Epidermal Growth Factor-induced DNA Synthesis

We have previously demonstrated that phosphatidylinositol 3-kinase (PI3-kinase) is necessary and sufficient to account for epidermal growth factor (EGF)-induced mitogenesis in rat primary hepatocytes. A cytosolic Gab2-containing complex accounts for >80% of the total EGF-induced PI3-kinase activity (Kong, M., Mounier, C., Wu, J., and Posner, B. I. (2000) J. Biol. Chem. 275, 36035–36042), suggesting a key role for Gab2 in EGF-induced mitogenesis. Here, we demonstrate that PP1, a selective inhibitor of Src family kinases, blocks the EGF-induced Gab2 tyrosine phosphorylation without inhibiting EGF-induced phosphorylation of the EGF receptor, ErbB3, or Shc. We also show that Gab2 phosphorylation is increased in Csk knockout cells in which Src family kinases are constitutively activated. Furthermore, PP1 blocks Gab2-associated downstream events including EGF-induced PI3-kinase activation, Akt phosphorylation, and DNA synthesis. We demonstrate that Gab2 and Src are constitutively associated. Since this association involves the proline-rich sequences of Gab2, it probably involves the Src homology 3 domain of Src kinase. Mutation of the proline-rich sequences in Gab2 prevented EGF-induced Gab2 phosphorylation, PI3-kinase/Akt activation, and DNA synthesis, demonstrating that Gab2 phosphorylation is critical for EGF-induced mitogenesis and is not complemented by ErbB3 or Shc phosphorylation. We also found that overexpression of a Gab2 mutant lacking SHP2 binding sites increased EGF-induced Gab2 phosphorylation and the activation of PI3-kinase but blocked activation of MAPK. In addition, we demonstrated that the Src-induced response was down-regulated by Gab2-associated SHP2. In summary, our results have defined the role for Src activation in EGF-induced hepatic mitogenesis through the phosphorylation of Gab2 and the activation of the PI3-kinase cascade. We have previously demonstrated that phosphatidylinositol 3-kinase (PI3-kinase) is necessary and sufficient to account for epidermal growth factor (EGF)-induced mitogenesis in rat primary hepatocytes. A cytosolic Gab2-containing complex accounts for >80% of the total EGF-induced PI3-kinase activity (Kong, M., Mounier, C., Wu, J., and Posner, B. I. (2000) J. Biol. Chem. 275, 36035–36042), suggesting a key role for Gab2 in EGF-induced mitogenesis. Here, we demonstrate that PP1, a selective inhibitor of Src family kinases, blocks the EGF-induced Gab2 tyrosine phosphorylation without inhibiting EGF-induced phosphorylation of the EGF receptor, ErbB3, or Shc. We also show that Gab2 phosphorylation is increased in Csk knockout cells in which Src family kinases are constitutively activated. Furthermore, PP1 blocks Gab2-associated downstream events including EGF-induced PI3-kinase activation, Akt phosphorylation, and DNA synthesis. We demonstrate that Gab2 and Src are constitutively associated. Since this association involves the proline-rich sequences of Gab2, it probably involves the Src homology 3 domain of Src kinase. Mutation of the proline-rich sequences in Gab2 prevented EGF-induced Gab2 phosphorylation, PI3-kinase/Akt activation, and DNA synthesis, demonstrating that Gab2 phosphorylation is critical for EGF-induced mitogenesis and is not complemented by ErbB3 or Shc phosphorylation. We also found that overexpression of a Gab2 mutant lacking SHP2 binding sites increased EGF-induced Gab2 phosphorylation and the activation of PI3-kinase but blocked activation of MAPK. In addition, we demonstrated that the Src-induced response was down-regulated by Gab2-associated SHP2. In summary, our results have defined the role for Src activation in EGF-induced hepatic mitogenesis through the phosphorylation of Gab2 and the activation of the PI3-kinase cascade. Upon ligand binding, the