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Insulin Receptor Substrate 2 And Shc Play Different Roles In Insulin-like Growth Factor I Signaling

1998; Elsevier BV; Volume: 273; Issue: 51 Linguagem: Inglês

10.1074/jbc.273.51.34543

ISSN

1083-351X

Autores

Bhumsoo Kim, Hsin-Lin Cheng, Benjamin Margolis, Eva L. Feldman,

Tópico(s)

Metabolism, Diabetes, and Cancer

Resumo

The major substrates for the type I insulin-like growth factor (IGF-I) receptor are Shc and insulin receptor substrate (IRS) proteins. In the current study, we report that IGF-I induces a sustained tyrosine phosphorylation of Shc and its association with Grb2 in SH-SY5Y human neuroblastoma cells. The time course of Shc tyrosine phosphorylation parallels the time course of IGF-I-stimulated activation of extracellular signal-regulated kinase (ERK). Transfection of SH-SY5Y cells with a p52 Shc mutant decreases Shc tyrosine phosphorylation and Shc-Grb2 association. This results in the inhibition of IGF-I-mediated ERK tyrosine phosphorylation and neurite outgrowth. In contrast, IGF-I induces a transient tyrosine phosphorylation of IRS-2 and an association of IRS-2 with Grb2. The time course of IRS-2 tyrosine phosphorylation and IRS-2-Grb2 and IRS-2-p85 association closely resembles the time course of IGF-I-mediated membrane ruffling. Treating cells with the phosphatidylinositol 3′-kinase inhibitors wortmannin and LY294002 blocks IGF-I-induced membrane ruffling. The ERK kinase inhibitor PD98059, as well as transfection with the p52 Shc mutant, has no effect on IGF-I-mediated membrane ruffling. Immunolocalization studies show IRS-2 and Grb2, but not Shc, concentrated at the tip of the extending growth cone where membrane ruffling is most active. Collectively, these results suggest that the association of Shc with Grb2 is essential for IGF-I-mediated neurite outgrowth, whereas the IRS-2-Grb2-phosphatidylinositol 3′-kinase complex may regulate growth cone extension and membrane ruffling. The major substrates for the type I insulin-like growth factor (IGF-I) receptor are Shc and insulin receptor substrate (IRS) proteins. In the current study, we report that IGF-I induces a sustained tyrosine phosphorylation of Shc and its association with Grb2 in SH-SY5Y human neuroblastoma cells. The time course of Shc tyrosine phosphorylation parallels the time course of IGF-I-stimulated activation of extracellular signal-regulated kinase (ERK). Transfection of SH-SY5Y cells with a p52 Shc mutant decreases Shc tyrosine phosphorylation and Shc-Grb2 association. This results in the inhibition of IGF-I-mediated ERK tyrosine phosphorylation and neurite outgrowth. In contrast, IGF-I induces a transient tyrosine phosphorylation of IRS-2 and an association of IRS-2 with Grb2. The time course of IRS-2 tyrosine phosphorylation and IRS-2-Grb2 and IRS-2-p85 association closely resembles the time course of IGF-I-mediated membrane ruffling. Treating cells with the phosphatidylinositol 3′-kinase inhibitors wortmannin and LY294002 blocks IGF-I-induced membrane ruffling. The ERK kinase inhibitor PD98059, as well as transfection with the p52 Shc mutant, has no effect on IGF-I-mediated membrane ruffling. Immunolocalization studies show IRS-2 and Grb2, but not Shc, concentrated at the tip of the extending growth cone where membrane ruffling is most active. Collectively, these results suggest that the association of Shc with Grb2 is essential for IGF-I-mediated neurite outgrowth, whereas the IRS-2-Grb2-phosphatidylinositol 3′-kinase complex may regulate growth cone extension and membrane ruffling. insulin-like growth factor I IGF-I receptor insulin receptor substrate extracellular signal-regulated kinase phosphatidylinositol 3′-kinase son of sevenless mitogen-activated protein epidermal growth factor. Binding of insulin-like growth factor I (IGF-I)1 to the extracellular α-subunits of the type I insulin-like growth factor receptor (IGF-IR) results in autophosphorylation of the cytoplasmic β-subunits (1White M.F. Kahn C.R. J. Biol. Chem. 1994; 269: 1-4Abstract Full Text PDF PubMed Google Scholar, 2Van Obberghen E. Diabetologia. 