat ggc ctc tgg ggc cct cca ctt aat3'; T407S: 5'cgg ggt acc gta gtt aat aac ctc tgg ggc tga cca ctt aat3'.
Off by 1 numbering for mutations in P06239
T407D
protein
substitution
true negative
The K405D, K405G, T407D, and T407R mutants all exhibited autokinase activities that were 500 1200 times less than that of Wt Lck (data not shown).
However, the four mutants in this region analysed in vitro (K405D, K405G, T407D, and T407R) were also inactive upon immunoprecipitation.
The upstream PCR oligo at the HincII site was 5'tgc tct aga cat ggc agc cca aat tgc3' while the downstream PCR oligos containing the point mutations are summarized as follows: G399A: 5'cgg ggt acc gta gtt aat ggc ctc tgg ggc cgt cca ctt aat ggg aaa ctt ggc ggc ctc3'; K401S: 5'cgg ggt acc gta gtt aat ggc ctc tgg ggc cgt cca ctt aat ggg aaa gct ggc tcc3'; F402I: 5'cgg ggt acc gta gtt aat ggc ctc tgg ggc cgt cca ctt aat ggg gat ctt gcc3'; F402A: 5'cgg ggt acc gta gtt aat ggc ctc tgg ggc cgt cca ctt aat ggg tgc ctt ggc3'; P403S: 5'cgg ggt acc gta gtt aat ggc ctc tgg ggc cgt cca ctt aat tga aaa ctt3'; P403T: 5'cgg ggt acc gta gtt aat ggc ctc tgg ggc cgt cca ctt aat tgt aaa ctt3'; W406Y: 5'cgg ggt acc gta gtt aat ggc ctc tgg ggc cgt gta ctt aat 3'; T407A: 5'cgg ggt acc gta gtt aat ggc ctc tgg ggc tgc cca ctt aat3'; T407D:5'cgg ggt acc gta gtt aat ggc ctc tgg ggc gtc cca ctt aat3'; T407M: 5'cgg ggt acc gta gtt ggc ctc tgg ggc cat cca ctt aat3'; T407R: 5'cgg ggt acc gta gtt aat ggc ctc tgg ggc cct cca ctt aat3'; T407S: 5'cgg ggt acc gta gtt aat aac ctc tgg ggc tga cca ctt aat3'.
Off by 1 numbering for mutations in P06239
T407R
protein
substitution
true negative
The K405D, K405G, T407D, and T407R mutants all exhibited autokinase activities that were 500 1200 times less than that of Wt Lck (data not shown).
However, the four mutants in this region analysed in vitro (K405D, K405G, T407D, and T407R) were also inactive upon immunoprecipitation.
The upstream PCR oligo at the HincII site was 5'tgc tct aga cat ggc agc cca aat tgc3' while the downstream PCR oligos containing the point mutations are summarized as follows: G399A: 5'cgg ggt acc gta gtt aat ggc ctc tgg ggc cgt cca ctt aat ggg aaa ctt ggc ggc ctc3'; K401S: 5'cgg ggt acc gta gtt aat ggc ctc tgg ggc cgt cca ctt aat ggg aaa gct ggc tcc3'; F402I: 5'cgg ggt acc gta gtt aat ggc ctc tgg ggc cgt cca ctt aat ggg gat ctt gcc3'; F402A: 5'cgg ggt acc gta gtt aat ggc ctc tgg ggc cgt cca ctt aat ggg tgc ctt ggc3'; P403S: 5'cgg ggt acc gta gtt aat ggc ctc tgg ggc cgt cca ctt aat tga aaa ctt3'; P403T: 5'cgg ggt acc gta gtt aat ggc ctc tgg ggc cgt cca ctt aat tgt aaa ctt3'; W406Y: 5'cgg ggt acc gta gtt aat ggc ctc tgg ggc cgt gta ctt aat 3'; T407A: 5'cgg ggt acc gta gtt aat ggc ctc tgg ggc tgc cca ctt aat3'; T407D:5'cgg ggt acc gta gtt aat ggc ctc tgg ggc gtc cca ctt aat3'; T407M: 5'cgg ggt acc gta gtt ggc ctc tgg ggc cat cca ctt aat3'; T407R: 5'cgg ggt acc gta gtt aat ggc ctc tgg ggc cct cca ctt aat3'; T407S: 5'cgg ggt acc gta gtt aat aac ctc tgg ggc tga cca ctt aat3'.
Off by 1 numbering for mutations in P06239
T407S
protein
substitution
true negative
The activity of the Lys401 to Ser (K401S) and Thr407 to Ser (T407S) mutants are most similar to Wt and appear to have primarily lost phosphorylation of a 31 33 kDa doublet while substitution of Thr407 to either Ala (T407A) or Met (T407M) disrupts mutually exclusive protein phosphorylations.
Off by 1 numbering for mutations in P06239
L388D
protein
substitution
true negative
Mutation of each site to bulkier (Met; E390M) or charged residues (Asp; L388D) measurably reduces kinase activity towards bacterial proteins (as does the double mutant L388D/E390M).
Wt Lck, Lck containing activation loop mutations below Y394 (L388D, L388N, E390M, E390T, and L388D/E390M), and corresponding Y505F double mutants were subcloned into the prokaryotic expression vector pGEX-2T.
Drug-resistant pools of BalbC 3T3 broblasts expressing either Wt Lck, Lck containing activation loop mutations N-terminal to Y394 (L388D, L388N, E390M, E390T, and L388D/E390M), and corresponding Y505F double mutants were analysed for dierences in PTyr content by Western blotting.
However, the L388D, L388N, and L388D/E390M mutants display increased PTyr levels compared to Wt Lck and have increased abilities to phosphorylate cellular proteins (more easily visible on longer exposures).
The subtle Oncogene Effects of activation loop mutations on Lck regulation LE Laham et al 3964 dierences in substrate specicity observed when the L388D, E390M, and L388D/E390M mutants were expressed in bacteria are not detectable upon expression in broblasts.
Similarly, most of the mutants show slightly decreased kinase activity in a Y505F background and also display a level of morphological transformation which is slightly down from the Y505F single mutant, with the L388D/E390M/Y505F triple mutant having the least transformed morphology.
We show that mutations in the conserved amino terminal region of the activation loop (L388D, L388N, and L388D/E390M) activate Lck in vivo, coincident with increased levels of PTyr on the kinase, but have little eect on substrate specicity.
To generate the activation loop mutations N-terminal to Y394, doublestranded NheI BssHII oligos (L388D: 5'cta gca cgc gat att gag gac aac gag tac aca g3'; L388N: 5'cta gca cgc aat att gag gac aac gag tac aca g3'; E390M: 5'cta gca cgc ctc att atg gac aac gag tac aca g3'; E390T: 5'cta gca cgc ctc att acc gac aac gag tac aca g3'; L388D/E390M: 5'cta gca cgc gat att atg gac aac gag tac aca g3') were subcloned into the NheI BssHII digested synk-pGEM3zf+ backbone.
Off by 1 numbering for mutations in P06239
W406Y
protein
substitution
true negative
Interestingly, substitution of an adjacent residue, W406Y, destroyed Lck's ability to phosphorylate exogenous substrates when expressed in bacteria yet the mutant retained ability to phosphorylate itself (Mukhopadhyay, unpublished work).
The upstream PCR oligo at the HincII site was 5'tgc tct aga cat ggc agc cca aat tgc3' while the downstream PCR oligos containing the point mutations are summarized as follows: G399A: 5'cgg ggt acc gta gtt aat ggc ctc tgg ggc cgt cca ctt aat ggg aaa ctt ggc ggc ctc3'; K401S: 5'cgg ggt acc gta gtt aat ggc ctc tgg ggc cgt cca ctt aat ggg aaa gct ggc tcc3'; F402I: 5'cgg ggt acc gta gtt aat ggc ctc tgg ggc cgt cca ctt aat ggg gat ctt gcc3'; F402A: 5'cgg ggt acc gta gtt aat ggc ctc tgg ggc cgt cca ctt aat ggg tgc ctt ggc3'; P403S: 5'cgg ggt acc gta gtt aat ggc ctc tgg ggc cgt cca ctt aat tga aaa ctt3'; P403T: 5'cgg ggt acc gta gtt aat ggc ctc tgg ggc cgt cca ctt aat tgt aaa ctt3'; W406Y: 5'cgg ggt acc gta gtt aat ggc ctc tgg ggc cgt gta ctt aat 3'; T407A: 5'cgg ggt acc gta gtt aat ggc ctc tgg ggc tgc cca ctt aat3'; T407D:5'cgg ggt acc gta gtt aat ggc ctc tgg ggc gtc cca ctt aat3'; T407M: 5'cgg ggt acc gta gtt ggc ctc tgg ggc cat cca ctt aat3'; T407R: 5'cgg ggt acc gta gtt aat ggc ctc tgg ggc cct cca ctt aat3'; T407S: 5'cgg ggt acc gta gtt aat aac ctc tgg ggc tga cca ctt aat3'.
Off by 1 numbering for mutations in P06239
Y505F
protein
substitution
true negative
Mutant enzymes isolated from bacterial and mammalian expression systems were assayed using both the wildtype (Wt) and activating (Y505F) backgrounds.
To investigate the contributions of this region to kinase regulation, we have analysed the eects of mutations at two sites in the activation loop (Leu388 and Glu390) on the activity of Wt Lck or the activated variant, Y505F, expressed in both bacteria and mammalian cells.
When Lck is expressed in the bacteria, both Wt and Y505F mutant proteins exhibit similar kinase activities as judged by autokinase activity and by overall levels of PTyr on bacterial substrates because the bacteria lack expression of the down regulatory kinase Csk (Figure 2a).
Mutations at Leu388 to Asn (L388N) and at Glu390 to Thr (E390T) have little aect on activity, both in the Wt and Y505F backgrounds.
An activation loop mutation was determined to aect substrate specicity if the PTyr pattern for the mutant diered from that of the Wt and Y505F lysates, either by adding new proteins to the phosphorylation pattern or by decreasing tyrosine phosphorylation on a subset of the proteins.
Wt Lck, Lck containing activation loop mutations below Y394 (L388D, L388N, E390M, E390T, and L388D/E390M), and corresponding Y505F double mutants were subcloned into the prokaryotic expression vector pGEX-2T.
Drug-resistant pools of BalbC 3T3 broblasts expressing either Wt Lck, Lck containing activation loop mutations N-terminal to Y394 (L388D, L388N, E390M, E390T, and L388D/E390M), and corresponding Y505F double mutants were analysed for dierences in PTyr content by Western blotting.
In contrast to the activity observed with expression in bacteria, Wt Lck is inactive towards proteins in mammalian cells while Y505F Lck is constitutively active and transforming (Figure 2b).
Mutations that activate Lck would be apparent in the Wt background while mutations that decrease kinase activity or change substrate selection would be revealed using the activated Y505F background.
When these mutants are placed in a Y505F background, they display activities that approach that of the single Y505F mutant.
Similarly, most of the mutants show slightly decreased kinase activity in a Y505F background and also display a level of morphological transformation which is slightly down from the Y505F single mutant, with the L388D/E390M/Y505F triple mutant having the least transformed morphology.
To analyse the eects of activation loop mutations on autophosphorylation and subsequent activation of the kinase, Lck Y394F and Y394-downstream mutants were expressed in both bacterial and mammalian expression systems in combination with Y505F mutations.
As expected, Wt and Y505F Lck exhibited similar levels of kinase activity when expressed in bacterial cells that lack Csk expression (Figure 3a).
The fact that similar kinase activities are observed for the mutants in the presence or absence of an additional Y505F mutation suggests that the decreases in activity are not the result of downregulation via phosphorylation of Y505.
When the same mutants are placed in broblasts in combination with the Y505F background, the Y394downstream mutants have decreased abilities to phosphorylate endogenous proteins.
Specically, the YCPK/Y505F and YNQQ/Y505F mutants were able to phosphorylate proteins more readily than the YGQQ/ Y505F and Y394F/Y505F mutants (Figure 3b).
The overall PTyr content in cells expressing the Lck Y394downstream mutants is much lower than that in the single Y505F mutant.
Moreover, in the Y505F background, the mutants all display much lower levels of tyrosine phosphorylation on Lck (presumably at Y394) and the decrease in PTyr content correlates with decreased ability to phosphorylate endogenous proteins.
Correspondingly, as the level of phosphorylation on endogenous proteins decreases, the degree of transformed, more rounded phenotype also decreases when assayed in the Y505F background (Figure 3c).
Indeed Y394 phosphorylation is not necessary for in vitro kinase activity as the Y394F and the Y394F/Y505F Lck mutants isolated from either mammalian cells (Figure 3d) or insect cells (data not shown) are still active.
Wt Lck, Lck containing activation loop mutations at or immediately downstream of Y394 (Y394F, YCPK, YGQQ, YNQQ), and corresponding Y505F double mutants were subcloned into the prokaryotic expression vector pGEX-2T.
Mouse broblasts expressing Wt Lck, Lck containing activation loop mutations at or immediately downstream to Y394 (Y394F, YCPK, YGQQ, YNQQ), and corresponding Y505F double mutants were analysed for dierences in PTyr and Lck as described in Figure 2.
Kinase inactive constructs were generated by making K273R mutations in the catalytic domain of Lck The mutant proteins were then expressed in broblasts and their activities were compared to that of Wt and Y505F Lck (Figure 5b).
When these mutants were expressed in a Y505F background, the order of activity levels mimicked that observed in bacteria with the K401S mutant being most similar to Y505F followed by the T407A and G399A mutants.
However, the similarity in PTyr blotting patterns between Y505F and G399A/Y505F stands in contrast to the relatively large dierence in the morphologies of cells expressing these proteins.
This suggests that the G399A/Y505F mutant has lost the ability to phosphorylate a key substrate necessary for changes in transformation.
However, the mutations do decrease kinases activity and revert the morphological transformed phenotype of Y505F mutants with the extent of reversion correlating with the levels of decreased Y394 phosphorylation.
Wt Lck, Lck containing activation loop mutations C-terminal to Y394 (G399A, K401S, T407A, T407M, T407S), and the corresponding Y505F double mutants were subcloned into the prokaryotic expression vector pGEX-2T.
Mouse broblasts expressing Wt Lck, Lck containing activation loop mutations C-terminal to Y394 (G399A, K401S, T407A, T407M, T407S), and the corresponding Y505F double mutants were analysed for dierences in PTyr content by Western blotting as described in Figure 2.
These mutants also display dierences in broblast monolayer morphologies from that of Y505F to suggest the subtle loss of key substrate phosphorylations in mammalian cells.
To generate the activating Cterminal mutations at Y505 in Lck, a 3' 287 bp KpnI EcoRI fragment of synk/Y505F was substituted for the fragment in the wildtype (Wt) and the activation loop mutant constructs.
For construction of Y505F, YCPK, YGQQ, YNQQ and Y394F mutants in mammalian expression vectors, Wt Lck and mutant cDNA's were subcloned into pLNCX (Clontech, Palo Alto, CA, USA).
To construct the remaining constructs, Lck HindIII EcoRI fragments containing the activation loop mutations were subcloned into the Wt Lck/pLNCX or Y505F/pLNCX backbones.
Kinase inactive Lck constructs were generated by creating K273R mutations in the pLNCX expression vectors by substituting the 739 bp BglII SalI fragments from K273R synk (Carrera et al., 1993) into each of the Wt, Y394F, YCPK, YNQQ, and Y505F pLNCX mutants.
Off by 1 numbering for mutations in P06239
P403S
protein
substitution
true negative
The upstream PCR oligo at the HincII site was 5'tgc tct aga cat ggc agc cca aat tgc3' while the downstream PCR oligos containing the point mutations are summarized as follows: G399A: 5'cgg ggt acc gta gtt aat ggc ctc tgg ggc cgt cca ctt aat ggg aaa ctt ggc ggc ctc3'; K401S: 5'cgg ggt acc gta gtt aat ggc ctc tgg ggc cgt cca ctt aat ggg aaa gct ggc tcc3'; F402I: 5'cgg ggt acc gta gtt aat ggc ctc tgg ggc cgt cca ctt aat ggg gat ctt gcc3'; F402A: 5'cgg ggt acc gta gtt aat ggc ctc tgg ggc cgt cca ctt aat ggg tgc ctt ggc3'; P403S: 5'cgg ggt acc gta gtt aat ggc ctc tgg ggc cgt cca ctt aat tga aaa ctt3'; P403T: 5'cgg ggt acc gta gtt aat ggc ctc tgg ggc cgt cca ctt aat tgt aaa ctt3'; W406Y: 5'cgg ggt acc gta gtt aat ggc ctc tgg ggc cgt gta ctt aat 3'; T407A: 5'cgg ggt acc gta gtt aat ggc ctc tgg ggc tgc cca ctt aat3'; T407D:5'cgg ggt acc gta gtt aat ggc ctc tgg ggc gtc cca ctt aat3'; T407M: 5'cgg ggt acc gta gtt ggc ctc tgg ggc cat cca ctt aat3'; T407R: 5'cgg ggt acc gta gtt aat ggc ctc tgg ggc cct cca ctt aat3'; T407S: 5'cgg ggt acc gta gtt aat aac ctc tgg ggc tga cca ctt aat3'.
Off by 1 numbering for mutations in P06239
P403T
protein
substitution
true negative
The upstream PCR oligo at the HincII site was 5'tgc tct aga cat ggc agc cca aat tgc3' while the downstream PCR oligos containing the point mutations are summarized as follows: G399A: 5'cgg ggt acc gta gtt aat ggc ctc tgg ggc cgt cca ctt aat ggg aaa ctt ggc ggc ctc3'; K401S: 5'cgg ggt acc gta gtt aat ggc ctc tgg ggc cgt cca ctt aat ggg aaa gct ggc tcc3'; F402I: 5'cgg ggt acc gta gtt aat ggc ctc tgg ggc cgt cca ctt aat ggg gat ctt gcc3'; F402A: 5'cgg ggt acc gta gtt aat ggc ctc tgg ggc cgt cca ctt aat ggg tgc ctt ggc3'; P403S: 5'cgg ggt acc gta gtt aat ggc ctc tgg ggc cgt cca ctt aat tga aaa ctt3'; P403T: 5'cgg ggt acc gta gtt aat ggc ctc tgg ggc cgt cca ctt aat tgt aaa ctt3'; W406Y: 5'cgg ggt acc gta gtt aat ggc ctc tgg ggc cgt gta ctt aat 3'; T407A: 5'cgg ggt acc gta gtt aat ggc ctc tgg ggc tgc cca ctt aat3'; T407D:5'cgg ggt acc gta gtt aat ggc ctc tgg ggc gtc cca ctt aat3'; T407M: 5'cgg ggt acc gta gtt ggc ctc tgg ggc cat cca ctt aat3'; T407R: 5'cgg ggt acc gta gtt aat ggc ctc tgg ggc cct cca ctt aat3'; T407S: 5'cgg ggt acc gta gtt aat aac ctc tgg ggc tga cca ctt aat3'.
Off by 1 numbering for mutations in P06239
F402I
protein
substitution
true negative
The upstream PCR oligo at the HincII site was 5'tgc tct aga cat ggc agc cca aat tgc3' while the downstream PCR oligos containing the point mutations are summarized as follows: G399A: 5'cgg ggt acc gta gtt aat ggc ctc tgg ggc cgt cca ctt aat ggg aaa ctt ggc ggc ctc3'; K401S: 5'cgg ggt acc gta gtt aat ggc ctc tgg ggc cgt cca ctt aat ggg aaa gct ggc tcc3'; F402I: 5'cgg ggt acc gta gtt aat ggc ctc tgg ggc cgt cca ctt aat ggg gat ctt gcc3'; F402A: 5'cgg ggt acc gta gtt aat ggc ctc tgg ggc cgt cca ctt aat ggg tgc ctt ggc3'; P403S: 5'cgg ggt acc gta gtt aat ggc ctc tgg ggc cgt cca ctt aat tga aaa ctt3'; P403T: 5'cgg ggt acc gta gtt aat ggc ctc tgg ggc cgt cca ctt aat tgt aaa ctt3'; W406Y: 5'cgg ggt acc gta gtt aat ggc ctc tgg ggc cgt gta ctt aat 3'; T407A: 5'cgg ggt acc gta gtt aat ggc ctc tgg ggc tgc cca ctt aat3'; T407D:5'cgg ggt acc gta gtt aat ggc ctc tgg ggc gtc cca ctt aat3'; T407M: 5'cgg ggt acc gta gtt ggc ctc tgg ggc cat cca ctt aat3'; T407R: 5'cgg ggt acc gta gtt aat ggc ctc tgg ggc cct cca ctt aat3'; T407S: 5'cgg ggt acc gta gtt aat aac ctc tgg ggc tga cca ctt aat3'.
Off by 1 numbering for mutations in P06239
F402A
protein
substitution
true negative
The upstream PCR oligo at the HincII site was 5'tgc tct aga cat ggc agc cca aat tgc3' while the downstream PCR oligos containing the point mutations are summarized as follows: G399A: 5'cgg ggt acc gta gtt aat ggc ctc tgg ggc cgt cca ctt aat ggg aaa ctt ggc ggc ctc3'; K401S: 5'cgg ggt acc gta gtt aat ggc ctc tgg ggc cgt cca ctt aat ggg aaa gct ggc tcc3'; F402I: 5'cgg ggt acc gta gtt aat ggc ctc tgg ggc cgt cca ctt aat ggg gat ctt gcc3'; F402A: 5'cgg ggt acc gta gtt aat ggc ctc tgg ggc cgt cca ctt aat ggg tgc ctt ggc3'; P403S: 5'cgg ggt acc gta gtt aat ggc ctc tgg ggc cgt cca ctt aat tga aaa ctt3'; P403T: 5'cgg ggt acc gta gtt aat ggc ctc tgg ggc cgt cca ctt aat tgt aaa ctt3'; W406Y: 5'cgg ggt acc gta gtt aat ggc ctc tgg ggc cgt gta ctt aat 3'; T407A: 5'cgg ggt acc gta gtt aat ggc ctc tgg ggc tgc cca ctt aat3'; T407D:5'cgg ggt acc gta gtt aat ggc ctc tgg ggc gtc cca ctt aat3'; T407M: 5'cgg ggt acc gta gtt ggc ctc tgg ggc cat cca ctt aat3'; T407R: 5'cgg ggt acc gta gtt aat ggc ctc tgg ggc cct cca ctt aat3'; T407S: 5'cgg ggt acc gta gtt aat aac ctc tgg ggc tga cca ctt aat3'.
Off by 1 numbering for mutations in P06239
I389A
protein
substitution
true negative
Mutation of a nearby residue, Ile389 to Ala, also increased the basal activity of Lck expressed in broblasts whereas the mutation Asn392 to Thr did not (LE Laham, unpublished results).
Off by 1 numbering for mutations in P06239
N392T
protein
substitution
true negative
Mutation of a nearby residue, Ile389 to Ala, also increased the basal activity of Lck expressed in broblasts whereas the mutation Asn392 to Thr did not (LE Laham, unpublished results).
Off by 1 numbering for mutations in P06239
11904433
full text
R273A
protein
substitution
true negative
Next, we directly tested the role of Lck kinase activity in ubiquitination assays by comparing WT Lck with its kinase active (Y505F) and kinase dead (R273A) mutants (Fig.
Kinase dead Lck (R273A) was essentially insensitive to Cbl-mediated ubiquitination or degradation (compare lane 6 with lanes 2 and 4).
(B) 293T cells were transfected with plasmids encoding HA-Ub (5 g), Lck (WT), kinase active (Y505F), and kinase dead (R273A) (0.2 g each), and GFP-Cbl or a GFP control ( ) (3 g).
W97A
protein
substitution
true negative
The Lck SH2 (R154K), SH3 (W97A), and double mutant were generated by using Quickchange Mutagenesis (Invitrogen).
(W97A), or SH3 SH2 double mutant with WT Cbl or the Cbl TKB domain mutant (G306E) followed by coimmunoprecipitation revealed that disruption of the Lck SH3 domain severely abrogated association with Cbl (Fig.
293T cells were transfected with plasmids encoding HA-Cbl (1 g) and Lck (WT), SH2 (R154K), SH3 (W97A), or double mutants (R154K W97A) (0.5 g each).
Y505F
protein
substitution
true negative
Next, we directly tested the role of Lck kinase activity in ubiquitination assays by comparing WT Lck with its kinase active (Y505F) and kinase dead (R273A) mutants (Fig.
In contrast, constitutively active Lck (Y505F) showed detectable ubiquitination even in the absence of cotransfected Cbl, and this ubiquitination was markedly enhanced when Cbl was coexpressed (compare lane 3 with lane 4).
(B) 293T cells were transfected with plasmids encoding HA-Ub (5 g), Lck (WT), kinase active (Y505F), and kinase dead (R273A) (0.2 g each), and GFP-Cbl or a GFP control ( ) (3 g).
Finally, an activated mutant of Lck (Y505F) was more susceptible, whereas a kinase dead Lck (D273A) was resistant to Cbl-dependent ubiquitination and degradation compared with WT Cbl (Fig.
G306E
protein
substitution
true negative
(W97A), or SH3 SH2 double mutant with WT Cbl or the Cbl TKB domain mutant (G306E) followed by coimmunoprecipitation revealed that disruption of the Lck SH3 domain severely abrogated association with Cbl (Fig.
