The Tumor Suppressor hTid1 Inhibits STAT5b Activity via Functional Interaction
2010; Elsevier BV; Volume: 286; Issue: 7 Linguagem: Inglês
10.1074/jbc.m110.155903
ISSN1083-351X
AutoresIsabelle Dhennin‐Duthille, Rémy Nyga, Saliha Yahiaoui, Valérie Gouilleux‐Gruart, Jean-Claude Régnier, Kaı̈ss Lassoued, Fabrice Gouilleux,
Tópico(s)Cancer-related gene regulation
ResumoSTAT5a and -5b (signal transducers and activators of transcription 5a and 5b) proteins play an essential role in hematopoietic cell proliferation and survival and are frequently constitutively active in hematologic neoplasms and solid tumors. Because STAT5a and STAT5b differ mainly in the carboxyl-terminal transactivation domain, we sought to identify new proteins that bind specifically to this domain by using a bacterial two-hybrid screening. We isolated hTid1, a human DnaJ protein that acts as a tumor suppressor in various solid tumors. hTid1 interacts specifically with STAT5b but not with STAT5a in hematopoietic cell lines. This interaction involves the cysteine-rich region of the hTid1 DnaJ domain. We also demonstrated that hTid1 negatively regulates the expression and transcriptional activity of STAT5b and suppresses the growth of hematopoietic cells transformed by an oncogenic form of STAT5b. Our findings define hTid1 as a novel partner and negative regulator of STAT5b. STAT5a and -5b (signal transducers and activators of transcription 5a and 5b) proteins play an essential role in hematopoietic cell proliferation and survival and are frequently constitutively active in hematologic neoplasms and solid tumors. Because STAT5a and STAT5b differ mainly in the carboxyl-terminal transactivation domain, we sought to identify new proteins that bind specifically to this domain by using a bacterial two-hybrid screening. We isolated hTid1, a human DnaJ protein that acts as a tumor suppressor in various solid tumors. hTid1 interacts specifically with STAT5b but not with STAT5a in hematopoietic cell lines. This interaction involves the cysteine-rich region of the hTid1 DnaJ domain. We also demonstrated that hTid1 negatively regulates the expression and transcriptional activity of STAT5b and suppresses the growth of hematopoietic cells transformed by an oncogenic form of STAT5b. Our findings define hTid1 as a novel partner and negative regulator of STAT5b. IntroductionSTAT transcription factors play a central role in cytokine-dependent survival, proliferation, and differentiation of a large spectrum of cells. Following cytokine addition, STAT proteins become tyrosine-phosphorylated and subsequently dimerize, forming homo- or heterodimers, and translocate into the nucleus, where they bind to specific elements in the promoter of target genes and activate transcription (1Leonard W.J. Int. J. Hematol. 2001; 73: 271-277Crossref PubMed Scopus (180) Google Scholar). The STAT protein family comprises seven members, including the two closely related STAT5a and STAT5b molecules (2Liu X. Robinson G.W. Gouilleux F. Groner B. Hennighausen L. Proc. Natl. Acad. Sci. U.S.A. 1995; 92: 8831-8835Crossref PubMed Scopus (455) Google Scholar, 3Buitenhuis M. Coffer P.J. Koenderman L. Int. J. Biochem. Cell Biol. 2004; 36: 2120-2124Crossref PubMed Scopus (66) Google Scholar). Mice in which stat5a and stat5b genes were deleted revealed redundant and specific functions of both proteins. stat5a−/− mice