Serine Phosphorylation of Insulin Receptor Substrate 1 by Inhibitor κB Kinase Complex

Insulin resistance contributes importantly to the pathophysiology of type 2 diabetes mellitus. One mechanism mediating insulin resistance may involve the phosphorylation of serine residues in insulin receptor substrate-1 (IRS-1), leading to impairment in the ability of IRS-1 to activate downstream phosphatidylinositol 3-kinase-dependent pathways. Insulin-resistant states and serine phosphorylation of IRS-1 are associated with the activation of the inhibitor κB kinase (IKK) complex. However, the precise molecular mechanisms by which IKK may contribute to the development of insulin resistance are not well understood. In this study, using phosphospecific antibodies against rat IRS-1 phosphorylated at Ser307 (equivalent to Ser312 in human IRS-1), we observed serine phosphorylation of IRS-1 in response to TNF-α or calyculin A treatment that paralleled surrogate markers for IKK activation. The phosphorylation of human IRS-1 at Ser312 in response to tumor necrosis factor-α was significantly reduced in cells pretreated with the IKK inhibitor 15 deoxy-prostaglandin J2 as well as in cells derived from IKK knock-out mice. We observed interactions between endogenous IRS-1 and IKK in intact cells using a co-immunoprecipitation approach. Moreover, this interaction between IRS-1 and IKK in the basal state was reduced upon IKK activation and increased serine phosphorylation of IRS-1. Data from in vitro kinase assays using recombinant IRS-1 as a substrate were consistent with the ability of IRS-1 to function as a direct substrate for IKK with multiple serine phosphorylation sites in addition to Ser312. Taken together, our data suggest that IRS-1 is a novel direct substrate for IKK and that phosphorylation of IRS-1 at Ser312 (and other sites) by IKK may contribute to the insulin resistance mediated by activation of inflammatory pathways. Insulin resistance contributes importantly to the pathophysiology of type 2 diabetes mellitus. One mechanism mediating insulin resistance may involve the phosphorylation of serine residues in insulin receptor substrate-1 (IRS-1), leading to impairment in the ability of IRS-1 to activate downstream phosphatidylinositol 3-kinase-dependent pathways. Insulin-resistant states and serine phosphorylation of IRS-1 are associated with the activation of the inhibitor κB kinase (IKK) complex. However, the precise molecular mechanisms by which IKK may contribute to the development of insulin resistance are not well understood. In this study, using phosphospecific antibodies against rat IRS-1 phosphorylated at Ser307 (equivalent to Ser312 in human IRS-1), we observed serine phosphorylation of IRS-1 in response to TNF-α or calyculin A treatment that paralleled surrogate markers for IKK activation. The phosphorylation of human IRS-1 at Ser312 in response to tumor necrosis factor-α was significantly reduced in cells pretreated with the IKK inhibitor 15 deoxy-prostaglandin J2 as well as in cells derived from IKK knock-out mice. We observed interactions between endogenous IRS-1 and IKK in intact cells using a co-immunoprecipitation approach. Moreover, this interaction between IRS-1 and IKK in the basal state was reduced upon IKK activation and increased serine phosphorylation of IRS-1. Data from in vitro kinase assays using recombinant IRS-1 as a substrate were consistent with the ability of IRS-1 to function as a direct substrate for IKK with multiple serine phosphorylation sites in addition to Ser312. Taken together, our data suggest that IRS-1 is a novel direct substrate for IKK and that phosphorylation