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Ras-independent and Wortmannin-sensitive Activation of Glycogen Synthase by Insulin in Chinese Hamster Ovary Cells

1995; Elsevier BV; Volume: 270; Issue: 19 Linguagem: Inglês

10.1074/jbc.270.19.11304

ISSN

1083-351X

Autores

Hiroshi Sakaue, Kenta Hara, Tetsuya Noguchi, Takashi Matozaki, Kei Kotani, Wataru Ogawa, Kazuyoshi Yonezawa, Masato Kasuga, Michael D. Waterfield,

Tópico(s)

Protein Tyrosine Phosphatases

Resumo

Activation of glycogen synthase is one of the major metabolic events triggered by exposure of cells to insulin. The molecular mechanism by which insulin activates glycogen synthase was investigated. The possible role of Ras and mitogen-activated protein kinase cascade was investigated with a stable cell line, CHO-IR-C/S 46, that overexpresses insulin receptors and a catalytically inactive SH-PTP 2 protein phosphatase and in which insulin does not induce the formation of the Ras-GTP complex or the subsequently activation of the mitogen-activated protein kinase cascade. Insulin activated glycogen synthase in this cell line to a similar extent as in parental CHO-IR cells. The importance of heteromeric phosphoinositide (PI) 3-kinase in insulin activation of glycogen synthase was examined in a stable cell line, CHO-IR/Δp85, that overexpresses insulin receptors and a dominant negative mutant (Δp85) of the 85-kDa subunit of PI 3-kinase that lacks the binding site for the catalytic 110-kDa subunit. Insulin-dependent activation of PI-3 kinase and glucose transport, but not the formation of the Ras-GTP complex, are markedly attenuated in this cell line. In CHO-IR/Δp85 cells, insulin activated glycogen synthase to a similar extent as in parental CHO-IR cells. The failure of overproduction of the mutant (Δp85) protein to inhibit insulin activation of glycogen synthase was also confirmed by transient expression in Rat 1 cells with the use of a recombinant vaccinia virus. However, wortmannin abolished insulin activation of glycogen synthase in all cell lines. These data suggest the existence of a Ras-independent and wortmannin-sensitive pathway for activation of glycogen synthase by insulin. Activation of glycogen synthase is one of the major metabolic events triggered by exposure of cells to insulin. The molecular mechanism by which insulin activates glycogen synthase was investigated. The possible role of Ras and mitogen-activated protein kinase cascade was investigated with a stable cell line, CHO-IR-C/S 46, that overexpresses insulin receptors and a catalytically inactive SH-PTP 2 protein phosphatase and in which insulin does not induce the formation of the Ras-GTP complex or the subsequently activation of the mitogen-activated protein kinase cascade. Insulin activated glycogen synthase in this cell line to a similar extent as in parental CHO-IR cells. The importance of heteromeric phosphoinositide (PI) 3-kinase in insulin activation of glycogen synthase was examined in a stable cell line, CHO-IR/Δp85, that overexpresses insulin receptors and a dominant negative mutant (Δp85) of the 85-kDa subunit of PI 3-kinase that lacks the binding site for the catalytic 110-kDa subunit. Insulin-dependent activation of PI-3 kinase and glucose transport, but not the formation of the Ras-GTP complex, are markedly attenuated in this cell line. In CHO-IR/Δp85 cells, insulin activated glycogen synthase to a similar extent as in parental CHO-IR cells. The failure of overproduction of the mutant (Δp85) protein to inhibit insulin activation of glycogen synthase was also confirmed by transient expression in Rat 1 cells with the use of a recombinant vaccinia virus. However, wortmannin abolished insulin activation of glycogen synthase in all cell lines. These data suggest the existence of a Ras-independent and wortmannin-sensitive pathway for activation of glycogen synthase by insulin. Insulin elicits divergent biological activities by interacting with its specific receptor, which belongs to a large family of transmembrane tyrosine kinase receptors. The activated insulin receptor phosphorylates a variety of cellular proteins on tyrosine residues. One of the best studied substrates of the insulin receptor is insulin receptor substrate-1 (IRS-1) 1The abbreviations used are: IRS-Iinsulin receptor substrate-ISHSrc homologyPIphosphoinositideMAPmitogen-activated proteinCHOChinese hamster ovaryG6Pglucose 6-phosphateTKthymidine kinaseGSK-3glycogensynthase kinase-B1The abbreviations used are: IRS-Iinsulin receptor substrate-ISHSrc homologyPIphosphoinositideMAPmitogen-activated proteinCHOChinese hamster ovaryG6Pglucose 6-phosphateTKthymidine kinaseGSK-3glycogensynthase kinase-B, a 170–190-kDa protein originally designated as pp185 (Sun et al., 1991Sun X.J. Rothenberg P. Kahn C.R. Backer J.M. Araki E. Wilden P.A. Canili D.A. Goldstein B.J. White M.F. Nature. 