Mutant of Insulin Receptor Substrate-1 Incapable of Activating Phosphatidylinositol 3-Kinase Did Not Mediate Insulin-stimulated Maturation of Xenopus laevis Oocytes
1996; Elsevier BV; Volume: 271; Issue: 45 Linguagem: Inglês
10.1074/jbc.271.45.28677
ISSN1083-351X
AutoresRitsuko Yamamoto‐Honda, Zen‐ichiro Honda, Kohjiro Ueki, Kazuyuki Tobe, Yasushi Kaburagi, Yoshihiko Takahashi, Hiroyuki Tamemoto, Takeshi Suzuki, Kohji Itoh, Yasuo Akanuma, Yoshio Yazaki, Takashi Kadowaki,
Tópico(s)Ovarian function and disorders
ResumoInsulin receptor substrate-1 (IRS-1) is rapidly phosphorylated on multiple tyrosine residues in response to insulin and binds several Src homology 2 domain-containing proteins, thereby initiating downstream signaling. To assess the tyrosine phosphorylation sites that mediate relevant downstream signaling and biological effects, we created site-directed mutants of IRS-1 and overexpressed them in the Xenopus laevis oocyte. In oocytes overexpressing IRS-1 or IRS-1-895F (Tyr-895 replaced with phenylalanine), insulin activated phosphatidylinositol (PI) 3-kinase, p70 S6 kinase, and mitogen-activated protein kinase and induced oocyte maturation. In contrast, in oocytes overexpressing IRS-1-4F (Tyr-460, Tyr-608, Tyr-939, and Tyr-987 of IRS-1 replaced with phenylalanine), insulin did not activate PI 3-kinase, p70 S6 kinase, and mitogen-activated protein kinase and failed to induce oocyte maturation. These observations indicate that in X. laevis oocytes overexpressing IRS-1, the association of PI 3-kinase rather than Grb2 (growth factor-bound protein 2) with IRS-1 plays a major role in insulin-induced oocyte maturation. Activation of PI 3-kinase may lie upstream of mitogen-activated protein kinase activation and p70 S6 kinase activation in response to insulin. Insulin receptor substrate-1 (IRS-1) is rapidly phosphorylated on multiple tyrosine residues in response to insulin and binds several Src homology 2 domain-containing proteins, thereby initiating downstream signaling. To assess the tyrosine phosphorylation sites that mediate relevant downstream signaling and biological effects, we created site-directed mutants of IRS-1 and overexpressed them in the Xenopus laevis oocyte. In oocytes overexpressing IRS-1 or IRS-1-895F (Tyr-895 replaced with phenylalanine), insulin activated phosphatidylinositol (PI) 3-kinase, p70 S6 kinase, and mitogen-activated protein kinase and induced oocyte maturation. In contrast, in oocytes overexpressing IRS-1-4F (Tyr-460, Tyr-608, Tyr-939, and Tyr-987 of IRS-1 replaced with phenylalanine), insulin did not activate PI 3-kinase, p70 S6 kinase, and mitogen-activated protein kinase and failed to induce oocyte maturation. These observations indicate that in X. laevis oocytes overexpressing IRS-1, the association of PI 3-kinase rather than Grb2 (growth factor-bound protein 2) with IRS-1 plays a major role in insulin-induced oocyte maturation. Activation of PI 3-kinase may lie upstream of mitogen-activated protein kinase activation and p70 S6 kinase activation in response to insulin. INTRODUCTIONAfter stimulation with insulin, insulin receptor, a receptor tyrosine kinase, is activated and phosphorylates several signaling molecules including insulin receptor substrate-1 (IRS-1). 