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• W1973400979 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: 8856-8864Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). SHP2 is a protein-tyrosine phosphatase with two SH2 domains and phosphorylation sites with affinity for Grb2 SH2 domains. Its catalytic activity is stimulated by engagement of its SH2 domains with specific phosphorylated motifs that, in the case of EGF signaling, are provided by Gab1. Gab1 contains an N-terminal pleckstrin homology domain, multiple Ser/Thr and Tyr phosphorylation sites, and two Grb2 SH3-binding motifs. In response to EGF, Gab1 is recruited through Grb2 in the vicinity of the activated EGFR and becomes phosphorylated. As a result, Gab1 attracts and activates two partners that are essential for EGFR-mediated biological responses: SHP2 (6Gu H. Neel B.G. Trends Cell Biol. 2003; 13: 122-130Abstract Full Text Full Text PDF PubMed Scopus (322) Google Scholar) and the p85/p110 subtype of phosphoinositide 3-kinase (PI3K), the lipid products of which are also involved in Gab1 recruitment (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: 8856-8864Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, 7Rodrigues G.A. Falasca M. Zhang Z. Ong S.H. Schlessinger J. Mol. Cell. Biol. 2000; 20: 1448-1459Crossref PubMed Scopus (285) Google Scholar). It is now well accepted that SHP2 is functionally an atypical phosphatase that plays a positive role in the biological responses to growth factors, and the Ras/Erk pathway represents the major signaling module positively regulated by this phosphatase (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: 8856-8864Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, 6Gu H. Neel B.G. Trends Cell Biol. 2003; 13: 122-130Abstract Full Text Full Text PDF PubMed Scopus (322) Google Scholar). Yet, the mechanisms underlying this regulation have remained elusive. SHP2 has been first proposed to act as an adaptor in the case of platelet-derived growth factor (PDGF) signaling, because it can bind to the PDGF receptor then become phosphorylated on its Grb2-binding sites (8Li W. Nishimura R. Kashishian A. Batzer A.G. Kim W.J. Cooper J.A. Schlessinger J. Mol. Cell. Biol. 1994; 14: 509-517Crossref PubMed Google Scholar). SHP2 could hence participate in Ras activation by contributing to the recruitment of Grb2/Sos in the vicinity of the PDGF receptor. However, this model has been recently challenged by several reports. First, the phosphorylation of SHP2, required to bind Grb2, seems marginal, if not undetectable, under EGF stimulation (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: 8856-8864Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, 9Araki T. Nawa H. Neel B.G. J. Biol. Chem. 2003; 278: 41677-41684Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar). In addition, the catalytic activity of SHP2 is important for Ras/Erk activation induced by EGF, insulin, or hepatocyte growth factor, which implies that SHP2 does not simply function as an adaptor protein in response to these growth factors (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: 8856-8864Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, 10Yamauchi K. Milarski K.L. Saltiel A.R. Pessin J.E. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 664-668Crossref PubMed Scopus (268) Google Scholar, 11Maroun C.R. Naujokas M.A. Holgado-Madruga M. Wong A.J. Park M. Mol. Cell. Biol. 2000; 20: 8513-8525Crossref PubMed Scopus (241) Google Scholar). This may be also true for PDGF signaling, because a recent study has identified SHP2 mutants with normal Grb2-binding ability but defective in mediating PDGF-induced Erk activation (9Araki T. Nawa H. Neel B.G. J. Biol. Chem. 2003; 278: 41677-41684Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar). Thus, the elucidation of SHP2 function in Ras activation seems to require the identification of novel substrate(s) for which phosphorylation is detrimental to Ras activation. Recently, SHP2 was found to dephosphorylate Cbp/PAG and paxillin, two docking proteins that, when phosphorylated, can restrain Src activation by promoting its interaction with its regulatory kinase Csk (12Ren Y. Meng S. Mei L. Zhao Z.J. Jove R. Wu J. J. Biol. Chem. 2004; 279: 8497-8505Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar, 13Zhang S.Q. Yang W. Kontaridis M.I. Bivona T.G. Wen G. Araki T. Luo J. Thompson J.A. Schraven B.L. Philips M.R. Neel B.G. Mol. Cell. 2004; 13: 341-355Abstract Full Text Full Text PDF PubMed Scopus (364) Google Scholar). Nevertheless, downstream of