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• W2102683312 abstract "Tumor necrosis factor α (TNFα) inhibits insulin action, in part, through serine phosphorylation of IRS proteins; however, the phosphorylation sites that mediate the inhibition are unknown. TNFα promotes multipotential signal transduction cascades, including the activation of the Jun NH2-terminal kinase (JNK). Endogenous JNK associates with IRS-1 in Chinese hamster ovary cells. Anisomycin, a strong activator of JNK in these cells, stimulates the activity of JNK bound to IRS-1 and inhibits the insulin-stimulated tyrosine phosphorylation of IRS-1. Serine 307 is a major site of JNK phosphorylation in IRS-1. Mutation of serine 307 to alanine eliminates phosphorylation of IRS-1 by JNK and abrogates the inhibitory effect of TNFα on insulin-stimulated tyrosine phosphorylation of IRS-1. These results suggest that phosphorylation of serine 307 might mediate, at least partially, the inhibitory effect of proinflammatory cytokines like TNFα on IRS-1 function. Tumor necrosis factor α (TNFα) inhibits insulin action, in part, through serine phosphorylation of IRS proteins; however, the phosphorylation sites that mediate the inhibition are unknown. TNFα promotes multipotential signal transduction cascades, including the activation of the Jun NH2-terminal kinase (JNK). Endogenous JNK associates with IRS-1 in Chinese hamster ovary cells. Anisomycin, a strong activator of JNK in these cells, stimulates the activity of JNK bound to IRS-1 and inhibits the insulin-stimulated tyrosine phosphorylation of IRS-1. Serine 307 is a major site of JNK phosphorylation in IRS-1. Mutation of serine 307 to alanine eliminates phosphorylation of IRS-1 by JNK and abrogates the inhibitory effect of TNFα on insulin-stimulated tyrosine phosphorylation of IRS-1. These results suggest that phosphorylation of serine 307 might mediate, at least partially, the inhibitory effect of proinflammatory cytokines like TNFα on IRS-1 function. insulin receptor substrate c-Jun NH2-terminal kinase mitogen-activated protein MAP kinase kinase MAPKK kinase tumor necrosis factor Chinese hamster ovary polyacrylamide gel electrophoresis glutathione S-transferase high pressure liquid chromatography phosphatidylinositol JNK-interacting protein JNK-binding domain in vitro kinase immunoprecipitate(s) phosphotyrosine binding domain The insulin signaling system is complex, and a common mechanism to explain the occurrence of insulin resistance during diabetes is difficult to resolve. So far, genetic approaches provide important insight into certain early onset forms of diabetes but fail to explain insulin resistance that is associated with common type 2 diabetes (1.Hani E.H. Suaud L. Boutin P. Chevre J.C. Durand E. Philippi A. Demenais F. Vionnet N. Furuta H. Velho G. Bell G.I. Laine B. Froguel P. J. Clin. Invest. 1998; 101: 521-526Crossref PubMed Scopus (105) Google Scholar, 2.Carboni J.M. Yan N. Cox A.D. Bustelo X. Graham S.M. Lynch M.J. Weinmann R. Seizinger B.R. Der C.J. Barbacid M. Oncogene. 1995; 10: 1905-1913PubMed Google Scholar, 3.Vaxillaire M. Rouard M. Yamagata K. Oda N. Kaisaki P.J. Boriraj V.V. Chevre J.C. Boccio V. Cox R.D. Lathrop G.M. Dussoix P. Philippe J. Timsit J. Charpentier G. Velho G. Bell G.I. Froguel P. Hum. Mol. Genet. 1997; 6: 583-586Crossref PubMed Scopus (122) Google Scholar, 4.Comb D.G. Roseman S. J. Biol. Chem. 1958; 232: 807-827Abstract Full Text PDF PubMed Google Scholar). Recent results support the notion that dysregulation of the insulin receptor and reduced tyrosine phosphorylation of the IRS1 proteins might contribute significantly to peripheral insulin resistance and β-cell failure (5.Withers D.J. Burks D.J. Towery H.H. Altamuro S.L. Flint C.L. White M.F. Nat. Genet. 