1994; 37 Suppl. 2: S125-S134Crossref PubMed Scopus (60) Google Scholar). This autophosphorylation of the IGF-IR initiates a cascade of cellular signal transduction pathways. One key event is the binding of insulin receptor substrates (IRS) 1 and 2 to phosphotyrosine residues on the receptor β-subunits (3Myers Jr., M.G. White M.F. Annu. Rev. Pharmacol. Toxicol. 1996; 36: 615-658Crossref PubMed Scopus (298) Google Scholar). After binding by activated receptors, IRS-1 and -2 are tyrosine phosphorylated and act as docking proteins for downstream signaling molecules containing Src homology 2 domains, such as the 85-kDa regulatory (p85) subunit of phosphatidylinositol 3′-kinase (PI3K) and the adapter protein Grb2 (reviewed in Refs. 3Myers Jr., M.G. White M.F. Annu. Rev. Pharmacol. Toxicol. 1996; 36: 615-658Crossref PubMed Scopus (298) Google Scholar and4Waters S.B. Pessin J.E. Trends Cell Biol. 1996; 6: 1-4Abstract Full Text PDF PubMed Scopus (63) Google Scholar). Recent reports suggest IRS proteins play important and distinctive roles in IGF and insulin signaling. For example, IRS-1 is essential for IGF-I-stimulated mitogenesis (5Bruning J.C. Winnay J. Cheatham B. Kahn C.R. Mol. Cell. Biol. 1997; 17: 1513-1521Crossref PubMed Scopus (191) Google Scholar). Mice that are made IRS-1 deficient have retarded growth and reduced glucose metabolism when stimulated by insulin or IGF-I (6Tamemoto H. Kadowaki T. Tobe K. Yagi T. Sakura H. Hayakawa T. Terauchi Y. Ueki K. Kaburagi Y. Satoh S. Sekihara H. Yoshioka S. Horikoshi H. Furuta Y. Ikawa Y. Kasuga M. Yazaki Y. Aizawa S. Nature. 1994; 372: 182-186Crossref PubMed Scopus (906) Google Scholar, 7Araki E. Lipes M.A. Patti M.-E. Bruning J.C. Haag III, B. Johnson R.S. Kahn C.R. Nature. 1994; 372: 186-190Crossref PubMed Scopus (1098) Google Scholar). In contrast, IRS-2 is more tightly linked to glucose regulation. Mice that are made IRS-2 deficient have the typical phenotype of type 2 diabetes (8Withers D.J. Gutierrez J.S. Towery H. Burks D.J. Ren J.-M. Previs S. Zhang Y. Bernal D. Pons S. Shulman G.I. Bonner-Weir S. White M.F. Nature. 1998; 391: 900-904Crossref PubMed Scopus (1346) Google Scholar). Another substrate for activated IGF-IR is Shc, which exists in three isoforms: p66, p52, and p46 (9Pelicci G. Lanfrancone L. Grignani F. McGlade J. Cavallo F. Forni G. Nicoletti I. Pawson T. Pelicci P.G. Cell. 1992; 70: 93-104Abstract Full Text PDF PubMed Scopus (1140) Google Scholar). Shc is composed of a C terminus Src homology 2 domain, an N terminus phosphotyrosine binding domain, and a central collagen homologous domain (10Bonfini L. Migliaccio E. Pelicci G. Lanfrancone L. Pelicci P.G. Trends Biochem. Sci. 1996; 21: 257-261Abstract Full Text PDF PubMed Scopus (235) Google Scholar). Like IRS-1 and -2, Shc proteins are tyrosine phosphorylated upon binding to IGF-IR (11Giorgetti S. Pelicci P.G. Pelicci G. Van Obberghen E. Eur. J. Biochem. 1994; 223: 195-202Crossref PubMed Scopus (98) Google Scholar, 12Pronk G.J. McGlade J. Pelicci G. Pawson T. Bos J.L. J. Biol. Chem. 1993; 268: 5748-5753Abstract Full Text PDF PubMed Google Scholar) and can associate with Grb2 (13Rozakis-Adcock M. McGlade J. Mbamalu G. Pelicci G. Daly R. Li W. Batzer A. Thomas S. Brugge J. Pelicci P.G. Schlessinger J. Pawson T. Nature. 1992; 360: 689-692Crossref PubMed Scopus (828) Google Scholar, 14Skolnik E.Y. Lee C.