SOCS/CIS Protein Inhibition of Growth Hormone-stimulated STAT5 Signaling by Multiple Mechanisms
1999; Elsevier BV; Volume: 274; Issue: 50 Linguagem: Inglês
10.1074/jbc.274.50.35553
ISSN1083-351X
AutoresPrabha A. Ram, David J. Waxman,
Tópico(s)Protein Tyrosine Phosphatases
ResumoThe inhibition of growth hormone (GH) signaling by five members of the GH-inducible suppressor of cytokine signaling (SOCS/CIS) family was investigated in transfected COS cells. Complete inhibition of GH activation of the signal transducer STAT5b and STAT5b-dependent transcriptional activity was observed upon expression of SOCS-1 or SOCS-3, while partial inhibition (CIS, SOCS-2) or no inhibition (SOCS-6) was seen with other SOCS/CIS family members. SOCS-1, SOCS-2, SOCS-3, and CIS each strongly inhibited the GH receptor (GHR)-dependent tyrosine phosphorylation of JAK2 seen at low levels of transfected JAK2; however, only SOCS-1 strongly inhibited the GHR-independent tyrosine phosphorylation of JAK2 seen at higher JAK2 levels. To probe for interactions with GHR, in vitrobinding assays were carried out using glutathioneS-transferase-GHR fusion proteins containing variable lengths of GHR's COOH-terminal cytoplasmic domain. CIS and SOCS-2 bound to fusions containing as few as 80 COOH-terminal GHR residues, provided the fusion protein was tyrosine-phosphorylated. By contrast, SOCS-3 binding required tyrosine-phosphorylated GHR membrane-proximal sequences, SOCS-1 binding was tyrosine phosphorylation-independent, and SOCS-6 did not bind the GHR fusion proteins at all. Mutation of GHR's membrane-proximal tyrosine residues 333 and 338 to phenylalanine suppressed the inhibition by SOCS-3, but not by CIS, of GH signaling to STAT5b. SOCS/CIS proteins can thus inhibit GH signaling to STAT5b by three distinct mechanisms, distinguished by their molecular targets within the GHR-JAK2 signaling complex, as exemplified by SOCS-1 (direct JAK2 kinase inhibition), SOCS-3 (inhibition of JAK2 signaling via membrane-proximal GHR tyrosines 333 and 338), and CIS and SOCS-2 (inhibition via membrane-distal tyrosine(s)). The inhibition of growth hormone (GH) signaling by five members of the GH-inducible suppressor of cytokine signaling (SOCS/CIS) family was investigated in transfected COS cells. Complete inhibition of GH activation of the signal transducer STAT5b and STAT5b-dependent transcriptional activity was observed upon expression of SOCS-1 or SOCS-3, while partial inhibition (CIS, SOCS-2) or no inhibition (SOCS-6) was seen with other SOCS/CIS family members. SOCS-1, SOCS-2, SOCS-3, and CIS each strongly inhibited the GH receptor (GHR)-dependent tyrosine phosphorylation of JAK2 seen at low levels of transfected JAK2; however, only SOCS-1 strongly inhibited the GHR-independent tyrosine phosphorylation of JAK2 seen at higher JAK2 levels. To probe for interactions with GHR, in vitrobinding assays were carried out using glutathioneS-transferase-GHR fusion proteins containing variable lengths of GHR's COOH-terminal cytoplasmic domain. CIS and SOCS-2 bound to fusions containing as few as 80 COOH-terminal GHR residues, provided the fusion protein was tyrosine-phosphorylated. By contrast, SOCS-3 binding required tyrosine-phosphorylated GHR membrane-proximal sequences, SOCS-1 binding was tyrosine phosphorylation-independent, and SOCS-6 did not bind the GHR fusion proteins at all. Mutation of GHR's membrane-proximal tyrosine residues 333 and 338 to phenylalanine suppressed the inhibition by SOCS-3, but not by CIS, of GH signaling to STAT5b. SOCS/CIS proteins can thus inhibit GH signaling to STAT5b by three distinct mechanisms, distinguished by their molecular