1999; 23: 32-40Crossref PubMed Scopus (485) Google Scholar, 6.Dresner A. Laurent D. Marcucci M. Griffin M.E. Dufour S. Cline G.W. Slezak L.A. Andersen D.K. Hundal R.S. Rothman D.L. Petersen K.F. Shulman G.I. J. Clin. Invest. 1999; 103: 253-259Crossref PubMed Scopus (1039) Google Scholar). The principal insulin receptor substrates, IRS-1 and IRS-2, are phosphorylated on multiple tyrosine residues by the activated receptors for insulin, IGF-1, and various other cytokines (7.Yenush L. White M.F. BioEssays. 1997; 19: 491-500Crossref PubMed Scopus (257) Google Scholar). Tyrosine phosphorylation of IRS-1 and IRS-2 promotes their binding to the Src homology 2 domains in various downstream signaling proteins, including the phosphatidylinositol 3-kinase (PI 3-kinase), Grb-2, SHP2, and others (7.Yenush L. White M.F. BioEssays. 1997; 19: 491-500Crossref PubMed Scopus (257) Google Scholar, 8.Myers Jr., M.G. White M.F. Diabetes. 1993; 42: 643-650Crossref PubMed Google Scholar). During association with IRS proteins, PI 3-kinase is activated, and its phospholipid products promote the recruitment of various serine kinases to the plasma membrane, where they are activated by phosphorylation (9.Alessi D.R. Cohen P. Curr. Opin. Gene. 1998; 8: 55-62Crossref PubMed Scopus (677) Google Scholar). One of the membrane-associated kinases, protein kinase B/Akt, phosphorylates multiple downstream effectors that promote diverse biological responses, including stimulation of glucose transport, protein and glycogen synthesis, and the regulation of gene expression, which affects cellular proliferation and survival (10.Alessi D.R. Cohen P. Curr. Opin. Genet. Dev. 1998; 8: 55-62Crossref PubMed Scopus (662) Google Scholar,11.Brunet A. Bonni A. Zigmond M.J. Lin M.Z. Juo P. Hu L.S. Anderson M.J. Arden K.C. Blenis J. Greenberg M.E. Cell. 1999; 96: 857-868Abstract Full Text Full Text PDF PubMed Scopus (5454) Google Scholar). The insulin receptor and the IRS proteins might be counterregulated by degradation, differential expression, or modification by serine/threonine phosphorylation (12.Sun X.J. Goldberg J.L. Qiao L.Y. Mitchell J.J. Diabetes. 1999; 48: 1359-1364Crossref PubMed Scopus (197) Google Scholar, 13.Qiao L. Goldberg J.L. Russell J.C. Sun X.J. J. Biol. Chem. 1999; 274: 10625-10632Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 14.Li J. De Fea K. Roth R.A. J. Biol. Chem. 1999; 274: 9351-9356Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar, 15.De Fea K. Roth R.A. J. Biol. Chem. 1997; 272: 31400-31406Abstract Full Text Full Text PDF PubMed Scopus (267) Google Scholar). Increased serine phosphorylation of IRS-1 is a common finding during insulin resistance and type 2 diabetes (16.Virkamaki A. Ueki K. Kahn C.R. J. Clin. Invest. 1999; 103: 931-943Crossref PubMed Scopus (726) Google Scholar). However, the mechanism by which serine phosphorylation inhibits insulin signaling is difficult to establish, because IRS-1 contains more than 70 potential serine/threonine residues in consensus sequences for many protein kinases, including casein kinase II, cAMP-dependent protein kinase, protein kinase C, Cdc2 kinase, MAP kinase, and protein kinase B/Akt (14.Li J. De Fea K. Roth R.A. J. Biol. Chem. 1999; 274: 9351-9356Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar, 15.De Fea K. Roth R.A. J. Biol. Chem. 1997; 272: 31400-31406Abstract Full Text Full Text PDF PubMed Scopus (267) Google Scholar, 17.Sun X.J. Rothenberg P.L. Kahn C.R. Backer J.M. Araki E. Wilden P.A. Cahill D.A. Goldstein B.J. White M.F. Nature. 1991; 352: 73-77Crossref PubMed Scopus (1242) Google Scholar, 18.Sun X.J. Wang L.M. Zhang Y. Yenush L. Myers Jr., M.G. Glasheen E.M. Lane W.S. Pierce J.H. White M.F. Nature. 