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The SOCS Box of SOCS-1 Accelerates Ubiquitin-dependent Proteolysis of TEL-JAK2

2001; Elsevier BV; Volume: 276; Issue: 16 Linguagem: Inglês

10.1074/jbc.m010074200

ISSN

1083-351X

Autores

Shintaro Kamizono, Toshikatsu Hanada, Hideo Yasukawa, Shigeru Minoguchi, Reiko Kato, Mayu Minoguchi, Kimihiko Hattori, Shigetsugu Hatakeyama, Masayoshi Yada, Sumiyo Morita, Toshio Kitamura, Hirohisa Kato, Kei-ichi Nakayama, Akihiko Yoshimura,

Tópico(s)

interferon and immune responses

Resumo

Fusion of the TEL gene on 12p13 to the JAK2 tyrosine kinase gene on 9p24 has been found in human leukemia.TEL-mediated oligomerization of JAK2 results in constitutive activation of the tyrosine kinase (JH1) domain and confers cytokine-independent proliferation on interleukin-3-dependent Ba/F3 cells. Forced expression of the JAK inhibitor gene SOCS1/JAB/SSI-1 induced apoptosis of TEL-JAK2-transformed Ba/F3 cells. This suppression of TEL-JAK2 activity was dependent on SOCS box-mediated proteasomal degradation of TEL-JAK2 rather than on kinase inhibition. Degradation of JAK2 depended on its phosphorylation and its high affinity binding with SOCS1 through the kinase inhibitory region and the SH2 domain. It has been demonstrated that von Hippel-Lindau disease (VHL) tumor-suppressor gene product possesses the SOCS box that forms a complex with Elongin B and C and Cullin-2, and it functions as a ubiquitin ligase. The SOCS box of SOCS1/JAB has also been shown to interact with Elongins; however, ubiquitin ligase activity has not been demonstrated. We found that the SOCS box interacted with Cullin-2 and promoted ubiquitination of TEL-JAK2. Furthermore, overexpression of dominant negative Cullin-2 suppressed SOCS1-dependent TEL-JAK2 degradation. Our study demonstrates the substrate-specific E3 ubiquitin-ligase-like activity of SOCS1 for activated JAK2 and may provide a novel strategy for the suppression of oncogenic tyrosine kinases. Fusion of the TEL gene on 12p13 to the JAK2 tyrosine kinase gene on 9p24 has been found in human leukemia.TEL-mediated oligomerization of JAK2 results in constitutive activation of the tyrosine kinase (JH1) domain and confers cytokine-independent proliferation on interleukin-3-dependent Ba/F3 cells. Forced expression of the JAK inhibitor gene SOCS1/JAB/SSI-1 induced apoptosis of TEL-JAK2-transformed Ba/F3 cells. This suppression of TEL-JAK2 activity was dependent on SOCS box-mediated proteasomal degradation of TEL-JAK2 rather than on kinase inhibition. Degradation of JAK2 depended on its phosphorylation and its high affinity binding with SOCS1 through the kinase inhibitory region and the SH2 domain. It has been demonstrated that von Hippel-Lindau disease (VHL) tumor-suppressor gene product possesses the SOCS box that forms a complex with Elongin B and C and Cullin-2, and it functions as a ubiquitin ligase. The SOCS box of SOCS1/JAB has also been shown to interact with Elongins; however, ubiquitin ligase activity has not been demonstrated. We found that the SOCS box interacted with Cullin-2 and promoted ubiquitination of TEL-JAK2. Furthermore, overexpression of dominant negative Cullin-2 suppressed SOCS1-dependent TEL-JAK2 degradation. Our study demonstrates the substrate-specific E3 ubiquitin-ligase-like activity of SOCS1 for activated JAK2 and may provide a novel strategy for the suppression of oncogenic tyrosine kinases. Cytokines induce the activation of the JAK family tyrosine kinases (JAKs) 1The abbreviations used are:JAKJAK family tyrosine kinasesSTATsignal transducers and activators of transcriptionPDGFplatelet-derived growth factorIL-3interleukin-3IFNγinterferon γCul-2Cullin-2VHLvon Hippel-Lindau diseaseElongin BC, Elongin B and C complexPCRpolymerase chain reactionHAhemagglutininWTwild typedC40deletion mutant lacking 40 amino acids at the C terminusGSTglutathione S-transferaseEPORerythropoietin receptorEGFPexpressed green fluorescence protein and the subsequent recruitment of various signaling proteins to the receptor complex, including the STAT family of transcription factors. Constitutive activation of the JAK/STAT pathway has been found in many leukemic cell lines, including cells transformed with Bcr-Abl (1Shuai K. Halpern J. ten Hoeve J. Rao X. Sawyers C.L. Oncogene. 