have a profound defect in mammary gland development and in prolactin response, whereas stat5b−/− mice display a defect in growth hormone response (4Liu X. Robinson G.W. Wagner K.U. Garrett L. Wynshaw-Boris A. Hennighausen L. Genes Dev. 1997; 11: 179-186Crossref PubMed Scopus (907) Google Scholar, 5Udy G.B. Towers R.P. Snell R.G. Wilkins R.J. Park S.H. Ram P.A. Waxman D.J. Davey H.W. Proc. Natl. Acad. Sci. U.S.A. 1997; 94: 7239-7244Crossref PubMed Scopus (822) Google Scholar). Simultaneous inactivation of stat5a/b genes demonstrated the requirement of both proteins in myeloid and lymphoid cell proliferation (6Teglund S. McKay C. Schuetz E. van Deursen J.M. Stravopodis D. Wang D. Brown M. Bodner S. Grosveld G. Ihle J.N. Cell. 1998; 93: 841-850Abstract Full Text Full Text PDF PubMed Scopus (1067) Google Scholar, 7Cui Y. Riedlinger G. Miyoshi K. Tang W. Li C. Deng C.X. Robinson G.W. Hennighausen L. Mol. Cell Biol. 2004; 24: 8037-8047Crossref PubMed Scopus (398) Google Scholar). Indeed, erythroblasts, myeloid cells, mast cells, peripheral T cells, NK cells, and B cells display impaired proliferation and/or survival in mice lacking expression of STAT5 proteins (8Shelburne C.P. McCoy M.E. Piekorz R. Sexl V. Roh K.H. Jacobs-Helber S.M. Gillespie S.R. Bailey D.P. Mirmonsef P. Mann M.N. Kashyap M. Wright H.V. Chong H.J. Bouton L.A. Barnstein B. Ramirez C.D. Bunting K.D. Sawyer S. Lantz C.S. Ryan J.J. Blood. 2003; 102: 1290-1297Crossref PubMed Scopus (113) Google Scholar, 9Socolovsky M. Fallon A.E. Wang S. Brugnara C. Lodish H.F. Cell. 1999; 98: 181-191Abstract Full Text Full Text PDF PubMed Scopus (613) Google Scholar, 10Yao Z. Cui Y. Watford W.T. Bream J.H. Yamaoka K. Hissong B.D. Li D. Durum S.K. Jiang Q. Bhandoola A. Hennighausen L. O'Shea J.J. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 1000-1005Crossref PubMed Scopus (286) Google Scholar, 11Kieslinger M. Woldman I. Moriggl R. Hofmann J. Marine J.C. Ihle J.N. Beug H. Decker T. Genes Dev. 2000; 14: 232-244PubMed Google Scholar). STAT5 promotes cell survival and/or proliferation by regulating the expression of genes involved in the control of cell cycle and survival like bcl-xL, cyclins D1 and D2, p21waf1, and the proto-oncogene pim-1 (12Dumon S. Santos S.C. Debierre-Grockiego F. Gouilleux-Gruart V. Cocault L. Boucheron C. Mollat P. Gisselbrecht S. Gouilleux F. Oncogene. 1999; 18: 4191-4199Crossref PubMed Scopus (134) Google Scholar, 13Moriggl R. Topham D.J. Teglund S. Sexl V. McKay C. Wang D. Hoffmeyer A. van Deursen J. Sangster M.Y. Bunting K.D. Grosveld G.C. Ihle J.N. Immunity. 1999; 10: 249-259Abstract Full Text Full Text PDF PubMed Scopus (475) Google Scholar, 14Nosaka T. Kawashima T. Misawa K. Ikuta K. Mui A.L. Kitamura T. EMBO J. 1999; 18: 4754-4765Crossref PubMed Scopus (434) Google Scholar). Besides the physiological role of STAT5 in hematopoietic cell development, there is increasing evidence suggesting that inappropriate activation of STAT5 may contribute to the development of leukemias and solid cancers (15Benekli M. Baer M.R. Baumann H. Wetzler M. Blood. 2003; 101: 2940-2954Crossref PubMed Scopus (283) Google Scholar, 16Tan S.H. Nevalainen M.T. Endocr. Relat. Cancer. 