Voltar
Artigo Acesso aberto Revisado por pares
BIBTEX RIS

Epidermal Growth Factor-induced DNA Synthesis

2003; Elsevier BV; Volume: 278; Issue: 8 Linguagem: Inglês

10.1074/jbc.m208286200

ISSN

1083-351X

Autores

Mei Kong, Cathérine Mounier, Víctor Dumas, Barry I. Posner,

Tópico(s)

Ubiquitin and proteasome pathways

Resumo

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, Src, IRS-2, and hemagglutinin were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The anti-p85, IRS-1, Shc, ErbB3, Myc, and Gab2 (for immunoblotting) antibodies were from Upstate Biotechnology, Inc. (Lake Placid, NY). Anti-phospho-Akt473, Akt, phospho-Src416, phospho-Erk1/2, and Erk1/2 antibodies were from New England Biolabs (Beverly, MA). Anti-Gab2 antibody (for immunoprecipitation) was raised by immunizing rabbits with a glutathione S-transferase-Gab2 fusion protein containing amino acids 376–552 of the rat Gab2 sequence. All other reagents were obtained from Sigma and were of the highest grade available. The ΔSHP2Gab2 was generated by replacing Tyr-603 and Tyr-632 with phenylalanine using the Chameleon double-stranded site-directed mutagenesis kit (Stratagene, La Jolla, CA) according to the manufacturer's instructions. The mutant was verified by DNA sequencing. A plasmid containing ΔProGab2 with a deletion of 348–355 amino acids and mutations of P500A and R504A was kindly provided by Dr. Morag Park (McGill University, Montréal, Canada). In order to produce the adenoviruses, Gab2 mutants as well as the full-length cDNA (to generate the WTGab2 construct) were subcloned into pShuttle between the NheI and NotI sites using 5′-oligonucleotides containing an NheI site and the sequences encoding different tags (hemagglutinin for WTGab2, FLAG for ΔProGab2, and Myc for ΔSHP2Gab2) and 3′-oligonucleotides containing a NotI site. The recombinants of adenoviral DNA were further generated using the Adeno-X Expression System (Clontech, Palo Alto, CA) according to the kit's user manual. Large scale production of recombinant viral particles was performed by infecting 293A cells. The titer of viral particles was determined using the Tissue Culture Infectious Dose 50 (TCID50) method as described in the protocol of the Ad-easy vector system (Qbiogene, Carlsbad, CA). Primary hepatocytes were prepared as previously described (12Kong M. Mounier C. Wu J. Posner B.I. J. Biol. Chem. 2000; 275: 36035-36042Google Scholar). Before infection, cells were bathed for 24 h in Dulbecco's modified Eagle's medium/F-12 medium containing 10% fetal bovine serum, 10 mm HEPES, 20 mm NaHCO3, 500 IU/ml penicillin, and 500 μg/ml streptomycin. Wild type and Csk−/− cells, kindly provided by Dr. Philippe Soriano (Fred Hutchinson Cancer Research Center, Seattle, WA) (22Imamoto A. Soriano P. Cell. 1993; 73: 1117-1124Google Scholar) were maintained in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum. All cells were then infected with 10 MOI of different recombinant adenoviruses for 3 h at 37 °C. The infected cells were then serum-starved for 48 h before harvesting. Cell lysates, prepared as previously described (12Kong M. Mounier C. Wu J. Posner B.I. J. Biol. Chem. 2000; 275: 36035-36042Google Scholar), from EGF-treated or nontreated cells were precleared with nonimmune rabbit IgG (Sigma) and protein A-Sepharose for 1 h at 4 °C. After centrifugation, the resulting supernatants were incubated for 2 h at 4 °C with the antibody indicated in the figure legends. Protein A-Sepharose (50 μl of a 50% slurry) was added to each sample, and the incubation was continued for an additional 1 h. Immune complexes were isolated by centrifugation, washed three times in PBS, and boiled