1The abbreviations used are: IRS-1insulin receptor substrate-1SH2 proteinsSrc homology 2 domain-containing proteinsIGF-1insulin-like growth factor 1PI 3-kinasephosphatidylinositol 3-kinaseAsh/Grb2abundant Src homology/growth factor receptor bound protein 2p85 of PI 3-kinasethe 85-kDa regulatory subunit of PI 3-kinaseSosson of sevenlessShcSrc homology 2/α-collagen-relatedMAP kinasemitogen-activated protein kinaseMBPmyelin basic proteinα-PYantibodies specific for phosphotyrosinePAGEpolyacrylamide gel electrophoresisGSTglutathione S-transferaseGVBDgerminal vesicle breakdown. The tyrosine-phosphorylated proteins become associated with several Src homology 2 domain-containing proteins (SH2 proteins) and thereby transduce downstream signals (1White M.F. Kahn C.R. J. Biol. Chem. 1994; 269: 1-4Abstract Full Text PDF PubMed Google Scholar). IRS-1 is a substrate for insulin and IGF-1 receptors and is known to associate with several SH2 proteins, including PI 3-kinase and Ash/Grb2, through distinct phosphorylation sites (2Skolnik E.Y. Lee C.H. Batzer A.G. Vicentini L.M. Zhov M. Daly R.J. Myers Jr., M.G. Backer J.M. Ullirich A. White M.F. Schlessinger J. EMBO J. 1993; 12: 1929-1936Crossref PubMed Scopus (604) Google Scholar, 3Tobe K. Matuoka K. Tamemoto H. Ueki K. Kaburagi Y. Asai S. Noguchi T. Matsuda M. Tanaka S. Hattori S. Fukui Y. Akanuma Y. Yazaki Y. Takenawa T. Kadowaki T. J. Biol. Chem. 1993; 268: 11167-11171Abstract Full Text PDF PubMed Google Scholar, 4Backer J.M. Shoelson S.E. Chi D.J. Sun X.J. Miralpeix M. Hu P. Margolis B. Skolnik E.Y. Schlessinger J. White M.F. EMBO J. 1992; 11: 3469-3479Crossref PubMed Scopus (812) Google Scholar, 5Myers Jr., M.G. Backer J.M. Sun X.J. Shoelson S. Hu P. Schlessinger J. Yoakim M. Scaffhousen B. White M.F. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10350-10354Crossref PubMed Scopus (381) Google Scholar). Evidence has been shown to demonstrate the importance of IRS-1 on the pleotropic effects of insulin, such as mitogenesis of 32D cells and maturation of Xenopus laevis oocyte (6Myers Jr., M.G. Grammer T.C. Wang L.-M. Sun X.J. Pierce J.H. Blenis D.J. White M.F. J. Biol. Chem. 1994; 269: 28783-28789Abstract Full Text PDF PubMed Google Scholar, 7Chuang L-M Myers Jr., M.G. Seidner G.A. Birnbaum M.J. White M.F. Kahn C.R. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5172-5175Crossref PubMed Scopus (88) Google Scholar). IRS-1-deficient mice exhibit growth retardation and insulin resistance (8Tamemoto 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 (898) Google Scholar, 9Araki 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 (1086) Google Scholar). IRS-1 has multiple tyrosine phosphorylation sites, some of which could be redundantly associated with the same SH2 proteins, and it is not clear which phosphorylation sites of IRS-1 mediate relevant downstream signaling and biological effects. Furthermore, ubiquitous expression of IRS-1 makes it difficult to examine the functions of overexpressed IRS-1 and its mutated forms. In the present study, we created site-directed mutants of IRS-1 in which Tyr-895 or a group of four tyrosine residues (Tyr-460, Tyr-608, Tyr-939, and Tyr-987) was replaced with phenylalanine (IRS-1-895F and IRS-1-4F, respectively) and overexpressed them in unprimed the X. laevis oocyte. This system is suitable to study functions of IRS-1 and its mutants, since X. laevis oocytes possess receptors for IGF-1 and signaling molecules such as Ash/Grb2, p85 subunit of PI 3-kinase, Sos, and Shc-like proteins (10Janicot M. Flores-Riveros J.R. Lane M.D. J. Biol. Chem. 1991; 266: 9382-9391Abstract Full Text PDF PubMed Google Scholar, 11Grigorescu F. Andrieu J.M. Rouard M. Diabetes. 1994; 43: 3AGoogle Scholar), while the level of endogenous IRS-1 expression is very low (7Chuang L-M Myers Jr., M.G. Seidner G.A. Birnbaum M.J. White M.F. Kahn C.R. