Direct Cross-talk of Interleukin-6 and Insulin Signal Transduction via Insulin Receptor Substrate-1 in Skeletal Muscle Cells
2006; Elsevier BV; Volume: 281; Issue: 11 Linguagem: Inglês
10.1074/jbc.m509782200
ISSN1083-351X
AutoresCora Weigert, Anita M. Hennige, Rainer Lehmann, Katrin Brodbeck, Frank Baumgärtner, Myriam Schaüble, Hans Häring, Erwin Schleicher,
Tópico(s)Adipose Tissue and Metabolism
ResumoThe exercise-induced interleukin (IL)-6 production and secretion within skeletal muscle fibers has raised the question of a putative tissue-specific function of IL-6 in the energy metabolism of the muscle during and after the exercise. In the present study, we followed the hypothesis that IL-6 signaling may directly interact with insulin receptor substrate (IRS)-1, a keystone in the insulin signaling cascade. We showed that IL-6 induces a rapid recruitment of IRS-1 to the IL-6 receptor complex in cultured skeletal muscle cells. Moreover, IL-6 induced a rapid and transient phosphorylation of Ser-318 of IRS-1 in muscle cells and in muscle tissue, but not in the liver of IL-6-treated mice, probably via the IL-6-induced co-recruitment of protein kinase C-δ. This Ser-318 phosphorylation improved insulin-stimulated Akt phosphorylation and glucose uptake in myotubes since transfection with an IRS-1/Glu-318 mutant simulating a permanent phospho-Ser-318 modification increased Akt phosphorylation and glucose uptake. Noteworthily, two inhibitory mechanisms of IL-6 on insulin action, phosphorylation of the inhibitory Ser-307 residue of IRS-1 and induction of SOCS-3 expression, were only found in liver but not in muscle of IL-6-treated mice. Thus, the data provided evidence for a possible molecular mechanism of the physiological metabolic effects of IL-6 in skeletal muscle, thereby exerting short term beneficial effects on insulin action. The exercise-induced interleukin (IL)-6 production and secretion within skeletal muscle fibers has raised the question of a putative tissue-specific function of IL-6 in the energy metabolism of the muscle during and after the exercise. In the present study, we followed the hypothesis that IL-6 signaling may directly interact with insulin receptor substrate (IRS)-1, a keystone in the insulin signaling cascade. We showed that IL-6 induces a rapid recruitment of IRS-1 to the IL-6 receptor complex in cultured skeletal muscle cells. Moreover, IL-6 induced a rapid and transient phosphorylation of Ser-318 of IRS-1 in muscle cells and in muscle tissue, but not in the liver of IL-6-treated mice, probably via the IL-6-induced co-recruitment of protein kinase C-δ. This Ser-318 phosphorylation improved insulin-stimulated Akt phosphorylation and glucose uptake in myotubes since transfection with an IRS-1/Glu-318 mutant simulating a permanent phospho-Ser-318 modification increased Akt phosphorylation and glucose uptake. Noteworthily, two inhibitory mechanisms of IL-6 on insulin action, phosphorylation of the inhibitory Ser-307 residue of IRS-1 and induction of SOCS-3 expression, were only found in liver but not in muscle of IL-6-treated mice. Thus, the data provided evidence for a possible molecular mechanism of the physiological metabolic effects of IL-6 in skeletal muscle, thereby exerting short term beneficial effects on insulin action. Over the last two decades, the pleiotropic cytokine interleukin-6 (IL-6) 2The abbreviations used are: IL-6, interleukin-6; IL-6R, IL-6 receptor-α (CD-126); GLUT-4, glucose transporter-4; IRS, insulin receptor substrate; PI3K, phosphatidylinositol 3-kinase; PKC, protein kinase C; SOCS, suppressor of cytokine signaling; STAT, signal transducer and activator of transcription; siRNA, small interfering RNA; FCS, fetal calf serum; WT, wild type.2The abbreviations used are: IL-6, interleukin-6; IL-6R, IL-6 receptor-α (CD-126); GLUT-4, glucose transporter-4; IRS, insulin receptor substrate; PI3K, phosphatidylinositol 3-kinase; PKC, protein kinase C; SOCS, suppressor of cytokine signaling; STAT, signal transducer and activator of transcription; siRNA, small interfering RNA; FCS, fetal calf serum; WT, wild type. has been looked upon as a major component in the inflammatory network and the acute immune response (1Akira S. Taga T. Kishimoto T. Adv. Immunol. 