SOCS3 Exerts Its Inhibitory Function on Interleukin-6 Signal Transduction through the SHP2 Recruitment Site of gp130
2000; Elsevier BV; Volume: 275; Issue: 17 Linguagem: Inglês
10.1074/jbc.275.17.12848
ISSN1083-351X
AutoresJochen Schmitz, Manuela Weißenbach, Serge Haan, Peter C. Heinrich, Fred Schaper,
Tópico(s)Bioactive Compounds and Antitumor Agents
ResumoInterleukin-6 is involved in the regulation of many biological activities such as gene expression, cell proliferation, and differentiation. The control of the termination of cytokine signaling is as important as the regulation of initiation of signal transduction pathways. Three families of proteins involved in the down-regulation of cytokine signaling have been described recently: (i) SH2 domain-containing protein-tyrosine phosphatases (SHP), (ii) suppressors of cytokine signaling (SOCS), and (iii) protein inhibitors of activated STATs (PIAS). We have analyzed the interplay of two inhibitors in the signal transduction pathway of interleukin-6 and demonstrate that the tyrosine phosphatase SHP2 and SOCS3 do not act independently but are functionally linked. The activation of one inhibitor modulates the activity of the other; Inhibition of SHP2 activation leads to increased SOCS3-mRNA levels, whereas increased expression of SOCS3 results in a reduction of SHP2 phosphorylation after activation of the interleukin-6 signal transduction pathway. Furthermore, we show that tyrosine 759 in gp130 is essential for both SHP2 and SOCS3 but not for SOCS1 to exert their inhibitory activities on interleukin-6 signal transduction. Besides SHP2, SOCS3 also interacts with the Tyr(P)-759 peptide of gp130. Taken together, our results suggest differences in the function of SOCS1 and SOCS3 and a link between SHP2 and SOCS3. Interleukin-6 is involved in the regulation of many biological activities such as gene expression, cell proliferation, and differentiation. The control of the termination of cytokine signaling is as important as the regulation of initiation of signal transduction pathways. Three families of proteins involved in the down-regulation of cytokine signaling have been described recently: (i) SH2 domain-containing protein-tyrosine phosphatases (SHP), (ii) suppressors of cytokine signaling (SOCS), and (iii) protein inhibitors of activated STATs (PIAS). We have analyzed the interplay of two inhibitors in the signal transduction pathway of interleukin-6 and demonstrate that the tyrosine phosphatase SHP2 and SOCS3 do not act independently but are functionally linked. The activation of one inhibitor modulates the activity of the other; Inhibition of SHP2 activation leads to increased SOCS3-mRNA levels, whereas increased expression of SOCS3 results in a reduction of SHP2 phosphorylation after activation of the interleukin-6 signal transduction pathway. Furthermore, we show that tyrosine 759 in gp130 is essential for both SHP2 and SOCS3 but not for SOCS1 to exert their inhibitory activities on interleukin-6 signal transduction. Besides SHP2, SOCS3 also interacts with the Tyr(P)-759 peptide of gp130. Taken together, our results suggest differences in the function of SOCS1 and SOCS3 and a link between SHP2 and SOCS3. interleukin Janus kinase signal transducer and activator of transcription SH2-containing protein-tyrosine phosphatase 2 suppressor of cytokine signaling Src homology domain 2 glutathione S-transferase acute phase protein cytokine-inducible SH2 protein mitogen-activated protein kinase protein inhibitor of activated STATS Interleukin-6 exerts its biological activities through a receptor complex composed of the IL-61binding subunit gp80 and a dimer of the signal transducing receptor subunit gp130 (for review see Ref. 1.Heinrich P.C. Behrmann