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• W1970082050 abstract "Suppressors of cytokine signaling (SOCS) are negative feedback inhibitors of cytokine and growth factor signal transduction. Although the affect of SOCS proteins on the Jak-STAT pathway has been well characterized, their role in the regulation of other signaling modules is not well understood. In the present study, we demonstrate that SOCS3 physically interacts with the SH2/SH3-containing adapter proteins Nck and Crk-L, which are known to couple activated receptors to multiple downstream signaling pathways and the actin cytoskeleton. Our data show that the SOCS3/Nck and SOCS3/Crk-L interactions depend on tyrosine phosphorylation of SOCS3 Tyr221 within the conserved SOCS box motif and intact SH2 domains of Nck and Crk-L. Furthermore, SOCS3 Tyr221 forms a YXXP motif, which is a consensus binding site for the Nck and Crk-L SH2 domains. Expression of SOCS3 in NIH3T3 cells induces constitutive recruitment of a Nck-GFP fusion protein to the plasma membrane and constitutive tyrosine phosphorylation of endogenous Nck. Our findings suggest that SOCS3 regulates multiple cytokine and growth factor-activated signaling pathways by acting as a recruitment factor for adapter proteins. Suppressors of cytokine signaling (SOCS) are negative feedback inhibitors of cytokine and growth factor signal transduction. Although the affect of SOCS proteins on the Jak-STAT pathway has been well characterized, their role in the regulation of other signaling modules is not well understood. In the present study, we demonstrate that SOCS3 physically interacts with the SH2/SH3-containing adapter proteins Nck and Crk-L, which are known to couple activated receptors to multiple downstream signaling pathways and the actin cytoskeleton. Our data show that the SOCS3/Nck and SOCS3/Crk-L interactions depend on tyrosine phosphorylation of SOCS3 Tyr221 within the conserved SOCS box motif and intact SH2 domains of Nck and Crk-L. Furthermore, SOCS3 Tyr221 forms a YXXP motif, which is a consensus binding site for the Nck and Crk-L SH2 domains. Expression of SOCS3 in NIH3T3 cells induces constitutive recruitment of a Nck-GFP fusion protein to the plasma membrane and constitutive tyrosine phosphorylation of endogenous Nck. Our findings suggest that SOCS3 regulates multiple cytokine and growth factor-activated signaling pathways by acting as a recruitment factor for adapter proteins. The suppressors of cytokine signaling (SOCS) 1The abbreviations used are: SOCS, suppressors of cytokine signaling; STAT, signal transducers and activators of transcription; GST, glutathione S-transferase; WT, wild type; ERK, extracellular signal-regulated kinase; MAP, mitogen-activated protein; Jak, Janus kinase; PDGF, platelet-derived growth factor; IRS, insulin receptor substrate; GFP, green fluorescent protein; RTK, receptor-tyrosine kinase; SH, Src homology domain; GEF, guanine nucleotide exchange factor; IL, interleukin; HA, hemagglutinin; pY, phosphorylated tyrosine. represent a heterogeneous family of proteins characterized by an N-terminal protein-protein interaction domain followed by a conserved 40 amino acid motif known as the SOCS box (1Krebs D.L. Hilton D.J. Stem Cells. 2001; 19: 378-387Crossref PubMed Scopus (661) Google Scholar, 2Yasukawa H. Sasaki A. Yoshimura A. Annu. Rev. Immunol. 2000; 18: 143-164Crossref PubMed Scopus (516) Google Scholar, 3Hilton D.J. Richardson R.T. Alexander W.S. Viney E.M. Willson T.A. Sprigg N.S. Starr R. Nicholson S.E. Metcalf D. Nicola N.A. Proc. Natl. Acad. Sci. 1998; 95: 114-119Crossref PubMed Scopus (620) Google Scholar, 4Kile B.T. Schulman B.A. Alexander W.S. Nicola N.A. Martin H.M. Hilton D.J. Trends Biochem. Sci. 2002; 2: 235-241Abstract Full Text Full Text PDF Scopus (366) Google Scholar). The N termini of SOCS proteins are divergent and can contain SH2 domains, WD40 repeats, SPRY domains, or ankyrin repeats (3Hilton D.J. Richardson R.T. Alexander W.S. Viney