Modulation of Insulin Receptor Substrate-1 Tyrosine Phosphorylation and Function by Mitogen-activated Protein Kinase
1997; Elsevier BV; Volume: 272; Issue: 50 Linguagem: Inglês
10.1074/jbc.272.50.31400
ISSN1083-351X
AutoresKathryn De Fea, Richard A. Roth,
Tópico(s)Metabolism, Diabetes, and Cancer
ResumoIncreased serine phosphorylation of insulin receptor substrate-1 (IRS-1) has been observed in several systems to correlate with a decreased ability of the insulin receptor to tyrosine-phosphorylate this endogenous substrate and to inhibit its subsequent association with phosphatidylinositol 3-kinase. In the present studies we have examined the potential role of the mitogen-activated protein (MAP) kinase in the increased serine phosphorylation of IRS-1 observed in human embryonic kidney cells treated with an activator of protein kinase C, phorbol 12-myristate 13-acetate. First, recombinantly produced kinase was shown to phosphorylate intact IRS-1 in a way that decreased the ability of isolated insulin receptor to phosphorylate the tyrosines recognized by the SH2 domains of the phosphatidylinositol 3-kinase. Second, an inhibitor of MAP kinase activation, PD98059, blocked the phorbol 12-myristate 13-acetate-induced inhibition of the insulin-stimulated increase in IRS-1 associated phosphatidylinositol 3-kinase. Third, activation of MAP kinase in intact cells via a regulatable upstream kinase, a RAF:estrogen receptor construct, could also inhibit the insulin-stimulated increase in IRS-1-associated phosphatidylinositol 3-kinase. Fourth, an in gel kinase assay showed that MAP kinase was the primary renaturable kinase in cell extracts capable of phosphorylating an IRS-1 fusion protein. Finally, IRS-1 was found to associate in coprecipitation studies with endogenous MAP kinase. These studies implicate MAP kinase as one of the kinases capable of phosphorylating and regulating IRS-1 tyrosine phosphorylation. Increased serine phosphorylation of insulin receptor substrate-1 (IRS-1) has been observed in several systems to correlate with a decreased ability of the insulin receptor to tyrosine-phosphorylate this endogenous substrate and to inhibit its subsequent association with phosphatidylinositol 3-kinase. In the present studies we have examined the potential role of the mitogen-activated protein (MAP) kinase in the increased serine phosphorylation of IRS-1 observed in human embryonic kidney cells treated with an activator of protein kinase C, phorbol 12-myristate 13-acetate. First, recombinantly produced kinase was shown to phosphorylate intact IRS-1 in a way that decreased the ability of isolated insulin receptor to phosphorylate the tyrosines recognized by the SH2 domains of the phosphatidylinositol 3-kinase. Second, an inhibitor of MAP kinase activation, PD98059, blocked the phorbol 12-myristate 13-acetate-induced inhibition of the insulin-stimulated increase in IRS-1 associated phosphatidylinositol 3-kinase. Third, activation of MAP kinase in intact cells via a regulatable upstream kinase, a RAF:estrogen receptor construct, could also inhibit the insulin-stimulated increase in IRS-1-associated phosphatidylinositol 3-kinase. Fourth, an in gel kinase assay showed that MAP kinase was the primary renaturable kinase in cell extracts capable of phosphorylating an IRS-1 fusion protein. Finally, IRS-1 was found to associate in coprecipitation studies with endogenous MAP kinase. These studies implicate MAP kinase as one of the kinases capable of phosphorylating and regulating IRS-1 tyrosine phosphorylation. Insulin receptor substrate-1 (IRS-1), 1The abbreviations used are: IRS, insulin receptor substrate; SH, Src homology; PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate; IR, insulin