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Suppressor of Cytokine Signaling 6 Associates with KIT and Regulates KIT Receptor Signaling

2004; Elsevier BV; Volume: 279; Issue: 13 Linguagem: Inglês

10.1074/jbc.m313381200

ISSN

1083-351X

Autores

J Bayle, Sébastien Létard, Ronald Frank, Patrice Dubreuil, Paulo De Sepulveda,

Tópico(s)

Cell Adhesion Molecules Research

Resumo

Suppressor of cytokine signaling (SOCS) proteins are a family of Src homology 2-containing adaptor proteins. Cytokine-inducible Src homology domain 2-containing protein, SOCS1, SOCS2, and SOCS3 have been implicated in the down-regulation of cytokine signaling. The function of SOCS4, 5, 6, and 7 are not known. KIT receptor signaling is regulated by protein tyrosine phosphatases and adaptor proteins. We previously reported that SOCS1 inhibited cell proliferation in response to stem cell factor (SCF). By screening the other members of SOCS family, we identified SOCS6 as a KIT-binding protein. Using KIT mutants and peptides, we demonstrated that SOCS6 bound directly to KIT tyrosine 567 in the juxtamembrane domain. To investigate the function of this interaction, we constitutively expressed SOCS6 in cell lines. Ectopic expression of SOCS6 in Ba/F3-KIT cell line decreased cell proliferation in response to SCF but not SCF-induced chemotaxis. SOCS6 reduced SCF-induced activation of ERK1/2 and p38 but not activation of AKT or STATs in Ba/F3, murine embryonic fibroblast (MEF), or COS-7 cells. SOCS6 did not impair ERK and p38 activation by other stimuli. These results indicate that SOCS6 binds to KIT juxtamembrane region, which affects upstream signaling components leading to MAPK activation. Our results indicate that KIT signaling is regulated by several SOCS proteins and suggest a putative function for SOCS6 as a negative regulator of receptor tyrosine kinases. Suppressor of cytokine signaling (SOCS) proteins are a family of Src homology 2-containing adaptor proteins. Cytokine-inducible Src homology domain 2-containing protein, SOCS1, SOCS2, and SOCS3 have been implicated in the down-regulation of cytokine signaling. The function of SOCS4, 5, 6, and 7 are not known. KIT receptor signaling is regulated by protein tyrosine phosphatases and adaptor proteins. We previously reported that SOCS1 inhibited cell proliferation in response to stem cell factor (SCF). By screening the other members of SOCS family, we identified SOCS6 as a KIT-binding protein. Using KIT mutants and peptides, we demonstrated that SOCS6 bound directly to KIT tyrosine 567 in the juxtamembrane domain. To investigate the function of this interaction, we constitutively expressed SOCS6 in cell lines. Ectopic expression of SOCS6 in Ba/F3-KIT cell line decreased cell proliferation in response to SCF but not SCF-induced chemotaxis. SOCS6 reduced SCF-induced activation of ERK1/2 and p38 but not activation of AKT or STATs in Ba/F3, murine embryonic fibroblast (MEF), or COS-7 cells. SOCS6 did not impair ERK and p38 activation by other stimuli. These results indicate that SOCS6 binds to KIT juxtamembrane region, which affects upstream signaling components leading to MAPK activation. Our results indicate that KIT signaling is regulated by several SOCS proteins and suggest a putative function for SOCS6 as a negative regulator of receptor tyrosine kinases. Cytokines and growth factors regulate the survival, proliferation, differentiation, and migration of hematopoietic cells. Binding of these factors to transmembrane receptors induces receptor activation, which in turn results