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• W2092777252 abstract "Inhibitory serine phosphorylation is a potential molecular mechanism for insulin resistance. We have developed a new variant of the yeast two-hybrid method, referred to as disruptive yeast tri-hybrid (Y3H), to identify inhibitory kinases and sites of phosphorylation in insulin receptors (IR) and IR substrates, IRS-1. Using IR and IRS-1 as bait and prey, respectively, and c-Jun NH2-terminal kinase (JNK1) as the disruptor, we now show that phosphorylation of IRS-1 Ser-307, a previously identified site, is necessary but not sufficient for JNK1-mediated disruption of IR/IRS-1 binding. We further identify a new phosphorylation site, Ser-302, and show that this too is necessary for JNK1-mediated disruption. Seven additional kinases potentially linked to insulin resistance similarly block IR/IRS-1 binding in the disruptive Y3H, but through distinct Ser-302- and Ser-307-independent mechanisms. Phosphospecific antibodies that recognize sequences surrounding Ser(P)-302 or Ser(P)-307 were used to determine whether the sites were phosphorylated under relevant conditions. Phosphorylation was promoted at both sites in Fao hepatoma cells by reagents known to promote Ser/Thr phosphorylation, including the phorbol ester phorbol 12-myristate 13-acetate, anisomycin, calyculin A, and insulin. The antibodies further showed that Ser(P)-302 and Ser(P)-307 are increased in animal models of obesity and insulin resistance, including genetically obese ob/ob mice, diet-induced obesity, and upon induction of hyperinsulinemia. These findings demonstrate that phosphorylation at both Ser-302 and Ser-307 is necessary for JNK1-mediated inhibition of the IR/IRS-1 interaction and that Ser-302 and Ser-307 are phosphorylated in parallel in cultured cells and in vivo under conditions that lead to insulin resistance. Inhibitory serine phosphorylation is a potential molecular mechanism for insulin resistance. We have developed a new variant of the yeast two-hybrid method, referred to as disruptive yeast tri-hybrid (Y3H), to identify inhibitory kinases and sites of phosphorylation in insulin receptors (IR) and IR substrates, IRS-1. Using IR and IRS-1 as bait and prey, respectively, and c-Jun NH2-terminal kinase (JNK1) as the disruptor, we now show that phosphorylation of IRS-1 Ser-307, a previously identified site, is necessary but not sufficient for JNK1-mediated disruption of IR/IRS-1 binding. We further identify a new phosphorylation site, Ser-302, and show that this too is necessary for JNK1-mediated disruption. Seven additional kinases potentially linked to insulin resistance similarly block IR/IRS-1 binding in the disruptive Y3H, but through distinct Ser-302- and Ser-307-independent mechanisms. Phosphospecific antibodies that recognize sequences surrounding Ser(P)-302 or Ser(P)-307 were used to determine whether the sites were phosphorylated under relevant conditions. Phosphorylation was promoted at both sites in Fao hepatoma cells by reagents known to promote Ser/Thr phosphorylation, including the phorbol ester phorbol 12-myristate 13-acetate, anisomycin, calyculin A, and insulin. The antibodies further showed that Ser(P)-302 and Ser(P)-307 are increased in animal models of obesity and insulin resistance, including genetically obese ob/ob mice, diet-induced obesity, and upon induction of hyperinsulinemia. These findings demonstrate