receptor; PI, phosphatidylinositol; ERK, extracellular regulated kinase; MAP, mitogen-activated protein; HEK, human embryonic kidney; HT, hydroxytamoxifen; ER, estrogen receptor; CHO, Chinese hamster ovary; PBS, phosphate-buffered saline; GST, glutathioneS-transferase; FCS, fetal calf serum; PAGE, polyacrylamide gel electrophoresis; MEK, MAP kinase kinase.1The abbreviations used are: IRS, insulin receptor substrate; SH, Src homology; PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate; IR, insulin receptor; PI, phosphatidylinositol; ERK, extracellular regulated kinase; MAP, mitogen-activated protein; HEK, human embryonic kidney; HT, hydroxytamoxifen; ER, estrogen receptor; CHO, Chinese hamster ovary; PBS, phosphate-buffered saline; GST, glutathioneS-transferase; FCS, fetal calf serum; PAGE, polyacrylamide gel electrophoresis; MEK, MAP kinase kinase. a major endogenous substrate of the insulin receptor kinase, undergoes tyrosine phosphorylation upon insulin stimulation, after which it serves as a docking protein for a number of Src homology (SH) 2 domain containing proteins such as Grb2, Syp, Nck, and two isoforms of the regulatory subunit of PI 3-kinase (p85) (1Lee J. Pilch P.F. Am. J. Physiol. 1994; 266: C319-C334Crossref PubMed Google Scholar, 2White M.F. Kahn C.R. J. Biol. Chem. 1994; 269: 1-4Abstract Full Text PDF PubMed Google Scholar). Binding of tyrosine-phosphorylated IRS-1 to p85 leads to a 3–5-fold stimulation of PI 3-kinase activity (3Rordorf-Nikolic T. Van Horn D.J. Chen D. White M.F. Backer J.M. J. Biol. Chem. 1995; 270: 3662-3666Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar), an increase in the PI 3,4-bisphosphate and 3,4,5-trisphosphate in the cell (4Ruderman N.B. Kapeller R. White M.F. Cantley L.C. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 1411-1415Crossref PubMed Scopus (393) Google Scholar), and possibly through the stimulation of the Ser/Thr kinase AKT/PKB (5Kohn A.D. Summers S.A. Birnbaum M.J. Roth R.A. J. Biol. Chem. 1996; 271: 31372-31378Abstract Full Text Full Text PDF PubMed Scopus (1086) Google Scholar), to the final effects of insulin such as stimulation of glucose uptake and glycogen synthesis. Although IRS-1 contains at least 20 potential tyrosine phosphorylation sites (1Lee J. Pilch P.F. Am. J. Physiol. 1994; 266: C319-C334Crossref PubMed Google Scholar, 2White M.F. Kahn C.R. J. Biol. Chem. 1994; 269: 1-4Abstract Full Text PDF PubMed Google Scholar), one (tyrosine 608) appears to be a principal site of interaction with the SH2 domain of PI 3-kinase, while three others (tyrosines 460, 939, and 987) play additional roles (6Rocchi S. Tartare-Deckert S. Mothe I. Van Obberghen E. Endocrinology. 1995; 136: 5291-5296Crossref PubMed Scopus (38) Google Scholar). Under several conditions resulting in cellular insulin resistance, such as activation of protein kinase C (PKC) (7Chin J.E. Dickens M. Tavare J.M. Roth R.A. J. Biol. Chem. 1993; 268: 6338-6347Abstract Full Text PDF PubMed Google Scholar,8Takayama S. White M.F. Kahn C.R. J. Biol. Chem. 1988; 263: 3440-3447Abstract Full Text PDF PubMed Google Scholar), addition of the Ser/Thr phosphatase inhibitor okadaic acid (9Tanti J.-F. Gremeaux T. Van Obberghen E. Le Marchand-Brustel Y. J. Biol. Chem. 1994; 269: 6051-6057Abstract Full Text PDF PubMed Google Scholar), activation of cellular stress pathways by tumor necrosis factor and other cytokines (10Hotamisligil G.S. Peraldi P. Budavari A. Ellis R. White M.F. Spiegelman B.M. Science. 