E.M. Willson T.A. Sprigg N.S. Starr R. Nicholson S.E. Metcalf D. Nicola N.A. Proc. Natl. Acad. Sci. 1998; 95: 114-119Crossref PubMed Scopus (620) Google Scholar, 4Kile B.T. Schulman B.A. Alexander W.S. Nicola N.A. Martin H.M. Hilton D.J. Trends Biochem. Sci. 2002; 2: 235-241Abstract Full Text Full Text PDF Scopus (366) Google Scholar). The most well characterized SOCS subfamily is represented by CIS, SOCS1, SOCS2, and SOCS3, which contain a short (∼40 amino acids) N-terminal domain and an internal SH2 domain. These proteins have been shown to inhibit Janus kinase (Jak) signaling through SH2-dependent interactions with phosphotyrosine residues on cytokine receptors or in the catalytic loop of Jak kinases, to block downstream activation of the signal transducers and activators of transcription (STATs) (5Naka 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, 6Endo 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, 7Starr 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, 8Yoshimura 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 (625) Google Scholar, 9Yasukawa 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, 10Nicholson 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, 11Cohney S.J. Sanden D. Cacalano N. Johnston J.A. Mol. Cell. Biol. 1999; 19: 4980-4988Crossref PubMed Scopus (212) Google Scholar, 12Lehmann U. Schmitz J. Weissenbach M. Sobota R.M. Hortner M. Friederichs K. Behrmann I. Tsiaris W. Sasaki A. Schneider-Mergener J. Yoshimura A. Neel B.G. Heinrich P.C. Schaper F. J. Biol. Chem. 2003; 278: 661-671Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar, 13Sasaki A. Yasukawa H. Shouda T. Kitamura T. Dikic I. Yoshimura A. J. Biol. Chem. 2000; 275: 29338-29347Abstract Full Text Full Text PDF PubMed Scopus (271) Google Scholar). The SOCS box is also a protein-protein interaction domain and mediates binding to elongin C, a component of a multisub-unit E3 ubiquitin ligase. It has been shown to regulate the stability of SOCS proteins as well as SOCS-associated signaling molecules (4Kile B.T. Schulman B.A. Alexander W.S. Nicola N.A. Martin H.M. Hilton D.J. Trends Biochem. Sci. 2002; 2: 235-241Abstract Full Text Full Text PDF Scopus (366) Google Scholar, 14Kamura 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, 15Zhang J.-G. Farley A. Nicholson S.E. Willson T.A. Zugaro L.M. Simpson R.J. Moritz R.L. Cary D. Richardson R. Hausman G. Kile B.J. Kent S.B.H. Alexander W.S. Metcalf D. Hilton D.J. Nicola N.A. Baca M. Proc. Natl. Acad. Sci. 1999; 96: 2071-2076Crossref PubMed Scopus (531) Google Scholar). The current models for the mechanisms of SOCS protein function include direct inhibition of Jak-STAT signaling as well as targeting of signal transduction molecules for degradation through the ubiquitination machinery via its association with elongin C (16Ungureaunu D. Saharinen P. Junttila I. Hilton D.J. Silvennoinen O. Mol. Cell. Biol. 2002; 22: 3316-3326Crossref PubMed Scopus (213) Google Scholar, 17Frantsve J. Schwaller J. Sternberg D.W. Kutok J. Gilliland D.G. Mol. Cell. Biol. 2001; 21: 3547-3557Crossref PubMed Scopus (144) Google Scholar, 18Kamizono 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.-I. Yoshimura A. J. Biol. Chem. 2001; 276: 12530-12538Abstract Full Text Full Text PDF PubMed Scopus (277) Google Scholar, 19Rui 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, 20De Sepulveda P. Ilangumaran S. Rottapel R. J. Biol. Chem. 2000; 275: 14005-14008Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar, 21Liu E. Cote J.F. Vuori K. EMBO J. 2003; 19: 5036-5046Crossref Scopus (94) Google Scholar). A well studied example of the latter mechanism is inhibition of transformation by the TEL-Jak2 oncogenic fusion protein by SOCS1. SOCS1 binds Tel-Jak2 through its SH2 domain and induces its proteasome-mediated destruction (16Ungureaunu D. Saharinen P. Junttila I. Hilton D.J. Silvennoinen O. Mol. Cell. Biol. 2002; 22: 3316-3326Crossref PubMed Scopus (213) Google Scholar, 17Frantsve J. Schwaller J. Sternberg D.W. Kutok J. Gilliland D.G. Mol. Cell. Biol. 2001; 21: 3547-3557Crossref PubMed Scopus (144) Google Scholar, 18Kamizono 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.