that phosphorylation at both Ser-302 and Ser-307 is necessary for JNK1-mediated inhibition of the IR/IRS-1 interaction and that Ser-302 and Ser-307 are phosphorylated in parallel in cultured cells and in vivo under conditions that lead to insulin resistance. Insulin resistance is the condition in which target tissues fail to respond appropriately to circulating insulin. Although genetics may play a role in the pathogenesis of type 2 diabetes, it has become increasingly clear that acquired, non-genetic causes of insulin resistance represent a critical link between the rapidly growing national and worldwide epidemics in obesity and type 2 diabetes (1Group National Diabetes Data Diabetes in America. 2nd Ed. NIDDK, National Institutes of Health, Bethesda, MD1994Google Scholar, 2Mokdad A.H. Ford E.S. Bowman B.A. Nelson D.E. Engelgau M.M. Vinicor F. Marks J.S. Diabetes Care. 2000; 23: 1278-1283Crossref PubMed Scopus (940) Google Scholar, 3Mokdad A.H. Serdula M.K. Dietz W.H. Bowman B.A. Marks J.S. Koplan J.P. J. Am. Med. Assoc. 2000; 284: 1650-1651Crossref PubMed Scopus (310) Google Scholar, 4Mokdad A.H. Bowman B.A. Ford E.S. Vinicor F. Marks J.S. Koplan J.P. J. Am. Med. Assoc. 2001; 286: 1195-1200Crossref PubMed Scopus (2241) Google Scholar, 5Mokdad A.H. Ford E.S. Bowman B.A. Dietz W.H. Vinicor F. Bales V.S. Marks J.S. J. Am. Med. Assoc. 2003; 289: 76-79Crossref PubMed Scopus (4513) Google Scholar). Obesity, fatty diet, and sedentary lifestyle directly promote insulin resistance, and exercise and weight loss reverse it. Obesity and insulin resistance are also associated with and exacerbate hypertension and hyperlipidemia in addition to predisposing to the development of type 2 diabetes. This constellation of conditions, referred to collectively as either the metabolic or dysmetabolic syndrome, represents a third interrelated epidemic with a prevalence of ∼24% of adults in the United States (6Ford E.S. Giles W.H. Dietz W.H. J. Am. Med. Assoc. 2002; 287: 356-359Crossref PubMed Scopus (5712) Google Scholar). Individuals with the metabolic syndrome have a seriously increased risk of developing atherosclerotic cardiovascular disease. Elucidating the molecular pathways that connect obesity to pathogenesis of insulin resistance clearly has great public health importance.Of hypothesized mediators of insulin resistance, recent findings have profiled potential roles for inflammation and proinflammatory cytokines, other fat cell-derived cytokines, free fatty acids, and inhibitory serine/threonine (Ser/Thr) phosphorylation of upstream elements of insulin signaling (7Ridker P.M. Buring J.E. Cook N.R. Rifai N. Circulation. 2003; 107: 391-397Crossref PubMed Scopus (1986) Google Scholar, 8Fried S.K. Bunkin D.A. Greenberg A.S. J. Clin. Endocrinol. Metab. 1998; 83: 847-850Crossref PubMed Scopus (1411) Google Scholar, 9Pickup J.C. Mattock M.B. Chusney G.D. Burt D. Diabetologia. 1997; 40: 1286-1292Crossref PubMed Scopus (1040) Google Scholar, 10Kern P.A. Saghizadeh M. Ong J.M. Bosch R.J. Deem R. Simsolo R.B. J. Clin. Investig. 1995; 95: 2111-2119Crossref PubMed Scopus (1157) Google Scholar, 11Hotamisligil G.S. Int. J. Obes. 