in the recruitment of signaling complexes in the vicinity of the plasma membrane. The kinetics and magnitude of signal transduction are tightly regulated by multiple mechanisms. Among proteins that modulate signaling, members of the suppressor of cytokine signaling (SOCS) 1The abbreviations used are: SOCS, suppressor of cytokine signaling; SH2, Src homology 2; CIS, cytokine-inducible SH2-containing protein; STAT, signal transducers and activators of transcription; E3, ubiquitin-protein isopeptide ligase; RTK, receptor tyrosine kinases; IR, insulin receptor; EGF, epidermal growth factor; EGFR, EGF receptor; PMA, phorbol 12-myristate 13-acetate; GST, glutathione S-transferase; GFP, green fluorescent protein; EGFP, enhanced GFP; p, phosphorylated; WT, wild type; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; FBS, fetal bovine serum; MEF, murine embryonic fibroblast; IL, interleukin; JM, juxtamembrane domain; KI, kinase insert; SCF, stem cell factor; IRS, insulin receptor substrate; JAK, Janus kinase; PH, pleckstrin homology. 1The abbreviations used are: SOCS, suppressor of cytokine signaling; SH2, Src homology 2; CIS, cytokine-inducible SH2-containing protein; STAT, signal transducers and activators of transcription; E3, ubiquitin-protein isopeptide ligase; RTK, receptor tyrosine kinases; IR, insulin receptor; EGF, epidermal growth factor; EGFR, EGF receptor; PMA, phorbol 12-myristate 13-acetate; GST, glutathione S-transferase; GFP, green fluorescent protein; EGFP, enhanced GFP; p, phosphorylated; WT, wild type; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; FBS, fetal bovine serum; MEF, murine embryonic fibroblast; IL, interleukin; JM, juxtamembrane domain; KI, kinase insert; SCF, stem cell factor; IRS, insulin receptor substrate; JAK, Janus kinase; PH, pleckstrin homology. family have been shown to down-regulate the function of cytokines or growth factors (1Krebs D.L. Hilton D.J. Stem Cells. 2001; 19: 378-387Crossref PubMed Scopus (650) Google Scholar, 2Naka T. Fujimoto M. Kishimoto T. Trends Biochem. Sci. 1999; 24: 394-398Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, 3Yasukawa H. Sasaki A. Yoshimura A. Annu. Rev. Immunol. 2000; 18: 143-164Crossref PubMed Scopus (509) Google Scholar).The eight members of the SOCS family, SOCS1–7 and CIS (cytokine-inducible Src homology domain (SH2)-containing protein), are structurally characterized by a SH2 domain followed by a conserved C-terminal motif, the SOCS box (4Hilton 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. U. S. A. 1998; 95: 114-119Crossref PubMed Scopus (609) Google Scholar). The N-terminal region of SOCS proteins is variable both in length and in the primary amino acid sequence. Although many reports including knock-out studies shed light on the function of CIS (5Yoshimura 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 (617) Google Scholar, 6Matsumoto A. Seki Y. Kubo M. Ohtsuka S. Suzuki A. Hayashi I. Tsuji K. Nakahata T. Okabe M. Yamada S. Yoshimura A. Mol. Cell. Biol. 1999; 19: 6396-6407Crossref PubMed Scopus (217) Google Scholar), SOCS1 (7Naka T. Matsumoto T. Narazaki M. Fujimoto M. Morita Y. Ohsawa Y. Saito H. Nagasawa T. Uchiyama Y. Kishimoto T. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 15577-15582Crossref PubMed Scopus (258) Google Scholar, 8Starr R. Metcalf D. Elefanty A.G. Brysha M. Willson T.A. Nicola N.A. Hilton D.J. Alexander W.S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14395-14399Crossref PubMed Scopus (377) Google Scholar), SOCS2 (9Metcalf D. Greenhalgh C.J. Viney E. Willson T.A. Starr R. Nicola N.A. Hilton D.J. Alexander W.S. Nature. 