I. Müller-Newen G. Schaper F. Graeve L. Biochem. J. 1998; 334: 297-314Crossref PubMed Scopus (1757) Google Scholar). After ligand binding and gp130 dimer formation, constitutively associated kinases of the Janus family Jak1, Jak2, and tyrosine kinase 2 become activated by autophosphorylation. gp130, subsequently tyrosine phosphorylated on its cytoplasmic tail, recruits the transcription factors of the family of signal transducers and activators of transcription (STAT1 and STAT3) (2.Lütticken C. Wegenka U.M. Yuan J. Buschmann J. Schindler C. Ziemiecki A. Harpur A.G. Wilks A.F. Yasukawa K. Taga T. Kishimoto T. Barbieri G. Pellegrini S. Sendtner M. Heinrich P.C. Horn F. Science. 1994; 263: 89-92Crossref PubMed Scopus (713) Google Scholar, 3.Stahl N. Boulton T.G. Farruggella T. Ip N.Y. Davis S. Witthuhn B.A. Quelle F.W. Silvennoinen O. Barbieri G. Pellegrini S. Ihle J.N. Yancopoulos G.D. Science. 1994; 263: 92-95Crossref PubMed Scopus (849) Google Scholar) and the protein-tyrosine phosphatase SHP2 (4.Stahl N. Farruggella T.J. Boulton T.G. Zhong Z. Darnell Jr., J.E. Yancopoulos G.D. Science. 1995; 267: 1349-1353Crossref PubMed Scopus (869) Google Scholar) via specific phosphotyrosine-SH2 domain interactions (5.Heim M.H. Kerr I.M. Stark G.R. Darnell Jr., J.E. Science. 1995; 267: 1347-1349Crossref PubMed Scopus (352) Google Scholar, 6.Hemmann U. Gerhartz C. Heesel B. Sasse J. Kurapkat G. Grötzinger J. Wollmer A. Zhong Z. Darnell Jr., J.E. Graeve L. Heinrich P.C. Horn F. J. Biol. Chem. 1996; 271: 12999-13007Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar). In turn, these signaling components become tyrosine-phosphorylated also. Jak1 has been described to be crucial for the activation of gp130, the STAT factors (7.Guschin D. Rogers N. Briscoe J. Witthuhn B. Watling D. Horn F. Pellegrini S. Yasukawa K. Heinrich P. Stark G.R. Ihle J.N. Kerr I.M. EMBO J. 1995; 14: 1421-1429Crossref PubMed Scopus (365) Google Scholar), and SHP2 (8.Schaper F. Gendo C. Eck M. Schmitz J. Grimm C. Anhuf D. Kerr I.M. Heinrich P.C. Biochem. J. 1998; 335: 557-565Crossref PubMed Scopus (143) Google Scholar). The tyrosine-phosphorylated STATs form homo- and/or heterodimers (9.Zhong Z. Wen Z. Darnell Jr., J.E. Science. 1994; 264: 95-98Crossref PubMed Scopus (1735) Google Scholar) and translocate to the nucleus where they bind to enhancer elements of interleukin-6 inducible genes (10.Wegenka U.M. Buschmann J. Lütticken C. Heinrich P.C. Horn F. Mol. Cell. Biol. 1993; 13: 276-288Crossref PubMed Scopus (490) Google Scholar). The Jak/STAT signal transduction pathway is under negative control by several different mechanisms. The presence of a nuclear phosphatase leading to dephosphorylation of activated STAT1 has been proposed by Haspel et al. (11.Haspel R.L. Salditt-Georgieff M. Darnell Jr., J.E. EMBO J. 1996; 15: 6262-6268Crossref PubMed Scopus (272) Google Scholar). These authors observed a quantitative recycling of dephosphorylated STAT1 from the nucleus to the cytoplasm implicating a circulation of STAT factors between the cytoplasm and the nucleus. These data contradict those of Kim and Maniatis (12.Kim T.K. Maniatis T. Science. 1996; 273: 1717-1719Crossref PubMed Scopus (361) Google Scholar) who demonstrated a proteasome-dependent loss of activated STAT1 in the nucleus. Recently, another group of IL-6 signaling inhibitors has been described, STAT-binding proteins, known as protein inhibitors of activated STATs (PIAS) (13.Chung C.D. Liao J.Y. Liu B. Rao X.P. Jay P. Berta P. Shuai K. Science. 1997; 278: 1803-1805Crossref PubMed Scopus (809) Google Scholar, 14.Liu B. Liao J.Y. Rao X.P. Kushner S.A. Chung C.D. Chang D.D. Shuai K. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 10626-10631Crossref PubMed Scopus (636) Google Scholar). Although the PIAS do not contain phosphotyrosine binding domains such as SH2 or PTB domains, they associate with activated, tyrosine-phosphorylated STATs, leading to a loss of STAT-DNA binding activity. The mechanism of this highly specific interaction of protein inhibitor of activated STATs with activated STAT factors remains to be elucidated. Another new family of inhibitors of cytokine signaling has been discovered in three different laboratories, recently. These proteins are referred to as suppressors of cytokine signaling (SOCS) (15.Starr R. Willson T.A. Viney E.M. Murray L.J. Rayner J.R. Jenkins B.J. Gonda T.J. Alexander W.S. Metcalf D. Nicola N.A. Hilton D.J. Nature. 1997; 387: 917-921Crossref PubMed Scopus (1816) Google Scholar), Jak-binding proteins (16.Endo T.A. Masuhara M. Yokouchi M. Suzuki R. Sakamoto H. Mitsui K. Matsumoto A. Tanimura S. Ohtsubo M. Misawa H. Miyazaki T. Leonor N. Taniguchi T. Fujita T. Kanakura Y. Komiya S. Yoshimura A. Nature. 1997; 387: 921-924Crossref PubMed Scopus (1234) Google Scholar), or STAT-induced STAT inhibitors (17.Naka T. Narazaki M. Hirata M. Matsumoto T. Minamoto S. Aono A. Nishimoto N. Kajita T. Taga T. Yoshizaki K. Akira S. Kishimoto T. Nature. 1997; 387: 924-929Crossref PubMed Scopus (1139) Google Scholar). The members of this family contain a central SH2 domain as well as a carboxyl-terminal domain called the SOCS box. Depending on the cell type examined, SOCS1, SOCS2, and SOCS3 expression was found to be rapidly induced by IL-6. Because the SOCS proteins inhibit the IL-6-induced phosphorylation of Janus kinases, gp130 and STAT factors, they are regarded as feedback inhibitors of IL-6 signaling. SOCS1 inhibits the kinase activity of the three Janus kinases Jak1, Jak2, and tyrosine kinase 2 involved in IL-6 signaling (15.Starr R. Willson T.A. Viney E.M. Murray L.J. Rayner J.R. Jenkins B.J. Gonda T.J. Alexander W.S. Metcalf D. Nicola N.A. Hilton D.J. Nature. 1997; 387: 917-921Crossref PubMed Scopus (1816) Google Scholar, 16.Endo T.A. Masuhara M. Yokouchi M. Suzuki R. Sakamoto H. Mitsui K. Matsumoto A. Tanimura S. Ohtsubo M. Misawa H. Miyazaki T. Leonor N. Taniguchi T. Fujita T. Kanakura Y. Komiya S. Yoshimura A. Nature. 1997; 387: 921-924Crossref PubMed Scopus (1234) Google Scholar, 17.Naka T. Narazaki M. Hirata M. Matsumoto T. Minamoto S. Aono A. Nishimoto N. Kajita T. Taga T. Yoshizaki K. Akira S. Kishimoto T. Nature. 1997; 387: 924-929Crossref PubMed Scopus (1139) Google Scholar). Recently, it has been described that SOCS1 binds to phosphotyrosine 1007 within the kinase domain of activated Jak2 (18.Yasukawa H. Misawa H. Sakamoto H. Masuhara M. Sasaki A. Wakioka T. Ohtsuka S. Imaizumi T. Matsuda T. Ihle J.N. Yoshimura A. EMBO J. 1999; 18: 1309-1320Crossref PubMed Scopus (606) Google Scholar). Also, the protein-tyrosine phosphatase SHP2 was found to inhibit IL-6 signal transduction. Activation of the IL-6 receptor complex leads to a recruitment of SHP2 to tyrosine 759 in gp130 and to its subsequent tyrosine phosphorylation (4.Stahl N. Farruggella T.J. Boulton T.G. Zhong Z. Darnell Jr., J.E. Yancopoulos G.D. Science. 1995; 267: 1349-1353Crossref PubMed Scopus (869) Google Scholar). SHP2 activation is a crucial event for the induction of the mitogen-activated protein kinase (MAPK) pathway upon IL-6 stimulation (19.Fukada T. Hibi M. Yamanaka Y. Takahashitezuka M. Fujitani Y. Yamaguchi T. Nakajima K. Hirano T. Immunity. 