of IRS-1 at Ser312 (and other sites) by IKK may contribute to the insulin resistance mediated by activation of inflammatory pathways. Many factors implicated in the development of insulin resistance such as TNF-α 1The abbreviations used are: TNF-α, tumor necrosis factor-α: NFκB, nuclear factor κB; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; MEKK, MEK kinase; JNK, c-Jun N-terminal kinase; ERK, extracellular signal-regulated kinase; HEK, human embryonic kidney; HA, hemagglutinin; IRS, insulin receptor substrate; IP, immunoprecipitation; GST, glutathione S-transferase; pIRS-1, serine-phosphorylated IRS-1; 15dPGJ2, 15-deoxy-prostaglandin J2; NEMO, NFκB essential modulator. (1Peraldi P. Spiegelman B. Mol. Cell. Biochem. 1998; 182: 169-175Google Scholar, 2Hotamisligil G.S. J. Intern. Med. 1999; 245: 621-625Google Scholar), free fatty acids (3Boden G. Diabetes. 1997; 46: 3-10Google Scholar, 4Shulman G.I. J. Clin. Invest. 2000; 106: 171-176Google Scholar), and serine phosphatase inhibitors (5Jullien D. Tanti J.F. Heydrick S.J. Gautier N. Gremeaux T. Van Obberghen E. 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Adiponectin, a cytokine secreted by adipose cells whose plasma levels are negatively correlated with insulin resistance (12Maeda K. Okubo K. Shimomura I. Funahashi T. Matsuzawa Y. Matsubara K. Biochem. Biophys. Res. Commun. 1996; 221: 286-289Google Scholar), inhibits IKK activity in cells (13Ouchi N. Kihara S. Arita Y. Okamoto Y. Maeda K. Kuriyama H. Hotta K. Nishida M. Takahashi M. Muraguchi M. Ohmoto Y. Nakamura T. Yamashita S. Funahashi T. Matsuzawa Y. Circulation. 2000; 102: 1296-1301Google Scholar). Moreover, diet-induced insulin resistance is ameliorated in IKK2-deficient mice (14Yuan M. Konstantopoulos N. Lee J. Hansen L. Li Z.W. Karin M. Shoelson S.E. Science. 2001; 293: 1673-1677Google Scholar). Because IKK and NFκB are major components of the intracellular inflammatory pathway, a cross-talk between metabolic and inflammatory signaling pathways may play an important role in the development of insulin resistance and the pathophysiology of major public health problems such as diabetes and obesity. However, molecular mechanisms by which IKK may specifically interact with metabolic insulin signaling pathways are not well understood. IKK is a serine kinase that controls the activation of NFκB, a ubiquitous transcription factor closely associated with inflammation (15Karin M. J. Biol. Chem. 1999; 274: 27339-27342Google Scholar, 16Baeuerle P.A. Henkel T. Annu. Rev. Immunol. 1994; 12: 141-179Google Scholar, 17Baldwin Jr., A.S. Annu. Rev. Immunol. 1996; 14: 649-683Google Scholar, 18Ghosh S. Karin M. Cell. 2002; 109 (Suppl.): S81-S96Google Scholar). In addition to inflammation, NFκB also involves in other biological actions including apoptosis, oncogenesis, and cell differentiation (16Baeuerle P.A. Henkel T. Annu. Rev. Immunol. 1994; 12: 141-179Google Scholar, 17Baldwin Jr., A.S. Annu. Rev. Immunol. 1996; 14: 649-683Google Scholar). Before activation, NFκB is bound to the inhibitor κB (IκB) protein whose isoforms include IκBα, IκBβ, and IκBγ (16Baeuerle P.A. Henkel T. Annu. Rev. Immunol. 1994; 12: 141-179Google Scholar, 17Baldwin Jr., A.S. Annu. Rev. Immunol. 1996; 14: 649-683Google Scholar). This association between IκB and NFκB results in the cytosolic localization of NFκB. In response to inflammatory stimuli such as TNF-α and interleukin-1, IκB proteins are degraded in the proteasome, leading to nuclear translocation of NFκB. IκBα degradation is controlled by inducible phosphorylation of Ser32 and Ser36 in the IκBα protein. The phosphorylation of these two serines is directly catalyzed by the IKK complex, which is composed of at least three subunits (IKK1/α, IKK2/β, and NEMO/IKKγ) (18Ghosh S. Karin M. Cell. 2002; 109 (Suppl.): S81-S96Google Scholar). Although both IKK1 and IKK2 can phosphorylate IκB proteins in vitro, IKK2 is indispensable for NFκB activation in vivo (19Li Q. Van Antwerp D. Mercurio F. Lee K.F. Verma I.M. Science. 