1991; 352: 73-77Crossref PubMed Scopus (1265) Google Scholar. Tyrosine-phosphorylated IRS-1 binds several signaling molecules, including the 85-kDa subunit of phosphoinositide (PI) 3-kinase (Lavan et al., 1992Lavan B.E. Kuhne M.R. Garner C.W. Anderson D. Reedijik M. Pawson T. Lienhard G.E. J. Biol. Chem. 1992; 267: 11631-11636Abstract Full Text PDF PubMed Google Scholar; Backer et al., 1992Backer J.M. Myers Jr., M.G. Shoelson S.E. Chin D.J. Sun X.J. Miralpeix M. Hu P. Margolis B. Skolnik E.Y. Schlessinger J. White M.F. EMBO J. 1992; 9: 3469-3479Crossref Scopus (811) Google Scholar; Yonezawa et al., 1992Yonezawa K. Ueda H. Hara K. Nishida K. Ando A. Chavanieu A. Matsuba H. Shii K. Yokono K. Fukui Y. Calas B. Grigorescue F. Dhand R. Gout I. Otsu M. Waterfield M.D. Kasuga M. J. Biol. Chem. 1992; 267: 25958-25966Abstract Full Text PDF PubMed Google Scholar, Grb2 (Skolnik et al., 1993Skolnik E.Y. Lee C.-H. Batzer A. Vicentini 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 (601) Google Scholar, the protein-tyrosine phosphatase SH-PTP2 (Kuhne et al., 1993Kuhne M.R. Pawson T. Lienhard G.E. Feng G.-S. J. Biol. Chem. 1993; 268: 11479-11481Abstract Full Text PDF PubMed Google Scholar, and Nck (Lee et al., 1993Lee C.-H. Li W. Nishimura R. Zhou M. Batzer A.G. Myers Jr., M.G. White M.F. Schlessinger J. Skolnik E.Y. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 11713-11717Crossref PubMed Scopus (196) Google Scholar, via their Src homology 2 (SH2) domains. insulin receptor substrate-I Src homology phosphoinositide mitogen-activated protein Chinese hamster ovary glucose 6-phosphate thymidine kinase glycogensynthase kinase-B insulin receptor substrate-I Src homology phosphoinositide mitogen-activated protein Chinese hamster ovary glucose 6-phosphate thymidine kinase glycogensynthase kinase-B The GTP-binding protein Ras plays a pivotal role in signal transduction for many growth factors (Barbacid, 1987Barbacid M. Annu. Rev. Biochem. 1987; 56: 779-827Crossref PubMed Scopus (3748) Google Scholar). The Ras protein oscillates between GTP-bound active and GDP-bound inactive conformations and thereby turns on or turns off the signal transduction process (Barbacid, 1987Barbacid M. Annu. Rev. Biochem. 1987; 56: 779-827Crossref PubMed Scopus (3748) Google Scholar; Kaziro et al., 1991Kaziro Y. Itoh H. Kozasa T. Nakafuku M. Satoh T. Annu. Rev. Biochem. 1991; 60: 349-400Crossref PubMed Scopus (547) Google Scholar. Insulin stimulates the formation of the Ras-GTP complex (Burgering et al., 1991Burgering B.M.T. Medema R.H. Maassen J.A. Wetering M.L. Eb A.J. McCormick F. Bos J.L. EMBO J. 1991; 10: 1103-1109Crossref PubMed Scopus (205) Google Scholar; Yonezawa et al., 1994Yonezawa 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. Recent studies suggest that the Grb2-Sos complex (Skolnik et al., 1993Skolnik E.Y. Lee C.-H. Batzer A. Vicentini 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 (601) Google Scholar; Sakaue et al., 1995Sakaue M. Bowtell D. Kasuga M. Mol. Cell. Biol. 1995; 15: 379-388Crossref PubMed Scopus (60) Google Scholar or SH-PTP2 (Noguchi et al., 1994Noguchi T. Matozaki T. Horita K. Fujioka Y. Kasuga M. Mol. Cell. Biol. 1994; 14: 6674-6682Crossref PubMed Scopus (346) Google Scholar bound to tyrosine-phosphorylated IRS-1, or the Grb2-Sos complex bound to Shc (Pronk et al., 1993Pronk G.J. McGlade J. Pelicci G. Pawson T. Boss J. L J. Biol. Chem. 1993; 268: 5748-5753Abstract Full Text PDF PubMed Google Scholar, may be important in insulin-stimulated formation of Ras-GTP. GTP-bound Ras then initiates a cascade of sequential phosphorylation events in which the serine-threonine kinase Raf phosphorylates and activates mitogen-activated protein (MAP) kinase kinase, which in turn phosphorylates and activates MAP kinase (Crews and Erikson, 1993Crews C.M. Erikson R.L. Cell. 1993; 74: 215-217Abstract Full Text PDF PubMed Scopus (296) Google Scholar; Eagan and Weinberg, 1993Eagan S.E. Weinberg R.A. Nature. 1993; 365: 781-782Crossref PubMed Scopus (523) Google Scholar). MAP kinase phosphorylates other serine-threonine kinases such as p90rsk (Sturgill et al., 1988Sturgill T.W. Ray L.B. Erikson E. Maller J.L. Nature. 1988; 334: 715-718Crossref PubMed Scopus (751) Google Scholar; Ahn and Krebs, 1990Ahn N.G. Krebs E. J. Biol. Chem. 1990; 265: 11495-11501Abstract Full Text PDF PubMed Google Scholar). In contrast, MAP kinase and p70 S6 kinase lie on distinct signaling pathways (Ballou et al., 1991Ballou L.M. Luther H. Thomas G. Nature. 1991; 349: 348-350Crossref PubMed Scopus (152) Google Scholar; Price et al., 1992Price D.J. Grove J.R. Calvo V. Avruch J. Bierer B.E. Science. 1992; 257: 973-977Crossref PubMed Scopus (581) Google Scholar. The immunosuppressant rapamycin blocks activation of p70 S6 kinase by insulin and many growth factors but does not inhibit MAP kinase or pp90rsk (Chung et al., 1992Chung J. Kuo C.J. Crabtree G.R. Blenis J. Cell. 1992; 69: 1227-1236Abstract Full Text PDF PubMed Scopus (1011) Google Scholar; Price et al., 1992Price D.J. Grove J.R. Calvo V. Avruch J. Bierer B.E. Science. 1992; 257: 973-977Crossref PubMed Scopus (581) Google Scholar; Calvo et al., 1992Calvo V. Crews C.M. Vik T.A. Bierer B.E. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 7571-7575Crossref PubMed Scopus (165) Google Scholar. Heteromeric PI 3-kinase is a dimer that constists of an 85-kDa (p85) adaptor subunit and a 110-kDa (p110) catalytic subunit. Insulin induces the formation of a complex between tyrosine-phosphorylated IRS-1 and heteromeric PI 3-kinase via the SH2 domain of p85 and thereby activates the enzyme (Backer et al., 1992Backer J.M. Myers Jr., M.G. Shoelson S.E. Chin D.J. Sun X.J. Miralpeix M. Hu P. Margolis B. Skolnik E.Y. Schlessinger J. White M.F. EMBO J. 1992; 9: 3469-3479Crossref Scopus (811) Google Scholar. To study the role of PI 3-kinase, we established Chinese hamster ovary (CHO) cell lines that overexpress human insulin receptors and a mutant bovine p85 (Δp85) that lacks the binding site for p110 (CHO-IR/Δp85 cells) (Hara et al., 1994Hara K. Yonezawa K. Sakaue H. Ando A. Kotani K. Kitamura T. Kitamura Y. Ueda H. Stephens L. Jackson T.R. Hawkins P.T. Dhand R. Clark A.E. Holman G.D. Waterfield M.D. Kasuga M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7415-7419Crossref PubMed Scopus (416) Google Scholar. We showed that the insulin-induced association of PI 3-kinase activity with IRS-1 was markedly inhibited in these cell lines as a result of the competitive binding of the overexpressed Δp85. Displacement of the endogenous PI 3-kinase from the activated IRS-1 complex by Δp85 resulted in marked inhibition of both the insulin-induced accumulation of phosphatidylinositol 3,4,5-trisphosphate and insulin-induced glucose uptake, but not of insulin-induced formation of the Ras-GTP complex (Hara et al., 1994Hara K. Yonezawa K. Sakaue H. Ando A. Kotani K. Kitamura T. Kitamura Y. Ueda H. Stephens L. Jackson T.R. Hawkins P.T. Dhand R. Clark A.E. Holman G.D. Waterfield M.D. Kasuga M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7415-7419Crossref PubMed Scopus (416) Google Scholar. Wortmannin, a fungal metabolite that had previously been shown to inhibit myosin light chain kinase with a 50% inhibitory concentration of 500 nM (Nakanishi et al., 1992Nakanishi S. Kakita S. Takahashi I. Kawahara K. Tsukuda E. Sano T. Yamada K. Yoshida M. Kase H. Matsuda Y. Hashimoto Y. Nonomura Y. J. Biol. Chem. 1992; 267: 2157-2163Abstract Full Text PDF PubMed Google Scholar, was recently found to inhibit heteromeric PI 3-kinase activity with a 50% inhibitory concernation of 5 nM (Yano et al., 1993Yano H. Nakanishi S. Kimura K. Hanai N. Saitoh Y. Fukui Y. Nonomura Y. Matsuda Y. J. Biol. Chem. 1993; 268: 25846-25856Abstract Full Text PDF PubMed Google Scholar; Arcaro and Wymann, 1993Arcaro A. Wymann M.P. Biochem. J. 1993; 296: 297-301Crossref PubMed Scopus (1041) Google Scholar; Okada et al., 1994Okada T. Kawano T. Sakakibara T. Hazeki O. Ui M. J. Biol. Chem. 1994; 269: 3568-3573Abstract Full Text PDF PubMed Google Scholar. In addition, wortmannin inhibits insulin-stimulated glucose uptake in rat adipocytes (Okada et al., 1994Okada T. Kawano T. Sakakibara T. Hazeki O. Ui M. J. Biol. Chem. 1994; 269: 3568-3573Abstract Full Text PDF PubMed Google Scholar. Glycogen synthesis is one of the major metabolic processes stimulated by insulin. Although the precise molecular mechanism is not known, the activation of glycogen synthase by insulin has been shown to be mediated by enzyme dephosphorylation (Dent et al., 1990Dent P. Lavoinne A. Nakielny S. Caudwell F.B. Watt P. Cohen P. Nature. 1990; 348: 302-308Crossref PubMed Scopus (401) Google Scholar. Because MAP kinase is an upstream activator of p90rsk (Sturgill et al., 1988Sturgill T.W. Ray L.B. Erikson E. Maller J.L. Nature. 1988; 334: 715-718Crossref PubMed Scopus (751) Google Scholar; Ahn and Krebs, 1990Ahn N.G. Krebs E. J. Biol. Chem. 1990; 265: 11495-11501Abstract Full Text PDF PubMed Google Scholar), the observation that protein phosphatase 1, which dephosphorylates glycogen synthase, is activated in response to phosphorylation by an insulin-stimulated protein kinase, originally designated ISPK but later found to be p90rsk (Dent et al., 1990Dent P. Lavoinne A. Nakielny S. Caudwell F.B. Watt P. Cohen P. Nature. 1990; 348: 302-308Crossref PubMed Scopus (401) Google Scholar: Sutherland et al., 1993Sutherland C. Campbell D.G. Cohen P. Eur. J Biochem. 1993; 212: 581-588Crossref PubMed Scopus (113) Google Scholar, suggested that activation of the Ras-MAP kinase cascade is required for activation of glycogen synthase. To elucidate the molecular mechanism of insulin activation of glycogen synthase, we have now investigated the roles of two major insulin signaling pathways, the Ras-MAP kinase cascade and the PI 3-kinase pathway. We previously established CHO cell lines that overexpress human insulin receptors and a catalytically inactive SH-PTP2 (CHO-IR-C/S 46 cells) (Noguchi et al., 1994Noguchi T. Matozaki T. Horita K. Fujioka Y. Kasuga M. Mol. Cell. Biol. 1994; 14: 6674-6682Crossref PubMed Scopus (346) Google Scholar. The insulin-induced formation of the Ras-GTP complex and MAP kinase activation are markedly inhibited in these cell lines as a result of the competitive binding of the catalytically inactive SH-PTP2 to IRS-1 (Noguchi et al., 1994Noguchi T. Matozaki T. Horita K. Fujioka Y. Kasuga M. Mol. Cell. Biol. 1994; 14: 6674-6682Crossref PubMed Scopus (346) Google Scholar. CHO-IR-C/S 46 cells were thus used to investigate the contribution of the Ras-MAP kinase cascade to the insulin-induced activation of glycogen synthase. Overexpression of Δp85 and wortmannin were used to assess the contribution of heteromeric PI 3-kinase. Cell Culture and Antibodies—A CHO-IR/Δp85 cell line overexpressing human insulin receptors and a dominant negative mutant of the 85-kDa regulatory subunit of PI 3-kinase was established as described (Hara et al., 1994Hara K. Yonezawa K. Sakaue H. Ando