activated epidermal growth factor receptor (EGFR) 1The abbreviations used are: EGFR, epidermal growth factor receptor; EGF, epidermal growth factor; PI3-kinase, phosphatidylinositol 3-kinase; MAPK, mitogen-activated protein kinase; MOI, multiplicity of infection; SH2 and -3, Src homology 2 and 3, respectively; IRS, insulin receptor substrate; WTGab2, wild type Gab2; IP, immunoprecipitation; WB, Western blot; ERK, extracellular signal-regulated kinase; αPY, anti-phosphotyrosine mediates a number of important biological responses, including the stimulation of cell proliferation, migration, and differentiation (1Ullrich A. Schlessinger J. Cell. 1990; 61: 203-212Google Scholar, 2Boonstra J. Rijken P. Humbel B. Cremers F. Verkleij A. van Bergen en Henegouwen P. Cell Biol. Int. 1995; 19: 413-430Google Scholar, 3Wells A. Gupta K. Chang P. Swindle S. Glading A. Shiraha H. Microsc. Res. Tech. 1998; 43: 395-411Google Scholar). Src family kinases are nonreceptor tyrosine kinases and have been described as essential mediators of EGF signaling (4Wilde A. Beattie E.C. Lem L. Riethof D.A. Liu S.H. Mobley W.C. Soriano P. Brodsky F.M. Cell. 1999; 96: 677-687Google Scholar). Like most nonreceptor tyrosine kinases, Src family kinases contain an Src homology 2 (SH2) domain, which binds phosphotyrosine residues, and an Src homology 3 (SH3) domain, which binds proline rich sequences (reviewed in Ref. 5Pellicena P. Miller W.T. Front. Biosci. 2002; 7: d256-d267Google Scholar). Several studies have established that Src kinases are required for growth factor-induced mitogenesis such as that effected via receptors for EGF (6Roche S. Koegl M. Barone M.V. Roussel M.F. Courtneidge S.A. Mol. Cell. Biol. 1995; 15: 1102-1109Google Scholar, 7Luttrell D.K. Luttrell L.M. Parsons S.J. Mol. Cell. Biol. 1988; 8: 497-501Google Scholar, 8Wilson L.K. Luttrell D.K. Parsons J.T. Parsons S.J. Mol. Cell. Biol. 1989; 9: 1536-1544Google Scholar), the platelet-derived growth factor (9Broome M.A. Hunter T. J. Biol. Chem. 1996; 271: 16798-16806Google Scholar, 10Erpel T. Alonso G. Roche S. Courtneidge S.A. J. Biol. Chem. 1996; 271: 16807-16812Google Scholar), and colony stimulation factor-1 (6Roche S. Koegl M. Barone M.V. Roussel M.F. Courtneidge S.A. Mol. Cell. Biol. 1995; 15: 1102-1109Google Scholar). To date, the manner in which Src family kinases participate in effecting the mitogenic response is unclear. Several reports have identified Gab (Grb2-associated binder) family proteins as key molecules for EGF-induced mitogenesis (11Winnay J.N. Bruning J.C. Burks D.J. Kahn C.R. J. Biol. Chem. 2000; 275: 10545-10550Google Scholar, 12Kong M. Mounier C. Wu J. Posner B.I. J. Biol. Chem. 2000; 275: 36035-36042Google Scholar). Gab proteins, which include mammalian Gab1, Gab2, and Gab3, theDrosophila homolog DOS (daughter ofsevenless), and the Caenorhabditis eleganshomolog Soc1 (Suppressor of clear), belong to a family of scaffolding proteins closely related to insulin receptor substrates (IRS-1, IRS-2, and IRS-3), FRS2 (fibroblast growth factor substrate), LAT (linker of T cell), and Dok (downstream of kinase) (reviewed in Refs. 13White M.F. Mol. Cell. Biochem. 1998; 182: 3-11Google Scholar, 14Zhang W. Samelson L.E. Semin. Immunol. 2000; 12: 35-41Google Scholar, 15Liu Y. Rohrschneider L.R. FEBS Lett. 2002; 515: 1-7Google Scholar). They have in common a central proline-rich domain and multiple potential binding sites for the SH2 domains of p85, SHP2 (Srchomology 2 domain-containing protein-tyrosine phosphatase-2), phospholipase Cγ, or Crk. Gab2 is tyrosine-phosphorylated upon stimulation of hepatocytes by EGF (12Kong M. Mounier C. Wu J. Posner B.I. J. Biol. Chem. 2000; 275: 36035-36042Google Scholar) and T cells by cytokines (16Brockdorff J.L. Gu H. Mustelin T. Kaltoft K. Geisler C. Ropke C. Odum N. Exp. Clin. Immunogenet. 