in Laemmli sample buffer. Immunoprecipitates or intact samples were subjected to SDS-PAGE, transferred to Immobilon-P membranes (Millipore Ltd., Mississauga, Canada), and immunoblotted with the indicated first antibody for 90 min followed by a 1-h incubation with horseradish peroxidase-, or 125I-labeled goat anti-rabbit antibody or goat anti-mouse antibody IgG. Immunoreactive proteins were detected by autoradiography or by ECL system (Amersham Biosciences). Densitometric quantification of the signals was performed using a Bio-Rad densitometer, model GS-700. After viral exposure, infected cells were serum-starved for 20 h in serum-free medium, and then 100 ng/ml EGF and [3H]methylthymidine (5 μCi/ml) were added to the medium. After an 18-h incubation, cells were rinsed three times with 3 ml of cold PBS, incubated for 15 min at 4 °C in 10% trichloroacetic acid, solubilized at room temperature in 1 ml of 1 n NaOH, neutralized with 1 ml of 1n HCl, and then transferred to scintillation vials and counted for 3H. Lysates (500 μg of protein) from EGF-treated (100 ng/ml EGF for 1 min) or nontreated cells were immunoprecipitated in the presence of protein A-Sepharose, using different antibodies as indicated in the figure legends. Immunoprecipitates were extensively washed, and the Protein A-Sepharose pellets were resuspended in 50 μl of kinase assay buffer (20 mm Tris-HCl, pH 7.5, 100 mm NaCl, 0.5 mm EGTA) containing 0.5 mg/mll-α-phosphatidylinositol (Avanti Polar Lipids, Inc., Alabaster, AL) and assayed for PI3-kinase activity as previously described (20Band C.J. Mounier C. Posner B.I. Endocrinology. 1999; 140: 5626-5634Google Scholar). In the liver, tyrosine-phosphorylated Gab2 is largely localized in the cytosol, could not be demonstrated to associate with the EGFR, and is phosphorylated in a pleckstrin homology domain-independent manner.2 EGF has been shown to activate 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 linked their activation to cell proliferation (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, 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). We tested the hypothesis that Gab2 is a Src substrate by examining the effect of PP1, a selective inhibitor of Src family kinases (23Hanke J.H. Gardner J.P. Dow R.L. Changelian P.S. Brissette W.H. Weringer E.J. Pollok B.A. Connelly P.A. J. Biol. Chem. 1996; 271: 695-701Google Scholar), on EGF-induced Gab2 tyrosine phosphorylation in rat primary hepatocytes. As shown in Fig.1 A (top panels), PP1 reduced EGF-induced tyrosine phosphorylation of Gab2 in a dose-dependent manner. Compared with cells incubated with vehicle only, 1 μm PP1 decreased Gab2 phosphorylation by 50% and 20 μm PP1 by more than 90%. In contrast, the same doses of PP1 had no effect on EGF-induced tyrosine phosphorylation of EGFR (Fig. 1 A, middle panels), ErbB3, or Shc (Fig. 1 A, bottom panels). Nor did these doses of PP1 affect insulin-induced tyrosine phosphorylation of IRS-1 or IRS-2 (Fig. 1 B). PP2, another selective inhibitor of Src family kinases, also inhibited the stimulatory effect of EGF on Gab2 tyrosine phosphorylation (data not shown). The specificity of inhibition by PP1 and PP2 of EGF-induced tyrosine phosphorylation of Gab2 suggests that, in rat hepatocytes, this molecule, but not other tyrosine-phosphorylated docking proteins, is a substrate for Src family kinase(s). All Src family tyrosine kinases are negatively regulated by phosphorylation at a carboxyl-terminal tyrosine (Tyr-527 in Src kinase) carried out by another nonreceptor tyrosine kinase, Csk (C-terminal Src kinase) (24Okada M. Nakagawa H. Biochem. Biophys. Res. Commun. 