1993; 54: 1-78Crossref PubMed Google Scholar). Since 1997, when the adipose tissue was recognized as a relevant IL-6 producing organ, accounting for 10–35% of circulating IL-6 plasma levels in humans (2Mohamed-Ali V. Goodrick S. Rawesh A. Katz D.R. Miles J.M. Yudkin J.S. Klein S. Coppack S.W. J. Clin. Endocrinol. Metab. 1997; 82: 4196-4200Crossref PubMed Google Scholar), this view was renewed and broadened. It has been established in numerous clinical trials and association studies that IL-6 plasma concentrations increased with weight gain and are associated with the development of insulin resistance (3Fernandez-Real J.M. Vayreda M. Richart C. Gutierrez C. Broch M. Vendrell J. Ricart W. J. Clin. Endocrinol. Metab. 2001; 86: 1154-1159Crossref PubMed Scopus (412) Google Scholar, 4Pradhan A.D. Manson J.E. Rifai N. Buring J.E. Ridker P.M. J. Am. Med. Assoc. 2001; 286: 327-334Crossref PubMed Scopus (3295) Google Scholar, 5Vozarova B. Weyer C. Hanson K. Tataranni P.A. Bogardus C. 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Noteworthily, the IL-6 production in the muscle fibers is markedly increased by exercise, reaching interstitial IL-6 concentrations >1 ng/ml (i.e. 100-fold above physiological plasma concentrations) and, depending on the duration and intensity of the exercise, leading to elevated IL-6 plasma concentrations (12Steensberg A. Keller C. Starkie R.L. Osada T. Febbraio M.A. Pedersen B.K. Am. J. Physiol. 2002; 283: E1272-E1278Crossref PubMed Scopus (322) Google Scholar, 13Rosendal L. Sogaard K. Kjaer M. Sjogaard G. Langberg H. Kristiansen J. J. Appl. Physiol. 2004; 182: 379-388Google Scholar). Since exercise is one of the most important therapeutic interventions to cope with weight gain and insulin resistance (14Hawley J.A. Diabetes-Metab. Res. Rev. 2004; 20: 383-393Crossref PubMed Scopus (236) Google Scholar), it is obvious to suggest that exercise-induced IL-6 expression may exert positive effects on insulin signal transduction in the skeletal muscle. In fact, moderate exercise has been shown to improve insulin sensitivity, glucose uptake, and glycogen synthesis in muscle (15Wojtaszewski J.F. Hansen B.F. Gade Kiens B. Markuns J.F. Goodyear L.J. Richter E.A. Diabetes. 2000; 49: 325-331Crossref PubMed Scopus (285) Google Scholar, 16Wojtaszewski J.F. Jorgensen S.B. Frosig C. MacDonald C. Birk J.B. Richter E.A. Acta Physiol. Scand. 2003; 178: 321-328Crossref PubMed Scopus (58) Google Scholar, 17Sakamoto K. Arnolds D.E. Ekberg I. Thorell A. Goodyear L.J. Biochem. Biophys. Res. Commun. 2004; 319: 419-425Crossref PubMed Scopus (77) Google Scholar). An increasing number of reports suggest that IL-6 could act as a myocyte-derived "exercise factor" that improves the energy supply of the muscle by increasing the output of glucose and fatty acids from the energy-storing organs liver and fat and by enhancing glucose uptake, fatty acid oxidation, and glycogen synthesis in muscle (18Pedersen B.K. Steensberg A. Fischer C. Keller C. Keller P. Plomgaard P. Febbraio M. Saltin B. J. Muscle Res. Cell Motil. 2003; 24: 113-119Crossref PubMed Scopus (347) Google Scholar, 19Febbraio M.A. Hiscock N. Sacchetti M. Fischer C.P. Pedersen B.K. Diabetes. 2004; 53: 1643-1648Crossref PubMed Scopus (301) Google Scholar, 20Tsigos C. Papanicolaou D.A. Kyrou I. Defensor R. Mitsiadis C.S. Chrousos G.P. J. Clin. Endocrinol. Metab. 1997; 82: 4167-4170Crossref PubMed Google Scholar, 21van Hall G. Steensberg A. Sacchetti M. Fischer C. Keller C. Schjerling P. Hiscock N. Moller K. Saltin B. Febbraio M.A. Pedersen B.K. J. Clin. Endocrinol. Metab. 