2001; 18: 86-95Google Scholar) and following activation of T- and B-cell antigen receptors (17Yamasaki S. Nishida K. Hibi M. Sakuma M. Shiina R. Takeuchi A. Ohnishi H. Hirano T. Saito T. J. Biol. Chem. 2001; 276: 45175-45183Google Scholar, 18Nishida K. Yoshida Y. Itoh M. Fukada T. Ohtani T. Shirogane T. Atsumi T. Takahashi-Tezuka M. Ishihara K. Hibi M. Hirano T. Blood. 1999; 93: 1809-1816Google Scholar). Phosphorylated Gab2 has been shown to bind PI3-kinase via its 85-kDa (p85) regulatory subunit (19Carpenter C.L. Duckworth B.C. Auger K.R. Cohen B. Schaffhausen B.S. Cantley L.C. J. Biol. Chem. 1990; 265: 19704-19711Google Scholar) as well as Grb2 (growth factor receptor-bound 2) and SHP2. In previous studies, we demonstrated that the activation of PI3-kinase and not mitogen-activated protein kinase (MAPK), is necessary and sufficient to account for EGF-induced mitogenesis (12Kong M. Mounier C. Wu J. Posner B.I. J. Biol. Chem. 2000; 275: 36035-36042Google Scholar, 20Band C.J. Mounier C. Posner B.I. Endocrinology. 1999; 140: 5626-5634Google Scholar). Although activated PI3-kinase was shown to associate with three phosphotyrosine-phosphorylated proteins (ErbB3, Shc, and Gab2), over 80% was found in a multimeric complex consisting of Gab2-p85-SHP2-Grb2 (12Kong M. Mounier C. Wu J. Posner B.I. J. Biol. Chem. 2000; 275: 36035-36042Google Scholar). Confirming the key role of Gab2 was our finding that overexpression of wild type Gab2 (WTGab2) augmented EGF-induced PI3-kinase activity and DNA synthesis, whereas the Gab2 mutant (Gab2Δp85) lacking p85 binding sites (pYXXM motifs as reviewed in Ref. 21Wymann M.P. Pirola L. Biochim. Biophys. Acta. 1998; 1436: 127-150Google Scholar, where pY represents phosphotyrosine) effected no such augmentation. Furthermore, we showed that following EGF treatment, the phosphorylated multimeric Gab2 complex was exclusively cytosolic and did not associate with membranes. Nor did overexpression of the pleckstrin homology domain of Gab2 interfere with EGF-induced Gab2 phosphorylation or mitogenesis. 2M. Kong, C. Mounier, A. Balbis, G. Baquiran, and B. I. Posner, submitted for publication. In the present study, we considered the possibility that Gab2 is phosphorylated by a tyrosine kinase other than the EGFR and thus sought to elucidate a link between Src family kinases and Gab2 in the regulation of EGF-induced mitogenesis in primary hepatocytes. Our study demonstrates that Src kinase(s) promote EGF-induced PI3-kinase activation and DNA synthesis through effecting the tyrosine phosphorylation of Gab2. In addition, we found that the proline-rich domains of Gab2 are essential for constitutive Src association with and tyrosine phosphorylation of Gab2. Finally, we demonstrate that these Src-dependent responses are down-regulated by the association of SHP2 with Gab2 and that this latter association is critical to EGF-induced MAPK activation. Mouse EGF was obtained from Collaborative Biomedical Products (Bedford, MA). Collagenase was from Worthington). Cell culture medium and antibiotics were from Invitrogen. Vitrogen-100 was from Collagen Corp. (Toronto, Canada). [3H]methylthymidine, 125I- labeled goat anti-rabbit antibody, 125I-labeled goat anti-mouse antibody, and [γ-32P]ATP were purchased from PerkinElmer Life Sciences. Protein A-Sepharose was from AmershamBiosciences. PP1 was purchased from Calbiochem. The anti-phosphotyrosine antibody (PY99) and antibodies to SHP2, IRS-2, and were from The Shc, ErbB3, and Gab2 antibodies were from and antibodies were from MA). antibody was by with a protein of the rat Gab2 other were obtained from and were of the The was by and with the to the The mutant was by DNA A with a of and of and was by Canada). In to the Gab2 as well as the the were between the and sites an and the sequences