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• W2017584173 abstract "Serine phosphorylation of insulin receptor substrate-1 (IRS-1) reduces its ability to act as an insulin receptor substrate and inhibits insulin receptor signal transduction. Here, we report that serine phosphorylation of IRS-1 induced by either okadaic acid (OA) or chronic insulin stimulation prevents interferon-α (IFN-α)-dependent IRS-1 tyrosine phosphorylation and IFN-α-dependent IRS-1/phosphatidylinositol 3′-kinase (PI3K) association. In addition, we demonstrate that serine phosphorylation of IRS-1 renders it a poorer substrate for JAK1 (Janus kinase-1). We found that treatment of U266 cells with OA induced serine phosphorylation of IRS-1 and completely blocked IFN-α-dependent tyrosine phosphorylation of IRS-1 and IFN-α-dependent IRS-1/PI3K association. Additionally, IRS-1 from OA-treated cells could not be phosphorylated in vitro by IFN-α-activated JAK1. Chronic treatment of U266 cells with insulin led to a 50% reduction in IFN-α-dependent tyrosine phosphorylation of IRS-1 and IRS-1/PI3K association. More importantly, serine-phosphorylated IRS-1-(511–722) could not be phosphorylated in vitro by IFN-α-activated JAK1. Taken together, these data indicate that serine phosphorylation of IRS-1 prevents its subsequent tyrosine phosphorylation by JAK1 and suggest that IRS-1 serine phosphorylation may play a counter-regulatory role in pathways outside the insulin signaling system. Serine phosphorylation of insulin receptor substrate-1 (IRS-1) reduces its ability to act as an insulin receptor substrate and inhibits insulin receptor signal transduction. Here, we report that serine phosphorylation of IRS-1 induced by either okadaic acid (OA) or chronic insulin stimulation prevents interferon-α (IFN-α)-dependent IRS-1 tyrosine phosphorylation and IFN-α-dependent IRS-1/phosphatidylinositol 3′-kinase (PI3K) association. In addition, we demonstrate that serine phosphorylation of IRS-1 renders it a poorer substrate for JAK1 (Janus kinase-1). We found that treatment of U266 cells with OA induced serine phosphorylation of IRS-1 and completely blocked IFN-α-dependent tyrosine phosphorylation of IRS-1 and IFN-α-dependent IRS-1/PI3K association. Additionally, IRS-1 from OA-treated cells could not be phosphorylated in vitro by IFN-α-activated JAK1. Chronic treatment of U266 cells with insulin led to a 50% reduction in IFN-α-dependent tyrosine phosphorylation of IRS-1 and IRS-1/PI3K association. More importantly, serine-phosphorylated IRS-1-(511–722) could not be phosphorylated in vitro by IFN-α-activated JAK1. Taken together, these data indicate that serine phosphorylation of IRS-1 prevents its subsequent tyrosine phosphorylation by JAK1 and suggest that IRS-1 serine phosphorylation may play a counter-regulatory role in pathways outside the insulin signaling system. Tyrosine phosphorylation of IRS-1 1The abbreviations used are:IRSinsulin receptor substrateILinterleukinIFNinterferonPI3Kphosphatidylinositol 3′-kinaseOAokadaic acidPAS kinasePI3K-associated serine kinasePAGEpolyacrylamide gel electrophoresisGSTglutathione S-transferase is common to the signal transduction pathways of a variety of growth factors, hormones, and cytokines (1Yenush L. White M.F. Bioessays. 1997; 19: 491-500Crossref PubMed Scopus (257) Google Scholar). Since the first description of IRS-1 tyrosine phosphorylation in insulin-stimulated Fao hepatoma cells (2White M.F. Maron R. Kahn C.R. Nature. 1985; 318: 183-186Crossref PubMed Scopus (451) Google Scholar), IRS-1 phosphorylation has been shown to occur after insulin-like growth factor-1, IL-2, IL-4, IL-10, IL-15, growth hormone, leukemia inhibitory factor, and oncostatin M stimulation (3Wang L.M. Keegan A.D. Paul W.E. Heidaran M.A. Gutkind J.S. Pierce J.H. EMBO J. 1992; 11: 4899-4908Crossref PubMed Scopus (159) Google Scholar, 4Wang L.M. Myers Jr., M.G. Sun X.J. Aaronson S.A. White M. Pierce J.H. Science. 1993; 261: 1591-1594Crossref PubMed Scopus (371) Google Scholar, 5Johnston J.A. Kawamura M. Kirken R.A. Chen Y.Q. Blake T.B. Shibuya K. Ortaldo J.R. McVicar D.W. O'Shea J.J. Nature. 1994; 370: 151-153Crossref PubMed Scopus (510) Google Scholar, 6Burfoot M.S. Rogers N.C. Watling D. Smith J.M. Pons S. Paonessaw G. Pellegrini S. White M.F. Kerr I.M. J. Biol. Chem. 1997; 272: 24183-24190Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). Recently, type I interferons were demonstrated to induce tyrosine phosphorylation of IRS-1 (7Uddin S. Yenush L. Sun X.J. Sweet M.E. White M.F. Platanias L.C. J. Biol. Chem. 1995; 270: 15938-15941Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar, 8Platanias L.C. Uddin S. Yetter A. Sun X.J. White M.F. J. Biol. Chem. 1996; 271: 278-282Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar, 9Uddin S. Fish E.N. Sher D. Gardziola C. Colamonici O.R. Kellum M. Pitha P.M. White M.F. Platanias L.C. Blood. 1997; 90: 2574-2582PubMed Google Scholar). This class of interferons is composed of α-, β-, and ω-subtypes, which all activate the type I IFN receptor (10Domanski P. Colamonici O.R. Cytokine Growth Factor Rev. 1996; 7: 143-151Crossref PubMed Scopus (114) Google Scholar). After IFN-α binds to the type I IFN receptor, it induces receptor oligomerization, which, in turn, activates the Janus kinase family members Tyk2 and JAK1 (11Pellegrini S. John J. Shearer M. Kerr I.M. Stark G.R. Mol. Cell. Biol. 1989; 9: 4605-4612Crossref PubMed Scopus (317) Google Scholar, 12Velazquez L. Fellous M. Stark G.R. Pellegrini S. Cell. 1992; 70: 313-322Abstract Full Text PDF PubMed Scopus (714) Google Scholar, 13Muller M. Briscoe J. Laxton C. Guschin D. Ziemiecki A. Silvennoinen O. Harpur A.G. Barbieri G. Witthuhn B.A. Schindler C. Pellefrini S. Wilks A.F. Ihle J.N. Stark G.R. Kerr I.M. Nature. 