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In fact, inflammation- and free fatty acid-mediated mechanisms may converge at the level of Ser/Thr phosphorylation of insulin receptors (IRs) 1The abbreviations used are: IR, insulin receptor; IRS, IR substrate; MAPK, mitogen-activated protein kinase; PMA, phorbol 12-myristate 13-acetate; GSK, glycogen synthase kinase; PKA and PKC, protein kinase A and C, respectively; JNK, c-Jun NH2-terminal kinase; Y3H, yeast tri-hybrid; WT, wild type; IKK, IκB kinase complex.1The abbreviations used are: IR, insulin receptor; IRS, IR substrate; MAPK, mitogen-activated protein kinase; PMA, phorbol 12-myristate 13-acetate; GSK, glycogen synthase kinase; PKA and PKC, protein kinase A and C, respectively; JNK, c-Jun NH2-terminal kinase; Y3H, yeast tri-hybrid; WT, wild type; IKK, IκB kinase complex. and insulin receptor substrates (IRSs) to provide potentially unifying mechanisms for insulin resistance (15Yuan M. Konstantopoulos N. Lee J. Hansen L. Li Z.W. Karin M. Shoelson S.E. Science. 2001; 293: 1673-1677Crossref PubMed Scopus (1615) Google Scholar, 16Kim J.K. Kim Y.J. Fillmore J.J. Chen Y. Moore I. Lee J. Yuan M. Li Z.W. Karin M. Perret P. Shoelson S.E. Shulman G.I. J. Clin. Investig. 2001; 108: 437-446Crossref PubMed Scopus (611) Google Scholar, 17Aguirre V. Uchida T. Yenush L. Davis R. White M.F. J. Biol. Chem. 2000; 275: 9047-9054Abstract Full Text Full Text PDF PubMed Scopus (1157) Google Scholar, 18Aguirre V. Werner E.D. Giraud J. Lee Y.H. Shoelson S.E. White M.F. J. Biol. Chem. 2002; 277: 1531-1537Abstract Full Text Full Text PDF PubMed Scopus (757) Google Scholar, 19Hotamisligil G.S. Budavari A. Murray D. Spiegelman B.M. J. Clin. Investig. 1994; 94: 1543-1549Crossref PubMed Scopus (721) Google Scholar, 20Hirosumi J. Tuncman G. Chang L. Gorgun C.Z. Uysal K.T. Maeda K. Karin M. Hotamisligil G.S. Nature. 2002; 420: 333-336Crossref PubMed Scopus (2594) Google Scholar). Consistent with this, insulin-sensitizing, anti-inflammatory salicylates reverse Ser/Thr phosphorylation of IR and IRSs in insulin-responsive tissues in obesity-, diet- and free fatty acid-induced models of insulin resistance (15Yuan M. Konstantopoulos N. Lee J. Hansen L. Li Z.W. Karin M. Shoelson S.E. Science. 2001; 293: 1673-1677Crossref PubMed Scopus (1615) Google Scholar, 16Kim J.K. Kim Y.J. Fillmore J.J. Chen Y. Moore I. Lee J. Yuan M. Li Z.W. Karin M. Perret P. Shoelson S.E. Shulman G.I. J. Clin. Investig. 2001; 108: 437-446Crossref PubMed Scopus (611) Google Scholar).Although IR is a tyrosine kinase, insulin also stimulates Ser/Thr phosphorylation of numerous signaling enzymes and other proteins (21Czech M.P. Klarlund J.K. Yagaloff K.A. Bradford A.P. Lewis R.E. J. Biol. Chem. 1988; 263: 11017-11020Abstract Full Text PDF PubMed Google Scholar, 22Saltiel A.R. FASEB J. 1994; 8: 1034-1040Crossref PubMed Scopus (45) Google Scholar). Many are Ser/Thr kinases involved in kinase cascades. Those mediating some of the insulin cellular actions include Raf, MEK (mitogen-activated protein kinase/extracellular signal-regulated kinase kinase), MAPK, p90RSK, phosphatidylinositol 3-kinase, phosphoinositide-dependent protein kinase 1, protein kinase B/AKT, mTOR, p70 S6 kinase, GSK3β, PKCβ1, PKCζ, and PKCλ. Overexpression of many of these kinases paradoxically inhibits insulin signaling as opposed to activating it, suggesting that the same kinases that mediate insulin signaling might also play roles in negative feedback of it (23Zick Y. Trends Cell Biol. 2001; 11: 437-441Abstract Full Text PDF PubMed Scopus (186) Google Scholar). In fact, IR and IRSs are themselves Ser/Thr-phosphorylated in response to insulin, providing a potential mechanism for negative feedback.Basal levels of IRS-1 Ser/Thr phosphorylation are increased in cells under various conditions, leading to observable shifts in electrophoretic mobility (15Yuan M. Konstantopoulos N. Lee J. Hansen L. Li Z.W. Karin M. Shoelson S.E. Science. 