-I. Yoshimura A. J. Biol. Chem. 2001; 276: 12530-12538Abstract Full Text Full Text PDF PubMed Scopus (277) Google Scholar). Other signaling proteins that are known to be degraded by SOCS include insulin receptor substrate (IRS)-1 and IRS-2, the guanine nucleotide exchange factor (GEF) Vav, and focal adhesion kinase (FAK) (19Rui 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, 20De Sepulveda P. Ilangumaran S. Rottapel R. J. Biol. Chem. 2000; 275: 14005-14008Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar, 21Liu E. Cote J.F. Vuori K. EMBO J. 2003; 19: 5036-5046Crossref Scopus (94) Google Scholar). Gene-targeting studies have demonstrated the essential role of SOCS1 and SOCS3 in cytokine responses (22Marine J.-C. Topham D.J. McKay C. Wang D. Parganas E. Stravopodis D. Yoshimura A. Ihle J. Cell. 1999; 98: 609-616Abstract Full Text Full Text PDF PubMed Scopus (454) Google Scholar, 23Alexander W.S. Starr R. Fenner J.E. Scott C.L. Handman E. Sprigg N.S. Corbin J.E. Cornish A.L. Darwiche R. Owczarek C.M. Kay T.W. Nicola N.A. Hertzog P.J. Metcalf D. Hilton D.J. Cell. 1999; 98: 597-608Abstract Full Text Full Text PDF PubMed Scopus (657) Google Scholar, 24Roberts A.W. Robb L. Rakar S. Hartley L. Cluse L. Nicola N.A. Metcalf D. Hilton D.J. Alexander W.S. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 9324-9329Crossref PubMed Scopus (260) Google Scholar, 25Marine J.C. McKay C. Wang D. Topham D.J. Parganas E. Nakajima H. Pandeville H. Yasukawa H. Sasaki A. Yoshimura A. Ihle J.N. Cell. 1999; 98: 617-627Abstract Full Text Full Text PDF PubMed Scopus (314) Google Scholar, 26Takahashi Y. Carpino N. Cross J.C. Torres M. Parganas E. Ihle J.N. EMBO J. 2003; 22: 372-384Crossref PubMed Scopus (174) Google Scholar). SOCS1 deficiency results in perinatal lethality due to hyper-responsiveness to interferon (IFN)-γ (22Marine J.-C. Topham D.J. McKay C. Wang D. Parganas E. Stravopodis D. Yoshimura A. Ihle J. Cell. 1999; 98: 609-616Abstract Full Text Full Text PDF PubMed Scopus (454) Google Scholar, 23Alexander W.S. Starr R. Fenner J.E. Scott C.L. Handman E. Sprigg N.S. Corbin J.E. Cornish A.L. Darwiche R. Owczarek C.M. Kay T.W. Nicola N.A. Hertzog P.J. Metcalf D. Hilton D.J. Cell. 1999; 98: 597-608Abstract Full Text Full Text PDF PubMed Scopus (657) Google Scholar). Likewise, mice deficient in SOCS3 die embryonically because of combined defects in trophoblast implantation and uncontrolled proliferation of hematopoietic cells (24Roberts A.W. Robb L. Rakar S. Hartley L. Cluse L. Nicola N.A. Metcalf D. Hilton D.J. Alexander W.S. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 9324-9329Crossref PubMed Scopus (260) Google Scholar, 25Marine J.C. McKay C. Wang D. Topham D.J. Parganas E. Nakajima H. Pandeville H. Yasukawa H. Sasaki A. Yoshimura A. Ihle J.N. Cell. 1999; 98: 617-627Abstract Full Text Full Text PDF PubMed Scopus (314) Google Scholar, 26Takahashi Y. Carpino N. Cross J.C. Torres M. Parganas E. Ihle J.N. EMBO J. 2003; 22: 372-384Crossref PubMed Scopus (174) Google Scholar). Furthermore, signaling studies have shown that SOCS3 can regulate responses to many cytokines and growth factors including interleukin (IL)-2, erythropoietin (Epo), IL-6 family cytokines, granulocyte-macrophage colony stimulating factor (G-CSF), leptin, growth hormone, and IL-12 (11Cohney S.J. Sanden D. Cacalano N. Johnston J.A. Mol. Cell. Biol. 1999; 19: 4980-4988Crossref PubMed Scopus (212) Google Scholar, 12Lehmann U. Schmitz J. Weissenbach M. Sobota R.M. Hortner M. Friederichs K. Behrmann I. Tsiaris W. Sasaki A. Schneider-Mergener J. Yoshimura A. Neel B.G. Heinrich P.C. Schaper F. J. Biol. Chem. 2003; 278: 661-671Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar, 13Sasaki A. Yasukawa H. Shouda T. Kitamura T. Dikic I. Yoshimura A. J. Biol. Chem. 2000; 275: 29338-29347Abstract Full Text Full Text PDF PubMed Scopus (271) Google Scholar, 27Hortner M. Nielsch U. Mayr L.M. Johnston J.A. Heinrich P.C. Haan S. J. Immunol. 