1993; 366: 129-135Crossref PubMed Scopus (646) Google Scholar, 14Colamonici O.R. Uyttendaele H. Domanski P. Yan H. Krolewski J.J. J. Biol. Chem. 1994; 269: 3518-3522Abstract Full Text PDF PubMed Google Scholar, 15Novick D. Cohen B. Rubinstein M. Cell. 1994; 77: 391-400Abstract Full Text PDF PubMed Scopus (585) Google Scholar, 16Barbieri G. Velazquez L. Scrobogna M. Fellous M. Pellegrini S. Eur. J. Biochem. 1994; 223: 427-435Crossref PubMed Scopus (56) Google Scholar, 17Domanski P. Witte M. Kellum M. Rubinstein M. Hackett R. Pitha P. Colamonici O.R. J. Biol. Chem. 1995; 270: 21606-21611Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar). Activation of JAK1 induces IFN receptor tyrosine phosphorylation and leads to subsequent JAK1-dependent tyrosine phosphorylation of STAT1, STAT2, STAT3, IRS-1, and IRS-2 (6Burfoot M.S. Rogers N.C. Watling D. Smith J.M. Pons S. Paonessaw G. Pellegrini S. White M.F. Kerr I.M. J. Biol. Chem. 1997; 272: 24183-24190Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 10Domanski P. Colamonici O.R. Cytokine Growth Factor Rev. 1996; 7: 143-151Crossref PubMed Scopus (114) Google Scholar) and the association of STAT3, IRS-1, and IRS-2 with PI3K (7Uddin S. Yenush L. Sun X.J. Sweet M.E. White M.F. Platanias L.C. J. Biol. Chem. 1995; 270: 15938-15941Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar, 8Platanias L.C. Uddin S. Yetter A. Sun X.J. White M.F. J. Biol. Chem. 1996; 271: 278-282Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar, 9Uddin S. Fish E.N. Sher D. Gardziola C. Colamonici O.R. Kellum M. Pitha P.M. White M.F. Platanias L.C. Blood. 1997; 90: 2574-2582PubMed Google Scholar, 18Pfeffer L.M. Mullersman J.E. Pfeffer S.R. Murti A. Shi W. Yang C.H. Science. 1997; 276: 1418-1420Crossref PubMed Scopus (237) Google Scholar). insulin receptor substrate interleukin interferon phosphatidylinositol 3′-kinase okadaic acid PI3K-associated serine kinase polyacrylamide gel electrophoresis glutathione S-transferase Tyrosine-phosphorylated IRS-1 coordinates the intracellular signaling of various growth factor, hormone, and cytokine receptors in part by binding to SH2 domain-containing proteins (1Yenush L. White M.F. Bioessays. 1997; 19: 491-500Crossref PubMed Scopus (257) Google Scholar). The association of PI3K with IRS-1 is the best characterized of these IRS-1/SH2 domain interactions and, in signaling pathways that require IRS-1, appears to be a critical step in effecting post-receptor function (1Yenush L. White M.F. Bioessays. 1997; 19: 491-500Crossref PubMed Scopus (257) Google Scholar, 19Kapeller R. Cantley L.C. Bioessays. 1994; 16: 565-576Crossref PubMed Scopus (553) Google Scholar, 20Backer 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; 11: 3469-3479Crossref PubMed Scopus (822) Google Scholar, 21Shoelson S.E. Sivaraja M. Williams K.P. Hu P. Schlessinger J. Weiss M.A. EMBO J. 1993; 12: 795-802Crossref PubMed Scopus (141) Google Scholar, 22Sun X.J. Crimmins D.L. Myers Jr., M.G. Miralpeix M. White M.F. Mol. Cell. Biol. 1993; 13: 7418-7428Crossref PubMed Google Scholar). PI3K is a heterodimeric protein composed of a regulatory 85-kDa subunit (p85) and a catalytic 110-kDa subunit (p110) (19Kapeller R. Cantley L.C. Bioessays. 1994; 16: 565-576Crossref PubMed Scopus (553) Google Scholar). p85 contains N- and C-terminal SH2 domains that bind to tyrosine-phosphorylated IRS-1 and induce PI3K activation (20Backer 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; 11: 3469-3479Crossref PubMed Scopus (822) Google Scholar, 21Shoelson S.E. Sivaraja M. Williams K.P. Hu P. Schlessinger J. Weiss M.A. EMBO J. 1993; 12: 795-802Crossref PubMed Scopus (141) Google Scholar, 22Sun X.J. Crimmins D.L. Myers Jr., M.G. Miralpeix M. White M.F. Mol. Cell. Biol. 1993; 13: 7418-7428Crossref PubMed Google Scholar). p110 phosphorylates phosphoinositides on the D-3 position of the inositol ring, generating 3,4-bis- and 3,4,5-trisphosphates (23Cantley L.C. Auger K.R. Carpenter C. Duckworth B. Graziani A. Kapeller R. Soltoff S. Cell. 1991; 64: 281-302Abstract Full Text PDF PubMed Scopus (2187) Google Scholar, 24Hiles I.D. Otsu M. Volinia S. Fry M.J. Gout I. Dhand R. Panayotou G. Ruiz-Larrea F. Thompson A. Totty N.F. Hsuan J.J. Courtneidge S.A. Parker P.J. Waterfield M.D. Cell. 1992; 70: 419-429Abstract Full Text PDF PubMed Scopus (541) Google Scholar). In the IFN-α signaling cascade, PI3K is recruited to and activated by tyrosine-phosphorylated IRS-1 (7Uddin S. Yenush L. Sun X.J. Sweet M.E. White M.F. Platanias L.C. J. Biol. Chem. 1995; 270: 15938-15941Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar, 8Platanias L.C. Uddin S. Yetter A. Sun X.J. White M.F. J. Biol. Chem. 1996; 271: 278-282Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar, 9Uddin S. Fish E.N. Sher D. Gardziola C. Colamonici O.R. Kellum M. Pitha P.M. White M.F. Platanias L.C. Blood. 1997; 90: 2574-2582PubMed Google Scholar,18Pfeffer L.M. Mullersman J.E. Pfeffer S.R. Murti A. Shi W. Yang C.H. Science. 1997; 276: 1418-1420Crossref PubMed Scopus (237) Google Scholar). Inhibition of PI3K activity with wortmannin leads to decreased IFN-α-dependent STAT3 serine phosphorylation and decreased IFN-α-dependent transcriptional activation (18Pfeffer L.M. Mullersman J.E. Pfeffer S.R. Murti A. Shi W. Yang C.H. Science. 