-H. Batzer A. Vincentini L.M. Zhou M. Daly R. Myers Jr., M.G. Backer J.M. Ullrich A. White M.F. Schlessinger J. EMBO J. 1993; 12: 1929-1936Crossref PubMed Scopus (607) Google Scholar). Shc has been implicated in the mitogenic signaling of IGF-I (15Sasaoka T. Rose D.W. Jhun B.H. Saltiel A.R. Draznin B. Olefsky J.M. J. Biol. Chem. 1994; 269: 13689-13694Abstract Full Text PDF PubMed Google Scholar), the maintenance of cell-cell interactions, and transformation in breast cancer cells (16Nolan M.K. Jankowska L. Prisco M. Xu S.-Q. Guvakova M.A. Surmacz E. Int. J. Cancer. 1997; 72: 828-834Crossref PubMed Scopus (69) Google Scholar). The binding of IRS or Shc proteins to Grb2 induces the Grb2-associated son of sevenless (SOS) protein to activate p21ras by stimulating GDP/GTP exchange (14Skolnik E.Y. Lee C.-H. Batzer A. Vincentini L.M. Zhou M. Daly R. Myers Jr., M.G. Backer J.M. Ullrich A. White M.F. Schlessinger J. EMBO J. 1993; 12: 1929-1936Crossref PubMed Scopus (607) Google Scholar, 17Baltensperger K. Kozma L.M. Cherniack A.D. Klarlund J. Chawla A. Banarjee U. Czech M.P. Science. 1993; 260: 1950-1952Crossref PubMed Scopus (232) Google Scholar, 18Skolnik E.Y. Batzer A. Li N. Lee C.-H. Lowenstein E. Mohammadi M. Margolis B. Schlessinger J. Science. 1993; 260: 1953-1955Crossref PubMed Scopus (505) Google Scholar). Stimulation of p21ras leads to activation of the mitogen-activated protein (MAP) kinase pathway, which plays an important role in cellular differentiation and growth (19Davis R.J. J. Biol. Chem. 1993; 268: 14553-14556Abstract Full Text PDF PubMed Google Scholar, 20Pelech S.L. Sanghera J.S. Trends Biochem. Sci. 1992; 17: 233-238Abstract Full Text PDF PubMed Scopus (338) Google Scholar). Activation of one of the MAP kinases, the extracellular signal-regulated kinase (ERK), is required for IGF-I-mediated neurite outgrowth in SH-SY5Y human neuroblastoma cells (21Kim B. Leventhal P.S. Saltiel A.R. Feldman E.L. J. Biol. Chem. 1997; 272: 21268-21273Crossref PubMed Scopus (143) Google Scholar). There is continued controversy surrounding the relative importance of Shc compared with the IRS proteins in activation of the MAP kinase pathway. Sasaoka et al. (22Sasaoka T. Draznin B. Leitner J.W. Langlois W.J. Olefsky J.M. J. Biol. Chem. 1994; 269: 10734-10738Abstract Full Text PDF PubMed Google Scholar) reported that the majority of p21ras guanine nucleotide exchange activity was present in Shc immunoprecipitates, whereas only negligible amounts were found in IRS-1 immunoprecipitates. Studies with mutant insulin receptors show that insulin can mediate the tyrosine phosphorylation of Shc, the association of Shc and Grb2, and p21ras-GTP formation, all without detectable tyrosine phosphorylation of IRS-1 or enhancement of the interaction between IRS-1 and Grb2 (23Ouwens D.M. van der Zon G.C.M. Pronk G.J. Bos J.L. Moller W. Cheatham B. Kahn C.R. Maassen J.A. J. Biol. Chem. 1994; 269: 33116-33122Abstract Full Text PDF PubMed Google Scholar). Taken together, these studies suggest that the Shc-Grb2 complex is the key intermediate in downstream signaling of the MAP kinase pathway. In contrast, several reports implicate a central role for IRS proteins in MAP kinase signaling. SOS can be found in IRS-1 immunoprecipitates (17Baltensperger K. Kozma L.M. Cherniack A.D. Klarlund J. Chawla A. Banarjee U. Czech M.P. Science. 