for WTGab2, for and for and a The of DNA were the to the of was by The of was the as described in the of the hepatocytes were as previously described (12Kong M. Mounier C. Wu J. Posner B.I. J. Biol. Chem. 2000; 275: 36035-36042Google Scholar). cells were for in medium and type and by Soriano A. Soriano P. Cell. Scholar) were in medium cells were with of for 3 The cells were for Cell as previously described (12Kong M. Mounier C. Wu J. Posner B.I. J. Biol. Chem. 2000; 275: 36035-36042Google Scholar), from or cells were with and protein A-Sepharose for the were for 2 with the antibody in the Protein A-Sepharose of a was to and the was for an were by three in and in or were to to and with the antibody for by a with or 125I-labeled goat anti-rabbit antibody or goat anti-mouse antibody proteins were by or by of the was a cells were for in and EGF and were to the an cells were three with 3 of for in in of with of and to and for of from EGF for or cells were in the of protein antibodies as in the were and the Protein A-Sepharose were in of kinase and for PI3-kinase activity as previously described C.J. Mounier C. Posner B.I. Endocrinology. 1999; 140: 5626-5634Google Scholar). In the tyrosine-phosphorylated Gab2 is in the not demonstrated to associate with the EGFR, and is phosphorylated in a pleckstrin homology EGF has been shown to tyrosine kinases of the Src family (4Wilde A. Beattie E.C. Lem L. Riethof D.A. Liu S.H. Mobley W.C. Soriano P. Brodsky F.M. Cell. 1999; 96: 677-687Google Scholar), and studies have activation to cell (6Roche S. Koegl M. Barone M.V. Roussel M.F. Courtneidge S.A. Mol. Cell. Biol. 1995; 15: 1102-1109Google Scholar, 7Luttrell D.K. Luttrell L.M. Parsons S.J. Mol. Cell. Biol. 1988; 8: 497-501Google Scholar, 8Wilson L.K. Luttrell D.K. Parsons J.T. Parsons S.J. Mol. Cell. Biol. 1989; 9: 1536-1544Google Scholar, M.A. Hunter T. J. Biol. Chem. 1996; 271: 16798-16806Google Scholar, 10Erpel T. Alonso G. Roche S. Courtneidge S.A. J. Biol. Chem. 1996; 271: 16807-16812Google Scholar). We the that Gab2 is a Src by the of PP1, a selective inhibitor of Src family kinases J. Biol. Chem. 1996; 271: Scholar), EGF-induced Gab2 tyrosine phosphorylation in rat primary hepatocytes. shown in A PP1 EGF-induced tyrosine phosphorylation of Gab2 in a with cells with PP1 Gab2 phosphorylation by and PP1 by than In the of PP1 no EGF-induced tyrosine phosphorylation of EGFR ErbB3, or Shc Nor did these of PP1 tyrosine phosphorylation of or selective inhibitor of Src family kinases, also the of EGF Gab2 tyrosine phosphorylation not The of by PP1 and of EGF-induced tyrosine phosphorylation of Gab2 in rat this but not other tyrosine-phosphorylated proteins, is a for Src family Src family tyrosine kinases are by phosphorylation a tyrosine in Src kinase) by nonreceptor tyrosine Csk Src kinase) M. H. Biochem. Biophys. Res. 1988; Scholar). from an of in activity of Src and the related kinase A. Soriano P. Cell. Scholar, S. T. H. T. H. Y. M. S. Cell. Scholar). To the role of Src family kinases Gab2 phosphorylation, we and wild type with Gab2 and the cells for Western a antibody W. A. M. Mol. Cell. 1999; Scholar), demonstrated that Src kinase is constitutively activated in cells Gab2 tyrosine phosphorylation was in the of EGF with and Western with anti-phosphotyrosine demonstrated that Gab2 phosphorylation is increased in with wild type 2 study the that Gab2 is a for Src family kinases in In previous we showed that over 80% of PI3-kinase, activated following EGF treatment, was with tyrosine-phosphorylated Gab2 (12Kong M. Mounier C. Wu J. Posner B.I. J. Biol. Chem. 2000; 275: 36035-36042Google Scholar) and that overexpression of Gab2 was sufficient to EGF-induced DNA In this study, we hepatocytes with or PP1 for by a stimulation with shown in EGF a in Gab2-associated