1997; 276: 1418-1420Crossref PubMed Scopus (237) Google Scholar). Growth factors, hormones, and cytokines can also induce serine phosphorylation of IRS-1 (3Wang L.M. Keegan A.D. Paul W.E. Heidaran M.A. Gutkind J.S. Pierce J.H. EMBO J. 1992; 11: 4899-4908Crossref PubMed Scopus (159) Google Scholar, 4Wang L.M. Myers Jr., M.G. Sun X.J. Aaronson S.A. White M. Pierce J.H. Science. 1993; 261: 1591-1594Crossref PubMed Scopus (371) Google Scholar, 25Uddin S. Fish E.N. Sher D.A. Gardziola C. White M.F. Platanias L.C. J. Immunol. 1997; 158: 2390-2397PubMed Google Scholar, 26Tanti J.F. Gremeaux T. van Obberghen E. Le Marchand-Brustel Y. J. Biol. Chem. 1994; 269: 6051-6057Abstract Full Text PDF PubMed Google Scholar, 27Kanety 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, 28Hotamisligil G.S. Peraldi P. Budavari A. Ellis R. White M.F. Spiegelman B.M. Science. 1996; 271: 665-668Crossref PubMed Scopus (2223) Google Scholar), and in the insulin signaling system, serine phosphorylation of IRS-1 blocks insulin action (26Tanti J.F. Gremeaux T. van Obberghen E. Le Marchand-Brustel Y. J. Biol. Chem. 1994; 269: 6051-6057Abstract Full Text PDF PubMed Google Scholar, 27Kanety 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, 28Hotamisligil G.S. Peraldi P. Budavari A. Ellis R. White M.F. Spiegelman B.M. Science. 1996; 271: 665-668Crossref PubMed Scopus (2223) Google Scholar, 29De Fea K. Roth R.A. Biochemistry. 1997; 36: 12939-12947Crossref PubMed Scopus (223) Google Scholar, 30De Fea K. Roth R.A. J. Biol. Chem. 1997; 272: 31400-31406Abstract Full Text Full Text PDF PubMed Scopus (267) Google Scholar, 31Ricort J.M. Tanti J.F. van Obberghen E. Le Marchand-Brustel Y. J. Biol. Chem. 1997; 272: 19814-19818Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 32Folli F. Kahn C.R. Hansen H. Bouchie J.L. Feener E.P. J. Clin. Invest. 1997; 100: 2158-2169Crossref PubMed Scopus (405) Google Scholar, 33Paz K. Hemi R. LeRoith D. Karasik A. Elhanany E. Kanety H. Zick Y. J. Biol. Chem. 1997; 272: 29911-29918Abstract Full Text Full Text PDF PubMed Scopus (452) Google Scholar, 34Cengel K.A. Kason R.E. Freund G.G. Biochem. J. 1998; 335: 397-404Crossref PubMed Scopus (11) Google Scholar, 35Sun X. Qiao L. Goldberg J. Diabetes. 1998; 47 (abstr.): A35Google Scholar). Treatment of 3T3-L1 adipocytes with the serine phosphatase inhibitor okadaic acid (OA) results in increased IRS-1 serine phosphorylation, reduced IRS-1/insulin receptor association, and decreased insulin receptor-dependent tyrosine phosphorylation of IRS-1 (26Tanti J.F. Gremeaux T. van Obberghen E. Le Marchand-Brustel Y. J. Biol. Chem. 1994; 269: 6051-6057Abstract Full Text PDF PubMed Google Scholar). Similarly, IRS-1 serine phosphorylation induced by phorbol esters, tumor necrosis factor-α, platelet-derived growth factor, angiotensin II, PI3Kassociated serine kinase (PAS kinase), and insulin reduces subsequent insulin receptor-dependent IRS-1 tyrosine phosphorylation and IRS-1/PI3K association (27Kanety 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, 28Hotamisligil G.S. Peraldi P. Budavari A. Ellis R. White M.F. Spiegelman B.M. Science. 1996; 271: 665-668Crossref PubMed Scopus (2223) Google Scholar, 29De Fea K. Roth R.A. Biochemistry. 1997; 36: 12939-12947Crossref PubMed Scopus (223) Google Scholar, 30De Fea K. Roth R.A. J. Biol. Chem. 1997; 272: 31400-31406Abstract Full Text Full Text PDF PubMed Scopus (267) Google Scholar, 31Ricort J.M. Tanti J.F. van Obberghen E. Le Marchand-Brustel Y. J. Biol. Chem. 1997; 272: 19814-19818Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 32Folli F. Kahn C.R. Hansen H. Bouchie J.L. Feener E.P. J. Clin. Invest. 1997; 100: 2158-2169Crossref PubMed Scopus (405) Google Scholar, 33Paz K. Hemi R. LeRoith D. Karasik A. Elhanany E. Kanety H. Zick Y. J. Biol. Chem. 1997; 272: 29911-29918Abstract Full Text Full Text PDF PubMed Scopus (452) Google Scholar, 34Cengel K.A. Kason R.E. Freund G.G. Biochem. J. 1998; 335: 397-404Crossref PubMed Scopus (11) Google Scholar, 35Sun X. Qiao L. Goldberg J. Diabetes. 1998; 47 (abstr.): A35Google Scholar). Although much is known about the effect of IRS-1 serine phosphorylation on insulin signaling, nothing is known about the impact of IRS-1 serine phosphorylation on cytokine signaling. Here, we report that serine phosphorylation of IRS-1 induced by either OA or chronic insulin stimulation inhibits IFN-α-dependent tyrosine phosphorylation of IRS-1 by JAK1 and blocks subsequent IRS-1/PI3K association. These findings indicate that IRS-1 serine phosphorylation may play a counter-regulatory role in signaling pathways outside the insulin system and suggest that hyperinsulinemia may alter signaling of JAK1- dependent cytokine receptors. The myeloma cell line U266 was purchased from American Type Culture Collection (Manassas, VA). [γ-32P]ATP and 32Pi were purchased from Amersham Pharmacia Biotech. Anti-PI3K p85 (catalog no. 06-195), anti-phosphotyrosine (catalog no. 05-321), and anti-IRS-1 (catalog no. 06-248C) antisera were purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). Anti-JAK1 antiserum (catalog no. 15206E) was purchased from Pharmingen (San Diego, CA). Neonatal bovine serum was purchased from Biocell (Rancho Dominguez, CA). Protein G-Sepharose, glutathione-Sepharose, Precission protease, and pGex6P3 vector were purchased from Amersham Pharmacia Biotech (Uppsala, Sweden). Recombinant human