1988; 154: 796-802Google Scholar). Cells derived from Csk-deficient embryos exhibit an order of magnitude increase in activity of Src and the related Fyn kinase (22Imamoto A. Soriano P. Cell. 1993; 73: 1117-1124Google Scholar, 25Nada S. Yagi T. Takeda H. Tokunaga T. Nakagawa H. Ikawa Y. Okada M. Aizawa S. Cell. 1993; 73: 1125-1135Google Scholar). To further confirm the role of Src family kinases on Gab2 phosphorylation, we infected Csk-deficient and wild type mouse embryo fibroblasts with recombinant Gab2 adenovirus and starved the cells for 48 h. Western blot analysis, using a specific phospho-Src antibody (p-Src416) (26Xu W. Doshi A. Lei M. Eck M.J. Harrison S.C. Mol. Cell. 1999; 3: 629-638Google Scholar), demonstrated that Src kinase is constitutively activated in Csk−/− cells (Fig.2 A, top panel). Gab2 tyrosine phosphorylation was then investigated in the absence of EGF stimulation. Immunoprecipitation with αGab2 and Western blotting with anti-phosphotyrosine demonstrated that Gab2 phosphorylation (Fig.2 A, middle panel) is increased 1.8-fold in Csk−/− compared with wild type fibroblasts (Fig. 2 B). This study supports the view that Gab2 is a substrate for Src family kinases in vivo. In previous work, we showed that over 80% of PI3-kinase, activated following EGF treatment, was associated 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 augment EGF-induced DNA synthesis.2 In this study, we pretreated hepatocytes with either vehicle (Me2SO) or 20 μm PP1 for 30 min followed by a stimulation with 100 ng/ml EGF. As shown in Fig.3 A, EGF induced a 20-fold increase in Gab2-associated PI3-kinase activity in control cells. Pretreatment with PP1 abolished this activation, that of Akt, as reflected in Akt-Ser-473 phosphorylation (Fig. 3 B), and DNA synthesis (Fig. 3 C). Similar results were obtained by treatment of the cells with PP2 (data not shown). Our results demonstrate that EGF-induced tyrosine phosphorylation of Gab2, mediated 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 unaffected by PP1 (Fig. 1), cannot supplant the critical requirement for tyrosine-phosphorylated Gab2 for this response. In our previous experiments, demonstrating that Gab2 is a substrate for Src family kinases, we observed a 60-kDa band in anti-Gab2 immunoprecipitates (data not shown). We therefore examined the possibility that Src kinase was associated with its substrate, Gab2. As shown in Fig.4 A (top panel), Src family kinases are constitutively associated with endogenous Gab2, and EGF treatment did not appear to augment this association. This suggested that the association may result from the binding of the SH3 domain of Src with proline-rich sequences in Gab2. To test this hypothesis, we generated a recombinant adenovirus containing a Gab2 mutant in which key proline-rich sequences were mutated (ΔProGab2) (see "Experimental Procedures"). Hepatocytes were infected with either WTGab2 or ΔProGab2 recombinant adenovirus and starved for 48 h before EGF treatment. In contrast to WTGab2, overexpressed ΔProGab2 manifested no association with Src kinase (Fig.4 B, top panel, lanes 1 and2 versus lanes 3 and4), indicating that the proline-rich sequences are essential for mediating the binding of Src family kinases to Gab2. Src kinase phosphorylation was analyzed in anti-Gab2 immunoprecipitates. A tyrosine-phosphorylated band migrating at the same position as Src kinase (60 kDa) was detected in EGF-treated cells infected with WTGab2 but not in EGF-treated cells infected with ΔProGab2 (Fig.4 C, top panel). These results suggest that Src kinase is activated upon EGF treatment, although it is constitutively associated with Gab2. We sought to determine the extent to which