2000; 405: 1069-1073Crossref PubMed Scopus (400) Google Scholar, 10Leung K.C. Doyle N. Ballesteros M. Sjogren K. Watts C.K. Low T.H. Leong G.M. Ross R.J. Ho K.K. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 1016-1021Crossref PubMed Scopus (183) Google Scholar), and SOCS3 (11Marine J.C. McKay C. Wang D. Topham D.J. Parganas E. Nakajima H. Pendeville H. Yasukawa H. Sasaki A. Yoshimura A. Ihle J.N. Cell. 1999; 98: 617-627Abstract Full Text Full Text PDF PubMed Scopus (309) Google Scholar, 12Roberts 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 (256) Google Scholar, 13Takahashi Y. Carpino N. Cross J.C. Torres M. Parganas E. Ihle J.N. EMBO J. 2003; 22: 372-384Crossref PubMed Scopus (164) Google Scholar), very little is known regarding the function of SOCS4, SOCS5, SOCS6, and SOCS7.The mechanisms whereby CIS, SOCS1, and SOCS3 inhibit signaling by classical cytokine receptor (i.e. receptors without catalytic activity that associate with JAK tyrosine kinases) are the best characterized. All three are involved in the down-regulation of the JAK/STAT pathway. SOCS1 has a dual function as a direct potent JAK kinase inhibitor (14Starr 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 (1793) Google Scholar, 15Naka 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 (1128) Google Scholar, 16Endo 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 (1221) Google Scholar, 17Yasukawa 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) and as a component of an E3 ubiquitin-ligase complex recruiting substrates to the protein degradation machinery (18Kamura T. Sato S. Haque D. Liu L. Kaelin Jr., W.G. Conaway R.C. Conaway J.W. Genes Dev. 1998; 12: 3872-3881Crossref PubMed Scopus (499) 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 (519) Google Scholar, 20Ungureanu D. Saharinen P. Junttila I. Hilton D.J. Silvennoinen O. Mol. Cell. Biol. 2002; 22: 3316-3326Crossref PubMed Scopus (211) Google Scholar). SOCS3 also inhibits JAK activity but indirectly through recruitment to the cytokine receptors (1Krebs D.L. Hilton D.J. Stem Cells. 2001; 19: 378-387Crossref PubMed Scopus (650) Google Scholar, 21Sasaki 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 (266) Google Scholar). More recently, SOCS3 has been suggested to compete with SHP2 for the same binding sites on glycoprotein 130 (22Schmitz J. Weissenbach M. Haan S. Heinrich P.C. Schaper F. J. Biol. Chem. 2000; 275: 12848-12856Abstract Full Text Full Text PDF PubMed Scopus (330) Google Scholar, 23Nicholson 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 (394) Google Scholar), erythropoietin receptor (21Sasaki 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 (266) Google Scholar), and leptin receptor (24Bjorbak C. Lavery H.J. Bates S.H. Olson R.K. Davis S.M. Flier J.S. Myers Jr., M.G. J. Biol. Chem. 2000; 275: 40649-40657Abstract Full Text Full Text PDF PubMed Scopus (431) Google Scholar). CIS binds to cytokine receptors at STAT5-docking sites, which impairs recruitment of STAT5 to the receptor signaling complex and results in the down-regulation of STAT5 activation (6Matsumoto A. Seki Y. Kubo M. Ohtsuka S. Suzuki A. Hayashi I. Tsuji K. Nakahata T. Okabe M. Yamada S. Yoshimura A. Mol. Cell. Biol. 1999; 19: 6396-6407Crossref PubMed Scopus (217) Google Scholar, 25Matsumoto A. Masuhara M. Mitsui K. Yokouchi M. Ohtsubo M. Misawa H. Miyajima A. Yoshimura A. Blood. 