1995; 377: 173-177Crossref PubMed Google Scholar, 19.Barthel A. Nakatani K. Dandekar A.A. Roth R.A. Biochem. Biophys. Res. Commun. 1998; 243: 509-513Crossref PubMed Scopus (53) Google Scholar, 20.De Fea K. Roth R.A. Biochemistry. 1997; 36: 12939-12947Crossref PubMed Scopus (223) Google Scholar). The action of proinflammatory cytokines like TNFα or interleukin-1β to promote serine phosphorylation of IRS-1 might provide a common mechanism for insulin/IGF-1 resistance observed during acute trauma and chronic obesity (21.Feuerstein G.Z. Liu T. Barone F.C. Cerebrovasc. Brain Metab. Rev. 1994; 6: 341-360PubMed Google Scholar, 22.Barone F.C. Arvin B. White R.F. Miller A. Webb C.L. Willette R.N. Lysko P.G. Feuerstein G.Z. Stroke. 1997; 28: 1233-1244Crossref PubMed Scopus (731) Google Scholar, 23.Hotamisligil G.S. Spiegelman B.M. Diabetes. 1994; 43: 1271-1278Crossref PubMed Google Scholar, 24.Hotamisligil G.S. Shargill N.S. Spiegelman B.M. Science. 1993; 259: 87-91Crossref PubMed Google Scholar, 25.Nawashiro H. Martin D. Hallenbeck J.M. J. Cereb. Blood Flow Metab. 1997; 17: 229-232Crossref PubMed Scopus (166) Google Scholar, 26.Lavine S.D. Hofman F.M. Zlokovic B.V. J. Cereb. Blood Flow Metab. 1998; 18: 52-58Crossref PubMed Scopus (175) Google Scholar). TNFα is produced systemically by macrophages and lymphocytes after inflammatory stimulation or trauma and increases rapidly during experimental injury induced by cerebral ischemic, excitotoxic, and traumatic injury (27.Loddick S.A. Rothwell N.J. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 9449-9451Crossref PubMed Scopus (47) Google Scholar). Moreover, obese animals and humans also produce TNFα in positive correlation to body mass index and hyperglycemia, an indirect measure of insulin resistance (23.Hotamisligil G.S. Spiegelman B.M. Diabetes. 1994; 43: 1271-1278Crossref PubMed Google Scholar, 24.Hotamisligil G.S. Shargill N.S. Spiegelman B.M. Science. 1993; 259: 87-91Crossref PubMed Google Scholar). During chronic obesity or sudden trauma, insulin receptor kinase activity and tyrosine phosphorylation of IRS-1 are reduced in skeletal muscle; however, in each case dephosphorylation of IRS-1 by incubation in vitro with alkaline phosphatase restores its ability to undergo tyrosine phosphorylation by the activated insulin receptor (28.Hotamisligil G.S. Peraldi P. Budvari A. Ellis R.W. White M.F. Spiegelman B.M. Science. 1996; 271: 665-668Crossref PubMed Scopus (2223) Google Scholar, 29.Peraldi P. Hotamisligil G.S. Buurman W.A. White M.F. Spiegelman B.M. J. Biol. Chem. 1996; 271: 13018-13022Abstract Full Text Full Text PDF PubMed Scopus (368) Google Scholar, 30.Ikezu T. Okamoto T. Yonezawa K. Tompkins R.G. Martyn J.A. J. Biol. Chem. 1997; 272: 25289-25295Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). TNFα receptor null mice display less insulin resistance during diet-induced obesity, suggesting that TNFα signals promote, at least partially, the inhibition of IRS-1 function in mice (31.Hotamisligil G.S. J. Intern. Med. 1999; 245: 621-625Crossref PubMed Scopus (701) Google Scholar, 32.Uysal K.T. Wiesbrock S.M. Marino M.W. Hotamisligil G.S. Nature. 1997; 389: 610-614Crossref PubMed Scopus (1917) Google Scholar). Moreover, TNFα treatment of adipocytes increases serine phosphorylation of IRS-proteins, which inhibits insulin-stimulated tyrosine phosphorylation and impairs insulin signaling (28.Hotamisligil G.S. Peraldi P. Budvari A. Ellis R.W. White M.F. Spiegelman B.M. Science. 