1996; 271: 665-668Crossref PubMed Scopus (2178) Google Scholar, 11Kanety 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, 12Haddad T.C. Conover C.A. J. Biol. Chem. 1997; 272: 19525-19531Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar), a decrease in insulin-stimulated tyrosine phosphorylation of IRS-1 is observed; however, the mechanism(s) by which these inhibitory effects occur has been difficult to elucidate.Under many of the conditions mentioned above, Ser/Thr kinases are activated, and one hypothesis has been that increased serine phosphorylation of IRS-1 can result in subsequent inhibition of its tyrosine phosphorylation by the insulin receptor (9Tanti J.-F. Gremeaux T. Van Obberghen E. Le Marchand-Brustel Y. J. Biol. Chem. 1994; 269: 6051-6057Abstract Full Text PDF PubMed Google Scholar, 10Hotamisligil G.S. Peraldi P. Budavari A. Ellis R. White M.F. Spiegelman B.M. Science. 1996; 271: 665-668Crossref PubMed Scopus (2178) Google Scholar, 11Kanety 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, 12Haddad T.C. Conover C.A. J. Biol. Chem. 1997; 272: 19525-19531Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). In fact, IRS-1 contains numerous serine phosphorylation sites and is extensively serine-phosphorylated after insulin stimulation, possibly in part by casein kinase II (13Sun Z.J. Rothenberg P. Kahn C.R. Backer J.M. Araki E. Wilden P.A. Cahill D.A. Goldstein B.J. White M.F. Nature. 1991; 352: 73-78Crossref PubMed Scopus (1276) Google Scholar, 14Tanasijevic M.J. Myers Jr M.G. Thoma R.S. Crimmins D.L. White M.F. Sacks D.B. J. Biol. Chem. 1993; 268: 18157-18166Abstract Full Text PDF PubMed Google Scholar). In addition, IRS-1 contains 4 serines in the motif, Pro-X-Ser-Pro, a consensus sequence recognized by members of the MAP kinase family (15Kennelly P.J. Krebs E.G. J. Biol. Chem. 1991; 266: 15555-15558Abstract Full Text PDF PubMed Google Scholar). All 4 of these serines are located exactly 4 amino acids downstream of potential tyrosine phosphorylation sites, and one (serine 612) is close to the major PI 3-kinase binding site, tyrosine 608. Moreover, serine 612 is phosphorylated upon PKC activation in a human kidney fibroblasts cell line (293 cells) (16DeFea K. Roth R.A. Biochemistry. 1997; 36: 181-189Crossref PubMed Scopus (31) Google Scholar), and a mutant IRS-1 lacking this site is less inhibited by PKC activation. Since p42 and p44 MAP kinase activity are known to be stimulated by PKC (via the sequential activation of Raf and the MAP kinase kinase I (MEK1)), these kinases are potential candidates for mediating the phosphorylation of IRS-1 at this site (17Blumer K.J. Johnson G.L. Trends Biochem. Sci. 1994; 19: 236-240Abstract Full Text PDF PubMed Scopus (420) Google Scholar, 18Robinson M.J. Cobb M.H. Curr. Opin. Cell Biol. 1997; 9: 180-186Crossref PubMed Scopus (2275) Google Scholar, 19Seger R. Krebs E.G. FASEB J. 1995; 9: 726-735Crossref PubMed Scopus (3188) Google Scholar). Two other subfamilies of MAP kinases, the stress-activated protein kinases (also called c-Jun N-terminal kinases) and p38 MAP kinase, are activated by a number of conditions causing a cellular stress response, such as osmotic shock, ultraviolet irradiation, transcriptional and translational inhibitors, and inflammatory cytokines (17Blumer K.J. Johnson G.L. Trends Biochem. Sci. 1994; 19: 236-240Abstract Full Text PDF PubMed Scopus (420) Google Scholar, 18Robinson M.J. Cobb M.H. Curr. Opin. Cell Biol. 1997; 9: 180-186Crossref PubMed Scopus (2275) Google Scholar, 19Seger R. Krebs E.G. FASEB J. 1995; 9: 726-735Crossref PubMed Scopus (3188) Google Scholar). Thus, some of the same factors known to activate the various members of the MAP kinase family have been implicated in cellular insulin resistance.Previous studies have shown that PKC activation, both in CHO cells tranfected with PKCα (CHO/PKC cells) and in 293 cells stably expressing IRS-1 (293/IRS), results in decreased insulin-stimulated tyrosine phosphorylation of IRS-1 and a decrease in its subsequent association with PI 3-kinase in whole cells. In the same studies, no decrease in IR autophosphorylation or in vitro kinase activity was observed (7Chin J.E. Dickens M. Tavare J.M. Roth R.A. J. Biol. Chem. 1993; 268: 6338-6347Abstract Full Text PDF PubMed Google Scholar, 16DeFea K. Roth R.A. Biochemistry. 