2001; 293: 1673-1677Crossref PubMed Scopus (1615) Google Scholar, 24Mothe I. Van Obberghen E. J. Biol. Chem. 1996; 271: 11222-11227Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar, 25Tanti J.F. Gremeaux T. Van Obberghen E. Marchand-Brustel Y. J. Biol. Chem. 1994; 269: 6051-6057Abstract Full Text PDF PubMed Google Scholar, 26Kanety H. Feinstein R. Papa M.Z. Hemi R. Karasik A. J. Biol. Chem. 1995; 270: 23780-23784Abstract Full Text Full Text PDF PubMed Scopus (381) Google Scholar, 27Paz 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 (447) Google Scholar). The magnitudes of the shifts demonstrate that multiple sites are phosphorylated. In fact, IRS-1 contains 232 serines and threonines, nearly 19% of its 1231 residues, providing great potential for multisite phosphorylation. The rapid accumulation of “hyper-phosphorylated” IRS-1 upon treatment with the Ser/Thr phosphatase inhibitors okadaic acid or calyculin A further indicates that it is constantly being Ser/Thr-phosphorylated. Normally this is countered by phosphatases like PP2A and PP1, such that under steady-state conditions there should be a given “responsiveness” to the system. The balance might be shifted by activating Ser/Thr kinases that phosphorylate IRS or by inhibiting a relevant phosphatase. The net result in these cases would be the same; that is, Ser/Thr phosphorylation of IRS-1, diminished insulin signaling, and the development of insulin resistance. Conversely, insulin signaling should be sensitized either by inhibiting the relevant Ser/Thr kinase(s) or activating the appropriate phosphatase(s).A host of Ser/Thr kinases can be shown to attenuate upstream insulin action in cultured cells, including PKA (28Stadtmauer L. Rosen O.M. J. Biol. 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Chem. 2000; 275: 9047-9054Abstract Full Text Full Text PDF PubMed Scopus (1157) Google Scholar, 18Aguirre V. Werner E.D. Giraud J. Lee Y.H. Shoelson S.E. White M.F. J. Biol. Chem. 2002; 277: 1531-1537Abstract Full Text Full Text PDF PubMed Scopus (757) Google Scholar, 20Hirosumi J. Tuncman G. Chang L. Gorgun C.Z. Uysal K.T. Maeda K. Karin M. Hotamisligil G.S. Nature. 2002; 420: 333-336Crossref PubMed Scopus (2594) Google Scholar, 42Lee Y.H. Giraud J. Davis R.J. White M.F. J. Biol. Chem. 2003; 278: 2896-2902Abstract Full Text Full Text PDF PubMed Scopus (338) Google Scholar, 51Greene M.W. Sakaue H. Wang L. Alessi D.R. Roth R.A. J. Biol. Chem. 2003; 278: 8199-8211Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar, 54Rui L. Aguirre V. Kim J.K. Shulman G.I. Lee A. Corbould A. Dunaif A. White M.F. J. Clin. Investig. 2001; 107: 181-189Crossref PubMed Scopus (477) Google Scholar, 55Jiang G. Dallas-Yang Q. Liu F. Moller D.E. Zhang B.B. J. Biol. 