Src, how Ras can benefit from this activation remains poorly documented. In addition, SHP2 was reported to promote Erk activation in response to fibroblast growth factor by dephosphorylating Sprouty2, a protein that, when phosphorylated, can act as a trap for Grb2 SH2 domain (14Hanafusa H. Torii S. Yasunaga T. Matsumoto K. Nishida E. J. Biol. Chem. 2004; 279: 22992-22995Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). Nevertheless, it seems unlikely that this mechanism could also occur under EGF stimulation, because SHP2 is not known to maintain the interaction 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: 8856-8864Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). Evidences that Gab1 itself is a SHP2 substrate have been reported, suggesting that this interaction might participate in Ras activation. First, in Drosophila, genetic analysis have led to the identification of Dos, a Gab1 homolog, as a substrate of Csw, the SHP2 homolog (15Herbst R. Carroll P.M. Allard J.D. Schilling J. Raabe T. Simon M.A. Cell. 1996; 85: 899-909Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar, 16Herbst R. Zhang X. Qin J. Simon M.A. EMBO J. 1999; 18: 6950-6961Crossref PubMed Scopus (40) Google Scholar). More recently, a “substrate-trapping” approach in mammalian cells has shown that Gab1 can form stable complexes with SHP2 catalytically inactive mutants, which is a strong indication that Gab1 can be also a SHP2 substrate (17Agazie Y.M. Hayman M.J. J. Biol. Chem. 2003; 278: 13952-13958Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). In agreement, SHP2 was found to control PI3K activation in response to EGF by dephosphorylating the PI3K-binding sites of Gab1 (18Zhang S.Q. Tsiaras W.G. Araki T. Wen G. Minichiello L. Klein R. Neel B.G. Mol. Cell. Biol. 2002; 22: 4062-4072Crossref PubMed Scopus (209) Google Scholar). However, the multiplicity of Gab1 phosphorylation sites (19Holgado-Madruga M. Emlet D.R. Moscatello D.K. Godwin A.K. Wong A.J. Nature. 1996; 379: 560-564Crossref PubMed Scopus (601) Google Scholar) suggests that the phosphatase could dephosphorylate Gab1 phosphotyrosine residues, which could be unfavorable for Ras activation. For example, these sites could provide docking signals for Ras negative regulatory proteins, and then SHP2 function would be to control their recruitment in EGFR signaling complexes by dephosphorylating Gab1. A good candidate for such a Ras negative regulator controlled through dephosphorylation of its docking site(s) is p120-Ras-GAP, the GTPase-activating protein that inactivates Ras by turning on its intrinsic enzymatic activity. RasGAP contains two SH2 domains that have binding affinity for the phosphorylated YXXP motif. The major binding partners of RasGAP are DOK proteins, a family of adaptors preferentially expressed in lymphoid tissues. Their prototype, DOK1, contains six YXXP motifs (20Yamanashi Y. Baltimore D. Cell. 1997; 88: 205-211Abstract Full Text Full Text PDF PubMed Scopus (311) Google Scholar). Strikingly, Gab1 also contains six of these motifs. Yet, it is not known whether Gab1 can associate with RasGAP. Various candidates can potentially recognize Gab1 YXXP motifs, including the adaptors Nck and Crk, and the phospholipase Cγ. However, thus far, these proteins have been identified as Gab1 binding partners only in response to hepatocyte growth factor stimulation (21Sakkab D. Lewitzky M. Posern G. Schaeper U. Sachs M. Birchmeier W. Feller S.M. J. Biol. Chem. 2000; 275: 10772-10778Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, 22Gual P. Giordano S. Williams T.A. Rocchi S. Van Obberghen E. Comoglio P.M. Oncogene. 2000; 19: 1509-1518Crossref PubMed Scopus (138) Google Scholar). Under EGF stimulation, these sites have not been allocated to any particular effector, even though some of them appear to be phosphorylated in vitro by the EGFR (22Gual P. Giordano S. Williams T.A. Rocchi S. Van Obberghen E. Comoglio P.M. Oncogene. 2000; 19: 1509-1518Crossref PubMed Scopus (138) Google Scholar, 23Lehr S. Kotzka J. Herkner A. Klein E. Siethoff C. Knebel B. Noelle V. Bruning J. Klein H. Meyer H. Krone W. Muller-Wieland D. Biochemistry. 