1997; 89: 3148-3154Crossref PubMed Google Scholar).Mice lacking SOCS6 have been generated, and they developed normally with the exception of a 10% reduction in weight compared with wild-type littermates (26Krebs D.L. Uren R.T. Metcalf D. Rakar S. Zhang J.G. Starr R. De Souza D.P. Hanzinikolas K. Eyles J. Connolly L.M. Simpson R.J. Nicola N.A. Nicholson S.E. Baca M. Hilton D.J. Alexander W.S. Mol. Cell. Biol. 2002; 22: 4567-4578Crossref PubMed Scopus (122) Google Scholar). SOCS6 mRNA was induced by erythropoietin in cell lines (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) and was ubiquitously expressed in murine tissues (26Krebs D.L. Uren R.T. Metcalf D. Rakar S. Zhang J.G. Starr R. De Souza D.P. Hanzinikolas K. Eyles J. Connolly L.M. Simpson R.J. Nicola N.A. Nicholson S.E. Baca M. Hilton D.J. Alexander W.S. Mol. Cell. Biol. 2002; 22: 4567-4578Crossref PubMed Scopus (122) Google Scholar). SOCS6 does not interact with JAKs, but the interaction with elongins B and C suggests that, as all SOCS proteins, it might be part of an E3 ubiquitin-ligase complex (28Kile B.T. Schulman B.A. Alexander W.S. Nicola N.A. Martin H.M. Hilton D.J. Trends Biochem. Sci. 2002; 27: 235-241Abstract Full Text Full Text PDF PubMed Scopus (360) Google Scholar). Yet, there is no evidence so far suggesting that SOCS6 might be involved in the degradation of proteins.Regulation of receptor tyrosine kinases (RTK) by SOCS proteins is much less understood. In vivo interaction between SOCS proteins and RTK has been previously reported. SOCS1 binds to KIT, FLT3 (29De Sepulveda P. Okkenhaug K. Rose J.L. Hawley R.G. Dubreuil P. Rottapel R. EMBO J. 1999; 18: 904-915Crossref PubMed Scopus (180) Google Scholar), and FMS (30Bourette R.P. De Sepulveda P. Arnaud S. Dubreuil P. Rottapel R. Mouchiroud G. J. Biol. Chem. 2001; 276: 22133-22139Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). SOCS2 binds to the insulin-like growth factor receptor (31Dey B.R. Spence S.L. Nissley P. Furlanetto R.W. J. Biol. Chem. 1998; 273: 24095-24101Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar). SOCS1 and SOCS3 bind to EGFR and may down-regulate activation of STATs by EGFR (32Xia L. Wang L. Chung A.S. Ivanov S.S. Ling M.Y. Dragoi A.M. Platt A. Gilmer T.M. Fu X.Y. Chin Y.E. J. Biol. Chem. 2002; 277: 30716-30723Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). SOCS3 interacts with insulin receptor (IR) (33Emanuelli B. Peraldi P. Filloux C. Sawka-Verhelle D. Hilton D. Van Obberghen E. J. Biol. Chem. 2000; 275: 15985-15991Abstract Full Text Full Text PDF PubMed Scopus (382) Google Scholar). More recently, SOCS1 and SOCS6 have also been shown to associate with and inhibit IR downstream signaling events such as the activation of ERK1/2, AKT, and IRS-1 when expressed in hepatoma cells (34Mooney R.A. Senn J. Cameron S. Inamdar N. Boivin L.M. Shang Y. Furlanetto R.W. J. Biol. Chem. 2001; 276: 25889-25893Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar).Binding of stem cell factor (SCF) to KIT RTK activates multiple signal transduction components, leading to the activation of all three MAPKs pathways, phosphatidylinositol 3-kinase, and STAT1, STAT3, and STAT5. We have previously identified SOCS1 protein as downstream component of the KIT receptor signaling pathway (29De Sepulveda P. Okkenhaug K. Rose J.L. Hawley R.G. Dubreuil P. Rottapel R. EMBO J. 1999; 18: 904-915Crossref PubMed Scopus (180) Google Scholar). We have demonstrated that SOCS1 bound to KIT via its SH2 domain. Constitutive expression of SOCS1 strongly suppressed the proliferative signals transduced by KIT without suppression of KIT catalytic activity. Neither SOCS1 docking site nor the mechanism of