Tyrosine Phosphorylation Disrupts Elongin Interaction and Accelerates SOCS3 Degradation
2003; Elsevier BV; Volume: 278; Issue: 34 Linguagem: Inglês
10.1074/jbc.m303170200
ISSN1083-351X
AutoresSerge Haan, Paul Ferguson, Ülrike Sommer, Meena Hiremath, Daniel W. McVicar, Peter C. Heinrich, James A. Johnston, Nicholas A. Cacalano,
Tópico(s)Medicinal Plant Pharmacodynamics Research
ResumoThe suppressors of cytokine signaling (SOCS) are negative feedback inhibitors of cytokine and growth factor-induced signal transduction. The C-terminal SOCS box region is thought to regulate SOCS protein stability most likely via an elongin C interaction. In the present study, we have found that phosphorylation of SOCS3 at two tyrosine residues in the conserved SOCS box, Tyr204 and Tyr221, can inhibit the SOCS3-elongin C interaction and activate proteasome-mediated SOCS3 degradation. Jak-mediated phosphorylation of SOCS3 decreased SOCS3 protein half-life, and phosphorylation of both Tyr204 and Tyr221 was required to fully destabilize SOCS3. In contrast, a phosphorylation-deficient mutant of SOCS3, Y204F,Y221F, remained stable in the presence of activated Jak2 and receptor tyrosine kinases. SOCS3 stability correlated with the relative amount that bound elongin C, because in vitro phosphorylation of a SOCS3-glutathione S-transferase fusion protein abolished its ability to interact with elongin C. In addition, a SOCS3/SOCS1 chimera that co-precipitates with markedly increased elongin C, was significantly more stable than wild-type SOCS3. The data suggest that interaction with elongin C stabilizes SOCS3 protein expression and that phosphorylation of SOCS box tyrosine residues disrupts the complex and enhances proteasome-mediated degradation of SOCS3. The suppressors of cytokine signaling (SOCS) are negative feedback inhibitors of cytokine and growth factor-induced signal transduction. The C-terminal SOCS box region is thought to regulate SOCS protein stability most likely via an elongin C interaction. In the present study, we have found that phosphorylation of SOCS3 at two tyrosine residues in the conserved SOCS box, Tyr204 and Tyr221, can inhibit the SOCS3-elongin C interaction and activate proteasome-mediated SOCS3 degradation. Jak-mediated phosphorylation of SOCS3 decreased SOCS3 protein half-life, and phosphorylation of both Tyr204 and Tyr221 was required to fully destabilize SOCS3. In contrast, a phosphorylation-deficient mutant of SOCS3, Y204F,Y221F, remained stable in the presence of activated Jak2 and receptor tyrosine kinases. SOCS3 stability correlated with the relative amount that bound elongin C, because in vitro phosphorylation of a SOCS3-glutathione S-transferase fusion protein abolished its ability to interact with elongin C. In addition, a SOCS3/SOCS1 chimera that co-precipitates with markedly increased elongin C, was significantly more stable than wild-type SOCS3. The data suggest that interaction with elongin C stabilizes SOCS3 protein expression and that phosphorylation of SOCS box tyrosine residues disrupts the complex and enhances proteasome-mediated degradation of SOCS3. The suppressors of cytokine signaling (SOCS) 1The abbreviations used are: SOCS, suppressors of cytokine signaling; STAT, signal transducers and activators of transcription; IL, interleukin; VHL, Von Hippel-Lindau; HA, hemagglutinin; GST, glutathione S-transferase; EGF, epidermal growth factor; EGFR, EGF receptor; LPS, lipopolysaccharide; E2, ubiquitin carrier protein; E3, ubiquitin-protein isopeptide ligase. family of proteins can be transcriptionally activated by a broad range of extracellular ligands and functions in a classical feedback loop to regulate signal transduction through multiple cytokine and growth factor receptors (1Gadina M. Hilton D. Johnston J.A. Morinobu A. Lighvani A. Zhou Y.J. Visconti R. O'Shea J.J. Curr. Opin. 