2003; 88: 3005-3010Crossref PubMed Scopus (518) Google Scholar). Apparently, this argues for some muscle-specific insulin-sensitizing effects of IL-6, but the underlying molecular mechanism is unclear. IRS-1 is a key player in insulin signaling in muscle, and its cross-talk with metabolic and mitogenic pathways is modulated in a very subtle and complex manner using the phosphorylation pattern of the Ser/Thr-phosphorylation sites of IRS-1 as a regulatory mechanism for the interaction with its important downstream mediators, e.g. phosphatidylinositol-3 kinase (PI3K) (22Gual P. Marchand-Brustel Y. Tanti J.F. Biochimie (Paris). 2005; 87: 99-109Crossref PubMed Scopus (648) Google Scholar, 23Rui 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 (472) Google Scholar, 24Moeschel K. Beck A. Weigert C. Lammers R. Kalbacher H. Voelter W. Schleicher E.D. Haring H.U. Lehmann R. J. Biol. Chem. 2004; 279: 25157-25163Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 25Werner E.D. Lee J. Hansen L. Yuan M. Shoelson S.E. J. Biol. 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J. 2003; 374: 1-20Crossref PubMed Scopus (2442) Google Scholar), we hypothesize that IL-6 could cross-talk with the insulin signaling cascade by phosphorylation of IRS-1. We showed in the present study that IL-6 induces the Ser-318 phosphorylation of IRS-1 in various muscle cell lines and in muscle tissue of mice but not in the liver, that IRS-1 coimmunoprecipitates with the IL-6 receptor (IL-6R) and protein kinase C (PKC)-δ in an IL-6-dependent manner, and that Ser-318 phosphorylation may improve insulin action. Materials—C2C12 cells were from ATCC (Manassas, VA). Parental L6 cells and L6 GLUT4Myc cells were kindly provided from A. Klip, Hospital for Sick Children, Toronto, Canada. Cell culture media and supplements were from Invitrogen (Eggenstein, Germany). Human, mouse, and rat recombinant IL-6 were from Sigma (Munich, Germany) and R&D Systems (Wiesbaden, Germany). Oligonucleotides were synthesized by Invitrogen (Karlsruhe, Germany); reagents for cDNA synthesis and the LightCycler system were from Roche Applied Science (Mannheim, Germany). Antibodies against phospho-STAT-3 Tyr-705, STAT-3, and phospho-Akt Ser-473 were from Cell Signaling (Frankfurt, Germany); antibodies against IRS-1 (C terminus) and phosphotyrosine (4G10) were from Upstate Biotechnology (Lake Placid, NY), antibodies against PKC-δ were from BD Biosciences, antibodies against IL-6R used for immunoblotting were from Santa Cruz Biotechnology (Santa Cruz, CA), and the antibody against IL-6R used for immunoprecipitation was from Biolegend (San Diego, CA). The phospho-IRS-1 Ser-307 antibody was a kind gift from M. F. White, Harvard Medical School, Boston, MA. Polyclonal anti-phospho-Ser-318 antiserum was raised against a synthetic peptide (SMVGGKPGpSFRVRASSD) flanking Ser-318 in IRS-1, conserved among mouse, rat, and humans, and characterized as described in Ref. 24Moeschel K. Beck A. Weigert C. Lammers R. Kalbacher H. Voelter W. Schleicher E.D. Haring H.U. Lehmann R. J. Biol. Chem. 2004; 279: 25157-25163Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar. 2-[3H(G)]Deoxy-d-glucose (185–370 GBq/mmol) was from PerkinElmer Life Sciences. The cytomegalovirus promoter-based expression vector for rat IRS-1 was described in Ref. 30Kellerer M. Mushack J. Seffer E. Mischak H. Ullrich A. Haring H.U. Diabetologia. 1998; 41: 833-838Crossref PubMed Scopus (99) Google Scholar, and the cytomegalovirus promoter-based expression vectors for murine PKC-δ and dominant kinase-negative PKC-δ were described in Ref. 31Eitel K. Staiger H. Rieger J. Mischak H. Brandhorst H. Brendel M.D. Bretzel R.G. Haring H.U. Kellerer M. Diabetes. 