Interaction of Scaffolding Adaptor Protein Gab1 with Tyrosine Phosphatase SHP2 Negatively Regulates IGF-I-dependent Myogenic Differentiation via the ERK1/2 Signaling Pathway
2008; Elsevier BV; Volume: 283; Issue: 35 Linguagem: Inglês
10.1074/jbc.m803907200
ISSN1083-351X
AutoresTatsuya Koyama, Yoshikazu Nakaoka, Yasushi Fujio, Hisao Hirota, Keigo Nishida, Shoko Sugiyama, Kitaro Okamoto, Keiko Yamauchi‐Takihara, Michihiro Yoshimura, Seibu Mochizuki, Masatsugu Hori, Toshio Hirano, Naoki Mochizuki,
Tópico(s)Cytokine Signaling Pathways and Interactions
ResumoGrb2-associated binder 1 (Gab1) coordinates various receptor tyrosine kinase signaling pathways. Although skeletal muscle differentiation is regulated by some growth factors, it remains elusive whether Gab1 coordinates myogenic signals. Here, we examined the molecular mechanism of insulin-like growth factor-I (IGF-I)-mediated myogenic differentiation, focusing on Gab1 and its downstream signaling. Gab1 underwent tyrosine phosphorylation and subsequent complex formation with protein-tyrosine phosphatase SHP2 upon IGF-I stimulation in C2C12 myoblasts. On the other hand, Gab1 constitutively associated with phosphatidylinositol 3-kinase regulatory subunit p85. To delineate the role of Gab1 in IGF-I-dependent signaling, we examined the effect of adenovirus-mediated forced expression of wild-type Gab1 (Gab1WT), mutated Gab1 that is unable to bind SHP2 (Gab1ΔSHP2), or mutated Gab1 that is unable to bind p85 (Gab1Δp85), on the differentiation of C2C12 myoblasts. IGF-I-induced myogenic differentiation was enhanced in myoblasts overexpressing Gab1ΔSHP2, but inhibited in those overexpressing either Gab1WT or Gab1Δp85. Conversely, IGF-I-induced extracellular signal-regulated kinase 1/2 (ERK1/2) activation was significantly repressed in myoblasts overexpressing Gab1ΔSHP2 but enhanced in those overexpressing either Gab1WT or Gab1Δp85. Furthermore, small interference RNA-mediated Gab1 knockdown enhanced myogenic differentiation. Overexpression of catalytic-inactive SHP2 modulated IGF-I-induced myogenic differentiation and ERK1/2 activation similarly to that of Gab1ΔSHP2, suggesting that Gab1-SHP2 complex inhibits IGF-I-dependent myogenesis through ERK1/2. Consistently, the blockade of ERK1/2 pathway reversed the inhibitory effect of Gab1WT overexpression on myogenic differentiation, and constitutive activation of the ERK1/2 pathway suppressed the enhanced myogenic differentiation by overexpression of Gab1ΔSHP2. Collectively, these data suggest that the Gab1-SHP2-ERK1/2 signaling pathway comprises an inhibitory axis for IGF-I-dependent myogenic differentiation. Grb2-associated binder 1 (Gab1) coordinates various receptor tyrosine kinase signaling pathways. Although skeletal muscle differentiation is regulated by some growth factors, it remains elusive whether Gab1 coordinates myogenic signals. Here, we examined the molecular mechanism of insulin-like growth factor-I (IGF-I)-mediated myogenic differentiation, focusing on Gab1 and its downstream signaling. Gab1 underwent tyrosine phosphorylation and subsequent complex formation with protein-tyrosine phosphatase SHP2 upon IGF-I stimulation in C2C12 myoblasts. On the other hand, Gab1 constitutively associated with phosphatidylinositol 3-kinase regulatory subunit p85. To delineate the role of Gab1 in IGF-I-dependent signaling, we examined the effect of adenovirus-mediated forced expression of wild-type Gab1 (Gab1WT), mutated Gab1 that is unable to bind SHP2 (Gab1ΔSHP2), or mutated Gab1 that is unable to bind p85 (Gab1Δp85), on the differentiation of C2C12 myoblasts. IGF-I-induced myogenic differentiation was