1996; 5: 449-460Abstract Full Text Full Text PDF PubMed Scopus (584) Google Scholar). Mutation of Tyr-759 in gp130 results in an enhanced and prolonged STAT1 and STAT3 activation and in an increased gene induction (8.Schaper F. Gendo C. Eck M. Schmitz J. Grimm C. Anhuf D. Kerr I.M. Heinrich P.C. Biochem. J. 1998; 335: 557-565Crossref PubMed Scopus (143) Google Scholar, 20.Symes A. Stahl N. Reeves S.A. Farruggella T. Servidei T. Gearan T. Yancopoulos G. Fink J.S. Curr. Biol. 1997; 7: 697-700Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar, 21.Kim H.K. Hawley T.S. Hawley R.G. Baumann H. Mol. Cell. Biol. 1998; 18: 1525-1533Crossref PubMed Scopus (104) Google Scholar). Because SOCS3 is induced by IL-6 (15.Starr R. Willson T.A. Viney E.M. Murray L.J. Rayner J.R. Jenkins B.J. Gonda T.J. Alexander W.S. Metcalf D. Nicola N.A. Hilton D.J. Nature. 1997; 387: 917-921Crossref PubMed Scopus (1816) Google Scholar, 16.Endo T.A. Masuhara M. Yokouchi M. Suzuki R. Sakamoto H. Mitsui K. Matsumoto A. Tanimura S. Ohtsubo M. Misawa H. Miyazaki T. Leonor N. Taniguchi T. Fujita T. Kanakura Y. Komiya S. Yoshimura A. Nature. 1997; 387: 921-924Crossref PubMed Scopus (1234) Google Scholar, 17.Naka T. Narazaki M. Hirata M. Matsumoto T. Minamoto S. Aono A. Nishimoto N. Kajita T. Taga T. Yoshizaki K. Akira S. Kishimoto T. Nature. 1997; 387: 924-929Crossref PubMed Scopus (1139) Google Scholar) and SHP2 is simultaneously activated (4.Stahl N. Farruggella T.J. Boulton T.G. Zhong Z. Darnell Jr., J.E. Yancopoulos G.D. Science. 1995; 267: 1349-1353Crossref PubMed Scopus (869) Google Scholar), we asked whether these proteins influence each other in respect to expression (SOCS3) or tyrosine phosphorylation (SHP2). We observed that an inhibition of SHP2 activation led to an enhanced induction of SOCS3 mRNA. On the other hand the expression of the SOCS3 protein decreased the level of tyrosine-phosphorylated SHP2 after IL-6 stimulation. Furthermore, we found that SOCS3, but not SOCS1, requires the SHP2 recruitment site in gp130 to exert its negative function on the IL-6 signal transduction pathway. Finally, it is demonstrated in the present study that both SHP2 and SOCS3 interact with a phosphotyrosine peptide containing the Tyr-759 motif of gp130. Although we demonstrate an SHP2-SOCS3 protein-protein interaction, we were also able to show that binding of SOCS3 to Tyr-759 of gp130 does not depend on the presence of SHP2. Restriction enzymes were purchased from Roche Molecular Biochemicals and AGS (Heidelberg, Germany), and oligonucleotides were synthesized by MWG-Biotech (Ebersberg, Germany). Vent polymerase was obtained from New England BioLabs (Beverly, MA). Recombinant erythropoietin was a generous gift of Drs. J. Burg and K. H. Sellinger of Roche Molecular Biochemicals. Peptides were kindly provided by Dr. J. Schneider-Mergener, JERINI (Berlin, Germany). The peptides used had the following amino acid sequences Tyr(P)-683, biotin-βA-NSKDQMpYSDGNFTD; Tyr-759, biotin-βA-TSSTVQYSTVVHSG; Tyr(P)-759, biotin-βA-TSSTVQpYSTVVHSG; Tyr(P)-767, biotin-βA-TVVHSGpYRHQVPSV; Tyr(P)-814, biotin-βA-ILPRQQpYFKQNCSQ; Tyr(P)-905, biotin-βA-EGMPKSpYLPQTVRQ, and Tyr(P)-915 biotin-βA- PQTVRQGGpYMPQ. Antibodies to gp130 (B-P4) were gifts from Dr. J. Wijdenes (Besançon, France). Antibodies to the Tyr-705-phosphorylated STAT3 and Tyr-701-phosphorylated STAT1 were purchased from New England BioLabs (Beverly, MA), and antibodies to SHP2 and GST from Santa Cruz Biotechnology (Santa Cruz, CA). Phosphotyrosine antibodies (4G10) were from Upstate Laboratories (Lake Placid, NY), and anti-Flag-antibodies were from Sigma. Recombinant IL-6 and sIL-6R were prepared as described (22.Weiergräber O. Hemmann U. Küster A. Müller-Newen G. Schneider J. Rose-John S. Kurschat P. Brakenhoff J.P.J. Hart M.H.L. Stabel S. Heinrich P.C. Eur. J. Biochem. 1995; 234: 661-669Crossref PubMed Scopus (85) Google Scholar, 23.Arcone R. Pucci P. Zappacosta F. Fontaine V. Marloni A. Marino G. Ciliberto G. Eur. J. Biochem. 