1993; 260: 1950-1952Crossref PubMed Scopus (232) Google Scholar), and the expression of IRS-1 induces cellular transformation with the activation of MAP kinases (24Ito T. Sasaki Y. Wands J.R. Mol. Cell. Biol. 1996; 16: 943-951Crossref PubMed Scopus (81) Google Scholar). Furthermore, mutated insulin receptors that tyrosine phosphorylate IRS-1 but not Shc have normal activation of p21ras (25Yonezawa K. Ando A. Kaburagi Y. Yamamoto-Honda R. Kitamura T. Hara K. Nakafuku M. Okabayashi Y. Kadowaki T. Kaziro Y. Kasuga M. J. Biol. Chem. 1994; 269: 4634-4640Abstract Full Text PDF PubMed Google Scholar). These reports suggest that the relative roles of Shc and IRS proteins for mediating insulin and IGF-I signaling depend more on the nature of the signaling molecules and the individual cell types (26Yamauchi K. Pessin J.E. Mol. Cell. Biol. 1994; 14: 4427-4434Crossref PubMed Scopus (50) Google Scholar). Unlike Shc, which seems to associate only with Grb2, IRS proteins can associate with a variety of other downstream signaling molecules, such as PI3K (3Myers Jr., M.G. White M.F. Annu. Rev. Pharmacol. Toxicol. 1996; 36: 615-658Crossref PubMed Scopus (298) Google Scholar, 4Waters S.B. Pessin J.E. Trends Cell Biol. 1996; 6: 1-4Abstract Full Text PDF PubMed Scopus (63) Google Scholar). Among them, PI3K is implicated in insulin- and IGF-I-mediated membrane ruffling. We have demonstrated that IGF-I induces membrane ruffling and growth cone extension in SH-SY5Y cells (27Leventhal P.S. Shelden E.A. Kim B. Feldman E.L. J. Biol. Chem. 1997; 272: 5214-5218Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). In parallel, the microinjection of a mutant p85 subunit of PI3K into cells inhibits IRS-1-PI3K association and insulin- and IGF-I-induced membrane ruffling (28Kotani K. Yonezawa K. Hara K. Ueda H. Kitamura Y. Sakaue H. Ando A. Chavanieu A. Calas B. Grigorescu F. Nishiyama M. Waterfield M.D. Kasuga M. EMBO J. 1994; 13: 2313-2321Crossref PubMed Scopus (328) Google Scholar). PI3K may mediate growth factor-induced membrane ruffling by acting as an upstream regulator of the small GTPase protein Rac (29Parker P.J. Curr. Biol. 1995; 5: 577-579Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 30Hawkins P.T. Eguinoa A. Qui R.G. Stokoe D. Cooke F.T. Wakter R. Wennström S. Claesson W.L. Evans T. Symons M. Stephens L. Curr. Biol. 1995; 5: 393-403Abstract Full Text Full Text PDF PubMed Scopus (496) Google Scholar, 31Wennström S. Hawkins P. Cooke F. Hara K. Yonezawa K. Kasuga M. Jackson T. Claesson-Welsh L. Stephens L. Curr. Biol. 1994; 4: 385-393Abstract Full Text Full Text PDF PubMed Scopus (395) Google Scholar, 32Nishiyama T. Sasaki T. Takaishi K. Kato M. Yaku H. Araki K. Matsuura Y. Takai Y. Mol. Cell. Biol. 1994; 14: 2447-2456Crossref PubMed Scopus (160) Google Scholar). In our laboratory, we are studying the pleiotrophic effects of IGF-I:IGF-IR signaling in the nervous system (21Kim B. Leventhal P.S. Saltiel A.R. Feldman E.L. J. Biol. Chem. 1997; 272: 21268-21273Crossref PubMed Scopus (143) Google Scholar, 27Leventhal P.S. Shelden E.A. Kim B. Feldman E.L. J. Biol. Chem. 1997; 272: 5214-5218Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar, 33Singleton J.R. Dixit V.M. Feldman E.L. J. Biol. Chem. 1996; 271: 31791-31794Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar, 34Leventhal P.S. Feldman E.L. Trends Endocrinol. Metab. 1997; 8: 1-6Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 35Feldman E.L. Sullivan K.A. Kim B. Russell J.W. Neurobiol. Dis. 1997; 4: 201-214Crossref PubMed Scopus (175) Google Scholar, 36Cheng H.-L. Feldman E.L. J. Biol. Chem. 1998; 273: 14560-14565Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 37Kim B. Leventhal P.S. White M.F. Feldman E.L. Endocrinology. 