targets within the GHR-JAK2 signaling complex, as exemplified by SOCS-1 (direct JAK2 kinase inhibition), SOCS-3 (inhibition of JAK2 signaling via membrane-proximal GHR tyrosines 333 and 338), and CIS and SOCS-2 (inhibition via membrane-distal tyrosine(s)). growth hormone GH receptor Janus tyrosine kinase 2 suppressor of cytokine signaling cytokine-inducible SH2 protein signal transducer and activator of transcription electrophoretic mobility shift assay STAT5-activated sodium-dependent taurocholate cotransporter gene glutathione S-transferase Src homology 2 Growth hormone (GH)1regulates important physiological processes, including somatic growth and development, carbohydrate and lipid metabolism, and liver metabolic functions, in part through its effects on gene transcription. GH signaling is initiated by hormone-induced receptor dimerization (1Cunningham B.C. Ultsch M. de Vos A.M. Mulkerrin M.G. Clauser K.R. Wells J.A. Science. 1991; 254: 821-825Crossref PubMed Scopus (780) Google Scholar), which directly leads trans-phosphorylation associated with activation of the GH receptor (GHR)-associated tyrosine kinase JAK2 (2Argetsinger L.S. Campbell G.S. Yang X. Witthuhn B.A. Silvennoinen O. Ihle J.N. Carter-Su C. Cell. 1993; 74: 237-244Abstract Full Text PDF PubMed Scopus (815) Google Scholar). JAK2, in turn, phosphorylates GHR on multiple intracellular tyrosine residues, which form docking sites for the transcriptional activator STAT5b and for other GH-activated intracellular signaling molecules (3Moutoussamy S. Kelly P.A. Finidori J. Eur. J. Biochem. 1998; 255: 1-11Crossref PubMed Scopus (104) Google Scholar, 4Argetsinger L.S. Carter-Su C. Physiol. Rev. 1996; 76: 1089-1107Crossref PubMed Scopus (246) Google Scholar). STAT5b, as well as the closely related STAT5a, responds directly and repeatedly to the pulsate plasma pattern of pituitary GH secretion (5Tannenbaum G.S. Martin J.B. Endocrinology. 1976; 98: 562-570Crossref PubMed Scopus (649) Google Scholar) that characterizes adult male rats (6Waxman D.J. Ram P.A. Park S.H. Choi H.K. J. Biol. Chem. 1995; 270: 13262-13270Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar, 7Choi H.K. Waxman D.J. Endocrinology. 1999; 140: 5126-5135Crossref PubMed Scopus (75) Google Scholar). Biochemical and genetic studies strongly suggest that STAT5b is a key intracellular mediator of the effects of plasma GH pulses on several liver-expressed genes, including cytochrome P450 genes expressed in a male-specific manner (6Waxman D.J. Ram P.A. Park S.H. Choi H.K. J. Biol. Chem. 1995; 270: 13262-13270Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar,8Davey H.W. Wilkins R.J. Waxman D.J. Am. J. Hum. Genet. 1999; 65: 959-965Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). STAT5b gene disruption leads to an apparent GH pulse insensitivity of liver tissue (55Davey H.W. Park S.H. Grattan D.R. McLachlan M.J. Waxman D.J. J. Biol. Chem. 1999; 274: 35331-35336Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar) associated with a decrease in male-characteristic pubertal body growth rate and a loss of male-specific liver gene expression (9Udy G.B. Towers R.P. Snell R.G. Wilkins R.J. Park S.H. Ram P.A. Waxman D.J. Davey H.W. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7239-7244Crossref PubMed Scopus (819) Google Scholar, 10Park S.H. Liu X. Hennighausen L. Davey H.W. Waxman D.J. J. Biol. Chem. 1999; 274: 7421-7430Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). The absence of the male-specific pattern of GH pulse-induced liver gene transcription in adult female rats is linked to the down-regulation of signaling to STAT5b by the female plasma pattern of near-continuous GH exposure (6Waxman D.J. Ram P.A. Park S.H. Choi H.K. J. Biol. Chem. 1995; 270: 13262-13270Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar, 11Gebert C.A. Park S.H. Waxman D.J. Mol. Endocrinol. 