2003; 52: 991-997Crossref PubMed Scopus (117) Google Scholar. Mutation of Ser-318 of IRS-1 to alanine or glutamate was made by oligonucleotide-mediated mutagenesis with the mutagenic upstream primers IRS-1/Ala-318 5′-ccagtatggtgggtgggaaaccaggtgccttcagggtgcgtgcctccagc-3′ and IRS-1/Glu-318 5′-ccagtatggtgggtgggaaaccaggtgagttcagggtgcgtgcctccagc-3′ (32Weigert C. Hennige A.M. Brischmann T. Beck A. Moeschel K. Schauble M. Brodbeck K. Haring H.U. Schleicher E.D. Lehmann R. J. Biol. Chem. 2005; 280: 37393-37399Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). In Vivo IL-6 Treatment of Mice—Ten-week-old male C57Bl/6 mice were obtained from The Jackson Laboratory and studied after 2 weeks of acclimatization. They were maintained on a normal light/dark cycle and kept on a regular diet. For short term IL-6 effects, mice were anesthetized with an intraperitoneal injection of ketamine (100 mg/kg of body weight) and xylazine (10 mg/kg of body weight), and a total of 2.5 μg/kg of mouse recombinant IL-6 or 5 IU of insulin was injected into the inferior vena cava. Controls received a comparable amount of diluent. Tissues (liver, muscle) were removed at the indicated time points, and protein extracts were prepared as described (33Hennige A.M. Burks D.J. Ozcan U. Kulkarni R.N. Ye J. Park S. Schubert M. Fisher T.L. Dow M.A. Leshan R. Zakaria M. Mossa-Basha M. White M.F. J. Clin. Investig. 2003; 112: 1521-1532Crossref PubMed Scopus (222) Google Scholar). All procedures were approved by the local Animal Care and Use Committee. Cell Culture and Transfection—Human primary skeletal muscle cells were grown from satellite cells obtained from percutaneous needle biopsies performed on the lateral portion of the quadriceps femoris (vastus lateris) muscle as recently described (34Krutzfeldt J. Kausch C. Volk A. Klein H.H. Rett K. Haring H.U. Stumvoll M. Diabetes. 2000; 49: 992-998Crossref PubMed Scopus (60) Google Scholar). When myoblasts reached 80–90% confluence, the cells were fused for 5 days in α-MEM containing 5.5 mm glucose with 2% FCS and 2% antibiotic antimycotic solution (fusion medium). Stimulation of the myotubes with IL-6 was performed in fusion medium. C2C12 myoblasts were cultured in Dulbecco's modified Eagle's medium containing 25 mm glucose, 10% FCS, 2 mm glutamine, 100 units/ml penicillin, and 100 μg/ml streptomycin. 1.0 × 105 cells/well of a 6-well plate were transfected using the Ca3(PO4)2-DNA-co-precipitation-method. Cells were lysed with 150 μl of lysis buffer/well (50 mm Tris, pH 7.6, 150 mm NaCl, 1% Triton X-100, containing protease and phosphatase inhibitors). Total protein (400 μg) was used for immunoprecipitation. L6 myoblasts were cultured in α-MEM containing 5.5 mm glucose, 10% FCS, 100 units/ml penicillin, and 100 μg/ml streptomycin as described (35Mitsumoto Y. Klip A. J. Biol. Chem. 1992; 267: 4957-4962Abstract Full Text PDF PubMed Google Scholar). For differentiation, 0.5 × 105 cells/well of a 12-well plate or 2.0 × 105/well of a 6-well plate were seeded in differentiation medium (α-MEM containing 5.5 mm glucose, 2% FCS, 100 units/ml penicillin and, 100 μg/ml streptomycin). Myotubes were used 7–8 days after cell seeding. To obtain stable expression of IRS-1 WT, Ala-318, and Glu-318, L6 Glut4Myc myoblasts were transfected with Effectene (Qiagen, Hilden, Germany) according to the instructions of the supplier. 2.0 × 105 cells/well in a 6-well plate received 0.8 μg of IRS-1 expression vector and 0.2 μg of pSVneo encoding neomycin resistance. Transfected cells were selected for their resistance to the antibiotic G418. G418-resistant clones were screened for IRS-1 expression and Ser-318 phosphorylation by Western blotting. Western Blotting—Cells were lysed with 600 μl of lysis buffer/10-cm dish (50 mm Tris, pH 7.6, 150 mm NaCl, 1% Triton X-100, containing protease and phosphatase inhibitors). Cytosolic extracts of myotubes were separated by sodium dodecyl sulfate polyacrylamide (7.5%) gel electrophoresis (SDS-PAGE). Proteins were transferred to nitrocellulose by semi-dry-electroblotting, and immunodetection was performed as described in Ref. 36Weigert C. Brodbeck K. Staiger H. Kausch C. Machicao F. Haring H.U. Schleicher E.D. J. Biol. Chem. 2004; 279: 23942-23952Abstract Full Text Full Text PDF PubMed Scopus (235) Google Scholar. 