the loss of constitutive Src binding influenced Gab2 phosphorylation by Src family kinases. Primary hepatocytes were infected with recombinant WTGab2 or ΔProGab2 adenoviruses. As shown in Fig. 5(top panel), EGF treatment resulted in substantial tyrosine phosphorylation of WTGab2, whereas the phosphorylation of ΔProGab2 is reduced by more than 70% (Fig. 5, bottom panel). These results demonstrate that, in hepatocytes, the proline-rich sequences of Gab2 are important for Src kinase-mediated Gab2 phosphorylation. Since overexpressing ΔProGab2 dramatically reduced EGF-induced Gab2 tyrosine phosphorylation, we examined whether this resulted in a decrease of Gab2-dependent downstream signaling. Cells were infected with either control recombinant adenovirus (LacZ) or Gab2 constructs (WTGab2 or ΔProGab2). As previously observed, overexpression of WTGab2 potentiated EGF-induced PI3-kinase activation by 25-fold (Fig. 6 A, top panel, lane 2 versus lane 4), whereas overexpression of ΔProGab2 reduced EGF-induced PI3-kinase activity by more than 70% (Fig.6 A, top panel, lane 4 versus lane 6). This parallels the association of Gab2 with p85 (Fig. 6 A, middle panel). Interestingly, ΔProGab2 did not exert a dominant negative effect in that it did not reduce PI3 kinase activity to less than control levels despite being expressed at 20-fold the level of endogenous Gab2. Corresponding to the PI3-kinase activity, we found that EGF treatment augmented Akt serine 473 phosphorylation in cells overexpressing WTGab2 but not in those overexpressing ΔProGab2 (WTGab2- versus ΔProGab2-infected cells, expressed as a percentage of LacZ-infected cells was 146 ± 4.7 versus110 ± 4.1 (n = 3, mean ± S.E.,p < 0.01)) (Fig. 6 B). Parallel results were obtained when we measured EGF-induced DNA synthesis (Fig.6 C). The observations in Fig. 6 are consistent with a greater sensitivity to EGF of PI3-kinase activation and p85 recruitment than to Akt phosphorylation and DNA synthesis. This could explain the closer correlation between Akt activation and the extent of stimulation of DNA synthesis. These findings indicate that the proline-rich sequences in Gab2, which mediate Src binding, are important for EGF-induced PI3-kinase activation and DNA synthesis. We then examined the effect of WTGab2 and ΔProGab2 on ERK phosphorylation. As shown in Fig. 6 D, ERK phosphorylation was barely observed with 0.2 ng/ml EGF in LacZ-infected cells, whereas, at this dose, there was clear phosphorylation of ERK in cells infected with WTGab2. We also demonstrated that ERK1/2 phosphorylation at higher doses of EGF (10 or 100 ng/ml) is much greater than that at 0.2 and 1.0 ng/ml. At these concentrations, we did not observe any effect of overexpressed Gab2 (data not shown). This suggests that overexpressed Gab2 increases the sensitivity of ERK to EGF, which correlated with previous studies showing that at a low dose of EGF (0.25 ng/ml), overexpression of Gab1 potentiated EGF-induced ERK activation in HEK293 cells (27Cunnick J.M. Dorsey J.F. Munoz-Antonia T. Mei L. Wu J. J. Biol. Chem. 2000; 275: 13842-13848Google Scholar). These results may indicate that, at higher doses of EGF, the influences of Gab2 could be obscured by input(s) from Gab2-independent signaling pathways. Alternatively, a high sensitivity of ERK phosphorylation to EGF may result in its saturation at the 10 ng/ml dose of EGF. As shown in Fig. 6 D, ΔProGab2 comparably potentiated EGF-induced ERK1/2 activation as WTGab2 at the low dose of EGF (i.e. following EGF (0.2 ng/ml) treatment, ERK activation in WTGab2 and ΔProGab2 was 218 ± 4.2 and 236 ± 12, respectively, when expressed as a percentage of values in