IFN-α-1 was purchased from Intergen Co. (Purchase, NY). Polyvinylidene difluoride membrane was purchased from Bio-Rad. Cellulose-coated TLC plates were purchased from Analtech Inc. (Newark, DE). Minifilter columns (catalog no. QSQ) were purchased from Midwest Scientific (Valley Park, MO). All other cell culture reagents and chemicals were purchased from Sigma. Oligonucleotide primers were purchased from Operon Technologies, Inc. (Alameda, CA). All other molecular biology reagents and chemicals were purchased from Promega. U266 cells were grown in growth medium (RPMI 1640 medium supplemented with 10% neonatal bovine serum, 2.0 g/liter glucose, 100,000 units/liter penicillin, and 100 mg/liter streptomycin). Cells were passaged 1:1 with fresh medium every 3 days. For OA treatment, cells were washed twice and resuspended in growth medium supplemented with 1 μm OA. For insulin treatment, cells were washed twice and resuspended in growth medium supplemented with 1 nm insulin. PI3K assays were performed as described previously (36Freund G.G. Kulas D.T. Way B.A. Mooney R.A. Cancer Res. 1994; 54: 3179-3185PubMed Google Scholar). In brief, 20 × 106 cells/ml were treated as indicated and lysed in 1 ml of ice-cold lysis buffer (1% Nonidet P-40, 100 mm NaCl, 50 mm NaF, 1 mm dithiothreitol, 25 mm benzamidine, 1 mm phenylmethylsulfonyl fluoride, 2 μg/ml aprotinin, 2 μg/ml leupeptin, 2 mm sodium orthovanadate, 250 nm okadaic acid, and 50 mm Tris, pH 7.4). IRS-1 was immunoprecipitated from lysates with 4 μl of anti-IRS-1 antiserum/test, and the resultant immune complexes were washed extensively. Kinase reactions were performed in 100 μl of buffer containing 0.33 mg/ml l-α-phosphatidylinositol, 7.5 mm MgCl2, 0.4 mm EGTA, 0.4 mm NaPO4, 7.5 μm[γ-32P]ATP (13 μCi/nmol), and 20 mmHEPES, pH 7.1, at 22 °C for 15 min. The assay conditions used were linear with respect to time and amount of kinase. Phospholipids were extracted with 1:1 chloroform/methanol and resolved on silica gel plates by TLC in chloroform/methanol/4 m ammonium hydroxide (75:58:17). Results were analyzed by autoradiography on a Molecular Dynamics PhosphorImager system. Western analysis was performed as described previously (37Cengel K.A. Godbout J.G. Freund G.G. Biochem. Biophys. Res. Commun. 1998; 242: 513-517Crossref PubMed Scopus (6) Google Scholar). In brief, 20 × 106cells/ml were treated as indicated and lysed in 1 ml of ice-cold lysis buffer. Proteins of interest were immunoprecipitated with the indicated antiserum (4 μl/test), and the resultant immune complexes were washed extensively. Proteins were resolved by SDS-PAGE under reducing conditions on 10% gels, electrotransferred to polyvinylidene difluoride membrane, and probed with the indicated antiserum. Immunoreactive proteins were visualized by secondary detection using an125I-labeled goat anti-rabbit antibody, followed by autoradiography and densitometry. Cells (20 × 106/ml) were treated as indicated and lysed in 1 ml of ice-cold lysis buffer. JAK1 was immunoprecipitated from lysates with 4 μl of anti-JAK1 antiserum/test, and the resultant immune complexes were washed extensively. Kinase reactions were performed in 100 μl of buffer containing 7.5 mm MgCl2, 2.5 mmMnCl2, 20 μm [γ-32P]ATP (10 μCi/nmol), and 20 mm HEPES, pH 7.5, at 22 °C for 20 min with 5 μg/ml IRS-1-(511–772) or eluted IRS-1 or with no substrate as indicated. In reactions using eluted IRS-1 from U266 cells, IRS-1 was immunoprecipitated from 20 × 106cells. The resultant immune complexes were eluted for 60 min at 37 °C in 10 μl of 100 mm dithiothreitol, 0.5% SDS, 1 mg/ml bovine serum, and 20 mm HEPES, pH 7.4, and used in kinase reactions at a 1:10 dilution. Kinase reactions were terminated by addition of SDS-PAGE loading buffer, and the assay conditions used were linear with respect to time and amount of kinase. Resultant phosphoproteins were resolved by SDS-PAGE under reducing conditions on 7–20% gradient gels. Serine and threonine phosphoamino acids were base-hydrolyzed (39Dulcos B. Marcandier S. Cozzone A.J. Methods Enzymol. 1991; 201: 10-21Crossref PubMed Scopus (210) Google Scholar), and phosphotyrosine-containing proteins were examined by autoradiography and densitometry. The coding sequence for amino acids 511–772 of IRS-1 was amplified from rat IRS-1 sequence (a kind gift of Morris F. White) by previously described methods (34Cengel K.A. Kason R.E. Freund G.G. Biochem. J. 1998; 335: 397-404Crossref PubMed Scopus (11) Google Scholar) using the forward primer 5′-CAGGATCCGATCTGGATAACCGGTTTC-3′ and the reverse primer 5′-GAGAATTCGCGCTGGGTGTGCTAAAAG-3′. This 799-base pair product was introduced into the pGex6P3 plasmid using the BamHI andEcoRI restriction sites. GST-IRS-1-(511–772) was produced as described previously (38Smith D.B. Johnson K.S. Gene ( Amst. ). 1988; 67: 31-40Crossref PubMed Scopus (5047) Google Scholar). In brief, protein expression was induced in Escherichia coli strain BL21 by addition of isopropyl-β-d-thiogalactopyranoside to a final concentration of 1 mm. After 1 h, bacteria were lysed by mild sonication at 4 °C in phosphate-buffered saline (140 mm NaCl, 2.7 mm KCl, 10 mmNa2HPO4, and 1.8 mmKH2PO4, pH 7.4) supplemented with 1% Triton X-100, 1 mm phenylmethylsulfonyl fluoride, and 10 mm dithiothreitol. GST fusion proteins were affinity-purified from clarified lysates using glutathione-Sepharose, and GST was removed by digestion with 5 units of Precission protease at 4 °C. Whole cell phosphorylation was performed as described previously (37Cengel K.A. Godbout J.G. Freund G.G. Biochem. Biophys. Res. Commun. 