2-Deoxy-glucose Uptake—L6 Glut4Myc myoblasts were seeded in 12-well plates and were grown to confluence or differentiated to myotubes. Cells were serum-starved for 3 h in α-MEM prior to experiment (37Wang Q. Somwar R. Bilan P.J. Liu Z. Jin J. Woodgett J.R. Klip A. Mol. Cell. Biol. 1999; 19: 4008-4018Crossref PubMed Scopus (499) Google Scholar). After stimulation with IL-6 and insulin, cells were washed with Hepes-buffered saline (containing 140 mm NaCl, 20 mm Hepes-sodium, pH 7.4, 5 mm KCl, 2.5 mm MgSO4, 1 mm CaCl2), and 300 μl/well of 2-deoxy-glucose mix (Hepes-buffered saline containing 0.25 μCi/ml 2-[3H]deoxy-glucose and 10 μm 2-deoxy-glucose) was added. After incubation at 37 °C, 5% CO2 for 7 min, cells were washed three times with 0.9% NaCl and lysed with 250 μl of lysis buffer. Total amounts of cell lysates were counted by liquid scintillation counting. Protein content of lysates generally varied <10%. Reverse Transcription-PCR and Real-time Quantitative PCR Analysis—Total RNA of muscle and liver was isolated with PeqGOLD TriFast (Peqlab, Erlangen, Germany). Reverse transcription of total RNA (1 μg) was performed using avian myeloblastosis virus reverse transcriptase with the first-strand cDNA synthesis kit for reverse transcription-PCR. Aliquots (2 μl) of the reverse transcription reactions were then submitted in duplicate to online quantitative PCR with the LightCycler system with SYBR® green using the FastStart DNA-MasterSYBR Green I as described (36Weigert C. Brodbeck K. Staiger H. Kausch C. Machicao F. Haring H.U. Schleicher E.D. J. Biol. Chem. 2004; 279: 23942-23952Abstract Full Text Full Text PDF PubMed Scopus (235) Google Scholar). The following SOCS-3 primer pair was used: sense, 5′-gctggccaaagaaataacca-3′; antisense, 5′-agctcaccagcctcatctgt-3′, product of 224 bp. The PCR was performed in a volume of 20 μl: 2 μlof FastStart DNA-MasterSYBR Green I, 3 mm MgCl2, and primers according to a primer concentration of 1 μm. The instrument settings were: Denaturing at 95 °C for 10 min, 45× denaturing at 95 °C for 15 s, annealing at 66 °C for 10 s, elongation for 9 s. Small Interfering RNA (siRNA)—siRNA oligonucleotides targeting PKC-δ were designed and synthesized and annealed at Dharmacon Research, Lafayette, CO. An unrelated siRNA targeting firefly luciferase was used as control in all experiments. Transfection was performed with CellPhect (Amersham Biosciences, Buckinghamshire, UK) with 100 pmol of siRNA according to the instructions of the manufacturer. Briefly, 1 × 105 of C2C12 cells/well were seeded in 6-well plates and transfected in Dulbecco's modified Eagle's medium containing 25 mm glucose, 10% FCS without antibiotics. 24 h after the glycerol shock, cells were stimulated as indicated. Statistical Analysis—Results presented are derived from at least three independent experiments. Means ± S.E. were calculated, and groups of data were compared using Student's t test. Statistical significance was set at p < 0.05. IL-6 Induces Ser-318 Phosphorylation of IRS-1 in Muscle and in Muscle Cell Culture Models—Since previous studies showed that serine phosphorylation of IRS-1 modulates insulin signal transduction, we studied whether IL-6 treatment of mice (2.5 μg/kg intravenously for 5 min) induces Ser phosphorylation of IRS-1 using phospho-site-specific antibodies against inhibitory serine residue 307 (23Rui 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 (472) Google Scholar) and the Ser-318 phosphorylation previously reported from our group (24Moeschel K. Beck A. Weigert C. Lammers R. Kalbacher H. Voelter W. Schleicher E.D. Haring H.U. Lehmann R. J. Biol. Chem. 2004; 279: 25157-25163Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 32Weigert C. Hennige A.M. Brischmann T. Beck A. Moeschel