Title: S6K Directly Phosphorylates IRS-1 on Ser-270 to Promote Insulin Resistance in Response to TNF-α Signaling through IKK2
Abstract: S6K1 (p70S6K) is a serine kinase downstream from Akt in the insulin signaling pathway that is involved in negative feedback regulation of insulin action. S6K1 is also activated by TNF-α, a pro-inflammatory cytokine. However, its role remains to be characterized. In the current study, we elucidated a mechanism for S6K1 to mediate TNF-α-induced insulin resistance in adipocytes and hepatocytes. S6K1 was phosphorylated at Thr-389 in response to TNF-α. This led to phosphorylation of IRS-1 by S6K1 at multiple serine residues including Ser-270, Ser-307, Ser-636, and Ser-1101 in human IRS-1 (Ser-265, Ser-302, Ser-632, and Ser-1097, in rodent IRS-1). Direct phosphorylation of these sites by S6K1 was observed in an in vitro kinase assay using purified IRS-1 and S6K1. Phosphorylation of all these serines was increased in the adipose tissue of obese mice. RNAi knockdown demonstrated an important role for S6K1 in mediating TNF-α-induced IRS-1 inhibition that led to impaired insulin-stimulated glucose uptake in adipocytes. A point mutant of IRS-1 (S270A) impaired association of IRS-1 with S6K1 resulting in diminished phosphorylation of IRS-1 at three other S6K1 phosphorylation sites (Ser-307, Ser-636, and Ser-1101). Expression of a dominant negative S6K1 mutant prevented TNF-induced Ser-270 phosphorylation and IRS-1 protein degradation. Moreover, in IKK2 (but not IKK1)-null cells, TNF-α treatment did not result in Thr-389 phosphorylation of S6K1. We present a new mechanism for TNF-α to induce insulin resistance that involves activation of S6K by an IKK2-dependent pathway. S6K directly phosphorylates IRS-1 on multiple serine residues to inhibit insulin signaling. S6K1 (p70S6K) is a serine kinase downstream from Akt in the insulin signaling pathway that is involved in negative feedback regulation of insulin action. S6K1 is also activated by TNF-α, a pro-inflammatory cytokine. However, its role remains to be characterized. In the current study, we elucidated a mechanism for S6K1 to mediate TNF-α-induced insulin resistance in adipocytes and hepatocytes. S6K1 was phosphorylated at Thr-389 in response to TNF-α. This led to phosphorylation of IRS-1 by S6K1 at multiple serine residues including Ser-270, Ser-307, Ser-636, and Ser-1101 in human IRS-1 (Ser-265, Ser-302, Ser-632, and Ser-1097, in rodent IRS-1). Direct phosphorylation of these sites by S6K1 was observed in an in vitro kinase assay using purified IRS-1 and S6K1. Phosphorylation of all these serines was increased in the adipose tissue of obese mice. RNAi knockdown demonstrated an important role for S6K1 in mediating TNF-α-induced IRS-1 inhibition that led to impaired insulin-stimulated glucose uptake in adipocytes. A point mutant of IRS-1 (S270A) impaired association of IRS-1 with S6K1 resulting in diminished phosphorylation of IRS-1 at three other S6K1 phosphorylation sites (Ser-307, Ser-636, and Ser-1101). Expression of a dominant negative S6K1 mutant prevented TNF-induced Ser-270 phosphorylation and IRS-1 protein degradation. Moreover, in IKK2 (but not IKK1)-null cells, TNF-α treatment did not result in Thr-389 phosphorylation of S6K1. We present a new mechanism for TNF-α to induce insulin resistance that involves activation of S6K by an IKK2-dependent pathway. S6K directly phosphorylates IRS-1 on multiple serine residues to inhibit insulin signaling. TNF-α is a pro-inflammatory cytokine implicated in development of insulin resistance in obesity (1Hotamisligil G.S. Exp. Clin. Endocrinol. Diabetes. 1999; 107: 119-125Crossref PubMed Scopus (382) Google Scholar, 2Peraldi P. Spiegelman B. Mol. Cell Biochem. 1998; 182: 169-175Crossref PubMed Scopus (243) Google Scholar). 