1998; (in press)Google Scholar). In the current study, we examined the roles of Shc and IRS-2 in IGF-I signaling in human SH-SY5Y neuroblastoma cells. IGF-I induced distinct temporal patterns of Shc and IRS-2 tyrosine phosphorylation. The transfection of a p52 Shc tyrosine mutant, p52 Shc Y239F/Y240F/Y317F, greatly reduced the IGF-I-mediated tyrosine phosphorylation of ERK and subsequent neurite outgrowth. In contrast, IRS-2-PI3K was required for IGF-I-mediated membrane ruffling. Finally, immunoreactive Grb2 and IRS-2 were localized to membrane ruffles, whereas Shc displayed a more ubiquitous staining. Our data suggest that the IGF-I-mediated association of Shc and Grb2 plays an important role in ERK activation and neurite outgrowth, whereas the IGF-I activation of the association of IRS-2 with PI3K is necessary for IGF-I-mediated membrane ruffling and growth cone extension. These studies imply distinct roles for Shc and IRS proteins, the two most prominent downstream mediators of IGF-IR signaling. Anti-phosphotyrosine antibodies were purchased from Transduction Laboratories (PY20; Lexington, KY) and Upstate Biochemicals, Inc. (4G10; Lake Placid, NY). Anti-Grb2 polyclonal antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-Shc polyclonal antibody was obtained from Transduction Laboratories. Anti-active MAP kinase antibody was obtained from Promega (Madison, WI). Anti-IGF-IR α-subunit antibody (α-IR3) and epidermal growth factor (EGF) were obtained from Oncogene Science (Uniondale, NY). Monoclonal antibody against Myc-tag was used as described previously (38Blaikie P.A. Fournier E. Dilworth S.N. Birnbaum D. Borg J.-P. Margolis B. J. Biol. Chem. 1997; 272: 20671-20677Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). Anti-IRS-2 and anti-p85pan antiserums were generous gifts from Dr. M. F. White (Joslin Diabetes Center, Harvard Medical School, Boston, MA). LY294002 and wortmannin were purchased from Biomol (Plymouth Meeting, PA). MAP/ERK kinase inhibitor PD98059 was kindly provided by Dr. A. Saltiel of Parke-Davis Pharmaceutical Research (Ann Arbor, MI). IGF-I was a gift from Cephalon Corp. (West Chester, PA). Dulbecco's modified Eagle's medium with high glucose, l-glutamine, and sodium pyruvate was from Life Technologies, Inc. Other reagents were purchased from Sigma or Boehringer Mannheim. SH-SY5Y human neuroblastoma cells were grown in Dulbecco's modified Eagle's medium containing 10% calf serum and maintained at 37 °C in a humidified atmosphere with 10% CO2. 18–24 h before the experiments, the media were replaced with Dulbecco's modified Eagle's medium without serum. For neurite outgrowth experiments, serum-starved cells were incubated in serum-free media for 24 h with or without IGF-I. Processes longer than the cell body were considered as neurites, as described previously (21Kim B. Leventhal P.S. Saltiel A.R. Feldman E.L. J. Biol. Chem. 1997; 272: 21268-21273Crossref PubMed Scopus (143) Google Scholar). The p52 Shc tyrosine mutant construct p52 Shc Y239F/Y240F/Y317F, which has a Myc-tag, has been described previously (38Blaikie P.A. Fournier E. Dilworth S.N. Birnbaum D. Borg J.-P. Margolis B. J. Biol. Chem. 1997; 272: 20671-20677Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). Shc cDNA was subcloned into the mammalian expression vector pSFFV (39Singleton J.R. Randolph A.E. Feldman E.L. Cancer Res. 1996; 56: 4522-4529PubMed Google Scholar). Plasmid DNA was prepared using a plasmid maxi kit from Qiagen (Chatsworth, CA). SH-SY5Y cells were transfected using LipofectAMINE reagent (Life Technologies, Inc.) according to the manufacturer's protocol. For stable transfection, cells were selected by growing in media containing 0.2 mg/ml G418. Immunoprecipitation and immunoblotting were performed as described previously (21Kim B. Leventhal P.S. Saltiel A.R. Feldman E.L. J. Biol. Chem. 1997; 272: 21268-21273Crossref PubMed Scopus (143) Google Scholar). All experiments were repeated at least twice, and typical representative results are shown in the figures. Immunocytochemistry was performed as described previously (27Leventhal P.S. Shelden E.A. Kim B. Feldman E.L. J. Biol. Chem. 1997; 272: 5214-5218Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). Polyclonal