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Zhande R. Jetton T.L. Zhou G. Sun X.J. J. Biol. Chem. 2002; 277: 26530-26539Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). Of these, Ser-307 2Numbering of IRS-1 in this manuscript refers to rodent (r) sequences. Human (h) sequences are identical in the Ser-302/Ser-307 region, although the numbering is offset by 5 residues (rSer-307 = hSer-312; rSer-302 = hSer-307). hIRS-1, 295SQVGLTRRSRTES307ITATS312PASMVGGKPGSFRVRAS334; rIRS-1, 290SQVGLTRRSRTES302ITATS307PASMVGGKPGSFRVRAS329.2Numbering of IRS-1 in this manuscript refers to rodent (r) sequences. Human (h) sequences are identical in the Ser-302/Ser-307 region, although the numbering is offset by 5 residues (rSer-307 = hSer-312; rSer-302 = hSer-307). hIRS-1, 295SQVGLTRRSRTES307ITATS312PASMVGGKPGSFRVRAS334; rIRS-1, 290SQVGLTRRSRTES302ITATS307PASMVGGKPGSFRVRAS329. phosphorylation has been studied most intensively as a mechanism for disrupting IR/IRS-1 interactions (17Aguirre V. Uchida T. Yenush L. Davis R. White M.F. J. Biol. Chem. 2000; 275: 9047-9054Abstract Full Text Full Text PDF PubMed Scopus (1157) Google Scholar, 18Aguirre V. Werner E.D. Giraud J. Lee Y.H. Shoelson S.E. White M.F. J. Biol. Chem. 2002; 277: 1531-1537Abstract Full Text Full Text PDF PubMed Scopus (757) Google Scholar, 42Lee Y.H. Giraud J. Davis R.J. White M.F. J. Biol. Chem. 2003; 278: 2896-2902Abstract Full Text Full Text PDF PubMed Scopus (338) Google Scholar, 51Greene M.W. Sakaue H. Wang L. Alessi D.R. Roth R.A. J. Biol. Chem. 2003; 278: 8199-8211Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar, 54Rui L. Aguirre V. Kim J.K. Shulman G.I. Lee A. Corbould A. Dunaif A. White M.F. J. Clin. Investig. 2001; 107: 181-189Crossref PubMed Scopus (477) Google Scholar, 55Jiang G. Dallas-Yang Q. Liu F. Moller D.E. Zhang B.B. J. Biol. Chem. 2003; 278: 180-186Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar, 56Yu C. Chen Y. Cline G.W. Zhang D. Zong H. Wang Y. Bergeron R. Kim J.K. Cushman S.W. Cooney G.J. Atcheson B. White M.F. Kraegen E.W. Shulman G.I. J. Biol. Chem. 2002; 277: 50230-50236Abstract Full Text Full Text PDF PubMed Scopus (1186) Google Scholar, 59Gao Z. Hwang D. Bataille F. Lefevre M. York D. Quon M.J. Ye J. J. Biol. Chem. 2002; 277: 48115-48121Abstract Full Text Full Text PDF PubMed Scopus (585) Google Scholar, 60Gao Z. Zuberi A. Quon M.J. Dong Z. Ye J. J. Biol. Chem. 2003; 278: 24944-24950Abstract Full Text Full Text PDF PubMed Scopus (225) Google Scholar, 61Gual P. Gonzalez T. Gremeaux T. Barres R. Marchand-Brustel Y. Tanti J.F. J. Biol. Chem. 2003; 278: 26550-26557Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). Originally identified as a target of JNK in cells (17Aguirre V. Uchida T. Yenush L. Davis R. White M.F. J. Biol. Chem. 2000; 275: 9047-9054Abstract Full Text Full Text PDF PubMed Scopus (1157) Google Scholar), Ser-307 was subsequently found to be phosphorylated as well in cells treated with tumor necrosis factor-α, PMA, insulin, or insulin-like growth factor-1 (42Lee Y.H. Giraud J. Davis R.J. White M.F. J. Biol. Chem. 2003; 278: 2896-2902Abstract Full Text Full Text PDF PubMed Scopus (338) Google Scholar, 51Greene M.W. Sakaue H. Wang L. Alessi D.R. Roth R.A. J. Biol. Chem. 2003; 278: 8199-8211Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar, 54Rui L. Aguirre V. Kim J.K. Shulman G.I. Lee A. Corbould A. Dunaif A. White M.F. J. Clin. Investig. 