The association data correlated with the ability of Cbl or its TKB domain mutant (G306E) to mediate Lck degradation, as assessed by quantification of Lck protein levels by densitometry (Fig.
R154K
protein
substitution
true negative
The Lck SH2 (R154K), SH3 (W97A), and double mutant were generated by using Quickchange Mutagenesis (Invitrogen).
Coexpression of WT Lck, SH2 mutant (R154K), SH3 mutant Rao et al.
293T cells were transfected with plasmids encoding HA-Cbl (1 g) and Lck (WT), SH2 (R154K), SH3 (W97A), or double mutants (R154K W97A) (0.5 g each).
D273A
protein
substitution
true negative
Finally, an activated mutant of Lck (Y505F) was more susceptible, whereas a kinase dead Lck (D273A) was resistant to Cbl-dependent ubiquitination and degradation compared with WT Cbl (Fig.
11606538
full text
15626738
full text
D835Y
protein
substitution
true positive
P36888
In vitro mutagenesis The FLT3-D835Y/N and FLT3-V592A point mutations were introduced into either the FLT3-WT, FLT3-V592A, FLT3-ITD-W51, or the FLT3-ITDNPOS constructs using the QuikChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA) according to the manufacturer's instructions.
Our results clearly demonstrate that Ba/F3 FLT3-ITDD835Y/N cells show a 5-fold higher cellular resistance to daunorubicin than FLT3-ITD expressing Ba/F3 cells in proliferation assays (IC50 0.1 and 0.5 g/mL, respectively) (Figure 2A).
For this purpose we mixed Ba/F3 cells expressing the FLT3-ITD-D835Y/N dual construct (YFP ) with cells carrying a FLT3-ITD mutation alone (GFP ) in a ratio of 1:10 in the presence of low concentrations of daunorubicin (0.01 g/mL).
Our results show a significant competitive growth advantage of Ba/F3 FLT3-ITD-D835Y/N cells in the presence of daunorubicin compared to FLT3-ITD cells (Figure 2B).
(A) The phosphorylation status of STAT5 in extracts of Ba/F3 FLT3WT, FLT3-ITD, and FLT3-ITD-D835Y/N cells was determined by Western blot analysis using the polyclonal anti-pSTAT5 antibody.
Our data clearly show that only the combination of an ITD and a TKD mutation (FLT3-ITD-D835Y/N dual mutations), but not the TKD mutation alone, caused drug resistance to the FLT3 PTK inhibitor SU5614.
V592A
protein
substitution
true positive
P36888
In vitro mutagenesis The FLT3-D835Y/N and FLT3-V592A point mutations were introduced into either the FLT3-WT, FLT3-V592A, FLT3-ITD-W51, or the FLT3-ITDNPOS constructs using the QuikChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA) according to the manufacturer's instructions.
For this purpose we generated Ba/F3 cells transformed with FLT3-V592A-D835N and FLT3-NPOS-D835N mutants.
The V592A mutation can be found in the AML cell lines Mono Mac1/6 and in AML patient samples and represents the first activating point mutation in the JM region of FLT3.32,33 The FLT3-ITD-NPOS mutant contains a 28-amino acid duplicated sequence inserted between amino acids 611/612 in contrast to the FLT3-ITD-W51 BLOOD, 1 MAY 2005 VOLUME 105, NUMBER 9 FLT3-ITD-TKD DUAL MUTANTS INDUCE DRUG RESISTANCE 3681 Figure 1.
FLT3-ITD-TKD dual mutants induce hyperactivation of STAT5 and up-regulation of its downstream targets Bcl-x(L) and RAD51 in Ba/F3 cells mutant that contains only a 7-amino acid duplicated sequence inserted between amino acids 601/602.12 We found that all these dual mutants show partial resistance to SU5614 although to a different degree (IC50 0.3 M for the V592A-D835N and 1 M for the NPOS-D835N mutant) (data not shown).
Furthermore, the combination of different kinds of activating mutations in the JM domain (different length and localization of ITDs and the point mutant V592A) and in the TKD region (D835Y or D835N) resulted in a similar SU5614-resistant phenotype.
D835N
protein
substitution
true positive
P36888
For this purpose we generated the following Ba/F3 cell lines: (1) FLT3-ITD-D835Y and FLT3-ITD-D835N (both mutations on the same "allele") by retroviral transduction with an MIY FLT3-ITD-TKD dual mutant cDNA, (2) FLT3-ITD/D835N and FLT3-ITD/WT (carrying the FLT3 mutations on different "alleles") by transduction of Ba/F3 MIG FLT3-ITD carrying cells (green) with MIY (yellow) FLT3-D835N or FLT3-WT cDNA, respectively, and sorting of double-positive (YFP and GFP ) cells.
We performed proliferation assays in the presence of different concentrations of the FLT3 PTK inhibitor SU5614 and found that Ba/F3 FLT3-ITD-D835Y (Figure 1A) and FLT3-ITD-D835N (data not shown) dual mutationexpressing cells were partially resistant to the inhibitor.
In contrast, our proliferation assay data clearly show that cells transformed with FLT3 constructs located on different "alleles" (FLT3-ITD/D835N and FLT3-ITD/ WT) were as sensitive to the inhibitor treatment as FLT3-ITD cells (Figure 1B).
To further analyze the mechanisms of PTK inhibitor resistance, we asked whether structurally different length mutations in the JM region can lead to the differences in the sensitivity to the FLT3 PTK inhibitor SU5614 in the background of the FLT3-D835N construct.
For this purpose we generated Ba/F3 cells transformed with FLT3-V592A-D835N and FLT3-NPOS-D835N mutants.
(B) Dose-response curves of the inhibitory activity of SU5614 in Ba/F3 FLT3-ITD (F), FLT3-ITD/WT (E), and FLT3-ITD/D835N cells () after 72 hours of incubation.
(C) Ba/F3 cells transduced with the FLT3-ITD (F), FLT3-ITD-D835N (E), or FLT3-ITD-D835Y constructs () were incubated with different concentrations of SU5614 for 24 hours and were analyzed by flow cytometry after staining with annexin VPE and 7-AAD.
(D) MIG FLT3-ITD (GFP ; E) and MIY FLT3-ITD-D835N (YFP )expressing Ba/F3 cells (F) were mixed in a ratio of 10:1, and the percentage of GFP and YFP cells was measured every 3 to 4 days for a time period of 12 days by FACS analysis in the presence of 0.2 M of SU5614.
FLT3-ITD-TKD dual mutants induce hyperactivation of STAT5 and up-regulation of its downstream targets Bcl-x(L) and RAD51 in Ba/F3 cells mutant that contains only a 7-amino acid duplicated sequence inserted between amino acids 601/602.12 We found that all these dual mutants show partial resistance to SU5614 although to a different degree (IC50 0.3 M for the V592A-D835N and 1 M for the NPOS-D835N mutant) (data not shown).
Furthermore, the combination of different kinds of activating mutations in the JM domain (different length and localization of ITDs and the point mutant V592A) and in the TKD region (D835Y or D835N) resulted in a similar SU5614-resistant phenotype.
9516479
full text
Y1243F
protein
substitution
true negative
Tyrosine to phenylalanine mutations in erbB3 were performed using the QuickChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) to produce erbB3-Y1180F, erbB3-Y1243F, and erbB3-Y1180,1243F.
However, mutation of Tyr-1180 or Tyr-1243 to Phe (Y1180F and Y1243F) reduced this association to 35% and 80% of the wild-type value, respectively (Fig.
R1183Q
protein
substitution
true negative
(Melbourne, Victoria, Australia) and were as follows; Tyr-1139, PQPEpYVNQPD; Q1142R, PQPEpYVNRPD; Tyr-1196, VENPEpYLTPQG; Tyr-1180, DEEYEpYMNRRR; R1183Q, DEEYEpYMNQRR; Tyr1243, DEDYEpYMNRQR, where pY represents a phosphotyrosine residue.
7, R1183Q peptide).
In particular, although R1183Q and not Tyr-1180 bound the Grb2 SH2, introduction of an Arg residue at 3 into the Tyr-1139 phosphopeptide did not eliminate binding (Fig.
Y1180F
protein
substitution
true negative
Tyrosine to phenylalanine mutations in erbB3 were performed using the QuickChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) to produce erbB3-Y1180F, erbB3-Y1243F, and erbB3-Y1180,1243F.
However, mutation of Tyr-1180 or Tyr-1243 to Phe (Y1180F and Y1243F) reduced this association to 35% and 80% of the wild-type value, respectively (Fig.
Q1142R
protein
substitution
true negative
(Melbourne, Victoria, Australia) and were as follows; Tyr-1139, PQPEpYVNQPD; Q1142R, PQPEpYVNRPD; Tyr-1196, VENPEpYLTPQG; Tyr-1180, DEEYEpYMNRRR; R1183Q, DEEYEpYMNQRR; Tyr1243, DEDYEpYMNRQR, where pY represents a phosphotyrosine residue.
7, Q1142R peptide).
7, Q1142R).
These experiments also highlighted the overlapping but distinct specificity of the Grb7 SH2 domain, which bound strongly to the erbB3-derived peptides; however, when compared with the Grb2 SH2, it bound less well to Q1142R.
10500177
full text
S222E
protein
substitution
true negative
In contrast, overexpression of a wild-type or the S222E mutant kinase had little or no effect on the progression through G2 M.
Cell lines expressing either wild-type or another mutant form of MAPKK1 (S222E) as well as mock-transfected cells were examined as controls.
The S222E mutant behaved essentially like wild-type kinase because it still requires phosphorylation of serine 218 for activation (3).
Summary of cell-cycle kinetics of MAPKK-mutant expressing cell lines Cell line Mock-1 Mock 2 Mock-9 S222A-13* S222A-21 K97A-2 S222E-1 S22E-8 WT-31 WT-32 S phase, hr 6.2 5.9 5.9 6.0 6.5 7.0 0.2 0.1 0.1 0.2 0.3 0.0 G2 M phase, hr 3.0 3.2 3.5 5.9 6.2 6.7 3.9 3.5 4.3 3.7 0.3 0.3 0.5 0.1 0.1 0.1 0.4 0.2 0.2 G1 phase, hr 6.0 6.5 5.9 0.5 0.3 Proc.
Table 1 shows that similar results were obtained with multiple independently selected clones of mock-transfected controls or cells transfected with either S222A or S222E, arguing that differences were not caused simply by clonal variation.
2B Right), the S222E mutant, which showed normal G2 M kinetics by PI staining (Table 1), FIG.
MAPKK S222E (black squares) and S222A dominant-negative (white squares) MAPKK mutant-expressing cell lines are compared (Right).
S222A
protein
substitution
true negative
The two different dominantnegative forms of MAPKK1 used included mutation of the ATP-binding lysine (K97A) and mutation of one of the two regulatory phosphorylation sites (S222A).
``Mock'' is a cell line transfected with the pcDNA3 vector; ``S222A'' is a cell line transfected with the S222A-MAPKK1 mutant; ``K97A'' is a cell line transfected with the K97A-MAPKK1 mutant.
(B) MAPK phosphorylation in NIH 3T3 cell lines expressing MAPKK1 mutants was measured by gel mobility shift assays of synchronized extracts from mock-transfected and S222A MAPKK-mutant transfected cell lines.
Summary of cell-cycle kinetics of MAPKK-mutant expressing cell lines Cell line Mock-1 Mock 2 Mock-9 S222A-13* S222A-21 K97A-2 S222E-1 S22E-8 WT-31 WT-32 S phase, hr 6.2 5.9 5.9 6.0 6.5 7.0 0.2 0.1 0.1 0.2 0.3 0.0 G2 M phase, hr 3.0 3.2 3.5 5.9 6.2 6.7 3.9 3.5 4.3 3.7 0.3 0.3 0.5 0.1 0.1 0.1 0.4 0.2 0.2 G1 phase, hr 6.0 6.5 5.9 0.5 0.3 Proc.
USA 96 (1999) 11337 ND ND ND 4.6 4.5 7.0 7.0 0.3 5.8 0.2 6.0 6.0 6.0 0.0 0.0 *Each clone is labeled with the letters of the mutation, followed by the cell clone number assigned in the original isolation (e.g., S222A-13).
Table 1 shows that similar results were obtained with multiple independently selected clones of mock-transfected controls or cells transfected with either S222A or S222E, arguing that differences were not caused simply by clonal variation.
In comparison, the ERK phosphorylation was significantly attenuated in the cells expressing the S222A-MAPKK (Fig.
At time points after release into S phase, synchronized S222A MAPKK-transfected cells and control cells were stained with the MPM-2 monoclonal antibody as well as with PI for DNA content.
The mock-transfected cell line peaked in MPM-2 reactivity at 6.5 hr after release, whereas the dominantnegative S222A mutant peaked in MPM-2 signal at 8 hr.
(A) Two-dimensional FACS analysis of cells stained with PI and MPM-2 antibody FITC secondary antibody were compared from mock and MAPKK-S222A mutant transfected cell lines.
time from two data sets, one from the data set above (Left) comparing mocktransfected (black circles) and MAPKK S222A mutant-expressing (white squares) cell lines.
MAPKK S222E (black squares) and S222A dominant-negative (white squares) MAPKK mutant-expressing cell lines are compared (Right).
(C) Comparison of the timing and magnitude of CDC2 activation in MAPKK S222A mutant and mocktransfected cell lines.
also peaked in MPM-2 signal at 6.5 hr, whereas the S222A mutant again showed delayed appearance of MPM-2 antigens.
Further evidence that cells were delayed in G2 is that S222A mutant cells had a spread morphology and intact nuclear envelopes at 6.5 hr after release, whereas controls had peak populations of rounded cells without intact nuclei (data not shown).
Comparison of mock- and S222A-transfected cell lines revealed that the peak of CDC2 kinase activity in the S222A mutant was both 11338 Cell Biology: Wright et al.
K97A
protein
substitution
true negative
All the cell lines demonstrated similar rates of progression through S phase, except the K97A mutant expressing cells that showed a 1-hr elongation of S phase.
The two different dominantnegative forms of MAPKK1 used included mutation of the ATP-binding lysine (K97A) and mutation of one of the two regulatory phosphorylation sites (S222A).
``Mock'' is a cell line transfected with the pcDNA3 vector; ``S222A'' is a cell line transfected with the S222A-MAPKK1 mutant; ``K97A'' is a cell line transfected with the K97A-MAPKK1 mutant.
Summary of cell-cycle kinetics of MAPKK-mutant expressing cell lines Cell line Mock-1 Mock 2 Mock-9 S222A-13* S222A-21 K97A-2 S222E-1 S22E-8 WT-31 WT-32 S phase, hr 6.2 5.9 5.9 6.0 6.5 7.0 0.2 0.1 0.1 0.2 0.3 0.0 G2 M phase, hr 3.0 3.2 3.5 5.9 6.2 6.7 3.9 3.5 4.3 3.7 0.3 0.3 0.5 0.1 0.1 0.1 0.4 0.2 0.2 G1 phase, hr 6.0 6.5 5.9 0.5 0.3 Proc.
S22E
protein
substitution
true negative
Summary of cell-cycle kinetics of MAPKK-mutant expressing cell lines Cell line Mock-1 Mock 2 Mock-9 S222A-13* S222A-21 K97A-2 S222E-1 S22E-8 WT-31 WT-32 S phase, hr 6.2 5.9 5.9 6.0 6.5 7.0 0.2 0.1 0.1 0.2 0.3 0.0 G2 M phase, hr 3.0 3.2 3.5 5.9 6.2 6.7 3.9 3.5 4.3 3.7 0.3 0.3 0.5 0.1 0.1 0.1 0.4 0.2 0.2 G1 phase, hr 6.0 6.5 5.9 0.5 0.3 Proc.
11526226
full text
12482964
full text
Y447F
protein
substitution
true positive
Q64434
The plasmids expressing wild-type and mutant forms of the Sik/BRK kinase, pcDNA3-Sikwt (pHR2531), pcDNA3-Sik(Y447F) (pHR2533), and pcDNA3-Sik(K219M) (pHR2533), were kind gifts from Angela Tyner (9).
Coexpression of the constitutively active Sik(Y447F) mutant protein with Sam68 inhibits Sam68-mediated enhancement of CTE function.
(A) Five micrograms of pCMVGag-Pol-CTE reporter (pHR1361) and 0.25 g of pCMVSEAP (pHR1831) were transfected into 293T cells with 1 g of a plasmid that expresses either the wild-type (pcDNA3-Sikwt, pHR2531), the kinase-inactive [pcDNA3-Sik(K219M), pHR2533], or the constitutively active [pcDNA3Sik(Y447F), pHR2533] form of Sik.
(B) Increasing amounts of pcDFSam68 or pcDNA3-Sik(Y447F) were cotransfected with 5 g of pCMVGag-Pol-CTE reporter (pHR1361) and 0.25 g of pCMVSEAP (pHR1831) plasmid into 293T cells.
To investigate whether expression of Sik/BRK would affect Sam68-mediated enhancement of CTE function in 293T cells, cells were transfected with the Gag-Pol-CTE vector with or without Sam68 in the presence of plasmids expressing three different Sik/BRK proteins [wild-type, constitutively active mutant Sik/BRK(Y447F), or kinase-inactive mutant Sik/BRK (K219M)].
However, in the Sam68 cotransfections, a significant decrease in the level of p24 expression was observed in the presence of constitutively active Sik/BRK (Y447F).
To further investigate the effect of Sik(Y447F) on Sam68mediated enhancement of CTE function, we performed a dose-response experiment in which the ratio of the plasmid expressing Sik(Y447F) to the plasmid expressing Sam68 was varied.
23, 2003 Sam68 ENHANCES CYTOPLASMIC UTILIZATION OF RNA 97 Sik(Y447F) reduced p24 expression in a dose-responsive fashion for a given amount of Sam68 (1 g of transfected plasmid).
The lower part of the figure illustrates that the inhibitory effect seen for a given amount of Sik(Y447F) (1 g of transfected plasmid) could readily be titrated out by increasing the levels of the Sam68 plasmid.
These results demonstrate that the enhancement function of Sam68 can be modulated by Sik(Y447F) in a dose-dependent manner and thus lend further support to the hypothesis that Sik serves to functionally regulate the effects of Sam68 in this system.
Sam68 is hyperphosphorylated in the presence of Sik(Y447F) in 293T cells.
To directly analyze if Sik(Y447F) affected Sam68 phosphorylation in our system, we transfected 293T cells with pcDFSam68 in the absence or presence of the plasmid expressing Sik(Y447F) and labeled these cells with [32P]orthophosphate.
Similar amounts of Sam68 were detected in the absence or presence of Sik(Y447F) by Western blot analysis (Fig.
In contrast, the amount of 32P label detected in this protein was dramatically increased in the presence of Sik(Y447F) (Fig.
Several novel phosphopeptides were detected in the presence of Sik(Y447F), indicating that Sam68 is phosphorylated at multiple novel sites in response to the expression of this kinase.
Expression of the Sik(Y447F) mutant increases phosphorylation of Sam68 at multiple residues.
293T cells were transfected with plasmids expressing Flag-Sam68 either alone or with a plasmid expressing Sik(Y447F) and labeled with [32P]orthophosphate.
(A, right side) Western blot analysis of Sam68 in the absence or presence of Sik(Y447F).
(B) Two-dimensional phosphopeptide analysis of Sam68 expressed in the presence or absence of Sik(Y447F).
Arrows indicate peptides that appeared de novo or increased in intensity in the presence of Sik(Y447F).
K219M
protein
substitution
true positive
Q64434
The plasmids expressing wild-type and mutant forms of the Sik/BRK kinase, pcDNA3-Sikwt (pHR2531), pcDNA3-Sik(Y447F) (pHR2533), and pcDNA3-Sik(K219M) (pHR2533), were kind gifts from Angela Tyner (9).
(A) Five micrograms of pCMVGag-Pol-CTE reporter (pHR1361) and 0.25 g of pCMVSEAP (pHR1831) were transfected into 293T cells with 1 g of a plasmid that expresses either the wild-type (pcDNA3-Sikwt, pHR2531), the kinase-inactive [pcDNA3-Sik(K219M), pHR2533], or the constitutively active [pcDNA3Sik(Y447F), pHR2533] form of Sik.
To investigate whether expression of Sik/BRK would affect Sam68-mediated enhancement of CTE function in 293T cells, cells were transfected with the Gag-Pol-CTE vector with or without Sam68 in the presence of plasmids expressing three different Sik/BRK proteins [wild-type, constitutively active mutant Sik/BRK(Y447F), or kinase-inactive mutant Sik/BRK (K219M)].
In contrast, neither the wild-type nor the K219M mutated form of the protein had any significant effect on Sam68 enhancement.
Y527F
protein
substitution
true positive
P12931-2
The plasmids expressing c-Src proteins (pcDNA3-K-Src [pHR2228] and pcDNA3-SrcY527F [pHR2229]) (2) were kind gifts from Sally Parsons.
To do this, we transfected 293 cells with plasmids expressing c-Src proteins that were either kinase dead or constitutively activated, respectively (pcDNA3-K-Src and pcDNA3- SrcY527F).
G178E
protein
substitution
true negative
Enhancement of CTE function was not seen when a Sam68 point mutant (G178E) that is defective for RNA binding was used.
The plasmids expressing wild-type and mutant forms of mouse Sam68, pcDFSam68 (pHR2208), pcDHSam68 KH (pHR2212), and pcDHSam68 (G178E) (pHR2210), have also been previously described (40).
Five micrograms of the pCMVGag-Pol-CTE reporter plasmid (pHR1361) and 0.25 g of pCMVSEAP (pHR1831) were transfected into 100-mm-diameter dishes of either 293 cells (A) or COS cells (B) together with 1 g each of pcDFSam68 (pHR2208), pcDHSam68 KH (pHR2212), pcDHSam68(G178E) (pHR2210), or pcDNA3 with no insert (pHR2214).
In contrast, very little enhancement was observed in transfections with two different Sam68 mutants (Sam68 KH and Sam68G178E).
Sam68 KH has a large deletion in the RNA binding KH domain, whereas Sam68G178E corresponds to a mutation in the KH domain of the C.
In either case, the fact that the G178E mutant, which has previously been shown to be defective for RNA binding (40), failed to enhance CTE function suggests that RNA binding is essential for the observed phenotype.
10207078
full text
S222D
protein
substitution
true negative
Wild-type or K52R mutant (His)6-tagged rat ERK2 (a gift of Melanie Cobb) was expressed in bacteria, purified by Ni2 -nitrilotriacetic acid (NTA) metal affinity chromatography (Qiagen), and activated with MKK1-G7B ( N4/S218D/M219D/N221D/S222D) (35), which was expressed in bacteria and subjected to proteolysis with enterokinase to remove the (His)6 tag.
A (His)6-tagged ERK2 mutant deficient in dimerization (ERK2-H176E/L4A [H176E/ L333,336,341,344A] [see reference 23]) was coexpressed in bacteria with untagged, constitutively active MKK1 (MKK1-R4F [ N3/S218E/S222D] [36]), yielding partially phosphorylated ERK2-H176E/L4A, which was further phosphorylated as described above for wild-type ERK.
The cDNA constructs for expression of wild-type MKK1 and constitutively active MKK1 (MKK1-G1C [ N4/S218E/S222D]) were previously described (35, 58).
N221D
protein
substitution
true negative
Wild-type or K52R mutant (His)6-tagged rat ERK2 (a gift of Melanie Cobb) was expressed in bacteria, purified by Ni2 -nitrilotriacetic acid (NTA) metal affinity chromatography (Qiagen), and activated with MKK1-G7B ( N4/S218D/M219D/N221D/S222D) (35), which was expressed in bacteria and subjected to proteolysis with enterokinase to remove the (His)6 tag.
S218E
protein
substitution
true negative
A (His)6-tagged ERK2 mutant deficient in dimerization (ERK2-H176E/L4A [H176E/ L333,336,341,344A] [see reference 23]) was coexpressed in bacteria with untagged, constitutively active MKK1 (MKK1-R4F [ N3/S218E/S222D] [36]), yielding partially phosphorylated ERK2-H176E/L4A, which was further phosphorylated as described above for wild-type ERK.
The cDNA constructs for expression of wild-type MKK1 and constitutively active MKK1 (MKK1-G1C [ N4/S218E/S222D]) were previously described (35, 58).
L4A
protein
substitution
true negative
A (His)6-tagged ERK2 mutant deficient in dimerization (ERK2-H176E/L4A [H176E/ L333,336,341,344A] [see reference 23]) was coexpressed in bacteria with untagged, constitutively active MKK1 (MKK1-R4F [ N3/S218E/S222D] [36]), yielding partially phosphorylated ERK2-H176E/L4A, which was further phosphorylated as described above for wild-type ERK.
To test the influence of ERK dimerization on topoisomerase II activation, the mutant ERK2-H176E/ L4A, which is impaired in terms of dimerization ability (23), was phosphorylated and tested in relaxation assays.
Diphosphorylated ERK2-H176E/L4A in fact enhanced topoisomerase activity (Fig.
This difference could not be accounted for by differences in ERK phosphorylation, since diphosphorylated ERK2-H176E/L4A and wild-type ERK2 showed similar degrees of gel mobility retardation on immunoblots (Fig.
An incompletely phosphorylated form of ERK2-H176E/L4A (Fig.