2008; 15: 367-390Crossref PubMed Scopus (93) Google Scholar). STAT5 is frequently hyperactivated in cancer and leukemias, most probably by alterations of tyrosine kinase activities. Importantly, STAT5 is a common and crucial target for different oncoproteins with tyrosine kinase activity, like Tel-Jak2, Bcr-Abl, the mutated forms of Flt3 and c-Kit, and the Jak2V617F mutant (17Lacronique V. Boureux A. Monni R. Dumon S. Mauchauffé M. Mayeux P. Gouilleux F. Berger R. Gisselbrecht S. Ghysdael J. Bernard O.A. Blood. 2000; 95: 2076-2083Crossref PubMed Google Scholar, 18Nieborowska-Skorska M. Wasik M.A. Slupianek A. Salomoni P. Kitamura T. Calabretta B. Skorski T. J. Exp. Med. 1999; 189: 1229-1242Crossref PubMed Scopus (241) Google Scholar, 19Mizuki M. Fenski R. Halfter H. Matsumura I. Schmidt R. Müller C. Grüning W. Kratz-Albers K. Serve S. Steur C. Büchner T. Kienast J. Kanakura Y. Berdel W.E. Serve H. Blood. 2000; 96: 3907-3914Crossref PubMed Google Scholar, 20Harir N. Boudot C. Friedbichler K. Sonneck K. Kondo R. Martin-Lannerée S. Kenner L. Kerenyi M. Yahiaoui S. Gouilleux-Gruart V. Gondry J. Bénit L. Dusanter-Fourt I. Lassoued K. Valent P. Moriggl R. Gouilleux F. Blood. 2008; 112: 2463-2473Crossref PubMed Scopus (87) Google Scholar, 21Levine R.L. Wadleigh M. Cools J. Ebert B.L. Wernig G. Huntly B.J. Boggon T.J. Wlodarska I. Clark J.J. Moore S. Adelsperger J. Koo S. Lee J.C. Gabriel S. Mercher T. D'Andrea A. Fröhling S. Döhner K. Marynen P. Vandenberghe P. Mesa R.A. Tefferi A. Griffin J.D. Eck M.J. Sellers W.R. Meyerson M. Golub T.R. Lee S.J. Gilliland D.G. Cancer Cell. 2005; 7: 387-397Abstract Full Text Full Text PDF PubMed Scopus (2425) Google Scholar). Furthermore, it has been shown that STAT5 plays a critical role in Bcr-Abl- and Tel-Jak2-induced myeloproliferative disease (22Hoelbl A. Kovacic B. Kerenyi M.A. Simma O. Warsch W. Cui Y. Beug H. Hennighausen L. Moriggl R. Sexl V. Blood. 2006; 107: 4898-4906Crossref PubMed Scopus (172) Google Scholar, 23Schwaller J. Parganas E. Wang D. Cain D. Aster J.C. Williams I.R. Lee C.K. Gerthner R. Kitamura T. Frantsve J. Anastasiadou E. Loh M.L. Levy D.E. Ihle J.N. Gilliland D.G. Mol. Cell. 2000; 6: 693-704Abstract Full Text Full Text PDF PubMed Scopus (269) Google Scholar). The most direct evidence that constitutive activation of STAT5 is an important causative event in cell transformation came from the analysis of the STAT5 mutants, STAT5a1*6 and STAT5b1*6, and cS5F. These proteins with mutations at residues His299 → Arg and Ser711/716 → Phe (STAT5a1*6 or STAT5b1*6) or with the single mutation Ser711 → Phe (cS5F) possess constitutive tyrosine phosphorylation and are capable of inducing leukemias in mice (23Schwaller J. Parganas E. Wang D. Cain D. Aster J.C. Williams I.R. Lee C.K. Gerthner R. Kitamura T. Frantsve J. Anastasiadou E. Loh M.L. Levy D.E. Ihle J.N. Gilliland D.G. Mol. Cell. 2000; 6: 693-704Abstract Full Text Full Text PDF PubMed Scopus (269) Google Scholar, 24Moriggl R. Sexl V. Kenner L. Duntsch C. Stangl K. Gingras S. Hoffmeyer A. Bauer A. Piekorz R. Wang D. Bunting K.D. Wagner E.F. Sonneck K. Valent P. Ihle J.N. Beug H. Cancer Cell. 2005; 7: 87-99Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar). In addition, STAT5b plays an important role in the proliferation and/or survival of tumor cells from head and neck cancer, glioblastomas, and prostate cancer (16Tan S.H. Nevalainen M.T. Endocr. Relat. Cancer. 