SOCS1 inhibition could be determined in these studies. However, we showed that SOCS1 not only interacted with KIT and JAK kinases but also interacted with SH3 domain-containing proteins, other receptor tyrosine kinases, and with VAV proteins.Here, we have screened the putative interactions of SOCS proteins with the KIT receptor in response to SCF stimulation. Our findings revealed that SOCS6 protein interacts with KIT following SCF-stimulated tyrosine phosphorylation. The SH2 domain of SOCS6 binds directly to tyrosine 567 in KIT juxtamembrane domain. To determine the functional consequences of this interaction, we have studied the effect of SOCS6 ectopic expression on the cell proliferation and migration induced by SCF and investigated which signaling pathways are regulated by SOCS6.EXPERIMENTAL PROCEDURESCell Culture—Ba/F3 were cultured in RPMI 1640 medium with 10% fetal bovine serum (FBS) in the presence of 0.1% of conditioned medium from X63-IL-3 cells (35Karasuyama H. Melchers F. Eur. J. Immunol. 1988; 18: 97-104Crossref PubMed Scopus (1077) Google Scholar). EML-C1 cells were grown in RPMI 1640 medium supplemented with 20% FBS and 250 ng/ml murine SCF. COS-7, R4 MEFs, Phoenix A, and GP+E-86 retrovirus-packaging cell lines were maintained in Dulbecco's modified Eagle's medium supplemented with 10% FBS and 0.5 mg/ml sodium pyruvate. All of the media and sera were purchased from Invitrogen.Reverse Transcription and PCR—Total RNAs were extracted from cell lines and bone marrow-derived mast cells using TRIzol reagent (Invitrogen). First-strand cDNAs were synthesized with 2 μg of RNA using oligo(dT) primers and Superscript II reverse transcriptase (Invitrogen), and one-tenth of the cDNA was then subjected to 35 cycles of PCR amplification using SOCS6 primers (sense, 5′-CTCTCACCATTGCTACCTCCAA-3′; antisense, 5′-TCTCGCCCCCAGAATAGATGTAG-3′, 468-bp product) and 2 units of Taq polymerase (Invitrogen). Amplification of murine and human β2-microglobulin RNAs were used as controls.Expression Constructs—pEF-FLAG-SOCS6 construct was kindly provided by D. Hilton (The Walter and Eliza Hall Institute). To convert tyrosine residues (at codons 544-546-552-567-569-577-702-719-728-745 and 934) to phenylalanine residues in KIT, point mutations were generated by site-directed mutagenesis using Stratagene Chameleon double-stranded mutagenesis kit. The reactions were performed on mouse KIT cDNA in the pECE-KIT vector. All of the cDNAs were entirely sequenced. The KIT-JM construct contained all six Y544F, Y546F, Y552F, Y567F, Y569F, and Y577F mutations in the juxtamembrane domain. The KIT-KI construct contained all four Y702F, Y719F, Y728F, and Y745F mutations in the interkinase domain. Retroviral vectors pMIEV and pMIEV-hemagglutinin-SOCS1 were described previously (29De Sepulveda P. Okkenhaug K. Rose J.L. Hawley R.G. Dubreuil P. Rottapel R. EMBO J. 1999; 18: 904-915Crossref PubMed Scopus (180) Google Scholar). The FLAG-SOCS6 cDNA was cloned in pMIEV using the Gateway system (Invitrogen). The yeast two-hybrid vectors are described below.Reagents and Antibodies—Rabbit polyclonal anti-KIT serum was raised against glutathione S-transferase (GST) fusion protein containing the kinase insert. The anti-FLAG M2 monoclonal antibody was obtained from Sigma. Monoclonal anti-phosphotyrosine antibody (4G10) was purchased from Upstate Biotechnology. Polyclonal rabbit anti-SOCS6, anti-STAT5b, anti-ERK2 antibodies were from Santa Cruz Biotechnology. Phospho-p44/42 ERK (Thr-202, Tyr-204) and phospho-p38 (Thr-180, Tyr-182) antibodies were obtained