1999; 284: 321-325Google Scholar). Dominant negative mutants of IKK2 are frequently used to block NFκB pathways in intact cells. Activation of IKK can be initiated by a variety of kinases including protein kinase C (20Tojima Y. Fujimoto A. Delhase M. Chen Y. Hatakeyama S. Nakayama K. Kaneko Y. Nimura Y. Motoyama N. Ikeda K. Karin M. Nakanishi M. Nature. 2000; 404: 778-782Google Scholar, 21Sun Z. Arendt C.W. Ellmeier W. Schaeffer E.M. Sunshine M.J. Gandhi L. Annes J. Petrzilka D. Kupfer A. Schwartzberg P.L. Littman D.R. Nature. 2000; 404: 402-407Google Scholar, 22Su T.T. Guo B. Kawakami Y. Sommer K. Chae K. Humphries L.A. Kato R.M. Kang S. Patrone L. Wall R. Teitell M. Leitges M. Kawakami T. Rawlings D.J. Nat. Immunol. 2002; 3: 780-786Google Scholar), NFκB-inducing kinase (23Malinin N.L. Boldin M.P. Kovalenko A.V. Wallach D. Nature. 1997; 385: 540-544Google Scholar), and MEK kinase 1 (MEKK1) (24Lange-Carter C.A. Pleiman C.M. Gardner A.M. Blumer K.J. Johnson G.L. Science. 1993; 260: 315-319Google Scholar). In some cases, NFκB can be activated in the absence of IκB protein degradation (e.g.vanadium-induced NFκB activation) (25Imbert V. Rupec R.A. Livolsi A. Pahl H.L. Traenckner E.B. Mueller-Dieckmann C. Farahifar D. Rossi B. Auberger P. Baeuerle P.A. Peyron J.F. Cell. 1996; 86: 787-798Google Scholar). Insulin receptor substrate (IRS) proteins are crucial signaling molecules mediating metabolic actions of insulin (26White M.F. Yenush L. Curr. Top. Microbiol. Immunol. 1998; 228: 179-208Google Scholar, 27Saltiel A.R. Kahn C.R. Nature. 2001; 414: 799-806Google Scholar). Four IRS isoforms (IRS-1, IRS-2, IRS-3, and IRS-4) have been identified that are expressed in a tissue-specific manner (26White M.F. Yenush L. Curr. Top. Microbiol. Immunol. 1998; 228: 179-208Google Scholar). The activated insulin receptor phosphorylates IRS proteins on multiple tyrosine residues (28Kahn B.B. Flier J.S. J. Clin. Invest. 2000; 106: 473-481Google Scholar) that serve as docking sites for downstream mediators of metabolic actions including phosphatidylinositol-3 kinase (26White M.F. Yenush L. Curr. Top. Microbiol. Immunol. 1998; 228: 179-208Google Scholar). IRS proteins also undergo serine phosphorylation, which regulates its function (26White M.F. Yenush L. Curr. Top. Microbiol. Immunol. 1998; 228: 179-208Google Scholar, 29Saltiel A.R. Cell. 2001; 104: 517-529Google Scholar). For example, the phosphorylation of rodent IRS-1 at Ser307or Ser612 (Ser312 and Ser616 in human IRS-1, respectively) results in the impairment of metabolic insulin signaling pathways (30Aguirre V. Werner E.D. Giraud J. Lee Y.H. Shoelson S.E. White M.F. J. Biol. Chem. 2002; 277: 1531-1537Google Scholar, 31De Fea K. Roth R.A. Biochemistry. 1997; 36: 12939-12947Google Scholar). IRS-1 is a substrate for multiple serine kinases including JNK (32Aguirre V. Uchida T. Yenush L. Davis R. White M.F. J. Biol. Chem. 2000; 275: 9047-9054Google Scholar, 33Rui L. Aguirre V. Kim J.K. Shulman G.I. Lee A. Corbould A. Dunaif A. White M.F. J. Clin. Invest. 2001; 107: 181-189Google Scholar), ERK (31De Fea K. Roth R.A. Biochemistry. 1997; 36: 12939-12947Google Scholar, 34De Fea K. Roth R.A. J. Biol. Chem. 1997; 272: 31400-31406Google Scholar, 35Engelman J.A. Berg A.H. Lewis R.Y. Lisanti M.P. Scherer P.E. Mol. Endocrinol. 