1991; 198: 541-547Crossref PubMed Scopus (129) Google Scholar). The specific activity of IL-6 was 2 × 106 units of B cell stimulatory factor 2/mg of protein. Constructions were carried out by standard procedures (24.Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning : A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). pGL3α2M-215Luc contains the promoter region −215 to +8 of the rat α2-macroglobulin gene fused to the luciferase encoding sequence and was described previously (8.Schaper F. Gendo C. Eck M. Schmitz J. Grimm C. Anhuf D. Kerr I.M. Heinrich P.C. Biochem. J. 1998; 335: 557-565Crossref PubMed Scopus (143) Google Scholar). The expression vector pSVL-EG encoding the chimeric EpoR/gp130 receptor (25.Gerhartz C. Heesel B. Sasse J. Hemmann U. Landgraf C. Schneider-Mergener J. Horn F. Heinrich P.C. Graeve L. J. Biol. Chem. 1996; 271: 12991-12998Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar) was modified by polymerase chain reaction mutagenesis to code for a chimeric EpoR/gp130 receptor (pSVL-EG(YYYYYY)) and a variant where tyrosine 759 in the cytoplasmic domain of gp130 was exchanged for phenylalanine (pSVL-EG(YFYYYY)) (8.Schaper F. Gendo C. Eck M. Schmitz J. Grimm C. Anhuf D. Kerr I.M. Heinrich P.C. Biochem. J. 1998; 335: 557-565Crossref PubMed Scopus (143) Google Scholar). The DNA fragments encoding the EpoR/gp130 chimeric receptors were also transferred into pRc/CMV-EG and used for expression in HepG2 cells (pRc/CMV-EG(YYYYYY) and pRc/CMV-EG(YFYYYY)). The DNA fragments coding for the transmembrane and cytoplasmic domains of the chimeric receptors were introduced into pSVL-gp130 resulting in expression vectors for wild type gp130 (pSVL-gp130(YYYYYY)) and for gp130 with a tyrosine to phenylalanine exchange at position 759 (pSVL-gp130(YFYYYY)) (8.Schaper F. Gendo C. Eck M. Schmitz J. Grimm C. Anhuf D. Kerr I.M. Heinrich P.C. Biochem. J. 1998; 335: 557-565Crossref PubMed Scopus (143) Google Scholar). pSVL-gp130(FYFFFF) was generated by mutation of all but not Tyr-759 cytoplasmic tyrosine residues to phenylalanine. The latter constructs were used for generating stably transfected Ba/F3 cells. The expression vectors for the SOCS or CIS proteins were pEF-Flag-I/mSOCS1, pEF-Flag-I/mSOCS2, pEF-Flag-I/mSOCS3, and pEF-Flag-I/hCIS (15.Starr R. Willson T.A. Viney E.M. Murray L.J. Rayner J.R. Jenkins B.J. Gonda T.J. Alexander W.S. Metcalf D. Nicola N.A. Hilton D.J. Nature. 1997; 387: 917-921Crossref PubMed Scopus (1816) Google Scholar). The expression vector for GST-SOCS3-(23–151) was generated by insertion of the DNA fragment encoding amino acids 23–151 of SOCS3 into the GST fusion protein expression vector pGEX-5X-3 (Amersham Pharmacia Biotech). Total RNA was isolated from cultured Ba/F3 cells by using the Qiagen total RNA kit (Qiagan, Hilden, Germany) according to manufacturer's instructions. Gel electrophoresis and Northern blot analysis were performed with 10 μg of total RNA as described previously (26.Gatsios P. Haubeck H.D. van der Leur E. Frisch W. Apte S.S. Greiling H. Heinrich P.C. Graeve L. Eur. J. Biochem. 1996; 241: 56-63Crossref PubMed Scopus (54) Google Scholar) using a 32P-labeled murine SOCS3-cDNA (15.Starr R. Willson T.A. Viney E.M. Murray L.J. Rayner J.R. Jenkins B.J. Gonda T.J. Alexander W.S. Metcalf D. Nicola N.A. Hilton D.J. Nature. 