Title: Mechanisms of SOCS3 Phosphorylation upon Interleukin-6 Stimulation
Abstract: The suppressors of cytokine signaling (SOCS) are negative feedback inhibitors of cytokine signal transduction. SOCS3 is a key negative regulator of interleuking-6 (IL-6) signal transduction. Furthermore, SOCS3 was shown to be phosphorylated upon treatment of cells with IL-2, and this has been reported to regulate its function and half-life. We set out to investigate whether SOCS3 phosphorylation may play a role in IL-6 signaling. Tyrosine-phosphorylated SOCS3 was detected upon treatment of mouse embryonic fibroblasts with IL-6. Interestingly, the observed SOCS3 phosphorylation does not require SOCS3 recruitment to phosphotyrosine (Tyr(P)) 759 of gp130, and the kinetics of SOCS3 phosphorylation do not match the activation kinetics of the Janus kinases. This suggests that other kinases may be involved in SOCS3 phosphorylation. Using Src and Janus kinase inhibitors as well as Src kinase-deficient mouse embryonic fibroblasts, we provide evidence that Src kinases, which we found to be constitutively active in these cells, are involved in the phosphorylation of IL-6-induced SOCS3. In addition, we found that receptor-tyrosine kinases such as platelet-derived growth factor receptor or epidermal growth factor receptor can very potently phosphorylate IL-6-induced SOCS3. Taken together, these results suggest that SOCS3 phosphorylation is not a JAK-mediated phenomenon but is dependent on the activity of other kinases such as Src kinases or receptor-tyrosine kinases, which can either be constitutively active or activated by an additional stimulus. The suppressors of cytokine signaling (SOCS) are negative feedback inhibitors of cytokine signal transduction. SOCS3 is a key negative regulator of interleuking-6 (IL-6) signal transduction. Furthermore, SOCS3 was shown to be phosphorylated upon treatment of cells with IL-2, and this has been reported to regulate its function and half-life. We set out to investigate whether SOCS3 phosphorylation may play a role in IL-6 signaling. Tyrosine-phosphorylated SOCS3 was detected upon treatment of mouse embryonic fibroblasts with IL-6. Interestingly, the observed SOCS3 phosphorylation does not require SOCS3 recruitment to phosphotyrosine (Tyr(P)) 759 of gp130, and the kinetics of SOCS3 phosphorylation do not match the activation kinetics of the Janus kinases. This suggests that other kinases may be involved in SOCS3 phosphorylation. Using Src and Janus kinase inhibitors as well as Src kinase-deficient mouse embryonic fibroblasts, we provide evidence that Src kinases, which we found to be constitutively active in these cells, are involved in the phosphorylation of IL-6-induced SOCS3. In addition, we found that receptor-tyrosine kinases such as platelet-derived growth factor receptor or epidermal growth factor receptor can very potently phosphorylate IL-6-induced SOCS3. Taken together, these results suggest that SOCS3 phosphorylation is not a JAK-mediated phenomenon but is dependent on the activity of other kinases such as Src kinases or receptor-tyrosine kinases, which can either be constitutively active or activated by an additional stimulus. Interleukin-6 (IL-6) 1The abbreviations used are: IL, interleukin; sIL-6Rα, soluble IL-6 receptor α; EGF, epidermal growth factor; Epo, erythropoietin; GAP, GTPase-activating protein; gp, glycoprotein; JAK, Janus kinase; JH1, JAK homology region 1; Lck, lymphocyte kinase; MEF, mouse embryonic fibroblasts; PDGF, platelet-derived growth factor; RTK, receptortyrosine kinase; SHP, SH2 domain containing protein-tyrosine phosphatase 2; SOCS, suppressors of cytokine signaling; Src, Rous sarcoma; STAT, signal transducers and activators of transcription; OSM, oncostatinM; SYF cells, Src/Yes/Fyn-deficient MEF cells; YFP, yellow fluorescent protein.1The abbreviations used are: IL, interleukin; sIL-6Rα, soluble IL-6 receptor α; EGF, epidermal