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5172-5175Crossref PubMed Scopus (88) Google Scholar). Thus, overexpressed IRS-1 is presumed to amplify signals evoked by IGF-1 or by high concentrations of insulin to downstream effectors. We found that IRS-1 overexpression in oocyte actually augmented PI 3-kinase activation in response to insulin, whereas IRS-1-4F expression did not. Overexpressing IRS-1-4F did not augment the activities of downstream signals including MAP kinase, p70 S6 kinase, and maturation of oocytes. These observation suggests that PI 3-kinase activity associated with IRS-1 is necessary for these signaling events. We also noted that oocytes overexpressing IRS-895F, a putative Ash/Grb2 binding site mutant, efficiently transmit signals leading to oocyte maturation, suggesting that the association of overexpressed IRS-1 with Ash/Grb2 may play a minor roles in these signals.DISCUSSIONIn the present study, we attempted to evaluate the relative importance of IRS-1 phosphorylation sites that could bind PI 3-kinase or Ash/Grb2 for downstream signals by comparing the signaling activities of wild-type IRS-1, mutated IRS-1 unable to activate PI 3-kinase, and that unable to bind to Ash/Grb2. This strategy enabled us to specify signals derived from IRS-1 and to assess the roles of each phosphorylation site of IRS-1 on downstream signaling.In the X. laevis oocyte, insulin-induced maturation was achieved either by pretreatment with gonadotropin (priming) or by overexpressing IRS-1 to unprimed oocytes (7Chuang L-M Myers Jr., M.G. Seidner G.A. Birnbaum M.J. White M.F. Kahn C.R. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5172-5175Crossref PubMed Scopus (88) Google Scholar). The present study was undertaken to define mechanisms of overexpressed IRS-1 that could induce maturation in unprimed X. laevis oocytes. Previous studies have shown the importance of p21ras-MAP kinase cascade in insulin-induced oocyte maturation (21Maller J.L. Biochemistry. 1990; 29: 3157-3166Crossref PubMed Scopus (127) Google Scholar). Activation of p21ras is necessary (22Korn L.J. Siebel C.W. McCormic F. Roth R.A. Science. 1987; 236: 1502-1503Crossref Scopus (141) Google Scholar, 23Deshpande A.K. Kung H.F. Mol. Cell. Biol. 1987; 7: 1285-1288Crossref PubMed Scopus (141) Google Scholar) and sufficient (24Birchmeier C. Broek D. Wiegler M. Cell. 1985; 43: 615-621Abstract Full Text PDF PubMed Scopus (196) Google Scholar) for insulin-induced germinal vesicle breakdown. MAP kinase cascade lies downstream of p21ras. Recent experiments exhibited that MAP kinase activation is also necessary (25Kosako H. Gotoh Y. Nishida E. EMBO J. 1994; 13: 2131-2138Crossref PubMed Scopus (189) Google Scholar, 26Fukuda M. Gotoh Y. Kosako H. Hattori S. Nishida E. J. Biol. Chem. 1994; 269: 33097-33101Abstract Full Text PDF PubMed Google Scholar) and sufficient (27Gotoh Y. Masuyama N. Dell K. Shirakabe K. Nishida E. J. Biol. Chem. 1995; 270: 25898-25904Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar, 28Huang W. Kessler D.S. Erickson R.L. Mol. Biol. Cell. 1995; 6: 237-245Crossref PubMed Scopus (110) Google Scholar, 29Haccard O. Lewellyn A. Hartley R.S. Erickson E. Maller J.L. Dev. Biol. 1995; 168: 677-682Crossref PubMed Scopus (147) Google Scholar) to induce oocyte maturation. Ash/Grb2 is one of the SH2 proteins associated with IRS-1 or Shc that mediates p21ras activation through Sos in mammalian cells (2Skolnik E.Y. Lee C.H. Batzer A.G. Vicentini L.M. Zhov M. Daly R.J. Myers Jr., M.G. Backer J.M. Ullirich A. White M.F. Schlessinger J. EMBO J. 1993; 12: 1929-1936Crossref PubMed Scopus (604) Google Scholar, 3Tobe K. Matuoka K. Tamemoto H. Ueki K. Kaburagi Y. Asai S. Noguchi T. Matsuda M. Tanaka S. Hattori S. Fukui Y. Akanuma Y. Yazaki Y. Takenawa T. Kadowaki T. J. Biol. Chem. 1993; 268: 11167-11171Abstract Full Text PDF PubMed Google Scholar, 30Baltensperger K. Kozma L.M. Cherniak A.D. Klurlund J.K. Chawla A. Banerjee V. Czech M.P. Science. 