1999; 13: 213-227Crossref PubMed Scopus (61) Google Scholar). Activation of the GHR by GH initiates negative regulatory pathways important for termination of GH signaling (11Gebert C.A. Park S.H. Waxman D.J. Mol. Endocrinol. 1999; 13: 213-227Crossref PubMed Scopus (61) Google Scholar, 12Gebert C.A. Park S.H. Waxman D.J. Mol. Endocrinol. 1999; 13: 38-56Crossref PubMed Scopus (73) Google Scholar, 13Fernandez L. Flores-Morales A. Lahuna O. Sliva D. Norstedt G. Haldosen L.A. Mode A. Gustafsson J.A. Endocrinology. 1998; 139: 1815-1824Crossref PubMed Google Scholar). One negative regulatory pathway is proposed to involve tyrosine phosphatases, such as SHP-1 and SHP-2 (14Byon J.C. Kenner K.A. Kusari A.B. Kusari J. Proc. Soc. Exp. Biol. Med. 1997; 216: 1-20Crossref PubMed Scopus (41) Google Scholar), which bind to tyrosine-phosphorylated residues on GHR, JAK2 kinase, and STAT5b via their SH2 domains and may catalyze dephosphorylation leading to inactivation of these signaling molecules (15Hackett R.H. Wang Y.D. Sweitzer S. Feldman G. Wood W.I. Larner A.C. J. Biol. Chem. 1997; 272: 11128-11132Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 16Ram P.A. Waxman D.J. J. Biol. Chem. 1997; 272: 17694-17702Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar, 17Yin T. Shen R. Feng G.S. Yang Y.C. J. Biol. Chem. 1997; 272: 1032-1037Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 18Kim S.O. Jiang J. Yi W. Feng G.S. Frank S.J. J. Biol. Chem. 1998; 273: 2344-2354Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar, 19Stofega M.R. Wang H. Ullrich A. Carter-Su C. J. Biol. Chem. 1998; 273: 7112-7117Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). A second negative regulatory pathway of GH signaling, which is spontaneously activated in liver cells beginning ∼40 min after initiation of a GH pulse, requires new protein synthesis (12Gebert C.A. Park S.H. Waxman D.J. Mol. Endocrinol. 1999; 13: 38-56Crossref PubMed Scopus (73) Google Scholar). This pathway is proposed to involve “immediate early” response genes whose protein products down-regulate signaling to STAT5b by the GHR-JAK2 signaling complex, thereby preventing the activation of additional STAT5b molecules. Precedent for such an inhibitory mechanism is provided by the discovery that cytokine receptor-JAK kinase signal inhibitory molecules, termed SOCS or CIS proteins, are immediate-early gene products that can be induced by a variety of cytokines via a STAT-induced transcriptional mechanism (Refs. 20Endo 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 (1220) Google Scholar, 21Naka 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 (1126) Google Scholar, 22Starr 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 (1789) Google Scholar; for review, see Ref. 23Yoshimura A. Cytokine Growth Factor Rev. 1998; 9: 197-204Crossref PubMed Scopus (57) Google Scholar). The cytokine signaling inhibitory protein termed SOCS-1 contains an SH2 domain through which it binds to, and thereby inhibits, the JH1 kinase domain of activated, tyrosine-phosphorylated JAK2 kinase (24Yasukawa 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 (598) Google Scholar). By contrast, CIS, a member of the same cytokine inhibitor protein family, is a STAT5-inducible negative feedback regulator that binds to the tyrosine-phosphorylated receptors for erythropoietin and interleukin-3 to block STAT5 phosphorylation and downstream STAT5-dependent transcriptional responses (25Yoshimura A. Ohkubo T. Kiguchi T. Jenkins N.A. Gilbert D.J. Copeland N.G. Hara T. Miyajima A. EMBO J. 1995; 14: 2816-2826Crossref PubMed Scopus (615) Google Scholar, 26Matsumoto A. Masuhara M. Mitsui K. Yokouchi M. Ohtsubo M. Misawa H. Miyajima A. Yoshimura A. Blood. 