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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, 8Endo T.A. Masuhara M. Yokouchi M. Suzuki R. Sakamoto H. Mitsui K. Mastumoto 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, 9Starr R. Willson T.A. Viney E.M. Murray L.J.L. 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). CIS regulates signaling through the erythropoietin and IL-3 receptors, whereas SOCS1 has been shown to be essential for proper regulation of IL-6 and interferon γ responses (10Yoshimura A. Ohkubo T. Kiguchi T. Jenkins N.A. Gilbert D.J. Copel N.G. Hara T. Miyajima A. EMBO J. 1995; 14: 2816-2826Crossref PubMed Scopus (625) Google Scholar, 11Marine J.C. et al.Cell. 1999; 98: 609-616Abstract Full Text Full Text PDF PubMed Scopus (454) Google Scholar). These two molecules represent a subfamily of SOCS proteins that contain a central SH2 domain and inhibit cytokine-induced STAT activation by binding phosphotyrosine STAT docking sites on activated receptors or by interacting with phosphorylated tyrosine residues in the catalytic loop of receptor-associated Jaks to occlude the catalytic cleft and inhibit kinase activity and downstream signal transduction (12Cohney S.J. Sanden D. Cacalano N. et al.Mol. Cell Biol. 1999; 19: 4980-4988Crossref PubMed Scopus (212) Google Scholar, 13Nicholson 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, 14Yasukawa 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). Another member of the family, SOCS-3, was initially reported to inhibit signal transduction by binding to the activation loop of the Janus kinases. But SOCS-3 also exerts at least part of its effect by directly binding to specific phosphotyrosine motifs of activated cytokine receptor subunits (12Cohney S.J. Sanden D. Cacalano N. et al.Mol. Cell Biol. 1999; 19: 4980-4988Crossref PubMed Scopus (212) Google Scholar, 15Nicholson S.E. De Souza D. Fabri L.J. Corbin J. Willson T.A. Zhang J.G. Silva A. Asimakis M. Farley A. Nash A.D. Metcalf D. Hilton D.J. Nicola N.A. Baca M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6493-6498Crossref PubMed Scopus (398) Google Scholar, 16Schmitz J. Weissenbach M. Haan S. Heinrich P.C. Schaper F. J. Biol. Chem. 2000; 275: 12848-12856Abstract Full Text Full Text PDF PubMed Scopus (332) Google Scholar). Overexpression studies have implicated SOCS3 in the negative regulation of a number of cytokine signaling pathways. Mice nullizygous for SOCS3 demonstrated embryonic lethality caused by impaired placental development (17Takahashi Y. Carpino N. Cross J.C. Torres M. Parganas E. Ihle J.N. EMBO J. 2003; 22: 372-384Crossref PubMed Scopus (174) Google Scholar). Analysis of these mice indicated a role for SOCS3 in modulating leukemia inhibitory factor signaling during trophoblast giant cell differentiation. Furthermore, tetraploid rescued SOCS3–/– mice died within 3 weeks of birth because of heart failure. Phenotypically these mice displayed growth retardation and lethargy. Post-mortem analysis revealed cardiac monocyte hypertrophy presumably caused by a loss of SOCS3-mediated inhibition of leukemia inhibitory factor and CT-1 signaling. The presence of a C-terminal SOCS box homology region in all SOCS proteins suggests that this domain can play an essential role in SOCS