A Novel Role for Gab1 and SHP2 in Epidermal Growth Factor-induced Ras Activation

Abstract

SHP2 was recently found to down-regulate PI3K activation by dephosphorylating Gab1 but the mechanisms explaining the positive role of the Gab1/SHP2 pathway in EGF-induced Ras activation remain ill defined. Substrate trapping experiments now suggest that SHP2 dephosphorylates other Gab1 phosphotyrosines located within a central region displaying four YXXP motifs. Because these sites are potential docking motifs for Ras-GAP, we tested whether SHP2 dephosphorylates them to facilitate Ras activation. We observed that a Gab1 construct preventing SHP2 recruitment promoted membrane relocation of RasGAP. Moreover, a RasGAP-inactive mutant restored the activation of Ras in cells transfected with SHP2-inactivating Gab1 mutant or in SHP2-deficient fibroblasts, supporting the hypothesis that RasGAP is a downstream target of SHP2. To determine whether Gab1 is a RasGAP-binding partner, a Gab1 mutant deleted of four YXXP motifs was produced. The deletion suppressed RasGAP redistribution and restored the defective Ras activation caused by SHP2-inactivating mutations. Moreover, Gab1 was found to interact with RasGAP SH2 domains, only under conditions where SHP2 is not activated. To identify Ras-GAP-binding sites, Tyr to Phe mutants of Gab1 YXXP motifs were produced. Gab1 constructs mutated on Tyr317 were severely affected in RasGAP binding and were the most active in compensating for Ras-defective activation and blocking RasGAP redistribution induced by SHP2 inactivation. We have thus localized on Gab1 a Ras-negative regulatory tyrosine phosphorylation site involved in RasGAP binding and showed that an important SHP2 function is to down-regulate its phosphorylation to disengage RasGAP and sustain Ras activation. SHP2 was recently found to down-regulate PI3K activation by dephosphorylating Gab1 but the mechanisms explaining the positive role of the Gab1/SHP2 pathway in EGF-induced Ras activation remain ill defined. Substrate trapping experiments now suggest that SHP2 dephosphorylates other Gab1 phosphotyrosines located within a central region displaying four YXXP motifs. Because these sites are potential docking motifs for Ras-GAP, we tested whether SHP2 dephosphorylates them to facilitate Ras activation. We observed that a Gab1 construct preventing SHP2 recruitment promoted membrane relocation of RasGAP. Moreover, a RasGAP-inactive mutant restored the activation of Ras in cells transfected with SHP2-inactivating Gab1 mutant or in SHP2-deficient fibroblasts, supporting the hypothesis that RasGAP is a downstream target of SHP2. To determine whether Gab1 is a RasGAP-binding partner, a Gab1 mutant deleted of four YXXP motifs was produced. The deletion suppressed RasGAP redistribution and restored the defective Ras activation caused by SHP2-inactivating mutations. Moreover, Gab1 was found to interact with RasGAP SH2 domains, only under conditions where SHP2 is not activated. To identify Ras-GAP-binding sites, Tyr to Phe mutants of Gab1 YXXP motifs were produced. Gab1 constructs mutated on Tyr317 were severely affected in RasGAP binding and were the most active in compensating for Ras-defective activation and blocking RasGAP redistribution induced by SHP2 inactivation. We have thus localized on Gab1 a Ras-negative regulatory tyrosine phosphorylation site involved in RasGAP binding and showed that an important SHP2 function is to down-regulate its phosphorylation to disengage RasGAP and sustain Ras activation. The GTPase Ras is a crucial signaling relay of receptor tyrosine kinases, and the mechanisms controlling its activation by growth factors appear well understood. For example, the activated epidermal growth factor (EGF) 1The abbreviations used are: EGF, epidermal growth factor; EGFR, EGF receptor; Gab1-ΔPQ, Gab1 deleted between Pro161 and Gln318; GAP, GTPase-activating protein; GAP3S, SH2-SH3-SH2 domains of RasGAP; GST, glutathione S-transferase; HA, hemagglutinin epitope tag; IB, immunoblot; IP, immunoprecipitation; PDGF, platelet-derived growth factor; PI3K, phosphoinositide 3-kinase; RBD, Ras-GTP binding domain; SH, Src homology; SHP2-C/S, SHP2 catalytically inactive mutant; WT, wild-type; Erk1/2, extracellular signal-regulated kinases 1 and 2; MEF, mouse embryo fibroblast; C/S, C459S mutant; 2RQ, R789Q/R903Q double mutant. receptor (EGFR) autophosphorylates on tyrosine residues, which creates docking sites for