K. Schauble M. Brodbeck K. Haring H.U. Schleicher E.D. Lehmann R. J. Biol. Chem. 2005; 280: 37393-37399Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). We detected a rapid and significant increase in the phosphorylation of Ser-307 in liver but not in muscle of IL-6-treated mice (Fig. 1, A and C). Phosphorylation of Tyr-705 of STAT-3 demonstrated the activation of IL-6-dependent pathways. Insulin treatment resulted in a clear phosphorylation of Ser-307 in muscle (Fig. 1A) as reported previously (23Rui 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 (472) Google Scholar). When we studied the phosphorylation of Ser-318 of IRS-1 in these animals, we found just the opposite; IL-6 induced a rapid (5 min) and significant phosphorylation of this serine residue in muscle but not in liver (Fig. 1, B and C). Noteworthily, we observed a clear although less pronounced phosphorylation of STAT-3 after IL-6 treatment in muscle when compared with liver tissue, indicating that the muscle responds to IL-6. To investigate the rapid and unexpected IL-6-induced phosphorylation of Ser-318 in skeletal muscle, we studied this effect in cultured skeletal muscle cells. Using three different cell models, we observed a rapid (2 min) and transient IL-6-induced phosphorylation of Ser-318 in human myotubes, murine C2C12 myoblasts, and rat L6 myotubes, which reached a maximum after 5–10 min of IL-6 stimulation, whereas the phosphorylation of STAT-3 proceeded (Fig. 2, A–C). Together the data show that IL-6 induces a very rapid phosphorylation of a distinct serine residue (i.e. Ser-318 in muscle) on IRS-1, an adaptor molecule previously described as an essential mediator in the signal transduction of insulin and IGF-1 (38White M.F. Am. J. Physiol. 2002; 283: E413-E422Crossref PubMed Scopus (44) Google Scholar). The following questions arise from this novel finding. First, how are the IL-6 and the insulin signaling pathways linked to each other; second, which kinase is responsible for the IL-6-stimulated Ser-318 phosphorylation, and third, what is the physiological function of this serine phosphorylation of IRS-1 in muscle? IL-6 Induces IRS-1 Association with IL-6 Receptor Complex—Our results showing an unexpectedly rapid, IL-6-induced Ser phosphorylation of IRS-1 indicated a possible interaction of proximal IL-6 signaling molecules and IRS-1. To test this hypothesis, we transfected C2C12 cells with IRS-1 and performed co-immunoprecipitation studies using antibodies both against IL-6 receptor subunit gp130, which is not unique for IL-6 signal transduction, and against the specific IL-6 receptor subunit IL-6R. IL-6 signal transduction as assessed by STAT-3 phosphorylation was similar in wild-type and IRS-1-transfected cells (Fig. 3A). In nontransfected cells, we detected a faint signal for endogenous IRS-1 in the gp130 immunoprecipitate that increased after short term IL-6 stimulation (Fig. 3B). In IRS-1-transfected cells, the basal and IL-6-stimulated IRS-1 association was much more pronounced (Fig. 3B). By immunoprecipitation of IL-6R, the amount of co-precipitated IRS-1 is also markedly enhanced in IRS-1-transfected cells after 10 min of IL-6 stimulation (Fig. 3C). The results indicate that ligand-induced IL-6 receptor complex stimulation rapidly recruits IRS-1. Co-recruited PKC-δ Phosphorylates Ser-318—The serine 318 residue of IRS-1 was previously identified by in vitro phosphorylation of IRS-1-GST fusion proteins and subsequent mass spectrometry-based sequencing as a PKC-dependent phosphorylation site (24Moeschel K. Beck A. Weigert C. Lammers R. Kalbacher H. Voelter W. Schleicher E.D. Haring H.U. Lehmann R. J. Biol. Chem. 2004; 279: 25157-25163Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 39Beck A. Moeschel K. Deeg M. Haring H.U. Voelter W. Schleicher E.D. Lehmann R. J. Am. Soc. Mass Spectrom. 