1997; 36: 181-189Crossref PubMed Scopus (31) Google Scholar). More recently this inhibition was found to correspond to the activation of a cytosolic serine kinase capable of phosphorylating IRS-1 and a synthetic peptide containing the sequence corresponding to serine 612 (16DeFea K. Roth R.A. Biochemistry. 1997; 36: 181-189Crossref PubMed Scopus (31) Google Scholar). The presence of a phosphate on serine 612 in this peptide also appears to inhibit the ability of the IR to phosphorylate the tyrosine in this peptide which corresponds to tyrosine 608 in IRS-1 (16DeFea K. Roth R.A. Biochemistry. 1997; 36: 181-189Crossref PubMed Scopus (31) Google Scholar), the major PI 3-kinase binding site.The present studies were directed at identifying the Ser/Thr kinase responsible for phosphorylating IRS-1 and this IRS-1 peptide after phorbol 12-myristate 13-acetate (PMA) stimulation. Since the serine phosphorylation site in this peptide corresponds to a MAP kinase consensus phosphorylation site, we focused on the possibility that MAP kinase was responsible for this activity. A variety of approaches were utilized to investigate this question, including the use of a specific inhibitor of the upstream regulator of MAP kinase, MEK1, in vitro phosphorylation of IRS-1 and its peptide by recombinant ERK2, as well as expression of a regulatable Raf so that MAP kinase could be independently activated in cells.EXPERIMENTAL PROCEDURESMaterialsMonoclonal anti-Myc 9E10 antibody for immunoprecipitation was purchased from Berkeley Antibody Co. Monoclonal anti-p85 hybridoma supernatant was a gift from Dr. Kozui Shii. Polyclonal anti-IRS-1 (C terminus) antibody for Western blot analysis was purchased from Upstate Biotechnology Inc. Anti-phosphotyrosine antibody (RC20) coupled to horseradish peroxidase was obtained from Transduction Laboratories. Protein G-agarose was purchased from Pharmacia Biotech Inc. The antibody to active MAP kinase was from Promega. A polyclonal antisera to the estrogen receptor (HC-20) was purchased from Santa Cruz Biochemicals. SB203580 was a gift of Dr. John Lee, SmithKline Beecham. [γ-32P]ATP was purchased from DuPont NEN. Purified PI was obtained from Avanti Biochemicals. Immobilon membranes were obtained from Bio-Rad. PMA was purchased from LCI. Plasmid preparation and DNA purification kits were purchased from QIAGEN. pcDNAzeo plasmid was obtained from INVITROGEN. Enzymes for molecular biology were obtained from New England Biolabs and Life Technologies, Inc. SDS-PAGE reagents were purchased from Life Technologies, Inc. IRS-1 peptides were synthesized by the Beckman PAN facility. All other reagents were purchased from Sigma.MethodsPlasmid Construction and Bacterial Protein ExpressionFor the transient transfection studies, the 2-kilobase pair insert of pBABE-ΔRaf:ER (a gift of Dr. Martin McMahon, DNAX) (20McCarthy S.A. Chen D. Yang B.S. Garcia Ramirez J.J. Cherwinski H. Chen X.R. Klagsbrun M. Hauser C.A. Ostrowski M.C. McMahon M. Mol. Cell. Biol. 1997; 17: 2401-2412Crossref PubMed Scopus (151) Google Scholar) was subcloned into pcDNAzeo. To make the GST-IRS-1 fusion protein, anEcoRI fragment from pucIRS (16DeFea K. Roth R.A. Biochemistry. 