A. Kotani K. Kitamura T. Kitamura Y. Ueda H. Stephens L. Jackson T.R. Hawkins P.T. Dhand R. Clark A.E. Holman G.D. Waterfield M.D. Kasuga M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7415-7419Crossref PubMed Scopus (416) Google Scholar. A CHO-IR-C/S46 cell line overexpressing human insulin receptors and a catalytically inactive mutant of SH-PTP2 was also established as described previously (Noguchi et al., 1994Noguchi T. Matozaki T. Horita K. Fujioka Y. Kasuga M. Mol. Cell. Biol. 1994; 14: 6674-6682Crossref PubMed Scopus (346) Google Scholar. Rat1 cells overexpressing human insulin receptors (Rat1-IR cells) were kindly provided by Dr. J. Olefsky (University of California, San Diego). Monoclonal antibodies to phosphotyrosine (PY20) were obtained from ICN (Costa Mesa, CA). Polyclonal antibodies prepared against a synthetic peptide of p70 S6 kinase (amino acids 2–23) were kindly provided by Dr. Y. Okabayashi (Kobe University School of Medicine). Treatment of Cells with Wortmannin and Rapamycin—Wortmannin (kindly provided by Dr. Y. Matsuda, Kyowa Hakko Kogyo, Matchida, Japan) was dissolved in dimethyl sulfoxide at a final concentration of 1 mm, stored at –20 ºC in the dark, and diluted with distilled water immediately before use. Wortmannin was added to culture medium at the indicated concentrations, and cells were incubated with the agent for 10 min at 37 °C before insulin stimulation. The final concentration of dimethyl sulfoxide was 0.1%; the same concentration of dimethyl sulfoxide alone was added to medium as a control. Rapamycin (kindly provided by Wyeth-Ayerst Research, Princeton, NJ) was dissolved in ethanol at a final concentration of 2 mg/ml, stored at –20 °C in the dark, and diluted with distilled water immediately before use. Rapamycin was also added to culture medium at the indicated concentrations and cells were incubated with the drug for 30 min at 37 °C before insulin stimulation. The final concentration of ethanol was 0.01%; the same concentration of ethanol alone was added to medium as a control. Assay of Glycogen Synthase Activity—Glycogen synthase activity was assayed as described (Thomas et al., 1968Thomas L.A. Schlender K.K. Larnar J. Anal. Biochem. 1968; 25: 486-499Crossref PubMed Scopus (947) Google Scholar with modifications. In brief, serum-deprived confluent cell monolayers in 60-mM culture dishes were incubated with the indicated concentrations of insulin for 30 min at 37 °C, immedieately frozen with liquid nitrogen, and then lysed in 120 μl of lysis buffer containing 50 mm Tris-HCl (pH 7.6), 100 mm KF, 10 mm phenylmethylsulfonyl fluoride, and 30% (v/v) glycerol. After removing insoluble material by centrifugation (12,000 × g for 30 min), 30 μl of the supernatant were mixed with 60 µl of assay solution composed of 50 mm Tris-HCl (pH 7.6), 20 mm EDTA, 25 mm KF, glycogen (10 mg/ml), 6.7 mm uridine diphospho-D-[U-14C]glucose (0.05 µCi/sample), and plus or minus 10 mm glucose 6-phosphate (G6P). After 30 min at 30 °C, the reaction was terminated by spotting the reaction mixture on filter paper, and glycogen was precipitated on the paper by soaking in ice-chilled 70% (v/v) ethanol. Each filter paper was washed four times with ice-chilled 70% ethanol, and radioactivity remaining on the paper was measured with a liquid scintillation counter. The activity ratio (-G6P/+G6P) was calculated. PI 3-Kinase Assay—Immunoprecipitation was performed (Yonezawa et al., 1992Yonezawa K. Ueda H. Hara K. Nishida K. Ando A. Chavanieu A. Matsuba H. Shii K. Yokono K. Fukui Y. Calas B. Grigorescue F. Dhand R. Gout I. Otsu M. Waterfield M.D. Kasuga M. J. Biol. Chem. 1992; 267: 25958-25966Abstract Full Text PDF PubMed Google Scholar, and PI 3-kinase activity was assayed (Endemann et al., 1990Endemann G. Yonezawa K. Roth R.A. J. Biol. Chem. 1990; 265: 396-400Abstract Full Text PDF PubMed Google Scholar as described previously. In brief, serum-deprived confluent monolayers were incubated with the indicated concentration of insulin for 1 min at 37 °C, immediately frozen with liquid nitrogen, and lysed on ice in a buffer containing 20 mm Tris-HCl (pH 7.6), 1% Nonidet P-40, 10% (v/v) glycerol, 137 mm NaCl, 1 mm MgCl2, 1 mm CaCl2, 1 mm dithiothreitol, 1 mm phenylmethylsulfonyl fluoride, and 1 mm sodium orthovana-date. The lysate was centrifuged, and the supernatant was subjected to immunoprecipitation with PY20 monoclonal antibodies to phosphotyrosine. PI 3-kinase activity was assayed in the immunoprecipitates. p70 S6 Kinase Assay—The p70 S6 kinase assay was performed as described (Flotow and Thomas, 1992Flotow H. Thomas G. J. Biol. Chem. 1992; 267: 3074-3078Abstract Full Text PDF PubMed Google Scholar). In brief, serum-deprived confluent cell monolayers were incubated in the absence or presence of insulin for 10 min at 37 ºC, immediately frozen with liquid nitrogen, and lysed on ice in a buffer containing 50 mm Tris-HCl (pH 8.0), 120 mm NaCl, 20 mm NaF, 1 mm benzamidine, 1 mm EDTA, 6 mm EGTA, 15 mm sodium pyrophosphate, 1% Nonidet P-40, 30 mm p-nitrophenyl phosphate, 0.5 mm dithiothreitol, and 1.1 mm phenylmethylsulfonyl fluoride. The lysate was centrifuged, and the supernatant was subjected to immunoprecipitate with antibodies to p70 S6 kinase. After extensive washing, the immunoprecipitate was assayed for p70 S6 kinase activity with an S6 synthetic peptide (KRRRLSSLRASTSKSESSQK) as substrate. After 30 min at 37 °C, the reaction was terminated by the addition of a solution containing 20 mm EDTA and 1.5 mm adenosine, the mixture was centrifuged, and the supernatant was spotted on P81 paper (Whatmann). After washing the paper with 0.5% phosphoric acid, the radioactivity remaining on the paper was determed. Transient Expression of Δp85 by Vaccinia Virus—Transfer plasmid vector pAK10 (Hoshikawa et al., 1991Hoshikawa N. Kojima A. Yasuda A. Takayashiki E. Masuko S. Chiba J. Kurata T. J. Gen. Virol. 