growth factor; Epo, erythropoietin; GAP, GTPase-activating protein; gp, glycoprotein; JAK, Janus kinase; JH1, JAK homology region 1; Lck, lymphocyte kinase; MEF, mouse embryonic fibroblasts; PDGF, platelet-derived growth factor; RTK, receptortyrosine kinase; SHP, SH2 domain containing protein-tyrosine phosphatase 2; SOCS, suppressors of cytokine signaling; Src, Rous sarcoma; STAT, signal transducers and activators of transcription; OSM, oncostatinM; SYF cells, Src/Yes/Fyn-deficient MEF cells; YFP, yellow fluorescent protein. is a cytokine that plays important roles in the coordination and regulation of immune responses. It was first described as B-cell stimulating factor-2, which induces differentiation and proliferation of B and T cells (1Hirano T. Yasukawa K. Harada H. Taga T. Watanabe Y. Matsuda T. Kashiwamura S. Nakajima K. Koyama K. Iwamatsu A. Tsunasawa S. Sakiyama F. Matsui H. Takahara Y. Taniguchi T. Kishimoto T. Nature. 1986; 324: 73-76Crossref PubMed Scopus (1666) Google Scholar). Furthermore, IL-6 is the major mediator of acute phase proteins in rat hepatocytes (2Andus T. Geiger T. Hirano T. Northoff H. Ganter U. Bauer J. Kishimoto T. Heinrich P.C. FEBS Lett. 1987; 221: 18-22Crossref PubMed Scopus (244) Google Scholar, 3Gauldie J. Richards C. Harnish D. Lansdorp P. Baumann H. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 7251-7255Crossref PubMed Scopus (1369) Google Scholar, 4Castell J.V. Gomez-Lechon M. David M. Hirano T. Kishimoto T. Heinrich P.C. FEBS Lett. 1988; 232: 347-350Crossref PubMed Scopus (374) Google Scholar). 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The members of this protein family (CIS and SOCS1 to SOCS7) contain a central SH2 domain and a C-terminal SOCS box. SOCS3 is rapidly induced upon IL-6 stimulation and inhibits IL-6-mediated signaling in a classic feedback loop (18Schmitz J. Weissenbach M. Haan S. Heinrich P.C. Schaper F. J. Biol. Chem. 2000; 275: 12848-12856Abstract Full Text Full Text PDF PubMed Scopus (331) Google Scholar, 25Nicholson S.E. Willson T.A. Farley A. Starr R. Zhang J.G. Baca M. Alexander W.S. Metcalf D. Hilton D.J. Nicola N.A. EMBO J. 1999; 18: 375-385Crossref PubMed Scopus (365) Google Scholar). The crucial role of SOCS3 for the inhibition of IL-6 signaling was recently confirmed in SOCS3-deficient macrophages (26Croker B.A. Krebs D.L. Zhang J.G. Wormald S. Willson T.A. Stanley E.G. Robb L. Greenhalgh C.J. Forster I. Clausen B.E. Nicola N.A. Metcalf D. Hilton D.J. Roberts A.W. Alexander W.S. Nat. Immunol. 2003; 4: 540-545Crossref PubMed Scopus (657) Google Scholar, 27Yasukawa H. Ohishi M. Mori H. 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Yoshimura A. Mui A. Migone T.S. Johnston J.A. Mol. Cell. Biol. 1999; 19: 4980-4988Crossref PubMed Scopus (207) Google Scholar, 41Cacalano N.A. Sanden D. Johnston J.A. Nat. Cell Biol. 2001; 3: 460-465Crossref PubMed Scopus (174) Google Scholar, 42Peraldi P. Filloux C. Emanuelli B. Hilton D.J. Van Obberghen E. J. Biol. Chem. 2001; 276: 24614-24620Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). This modification occurs on Tyr-204 and Tyr-221 within the SOCS box and seems to be involved in regulation of the stability of SOCS3 (41Cacalano N.A. Sanden D. Johnston J.A. Nat. Cell Biol. 2001; 3: 460-465Crossref PubMed Scopus (174) Google Scholar, 43Haan S. Ferguson P. Sommer U. Hiremath M. McVicar D.W. Heinrich P.C. Johnston J.A. Cacalano N.A. J. Biol. Chem. 2003; 278: 31972-31979Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). 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However, the kinase that triggers SOCS3 phosphorylation upon cytokine stimulation has not been identified so far. We set out to investigate SOCS3 phosphorylation in the context of IL-6 signal transduction. We found IL-6-induced SOCS3 to be phosphorylated in MEF cells but that Janus kinases are not responsible for the observed phosphorylation. We provide evidence that IL-6-induced SOCS3 can be phosphorylated by other kinases such as Src kinases or receptor-tyrosine kinases (e.g. EGF receptor or PDGF receptor) in cells where these kinases are either constitutively active or have been activated by an additional stimulus. Materials—The preparation of recombinant human IL-6 was performed by the method of Arcone et al. (44Arcone R. Pucci P. Zappacosta F. Fontaine V. Malorni A. Marino G. Ciliberto G. Eur. J. Biochem. 