PI3-kinase activity in with PP1 this activation, that of as in phosphorylation 3 and DNA synthesis 3 results were obtained by of the cells with not Our results demonstrate that EGF-induced tyrosine phosphorylation of Gab2, by Src family kinases, is necessary for EGF-induced DNA synthesis in primary rat hepatocytes and that the tyrosine phosphorylation of ErbB3 and Shc, which is by PP1 the critical for tyrosine-phosphorylated Gab2 for this In our previous demonstrating that Gab2 is a for Src family kinases, we a in not We the possibility that Src kinase was with its Gab2. shown in A Src family kinases are constitutively with Gab2, and EGF did not to this that the association from the binding of the domain of Src with proline-rich sequences in Gab2. To this we a a Gab2 mutant in which key proline-rich sequences were were with or and for EGF In to WTGab2, no association with Src kinase 3 that the proline-rich sequences are essential for the binding of Src family kinases to Gab2. Src kinase phosphorylation was in A tyrosine-phosphorylated the as Src kinase was in cells with but not in cells with results that Src kinase is activated upon EGF treatment, it is constitutively with Gab2. We sought to the to which the of constitutive Src binding Gab2 phosphorylation by Src family hepatocytes were with or shown in EGF in tyrosine phosphorylation of WTGab2, whereas the phosphorylation of is by than results demonstrate in the proline-rich sequences of Gab2 are important for Src Gab2 phosphorylation. Since EGF-induced Gab2 tyrosine phosphorylation, we this in a of downstream were with or Gab2 or previously overexpression of EGF-induced PI3-kinase activation by 2 whereas overexpression of EGF-induced PI3-kinase activity by than the association of Gab2 with p85 did not a in that it did not kinase activity to than the of Gab2. to the PI3-kinase we found that EGF augmented Akt phosphorylation in cells but not in as a of cells was 3, results were obtained we EGF-induced DNA synthesis The in are with a to EGF of PI3-kinase activation and p85 than to Akt phosphorylation and DNA synthesis. the between Akt activation and the of stimulation of DNA synthesis. that the proline-rich sequences in Gab2, which Src binding, are important for EGF-induced PI3-kinase activation and DNA synthesis. We the of and phosphorylation. shown in phosphorylation was with EGF in this was phosphorylation of in cells with We also demonstrated that phosphorylation of EGF or is than that and these we did not of Gab2 not that Gab2 the of to EGF, which with previous studies that a of EGF overexpression of EGF-induced activation in cells T. L. Wu J. J. Biol. Chem. 2000; 275: Scholar). results of EGF, the of Gab2 by from signaling a of phosphorylation to EGF in its the of shown in EGF-induced activation as the of EGF following EGF treatment, activation in and was and as a of in cells 3, the of tyrosine phosphorylation of to sufficient to but not Upon EGF treatment, Src activation to tyrosine phosphorylation of Gab2 and activation of DNA synthesis. We previously demonstrated that phosphorylated Gab2 with the protein-tyrosine SHP2 (12Kong M. Mounier C. Wu J. Posner B.I. J. Biol. Chem. 2000; 275: 36035-36042Google Scholar). We considered that this binding of Gab2 and a Gab2 mutant lacking the SHP2 binding sites of in hepatocytes SHP2 binding to Gab2 2 and was tyrosine-phosphorylated to an than is the finding that overexpression of EGF-induced PI3-kinase activation with that in cells of this with augmented association In we found that following EGF treatment, Akt phosphorylation, as a of was in 3, results were we the EGF-induced DNA synthesis by SHP2 binding to tyrosine-phosphorylated Gab2 as a of EGF-induced PI3-kinase activation and DNA synthesis. In to these whereas overexpression of increased the of