1998; 242: 513-517Crossref PubMed Scopus (6) Google Scholar). In brief, 20 × 106 cells were suspended in 1 ml of phosphate-free RPMI 1640 medium supplemented with 0.75 mCi/ml32Pi and 20 mm HEPES, pH 7.4, at 37 °C for 1.5 h. For OA treatment, 1 μm OA was added at 1.5 h for 30 min. Cells were lysed, and IRS-1 was immunoprecipitated as described above. Phosphoproteins were resolved by SDS-PAGE under reducing conditions on 8% gels. For phosphoamino acid analysis, phosphoproteins were electrotransferred to polyvinylidene difluoride membrane. Bands of interest were excised and acid-hydrolyzed in 6 n constant boiling HCl for 2 h at 110 °C. Samples were treated with three cycles of water resuspension and evaporation and then resuspended in H2O/acetic acid/pyridine (89:10:1) running buffer containing 0.3 mg/ml phosphoserine, phosphothreonine, and phosphotyrosine standards. Phosphoamino acids were separated on cellulose-coated plates by high voltage TLC, and standards were visualized with ninhydrin. Results were analyzed by autoradiography and densitometry. PAS kinase assays were performed as described previously (37Cengel K.A. Godbout J.G. Freund G.G. Biochem. Biophys. Res. Commun. 1998; 242: 513-517Crossref PubMed Scopus (6) Google Scholar). In brief, PAS kinase was purified from 20 × 106 cells by affinity chromatography using glutathione-Sepharose-bound GST-p85 protein. Kinase reactions were performed in 100 μl of reaction buffer containing 5 μg/ml IRS-1-(511–772), 0.4 mm EGTA, 0.4 mmNaPO4, 1 μm [γ-32P]ATP (100 μCi/nmol), and 20 mm HEPES, pH 7.1, at 22 °C with or without 10 mm MgCl2 as a cofactor. For JAK1 kinase assays, IRS-1-(511–772) was prephosphorylated in the absence of [γ-32P]ATP and recovered by filtration through minifilter columns. IFN-α-activated JAK1 was isolated as described above, and JAK1 kinase assays using prephosphorylated IRS-1-(511–772) as a substrate were performed as described above. Reactions were terminated using SDS-PAGE loading buffer and were linear with respect to time and amount of kinase. Phosphoproteins were resolved by SDS-PAGE under reducing conditions on 7–20% gradient gels and examined by autoradiography and densitometry. Cells (10 × 106/10 ml) were treated with 1 nm insulin for 18 h and then pelleted at 500 × g for 5 min. The cell pellet was lysed in 100 μl of ice-cold 1 mm phenylmethylsulfonyl fluoride and 50 mm Tris, pH 7.4, by 10 passages through a 25-gauge needle. Lysates were centrifuged at 16,000 ×g for 10 min, and the supernatant fraction was adjusted to 0.4 mm EGTA, 0.4 mm NaPO4, 0.9 mm phenylmethylsulfonyl fluoride, 1 μm[γ-32P]ATP (100 μCi/nmol), 45 mm Tris, and 20 mm HEPES, pH 7.1, at 22 °C with or without 10 mm MgCl2 as a cofactor. This kinase mixture was then added to affinity-purified GST-IRS-1-(511–772) (bound to glutathione-Sepharose), and the reaction was allowed to proceed for 15 min at 22 °C. Reactions were terminated by addition of phosphate-buffered saline with 1 mm EDTA and were linear with respect to time and amount of kinase. Phosphorylated IRS-1-(511–772) was then removed from the solid phase with Precision protease as described above. For JAK1 kinase assays, IRS-1-(511–772) was prephosphorylated in the absence of [γ-32P]ATP and then used in JAK1 kinase assays as described above at a concentration of 5 μg/ml. Serine phosphorylation of IRS-1 induced by OA treatment of 3T3-L1 adipocytes stops insulin-dependent activation of PI3K (26Tanti J.F. Gremeaux T. van Obberghen E. Le Marchand-Brustel Y. J. Biol. Chem. 1994; 269: 6051-6057Abstract Full Text PDF PubMed Google Scholar). To determine if IFN-α-mediated PI3K activation was prevented by OA, IRS-1-associated PI3K activity was examined in U266 cells pretreated with 1 μm OA for 30 min. Fig. 1 A demonstrates that 1000 units/ml IFN-α induced 20-, 22-, and 10-fold increases in IRS-1-associated PI3K activity at 5, 10, and 30 min, respectively, and that pretreatment of cells with OA blocked this response. OA did not inhibit PI3K activity directly because PI3K activity in PI3K p85 immune complexes from OA-treated cells was no different than that from non-OA-treated cells (data not shown). To determine if this failure to activate PI3K was due to a loss of IFN-α-dependent IRS-1/PI3K association, Western analysis was performed. Fig.1 B shows that IFN-α-dependent IRS-1/PI3K association was increased at 5, 10, and 30 min (as measured by Western detection of PI3K p85) and that OA inhibited this association. To determine if this OA-dependent decline in IRS-1/PI3K association was due to an inhibition of IFN-α-dependent tyrosine phosphorylation of IRS-1, Western analysis was again performed. Fig. 1 C shows that IFN-α increased IRS-1 tyrosine phosphorylation at 5, 10, and 30 min and that OA blocked detectable tyrosine phosphorylation of IRS-1. To confirm that OA did not measurably alter IRS-1, p85, and JAK1 protein levels and the ability of these proteins to be immunoprecipitated by their respective antibodies, Western analysis was performed. Fig. 1 Ddemonstrates that IRS-1, p85, and JAK1 protein levels and their ability to be immunoprecipitated were unaffected by OA. Finally, to show that OA did not affect JAK1 autophosphorylation or its ability to phosphorylate in vitro substrates, JAK1 kinase assays were performed. Fig. 1 E demonstrates that JAK1 isolated from OA-treated cells phosphorylated recombinant IRS-1-(511–772) as well as JAK1 recovered from non-OA-treated cells and that JAK1 autophosphorylation was unchanged. Taken together, these findings indicate that OA blocks IFN-α-dependent IRS-1/PI3K association by a mechanism that inhibits tyrosine phosphorylation of IRS-1, but does not alter JAK1 kinase activity. Serine phosphorylation of IRS-1 blocks insulin receptor-dependent tyrosine phosphorylation of IRS-1 (26Tanti J.F. Gremeaux T. van Obberghen E. Le Marchand-Brustel Y. J. Biol. Chem. 1994; 269: 6051-6057Abstract Full Text PDF PubMed Google Scholar, 27Kanety 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, 28Hotamisligil G.S. Peraldi P. Budavari A. Ellis R. White M.F. Spiegelman B.M. Science. 