from Promega. Antiphospho STAT5, anti-phospho-AKT, anti-AKT, and anti-p38-specific antibodies were used according to the manufactured instructions (Cell Signaling). Anti-hemagglutinin antibody 12CA5 was from hybridoma culture supernatant. Protein A-horseradish peroxidase (ICN Biomedicals), goat anti-mouse IgG-horseradish peroxidase (Jackson Immunoresearch Laboratories), and anti-rabbit IgG-horseradish peroxidase (Cell Signaling Technology) were used as secondary antibodies for Western blots. Recombinant EGF was obtained from Invitrogen, phorbol 12-myristate 13-acetate (PMA) was from Sigma, and anisomycin was from Calbiochem.Transfection Procedure—Transfection of COS-7 cells was carried out in 60-mm plates. Cells were transfected with FuGENE 6 (Roche Applied Science) as recommended by the manufacturer's instructions with 1 μg of expression vector. Cells were serum-starved overnight 24 h after transfection in Dulbecco's modified Eagle's medium with 0.5% FBS and then stimulated for 5 min with 250 ng/ml murine SCF.Immunoprecipitation and Immunoblotting—Stimulated cells were washed in ice-cold phosphate-buffered saline prior to lysis, pelleted, and lysed in HNTG buffer (50 mm HEPES, pH 7, 50 mm NaF, 1 mm EGTA, 150 mm NaCl, 1% Triton X-100, 10% glycerol, 1.5 mm MgCl2) containing protease inhibitor mixture (Roche Applied Science) and 100 μm Na3VO4. Clarified whole cell lysates were mixed for 18 h with 2 μg of antibodies and a bed volume of 10 μl of protein A or protein G-Sepharose (Amersham Biosciences) for immunoprecipitation. The immunoprecipitates were washed three times with HNTG buffer and dissolved in SDS sample buffer. Following SDS-PAGE, proteins were transferred to polyvinylidene difluoride membranes (Immobilon-P, Millipore). Membranes were saturated with 5% bovine serum albumin (Sigma) and probed with different antibodies as specified in the text and figure legends. Blots were revealed using horseradish peroxidase-conjugated secondary antibodies and an ECL detection kit (Amersham Biosciences).Yeast Two-hybrid System—The entire cytoplasmic domains of wild-type murine KIT, KIT-JM, or KIT-KI mutants (nucleotides 1646–2988) were fused to LexA DNA-binding domain in the yeast expression vector pBTM116. SOCS6 cDNA coding for SH2 and C-terminal domains (amino acids 310–533), SOCS6 SH2 domain alone (amino acids 310–479), and SOCS1 full-length cDNA were cloned in the vector pACT2 using the Gateway system (Invitrogen). The vectors pLexA-lamin and pACT2 were used as controls for each yeast two-hybrid experiment.GST Pull-down Experiments—The GST-SOCS6 construct in pD-EST15 vector-expressing SOCS6 amino acids 310–533 was introduced in the Escherichia coli Rosetta (DE3) pLacI strain (Novagen). Bacterial cultures grown to log phase were induced with 0.1 mm isopropyl-β-d-galactopyranoside (Invitrogen) for 4 h at 30 °C. Bacteria were then lysed in HNTG buffer, and the GST fusion proteins were purified on glutathione-Sepharose beads (Amersham Biosciences). Lysates from non-stimulated and SCF-stimulated Ba/F3-KIT cells were incubated overnight at 4 °C with 5 μg of the GST fusion construct or with 5 μg of GST-β-catenin control immobilized on gluthatione-Sepharose beads. The beads were washed three times in HNTG buffer, and bound fractions were separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes. Bound KIT protein was visualized by Western blotting using the 4G10 antibody.Peptide Binding Assay—Forty-four 15-mer peptides corresponding to the 22 tyrosine motifs of KIT intracellular domain either phosphorylated or