1996; 13: 247-254PubMed Google Scholar, 2Chai S.K. Nichols G.L. Rothman P. J. Immunol. 1997; 15: 4720-4728Google Scholar), as well as in human T-cell lymphotrophic virus-1-transformed T cells (3Migone T.S. Lin J.X. Cereseto A. Mulloy J.C. O'Shea J.J. Franchini G. Leonard W.J. Science. 1995; 269: 79-81Crossref PubMed Scopus (511) Google Scholar, 4Xu X. Kang S.H. Heidenreich O. Brown D.A. Nerenberg M.I. J. Clin. Invest. 1995; 96: 1548-1555Crossref PubMed Scopus (103) Google Scholar). A constitutively activated form of STAT5 conferred factor-independent growth on Ba/F3 cells (5Onishi M. Nosaka T. Misawa K. Mui A.L. Gorman D. McMahon M. Miyajima A. Kitamura T. Mol. Cell. Biol. 1998; 18: 3871-3879Crossref PubMed Scopus (352) Google Scholar), and that of STAT3 has also been shown to function as an oncogene (6Bromberg J.F. Wrzeszczynska M.H. Devgan G. Zhao Y. Pestell R.G. Albanese C. Darnell Jr., J.E. Cell. 1999; 98: 295-303Abstract Full Text Full Text PDF PubMed Scopus (2553) Google Scholar). Moreover, a constitutively activated JAK kinase generated by chromosome translocation between the TELgene on 12p13 and the JAK2 gene on 9p24 has been shown to be associated with human leukemia (7Lacronique V. Boureux A. Valle V.D. Poirel H. Quang C.T. Mauchauffe M. Berthou C. Lessard M. Berger R. Ghysdael J. Bernard O.A. Science. 1997; 278: 1309-1312Crossref PubMed Scopus (688) Google Scholar, 8Peeters P. Raynaud S.D. Cools J. Wlodarska I. Grosgeorge J. Philip P. Monpoux F. Van Rompaey L. Baens M. Van den Berghe H. Marynen P. Blood. 1997; 90: 2535-2540Crossref PubMed Google Scholar). TEL, a subset of the ETS family of transcription factors, contains a conserved oligomerization domain, known as the PNT domain, in the N-terminal region. Like other TEL-tyrosine kinase fusion proteins such as the TEL-PDGF receptor β chain and TEL-Abl, the JAK2 tyrosine kinase domain is constitutively activated by oligomerization mediated by the PNT domain. Stable expression of TEL-JAK2 confers factor-independent growth on IL-3-dependent Ba/F3 cells and induces myeloproliferative and T-cell lymphoproliferative diseases in mice (9Schwaller J. Frantsve J. Aster J. Williams I.R. Tomasson M.H. Ross T.S. Peeters P. Van Rompaey L. Van Etten R.A. Ilaria Jr., R. Marynen P. Gilliland D.G. EMBO J. 1998; 17: 5321-5333Crossref PubMed Scopus (226) Google Scholar). JAK family tyrosine kinases signal transducers and activators of transcription platelet-derived growth factor interleukin-3 interferon γ Cullin-2 von Hippel-Lindau disease C, Elongin B and C complex polymerase chain reaction hemagglutinin wild type deletion mutant lacking 40 amino acids at the C terminus glutathione S-transferase erythropoietin receptor expressed green fluorescence protein The JAK/STAT pathway is regulated by several mechanisms, including dephosphorylation by protein phosphatases and degradation by the ubiquitin/proteasome system (see review; Yasukawa et al.(10Yasukawa H. Sasaki A. Yoshimura A. Annu. Rev. Immunol. 