1991; 72: 2509-2517Crossref PubMed Scopus (67) Google Scholar, in which the promoter of a gene encoding a 7.5-kDa polypeptide of vaccinia virus (P7.6) flanked by polylinker sequences had been inserted into the vaccinia virus thymidine kinase (TK) gene, was modified by digestion with SalI and XhoI and subsequent self-ligation to remove a SalI-XhoI fragment; the modified plasmid was designated pAK10 s/x. The cDNA encoding Δp85 (Dhand et al., 1994Dhand R. Hara K. Hiles I. Bax B. Gout I. Vicendo P. Fry M.J. Panayotou G. Yonezawa K. Kasuga M. Waterfield M.D. EMBO J. 1994; 13: 511-521Crossref PubMed Scopus (295) Google Scholar was ligated into the BamHI-EcoRI site of pAK10 s/x to generate pAK10 s/x-Δp85. TK− recombinant vaccinia virus in which the Δp85 cDNA had been inserted (LCΔp85) was isolated by plaque assay on 143TK− cells in the presence of 5-bromo-2′-deoxyuridine at a concentration of 25 mg/ml and screened for recombinants of interest by dot hybridization as described previously (Hoshikawa et al., 1991Hoshikawa N. Kojima A. Yasuda A. Takayashiki E. Masuko S. Chiba J. Kurata T. J. Gen. Virol. 1991; 72: 2509-2517Crossref PubMed Scopus (67) Google Scholar. Wild-type vaccinia virus strain LC16 mO (LCmO) (Sugimoto et al., 1985Sugimoto M. Yasuda A. Miki K. Morita M. Suzuki K. Uchida N. Hashizume S. Microbiol. Immunol. 1985; 29: 421-428Crossref PubMed Scopus (26) Google Scholar and the recombinant LCΔp85 strain were propagated in RK13 cells and purified from cytoplasmic extracts. Subconfluent Ratl-IR cells were infected with 10 plaque-forming units of recombinant vaccinia virus (LCΔp85) in serum-free medium. After 1 h, the virus-containing medium was replaced with fresh serum-free medium. At 7 h after infection, cells were lysed and subjected to the PI 3-kinase assay and the glycogen synthase assay. Ratl-IR cells infected with wild-type vaccinia virus (LCmO) were used as controls. Effect of Insulin on Glycogen Synthase Activity in CHO-IRC/S 46 and CHO-IR/Δp85 Cells—To elucidate the role of the Ras-MAP kinase cascade in insulin activation of glycogen synthase, we investigated the effect of insulin on glycogen synthase activity in CHO-IR-C/S 46 cells. Cells were incubated with various concentrations of insulin for 30 min, and glycogen synthase activity was measured. Although the formation of the Ras-GTP complex and activation of MAP kinase in response to insulin are markedly decreased in CHO-IR-C/S 46 cells (Noguchi et al., 1994Noguchi T. Matozaki T. Horita K. Fujioka Y. Kasuga M. Mol. Cell. Biol. 1994; 14: 6674-6682Crossref PubMed Scopus (346) Google Scholar, insulin stimulated glycogen synthase activity in these cells with a dose-response curve similar to that for control cells (Fig. 1A). To elucidate the role of heteromeric PI 3-kinase activity in insulin activation of glycogen synthase, we investigated the effect of insulin on glycogen synthase activity in CHO-IR/Δp85 cells, in which insulin-stimulated PI 3-kinase activity in immunoprecipitates prepared with anti-phosphotyrosine antibodies (Fig. 1B) or anti-IRS-1 antibodies (Hara et al., 1995Hara K. Yonezawa K. Sakaue H. Kotani K. Kotani K. Kojima A. Waterfield M.D. Kasuga M. Biochem. Biophys. Res. Commun. 1995; 208: 735-741Crossref PubMed Scopus (45) Google Scholar was markedly decreased. Insulin stimulated glycogen synthase activity in CHO-IR/Δp85 cells with a dose-response curve similar to that for control cells (Fig. 1A). Effect of Wortmannin on Insulin Activation of Glycogen Synthase—Because wortmannin inhibits PI 3-kinase activity (Yano et al., 1993Yano H. Nakanishi S. Kimura K. Hanai N. Saitoh Y. Fukui Y. Nonomura Y. Matsuda Y. J. Biol. Chem. 1993; 268: 25846-25856Abstract Full Text PDF PubMed Google Scholar; Arcaro and Wymann, 1993Arcaro A. Wymann M.P. Biochem. J. 1993; 296: 297-301Crossref PubMed Scopus (1041) Google Scholar; Okada et al., 1994Okada T. Kawano T. Sakakibara T. Hazeki O. Ui M. J. Biol. Chem. 1994; 269: 3568-3573Abstract Full Text PDF PubMed Google Scholar, we next treated CHO-IR cells with various concentrations of wortmannin for 10 min before stimulation with 10−7 m of insulin and assay of glycogen synthase activity. Wortmannin inhibited insulin-stimulated PI 3-kinase activity in immunoprecipitates prepared with anti-phosphotyrosine antibodies (Fig. 2A) or anti-IRS-1 antibodies (Hara et al., 1995Hara K. Yonezawa K. Sakaue H. Kotani K. Kotani K. Kojima A. Waterfield M.D. Kasuga M. Biochem. Biophys. Res. Commun. 