2008; 15: 367-390Crossref PubMed Scopus (93) Google Scholar, 25Xi S. Zhang Q. Gooding W.E. Smithgall T.E. Grandis J.R. Cancer Res. 2003; 63: 6763-6771PubMed Google Scholar, 26Liang Q.C. Xiong H. Zhao Z.W. Jia D. Li W.X. Qin H.Z. Deng J.P. Gao L. Zhang H. Gao G.D. Cancer Lett. 2009; 273: 164-171Crossref PubMed Scopus (67) Google Scholar, 27Gu L. Dagvadorj A. Lutz J. Leiby B. Bonuccelli G. Lisanti M.P. Addya S. Fortina P. Dasgupta A. Hyslop T. Bubendorf L. Nevalainen M.T. Am. J. Pathol. 2010; 176: 1959-1972Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). STAT5b acts downstream of epidermal growth factor receptor, which is frequently overexpressed or hyperactivated in these tumors (28Kloth M.T. Laughlin K.K. Biscardi J.S. Boerner J.L. Parsons S.J. Silva C.M. J. Biol. Chem. 2003; 278: 1671-1679Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). Furthermore, STAT5b is specifically activated in T-cell lymphomas transformed by the oncogenic fusion NPM1-ALK and contributes to the NPM1-ALK oncogenesis by promoting cell growth and survival, whereas STAT5a acts as a tumor suppressor in these malignant cells (29Zhang Q. Wang H.Y. Liu X. Wasik M.A. Nat. Med. 2007; 13: 1341-1348Crossref PubMed Scopus (100) Google Scholar). This suggests that STAT5a and STAT5b may have some non overlapping and opposite functions in the transformation of similar target cells.Like other STAT family members, STAT5a and STAT5b proteins contain in their carboxyl-terminal part a transactivation domain that is required for transcriptional activation (30Moriggl R. Gouilleux-Gruart V. Jähne R. Berchtold S. Gartmann C. Liu X. Hennighausen L. Sotiropoulos A. Groner B. Gouilleux F. Mol. Cell Biol. 1996; 16: 5691-5700Crossref PubMed Scopus (246) Google Scholar). In some early hematopoietic progenitors and in peripheral T cells, cleavage of full-length STAT5 proteins by proteases generates carboxyl-terminally truncated STAT5 proteins called STAT5γ that lack the transactivation domain and function as dominant negative proteins (3Buitenhuis M. Coffer P.J. Koenderman L. Int. J. Biochem. Cell Biol. 2004; 36: 2120-2124Crossref PubMed Scopus (66) Google Scholar). Mutagenesis analyses have shown that a small amphipathic α-helical region within this domain is required not only for transcriptional activation of STAT5 proteins but also for the rapid proteasome-dependent turnover of the molecules (31Wang D. Moriggl R. Stravopodis D. Carpino N. Marine J.C. Teglund S. Feng J. Ihle J.N. EMBO J. 2000; 19: 392-399Crossref PubMed Google Scholar). This region is also involved in the recruitment of the cofactors CBP/P300 and NCoA1/SRC-1 (32Litterst C.M. Kliem S. Marilley D. Pfitzner E. J. Biol. Chem. 2003; 278: 45340-45351Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 33Pfitzner E. Jähne R. Wissler M. Stoecklin E. Groner B. Mol. Endocrinol. 1998; 12: 1582-1593Crossref PubMed Google Scholar). Thus, transcriptional activation and down-regulation of STAT5 proteins are mediated via a similar region located in the transactivation domain. STAT5a and STAT5b share 96% homology at the amino acid level and differ mainly in the carboxyl-terminal region. Importantly, a serine residue at position 779 that is phosphorylated in STAT5a is absent at a similar position in STAT5b (34Beuvink I. Hess D. Flotow H. Hofsteenge J. Groner B. Hynes N.E. J. Biol. Chem. 2000; 275: 10247-10255Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). There is evidence that STAT5b is phosphorylated on tyrosine residues in the carboxyl terminus distinct from the residue Tyr699, which is necessary for STAT5b dimerization and activation (35Kloth M.T. Catling A.D. Silva C.M. J. Biol. Chem. 2002; 277: 8693-8701Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). Such phosphorylations may eventually affect STAT5b intracellular trafficking or interaction with cellular proteins (36Kazansky A.V. Rosen J.M. Cell Growth Differ. 