activation to EGF and overexpression of no to EGF-induced activation of following EGF activity in as of was 3, that SHP2 binding to tyrosine-phosphorylated Gab2 EGF downstream signaling by PI3-kinase activation and activation of the MAPK The the by which Src EGF-induced mitogenesis in hepatocytes. Upon EGF binding, Gab2-associated Src is to activation of the PI3-kinase and DNA synthesis. to by SHP2 through of Gab2. In response to the binding of EGF to its receptor, Gab proteins tyrosine-phosphorylated and bind SH2 domain-containing proteins, including p85, the PI3-kinase and SHP2 (12Kong M. Mounier C. Wu J. Posner B.I. J. Biol. Chem. 2000; 275: 36035-36042Google Scholar, T. L. Wu J. J. Biol. Chem. 2000; 275: Scholar, Liu Cantley J. Biol. Chem. 2002; Scholar, M. D.K. 1996; Scholar, L. Wu J. J. Biol. Chem. 2001; 276: Scholar). The by which EGF the phosphorylation of Gab proteins is key to EGF-induced Several reports that EGFR is the kinase that M. S.H. Schlessinger J. Mol. Cell. Biol. 2000; Scholar, H. S.J. J.C. W.C. Cell 2001; 12: Scholar, S. J. A. C. B. Bruning J.C. W. 1999; Scholar). Furthermore, studies have demonstrated that the pleckstrin homology domain of is required for its to thus its phosphorylation by the EGFR kinase M. S.H. Schlessinger J. Mol. Cell. Biol. 2000; Scholar, C.R. M. I. M.A. M. Mol. Cell. Biol. 1999; 19: Scholar, C.R. D.K. M.A. M. M. J. Biol. Chem. 1999; Scholar). the in Gab2 to as we have previously Gab2 is phosphorylated in a pleckstrin homology was it to associate with the the multimeric Gab2 complex was found exclusively in the of rat these we the possibility that Gab2 is tyrosine-phosphorylated by a cytosolic kinase and not the Although previous reports have established that Src family kinases are required for EGF-induced mitogenesis (6Roche S. Koegl M. Barone M.V. Roussel M.F. Courtneidge S.A. Mol. Cell. Biol. 1995; 15: 1102-1109Google Scholar, 7Luttrell D.K. Luttrell L.M. Parsons S.J. Mol. Cell. Biol. 1988; 8: 497-501Google Scholar, 8Wilson L.K. Luttrell D.K. Parsons J.T. Parsons S.J. Mol. Cell. Biol. 1989; 9: 1536-1544Google Scholar), the by which Src family kinases has unclear. In the present study, we demonstrate that EGF of rat hepatocytes to Gab2 tyrosine phosphorylation, which Gab2 SHP2, and p85, thus a multimeric cytosolic The that Src family kinases are in Gab2 tyrosine phosphorylation is the of PP1 and used of Src family kinases Hunter S. P. J. Biol. Chem. 2002; Scholar, J. S. J. Biol. Chem. 2002; Scholar). in the present study, we that the of Gab2 tyrosine phosphorylation was and in the of of tyrosine phosphorylation of EGFR, ErbB3, or the proteins Furthermore, in which Src family kinases are constitutively we a key role for these kinase(s) in the phosphorylation of Gab2. with our results are previous studies demonstrating that the in Src-dependent mitogenic activity is a cytosolic to Src lacking the of the including the binding domain, were shown to cell G. R. T. H. J. 61: Scholar). has been for that EGF signaling involves substrates with of and L.K. Parsons S.J. 1990; Scholar). studies have identified as L.K. Parsons J.T. Parsons S.J. 7: Scholar) and as proteins, including B.S. Parsons J.T. S. A. Scholar) and R. A. Hirano N. S. T. H. Y. H. J. Scholar). the of has Our results to the Src kinase as Gab2. To elucidate the by which Gab2 is phosphorylated by Src family kinases, we the association between these In most is by with of the Src family kinases such as SH2 and domains (reviewed in Ref. 5Pellicena P. Miller W.T. Front. Biosci. 