(A) Human topoisomerase II was preincubated in the presence of ATP with no ERK2 (lanes 1 and 2), with diphosphorylated dimerization-defective mutant ERK2 (ppERK H176E/L4A) (lanes 3 and 4), with phosphorylated wild-type ERK2 (ppERK WT) (lanes 5 and 6), or with incompletely phosphorylated mutant ERK2 [(p)ERK H176E/L4A] (lanes 7 and 8).
Note that diphosphorylated ppERK2 H176E/L4A and ppERK WT show nearly complete shifts toward slower-migrating form whereas incompletely phosphorylated (p)ERK H176E/L4A shows significant levels of the faster-migrating unphosphorylated ERK.
H176E
protein
substitution
true negative
A (His)6-tagged ERK2 mutant deficient in dimerization (ERK2-H176E/L4A [H176E/ L333,336,341,344A] [see reference 23]) was coexpressed in bacteria with untagged, constitutively active MKK1 (MKK1-R4F [ N3/S218E/S222D] [36]), yielding partially phosphorylated ERK2-H176E/L4A, which was further phosphorylated as described above for wild-type ERK.
To test the influence of ERK dimerization on topoisomerase II activation, the mutant ERK2-H176E/ L4A, which is impaired in terms of dimerization ability (23), was phosphorylated and tested in relaxation assays.
Diphosphorylated ERK2-H176E/L4A in fact enhanced topoisomerase activity (Fig.
This difference could not be accounted for by differences in ERK phosphorylation, since diphosphorylated ERK2-H176E/L4A and wild-type ERK2 showed similar degrees of gel mobility retardation on immunoblots (Fig.
An incompletely phosphorylated form of ERK2-H176E/L4A (Fig.
(A) Human topoisomerase II was preincubated in the presence of ATP with no ERK2 (lanes 1 and 2), with diphosphorylated dimerization-defective mutant ERK2 (ppERK H176E/L4A) (lanes 3 and 4), with phosphorylated wild-type ERK2 (ppERK WT) (lanes 5 and 6), or with incompletely phosphorylated mutant ERK2 [(p)ERK H176E/L4A] (lanes 7 and 8).
Note that diphosphorylated ppERK2 H176E/L4A and ppERK WT show nearly complete shifts toward slower-migrating form whereas incompletely phosphorylated (p)ERK H176E/L4A shows significant levels of the faster-migrating unphosphorylated ERK.
M219D
protein
substitution
true negative
Wild-type or K52R mutant (His)6-tagged rat ERK2 (a gift of Melanie Cobb) was expressed in bacteria, purified by Ni2 -nitrilotriacetic acid (NTA) metal affinity chromatography (Qiagen), and activated with MKK1-G7B ( N4/S218D/M219D/N221D/S222D) (35), which was expressed in bacteria and subjected to proteolysis with enterokinase to remove the (His)6 tag.
S218D
protein
substitution
true negative
Wild-type or K52R mutant (His)6-tagged rat ERK2 (a gift of Melanie Cobb) was expressed in bacteria, purified by Ni2 -nitrilotriacetic acid (NTA) metal affinity chromatography (Qiagen), and activated with MKK1-G7B ( N4/S218D/M219D/N221D/S222D) (35), which was expressed in bacteria and subjected to proteolysis with enterokinase to remove the (His)6 tag.
K52R
protein
substitution
true negative
Wild-type or K52R mutant (His)6-tagged rat ERK2 (a gift of Melanie Cobb) was expressed in bacteria, purified by Ni2 -nitrilotriacetic acid (NTA) metal affinity chromatography (Qiagen), and activated with MKK1-G7B ( N4/S218D/M219D/N221D/S222D) (35), which was expressed in bacteria and subjected to proteolysis with enterokinase to remove the (His)6 tag.
(A) Plasmid relaxation by Drosophila topoisomerase II following preincubation with active phosphorylated wild-type ERK2 (closed triangles), inactive unphosphorylated wild-type ERK2 (open triangles), unphosphorylated catalytically inactive mutant ERK2-K52R (closed circles), or no ERK2 ( ) (open circles).
Control incubations with a nonactivable ERK2K52R mutant (Fig.
10590123
full text
R53I
protein
substitution
true negative
Site-directed mutagenesis was carried out with the Altered Sites mutagenesis kit (Promega) by using mutant oligonucleotides to introduce specific mutations (Tat D80E, 5 CCC GAG GGG AAC CGA CAG GCC 3 ; Tat R78K/D80E, 5 CCC GAG GGG AAC CGA CAG CC 3 and 5 ACC TCC CAA TCC AAA GGG GAA CCG AC 3 ; Tat R49G/K50I, 5 CTC CTA TGG CGG GAT CAA GCG GAG AC 3 ; Tat R49G/K50I/R52L/R53I, 5 CTC CTA TGG CGG GAT CAA GCG GAG AC and 5 CGG GAT CAA GCT AAT ACA GCG ACG AAG 3 ).
MBP, GST, Tat86, *Tat86, Tat R78K/D80E, Tat R49G/K50I/R52L/R53I, or *Tat C(22,25,27)A (2- g samples) were dissolved in 200 l of 20 mM sodium phosphate buffer (pH 7.4) without dithiothreitol and transferred in iodogen-coated tubes (50 g/ml) (Pierce Europe B.V., Oud Beijerland, The Netherlands), where proteins were iodinated (5 min at 4C) with 0.2 mCi of 125I (Amersham Pharmacia Biotech, Little Chalfont, United Kingdom).
The specific activities of the tracers were as follows: MBP, 1 Ci/118 fmol; GST, 1 Ci/134 fmol; Tat86, 1 Ci/108 fmol; *Tat86, 1 Ci/100 fmol; Tat R78K/D80E, 1 Ci/128 fmol; Tat R49G/K50I/R52L/R53I, 1 Ci/112 fmol; and *Tat C(22,25,27)A, 1 Ci/103 fmol.
Tat R49G/K50I, Tat R49G/K50I/R52L/R53I, and *Tat R(49,52,53,55,56,57)A had a reduced effect in terms of migration in the Boyden chamber (Fig.
3) of human EC compared to wild-type molecules (Tat86 or *Tat101) (P 0.005), with Tat R49G/K50I being more active than Tat R49G/K50I/R52L/R53I and *Tat R(49,52,53,55, 56,57)A (P 0.005).
Finally, the adhesion of EC to Tat R49G/ K50I/R52L/R53I and *Tat R(49,52,53,55,56,57)A was markedly reduced with respect to the adhesion to Tat86 or Tat R49G/K50I (P 0.005) (Fig.
In an effort to better define the activation of EC by the different Tat domains, the migration of EC was triggered by different concentrations of Tat86, Tat R49G/K50I/R52L/R53I, Tat R78K/D80E, and *Tat C(22,25,27)A.
Saturation binding curves of [125I]Tat R49G/K50I/R52L/R53I and of [125I]*Tat C(22,25,27)A (Fig.
The number of low-affinity sites bound by [125I]Tat R49G/K50I/R52L/R53I and [125I]*Tat C(22,25,27)A was increased compared to that bound by [125I]Tat86 (Table 2).
In contrast, mutants with mutations in the basic domain [(Tat R49G/K50I, Tat R49G/K50I/R52L/R53I, and *Tat R(49,52,53,55,56,57)A] showed a reduced ability to phosphorylate the receptor.
This feature depends on the number of basic residues mutated, with Tat R49G/K50I being more active than Tat R49G/K50I/R52L/R53I and *Tat R(49,52,53, 55,56,57)A.
Control Tat86 Tat72 *Tat101 Tat D80E Tat R78K/D80E Tat R49G/K50I Tat R49G/K50I/R52L/R53I *Tat C(22,25,27)A MBP GST 3/13 10/11 2/7 3/3 4/4 3/9 9/13 1/8 1/5 1/5 1/5 6 56 13 56 49 5 36 7 11 9 8 3 7 3 13 8 3 9 8 7 7 3 FIG.
To support the notion that the basic domain of Tat86 is involved in the signalling pathways downstream of VEGFR-2, we tested the activity of PI 3-kinase in EC stimulated with Tat R49G/K50I/R52L/R53I and Tat R78K/D80E.
8 show that Tat R49G/K50I/R52L/R53I activated PI 3-kinase activity to a lesser extent than Tat86 or Tat R78K/D80E did.
Dose-dependent effect of Tat86 (F), Tat R49G/K50I/R52L/R53I ( ), TatR78K/D80E (OE), and *Tat C(22,25,27)A (E) on EC migration.
Specific binding at equilibrium and Scatchard plot of [125I]Tat86 (A and B), [125I]Tat R78K/D80E (C and D), [125I] Tat R49G/K50I/R52L/R53I (E and F), and [125I] *Tat C(22,25,27)A (G and H) to EC.
Tat 4.21 nM; Bmax (site 2) R49G/K50I/R52L/R53I: Kd (site 1) 4.58 nM; Bmax (site 1) 3.51 pmol.
IC50c (nM) Tat86 Tat R78K/D80E Tat R49G/K50I/R52L/R53I *Tat C(22,25,27)A a b c 15.7 7.3 25.6 1.6 NDd ND 31.9 7.2 4.6 1.2 ND ND 12.3 7.1 10.2 4.1 ND ND 8.5 11.2 5.6 4.8 3.2 4.3 2.1 2.8 2.6 4.3 5.5 3.1 1.4 2.1 2.4 0.2 17.0 8.5 12.3 10.7 6.2 3.2 6.3 3.5 Mean standard deviation of three experiments.
Tat R49G/K50I/R52L/R53I and *Tat R(49,52,53,55,56,57)A failed to activate the receptor.
Spots corresponding to PIP3 were recovered and counted (n 3): control, 420 231; Tat R78K/D80E, 1,280 280; Tat R49G/K50I/ 132 cpm; Tat86, 1,342 R52L/R53I, 750 201.
D80E
protein
substitution
true negative
Site-directed mutagenesis was carried out with the Altered Sites mutagenesis kit (Promega) by using mutant oligonucleotides to introduce specific mutations (Tat D80E, 5 CCC GAG GGG AAC CGA CAG GCC 3 ; Tat R78K/D80E, 5 CCC GAG GGG AAC CGA CAG CC 3 and 5 ACC TCC CAA TCC AAA GGG GAA CCG AC 3 ; Tat R49G/K50I, 5 CTC CTA TGG CGG GAT CAA GCG GAG AC 3 ; Tat R49G/K50I/R52L/R53I, 5 CTC CTA TGG CGG GAT CAA GCG GAG AC and 5 CGG GAT CAA GCT AAT ACA GCG ACG AAG 3 ).
MBP, GST, Tat86, *Tat86, Tat R78K/D80E, Tat R49G/K50I/R52L/R53I, or *Tat C(22,25,27)A (2- g samples) were dissolved in 200 l of 20 mM sodium phosphate buffer (pH 7.4) without dithiothreitol and transferred in iodogen-coated tubes (50 g/ml) (Pierce Europe B.V., Oud Beijerland, The Netherlands), where proteins were iodinated (5 min at 4C) with 0.2 mCi of 125I (Amersham Pharmacia Biotech, Little Chalfont, United Kingdom).
The specific activities of the tracers were as follows: MBP, 1 Ci/118 fmol; GST, 1 Ci/134 fmol; Tat86, 1 Ci/108 fmol; *Tat86, 1 Ci/100 fmol; Tat R78K/D80E, 1 Ci/128 fmol; Tat R49G/K50I/R52L/R53I, 1 Ci/112 fmol; and *Tat C(22,25,27)A, 1 Ci/103 fmol.
Tat R78K/D80E showed reduced proliferation and migration activities compared to Tat86 (P 0.005).
Tat D80E had an effect similar to Tat86, indicating that a unique mutation is insufficient to impair the activities of Tat86 on EC.
Deletion of the sequence encoded by exon 2 (*Tat72) resulted in a molecule with biological activities (migration and proliferation) similar to those of Tat R78K/D80E (Fig.
Similarly, *Tat72 and Tat R78K/D80E were not permissive for EC adhesion (Fig.
In an effort to better define the activation of EC by the different Tat domains, the migration of EC was triggered by different concentrations of Tat86, Tat R49G/K50I/R52L/R53I, Tat R78K/D80E, and *Tat C(22,25,27)A.
However, the highest concentrations of Tat R78K/D80E (50 to 100 ng/ml) tended to reach the activity of Tat86 (Fig.
The same experimental approaches showed that Tat R78K/D80E bound endothelium in almost the same manner as did Tat86 (Fig.
Tat86, *Tat72, Tat R78K/D80E, and TatD80E induced the phosphorylation of VEGFR-2.
Moreover, we consistently observed that Tat R78K/D80E phosphorylated the receptor more efficiently than Tat86 did.
Control Tat86 Tat72 *Tat101 Tat D80E Tat R78K/D80E Tat R49G/K50I Tat R49G/K50I/R52L/R53I *Tat C(22,25,27)A MBP GST 3/13 10/11 2/7 3/3 4/4 3/9 9/13 1/8 1/5 1/5 1/5 6 56 13 56 49 5 36 7 11 9 8 3 7 3 13 8 3 9 8 7 7 3 FIG.
To support the notion that the basic domain of Tat86 is involved in the signalling pathways downstream of VEGFR-2, we tested the activity of PI 3-kinase in EC stimulated with Tat R49G/K50I/R52L/R53I and Tat R78K/D80E.
8 show that Tat R49G/K50I/R52L/R53I activated PI 3-kinase activity to a lesser extent than Tat86 or Tat R78K/D80E did.
Dose-dependent effect of Tat86 (F), Tat R49G/K50I/R52L/R53I ( ), TatR78K/D80E (OE), and *Tat C(22,25,27)A (E) on EC migration.
Specific binding at equilibrium and Scatchard plot of [125I]Tat86 (A and B), [125I]Tat R78K/D80E (C and D), [125I] Tat R49G/K50I/R52L/R53I (E and F), and [125I] *Tat C(22,25,27)A (G and H) to EC.
Tat R78K/D80E: Kd (site 1) 23.8 pM; Bmax (site 1) 38.4 fmol; Kd (site 2) 13.2 nM; Bmax (site 2) 1.50 pmol.
IC50c (nM) Tat86 Tat R78K/D80E Tat R49G/K50I/R52L/R53I *Tat C(22,25,27)A a b c 15.7 7.3 25.6 1.6 NDd ND 31.9 7.2 4.6 1.2 ND ND 12.3 7.1 10.2 4.1 ND ND 8.5 11.2 5.6 4.8 3.2 4.3 2.1 2.8 2.6 4.3 5.5 3.1 1.4 2.1 2.4 0.2 17.0 8.5 12.3 10.7 6.2 3.2 6.3 3.5 Mean standard deviation of three experiments.
To ascertain whether the RGD sequence is required for Tat binding or activity, mutants with single (D80E) and double (R78K/D80E) mutations and a variant truncated after the residue 72 (Tat72) were obtained.
Tat R78K/D80E and Tat72 had reduced biological activity in vitro and in vivo, which is consistent with previous reports showing that v 3-, v 5-, and 5 1integrin participate in Tat-induced activation of EC (5, 13) as well as of other cell types (6, 7, 35, 48, 62, 70).
Moreover, the RGD sequence and the C-terminal domain are not required for VEGFR-2 activation, as shown by the ability of Tat R78K/ D80E and Tat72 to induce VEGFR-2 phosphorylation.
In our experiments, we have observed that the activity of Tat R78K/ D80E on VEGFR-2 phosphorylation was greater than that elicited by Tat86.
Spots corresponding to PIP3 were recovered and counted (n 3): control, 420 231; Tat R78K/D80E, 1,280 280; Tat R49G/K50I/ 132 cpm; Tat86, 1,342 R52L/R53I, 750 201.
Except for Tat D80E, all mutants studied showed a reduced ability to activate EC functions, but their effects are only partially overlapping.
K50I
protein
substitution
true negative
Site-directed mutagenesis was carried out with the Altered Sites mutagenesis kit (Promega) by using mutant oligonucleotides to introduce specific mutations (Tat D80E, 5 CCC GAG GGG AAC CGA CAG GCC 3 ; Tat R78K/D80E, 5 CCC GAG GGG AAC CGA CAG CC 3 and 5 ACC TCC CAA TCC AAA GGG GAA CCG AC 3 ; Tat R49G/K50I, 5 CTC CTA TGG CGG GAT CAA GCG GAG AC 3 ; Tat R49G/K50I/R52L/R53I, 5 CTC CTA TGG CGG GAT CAA GCG GAG AC and 5 CGG GAT CAA GCT AAT ACA GCG ACG AAG 3 ).
MBP, GST, Tat86, *Tat86, Tat R78K/D80E, Tat R49G/K50I/R52L/R53I, or *Tat C(22,25,27)A (2- g samples) were dissolved in 200 l of 20 mM sodium phosphate buffer (pH 7.4) without dithiothreitol and transferred in iodogen-coated tubes (50 g/ml) (Pierce Europe B.V., Oud Beijerland, The Netherlands), where proteins were iodinated (5 min at 4C) with 0.2 mCi of 125I (Amersham Pharmacia Biotech, Little Chalfont, United Kingdom).
The specific activities of the tracers were as follows: MBP, 1 Ci/118 fmol; GST, 1 Ci/134 fmol; Tat86, 1 Ci/108 fmol; *Tat86, 1 Ci/100 fmol; Tat R78K/D80E, 1 Ci/128 fmol; Tat R49G/K50I/R52L/R53I, 1 Ci/112 fmol; and *Tat C(22,25,27)A, 1 Ci/103 fmol.
Tat R49G/K50I, Tat R49G/K50I/R52L/R53I, and *Tat R(49,52,53,55,56,57)A had a reduced effect in terms of migration in the Boyden chamber (Fig.
3) of human EC compared to wild-type molecules (Tat86 or *Tat101) (P 0.005), with Tat R49G/K50I being more active than Tat R49G/K50I/R52L/R53I and *Tat R(49,52,53,55, 56,57)A (P 0.005).
Finally, the adhesion of EC to Tat R49G/ K50I/R52L/R53I and *Tat R(49,52,53,55,56,57)A was markedly reduced with respect to the adhesion to Tat86 or Tat R49G/K50I (P 0.005) (Fig.
In an effort to better define the activation of EC by the different Tat domains, the migration of EC was triggered by different concentrations of Tat86, Tat R49G/K50I/R52L/R53I, Tat R78K/D80E, and *Tat C(22,25,27)A.
Saturation binding curves of [125I]Tat R49G/K50I/R52L/R53I and of [125I]*Tat C(22,25,27)A (Fig.
The number of low-affinity sites bound by [125I]Tat R49G/K50I/R52L/R53I and [125I]*Tat C(22,25,27)A was increased compared to that bound by [125I]Tat86 (Table 2).
In contrast, mutants with mutations in the basic domain [(Tat R49G/K50I, Tat R49G/K50I/R52L/R53I, and *Tat R(49,52,53,55,56,57)A] showed a reduced ability to phosphorylate the receptor.
This feature depends on the number of basic residues mutated, with Tat R49G/K50I being more active than Tat R49G/K50I/R52L/R53I and *Tat R(49,52,53, 55,56,57)A.
Control Tat86 Tat72 *Tat101 Tat D80E Tat R78K/D80E Tat R49G/K50I Tat R49G/K50I/R52L/R53I *Tat C(22,25,27)A MBP GST 3/13 10/11 2/7 3/3 4/4 3/9 9/13 1/8 1/5 1/5 1/5 6 56 13 56 49 5 36 7 11 9 8 3 7 3 13 8 3 9 8 7 7 3 FIG.
To support the notion that the basic domain of Tat86 is involved in the signalling pathways downstream of VEGFR-2, we tested the activity of PI 3-kinase in EC stimulated with Tat R49G/K50I/R52L/R53I and Tat R78K/D80E.
8 show that Tat R49G/K50I/R52L/R53I activated PI 3-kinase activity to a lesser extent than Tat86 or Tat R78K/D80E did.
Dose-dependent effect of Tat86 (F), Tat R49G/K50I/R52L/R53I ( ), TatR78K/D80E (OE), and *Tat C(22,25,27)A (E) on EC migration.
Specific binding at equilibrium and Scatchard plot of [125I]Tat86 (A and B), [125I]Tat R78K/D80E (C and D), [125I] Tat R49G/K50I/R52L/R53I (E and F), and [125I] *Tat C(22,25,27)A (G and H) to EC.
Tat 4.21 nM; Bmax (site 2) R49G/K50I/R52L/R53I: Kd (site 1) 4.58 nM; Bmax (site 1) 3.51 pmol.
IC50c (nM) Tat86 Tat R78K/D80E Tat R49G/K50I/R52L/R53I *Tat C(22,25,27)A a b c 15.7 7.3 25.6 1.6 NDd ND 31.9 7.2 4.6 1.2 ND ND 12.3 7.1 10.2 4.1 ND ND 8.5 11.2 5.6 4.8 3.2 4.3 2.1 2.8 2.6 4.3 5.5 3.1 1.4 2.1 2.4 0.2 17.0 8.5 12.3 10.7 6.2 3.2 6.3 3.5 Mean standard deviation of three experiments.
Tat R49G/K50I/R52L/R53I and *Tat R(49,52,53,55,56,57)A failed to activate the receptor.
Tat R49G/K50I induced VEGFR-2 phosphorylation but to a lesser extent.
Spots corresponding to PIP3 were recovered and counted (n 3): control, 420 231; Tat R78K/D80E, 1,280 280; Tat R49G/K50I/ 132 cpm; Tat86, 1,342 R52L/R53I, 750 201.
Tat R49G/K50I promoted cell adhesion as Tat86, but its migratory and proliferative capacities were consistently lower than those of Tat86.
R49G
protein
substitution
true negative
Site-directed mutagenesis was carried out with the Altered Sites mutagenesis kit (Promega) by using mutant oligonucleotides to introduce specific mutations (Tat D80E, 5 CCC GAG GGG AAC CGA CAG GCC 3 ; Tat R78K/D80E, 5 CCC GAG GGG AAC CGA CAG CC 3 and 5 ACC TCC CAA TCC AAA GGG GAA CCG AC 3 ; Tat R49G/K50I, 5 CTC CTA TGG CGG GAT CAA GCG GAG AC 3 ; Tat R49G/K50I/R52L/R53I, 5 CTC CTA TGG CGG GAT CAA GCG GAG AC and 5 CGG GAT CAA GCT AAT ACA GCG ACG AAG 3 ).
MBP, GST, Tat86, *Tat86, Tat R78K/D80E, Tat R49G/K50I/R52L/R53I, or *Tat C(22,25,27)A (2- g samples) were dissolved in 200 l of 20 mM sodium phosphate buffer (pH 7.4) without dithiothreitol and transferred in iodogen-coated tubes (50 g/ml) (Pierce Europe B.V., Oud Beijerland, The Netherlands), where proteins were iodinated (5 min at 4C) with 0.2 mCi of 125I (Amersham Pharmacia Biotech, Little Chalfont, United Kingdom).
The specific activities of the tracers were as follows: MBP, 1 Ci/118 fmol; GST, 1 Ci/134 fmol; Tat86, 1 Ci/108 fmol; *Tat86, 1 Ci/100 fmol; Tat R78K/D80E, 1 Ci/128 fmol; Tat R49G/K50I/R52L/R53I, 1 Ci/112 fmol; and *Tat C(22,25,27)A, 1 Ci/103 fmol.
Tat R49G/K50I, Tat R49G/K50I/R52L/R53I, and *Tat R(49,52,53,55,56,57)A had a reduced effect in terms of migration in the Boyden chamber (Fig.
3) of human EC compared to wild-type molecules (Tat86 or *Tat101) (P 0.005), with Tat R49G/K50I being more active than Tat R49G/K50I/R52L/R53I and *Tat R(49,52,53,55, 56,57)A (P 0.005).
Finally, the adhesion of EC to Tat R49G/ K50I/R52L/R53I and *Tat R(49,52,53,55,56,57)A was markedly reduced with respect to the adhesion to Tat86 or Tat R49G/K50I (P 0.005) (Fig.
In an effort to better define the activation of EC by the different Tat domains, the migration of EC was triggered by different concentrations of Tat86, Tat R49G/K50I/R52L/R53I, Tat R78K/D80E, and *Tat C(22,25,27)A.
Saturation binding curves of [125I]Tat R49G/K50I/R52L/R53I and of [125I]*Tat C(22,25,27)A (Fig.
The number of low-affinity sites bound by [125I]Tat R49G/K50I/R52L/R53I and [125I]*Tat C(22,25,27)A was increased compared to that bound by [125I]Tat86 (Table 2).
In contrast, mutants with mutations in the basic domain [(Tat R49G/K50I, Tat R49G/K50I/R52L/R53I, and *Tat R(49,52,53,55,56,57)A] showed a reduced ability to phosphorylate the receptor.
This feature depends on the number of basic residues mutated, with Tat R49G/K50I being more active than Tat R49G/K50I/R52L/R53I and *Tat R(49,52,53, 55,56,57)A.
Control Tat86 Tat72 *Tat101 Tat D80E Tat R78K/D80E Tat R49G/K50I Tat R49G/K50I/R52L/R53I *Tat C(22,25,27)A MBP GST 3/13 10/11 2/7 3/3 4/4 3/9 9/13 1/8 1/5 1/5 1/5 6 56 13 56 49 5 36 7 11 9 8 3 7 3 13 8 3 9 8 7 7 3 FIG.