1993; 260: 1950-1952Crossref PubMed Scopus (230) Google Scholar). Previous studies showed that microinjection of the SH2 domain of Ash/Grb2 inhibited insulin-induced X. laevis oocyte maturation (36Chuang L-M Hausdorff S.F. Myers Jr., M.G. White M.F. Birnbaum M.J. Kahn C.R. J. Biol. Chem. 1994; 269: 27645-27649Abstract Full Text PDF PubMed Google Scholar). Here we show that overexpressed IRS-1-895F, a mutant that could not bind Ash/Grb2 (4Backer J.M. Shoelson S.E. Chi D.J. Sun X.J. Miralpeix M. Hu P. Margolis B. Skolnik E.Y. Schlessinger J. White M.F. EMBO J. 1992; 11: 3469-3479Crossref PubMed Scopus (812) Google Scholar), was able to transmit signals to MAP kinase activation and oocyte maturation as well as overexpressed IRS-1. It could be that in oocytes overexpressing IRS-1-895F insulin-induced signals might well be compensated by interaction between Ash/Grb2 and phosphoproteins other than IRS-1, such as Shc (31Yamauchi K. Holt K. Pessin J.E. J. Biol. Chem. 1993; 268: 14597-14600Abstract Full Text PDF PubMed Google Scholar). Although at present reports on X. laevis Shc-like protein are limited (11Grigorescu F. Andrieu J.M. Rouard M. Diabetes. 1994; 43: 3AGoogle Scholar) and cDNA encoding this protein has not been reported, the role of Shc in insulin-induced maturation of X. laevis oocytes should be investigated in future experiments. In 32D cells, overexpressed IRS-1-895F was reported to affect insulin-induced mitogenesis and MAP kinase activation (4Backer J.M. Shoelson S.E. Chi D.J. Sun X.J. Miralpeix M. Hu P. Margolis B. Skolnik E.Y. Schlessinger J. White M.F. EMBO J. 1992; 11: 3469-3479Crossref PubMed Scopus (812) Google Scholar). The discrepancy between our observations and the results in 32D cells could be that the sets of phosphoproteins that bound to Ash/Grb2 might be different in these cells.Insulin-induced activation of PI 3-kinase also contributes to MAP kinase activation in various degrees in both p21ras-dependent and -independent pathways (31Yamauchi K. Holt K. Pessin J.E. J. Biol. Chem. 1993; 268: 14597-14600Abstract Full Text PDF PubMed Google Scholar, 32Welsh G.I. Foulstone E.J. Young S.W. Tavare J.M. Proud C.G. Biochem. J. 1994; 303: 15-20Crossref PubMed Scopus (182) Google Scholar, 33Cross D.A. Alessi D.R. Vandenheede J.R. McDowell H.E. Hundal H.S. Cohen P. Biochem. J. 1994; 303: 21-26Crossref PubMed Scopus (419) Google Scholar). Recently, the importance of PI 3-kinase in p21ras-dependent oocyte maturation has also been proposed. Constitutive active mutant of p85-p110 fusion subunit of PI 3-kinase was able to induce oocyte maturation, and the oocyte maturation was inhibited by dominant negative p21ras (34Hu Q. Klippel A. Muslin A.J. Fautl W.J. Williams L.T. Science. 1995; 268: 100-102Crossref PubMed Scopus (516) Google Scholar), indicating that activation of PI 3-kinase seems to be sufficient for oocyte maturation and that PI 3-kinase may act upstream of p21ras. Activation of PI 3-kinase was also necessary for insulin-induced germinal vesicle breakdown, since pretreatment with wortmannin (35Liu X.J. Sorisky A. Zhu L. Pawson T. Mol. Cell. Biol. 1995; 15: 3563-3570Crossref PubMed Scopus (70) Google Scholar) or microinjected fusion protein for SH2 domains of p85α subunit of PI 3-kinase (36Chuang L-M Hausdorff S.F. Myers Jr., M.G. White M.F. Birnbaum M.J. Kahn C.R. J. Biol. Chem. 1994; 269: 27645-27649Abstract Full Text PDF PubMed Google Scholar, 37Chuang L-M Myers Jr., M.G. Backer J.M. Shoelson S.E. White M.F. 