1997; 89: 3148-3154Crossref PubMed Google Scholar). Individual SOCS/CIS mRNAs exhibit unique tissue-specific and time-dependent responses to a broad range of cytokines (22Starr 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 (1789) Google Scholar,27Masuhara M. Sakamoto H. Matsumoto A. Suzuki R. Yasukawa H. Mitsui K. Wakioka T. Tanimura S. Sasaki A. Misawa H. Yokouchi M. Ohtsubo M. Yoshimura A. Biochem. Biophys. Res. Commun. 1997; 239: 439-446Crossref PubMed Scopus (216) Google Scholar). SOCS/CIS mRNA expression can be induced in liver cells in response to a pulse of GH (28Adams T.E. Hansen J.A. Starr R. Nicola N.A. Hilton D.J. Billestrup N. J. Biol. Chem. 1998; 273: 1285-1287Abstract Full Text Full Text PDF PubMed Scopus (271) Google Scholar, 29Tollet-Egnell P. Flores-Morales A. Stavreus-Evers A. Sahlin L. Norstedt G. Endocrinology. 1999; 140: 3693-3704Crossref PubMed Google Scholar), raising the possibility that one or more of these proteins may contribute to negative feedback regulation of GHR-JAK2 signaling to STAT5b. Indeed, both SOCS-1 and SOCS-3 can inhibit GH-induced STAT5-dependent transcriptional responses in transfected cells (28Adams T.E. Hansen J.A. Starr R. Nicola N.A. Hilton D.J. Billestrup N. J. Biol. Chem. 1998; 273: 1285-1287Abstract Full Text Full Text PDF PubMed Scopus (271) Google Scholar, 30Favre H. Benhamou A. Finidori J. Kelly P.A. Edery M. FEBS Lett. 1999; 453: 63-66Crossref PubMed Scopus (113) Google Scholar). Although GH-activated STATs can transcriptionally activate expression of SOCS/CIS genes, the precise roles of SOCS/CIS proteins in terminating GHR/JAK2 signaling are only partially understood. The present studies were carried out to investigate the mechanisms whereby individual GH-inducible SOCS/CIS proteins inhibit signaling from the GHR-JAK2 receptor-kinase complex to STAT5b. Our findings reveal that SOCS/CIS proteins inhibit GH-stimulated tyrosine phosphorylation of STAT5b and STAT5b-dependent gene transcription by three distinct mechanisms, distinguished by their targets within the GHR-JAK2 signaling complex. GHR tyrosine residues 333 and 338, which are phosphorylated in response to GH stimulation (31VanderKuur J.A. Wang X. Zhang L. Allevato G. Billestrup N. Carter-Su C. J. Biol. Chem. 1995; 270: 21738-21744Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar) but are not obligatory for GH-stimulated STAT5b activation (32Lobie P.E. Allevato G. Nielsen J.H. Norstedt G. Billestrup N. J. Biol. Chem. 1995; 270: 21745-21750Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar), are presently shown to play a key role in mediating the inhibitory effects of SOCS-3 on GH signaling. These and related findings are discussed in the context of the role of SOCS/CIS proteins in determining the responsiveness of STAT5b to the sex-dependent temporal patterns of plasma GH stimulation. Human CIS, SOCS-1, SOCS-2, SOCS-3, and SOCS-6 cDNAs cloned into the expression plasmid pcDNA3 with an NH2-terminal fused myc tag (27Masuhara M. Sakamoto H. Matsumoto A. Suzuki R. Yasukawa H. Mitsui K. Wakioka T. Tanimura S. Sasaki A. Misawa H. Yokouchi M. Ohtsubo M. Yoshimura A. Biochem. Biophys. Res. Commun. 