2001; 12: 1-7PubMed Google Scholar). The carboxyl-terminal regions of STAT5a and STAT5b may therefore confer distinct functions to these two molecules that might be related in part to interactions with distinct molecular partners. In this work, we aimed to identify new STAT5 transactivation domain-interacting proteins that could differentially regulate STAT5a or STAT5b activity. For this purpose, we used a bacterial two-hybrid screening approach. We have identified hTid1, a human DnaJ protein, homolog of the Drosophila tumor suppressor Tid56, as a novel and specific negative regulator of STAT5b activity and expression in hematopoietic cell lines.DISCUSSIONSTAT Transcription Factors Are Important Mediators of Cytokine-induced Cell Survival and Proliferation. They transmit signals emanating from activated receptors to the nucleus in a rapid and transient manner (1Leonard W.J. Int. J. Hematol. 2001; 73: 271-277Crossref PubMed Scopus (180) Google Scholar). STAT activities are switched off subsequently by several and distinct negative regulatory mechanisms. These include the activities of phosphatases; inhibition by SOCS proteins (suppressors of cytokine signaling); interaction of inhibitory proteins, such as PIAS (protein inhibitor of activated STATs); and targeted proteasome-dependent degradation of active STATs (45Hilton D.J. Cell Mol. Life Sci. 1999; 55: 1568-1577Crossref PubMed Scopus (188) Google Scholar, 46Kile B.T. Nicola N.A. Alexander W.S. Int. J. Hematol. 2001; 73: 292-298Crossref PubMed Scopus (63) Google Scholar, 47Tanaka T. Soriano M.A. Grusby M.J. Immunity. 2005; 22: 729-736Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar). For instance, it was shown that tyrosine dephosphorylation of STAT5 is induced by distinct tyrosine phosphatases like SHP-2, PTP1B, and LMW-PTP (48Yu C.L. Jin Y.J. Burakoff S.J. J. Biol. Chem. 2000; 275: 599-604Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar, 49Aoki N. Matsuda T. J. Biol. Chem. 2000; 275: 39718-39726Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar, 50Rigacci S. Talini D. Berti A. Biochem. Biophys. Res. Commun. 2003; 312: 360-366Crossref PubMed Scopus (30) Google Scholar). In addition, targeted proteasome degradation of STAT5 is mediated via their ubiquitination (47Tanaka T. Soriano M.A. Grusby M.J. Immunity. 2005; 22: 729-736Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar). Interestingly, The carboxyl-terminal region of Sat5 containing the transactivation domain is involved in the dephosphorylation induced by the phosphatase LMW-PTP (50Rigacci S. Talini D. Berti A. Biochem. Biophys. Res. Commun. 2003; 312: 360-366Crossref PubMed Scopus (30) Google Scholar). Similarly, nuclear ubiquitination and degradation of STAT5a by an as yet unidentified E3-ubiquitin ligase requires the α-amphipathic helix present in the transactivation domain of STAT5 (51Chen Y. Dai X. Haas A.L. Wen R. Wang D. Blood. 