2002; 169: 1219-1227Crossref PubMed Scopus (111) Google Scholar, 28Croker B.A. Metcalf D. Robb L. Wei W. Mifsud S. DiRago L. Cluse L.A. Sutherland K.D. Hartley L. Williams E. Zhang J.G. Hilton D.J. Nicola N.A. Alexander W.S. Roberts A.W. Immunity. 2004; 20: 153-165Abstract Full Text Full Text PDF PubMed Scopus (231) Google Scholar, 29Kimura A. Kinjyo I. Matsumura Y. Mori H. Mashima R. Harada M. Chien K.R. Yasukawa H. Yoshimura A. J. Biol. Chem. 2004; 279: 6905-6910Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, 30Bjorbaek C. El-Haschimi K. Frantz J.D. Flier J.S. J. Biol. Chem. 1999; 274: 30059-30065Abstract Full Text Full Text PDF PubMed Scopus (542) Google Scholar, 31Bjorbaek C. Elmquist J.K. Frantz J.D. Shoelson S.E. Flier J.S. Mol. Cell. 1998; 1: 619-625Abstract Full Text Full Text PDF PubMed Scopus (855) Google Scholar, 32Ronn S.G. Hansen J.A. Lindberg K. Karlsen A.E. Billestrup N. Mol. Endocrinol. 2002; 16: 2124-2134Crossref PubMed Scopus (29) Google Scholar, 33Yamamoto K. Yamaguchi M. Miyasaka N. Miura O. Biochem. Biophys. Res. Commun. 2003; 310: 1188-1193Crossref PubMed Scopus (101) Google Scholar, 34Seki Y. Inoue H. Nagata N. Hayashi K. Fukuyama S. Matsumoto K. Komine O. Hamano S. Himeno K. Inagaki-Ohara K. Cacalano N. O'Garra A. Oshida T. Saito H. Johnston J.A. Yoshimura A. Kubo M. Nat. Med. 2003; 9: 1047-1054Crossref PubMed Scopus (327) Google Scholar). Most of these studies have focused on the role of SOCS3 as an inhibitor of the Jak-STAT pathway. However, the finding that SOCS1 and SOCS3 regulate the stability of other families of signaling proteins such as GEFs and IRS proteins suggest that they have a much broader spectrum of action. Our laboratory has identified a function for SOCS3 beyond its role as a STAT inhibitor. We have previously shown that SOCS3 is phosphorylated by activated Jaks, Src family kinases, and receptor-tyrosine kinases (RTKs) on Tyr204 and Tyr221 in the conserved SOCS box motif (35Cacalano N.A. Sanden D. Johnston J.A. Nat. Cell Biol. 2001; 3: 460-465Crossref PubMed Scopus (175) Google Scholar). Tyrosine 221 of SOCS3 forms part of a YXXP motif (where X is any amino acid), which is a consensus binding site for the SH2 domains of the Ras inhibitor p120 RasGAP. Phosphorylated SOCS3 inactivates RasGAP and allows for sustained MAP kinase activation (35Cacalano N.A. Sanden D. Johnston J.A. Nat. Cell Biol. 2001; 3: 460-465Crossref PubMed Scopus (175) Google Scholar). Tyrosine phosphorylation of SOCS3 is therefore important for its biological function. In the present study, we identify the Nck and Crk-L adapter proteins as SOCS3 binding partners. We demonstrate that phospho-SOCS3 recruits Nck to activated RTKs and modulates Nck tyrosine phosphorylation in fibroblasts, suggesting that SOCS3 regulates adapter protein signal transduction. Cell Culture—All of the tissue culture media were supplemented with 10% fetal bovine serum (Hyclone, Logan, UT), penicillin/streptomycin, l-glutamine, and 10 mm HEPES (Mediatech, Herndon, VA). 293T, NIH3T3, and the retroviral packaging cell line PlatE were grown in Dulbecco's modified Eagle's medium (Mediatech). Plasmids, Antibodies, and Fusion Proteins—C-terminally FLAG-tagged or HA-tagged wild-type and mutant SOCS3 cDNAs cloned into the mammalian expression vector pME18S and the retroviral expression vector pMX-IRES-GFP have been described previously (36Cacalano 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, 37Haan 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 (92) Google Scholar). SOCS3 pY204/pY221 peptide spanning amino acids 200-225 (DSpYEKVTQLPGPIREFLDQpYDAPL) as well as pY204 and pY221 singly phosphorylated peptides and unphosphorylated controls were supplied by Research Genetics. Mammalian expression vector pEBB encoding wild-type (WT) Nckα and R308K SH2 mutant Nckα cDNAs, tagged