1997; 239: 439-446Crossref PubMed Scopus (216) Google Scholar) were provided from Dr. A. Yoshimura, Kurume University (Kurume, Japan). Other designations for these cDNAs include CIS = CIS-1; SOCS-1 = JAB, SSI-1; SOCS-2 = CIS-2, SSI-2; SOCS-3 = CIS-3, SSI-3; and SOCS-6 = CIS-4. Mouse STAT5a and STAT5b cloned into the expression plasmid pME18S (33Mui A.L.F. Wakao H. O'Farrell A.M. Harada N. Miyajima A. EMBO J. 1995; 14: 1166-1175Crossref PubMed Scopus (534) Google Scholar) were obtained from Dr. A. Mui (DNAX Corp., Palo Alto, CA). Rat GHR cloned into pCDNAI/Amp or into pLM108, a plasmid that contains a metallothionein promoter and an SV40 enhancer (34Billestrup N. Moldrup A. Serup P. Mathews L.S. Norstedt G. Nielsen J.H. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 7210-7214Crossref PubMed Scopus (59) Google Scholar), and derivatives of pLM108 containing tyrosine to phenylalanine point mutations at residues 333, 338, and both 333 and 338 (32Lobie P.E. Allevato G. Nielsen J.H. Norstedt G. Billestrup N. J. Biol. Chem. 1995; 270: 21745-21750Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar) were kindly provided by Dr. Nils Billestrup (Hagedorn Research Institute, Gentofte, Denmark). Tyrosine to phenylalanine codon replacements were confirmed by DNA sequencing. JAK2 cloned into the expression plasmid pRK5 was obtained from Dr. J. Ihle (St. Jude's, Memphis TN). The STAT5 reporter plasmid 4X-pT109-Luc, which contains four copies of a STAT5 response element from the rat ntcp gene (35Ganguly T.C. O'Brien M.L. Karpen S.J. Hyde J.F. Suchy F.J. Vore M. J. Clin. Invest. 1997; 99: 2906-2914Crossref PubMed Scopus (88) Google Scholar), was provided by Dr. M. Vore (University of Kentucky, Lexington, KY). Male Fischer 344 rats, untreated or hypophysectomized at 8 weeks of age, were purchased from Taconic Farms Inc. (Germantown, NY) and were maintained on a 12-h light-dark cycle. Hypophysectomized rats were administered rat GH (NIDDK-rGH-B-14-SIAFP) at 12.5 μg/100 g body weight by intraperitoneal injection. Longer term GH treatment was by hormone infusion at 2 μg/100 g body weight/h for 1 or 3 days using an Alzet osmotic minipump. Minipumps were implanted subcutaneously as described earlier (6Waxman D.J. Ram P.A. Park S.H. Choi H.K. J. Biol. Chem. 1995; 270: 13262-13270Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar). Rats were killed at different time intervals, livers excised, and stored at −80 °C. CWSV-1 cells were grown and stimulated with a pulse of GH (500 ng/ml) using methods described previously (36Gebert C.A. Park S.H. Waxman D.J. Mol. Endocrinol. 1997; 11: 400-414Crossref PubMed Scopus (111) Google Scholar). COS-1 cells were cultured in Dulbecco's modified Eagle's medium containing 10% fetal calf serum. Plasmids and reporter genes were introduced into cells grown in six-well (35 mm diameter) tissue culture plates using the calcium-phosphate method. To obtain similar levels of protein expression, as judged by Western blotting with anti-myc antibody, the following amounts of SOCS/CIS plasmid DNA were used per well: SOCS-1, 0.2 μg; SOCS-3, 1 μg; CIS, SOCS-2, and SOCS-6, 2 μg each. In addition, 0.5 μg of GHR expression plasmid and 0.5 μg of STAT5a or STAT5b expression plasmid were used per well of six-well plates. JAK2 expression plasmid (25 or 70 ng) was used, where indicated. The total amount of transfected DNA was adjusted to 3 μg/well using salmon sperm DNA (Stratagene, La Jolla, CA). At 10–12 h after addition of the DNA-calcium phosphate precipitates, the cells were washed and incubated in Dulbecco's modified Eagle's medium without serum for 36 h. Cells were then stimulated for 30 min with rat GH at a concentration of 500 ng/ml. Whole cell extracts were prepared using lysis buffer (Tropix, Inc.) and stored at −80 °C until ready for STAT5b EMSA and Western blot analysis. In experiments where STAT5-dependent