2006; 108: 566-574Crossref PubMed Scopus (22) Google Scholar). To identify specific binding of proteins to the carboxyl-terminal region of STAT5a and STAT5b, we used a bacterial two-hybrid screening and identified hTid1 as an interacting partner of STAT5b but not STAT5a. hTid1 has a high homology to Tid56, the protein encoded by the Drosophila melanogaster tumor suppressor gene l(2)tid (52Kaymer M. Debes A. Kress H. Kurzik-Dumke U. Gene. 1997; 204: 91-103Crossref PubMed Scopus (7) Google Scholar). Tid56 and hTid1 have 65.8 and 54.9% amino acid similarity and identity, respectively, and thus have been well conserved through evolution (53Yin X. Rozakis-Adcock M. Gene. 2001; 278: 201-210Crossref PubMed Scopus (25) Google Scholar). Tid56 and hTid1 are members of the DnaJ chaperone protein family, which contains the J domain, a highly conserved domain that binds to Hsp70 molecular chaperones (54Cyr D.M. Langer T. Douglas M.G. Trends Biochem. Sci. 1994; 19: 176-181Abstract Full Text PDF PubMed Scopus (398) Google Scholar). The DnaJ-Hsp70 complexes are involved in protein folding, protein degradation, and the assembly or disassembly of multiprotein complexes. The htid1 gene encodes two spliced variants of hTid1, hTid1L and hTid1S, with opposite functions (40Syken J. De-Medina T. Münger K. Proc. Natl. Acad. Sci. U.S.A. 1999; 96: 8499-8504Crossref PubMed Scopus (121) Google Scholar). We showed in this study that both forms of hTid1 were able to interact with STAT5b in different murine and human hematopoietic cell lines and in COS cells co-expressing STAT5b and hTid1. Lu et al. (55Lu B. Garrido N. Spelbrink J.N. Suzuki C.K. J. Biol. Chem. 2006; 281: 13150-13158Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar) reported that only the long form of hTid1 interacts with STAT1 and STAT3 in the U2OS osteosarcoma cells. Our data showed that both forms of hTid1 interact with STAT1 but not STAT3 in hematopoietic cell lines. The reason for these discrepancies is not known, but they may be due to the requirement of an unidentified additional partner that is missing in some types of cells. In sharp contrast to STAT1, association of hTid1 isoforms with STAT5b is no longer detected following cell stimulation with cytokines. Phosphorylation of STAT5b on tyrosine residue Tyr699 was further shown to be involved in the dissociation of STAT5b from hTid1 upon cytokine stimulation. Nevertheless, we observed that hTid1 inhibits the transcriptional activity of STAT5b but not STAT5a in transfected COS cells, providing evidence for a specific and functional interaction between hTid1 and STAT5b. Importantly, we found evidence that hTid1 had the ability to inhibit the expression of STAT5b. Many studies have emphasized the important role of hTid1 in the regulation of stability and/or degradation of cellular proteins. For example, it was shown that hTid1 allowed the degradation of the transcription factor HIF1α by the Von Hippel-Lindau protein (43Bae M.K. Jeong J.W. Kim S.H. Kim S.Y. Kang H.J. Kim D.M. Bae S.K. Yun I. Trentin G.A. Rozakis-Adcock M. Kim K.W. Cancer Res. 2005; 65: 2520-2525Crossref PubMed Scopus (37) Google Scholar). Similarly, hTid1 may prolong the half-life of IκB kinases or act on the degradation of the receptor Erb2 in breast carcinoma cells (42Cheng H. Cenciarelli C. Nelkin G. Tsan R. Fan D. Cheng-Mayer C. Fidler I.J. Mol. Cell Biol. 2005; 25: 44-59Crossref PubMed Scopus (37) Google Scholar, 