LacZ-infected cells (n = 3, mean ± S.E.,p < 0.05 LacZ versus either construct)). Thus, the reduced level of tyrosine phosphorylation of ΔProGab2 would appear to be sufficient to activate ERK1/2 but not PI3-kinase. Upon EGF treatment, Src activation leads to substantial tyrosine phosphorylation of Gab2 and activation of DNA synthesis. We previously demonstrated that phosphorylated Gab2 associates with the SH2-containing protein-tyrosine phosphatase, SHP2 (12Kong M. Mounier C. Wu J. Posner B.I. J. Biol. Chem. 2000; 275: 36035-36042Google Scholar). We considered that this binding might effect dephosphorylation of Gab2 and therefore generated a Gab2 mutant lacking the SHP2 binding sites (ΔSHP2Gab2; see "Experimental Procedures"). Overexpression of ΔSHP2Gab2 in hepatocytes totally abolished SHP2 binding to Gab2 (Fig.7 A, top panel,lane 2 versus lane 4) and was tyrosine-phosphorylated (Fig. 7 B,lane 4 versus lane 6) to an extent 25% greater than WTGab2 (Fig.7 C). Of interest is the finding that overexpression of ΔSHP2Gab2 potentiated EGF-induced PI3-kinase activation 2.6-fold compared with that observed in cells overexpressing comparable levels of WTGab2 (Fig.8 A, top panel,lane 4 versus lane 6). As expected, this correlated with augmented p85-Gab2 association (Fig. 8 A, middle panel,lane 4 versus lane 6). In parallel, we found that following EGF treatment, Akt serine 473 phosphorylation, expressed as a percentage of LacZ-infected cells, was 146 ± 4.7 versus 201 ± 25, in WTGab2versus ΔSHP2Gab2 (n = 3, mean ± S.E., p < 0.01) (Fig. 8 B, top panel, lane 4 versus lane 6). Similar results were observed when we looked at the EGF-induced DNA synthesis by -fold (Fig. 8 C). Thus, SHP2 binding to tyrosine-phosphorylated Gab2 acts as a negative regulator of EGF-induced PI3-kinase activation and DNA synthesis. In contrast to these findings, whereas overexpression of WTGab2 increased the sensitivity of ERK1/2 activation to EGF (Figs.6 D and 8 D, lanes 4–6), overexpression of ΔSHP2Gab2 had no capacity to effect EGF-induced activation of ERK1/2 (Fig. 8 D, lanes 7–9) (i.e. following EGF treatment (0.2 ng/ml), ERK activity in WTGab2- versus ΔSHP2Gab2-infected cells, expressed as percentage of LacZ-infected cells, was 218 ± 4.2versus 130 ± 9 (n = 3, mean ± S.E., p < 0.05)) (Fig. 8 D). These data indicate that SHP2 binding to tyrosine-phosphorylated Gab2 influences EGF downstream signaling by negatively affecting EGF-dependent PI3-kinase activation and positively affecting activation of the MAPK pathway. The above observations identify the mechanism by which Src regulates EGF-induced mitogenesis in hepatocytes. Upon EGF binding, Gab2-associated Src is activated, leading to activation of the PI3-kinase cascade and eventually DNA synthesis. This signal appears to be attenuated by SHP2 through dephosphorylation of Gab2. In response to the binding of EGF to its receptor, Gab proteins become tyrosine-phosphorylated and bind SH2 domain-containing proteins, including p85, the PI3-kinase subunit, and SHP2 (12Kong M. Mounier C. Wu J. Posner B.I. J. Biol. Chem. 2000; 275: 36035-36042Google Scholar, 27Cunnick J.M. Dorsey J.F. Munoz-Antonia T. Mei L. Wu J. J. Biol. Chem. 2000; 275: 13842-13848Google Scholar, 28Yu C.F. Liu Z.X. Cantley L.G. J. Biol. Chem. 2002; 272: 19382-19388Google Scholar, 29Holgado-Madruga M. Emlet D.R. Moscatello D.K. Godwin A.K. Wong A.J. Nature. 1996; 379: 560-564Google Scholar, 30Cunnick J.M. Mei L. Doupnik C.A. Wu J. J. Biol. Chem. 2001; 276: 24380-24387Google Scholar). The mechanism by which EGF induces the phosphorylation of Gab docking proteins is key to understanding EGF-induced signaling. Several reports indicate that EGFR is the kinase that phosphorylates Gab1 (31Rodrigues G.A. Falasca M. Zhang Z. Ong S.H. Schlessinger J. Mol. Cell. Biol. 2000; 20: 1448-1459Google Scholar, 32Kameda H. Risinger J.I. Han B.B. Baek S.J. Barrett J.C. Glasgow W.C. Eling T.E. Cell Growth Differ. 