function. More than 50 proteins have been identified in mammals, Drosophila, and Caenorhabditis elegans that possess diverse protein-protein interaction motifs such as SH2 domains, SPRY domains, WD40 motifs, and ankyrin repeats, linked to this common homologous region known as the SOCS box, a C-terminal domain of ∼40 amino acids (5Kile B.T. Schulman B.A. Alexander W.S. Nicola N.A. Martin H.M.E. Hilton D.J. Trends Biochem. Sci. 2002; 27: 235-241Abstract Full Text Full Text PDF PubMed Scopus (366) Google Scholar, 6Hilton D.J. Richardson R.T. Alexander W.S. et al.Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 114-119Crossref PubMed Scopus (620) Google Scholar). It has recently been shown that the SOCS box is a protein-protein interaction domain that mediates complex formation with elongin C (18Kamura T. Sato S. Haque D. Liu L. Kaelin W.G. Conaway R.C. Conaway J.W. Genes Dev. 1998; 12: 3872-3881Crossref PubMed Scopus (506) Google Scholar, 19Zhang J.G. Farley A. Nicholson S.E. Willson T.A. Zugaro L.M. Simpson R.J. Moritz R.L. Cary D. Richardson R. Hausmann G. Kile B.J. Kent S.B. Alexander W.S. Metcalf D. Hilton D.J. Nicola N.A. Baca M. Proc Natl Acad Sci. U. S. A. 1999; 96: 2071-2076Crossref PubMed Scopus (531) Google Scholar). Elongin C is a component of ubiquitin ligases that include elongin B, the ring finger protein Roc1, and one of the scaffold proteins Cul2 or Cul5 (5Kile B.T. Schulman B.A. Alexander W.S. Nicola N.A. Martin H.M.E. Hilton D.J. Trends Biochem. Sci. 2002; 27: 235-241Abstract Full Text Full Text PDF PubMed Scopus (366) Google Scholar, 6Hilton D.J. Richardson R.T. Alexander W.S. et al.Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 114-119Crossref PubMed Scopus (620) Google Scholar, 19Zhang J.G. Farley A. Nicholson S.E. Willson T.A. Zugaro L.M. Simpson R.J. Moritz R.L. Cary D. Richardson R. Hausmann G. Kile B.J. Kent S.B. Alexander W.S. Metcalf D. Hilton D.J. Nicola N.A. Baca M. Proc Natl Acad Sci. U. S. A. 1999; 96: 2071-2076Crossref PubMed Scopus (531) Google Scholar, 20Tyers M. Rottapel R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12230-12232Crossref PubMed Scopus (46) Google Scholar). The function of the SOCS-elongin C interaction, however, is not well understood. Some recent data suggest that elongin C links SOCS proteins to the proteasome and targets them for degradation. Consistent with this model are the findings that SOCS1 induces the degradation of Jak2, the TEL-Jak2 oncogenic fusion proteins, and IRS-1/2 (21Frantsve J. Schwaller J. Sternberg D.W. Kutok J. Gilliland D.G. Mol. Cell. Biol. 2001; 21: 3547-3557Crossref PubMed Scopus (144) Google Scholar, 22Kamizono S. Hanada T. Yasukawa H. Minoguchi S. Kato R. Minoguchi M. Hattori K. Hatakeyama S. Yada M. Morita S. Kitamura T. Kato H. Nakayama K. Yoshimura A. J. Biol. Chem. 2001; 276: 12530-12538Abstract Full Text Full Text PDF PubMed Scopus (277) Google Scholar, 23Sasaki 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, 24Ungureaunu D. Saharinen P. Junttila I. Hilton D.J. Silvennoinen O. Mol. Cell. Biol. 2002; 22: 3316-3326Crossref PubMed Scopus (213) Google Scholar, 25Rui L. Yuan M. Frantz D. Shoelson S. White M.F. J. Biol. Chem. 2002; 277: 42394-42398Abstract Full Text Full Text PDF PubMed Scopus (726) Google Scholar). In addition, mutations or post-translational modifications of SOCS1 that disrupt the elongin C interaction stabilize the protein and increase its half-life (18Kamura T. Sato S. Haque D. Liu L. Kaelin W.G. Conaway R.C. Conaway J.W. Genes Dev. 1998; 12: 3872-3881Crossref PubMed Scopus (506) Google Scholar, 26Chen X.P. Losman J.A. Cowan S. Donahue E. Fay S. Vuong B.Q. Nawijn M.C. Capece D. Cohan V.L. Rothman P. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 2175-2180Crossref PubMed Scopus (163) Google Scholar). However, there is also evidence that elongin C can stabilize SOCS protein expression and that disruption of this interaction leads to proteasome-mediated SOCS destruction (19Zhang J.G. Farley A. Nicholson S.E. Willson T.A. Zugaro L.M. Simpson R.J. Moritz R.L. Cary D. Richardson R. Hausmann G. Kile B.J. Kent S.B. Alexander W.S. Metcalf D. Hilton D.J. Nicola N.A. Baca M. Proc Natl Acad Sci. U. S. A. 1999; 96: 2071-2076Crossref PubMed Scopus (531) Google Scholar, 27Hanada T. Yoshida T. Kinjyo I. Minoguchi S. Yasukawa H. Kato S. Mimata H. Nomura Y. Seki Y. Kubo M. Yoshimura A. J. Biol. Chem. 2001; 276: 40746-40754Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). A well characterized elongin C-binding partner, the Von Hippel-Lindau (VHL) tumor suppressor, regulates the stability of a hypoxia-induced transcription factor HIF-1α. Mutations in the VHL SOCS box that interfere with its interaction with elongin C destabilize the protein result in marked reduction in protein levels and are responsible for the loss of function phenotype in human VHL syndrome (28Kaelin W.G. Nat. Rev. Cancer. 2002; 2: 673-682Crossref PubMed Scopus (698) Google Scholar, 29Ohh M. Tagaki Y. Aso T. Stebbins C.E. Pavletich N.P. Zbar B. Conaway R.C. Conaway J.W. Kaelin W.G. J Clin. Invest. 1999; 104: 1583-1591Crossref PubMed Scopus (83) Google Scholar, 30Stebbins C.E. Kaelin W.G. Pavletich N.P. Science. 1999; 284: 455-461Crossref PubMed Scopus (695) Google Scholar, 31Kamura T. Brower C.S. Conaway R.C. Conaway J.W. J. Biol. Chem. 2002; 277: 30388-30393Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). Thus, the binding of elongin C to SOCS proteins may regulate protein stability. We have previously demonstrated that SOCS3 is phosphorylated by Jaks and receptor tyrosine kinases at two tyrosine residues, Tyr204 and Tyr221, within the conserved SOCS box region and that phosphorylated Tyr221 interacts with the SH2 domains of the Ras inhibitor p120 RasGAP (32Cacalano N.A. Sanden D. Johnston J.A. Nat. Cell Biol. 2001; 3: 460-465Crossref PubMed Scopus (175) Google Scholar). Here, we demonstrate that SOCS3 tyrosine phosphorylation can regulate protein stability and elongin C interaction. Tyrosine phosphorylation decreased SOCS3 protein half-life by disrupting the interaction between SOCS3 and elongin C. Furthermore, the data demonstrated that a SOCS3/SOCS1 chimera (3/1/3), which bound much more strongly to elongin C, was significantly more stable than wild-type SOCS3. Cell Culture—All of the tissue culture media were supplemented with 10% fetal bovine serum (Hyclone, Logan, UT), penicillin/streptomycin, l-glutamine (Biowhittaker, Walkersville, MD), and 10 mm HEPES (Mediatech, Herndon, VA). 293T, RAW264, A431, COS-7, and the retroviral packaging cell lines Phoenix A and PlatE (33Grignani F. Kinsella T. Mencarelli A. Valtieri M. Riganelli D. Grignani F. Lanfrancone L. Peschle C. Nolan G.P. Pelicci P.G. Cancer Res. 1998; 58: 14-19PubMed Google Scholar, 34Onishi M. Kinoshita S. Morikawa Y. Shibuya A. Phillips J. Lanier L.L. Gorman D.M. Nolan G.P. Miyajima A. Kitamura T. Exp. Hematol. 