2000; 14: 1557-1569Google Scholar), mammalian target of rapamycin (36Haruta T. Uno T. Kawahara J. Takano A. Egawa K. Sharma P.M. Olefsky J.M. Kobayashi M. Mol. Endocrinol. 2000; 14: 783-794Google Scholar, 37Ozes O.N. Akca H. Mayo L.D. Gustin J.A. Maehama T. Dixon J.E. Donner D.B. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 4640-4645Google Scholar, 38Li J. DeFea K. Roth R.A. J. Biol. Chem. 1999; 274: 9351-9356Google Scholar), Akt/protein kinase B (39Ogihara T. Isobe T. Ichimura T. Taoka M. Funaki M. Sakoda H. Onishi Y. Inukai K. Anai M. Fukushima Y. Kikuchi M. Yazaki Y. Oka Y. Asano T. J. Biol. Chem. 1997; 272: 25267-25274Google Scholar, 40Paz K. Liu Y.F. Shorer H. Hemi R. LeRoith D. Quan M. Kanety H. Seger R. Zick Y. J. Biol. Chem. 1999; 274: 28816-28822Google Scholar), protein kinase Cζ (41Ravichandran L.V. Esposito D.L. Chen J. Quon M.J. J. Biol. Chem. 2001; 276: 3543-3549Google Scholar, 42Liu Y.F. Paz K. Herschkovitz A. Alt A. Tennenbaum T. Sampson S.R. Ohba M. Kuroki T. LeRoith D. Zick Y. J. Biol. Chem. 2001; 276: 14459-14465Google Scholar), glycogen synthase kinase-3 (43Eldar-Finkelman H. Krebs E.G. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9660-9664Google Scholar), and casein kinase II (44Tanasijevic M.J. Myers Jr., M.G. Thoma R.S. Crimmins D.L. White M.F. Sacks D.B. J. Biol. Chem. 1993; 268: 18157-18166Google Scholar). However, IRS-1 has not previously been identified as a direct substrate for kinases that participate in inflammatory pathways related to activation of NFκB. In the this study, we evaluate the possibility that IRS-1 is a direct substrate for IKK that can provide a mechanism for cross-talk between metabolic and inflammatory signaling pathways. Cell lines including human hepatoma HepG2 (HB-8065), human embryo kidney (HEK) 293 (CRL-1573) and mouse fibroblast 3T3-L1 (CL-173) were purchased from the American Type Culture Collection. All cells were maintained in Dulbecco's modified Eagle's culture medium supplemented with 10% fetal calf serum. For 3T3 cells, 4 mm glutamine was used in the culture medium. Wild-type and IKK1/2 double knock-out (IKK1/2−/−) cell lines were a gift from Dr. Inder Verma at the Salk Institute (La Jolla, CA). Antibodies against phospho-IRS-1 (Ser307) (number 07-247) and IKK2 (number 05-535), and λ-protein phosphatase number 14-405) were obtained from Upstate Biotechnology (Lake Placid, NY). Antibodies against IRS-1 (sc-7200), IRS-2 (sc-8299), IKK1 (sc-7182), HA (sc-7392), IκBα (sc-371), IκBβ (sc-945), and pJUN (sc-822) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). β-actin antibody (ab6276) was obtained from Abcam (Cambridge, United Kingdom). Calyculin A (EI-192) and SP600125 (EI-305) were from Biomol (Plymouth Meeting, PA). TNF-α (210-TA-010) was obtained from R&D systems (Minneapolis, MN). 15-Deoxy-prostaglandin J2(15dPGJ2, 538927) was from Calbiochem. Whole cell lysates were made by sonication in lysis buffer (1% Triton X-100, 50 mm KCl, 25 mm Hepes, pH 7.8, 10 μg/ml leupeptin, 20 μg/ml aprotinin, 125 μm dithiothreitol, 1 mmphenylmethylsulfonyl fluoride, 1 mm sodium orthovanadate). Samples (100 μg of total protein) in 50 μl of reducing sample buffer were boiled for 3 min and resolved on 6% mini-SDS-PAGE for 90 min at 100 v. The contents of the gel were transferred onto polyvinylidene difluoride membrane (162–0184, Bio-Rad) at 21 v for 120 min. The membrane was pre-blotted in milk buffer for 20 min and then immunoblotted with primary antibody for 1–24 h followed by secondary antibody for 30 min. Horseradish peroxidase-conjugated secondary antibodies (NA934V or NA931, Amersham Biosciences) were used in conjunction with chemiluminescence reagent (NEL-105, PerkinElmer Life Sciences). To detect multiple signals from a single membrane, membranes were treated with a stripping buffer (59 mmTris-HCI, 2% SDS, 0.75% 2-mercaptoethanol) for 20 min at 37 °C prior to re-blotting with a different antibody. λ-Protein phosphatase (number 14-405) was used to dephosphorylate IRS-1 in whole cell lysates of calyculin-treated HepG2 cells. The reaction was performed according to the manufacturer's instruction. 