2002; 7: d256-d267Google Scholar). A constitutive association of Src family kinases with Gab2 was and this association the key proline-rich Gab2 were that the domain of Src is probably in this is with previous reports demonstrating that the domain of Src is required for EGF mitogenic signaling (9Broome M.A. Hunter T. J. Biol. Chem. 1996; 271: 16798-16806Google Scholar, 10Erpel T. Alonso G. Roche S. Courtneidge S.A. J. Biol. Chem. 1996; 271: 16807-16812Google Scholar) and that this domain bind to Gab2 in an in binding C. R. J. Biol. Chem. 1999; Scholar). The domains of Src family kinases proline-rich sequences found in a number of substrates of Src family kinases T. R. K. Y. H. J. Biol. Chem. 1996; 271: Scholar, F. S. P. F. P. P. F. S. A. Scholar, S.J. Scholar). The SH2 domains of Src family kinase the with a an important of binding M. G. T. F. T. S. H. Cantley L.C. Cell. Scholar). the Gab2 not contain a suggesting that Src not bind to Gab2 through its SH2 Although Src family kinases are constitutively with Gab2, the activation of Src kinase is EGF The by which Src kinase activation is effected by the binding of EGF to the EGFR to In and are the three Src family Cell Biol. Scholar). in the present we not which Gab2. the of PP1 we used three Src Furthermore, the Src antibody we used for Western and as well as In this we show that in rat of Src family kinases EGF-induced phosphorylation of Gab2 and activation of PI3-kinase/Akt as well as DNA synthesis and Several studies have shown that activation of Src family kinases is in by PI3-kinase to differentiation by effecting Src-dependent tyrosine phosphorylation of and the activation of PI3-kinase to the receptor Y. T. A. Ohnishi H. Y. M. T. H. J. 2001; Scholar). in was shown to promote cell via a Src-dependent activation of PI3-kinase G. A. A. M. A. M. R. L. Barone M.V. F. J. 2001; Scholar). Finally, in EGF activation of Akt was blocked in with of Src activation Hunter S. P. J. Biol. Chem. 2002; Scholar). In rat Gab2 phosphorylation to downstream PI3-kinase/Akt activation and DNA synthesis and is effected by the activation of Src constitutively with Gab2 SHP2 binds with Gab family proteins through a binding in the of Gab proteins Y. Rohrschneider L.R. FEBS Lett. 2002; 515: 1-7Google Scholar). The of the has been of Gab family proteins lacking SHP2 binding studies a role of SHP2 in SHP2 association with is required for C.R. M.A. M. M. Mol. Cell. Biol. 2000; Scholar) as well as EGF and MAPK activation T. L. Wu J. J. Biol. Chem. 2000; 275: Scholar, L. Wu J. J. Biol. Chem. 2001; 276: Scholar). was also found that SHP2 association with Gab2 is essential for factor-induced differentiation Y. B. J.L. Rohrschneider L.R. Mol. Cell. Biol. 2001; Scholar). In the present study, we identified a role for SHP2 in EGF-induced MAPK activation reports have also identified a of SHP2 binding Gab protein tyrosine phosphorylation. and Gab2 have been identified as substrates for SHP2 K. Yoshida Y. Itoh M. Fukada T. Ohtani T. Shirogane T. Atsumi T. Takahashi-Tezuka M. Ishihara K. Hibi M. Hirano T. Blood. 1999; 93: 1809-1816Google Scholar). studies have shown that of SHP2 increased tyrosine phosphorylation and PI3-kinase activity Liu Cantley J. Biol. Chem. 2002; Scholar, T. G. L. R. Mol. Cell. Biol. 2002; Scholar). The regulation of PI3-kinase activation by SHP2 to through the of p85 binding sites T. G. L. R. Mol. Cell. Biol. 2002; Scholar). In with this study, we have found in rat a of phosphorylation with was by a of PI3-kinase activity and DNA synthesis, suggesting that SHP2 is in the of p85 binding sites Gab2 the that the mutant blocks EGF-induced MAPK activation and PI3-kinase and DNA synthesis in primary the key role of PI3-kinase and not MAPK in EGF-induced DNA synthesis. In summary, we have found in rat Src family kinases EGF-induced mitogenesis through association to and phosphorylation of the cytosolic Gab2. Furthermore, we demonstrate that the association of SHP2 with tyrosine-phosphorylated Gab2 to of the latter and a in EGF-induced DNA synthesis.

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