enhanced in myoblasts overexpressing Gab1ΔSHP2, but inhibited in those overexpressing either Gab1WT or Gab1Δp85. Conversely, IGF-I-induced extracellular signal-regulated kinase 1/2 (ERK1/2) activation was significantly repressed in myoblasts overexpressing Gab1ΔSHP2 but enhanced in those overexpressing either Gab1WT or Gab1Δp85. Furthermore, small interference RNA-mediated Gab1 knockdown enhanced myogenic differentiation. Overexpression of catalytic-inactive SHP2 modulated IGF-I-induced myogenic differentiation and ERK1/2 activation similarly to that of Gab1ΔSHP2, suggesting that Gab1-SHP2 complex inhibits IGF-I-dependent myogenesis through ERK1/2. Consistently, the blockade of ERK1/2 pathway reversed the inhibitory effect of Gab1WT overexpression on myogenic differentiation, and constitutive activation of the ERK1/2 pathway suppressed the enhanced myogenic differentiation by overexpression of Gab1ΔSHP2. Collectively, these data suggest that the Gab1-SHP2-ERK1/2 signaling pathway comprises an inhibitory axis for IGF-I-dependent myogenic differentiation. Skeletal muscle differentiation is a multistep process in which multipotent mesodermal cells give rise to myoblasts that subsequently withdraw from the cell cycle and differentiate into multinucleated myotubes. Most skeletal muscle cell lines from rodents proliferate in high serum conditions containing various mitogens, and post-confluent cells spontaneously differentiate after several days in low serum conditions (1Bach L.A. Salemi R. Leeding K.S. Endocrinology. 1995; 136: 5061-5069Crossref PubMed Scopus (59) Google Scholar, 2Ewton D.Z. Roof S.L. Magri K.A. McWade F.J. Florini J.R. J. Cell Physiol. 1994; 161: 277-284Crossref PubMed Scopus (94) Google Scholar). Among various growth factors, the insulin-like growth factors (IGFs), 3The abbreviations used are:IGFinsulin-like growth factorGab1Grb2-associated binder 1Gab2Grb2-associated binder 2WTwild-typePI3Kphosphatidylinositol 3-kinaseMAPKmitogen-activated protein kinaseMEK1/2MAPK/extracellular signal-regulated kinase kinase 1/2ERK1/2extracellular signal-regulated kinase 1/2SH2Src homology 2SHP2SH2-containing protein-tyrosine phosphatase 2EGFepidermal growth factorVEGFvascular endothelial growth factorIRS-1insulin receptor substrate-1MHCmyosin heavy chainDMEMDulbecco's modified Eagle's mediumFBSfetal bovine serumβ-galβ-galactosidaseHShorse serumFGF2fibroblast growth factor 2IPimmunoprecipitationANOVAanalysis of variancesiRNAsmall interference RNARNAiRNA interference. 3The abbreviations used are:IGFinsulin-like growth factorGab1Grb2-associated binder 1Gab2Grb2-associated binder 2WTwild-typePI3Kphosphatidylinositol 3-kinaseMAPKmitogen-activated protein kinaseMEK1/2MAPK/extracellular signal-regulated kinase kinase 1/2ERK1/2extracellular signal-regulated kinase 1/2SH2Src homology 2SHP2SH2-containing protein-tyrosine phosphatase 2EGFepidermal growth factorVEGFvascular endothelial growth factorIRS-1insulin receptor substrate-1MHCmyosin heavy chainDMEMDulbecco's modified Eagle's mediumFBSfetal bovine serumβ-galβ-galactosidaseHShorse serumFGF2fibroblast growth factor 2IPimmunoprecipitationANOVAanalysis of variancesiRNAsmall interference RNARNAiRNA interference. including IGF-I and IGF-II, have been reported to be quite unique in that they stimulate both proliferation and differentiation of muscle cells in culture (3Coolican S.A. 