To support the notion that the basic domain of Tat86 is involved in the signalling pathways downstream of VEGFR-2, we tested the activity of PI 3-kinase in EC stimulated with Tat R49G/K50I/R52L/R53I and Tat R78K/D80E.
8 show that Tat R49G/K50I/R52L/R53I activated PI 3-kinase activity to a lesser extent than Tat86 or Tat R78K/D80E did.
Dose-dependent effect of Tat86 (F), Tat R49G/K50I/R52L/R53I ( ), TatR78K/D80E (OE), and *Tat C(22,25,27)A (E) on EC migration.
Specific binding at equilibrium and Scatchard plot of [125I]Tat86 (A and B), [125I]Tat R78K/D80E (C and D), [125I] Tat R49G/K50I/R52L/R53I (E and F), and [125I] *Tat C(22,25,27)A (G and H) to EC.
Tat 4.21 nM; Bmax (site 2) R49G/K50I/R52L/R53I: Kd (site 1) 4.58 nM; Bmax (site 1) 3.51 pmol.
IC50c (nM) Tat86 Tat R78K/D80E Tat R49G/K50I/R52L/R53I *Tat C(22,25,27)A a b c 15.7 7.3 25.6 1.6 NDd ND 31.9 7.2 4.6 1.2 ND ND 12.3 7.1 10.2 4.1 ND ND 8.5 11.2 5.6 4.8 3.2 4.3 2.1 2.8 2.6 4.3 5.5 3.1 1.4 2.1 2.4 0.2 17.0 8.5 12.3 10.7 6.2 3.2 6.3 3.5 Mean standard deviation of three experiments.
Tat R49G/K50I/R52L/R53I and *Tat R(49,52,53,55,56,57)A failed to activate the receptor.
Tat R49G/K50I induced VEGFR-2 phosphorylation but to a lesser extent.
Spots corresponding to PIP3 were recovered and counted (n 3): control, 420 231; Tat R78K/D80E, 1,280 280; Tat R49G/K50I/ 132 cpm; Tat86, 1,342 R52L/R53I, 750 201.
Tat R49G/K50I promoted cell adhesion as Tat86, but its migratory and proliferative capacities were consistently lower than those of Tat86.
R52L
protein
substitution
true negative
Site-directed mutagenesis was carried out with the Altered Sites mutagenesis kit (Promega) by using mutant oligonucleotides to introduce specific mutations (Tat D80E, 5 CCC GAG GGG AAC CGA CAG GCC 3 ; Tat R78K/D80E, 5 CCC GAG GGG AAC CGA CAG CC 3 and 5 ACC TCC CAA TCC AAA GGG GAA CCG AC 3 ; Tat R49G/K50I, 5 CTC CTA TGG CGG GAT CAA GCG GAG AC 3 ; Tat R49G/K50I/R52L/R53I, 5 CTC CTA TGG CGG GAT CAA GCG GAG AC and 5 CGG GAT CAA GCT AAT ACA GCG ACG AAG 3 ).
MBP, GST, Tat86, *Tat86, Tat R78K/D80E, Tat R49G/K50I/R52L/R53I, or *Tat C(22,25,27)A (2- g samples) were dissolved in 200 l of 20 mM sodium phosphate buffer (pH 7.4) without dithiothreitol and transferred in iodogen-coated tubes (50 g/ml) (Pierce Europe B.V., Oud Beijerland, The Netherlands), where proteins were iodinated (5 min at 4C) with 0.2 mCi of 125I (Amersham Pharmacia Biotech, Little Chalfont, United Kingdom).
The specific activities of the tracers were as follows: MBP, 1 Ci/118 fmol; GST, 1 Ci/134 fmol; Tat86, 1 Ci/108 fmol; *Tat86, 1 Ci/100 fmol; Tat R78K/D80E, 1 Ci/128 fmol; Tat R49G/K50I/R52L/R53I, 1 Ci/112 fmol; and *Tat C(22,25,27)A, 1 Ci/103 fmol.
Tat R49G/K50I, Tat R49G/K50I/R52L/R53I, and *Tat R(49,52,53,55,56,57)A had a reduced effect in terms of migration in the Boyden chamber (Fig.
3) of human EC compared to wild-type molecules (Tat86 or *Tat101) (P 0.005), with Tat R49G/K50I being more active than Tat R49G/K50I/R52L/R53I and *Tat R(49,52,53,55, 56,57)A (P 0.005).
Finally, the adhesion of EC to Tat R49G/ K50I/R52L/R53I and *Tat R(49,52,53,55,56,57)A was markedly reduced with respect to the adhesion to Tat86 or Tat R49G/K50I (P 0.005) (Fig.
In an effort to better define the activation of EC by the different Tat domains, the migration of EC was triggered by different concentrations of Tat86, Tat R49G/K50I/R52L/R53I, Tat R78K/D80E, and *Tat C(22,25,27)A.
Saturation binding curves of [125I]Tat R49G/K50I/R52L/R53I and of [125I]*Tat C(22,25,27)A (Fig.
The number of low-affinity sites bound by [125I]Tat R49G/K50I/R52L/R53I and [125I]*Tat C(22,25,27)A was increased compared to that bound by [125I]Tat86 (Table 2).
In contrast, mutants with mutations in the basic domain [(Tat R49G/K50I, Tat R49G/K50I/R52L/R53I, and *Tat R(49,52,53,55,56,57)A] showed a reduced ability to phosphorylate the receptor.
This feature depends on the number of basic residues mutated, with Tat R49G/K50I being more active than Tat R49G/K50I/R52L/R53I and *Tat R(49,52,53, 55,56,57)A.
Control Tat86 Tat72 *Tat101 Tat D80E Tat R78K/D80E Tat R49G/K50I Tat R49G/K50I/R52L/R53I *Tat C(22,25,27)A MBP GST 3/13 10/11 2/7 3/3 4/4 3/9 9/13 1/8 1/5 1/5 1/5 6 56 13 56 49 5 36 7 11 9 8 3 7 3 13 8 3 9 8 7 7 3 FIG.
To support the notion that the basic domain of Tat86 is involved in the signalling pathways downstream of VEGFR-2, we tested the activity of PI 3-kinase in EC stimulated with Tat R49G/K50I/R52L/R53I and Tat R78K/D80E.
8 show that Tat R49G/K50I/R52L/R53I activated PI 3-kinase activity to a lesser extent than Tat86 or Tat R78K/D80E did.
Dose-dependent effect of Tat86 (F), Tat R49G/K50I/R52L/R53I ( ), TatR78K/D80E (OE), and *Tat C(22,25,27)A (E) on EC migration.
Specific binding at equilibrium and Scatchard plot of [125I]Tat86 (A and B), [125I]Tat R78K/D80E (C and D), [125I] Tat R49G/K50I/R52L/R53I (E and F), and [125I] *Tat C(22,25,27)A (G and H) to EC.
Tat 4.21 nM; Bmax (site 2) R49G/K50I/R52L/R53I: Kd (site 1) 4.58 nM; Bmax (site 1) 3.51 pmol.
IC50c (nM) Tat86 Tat R78K/D80E Tat R49G/K50I/R52L/R53I *Tat C(22,25,27)A a b c 15.7 7.3 25.6 1.6 NDd ND 31.9 7.2 4.6 1.2 ND ND 12.3 7.1 10.2 4.1 ND ND 8.5 11.2 5.6 4.8 3.2 4.3 2.1 2.8 2.6 4.3 5.5 3.1 1.4 2.1 2.4 0.2 17.0 8.5 12.3 10.7 6.2 3.2 6.3 3.5 Mean standard deviation of three experiments.
Tat R49G/K50I/R52L/R53I and *Tat R(49,52,53,55,56,57)A failed to activate the receptor.
Spots corresponding to PIP3 were recovered and counted (n 3): control, 420 231; Tat R78K/D80E, 1,280 280; Tat R49G/K50I/ 132 cpm; Tat86, 1,342 R52L/R53I, 750 201.
E80D
protein
substitution
true negative
Mutants with mutations in basic and in cysteine-rich domains had similar kinetic binding parameters, which differed from those of TatR78K/E80D.
Furthermore, Tat E80D was similar to Tat86 or *Tat101 in all assays performed.
R78K
protein
substitution
true negative
Site-directed mutagenesis was carried out with the Altered Sites mutagenesis kit (Promega) by using mutant oligonucleotides to introduce specific mutations (Tat D80E, 5 CCC GAG GGG AAC CGA CAG GCC 3 ; Tat R78K/D80E, 5 CCC GAG GGG AAC CGA CAG CC 3 and 5 ACC TCC CAA TCC AAA GGG GAA CCG AC 3 ; Tat R49G/K50I, 5 CTC CTA TGG CGG GAT CAA GCG GAG AC 3 ; Tat R49G/K50I/R52L/R53I, 5 CTC CTA TGG CGG GAT CAA GCG GAG AC and 5 CGG GAT CAA GCT AAT ACA GCG ACG AAG 3 ).
MBP, GST, Tat86, *Tat86, Tat R78K/D80E, Tat R49G/K50I/R52L/R53I, or *Tat C(22,25,27)A (2- g samples) were dissolved in 200 l of 20 mM sodium phosphate buffer (pH 7.4) without dithiothreitol and transferred in iodogen-coated tubes (50 g/ml) (Pierce Europe B.V., Oud Beijerland, The Netherlands), where proteins were iodinated (5 min at 4C) with 0.2 mCi of 125I (Amersham Pharmacia Biotech, Little Chalfont, United Kingdom).
The specific activities of the tracers were as follows: MBP, 1 Ci/118 fmol; GST, 1 Ci/134 fmol; Tat86, 1 Ci/108 fmol; *Tat86, 1 Ci/100 fmol; Tat R78K/D80E, 1 Ci/128 fmol; Tat R49G/K50I/R52L/R53I, 1 Ci/112 fmol; and *Tat C(22,25,27)A, 1 Ci/103 fmol.
Tat R78K/D80E showed reduced proliferation and migration activities compared to Tat86 (P 0.005).
Deletion of the sequence encoded by exon 2 (*Tat72) resulted in a molecule with biological activities (migration and proliferation) similar to those of Tat R78K/D80E (Fig.
Similarly, *Tat72 and Tat R78K/D80E were not permissive for EC adhesion (Fig.
In an effort to better define the activation of EC by the different Tat domains, the migration of EC was triggered by different concentrations of Tat86, Tat R49G/K50I/R52L/R53I, Tat R78K/D80E, and *Tat C(22,25,27)A.
However, the highest concentrations of Tat R78K/D80E (50 to 100 ng/ml) tended to reach the activity of Tat86 (Fig.
The same experimental approaches showed that Tat R78K/D80E bound endothelium in almost the same manner as did Tat86 (Fig.
Tat86, *Tat72, Tat R78K/D80E, and TatD80E induced the phosphorylation of VEGFR-2.
Moreover, we consistently observed that Tat R78K/D80E phosphorylated the receptor more efficiently than Tat86 did.
Control Tat86 Tat72 *Tat101 Tat D80E Tat R78K/D80E Tat R49G/K50I Tat R49G/K50I/R52L/R53I *Tat C(22,25,27)A MBP GST 3/13 10/11 2/7 3/3 4/4 3/9 9/13 1/8 1/5 1/5 1/5 6 56 13 56 49 5 36 7 11 9 8 3 7 3 13 8 3 9 8 7 7 3 FIG.
To support the notion that the basic domain of Tat86 is involved in the signalling pathways downstream of VEGFR-2, we tested the activity of PI 3-kinase in EC stimulated with Tat R49G/K50I/R52L/R53I and Tat R78K/D80E.
8 show that Tat R49G/K50I/R52L/R53I activated PI 3-kinase activity to a lesser extent than Tat86 or Tat R78K/D80E did.
Dose-dependent effect of Tat86 (F), Tat R49G/K50I/R52L/R53I ( ), TatR78K/D80E (OE), and *Tat C(22,25,27)A (E) on EC migration.
Specific binding at equilibrium and Scatchard plot of [125I]Tat86 (A and B), [125I]Tat R78K/D80E (C and D), [125I] Tat R49G/K50I/R52L/R53I (E and F), and [125I] *Tat C(22,25,27)A (G and H) to EC.
Tat R78K/D80E: Kd (site 1) 23.8 pM; Bmax (site 1) 38.4 fmol; Kd (site 2) 13.2 nM; Bmax (site 2) 1.50 pmol.
IC50c (nM) Tat86 Tat R78K/D80E Tat R49G/K50I/R52L/R53I *Tat C(22,25,27)A a b c 15.7 7.3 25.6 1.6 NDd ND 31.9 7.2 4.6 1.2 ND ND 12.3 7.1 10.2 4.1 ND ND 8.5 11.2 5.6 4.8 3.2 4.3 2.1 2.8 2.6 4.3 5.5 3.1 1.4 2.1 2.4 0.2 17.0 8.5 12.3 10.7 6.2 3.2 6.3 3.5 Mean standard deviation of three experiments.
To ascertain whether the RGD sequence is required for Tat binding or activity, mutants with single (D80E) and double (R78K/D80E) mutations and a variant truncated after the residue 72 (Tat72) were obtained.
Tat R78K/D80E and Tat72 had reduced biological activity in vitro and in vivo, which is consistent with previous reports showing that v 3-, v 5-, and 5 1integrin participate in Tat-induced activation of EC (5, 13) as well as of other cell types (6, 7, 35, 48, 62, 70).
Moreover, the RGD sequence and the C-terminal domain are not required for VEGFR-2 activation, as shown by the ability of Tat R78K/ D80E and Tat72 to induce VEGFR-2 phosphorylation.
In our experiments, we have observed that the activity of Tat R78K/ D80E on VEGFR-2 phosphorylation was greater than that elicited by Tat86.
Spots corresponding to PIP3 were recovered and counted (n 3): control, 420 231; Tat R78K/D80E, 1,280 280; Tat R49G/K50I/ 132 cpm; Tat86, 1,342 R52L/R53I, 750 201.
Mutants with mutations in basic and in cysteine-rich domains had similar kinetic binding parameters, which differed from those of TatR78K/E80D.
14627992
full text
Y1235D
protein
substitution
true negative
As summarized in Table 2, none of the parameters was significantly linked Met Y1253D in oropharyngeal cancer DM Aebersold et al 8521 a 0.34 wild type 100% 0.30 0.26 0.22 0.18 0.14 0.10 44 46 48 50 52 54 56 58 60 62 64 C 0.06 Tm = 52.1 sample water control b 0.34 0.30 0.26 0.22 0.18 0.14 0.10 Y1235D 100% c 0.34 0.30 0.26 0.22 Y1235D/wild type 50% Fluorescence (F2/F1) sample water control 0.18 0.14 0.10 sample water control 44 46 48 50 52 54 56 58 60 62 64 C 0.04 0.03 Tm = 59.6 44 46 48 50 52 54 56 58 60 62 64 C 0.03 Tm = 52.1 0.02 0.01 0.00 -0.01 Tm = 59.6 Fluorescence -d(F2/F1)/dT 0.04 0.02 -0.00 -0.02 0.02 0.01 0.00 -0.01 -0.02 44 46 48 50 52 54 56 58 60 62 64 C 44 46 48 50 52 54 56 58 60 62 64 C -0.02 44 46 48 50 52 54 56 58 60 62 64 C d 0.34 Y1235D/wild type 10% 0.30 0.26 0.22 0.18 0.14 0.10 44 46 48 50 52 54 56 58 60 62 64 C sample water control e 0.34 0.30 0.26 0.22 0.18 0.14 0.10 Y1235D/wild type 2% f 0.21 0.19 0.17 0.15 Y1235D/wild type 2% + PNA Fluorescence (F2/F1) sample water control 0.13 0.11 sample water control 44 46 48 50 52 54 56 58 60 62 64 C 0.06 0.04 Tm = 52.1 0.020 0.016 44 46 48 50 52 54 56 58 60 62 64 C Tm = 59.6 0.05 Tm = 52.1 Fluorescence -d(F2/F1)/dT 0.04 0.03 0.02 0.01 0.00 -0.01 -0.02 44 46 48 50 52 54 56 58 60 62 64 C Tm = 59.6 0.012 0.008 0.004 0.02 -0.00 -0.02 44 46 48 50 52 54 56 58 60 62 64 C 0.000 -0.004 44 46 48 50 52 54 56 58 60 62 64 C Temperature Temperature s Temperature Figure 1 Detection of the Met Y1253D mutation by LightCycler real-time PCR in combination with PNA-directed DNA clamping and melting curve analysis.
Typo
T3757G
protein
substitution
true negative
In the present retrospective study, we aimed to determine the prevalence and clinical impact of the Met-activating mutation Y1253D (corresponding to T3757G at the genomic level) in patients with squamous cell cancer of the oropharynx treated by radical radiotherapy.
The sensor probe that was designed to fully match the allele that harbours the T3757G substitution was 30 -terminally labelled with fluorescein: 50 -GTTGTGTACACTATCGTATTCTTT-F (mutated base is underlined).
DNA mutation
Y1253D
protein
substitution
P08581-2
true positive
Oncogene (2003) 22, 85198523 & 2003 Nature Publishing Group All rights reserved 0950-9232/03 $25.00 www.nature.com/onc SHORT REPORTS Prevalence and clinical impact of Met Y1253D-activating point mutation in radiotherapy-treated squamous cell cancer of the oropharynx Daniel M Aebersold1,3, Olfert Landt2, Sylvie Berthou3, Gunther Gruber1, Karl T Beer1, Richard H Greiner1 and Yitzhak Zimmer*,3 1 Department of Radiation Oncology, Inselspital Bern, Switzerland; 2TIB MOLBIOL, Berlin, Germany; 3Department of Clinical Research, University of Bern, Switzerland Aberrant signalling through the hepatocyte growth factor/ scatter factor receptor Met has been implicated in various aspects of the development of human cancer including the promotion of tumour invasion, angiogenesis and metastasis.
We determined the prevalence and clinical impact of the Met-activating mutation Y1253D in patients with squamous cell cancer of the oropharynx treated by radical radiotherapy.
By this approach, Met Y1253D was detected in tumours of 15 out of 138 patients (10.9%).
Both univariate and multivariate survival analysis revealed Met Y1253D to be significantly associated with impaired local tumour control.
Our results provide evidence that the Met-activating mutation Y1253D is present in a notable subset of patients with oropharyngeal cancer and indicate that it may interfere with radioresponsiveness of these tumours, supporting the notion of aberrant Met signalling as a potential target for radiosensitization.
These mutations were initially reported in hereditary and sporadic cases of papillary renal carcinoma (Schmidt et al., 1999), but later also in hepatocellular carcinoma (Park et al., 1999) and squamous cell carcinomas of the head and neck, where the Met Y1253D mutation has first been identified (Di Renzo et al., 2000; Lorenzato et al., 2002).
In the present retrospective study, we aimed to determine the prevalence and clinical impact of the Met-activating mutation Y1253D (corresponding to T3757G at the genomic level) in patients with squamous cell cancer of the oropharynx treated by radical radiotherapy.
Nevertheless, the ability to Met Y1253D in oropharyngeal cancer DM Aebersold et al 8520 detect low-abundant mutations is of immense importance since cells harbouring these alleles can be associated with a clonal expansion, giving rise to a population with a more aggressive tumour phenotype.
(2000) have shown that the abundance of the Met Y1253D allele is about 2% in primary tumours of patients with squamous cell carcinoma of the head and neck.
To screen archival pretherapeutic biopsy material for the presence of Met Y1253D, we have therefore established a novel method using LightCyclers-based real-time PCR in combination with peptide nucleic acid (PNA)-directed clamping.
In our case, we distinguished between amplified wild-type Met and mutated Met Y1253D using melting curve analysis with specific fluorescent hybridization probes.
Figure 1 shows the principle of the method as it was established using plasmids carrying the Met wild-type and Y1253D alleles.
These results indicate that our screening approach is able to detect the presence of the Y1253D Met mutation within a background of at least 98% wildtype alleles, corresponding to the low abundance of the Y1253D mutation within primary tumours as described by Di Renzo et al.
To validate the results obtained with the plasmids, genomic DNA from a tumour sample with confirmed Y1253D mutation (kindly provided by MF Die Renzo) was tested and found to yield exactly the same two melting temperatures (data not shown).
The method was then used to screen the pretherapeutic tumour biopsies for the presence of the Y1253D Met mutation.
Again, the Tm values for Met wild type and, in our positive cases, for the Met Y1253D were equal to the corresponding Tm values in the plasmid experiments (Figure 2).
Met Y1253D was detected in 15 of 138 pretherapeutic tumour samples (10.9%), indicating its presence in a notable subset of patients with oropharyngeal cancer.
Met Y1253D did not correlate with any of the clinicopathological parameters (Table 1).
Assessing the impact of Met Y1253D for treatment outcome, we found a significant decrease of the local tumour control probability by radiotherapy (P 0.009, log rank; Figure 3), the risk ratio for local tumour progression being 2.44 in the presence of the mutation (P 0.01, Cox regression; Table 2).
The retrospective property of the study and the relatively low number of Met Y1253D-positive patients limit the statistical power of these results.
However, the multivariate analysis proved the effect of Met Y1253D on local tumour progression to be independent from advanced T stage and tobacco exposure, which are known adverse predictive factors in this tumour entity (Table 2), strongly supporting the validity of the adverse effect exerted by Met Y1253D.
Besides such a direct interference with radiation-induced cell killing, impaired local control in the presence of the Met Y1253D mutation may also be due to increased cellular proliferation, enhanced cell motility and local tumour invasiveness, which have all been described to result from aberrant Met activity in various tumour systems (To and Tsao, 1998; Danilkovitch-Miagkova and Zbar, 2002; Lorenzato et al., 2002).
Since aberrant Met signalling has also been found to be involved in the promotion of distant metastasis (for a review, see To and Tsao, 1998), we assessed whether the presence of the Met Y1253D mutation was associated with the development of systemic spread and/or with overall survival in our study group.
As summarized in Table 2, none of the parameters was significantly linked Met Y1253D in oropharyngeal cancer DM Aebersold et al 8521 a 0.34 wild type 100% 0.30 0.26 0.22 0.18 0.14 0.10 44 46 48 50 52 54 56 58 60 62 64 C 0.06 Tm = 52.1 sample water control b 0.34 0.30 0.26 0.22 0.18 0.14 0.10 Y1235D 100% c 0.34 0.30 0.26 0.22 Y1235D/wild type 50% Fluorescence (F2/F1) sample water control 0.18 0.14 0.10 sample water control 44 46 48 50 52 54 56 58 60 62 64 C 0.04 0.03 Tm = 59.6 44 46 48 50 52 54 56 58 60 62 64 C 0.03 Tm = 52.1 0.02 0.01 0.00 -0.01 Tm = 59.6 Fluorescence -d(F2/F1)/dT 0.04 0.02 -0.00 -0.02 0.02 0.01 0.00 -0.01 -0.02 44 46 48 50 52 54 56 58 60 62 64 C 44 46 48 50 52 54 56 58 60 62 64 C -0.02 44 46 48 50 52 54 56 58 60 62 64 C d 0.34 Y1235D/wild type 10% 0.30 0.26 0.22 0.18 0.14 0.10 44 46 48 50 52 54 56 58 60 62 64 C sample water control e 0.34 0.30 0.26 0.22 0.18 0.14 0.10 Y1235D/wild type 2% f 0.21 0.19 0.17 0.15 Y1235D/wild type 2% + PNA Fluorescence (F2/F1) sample water control 0.13 0.11 sample water control 44 46 48 50 52 54 56 58 60 62 64 C 0.06 0.04 Tm = 52.1 0.020 0.016 44 46 48 50 52 54 56 58 60 62 64 C Tm = 59.6 0.05 Tm = 52.1 Fluorescence -d(F2/F1)/dT 0.04 0.03 0.02 0.01 0.00 -0.01 -0.02 44 46 48 50 52 54 56 58 60 62 64 C Tm = 59.6 0.012 0.008 0.004 0.02 -0.00 -0.02 44 46 48 50 52 54 56 58 60 62 64 C 0.000 -0.004 44 46 48 50 52 54 56 58 60 62 64 C Temperature Temperature s Temperature Figure 1 Detection of the Met Y1253D mutation by LightCycler real-time PCR in combination with PNA-directed DNA clamping and melting curve analysis.
(a, b) Melting curves of Met wild type and Met Y1253D expressing plasmids.
(ce) Dilution series of Met wild type and Met Y1253D expressing plasmids without PNA.
(f) 2% Met Y1253D plasmid on a background of 98% of Met wild-type plasmid with PNA.
A 154 bp fragment corresponding to Met exon 19, where Y1253D is located, was amplified and analysed by running a melting curve.
However, the risk ratio for distant metastasis was considerably enhanced (2.41) in tumours with the Y1253D mutation.
The lack of statistical significance may be due to the low number of patients with distant metastasis (14.5%) along with the relatively low number of Met Y1253D-positive tumours.
Low statistical power along with uncontrolled imbalances in the context of a retrospective study may also account for the absence of statistical significance of the association of the Met Y1253D mutation with overall survival, the risk ratio for death being 1.29 in the presence of the mutation (Table 2).