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Recent studies demonstrate that S6K mediates mTOR signaling to phosphorylate IRS-1 at serine residues (rodent/human) including Ser-302/307 (23Harrington L.S. Findlay G.M. Gray A. Tolkacheva T. Wigfield S. Rebholz H. Barnett J. Leslie N.R. Cheng S. Shepherd P.R. Gout I. Downes C.P. Lamb R.F. J. Cell Biol. 2004; 166: 213-223Crossref PubMed Scopus (932) Google Scholar), Ser-307/312 (24Carlson C.J. White M.F. Rondinone C.M. Biochem. Biophys. Res. Commun. 2004; 316: 533-539Crossref PubMed Scopus (122) Google Scholar), Ser-632/636 (17Ozes 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: 4640-4645Crossref PubMed Scopus (326) Google Scholar), and Ser-1097/Ser-1101 (25Tremblay F. Brule S. Hee Um S. Li Y. Masuda K. Roden M. Sun X.J. Krebs M. Polakiewicz R.D. Thomas G. Marette A. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 14056-14061Crossref PubMed Scopus (352) Google Scholar). S6K knock-out mice are protected against diet-induced insulin resistance, and this phenotype is associated with reduced phosphorylation of IRS-1 Ser-636 (Ser-632 in rodent) (26Um S.H. Frigerio F. Watanabe M. Picard F. Joaquin M. Sticker M. Fumagalli S. Allegrini P.R. Kozma S.C. Auwerx J. Thomas G. Nature. 2004; 431: 200-205Crossref PubMed Scopus (1376) Google Scholar). However, it remains possible that S6K phosphorylates IRS-1 at additional sites. Additionally, direct phosphorylation of IRS-1 by S6K has not been previously demonstrated in a kinase assay. tumor necrosis factor hemagglutinin dominant negative, S6K/IRS-1 interaction is involved in regulation of insulin sensitivity by amino acids and insulin (27Tremblay F. Marette A. J. Biol. Chem. 2001; 276: 38052-38060Abstract Full Text Full Text PDF PubMed Google Scholar, 28Tremblay F. Krebs M. Dombrowski L. Brehm A. Bernroider E. Roth E. Nowotny P. Waldhausl W. Marette A. Roden M. 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However, the molecular mechanisms underlying the elevated S6K activity are not clear in obesity. Two studies suggest that S6K may be activated by TNF-α (7Gao Z. Zuberi A. Quon M. Dong Z. Ye J. J. Biol. Chem. 2003; 278: 24944-24950Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar, 17Ozes 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: 4640-4645Crossref PubMed Scopus (326) Google Scholar). However, the molecular mechanism by which TNF-α activates mTOR/S6K is controversial. In an initial study, Ozes et al. (17Ozes 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: 4640-4645Crossref PubMed Scopus (326) Google Scholar) reported that the PI3K/Akt/mTOR pathway mediated the TNF-α signal. In a more recent study, Lee et al. (32Lee D.-F. Kuo H.-P. Chen C.-T. Hsu J.-M. Chou C.-K. Wei Y. Sun H.-L. Li L.-Y. Ping B. Huang W.-C. He X. Hung J.-Y. Lai C.-C. Ding Q. Su J.-L. Yang J.-Y. Sahin A.A. Hortobagyi G.N. Tsai F.-J. Tsai C.-H. Hung M.-C. Cell. 2007; 130: 440-455Abstract Full Text Full Text PDF PubMed Scopus (542) Google Scholar) demonstrated that S6K can be activated by TNF-α through a PI3K-independent pathway. It remains to be determined which pathway mediates the S6K activation by TNF-α. Given the potentially important role of S6K and TNF-α in the control of insulin sensitivity, it is of interest to determine if S6K mediates TNF signaling related to insulin resistance. It is also important to determine the relationship between S6K and other serine kinases that are activated by TNF-α for insulin resistance. The association of IRS-1 Ser-636 phosphorylation with TNF-α treatment suggests a potential mechanism for S6K to mediate TNF signals for insulin resistance (7Gao Z. Zuberi A. Quon M. Dong Z. Ye J. J. Biol. Chem. 2003; 278: 24944-24950Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar, 17Ozes 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: 4640-4645Crossref PubMed Scopus (326) Google Scholar). In the present study, we conducted a systematic analysis of the role of S6K in mediating TNF-α-induced insulin resistance. Our data suggest that TNF-α activates S6K