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Minami M. Watanabe T. Seyama Y. Okado H. Toh H. Itoh K. Miyamoto T. Shimizu T. Nature. 1991; 349: 342-346Crossref PubMed Scopus (521) Google Scholar, 38Rodriguez-Viciana P. Warne P.H. Dhand R. VanHaesebroek B. Gout I. Fry M.J. Waterfield M.D. Downward D.J. Nature. 1994; 370: 527-532Crossref PubMed Scopus (1716) Google Scholar). Therefore, it was difficult to conclude that PI 3-kinase activity associated with overexpressed IRS-1 was crucial in insulin-induced oocyte maturation. To circumvent these issues and to examine more directly the contribution of PI 3-kinase associated with IRS-1, we overexpressed wild-type IRS-1 and IRS-1 mutated at possible binding sites for PI 3-kinase (IRS-1-4F) in the X. laevis oocyte. We found that IRS-1-4F failed to stimulate PI 3-kinase, MAP kinase, and oocyte maturation in response to insulin. The following conclusions can be drawn from the results presented in this paper. First, as predicted by cell-free experiments (39Sun X.J. Crimmins D.L. Myers Jr., M.G. Miralpeix M. White M.F. Mol. Cell. Biol. 1993; 13: 7418-7428Crossref PubMed Google Scholar, 40Pons S. Asano T. Glasheen E. Miralpeix M. Zhang Y. Fisher T.L. Myers Jr., M.G. Sun X.J. White M.F. Mol. Cell. Biol. 1995; 15: 4453-4465Crossref PubMed Scopus (229) Google Scholar), we proved that Tyr-X-X-Met motifs of some of Tyr-460, −608, −939, and −987 of IRS-1 might be necessary for association and full activation of PI 3-kinase in intact cell systems. Similar observations that insulin-induced activation of PI 3-kinase was impaired in 293 cells overexpressed with IRS-1 mutated at Tyr-608 were recently reported by Rocci et al. (41Rocci S. Tartare-Deckert S. Mothe I. van Obberghen E. Endocrinology. 1995; 136: 5291-5297Crossref PubMed Scopus (38) Google Scholar). Under our present experimental conditions, in which a high concentration of insulin is required to induce effects, IRS-1-4F failed to activate PI 3-kinase in response to insulin. However, a significant amount of PI 3-kinase activity in response to insulin was observed when IRS-1-4F was overexpressed to primary hepatocytes. 2K. Ueki, H. Tamemoto, and T. Kadowaki, unpublished observations. Thus in insulin-sensitive tissues, Tyr-X-X-Met motifs other than Tyr-460, −608, −939, and −987 of IRS-1 might bind and activate PI 3-kinase in response to insulin. As the present study does not address whether Tyr-X-X-Met motifs of Tyr-460, −608, −939, and −987 of IRS-1 are sufficient for activating PI 3-kinase, PI 3-kinase activity of added-back IRS-1 mutant possessing only Tyr-X-X-Met motifs should next be examined. Second, the interaction between some of Tyr-X-X-Met motifs of Tyr-460, −608, −939, and −987 of IRS-1 and the SH2 proteins bound to these sites was required for insulin-induced activation of MAP kinase and oocyte maturation. Molecules that bound to tyrosine-phosphorylated IRS-1 reported to date include p85 subunit of PI 3-kinase (4Backer J.M. Shoelson S.E. Chi D.J. Sun X.J. Miralpeix M. Hu P. Margolis B. Skolnik E.Y. Schlessinger J. White M.F. EMBO J. 1992; 11: 3469-3479Crossref PubMed Scopus (812) Google Scholar, 5Myers Jr., M.G. Backer J.M. Sun X.J. Shoelson S. Hu P. Schlessinger J. Yoakim M. Scaffhousen B. White M.F. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10350-10354Crossref PubMed Scopus (381) Google Scholar), Ash/Grb2 (2Skolnik E.Y. Lee C.H. Batzer A.G. Vicentini L.M. Zhov M. Daly R.J. Myers Jr., M.G. Backer J.M. Ullirich A. White M.F. Schlessinger J. EMBO J. 1993; 12: 1929-1936Crossref PubMed Scopus (604) Google Scholar, 3Tobe K. Matuoka K. Tamemoto H. Ueki K. Kaburagi Y. Asai S. Noguchi T. Matsuda M. Tanaka S. Hattori S. Fukui Y. Akanuma Y. Yazaki Y. Takenawa T. Kadowaki T. J. Biol. Chem. 1993; 268: 11167-11171Abstract Full Text PDF PubMed Google Scholar), SHPTP2 (42Kuhne M.J. Pawson T. Lienhard G.E. Feng G.-S. J. Biol. Chem. 1993; 268: 11479-11481Abstract Full Text PDF PubMed Google Scholar), p55PIK (40Pons S. Asano T. Glasheen E. Miralpeix M. Zhang Y. Fisher T.L. Myers Jr., M.G. Sun X.J. White M.F. Mol. Cell. Biol. 1995; 15: 4453-4465Crossref PubMed Scopus (229) Google Scholar), and Nck (43Lee C.H. Nishimura R. Zhou M. Batzer A.G. Myers Jr., M.G. White M.F. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 11713-11717Crossref PubMed Scopus (196) Google Scholar). Although Nck was reported to bind to Tyr-751 of platelet-derived growth factor receptor (44Nishimura R. Li W. Kashishian A. Mondino A. Zhou M. Cooper J. Schlessinger J. Mol. Cell. Biol. 1993; 13: 6889-6896Crossref PubMed Scopus (152) Google Scholar), one of the binding sites of p85 subunit of PI 3-kinase, only p85 subunit of PI 3-kinase and p55PIK, a variant form of the regulatory subunit of PI 3-kinase, are able to bind to phosphopeptides containing Tyr-460, −608, −939, or −987 of IRS-1 (39Sun X.J. Crimmins D.L. Myers Jr., M.G. Miralpeix M. White M.F. Mol. Cell. Biol. 1993; 13: 7418-7428Crossref PubMed Google Scholar, 40Pons S. Asano T. Glasheen E. Miralpeix M. Zhang Y. Fisher T.L. Myers Jr., M.G. Sun X.J. White M.F. Mol. Cell. Biol. 1995; 15: 4453-4465Crossref PubMed Scopus (229) Google Scholar). Thus the present observation suggested that activation of IRS-1-associated PI 3-kinase was the upstream mechanism for activation of p21ras-MAP kinase cascade and induction of oocyte maturation. In the present system, however, the possible interaction of any one of the four Tyr-X-X-Met motifs with some unidentified SH2 domain-containing molecules cannot be completely ruled out. To examine the possibility that phosphorylation of any one of the Tyr-X-X-Met motifs could be required for oocyte maturation, we carried out experiments for insulin-induced maturation of oocytes overexpressing IRS-1-460F, IRS-1-608F, or IRS-939F-987F. The result indicated that not any one of the four Tyr-X-X-Met motifs played a predominant role in oocyte maturation. Finally, Yamauchi and Pessin reported that overexpressed IRS-1 resulted in a concomitant reduction of Ash/Grb2 associated with Shc and inhibited insulin signaling (45Yamauchi K. Pessin J.E. J. Biol. Chem. 1994; 269: 31107-31114Abstract Full Text PDF PubMed Google Scholar). These observations hence raised the possibility that overexpression of mutant IRS-1 could somehow affect the insulin signaling of endogenous IRS-1-independent pathway. However, differences in IRS-1-induced and mutated IRS-1-induced signaling are most likely caused by the mutation itself, since overexpression of wild-type IRS-1 might also affect this putative IRS-1-independent pathway in a similar fashion.p70 S6 kinase is known to be involved in G1 progression (46Lane H.A. Fernandez A. Lamb N.J.C. Thomas G. Nature. 1993; 363: 170-172Crossref PubMed Scopus (318) Google Scholar) and protein synthesis by phosphorylating ribosomal protein S6 and translation initiation factors in mammalian cells (47Lin T.