2000; 18: 143-164Crossref PubMed Scopus (519) Google Scholar)). The CIS family (also referred to as the SOCS or SSI family) has been shown to play an important role in regulating cytokine signal transduction. CIS1, the first member of this family to be cloned, suppresses STAT5 activation by binding to cytokine receptors (11Yoshimura 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 (637) Google Scholar, 12Matsumoto 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 (225) Google Scholar). The second family member found, JAB/SOCS1/SSI-1, directly binds to the JAK2 kinase (JH1) domain, thereby inhibiting tyrosine kinase activity (13Endo 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: 92-921Crossref Scopus (1243) Google Scholar, 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 (1834) 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 (1147) Google Scholar). Mutational analysis and biochemical characterization revealed a novel type of inhibition of JAK2 tyrosine kinase activity through the two independent binding sites of SOCS1/JAB: the N-terminal kinase inhibitory region binds to the catalytic groove of JH1, and the SH2 domain binds to the phosphorylated tyrosine residue Tyr-1007 in the activation loop (16Yasukawa 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 (613) Google Scholar, 17Nicholson 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 (373) Google Scholar). Gene disruption studies have suggested that one of the major physiological functions of SOCS1 is the negative regulation of the IFNγ/STAT1 pathway (18Marine J.C. Topham D.J. McKay C. Wang D. Parganas E. Stravopodis D. Yoshimura A. Ihle J.N. Cell. 1999; 98: 609-616Abstract Full Text Full Text PDF PubMed Scopus (458) Google Scholar,19Alexander 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 (664) Google Scholar). Six additional CIS/SOCS/SSI family members were cloned from a data base search (20Masuhara 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, 21Minamoto S. Ikegame K. Ueno K. Narazaki M. Naka T. Yamamoto H. Matsumoto T. Saito H. Hosoe S. Kishimoto T. Biochem. Biophys. Res. Commun. 1997; 237: 79-83Crossref PubMed Scopus (143) Google Scholar, 22Hilton 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 (628) Google Scholar). In this family, the SH2 domain and the C-terminal region of about 40 amino acids, referred to as the SOCS box, are conserved. The data base search also revealed that a similar SOCS box is present in several proteins containing ankyrin-like repeats, Ras-like GTPases, or WD40 domains (20Masuhara 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, 22Hilton 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 (628) Google Scholar). The SOCS box has been implicated in protein stability or degradation of associated molecules, because it was found to interact with the Elongin B and Elongin C (Elongin B,C) complex, which may recruit Cullin-2 (Cul-2), Rbx1, and the E2 ubiquitin-conjugating enzyme (23Kamura 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 (511) Google Scholar, 24Zhang 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 (535) Google Scholar, 25Tyers M. Willems A.R. Science. 1999; 284: 601-604Crossref PubMed Scopus (142) Google Scholar). Therefore, CIS family members are hypothesized to function as E3-like ubiquitin-ligase complexes against target molecules from an analogy with the von Hippel-Lindau (VHL) tumor-suppressor gene product. However, no evidence in support of this hypothesis has yet been reported. Kamura et al. (23Kamura 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 (511) Google Scholar) reported that the protein levels of full-length JAK2 were not affected by coexpression of SOCS1, and, rather, that coexpression of Elongin B,C stabilized the SOCS1 protein. Furthermore, it has been shown that the SOCS box is not essential for the inhibition of cytokine-induced JAK/STAT activation by SOCS1 (16Yasukawa 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 (613) Google Scholar, 17Nicholson 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 (373) Google Scholar, 23Kamura 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 (511) Google Scholar, 26Narazaki M. Fujimoto M. Matsumoto T. Morita Y. Saito H. Kajita T. Yoshizaki K. Naka T. Kishimoto T. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13130-13134Crossref PubMed Scopus (217) Google Scholar). Therefore, the role of the SOCS box of SOCS1 still remains to be elucidated. To suppress the oncogenic potential of activated tyrosine kinases, we introduced the SOCS1 gene into Ba/F3 cells transformed with TEL-JAK2 using a retrovirus system. Overexpression of SOCS1 could efficiently suppress the transforming potential of TEL-JAK2. However, simple inhibition of kinase activity by SOCS1 could not explain the suppression of TEL-JAK2. We found that SOCS1 promoted ubiquitin-proteasome-dependent degradation of TEL-JAK2 and full-length JAK2, and that this process required the C-terminal SOCS box of SOCS1 as well as the phosphorylation of JAK2. Murine IL-3-dependent Ba/F3 cells were maintained in RPMI medium supplemented with 10% fetal calf serum and 10% conditioned medium from WEHI-3B cells as a source of IL-3. Ba/F3 cells were transformed with pCDNA3-TEL-JAK2 or pCDNA3-Bcr-Abl as described previously (11Yoshimura 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 (637) Google Scholar). After selection with G418 (1 mg/ml), cells that could grow without IL-3 were subsequently selected. Transient transfection and the luciferase assay in 293 cells have been described previously (20Masuhara 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). Deletion, substitution, and chimeric mutants were generated by standard PCR methods as described previously (16Yasukawa 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 (613) Google Scholar, 27Sasaki A. Yasukawa H. Suzuki A. Kamizono S. Syoda T. Kinjyo I. Sasaki M. Johnston J.A. Yoshimura A. Genes Cells. 1999; 4: 339-351Crossref PubMed Scopus (314) Google Scholar). Some of the mutants and wild-type SOCS1 were subcloned into a pMX-IRES-EGFP vector (28Nosaka T. Kawashima T. Misawa K. Ikuta K. Mui A.L. Kitamura T. EMBO J. 1999; 18: 4754-4765Crossref PubMed Scopus (442) Google Scholar). For swapping of the SOCS box, the SOCS boxes of SOCS1 (codon 167–212), CIS3/SOCS3 (codon 180–225), and CIS1 (codon 213–257) were interchanged by introducing an SplI site at the joint. All constructs contained an N-terminal Myc- or FLAG-tag (13Endo 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: 92-921Crossref Scopus (1243) Google Scholar). For TEL-JAK2 fusion, the human TEL (codon 1–162) part was obtained by PCR and fused to the mouse JAK2 JH1 domain (codon 839–1127). This fusion gene corresponds to the TEL-JAK2 found in B-cell lymphoblastic leukemia patients (9Schwaller J. Frantsve J. Aster J. Williams I.R. Tomasson M.H. Ross T.S. Peeters P. Van Rompaey L. Van Etten R.A. Ilaria Jr., R. Marynen P. Gilliland D.G. EMBO J. 1998; 17: 5321-5333Crossref PubMed Scopus (226) Google Scholar). Murine Cul-2 cDNA was obtained by PCR from a brain cDNA library and cloned into a pCDNA3 vector containing an N-terminal HA-tag. The R452C mutant was created by site-directed mutagenesis. Retroviruses were produced by transient transfection of the PLAT-E packaging cell line with cDNAs in pMX-IRES-EGFP (28Nosaka T. Kawashima T. Misawa K. Ikuta K. Mui A.L. Kitamura T. EMBO J. 1999; 18: 4754-4765Crossref PubMed Scopus (442) Google Scholar). Forty-eight hours after transfection, the culture supernatant was harvested and stored at −80 °C. BF/TEL-JAK or BF/Bcr-Abl cells (2 × 105cells) were infected with appropriately diluted PLAT-E supernatants containing 10 μg/ml Polybrene for 24 h. After being washed, cells were further cultured in an RPMI medium for an additional 24 or 48 h. Then, aliquots of cells (1 × 104) were analyzed using a Coulter EPICS-XL flow cytometer. All experiments