1995; 208: 735-741Crossref PubMed Scopus (45) Google Scholar in a dose-dependent manner. Wortmannin also inhibited insulin-stimulated glycogen synthase activity in CHO-IR cells in a dose-dependent manner (Fig. 2B). At a concerntration of 50 nM, wortmannin abolished the activation of glycogen synthase in response to insulin at all doses tested in CHO-IR cells (Fig. 2C). Furthermore, 50 nM wortmannin abolished 10−7 M insulin-stimulated activation of glycogen synthase not only in CHO-IR cells but also in CHO-IR/Δp85 and CHO-IR-C/S 46 cells (Fig. 2D). Effect of Wortmannin or Transient Overexpression of Δp85 on Insulin Activation of Glycogen Synthase in Rat1-IR Cells—The Δp85 protein was transiently overexpressed in intact cells with the use of the vaccinia virus expression system. We found that Rat1 cells and Hela cells, but not CHO cells, were good target cells for vaccinia virus. Rat1 cells overexpressing human insulin receptors (Ratl-IR cells) were infected with wild-type vaccinia virus (LCmO) or a recombinant vaccinia virus (LC Δp85) that encodes Δp85 (Rat1-IR/mO cells and Ratl-IR/Δp85 cells, respectively). Immunoblot analysis revealed that the amount of Δp85 in Ratl-IR/Δp85 cells 7 h after infection was ~10–15 times the amount of endogenous p85α in Ratl-IR cells (Hara et al., 1995Hara K. Yonezawa K. Sakaue H. Kotani K. Kotani K. Kojima A. Waterfield M.D. Kasuga M. Biochem. Biophys. Res. Commun. 1995; 208: 735-741Crossref PubMed Scopus (45) Google Scholar. Insulin-stimulated PI 3-kinase activity in anti-IRS-1 immunoprecipitates (Hara et al., 1995Hara K. Yonezawa K. Sakaue H. Kotani K. Kotani K. Kojima A. Waterfield M.D. Kasuga M. Biochem. Biophys. Res. Commun. 1995; 208: 735-741Crossref PubMed Scopus (45) Google Scholar or anti-phosphotyrosine immunoprecipitates (Fig. 3A) was markedly attenuated in Rat1-IR/Δp85 cells 7 h after infection relative to that apparent in Rat1-IR/mO cells or noninfected Ratl-IR cells. As with CHO-IR cells, treatment of Ratl-IR cells or Rat1-IR/mO cells with 50 nM wortmannin for 10 min before insulin stimulation resulted in a marked decrease in the amount of PI 3-kinase activity associated with tyrosine-phosphorylated proteins (Fig. 3A). Wortmannin also induced a further decrease in PI 3-kinase activity associated with tyrosine-phosphorylated proteins in insulin-stimulated Rat1-IR/Δp85 cells (Fig. 3A). No differences in the extent of tyrosine phosphorylation of the insulin receptor or IRS-1 were detected among Ratl-IR, Rat1-IR/mO, and Ratl-LR/Δp85 cells by immunoblotting with antibodies to phosphotyrosine (data not shown). The basal activity of glycogen synthase in both Rat1-IR/Δp85 cells and Rat1-IR/mO cells did not differ from that in noninfected Ratl-IR cells. Insulin stimulated glycogen synthase activity approximately 2-fold in all three cell lines (Fig. 3B). These data are consistent with those from CHO-IR/Δp85 cells and indicate that the transient overexpression of Δp85 did not inhibit insulin activation of glycogen synthase. Wortmannin (50 nM) markedly inhibited insulin activation of glycogen synthase in Ratl-IR, Rat1-IR/mO, and Rat1-IR/Δp85 cells (Fig. 3B). Effect of Rapamycin on the Activation of Glycogen Synthase by Insulin in CHO-IR Cells—To examine the possible participation of the p70 S6 kinase pathway in insulin activation of glycogen synthase, we exposed CHO-IR cells to various concentrations of rapamycin for 30 min before stimulation with insulin and assay of glycogen synthase activity. At all concentrations tested, rapamycin did not inhibit the activation of glycogen synthase induced by 10−7 M insulin (Fig. 4A). Rapamycin (20 ng/ml) completely abolished the activation of p70 S6 kinase induced by 10−7 m insulin (Fig. 4B) We have investigated the mechanism of insulin activation of glycogen synthase. Because Ras is a key element in signal transduction for many growth factors, we first examined the possible participation of this protein in insulin-stimulated glycogen synthase activation. Whereas insulin-induced Ras activation and the consequent stimulation of MAP kinase are almost completely abolished in CHO cells overexpressing insulin receptors and a catalytically inactive SH-PTP2 (CHO-IR-C/S 46 cells) (Noguchi et al., 1994Noguchi T. Matozaki T. Horita K. Fujioka Y. Kasuga M. Mol. Cell. Biol. 1994; 14: 6674-6682Crossref PubMed Scopus (346) Google Scholar, we showed that insulin fully activated glycogen synthase in these cells. It is possible that insulin activation of glycogen synthase is normally Ras-dependent, but was somehow rescued by overexpression of the catalytically inactive SH-PTP2 in these cells. However, this possibility is unlikely, given that insulin-stimulated glycogen synthase activation is also normal in CHO-IR/ΔSOS cells, in which insulin-induced Ras activation is markedly impaired by overexpression of a dominant negative mutant Sos (Sakaue et al., 1995Sakaue M. Bowtell D. Kasuga M. Mol. Cell. Biol. 1995; 15: 379-388Crossref PubMed Scopus (60) Google Scholar. These results indicate that the activation of Ras is not required for activation of glycogen synthase. This conclusion appears inconsistent with the observation that an insulin-stimulated protein kinase, originally designated ISPK but later found to be p90rsk (Sutherland et al., 1993Sutherland C. Campbell D.G. Cohen P. Eur. J Biochem. 1993; 212: 581-588Crossref PubMed Scopus (113) Google Scholar, phosphorylates and activates PP1G, the glycogen-bound form of type 1 protein phosphatase (Dent et al., 1990Dent P. Lavoinne A. Nakielny S. Caudwell F.B. Watt P. Cohen P. Nature. 