Oncogene Met Y1253D in oropharyngeal cancer DM Aebersold et al 8522 0.9 0.8 Case 32 w/o PNA with PNA water control 0.6 0.5 0.4 0.3 0.2 0.1 Case 88 w/o PNA with PNA water control Fluorescence (F2/F1) 0.7 0.6 0.5 0.4 0.3 0.2 0.1 44 46 48 50 52 54 56 58 60 62 64 C 0.12 Tm = 52.1 0.12 44 46 48 50 52 54 56 58 60 62 64 C Fluorescence -d(F2/F1)/dT Tm = 52.1 0.1 0.08 0.06 Tm = 59.6 0.04 0.02 0 -0.02 Tm = 59.6 0.1 0.08 0.06 0.04 0.02 0 -0.02 44 46 48 50 52 54 56 58 60 62 64 C Temperature 44 46 48 50 52 54 56 58 60 62 64 C Temperature Figure 2 Detection of the Met Y1253D mutation by LightCyclers real-time PCR in combination with PNA-directed DNA clamping and melting curve analysis in two cases of oropharyngeal cancer.
The presence of squamous cell cancer was verified by a pathologist Table 1 Patients characteristics Met Y1253D (no.
of patients (%)) Parameter Total Gender Male Female Age (years) (median: 57) o57 X57 Tobacco exposure No Yes T stage T1/2 T3/4 N stage N0 N13 Grade of differentiation G1 G2/3 Chemotherapy No Yes Total 138 (100) 111 (80) 27 (20) 69 (50) 69 (50) 16 (12) 122 (88) 25 (18) 113 (82) 47 (34) 91 (66) 16 (12) 122 (88) 106 (77) 32 (23) Negative 123 (89) 98 (88) 25 (93) 60 (87) 63 (91) 14 (88) 109 (89) 23 (92) 100 (89) 43 (92) 80 (88) 16 (100) 107 (88) 94 (89) 29 (91) Positive 15 (11) 13 (12) 2 (7) 9 (13) 6 (9) 2 (12) 13 (11) 2 (8) 13 (11) 4 (8) 11 (12) 0 (0) 15 (12) 12 (11) 3 (9) NS NS NS NS NS NS NS P-value Probability of local progression-free survival 1.0 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0 0 1 2 3 4 5 Years 6 7 8 9 p = 0.009, log rank Met Y1253D positive (n = 15) Met Y1253D negative (n = 123) The 138 patients with histologically proven squamous cell cancer of the oropharynx have undergone radical external-beam radiotherapy between 1991 and 1997.
Correlations between patients parameters and presence of the Met Y1253D were analysed by Fisher's exact test Oncogene Figure 3 Local progression-free survival according to the presence or absence of the Met Y1253D-activating point mutation.
To individualize treatment strategies, identification of those patients who might benefit most Met Y1253D in oropharyngeal cancer DM Aebersold et al 8523 Table 2 Treatment outcome analysis following radical radiotherapy of oropharyngeal cancer Local progression-free survival Variable Risk ratio Confidence interval (95%) 0.703.64 0.601.78 1.3973.08 1.4414.89 0.501.51 0.472.99 0.341.37 1.224.88 1.5682.39 1.4615.06 1.265.05 P Risk ratio Distant metastasis Confidence interval (95%) 0.5311.13 0.312.05 0.255.79 0.6237.74 0.564.88 0.182.72 0.725.55 0.688.49 -- -- -- P Risk ratio Overall survival Confidence interval (95%) 0.902.82 0.611.41 1.296.79 1.013.28 0.842.01 0.461.64 0.491.36 0.672.51 1.316.91 1.033.35 -- P Univariate analysis Gender, male vs female Age (years), o57 vs X57 (median) Smoking, no vs yes T stage, T1/2 vs T3/4 N stage, N0 vs N13 Grade, G1 vs G2/3 Chemotherapy, no vs yes Met Y1253D, no vs yes Multivariate analysis Smoking, no vs yes T stage, T1/2 vs T3/4 Met Y1253D, no vs yes 1.60 1.04 10.08 4.64 0.87 1.19 0.69 2.44 11.35 4.68 2.52 NS NS 0.02 0.01 NS NS NS 0.01 0.02 0.01 0.009 2.42 0.79 1.21 4.83 1.66 0.70 2.00 2.41 -- -- -- NS NS NS NS NS NS NS NS -- -- -- 1.59 0.93 2.96 1.82 1.30 0.87 0.82 1.29 3.01 1.86 -- NS NS 0.01 0.05 NS NS NS NS 0.01 0.04 -- Cox regressions were calculated to determine the risk ratios of local progression-free survival and overall survival.
Acknowledgements We thank MF Di Renzo for providing the Met wild type and Met Y1253D containing expression vectors as well as genomic DNA from a Y1253D-carrying tumour and J Laissue for pathological review of the tumour samples.
12620993
full text
I47Q
protein
substitution
true negative
An artificial I47A substitution in engrailed reduces DNA binding affinity 10- to 20-fold in vitro (Ades and Sauer, 1995), while an I47Q substitution in the Para-Hox protein IPF1 abolishes DNA binding in vitro and greatly reduces transcriptional activation in transfected cells (Lu et al., 1996).
R31W
protein
substitution
true negative
Three families harbour different frameshifting deletions in HOXD13 (Goodman et al., 1998; Calabrese et al., 2000), which are predicted to result in truncated proteins unable to bind DNA, while the fourth family harbours a missense mutation in helix II of the HOXD13 homeodomain (R31W), which is predicted to destabilise the homeodomain (Debeer et al., 2002).
I47A
protein
substitution
true negative
An artificial I47A substitution in engrailed reduces DNA binding affinity 10- to 20-fold in vitro (Ades and Sauer, 1995), while an I47Q substitution in the Para-Hox protein IPF1 abolishes DNA binding in vitro and greatly reduces transcriptional activation in transfected cells (Lu et al., 1996).
I47L
protein
substitution
true negative
CORRIGENDUM An I47L substitution in the HOXD13 homeodomain causes a novel human limb malformation by producing a selective loss of function Caronia, G., Goodman, F.
Development 130, 1701-1712 2003 The Company of Biologists Ltd doi:10.1242/dev.00396 1701 DEVELOPMENT AND DISEASE An I47L substitution in the HOXD13 homeodomain causes a novel human limb malformation by producing a selective loss of function Giuliana Caronia1,*, Frances R.
We compared the HOXD13(I47L) mutant protein both in vitro and in vivo to the wild-type protein and to an artificial HOXD13 mutant, HOXD13(IQN), which is completely unable to bind DNA.
Using retrovirus-mediated misexpression in developing chick limbs, we showed that wild-type HOXD13 could upregulate chick EphA7 in the autopod, but that HOXD13(I47L) could not.
In the zeugopod, however, HOXD13(I47L) produced striking changes in tibial morphology and ectopic cartilages, which were never produced by HOXD13(IQN), consistent with a selective rather than generalised loss of function.
Intriguingly, both HOXD13(I47L) and HOXD13(IQN) produced more severe shortening in proximal limb regions than did wildtype HOXD13, suggesting that functional suppression of anterior Hox genes by more posterior ones does not require DNA binding and is mediated by protein:protein interactions.
We describe a six-generation family in which a unique combination of brachydactyly and central polydactyly co-segregates with a missense mutation in helix III of the HOXD13 homeodomain (I47L).
We have compared the functions of the HOXD13(I47L) mutant protein with those of wild-type HOXD13 and a HOXD13 mutant unable to bind DNA.
We show that the I47L substitution does not produce a dominant-negative effect or a gain of function, but instead impairs DNA binding at a subset of sites recognised by wild-type HOXD13, causing a selective loss of function.
Consistently, retrovirus-mediated overexpression of wild-type HOXD13 in the chick autopod upregulates chick EphA7, a putative downstream target of Hoxa13, but overexpression of HOXD13(I47L) does not.
To generate expression constructs for HOXD13 and HOXD13(I47L), the respective ORFs were cloned in frame with a hemagglutinin (HA) tag into the SV40-based vector pSG-5 (Stratagene).
Expression constructs for glutathione-Stransferase (GST)-homeodomain fusion proteins were produced by cloning restriction fragments containing the homeodomains of HOXD13 (HOXD13HD), HOXD13(I47L) [HOXD13HD(I47L)] and HOXD13(IQN) [HOXD13HD(IQN)] into the bacterial expression vector pGEX4T-1 (Amersham Biosciences).
Avian retroviral expression constructs containing HOXD13, HOXD13(I47L) and HOXD13(IQN) were generated by cloning the appropriate restriction fragments into the shuttle vector pSLAX13 and then subcloning into the retroviral vector pRCAS(BP)A (Morgan and Fekete, 1996).
Immunoblots of extracts from transfected cells showed that the expression constructs produced identical amounts of HOXD13 and HOXD13(I47L) (data not shown).
Electrophoretic mobility shift assays (EMSAs) Full-length HA-tagged HOXD13 and HOXD13(I47L) proteins were synthesised in vitro using the TNT-coupled transcription/translation system (Promega), diluted in 13 l of -buffer (20% glycerol, 20 mM KCl, 2 mM MgCl2, 0.2 mM EDTA, 0.5 mM DTT) and pre-incubated with 100 ng poly-(dI-dC) in a total volume of 20 l 1 binding buffer (0.1 M KCl, 2 mM MgCl2, 4 mM spermidine, 0.1 mg/ml BSA) for 15 minutes on ice.
GST-HOXD13HD, GSTHOXD13HD(I47L) and GST-HOXD13HD(IQN) fusion proteins were expressed in E.
DNA binding site selection assays Bacterially expressed, purified GST-HOXD13HD and GSTHOXD13HD(I47L) fusion proteins were loaded onto glutathioneSepharose 4B resin (Amersham Biosciences) and incubated with the SelD13 oligonucleotide pool in 1 binding buffer for 30 minutes at 4C.
Avian retrovirus production, microinjection, cartilage staining and whole-mount in situ hybridisation CEF cells were transfected by CaPO4 precipitation with 20 g of the RCAS-HOXD13, RCAS-HOXD13(I47L) or RCAS-HOXD13(IQN) retroviral constructs to generate virus stocks, which were harvested, concentrated and titrated on CEF cells (Morgan et al., 1992).
A new missense mutation (I47L) in the homeodomain of HOXD13 Direct sequencing of HOXD13 in the mother of the proband (V.7) revealed a heterozygous A-to-C transition in exon 2 at position 940 of the coding sequence.
The I47L mutation impairs HOXD13's ability to activate transcription at the HCR element No naturally occurring HOXD13 DNA-binding sites have been characterised, as none of the target genes of HOXD13 have yet been identified.
To examine whether the I47L substitution affects the ability of HOXD13 to control transcription, we used the HCR sequence (Fig.
To compare HOXD13(I47L) with a HOXD13 mutant that is completely unable to bind DNA, we also generated an artificial mutant, HOXD13(IQN), carrying alanine substitutions at positions 47(I), 50(Q) and 51(N) of the homeodomain.
We transiently co-transfected P19 cells with a luciferase reporter construct driven by the HCR sequence (pTHCR), together with increasing amounts of SV40driven constructs expressing HOXD13, HOXD13(I47L) or HOXD13(IQN).
Although HOXD13 increased basal reporter activity five- to sixfold, HOXD13(I47L) increased it only about Fig.
Transcriptional activity of A B HOXD13, HOXD13(I47L) and 600 800 HOXD13(IQN) at the HCR.
(A) Transcriptional activity mediated by increasing amounts of pSG-HOXD13, pSG200 HOXD13(I47L) or pSG200 HOXD13(IQN) assayed 100 separately.
(B) Transcriptional activity mediated by 1.0 g pSGHOXD13 in the presence of 0 0 increasing amounts of pSG1g D13 HOXD13(I47L) or pSG0.5 1.25 2.5 0.5 1.25 2.5 0.5 1.25 2.5 g 1 1.5 2 2.5 0.5 1 1.5 0.5 1 1.5 g HOXD13(IQN), with the activity D13 D13(I47L) D13(IQN) D13 D13(I47L) D13(IQN) mediated by increasing amounts of pSG-HOXD13 shown for C comparison.
Thus, the I47L substitution severely compromises the ability of HOXD13 to activate transcription through the HCR element, suggesting that it significantly impairs the capacity of the protein to bind DNA.
HOXD13(I47L) does not interfere with transcriptional activation by wild-type HOXD13 To examine whether HOXD13(I47L) can act as a dominant negative, we co-transfected P19 cells with fixed amounts of pTHCR and the HOXD13 expression construct, together with increasing amounts of the HOXD13(I47L) expression construct.
4B, the levels of reporter activity obtained with 1.0 g of HOXD13 together with 0.5, 1.0 or 1.5 g of HOXD13(I47L) were in each case slightly higher than that obtained with 1.0 g of HOXD13 alone, although substantially lower than those obtained with 1.5, 2.0 or 2.5 g of HOXD13 alone.
Thus transcriptional activation by wild-type HOXD13 is not significantly inhibited by co-expression of either HOXD13(I47L) or HOXD13(IQN), suggesting that neither mutant acts as a dominant negative.
The I47L mutation impairs the ability of HOXD13 to bind DNA To investigate whether the weak transcriptional activation mediated by HOXD13(I47L) at the HCR reflects defective DNA binding, we performed EMSAs using oligonucleotide probes derived from this element.
5A, lanes 3-5), HOXD13(I47L) bound more weakly, producing detectable complexes only at higher protein concentrations (Fig.
5B, lane 3), whereas HOXD13(I47L) bound only weakly (Fig.
We subsequently used bacterially expressed, purified GST fusions of the HOXD13 and HOXD13(I47L) homeodomains [GSTHOXD13HD and GST-HOXD13HD(I47L)] to determine their dissociation constants (Kd) at this site in EMSAs, obtaining Kd values of 5108 M and 3107 M respectively (data not shown).
Thus, HOXD13(I47L) fails to recognise at least some sites bound by the wild-type protein.
This suggests that the I47L mutation produces a loss of function, but leaves open the possibility that it produces a gain of function by changing binding site specificity.
We expected that a straightforward switch in binding specificity would result in equally efficient binding by HOXD13 and HOXD13 (I47L), as the different optimal binding sequences would be equally represented in the pool.
HOXD13(I47L) displays impaired DNAbinding ability in EMSAs.
(A) Binding of in vitro translated HOXD13 (lanes 3-6) and HOXD13(I47L) (lanes 7-10) to 32P-labelled HCR (lane 1).
(B) Binding of in vitro translated HOXD13 (lane 3) and HOXD13(I47L) (lane 4) to 32P-labelled HCR (lane 1).
(C) Binding of bacterially expressed, purified GSTHOXD13HD (lanes 2-6), GST-HOXD13HD(I47L) (lanes 711) and GST-HOXD13HD(IQN) (lanes 12-16) to 32P-labelled SelD13 (lane 1).
with increasing amounts of the purified GST-homeodomain fusion proteins, GST-HOXD13HD bound efficiently at all concentrations tested (lanes 2-6) whereas GSTHOXD13HD(I47L) bound only at higher concentrations (lanes 7-11).
The intermediate binding levels seen with HOXD13HD(I47L) are not consistent with a straightforward switch in binding specificity, but could have occurred either because the mutant protein binds more weakly than the wild-type protein at all sites in the pool, or because the mutant binds a smaller subset of sites in the pool.
HOXD13(I47L) binds a subset of the sites recognised by HOXD13 To distinguish between these possibilities, we performed binding site selection assays with purified GSTHOXD13HD and GST-HOXD13HD(I47L) using the SelD13 oligonucleotide pool.
By contrast, HOXD13(I47L) recognised a more restricted set of sites.
HOXD13(I47L) therefore appears to recognise one of the two sites selected by wild-type HOXD13 (site 2), while almost completely failing to recognise the other (site 1) (Fig.
HOXD13(I47L) bound the TTAT-containing probe much more weakly than the wild- Selective loss-of-function mutation in HOXD13 1707 A HOXD13 Site 1 TTTTATTGG Core TTAT (50) (48) TTTTATT TTTTATTG (43) (39) Site 2 TTT(T/A)ACGAG Core TTAC (50) (32) TTTTACG TTTTACGA TTTTACGAG Core TAAC (18) TTTAACGAG (29) (28) (25) (17) TTTTATTGG (36) Core TAAT (2) HOXD13(I47L) Site 1 TTTTATTGG Core TTAT (3) (1) Site 2 TTT(T/A)ACGAG Core TTAC (93) (73) TTTTACG TTTTACGA TTTTACGAG Core TAAT (2) Core TAAC (20) TTTAACGAG (59) (52) (47) (11) B HOXD13 site 1 100% 80% 60% 40% 20% 0% 1 2 3 4 5 6 7 8 9 HOXD13 site 2 AC 100% HOXD13(I47L) C T 100% 80% 60% A A 80% 60% A A A A T T T T T A T T GG 40% 20% 0% T T T T A C GAG 40% 20% 0% T T T T A C GAG 1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8 9 Fig.
HOXD13HD(I47L) binds a subset of the sites recognised by HOXD13HD.
Those selected by HOXD13HD fell into two equalsized groups, one with a TTAT core (site 1) and the other with a T(T/A)AC core (site 2), whereas those selected by HOXD13HD(I47L) formed a single group with a T(T/A)AC core.
(C) Binding of GST-HOXD13HD and GST-HOXD13HD(I47L) to site 1 (TTATTGG, lanes 1-7) and the two variants of site 2 (TTACGAG, lanes 8-14, and TAACGAG, lanes 15-21).
Thus, the I47L mutation does not result in recognition of a novel DNA-binding sequence.
Misexpression of HOXD13(I47L) in developing chick limbs does not affect the digits but produces more severe proximal abnormalities than HOXD13 To explore the effects of the I47L mutation further in vivo, we used the developing chick limb, a well-established model system for studying limb development in vertebrates.
Caronia and others full-length HOXD13, HOXD13(I47L) or HOXD13(IQN), and injected concentrated retroviral suspensions of comparable titres into the prospective right hindlimb field of stage 10 chick embryos in ovo.
Embryos misexpressing HOXD13(I47L) (Fig.
Phenotypes produced by misexpressing HOXD13, HOXD13(I47L) and HOXD13(IQN) in developing chick limbs.
(B,C) Embryos injected with RCAS-HOXD13(I47L), showing normal phalanges (the embryo in C was the only one out of 38 with a rudimentary extra digit (asterisked), but marked shortening of the proximal cartilages, particularly the tibia, which has a rounded morphology, and extra cartilages in the zeugopod (arrowheads).
Cartilage shortening in chick hindlimbs injected with RCAS-HOXD13, RCAS-HOXD13(I47L) or RCAS-HOXD13(IQN) Zeugopod Mutation D13 (n=30) D13(I47L) (n=38) D13(IQN) (n=102) Stylopod 0.780.09 0.720.11 0.700.10 Tibia 0.700.14 0.540.14 0.630.13 Fibula 0.830.28 0.720.14 0.780.22 Autopod Tarsometatarsals 0.870.24 0.800.12 0.720.14 Phalanges 1 1 1 Figures represent the ratio of cartilage lengths in the injected limb to the corresponding cartilage lengths in the uninjected opposite limb.
7D,E) again had no phalangeal abnormalities, but the proximal cartilages, especially the femur and tibia, were more severely shortened than in embryos misexpressing HOXD13, and the metatarsals were shorter than in embryos misexpressing either HOXD13 or HOXD13(I47L) (Table 1).
Overexpression of HOXD13 upregulates EphA7, while overexpression of HOXD13(I47L) does not In Hoxa13/ mice, EphA7 expression is significantly reduced in the condensing mesenchyme of the digits, carpals and tarsals, but not completely absent, suggesting that this ephrin receptor is a downstream target not only of Hoxa13 but perhaps also of other Hox proteins expressed in the developing autopod, like Hoxd13 (Stadler et al., 2001).
To investigate whether HOXD13 controls EphA7 expression, and, if so, whether the I47L mutation affects this activity, we analysed chick EphA7 expression in chick limbs overexpressing HOXD13, HOXD13(I47L) or HOXD13(IQN).
Overexpression of HOXD13(I47L) or HOXD13(IQN), however, had no effect upon EphA7 levels (Fig.
Thus, while HOXD13, like Hoxa13, upregulates EphA7 expression in the autopod, neither HOXD13(I47L) nor HOXD13(IQN) retains this activity.
Overexpression of HOXD13 upregulates expression of chick EphA7, but overexpression of HOXD13(I47L) does not.
Wholemounts of stage 28 chick embryos expressing HOXD13, HOXD13(I47L) or HOXD13(IQN) in the right hindlimb.
This expression is increased in the right limbs overexpressing HOXD13 (B), but unaltered in limbs overexpressing HOXD13(I47L) (D) or HOXD13(IQN) (F).
No I47L substitution, however, has yet been analysed.
HOXD13(I47L) exhibits a selective loss of DNAbinding ability Previous studies have shown that the most 5 Hox protein in Drosophila, Abd-B, selects sites with a TTAT core (Ekker et al., 1994), whereas the 5 vertebrate Hox proteins select sites with a TTAT or TTAC core (Benson et al., 1995; Shen et al., 1997).
HOXD13(I47L) selected only the second of these sites (TTT(T/A)ACGAG), perhaps reflecting the inability of L47 to make the contacts normally made by I47 with a T residue in the fourth core position.
Consistent with these findings, HOXD13(I47L) bound the TTTTACGAG and TTTAACGAG sequences with the same affinity as wild-type HOXD13 in EMSAs, but displayed a significantly lower affinity for the TTTTATTGG sequence.
The impaired capacity of HOXD13(I47L) to recognise TTAT- and TAAT-containing sites was further confirmed using the HCR element, which contains multiple TTAT and TAAT motifs bound by several 5 HOXD proteins, but only one TTAC motif (Zappavigna et al., 1991).
Although HOXD13 bound strongly and specifically to several of these sites in EMSAs, HOXD13(I47L) bound only weakly, with an affinity for one of them (HCR I, TTTTATTAG) about sixfold lower than that of the wild-type protein.
Moreover, while HOXD13 activated transcription through the full-length HCR, HOXD13(I47L) did not.
These observations suggest that HOXD13(I47L) is unable to regulate some of the genes regulated by wild-type HOXD13.
HOXD13 upregulates EphA7 expression but HOXD13(I47L) does not Signalling between Eph receptors and their ephrin ligands plays a key role in regulating many developmental processes and is probably a downstream effector of several 3 Hoxa and Hoxb genes [reviewed in (Frisen et al., 1999)].
Misexpression of HOXD13(I47L), however, had no effect on chick EphA7 levels, showing that the I47L mutation indeed impairs the capacity of HOXD13 to regulate one of its downstream targets.
Although no other HOXD13 targets have yet been identified, our finding that HOXD13(I47L) retains the ability in vitro to bind some sites bound by HOXD13 suggests that it can still regulate at least some of these targets.
Misexpression of HOXD13(I47L) in chick limbs produces a phenotype both quantitatively and qualitatively different to that produced by HOXD13 To analyse the consequences of the I47L substitution in vivo, we used retrovirus-mediated expression in the developing chick limb.
When overexpressed in the phalanges, where Hoxd13 is normally expressed, HOXD13(I47L), like wild-type HOXD13 (Goff and Tabin, 1997) (this work) and HOXD13(IQN), caused no defects.
Thus, HOXD13(I47L) does not interfere with the functions of endogenous Hoxd13 in vivo, or with distal autopod development, indicating that it does not act by a dominant-negative or other gain-of-function mechanism.
Consistently, HOXD13(I47L) did not interfere with transcriptional activation by HOXD13 at the HCR element in transiently transfected cells, and showed no gain or switch in DNA-binding activity in vitro.
In proximal limb regions, however, whereas HOXD13 caused only mild shortening of the long bone cartilages, HOXD13(I47L) produced severe shortening, as well as striking abnormalities of zeugopod morphology, including a change in the shape of the tibia from long to rounded cartilage and the formation of ectopic cartilages.
Misexpression of HOXD13(IQN) likewise caused severe shortening of the proximal cartilages, but never produced the abnormal zeugopod morphology observed with HOXD13(I47L).
The phenotype caused by HOXD13(I47L) in proximal limb regions Selective loss-of-function mutation in HOXD13 1711 is thus qualitatively as well as quantitatively different from that produced by wild-type HOXD13, but is also qualitatively different from that produced by HOXD13(IQN), which is completely unable to bind DNA.
This further strengthens the hypothesis that the I47L substitution results in a selective rather than a generalised loss of function.
The additional zeugopod abnormalities caused by HOXD13(I47L) probably reflect its ability to control only a subset of the genes normally controlled by HOXD13, leading to an imbalance in the regulation of downstream targets.
Strikingly, we found that the HOXD13(I47L) and HOXD13(IQN) mutants, the DNA-binding abilities of which are selectively and generally (respectively) impaired, caused more severe shortening of proximal limb cartilages than wildtype HOXD13.
9813065
full text
K31Q
protein
substitution
true negative
However, a mutation within the N-terminal calmodulin binding domain, W30A/K31Q, which caused a 10-fold decrease in affinity of this site for calmodulin, had no effect on calmodulin inhibition of rhodopsin phosphorylation by GRK5 (18).
Similarly, the W30A/K31Q mutation had no effect on calmodulin-stimulated autophosphorylation of GRK5 (Fig.
W30A
protein
substitution
true negative
However, a mutation within the N-terminal calmodulin binding domain, W30A/K31Q, which caused a 10-fold decrease in affinity of this site for calmodulin, had no effect on calmodulin inhibition of rhodopsin phosphorylation by GRK5 (18).
Similarly, the W30A/K31Q mutation had no effect on calmodulin-stimulated autophosphorylation of GRK5 (Fig.