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This Gab1 mutant is defective in delivering signals for hepatocyte growth factor-c-Met-dependent morphogenesis, epidermal growth factor (EGF)-dependent epidermal proliferation, and leukemia inhibitory factor-gp130-dependent cardiomyocyte hypertrophy (20Cunnick 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 (117) Google Scholar, 21Cunnick J.M. Mei L. Doupnik C.A. Wu J. J. Biol. Chem. 2001; 276: 24380-24387Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar, 23Schaeper 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). These findings underscore the importance of Gab1-SHP2 interaction and strongly suggest that the primary role of Gab1 might be to recruit SHP2 (24Cunnick J.M. Meng S. Ren Y. Desponts C. Wang H.G. Djeu J.Y. Wu J. J. Biol. Chem. 2002; 277: 9498-9504Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar). On the other hand, it has been reported that Gab1 also regulates the PI3K-AKT signaling pathway through association with p85 downstream of various growth factors (25Dance M. Montagner A. Yart A. Masri B. Audigier Y. Perret B. Salles J.P. Raynal P. J. Biol. Chem. 2006; 281: 23285-23295Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 26Jin Z.G. Wong C. Wu J. Berk B.C. J. Biol. Chem. 2005; 280: 12305-12309Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 27Laramee M. Chabot C. Cloutier M. Stenne R. Holgado-Madruga M. Wong A.J. Royal I. J. Biol. Chem. 2007; 282: 7758-7769Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 28Mattoon D.R. Lamothe B. Lax I. Schlessinger J. BMC. Biol. 2004; 2: 24Crossref PubMed Scopus (141) Google Scholar, 29Schaeper U. Vogel R. Chmielowiec J. Huelsken J. Rosario M. Birchmeier W. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 15376-15381Crossref PubMed Scopus (47) Google Scholar). Gab1 has been reported to be required for both EGF-dependent activation of PI3K-AKT signaling pathway and migration of keratinocytes (28Mattoon D.R. Lamothe B. Lax I. Schlessinger J. BMC. Biol. 2004; 2: 24Crossref PubMed Scopus (141) Google Scholar, 29Schaeper U. Vogel R. Chmielowiec J. Huelsken J. Rosario M. Birchmeier W. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 15376-15381Crossref PubMed Scopus (47) Google Scholar, 30Rodrigues G.A. Falasca M. Zhang Z. Ong S.H. Schlessinger J. Mol. Cell Biol. 2000; 20: 1448-1459Crossref PubMed Scopus (282) Google Scholar). In addition, Gab1 plays a key role for vascular endothelial growth factor-dependent activation of the PI3K signaling pathway and is required for endothelial cell migration and capillary formation (25Dance M. Montagner A. Yart A. Masri B. Audigier Y. Perret B. Salles J.P. Raynal P. J. Biol. Chem. 2006; 281: 23285-23295Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 27Laramee M. Chabot C. Cloutier M. Stenne R. Holgado-Madruga M. Wong A.J. Royal I. J. Biol. Chem. 2007; 282: 7758-7769Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar).Gab1knockout (Gab1KO) mice died in utero and displayed developmental defects in the heart, placenta, liver, skin, and skeletal muscle (31Itoh M. Yoshida Y. Nishida K. Narimatsu M. Hibi M. Hirano T. Mol. Cell Biol. 2000; 20: 3695-3704Crossref PubMed Scopus (226) Google Scholar, 32Sachs M. Brohmann H. Zechner D. Muller T. Hulsken J. Walther I. Schaeper U. Birchmeier C. Birchmeier W. J. Cell Biol. 2000; 150: 1375-1384Crossref PubMed Scopus (230) Google Scholar). Furthermore, Gab1KO mice displayed reduced and delayed migration of muscle progenitor cells into the limbs and diaphragm, resulting in the immature formation of limb muscles. These data suggest that Gab1 might have a key role in skeletal muscle development (32Sachs M. Brohmann H. Zechner D. Muller T. Hulsken J. Walther I. Schaeper U. Birchmeier C. Birchmeier W. J. Cell Biol. 2000; 150: 1375-1384Crossref PubMed Scopus (230) Google Scholar). We created cardiomyocyte-specific Gab1/Gab2 double knock-out mice and revealed that Gab1 and Gab2 play redundant, but essential roles in postnatal maintenance of cardiac function via the neuregulin-1/ErbB signaling pathway (33Nakaoka Y. Nishida K. Narimatsu M. Kamiya A. Minami T. Sawa H. Okawa K. Fujio Y. Koyama T. Maeda M. Sone M. Yamasaki S. Arai Y. Koh G.Y. Kodama T. Hirota H. Otsu K. Hirano T. Mochizuki N. J. Clin. Invest. 