1990; 348: 302-308Crossref PubMed Scopus (401) Google Scholar in muscle, given that Ras and MAP kinase are upstream regulators of p90rsk (Sturgill et al., 1988Sturgill T.W. Ray L.B. Erikson E. Maller J.L. Nature. 1988; 334: 715-718Crossref PubMed Scopus (751) Google Scholar; Ahn and Krebs, 1990Ahn N.G. Krebs E. J. Biol. Chem. 1990; 265: 11495-11501Abstract Full Text PDF PubMed Google Scholar). Although we cannot exclude the possibility that glycogen synthase is regulated differently in muscle cells and other types of cells (CHO-IR cells), the activation of MAP kinase by epidermal growth factor does not result in stimulation of glycogen synthase in 3T3-L1 adipocytes (Robinson et al., 1993Robinson L.J. Razzack Z.F. Lawrence Jr., J.C. James D.E. J. Biol. Chem. 1993; 268: 26422-26427Abstract Full Text PDF PubMed Google Scholar or isolated rat adipocytes (Lin and Lawrence, 1994Lin F.-A. Lawrence Jr., J.C. J. Biol. Chem. 1994; 269: 21255-21261Abstract Full Text PDF PubMed Google Scholar). Taken together, these observations indicate that MAP kinase activation may not be required or sufficient for the activation of glycogen synthase. We next investigated the potential role of PI 3-kinase, another key element in signal transduction for many growth factors, in the activation of glycogen synthase in response to insulin. We previously established a CHO cell line (CHO-IR/Δp85) that overexpresses insulin receptors and a dominant negative mutant of the PI 3-kinase p85 adaptor subunit that does not bind the p110 catalytic subunit (Hara et al., 1994Hara K. Yonezawa K. Sakaue H. Ando A. Kotani K. Kitamura T. Kitamura Y. Ueda H. Stephens L. Jackson T.R. Hawkins P.T. Dhand R. Clark A.E. Holman G.D. Waterfield M.D. Kasuga M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7415-7419Crossref PubMed Scopus (416) Google Scholar. PI 3-kinase activity in anti-IRS-1 immunoprecipitates from insulin-stimulated CHO-IR/Δp85 cells was markedly decreased, as was the insulin-induced cellular accumulation of phosphatidylinositol 3,4,5-trisphosphate. Moreover, insulin-stimulated translocation of the glucose transporters GLUTI, but not Ras activation, was markedly inhibited in CHO-IR/Δp85 cells (Hara et al., 1994Hara K. Yonezawa K. Sakaue H. Ando A. Kotani K. Kitamura T. Kitamura Y. Ueda H. Stephens L. Jackson T.R. Hawkins P.T. Dhand R. Clark A.E. Holman G.D. Waterfield M.D. Kasuga M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7415-7419Crossref PubMed Scopus (416) Google Scholar. In the present study, we showed that insulin fully activates glycogen synthase in CHO-IR/Δp85 cells. To exclude the possibility that our stably transfected CHO-IR/Δp85 cells had acquired somatic mutations that affected the cellular activity under investigation, we also transiently overexpressed Δp85 in Ratl-IR cells with the use of a recombinant vaccinia virus. Transient overexpression of Δp85 in Ratl-IR cells markedly decreased PI 3-kinase activity in immunoprecipitates prepared from insulin-treated cells with anti-phosphotyrosine antibodies or anti-IRS-1 antibodies, but did not affect insulin stimulation of glycogen synthase activity. Thus, normal activation of glycogen synthase by insulin in CHO-IR/Δp85 cells does not appear to be attributable to somatic mutations acquired during cloning and culture. Therefore, neither stable overexpression (CHO-IR/Δp85 cells) nor transient overexpression (Ratl-IR/Δp85 cells) of dominant negative Δp85-inhibited insulin stimulation of glycogen synthase, even though the insulin-induced activation of PI 3-kinase activity was markedly reduced. However, wortmannin abolished the activation of glycogen synthase by insulin in both CHO-IR/Δp85 cells and Rat/IR/Δp85. One interpretation of our data is that insulin-stimulated PI 3-kinase activity is not required for insulin activation of glycogen synthase and that wortmannin inhibits the activation of glycogen synthase independently of its inhibitory effect on the heteromeric PI 3-kinase. This interpretation appears reasonable, given that the specificity of wortmannin is not clear. For example, wortmannin inhibits other enzymes related to the heteromeric PI 3-kinase such as the G protein βγ subunit-sensitive PI 3-kinase (Stephens et al., 1994aStephens L. Smrcka A. Cooke F.T. Jackson T.R. Sternweis P.C. Hawkins P.T. Cell. 1994; 77: 83-93Abstract Full Text PDF PubMed Scopus (515) Google Scholar and phosphatidylinositol-specific PI 3-kinase (Stephens et al., 1994bStephens L. Cooke F.T. Walters R. Jackson T. Volinia S. Gout I. Waterfield M.D. Hawkins P.T. Curr. Biol. 1994; 4: 203-214Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar. Thus, wortmannin may inhibit an unknown enzyme that is an upstream regulator of glycogen synthase. Another interpretation is that a small increase in PI 3-kinase activity is sufficient for the activation of glycogen synthase but not for glucose uptake. Thus, the dominant negative p85 mutant does not completely block the association of PI 3-kinase activity with tyrosine-phosphorylated proteins, whereas wortmannin inhibits p110 catalytic activity completely. For example, wortmannin decreased the small amount of insulin-stimulated PI 3-kinase activity associated with