1999; 38: 151-159Crossref PubMed Scopus (46) Google Scholar). In this report, we have addressed the question of whether Gab1 YXXP motifs provide docking sites for RasGAP and constitute target sites for dephosphorylation by SHP2. Using substrate trapping and site-directed mutagenesis, we have obtained evidence that Gab1 YXXP motifs can recruit RasGAP and that an essential function of SHP2 is to down-regulate this interaction, thus allowing an efficient activation of Ras in response to EGF. Materials—Human recombinant EGF was from Calbiochem. Monoclonal antibody against Myc epitope tag (clone 9E10) was from Santa Cruz Biotechnology, Inc. Monoclonal anti-EGFR (clone LA1) and polyclonal anti-Gab1 (Catalog number 06-579) and anti-RasGAP (Catalog number 06-157) were from Upstate Biotechnologies, Inc. The monoclonal anti-HA epitope tag (clone 12CA5) was from Roche Applied Science. The monoclonal anti-SHP2 antibody was from BD Transduction Laboratories (clone 79). The polyclonal antibody against phospho-Gab1-Tyr307 was from Cell Signaling Technology (Catalog number 3234). The monoclonal anti-phosphotyrosine antibody was produced from the culture medium of the 4G10 hybridoma (kindly provided by Dr. P. Mayeux, Paris, France). Cell culture reagents were from Bio-Whittaker, Inc. Expression Plasmids and Site-directed Mutagenesis—The plasmids encoding Gab1-Y627F, Gab1-YF3, SHP2-WT, SHP2-C/S, HA-tagged WT Ras (HA-Ras), and GST-p85 have been already described (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: 8856-8864Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, 24Laffargue M. Raynal P. Yart A. Peres C. Wetzker R. Roche S. Payrastre B. Chap H. J. Biol. Chem. 1999; 274: 32835-32841Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 25Yart A. Roche S. Wetzker R. Laffargue M. Tonks N.K. Mayeux P. Chap H. Raynal P. J. Biol. Chem. 2002; 277: 21167-21178Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). A pcDNA3 vector encoding RasGAP-WT was kindly provided by Dr. A. Yoshimura (Fukuoka, Japan). To produce the RasGAP inactive mutant, we changed Arg789 to Gln, alone or in combination with Arg903, using the QuikChange multisite-directed mutagenesis kit (Stratagene). Novel Gab1 constructs used in this study (YF3/Y627F, ΔPQ±Y627F, Y242F/Y259F±Y627F, Y307F/Y317F±Y627F, Y242F±Y627F, and Y317F± Y627F) were created using the regular QuikChange kit that was more reliable (sequences of mutagenic primers available on request). All mutants were verified by sequencing, expression, and EGF-induced phosphorylation and membrane relocation. Cell Culture, Transfection, and Stimulations—This study was performed both in Vero cells (a non-transformed monkey kidney cell line, ATCC CCL 81) and in two immortalized mouse embryo fibroblast (MEF) cell lines expressing either the endogenous SHP2 (WT) or a truncated, non-recruitable form of the phosphatase (ΔSHP2) (26Shi Z.Q. Lu W. Feng G.S. J. Biol. Chem. 1998; 273: 4904-4908Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar). Cells were maintained in Dulbecco's modified Eagle's medium supplemented with 7.5% fetal bovine serum and antibiotics. For transient transfection of Vero cells, subconfluent 60-mm plates were incubated 3 h with 2 ml of Dulbecco's modified Eagle's medium containing 2 μg of total DNA and 6 μl of each Lipofectamine and Plus reagent (Invitrogen). In MEF, two 100-mm plates per assay were used, each containing 4 ml of medium, 4 μg of total of DNA, and 20 μl of each Lipofectamine and Plus reagent. Before stimulation, cells were blocked overnight by serum starvation. Cells were stimulated for 5 min with 10 (Vero) or 30 (MEF) ng/ml EGF. Cell Lysis, Immunoprecipitation, and Immunoblotting—Cells were scrapped off in lysis buffer containing 20 mm Tris, pH 7.4, 150 mm NaCl, 10 mm EDTA, 10% glycerol, 1% Nonidet P-40, 10 μg of each aprotinin and leupeptin, and 1 mm orthovanadate. After shaking for 15 min at 4 °C followed by a 13,000 × g centrifugation for 15 min, soluble material was incubated with the appropriate antibody for 2 h at 4 °C. The antigen-antibody complexes were incubated with protein A- or protein G-Sepharose (Sigma) for 1 h, then collected by brief centrifugation and washed three times with lysis buffer containing 0.1% Nonidet P-40, 1 μg/ml each of aprotinin and leupeptin, 0.1 mm orthovanadate. Proteins were then resolved by SDS-PAGE and immunoblotting using a standard procedure. Blots were developed using chemiluminescence (Amersham Biosciences). For immunoblotting analysis of cell lysates, cells were directly scrapped off in electrophoresis sample buffer, then boiled and processed for immunoblotting. GTP-Ras Affinity Precipitation Assay in Transfected Vero and MEF Cells—The assay was performed essentially as described using the recombinant Ras-binding domain (RBD) of Raf1 as an affinity probe for GTP-Ras (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: 8856-8864Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, 27de Rooij J. Bos J.L. Oncogene. 