10202147
full text
Y315F
protein
substitution
true positive
P43403
Introduction of a ZAP(Y315F) mutant into wild-type Jurkat cells inhibits several TCR-dependent responses, including the tyrosine phosphorylation of Vav and the transcription of an NFAT-dependent reporter gene.
Furthermore, while ectopic expression of wild-type ZAP70 reconstitutes B-cell antigen receptor (BCR) signaling functions in Syk/ chicken B-cells, the ZAP(Y315F) mutant is almost completely defective in this model system.
In these experiments, P116 cells, which are deficient in both ZAP-70 and Syk (Williams et al., 1998), were transiently transfected with a pNFAT-Luc reporter plasmid, together with wild-type ZAP-70, ZAP(Y315F) or ZAP(Y319F).
Unexpectedly, the ZAP(Y315F) mutant, which lacks the major binding site for Vav (Wu et al., 1997), also reconstituted TCR-dependent NFAT activation in P116 cells.
P116 cells were transfected with 10 g plasmid DNA encoding no protein (mock), wild-type ZAP-70 (WT), ZAPY315F (Y315F), ZAP-Y319F (Y319F) or ZAP-YYFF (YYFF).
P116 cells were transfected with wild-type ZAP-70 (P116.WT-1), ZAP(Y319F) (Y319F) or ZAP(Y315F) (Y315F).
Although the autokinase activity of the ZAP(Y315F) mutant was also increased in response to TCR ligation, both the basal and stimulated kinase activities were consistently lower than those observed in samples containing equivalent levels of wildtype ZAP-70 (also see Figure 3B).
Moreover, in spite of the reduction in autokinase activity induced by the Tyr315Phe substitution, the ZAP(Y315F) mutant retains substantial signaling activity when expressed in P116 cells.
In contrast, the PTK activity of ZAP(Y315F) isolated from unstimulated or pervanadate-stimulated cells was reproducibly lower than those obtained with the corresponding wild-type ZAP-70 and ZAP(Y319F) immunoprecipitates.
The results demonstrate that TCR stimulation provokes comparable increases in the tyrosine phosphorylation of the wild-type ZAP-70, ZAP(Y319F) and ZAP(Y315F) proteins.
Once again, the ZAP(Y315F) mutant restored the normal increase in intracellular Ca2 in the activated cells, indicating that this response is not dependent on the phosphorylation of Tyr315 in ZAP-70.
This PLC1 phosphorylation defect was corrected by reconstitution of P116 cells with either wild-type ZAP-70 or the ZAP(Y315F) mutant.
Stable expression of either wild-type ZAP-70 or ZAP(Y315F) in P116 cells reversed the defects in TCR-dependent SLP-76 and LAT tyrosine phosphorylation.
The ZAP(Y315F) mutant supported a similar increase in PLC-1 phosphorylation when co-expressed with Lck.
Interestingly, expression of either of the ZAP-70 interdomain B region mutants, ZAP(Y319F) or ZAP(Y315F), also reversed the ERK activation defect in P116 cells.
A comparison of the ZAP(Y319F) and ZAP(Y315F) single mutants revealed that the ability of this PTK to restore TCRPLC-1 coupling in P116 cells was exclusively dependent on the integrity of the Tyr319 site.
The functional capabilities of the ZAP(Y315F) single mutant in P116 cells were unexpected, based on earlier evidence that Tyr315 is an important positive regulatory site in ZAP-70 (Wu et al., 1997).
We have confirmed that overexpression of ZAP(Y315F) in the parental (ZAP-70 positive) Jurkat cell line partially inhibits the increase in NFAT activity provoked by TCR crosslinkage (B.L.Williams and R.T.Abraham, unpublished observations).
Similarly, while introduction of wild-type ZAP-70 fully restored BCRmediated Ca2 mobilization and NFAT activation in Syk/ B cells, the ZAP(Y315F) mutant displayed severe defects in this model system (Wu et al., 1997).
Nonetheless, we observed that expression of ZAP(Y315F) in ZAP-70-deficient P116 cells fully restores several TCRdependent signaling events, including PLC-1 tyrosine phosphorylation, intracellular Ca2 mobilization and NFAT activation.
Similar results were obtained by Acuto and colleagues, who reported that overexpression of the ZAP(Y315F) mutant failed to interfere with either Vav tyrosine phosphorylation or IL-2 production in activated T-hybridoma cells (Michel et al., 1998).
The explanation for the conflicting results obtained with the ZAP(Y315F) mutant in different B- and T-cell lines remains unclear.
On the one hand, the dysfunctional state of ZAP(Y315F) in Syk/ B cells may be attributable to fundamental differences in the regulation of substrate phosphorylation by BCR-associated PTKs, such that Tyr315 in ZAP-70 is crucial for the activation of PLC-1 and other downstream signaling proteins in B cells.
On the other hand, the ZAP(Y315F) mutant may function as a partial antagonist of TCR signaling in settings where the mutant PTK is greatly overexpressed relative to the endogenous, wildtype ZAP-70.
Mutations of Tyr315 and and/or Tyr319 to phenylalanine were introduced into the pcDNA3-mZAP plasmid by site-directed mutagenesis using the Transformer kit (Clontech, Palo Alto, CA) and the following primers (mutagenic codons are underlined): GACACGAGCGTGTTTGAGAGCCCCTAC (Y315F); TATGAGAGC CCCTTCAGCGACCCAGAG (Y319F); GACACGAGCGTGTTTGAGAGCCCCTTCAGCGACCCAGAG (Y315,319F double mutant).
The resulting plasmids were designated pcDNA3-ZAP-Y315F, pcDNA3-ZAP-Y319F and pcDNA3ZAP-YYFF, respectively.
For stable expression of the ZAP(Y315F) and ZAP(Y319F) proteins, the transfected cells were selected in standard growth medium containing 2 mg/ml of G418, and the drug-resistant bulk populations were cloned by limiting dilution.
The amounts of the individual expression plasmids used in cotransfections were as follows: AU1-PLC-1, 10 g; wild-type Myc-ZAP-70, 5 g; Myc-ZAP (Y315F), 10 g; Myc-ZAP(Y319F), 10 g; Myc-ZAP-KD (catalytically inactive mutant), 25 g; Myc-Lck, 5 g.
ZAP-70 kinase assay P116 cells were transiently transfected with Myc epitope-tagged versions of wild-type ZAP-70, ZAP(Y315F), or ZAP(Y319F) expression 1842 Role of Tyr319 phosphorylation in ZAP-70 signaling plasmids.
Y319F
protein
substitution
true positive
P43403
Although mutation of Tyr319 to Phe had no effect on the tyrosine kinase activity of ZAP-70, the resulting ZAP(Y319F) mutant failed to reconstitute TCRdependent Ca2 mobilization, Ras activation, CD69 expression and NFAT-dependent transcription in ZAP70-deficient Jurkat cells.
Y292F
protein
substitution
true positive
P43403
Substitution of Tyr292 with Phe generates an activated ZAP-70 mutant [ZAP(Y292F)] that, when expressed in Jurkat T-leukemic cells, drives a NFAT-mediated transcriptional response in the absence of TCR ligands (Kong et al., 1996; Zhao and Weiss, 1996).
11040941
full text
D816V
protein
substitution
true positive
P10721
Clinical correlates of the presence of the Asp816Val c-kit mutation in the peripheral blood mononuclear cells of patients with mastocytosis.
Analysis of the surface expression of c-kit and occurrence of the c-kit Asp816Val activating mutation in T cells, B cells, and myelomonocytic cells in patients with mastocytosis.
10433944
full text
V1206L
protein
substitution
true positive
P08581-2
M1149T
protein
substitution
true positive
P08581-2
H1112R
protein
substitution
true positive
P08581-2
Y1248D
protein
substitution
true positive
P08581-2
M1268T
protein
substitution
true positive
P08581-2
Y1248C
protein
substitution
true positive
P08581-2
V1110I
protein
substitution
true positive
P08581-2
H1124D
protein
substitution
true positive
P08581-2
10861237
full text
W666A
protein
substitution
true negative
(2000) 349, 261266 (Printed in Great Britain) 261 A single amino acid substitution (Trp666 Ala) in the interbox1/2 region of the interleukin-6 signal transducer gp130 abrogates binding of JAK1, and dominantly impairs signal transduction Claude HAAN, Heike M.
9398735
full text
V804M
protein
substitution
P07949
true positive
In addition, three mutations in the intracellular domain of the protein have been described, one in exon 13, Glu768Asp, and two in exon 14, Val804Leu and Val804Met (8 10).
V804L
protein
substitution
P07949
true positive
In addition, three mutations in the intracellular domain of the protein have been described, one in exon 13, Glu768Asp, and two in exon 14, Val804Leu and Val804Met (8 10).
E768D
protein
substitution
P07949
true positive
In addition, three mutations in the intracellular domain of the protein have been described, one in exon 13, Glu768Asp, and two in exon 14, Val804Leu and Val804Met (8 10).
S891A
protein
substitution
P07949
true positive
Solid symbols indicate the individuals harboring the Ser891Ala mutation and affected with MTC; hatched symbols indicate gene mutation carriers; open symbols indicate unaffected individuals.
Shown is the relevant part of the sequence containing the Ser891Ala mutation.
M918T
protein
substitution
P07949
true positive
For MEN 2B, a single mutation, Met918Thr, has been found in the intracellular domain in 95% of cases (4 7).
10515866
full text
E665K
protein
substitution
true positive
Q62120
The site of the E665K mutation within JH2 that hyperactivates the catalytic activity31 of murine Jak2 and D melanogaster HOP is marked with a solid arrowhead.
11729154
full text
15769897
full text
I836del
protein
substitution
true positive
P36888
Although the presence of FL had hardly any effect on PKC412 sensitivity of cells expressing Flt3-D835Y and D835H, surprisingly, the presence of FL could provide a protective role on Flt3-I836delexpressing cells (Figure 7E).
Because the inhibitory mechanisms of PKC412 are not known at present, it is not clear from our data how FL partially rescued Flt3-I836del cells.
D835Y
protein
substitution
true positive
P36888
The mutation to D835Y or D835H or I836 was introduced into the previously described Flt3-WT or Flt3-ITD (NEYFYVDFREYE) cDNA.24 All mutations were confirmed by DNA sequencing.
Generation of the Flt3-expressing 32D cell lines Construction of human Flt3-WT, Flt3-ITD, and Flt3-TKD mutant expressing 32D cell lines was essentially performed as described previously.6 Several bulk cultures were generated for Flt3-D835Y mutant, 2 bulk cultures D835Y-1 and D835Y-2 were used as indicated; the D835Y-1 was Patients, materials, and methods The use of human material for scientific purposes was approved by the human ethics committee of each participating institution.
Results Surface expression and phosphorylation analysis of Flt3-WT, Flt3-D835Y, and Flt3-ITD mutants Figure 1.
32D cells were transfected with constructs encoding either Flt3-WT or Flt3-D835Y or Flt3-ITD.
The results The substitution D835Y is the most frequently found Flt3-TKD mutation in AML.
Therefore, we first compared the transforming properties of Flt3-ITD and Flt3-WT versus Flt3-D835Y that we stably expressed in 32D cells.
Pools of 32D cells expressing either Flt3-WT, Flt3-ITD, or Flt3-D835Y were generated as described previously.6,27 Flow cytometric analyses revealed equal surface expression of all receptor isoforms (Figure 1A).
Western blot analyses of Flt3 immunoprecipitates for phosphotyrosine revealed strong ligand-independent autophosphorylation of Flt3-ITD and Flt3-D835Y after a starvation period of the cells from IL-3 and serum for 12 hours.
Therefore, Flt3-ITD and Flt3-D835Y display comparable levels of constitutive Flt3 autophosphorylation.
Differential transforming potentials of Flt3-D835Y and Flt3-ITD mutant receptors It was reported that both Flt3-ITD and Flt3-TKD mutations confer factor-independent growth to IL-3dependent hematopoietic cells Figure 2.
Factor-independent growth of 32D cells expressing either Flt3-D835Y or Flt3-ITD.
(A) Ligand-independent 3H-thymidine incorporation by Flt3-ITD and Flt3-D835Y mutants.
3H-thymidine incorporation was analyzed in the presence of the tyrosine kinase inhibitor SU11248 in the presence of FCS only (Flt3-ITD, Flt3-D835Y) or FCS and FL (Flt3-WT).
Both Flt3-ITD and Flt3-D835Y mutations induce resistance to radiation-induced apoptosis of 32D cells Next, we analyzed whether Flt3-D835Y provides resistance against radiation-induced apoptosis in 32D cells.
Flt3-D835Y does not induce clonogenic growth of 32D cells in semisolid media.
(B) Low-power photograph of representative areas of the plates demonstrates the lack of colony growth in 32D/Flt3-D835Y in the absence of IL-3.
in vitro.12,16 Here, we directly compared the growth properties of 32D cells expressing either Flt3-WT, Flt3-ITD, or Flt3-D835Y.
Transfection of 32D cells either with Flt3-ITD or Flt3-D835Y led to factor-independent growth in suspension cultures, whereas 32D-Flt3-WT cells remained factor-dependent and proliferated only in the presence of FL (Figure 2A-B).
In comparison to Flt3-ITD, Flt3-D835Y induced significantly less pronounced autonomous growth of the cells, both in short-term (Figure 2A) as well as in long-term growth assays (Figure 2B).
Cells expressing Flt3-ITD receptors readily formed colonies in semisolid media, regardless of the presence or absence of growth factors (Figure 3), as we have previously reported.6 To our surprise, 32D-Flt3-D835Y cells were unable to form colonies in semisolid media, even in presence of FL.
This is reminiscent of the effects of Flt3-WT, which also was unable to support colony growth in presence of FL (Figure 3A).6 However, we repeatedly observed that Flt3-D835Y Figure 4.
Expression of Flt3-D835Y and Flt3-ITD induces radiation-induced apoptosis resistance in myeloid cells.
The observed differences of WT versus ITD versus D835Y (left) and ITD versus WT and D835Y (middle) were statistically significant (P .01).
In the absence of FL, Flt3-D835Y only partially protected 32D cells from apoptotic death following irradiation.
These results again corroborated distinct biologic effects of Flt3-ITD and Flt3-D835Y.
For direct comparison, 32D cells expressing Flt3-WT, Flt3-ITD, or Flt3-D835Y were starved from IL-3 and serum for 12 hours, and cell lysates were prepared and subjected to immunoblotting using activation-specific antibodies against signal transduction intermediates.
As shown in Figure 5A-B, we observed constitutive activation of Erk-1 and Erk-2, Akt, and of Shc by both Flt3-ITD and Flt3-D835Y.
Flt3-D835Yexpressing cells induced a somewhat weaker phosphorylation of Erk-1 and Erk-2, but constitutive phosphorylation of Shc and Akt was comparable with the Flt3-ITDexpressing cells.
Collectively, these results demonstrate that both the ITD and D835Y mutations induce constitutive activation of Flt3 signaling pathways.
Cells were grown for 36 hours in the absence of IL-3 and with (Flt3-WT) or without (Flt3-D835Y and Flt3-ITD) FL in medium supplemented with 10% FCS.
(C) Differential activation of STAT5, but not of Erk, by ITD versus D835Y in primary AML samples.
D835Y marginally activates STAT5 and weakly induces STAT5 downstream targets Figure 5.
Constitutive activation of MAP kinase, Akt, and Shc by Flt3-ITD and Flt3-D835Y.
In contrast, STAT5 was only marginally activated by Flt3-D835Y in the absence (Figure 6A) or presence of FL (data not shown).
The STAT5 target gene CIS, a member of the suppressor of cytokine signaling (SOCS) protein family, was highly induced by Flt3-ITD, whereas expression of Flt3-D835Y was followed by weak induction of CIS.
The serine-threonine kinase Pim-2 is a functionally relevant downstream target of STAT5.24 Here, we 270 CHOUDHARY et al BLOOD, 1 JULY 2005 VOLUME 106, NUMBER 1 observed only a weak induction of Pim-2 by Flt3-D835Y compared to the effects of Flt3-ITD (Figure 6A).
Taken together, Flt3-D835Y induced a very weak but measurable activation of STAT5, as analyzed by STAT phosphorylation and induction of STAT5 target genes.
Interestingly, STAT5 protein expression was lost in 2 of 5 analyzed samples expressing Flt3-D835Y and it was quite weak in another sample.
However, this effect was not observed in cells expressing Flt3-D835Y or Flt3-WT in the presence of FL.
The effects of D835Y are recapitulated by other Flt3-TKD mutations Recently, we have shown that Flt3-ITD mutations regulate the expression and function of c/EBP and Pu.1.24 We now compared their regulation by Flt3-ITD, Flt3-D835Y, and Flt3-WT.
The presence of Flt3-ITD led to suppression of both Although D835Y is the most frequent AML-associated mutation found in the TKD of Flt3, several other activating mutations were reported in this region including other substitutions of D835 and small deletion or insertion mutations.12-15 To analyze the signal transduction properties of ITD and TKD mutations and gain insight into the effects of Flt3-TKD mutations as a class, we introduced 2 additional mutations into Flt3, D835H and I836, and expressed both constructs in 32D cells.
However, similar to Flt3-D835Y, the other TKD mutations induced a less pronounced proliferative response in suspension cultures and also failed to support 32D clonogenic growth (data not shown).
BLOOD, 1 JULY 2005 VOLUME 106, NUMBER 1 SIGNALING PROPERTIES OF Flt3-ITD VERSUS Flt3-TKD 271 found a striking resemblance of the signaling properties of D835Y that was in contrast to Flt3-ITD (Figure 7C), including the lack of significant effects of the TKD mutations on STAT5 activation or repression of myeloid transcription factors c/EBP and Pu.1 (Figure 7C).
Interestingly, 32D/Flt3-D835Y cells were much more sensitive toward PKC412 than 32D/Flt3-ITD or 32D/Flt3-WT cells (Figure 7D).
The higher sensitivity of Flt3-TKD mutants toward PKC412 in comparison to Flt3-ITD was not merely due to a "weaker" signal emanating from the Flt3-D835Y mutant.
Also, we constructed a doublemutant receptor containing both an ITD-mutation and the D835Y substitution and included this receptor in our analyses.
Although the presence of FL had hardly any effect on PKC412 sensitivity of cells expressing Flt3-D835Y and D835H, surprisingly, the presence of FL could provide a protective role on Flt3-I836delexpressing cells (Figure 7E).
Our observation of a very weak activation of STAT-dependent signal transduction by Flt3-TKD is in contrast to some previous reports where Flt3-D835Y activated STAT5 similar to Flt3-ITD.
Also, in primary AML samples we found activation of the Erk kinase pathways by both ITD and D835Y mutant receptors, whereas STAT5 was preferentially activated by FLt3-ITD.
However, the lack of STAT5 activation by Flt3-D835Y is surprisingly consistent with our data in 32D cells as well as with concurrently published data by Grundler et al43 that show similar differences in the ability of Flt3-TKD and Flt3-ITD mutations to activate STAT5 in primary mouse bone marrow and Ba/F3 cells.
Acknowledgments The authors are grateful to Justus Duyster, MD, Technical University of Munich, Munich, Germany, for providing us with cDNA constructs encoding murine Flt3 D835Y and Flt3-ITD.
D835H
protein
substitution
true positive
P36888
The mutation to D835Y or D835H or I836 was introduced into the previously described Flt3-WT or Flt3-ITD (NEYFYVDFREYE) cDNA.24 All mutations were confirmed by DNA sequencing.
The presence of Flt3-ITD led to suppression of both Although D835Y is the most frequent AML-associated mutation found in the TKD of Flt3, several other activating mutations were reported in this region including other substitutions of D835 and small deletion or insertion mutations.12-15 To analyze the signal transduction properties of ITD and TKD mutations and gain insight into the effects of Flt3-TKD mutations as a class, we introduced 2 additional mutations into Flt3, D835H and I836, and expressed both constructs in 32D cells.
When we analyzed the signaling properties of D835H and I836, we also Figure 7.
Although the presence of FL had hardly any effect on PKC412 sensitivity of cells expressing Flt3-D835Y and D835H, surprisingly, the presence of FL could provide a protective role on Flt3-I836delexpressing cells (Figure 7E).
9826715
full text
D1246H
protein
substitution
true positive
P08581-2
foci g DNA) TRK-MET construct* Control Wild type M1268T L1213V Y1248H D1246H NGF ( ) 8 13 300 74 105 68 NGF ( ) 5 300 300 300 300 300 Tumorigenicity No.
mice injected 0 0 4 4 5 4 5 5 4 4 5 4 Mean tumor size in mm2 0 0 245 133 257 99 *The control construct is the empty pMex expression vector; the wild-type construct encodes murine Met; M1268T, L1213V, Y1248H, and D1246H encode mutationally activated Met.
Nevertheless, the relative biolog- ical activity of the individual mutations remains unchanged in the context of Trk-Met (Table 2) or nonchimeric Met (15) (i.e., M1268T Y1248H D1246H).
L1213V
protein
substitution
true positive
P08581-2
foci g DNA) TRK-MET construct* Control Wild type M1268T L1213V Y1248H D1246H NGF ( ) 8 13 300 74 105 68 NGF ( ) 5 300 300 300 300 300 Tumorigenicity No.
mice injected 0 0 4 4 5 4 5 5 4 4 5 4 Mean tumor size in mm2 0 0 245 133 257 99 *The control construct is the empty pMex expression vector; the wild-type construct encodes murine Met; M1268T, L1213V, Y1248H, and D1246H encode mutationally activated Met.
(C) A representative ``scattered'' (motile) colony induced by mutationally activated Met (L1213V).
M1268T
protein
substitution
true positive
P08581-2
We found that cells expressing mutationally activated Met (M1268T) are highly metastatic in this assay, creating a severe lung metastasis burden in five of five animals sacrificed 34 weeks after injection; in contrast, cells expressing wild-type Met are much less metastatic, inducing a low metastatic burden in three of nine animals sacrificed 79 weeks after injection (Table 1).
Metastatic activity of mutationally activated Met in NIH 3T3 cells Metastasis Met construct* Control Wild type M1268T No.
mice injected 05 39 55 *The control construct is the empty pMex expression vector; the wild-type construct encodes murine Met: M1268T encodes mutationally activated murine Met.
Animals were sacrificed after 79 weeks (control and wild type) or 34 weeks (M1268T) and examined for lung metastasis.
foci g DNA) TRK-MET construct* Control Wild type M1268T L1213V Y1248H D1246H NGF ( ) 8 13 300 74 105 68 NGF ( ) 5 300 300 300 300 300 Tumorigenicity No.
mice injected 0 0 4 4 5 4 5 5 4 4 5 4 Mean tumor size in mm2 0 0 245 133 257 99 *The control construct is the empty pMex expression vector; the wild-type construct encodes murine Met; M1268T, L1213V, Y1248H, and D1246H encode mutationally activated Met.
Our findings indicate that each of the three Met mutants examined generates a significantly higher percentage of scattered colonies than does wild-type Met, with the most activating Met mutation (M1268T) exhibiting a 3-fold increase.
We suspect that the relatively low percentage of colonies exhibiting the scattered phenotype after transfection with even the most strongly activated Met molecule (21% with Met mutant M1268T) may be due to the low percentage of G418-resistant colonies expressing exogenous Met after cotransfection.
To this end, two independent, strongly activating mutations (M1268T and Y1248H) were expressed in transgenic mice under the control of the metallothionein promoter (24).
Founder animals harboring each metallothioneinmutant Met construct were selected and mated with nontransgenic partners (two males and two females for construct Y1248H; two males and four females for construct M1268T).
However, two 10-month-old female founder animals, one expressing Met mutant M1268T and the other expressing mutant Y1248H, developed overt tumors that were diagnosed as type B mammary adenocarcinomas (Fig.
4 C F, respectively), and cecum (not shown), and the tumor from the M1268T founder metastasizing to the lung (not shown).
One male from each construct and one female from construct M1268T exhibited no significant pathology, while one female from construct M1268T exhibited mammary hyperplasia (not shown).
Tumorigenic activity of mutationally activated Met in NIH 3T3 cells under the control of the metallothionein promoter Tumorigenicity MET construct* Wild type M1268T Y1248H No.
The experimental metastasis assay we used is thought to *The wild-type construct encodes murine Met; M1268T and Y1248H encode mutationally activated murine Met.
Nevertheless, the relative biolog- ical activity of the individual mutations remains unchanged in the context of Trk-Met (Table 2) or nonchimeric Met (15) (i.e., M1268T Y1248H D1246H).
Y1248H
protein
substitution
true positive
P08581-2
foci g DNA) TRK-MET construct* Control Wild type M1268T L1213V Y1248H D1246H NGF ( ) 8 13 300 74 105 68 NGF ( ) 5 300 300 300 300 300 Tumorigenicity No.
mice injected 0 0 4 4 5 4 5 5 4 4 5 4 Mean tumor size in mm2 0 0 245 133 257 99 *The control construct is the empty pMex expression vector; the wild-type construct encodes murine Met; M1268T, L1213V, Y1248H, and D1246H encode mutationally activated Met.
To this end, two independent, strongly activating mutations (M1268T and Y1248H) were expressed in transgenic mice under the control of the metallothionein promoter (24).
Founder animals harboring each metallothioneinmutant Met construct were selected and mated with nontransgenic partners (two males and two females for construct Y1248H; two males and four females for construct M1268T).