not were synthesized on cellulose membranes as described previously (36Frank R. Overwin H. Methods Mol. Biol. 1996; 66: 149-169PubMed Google Scholar). The membranes were saturated with 10% FBS in TBST buffer (100 mm Tris-HCl, pH 7.5, 150 mm NaCl, 1% Tween 20) for 1 h at room temperature. It was then probed overnight at 4 °C with 32P-labeled GST-SOCS6 (amino acids 310–533) and washed in TBST buffer. The interactions were analyzed by autoradiography and quantified using a PhosphorImager (Molecular Dynamics). The probe was prepared in a reaction mixture containing 5 μg of GST-SOCS6, 20 μCi of [γ-32P]ATP, 0.2 μm ATP, and 2500 units of cAMP-dependent protein kinase catalytic subunit (New England Biolabs) in the manufacturer's kinase buffer (50 mm Tris-HCl, pH 7.5, 10 mm MgCl2). Unincorporated ATP was removed, and the labeled protein was eluted with 20 mm glutathione.Infection of Cells with Retroviral Vectors—Stable populations of GP+E-86 cells producing MIEV or MIEV-SOCS6 retroviruses were obtained as follow. Phoenix A cells were transfected using FuGENE 6 reagent with pMIEVSOCS6 or empty pMIEV. In the next 2 days, filtered retroviral culture supernatants were used to infect GP+E-86 cells. GP+E-86 populations were then sorted for GFP expression. Infections of Ba/F3 or EML cells were done by coculture in 100-mm plates using 106 GP+E-86 cells stably producing retroviruses and 106 Ba/F3-KIT or Ba/F3-EGFR or EML with the target cell culture media and 4 ng/ml Polybrene (Sigma). Non-adherent cells were collected 48 h later and sorted for expression of GFP using a cell sorter. Infected R4 MEFs were obtained through similar procedures but with filtered retroviral culture supernatant instead of coculture.Cell Proliferation Assay—A total of 5000 cells/well were plated in triplicate into 96-well plates in 100 μl of RPMI 1640 medium with 10% FBS and 250 ng/ml SCF. Cells were incubated for 24 h at 37 °C and pulsed for 6 h with 0.5 μCi of [methyl-3H]thymidine (Amersham Biosciences). Cells were then transferred onto glass fiber filters (Packard, Netherlands), and incorporation was measured using a β-counter Rackbeta Compact 1212-411 (LKB, Uppsala, Sweden).In Vitro Two-chamber Migration Assay—Chemotaxis was assayed by a modification of the Boyden chamber assay. 3 × 105 serum-starved Ba/F3-KIT cells in 100 μl of chemotaxis media (RPMI 1640 with 0.5% bovine serum albumin) were added to the upper chamber of a Costar Transwell 24-well plate (6.5 mm diameter, 5 μm pore size, Cambridge, MA). 600 μl of chemotaxis medium was added to the lower chamber. SCF was added in the lower chamber to create a positive gradient. Transwell plates were incubated at 37 °C for 1 h. SCF-induced cell migration into the lower chamber was measured by counting using FACScan for 20 s at high flow rate. Average cell number and mean ± S.D. were calculated from quadruplet wells.RESULTSSOCS6 Protein Associates with KIT Receptor in Response to SCF—We earlier reported that SOCS1 physically associated with KIT receptor through its SH2 domain in response to KIT-ligand (SCF)-induced activation (29De Sepulveda P. Okkenhaug K. Rose J.L. Hawley R.G. Dubreuil P. Rottapel R. EMBO J. 1999; 18: 904-915Crossref PubMed Scopus (180) Google Scholar). To determine whether the other SOCS proteins interacted with KIT, we transiently expressed FLAG-tagged SOCS proteins and KIT in COS-7 cells. Transfected cells were serum-starved overnight prior to stimulation with SCF for 5 min. SCF induced a strong association of SOCS6 with KIT (Fig. 1) and a weak association of SOCS4 and SOCS5 with KIT (data not shown). By contrast, CIS bound to KIT constitutively, whereas SOCS2 and SOCS3 did not interact (data not shown).Transcripts encoding CIS, SOCS1, SOCS2, and SOCS3 are up-regulated following cytokine signaling (1Krebs D.L. Hilton D.J. Stem Cells. 