100 μg of protein was incubated with 500 units of enzyme in λ-phosphatase reaction buffer at 30 °C for 20 min. The reaction was stopped by adding Western blot sample buffer and boiling for 3 min. The dephosphorylated proteins were analyzed by immunoblotting. Immunoprecipitation was carried out using whole cell lysate (400 μg of total protein), 2–4 μg of antibody, and 20 μl of protein A- or protein G-Sepharose beads (Amersham Biosciences). After treatment, cells were lysed by sonication in a cell lysis buffer (1% Nonidet P-40, 50 mm Hepes, pH 7.6, 250 mm NaCl, 10% glycerol, 1 mm EDTA, 20 mmβ-glycerophosphate, 1 mm sodium orthovanadate, 1 mm sodium metabisulfite, 1 mm benzamidine hydrochloride, 10 μg/ml leupeptin, 20 μg/ml aprotinin, 1 mm phenylmethylsulfonyl fluoride). IP was conducted by incubating whole cell lysates with antibody for 3–4 h at 4 °C. The immune complex was washed five times in cell lysis buffer before being used for immunoblotting or for the kinase assay. To conduct the kinase assay, the immune complex was washed two more times in a kinase assay buffer (20 mm Hepes, pH 7.6, 20 mmMgCl2, 20 mm β-glycerophosphate, 1 mm dithiothreitol, 10 μm ATP, 1 mm EDTA, 1 mm sodium orthovanadate, 0.4 mm phenylmethylsulfonyl fluoride, 20 mmcreatine phosphate). The kinase assay was conducted at room temperature for 30 min in 20 μl of kinase assay buffer containing 5 μCi of [γ-32P]ATP. The product was resolved by SDS-PAGE and transferred onto polyvinylidene difluoride membrane for autoradiography or immunoblotting. An oligonucleotide containing the NFκB binding sequence in the human interleukin-6 gene promoter (−74TGGGATTTTCCCATGAGTCT−54) was synthesized as a NFκB binding probe. Nuclear extracts were prepared with a three-step procedure (48Ye J. Cippitelli M. Dorman L. Ortaldo J.R. Young H.A. Mol. Cell. Biol. 1996; 16: 4744-4753Google Scholar). The protein concentration was determined using BCA protein assay reagent (Pierce, Rockford, IL). The DNA-protein binding reaction was conducted in a 24-μl reaction mixture including 1 μg of poly(dI·dC) (Sigma), 3 μg of nuclear protein extract, 3 μg of bovine serum albumin, 4 × 104 cpm of32P-labeled oligonucleotide probe, and 12 μl of reaction buffer (24% glycerol, 24 mm Hepes, pH 7.9, 8 mm Tris-HCl, 2 mm EDTA, 2 mmdithiothreitol). After the addition of radiolabeled probe, the mixture was incubated for 20 min at room temperature and then resolved on a 5% acrylamide gel that had been pre-run at 170 V for 30 min with 0.5 × Tris borate buffer. The loaded gel was run at 200 V for 90 min, dried, placed on Kodak X-Omat AR film (Eastman Kodak Co.), and the film was developed after overnight exposure at −70 °C. HA-tagged fusion proteins of IKK1, IKK2, IRS-1, IRS-2, and Akt were expressed in HEK 293 cells by transient transfection. The plasmids for HA-IKK1, HA-IKK2, and kinase-dead HA-IKK2 were a gift from Dr. Michael Karin (Department of Pharmacology, University of California, San Diego, CA). The expression plasmids for HA-IRS-1 and HA-IRS-2 were constructed by inserting human IRS-1 or IRS-2 cDNA into pCIS2 expression vector (41Ravichandran L.V. Esposito D.L. Chen J. Quon M.J. J. Biol. Chem. 