phosphotyrosine-binding domains (e.g. SH2) of adaptor proteins, including Grb2 and Shc. Because Grb2 is constitutively associated with Sos, a guanine nucleotide exchange factor of Ras, the binding of Grb2 to phosphorylated EGFR results in the recruitment of Sos to the plasma membrane and has been proposed as a model for activation of membrane-bound Ras. Once activated, Ras can stimulate several effectors, notably the protein Ser/Thr kinase Raf1 and the downstream activated mitogen-activated protein kinases Erk1/2, major regulators of cell proliferation, differentiation, and survival (1Schlessinger J. Cell. 2000; 103: 211-225Abstract Full Text Full Text PDF PubMed Scopus (3538) Google Scholar). Although this pathway has reached a canonical status, the docking protein Gab1 and one of its binding partners, SHP2, were recently found to participate in Ras/Erk activation in response to EGF and other growth factors (2Shi Z.Q. Yu D.H. Park M. Marshall M. Feng G.S. Mol. Cell. Biol. 2000; 20: 1526-1536Crossref PubMed Scopus (190) Google Scholar, 3Schaeper U. Gehring N.H. Fuchs K.P. Sachs M. Kempkes B. Birchmeier W. J. Cell Biol. 2000; 149: 1419-1432Crossref PubMed Scopus (292) Google Scholar, 4Cunnick J.M. Dorsey J.F. Munoz-Antonia T. Mei L. Wu J. J. Biol. Chem. 2000; 275: 13842-13848Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, 5Yart A. Laffargue M. Mayeux P. Chretien S. Peres C. Tonks N.K. Roche S. Payrastre B. Chap H. Raynal P. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar). SHP2 is a with SH2 domains and phosphorylation sites with for Grb2 SH2 is by of its SH2 domains with phosphorylated motifs in the of EGF are by Gab1 an Ser/Thr and Tyr phosphorylation sites, and Grb2 motifs. response to EGF, Gab1 is Grb2 in the of the activated EGFR and a Gab1 and that are for SHP2 H. Cell Biol. Full Text Full Text PDF PubMed Scopus Google and the of phosphoinositide the of which are involved in Gab1 recruitment A. Laffargue M. Mayeux P. Chretien S. Peres C. Tonks N.K. Roche S. Payrastre B. Chap H. Raynal P. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar, M. J. Mol. Cell. Biol. 2000; 20: PubMed Scopus Google Scholar). is now well that SHP2 is an that a positive role in the to growth and the Ras/Erk pathway the major signaling by this (2Shi Z.Q. Yu D.H. Park M. Marshall M. Feng G.S. Mol. Cell. Biol. 2000; 20: 1526-1536Crossref PubMed Scopus (190) Google Scholar, 3Schaeper U. Gehring N.H. Fuchs K.P. Sachs M. Kempkes B. Birchmeier W. J. Cell Biol. 2000; 149: 1419-1432Crossref PubMed Scopus (292) Google Scholar, 4Cunnick J.M. Dorsey J.F. Munoz-Antonia T. Mei L. Wu J. J. Biol. Chem. 2000; 275: 13842-13848Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, 5Yart A. Laffargue M. Mayeux P. Chretien S. Peres C. Tonks N.K. Roche S. Payrastre B. Chap H. Raynal P. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar, H. Cell Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). the mechanisms this have SHP2 has been proposed to as an adaptor in the of platelet-derived growth factor can to the receptor phosphorylated on its sites W. A. J. Mol. Cell. Biol. PubMed Google Scholar). SHP2 participate in Ras activation by to the recruitment of in the of the this model has been recently by several the phosphorylation of SHP2, to not under EGF (2Shi Z.Q. Yu D.H. Park M. Marshall M. Feng G.S. Mol. Cell. Biol. 2000; 20: 1526-1536Crossref PubMed Scopus (190) Google Scholar, 5Yart A. Laffargue M. Mayeux P. Chretien S. Peres C. Tonks N.K. Roche S. Payrastre B. Chap H. Raynal P. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar, T. H. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). the of SHP2 is important for Ras/Erk activation induced by EGF, or growth which that SHP2 not function as an adaptor protein in response to these growth factors A. Laffargue M. Mayeux P. Chretien S. Peres C. Tonks N.K. Roche S. Payrastre B. Chap H. Raynal P. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar, U. S. A. PubMed Scopus Google Scholar, M. Park M. Mol. Cell. Biol. 2000; 20: PubMed Scopus Google Scholar). for a has SHP2 mutants with but defective in activation T. H. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). the of SHP2 function in Ras activation to the of for which phosphorylation is to Ras activation. SHP2 was found to and docking can Src activation by its with its regulatory kinase S. Mei L. Wu J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, W. T. J. Mol. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). downstream of Ras can this activation SHP2 was to activation in response to growth factor by dephosphorylating a protein can as a for Grb2 SH2 H. S. T. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). that this under EGF SHP2 is not to the between the activated EGFR and Grb2 (2Shi Z.Q. Yu D.H. Park M. Marshall M. Feng G.S. Mol. Cell. Biol. 2000; 20: 1526-1536Crossref PubMed Scopus (190) Google Scholar, 5Yart A. Laffargue M. Mayeux P. Chretien S. Peres C. Tonks N.K. Roche S. Payrastre B. Chap H. Raynal P. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar). that Gab1 is a SHP2 have been that this participate in Ras activation. in have to the of a Gab1 as a of the SHP2 J. T. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar, J. J. PubMed Scopus Google Scholar). a in cells has that Gab1 can with SHP2 catalytically inactive which is a that Gab1 can a SHP2 J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). SHP2 was found to PI3K activation in response to EGF by dephosphorylating the sites of Gab1 T. L. Mol. Cell. Biol. PubMed Scopus Google Scholar). the of Gab1 phosphorylation sites M. PubMed Scopus Google that the Gab1 residues, which for Ras activation. For example, these sites docking for Ras regulatory proteins, and SHP2 function to recruitment in EGFR signaling by dephosphorylating for a Ras of its docking is the GTPase-activating protein that Ras by on its RasGAP SH2 domains that have binding for the phosphorylated YXXP The major binding of RasGAP are proteins, a of in YXXP motifs Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). Gab1 of these motifs. is not whether Gab1 can with RasGAP. can Gab1 YXXP including the and and the thus these have been as Gab1 binding only in response to growth factor M. U. Sachs M. Birchmeier W. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar, P. S. S. 2000; PubMed Scopus Google Scholar). EGF these sites have not been to of them appear to phosphorylated in by the EGFR P. S. S. 2000; PubMed Scopus Google Scholar, S. J. A. C. B. J. H. H. W. PubMed Scopus Google Scholar). this we have the of whether Gab1 YXXP motifs docking sites for RasGAP and target sites for by SHP2. trapping and we have that Gab1 YXXP motifs can RasGAP and that an function of SHP2 is to down-regulate this thus an activation of Ras in response to EGF was epitope was and and were The epitope was Roche The was The was Cell The was the of the by P. Cell were and SHP2-C/S, Ras and have been A. Laffargue M. Mayeux P. Chretien S. Peres C. Tonks N.K. Roche S. Payrastre B. Chap H. Raynal P. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar, M. Raynal P. A. Peres C. Roche S. Payrastre B. Chap H. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, A. Roche S. Laffargue M. Tonks N.K. Mayeux P. Chap H. Raynal P. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). was by A. To the RasGAP inactive we to or in with the Gab1 constructs used in this and were the that was of on mutants were by and EGF-induced phosphorylation and membrane Cell and was in cells cell and in mouse embryo cell the SHP2 or a of the Z.Q. W. Feng G.S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). were in with and For of were with of of and of and MEF, were of of of and of and cells were by were for with or Cell and were in of and and 1 for by a for was with the for The were with protein or protein for 1 by and with 1 of and were by and a were For of cell cells were in and for in and was as the of Raf1 as an for A. Laffargue M. Mayeux P. Chretien S. Peres C. Tonks N.K. Roche S. Payrastre B. Chap H. Raynal P. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar). The was as protein in and To Ras activation in were with and of and the we have used of and of Gab1 or RasGAP cells were in 1 of 1 of and were for with of to were in and in and were by by with the SH2-SH3-SH2 domains of RasGAP or only the SH2 have been M. B. PubMed Scopus Google by J. The were in and The was the that of protein were used and 1 with were as A. A. B. H. U. S. A. PubMed Scopus Google Scholar). were in of and and 1 and were The was for and the was in The was and the was in with The was and the was as the membrane results are of experiments in Gab1 SHP2 in Ras and SHP2 Gab1 was recently to down-regulate the EGF-induced pathway by dephosphorylating Gab1 motifs T. L. Mol. Cell. Biol. PubMed Scopus Google Scholar). with we observed an of PI3K with Gab1 and activation in cells transfected with a Gab1 mutant to SHP2 A. Laffargue M. Mayeux P. Chretien S. Peres C. Tonks N.K. Roche S. Payrastre B. Chap H. Raynal P. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google and in SHP2-deficient not Although was to that this positive for Ras activation. We thus the Tyr of the motifs to Phe in the construct to the mutant. The has a on EGF-induced Ras/Erk activation J.M. Dorsey J.F. Munoz-Antonia T. Mei L. Wu J. J. Biol. Chem. 2000; 275: 13842-13848Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, 5Yart A. Laffargue M. Mayeux P. Chretien S. Peres C. Tonks N.K. Roche S. Payrastre B. Chap H. Raynal P. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar). We that Gab1 motifs were SHP2 in Ras or the of the Ras activation which that of Gab1 motifs by SHP2 a positive on Ras activation. We that SHP2 Gab1 not only on To this we have trapping experiments the SHP2 catalytically inactive mutant C459S that can with potential SHP2 J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The was by with Gab1 mutants Tyr residues, and the of an between these was by by Gab1 mutants tested in trapping experiments were the to the binding of its SH2 that SHP2-C/S, but not with cells were with EGF, as was that SHP2 dephosphorylates Gab1 motifs T. L. Mol. Cell. Biol. PubMed Scopus Google Scholar). of sites in the mutant the between and SHP2-C/S, but only to a that Gab1 sites, that is not to PI3K SHP2 Gab1 on between Pro161 and to identify these sites by SHP2-C/S, that one of them involved in a of Ras activation. to or sites, Gab1 Tyr residues, which a a central region located between Pro161 and four of the potential RasGAP-binding YXXP motifs Because this region not for Gab1 we deleted as a to determine whether Tyr by deletion was in and in the to trapping To the of these mutants to we have membrane and phosphorylation in response to that these mutants to other Gab1 constructs in of EGF-induced relocation to membrane which that the deleted region is not for Gab1 the phosphorylation of mutants is in with that of or that the deleted region phosphorylation sites this the construct was with to a trapping in with in the deletion severely the between and that SHP2 dephosphorylates Tyr located between Pro161 and RasGAP the Gab1/SHP2 the Gab1 region potential RasGAP-binding the suggest that SHP2 Ras activation by a between Gab1 and that RasGAP a downstream target of Gab1 and SHP2. To this we have the of RasGAP with membrane as of RasGAP in cells are transfected with EGF a redistribution of RasGAP. The membrane redistribution of Gab1 was used to the of cell and membrane to of in an important membrane relocation of RasGAP in response to EGF, which that the Gab1/SHP2 pathway RasGAP recruitment under To this hypothesis we have a RasGAP-inactive mutant to determine whether its the for the Gab1/SHP2 pathway in Ras activation. The RasGAP mutant was to a protein to interact with Ras but its other domains, to W. L. L. Marshall J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, Biol. PubMed Scopus Google Scholar). We have thus to RasGAP to or in with To the of these on the constructs were in and to with Ras activation was with that of in of the mutant the activation of Ras induced by EGF, to the of that the mutant a of the double mutant R789Q/R903Q a on Ras which that this mutant has most of its the construct used as a RasGAP mutant to whether RasGAP is a downstream target of SHP2. was by the of to for the defective Ras activation caused by SHP2 inactivation. mutant was transfected in cells to determine whether the Ras of the in the mutant the EGF-induced activation of Ras by this mutant not the of in cells which that the of the EGF-induced response is not to an on Ras activation. Moreover, to that the mutant has of Gab1 or SHP2, we have that its not the EGF-induced Gab1 phosphorylation of the binding of to SHP2 not To determine whether the mutant the for SHP2 in a was in mouse embryo cells a of SHP2 cells a activation of Ras in response to EGF, with (2Shi Z.Q. Yu D.H. Park M. Marshall M. Feng G.S. Mol. Cell. Biol. 2000; 20: 1526-1536Crossref PubMed Scopus (190) Google Scholar). in of the mutant in the activation of Ras induced by EGF, the in these the hypothesis that RasGAP is a downstream target of the Gab1/SHP2 Gab1 to RasGAP SH2 the Gab1 potential RasGAP-binding we have the that target sites for by SHP2. The was to the of to with RasGAP membrane We that the Gab1 region the sites RasGAP relocation SHP2 is not its