1996; 271: 665-668Crossref PubMed Scopus (2223) Google Scholar, 29De Fea K. Roth R.A. Biochemistry. 1997; 36: 12939-12947Crossref PubMed Scopus (223) Google Scholar, 30De Fea K. Roth R.A. J. Biol. Chem. 1997; 272: 31400-31406Abstract Full Text Full Text PDF PubMed Scopus (267) Google Scholar, 31Ricort J.M. Tanti J.F. van Obberghen E. Le Marchand-Brustel Y. J. Biol. Chem. 1997; 272: 19814-19818Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 32Folli F. Kahn C.R. Hansen H. Bouchie J.L. Feener E.P. J. Clin. Invest. 1997; 100: 2158-2169Crossref PubMed Scopus (405) Google Scholar, 33Paz K. Hemi R. LeRoith D. Karasik A. Elhanany E. Kanety H. Zick Y. J. Biol. Chem. 1997; 272: 29911-29918Abstract Full Text Full Text PDF PubMed Scopus (452) Google Scholar, 34Cengel K.A. Kason R.E. Freund G.G. Biochem. J. 1998; 335: 397-404Crossref PubMed Scopus (11) Google Scholar, 35Sun X. Qiao L. Goldberg J. Diabetes. 1998; 47 (abstr.): A35Google Scholar). To determine if OA inhibited JAK1-dependent IRS-1 tyrosine phosphorylation, JAK1 kinase assays were performed. Fig.2 A demonstrates that when IRS-1 isolated from OA-treated cells was used as a substrate for JAK1, IFN-α-dependent JAK1 phosphorylation was not observed. In contrast, when IRS-1 from non-OA-treated cells was used as a substrate for JAK1, IFN-α induced a 5-fold increase in JAK1-dependent IRS-1 phosphorylation. To examine the phosphorylation state of IRS-1 isolated from OA-treated cells, phosphoamino acid analysis was performed. These experiments showed that IRS-1 was predominantly phosphorylated on serine residues and that no tyrosine phosphorylation was detected (Fig. 2 B). Taken together, these findings indicate that serine phosphorylation of IRS-1 induced by OA renders IRS-1 a poorer substrate for JAK1. Chronic hyperinsulinemia induces serine phosphorylation of IRS-1 and reduces insulin signaling (33Paz K. Hemi R. LeRoith D. Karasik A. Elhanany E. Kanety H. Zick Y. J. Biol. Chem. 1997; 272: 29911-29918Abstract Full Text Full Text PDF PubMed Scopus (452) Google Scholar, 35Sun X. Qiao L. Goldberg J. Diabetes. 1998; 47 (abstr.): A35Google Scholar, 40Mayor P. Maianu L. Garvey W.T. Diabetes. 1992; 41: 274-285Crossref PubMed Google Scholar, 41Saad M.J. Folli F. Kahn C.R. Endocrinology. 1995; 136: 1579-1588Crossref PubMed Google Scholar, 42Heller-Harrison R.A. Morin M. Czech M.P. J. Biol. Chem. 1995; 270: 24442-24450Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). To determine if chronic insulin treatment inhibited IFN-α-dependent activation of PI3K, IRS-1-associated PI3K activity was examined in U266 cells pretreated with 1 nminsulin for 18 h. Fig. 3 Ademonstrates that 1000 units/ml IFN-α induced a 10-fold increase in IRS-1-associated PI3K activity at 5 min and that pretreatment of cells with insulin reduced this response by 50%. Chronic insulin treatment did not inhibit PI3K activity directly because PI3K activity in PI3K p85 immune complexes from insulin-treated cells was no different than that from non-insulin-treated cells (data not shown). To determine if this reduction in PI3K activation was due to a loss of IFN-α-dependent IRS-1/PI3K association, Western analysis was performed. Fig. 3 B shows that IFN-α increased IRS-1/PI3K association at 5 min (as measured by detection of PI3K p85) and that chronic insulin treatment reduced this association by 50%. To further examine the impact of chronic insulin treatment on IRS-1/PI3K association, IRS-1 present in PI3K immune complexes was examined by Western analysis (Fig. 3 C). As in Fig. 3 B, chronic insulin treatment reduced IFN-α-dependent IRS-1/PI3K association, and comparable low amounts of IRS-1 were associated with PI3K before and after chronic insulin treatment in cells not treated with IFN-α. To determine if this insulin-dependent decline in IRS-1/PI3K association after IFN-α treatment was due to an inhibition of IFN-α-dependent tyrosine phosphorylation of IRS-1, Western analysis was again performed. Fig. 3 D shows that IFN-α increased IRS-1 tyrosine phosphorylation at 5 min and that chronic insulin treatment reduced IFN-α-dependent tyrosine phosphorylation of IRS-1 by 50%. Additionally, chronic insulin treatment did not alter JAK1 activity in that IFN-α-activated JAK1 isolated from chronically insulin-treated cells phosphorylated recombinant IRS-1-(511–772) as well as JAK1 recovered from non-insulin-treated cells (data not shown). Finally, to confirm that chronic insulin treatment did not measurably alter IRS-1, p85, and JAK1 protein levels and the ability of these proteins to be immunoprecipitated by their respective antibodies, Western analysis was performed. Fig. 3 E demonstrates that IRS-1, p85, and JAK1 protein levels and the ability to be immunoprecipitated were unaffected by chronic insulin treatment. Taken together, these findings indicate that chronic insulin treatment inhibits IFN-α-dependent IRS-1/PI3K association by a mechanism that reduces tyrosine phosphorylation of IRS-1, but does not alter JAK1 kinase activity. Phosphorylation of IRS-1 by serine kinases renders it a poorer substrate for the insulin receptor (27Kanety 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, 28Hotamisligil G.S. Peraldi P. Budavari A. Ellis R. White M.F. Spiegelman B.M. Science. 