2001; 276: 3543-3549Google Scholar). Kinase-dead HA-Akt expression plasmid was from Dr. Bin-Hua Jiang (BMC Cancer Center, West Virginia University). Transient transfection was conducted using LipofectAMINE as reported previously (45Ye J. Zeidler P. Young S.H. Martinez A. Robinson V.A. Jones W. Baron P. Shi X. Castranova V. J. Biol. Chem. 2001; 276: 5360-5367Google Scholar). GST-IRS-1 expression plasmid for rat IRS-1 (amino acids 117–513) was from Dr. Xiao-Jian Sun (Endocrinology Division, University of Vermont College of Medicine). The purified GST-IRS-1 was prepared using a protocol described previously (48Ye J. Cippitelli M. Dorman L. Ortaldo J.R. Young H.A. Mol. Cell. Biol. 1996; 16: 4744-4753Google Scholar). TNF-α treatment of cells causes increased serine phosphorylation of IRS-1 as well as activation of IKK and NFκB (1Peraldi P. Spiegelman B. Mol. Cell. Biochem. 1998; 182: 169-175Google Scholar,15Karin M. J. Biol. Chem. 1999; 274: 27339-27342Google Scholar). To explore the relationship between IRS-1 serine phosphorylation and IKK activation, HepG2 cells were treated with TNF-α, and IRS-1 phosphorylation at Ser312 was examined using the phosphospecific antibody developed against phosphoserine 307 of rat IRS-1 (equivalent to Ser312 in human IRS-1). The IRS-1 phosphorylation was induced by TNF-α at 5 min, and this phosphorylation was sustained over 120 min (Fig. 1A, top panel). This phosphorylation of IRS-1 correlated with the disappearance of IκBα at 5 min (Fig. 1A, third panel), which is an indication of IKK activation. After 30 min, the level of IκBα started to increase and returned to the basal levels by 60–120 min. Calyculin is an inhibitor of protein serine phosphatases PP1 and PP2A (49Ishihara H. Martin B.L. Brautigan D.L. Karaki H. Ozaki H. Kato Y. Fusetani N. Watabe S. Hashimoto K. Uemura D. Biochem. Biophys. Res. Commun. 1989; 159: 871-877Google Scholar) that induces NFκB activity by activating IKK (50Sun S.C. Maggirwar S.B. Harhaj E. J. Biol. Chem. 1995; 270: 18347-18351Google Scholar). In calyculin-treated HepG2 cells, both IκBα degradation and IRS-1 phosphorylation were observed after 15 min (Fig. 1B,panels 1 and 5). IκBβ expression was also reduced slightly after 30 min of calyculin treatment. In addition, a mobility shift in IKK1 and IKK2 was observed at 30 and 60 min that is consistent with the serine phosphorylation and activation of these two kinases by calyculin (Fig. 1B, panels 3 and4). The IKK1 signal also increased in a time-dependent manner after calyculin treatment. The serine-phosphorylated IRS-1 exhibited reduced mobility on the gel, and this change resulted in two bands detected by the phosphospecific antibody. One band migrated at a level of ∼180 kDa, whereas the other band migrated at the level of 165 kDa. The intensity of both bands increased in a time-dependent manner. In addition, of the two forms of serine-phosphorylated IRS-1 (pIRS-1) seen in Fig. 1B, only the bottom band, not the top band, was recognized by the regular IRS-1 antibody. The bottom band was reduced significantly at the time points of 30 and 60 min when the serine phosphorylation was increased dramatically. In calyculin-treated cells, the signal intensity of the total IRS-1 was reduced at 15, 30, and 60 min (Fig. 1B, panel 2). This reduction may result from decreased antibody affinity to the pIRS-1 or a reduction in IRS-1 protein abundance. To distinguish between these two possibilities, whole cell lysates derived from calyculin-treated HepG2 cells were incubated with λ-protein phosphatase to dephosphorylate pIRS-1. The result shows that the IRS-1 protein signal was restored along with