1991; 198: 541-547Crossref PubMed Scopus (129) Google Scholar). The soluble IL-6Rα (sIL-6Rα) was prepared as previously described by Weiergräber et al. (45Weiergräber O. Hemmann U. Küster A. Müller-Newen G. Schneider J. Rose-John S. Kurschat P. Brakenhoff J.P. Hart M.H. Stabel S. Heinrich P.C. Eur. J. Biochem. 1995; 234: 661-669Crossref PubMed Scopus (85) Google Scholar). Recombinant human Epo was a generous gift from Drs. B. Hilger and K. H. Sellinger (Roche Applied Science). Murine OncostatinM (OSM) was obtained from R&D Systems, and human EGF and human PDGF-AB was purchased from Peprotech (London, UK). The gp130-derived peptides were described previously (18Schmitz J. Weissenbach M. Haan S. Heinrich P.C. Schaper F. J. Biol. Chem. 2000; 275: 12848-12856Abstract Full Text Full Text PDF PubMed Scopus (331) Google Scholar) and were kindly provided by Dr. J. Schneider-Mergener (Charité, Berlin, Germany). The proteasome inhibitor MG132 was obtained from Calbiochem, the Src kinase inhibitor PP1 was from Biomol (Hamburg, Germany), and the Janus kinase inhibitor 1 was from Calbiochem. Plasmids—The plasmid pcDNA3-hSOCS3 was previously described (46Hörtner M. Nielsch U. Mayr L.M. Heinrich P.C. Haan S. Eur. J. Biochem. 2002; 269: 2516-2526Crossref PubMed Scopus (68) Google Scholar). pcDNA3-Myc-SOCS3 F25A and pCMV2-FLAG-SOCS3 were described previously (30Sasaki A. Yasukawa H. Suzuki A. Kamizono S. Syoda T. Kinjyo I. Sasaki M. Johnston J.A. Yoshimura A. Genes Cells. 1999; 4: 339-351Crossref PubMed Scopus (302) Google Scholar). Murine cDNAs encoding the glutathione S-transferase-tagged kinase domain of JAK2 (JAK2-JH1) and an inactive kinase domain where Tyr-1007 and Tyr-1008 are mutated to phenylalanine (JAK2-JH1FF) were kindly provided by Dr. A. Yoshimura (Kyushu University, Fukuoka, Japan). The N-terminal glutathione S-transferase tag was replaced by a yellow fluorescent protein (YFP) tag by transferring JAK2-JH1 into the previously described expression vector, pEF-JAK2-YFP (47Behrmann I. Smyczek T. Heinrich P.C. Schmitz-Van De Leur H. Komyod W. Giese B. Muller-Newen G. Haan S. Haan C. J. Biol. Chem. 2004; 279: 35486-35493Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). pSVL-JAK1, pSVL-JAK2, pSVL-JAK3, and pSVL-TYK2 were described previously (48Haan C. Is'harc H. Hermanns H.M. Schmitz-Van De Leur H. Kerr I.M. Heinrich P.C. Grötzinger J. Behrmann I. J. Biol. Chem. 2001; 276: 37451-37458Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 49Hermanns H.M. Radtke S. Haan C. Schmitz-Van de Leur H. Tavernier J. Heinrich P.C. Behrmann I. J. Immunol. 1999; 163: 6651-6658PubMed Google Scholar, 50Radtke S. Hermanns H.M. Haan C. Schmitz-Van De Leur H. Gascan H. Heinrich P.C. Behrmann I. J. Biol. Chem. 2002; 277: 11297-11305Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). pME18S-SOCS3-wt, pME18S-SOCS3-Y204F, pME18S-SOCS3–221F, and pME18S-SOCS3-FF were described previously (41Cacalano N.A. Sanden D. Johnston J.A. Nat. Cell Biol. 2001; 3: 460-465Crossref PubMed Scopus (174) Google Scholar). The preparation of cDNAs encoding different variants of the chimeric receptors EpoR/gp130 was described previously (15Schmitz J. Dahmen H. Grimm C. Gendo C. Müller-Newen G. Heinrich P.C. Schaper F. J. Immunol. 