were performed in the absence of IL-3. An in vitro kinase assay for TEL-JAK2 was performed as described previously (16Yasukawa 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 (613) Google Scholar). Briefly, FLAG-tagged TEL-JAK2 expressed in 293 cells (3.0 μg/transfection) grown in 10-cm dishes was immunoprecipitated with anti-FLAG antibody in 60 μl (50% v/v) of protein G-Sepharose. Then the resin was incubated with 1 ml of cell extracts from 293 cells transiently expressing wild-type SOCS1 (WT) or a deletion mutant lacking 40 amino acids at the C terminus (dC40) at 4 °C for 1 h. After being washed twice with kinase reaction buffer (50 mm Hepes-buffer, pH 7.5, 50 mm NaCl, 5 mm MgCl2, 5 mm MnCl2, 10 μm dithiothreitol, and 10 μm Na3VO4), the beads were resuspended in 20 μl of kinase reaction buffer containing the substrate polypeptide, GST-EPOR cytoplasmic domain (GST-YY) (16Yasukawa 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 (613) Google Scholar) (0.1 mg/ml), and ATP (50 μm) and incubated at 30 °C for 5 min. Kinase activity was analyzed by immunoblotting of GST-YY with anti-phosphotyrosine (4G10). Immunoprecipitation and immunoblotting were performed as described previously (11Yoshimura 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 (637) Google Scholar). Anti-JAK2 JH1 (αJAK2) rabbit polyclonal antibody, anti-Myc (αMYC) monoclonal and polyclonal antibodies, and anti-phosphotyrosine (αPY, 4G10) antibodies have been described previously (16Yasukawa 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 (613) Google Scholar). For pulse-chase experiments, 293 cells (1 × 106) grown in 10-cm dishes were transfected with TEL-JAK2 (2.0 μg of plasmid) and WT or dC40 cDNA (0.02 μg). After 18 h, the cells were pulse-labeled with Tran35S-label (ICN) at a concentration of 150 μCi/ml for 15 min and then scraped. After being divided into four parts, cells were replated into 3.5-cm dishes. Following the indicated chase periods, cells were lysed and immunoprecipitated with anti-JAK2 antibody followed by protein A-Sepharose, separated on SDS-polyacrylamide gel electrophoresis, exposed, and quantified by a BAS-2000 imaging system (Fuji). To see the effect of proteasome inhibitors, 293 cells transfected with TEL-JAK2 and WT-SOCS1 were treated with lactacystin or MG132 (25 μm each) for 30 min before labeling. The drugs were maintained throughout the pulse-chase period. For the cycloheximide treatment experiment, 293 cells (1 × 106) were transfected with 2.0 μg of TEL-JAK2 plus WT or mutant SOCS1 (0.02 μg). Eighteen hours after transfection, cells were trypsinized and divided into four parts. After a 5-h incubation period, cells were attached to the dishes and then treated with 50 μg/ml cycloheximide for the indicated periods. The cell extracts were prepared and immunoblotted with anti-JAK2, anti-STAT5, and anti-Myc antibodies as described (16Yasukawa 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 (613) Google Scholar). Band intensity was quantified by a densitometer as described (16Yasukawa 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 (613) Google Scholar). To determine whether the JAK inhibitor SOCS1/JAB can suppress oncogenic tyrosine kinases, SOCS1 cDNA was introduced into Ba/F3 cells transformed with either TEL-JAK2 (BF/TEL-JAK) or p210 Bcr-Abl (BF/Bcr-Abl) together with enhanced green fluorescence protein (EGFP) using the bicistronic retrovirus vector pMX-IRES-EGFP (28Nosaka T. Kawashima T. Misawa K. Ikuta K. Mui A.L. Kitamura T. EMBO J. 1999; 18: 4754-4765Crossref PubMed Scopus (442) Google Scholar). Because the infected cells expressed both EGFP and Myc-tagged SOCS1, the percentage of infected cells was determined as the EGFP-positive rate by flow cytometry. The same virus was shown to induce apoptosis of parental Ba/F3 