with the Myc epitope, were a generous gift from Dr. Bruce Mayer, University of Connecticut Health Center. GST-Nck fusion plasmids were constructed by engineering a 5′-NdeI site (CATATG) at the start codon, and a ClaI site at the 3′-end of WT Nck coding sequence, then cloning this fragment into the bacterial expression vector pGEX4T1 (Clontech, Palo Alto, CA). Transformation of DH5 competent Escherichia coli (Invitrogen), isopropyl-d-thiogalactopyranoside induction, and purification of GST fusion proteins were performed according to standard methods. The Nck coding sequence was also cloned into the green fluorescent protein fusion vector pEGFP-N1 (Clontech) to make the Nck-GFP expression vector. The Nck-GFP fusion coding sequence was then cloned into the retroviral expression vector pMX-IRES-Puro for production of stable cell lines. The Nck-GFP fusion expression plasmid was constructed by inserting a Nck cDNA PCR fragment into pEGFP-N1 (Clontech). Primers were designed to amplify the entire Nck cDNA sequence flanked by an EcoRI site on the 5′-end and a BamHI site in frame 3 immediately preceding the stop codon. PCR was carried out with Pfu polymerase, and the resulting fragment encoding full-length, WT Nck was ligated by the EcoRI and BamHI sites into pEGFP-N1, which contains the GFP coding sequence in-frame. All PCR products were sequenced in their entirety. PCDNA-Crk-L expression vector was provided by Dr. Ronald Herbst, DNAX Research Inc., Palo Alto, CA. The Crk-L coding sequence was cloned by PCR amplification using a 5′-primer encoding a NdeI site at the start codon and a 3′-primer encoding a ClaI restriction site in place of the stop codon. The PCR product was cloned into our pME18S C-terminal FLAG tag mammalian expression vector, fusing the Crk-L cDNA sequence to the FLAG tag at its 3′-end (36Cacalano 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). Crk-L R39A was generated using a fusion PCR approach with overlapping (sense and antisense) mutagenic oligonucleotide primers encoding the point mutation. The 5′-PCR primer spanned the 5′-coding sequence of Crk-L preceded by a NdeI site at the start codon. The 3′-primer spanned the unique NaeI site in the Crk-L cDNA. The mutant PCR product was swapped with the WT Crk-L NdeI-NaeI fragment from pME18S-Crk-L-FLAG vector. Transfections and Infections—293T 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 PlatE cells was mixed with polybrene (8 mg/ml final concentration)(Sigma) and added to NIH3T3 cells in 2 ml of media at 48 and 72 h after PlatE transfections. After 48 h of infection, the cells were washed once and resuspended in fresh culture medium. Cells stably expressing the GFP fusion constructs were sorted by flow cytometry for green fluorescent protein expression. Immunoprecipitations, GST Pull-down Experiments, and Western Blotting—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 (ICN, Aurora, OH), 10 μg/ml leupeptin (ICN), 1 mm phenylmethylsulfonyl fluoride (Sigma), 1 mm Na3VO4 (Sigma). 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 and Sepharoseconjugated M2 monoclonal antibody were purchased from Sigma. Monoclonal anti-Myc 9E10, rabbit anti-hemagglutinin tag, and rabbit anti-Myc were from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-phosphotyrosine 4G10 was purchased from Upstate Biotechnology (Lake Placid, NY). For GST pull-down experiments, the cells were lysed as described above, and 2 μg of the appropriate GST fusion protein were added to the lysates in combination with 30 μl of a 50% slurry of glutathione-Sepharose beads (Sigma). The lysates were incubated with fusion proteins for 4 h at 4 °C. The precipitates were washed three times 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, as described in the figure legends, followed by incubation with horseradish peroxidase-labeled anti-mouse or anti-rabbit secondary antibodies (Amersham Biosciences). The proteins were detected with chemiluminescent substrate (Pierce). Confocal