2007; 117: 1771-1781Crossref PubMed Scopus (57) Google Scholar). In addition, liver-specific Gab1 knock-out mice displayed enhanced hepatic insulin sensitivity with reduced glycemia and improved glucose tolerance as a result of insufficient insulin-elicited activation of ERK1/2 (34Bard-Chapeau E.A. Hevener A.L. Long S. Zhang E.E. Olefsky J.M. Feng G.S. Nat. Med. 2005; 11: 567-571Crossref PubMed Scopus (70) Google Scholar). However, it remains elusive whether Gab1 has a specific role in skeletal muscle differentiation. In this study, we demonstrate for the first time that Gab1-SHP2 interaction exerts an inhibitory effect on IGF-I-induced myogenic differentiation via activation of the ERK1/2 signaling pathway.EXPERIMENTAL PROCEDURESReagents and Antibodies—Anti-phospho-p44/42 ERK1/2 (Thr-202/Tyr-204), anti-phospho-AKT (Thr-308), anti-ERK1/2, and anti-AKT antibodies were purchased from Cell Signaling Technology. Anti-Gab1 and anti-Gab2 sera for immunoprecipitation were described previously (15Nishida 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: 1809-1816Crossref PubMed Google Scholar, 16Takahashi-Tezuka M. Yoshida Y. Fukada T. Ohtani T. Yamanaka Y. Nishida K. Nakajima K. Hibi M. Hirano T. Mol. Cell Biol. 1998; 18: 4109-4117Crossref PubMed Scopus (248) Google Scholar, 22Nakaoka Y. Nishida K. Fujio Y. Izumi M. Terai K. Oshima Y. Sugiyama S. Matsuda S. Koyasu S. Yamauchi-Takihara K. Hirano T. Kawase I. Hirota H. Circ. Res. 2003; 93: 221-229Crossref PubMed Scopus (79) Google Scholar). The antibodies against the following molecules used for immunoblotting, Gab1, Gab2, insulin receptor substrate-1 (IRS-1), and p85 PI3K were from Millipore; PY99, SHP2, MEK1, and myogenin were from Santa Cruz Biotechnology. Anti-myosin heavy chain (MHC) monoclonal antibody (MF20) was purchased from the Developmental Hybridoma Bank (Dr. D. A. Fischman, University of Iowa, Iowa City, IA). Hoechst 33342 nuclear dye was from Sigma. Horseradish peroxidase-conjugated anti-mouse and anti-rabbit antibodies were from GE Health Science. U0126 was from Promega (Madison, WI). Serum and cell culture reagents were from Invitrogen. Human recombinant IGF-I was kindly provided by Astellas Pharmaceuticals.Adenovirus Vector Construction—The generation of adenovirus vectors expressing human Gab1WT and Gab1ΔSHP2 (mutated on the two tyrosine residues responsible for binding with SHP2) were described previously (22Nakaoka Y. Nishida K. Fujio Y. Izumi M. Terai K. Oshima Y. Sugiyama S. Matsuda S. Koyasu S. Yamauchi-Takihara K. Hirano T. Kawase I. Hirota H. Circ. Res. 2003; 93: 221-229Crossref PubMed Scopus (79) Google Scholar). In this study, we constructed the adenovirus vectors expressing Gab1Δp85, which can't bind with p85 due to the substitution of tyrosine residues 447, 472, and 589 of human Gab1, corresponding to the YXXM motifs, to phenylalanines by PCR-based mutagenesis described previously (35Yamasaki S. Nishida K. Yoshida Y. Itoh M. Hibi M. Hirano T. Oncogene. 