through IKK2, and that S6K directly phosphorylates IRS-1 at four serine residues including Ser-265/270, Ser-302/307, Ser-632/636, and Ser-1097/1101 in rodents/humans. The Ser-265/270 is required for S6K to phosphorylate IRS-1 at other three serines. Animals—Male C57BL/6J-Lepob, and C57BL/6J mice were purchased from Jackson Laboratory (Bar Harbor, ME) at 5 weeks of age and used in the study according to an animal protocol approved by the institutional animal care and use committee. Mice were housed in a regular cage at 4 mice/cage with free access to water and standard chow unless noted. Epididymal fat from mice fasted overnight were collected, frozen in liquid nitrogen, and stored at –70 °C until further analysis. All procedures were performed in accordance with National Institutes of Health guidelines for the care and use of animals. Cells and Reagents—Cell lines including mouse NIH-3T3 (CRL-1658) and human embryonic kidney (HEK) 293 (CRL-1573) were purchased from the American Type Culture Collection (ATCC). IKK wild-type, IKK1, or IKK2 knock-out cell lines were described in a previous study (6Gao 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 (604) Google Scholar). The H4IIE cell line stably transfected with Flag-IRS1 wild type was a gift from Dr. Richard A. Roth at Stanford University Medical School, Stanford, CA 94305-5174 (33Greene 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 (171) Google Scholar). All cells were maintained in Dulbecco's modified Eagle's culture medium supplemented with 10! fetal calf serum. Phospho-IRS-1 (Ser-312/307) antibody (07-247) was from Upstate Biotechnology (Lake Placid, NY). Antibodies to phospho-Ser-307/302 (2384) and phospho-Ser-1101/1097 (2385) in IRS-1, phospho-Thr-308 (9275) and phospho-Ser-473 (9271) in Akt, phospho-Thr-389 (9205) in p70S6 were obtained from Cell Signaling (Beverly, MA). Antibodies to phospho-Ser-270 (sc-17192) in IRS-1, IRS-1 (sc-7200), and IκBα (sc-371) were from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies to S6K (ab9366), phospho-Ser-636/632 of IRS-1 (ab47764) and β-actin (ab6276) were from Abcam (Cambridge, UK). Rapamycin (A-275), LY294002 (ST420), and SP600125 (EI-305) were acquired from Biomol (Plymouth Meeting, PA). 15-Deoxyprostaglandin J2 (15dPGJ2, 538927), PD98059 (513000), and SB203580 (203580) were purchased from Calbiochem. Wortmannin (W-1628), Type II collagenase (C6885), and TNF-α (T6674) were from Sigma. Purified p70S6 kinase (T412E), active IKK2 (IKKβ), and PKCθ were obtained from Upstate Biotechnology (Lake Placid, NY). Generation of Adenovirus—Adenovirus carrying a dominant negative S6K1 (S6K-DN) was constructed using ViraPower Adenoviral Expression System (K4930-00), which was from Invitrogen (Carlsbad, CA). Briefly, S6K1-DN cDNA with HA tag was inserted into TOPO pENTR vector (K2400-20) and was recombined into the adenovirus expression plasmid pAd/CMV/V5-DEST. The pAd/CMV/V5-DEST plasmid with S6K1 cDNA was digested with the PacI endonuclease and transfected with 293A cells for production of adenovirus. The medium supernatant containing adenovirus was collected 3 days later and titrated according to the manufacturer's instructions. Immunoblotting—Adipose tissue was homogenized in cold lysis buffer followed by sonication (6Gao 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 (604) Google Scholar). Lysis buffer contains 1! Nonidet P-40, 50 mm Hepes, pH 7.6, 250 mm NaCl, 10! glycerol, 1 mm EDTA, 20 mm β-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. Cultured cells were kept in serum-free media overnight and treated with various reagents as indicated. After treatment, whole cell lysates were made in lysis buffer with sonication, and the supernatant was used for immunoblotting after centrifugation at 10,000 × g for 10 min at 4 °C. Total protein (100 μg) in 50 μl of reducing sample buffer was used for immunoblotting as described previously (34Gao Z. He