-A. Kong X. 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Chem. 1994; 269: 28783-28789Abstract Full Text PDF PubMed Google Scholar). p70 S6 kinase seems to lie downstream of one or some of the PI 3-kinase family molecules, because it is activated by autoactive fusion subunit of p85-p110 PI 3-kinase (50Weng Q.P. Andrabi K. Klippel A. Kozlowski M.T. Wiliams L.T. Avruch J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5744-5748Crossref PubMed Scopus (202) Google Scholar) and inhibited by wortmannin and LY294002, inhibitors of PI 3-kinase family molecules (51Cheatham B. Vlahos C.J. Cheatham L. Wang L. Blenis J. Kahn C.R. Mol. Cell. Biol. 1994; 14: 4902-4911Crossref PubMed Scopus (997) Google Scholar, 52Cung J. Grammer T.C. Lemon K.P. Kazlavskas A. Blenis J. Nature. 1994; 370: 71-75Crossref PubMed Scopus (656) Google Scholar). To examine more directly the contribution of Tyr-X-X-Met motifs of IRS-1 on insulin-induced p70 S6 kinase activation, we analyzed p70 S6 kinase activity in oocytes overexpressing normal and mutant IRS-1s. We observed that insulin-induced p70 S6 kinase activity was increased by overexpression of IRS-1 but not by that of IRS-1-4F (Fig. 4). These results indicated that interaction between some of the Tyr-460, −608, −939, and −987 of IRS-1 and the SH2 proteins bound to these sites, most likely PI 3-kinase, was necessary for insulin-induced activation of p70 S6 kinase in this system. Recently overexpression of truncated mutant of p85 subunit of PI 3-kinase was reported to antagonize platelet-derived growth factor-mediated activation of p70 S6 kinase (53Burgering B.M.T. Coffer P.J. Nature. 1995; 376: 599-602Crossref PubMed Scopus (1869) Google Scholar). The result is consistent with our present observations. p70 S6 kinase is apparently not involved in oocyte maturation, because rapamycin, an inhibitor of p70 S6 kinase, failed to antagonize oocyte maturation in response to insulin (data not shown).In conclusion, MAP kinase activation, p70 S6 kinase activation, and oocyte maturation in response to insulin appears to link with PI 3-kinase, which was associated with IRS-1. INTRODUCTIONAfter stimulation with insulin, insulin receptor, a receptor tyrosine kinase, is activated and phosphorylates several signaling molecules including insulin receptor substrate-1 (IRS-1). 1The abbreviations used are: IRS-1insulin receptor substrate-1SH2 proteinsSrc homology 2 domain-containing proteinsIGF-1insulin-like growth factor 1PI 3-kinasephosphatidylinositol 3-kinaseAsh/Grb2abundant Src homology/growth factor receptor bound protein 2p85 of PI 3-kinasethe 85-kDa regulatory subunit of PI 3-kinaseSosson of sevenlessShcSrc homology 2/α-collagen-relatedMAP kinasemitogen-activated protein kinaseMBPmyelin basic proteinα-PYantibodies specific for phosphotyrosinePAGEpolyacrylamide gel electrophoresisGSTglutathione S-transferaseGVBDgerminal vesicle breakdown. The tyrosine-phosphorylated proteins become associated with several Src homology 2 domain-containing proteins (SH2 proteins) and thereby transduce downstream signals (1White M.F. Kahn C.R. J. Biol. Chem. 1994; 269: 1-4Abstract Full Text PDF PubMed Google Scholar). IRS-1 is a substrate for insulin and IGF-1 receptors and is known to associate with several SH2 proteins, including PI 3-kinase and Ash/Grb2, through distinct phosphorylation sites (2Skolnik E.Y. Lee C.H. Batzer A.G. Vicentini L.M. Zhov M. Daly R.J. Myers Jr., M.G. Backer J.M. Ullirich A. White M.F. Schlessinger J. EMBO J. 1993; 12: 1929-1936Crossref PubMed Scopus (604) Google Scholar, 3Tobe K. Matuoka K. Tamemoto H. Ueki K. Kaburagi Y. Asai S. Noguchi T. Matsuda M. Tanaka S. Hattori S. Fukui Y. Akanuma Y. Yazaki Y. Takenawa T. Kadowaki T. J. Biol. Chem. 1993; 268: 11167-11171Abstract Full Text PDF PubMed Google Scholar, 4Backer J.M. Shoelson S.E. Chi D.J. Sun X.J. Miralpeix M. Hu P. Margolis B. Skolnik E.Y. Schlessinger J. White M.F. EMBO J. 1992; 11: 3469-3479Crossref PubMed Scopus (812) Google Scholar, 5Myers Jr., M.G. Backer J.M. Sun X.J. Shoelson S. Hu P. Schlessinger J. Yoakim M. Scaffhousen B. White M.F. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10350-10354Crossref PubMed Scopus (381) Google Scholar). Evidence has been shown to demonstrate the importance of IRS-1 on the pleotropic effects of insulin, such as mitogenesis of 32D cells and maturation of Xenopus laevis oocyte (6Myers Jr., M.G. Grammer T.C. Wang L.-M. Sun X.J. Pierce J.H. Blenis D.J. White M.F. J. Biol. Chem. 1994; 269: 28783-28789Abstract Full Text PDF PubMed Google Scholar, 7Chuang L-M Myers Jr., M.G. Seidner G.A. Birnbaum M.J. White M.F. Kahn C.R. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5172-5175Crossref PubMed Scopus (88) Google Scholar). IRS-1-deficient mice exhibit growth retardation and insulin resistance (8Tamemoto 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 (898) Google Scholar, 9Araki 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 (1086) Google Scholar). IRS-1 has multiple tyrosine phosphorylation sites, some of which could be redundantly associated with the same SH2 proteins, and it is not clear which phosphorylation sites of IRS-1 mediate relevant downstream signaling and biological effects. Furthermore, ubiquitous expression of IRS-1 makes it difficult to examine the functions of overexpressed IRS-1 and its mutated forms. In the present study, we created site-directed mutants of IRS-1 in which Tyr-895 or a group of four tyrosine residues (Tyr-460, Tyr-608, Tyr-939, and Tyr-987) was replaced with phenylalanine (IRS-1-895F and IRS-1-4F, respectively) and overexpressed them in unprimed the X. laevis oocyte. This system is suitable to study functions of IRS-1 and its mutants, since X. laevis oocytes possess receptors for IGF-1 and signaling molecules such as Ash/Grb2, p85 subunit of PI 3-kinase, Sos, and Shc-like proteins (10Janicot M. Flores-Riveros J.R. Lane M.D. J. Biol. Chem. 1991; 266: 9382-9391Abstract Full Text PDF PubMed Google Scholar, 11Grigorescu F. Andrieu J.M. Rouard M. Diabetes. 1994; 43: 3AGoogle Scholar), while the level of endogenous IRS-1 expression is very low (7Chuang L-M Myers Jr., M.G. Seidner G.A. Birnbaum M.J. White M.F. Kahn C.R. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5172-5175Crossref PubMed Scopus (88) Google Scholar). Thus, overexpressed IRS-1 is presumed to amplify signals evoked by IGF-1 or by high concentrations of insulin to downstream effectors. We found that IRS-1 overexpression in oocyte actually augmented PI 3-kinase activation in response to insulin, whereas IRS-1-4F expression did not. Overexpressing IRS-1-4F did not augment the activities of downstream signals including MAP kinase, p70 S6 kinase, and maturation of oocytes. These observation suggests that PI 3-kinase activity associated with IRS-1 is necessary for these signaling events. We also noted that oocytes overexpressing IRS-895F, a putative Ash/Grb2 binding site mutant, efficiently transmit signals leading to oocyte maturation, suggesting that the association of overexpressed IRS-1 with Ash/Grb2 may play a minor roles in these signals.
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