2003; 22: 1546-1556Crossref PubMed Scopus (59) Google Scholar). Substitution of these tyrosine residues by phenylalanine renders the molecule incapable of binding with p85. We also constructed adenovirus vectors expressing wild-type SHP2 (SHP2WT) and phosphatase-inactive SHP2 (SHP2C/S) using the plasmid vectors described previously (15Nishida 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: 1809-1816Crossref PubMed Google Scholar). For adenovirus production, the sequence encoding Gab1Δp85, SHP2WT, or SHP2C/S was subcloned into the shuttle plasmid pACCMVpLpA. Recombinant adenoviruses were then obtained according to the homologous recombination system described elsewhere (36Becker T.C. Noel R.J. Coats W.S. Gomez-Foix A.M. Alam T. Gerard R.D. Newgard C.B. Methods Cell Biol. 1994; 43: 161-189Crossref PubMed Scopus (561) Google Scholar). The adenovirus vectors expressing constitutive-active MEK1 and dominant-negative MEK1 were kindly provided by Dr. S. Kawashima (Kobe University) and described previously (37Ueyama T. Kawashima S. Sakoda T. Rikitake Y. Ishida T. Kawai M. Yamashita T. Ishido S. Hotta H. Yokoyama M. J. Mol. Cell Cardiol. 2000; 32: 947-960Abstract Full Text PDF PubMed Scopus (105) Google Scholar). The construction of adenovirus vector expressing human Gab2ΔSHP2, which can't bind with SHP2, is described in the supplemental data.Cell Culture, Stimulation, and Adenoviral Infection—C2C12 murine myoblast cells were maintained as subconfluent monolayers in Dulbecco's modified Eagle's medium (DMEM) containing 4.5 g/liter glucose, 0.58 g/liter l-glutamine, 100 units/ml penicillin, and 100 μg/ml streptomycin supplemented with 20% fetal bovine serum (FBS). Before stimulation, cells were serum-starved overnight. Stimulations were performed using 80 ng/ml IGF-I for 10 min, unless otherwise indicated. For adenoviral infection of C2C12 myoblasts, subconfluent cells were cultured in DMEM with 20% FBS at a multiplicity of infection of 50 for 24 h. In the dual infection of adenovirus vectors, C2C12 cells were cultured in DMEM with 20% FBS with each virus at a multiplicity of infection of 30. Then, the myoblasts were serum-starved overnight and stimulated with or without IGF-I for the experiments examining ERK1/2 and AKT phosphorylation. Infection efficiency, determined by lacZ gene expression in cultured myoblasts, is consistently >90% with this method. Adenovirus vector expressing β-galactosidase (β-gal) was used as a control. For the induction of myogenic differentiation, cultured medium was switched from DMEM containing 20% FBS to DMEM containing 2% horse serum (HS) or 80 ng/ml IGF-I, when cell density reached confluency. The differentiation medium containing 2% HS was exchanged every other day, and that containing IGF-I was exchanged every day.Cell Lysis, Immunoprecipitations, and Immunoblotting—Cells were scraped off in lysis buffer containing 20 mm Tris (pH 7.4), 150 mm NaCl, 3 mm EDTA, 1% Nonidet P-40, 2 mm sodium orthovanadate, and protease inhibitor mixture Complete (Roche Applied Science). Cell lysates were collected form confluent 6-cm dishes and precleared by 15,000 × g centrifugation for 15 min. For immunoprecipitation, the cleared lysates of 500 μl containing 1 mg of protein, were rotationally incubated with 1 μl of anti-Gab1 antiserum, or 1.2 μg of SHP2 antibody, or 5 μl of p85 antibody, or 10 μl of IRS-1 antibody and with 20 μl of protein A-Sepharose (GE Healthcare) overnight at 4 °C. The antigen-antibody complexes were collected by centrifugation, washed three times with lysis buffer without protease inhibitor mixture, and boiled in standard electrophoresis sample buffer. All the proteins immunoprecipitated were then resolved by SDS-PAGE and subjected to immunoblotting using a standard procedure. Blots were developed using ECL system (GE Healthcare). For direct immunoblotting analyses, the crude cell lysates were collected from 3.5-cm dishes and subjected to 15,000 × g centrifugation. The precleared lysates containing 30 μg of protein were loaded in each lane for immunoblotting.Immunocytochemistry—Cells cultured on 3.5-cm collagen type I-coated plastic dishes (Iwaki Asahi Glass Co.) were fixed with 2% formaldehyde in phosphate-buffered saline and permeabilized with 0.1% Triton X-100 for 10 min. Cells were blocked with phosphate-buffered saline containing 1% bovine serum albumin for 1 h and incubated with anti-MHC antibody (MF20), followed by incubation with Alexa 488-labeled goat anti-mouse secondary antibody (Molecular Probes). Cells were post-stained with Hoechst 33342 nuclear dye and viewed by fluorescence microscopy. siRNA-mediated Protein Knockdown—Stealth Select small interfering RNAs (siRNAs) targeted to murine Gab1 (#1, MSS204497; #2, MSS204499) and siRNA duplex with irrelevant sequences (Stealth™ RNAi negative control) as a control were purchased from Invitrogen. Stealth siRNAs targeted to murine SHP2 were purchased from Invitrogen. and the detailed sequences are described in the supplemental data. C2C12 myoblasts were transfected with 10 nm siRNA duplexes using Lipofectamine™ RNAiMAX reagent according to the manufacturer's instructions for reverse transfection. Briefly, C2C12 myoblasts (1.5 × 105 cells per each dish) were diluted in 860 μl of DMEM containing 20% FBS and plated on 3.5-cm collagen type I-coated plastic dishes. To each dish, 140 μl of RNAi duplex-Lipofectamine™ RNAiMAX complex diluted in Opti-MEM I medium was added. After incubation for 72 h, the cells were used for the experiments.Statistics—All data are expressed as mean ± S.D. Differences among multiple groups were compared by one-way ANOVA followed by a post hoc comparison tested with Scheffe's method. Values of p < 0.05 were considered significant.RESULTSGab1 Undergoes Tyrosine Phosphorylation and Subsequently Associates with SHP2 upon IGF-I Stimulation in C2C12 Myoblasts—We examined the effect of IGF-I on tyrosine-phosphorylation of Gab1 and its association with SH2 domain-containing signaling molecules in C2C12 myoblasts. IGF-I indeed induced tyrosine phosphorylation of Gab1 and subsequent association of Gab1 with SHP2 in C2C12 myoblasts (Fig. 1A, left panel). Furthermore, SHP2 was also tyrosine-phosphorylated and associated with Gab1 after stimulation with IGF-I (Fig. 1A, right panel). On the other hand, Gab1 constitutively associated with p85 both before and after IGF-I stimulation (Fig. 1A, left panel). In the IGF-I-dependent signaling pathway, IRS-1 has been reported to be a major binding partner of p85 and essential for IGF-I-dependent PI3K-AKT signaling in skeletal muscle cells (38Sarbassov D.D. Peterson C.A. Mol. Endocrinol. 1998; 12: 1870-1878Crossref PubMed Scopus (58) Google Scholar). Consistently, IRS-1 underwent strong tyrosine phosphorylation after stimulation with IGF-I in C2C12 myoblasts. In clear contrast to Gab1, IRS-1 associated with p85 in a manner dependent on IGF-I stimulation (Fig. 1B). In addition, we could not detect the complex formation of IRS-1 with SHP2 either before or after stimulation with IGF-I (Fig. 1B). These results demonstrate that IGF-I induces tyrosine phosphorylation of Gab1, leading to complex formation of Gab1 with SHP2 in C2C12 myoblasts.IGF-I-induced Tyrosine Phosphorylation of Gab1 and Association of Gab1 with SH2 Domain-containing Molecules in the C2C12 Myoblasts Infected with Adenovirus Vectors—IGFs have been reported to stimulate both proliferation and differentiation of cultured skeletal muscle cells (3Coolican S.A. Samuel D.S. Ewton D.Z. McWade F.J. Florini J.R. J. Biol. Chem. 1997; 272: 6653-6662Abstract Full Text Full