However, two 10-month-old female founder animals, one expressing Met mutant M1268T and the other expressing mutant Y1248H, developed overt tumors that were diagnosed as type B mammary adenocarcinomas (Fig.
Moreover, both of these tumors exhibited metastasis, with the tumor derived from the Y1248H founder metastasizing to the lung, lymph node, kidney, heart (Fig.
Tumorigenic activity of mutationally activated Met in NIH 3T3 cells under the control of the metallothionein promoter Tumorigenicity MET construct* Wild type M1268T Y1248H No.
The experimental metastasis assay we used is thought to *The wild-type construct encodes murine Met; M1268T and Y1248H encode mutationally activated murine Met.
Nevertheless, the relative biolog- ical activity of the individual mutations remains unchanged in the context of Trk-Met (Table 2) or nonchimeric Met (15) (i.e., M1268T Y1248H D1246H).
All samples shown are from a female founder expressing Met mutant Y1248H.
9950359
full text
P250R
protein
substitution
true positive
P22607
Downloaded from jmg.bmjjournals.com on 5 December 2005 Sex related expressivity of the phenotype in coronal craniosynostosis caused by the recurrent P250R FGFR3 mutation Elisabeth Lajeunie, Vincent El Ghouzzi, Martine Le Merrer, Arnold Munnich, Jacky Bonaventure and Dominique Renier J.
11739714
full text
15858182
full text
V617F
protein
substitution
true positive
O60674
PERSPECTIVE A Receptor dimer A Unifying Mutation in Chronic Myeloproliferative Disorders Ligand P JAK2 P SH2 P P Phosphorylation STAT Cytoplasm Nucleus P DNA P B Amino terminus FERM P SH2 V617F Pseudokinase P JH2 P Kinase PP JH1 Carboxyl terminus Involvement of Janus Kinases in Cytokine Signal Transduction (Panel A) and Structural Map of Janus Kinase 2 (Panel B).
The position of the mutated V617F codon is indicated by the arrow.
to-thymine mutation encoding a valine-to-phenylalanine substitution at position 617 (V617F) in the JH2, or autoinhibitory, domain of JAK2 (see diagram, Panel B).
A Boston group3 has reported that 121 of 164 patients with polycythemia vera had the V617F mutation in their granulocytes, as did 37 of 115 patients with essential thrombocythemia and 16 of 46 patients with myelofibrosis.
And a group in Cambridge, United Kingdom,4 found the V617F mutation in 71 of 73 patients with polycythemia vera, 29 of 51 patients with essential thrombocythemia, and 8 of 16 patients with myelofibrosis.
The remarkable finding of the V617F mutation by four independent research groups raises as many questions as it answers.
10951518
full text
P250R
protein
substitution
P22607
true positive
Up to 30% of such patients with coronal y Ala315Ser mutation in FGFR2 D Johnson et al 572 craniosynostosis have a specific mutation in FGFR3 (Pro250Arg) that is more reliably identified by genetic testing than by clinical features.57 Mutations of FGFR2 are much rarer in non-syndromic patients, but a small number of FGFR2 mutations have been identified in individuals with mild, atypical or more variable phenotypes.812 In the majority of non-syndromic patients, no genetic cause can be identified.
D321A
protein
substitution
true positive
P21803
The closest mutation downstream of Ala315Ser described to date is the substitution Asp321Ala, located in a short -helical (D) segment3436 and identified in four cases of Pfeiffer syndrome.22,27,37 No experimental studies have directly addressed the pathologic mechanism of this mutation, but disruption of the immunoglobulin fold leading to covalent dimerisation and constitutive activation of FGFR2 is the mechanism by which other mutations of the IgIIIc domain are believed to act.38 Several lines of evidence argue against the Ala315Ser mutation having a similarly gross disruptive effect.
A314S
protein
substitution
true positive
P21803
The G T transversion occurs at the fourth nucleotide of the alternatively spliced IgIIIc exon of FGFR2, which is the exon most commonly mutated in Crouzon and Pfeiffer syndromes.1,2 A cluster of mutations has been described in the intron just upstream of the IgIIIc exon (Figure 4); all these mutations are associated with either Pfeiffer or Apert syndrome.20,2229 These mutations are predicted to affect correct recognition of the IgIIIc acceptor splice site, and in three cases, alternative use of the IgIIIb exon has been demonstrated.20 Several G T transversions, all associated with Pfeiffer syndrome, have been described in the first nucleotide of the IgIIIc exon.23,30 These are predicted to encode an Ala314Ser substitution; alternatively, their predominant pathogenic effect may be on splicing.
A315S
protein
substitution
true positive
P21803
European Journal of Human Genetics (2000) 8, 571577 2000 Macmillan Publishers Ltd All rights reserved 10184813/00 $15.00 www.nature.com/ejhg y ARTICLE A novel mutation, Ala315Ser, in FGFR2: a geneenvironment interaction leading to craniosynostosis? David Johnson1,2, Steven A Wall2, Susan Mann2,3 and Andrew OM Wilkie1,2 1 Institute of Molecular Medicine, John Radcliffe Hospital, Oxford; 2Department of Plastic and Reconstructive Surgery and Oxford Craniofacial Unit, Radcliffe Infirmary, Oxford; 3Department of Anaesthetics, John Radcliffe Hospital, Oxford, UK Mutations in the fibroblast growth factor receptor 1, 2 and 3 (FGFR1, -2 and -3) and TWIST genes have been identified in several syndromic forms of craniosynostosis.
We describe a novel heterozygous mutation of FGFR2 (943G T, encoding the amino acid substitution Ala315Ser) in a girl with non-syndromic unicoronal craniosynostosis.
Up to 30% of such patients with coronal y Ala315Ser mutation in FGFR2 D Johnson et al 572 craniosynostosis have a specific mutation in FGFR3 (Pro250Arg) that is more reliably identified by genetic testing than by clinical features.57 Mutations of FGFR2 are much rarer in non-syndromic patients, but a small number of FGFR2 mutations have been identified in individuals with mild, atypical or more variable phenotypes.812 In the majority of non-syndromic patients, no genetic cause can be identified.
We describe a novel heterozygous mutation of FGFR2, Ala315Ser, in a patient with non-syndromic unicoronal craniosynostosis.
DNA sequencing identified a 943G T transversion (Figure 1B) corresponding to the amino acid substitution Ala315Ser.
European Journal of Human Genetics Ala315Ser mutation in FGFR2 D Johnson et al 573 y Figure 1 Identification of the Ala315Ser mutation in FGFR2.
European Journal of Human Genetics y Ala315Ser mutation in FGFR2 D Johnson et al 574 Figure 2 Anteroposterior and right oblique three-dimensional CT reconstruction of the proband's skull at the age of nine months, demonstrating synostosis of the right coronal suture.
Discussion The presence of the Ala315Ser mutation in FGFR2 in three members of this family, only one of whom has overt craniosynostosis, and none of whom has Crouzonoid facial features, raises the question as to whether this mutation has predisposed to the craniosynostosis or is a coincidence.
The closest mutation downstream of Ala315Ser described to date is the substitution Asp321Ala, located in a short -helical (D) segment3436 and identified in four cases of Pfeiffer syndrome.22,27,37 No experimental studies have directly addressed the pathologic mechanism of this mutation, but disruption of the immunoglobulin fold leading to covalent dimerisation and constitutive activation of FGFR2 is the mechanism by which other mutations of the IgIIIc domain are believed to act.38 Several lines of evidence argue against the Ala315Ser mutation having a similarly gross disruptive effect.
First, the substitution is relatively conservative: the side chains of alanine and serine have similar polarity and molecular volume.39 Second, this residue lies on the surface of the IgIII Ala315Ser mutation in FGFR2 D Johnson et al 575 y Figure 3 Clinical photographs of the proband (A, B, C) aged six months, her mother (D, E) and maternal grandfather (F, G).
Third, the alternatively spliced IgIIIb exon encodes a serine at the European Journal of Human Genetics y Ala315Ser mutation in FGFR2 D Johnson et al 576 Figure 4 Genomic sequence of FGFR2 showing the position the Ala315Ser mutation (circled) in the context of neighbouring mutations.
Given the above arguments, it might be concluded that Ala315Ser is a neutral polymorphism.
The proband, in addition to inheriting the Ala315Ser mutation, had an abnormal intrauterine history.
The importance of foetal head constraint in the aetiology of craniosynostosis is attested to both by anecdotal reports1316 and experimental studies in animals.17 This environmental factor, occurring in the context of an existing Ala315Ser mutation, offers a plausible explanation for why craniosynostosis occurred only in the proband, although the mutation had previously been transmitted over at least two preceding generations.
Given the small sample size and inherent ascertainment bias, it is impossible to provide an accurate estimate of the risk of craniosynostosis associated with inheritance of the Ala315Ser mutation.
11884411
full text
K608E
protein
substitution
true positive
Q62371
DDR2 / skin fibroblasts were reconstituted by retroviral infection with green fluorescent protein, wild type mouse DDR2, or a K608E point mutation to produce a kinase-dead DDR2 (9).
pMT21FcDDR2KE encodes a mutant FcDDR2 cDNA containing a K608E.
pMT21FcDDR2KEY471F encodes double mutant FcDDR2 cDNA containing K608E and Y471F.
Cell lysates were precipitated by protein A, followed by Western blotting with anti-phosphotyrosine, anti-DDR2, and anti-Shc as described under "Experimental Procedures." The Y471F mutant DDR2 (lanes 3 and 8) slightly decreased its phosphorylation; however, the K608E,Y471F DDR2 double mutants (lanes 5 and 10) dramatically decreased its phosphorylation (arrowhead).
Plasmids encoding a point mutation of Y471F and double point mutations of K608E and Y471F of FcDDR2 were cotransfected with or without wild type Src in COS7 cells.
However, the K608E and Y471F double mutation of FcDDR2 dramatically decreased phosphorylation by 90%.
The K608E/Y471F double mutations reduced Shc association by 90% (Fig.
8, a Y471F mutation and/or kinase-dead K608E dramatically decreased Shc association; however, it did not totally inhibit the association, which suggests there are still other minor association sites with Shc.
Y471F
protein
substitution
true positive
Q62371
pMT21FcDDR2Y471F encodes a mutant FcDDR2 cDNA containing a Y471F.
pMT21FcDDR2KEY471F encodes double mutant FcDDR2 cDNA containing K608E and Y471F.
A Y471F mutation reduces the association of DDR2 with Shc and Src.
FcDDR2 (lanes 2 and 7), FcDDR2Y471F (lanes 3 and 8), FcDDR2KE (lanes 4 and 9), or FcDDR2KEY471F (lanes 5 and 10) were transiently expressed with (lanes 6 10) or without (lanes 15) wild type Src in COS7 cells.
Cell lysates were precipitated by protein A, followed by Western blotting with anti-phosphotyrosine, anti-DDR2, and anti-Shc as described under "Experimental Procedures." The Y471F mutant DDR2 (lanes 3 and 8) slightly decreased its phosphorylation; however, the K608E,Y471F DDR2 double mutants (lanes 5 and 10) dramatically decreased its phosphorylation (arrowhead).
Furthermore, Shc association was dramatically decreased by the Y471F mutation (arrow).
To identify residues in DDR2 critical for Src/Shc interaction, we generated a Y471F mutant in the juxtamembrane region of DDR2 to investigate whether this site is critical for Src/Shc interaction based on the following reasons.
Plasmids encoding a point mutation of Y471F and double point mutations of K608E and Y471F of FcDDR2 were cotransfected with or without wild type Src in COS7 cells.
The Y471F mutation of FcDDR2 moderately decreased DDR2 phosphorylation with or without Src.
However, the K608E and Y471F double mutation of FcDDR2 dramatically decreased phosphorylation by 90%.
Although Shc association with DDR2 was dependent on Src, it was also decreased by the Y471F mutation.
The Y471F mutation decreased its association with Shc by 70%.
The K608E/Y471F double mutations reduced Shc association by 90% (Fig.
8, a Y471F mutation and/or kinase-dead K608E dramatically decreased Shc association; however, it did not totally inhibit the association, which suggests there are still other minor association sites with Shc.
10477765
full text
V789M
protein
substitution
true positive
Q61006
Genetic analysis in available relatives revealed, according to the recessive mode of inheritance in this family, that the insertion was transmitted by the individual I I (deceased father with no DNA available) to individuals II-1 and II-3 and the missense V790M mutation was transmitted by individual I-2 (mother) to affected individuals II-1 and II-3 and unaffected individual Functional evaluation of MUSK mutations In order to investigate whether the mutations detected did indeed cause the myasthenic syndrome observed in the patient, site-directed mutagenesis of a rat MuSK cDNA was used to generate two MuSK constructs that reproduced the two mutations: c.220insC and c.2365G ! A (V789M) detected in the patient.
This explains the numbering difference observed between the human (V790M) and the rat (V789M) corresponding mutation.
V790M
protein
substitution
true positive
Q61006
Gene analysis identified two heteroallelic mutations, a frameshift mutation (c.220insC) and a missense mutation (V790M).
The second mutation is a G ! A transition at nucleotide 2368 of the coding sequence in exon 14 (c.2368G ! A) leading to the missense V790M mutation.
Genetic analysis in available relatives revealed, according to the recessive mode of inheritance in this family, that the insertion was transmitted by the individual I I (deceased father with no DNA available) to individuals II-1 and II-3 and the missense V790M mutation was transmitted by individual I-2 (mother) to affected individuals II-1 and II-3 and unaffected individual Functional evaluation of MUSK mutations In order to investigate whether the mutations detected did indeed cause the myasthenic syndrome observed in the patient, site-directed mutagenesis of a rat MuSK cDNA was used to generate two MuSK constructs that reproduced the two mutations: c.220insC and c.2365G ! A (V789M) detected in the patient.
This explains the numbering difference observed between the human (V790M) and the rat (V789M) corresponding mutation.
In the proband, one allele bears the 220insC frameshifting mutation, the other bears the 2368G ! A missense mutation which induces V790M in MuSK.
DISCUSSION We report here the description of a case of CMS associated with heteroallelic mutations in the MuSK gene: a frameshift mutation (220insC) and a missense mutation (V790M).
9461530
full text
K721M
protein
substitution
true negative
Whereas EGF receptors incorporating Lys-721 Met [8] or Lys-721 Arg [12] substitutions were both shown to activate the MAPK signalling pathway, these receptors did not stimulate mitogenesis.
In order to understand better the effects that specific amino acid substitutions have on the catalytic and signal-transducing properties of PTKs, the nucleotide binding characteristics of recombinant EGF receptor PTK domains were examined by the Abbreviations used : TNP-ATP, 2h(3h)-O-(2,4,6-trinitrophenyl)adenosine 5h-triphosphate ; EGF, epidermal growth factor ; PTK, protein tyrosine kinase ; TKD61, recombinant EGF receptor cytosolic domain ; TKD61-K/M, TKD61 protein with a Lys-721 Met amino acid substitution ; TKD61-D/A, TKD61 protein with a Asp-813 Ala amino acid substitution ; GAT, a random co-polymer of glutamate, alanine and tyrosine [(Glu : Ala : Tyr)6:3:1] ; MAPK, mitogen-activated protein kinase ; FGF, fibroblast growth factor.
Both the wild-type protein and proteins incorporating either a Lys-721 Met or Asp-813 Ala mutation were studied.
Full-length EGF receptor cDNAs containing mutations corresponding to the Lys-721 Met [6] and Asp-813 Ala [11] substitutions have been previously described, and baculovirus vectors encoding the cytosolic domains of these proteins (TKD61-KM and TKD61-DA) were generated by subcloning PCR-amplified fragments of the mutant EGF receptor cDNAs into baculovirus transfer vectors.
Substrate RESULTS Generation of EGF receptor PTK domains In order to compare the nucleotide binding properties of the wild-type EGF receptor PTK with those of mutant EGF receptor proteins incorporating site-specific amino acid substitutions in the catalytic domain, the PTK domain of each of the proteins Nucleotide binding by EGF receptor 355 Figure 1 Expression and purification of recombinant EGF receptor cytosolic domain proteins The cytosolic domains of wild-type (TKD61) and mutant EGF receptor cytosolic domain proteins incorporating Lys-721 Met (TKD61-K/M) and Asp-813 Ala (TKD61-D/A) amino acid substitutions were expressed in the baculovirus/insect cell system and purified as previously described [20].
Here, the PTK activities of the Lys-721 Met (0n21p0n03 nmol:min-":mg-") and Asp-813 Ala (0n16p0n05 nmol:min-":mg-") mutant EGF receptor proteins were indistinguishable from the assay blank (0n21p0n09 nmol:min-":mg-"), which indicated that these proteins possessed activities less than 2 % of the wild-type EGF receptor protein (12n7p0n5 nmol:min-":mg-").
Together these assay results confirmed earlier observations that the Lys-721 Met and Asp-813 Ala substitutions both render the EGF receptor PTK inactive.
EGF receptor mutants with either Lys-721 Met or Asp-813 Ala substitutions appear to be devoid of intrinsic kinase activity, yet are still capable of mediating an EGF-dependent activation of the MAPK signalling pathway [8,11].
Whereas the Lys-721 Met mutant receptor protein could not relay a full mitogenic signal [8], an EGF-dependent stimulation of DNA synthesis was mediated by the Asp-813 Ala mutant receptor [11].
The results of these experiments indicated that mutant EGF receptor proteins incorporating either the Lys-721 Met (TKD61-KM) or Asp-813 Ala (TKD61-DA) amino acid substitution were still capable of binding ATP with near normal affinity (Table 1).
However, it was observed that the dissociation constant for binding of the Mn[ATP complex to the Lys-721 Met mutant protein (100 M) was significantly greater than that for the wild-type (6n1 M) and Asp-813 Ala mutant (3n7 M) proteins.
while the defect in the Asp-813 Ala mutant protein that abolishes catalytic activity did not significantly alter the nucleotide binding properties of the protein, the Lys-721 Met mutation did perturb the nucleotide binding site, with a pronounced effect seen in the case of Mn:ATP binding.
It is clear that EGF receptors harbouring either the Lys-721 Met or Asp-813 Ala substitution retained an ability to bind both ATP and a bivalent metal ionATP complex.
In the case of the Lys-721 Met substitution, the affinity of Mn[ATP binding was found to be significantly reduced relative to the wild-type protein.
This latter finding suggested that the Lys-721 Met mutation did alter the conformation of PTK catalytic domain.
The observed differences in the nucleotide binding properties of the Asp-813 Ala and Lys-721 Met mutant proteins, as was most evident in the binding of the divalent metal ionnucleotide complex, correlate with observed differences in the signal transduction potentials of full-length receptor proteins incorporating these mutations [8,11].
The attenuated signal transduction capabilities of the Lys-721 Met receptor mutant may reflect a significant perturbation of PTK structure, as was indicated here by an attenuation of intrinsic tryptophan fluorescence emission in TKD61-KM, or may result from an abnormal enzymesubstrate interaction, as was evidenced in TKD61-KM by a significant reduction in affinity for Mn:ATP and also by an attenuation of the fluorescence yield of enzyme-bound Mn[TNPATP (Figure 4B).
In the present study, the Lys-721 Met substitution in the EGF receptor PTK induced similar effects : a near abolition of catalytic activity, and a large reduction (16fold) in binding affinity for the bivalent metal ionATP substrate complex.
D61K
protein
substitution
true negative
In order to understand better the effects that specific amino acid substitutions have on the catalytic and signal-transducing properties of PTKs, the nucleotide binding characteristics of recombinant EGF receptor PTK domains were examined by the Abbreviations used : TNP-ATP, 2h(3h)-O-(2,4,6-trinitrophenyl)adenosine 5h-triphosphate ; EGF, epidermal growth factor ; PTK, protein tyrosine kinase ; TKD61, recombinant EGF receptor cytosolic domain ; TKD61-K/M, TKD61 protein with a Lys-721 Met amino acid substitution ; TKD61-D/A, TKD61 protein with a Asp-813 Ala amino acid substitution ; GAT, a random co-polymer of glutamate, alanine and tyrosine [(Glu : Ala : Tyr)6:3:1] ; MAPK, mitogen-activated protein kinase ; FGF, fibroblast growth factor.
Substrate RESULTS Generation of EGF receptor PTK domains In order to compare the nucleotide binding properties of the wild-type EGF receptor PTK with those of mutant EGF receptor proteins incorporating site-specific amino acid substitutions in the catalytic domain, the PTK domain of each of the proteins Nucleotide binding by EGF receptor 355 Figure 1 Expression and purification of recombinant EGF receptor cytosolic domain proteins The cytosolic domains of wild-type (TKD61) and mutant EGF receptor cytosolic domain proteins incorporating Lys-721 Met (TKD61-K/M) and Asp-813 Ala (TKD61-D/A) amino acid substitutions were expressed in the baculovirus/insect cell system and purified as previously described [20].
Figure 2 CD and intrinsic tryptophan fluorescence spectra of wild-type and mutant EGF receptor cytosolic domain proteins (A) Near-UV CD spectra of the wild-type TKD61 (----), TKD61-K/M (- - - - -) and TKD61-D/A (-- - --) proteins were recorded and analysed as described in the Experimental section.
Spectral decomposition analysis yielded the apparent percentages of -helical (), -sheet (s ), turns (t) and unordered (u) structural elements for TKD61 ( l 41 %, s l 35 %, t l 10 %, u l 14 %), TKD61-K/M ( l 28 %, s l 35 %, t l 13 %, u l 22 %), and TKD61-D/A ( l 26 %, s l 38 %, t l 12 %, u l 23 %).
(B) Intrinsic tryptophan emission spectra of the wild-type TKD61 (----), TKD61-K/M (- - - - -) and TKD61-D/A (-- - --) proteins are shown along with a solvent blank spectrum ( ).
Koland Figure 3 Autophosphorylation activities of wild-type and mutant EGF receptor cytosolic domain proteins TKD61, TKD61-K/M or TKD61-D/A (10 pmol) was incubated in the presence of 10 mM MnCl2 and 70 M [-32P]ATP for 1 min at room temperature, and autophosphorylation of the recombinant protein analysed by SDS/PAGE and autoradiography.
Figure 4 Binding of TNP-ATP and Mn:TNP-ATP complex to wild-type and mutant EGF receptor cytosolic domain proteins Solutions of either wild-type TKD61 (#), TKD61-K/M (]) or TKD61-D/A ( ) [0n5 M in (A) and 1n0 M in (B)] were titrated with increasing concentrations of TNP-ATP as the fluorescence of the nucleotide analogue was recorded.
KTNP-ATP TKD61 TKD61-D/A TKD61-K/M 0n75p0n24 0n35p0n12 0n45p0n22 KMnTNP-ATP 12n3p3n0 4n4p1n2 8n2p1n7 KATP 660p63 309p25 334p27 KMnATP 6n1p0n8 3n7p0n7 100p23 357 PTKs were determined by competition titrations in which the fluorescence of bound TNP-ATP was monitored as increasing concentrations of ATP or Mn[ATP were added (Figure 5).
Hence, Figure 5 Binding of ATP and Mn[ATP complex to wild-type and mutant EGF receptor cytosolic domain proteins Solutions of either TKD61 (0n5 M, #), TKD61-K/M (0n5 M, W), or TKD61-D/A (1n0 M, ) and TNP-ATP [0n4 M (#, W) or 0n1 M ( ) in A ; 2n0 M (#), 4n0 M (W) or 1n0 M ( ) in B] were titrated with increasing concentrations of ATP as the fluorescence of the nucleotide analogue was recorded.
K721R
protein
substitution
true negative
Whereas EGF receptors incorporating Lys-721 Met [8] or Lys-721 Arg [12] substitutions were both shown to activate the MAPK signalling pathway, these receptors did not stimulate mitogenesis.
D813A
protein
substitution
true negative
In contrast, an EGF receptor mutant with an Asp-813 Ala substitution was found to stimulate DNA synthesis, as well as MAPK activity [11].
In order to understand better the effects that specific amino acid substitutions have on the catalytic and signal-transducing properties of PTKs, the nucleotide binding characteristics of recombinant EGF receptor PTK domains were examined by the Abbreviations used : TNP-ATP, 2h(3h)-O-(2,4,6-trinitrophenyl)adenosine 5h-triphosphate ; EGF, epidermal growth factor ; PTK, protein tyrosine kinase ; TKD61, recombinant EGF receptor cytosolic domain ; TKD61-K/M, TKD61 protein with a Lys-721 Met amino acid substitution ; TKD61-D/A, TKD61 protein with a Asp-813 Ala amino acid substitution ; GAT, a random co-polymer of glutamate, alanine and tyrosine [(Glu : Ala : Tyr)6:3:1] ; MAPK, mitogen-activated protein kinase ; FGF, fibroblast growth factor.
Both the wild-type protein and proteins incorporating either a Lys-721 Met or Asp-813 Ala mutation were studied.
The nucleotide binding and structural characteristics of the Asp-813 Ala mutant protein were not appreciably altered, which indicated that this kinase-inactivating mutation effected a subtler defect in the catalysis of the phosphotransfer reaction.
Full-length EGF receptor cDNAs containing mutations corresponding to the Lys-721 Met [6] and Asp-813 Ala [11] substitutions have been previously described, and baculovirus vectors encoding the cytosolic domains of these proteins (TKD61-KM and TKD61-DA) were generated by subcloning PCR-amplified fragments of the mutant EGF receptor cDNAs into baculovirus transfer vectors.