Q. Peng B. Chiao P.J. Ye J. J. Biol. Chem. 2006; 281: 4540-4547Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). Immunoblots were quantified using a scanning densitometer in conjunction with NIH ImageJ software. Signals were normalized to loading controls. Plasmids and Transfection—Expression vectors for HA-tagged IRS-1 wild type and IRS-1 S270A mutant were constructed in pCIS2 expression vector as described (11Ravichandran L.V. Esposito D.L. Chen J. Quon M.J. J. Biol. Chem. 2001; 276: 3543-3549Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar). Plasmids for HA-S6K1 WT (8984) and HA-S6K1 dominant negative (8985) were obtained from Addgene (Cambridge, MA) (35Schalm S.S. Blenis J. Curr. Biol. 2002; 12: 632-639Abstract Full Text Full Text PDF PubMed Scopus (388) Google Scholar). The plasmids for HA-IKK2 WT, HA-IKK2 kinase dead mutant, and GST-IRS-1 were described previously (6Gao 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 (604) Google Scholar). Constructs were expressed in HEK293 cells by transient transfection using Lipofectamine. Purified GST-IRS-1 was described previously (6Gao 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 (604) Google Scholar). Immunoprecipitation—Immunoprecipitation was carried out using whole cell lysates (400 μg of total protein), 2–4 μgof antibody, and 20 μl of protein A- or protein G-Sepharose beads (Amersham Biosciences). After treatment, cell lysates were prepared by sonication in cell lysis buffer. IP was conducted by incubating the whole cell lysate 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 kinase assays. Kinase Assay—For each in vitro kinase assay, purified GST-IRS-1 or HA-IRS-1 protein was used as substrate. The proteins were diluted in kinase assay buffer (20 mm Hepes, pH 7.6, 20 mm MgCl2, 20 mm glycerophosphate, 1 mm dithiothreitol, 10 μm ATP, 1 mm EDTA, 1 mm sodium orthovanadate, 0.4 mm phenylmethylsulfonyl fluoride, 20 mm creatine phosphate). The kinase assay was conducted at 37 °C for 30 min in 20 μl of kinase assay buffer and 0.2 μg (2 μl) of kinase, such as S6K1, IKK2 (IKKβ), or PKCθ. The product was resolved by 8! SDS-PAGE and immunoblotted with phosphospecific IRS-1 antibodies. RNA Interference—Mouse S6K-specific shRNA was expressed in retroviral silencing plasmids. The shRNA plasmids were made under a contract service agreement with Origene (Rockville, MD). Expression vectors for four independent shRNA were cotransfected into NIH-3T3 cells using Lipofectamine 2000 (Invitrogen). Empty vector was used as a negative control. 60 h after transfection, cells were kept in serum-free medium overnight and then stimulated without or with TNF-α or insulin for 30 min. Primary Adipocytes—Preadipocytes were isolated from epididymal fat pads of C57BL/6J mice as described elsewhere (36Ye J. Gao Z. Yin J. He H. Am. J. Physiol. Endocrinol. Metab. 2007; 293: E1118-E1128Crossref PubMed Scopus (656) Google Scholar). The tissue was digested with Type II collagenase, and single cells were plated in a 100-mm flask in normal culture medium. After 24 h, cells attached to the flask were used as preadipocytes. The preadipocytes were plated into a 6-well plates and differentiated into adipocytes in standard adipogenic mixture (5 μg/ml insulin, 0.5 mm isobutylmethylxanthine, and 10 μm dexamethasone). Mature adipocytes were used at day 9 of differentiation. Insulin-induced Glucose Uptake—3T3-L1 preadipocytes (5 × 105/well) were differentiated into adipocytes in a 12-well plate, and used in the glucose uptake assay as described elsewhere (15Gao Z. Zhang X. Zuberi A. Hwang D. Quon M.J. Lefevre M. Ye J. Mol. Endocrinol. 