Text PDF PubMed Scopus (551) Google Scholar, 4Florini J.R. Ewton D.Z. Coolican S.A. Endocr. Rev. 1996; 17: 481-517PubMed Google Scholar). The effect of IGF-I on the proliferation of myoblasts has been reported to be attributed mainly to ERK1/2 pathway (3Coolican S.A. Samuel D.S. Ewton D.Z. McWade F.J. Florini J.R. J. Biol. Chem. 1997; 272: 6653-6662Abstract Full Text Full Text PDF PubMed Scopus (551) Google Scholar). In the present study, we tried to reveal the role of Gab1 in IGF-I-dependent differentiation from myoblasts into myotubes. To discern the role of Gab1-SHP2 interaction from that of Gab1-p85 interaction in IGF-I-dependent differentiation, we used recombinant adenovirus vectors carrying β-gal (control), Gab1WT, or Gab1ΔSHP2 as described previously (22Nakaoka Y. Nishida K. Fujio Y. Izumi M. Terai K. Oshima Y. Sugiyama S. Matsuda S. Koyasu S. Yamauchi-Takihara K. Hirano T. Kawase I. Hirota H. Circ. Res. 2003; 93: 221-229Crossref PubMed Scopus (79) Google Scholar) and created an adenovirus vector overexpressing Gab1Δp85. We examined tyrosine phosphorylation of Gab1 upon stimulation with IGF-I in myoblasts overexpressing β-gal, Gab1WT, Gab1ΔSHP2, or Gab1Δp85. As shown in Fig. 2A, tyrosine phosphorylation of Gab1 and the amount of co-immunoprecipitated SHP2 with Gab1 were increased after stimulation with IGF-I in control myoblasts expressing β-gal. On the other hand, tyrosine phosphorylation of Gab1 in the myoblasts overexpressing Gab1WT, Gab1ΔSHP2, or Gab1Δp85 increased much more at baseline compared with in those overexpressing β-gal. In these cells, tyrosine phosphorylation of Gab1 decreased after stimulation with IGF-I. The IGF-I-induced association of Gab1 with SHP2 increased in the C2C12 myoblasts overexpressing Gab1WT, or Gab1Δp85 compared with those overexpressing β-gal, but was almost abrogated in those overexpressing Gab1ΔSHP2 (Fig. 2A). The co-immunoprecipitation of Gab1 with p85 was increased in cells expressing Gab1WT or Gab1ΔSHP2 compared with those expressing β-gal, but was almost abrogated in those expressing Gab1Δp85 at the baseline. The association of Gab1 with p85 decreased in response to IGF-I in the cells overexpressing Gab1WT or Gab1ΔSHP2 (Fig. 2A). We observed much more dissociation of p85 from Gab1 in myoblasts overexpressing Gab1WT compared with those overexpressing Gab1ΔSHP2, which might be attributed to the increased activation of SHP2. Presumably, SHP2 dephosphorylates the tyrosine residues for p85 binding site of Gab1 consistent with the previous report on EGF-dependent signaling (39Zhang 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 (207) Google Scholar). These data demonstrate that overexpression of Gab1ΔSHP2 or Gab1Δp85 sufficiently suppresses the association of Gab1 with SHP2 or p85, respectively.FIGURE 2Overexpression of Gab1ΔSHP2 or Gab1Δp85 specifically perturbs the IGF-I-dependent molecular association of Gab1 with SHP2 or p85, respectively. C2C12 myoblasts were infected with adenovirus vectors expressing β-gal, Gab1WT, Gab1ΔSHP2, or Gab1Δp85. Serum-starved C2C12 cells were stimulated with vehicle (-) or IGF-I for 10 min, and cell lysates of 500 μl were collected from 6-cm dishes. Cell lysates were subjected to immunoprecipitation with anti-Gab1 serum (A) or with anti-IRS-1 antibody (B), followed by immunoblotting with anti-phosphotyrosine antibody (PY99). A, blots were reprobed with anti-Gab1, anti-p85, and anti-SHP2 antibodies. B, blots were reprobed with anti-IRS-1, anti-p85, and anti-SHP2 antibodies. Experiments were repeated three times with similar results.View Large Image Fig
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