1996; 24: 324-329PubMed Google Scholar, 35Cacalano N.A. Migone T.S. Bazan F. Hanson E.P. Chen M. Candotti F. O'Shea J.J. Johnston J.A. EMBO J. 1999; 18: 1549-1558Crossref PubMed Scopus (102) Google Scholar) were grown in Dulbecco's modified Eagle's medium (Mediatech). The murine B cell line 1881 was a generous gift from Dr. Naomi Rosenberg (Tufts University, Boston, MA) and was grown in RPMI (Mediatech) containing 50 μm 2-mercaptoethanol (Bio-Rad). The murine IL-2-dependent cell line CTLL-2 was grown in RPMI supplemented with 50 μm 2-mercaptoethanol and 100 units/ml murine IL-2 (R&D Systems, Minneapolis, MN). LPS (Sigma) was used at 10 ng/ml. Plasmids/Antibodies/Fusion Proteins—C-terminally FLAG-tagged wild-type and mutant SOCS3 cDNAs were cloned into the mammalian expression vector pME18S have been described previously (32Cacalano N.A. Sanden D. Johnston J.A. Nat. Cell Biol. 2001; 3: 460-465Crossref PubMed Scopus (175) Google Scholar, 35Cacalano N.A. Migone T.S. Bazan F. Hanson E.P. Chen M. Candotti F. O'Shea J.J. Johnston J.A. EMBO J. 1999; 18: 1549-1558Crossref PubMed Scopus (102) Google Scholar). C-terminally HA-tagged SOCS3 and 3/1/3 chimera were constructed by engineering a ClaI site at the 3′ end of the SOCS3 coding sequence by PCR-based mutagenesis, followed by cloning into pME18S encoding a 5′ in-frame ClaI site followed by the sequence for a hemagglutinin epitope tag. SOCS 3/1/3 and 3/V/3 chimeras were generated using a fusion PCR approach with overlapping (sense and antisense) mutagenic oligonucleotide primers encoding the SOCS1 or VHL BC boxes. The 5′ PCR primer spanned the unique SacII site in the SOCS3 cDNA and the 3′ primer covered the 3′ coding sequence of SOCS3 followed by a NotI restriction site. The PCR product containing the chimeric sequence was swapped with the SacII/NotI fragment from the WT SOCS3 expression vector. All of the constructs were confirmed by sequencing (Seqwright, Houston, TX). FLAG-tagged SOCS3 and 3/1/3 chimera were cloned into the retroviral vector pMX-IRES-GFP, which has been described elsewhere (36Staal F.J. Bakker A.Q. Verkuijlen M. van Oort E. Spits H. Cancer Gene Ther. 1996; 3: 345-351PubMed Google Scholar, 37Jaleco A.C. Stegmann A.P. Heemskerk M.H. Couwenberg F. Bakker A.Q. Weijer K. Spits H. Blood. 1999; 94: 2637-2646Crossref PubMed Google Scholar). GST fusion constructs were produced by cloning the SOCS3, 3/1/3 or 3/V/3 chimeras into the bacterial expression vector pGEX4T (Clontech, Palo Alto, CA). Transformation of DH5α competent Escherichia coli (Invitrogen), isopropyl-β-d-thiogalactopyranoside induction, and purification of GST fusion proteins were performed according to standard methods. N-terminally FLAG-tagged elongin B and N-terminally Myc-tagged elongin C constructs were generous gifts from Drs. Doug Hilton and Tracy Willson (Walter and Eliza Hall Institute, Parkville, Australia). The GST-JAK2-JH1 constructs were kindly provided by Dr. Akihiko Yoshimura (Fukuoka, Japan). All of the restriction endonucleases and DNA-modifying enzymes were purchased from New England Biolabs (Beverly, MA). FLAG antibody for FLAG-SOCS3 immunoprecipitation was purchased from Sigma. Transfections/Infections—293T, COS-7, and PlatE cells were transfected with Effectene lipid-based transfection reagent (Qiagen, Crawley, UK) according to the manufacturer's instructions. For retroviral infections, 2 ml of supernatant from transfected Phoenix A or PlatE cells was added to 2 ml of A431 or 1881 cells at a concentration of 2 × 105 cells/ml and incubated for 18 h at 37 °C. The cells were washed once and resuspended in fresh culture medium. Cells stably expressing the SOCS3-IRES-GFP retrovirus were sorted by flow cytometry for green fluorescent protein expression. Immunoprecipitations/GST Pull-down Experiments/Western Blotting—The cells were lysed in buffer