2000; 164: 848-854Crossref PubMed Scopus (73) Google Scholar). These constructs contain the extracellular part of the EpoR and the transmembrane and cytoplasmic parts of gp130. Chimeric receptor constructs EpoR/gp130 (YF4Y) and EpoR/gp130 (YY4F) contain tyrosine to phenylalanine substitutions within the cytoplasmic part of the receptor as indicated. The receptors were subcloned into the pM5 retroviral vector based on the myeloproliferative sarcoma virus containing the internal ribosome entry site (IRES) from the negative regulatory factor (NRF) followed by cDNA encoding a green fluorescent protein-Neo fusion protein. Antibodies—For immunoprecipitations a SOCS3 rabbit antibody FA1017 (Fusion Antibodies, Belfast, UK), a SOCS3 rabbit polyclonal antibody C005 (IBL, Hamburg, Germany), and a JAK1-HR785 rabbit polyclonal antibody (Santa Cruz Biotechnology, Heidelberg, Germany) were used. For immunodetection, the following primary antibodies were used unless otherwise noted: SOCS3-M20 goat polyclonal antibody (Santa Cruz Biotechnology), JAK1-HR785 rabbit polyclonal antibodies JAK2-C20 and JAK3-C21 (Santa Cruz Biotechnology), TYK2-T20220 (BD Biosciences), Lck-FA1072 antibody (Fusion Antibodies), phosphotyrosine-specific STAT3 (pY705STAT3) rabbit polyclonal antibody (Cell Signaling, Frankfurt, Germany), STAT3 mouse antibody (BD Biosciences), green fluorescent protein mouse antibody (Santa Cruz Biotechnology), phosphotyrosine-specific Src (pY-418) rabbit polyclonal antibody, and a mixture of the phosphotyrosine mouse antibodies 4G10 (Upstate Biotechnology, Hamburg, Germany) and pY99 (Santa Cruz Biotechnology). Horseradish peroxidase-conjugated secondary antibodies were obtained from DAKO (Hamburg, Germany). Cell Culture and Transfection—COS-7 cells, Yes/Fyn-deficient cells (Src++), Src/Yes/Fyn-deficient MEF cells (SYF), SYF cells reconstituted with c-Src (SYF+c-Src), MEF cells, A431 (epidermoid cancer cell), and NIH-3T3 fibroblasts were purchased from ATCC. NIH-3T3 fibroblasts stably expressing wild type SOCS3 or mutant SOCS3 (SOCS3-FF) were described previously (41Cacalano N.A. Sanden D. Johnston J.A. Nat. Cell Biol. 2001; 3: 460-465Crossref PubMed Scopus (174) Google Scholar). The cell lines were cultivated in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% fetal calf serum (Seromed, Wien, Austria) and 1% penicillin/streptomycin (Invitrogen). Stable MEF cells were cultivated additionally in 1% neomycin (Sigma). COS-7 cells were transfected by the diethylaminoethyldextran method as described previously (49Hermanns H.M. Radtke S. Haan C. Schmitz-Van de Leur H. Tavernier J. Heinrich P.C. Behrmann I. J. Immunol. 1999; 163: 6651-6658PubMed Google Scholar). For stimulation, the cells were starved in medium without fetal calf serum and treated with 20 ng/ml IL-6 and 1 μg/ml sIL-6R, 20 ng/ml OSM, 7 units/ml Epo, 50 ng/ml EGF, or 50 ng/ml PDGF-AB. The inhibitor MG132 was dissolved in Me2SO and used at a concentration of 10 μm. Retroviral Infection of Murine Embryonic Fibroblasts—Retroviral vectors pM5-EpoR/gp130 (6Y), pM5-EpoR/gp130 (YF4Y), and pM5-EpoR/gp130 (YY4F) were introduced into murine embryonic fibroblasts. Briefly, retroviral vectors were transfected together with Ecopack packaging vector and gag-pol vector into Hek293T cells, and after 48 h supernatants were collected and used in the presence of Polybrene (8 μg/ml) for infection of target cells. After 48 h transduced cells were subjected for G418 selection. The expression of the chimeric receptors was verified using Western blotting. A pool of positive cells was used for the experiments. Immunoprecipitation—For immunoprecipitation of endogenous proteins, about 3 × 107 cells were lysed in 500–800 μl of lysis buffer (1% Triton X-100, 20 mm Tris/HCl (pH 7.6), 150 mm NaCl, 10 mm NaF supplemented with 1 mm Na3VO4, 1 mm phenylmethylsulfonyl fluoride, 5 μg/ml aprotinin, 5 μg/ml leupeptin, 5 μg/ml pepstatin, and 10 μm MG132) for 30 min at 4 °C. Insoluble material was removed by centrifugation, and cell lysates were precleared with protein A-Sepharose (Amersham Biosciences) for 1 h at 4 °C. After removal of the Sepharose, the lysates (1–3 mg of protein) were incubated overnight with specific antibodies