Visualization—GFP fusion protein expressing NIH3T3 cells were plated in glass bottom microwell dishes (MatTek, Ashland, MA) 24 h before visualization. Cells were serum-starved for 6 h in 0.1% serum and stimulated with 50 ng/ml PDGF BB. Microscopy was preformed on a Leica TCS SP MP Inverted Confocal Microscope with a 488 nm excitation line provided by an argon laser. Image analysis was preformed with Leica Confocal Software. In a previous study, we determined that SOCS3 Tyr221 is phosphorylated by Jak1, Jak2, EGF receptor, and PDGF receptor and interacts with p120 RasGAP in IL-2-stimulated T cells (35Cacalano N.A. Sanden D. Johnston J.A. Nat. Cell Biol. 2001; 3: 460-465Crossref PubMed Scopus (175) Google Scholar). We have extended these findings to demonstrate that phosphorylated SOCS3 interacts with p120 RasGAP in PDGF-stimulated fibroblasts as well. As shown in Fig. 1, panels B and C, stimulation of murine NIH3T3 fibroblasts with 50 ng/ml PDGF BB resulted in robust SOCS3 expression and tyrosine phosphorylation at the 1- and 2-h time points. Furthermore, p120 RasGAP was found in SOCS3 immunoprecipitates from PDGF-treated cells (Fig. 1, panel A). We examined the sequences surrounding SOCS3 Tyr204 and Tyr221 and found that Tyr221 forms part of a YXXP motif, which is a consensus binding site for the RasGAP SH2 domains (Table I). The YXXP motif has also been shown to interact with the SH2 domains of the Nck and Crk-L adapter proteins (38Songyang Z. Shoelson S.E. Chaudhuri M. Gish G. Pawson T. Haser W.G. King F. Roberts T. Ratnofsky S. Lechleider R.J. Neel B.G. Birge R.B. Fajardo J.E. Chou M.M. Hanafusa H. Schaffhausen B. Cantley L.C. Cell. 1993; 72: 767-778Abstract Full Text PDF PubMed Scopus (2391) Google Scholar, 39McCarty J.H. Bioessays. 1998; 20: 913-921Crossref PubMed Scopus (80) Google Scholar, 40Feller S.M. Posern G. Voss J. Kardinal C. Sakkab D. Zheng J. Knudsen B.S. J. Cell. Physiol. 1998; 177: 535-552Crossref PubMed Scopus (124) Google Scholar, 41Feller S.M. Oncogene. 2001; 20: 6348-6371Crossref PubMed Scopus (419) Google Scholar, 42Li W. Fan J. Woodley D.T. Oncogene. 2001; 20: 6403-6417Crossref PubMed Scopus (134) Google Scholar, 43Tanaka M. Gupta R. Mayer B.J. Mol. Cell. Biol. 1995; 15: 6829-6837Crossref PubMed Scopus (221) Google Scholar). Therefore, we examined the ability of phosphorylated SOCS3 to interact with Nck and Crk-L. Lysates from NIH3T3 cells were precipitated with N-terminal biotinylated peptides spanning both SOCS3 phosphorylation sites (amino acids 200-225). In order to map the Nck and Crk-L binding sites, we measured Nck and Crk-L binding to peptides containing pY204/pY221, single pY204 or pY221 phosphorylation sites, or a control unphosphorylated peptide of identical sequence. As shown in Fig. 2A, both Nck and Crk-L bound the pY204/pY221 peptide (lanes 2 and 6), as well as the pY221 singly phosphorylated peptide (lanes 4 and 8). However, both molecules failed to interact with the pY204 peptide (lanes 3 and 7) or the unphosphorylated control (lanes 1 and 5).Table IPutative RasGAP binding sitesMoleculeSequencep62 DOKS P P A L Y A E P LS Q D S L Y S D P LK E D P I Y D E P EP P Q G L Y D L P RV K E E G Y E L P YP A T D D Y A V P Pp56 DOK-2P G T Q L Y D W P YA P E G E Y A V P EL P D H I Y D E P ESOCS3 Tyr221E F L D Q Y D A P LSOCS3 Tyr204aNon binding.G H L D S Y E K V TConsensus sequenceY X X Pa Non binding. Open table in a new tab Fig. 2Nck and Crk-L SH2 domains bind phosphorylated SOCS3 Tyr221.A, N-terminal biotinylated peptides spanning the SOCS3 phosphorylation sites (amino acids 200-225) were prebound to streptavidin beads. Lysates from 5 × 106 NIH3T3 cells were precipitated with streptavidin beads bound to pY(204, 221) double-phosphorylated peptides, singly phosphorylated pY204 or pY221 peptides, or an unphosphorylated control peptide of the same amino acid sequence. The precipitates were analyzed by SDS-PAGE followed by Western blotting for Nck (lanes 1-4) or Crk-L (lanes 5-8). B, 293T cells were transiently transfected with mammalian expression vector pEBB encoding N-terminal Myc-tagged WT Nck (lane 1); a Nck SH2 mutant, R308A (lane 2); or WT Nck SH2 domain (lane 3). 