containing 150 mm NaCl, 50 mm Tris-HCl, 2 mm EDTA, 0.875% Brij 97 (Sigma), 0.125% Nonidet P-40 (British Drug Houses, Poole, UK), 10 μg/ml aprotinin, 10 μg/ml leupeptin, 1 mm phenylmethylsulfonyl fluoride, 1 mm Na3VO4, and 1 mm NaF. The lysates were centrifuged at 12,000 × g for 5 min at 4 °C to remove nuclei. The lysates were immunoprecipitated with appropriate antibodies as described in the figure legends. Rabbit polyclonal anti FLAG antibodies were purchased from Zymed Laboratories Inc. (South San Francisco, CA). Sepharose-conjugated M2 monoclonal was from Sigma. Monoclonal anti-Myc 9E10, rabbit anti-hemagglutinin tag, rabbit anti-Myc, and rabbit anti-GST antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-RasGAP antibodies were from New England Biolabs (Beverly, MA). Anti-phosphotyrosine 4G10 was purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). For GST pull-down experiments, the cells were lysed as described above, and 5 μg of GST, GST-wild-type SOCS3-GST, or GST-3/1/3 were added to the lysates in combination with 30 μl of a 50% slurry of glutathione-Sepharose beads (Sigma). The lysates were incubated with the fusion proteins for 4 h at 4 °C. The precipitates were washed in lysis buffer, boiled, and resolved by SDS-PAGE. The proteins were electroblotted onto nylon membranes (Immobilon-P, Millipore, Bradford, MA). The membranes were probed with appropriate antibodies followed by incubation with horseradish peroxidase-labeled anti-mouse or anti-rabbit secondary antibodies (BioRad). The proteins were detected with chemiluminescent substrate (Pierce). Pulse-Chase Analysis/Cycloheximide Time Course—For pulse-chase analysis, transfected cells were washed twice in phosphate-buffered saline and incubated in cysteine/methionine-free MEM (ICN) supplemented with 0.2% bovine serum albumin (Serva) for 30 min at 37 °C. Approximately 100 μCi of 35S translabel (ICN) was added per 106 cells and incubated for 15 min at 37 °C. Labeling was chased by the addition of Dulbecco's modified Eagle's medium containing cysteine and methionine supplemented with 10% fetal calf serum. The cells were harvested at the time points post-chase indicated in the figure legends. For the cycloheximide time course, SOCS3-expressing cells were treated with 25 μm cycloheximide (Sigma) in the presence or absence of 25 μm pervanadate. Aliquots of cells were taken at the time points indicated in the figure legends and immunoblotted as described in the text. Molecular Modeling of the SOCS3 Box Region—For molecular modeling and graphic representation of the protein structures, the programs WHAT IF (53Vriend G. J. Mol. Graph. 1990; 8: 532-556Google Scholar) and pcRibbons were used. Energy minimizations were performed under vacuum conditions with the Gromos program library (W. F. van Gunsteren, distributed by BIOMOS Biomolecular Software B.V., Laboratory of Physical Chemistry, University of Groningen, the Netherlands). The structure of the VHL α-domain (Brookhaven data bank entry code 1VCB) was used as a template for the model structure of the SOCS3 box region (30Stebbins C.E. Kaelin W.G. Pavletich N.P. Science. 