1996; 271: 665-668Crossref PubMed Scopus (2223) Google Scholar, 29.Peraldi P. Hotamisligil G.S. Buurman W.A. White M.F. Spiegelman B.M. J. Biol. Chem. 1996; 271: 13018-13022Abstract Full Text Full Text PDF PubMed Scopus (368) Google Scholar, 34.Kanety H. Feinstein R. Papa M.Z. Hemi R. Karasik A. J. Biol. Chem. 1995; 270: 23780-23784Abstract Full Text Full Text PDF PubMed Scopus (385) Google Scholar). These results suggest that serine phosphorylation of IRS-1 promotes an inhibitory effect of proinflammatory cytokines on insulin receptor signaling (30.Ikezu T. Okamoto T. Yonezawa K. Tompkins R.G. Martyn J.A. J. Biol. Chem. 1997; 272: 25289-25295Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). The identification of the serine kinases that phosphorylate IRS proteins during acute trauma and chronic obesity is an essential step for learning how to reverse the insulin resistance that perturbs metabolic homeostasis and contributes to diabetes. The JNK signaling pathway is implicated in many biological responses, including mammalian embryogenesis and the response to stress (35.Kuan C.Y. Yang D.D. Samanta Roy D.R. Davis R.J. Rakic P. Flavell R.A. Neuron. 1999; 22: 667-676Abstract Full Text Full Text PDF PubMed Scopus (775) Google Scholar, 36.Rincon M. Whitmarsh A. Yang D.D. Weiss L. Derijard B. Jayaraj P. Davis R.J. Flavell R.A. J. Exp. Med. 1998; 188: 1817-1830Crossref PubMed Scopus (193) Google Scholar). During TNFα binding, the tumor necrosis factor receptor 1 trimerizes, which promotes the assembly of a multicomponent complex that activates the JNK signaling cascade (37.Yuasa T. Ohno S. Kehrl J.H. Kyriakis J.M. J. Biol. Chem. 1998; 273: 22681-22692Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar). Activated JNK phosphorylates many cellular proteins, including components of the AP-1 transcription factor complex (38.Ip Y.T. Davis R.J. Curr. Opin. Cell Biol. 1998; 10: 205-219Crossref PubMed Scopus (1386) Google Scholar). Here we provide evidence to support the hypothesis that JNK associates with IRS-1 and promotes the phosphorylation of Ser307 near the PTB domain. Our results suggest that phosphorylation of Ser307 by JNK or another related kinase might mediate the inhibitory effects of TNFα on insulin signal transduction. Phosphospecific antibodies were purchased from New England Biolabs. Antibodies against IRS-1, IRS-2, and p85 were described previously (39.Sun X.J. Wang L.M. Zhang Y. Yenush L. Myers Jr., M.G. Glasheen E.M. Lane W.S. Pierce J.H. White M.F. Nature. 1995; 377: 173-177Crossref PubMed Scopus (767) Google Scholar, 40.Pons S. Asano T. Glasheen E.M. 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 (232) Google Scholar). JNK1 antibody was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), and rabbit polyclonal antibodies against p38 have been described (41.Raingeaud J. Gupta S. Rogers J.S. Dickens M. Han J. Ulevitch R.J. Davis R.J. J. Biol. Chem. 1995; 270: 7420-7426Abstract Full Text Full Text PDF PubMed Scopus (2046) Google Scholar). Insulin, alkaline phosphatase, M2 antibody, and phosphoamino standards were purchased from Sigma. Constructs for JNK1, GST-ATF2, and GST-JIP-1-JBD have been described (42.Derijard B. Hibi M. Wu I.H. Barrett T. Deng T. Karin M. Davis R.J. Cell. 1994; 76: 1025-1037Abstract Full Text PDF PubMed Scopus (2957) Google Scholar, 43.Gupta S. Campbell D. Derijard B. Davis R.J. Science. 1995; 267: 389-393Crossref PubMed Scopus (1339) Google Scholar, 44.Dickens M. Rogers J.S. Cavanagh J. Raitano A. Xia Z. Halpern J.R. Greenberg M.E. Sawyers C.L. Davis R.J. Science. 1997; 277: 693-696Crossref PubMed Scopus (629) Google Scholar). Point mutants in IRS-1 were generated using the Stratagene Quikchange site-directed mutagenesis method. Chinese hamster ovary (CHO) cells overexpressing the human insulin receptor were described previously (45.Sun X.J. Miralpeix M. Myers Jr., M.G. Glasheen E.M. Backer J.M. Kahn C.R. White M.F. J. Biol. Chem. 1992; 267: 22662-22672Abstract Full Text PDF PubMed Google Scholar, 46.Sun X.J. Rothenberg P.L. Kahn C.R. Backer J.M. Araki E. Wilden P.A. Cahill D.A. Goldstein B.J. White M.F. Nature. 