1996; 271: 665-668Crossref PubMed Scopus (2223) Google Scholar, 29De Fea K. Roth R.A. Biochemistry. 1997; 36: 12939-12947Crossref PubMed Scopus (223) Google Scholar, 30De Fea K. Roth R.A. J. Biol. Chem. 1997; 272: 31400-31406Abstract Full Text Full Text PDF PubMed Scopus (267) Google Scholar, 31Ricort J.M. Tanti J.F. van Obberghen E. Le Marchand-Brustel Y. J. Biol. Chem. 1997; 272: 19814-19818Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 32Folli F. Kahn C.R. Hansen H. Bouchie J.L. Feener E.P. J. Clin. Invest. 1997; 100: 2158-2169Crossref PubMed Scopus (405) Google Scholar, 33Paz K. Hemi R. LeRoith D. Karasik A. Elhanany E. Kanety H. Zick Y. J. Biol. Chem. 1997; 272: 29911-29918Abstract Full Text Full Text PDF PubMed Scopus (452) Google Scholar, 34Cengel K.A. Kason R.E. Freund G.G. Biochem. J. 1998; 335: 397-404Crossref PubMed Scopus (11) Google Scholar, 35Sun X. Qiao L. Goldberg J. Diabetes. 1998; 47 (abstr.): A35Google Scholar). To determine if serine phosphorylation of IRS-1 inhibits its ability to act as a JAK1 substrate, kinase assays were performed. Fig.4 A shows that plasma membrane-depleted lysates from U266 cells treated with 1 nminsulin for 18 h contained kinase activity that phosphorylated IRS-1-(511–772) exclusively on serine residues. Fig. 4 Bdemonstrates that phosphorylation of IRS-1-(511–772) by plasma membrane-depleted serine kinase activity reduced by 50% the ability of IRS-1-(511–772) to serve as a substrate for IFN-α-activated JAK1. Fig. 4 C demonstrates that IRS-1-(511–772) was a substrate for the serine kinase PAS kinase (34Cengel K.A. Kason R.E. Freund G.G. Biochem. J. 1998; 335: 397-404Crossref PubMed Scopus (11) Google Scholar) and that phosphorylation of IRS-1-(511–772) by PAS kinase reduced by 75% the ability of IRS-1-(511–772) to serve as a substrate for IFN-α-activated JAK1 (Fig. 4 D). These results indicate that serine phosphorylation of IRS-1 inhibits its ability to act as a JAK1 substrate. These data establish that serine phosphorylation of IRS-1 renders it a poorer substrate for JAK1. Western analysis and PI3K assays demonstrated that IFN-α-dependent IRS-1 tyrosine phosphorylation and IRS-1/PI3K association and activity were blocked by OA treatment and that this was not due to an effect of OA on JAK1 (Fig.1, A–D). Isolation of IRS-1 from OA-treated cells showed that OA-dependent serine phosphorylation of IRS-1 completely inhibited the ability of IFN-α-activated JAK1 to phosphorylate IRS-1 (Fig. 2). Likewise, chronic insulin stimulation reduced by 50% the ability of IFN-α to stimulate IRS-1 tyrosine phosphorylation and IRS-1/PI3K association and activity (Fig. 3). More importantly, serine phosphorylation of IRS-1-(511–772) by serine kinases derived from chronically insulin-stimulated cells and by PAS kinase reduced by 50 and 75%, respectively, the ability of IFN-α-activated JAK1 to phosphorylate IRS-1-(511–772) (Fig. 4). Taken together, these findings indicate that serine phosphorylation of IRS-1 reduces the ability of IRS-1 to serve as a JAK1 substrate, that IRS-1 serine phosphorylation inhibits signal transduction in pathways outside the insulin system, and that hyperinsulinemia may alter signaling of JAK1-dependent cytokine receptors. Inhibition of PP1 and PP2A serine phosphatases by OA and calyculin A increase IRS-1 serine phosphorylation and leads to decreased insulin receptor-mediated IRS-1 tyrosine phosphorylation (26Tanti J.F. Gremeaux T. van Obberghen E. Le Marchand-Brustel Y. J. Biol. Chem. 1994; 269: 6051-6057Abstract Full Text PDF PubMed Google Scholar, 33Paz K. Hemi R. LeRoith D. Karasik A. Elhanany E. Kanety H. Zick Y. J. Biol. Chem. 1997; 272: 29911-29918Abstract Full Text Full Text PDF PubMed Scopus (452) Google Scholar). Chronic insulin treatment has also been shown to induce serine phosphorylation of IRS-1 and to inhibit insulin receptor-dependent phosphorylation of IRS-1 (33Paz K. Hemi R. LeRoith D. Karasik A. Elhanany E. Kanety H. Zick Y. J. Biol. Chem. 1997; 272: 29911-29918Abstract Full Text Full Text PDF PubMed Scopus (452) Google Scholar, 35Sun X. Qiao L. Goldberg J. Diabetes. 1998; 47 (abstr.): A35Google Scholar, 40Mayor P. Maianu L. Garvey W.T. Diabetes. 1992; 41: 274-285Crossref PubMed Google Scholar, 41Saad M.J. Folli F. Kahn C.R. Endocrinology. 1995; 136: 1579-1588Crossref PubMed Google Scholar, 42Heller-Harrison R.A. Morin M. Czech M.P. J. Biol. Chem. 1995; 270: 24442-24450Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). Recently, the region of IRS-1 susceptible to chronic insulin treatment-dependent serine phosphorylation has been reported, and it appears to reside between amino acids 530 and 843 (35Sun X. Qiao L. Goldberg J. Diabetes. 1998; 47 (abstr.): A35Google Scholar). The kinase responsible for this phosphorylation is unknown, but appears to be insensitive to inhibitors of protein kinases C and A, PI3K, and mitogen-activated protein kinase (35Sun X. Qiao L. Goldberg J. Diabetes. 