1997; 387: 917-921Crossref PubMed Scopus (1816) Google Scholar). Human hepatoma cells HepG2 were grown and transiently transfected by the calcium phosphate coprecipitation method as described previously (27.Yuan J. Wegenka U.M. Lütticken C. Buschmann J. Decker T. Schindler C. Heinrich P.C. Horn F. Mol. Cell. Biol. 1994; 14: 1657-1668Crossref PubMed Scopus (177) Google Scholar). Transfections were adjusted with control vectors to equal amounts of DNA. Cell lysis and luciferase assays were carried out using the luciferase kit (Promega, Madison, WI) as described by the manufacturer's instructions. All transient expression experiments were done at least in triplicate. Luciferase activity values were normalized to transfection efficiency monitored by the cotransfected β-galactosidase expression vector (pCR3lacZ, Amersham Pharmacia Biotech) (1.5 μg). COS7 cells were grown in Dulbecco's modified Eagle's medium with 10% fetal calf serum, 100 mg/liter streptomycin, and 60 mg/liter penicillin. Approximately 1.5 × 107COS7 cells were transiently transfected with 6–25 μg of DNA using the DEAE-dextran method. Briefly, cells were incubated in medium containing the DNA, 80 μm chloroquine, and 0.4 mg/ml DEAE-dextran for 80 min avoiding gas exchange. Afterward, cells were incubated for 1 min in phosphate-buffered saline containing 10% Me2SO. After 24 h cells were split 1:2, and after an additional 24 h in culture medium cells were stimulated. Ba/F3 cells were grown and stably transfected as described previously (28.Horsten U. Müller-Newen G. Gerhartz C. Wollmer A. Wijdenes J. Heinrich P.C. Grötzinger J. J. Biol. Chem. 1997; 272: 23748-23757Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). Equal surface expression levels of gp130 were verified by fluorescence-activated cell sorter analysis with the B-P4 antibody specific for the extracellular domain of gp130. The preparation of nuclear extracts, measurements of protein concentrations, and electrophoretic mobility shift assays have been described (28.Horsten U. Müller-Newen G. Gerhartz C. Wollmer A. Wijdenes J. Heinrich P.C. Grötzinger J. J. Biol. Chem. 1997; 272: 23748-23757Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). We used a STAT1- and STAT3-specific double-stranded 32P-labeled probe, a mutated SIE oligonucleotide of the c-fos promoter (m67 SIE, 5′-GATCCGGGAGGGATTTACGGGAAATGCTG-3′). Protein-DNA complexes were separated on a 4.5% polyacrylamide gel containing 7.5% glycerol in TBE (23 mm Tris, 23 mm boric acid, 0.5 mm EDTA, pH 8.0) at 20 V/cm for 4 h. Gels were fixed in 10% methanol, 10% acetic acid, and 80% water for 30 min, dried, and autoradiographed. For immunoprecipitation 2 × 107 cells were lysed in 500 μl of lysis buffer (50 mm HEPES, pH 7.5; 150 mm NaCl; 1% Triton X-100; 10% glycerol; 1 mmEGTA; 1.5 mm MgCl2) or for peptide/protein coprecipitation in Brij-lysis buffer (1% Brij96; 20 mmTris/HCl, pH 7.5; 150 mm NaCl; 1 mm EDTA). Buffers were supplemented with 10 μg/ml aprotinin, pepstatin, and leupeptin. Equal amounts of cellular protein or 10 μg of purified GST fusion proteins were incubated with the appropriate antibodies or with 2 μm of biotinylated peptides at 4 °C overnight and precipitated with 2.5 mg of protein A-Sepharose (Amersham Pharmacia Biotech) or NeutrAvidine-coupled agarose (Pierce), respectively. Immune complexes were separated by SDS-polyacrylamide gel electrophoresis and transferred to a polyvinylfluoride membrane. Antigens were detected by incubation with the appropriate primary antibodies (4G10, 1:1000; anti-SHP2, 1:1000; anti FlagM2-biotin, 1:500; anti-GST, 1:1000) and horseradish peroxidase-coupled secondary antibodies (1:2000) (Dako, Hamburg, Germany) or horseradish peroxidase-coupled streptavidine (1:5000) (Pierce). The membranes were developed with an enhanced chemoluminescence kit (Amersham Pharmacia Biotech). To verify application of equal amounts of protein, blots were stripped and reprobed. The expression GST-SOCS3-(23–151) was performed in Escherichia coli (BL21). Bacteria were grown at 37 °C in LB medium with ampicillin to an A 600 of 1.2 and treated with isopropyl-1-thio-β-d-galactopyranoside (1 mm) for 5 h at 20 °C to induce expression to the GST-SOCS3-(23–151) fusion protein. Cells were lysed by sonication, and