1991; 352: 73-77Crossref PubMed Scopus (1291) Google Scholar). Stable CHO cell lines expressing murine IRS-1 or murine IRS-2 were generated by Fugene-6-mediated transfection of pCMVHis containing the appropriate inserts (Roche Molecular Biochemicals); 32D cell transfectants were generated by electroporation. In each case, transfected cells were selected in histidinol as described previously (47.Yenush L. Zanella C. Uchida T. Bernal D. White M.F. Mol. Cell. Biol. 1998; 18: 6784-6794Crossref PubMed Scopus (73) Google Scholar). CHO cells were maintained in Ham's F-12 medium supplemented with 10% fetal bovine serum and 5 mm histidinol and made quiescent by serum starvation for 12 h, whereas 32D cells were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum, 5% WEHI conditioned medium (as a source of interleukin-3), and 5 mm histidinol and made quiescent by serum starvation for 4 h. HEK 293 cells were maintained in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and made quiescent by serum starvation for 12 h. CHO and 32D cells were lysed in 50 mm Tris (pH 7.4), containing 130 mm NaCl, 5 mm EDTA, 1.0% Nonidet P-40, 100 mm NaF, 50 mm β-glycerophosphate, 100 μm NaVO4, 1 mmphenylmethylsulfonyl fluoride, 5 μg/ml leupeptin, and 5 μg/ml aprotinin. Immunoprecipitations were performed for 2 h at 4 °C and collected on protein A-Sepharose. Lysates and immunoprecipitates were resolved by SDS-PAGE and transferred to nitrocellulose, and proteins were detected by immunoblotting/125I-labeled protein A and analysis on a Molecular Dynamics PhosphorImager. 293 cells were lysed in 20 mm Tris (pH 7.4) containing 137 mm NaCl, 25 mm β-glycerophosphate, 2 mm sodium pyrophosphate, 2 mm EDTA, 1% Triton X-100, 10% glycerol, 1 mm phenylmethylsulfonyl fluoride, 5 μg/ml leupeptin, 5 μg/ml aprotinin, 2 mm benzamidine, and 0.5 mm dithiothreitol. Cell lysates from CHOIR/IRS-1 cells treated with anisomycin were incubated with 20 units of alkaline phosphatase at 37 °C for 1 h. The dephosphorylation reaction was stopped by the addition of SDS sample buffer and boiling. Proteins were resolved by SDS-PAGE, transferred to nitrocellulose, and analyzed by Western blot using antibodies against IRS-1(47). GST fusion proteins containing portions of IRS-1 were made by subcloning the indicated residues into pGex-2TK (Amersham Pharmacia Biotech), expressed inEscherichia coli (BL-21) and purified using glutathione-agarose (Amersham Pharmacia Biotech). GST fusion proteins (111 pmol) were incubated with 293 cell lysates for 2 h at 4 °C. Where indicated, experiments were performed in the presence or absence of a 64 μg/ml concentration of a wild type JIP-1-JBD (residues 148–174) synthetic peptide or a random sequence control peptide (48.Yasuda J. Whitmarsh A.J. Cavanagh J. Sharma M. Davis R.J. Mol. Cell. Biol. 1999; 19: 7245-7254Crossref PubMed Scopus (409) Google Scholar). Proteins bound to the GST fusion proteins were fractionated by SDS-PAGE, transferred to nitrocellulose, and analyzed by Western blot with antibodies against JNK1 or p38 (48.Yasuda J. Whitmarsh A.J. Cavanagh J. Sharma M. Davis R.J. Mol. Cell. Biol. 1999; 19: 7245-7254Crossref PubMed Scopus (409) Google Scholar). HEK 293 cells were transiently transfected with either pCDNA3-FLAG-JNK1 or pCNDA3 using Fugene-6. Transient transfectants were made quiescent by serum starvation for 12 h and assayed at 36 h. Following stimulation with 10 μg/ml anisomycin and lysis, FLAG-JNK1 was immunoprecipitated with M2 antibody for 