293T cells were also transfected with pME18S encoding C-terminal FLAG-tagged WT Crk-L (lane 5) or the Crk-L SH2 mutant R39A (lane 6). Cell lysates were precipitated with biotinylated pY221 SOCS3 phosphopeptide bound to streptavidinagarose beads, and the precipitates were analyzed in Western blotting with anti-Myc antibodies to detect Nck (lanes 1-4) or anti-FLAG antibodies to detect Crk-L (lanes 5-6). C, GST-Nck fusion protein binds to phosphorylated full-length SOCS3. 293T cells were transiently transfected with SOCS3-HA tag pME18S either alone or in combination with pME18S encoding Jak1. Cell lysates were split and precipitated with GST-Nck or GST proteins bound to glutathione-agarose beads, and the precipitates were analyzed in Western blotting with anti-HA antibodies, to detect SOCS3 (panel a). Expression controls for SOCS3 (panel b), tyrosine-phosphorylated Jak1 (panel c), and phosphorylated SOCS3 (panel d) are shown.View Large Image Figure ViewerDownload (PPT) To further characterize the requirements for SOCS3/Nck and SOCS3/Crk-L interactions, we examined the binding of SOCS3 pY221 phosphopeptide to WT and SH2 domain mutants of Nck and Crk-L. 293T cells were transfected with Myc-tagged Nck or FLAG-tagged Crk-L expression vectors. Included were WT full-length Nck, a Nck SH2 domain mutant (R308A), WT Nck SH2 domain alone (lacking the N-terminal SH3 domains), as well as WT Crk-L and a Crk-L SH2 mutant (R39A). Lysates from transfected cells were precipitated with the SOCS3 phosphopeptide and analyzed by Western blotting with anti-Myc or anti-FLAG antibodies to detect Nck or Crk-L. As shown in Fig. 2B, the binding of Nck and Crk-L to the SOCS3 phosphopeptide was dependent on intact SH2 domains. WT Nck and Crk-L bound strongly to the peptide (lanes 2 and 5), while both SH2 mutants failed to bind (lanes 3 and 6). In addition, strong binding of the Nck SH2 domain to the SOCS3 peptide demonstrates that an intact SH2 domain is sufficient for the interaction, and the 3 N-terminal SH3 domains of Nck are not required for binding (lane 4). We next determined whether full-length phosphorylated SOCS3 was capable of interacting with Nck. 293T cells were transiently transfected with a pME18S HA-tagged SOCS3 expression construct either alone or in combination with pME18S-Jak1 vector, to induce SOCS3 tyrosine phosphorylation. Cell lysates were split and precipitated with either GST or GST-Nck proteins coupled to glutathione beads, followed by anti-HA tag Western blotting to detect SOCS3. As shown in Fig. 2C, full-length SOCS3 interacted with the GST-Nck fusion protein when tyrosine phosphorylated (lane 4), whereas unphosphorylated SOCS3 failed to interact with Nck (lane 3). In a previous report, we determined that stimulation of fibroblasts with PDGF for 1 h induces SOCS3 expression and tyrosine phosphorylation (35Cacalano N.A. Sanden D. Johnston J.A. Nat. Cell Biol. 2001; 3: 460-465Crossref PubMed Scopus (175) Google Scholar). Thus, in order to determine whether endogenous SOCS3/Nck and SOCS3/Crk-L complexes form in ligand-activated cells, we stimulated NIH3T3 cells with PDGF-BB for 1 h to induce SOCS3 expression. Cell lysates were immunoprecipitated with anti-SOCS3 antiserum followed by Western blotting with anti-Nck or anti-Crk-L antibodies. As shown in Fig. 3, PDGF stimulation of NIH3T3 cells induces SOCS3 expression and tyrosine phosphorylation (panels B and C). In addition, SOCS3 immunoprecipitates from stimulated cells contain Nck and Crk-L (panel A), demonstrating that endogenous SOCS3 interacts with these adapter protei" @default.
• W1970082050 created "2016-06-24" @default.
• W1970082050 creator A5014597861 @default.
• W1970082050 creator A5051760500 @default.
• W1970082050 creator A5078049576 @default.
• W1970082050 date "2004-09-01" @default.