2001; 107: 181-189Crossref PubMed Scopus (477) Google Scholar, 55Jiang G. Dallas-Yang Q. Liu F. Moller D.E. Zhang B.B. J. Biol. Chem. 2003; 278: 180-186Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar, 56Yu C. Chen Y. Cline G.W. Zhang D. Zong H. Wang Y. Bergeron R. Kim J.K. Cushman S.W. Cooney G.J. Atcheson B. White M.F. Kraegen E.W. Shulman G.I. J. Biol. Chem. 2002; 277: 50230-50236Abstract Full Text Full Text PDF PubMed Scopus (1186) Google Scholar, 59Gao Z. Hwang D. Bataille F. Lefevre M. York D. Quon M.J. Ye J. J. Biol. Chem. 2002; 277: 48115-48121Abstract Full Text Full Text PDF PubMed Scopus (585) Google Scholar, 61Gual P. Gonzalez T. Gremeaux T. Barres R. Marchand-Brustel Y. Tanti J.F. J. Biol. Chem. 2003; 278: 26550-26557Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). Importantly, Ser-307 has been found to be phosphorylated in vivo in insulin-resistant rodent models (20Hirosumi J. Tuncman G. Chang L. Gorgun C.Z. Uysal K.T. Maeda K. Karin M. Hotamisligil G.S. Nature. 2002; 420: 333-336Crossref PubMed Scopus (2594) Google Scholar) and in human skeletal muscle (54Rui L. Aguirre V. Kim J.K. Shulman G.I. Lee A. Corbould A. Dunaif A. White M.F. J. Clin. Investig. 2001; 107: 181-189Crossref PubMed Scopus (477) Google Scholar). We have shown previously that Ser-307 phosphorylation blocks IR/IRS-1 binding in disruptive yeast tri-hybrid (Y3H) experiments (18Aguirre V. Werner E.D. Giraud J. Lee Y.H. Shoelson S.E. White M.F. J. Biol. Chem. 2002; 277: 1531-1537Abstract Full Text Full Text PDF PubMed Scopus (757) Google Scholar). We have now used the disruptive Y3H method to identify an additional serine (Ser-302) in IRS-1 that is equally necessary for JNK-mediated disruption. We also show that Ser-302 and Ser-307 are phosphorylated in cells and in vivo under equivalent conditions of insulin resistance.MATERIALS AND METHODSYeast Two-hybrid—Matchmaker LexA two-hybrid reagents were purchased from Clontech. Saccharomyces cerevisiae strain EGY48 (Matα trp1, his3, ura3, 6LexAop-LEU2, LYS2), transformed with p8op-lacZ (kindly provided by T. A. Gustafson, Metabolex), was used as the host for interaction studies. EGY48/p8op-lacZ was sequentially transformed with plasmid constructs using polyethylene glycol/lithium acetate according to manufacturer's protocols. To determine protein-protein interactions as a function of leucine biosynthesis (LEU2), transformants were grown on synthetic dextrose agar plates for 3 days at 30 °C. Four independent colonies were streaked on synthetic dextrose glucose agar plates, grown overnight, replica-plated on synthetic galactose/raffinose agar plates, and regrown for 5 days at 30 °C to induce expression of B42 fusion proteins.Disruptive Yeast Tri-hybrid—To create the disruptive yeast tri-hybrid assay, we modified the existing LexA yeast two-hybrid method. S. cerevisiae EGY48 cells were sequentially transformed as above with pLexA expressing human insulin receptor kinase (residues 940–1343) as bait, pB42AD expressing various human IRS-1 constructs as prey, and in most cases JNK1α1 in the third pDis plasmid. pDis was derived from the p426:Gal1 plasmid (ATCC) by replacing its multiple cloning region with another having a nuclear localization signal under Gal1 promoter control. In selected experiments alternative kinases were expressed in the pDis plasmid, including GSK3β(S9A), IKKα, IKKβ(S177E/S181E), p38 MAPK, PKA, PKCα, PKCβ2, PKCϵ, and PKCθ. Transformants were grown on the appropriate synthetic dextrose plates for 3 days at 30 °C. Four independent colonies were streaked on synthetic dextrose plates, incubated overnight, and replicaplated on GR plates. The plates were immediately replica-cleaned, incubated overnight, replica-cleaned, and incubated at 30 °C for 5 days to induce expression of pDis and B42 fusion proteins.Cell Culture—Fao hepatoma cells were maintained in RPMI medium