2 h at 4 °C, and immune complexes were collected on anti-mouse agarose (Sigma). FLAG-JNK1 was eluted with FLAG peptide (100 μg/ml) in kinase buffer (25 mm Hepes (pH 7.4), 25 mm β-glycerophosphate, 25 mm MgCl2, 100 μm sodium orthovanadate, and 0.5 mm dithiothreitol) overnight at 4 °C. IRS-1 was immunopurified from quiescent CHOIR/IRS-1 cells. Kinase assays were initiated by the addition of kinase and 50 μm [γ-32P]ATP to IRS-1 immune complexes, 1 mg of ATF2GST, or 1 mg of NH2-JunGST in a final volume of 50 μl of kinase buffer. Where indicated, kinase assays were performed in the presence of 10 μm LY294002 (Calbiochem). Reactions were terminated after 30 min at room temperature with ice-cold PBS and the addition of SDS-sample buffer. Phosphorylation of substrate proteins was examined, after SDS-PAGE and transfer to nitrocellulose, by autoradiography and Cerenkov 32P counting. Metabolic labeling of CHO cells with [32P]orthophosphate was performed as described (49.Sun X.J. Crimmins D.L. Myers Jr., M.G. Miralpeix M. White M.F. Mol. Cell. Biol. 1993; 13: 7418-7428Crossref PubMed Google Scholar). Tryptic peptides were generated from IRS-1 on nitrocellulose and resolved on a Waters HPLC system equipped with a Hi-Pore reverse phase column (Bio-Rad) as described (49.Sun X.J. Crimmins D.L. Myers Jr., M.G. Miralpeix M. White M.F. Mol. Cell. Biol. 1993; 13: 7418-7428Crossref PubMed Google Scholar, 50.Feener E.P. Backer J.M. King G.L. Wilden P.A. Sun X.J. Kahn C.R. White M.F. J. Biol. Chem. 1993; 268: 11256-11264Abstract Full Text PDF PubMed Google Scholar). Phosphoamino acid analysis and manual radiosequencing by Edman degradation were performed as described (49.Sun X.J. Crimmins D.L. Myers Jr., M.G. Miralpeix M. White M.F. Mol. Cell. Biol. 1993; 13: 7418-7428Crossref PubMed Google Scholar, 50.Feener E.P. Backer J.M. King G.L. Wilden P.A. Sun X.J. Kahn C.R. White M.F. J. Biol. Chem. 1993; 268: 11256-11264Abstract Full Text PDF PubMed Google Scholar). Endoproteinase Glu-C (Promega) digestion of the initial tryptic HPLC peak was performed in 100 mm ammonium bicarbonate (pH 7.8) for 24 h at 37 °C. TNFα promotes many biological responses, including the activation of the c-Jun NH2-terminal kinase (JNK) and p38 MAP kinase, which might mediate the phosphorylation of IRS-1 (38.Ip Y.T. Davis R.J. Curr. Opin. Cell Biol. 1998; 10: 205-219Crossref PubMed Scopus (1386) Google Scholar, 41.Raingeaud J. Gupta S. Rogers J.S. Dickens M. Han J. Ulevitch R.J. Davis R.J. J. Biol. Chem. 1995; 270: 7420-7426Abstract Full Text Full Text PDF PubMed Scopus (2046) Google Scholar). To investigate the phosphorylation of IRS-proteins by these kinases, we activated JNK and p38 in Chinese hamster ovary cells expressing the insulin receptor (CHOIR) and either IRS-1 or IRS-2. Anisomycin is a protein synthesis inhibitor, but at low concentrations it strongly activates JNK and p38 without inhibition of protein synthesis (52.Cano E. Hazzalin C.A. Mahadevan L.C. Mol. Cell. Biol. 1994; 14: 7352-7362Crossref PubMed Scopus (271) Google Scholar, 53.Zinck R. Cahill M.A. Kracht M. Sachsenmaier C. Hipskind R.A. Nordheim A. Mol. Cell. Biol. 1995; 15: 4930-4938Crossref PubMed Scopus (238) Google Scholar). CHOIR/IRS-1 cells were treated for 30 min with anisomycin, and cell lysates were resolved by SDS-PAGE and screened with phosphospecific antibodies against c-Jun (a JNK1 substrate) and p38. Anisomycin induced the phosphorylation of each protein, confirming that JNK and p38 were activated by anisomycin in CHOIR/IRS-1 cells (Fig. 1 A). In addition, anisomycin reduced the rate of migration of IRS-1 during SDS-PAGE, and alkaline phosphatase treatment of cell lysates restored a normal rate of migration (Fig. 1 B). These