transcriptional activity was assayed, the STAT5-luciferase reporter plasmid 4X-pT109-Luc (0.4 μg) and the Renilla luciferase reporter plasmid pRL-TK (40 ng; Promega Corp., Madison, WI) were cotransfected with expression plasmids encoding STAT5b, GHR (wild-type or tyrosine to phenylalanine mutated, as specified), and a SOCS/CIS protein, as indicated. Cells were stimulated with rat GH (500 ng/ml) added 24 h after the initial addition of the DNA-calcium phosphate precipitates to the cells (see above). Cells were harvested and assayed for both firefly and Renilla luciferase activities using a dual luciferase assay kit (Promega, Madison, WI). Normalized luciferase activity values (firefly/Renilla) were calculated to adjust for transfection efficiencies between samples. Luciferase and EMSA activity data presented are mean ± S.D. values and are representative of at least two or three independent sets of experiments. Whole cell extracts (20 μg of protein/lane) were resolved on SDS-polyacrylamide gels, electrotransferred to nitrocellulose and then probed sequentially with anti-STAT and anti-myc antibodies. STAT5b-specific antibody sc-835 is a rabbit polyclonal antibody raised to mouse STAT5b amino acids 711–727 (Santa Cruz Biotechnology). Monoclonal antibody raised to the myc epitope tag (c-myc amino acids 408–439; Santa Cruz Biotechnology sc-40) was used at a dilution of 1/1000. Monoclonal anti-phosphotyrosine antibody 4G10 was from Upstate Biotechnology, Inc. (Lake Placid, NY). EMSA analysis of STAT5b DNA binding activity using a β-casein STAT5 response element probe was carried out using 20 μg of cell extract/well, as described previously (37Ram P.A. Park S.H. Choi H.K. Waxman D.J. J. Biol. Chem. 1996; 271: 5929-5940Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar). Gels were exposed to PhosphorImager plates for quantitation using a PhosphorImager (Molecular Dynamics, Sunnyvale, CA) and ImageQuant software. Cell cultures were rinsed twice with ice-cold phosphate-buffered saline; total RNA was extracted from cell cultures and liver samples using guanidium thiocyanate-acid phenol (38Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (62909) Google Scholar). Poly(A) RNA was isolated from the liver RNA samples using oligo(dT)-cellulose columns (Molecular Research Center, Inc., Cincinnati, OH). RNA was fractionated by electrophoresis through agarose-formaldehyde gels and transferred to nylon membranes. Membranes were hybridized at 42 °C overnight with random primed, radiolabeled probes derived from full-length cDNA insert encoding CIS. Membranes were washed at high stringency (0.1× SSC, 0.1% SDS at 42 °C) and exposed to PhosphorImager plates. Bacteria expressing human GHR cytoplasmic domain-GST fusion proteins cloned into pGEX2TRS and their phosphorylated forms (39Yi W. Kim S.O. Jiang J. Park S.H. Kraft A.S. Waxman D.J. Frank S.J. Mol. Endocrinol. 1996; 10: 1425-1443PubMed Google Scholar) were kindly provided by Dr. Stuart Frank (University of Alabama, Birmingham, AL). Bacteria were grown at 30 °C in LB medium with ampicillin to an OD600 nm of 0.9, treated with isopropyl-1-thio-β-d-galactopyranoside (1 mm) for 3 h to induce expression of the GST-GHR fusion proteins, and lysed by lysozyme treatment in 50 mm Tris-Cl, pH 8, buffer containing 150 mm NaCl, 5 mm EDTA, 4 mm benzamidine, 0.4 mm phenylmethylsulfonyl fluoride, 1 mm DTT, 1 mm sodium orthovanadate. Soluble bacterial extracts (12,000 × g × 15 min supernatants) containing GST-GHR fusion proteins were stored at −80 °C. To assay the binding of SOCS/CIS proteins or STAT5b to the GST-GHR fusion proteins, bacterial extracts containing equivalent amounts