cells in the presence of IL-3 (28Nosaka T. Kawashima T. Misawa K. Ikuta K. Mui A.L. Kitamura T. EMBO J. 1999; 18: 4754-4765Crossref PubMed Scopus (442) Google Scholar). As shown in Fig. 1 (A andB), the population of wild-type SOCS1 (WT)-infected BF/TEL-JAK cells decreased markedly, suggesting that WT-infected cells disappeared with cell death. Indeed, WT-infected BF/TEL-JAK cells underwent apoptosis characterized by DNA fragmentation (Fig.1 C, lane 2). Similar effects were observed even in the presence of IL-3 (data not shown). WT did not affect the growth of BF/Bcr-Abl cells (Fig. 1 B), which is consistent with the observation that SOCS1 did not inhibit Bcr-Abl tyrosine kinase activity (data not shown). Thus, the inhibitory effect of SOCS1 was shown to be specific to JAKs. We have shown that the N-terminal kinase inhibitory region and the SH2 domain, but not the C-terminal SOCS box, are essential for SOCS1 to inhibit JAK kinase activity in vitro and in vivo(16Yasukawa 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 (613) Google Scholar). However, unexpectedly, the dC40 mutant lacking the entire SOCS box could not suppress the growth of BF/TEL-JAK cells (Fig. 1,B and C). To clarify the reason for this discrepancy, we examined STAT5 tyrosine phosphorylation in BF/TEL-JAK cells after infection (Fig. 2; infection efficiency is listed as I.E.). Infection with WT and dC40, but not with a control virus, resulted in a decrease in the tyrosine phosphorylation of STAT5 (Fig. 2, αPY-STAT5). Reduction of STAT5 phosphorylation by WT was more profound than that by dC40. Tyrosine phosphorylation of TEL-JAK2 was partially reduced by dC40, suggesting that dC40 could reduce TEL-JAK2 kinase activity but was not sufficient to induce apoptosis of BF/TEL-JAK cells at the expression levels obtained by the retrovirus system. It should be noted that the infection efficiency of the WT virus to BF/TEL-JAK2 judged by flow cytometry was less than 55%, whereas those of dC40 and control viruses were more than 75%, even though virus titers were similar when assayed with NIH-3T3 cells. This is presumably because WT virus-infected cells die rapidly after infection. Thus, the infection efficiency of the WT virus will be underestimated. More drastically, we noticed that the protein levels of TEL-JAK2 decreased in WT-infected cells but not in dC40-infected cells (Fig. 2,αJAK2). WT-SOCS1 did not affect the protein level of Bcr-Abl (Fig. 2, αAbl). We found a 70–80% decrease of TEL-JAK2 and PY-STAT5 levels in WT virus-infected cells, whereas the infection efficiency was only 50%. This is probably because of the underestimation of the infection efficiency of WT-infected cells. These data suggest that kinase inhibition by SOCS1 is not sufficient to suppress the oncogenic potential of TEL-JAK2 and that the C-terminal SOCS box is necessary for complete suppression of the oncogenic potential of TEL-JAK2 by reducing the TEL-JAK2 protein level. We clarified the mechanism of reduction of the protein level of TEL-JAK2 by coexpression of SOCS1 using a transient expression system in 293 cells. As shown in Fig.3 A, WT, but not dC40-SOCS1, also reduced the level of TEL-JAK2 in a dose-dependent manner in 293 cells (Fig. 3 A,αJAK2). Thus, SOCS1 reduced the TEL-JAK2 protein level SOCS-box-dependently not only in Ba/F3 cells but also in 293 cells. Consequently, WT suppressed the TEL-JAK2-mediated STAT activation ∼50 times more efficiently than dC40 (Fig. 3 B). To confirm a similar kinase inhibitory activity of WT and dC40, we performed an in vitro kinase assay using the recombinant protein of the GST-tagged erythropoietin receptor (EPOR) cytoplasmic domain as substrate (16Yasukawa 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 (613) Google Scholar). Consistently, with the previous