2003; 14: 401-405Crossref PubMed Scopus (26) Google Scholar). Further studies using this in vitro approach revealed that all three classes of PKC isoforms, classical, novel and atypical PKCs, can phosphorylate Ser-318 (40Mussig K. Staiger H. Fiedler H. Moeschel K. Beck A. Kellerer M. Haring H.U. J. Biol. Chem. 2005; 280: 32693-32699Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). Since an important role for PKC-δ in IL-6 signaling has been demonstrated (41Novotny-Diermayr V. Zhang T. Gu L. Cao X. J. Biol. Chem. 2002; 277: 49134-49142Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar), we hypothesized that this PKC isoform could be responsible for IL-6-induced Ser-318 phosphorylation of IRS-1. To elucidate the possible participation of PKC-δ, we co-transfected active or kinasenegative PKC-δ and IRS-1 in C2C12 cells. Overexpression of active PKC-δ caused a very strong increase in Ser-318 phosphorylation in unstimulated cells, with no further obvious increase after IL-6 stimulation (Fig. 4A). In control cells, IL-6 treatment enhances Ser-318 phosphorylation as expected, whereas overexpression of the kinase-inactive PKC-δ caused no change in Ser-318 phosphorylation (Fig. 4A). The data suggest that this serine residue can be phosphorylated by PKC-δ in skeletal muscle cells. To strengthen our hypothesis, we investigated the effect of IL-6 on Ser-318 phosphorylation in C2C12 cells after knock-down of PKC-δ using a siRNA silencing approach (Fig. 4B). The IL-6-induced phosphorylation of STAT-3 remained unaffected in siRNA oligonucleotides targeting PKC-δ (si-PKC-δ)-transfected cells, but phosphorylation of Ser-318 was blocked completely (Fig. 4B). Thus, we conclude that PKC-δ mediates the effect of IL-6 on Ser-318 phosphorylation. We then studied whether IL-6 stimulates the association of both IRS-1 and PKC-δ with the IL-6R. As shown in Fig. 4C, the minor coprecipitation of endogenous PKC-δ with IL-6R in wild-type cells is increased by IL-6 treatment, and this increase is similar in IRS-1-transfected cells. Transfection of C2C12 cells with PKC-δ or both PKC-δ and IRS-1 led to a comparable stimulation; the amount of endogenous as well as co-transfected PKC-δ was increased in the IL-6R immunoprecipitates in an IL-6-dependent manner (Fig. 4, C and D). The amount of co-precipitated IRS-1 was not enhanced by transfection of PKC-δ (Fig. 4D) or influenced by transfection of kinase-negative PKC-δ (data not shown). Of note, IL-6-induced phosphorylation of STAT-3 was comparable in control cells and after transfection of IRS-1 and PKC-δ (Fig. 4, A and E) or IRS-1 and kinase-negative PKC-δ (Fig. 4A). Moreover, when PKC-δ expression is down-regulated in siRNA-transfected C2C12 cells (Fig. 4B), the IL-6-induced recruitment of IRS-1 in the IL-6R complex is not affected (Fig. 4F). Thus, IL-6 induced the association of IRS-1 and IL-6R independent of PKC-δ. The data also suggest that IRS-1 and PKC-δ were in close proximity, thus enabling PKC-δ to phosphorylate Ser-318 of IRS-1. IL-6 Treatment Does Not Influence Tyrosine Phosphorylation of IRS-1—To study the biological function of the IL-6-stimulated recruitment of IRS-1 to the IL-6R complex and the subsequent Ser-318 phosphorylation of IRS-1, we investigated the tyrosine phosphorylation of IRS-1 in IRS-1-transfected C2C12 cells after stimulation with IL-6 for 10 min or insulin for 5 min. Although IL-6 treatment of the IRS-1-transfected cells did not induce the tyrosine phosphorylation of total IRS-1 protein, stimulation with insulin led to the expected increase of this phosphorylation (Fig. 5A). Although a huge amount of IRS-1 was co-immunoprecipitated with the IL-6R, no tyrosine phosphorylation of this IRS-1 fraction was detectable in both IL-6- and insulin-stimulated cells (Fig. 5A). Similarly, we observed no tyrosine phosphorylation of co-precipitated IRS-1 in control cells (Fig. 5A). To further investigate the role of Ser-318, we transfe
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