antibodies against IRS-2, Grb2, or Shc and a monoclonal antibody against IGF-IR (α-IR3) were used to study the cellular distribution of these proteins. Actin filaments were stained by incubating fixed, permeabilized cells with 2 units/ml rhodamine-phalloidin (Molecular Probes, Eugene, OR). We recently reported that in SH-SY5Y human neuroblastoma cells, IGF-I induces the tyrosine phosphorylation of several intracellular proteins including IGF-IR, IRS-2, focal adhesion kinase, paxillin, and ERKs 1 and 2 (21Kim B. Leventhal P.S. Saltiel A.R. Feldman E.L. J. Biol. Chem. 1997; 272: 21268-21273Crossref PubMed Scopus (143) Google Scholar, 27Leventhal P.S. Shelden E.A. Kim B. Feldman E.L. J. Biol. Chem. 1997; 272: 5214-5218Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar, 37Kim B. Leventhal P.S. White M.F. Feldman E.L. Endocrinology. 1998; (in press)Google Scholar). In agreement with our previous studies (37Kim B. Leventhal P.S. White M.F. Feldman E.L. Endocrinology. 1998; (in press)Google Scholar), 10 nm IGF-I induced a rapid tyrosine phosphorylation of IRS-2, which was maintained for less than 30 min (Fig.1 A). Because it has been reported that Shc is another substrate for the IGF-IR and the insulin receptor (1White M.F. Kahn C.R. J. Biol. Chem. 1994; 269: 1-4Abstract Full Text PDF PubMed Google Scholar), we investigated the effect of IGF-I on Shc tyrosine phosphorylation. When SH-SY5Y cells were treated with 10 nmIGF-I, anti-Shc antibody immunoprecipitated three tyrosine phosphorylated proteins ranging between 45 and 70 kDa (Fig.1 B). These correspond to the reported mobilities of three isoforms of Shc, p66, p52, and p46. Anti-Shc immunoblotting confirmed that these proteins were Shc (see Fig. 3 D). Tyrosine phosphorylation of p52 and p66 Shc was detected as early as 5 min after the addition of IGF-I and, unlike IRS-2 tyrosine phosphorylation, was maintained for at least 2 h. p46 Shc showed constitutive tyrosine phosphorylation even in unstimulated cells, and IGF-I treatment had little effect on the level of tyrosine phosphorylation.Figure 3Transfection of the p52 Shc mutant inhibits IGF-I-induced tyrosine phosphorylation of IRS-2, and the association of IRS-2 with PI3K and IRS-2 with Grb2. Vector- or p52 Shc mutant-transfected SH-SY5Y cells were treated with 10 nmIGF-I for 5 min. The cell lysates were immunoprecipitated with anti-IRS-2 (A and B) or anti-Grb2 (C) antibodies followed by immunoblotting with anti-phosphotyrosine (A and C) or anti-p85pan(B) antibodies. Results are representative of at least two different experiments.View Large Image Figure ViewerDownload (PPT) It has been reported that Grb2 can bind to both IRS-1 and Shc (14Skolnik E.Y. Lee C.-H. Batzer A. Vincentini L.M. Zhou M. Daly R. Myers Jr., M.G. Backer J.M. Ullrich A. White M.F. Schlessinger J. EMBO J. 1993; 12: 1929-1936Crossref PubMed Scopus (607) Google Scholar). To study the association of Grb2 with IRS-2 and Shc, we immunoprecipitated cell lysates with anti-Grb2 antibody and immunoblotted with anti-phosphotyrosine antibodies. In agreement with our previous results (37Kim B. Leventhal P.S. White M.F. Feldman E.L. Endocrinology. 1998; (in press)Google Scholar), we observe the transient association of Grb2 with IRS-2 (Fig.1 C). In the same anti-phosphotyrosine blot, we also detect all three Shc proteins, confirming the association of Grb2 with both Shc and IRS-2. Unlike the association of Grb2 with IRS-2, the Grb2-Shc association showed a prolonged time course similar to that of Shc tyrosine phosphorylation (Fig. 1 C). We have reported that IGF-I induces sustained activation of ERK2 (21Kim B. Leventhal P.S. Saltiel A.R. Feldman E.L. J. Biol. Chem. 1997; 272: 21268-21273Crossref PubMed Scopus (143) Google Scholar). The time course of ERK tyrosine phosphorylation more closely resembles the time course of the association of Shc with Grb2 than that of the IRS-2-Grb2 association. To examine whether prolonged tyrosine phosphorylation of Shc is an intrinsic characteristic of SH-SY5Y cells, we treated the cells with 100 nm EGF. In anti-Shc immunoprecipitates, we detect strong tyrosine phosphorylation of Shc proteins after EGF stimulation (Fig. 1 D). Unlike IGF-I stimulation, EGF-induced Shc tyrosine phosphorylation was transient, lasting less than 30 min. We also detect a transient EGF-mediated association between Shc and Grb2 (data not shown). In parallel with EGF-stimulated Shc tyrosine phosphorylation and Shc-Grb2 association, EGF-stimulated ERK2 tyrosine phosphorylation was also transient (Fig. 1 E). EGF-stimulated ERK tyrosine phosphorylation was maximal at 5 min and rapidly decreased afterward. Unlike IGF-I (40Cooper M.J. Hutchins G.M. Cohen P.S. Helman L.J. Israel M.A. Prog. Clin. Biol. Res. 1991; 366: 343-350PubMed Google Scholar), EGF did not mediate neurite outgrowth (data not shown). Collectively, these results suggest that IGF-I induces distinct temporal patterns of Shc and IRS-2 tyrosine phosphorylation and their respective association with Grb2. Because the above results suggested the involvement of a Shc-Grb2 association in ERK tyrosine phosphorylation, we examined the role of Shc in the activation of the MAP kinase pathway. Recent reports have identified three major tyrosine residues important for Shc tyrosine phosphorylation and Shc-Grb2 association (38Blaikie P.A. Fournier E. Dilworth S.N. Birnbaum D. Borg J.-P. Margolis B. J. Biol. Chem. 1997; 272: 20671-20677Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar, 41Ishihara H. Sasaoka T. Ishiki M. Takata Y. Imamura T. Usui I. Langlois W.J. Sawa T. Kobayashi M. J. Biol. Chem. 1997; 272: 9581-9586Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). We stably transfected the cells with a Myc-tagged p52 Shc construct that has mutations at the three critical tyrosine residues (p52 Shc Y239F/Y240F/Y317F). We detected an increased expression in whole cell lysates of Shc proteins after stable transfection (Fig.2 A). Efficient transfection was confirmed by immunoblotting anti-Shc immunoprecipitates with an anti-Myc antibody. The anti-Myc antibody detected the p52 band from the p52 Shc mutants but not from the cells transfected with vector alone (Fig. 2 B). Next we examined the effects of IGF-I on Shc tyrosine phosphorylation in cells transfected with mutant p52 Shc or vector alone. IGF-I treatment of vector-transfected cells resulted in the tyrosine phosphorylation of Shc proteins (Fig. 2 C). In agreement with a previous report (38Blaikie P.A. Fournier E. Dilworth S.N. Birnbaum D. Borg J.-P. Margolis B. J. Biol. Chem. 1997; 272: 20671-20677Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar), transfection with mutant p52 Shc blocked the IGF-I-induced Shc tyrosine phosphorylation of SH-SY5Y cells (Fig.2 C). When the blot was stripped and reprobed with an anti-Shc antibody, the p66, p52, and p46 Shc bands were seen (Fig.2 D). In parallel with Shc tyrosine phosphorylation, IGF-I-mediated association of Shc with Grb2 was also markedly inhibited by transfection with the p52 Shc mutant (Fig. 2 E). These results show the efficient transfection of the p52 Shc mutant and suggest that Shc tyrosine phosphorylation is necessary for IGF-I-mediated Shc-Grb2 association. Because several studies have shown that there are distinct as well as overlapping domains of the IGF-IR and insulin receptor for Shc and IRS-1 binding (42Wolf G. Trüb T. Ottinger E. Groninga L. Lynch A. White M.F. Miyazaki M. Lee J. Shoelson S.E. J. Biol. Chem. 1995; 270: 