1997; 36: 181-189Crossref PubMed Scopus (31) Google Scholar) containing nucleotides 2112–3283 of IRS-1 plus a 17 nucleotide stretch of the puc18 polylinker was cloned into pGEX4T2 (Invitrogen) to give pGEX-IRS-1B/R. The expressed fusion protein is approximately 70 kDa, corresponding to GST and residues 509–890 of IRS-1. For the Far Western studies, a 1-liter culture of pGEX-p852SH2 (21Wood E.R. McDonald O.B. Sahyoun N. J. Biol. Chem. 1992; 267: 14138-14144Abstract Full Text PDF PubMed Google Scholar) (a gift of Dr. Edgar Wood, Burroughs Welcome) in BL21 cells was induced with 0.4 mmisopropyl-1-thio-β-d-galactopyranoside for 3 h at 30 °C. The cells were harvested, resuspended in PBS supplemented with 2 mm EDTA, 1 mm dithiothreitol, and protease inhibitors, sonicated on ice for a total of 3 min after which 1% Triton X-100 was added. Lysed cells were centrifuged for 20 min at 13,000 rpm, and supernatants were filtered through a 0.25-μm filter and incubated with glutathione agarose at 4 °C for 2 h. GST-p85 was eluted with 1 ml of 15 mm glutathione and stored at 4 °C. The final concentration of eluted protein was approximately 800 μg/ml. For in-gel kinase assays, 1-liter cultures of BL21 cells containing pGEX-IRS-1B/R were induced with 1 mmisopropyl-1-thio-β-d-galactopyranoside for 2 h at 30 °C. Cells were harvested, resuspended in 6 m urea, PBS, and centrifuged for 20 min at 13,000 rpm. The supernatants were dialyzed overnight against PBS to remove urea. They were then incubated with glutathione agarose for 2 h at 4 °C. The GST-IRS-1B/R was eluted with 2 ml of 40 mm glutathione and stored at 4 °C. The final concentration of eluted protein was approximately 500 μg/ml.Cell Culture and TransfectionsEpitope-tagged IRS-1 was transiently coexpressed with pcDNA-Raf:ER or pcDNAzeo (vector control) in 293T (a 293 line which constitutively expresses T antigen) or pBABE-Raf:ER/puro was stably expressed in 3T3-L1 cells by retroviral infection (16DeFea K. Roth R.A. Biochemistry. 1997; 36: 181-189Crossref PubMed Scopus (31) Google Scholar). Both cell lines were cultured in Dulbecco's modified Eagle's medium containing 10% fetal calf serum (FCS). Transient transfections were performed in 293T cells using the calcium phosphate precipitation method as described previously (16DeFea K. Roth R.A. Biochemistry. 1997; 36: 181-189Crossref PubMed Scopus (31) Google Scholar). Fourteen to 17 h after transfection, cells were incubated in Dulbecco's modified Eagle's medium containing 10% FCS for 24 h, at which point they were split 1:2 or 1:4 and grown for an additional 24–36 h. Retroviral infections were performed as described elsewhere (22Pear W.S. Nolan G.P. Scott M.L. Baltimore D. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8392-8396Crossref PubMed Scopus (2283) Google Scholar). Cells were split 1:10 and 1:20 in Dulbecco's modified Eagle's medium/FCS containing 1.5 μg/ml puromycin 48 h after infection, and after 7 days surviving cell populations were screened for expression of Raf:ER by Western blotting with antibodies to the estrogen receptor. Positive cell populations were identified and maintained in Dulbecco's modified Eagle's medium, 10% FCS.In Situ Tyrosine Phosphorylation of IRS-1 and p85 BindingTen- cm dishes of 293 cells stably expressing IRS-Myc (293/IRS) were serum-starved for 16 h and then treated with or without 1.6 μm PMA for 20 min followed by 5 min in the presence or absence of 1 μm insulin. For the 3T3-L1-Raf:ER cells, they were differentiated as described, serum-starved for 16 h, followed by treatment with or without 1 μm 4-hydroxytamoxifen (4-HT) for 2 h, then incubated for 5 min in the presence or absence of 1 μm insulin. 