of each fusion protein bound to glutathione-agarose beads (as determined by anti-GST Western blotting) were incubated for 1.5–2 h at 4 °C with total extracts of COS-1 cells transfected as described in the text (75–100 μg of COS-1 cell extract in 0.1 ml of fusion lysis buffer (Ref. 16Ram P.A. Waxman D.J. J. Biol. Chem. 1997; 272: 17694-17702Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar)). The beads were washed (16Ram P.A. Waxman D.J. J. Biol. Chem. 1997; 272: 17694-17702Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar), eluted in SDS gel sample buffer, and then analyzed on a Western blot probed sequentially with anti-STAT5b, anti-myc, and anti-GST antibodies. Tyrosine phosphorylation of JAK2 immunoprecipitated from JAK2-transfected COS-1 cells was carried out by anti-phosphotyrosine 4G10 Western blotting as described (16Ram P.A. Waxman D.J. J. Biol. Chem. 1997; 272: 17694-17702Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar). COS-1 cell extracts (150 μg) were incubated with anti-JAK2 serum (1/1000 dilution) for 3 h at 4 °C. The immunoprecipitates were bound to protein A-Sepharose beads, washed (16Ram P.A. Waxman D.J. J. Biol. Chem. 1997; 272: 17694-17702Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar), eluted with SDS sample buffer, and then analyzed by Western blotting with anti-phosphotyrosine antibody 4G10. JAK2 recovery was verified by reprobing with anti-JAK2 antibody. A physiologic pulse of GH given to hypophysectomized rats stimulates liver STAT5 tyrosine phosphorylation and nuclear translocation within 10 min (6Waxman D.J. Ram P.A. Park S.H. Choi H.K. J. Biol. Chem. 1995; 270: 13262-13270Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar). To determine whether CIS is activated under these conditions of in vivo GH treatment, hypophysectomized rats were treated with a pulse of GH, followed by Northern blot analysis of CIS mRNA levels. Fig. 1 A shows that hypophysectomy substantially decreases the endogenous CIS mRNA signal. Moreover, CIS mRNA was strongly induced by a single pulse of GH. The increase in CIS mRNA was first detected at 60 min, was maximal by 90 min, and was still detectable at 4 h. By contrast, GH given as a continuous infusion, a pattern that mimics the female plasma GH pattern, activated CIS gene expression at a lower level, which continued for several days (Fig. 1 A, lanes 7 and 8). In experiments carried out using the GH-responsive rat liver cell line CWSV-1 (36Gebert C.A. Park S.H. Waxman D.J. Mol. Endocrinol. 1997; 11: 400-414Crossref PubMed Scopus (111) Google Scholar), GH given in the form of a 1-h pulse induced CIS mRNA within 45 min (Fig. 1 B). The induced CIS mRNA was maintained for a full 1 h after removal of the GH stimulus (lanes 4 and5) but decayed substantially within 3 h (lane 6). This time course correlates with the time course for termination of GHR-JAK2 signaling to STAT5b, which becomes evident by ∼40 min after GH stimulation (12Gebert C.A. Park S.H. Waxman D.J. Mol. Endocrinol. 1999; 13: 38-56Crossref PubMed Scopus (73) Google Scholar). Moreover, the decay of CIS mRNA expression within 3 h after termination of the GH pulse corresponds to the time required to regain GH pulse-responsiveness of STAT5b activation in these cells (36Gebert C.A. Park S.H. Waxman D.J. Mol. Endocrinol. 