1997; 14: 623-625Crossref PubMed Scopus (420) Google Scholar). The RBD was expressed as GST fusion protein in Escherichia coli and extracted using glutathione-Sepharose beads (Sigma). To measure Ras activation in Vero cells, they were cotransfected with 0.2 and 1.8 μg of plasmid encoding HA-tagged RasWT and the indicated effector, respectively. In MEF cells, we have used 0.4 μg of HA-Ras vector and 3.6 μg of plasmid encoding Gab1 or RasGAP constructs. Following stimulation, cells were scrapped off in 1 ml of lysis buffer containing 50 mm Tris, pH 8.0, 150 mm NaCl, 10 mm MgCl2, 0.5% deoxycholate, 1% Nonidet P-40, 0.1% SDS, 1 mm phenylmethylsulfonyl fluoride, 10 μg/ml each of aprotinin and leupeptin. Cleared lysates were incubated at 4 °C for 30 min with 10 μg of GST-RBD bound to glutathione-Sepharose beads. Beads were washed three times in lysis buffer and then boiled in standard electrophoresis sample buffer, and proteins were resolved by SDS-PAGE, followed by immunoblotting with anti-HA antibody. GST-RasGAP Binding Assay—The GST fusion proteins containing the SH2-SH3-SH2 domains of RasGAP or only the C-terminal SH2 domain have been already described (28Duchesne M. Schweighoffer F. Parker F. Clerc F. Frobert Y. Thang M.N. Tocque B. Science. 1993; 259: 525-528Crossref PubMed Scopus (110) Google Scholar) (kindly provided by Dr. J. Nunés, Marseille, France). The fusion proteins were expressed in E. coli and extracted using glutathione-Sepharose beads (Sigma). The pull-down was conducted like the GTP-Ras assay, except that 3 μg of fusion protein were used and incubated during 1 h with cleared lysates. Membrane Fractions—Fractions containing membrane-associated proteins were prepared essentially as described (29August A. Sadra A. Dupont B. Hanafusa H. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11227-11232Crossref PubMed Scopus (150) Google Scholar). Cells were scrapped off in lysis buffer containing 20 mm Tris, pH 7.4, 150 mm NaCl, 1mm EDTA, 10 mm EGTA, 2 mm MgCl2, 10 μg/ml each of aprotinin and leupeptin, and 1 mm orthovanadate and were Dounce-homogenized. The lysate was spun at 13,000 × g for 30 min, and the pellet was resuspended in lysis buffer. The sample was then centrifuged again, and the pellet was washed once then dissolved in lysis buffer supplemented with 1.2% Triton X-100. The insoluble material was spun out, and the supernatant was taken as the solubilized membrane fraction. Statistics—The results shown are representative of at least two independent experiments performed in duplicates, unless otherwise indicated. Evidence That Gab1 PI3K-binding Sites Are Not SHP2 Targets in Ras Activation and That SHP2 Dephosphorylates Other Gab1 Sites—SHP2 was recently shown to down-regulate the EGF-induced PI3K/Akt pathway by dephosphorylating Gab1 YXXM p85-binding motifs (18Zhang S.Q. Tsiaras W.G. Araki T. Wen G. Minichiello L. Klein R. Neel B.G. Mol. Cell. Biol. 2002; 22: 4062-4072Crossref PubMed Scopus (209) Google Scholar). In agreement with this, we observed an increase of both PI3K association with Gab1 and Akt activation in Vero cells transfected with a Gab1 mutant (Y627F) unable to recruit SHP2 (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: 8856-8864Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar) and in SHP2-deficient fibroblasts (data not shown). Although unlikely, it was necessary to exclude that this dephosphorylation could be somehow positive for Ras activation. We thus changed the Tyr of the three YXXM motifs to Phe in the Gab1-Y627F construct to produce the so-called YF3/Y627F mutant. The Y627F mutation has a strong dominant negative effect on EGF-induced Ras/Erk activation (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: 8856-8864Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). We therefore assumed that if Gab1 YXXM motifs were SHP2 targets in Ras activation, their mutation should suppress or reduce the dominant negative effect of the Y627F mutation. However, Gab1-YF3/Y627F inhibits Ras activation even stronger than Gab1-Y627F (Fig. 1A), which excludes that dephosphorylation of Gab1 YXXM motifs by SHP2 could produce a positive effect on Ras activation. We next hypothesized that SHP2 could dephosphorylate Gab1 not only on PI3K-binding sites. To test this hypothesis, we have performed substrate trapping experiments using the SHP2 catalytically inactive mutant C459S (C/S) that can form stable complexes with potential SHP2 substrates (17Agazie Y.M. Hayman M.J. J. Biol. Chem. 2003; 278: 13952-13958Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). The