1999; 284: 455-461Crossref PubMed Scopus (695) Google Scholar). We have previously reported that SOCS3 is tyrosine-phosphorylated by several families of kinases, including Jaks and receptor tyrosine kinases (32Cacalano N.A. Sanden D. Johnston J.A. Nat. Cell Biol. 2001; 3: 460-465Crossref PubMed Scopus (175) Google Scholar). In mutagenesis studies, we have identified Tyr204 and Tyr221 within the conserved SOCS box motif as the major targets of phosphorylation by Jak1, Jak2, the platelet-derived growth factor receptor, and epidermal growth factor receptor (EGFR) and demonstrated that phosphorylated Tyr221 binds the Ras inhibitor p120 RasGAP, thus modulating the ERK/MAP kinase pathway (32Cacalano N.A. Sanden D. Johnston J.A. Nat. Cell Biol. 2001; 3: 460-465Crossref PubMed Scopus (175) Google Scholar). The previous observation that the SOCS box regulates protein stability (18Kamura T. Sato S. Haque D. Liu L. Kaelin W.G. Conaway R.C. Conaway J.W. Genes Dev. 1998; 12: 3872-3881Crossref PubMed Scopus (506) Google Scholar, 19Zhang J.G. Farley A. Nicholson S.E. Willson T.A. Zugaro L.M. Simpson R.J. Moritz R.L. Cary D. Richardson R. Hausmann G. Kile B.J. Kent S.B. Alexander W.S. Metcalf D. Hilton D.J. Nicola N.A. Baca M. Proc Natl Acad Sci. U. S. A. 1999; 96: 2071-2076Crossref PubMed Scopus (531) Google Scholar, 20Tyers M. Rottapel R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12230-12232Crossref PubMed Scopus (46) Google Scholar, 21Frantsve J. Schwaller J. Sternberg D.W. Kutok J. Gilliland D.G. Mol. Cell. Biol. 2001; 21: 3547-3557Crossref PubMed Scopus (144) Google Scholar, 22Kamizono S. Hanada T. Yasukawa H. Minoguchi S. Kato R. Minoguchi M. Hattori K. Hatakeyama S. Yada M. Morita S. Kitamura T. Kato H. Nakayama K. Yoshimura A. J. Biol. Chem. 2001; 276: 12530-12538Abstract Full Text Full Text PDF PubMed Scopus (277) Google Scholar, 23Sasaki 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, 24Ungureaunu D. Saharinen P. Junttila I. Hilton D.J. Silvennoinen O. Mol. Cell. Biol. 2002; 22: 3316-3326Crossref PubMed Scopus (213) Google Scholar, 25Rui L. Yuan M. Frantz D. Shoelson S. White M.F. J. Biol. Chem. 2002; 277: 42394-42398Abstract Full Text Full Text PDF PubMed Scopus (726) Google Scholar, 26Chen X.P. Losman J.A. Cowan S. Donahue E. Fay S. Vuong B.Q. Nawijn M.C. Capece D. Cohan V.L. Rothman P. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 2175-2180Crossref PubMed Scopus (163) Google Scholar, 27Hanada T. Yoshida T. Kinjyo I. Minoguchi S. Yasukawa H. Kato S. Mimata H. Nomura Y. Seki Y. Kubo M. Yoshimura A. J. Biol. Chem. 2001; 276: 40746-40754Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar) suggested that Tyr204 and Tyr221 phosphorylation might regulate this property of SOCS3. LPS is known to induce SOCS3 expression. The murine monocytic cell line RAW264 was treated with LPS to assess whether endogenous SOCS3 protein induced by LPS would become tyrosine-phosphorylated. LPS-induced SOCS3 was not tyrosine-phosphorylated (Fig. 1A, top panel), presumably because LPS does not trigger tyrosine kinase activity. In these cells treated with LPS we have observed SOCS3 expression for periods up to 24 h without turnover of SOCS3 (results not shown). However, SOCS3 tyrosine phosphorylation can be induced by treating cells with LPS in the presence of the proteintyrosine phosphatase inhibitor sodium pervanadate. RAW264 cells were stimulated with LPS for up to 4 h. The cells were also incubated with or without pervanadate during the final 15–60 min of the experiment. SOCS3 protein levels were markedly increased in cells that had been activated by LPS for 3 h (Fig. 1A, bottom panel, lanes 1 and 3), but there was no detectable tyrosine phosphorylation of SOCS3 under these conditions (Fig. 1A, top panel, lane 3). In contrast, pervanadate treatment combined with LPS stimulation resulted in SOCS3 tyrosine phosphorylation (Fig. 1A, top panel, compare lanes 3 and 4). Interestingly, SOCS3 protein levels were significantly reduced upon extended treatment