containing 25 mm glucose and 10% heat-inactivated fetal bovine serum (Sigma) under 5% CO2. Before experiments, Fao cells were serum-starved for 16 h in RPMI containing 0.1% bovine serum albumin. After treatment, cells were washed (phosphate-buffered saline containing 1.0 mm phenylmethylsulfonyl fluoride, 3.0 μm aprotinin, 10 μm leupeptin, 5.0 μm pepstatin A, 25 mm benzamidine, 25 mm sodium vanadate, 5.0 mm glycerol phosphate, 100 mm NaF, 1.0 mm ammonium molybdate, 30 mm tetrasodium pyrophosphate, 5 mm EGTA) and lysed (in 30 mm HEPES, 150 mm NaCl, 1.0 mm phenylmethylsulfonyl fluoride, 3.0 μm aprotinin, 10 μm leupeptin, 5.0 μm pepstatin A, 25 mm benzamidine, 25 mm sodium vanadate, 5.0 mm glycerol phosphate, 100 mm NaF, 1.0 mm ammonium molybdate, 30 mm tetrasodium pyrophosphate, 5.0 mm EGTA, 10% glycerol, 1% Triton X-100, and 0.5% sodium deoxycholate, pH 7.4) for immunoprecipitation and Western-blotting experiments. CHO-IR cells were maintained in F-12 medium supplemented with 10% fetal bovine serum in the presence of 0.4 mg/ml G418 and 2 mm glutamine in 5% CO2. Cells at 50–60% confluence were transfected using FuGENE 6 (Roche Applied Science) with pCMV(WT IRS-1), pCMV-(IRS-1 S307A), pCMV(IRS-1 S312A), or pCMV(IRS-1 S307A/S3012A). Cells were incubated for 24 h, serum-starved overnight, treated for 30 min with 20 μm anisomycin or 20 nm calyculin A (Bio-Mol) and for 5 min with 1 nm insulin and lysed as describe above.In Vivo Animal Studies—Fourteen week-old ob/ob (Lepob/ob) mice and congenic Lep+/+ controls were sacrificed after an overnight fast. For the “diet-induced obesity” study, 8 week-old C57BL/6 (Jackson Laboratories) mice were fed a high fat diet (Research Diet D12451, 45% of calories from fat) for 8 weeks; controls were fed regular chow that derives 17% of calories from fat. After an overnight fast the 16-week-old mice were sacrificed. To determine the effects of acute, high dose insulin, chow-fed 12-week-old C57BL/6 mice were fasted overnight, injected intraperitoneally with 1 units/kg of insulin, and sacrificed after 10 min. Harvested livers, stored until use in liquid N2, were pulverized and homogenized with a Polytron for 30 s in lysis buffer. Cleared lysates were used for immunoprecipitations and Western blotting.Antibody Preparation and Use—Phosphospecific antibodies against IRS-1 Ser(P)-302 were generated in rabbits. Phosphopeptide RRSRTEpSITATSP (p indicates phosphorylated serine) was coupled to keyhole limpet hemocyanin for use as antigen. Rabbit antisera were passed first over immobilized RRSRTESITATSP to remove antibodies that recognized the unphosphorylated sequence. Phosphospecific antibodies were affinity-isolated by passing the precleared sera over immobilized RRSRTEpSITATSP followed by a low pH elution (62Harlow E. Lane D. Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1988Google Scholar). Phosphospecific IRS-1 Ser(P)-307 antibody was either purchased from Cell Signaling or kindly provided by Morris White (" @default.
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• W2092777252 date "2004-08-01" @default.
• W2092777252 modified "2023-10-12" @default.
• W2092777252 title "Insulin Resistance Due to Phosphorylation of Insulin Receptor Substrate-1 at Serine 302" @default.
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• W2092777252 doi "https://doi.org/10.1074/jbc.m405203200" @default.
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