the gel mobility after dephosphosphorylation at serine residues (Fig. 1C), suggesting that the loss of IRS-1 signal is a result of reduced antibody affinity to the phosphorylated IRS-1. This is also supported by studies using a different IRS-1 antibody or using proteasome inhibitor (data not shown). Vanadate is a tyrosine phosphatase inhibitor that induces the DNA binding of NFκB through an alternative pathway in which IKK is not activated (25Imbert V. Rupec R.A. Livolsi A. Pahl H.L. Traenckner E.B. Mueller-Dieckmann C. Farahifar D. Rossi B. Auberger P. Baeuerle P.A. Peyron J.F. Cell. 1996; 86: 787-798Google Scholar). As expected, in vanadate-treated cells, IκBα protein levels and IKK mobility were not altered (Fig. 1B), whereas the DNA binding of NFκB was induced (Fig. 1D). Moreover, vanadate treatment did not result in phosphorylation of IRS-1 at Ser312 (Fig. 1B). To investigate the relationship between serine phosphorylation of IRS-1 and IKK activity more directly, we used the IKK inhibitor 15dPGJ2 (51Rossi A. Kapahi P. Natoli G. Takahashi T. Chen Y. Karin M. Santoro M.G. Nature. 2000; 403: 103-108Google Scholar). When HepG2 cells were pre-treated with 15dPGJ2, the phosphorylation of IRS-1 at Ser312in response to TNF-α treatment was inhibited (Fig. 2A). In line with previously published results (30Aguirre V. Werner E.D. Giraud J. Lee Y.H. Shoelson S.E. White M.F. J. Biol. Chem. 2002; 277: 1531-1537Google Scholar, 32Aguirre V. Uchida T. Yenush L. Davis R. White M.F. J. Biol. Chem. 2000; 275: 9047-9054Google Scholar), the JNK inhibitor SP600125 (52Bennett B.L. Sasaki D.T. Murray B.W. O'Leary E.C. Sakata S.T. Xu W. Leisten J.C. Motiwala A. Pierce S. Satoh Y. Bhagwat S.S. Manning A.M. Anderson D.W. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 13681-13686Google Scholar) also inhibited the phosphorylation of IRS-1 at Ser312 (Fig. 2A). Pre-treatment with both 15dPGJ2 and SP600125 resulted in more complete inhibition of serine phosphorylation on IRS-1 than treatment with either inhibitor alone (Fig. 2A). Consistent with the ability of 15dPGJ2 to inhibit IKK activity in a specific manner, IκBα degradation in response to TNF-α treatment was inhibited by 15dPGJ2 but not SP600125 (Fig. 2B). Similarly, SP600125 but not 15dPGJ2 inhibited phosphorylation of c-JUN in response to TNF-α (Fig. 2C). Thus, specific suppression of IKK activity is associated with the inhibition of IRS-1 phosphorylation at Ser312 in response to TNF-α. Based on the close relationship between IKK activity and serine phosphorylation of IRS-1 described above, we next inquired whether IRS-1 could serve as a direct substrate for IKK. As a first step to test this hypothesis, the physical association between IRS-1 and the IKK complex was examined using co-immunoprecipitation (Fig. 3). In HepG2 cells, IRS-1 was detected in the immune complex precipitated with anti-IKK-1 antibody (Fig. 3A). Moreover, the association between IKK-1 and IRS-1 observed in the basal state was reduced by calyculin treatment but not by vanadate treatment (Fig. 3A). Consistent with immunoblotting results shown in Fig. 1B, the intensity of the IKK1 signal was increased and the intensity of the IRS-1 signal was decreased by calyculin in a time-dependent manner. As expected, IKK2 was also detected in the IKK1 immunoprecipitate. Similar results were obtained when IRS-1 immunoprecipitates were probed with the IKK antibody (Fig. 3B). These results suggest an association between IRS-1 and IKK1 that was observed in the basal state and reduced dramatically after calyculin treatment. It is interesting to note that the dynamics of the association between IRS-1 and