results support the hypothesis that anisomycin stimulates phosphorylation of IRS-1 with kinetics similar to those observed for the activation of JNK and p38. The effect of anisomycin on insulin-stimulated tyrosine phosphorylation was investigated by immunoblotting CHOIR cell extracts expressing IRS-1 or IRS-2 with antibodies against phosphotyrosine. Before incubation with anisomycin, insulin (25 nm) strongly increased the tyrosine phosphorylation of IRS-1 and IRS-2, whereas treatment of these cells with anisomycin for 30 min before insulin stimulation inhibited tyrosine phosphorylation (Fig. 1, Cand D). Half-maximal inhibition occurred at 1 μg/ml anisomycin; inhibition was associated with a reduced migration of IRS-1 and IRS-2 during SDS-PAGE (Fig. 1, C and D). As expected, anisomycin inhibited the association of p85 with IRS-1 during insulin stimulation (Fig. 1 E). Thus, anisomycin appears to promote serine phosphorylation of IRS-1 and IRS-2, which is associated with inhibition of insulin-stimulated tyrosine phosphorylation. IRS-1 contains over 70 putative serine phosphorylation sites and is extensively serine-phosphorylated in the basal state (45.Sun X.J. Miralpeix M. Myers Jr., M.G. Glasheen E.M. Backer J.M. Kahn C.R. White M.F. J. Biol. Chem. 1992; 267: 22662-22672Abstract Full Text PDF PubMed Google Scholar). Consequently, the identification of serine phosphorylation sites that inhibit tyrosine phosphorylation is difficult. To identify the regions of IRS-1 that might be involved, various IRS-1 deletion mutants were expressed in CHOIRcells, and the effect of anisomycin on their migration during SDS-PAGE and tyrosine phosphorylation was determined (Fig.2 A). The effect of anisomycin (5 μg/ml) to reduce the migration of IRS-1 during SDS-PAGE and to inhibit insulin-stimulated tyrosine phosphorylation was retained upon deletion of the pleckstrin homology domain or deletion of residues 584–898 in the COOH terminus of IRS-1. By contrast, deletion of the PTB domain (residues 140–309) significantly reduced these effects of anisomycin (Fig. 2 B). Based on this analysis, a truncated IRS-1 protein was constructed, which contained the first 309 residues of IRS-1 (the pleckstrin homology and PTB domains) fused to residues 555–898 of the COOH-terminal tail (Fig. 2 A). This construct, called PPYIRS-1, is tyrosine-phosphorylated during insulin stimulation, binds PI 3-kinase, and mediates the expected downstream signals (54.Yenush L. Zanella C. Uchida T. Bernal D. White M.F. Mol. Cell. Biol. 1998; 18: 6784-6794Crossref PubMed Scopus (75) Google Scholar). Importantly, anisomycin decreased the electrophoretic mobility of PPYIRS-1 and inhibited its insulin-stimulated tyrosine phosphorylation, suggesting that PPYIRS-1 contains the elements by which anisomycin inhibits IRS-1 tyrosine phosphorylation (Fig. 3). Furthermore, TNFα inhibits the insulin-stimulated tyrosine phosphorylation of PPYIRS-1, but not the phosphorylation of an IRS-1 molecule deleted for the PTB domain, in 32D cells (data not shown).Figure 3PPYIRS-1 contains the elements necessary for the inhibition of insulin-stimulated tyrosine phosphorylation by anisomycin. Insulin-stimulated tyrosine phosphorylation of PPYIRS-1 is inhibited by anisomycin. CHOIR/PPYIRS-1 cells were treated for 30 min with (lanes b and d) or without (lanes a and c) 5.0 μg/ml anisomycin prior to stimulation with (lanes c andd) or without (lanes a andb) insulin. Lysates were analyzed by immunoblotting with antibodies against phosphotyrosine (αPY) and IRS-1.View Large Image Figure ViewerDownload Hi-res image Download (PPT) In several cases, protein phosphorylation by various MAP kinases is dependent on allosteric binding of the kinase to its substrate or an associated scaffold protein (55.Pawson T. Scott J.D. Science. 