2001; 12: 307-318Google Scholar, 33Lehr S. Kotzka J. Herkner A. Klein E. Siethoff C. Knebel B. Noelle V. Bruning J.C. Klein H.W. Meyer H.E. Krone W. Muller-Wieland D. Biochemistry. 1999; 38: 151-159Google Scholar). Furthermore, studies have demonstrated that the pleckstrin homology domain of Gab1 is required for its localization to membranes, thus facilitating its phosphorylation by the EGFR kinase (31Rodrigues G.A. Falasca M. Zhang Z. Ong S.H. Schlessinger J. Mol. Cell. Biol. 2000; 20: 1448-1459Google Scholar, 34Maroun C.R. Holgado-Madruga M. Royal I. Naujokas M.A. Fournier T.M. Wong A.J. Park M. Mol. Cell. Biol. 1999; 19: 1784-1799Google Scholar, 35Maroun C.R. Moscatello D.K. Naujokas M.A. Holgado-Madruga M. Wong A.J. Park M. J. Biol. Chem. 1999; 274: 31719-31726Google Scholar). However, the process involved in phosphorylating Gab2 appears to be different. Thus, as we have previously found, Gab2 is phosphorylated in a pleckstrin homology domain-independent manner; nor was it observed to associate with the EGFR. Moreover, the major multimeric Gab2 complex was found exclusively in the cytosol of rat liver.2 For these reasons, we examined the possibility that Gab2 is tyrosine-phosphorylated by a cytosolic kinase and not the EGFR. 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 mechanism by which Src family kinases act has remained unclear. In the present study, we demonstrate that EGF treatment of rat hepatocytes activates Src, leading to Gab2 tyrosine phosphorylation, after which Gab2 recruits SHP2, Grb2, and p85, thus forming a multimeric cytosolic complex.2 The evidence that Src family kinases are involved in Gab2 tyrosine phosphorylation is based on the inhibitory effects of PP1 and PP2, two widely used specific inhibitors of Src family kinases (36Kassenbrock C.K. Hunter S. Garl P. Johnson G.L. Anderson S.M. J. Biol. Chem. 2002; 277: 24967-24975Google Scholar, 37Kim J. Eckhart A.D. Eguchi S. Koch W.J. J. Biol. Chem. 2002; 277: 32116-32123Google Scholar). Thus, in the present study, we observed that the inhibition of Gab2 tyrosine phosphorylation was dose-dependent and occurred in the absence of inhibition of tyrosine phosphorylation of EGFR, ErbB3, or the IRS proteins (Fig. 1). Furthermore, using CSK−/− mouse embryonic cells, in which Src family kinases are constitutively activated, we confirmed a key role for these kinase(s) in the phosphorylation of Gab2. Consistent with our results are previous studies demonstrating that the target involved in Src-dependent mitogenic activity is a cytosolic molecule accessible to unmyristoylated Src. Thus, Src deletion mutants, lacking the amino-terminal one-third of the molecule, including the membrane binding domain, were shown to still induce cell proliferation (38Calothy G. Laugier D. Cross F.R. Jove R. Hanafusa T. Hanafusa H. J. Virol. 1987; 61: 1678-1681Google Scholar). It has been known for some time that EGF signaling involves c-Src substrates with molecular sizes of 120–130, 100, and 75 kDa (39Wilson L.K. Parsons S.J. Oncogene. 1990; 5: 1471-1480Google Scholar). Further studies have identified p75 as cortactin (40Maa M.C. Wilson L.K. Moyers J.S. Vines R.R. Parsons J.T. Parsons S.J. Oncogene. 1992; 7: 2429-2438Google Scholar) and p120–130 as comprising several proteins, including p125FAK (41Schaller M.D. Borgman C.A. Cobb B.S. Vines R.R. Reynolds A.B. Parsons J.T. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 5192-5196Google Scholar) and p130CAS (42Sakai R. Iwamatsu A. Hirano N. Ogawa S. Tanaka T. Mano H. Yazaki Y. Hirai H. EMBO J. 1994; 13: 3748-3756Google Scholar). However, the identity of p100 has hitherto remained unknown. Our results appear to establish the p100 Src kinase substrate as Gab2. To elucidate the mechanism by which Gab2 is phosphorylated by Src family kinases, we analyzed the association between these two proteins. In most cases, substrate recognition is dictated primarily by interactions with noncatalytic regions of the Src family kinases such as SH2 and SH3 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 observed, and this association disappeared when the two key proline-rich regions on Gab2 were mutated (Fig. 4), indicating that the SH3 domain of Src is probably involved in this interaction. This is consistent with two previous reports demonstrating that the SH3 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 can bind to Gab2 in an in vitro binding assay (43Zhao C. Yu D.H. Shen R. Feng G.S. J. Biol. Chem. 1999; 274: 19649-19654Google Scholar). The SH3 domains of Src family kinases recognize proline-rich sequences found in a large number of substrates of Src family kinases (44Nakamoto T. Sakai R. Ozawa K. Yazaki Y. Hirai H. J. Biol. Chem. 1996; 271: 8959-8965Google Scholar, 45Onofri F. Giovedi S. Vaccaro P. Czernik A.J. Valtorta F. De Camilli P. Greengard P. Benfenati F. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12168-12173Google Scholar, 46Taylor S.J. Shalloway D. Nature. 1994; 368: 867-871Google Scholar). The SH2 domains of Src family kinase selectively recognize the sequence pYEEI, with a hydrophobic residue at position +3 being an important determinant of binding (47Songyang Z. Shoelson S.E. Chaudhuri M. Gish G. Pawson T. Haser W.G. King F. Roberts T. Ratnofsky S. Lechleider R.J. Neel B.G. Birge R.B. Fajardo J.E. Chou M.M. Hanafusa H. Cantley L.C. Cell. 1993; 72: 767-778Google Scholar). Interestingly, the Gab2 sequence does not contain a pYEEI motif, suggesting that Src does not bind to Gab2 through its SH2 domain. Although Src family kinases are constitutively associated with Gab2, the activation of Src kinase is only observed after EGF treatment (Fig.4 C). The mechanism by which Src kinase activation is effected by the binding of EGF to the EGFR remains to be elucidated. In hepatocytes, Src, Fyn, and Yes are the three Src family members expressed (48Thomas S.M. Brugge J.S. Annu. Rev. Cell Dev. Biol. 1997; 13: 513-609Google Scholar). However, in the present analysis, we could not identify which isoform(s) phosphorylate Gab2. Indeed, the dose of PP1 we used inhibits all three Src isoforms. Furthermore, the Src antibody we used for Western blots recognizes Fyn and Yes as well as Src. In this paper, we show that in rat hepatocytes, inhibition of Src family kinases prevents EGF-induced phosphorylation of Gab2 and activation of PI3-kinase/Akt as well as DNA synthesis (Figs. 1 and 3). Several recent studies have shown that activation of Src family kinases is involved in signal transduction by regulating PI3-kinase activity. Thus, erythropoietin appears to effect erythroid differentiation by effecting Src-dependent tyrosine phosphorylation of and the recruitment plus activation of PI3-kinase to the erythropoietin receptor (49Kubota Y. Tanaka T. Kitanaka A. Ohnishi H. Okutani Y. Waki M. Ishida T. Kamano H. EMBO J. 2001; 20: 5666-5677Google Scholar). Also, in MCF-7 cells, estradiol was shown to promote cell cycle progression via a Src-dependent activation of PI3-kinase (50Castoria G. Migliaccio A. Bilancio A. Di Domenico M. de Falco A. Lombardi M. Fiorentino R. Varricchio