purification was performed according to the manufacturer's instructions (Amersham Pharmacia Biotech). To find out which of the IL-6-induced SOCS proteins interfere with the IL-6-stimulated induction of acute phase protein (APP) synthesis in liver cells, we tested whether SOCS1, SOCS2, SOCS3, or CIS expression affects APP gene promoter induction in human HepG2 hepatoma cells (Fig.1). The respective SOCS or CIS cDNAs were cotransfected together with a reporter gene construct harboring the promoter of the α2-macroglobulin gene linked to the luciferase reporter gene (pGL3α2M-215Luc) and an expression vector for a chimeric receptor containing the extracellular domain of the EpoR and the transmembrane and cytoplasmic domains of gp130 (EG(YYYYYY)), which allowed us to study the IL-6 signal transduction pathway independently from endogenous gp130 (25.Gerhartz C. Heesel B. Sasse J. Hemmann U. Landgraf C. Schneider-Mergener J. Horn F. Heinrich P.C. Graeve L. J. Biol. Chem. 1996; 271: 12991-12998Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar). Stimulation with erythropoietin led to a 20-fold induction of the reporter gene in the transiently transfected cells expressing the chimeric receptor in the absence of SOCS/CIS. Coexpression of SOCS1 or SOCS3 led to a dramatic reduction in APP gene promoter induction. Even the background level of reporter gene expression was reduced indicating that SOCS1 and SOCS3 also influence basal transcription in unstimulated cells. In contrast, SOCS2 and CIS had only moderate effects. Thus, SOCS1 and SOCS3 but neither SOCS2 nor CIS are potent inhibitors for acute phase protein gene induction by interleukin-6. SHP2 counteracts the IL-6-induced acute phase protein gene induction (8.Schaper F. Gendo C. Eck M. Schmitz J. Grimm C. Anhuf D. Kerr I.M. Heinrich P.C. Biochem. J. 1998; 335: 557-565Crossref PubMed Scopus (143) Google Scholar, 20.Symes A. Stahl N. Reeves S.A. Farruggella T. Servidei T. Gearan T. Yancopoulos G. Fink J.S. Curr. Biol. 1997; 7: 697-700Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar, 25.Gerhartz C. Heesel B. Sasse J. Hemmann U. Landgraf C. Schneider-Mergener J. Horn F. Heinrich P.C. Graeve L. J. Biol. Chem. 1996; 271: 12991-12998Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar). To study the interplay of the IL-6 signal transduction inhibitors SOCS1, SOCS3, and SHP2, we asked whether SHP2 might also negatively regulate the IL-6-induced expression of SOCS1 and SOCS3 (Fig. 2, A andB). Therefore, IL-3-dependent Ba/F3 cells, which do not express endogenous gp130 (29.Murakami M. Narazaki M. Hibi M. Yawata H. Yasukawa K. Hamaguchi M. Taga T. Kishimoto T. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 11349-11353Crossref PubMed Scopus (492) Google Scholar), were stably transfected with gp130 receptor mutant cDNAs. Receptor surface expression of the transfected Ba/F3 cells was monitored by fluorescence-activated cell sorter analysis with an antibody raised against the extracellular domain of gp130 and was found to be comparable for all transfectants (Fig. 2 C). Stimulation of cells expressing the wild type receptor gp130(YYYYYY) led to a rapid induction of SOCS3 mRNA as determined in Northern blot analysis (Fig. 2 A). The data were normalized to glyceraldehyde-3-phosphate dehydrogenase-mRNA levels (Fig. 2 B). There was no detectable induction of SOCS1 mRNA in Ba/F3 cells (data not shown). Stimulation of cells carrying a mutation of the SHP2 recruitment site in gp130 by a substitution of Tyr-759 to Phe (gp130(YFYYYY)) resulted in increased SOCS3 mRNA levels compared with the wild type receptor. The single tyrosine 759 in the cytoplasmic part of gp130 (gp130(FYFFFF)) was not sufficient to mediate induction of the SOCS3 gene. These