293T cells, transiently transfected with IRS-Myc and pcDNA-Raf:ER as described above, were serum-starved for 16 h and then treated with or without 1 μm 4-HT for 1 h, followed by treatment with or without 1 μm insulin. Cells were washed one time in 50 mm Hepes, pH 7.6, 150 mm NaCl (HBS) and lysed on ice for 20 min in lysis buffer A (50 mmHepes, pH 7.6, 150 mm NaCl, 1% Triton X-100, 2 mm vanadate, 100 mm NaF, 2.5 mmsodium pyrophosphate, 10 mm EDTA, 0.25 mmphenylmethylsulfonyl fluoride, 10 μg/ml aprotinin), and adsorbed with either anti-Myc or anti-p85 antibodies bound to protein G agarose to isolate total IRS-1 or p85-associated IRS-1, respectively. After 3 h at 4 °C, the agarose beads were washed two times in WGBT (50 mm Hepes, pH 7.6, 150 mm NaCl, 0.1% bovine serum albumin, and 0.1% Triton X-100) and one time in phosphate-buffered saline, and the bound proteins were eluted by boiling in gel loading buffer (40% glycerol, 1% SDS, 50 mm Tris, pH 6.8, 1 m dithiothreitol). After separation of the samples by SDS-PAGE and transfer to Immobilon, the membrane was probed with an anti-phosphotyrosine antibody coupled to horseradish peroxidase and analyzed by enhanced chemiluminescence. Membranes were stripped of anti-phosphotyrosine antibodies by incubation in 2% phenyl phosphate in Tris/NaCl for 3 h, washed two times, and reprobed with either the monoclonal anti-Myc antibody, a polyclonal antibody to p85, or a polyclonal antibody to IRS-1, followed by visualization by secondary antibodies coupled to alkaline phosphatase and development with chromagenic substrates. Quantitation of the bands was performed by scanning and the amount of p85 associated or tyrosine phosphorylated IRS-1 was normalized to the total amounts of p85 or IRS-1 present in each lane and fold-insulin stimulation was calculated as the level of tyrosine phosphorylation in the immunoprecipitates from insulin-treated cells versusuntreated cells.PI 3-Kinase Activity293 cells stably expressing IRS-Myc were serum-starved for 16 h and first treated with or without 50 μm PD98059 (a gift of Dr. Alan Saltiel, Warner Lambert), followed by treatment with or without 1 μm PMA, and then incubated with or without 1 μm insulin. Cells were lysed (20 mm Tris, pH 8.0, 137 mm NaCl, 1% Nonidet-P40, 10% glycerol, 1 mm CaCl2, 1 mm MgCl2, 200 μm vanadate, 1 mm phenylmethylsulfonyl fluoride, and 10 μg/ml aprotinin), and the lysates were immunoprecipitated with anti-Myc antibodies bound to protein G-agarose. The beads were washed, incubated with 10 μg of PI and 5 μCi of [γ-32P]ATP for 5 min at room temperature, the reaction was terminated, and the phosphorylated PI was analyzed by thin layer chromatography as described previously (7Chin J.E. Dickens M. Tavare J.M. Roth R.A. J. Biol. Chem. 1993; 268: 6338-6347Abstract Full Text PDF PubMed Google Scholar). Bands were excised from the plates and counted.In Vitro Phosphorylation of IRS-1 by Recombinant ERK2293 cells expressing IRS-Myc were serum-starved 16 h and lysed in buffer A. Cell lysates were immunoprecipitated with anti-Myc antibodies bound to protein G-agarose. After 2 h, beads were washed two times in WGBT and one time in HBS, 0.1% Triton X-100 (WGB). Either 1 μg of activated recombinant ERK2, activated with 0.25 μg of recombinant MEK in kinase buffer MEK (10 mm Hepes, 10 mmMgCl2, 100 μm ATP, and 1 mg/ml bovine serum albumin) for 1 h at 30 °C, or 1 μg of unactivated ERK2 was added in 100 μl of MAP kinase buffer (final concentrations: 20 mm Hepes, 12 mm MgCl2, 10 μm ATP and 2 μCi of [γ-32P]ATP) for 30 min at 24 °C. Beads were washed twice in 1 m NaCl, 100 mm Tris, pH 7.6, and once in PBS, boiled in gel loading buffer, and analyzed on 7.5% SDS-PAGE, followed by transfer to Immobilon. Blots were probed with antibody to IRS-1; bands were excised and counted.Phosphorylation by IR and Far Western Blots with GST-p85IRS-1 was phosphorylated with ERK2 as described above, except that 1 mm cold ATP was added to MAP kinase buffer in place of the radioactive ATP. IR was prepared from one confluent 10-cm plate of 293 