deletion RasGAP redistribution by of the and EGF in of only a on RasGAP relocation in with as this relocation is induced under conditions that SHP2 activation of the deletion in the the RasGAP of the the that the deleted region sites the by SHP2 of RasGAP To the between Gab1 and we an with or of the SH2-SH3-SH2 region of were on and used as with the were by We whether the Gab1 in of a cell that conditions to an by SHP2. the RasGAP SH2-SH3-SH2 region is used as Gab1 is a positive with PI3K SH2 domains were used to Gab1 cells which the conditions and that SHP2 PI3K with Gab1 T. L. Mol. Cell. Biol. PubMed Scopus Google Scholar). a Gab1 is the is with that only the SH2 of RasGAP not that phosphorylated Gab1 with RasGAP SH2 domains and that this is by SHP2. To Gab1 RasGAP-binding sites, we have used the mutant. in its to with RasGAP was the To the the was with transfected and constructs as the protein in the RasGAP region Gab1 the one the deletion of the a deletion of this region not and the between Gab1 and SH2 domains, an that to a between and binding to the deleted these results that the deletion Gab1 RasGAP-binding that with RasGAP SH2 domains, which is in of Gab1 by SHP2 on RasGAP-binding We these experiments that RasGAP SH2 domains with Gab1 motifs located within the Gab1 for RasGAP the for SHP2 in Ras the that SHP2 in Ras activation by controlling the recruitment of RasGAP. this Ras activation of SHP2 in conditions where the EGFR only Gab1 RasGAP-binding To this we have whether of in cells SHP2 function as a positive of Ras activation. in in the the deletion has a on Ras which is not the deleted region RasGAP-binding motifs. in the the deletion the Ras of the that the deleted region Ras sites by SHP2. To this a was in in of the defective Ras activation in these results the in the region of Ras sites that by SHP2 to a activation of Ras. of Gab1 RasGAP-binding the Gab1 Tyr involved in RasGAP of the Tyr of the region was with Phe to the and mutants were in the and were tested for to RasGAP the in the double a on the Gab1 binding to the construct mutated on the has an for RasGAP that these Tyr major Ras-GAP-binding We have the of the double mutants to with RasGAP membrane relocation induced by the and EGF that the mutant this the mutant has a which that and are for RasGAP The Gab1 double mutants were by to the defective Ras activation in of the but not of the Ras activation induced by EGF, which the of the RasGAP-binding site on and we the of the by the double Tyr mutants with that of the deletion a on and Ras activation The mutant to the mutant in of of RasGAP binding and of to Ras activation in these results that and major RasGAP-binding sites on Gab1 and the that these sites by SHP2 to an activation of Ras. we to the of the RasGAP-binding site to a Gab1 To and we have of an that the phosphorylated of Gab1 We have thus the EGF-induced phosphorylation of this site in cells or a RasGAP docking a target of SHP2, its phosphorylation in cells to SHP2. that this is not the phosphorylation was in cells results were by phosphorylation on Gab1 in and not Because these results to as a SHP2 we have a Phe mutant of Tyr317 in the to its to RasGAP. in the mutant has an for severely with or to the suppressed the membrane relocation of RasGAP induced by the SHP2-inactivating which that Tyr317 RasGAP we have the of to Ras activation in of this mutant the of Ras induced by EGF these results that Tyr317 is an important RasGAP-binding that by SHP2 to a activation of Ras. a of that Gab1 is not only an but a of SHP2, that a of the between the and the major signaling relay of the EGFR on of Ras activation. was only that SHP2 can Gab1 sites T. L. Mol. Cell. Biol. PubMed Scopus Google was to that of Gab1 motifs positive for Ras activation. motifs on the receptor were recently found to PI3K a regulatory function by the receptor to H. L. J.M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). PI3K activation participate in Ras activation A. Laffargue M. Mayeux P. Chretien S. Peres C. Tonks N.K. Roche S. Payrastre B. Chap H. Raynal P. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google one not an between PI3K and Ras, for to an recruitment of in the of we have found that of its motifs not but the activation of Ras to an SHP2-inactivating which that sites a positive on Ras activation. We have the that SHP2 Gab1 on other Tyr trapping as an to identify SHP2 J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google we have observed that catalytically inactive was to with a Gab1 mutant and the Tyr residues, were within YXXP motifs have a potential for signaling were as docking sites for the SH2 domains of and M. PubMed Scopus Google Scholar). these have not been to with Gab1 under EGF other binding and RasGAP to an The RasGAP was to the that its SH2 domains the found on the receptor for A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google but on with or the EGFR showed that the YXXP is to RasGAP P. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar, Mol. Cell. Biol. PubMed Scopus Google Scholar). was thus important to the role of Gab1 YXXP as potential RasGAP-binding motifs and target for SHP2. a we have deleted Gab1 of a region located between Pro161 and to four YXXP motifs in a of a that not Gab1 the of to these sites to mutated one the The experiments with this deletion mutant several in of the hypothesis that Gab1 RasGAP-binding motifs that by SHP2 to a activation of the mutant the to by in with suppressed the membrane of RasGAP induced by a Gab1 mutant preventing SHP2 the of Gab1 with RasGAP SH2 domains was in the its for was the deletion the for SHP2 in suppressed the of the in cells and restored the defective Ras activation in The was to the YXXP involved in RasGAP binding within the We a of Gab1 constructs Tyr to Phe on of the YXXP which to that Tyr317 is an important for binding RasGAP. of this in the and the double Gab1 with RasGAP SH2 Moreover, the constructs mutated on Tyr317 the membrane relocation of RasGAP induced by of Gab1 SHP2-inactivating mutant and restored Ras activation in The of in this SHP2 was not activated We thus these that Tyr317 an important target site for by SHP2 that can down-regulate RasGAP a on most by the phosphorylation sites of Gab1 between and SHP2-deficient cells and the of this signaling The that RasGAP can a downstream target of SHP2 in EGFR signaling has been recently on the that RasGAP has for a site on the EGFR that can by catalytically inactive SHP2 Mol. Cell. Biol. PubMed Scopus Google Scholar). Although we an to RasGAP with to the of a between SHP2 and RasGAP. we have been to that of a Gab1 mutant to SHP2 activation in a redistribution of RasGAP in membrane under EGF which was a that RasGAP is a downstream target of the Gab1/SHP2 Moreover, on the of that sites W. L. L. Marshall J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, Biol. PubMed Scopus Google we have for the a RasGAP-inactive mutant that as a RasGAP mutant. to restored the defective activation of Ras in of SHP2 of the of activated Ras of the signaling of SHP2, which to the of this RasGAP to an important downstream target of the on the pathway EGFR to Ras to SHP2 function downstream of other the or in A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, P. C. Cell. Google Scholar). to this is the that SHP2 the recruitment of RasGAP not to a receptor but to a downstream on the results in this and on the we the model in to the role of SHP2 Ras. We that the can PI3K activation by dephosphorylating Gab1 sites T. L. Mol. Cell. Biol. PubMed Scopus Google but the of this the positive role of SHP2 in we have located on Gab1 Ras tyrosine phosphorylation sites involved in the binding to RasGAP. We that an important function of SHP2 is to these sites to disengage RasGAP Ras activation model is with that SHP2 is involved in the of Ras activation in the of this activation (2Shi Z.Q. Yu D.H. Park M. Marshall M. Feng G.S. Mol. Cell. Biol. 2000; 20: 1526-1536Crossref PubMed Scopus (190) Google Scholar, Mol. Cell. Biol. PubMed Scopus Google Scholar). to SHP2 is not for the of to Ras not the binding of to the Gab1 phosphorylation RasGAP can SHP2 to down-regulate this recruitment and to this the of Ras activation. that to the of a signaling of a protein a and a protein that the binding of this Because Gab1 potential RasGAP-binding sites to of we that these sites a an of the Ras pathway induced by the that only a of EGFR and Gab1 to and to disengage Ras-GAP, can to a activation of Ras. to was in signaling P. C. Cell. Google this the of the and of the EGFR by SHP2. the other have that RasGAP have for in cell or activation P. M. T. J. Cell Biol. 2000; 149: PubMed Scopus Google Scholar, J. M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). important for EGF to in a of EGFR to disengage RasGAP to an of Ras activation. of the of RasGAP recruitment and associated downstream are to this We and for