Substrate RESULTS Generation of EGF receptor PTK domains In order to compare the nucleotide binding properties of the wild-type EGF receptor PTK with those of mutant EGF receptor proteins incorporating site-specific amino acid substitutions in the catalytic domain, the PTK domain of each of the proteins Nucleotide binding by EGF receptor 355 Figure 1 Expression and purification of recombinant EGF receptor cytosolic domain proteins The cytosolic domains of wild-type (TKD61) and mutant EGF receptor cytosolic domain proteins incorporating Lys-721 Met (TKD61-K/M) and Asp-813 Ala (TKD61-D/A) amino acid substitutions were expressed in the baculovirus/insect cell system and purified as previously described [20].
Here, the PTK activities of the Lys-721 Met (0n21p0n03 nmol:min-":mg-") and Asp-813 Ala (0n16p0n05 nmol:min-":mg-") mutant EGF receptor proteins were indistinguishable from the assay blank (0n21p0n09 nmol:min-":mg-"), which indicated that these proteins possessed activities less than 2 % of the wild-type EGF receptor protein (12n7p0n5 nmol:min-":mg-").
Together these assay results confirmed earlier observations that the Lys-721 Met and Asp-813 Ala substitutions both render the EGF receptor PTK inactive.
The Kd values for binding of Mn[ATP to the wild-type and Asp-813 Ala mutant proteins were similar to the Km for the substrate complex in the autophosphorylation reaction of the PTK (Km " 6 M [22]).
EGF receptor mutants with either Lys-721 Met or Asp-813 Ala substitutions appear to be devoid of intrinsic kinase activity, yet are still capable of mediating an EGF-dependent activation of the MAPK signalling pathway [8,11].
Whereas the Lys-721 Met mutant receptor protein could not relay a full mitogenic signal [8], an EGF-dependent stimulation of DNA synthesis was mediated by the Asp-813 Ala mutant receptor [11].
The results of these experiments indicated that mutant EGF receptor proteins incorporating either the Lys-721 Met (TKD61-KM) or Asp-813 Ala (TKD61-DA) amino acid substitution were still capable of binding ATP with near normal affinity (Table 1).
However, it was observed that the dissociation constant for binding of the Mn[ATP complex to the Lys-721 Met mutant protein (100 M) was significantly greater than that for the wild-type (6n1 M) and Asp-813 Ala mutant (3n7 M) proteins.
while the defect in the Asp-813 Ala mutant protein that abolishes catalytic activity did not significantly alter the nucleotide binding properties of the protein, the Lys-721 Met mutation did perturb the nucleotide binding site, with a pronounced effect seen in the case of Mn:ATP binding.
It is clear that EGF receptors harbouring either the Lys-721 Met or Asp-813 Ala substitution retained an ability to bind both ATP and a bivalent metal ionATP complex.
In contrast, the Asp-813 Ala mutation, which also essentially abolished catalytic activity, had only minor effects on the nucleotide-binding properties of the enzyme.
The observed differences in the nucleotide binding properties of the Asp-813 Ala and Lys-721 Met mutant proteins, as was most evident in the binding of the divalent metal ionnucleotide complex, correlate with observed differences in the signal transduction potentials of full-length receptor proteins incorporating these mutations [8,11].
The observation that the Asp-813 Ala substitution in the EGF receptor PTK domain did not perturb nucleotide binding is therefore consistent with the available structural data.
K116A
protein
substitution
true negative
Although less than 1 % of wild-type kinase activity was exhibited by the Lys-116 Ala mutant enzyme, the Km for Mg[ATP of this mutant could still be determined, and was found to be increased six-fold relative to the wild-type enzyme [28].
K273R
protein
substitution
true negative
Also, the Lys-273 Arg Lck mutant could be photoaffinity-labelled with 8-azidoATP, which suggested that the defect in catalysis of this mutant was not in the binding of nucleotide, but in the enzymic process of phospho-transfer.
8647804
full text
R112Q
protein
substitution
true negative
To further characterize the interaction between c and ShcN, we co-transfected wild type GST-Shc-N or mutant forms of GST-Shc-N (R175K or R112Q) with c656 and JAK2 (Fig.
In contrast, a mutation at another arginine residue (R112Q), which lies outside the phosphoprotein binding pocket, 12140 GMR- c Interacts with the Shc-PTB Domain followed by tyrosine phosphorylation of Shc.
R175K
protein
substitution
true negative
To further characterize the interaction between c and ShcN, we co-transfected wild type GST-Shc-N or mutant forms of GST-Shc-N (R175K or R112Q) with c656 and JAK2 (Fig.
While the wild type GST-Shc-N domain bound to c656, the R175K mutant failed to interact with c.
10551845
full text
K832R
protein
substitution
true negative
The plasmid for bacterial expression of GST-p85wt and other mammalian expression constructs has already been described: pGEX-p85wt (22), SR - p85 (23), pSG5- p110 wt (24), pRK5-p110 wt (25), pCMV-p110 K832R (17).
The accumulation of PI3,4P2 and PIP3 upon LPA was measured in Cos cells transiently transfected with a kinase-dead mutant of p110 (K832R) that inhibits the G -induced activation of the Ras/MAPK pathway (Fig.
A, Cos cells were transiently transfected with a kinase-dead mutant of p110 (K832R) or vector as a control.
The results represent the percentage of inhibition of PI polyphosphate production in transfected cells normalized for the percentage of transfection, as described under "Experimental Procedures." B, the effect of the K832R mutant on G -induced phosphorylation of hemagglutinin (HA)-tagged Erk1 was measured in Cos cells transfected with the indicated constructs (V, empty vector).
Moreover, PI3,4P2 production was not modified in Cos cells expressing the K832R p110 mutant.
10075926
full text
L98A
protein
substitution
true positive
P52333
(D) 293T cells transfected with plasmids encoding c and FLAG-tagged WT Jak3 (lane 1), or FLAG-tagged Jak3 constructs with mutations K855A (lane 2), Y100C (lane 3), Y100C/K855A double mutant (lane 4), Y100A (lane 5), Y100F (lane 6), L98A/L99A/I102A triple mutant (lane 7), L98A (lane 8), L99A (lane 9), I102A (lane 10) were lysed and immunoprecipitated with M2.
Y105A
protein
substitution
true positive
P52333
Mutations in highly conserved aromatic amino acids Y105A and W109A of Jak3 had no significant effect on c binding in 293T co-immunoprecipitation experiments (not shown).
I102A
protein
substitution
true positive
P52333
(D) 293T cells transfected with plasmids encoding c and FLAG-tagged WT Jak3 (lane 1), or FLAG-tagged Jak3 constructs with mutations K855A (lane 2), Y100C (lane 3), Y100C/K855A double mutant (lane 4), Y100A (lane 5), Y100F (lane 6), L98A/L99A/I102A triple mutant (lane 7), L98A (lane 8), L99A (lane 9), I102A (lane 10) were lysed and immunoprecipitated with M2.
We generated a triple mutant (L98A, L99A, I102A), and compared c association in 293T cells with wild-type Jak3.
L98A and I102A mutations disrupted the binding of Jak3 to c by Jak3 receptor interaction domain Fig.
Additionally we show that other mutations in this region of Jak3 (Y100A, L98A, I102A) block Jak3c interaction, suggesting that this region of Jak3 probably encodes a domain that is essential for receptor interaction.
Y105F
protein
substitution
true positive
P52333
To study the effect of the Y100C mutation on c interaction, we expressed wild-type Jak3, a kinase-inactive form of Jak3, a form of Jak3 with a Y105F mutation in a conserved tyrosine adjacent to Y100, and the Y100C SCID patient mutant, in 293T cells, and tested their ability to co-immunoprecipitate with c.
As shown in Figure 2A, lanes 2, 3 and 5, we observed co-immunoprecipitation of c with wild-type Jak3, the kinase inactive mutant, K855A, and the Y105F mutant.
However, we did not detect co-precipitation of c with Jak3 Y100C or the Jak3 Y100C/Y105F double mutant (Figure 2A, lanes 4 and 6), although equal levels of Jak3 and c were expressed in these cells (Figure 2A, lower panels), suggesting that the Y100C mutation disrupts receptor interaction.
Again, wild-type, kinase-inactive and Y105F Jak3 interacted strongly with c, while the Y100C mutant and the Y100C/Y105F double mutant were unable to bind the receptor, suggesting that Jak3 Y100C lacks the ability to interact with c (Figure 2B).
(A) Lysates from 293T cells transfected with plasmids encoding FLAG-tagged wild-type (WT) (lane 2), kinase-inactive mutant Jak3 K855A (lane 3), Y100C (lane 4), Y105F (lane 5), and Y100C/Y105F double mutant (lane 6) and with c were immunoprecipitated with the FLAG antibody M2.
L99A
protein
substitution
true positive
P52333
(D) 293T cells transfected with plasmids encoding c and FLAG-tagged WT Jak3 (lane 1), or FLAG-tagged Jak3 constructs with mutations K855A (lane 2), Y100C (lane 3), Y100C/K855A double mutant (lane 4), Y100A (lane 5), Y100F (lane 6), L98A/L99A/I102A triple mutant (lane 7), L98A (lane 8), L99A (lane 9), I102A (lane 10) were lysed and immunoprecipitated with M2.
W109A
protein
substitution
true positive
P52333
Mutations in highly conserved aromatic amino acids Y105A and W109A of Jak3 had no significant effect on c binding in 293T co-immunoprecipitation experiments (not shown).
K855A
protein
substitution
true positive
P52333
As shown in Figure 2A, lanes 2, 3 and 5, we observed co-immunoprecipitation of c with wild-type Jak3, the kinase inactive mutant, K855A, and the Y105F mutant.
A kinase-inactive form of the patient mutant, Jak3 Y100C/K855A, also did not interact with c, and antiphosphotyrosine Western blotting confirmed that Jak3 Y100C/K855A was not phosphorylated in 293T cells (Figure 2D, second panel, lane 4), suggesting that the defect in receptor binding is not due to increased or aberrant phosphorylation of the mutant kinase, and that neither kinase activity nor phosphorylation of Y100 is necessary for c interaction.
(A) Lysates from 293T cells transfected with plasmids encoding FLAG-tagged wild-type (WT) (lane 2), kinase-inactive mutant Jak3 K855A (lane 3), Y100C (lane 4), Y105F (lane 5), and Y100C/Y105F double mutant (lane 6) and with c were immunoprecipitated with the FLAG antibody M2.
(C) NIH 3T3 fibroblasts expressing IL-2 receptor were stably infected with vector (lanes 1 and 2), FLAG-tagged WT Jak3 (lanes 3 and 4), kinase-inactive Jak3 K855A (lanes 5 and 6), or Jak3 Y100C (lanes 7 and 8), and were untreated or stimulated for 15 min with 1000 U/ml IL-2.
(D) 293T cells transfected with plasmids encoding c and FLAG-tagged WT Jak3 (lane 1), or FLAG-tagged Jak3 constructs with mutations K855A (lane 2), Y100C (lane 3), Y100C/K855A double mutant (lane 4), Y100A (lane 5), Y100F (lane 6), L98A/L99A/I102A triple mutant (lane 7), L98A (lane 8), L99A (lane 9), I102A (lane 10) were lysed and immunoprecipitated with M2.
However, we have demonstrated that the inability of Jak3 Y100C to interact with c is not due to its constitutive phosphorylation, as a kinase-inactive derivative, Jak3 Y100C/K855A, that is not detectably tyrosine phosphorylated, also fails to interact with c in 293T cells.
Y100C
protein
substitution
true positive
P52333
Here we describe a naturally occurring Jak3 mutation from a patient with autosomal severe combined immunodeficiency (SCID), where a single amino acid substitution, Y100C, in Janus homology domain 7 (JH7) prevents kinasereceptor interaction.
Results Jak3 mutation (Y100C) blocks IL-2-induced tyrosine phosphorylation SCID can result from several distinct molecular defects (Candotti and Blaese, 1996); the X-linked form of the disease results from loss or mutation of c, and a clinical phenotype indistinguishable from XSCID, which is due to loss or mutation of Jak3, and results in impaired signaling through c-using cytokine receptors.
(B) EBV-transformed B-cell lines from normal donor (JY) or Jak3 Y100C-expressing patient (GM) were untreated or stimulated with 1000 U/ml IL-2 for 15 min at 37C.
The patient described in Figure 1 is homozygous for a single amino acid substitution, Y100C, in the JH7 domain.
1550 Jak3 receptor interaction domain In the SCID patient's cells (GM), no inducible tyrosine phosphorylation of Jak3 was detected in response to IL-2, although Jak3 Y100C was tyrosine phosphorylated even in the absence of cytokine treatment (Figure 1B, lanes 3 and 4).
The expression controls show that the levels of precipitated endogenous Jak3 Y100C are lower compared with WT Jak3 (Figure 1B, lower panel), demonstrating that the phosphorylation state of Jak3 Y100C in the absence of IL-2 does not result from Jak3 Y100C overexpression.
These results suggest that although Jak3 Y100C was constitutively phosphorylated in the patient's cells, it was not inducibly phosphorylated and was unable to initiate downstream signaling events in response to IL-2.
Jak3 Y100C mutant does not interact with c The N-terminal portion of Jak kinases has been implicated in receptor association, but the precise region responsible has not been reported (Chen et al., 1997).
To study the effect of the Y100C mutation on c interaction, we expressed wild-type Jak3, a kinase-inactive form of Jak3, a form of Jak3 with a Y105F mutation in a conserved tyrosine adjacent to Y100, and the Y100C SCID patient mutant, in 293T cells, and tested their ability to co-immunoprecipitate with c.
However, we did not detect co-precipitation of c with Jak3 Y100C or the Jak3 Y100C/Y105F double mutant (Figure 2A, lanes 4 and 6), although equal levels of Jak3 and c were expressed in these cells (Figure 2A, lower panels), suggesting that the Y100C mutation disrupts receptor interaction.
Again, wild-type, kinase-inactive and Y105F Jak3 interacted strongly with c, while the Y100C mutant and the Y100C/Y105F double mutant were unable to bind the receptor, suggesting that Jak3 Y100C lacks the ability to interact with c (Figure 2B).
Therefore, we investigated the ability of Jak3 Y100C to become tyrosine phosphorylated and recruited to the receptor in response to IL-2.
Fibroblasts that stably express the IL-2 receptor chains (3T3) (Chen et al., 1997) were infected with retrovirus expressing epitope-tagged wild-type Jak3, a kinaseinactive form of Jak3 or the Y100C mutant.
However, no IL-2-induced phosphorylation of Jak3 Y100C was observed, although we did detect high basal tyrosine phosphorylation, in agreement with our results with the GM patient's cells (Figure 2C, lanes 7 and 8).
The expression controls again showed that the high basal phosphorylation state of Y100C was not due to overexpression of the mutant Jak3, as Y100C was expressed at levels comparable with wild-type and kinase-inactive Jak3 (Figure 2C, middle panel).
However, the cytokine-induced recruitment of Jak3 to the receptor complex was not observed with Jak3 Y100C (Figure 2C, bottom panel, lanes 7 and 8).
These data confirm that the Y100C mutation results in defective interaction with c, providing an explanation for the SCID phenotype observed in this patient.
Other Jak3 mutations that affect receptor interaction As the Y100C mutation clearly blocks Jak3receptor association, we investigated whether other mutations in this region of Jak3 could disrupt receptor interaction.
A kinase-inactive form of the patient mutant, Jak3 Y100C/K855A, also did not interact with c, and antiphosphotyrosine Western blotting confirmed that Jak3 Y100C/K855A was not phosphorylated in 293T cells (Figure 2D, second panel, lane 4), suggesting that the defect in receptor binding is not due to increased or aberrant phosphorylation of the mutant kinase, and that neither kinase activity nor phosphorylation of Y100 is necessary for c interaction.
(A) Lysates from 293T cells transfected with plasmids encoding FLAG-tagged wild-type (WT) (lane 2), kinase-inactive mutant Jak3 K855A (lane 3), Y100C (lane 4), Y105F (lane 5), and Y100C/Y105F double mutant (lane 6) and with c were immunoprecipitated with the FLAG antibody M2.
(C) NIH 3T3 fibroblasts expressing IL-2 receptor were stably infected with vector (lanes 1 and 2), FLAG-tagged WT Jak3 (lanes 3 and 4), kinase-inactive Jak3 K855A (lanes 5 and 6), or Jak3 Y100C (lanes 7 and 8), and were untreated or stimulated for 15 min with 1000 U/ml IL-2.
(D) 293T cells transfected with plasmids encoding c and FLAG-tagged WT Jak3 (lane 1), or FLAG-tagged Jak3 constructs with mutations K855A (lane 2), Y100C (lane 3), Y100C/K855A double mutant (lane 4), Y100A (lane 5), Y100F (lane 6), L98A/L99A/I102A triple mutant (lane 7), L98A (lane 8), L99A (lane 9), I102A (lane 10) were lysed and immunoprecipitated with M2.
Defining a minimal receptor interaction domain on Jak3 The results above indicated that the Y100C mutation might be located in a receptor interaction domain situated in the N-terminus of Jak3.
(A) Lysates from 293T cells co-transfected with wild-type (WT) or mutant Jak3 and c in the following combinations: WT Jak3 and WT c (lane 1), Jak3 Y100C and WT c (lane 2), WT Jak3 and P266,269A c (lane 3), or WT Jak3 and P266,269G c (lane 4) were immunoprecipitated with anti-FLAG M2, and blots were probed with Jak3 antibody (upper panel).
Interestingly, we show that a single point mutation in Jak3 (Y100C) can also lead to a severe phenotype by blocking Jak3 interaction with c and hence downstream signaling responses to IL-2.
The mutation Y100A, like the SCID patient mutation, Y100C, markedly reduces Jak3 receptor binding.
Interestingly, the single amino acid substitution of Jak3 described here (Y100C) also results in a loss of proper regulation of basal tyrosine phosphorylation.
However, we have demonstrated that the inability of Jak3 Y100C to interact with c is not due to its constitutive phosphorylation, as a kinase-inactive derivative, Jak3 Y100C/K855A, that is not detectably tyrosine phosphorylated, also fails to interact with c in 293T cells.
Y100A
protein
substitution
true positive
P52333
In addition, a Jak3 Y100A mutant abrogates c binding while a conservative Jak3 Y100F mutant still interacted with c (Figure 2D, lanes 5 and 6), presumably by preserving secondary structure required for receptor binding.
(D) 293T cells transfected with plasmids encoding c and FLAG-tagged WT Jak3 (lane 1), or FLAG-tagged Jak3 constructs with mutations K855A (lane 2), Y100C (lane 3), Y100C/K855A double mutant (lane 4), Y100A (lane 5), Y100F (lane 6), L98A/L99A/I102A triple mutant (lane 7), L98A (lane 8), L99A (lane 9), I102A (lane 10) were lysed and immunoprecipitated with M2.
Additionally we show that other mutations in this region of Jak3 (Y100A, L98A, I102A) block Jak3c interaction, suggesting that this region of Jak3 probably encodes a domain that is essential for receptor interaction.
The mutation Y100A, like the SCID patient mutation, Y100C, markedly reduces Jak3 receptor binding.
Y100F
protein
substitution
true positive
P52333
In addition, a Jak3 Y100A mutant abrogates c binding while a conservative Jak3 Y100F mutant still interacted with c (Figure 2D, lanes 5 and 6), presumably by preserving secondary structure required for receptor binding.
(D) 293T cells transfected with plasmids encoding c and FLAG-tagged WT Jak3 (lane 1), or FLAG-tagged Jak3 constructs with mutations K855A (lane 2), Y100C (lane 3), Y100C/K855A double mutant (lane 4), Y100A (lane 5), Y100F (lane 6), L98A/L99A/I102A triple mutant (lane 7), L98A (lane 8), L99A (lane 9), I102A (lane 10) were lysed and immunoprecipitated with M2.
conservative change (Y100F) has no significant effect on Jak3c interaction, this suggests that tyrosine 100 in Jak3 forms part of the structure that directly contacts c, and that ablation of the aromatic residue by an alanine or cysteine substitution disrupts the domain fold.
L271Q
protein
substitution
true positive
P52333
Interestingly, a point mutation (L271Q) in the Box1 region of c disrupts Jak3c interaction, and causes X-linked combined immunodeficiency (XCID) (Russell et al., 1994; Schmalstieg, 1995) Therefore, although Jaks interact with Box1/Box2 in the cytoplasmic domains of hematopoietic receptors, the region of Jaks that interact with Box1 has not been well characterized.
As mentioned previously, a naturally occurring mutation of c (L271Q) from a patient with XCID decreases the association of c with Jak3.
Another naturally occurring point mutation in the cytoplasmic tail of c (L271Q) within Box1/Box2 partially disrupts binding to Jak3 and leads to a less severe phenotype (Russell et al., 1995; Schmalstieg et al., 1995).
15738541
full text
G719A
protein
substitution
true positive
P00533
There were three patients with EGFR mutations (two with L858R and one with G719A) whose CEA level increased after gefitinib treatment but did not have measurable diseases.
E709G
protein
substitution
true positive
P00533
Point mutations 719 G 740 750 760 * * * KIPVAIKELREATSPKANKEILD Codon 719 C KIPVAIKELREATSPKANKEILD FGLAKLLG +E709H FGLAKLLG 12 FGRAKLLG FGRAKLLG +A871G FGRAKLLG +E709G FGRAKQLG 1 1 1 10 1 1 A KIPVAIKELREATSPKANKEILD Codon 858 G G Fig 1.
A871G
protein
substitution
true positive
P00533
Point mutations 719 G 740 750 760 * * * KIPVAIKELREATSPKANKEILD Codon 719 C KIPVAIKELREATSPKANKEILD FGLAKLLG +E709H FGLAKLLG 12 FGRAKLLG FGRAKLLG +A871G FGRAKLLG +E709G FGRAKQLG 1 1 1 10 1 1 A KIPVAIKELREATSPKANKEILD Codon 858 G G Fig 1.
L858R
protein
substitution
true positive
P00533
Of note, del746-750 might be superior to L858R mutations for prediction of gefitinib response.
In Figure 2, patient L703, L1492, and L1362 had EGFR mutations (delE746-A750, L858R, and E746-S752insA, respectively).
There were three patients with EGFR mutations (two with L858R and one with G719A) whose CEA level increased after gefitinib treatment but did not have measurable diseases.
There were also two patients with EGFR mutations, one with L858R E709H and one with I744-K745 ins KIPVAI whose tumor progressed.
Gefitinib sensitivity was essentially the same in COS cells transfected with L858R and in cells transfected with del L747-P753insS.16 A more recent study showed that the tyrosine residue at codon 845 is highly phosphorylated in L858R mutants, but not in deletion mutants after epidermal growth factor binding.23 This might explain the difference in gefitinib response between tumors with L858R and those with deletions.
E709H
protein
substitution
true positive
P00533
Point mutations 719 G 740 750 760 * * * KIPVAIKELREATSPKANKEILD Codon 719 C KIPVAIKELREATSPKANKEILD FGLAKLLG +E709H FGLAKLLG 12 FGRAKLLG FGRAKLLG +A871G FGRAKLLG +E709G FGRAKQLG 1 1 1 10 1 1 A KIPVAIKELREATSPKANKEILD Codon 858 G G Fig 1.
There were also two patients with EGFR mutations, one with L858R E709H and one with I744-K745 ins KIPVAI whose tumor progressed.
10190908
full text
D461N
protein
substitution
true positive
P43403
p116 cells stably expressing a vesicular stomatitis virus (VSV) epitope-tagged wild-type (WT) or mutant ZAP-70 (Y319F or D461N [kinase dead]) have been previously described.41,42 CD4 T cells from a previously described ZAP-70deficient patient27 as well as control CD4 T cells were cultured in Yssel medium43 supplemented with 1% human AB serum and recombinant human interleukin-2 (IL-2) (100 U/mL).
Y319F
protein
substitution
true positive
P43403
p116 cells stably expressing a vesicular stomatitis virus (VSV) epitope-tagged wild-type (WT) or mutant ZAP-70 (Y319F or D461N [kinase dead]) have been previously described.41,42 CD4 T cells from a previously described ZAP-70deficient patient27 as well as control CD4 T cells were cultured in Yssel medium43 supplemented with 1% human AB serum and recombinant human interleukin-2 (IL-2) (100 U/mL).
We therefore assessed phosphorylation in p116 cells stably expressing a Y319F mutant of ZAP-70.
(C) TCR- phosphorylation was monitored in whole cell lysates of unstimulated ( ) or CD3-stimulated ( ) p116 cells stably expressing WT ZAP-70, Y319F ZAP-70, or a kinase-dead (KD) ZAP-70.
Although elimination of the entire interdomain of ZAP-70 (including regulatory tyrosines Y292, Y315, and Y319) has not been found to modulate phosphorylation,56 the importance of an Lck/ZAP-70 complex, mediated via Y319 of ZAP-70, is underlined by the finding that inhibiting this interaction results in reduced IL-2 secretion following TCR activation.42,57 Moreover, the individual mutation of Y315 and Y292 of ZAP-70 has been shown to result in attenuated and augmented chain phosphorylation, respectively.58,59 We observed defective basal, but not CD3-induced, chain phosphorylation in p116 cells into which a Y319F ZAP-70 mutant was introduced.
10554038
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11827982
full text