at 4 °C. The immune complexes were bound to protein A-Sepharose for 1 h at 4 °C. After centrifugation the beads were washed 3 times with washing buffer (0.1% Triton X-100, 20 mm Tris/HCl (pH 7.6), 150 mm NaCl, 10 mm NaF, 1 mm Na3VO4). The precipitated proteins were resolved by SDS-PAGE. Peptide Precipitation Assay—COS-7 cells were transfected with pcDNA3-hSOCS3 (2 μg) and pEF-YFP-JAK2-JH1 or pEF-YFP-JAK2-JH1 (3 μg). Approximately 3 nm concentrations of biotinylated peptides were immobilized by incubation with 5 mg of NeutrAvidin-coupled Sepharose (Pierce) in 100 μl of lysis buffer PP (150 mm NaCl, 50 mm Tris/HCl, 0.1 mm EDTA, 10% glycerin, 0.5% Nonidet P-40 (pH 8.0) per sample. For SOCS3 precipitation, transfected cells were lysed in lysis buffer PP supplemented with 1 mm Na3VO4, 1 mm phenylmethylsulfonyl fluoride, 5 μg/ml aprotinin, 5 μg/ml leupeptin, 5 μg/ml pepstatin, and 10 μm MG132, and the total cell lysates were incubated with the immobilized peptides at 4 °C overnight. Precipitates were washed three times with lysis buffer PP. The precipitated proteins were resolved by SDS-PAGE. Immunoblotting and Immunodetection—The electrophoretically separated proteins were transferred to a polyvinylidene difluoride membrane (Millipore, Eschborn, Germany) by semidry Western-blotting method. Antigens were detected by incubation with specific primary antibodies and horseradish-peroxidase-coupled secondary antibodies (DAKO). The membranes were developed using the ECL detection system (Amersham Biosciences). Quantification of Western blot signals was performed using the Quantity one software (Bio-Rad). SOCS3 Phosphorylation Is Observed upon Treatment of MEF Cells with IL-6 or OSM—To investigate whether endogenous SOCS3 can be phosphorylated upon IL-6 stimulation, we treated 2fTGH, 2C4, HepG2, 293T, and MEF cells with IL-6. In 2fTGH, 2C4, HepG2, and 293T cells, IL-6 potently induces the expression of SOCS3 (data not shown). However, we found detectable SOCS3 phosphorylation to occur only in MEF cells treated with IL-6 (Fig. 1A). Fig. 1A, upper panels, shows the kinetics of SOCS3 phosphorylation in MEF cells treated with IL-6 and sIL-6R. Because we found that phosphorylation of SOCS3 destabilizes the protein (43Haan S. Ferguson P. Sommer U. Hiremath M. McVicar D.W. Heinrich P.C. Johnston J.A. Cacalano N.A. J. Biol. Chem. 2003; 278: 31972-31979Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar), the proteasome inhibitor MG132 was added to facilitate the detection of phosphorylated SOCS3 (pY-SOCS3). SOCS3 phosphorylation was seen after 60 min and persisted up to 180 min. The kinetics of phosphorylation exactly match those of SOCS3 protein expression. A control for which the cells were only treated with MG132 for 180 min (Fig. 1A, lane 7) shows that the inhibition of proteasomal degradation does not by itself lead to the accumulation of SOCS3 protein. To ascertain that the antibody FA1017 (Fusion Antibodies) precipitates the phosphorylated and non-phosphorylated forms of SOCS3 equally well, we compared this antibody with a SOCS3 antibody directed against the 18 N-terminal amino acids of SOCS3 (C005; IBL) (Fig. 1A, lower panels). As can be seen in Fig. 1A, lower panels; lanes 4 and 5, both antibodies FA1017 and C005 precipitate equal amounts of pY-SOCS3. The antibody C005 does not recognize the shorter SOCS3 isoform, which lacks the 11 N-terminal amino acids (51Sasaki A. Inagaki-Ohara K. Yoshida T. Yamanaka A. Sasaki M. Yasukawa H. Koromilas A.E. Yoshimura A. J. Biol. Chem. 2003; 278: 2432-2436Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). We next checked whether SOCS3 phosphorylation could also be observed after stimulation of MEF cells with other IL-6-type cytokines. Because these cells express the OSM receptor on their cell surface, we compared IL-6 and OSM in their capacity to induce SOCS3 phosphorylation. As shown in Fig. 1B, pY-SOCS3 was also detected after treatment o