2004; 18: 2024-2034Crossref PubMed Scopus (268) Google Scholar). In experiments using dominant negative mutant of S6K, the differentiated 3T3-L1 cells were infected by the S6K-DN adenovirus for 24 h and glucose uptake was examined after an additional 24 h. Statistical Analysis—All experiments were repeated independently at least three times with consistent results. For most figures with immunoblots, a representative blot is shown for each experiment along with a bar graph representing the mean ± S.E. of multiple independent experiments determined by densitometric analysis normalized to appropriate controls. Student's t test or one-way analysis of variance was used as appropriate in statistical analyses of the data. p < 0.05 was considered to indicate statistical significance. Association between Increased S6K Phosphorylation and Serine Phosphorylation of IRS-1 in ob/ob Mice—We hypothesized that S6K1 participates in the TNF-α-induced insulin resistance of obesity. Therefore, we first examined the relationship between S6K1 phosphorylation at Thr-389 (a proxy for S6K1 activity) and serine phosphorylation of IRS-1 in adipose tissue from ob/ob mice (Fig. 1). As expected, phosphorylation of S6K1 was significantly elevated in adipose tissue from ob/ob mice when compared with samples from lean control mice (Fig. 1, A and B). Expression levels for S6K1 protein were comparable between lean and ob/ob mice. However, expression of IRS-1 protein was decreased in adipose tissue from ob/ob mice (Fig. 1, A and B). In several independent studies, S6K is reported to be involved with phosphorylation of IRS-1 at multiple serine residues including Ser-302/307, Ser-632/636, Ser-1097/1101, and Ser-307/312 in the mouse/human IRS-1 proteins (17Ozes 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: 4640-4645Crossref PubMed Scopus (326) Google Scholar, 23Harrington L.S. Findlay G.M. Gray A. Tolkacheva T. Wigfield S. Rebholz H. Barnett J. Leslie N.R. Cheng S. Shepherd P.R. Gout I. Downes C.P. Lamb R.F. J. Cell Biol. 2004; 166: 213-223Crossref PubMed Scopus (932) Google Scholar, 24Carlson C.J. White M.F. Rondinone C.M. Biochem. Biophys. Res. Commun. 2004; 316: 533-539Crossref PubMed Scopus (122) Google Scholar, 25Tremblay F. Brule S. Hee Um S. Li Y. Masuda K. Roden M. Sun X.J. Krebs M. Polakiewicz R.D. Thomas G. Marette A. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 14056-14061Crossref PubMed Scopus (352) Google Scholar, 26Um S.H. Frigerio F. Watanabe M. Picard F. Joaquin M. Sticker M. Fumagalli S. Allegrini P.R. Kozma S.C. Auwerx J. Thomas G. Nature. 2004; 431: 200-205Crossref PubMed Scopus (1376) Google Scholar). Each abbreviation for serines stands for the two corresponding serines in rodent (Ser-302) and human (Ser-307) IRS-1. The abbreviations are used in the same way in rest of our study unless specified. These previously published studies did not compare all of these sites in the same experimental preparation. Therefore, using phosphospecific antibodies, we demonstrated that serine phosphorylation at all of these sites was elevated in IRS-1 in adipose tissue from ob/ob mice when compared with samples from lean control mice (Fig. 1, C and D). In addition, we evaluated serine phosphorylation of Ser-265/270 (Fig. 1, C and D) because the amino acid sequence surrounding this site is homologous to known S6K phosphorylation sites (Fig. 3A). Interestingly, Ser-265/270 phosphorylation was also increased in the samples from ob/ob mice. Thus, increased S6K phosphorylation in adipose tissue from ob/ob mice is associated with increased phosphorylation of IRS-1 at multiple serine residues as well as diminished protein expression of IRS-1. This raises the possibility that S6K may be directly phosphorylating IRS-1 at these 5 serine residues resulting in accelerated degradation of IRS-1. Role of S6K in Mediating TNF-induced Insulin Resistance—To evaluate the potential role of S6K in mediating TNF-α-induced insulin resistance, we first examined the ability of TNF-α to stimulate phosphorylation of S6K at Thr-389 and IRS-1 at Ser-265/270 (Fig. 2A). In both primary adipocytes and 3T3-L1 adipocytes, TNF-α stimulated a time-dependent increase in phosphorylation of S6K at Thr-389 and IRS-1 