cells treated with 1.6 μm PMA, by incubation of the lysates with 60 μl of wheat germ agglutinin coupled to agarose and eluted with 60 μl of 0.3 m N-acetylglucosamine as described previously. Either 10 μl of eluted IR, or 10 μl of the elution buffer, were added to each IRS-1 sample in the presence of 1 μm insulin, 1 mm ATP, 10 mm MgCl2, and 10 mm MnCl2 and incubated at room temperature for 20 min. Beads were washed three times in WGB followed by one wash in PBS, and IRS-1 was eluted and analyzed on 7.5% SDS-PAGE, followed by transfer to Immobilon. Fifty μg of GST-p85 were preincubated with 100 μg of anti-GST antibody. After blocking, the membrane was incubated in a 10-ml solution containing a 1:500 dilution of the GST-p85/antibody complex in 100 mm Tris, pH 7.6, 50 mm NaCl, 0.2% Tween, 3% bovine serum albumin, 1% FCS, and 5% milk for 2 h at room temperature. The mixture was then incubated with secondary antibody coupled to horseradish peroxidase and developed by enhanced chemiluminescence as described for anti-phosphotyrosine blots. Blots were stripped with 200 mm glycine, pH 3.0, for 10 min, washed with TBS three times for 30 min each, and reprobed with anti-IRS-1 antibody. Quantitation was performed by scanning, and values were normalized to the total IRS-1 present in each lane.Phosphorylation of IRS-1 Peptide by Cytosolic Lysates and ERK2293T cells, transfected with IRS-Myc and ER-Raf, were serum-starved for 16 h, followed by treatment with or without 1 μm 4-HT for 1 h. Cells were scraped into 150 μl of buffer A (20 mm Tris, pH 7.6, 220 mm sucrose, 10 mm EDTA, 10 mm Na-PPi, 2 mm Na-vanadate, 10 mm NaF, 10 μg/ml aprotinin, 1 mm phenylmethylsulfonyl fluoride, 250 μm okadaic acid), homogenized by 15 strokes, sonicated three times for 15 s, and centrifuged at 200,000 ×g for 20 min. The supernatants (the cytosol) were removed, and Hepes (60 mm, final concentration) and MgCl2 (10 mm) were added. Sixty μl of cytosol were added to reaction tubes containing 100 μm IRS-1 peptide (KKHTDDGYMPMSPGVA) and 20 mm Tris, pH 7.6, 10 mm MgCl2, 15 μm ATP, and 5 μCi of [γ-32P]ATP. After 20 min at 24 °C, reactions were terminated by the addition of 100 μl of buffer U (125 mmTris, pH 6.8, 6 m urea), and the samples were electrophoresed on 40% PAGE. Phosphorylated peptide bands were identified by autoradiography, excised, and counted. HEK 293/IRS cells were treated with or without 1.6 μm PMA for 20 min and lysed in lysis buffer A. Lysates were incubated with anti-ERK2 antibodies coupled to agarose for 2 h, the beads were washed three times in WGB, and then the bound ERK2 was incubated with varying concentrations of IRS-1 peptide and 100 μl of kinase buffer (20 mm Tris, pH 7.6, 10 mm MgCl2, 15 μm ATP, and 5 μCi of [γ-32P]ATP) for 20 min at 24 °C. One hundred μl of buffer U were added to the peptide-containing supernatants, and the samples were analyzed as described for cytosolic lysates.Coprecipitation of IRS-1 and ERK2293/IRS cells and 293T cells transfected with IRS-Myc and ER-Raf were treated with and without either 1.6 μm PMA or 1 μm 4-HT, respectively and lysed, and the lysates were immunoprecipitated with anti-Myc antibodies bound to protein G-agarose for 2 h. IRS-1 immunoprecipitates were either incubated with 100 μl of kinase mix with or without 100 μm IRS-1 peptide as described above or analyzed by 10% SDS-PAGE, followed by transfer to Immobilon and Western blotting with antibodies to ERK2. Blots were stripped and probed with antibodies to IRS-1. IRS-1 beads that had been incubated with kinase mix and peptide were microcentrifuged; peptide-containing supernatants were added to 100 μl of buffer U and analyzed by electrophoresis on 40% PAGE, and IRS-1 beads were washed and analyzed by electrophoresis