study using GST-JH1 as a constitutively activated kinase, WT and dC40 could similarly suppress the in vitrokinase activity of TEL-JAK2 (Fig. 3 C, lanes 3 and4). These results indicate that, although the SOCS box of SOCS1 is not necessary for kinase inhibition, the inhibitory effect of WT-SOCS1 was strongly enhanced by inducing degradation of TEL-JAK2 (Fig. 3, A and B). The half-life of TEL-JAK2 was examined in a metabolic pulse labeling and chase experiment (Fig.4 A). The half-life of TEL-JAK2 was over 60 min, but coexpression of WT accelerated the decay of TEL-JAK2, reducing its half-life to less than 30 min. dC40 did not affect the half-life of TEL-JAK2. Accelerated degradation of TEL-JAK2 in the presence of WT, but not dC40, was also observed after cells were treated with a protein synthesis inhibitor, cycloheximide (Fig.4 B). Normalized levels of TEL-JAK2 are shown as the TJ/S ratio against the levels of STAT5, which is a very stable protein (Fig.4 B). As shown in Fig. 4 (A and C), the rapid degradation of TEL-JAK2 by coexpression of SOCS1 was significantly delayed by treatment of the cells with two proteasome inhibitors, lactacystin and MG132. These data suggest that SOCS1 promotes proteasome-dependent degradation of TEL-JAK2. Previously, Kamura et al. (20Masuhara 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) reported that SOCS1 overexpression did not affect wild-type JAK2 protein stability. We tried to resolve this discrepancy between TEL-JAK2 and full-length JAK2. As shown in Fig.5 A, full-length JAK2 was much less tyrosine-phosphorylated than TEL-JAK2 when expressed alone in 293 cells. Therefore, we suspect that the phosphorylation of the JH1 domain is necessary for SOCS1-mediated degradation. Because glutathioneS-transferase (GST) is a dimer, the JH1 domain fused to GST (GST-JH1) is another constitutively activated form of the JAK2 tyrosine kinase domain (16Yasukawa 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 (613) Google Scholar). Like TEL-JAK2, GST-JH1 was markedly decreased in its expression level when coexpressed with WT, but not with dC40 (Fig.5 B). Pulse-chase experiments revealed that WT-SOCS1 also shortened the half-life of GST-JH1 (data not shown). Moreover, the protein level of the phosphorylation-deficient mutant (FF) of GST-JH1 was not affected by SOCS1 (Fig. 5 B). Therefore, reduction in the GST-JH1 protein level by SOCS1 was dependent on tyrosine phosphorylation or the activation of the JH1 domain. Next, we examined whether SOCS1 promotes the degradation of activated full-length JAK2. To achieve the activation of JAK2, JAK2 was fused to gyrase B (Gyr-JAK2), and Gyr-JAK2 was dimerized by coumermycin (29Mohi M.G. Arai K. Watanabe S. Mol. Biol. Cell. 1998; 9: 3299-3308Crossref PubMed Scopus (32) Google Scholar). As shown in Fig. 6 A, coumermycin treatment enhanced the tyrosine phosphorylation of Gyr-JAK2. Without coumermycin, WT-SOCS1 did not affect the protein levels of JAK2 (Fig.6 B, left). However, WT, but not dC40, induced the degradation of Gyr-JAK2 in the presence of coumermycin (Fig.6 B, Coum. (+), αJAK2). These data suggest that the SOCS1-SOCS box can potentially induce the degradation of full-length JAK2 but that this process requires tyrosine phosphorylation (or activation) of JAK2. To examine the functional redundancy of the SH2 domain and the SOCS box for TEL-JAK2 degradation, we constructed chimeric mutants among CIS1, CIS3/SOCS3, and SOCS1 (Fig.7). The SH2 domain mutant R105E-SOCS1 exhibited a lesser effect on TEL-JAK2 protein stability. Thus, tight binding of SOCS1 to the JH1 domain through the SH2 domain is necessary for the degradation of TEL-JAK2. CIS1, which does not bind to JAK2, did not induce TEL-JAK2 degradation, although CIS1

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