at Ser-265/270. Similarly in rat hepatoma cells (H4IIE), S6K1 phosphorylation was induced by TNF-α as early as 10 min (Fig. 2B). Akt phosphorylation at Thr-308 and Ser-473 was also increased in response to TNF-α in a time-dependent manner that seemed some 20–60 min later than the S6K phosphorylation. This difference in time course between S6K1 and Akt activation in response to TNF-α suggests that S6K1 activation may be independent of Akt. However, it should be noted that the sensitivity of the phospho-antibodies used against different proteins may not be similar. Thus, firm conclusions regarding relative time courses of phosphorylation for different proteins are not warranted based on these data alone. In these experiments, we also assessed IkBα degradation as a control for TNF-α activity (Fig. 2B). We next evaluated insulin-stimulated glucose uptake in 3T3-L1 adipocytes without or with pretreatment with TNF-α in the absence or presence of expression of a dominant negative mutant of S6K (S6K-DN) (Fig. 2C). As expected, insulin-stimulated glucose uptake was significantly impaired when cells were pretreated with TNF-α. Importantly, the effect of TNF-α to cause this insulin resistance was substantially blunted in cells expressing S6K-DN. Moreover, insulin-stimulated phosphorylation of Akt (Thr-308), a key mediator of insulin-stimulated glucose uptake (37Cong L.N. Chen H. Li Y. Zhou L. McGibbon M.A. Taylor S.I. Quon M.J. Mol. Endocrinol. 1997; 11: 1881-1890Crossref PubMed Google Scholar), was also significantly diminished by TNF-α pretreatment (p < 0.001) and this effect of TNF-α was significantly abrogated by expression of S6K-DN (p < 0.02) (Fig. 2D). Taken together, these results suggest that S6K plays a key role in mediating impairment in metabolic insulin signaling caused by TNF-α that contributes to insulin resistance. Identification of IRS-1 Ser-265/270 as a Novel Phosphorylation Site for S6K1—To identify novel serine phosphorylation sites on IRS-1 for S6K1, we compared an amino acid motif surrounding the phosphorylation site on S6, an authentic substrate of S6K1, with the whole IRS-1 amino acid sequence (Fig. 3A). The S6 motif is characterized by the sequence RXRXXS, in which the distal serine residue is the kinase target. We identified similar motifs in four of the IRS-1 phosphoserine sites evaluated in our present study (Ser-265/270, Ser-302/307, Ser-632/636, and Ser-1097/1101). This motif was not present for Ser-307/312 (Fig. 3A). We next evaluated the ability of S6K1 to directly phosphorylate IRS-1 by conducting in vitro kinase assays with purified S6K1 and recombinant HA-tagged IRS-1 (human IRS-1) immunoprecipitated from transfected 293 cells (Fig. 3B). Phosphorylation of IRS-1 was detected with phosphospecific antibodies as in Fig. 1B. Using this method, we found that S6K1 directly phosphorylated Ser-265/270, Ser-302/307, Ser-632/636, and Ser-1097/1101 in IRS-1. Interestingly, specific phosphorylation of IRS-1 at Ser-307/312 by S6K1 was not observed. These results are consistent with our sequence analysis (Fig. 3A) and support the specificity of our in vitro kinase assay. Thus, we have identified Ser-265/270 as a novel S6K1 target in the IRS-1 protein. Our observations are unlikely to result from nonspecific interaction between S6K1 and IRS-1 since Ser-307/312 phosphorylation was not induced by S6K under identical assay conditions. Regulatory Role for Phosphorylation of IRS-1 at Ser-265/270 by S6K1—Serine phosphorylation of IRS-1 at multiple sites has been implicated in inhibition of insulin signaling. It is possible that phosphorylation of IRS-1 at one serine residue may regulate the ability of S6K1 to phosphorylate other sites on IRS-1. To evaluate this possibility, we substituted Ser-270 with alanine in the human IRS-1 protein (S270A mutant). This serine site was selected for further investigation because it represents a novel phosphorylation target