on 7.5% SDS-PAGE and transferred to Immobilon. Phosphorylation was determined by autoradiography. Membranes were probed with antibodies to IRS-1, and either IRS-1 bands or peptide bands were excised and counted.In Gel Kinase AssaysConfluent 10-cm plates of 293/IRS cells were treated with or without 1.6 μm PMA for 20 min following overnight serum starvation, lysed in 200 μl of 1% SDS, PBS, passaged 10 times through an 18-gauge needle followed by 10 passages through a 21-gauge needle. Equal volumes of SDS loading buffer were added, and samples were boiled for 5 min and analyzed on 10% SDS-PAGE gels containing either 0.1 mg/ml glutathione transferase or GST-IRS-1B/R. Gels were soaked for 4 h in 40 mm Hepes containing 2% isopropanol to remove SDS. Gels were incubated for 1 h in 30 ml of reaction buffer containing 20 mmHepes, 10 mm MgCl2, and 1 μCi/ml [γ-32P]ATP. Gels were washed three times in 40 mm Hepes for 1 h, followed by 1% Na-PPiin 5% trichloroacetic acid for 1 h, stained 10 min with Coomassie Blue in 25% MeOH, 10% acetic acid, destained overnight, dried, and autoradiographed.Detection of Activated MAP KinaseConfluent 10-cm plates of 293/IRS cells were treated with 1.6 μm PMA for 20 min, or 3T3-L1 cells stably expressing ΔRaf:ER were treated with 1 μm 4-HT for 1 h. Cells were lysed as described previously; 10-μl samples (corresponding to 1/50 of a plate) were analyzed on 12.5% SDS-PAGE followed by transfer to Immobilon and immunoblotting with anti-active MAP kinase antibodies (Promega).DISCUSSIONPrior studies have indicated that increased Ser/Thr phosphorylation of IRS-1 stimulated by a variety of factors interferes with both the ability of this molecule to be tyrosine-phosphorylated after insulin treatment and its ability to subsequently associate with the PI 3-kinase (7Chin J.E. Dickens M. Tavare J.M. Roth R.A. J. Biol. Chem. 1993; 268: 6338-6347Abstract Full Text PDF PubMed Google Scholar, 8Takayama S. White M.F. Kahn C.R. J. Biol. Chem. 1988; 263: 3440-3447Abstract Full Text PDF PubMed Google Scholar, 9Tanti J.-F. Gremeaux T. Van Obberghen E. Le Marchand-Brustel Y. J. Biol. Chem. 1994; 269: 6051-6057Abstract Full Text PDF PubMed Google Scholar, 10Hotamisligil G.S. Peraldi P. Budavari A. Ellis R. White M.F. Spiegelman B.M. Science. 1996; 271: 665-668Crossref PubMed Scopus (2178) Google Scholar, 11Kanety 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, 12Haddad T.C. Conover C.A. J. Biol. Chem. 1997; 272: 19525-19531Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). Recent studies have implicated several serines in the IRS-1 molecule with this inhibitory response, including serines 612, 632, 662 and/or 731 (16DeFea K. Roth R.A. Biochemistry. 1997; 36: 181-189Crossref PubMed Scopus (31) Google Scholar, 30Mothe I. Van Obberghen E. J. Biol. Chem. 1996; 271: 11222-11227Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar). All 4 of these serine residues are in the consensus motif for MAP kinase phosphorylation sites (15Kennelly P.J. Krebs E.G. J. Biol. Chem. 1991; 266: 15555-15558Abstract Full Text PDF PubMed Google Scholar). In the present studies we provide evidence that MAP kinase can phosphorylate IRS-1 and is involved in the subsequent inhibition of insulin signaling. First, we have demonstrated that both recombinant ERK2 and ERK2 immunoprecipitated from PMA-treated 293/IRS cells have the ability to phosphorylate both full-length IRS-1 and a synthetic peptide corresponding to the serine 612 of IRS-1 (Fig. 2, Aand C). Second, we show that the major renaturable kinases in cell lysates from PMA-treated cells which can phosphorylate an IRS-1 fusion protein (containing residues 509–890) comigrate with ERK1 and 2 (Fig. 2
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