experiment was performed by cotransfecting SHP2-C/S with Gab1 mutants lacking different Tyr residues, and the existence of an interaction between these proteins was determined by coimmunoprecipitation followed by immunoblotting analysis. Of course, all Gab1 mutants tested in substrate trapping experiments were prepared from the Gab1-Y627F template to eliminate the binding of SHP2-C/S through its SH2 domains. Fig. 1B shows that SHP2-C/S, but not SHP2-WT, coimmunoprecipitates with Gab1-Y627F when cells were stimulated with EGF, as expected, because it was already demonstrated that SHP2 dephosphorylates Gab1 PI3K-binding motifs (18Zhang S.Q. Tsiaras W.G. Araki T. Wen G. Minichiello L. Klein R. Neel B.G. Mol. Cell. Biol. 2002; 22: 4062-4072Crossref PubMed Scopus (209) Google Scholar). More interestingly, disruption of all PI3K-binding sites in the Gab1-YF3/Y627F mutant reduces the interaction between Gab1-Y627F and SHP2-C/S, but only to a limited extent. This suggests that SHP2-C/S traps Gab1 residues separately from PI3K-binding sites, implying that SHP2-Gab1 interaction is not limited to PI3K regulation. SHP2 Dephosphorylates Gab1 on Residue(s) Located between Pro161 and Gln318—We attempted to identify these novel sites recognized by SHP2-C/S, assuming that one of them could be involved in recruiting a negative regulator of Ras activation. In addition to PI3K- or SHP2-binding sites, Gab1 sequence contains 15 Tyr residues, which virtually excludes a systematic mutagenesis approach. However, a central region located between Pro161 and Gln318 displays four out of the six potential RasGAP-binding YXXP motifs (Fig. 2A). Because this region does not include any known domain required for Gab1 recruitment, we deleted it as a first approach to determine whether it contained Tyr residues trapped by SHP2-C/S. This deletion was introduced in Gab1-WT and in the Gab1-Y627F background to perform substrate trapping experiments. To verify first the ability of these so-called ΔPQ mutants to be recruited, we have examined both their membrane translocation and phosphorylation in response to EGF. Fig. 2B shows that these mutants behave similarly to other Gab1 constructs in terms of EGF-induced relocation to membrane fractions, which confirmed that the deleted region is not required for Gab1 recruitment. In contrast, the phosphorylation of ΔPQ mutants is strongly reduced in comparison with that of Gab1-WT or Y627F, suggesting that the deleted region contains significant phosphorylation sites (Fig. 2C). Following this characterization, the Gab1-Y627F/ΔPQ construct was cotransfected with SHP2-C/S to perform a substrate trapping experiment, in comparison with Gab1-Y627F. As shown in Fig. 2D, the deletion severely reduces the interaction between SHP2-C/S and Gab1, strongly suggesting that SHP2 dephosphorylates Tyr residue(s) located between Pro161 and Gln318. A RasGAP Dominant Negative Mutant Bypasses the Gab1/SHP2 Pathway—Because the Gab1 Pro161-Gln318 region displays potential RasGAP-binding motifs, the previous data suggest that SHP2 facilitates Ras activation by down-regulating a putative association between Gab1 and RasGAP, implying that RasGAP should be a downstream target of Gab1 and SHP2. To test this hypothesis, we have first studied the association of RasGAP with membrane fractions as readout of RasGAP mobilization. As shown in Fig. 3A, when cells are transfected with Gab1-WT, EGF induces at best a minor redistribution of RasGAP. The membrane redistribution of Gab1 itself was used to control the quality of cell stimulation and membrane extraction. In contrast to Gab1-WT, transfection of Gab1-Y627F resulted in an important membrane relocation of RasGAP in response to EGF, which strongly suggests that the Gab1/SHP2 pathway down-regulates RasGAP recruitment under normal conditions. To test this hypothesis further, we have produced a RasGAP-inactive mutant to determine whether its overexpression could attenuate the requirement for the Gab1/SHP2 pathway in Ras activation. The RasGAP mutant was designed to produce a protein unable to interac" @default.
• W1973400979 created "2016-06-24" @default.
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• W1973400979 date "2005-02-01" @default.
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• W1973400979 title "A Novel Role for Gab1 and SHP2 in Epidermal Growth Factor-induced Ras Activation" @default.
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• W1973400979 doi "https://doi.org/10.1074/jbc.m410012200" @default.
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