observations indicate that SHP2 activation counteracts SOCS3 gene expression. To test whether SOCS3 counteracts SHP2 phosphorylation, COS7 cells were transiently transfected with SOCS3 expression vectors. SHP2 phosphorylation after stimulation with IL-6·sIL6R-complexes was analyzed by Western blotting (Fig. 3). Coexpression of SOCS3 led to a reduced SHP2 phosphorylation compared with cells not transfected with SOCS3-cDNA. We conclude from these data that SOCS3 also regulates signaling components, which themselves are negative regulators of the IL-6 signal transduction pathway. SOCS1 interacts with the kinase domain of activated Jak2 (16.Endo T.A. Masuhara M. Yokouchi M. Suzuki R. Sakamoto H. Mitsui K. Matsumoto A. Tanimura S. Ohtsubo M. Misawa H. Miyazaki T. Leonor N. Taniguchi T. Fujita T. Kanakura Y. Komiya S. Yoshimura A. Nature. 1997; 387: 921-924Crossref PubMed Scopus (1234) Google Scholar, 18.Yasukawa H. Misawa H. Sakamoto H. Masuhara M. Sasaki A. Wakioka T. Ohtsuka S. Imaizumi T. Matsuda T. Ihle J.N. Yoshimura A. EMBO J. 1999; 18: 1309-1320Crossref PubMed Scopus (606) Google Scholar, 30.Nicholson 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 (370) Google Scholar). On the other hand, only a weak association of Jak2 with SOCS3 has been described by Suzuki et al. (31.Suzuki R. Sakamoto H. Yasukawa H. Masuhara M. Wakioka T. Sasaki A. Yuge K. Komiya S. Inoue A. Yoshimura A. Oncogene. 1998; 17: 2271-2278Crossref PubMed Scopus (75) Google Scholar). Unlike SOCS1, SOCS3 does not inhibit Jak kinase activity in vitro(30.Nicholson 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 (370) Google Scholar). To learn more about the mechanism of action of SOCS3, we tested whether SOCS3 activity depends on the activation of SHP2 and examined the potential of SOCS1 and SOCS3 to inhibit acute phase protein gene induction in the presence or absence of tyrosine 759 in gp130. Therefore, chimeric EpoR/gp130 wild type or mutant receptors were expressed in HepG2 cells together with or without SOCS1/SOCS3. Reporter gene assays similar to those described above were performed (Fig.4). Mutation of Tyr-759 in gp130 to Phe led to an enhanced APP promoter/reporter gene induction as expected from previously described experiments (8.Schaper F. Gendo C. Eck M. Schmitz J. Grimm C. Anhuf D. Kerr I.M. Heinrich P.C. Biochem. J. 1998; 335: 557-565Crossref PubMed Scopus (143) Google Scholar) (compare YYYYYY with YFYYYY). Both the expression of SOCS1 (Fig. 4 A,left part) or SOCS3 (Fig. 4 B, left part) led to reduced levels of the reporter luciferase activity upon stimulation of the wild type receptor EG(YYYYYY). Interestingly, unlike SOCS1, SOCS3 was unable to reduce APP gene promoter induction in the absence of Tyr-759 (EG(YFYYYY)) (Fig. 4 B,right part). Rather a slightly enhanced luciferase activity was measured after expression of SOCS3. Thus, SOCS3 but not SOCS1 requires the SHP2 recruitment site, i.e. Tyr-759 in gp130 to exert its inhibitory effect on acute phase protein gene induction. To further confirm the requirement of Tyr-759 in gp130 for SOCS3 action, we asked whether the SOCS3-mediated reduction of STAT1/STAT3 activation (15.Starr R. Willson T.A. Viney E.M. Murray L.J. Rayner J.R. Jenkins B.J. Gonda T.J. Alexander W.S. Metcalf D. Nicola N.A. Hilton D.J. Nature. 1997; 387: 917-921Crossref PubMed Scopus (1816) Google Scholar, 16.Endo T.A. Masuhara M. Yokouchi M. Suzuki R. Sakamoto H. Mitsui K. Matsumoto A. Tanimura S. Ohtsubo M. Misawa H. Miyazaki T. Leonor N. Taniguchi T. Fujita T. Kanakura Y. Komiya S. Yoshimura A. Nature. 1997; 387: 921-924Crossref PubMed Scopus (1234) Google Scholar, 17.Naka T. Narazaki M. Hirata M. Matsumoto T. Minamoto S. Aono A. Nishi
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