with pervanadate (Fig. 1A, bottom panel, lanes 3–7). These results suggested that tyrosine phosphorylation can markedly enhance SOCS3 degradation. To demonstrate whether phosphorylation-induced SOCS3 destabilization occurred in a ligand-dependent manner, we used the human tumor cell line A431, which is transformed by the overexpression of EGFR (38Yarden Y. Sliwkowski M.X. Nat. Rev. Mol. Biol. 2001; 2: 127-137Crossref PubMed Scopus (5670) Google Scholar). Stimulation of A431 cells with EGF after serum starvation results in phosphorylation of known EGFR substrates, such as STAT3, Shc, Grb2, and the EGFR itself (38Yarden Y. Sliwkowski M.X. Nat. Rev. Mol. Biol. 2001; 2: 127-137Crossref PubMed Scopus (5670) Google Scholar). Using retroviral-mediated gene transfer, we stably expressed SOCS3 in A431 cells. To demonstrate that direct phosphorylation of SOCS3 was required to destabilize the protein, we also expressed a phosphorylation-deficient SOCS3 mutant, Y204F,Y221F, which we have previously shown not to be phosphorylated by Jak1, Jak2, and platelet-derived growth factor receptor (32Cacalano N.A. Sanden D. Johnston J.A. Nat. Cell Biol. 2001; 3: 460-465Crossref PubMed Scopus (175) Google Scholar). A431 cells were serum-starved for 8 h and incubated with 50 ng/ml EGF for 30 or 60 min, and SOCS3 protein levels as well as tyrosine phosphorylation were determined by immunoblotting. As shown in Fig. 1B, EGF stimulation of A431 cells resulted in strong tyrosine phosphorylation of wild-type SOCS3 (top panel, lanes 4–6), whereas EGF-induced phosphorylation of SOCS3 Y204F, Y221F was barely detectable (lanes 7–9). Interestingly, phosphorylation of wild-type SOCS3 resulted in a marked decrease in SOCS3 protein levels, but protein levels of the phosphorylation mutant remained unchanged after EGF stimulation (Fig. 1B, bottom panel, lanes 4–6 and 7–9). To determine the half-life of SOCS3 in the phosphorylated and unphosphorylated states, we stably expressed SOCS3 in a murine B cell line, 1881. These cells were treated with cycloheximide in the absence or presence of pervanadate to induce SOCS3 tyrosine phosphorylation. As shown in Fig. 1C, the half-life of unphosphorylated SOCS3 in this experiment was 8 h, whereas the half-life of phosphorylated SOCS3 was ∼4 h. Significant amounts of unphosphorylated SOCS3 were detectable at 6 and 8 h following cycloheximide treatment. In pervanadate-treated cells, however, SOCS3 protein was barely detectable 6 h following cycloheximide treatment, and no SOCS3 was present at the 8-h time point. These data suggest that tyrosine phosphorylation of SOCS3 in the conserved SOCS box region results in protein destabilization and accelerated degradation. We next examined the half-life of phosphorylated and unphosphorylated SOCS3 in pulse-chase experiments. COS7 cells were transfected with FLAG-tagged wild-type or Y204F,Y221F SOCS3 in combination with wild-type or kinase-inactive (FF) Jak2 catalytic domain (JH1) constructs. After [35S]cysteine-methionine labeling, the cells were lysed at time points from 0–2 h post-chase, and SOCS3 was immunoprecipitated with anti-FLAG antibodies. As shown in Fig. 2, the half-life of SOCS3, phosphorylated by the active Jak2 JH1 domain, was ∼15–30 min (Fig. 2A). In contrast, wild-type SOCS3 co-expressed with the catalytically inactive Jak2 mutant was significantly more stable than phospho-SOCS3 (half-life of 60 min; Fig. 2B). The phosphorylation-deficient Y204F,Y221F SOCS3 mutant had a half-life of >90 min (Fig. 2C), eve
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