27407-27410Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar, 43Tartare-Deckert S. Sawka-Verhelle D. Murdaca J. Van Obberghen E. J. Biol. Chem. 1995; 270: 23456-23460Crossref PubMed Scopus (104) Google Scholar, 44Craparo A. O'Neill T.J. Gustafson T.A. J. Biol. Chem. 1995; 270: 15639-15643Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar), we next examined the effects of the Shc mutant on IRS-2 tyrosine phosphorylation. As expected, treatment with IGF-I induced tyrosine phosphorylation of IRS-2 in vector-transfected cells (Fig.3 A). Interestingly, transfection of the cells with the p52 Shc mutant reduced IGF-I-stimulated IRS-2 tyrosine phosphorylation by 40% (measured by densitometry; data not shown). These data suggest that an overexpression of Shc alters the normal binding of IRS-2 to IGF-IR, supporting the contention that these two proteins share IGF-IR binding domains. We have previously reported that IRS-2 transiently binds to Grb2 and the p85 subunit of PI3K after IGF-I stimulation (37Kim B. Leventhal P.S. White M.F. Feldman E.L. Endocrinology. 1998; (in press)Google Scholar). In agreement with these results, the treatment of vector-transfected cells with IGF-I resulted in the association of IRS-2 with Grb2 and IRS-2 with p85 (Fig.3, B and C). In parallel with the observed decrease in IRS-2 tyrosine phosphorylation, IGF-I treatment of the Shc mutant-transfected cells resulted in a decreased association of IRS-2 with p85 and Grb2. We have reported that IGF-I mediates ERK activation in SH-SY5Y cells (21Kim B. Leventhal P.S. Saltiel A.R. Feldman E.L. J. Biol. Chem. 1997; 272: 21268-21273Crossref PubMed Scopus (143) Google Scholar). Because the association of Shc with Grb2 can lead to activation of the MAP kinase pathway (14Skolnik E.Y. Lee C.-H. Batzer A. Vincentini L.M. Zhou M. Daly R. Myers Jr., M.G. Backer J.M. Ullrich A. White M.F. Schlessinger J. EMBO J. 1993; 12: 1929-1936Crossref PubMed Scopus (607) Google Scholar,17Baltensperger K. Kozma L.M. Cherniack A.D. Klarlund J. Chawla A. Banarjee U. Czech M.P. Science. 1993; 260: 1950-1952Crossref PubMed Scopus (232) Google Scholar, 18Skolnik E.Y. Batzer A. Li N. Lee C.-H. Lowenstein E. Mohammadi M. Margolis B. Schlessinger J. Science. 1993; 260: 1953-1955Crossref PubMed Scopus (505) Google Scholar), we examined the effects of transfecting the p52 Shc mutant on ERK activation. First, we examined the effects of IGF-I treatment of the p52 Shc mutant-transfected cells by anti-phosphotyrosine immunoblotting of whole cell lysates. When vector-transfected cells were stimulated with 10 nm IGF-I, we detected prominent tyrosine phosphorylation of a 95-kDa protein (Fig.4 A) that we have confirmed as the IGF-IR (21Kim B. Leventhal P.S. Saltiel A.R. Feldman E.L. J. Biol. Chem. 1997; 272: 21268-21273Crossref PubMed Scopus (143) Google Scholar). Transfection of the Shc mutant did not affect IGF-I-stimulated IGF-IR tyrosine phosphorylation (Fig. 4 A). IGF-I also induced the tyrosine phosphorylation of an approximately 42-kDa protein in vector-transfected cells (Fig. 4 A). We have shown that this protein represents ERK2 (21Kim B. Leventhal P.S. Saltiel A.R. Feldman E.L. J. Biol. Chem. 1997; 272: 21268-21273Crossref PubMed Scopus (143) Google Scholar). When the cells were transfected with the p52 Shc mutant, IGF-I-stimulated ERK2 tyrosine phosphorylation was markedly inhibited. We confirmed the decrease in ERK2 activation by reprobing the blot with an antibody specific for activated MAP kinase (Fig. 4 B) and by observing a decrease in MAP kinase activity using the MAP kinase assay kit (New England Biolabs, Beverly, MA; data not shown). Because we have previously demonstrated that ERK2 activation is requi

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