2001; 19: 378-387Crossref PubMed Scopus (650) Google Scholar, 2Naka T. Fujimoto M. Kishimoto T. Trends Biochem. Sci. 1999; 24: 394-398Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, 3Yasukawa H. Sasaki A. Yoshimura A. Annu. Rev. Immunol. 2000; 18: 143-164Crossref PubMed Scopus (509) Google Scholar). As shown Fig. 2A, expression of socs6 gene was induced after 1 h of SCF stimulation in primary cultures of bone marrow-derived mast cells. Additionally, socs6 mRNA was present in cells that express KIT, including the cell lines UT-7, TF1, and MO7e and ES cells (Fig. 2B). These data indicate that socs6 is expressed in KIT-positive cells and that KIT signaling induces socs6 mRNA expression.Fig. 2Induction of socs6 gene expression by SCF. A, KIT receptor signaling induces socs6 mRNA in bone marrow-derived mast cells. Cells were serum-starved overnight, they were then either left untreated (lane 1) or stimulated with SCF (250 ng/ml) for 1 h (lanes 2) and 4 h (lanes 3), or cultured in the presence of IL-3 (lane 4). Total RNAs were isolated, and the expression of socs6 mRNA and β2-microglobulin were analyzed by RT-PCR as described under “Experimental Procedures.” B, socs6 mRNA is expressed in positive KIT cell line. RNAs from thymus and spleen were obtained from Stratagene. MO7e, UT-7, and TF1 cells were cultured in the media containing 1 and 5 ng/ml granulocyte macrophage colony-stimulating factor, respectively. Murine embryonic stem cells were cultured in the presence of leukemia inhibitory factor (1000 units/ml). Total RNAs were treated as in panel A.View Large Image Figure ViewerDownload Hi-res image Download (PPT)We set out to analyze the interaction of SOCS6 with KIT. As shown Fig. 1, KIT was detected in SOCS6 immunoprecipitates following KIT activation by SCF (Fig. 1, panel A, lane 7). Conversely, SOCS6 was detected in KIT immunoprecipitates (Fig. 1, panel C, lane 7). The interaction was dependent on SCF stimulation and tyrosine phosphorylation of KIT. Protein expression in the transfected cells was confirmed by immunoblotting of immunoprecipitates with the anti-FLAG (Fig. 1, panel B) and anti-KIT (Fig. 1, panel D) antibodies, respectively. Tyrosine phosphorylation of KIT was controlled by using an antiphosphotyrosine monoclonal antibody (Fig. 1, panel E). The binding of SOCS6 to KIT was confirmed by two other experimental procedures, the yeast two-hybrid system (Fig. 4A) and GST pull-down experiments (Fig. 5B).Fig. 4SOCS6 interacts with juxtamembrane domain of KIT receptor. A, SOCS6 SH2 domain interacts with KIT in the yeast two-hybrid system, full-length SOCS6, or the SH2 domain of SOCS6 as GAL4 activation domain fusion proteins in the pACT2 vector in the yeast strain L40. Wild-type KIT or lamin was expressed in the pBTM116 vector. Yeast were stained for β-galactosidase activity with 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (X-gal). B, the schematic structure of murine KIT is depicted, and the position of the 22 tyrosine residues of the cytoplasmic region are indicated. TM, transmembrane region; TK, tyrosine kinase domain. C and D, SOCS6 binds to the juxtamembrane of KIT in COS cells. SOCS6 and KIT-KI mutant (C) or SOCS6 and KIT-JM mutant (D) were coexpressed in COS cells, a

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