1997; 11: 400-414Crossref PubMed Scopus (111) Google Scholar). GH-stimulated expression of other SOCS/CIS family RNAs has been described in a mouse liver model (28Adams T.E. Hansen J.A. Starr R. Nicola N.A. Hilton D.J. Billestrup N. J. Biol. Chem. 1998; 273: 1285-1287Abstract Full Text Full Text PDF PubMed Scopus (271) Google Scholar). To study the functional role of SOCS/CIS proteins in the termination of GH signaling to STAT5, we utilized a COS-1 cell transfection model, where the effects of individual SOCS/CIS proteins on STAT5b activation and their functional interactions with GHR and JAK2 kinase could be evaluated. These studies utilized expression plasmids encoding five SOCS/CIS proteins, each fused to an amino-terminal myc epitope tag (27Masuhara M. Sakamoto H. Matsumoto A. Suzuki R. Yasukawa H. Mitsui K. Wakioka T. Tanimura S. Sasaki A. Misawa H. Yokouchi M. Ohtsubo M. Yoshimura A. Biochem. Biophys. Res. Commun. 1997; 239: 439-446Crossref PubMed Scopus (216) Google Scholar). COS-1 cells were transiently transfected with expression plasmids encoding GHR and STAT5b, in the presence or absence of a SOCS/CIS expression plasmid. Treatment of the cells with a pulse of GH for 30 min strongly activated STAT5b DNA binding activity, as shown by EMSA using a DNA probe containing a STAT5 response element derived from the rat β-casein gene (Fig. 2 A,lane 2 versus lane 1). Co-expression of CIS or SOCS-2 partially inhibited STAT5b activation (Fig. 2 A, lanes 4,5, 7, and 8 versus lane 2; Fig. 2 C), while SOCS-3 and SOCS-1 fully blocked GH-stimulated STAT5b activation (Fig. 2 A, lanes 10, 11,13, and 14). STAT5b activation was not inhibited in COS-1 cells expressing SOCS-6 (Fig. 2 C). SOCS/CIS expression led to a corresponding pattern of inhibition of STAT5b transcriptional activity, which was assayed using a highly responsive STAT5b-luciferase reporter gene containing four copies of a STAT5b binding site derived from the liver-expressed gene ntcp(35Ganguly T.C. O'Brien M.L. Karpen S.J. Hyde J.F. Suchy F.J. Vore M. J. Clin. Invest. 1997; 99: 2906-2914Crossref PubMed Scopus (88) Google Scholar). GH treatment activated the ntcp reporter ∼20-fold but had no effect on the empty vector pT109 (Fig. 2 D). SOCS-1 and SOCS-3 were highly inhibitory toward GH induction of ntcp reporter activity. By contrast, SOCS-2 was partially inhibitory and SOCS-6 was non-inhibitory, in agreement with the effects of these SOCS proteins on STAT5b EMSA activity. CIS inhibited GH induction of ntcp reporter gene activity to a greater extent than would be anticipated based on the degree of STAT5b EMSA inhibition (e.g. Fig. 2 D versus Fig. 2 C); this differential effect was observed over a range of CIS plasmid levels (data not shown). The inhibitory effects of the SOCS/CIS proteins were also seen when GH activation of STAT5a was evaluated. Thus, SOCS-1 (data not shown) and SOCS-3 were highly inhibitory to STAT5a EMSA activity (Fig. 2 B, lanes 16 and 17 versus lanes 4 and 5), while CIS and SOCS-2 were partially inhibitory (lanes 8 and 9 and lanes 12 and13, respectively). STAT5a is closely related to STAT5b (>90% identical coding sequence) and responds to GH in a manner similar to STAT5b but exhibits differences from STAT5b with respect to its DNA binding specificity (40Verdier F. Rabionet R. Gouilleux F. Beisenherz-Huss C. Varlet P. Muller O. Mayeux P. Lacombe C. Gisselbrecht S. Chretien S. Mol. Cell. Biol. 1998; 18: 5852-5860Crossref PubMed Scopus (130) Google Scholar, 41Boucheron C. Dumon S. Santos S.C. Moriggl R. Hennighausen L. Gisselbrecht S. Gouilleux F. J. Biol. Chem. 1998; 273: 33936-33941Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar) and its role in the sex-dependent and gene-specific effects that GH has on the liver (10Park S.H. Liu X. Hennighausen L. Davey H.W. Waxman D.J. J. Biol. Chem. 1999; 274: 7421-7430Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). GH activation of STAT5b can be monitored by the conversion of STAT5b to a slower migrating form
Referência(s)