tyrosine-phosphorylated proteins in CHO-IR/Δp85 cells (data not shown) and Rat 1-IR/Δp85 cells. A third interpretation is that the stably or transiently overexpressed Δp85 may interact with other proteins that bypass PI 3-kinase activity and rescue the signal transduction pathway from the insulin receptor to glycogen synthase. If this is the case, wortmannin must inhibit enzymes other than heteromeric PI 3-kinase, because this agent completely blocked the activation of glycogen synthase in cells overexpressing Δp85. Insulin fully stimulates p70 S6 kinase in CHO-IR/Δp85 and Ratl-IR/Δp85 cells, whereas wortmannin completely inhibits insulin-dependent activation of p70 S6 kinase in these cells. Furthermore, both p90rsk and p70 S6 kinase phosphorylate and inactivate glycogen synthase kinase-3 (GSK-3), a kinase thought to have a major role in regulating glycogen synthase activity, in a cell-free system (Sutherland et al., 1993Sutherland C. Campbell D.G. Cohen P. Eur. J Biochem. 1993; 212: 581-588Crossref PubMed Scopus (113) Google Scholar. These data prompted us to examine the role of p70 S6 kinase in the insulin-induced activation of glycogen synthase in intact cells. We examined the effects of the immunosuppressant rapamycin, which blocks the activation of p70 S6 kinase by insulin and many growth factors, but does not inhibit MAP kinase or p90rsk (Chung et al., 1992Chung J. Kuo C.J. Crabtree G.R. Blenis J. Cell. 1992; 69: 1227-1236Abstract Full Text PDF PubMed Scopus (1011) Google Scholar; Price et al., 1992Price D.J. Grove J.R. Calvo V. Avruch J. Bierer B.E. Science. 1992; 257: 973-977Crossref PubMed Scopus (581) Google Scholar; Calvo et al., 1992Calvo V. Crews C.M. Vik T.A. Bierer B.E. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 7571-7575Crossref PubMed Scopus (165) Google Scholar. We showed that rapamycin completely inhibited insulin activation of p70 S6 kinase at a concentration of 20 ng/ml, whereas no significant decrease in insulin-stimulated glycogen synthase activity was observed when CHO-IR cells were treated with rapamycin at concentrations up to 200 ng/ml. These observations suggest that the p70 S6 kinase pathway may not be important in the activation of glycogen synthase, consistent with data from rat adipocytes (Lin and Lawrence, 1994Lin F.-A. Lawrence Jr., J.C. J. Biol. Chem. 1994; 269: 21255-21261Abstract Full Text PDF PubMed Google Scholar). Recently, rapamycin was shown to be without effect on the inactivation of GSK-3 by insulin in CHO cells (Welsh et al., 1994Welsh 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 and the rat skeletal muscle cell line L6 (Cross et al., 1994Cross D.A.E. Alessi D.R. Vanddenheeden J.R. McDowell H.E. Hundal H.S. Cohen P. Biochem. J. 1994; 303: 21-26Crossref PubMed Scopus (419) Google Scholar. These studies also suggested that GSK-3 activity is regulated by a cascade involving MAP kinase and p90rsk and that wortmannin only partially blocks MAP kinase activation by insulin but almost completely blocks the effects of insulin on GSK-3 (Welsh et al., 1994Welsh 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: Cross et al., 1994Cross D.A.E. Alessi D.R. Vanddenheeden J.R. McDowell H.E. Hundal H.S. Cohen P. Biochem. J. 1994; 303: 21-26Crossref PubMed Scopus (419) Google Scholar. We also showed that wortmannin inhibited insulin activation of MAP kinase by only 20% (data not shown), whereas it abolished the insulin-induced activation of glycogen synthase in CHO-IR cells. As previously pointed out (Cross et al., 1994Cross D.A.E. Alessi D.R. Vanddenheeden J.R. McDowell H.E. Hundal H.S. Cohen P. Biochem. J. 1994; 303: 21-26Crossref PubMed Scopus (419) Google Scholar, these observations can be explained in two ways. First, only a small degree of activation of p90rsk may be required for maximal inhibition of GSK-3, because of the large potential of protein kinase cascades for amplification. Second, GSK-3 may be inhibited by a distinct insulin-stimulated protein kinase whose activation is also blocked by wortmannin. Given that activation of p90rsk appears insufficient to activate glycogen synthase (Lin and Lawrence, 1994Lin F.-A. Lawrence Jr., J.C. J. Biol. Chem. 1994; 269: 21255-21261Abstract Full Text PDF PubMed Google Scholar), the latter explanation may be more reasonable. Although it is possible that a small increase in heteromeric PI 3-kinase activity associated with tyrosine-phosphorylated protein in a specific compartment of cells is sufficient for the activation of glycogen synthase but not for glucose uptake, we favor the notion that a wortmannin-sensitive molecule other than the heteromeric PI 3-kinase is an upstream regulator of glycogen synthase. In conclusion, we demonstrated that neither Ras-MAP kinase cascade nor PI3-kinase may be required for insulin-stimulated glycogen synthase activation in CHO cell lines and p70S6 kinase is not likely to be involved in this event. However, the present studies do not exclude the possible role of these molecules on glycogen synthase activation in other tissues, such as liver and skeletal muscle. We thank Drs. A. Ando, H. Ueda, Kei Kotani, Ko Kotani, Y. Kitamura, T. Kitamura, and T. Maeda in our department for their assistance in performing experiments.

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