IKK1 were different depending on whether anit-IKK1 or anti-IRS-1 antibody was used for immunoprecipitation. With the anti-IKK1 antibody, the association of IRS-1 disappeared at 60 min (Fig. 3A). By contrast, with the anti-IRS-1 antibody, the association of IKK1 became undetectable at 5 min (Fig. 3B). These differences may be attributed to a change in antibody affinity for the phosphorylated IRS-1 as described above (Fig. 1). Thus, less IRS-1 may be immunoprecipitated with anti-IRS-1 antibody after calyculin treatment. Consistent with this explanation is the fact that the intensity of the IRS-1 signal detected by the regular IRS-1 antibody was decreased in the IRS-1 immunoprecipitate after calyculin treatment (Fig. 3B). In 293 cells transiently transfected with HA-tagged IKK2, both IRS-1 and IRS-2 were detected in the anti-HA immunoprecipitate (Fig. 3C). Similar to results from HepG2 cells, this association between IKK2 and IRS proteins was abolished after calyculin treatment of the cells. TNF-α treatment also abolished the interaction between IKK and IRS proteins (data not shown). To demonstrate that the immunoprecipitation results are specific, non-immune IgG as well as an unrelated antibody against p38 was used to immunoprecipitate samples in control experiments (Fig. 3D). These two controls did not give the results observed with the IRS-1 or IKK1 antibody. Taken together, these data suggest that IRS-1 and IKK directly interact in intact cells and that this interaction is reduced by activation of IKK and serine phosphorylation of IRS-1. To further examine the possibility that IRS-1 is a novel substrate for IKK, we performed immune complex kinase assays using recombinant IKK2 derived from transiently transfected 293 cells. Expression vectors for HA-tagged IRS-1 and HA-tagged IKK2 were co-transfected into HEK 293 cells, and then recombinant proteins were recovered by immunoprecipitating with the anti-HA antibody. After being extensively washed, the immune complex was used in an in vitro kinase assay (46Chen G. Cao P. Goeddel D.V. Mol. Cell. 2002; 9: 401-410Google Scholar). Three major phosphoproteins were observed in the HA immunoprecipitates when wild-type IKK2 was used (Fig. 4A). The identity of these phosphoproteins was determined by immunoblotting. They are HA-IRS-1 with a mobility of 160 kDa, HA-IKK2 with a mobility of 89 kDa, and NEMO/IKKγ with a mobility of 48 kDa. Importantly, these phosphoproteins were absent or significantly reduced when an empty vector or the kinase-dead IKK2 was used instead of wild-type IKK2 (Fig. 4A). Similar results were obtained with the wild type IKK1 with the exception that phosphorylation of IKK2 and NEMO was much weaker (Fig. 4A, lane 4). The phosphorylation of IKK2 is consistent with autophosphorylation (53Delhase M. Hayakawa M. Chen Y. Karin M. Science. 1999; 284: 309-313Google Scholar), and NEMO is known to be a substrate for IKK2 (54Prajapati S. Gaynor R.B. J. Biol. Chem. 2002; 277: 24331-24339Google Scholar). More importantly, these results suggest that IRS-1 is capable of functioning as a direct substrate for IKKin vitro because IRS-1 phosphorylation was only observed in the immune complexes with wild-type IKK2 but not with kinase-dead IKK2 or the empty vector. The effect of calyculin treatment on the ability of IRS-1 to undergo phosphorylation by IKK was also investigated (Fig. 4B). Calyculin treatment resulted in increased IRS-1 phosphorylation by wild-type but not kinase-dead IKK2. This finding suggests that calyculin increased the activity of IKK2 to phosphorylate IRS-1. Consistent with this possibility, the autophosphorylation of IKK2 was incr