• W1970082050 modified "2023-10-18" @default.
• W1970082050 title "Tyrosine-phosphorylated SOCS3 Interacts with the Nck and Crk-L Adapter Proteins and Regulates Nck Activation" @default.
• W1970082050 cites W1480179919 @default.
• W1970082050 cites W1488478985 @default.
• W1970082050 cites W1507160997 @default.
• W1970082050 cites W1535599521 @default.
• W1970082050 cites W1555692789 @default.
• W1970082050 cites W1556909023 @default.
• W1970082050 cites W1623683757 @default.
• W1970082050 cites W1662447390 @default.
• W1970082050 cites W1668039320 @default.
• W1970082050 cites W1855620956 @default.
• W1970082050 cites W1896838955 @default.
• W1970082050 cites W1965464733 @default.
• W1970082050 cites W1967258978 @default.
• W1970082050 cites W1967486091 @default.
• W1970082050 cites W1978816877 @default.
• W1970082050 cites W1979299941 @default.
• W1970082050 cites W1980000777 @default.
• W1970082050 cites W1984715042 @default.
• W1970082050 cites W1987399378 @default.
• W1970082050 cites W1989987282 @default.
• W1970082050 cites W1994316906 @default.
• W1970082050 cites W1997878751 @default.
• W1970082050 cites W2000583211 @default.
• W1970082050 cites W2005082414 @default.
• W1970082050 cites W2006398267 @default.
• W1970082050 cites W2007458155 @default.
• W1970082050 cites W2007746616 @default.
• W1970082050 cites W2008725579 @default.
• W1970082050 cites W2012198462 @default.
• W1970082050 cites W2016313989 @default.
• W1970082050 cites W2020666846 @default.
• W1970082050 cites W2022966849 @default.
• W1970082050 cites W2025236720 @default.
• W1970082050 cites W2027850624 @default.
• W1970082050 cites W2029389376 @default.
• W1970082050 cites W2031381715 @default.
• W1970082050 cites W2031431314 @default.
• W1970082050 cites W2032407944 @default.
• W1970082050 cites W2033881557 @default.
• W1970082050 cites W2037015481 @default.
• W1970082050 cites W2037127929 @default.
• W1970082050 cites W2040810264 @default.
• W1970082050 cites W2041872947 @default.
• W1970082050 cites W2052222199 @default.
• W1970082050 cites W2053656098 @default.
• W1970082050 cites W2054172043 @default.
• W1970082050 cites W2061013276 @default.
• W1970082050 cites W2067415612 @default.
• W1970082050 cites W2069571945 @default.
• W1970082050 cites W2069711307 @default.
• W1970082050 cites W2070190928 @default.
• W1970082050 cites W2071102056 @default.
• W1970082050 cites W2075099549 @default.
• W1970082050 cites W2076808220 @default.
• W1970082050 cites W2082390264 @default.
• W1970082050 cites W2082494746 @default.
• W1970082050 cites W2085580043 @default.
• W1970082050 cites W2086656606 @default.
• W1970082050 cites W2086971705 @default.
• W1970082050 cites W2087140878 @default.
• W1970082050 cites W2091484442 @default.
• W1970082050 cites W2097137746 @default.
• W1970082050 cites W2098437309 @default.
• W1970082050 cites W2104459535 @default.
• W1970082050 cites W2104746754 @default.
• W1970082050 cites W2108486371 @default.
• W1970082050 cites W2117036763 @default.
• W1970082050 cites W2118598606 @default.
• W1970082050 cites W2119596771 @default.
• W1970082050 cites W2124489640 @default.
• W1970082050 cites W2125753995 @default.
• W1970082050 cites W2134855648 @default.
• W1970082050 cites W2138747143 @default.
• W1970082050 cites W2139380465 @default.
• W1970082050 cites W2140010294 @default.
• W1970082050 cites W2142543015 @default.
• W1970082050 cites W2142574099 @default.
• W1970082050 cites W2152000177 @default.
• W1970082050 cites W2154893066 @default.
• W1970082050 cites W2158568391 @default.
• W1970082050 cites W2163862459 @default.
• W1970082050 cites W2164354034 @default.
• W1970082050 cites W2164682544 @default.
• W1970082050 cites W2164967233 @default.
• W1970082050 cites W2170910759 @default.
• W1970082050 cites W2319271253 @default.
• W1970082050 cites W2400188205 @default.
• W1970082050 cites W2542515380 @default.
• W1970082050 cites W63837087 @default.
• W1970082050 cites W74254474 @default.

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