1998; 47 (abstr.): A35Google Scholar). We have identified a kinase (PAS kinase) that can serine phosphorylate IRS-1 and inhibit the ability of IRS-1 to serve as an insulin receptor substrate (34Cengel K.A. Kason R.E. Freund G.G. Biochem. J. 1998; 335: 397-404Crossref PubMed Scopus (11) Google Scholar, 37Cengel K.A. Godbout J.G. Freund G.G. Biochem. Biophys. Res. Commun. 1998; 242: 513-517Crossref PubMed Scopus (6) Google Scholar). This kinase associates with the p85 subunit of PI3K through SH2 domain interactions and phosphorylates IRS-1 in IRS-1/PI3K complexes after insulin stimulation (37Cengel K.A. Godbout J.G. Freund G.G. Biochem. Biophys. Res. Commun. 1998; 242: 513-517Crossref PubMed Scopus (6) Google Scholar). Here, we show that PAS kinase can phosphorylate IRS-1-(511–772) and that this phosphorylation inhibits the ability of IFN-α-activated JAK1 to subsequently phosphorylate IRS-1-(511–772). Although serine phosphorylation of IRS-1 decreases the ability of the insulin receptor and now JAK1 to phosphorylate IRS-1, the mechanism of this effect is not clearly delineated. In the insulin signaling system, serine phosphorylation of IRS-1 within the IH1 phosphotyrosine-binding domain appears to impair NPXY-mediated IRS-1/insulin receptor association (33Paz K. Hemi R. LeRoith D. Karasik A. Elhanany E. Kanety H. Zick Y. J. Biol. Chem. 1997; 272: 29911-29918Abstract Full Text Full Text PDF PubMed Scopus (452) Google Scholar), thus abrogating direct IRS-1/insulin receptor interaction. Like the insulin receptor, the IL-4 receptor contains an NPXY motif, and this motif appears to coordinate the formation of receptor/JAK/IRS-1 complexes, which result in IRS-1 tyrosine phosphorylation (43Keegan A.D. Nelms K. White M. Wang L.M. Pierce J.H. Paul W.E. Cell. 1994; 76: 811-820Abstract Full Text PDF PubMed Scopus (288) Google Scholar). The IFN-α receptor does not contain an NPXY motif and may rely on the IRS-1 IH1 pleckstrin homology domain to coordinate receptor/JAK/IRS-1 association and subsequent IRS-1 phosphorylation (8Platanias L.C. Uddin S. Yetter A. Sun X.J. White M.F. J. Biol. Chem. 1996; 271: 278-282Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). This suggests that serine phosphorylation within the IRS-1 IH1 pleckstrin homology domain might be important for preventing IFN-α-activated JAK1-dependent tyrosine phosphorylation of IRS-1. We show here, however, that IFN-α-activated JAK1 can phosphorylate IRS-1-(511–772) and that serine phosphorylation of IRS-1-(511–772) inhibits this effect. This is important in that IRS-1-(511–772) does not contain either the IRS-1 IH1 pleckstrin homology or IH2 phosphotyrosine-binding domain and suggests that other regions of IRS-1 may be important in IRS-1/JAK1 interactions. Hyperinsulinemia and insulin resistance are prominent features in both syndrome X and the development of type 2 diabetes mellitus (44Reaven G.M. Physiol. Rev. 1995; 75: 473-486Crossref PubMed Scopus (1058) Google Scholar). However, the pathogenesis of the multiple complications and conditions associated with these diseases is not yet understood (45Horton E.S. Diabetes Res. Clin. Pract. 1995; 28 (suppl.): S3-S11Abstract Full Text PDF PubMed Scopus (34) Google Scholar). We show here that chronic insulin treatment and IRS-1 serine phosphorylation decrease JAK1-mediated IRS-1 tyrosine phosphorylation and IRS-1/PI3K association, suggesting that cytokine signal transduction may be altered during hyperinsulinemia. Currently, a rapidly growing number of hormone and cytokine receptors appear to signal through JAK and IRS family members, and this appears to be critical to hormone/cytokine function (1Yenush L. White M.F. Bioessays. 1997; 19: 491-500Crossref PubMed Scopus (257) Google Scholar). This is most clearly understood in IL-4 signaling, where IRS function has been shown to be critical to IL-4-dependent mitogenesis and anti-apoptosis (4Wang L.M. Myers Jr., M.G. Sun X.J. Aaronson S.A. White M. Pierce J.H. Science. 1993; 261: 1591-1594Crossref PubMed Scopus (371) Google Scholar, 46Zamorano J. Wang H.Y. Wang L.M. Pierce J.H. Keegan A.D. J. Immunol. 1996; 157: 4926-4934PubMed Google Scholar). Additionally, site-specific mutagenesis of the phosphotyrosine-binding domain-binding motif in the IL-4 receptor reduces both IRS and STAT6 tyrosine phosphorylation and abolishes the effect of IL-4 on the induction of DNA binding activity and CD23 induction (47Wang H.Y. Zamorano J. Keegan A.J. J. Biol. Chem. 1998; 273: 989-996Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). Thus, by inducing IRS serine phosphorylation, hyperinsulinemia may potentially contribute to the pathogenesis of syndrome X/type 2 diabetes mellitus complications by disrupting JAK-mediated cytokine and hormone signaling pathways that use IRS. In summary, we show that OA and chronic insulin treatment inhibit IFN-α-dependent IRS-1 tyrosine phosphorylation and IRS-1/PI3K association and activity. More importantly, we show that these effects are mediated by serine phosphorylation of IRS-1. We conclude that IRS-1 serine phosphorylation plays an inhibitory role in signaling pathways outside the insulin system and suggest that hyperinsulinemia may alter signaling of JAK1-dependent cytokine receptors through serine phosphorylation of IRS-1." @default.
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• W2017584173 title "JAK1-dependent Phosphorylation of Insulin Receptor Substrate-1 (IRS-1) Is Inhibited by IRS-1 Serine Phosphorylation" @default.
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