L. Barone M.V. Auricchio F. EMBO J. 2001; 20: 6050-6059Google Scholar). Finally, in T47D cells, EGF activation of Akt was blocked in parallel with inhibition of Src activation (36Kassenbrock C.K. Hunter S. Garl P. Johnson G.L. Anderson S.M. J. Biol. Chem. 2002; 277: 24967-24975Google Scholar). In rat hepatocytes, EGF-dependent Gab2 phosphorylation leads to downstream PI3-kinase/Akt activation and DNA synthesis and is effected by the activation of Src constitutively associated with Gab2 (Figs. 4and 6). SHP2 binds with Gab family proteins through a consensus binding motif (YXX(V/I/L)) located in the C-terminal ends of Gab proteins (15Liu Y. Rohrschneider L.R. FEBS Lett. 2002; 515: 1-7Google Scholar). The function of the Gab-SHP2 interaction has been extensively studied using mutants of Gab family proteins lacking SHP2 binding sites. These studies reveal a general positive role of SHP2 in Gab-mediated signal transduction. For example, SHP2 association with Gab1 is required for Met-dependent morphogenesis (51Maroun C.R. Naujokas M.A. Holgado-Madruga M. Wong A.J. Park M. Mol. Cell. Biol. 2000; 20: 8513-8525Google Scholar) as well as EGF and lysophosphatidic acid-induced MAPK activation (27Cunnick J.M. Dorsey J.F. Munoz-Antonia T. Mei L. Wu J. J. Biol. Chem. 2000; 275: 13842-13848Google Scholar, 30Cunnick J.M. Mei L. Doupnik C.A. Wu J. J. Biol. Chem. 2001; 276: 24380-24387Google Scholar). It was also found that SHP2 association with Gab2 is essential for macrophage colony-stimulating factor-induced macrophage differentiation (52Liu Y. Jenkins B. Shin J.L. Rohrschneider L.R. Mol. Cell. Biol. 2001; 21: 3047-3056Google Scholar). In the present study, we identified a positive role for SHP2 in Gab2-potentiated EGF-induced MAPK activation (Fig.8 D). Recent reports have also identified a negative effect of SHP2 binding on Gab protein tyrosine phosphorylation. Thus, both Gab1 and Gab2 have been identified as substrates for SHP2 using anin vitro phosphatase assay (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). More recently, two studies have shown that inactive forms of SHP2 markedly increased EGF-stimulated Gab1 tyrosine phosphorylation and PI3-kinase activity (28Yu C.F. Liu Z.X. Cantley L.G. J. Biol. Chem. 2002; 272: 19382-19388Google Scholar, 53Zhang S.Q. Tsiaras W.G. Araki T. Wen G. Minichiello L. Klein R. Neel B.G. Mol. Cell. Biol. 2002; 22: 4062-4072Google Scholar). The negative regulation of EGF-dependent PI3-kinase activation by SHP2 appears to be through the dephosphorylation of Gab1 p85 binding sites (53Zhang S.Q. Tsiaras W.G. Araki T. Wen G. Minichiello L. Klein R. Neel B.G. Mol. Cell. Biol. 2002; 22: 4062-4072Google Scholar). In agreement with this study, we have found that, in rat hepatocytes, a relatively minor increase of ΔSHP2Gab2 phosphorylation compared with WTGab2 (Fig.7 B) was accompanied by a more noticeable augmentation of PI3-kinase activity and DNA synthesis, suggesting that SHP2 is involved in the dephosphorylation of specific p85 binding sites on Gab2 (Fig. 8,A–C). Moreover, the fact that the ΔSHP2Gab2 mutant blocks EGF-induced MAPK activation and augments PI3-kinase and DNA synthesis further confirms, in primary hepatocytes, the key role of PI3-kinase and not MAPK in EGF-induced DNA synthesis. In summary, we have found that, in rat hepatocytes, Src family kinases regulate EGF-induced mitogenesis through association to and phosphorylation of the major cytosolic docking protein, Gab2. Furthermore, we demonstrate that the association of SHP2 with tyrosine-phosphorylated Gab2 leads to dephosphorylation of the latter and a corresponding decrease in EGF-induced DNA synthesis.

Referência(s)