1997; 278: 2075-2080Crossref PubMed Scopus (1904) Google Scholar). Similarities between the binding motifs of several JNK substrates reveals a putative consensus binding motif defined by the sequence (R/K)XXXXLXL (Fig.4 A) (44.Dickens M. Rogers J.S. Cavanagh J. Raitano A. Xia Z. Halpern J.R. Greenberg M.E. Sawyers C.L. Davis R.J. Science. 1997; 277: 693-696Crossref PubMed Scopus (629) Google Scholar, 56.Yang S.H. Whitmarsh A.J. Davis R.J. Sharrocks A.D. EMBO J. 1998; 17: 1740-1749Crossref PubMed Scopus (268) Google Scholar). Interestingly, the IRS-1 primary sequence contains 14 similar motifs, 11 of which are found in the COOH terminus, including two likely sites beginning at Arg852 and Arg780 (Fig. 4 A). Since PPYIRS-1 retains these putative JNK-binding domains, PPYIRS-1 was immunoprecipitated from quiescent CHOIR/PPYIRS-1 cells and analyzed by immunoblotting with antibodies against JNK1. JNK1 was detected in PPYIRS-1 immunoprecipitates but not in preimmune complexes, suggesting that JNK1 associated with PPYIRS-1 in vivo (Fig. 4 B). By contrast, JNK1 did not associate with PPIRS-1, a truncated IRS-1 molecule consisting of only the pleckstrin homology and PTB domains (Fig. 4 B). Immunoblots with p38-specific antibodies revealed that PPYIRS-1 and PPIRS-1 did not associate with p38. Furthermore, an inhibitor of p38 activity (SB 230580 (58.Enslen H. Raingeaud J. Davis R.J. J. Biol. Chem. 1998; 273: 1741-1748Abstract Full Text Full Text PDF PubMed Scopus (476) Google Scholar)) did not block the inhibitory effect of anisomycin on insulin-stimulated tyrosine phosphorylation of IRS-1 (data not shown). Thus, JNK but not p38, might bind to an (R/K)XXXXLXL motif between amino acids residues 555 and 898 of IRS-1 and mediate serine phosphorylation. The location of the preferred JNK binding region in IRS-1 was further verified with GST fusion proteins containing various regions of IRS-1, including residues 1–309, 140–581, 555–898, or 1064–1235; the JNK-binding domain in JNK-interacting protein (JIP)-1 was used as a positive control in these experiments, since it was shown previously to strongly bind JNK (48.Yasuda J. Whitmarsh A.J. Cavanagh J. Sharma M. Davis R.J. Mol. Cell. Biol. 1999; 19: 7245-7254Crossref PubMed Scopus (409) Google Scholar). HEK 293 cell lysates were incubated with these immobilized GST fusion proteins, and the binding of JNK1 to these proteins was detected by specific immunoblotting with antibodies against JNK1. Consistent with previous reports, JNK1 strongly bound to the JIP fragment; however, JNK also associated specifically with residues 555–898 of IRS-1, whereas it failed to bind to GST or GST fusion proteins containing other regions of IRS-1 (Fig. 4 C). The binding of JNK1 was blocked by a synthetic peptide that corresponds to the JNK-binding domain of JIP-1 (Fig. 4 D). The activity of JNK associated with IRS-1 was determined in immunocomplexes prepared from cell lysates before and after anisomycin treatment (Fig. 4 E). The assays were conducted in the presence of the PI 3-kinase inhibitor LY294002 to eliminate background activities due to PI 3-kinase-dependent kinases (14.Li J. De Fea K. Roth R.A. J. Biol. Chem. 1999; 274: 9351-9356Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar, 59.Vlahos C.J. Matter W.F. Hui K.Y. Brown R.F. J. Biochem. 1994; 269: 5241-5248Google Scholar). During incubation of the αIRS-1 immunocomplexes with [32P]ATP, serine phosphorylation of IRS-1 was significantly greater in samples from anisomycin-treated cells, suggesting that JNK might be involved in IRS-1 phospho" @default.
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