56Kim S.W. Chao T.H. Xiang R. Lo J.F. Campbell M.J. Fearns C. Lee J.D. Cancer Res. 2004; 64: 7732-7739Crossref PubMed Scopus (46) Google Scholar). It is therefore conceivable that hTid1 can regulate the stability/degradation of STAT5b. hTid1 interacts with the chaperone molecules Hsc70 and Hsp70 (40Syken J. De-Medina T. Münger K. Proc. Natl. Acad. Sci. U.S.A. 1999; 96: 8499-8504Crossref PubMed Scopus (121) Google Scholar, 55Lu B. Garrido N. Spelbrink J.N. Suzuki C.K. J. Biol. Chem. 2006; 281: 13150-13158Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). Interestingly, it was previously shown that overexpression of Hsp70 protein in leukemic cells increases the expression of STAT5 (57Guo F. Sigua C. Bali P. George P. Fiskus W. Scuto A. Annavarapu S. Mouttaki A. Sondarva G. Wei S. Wu J. Djeu J. Bhalla K. Blood. 2005; 105: 1246-1255Crossref PubMed Scopus (152) Google Scholar). Thus, it is likely that hTid1 may act on STAT5b expression via interaction with Hsp70, a hypothesis that awaits further experiments.Our mutagenesis experiments clearly demonstrated that the DnaJ cysteine-rich region of hTid1 is essential in the down-regulation of STAT5b expression and activity. This central cysteine-rich domain resembles a zinc finger structure and contains four repeats of the motif CXXCXGXG. Previous reports have shown that this region also interacts with the human T-cell lymphotrophic virus viral protein Tax and the transcription factor Smad7, a downstream signaling effector of BMP (bone morphogenetic protein) (58Cheng H. Cenciarelli C. Shao Z. Vidal M. Parks W.P. Pagano M. Cheng-Mayer C. Curr. Biol. 2001; 11: 1771-1775Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 59Torregroza I. Evans T. Biochem. J. 2006; 393: 311-320Crossref PubMed Scopus (7) Google Scholar). Interestingly, interaction with Smad7 was shown to alter the development of chicken embryos by inhibiting the signaling induced by BMP. Although the DnaJ cysteine-rich domain is essential for the hTid1/STAT5b interaction, we cannot rule out the contribution of the DnaJ domain amino terminus in regulating the expression of STAT5b because this domain plays a role in the targeted degradation of intracellular proteins, as has been observed for the IκB-IKK complex (42Cheng H. Cenciarelli C. Nelkin G. Tsan R. Fan D. Cheng-Mayer C. Fidler I.J. Mol. Cell Biol. 2005; 25: 44-59Crossref PubMed Scopus (37) Google Scholar). Although the physiological function of hTid1 in hematopoietic cells remains currently poorly documented, our findings and published reports suggest that hTid1 may regulate the proliferation and/or survival of hematopoietic cells (60Syken J. Macian F. Agarwal S. Rao A. Münger K. Oncogene. 2003; 22: 4636-4641Crossref PubMed Scopus (20) Google Scholar). Importantly, we showed that hTid1 can inhibit the growth of Ba/F3 cells transformed by an oncogenic STAT5b but not a STAT5a mutant. There is a body of evidence indicating that hTid1 plays an important role in apoptosis and/or cell proliferation in various cell types (38Edwards K.M. Münger K. Oncogene. 2004; 23: 8419-8431Crossref PubMed Scopus (26) Google Scholar, 40Syken J. De-Medina T. Münger K. Proc. Natl. Acad. Sci. U.S.A. 1999; 96: 8499-8504Crossref PubMed Scopus (121) Google Scholar). In addition, it was shown that hTid regulates apoptosis of glioma cells and suppresses growth of head and neck cancer cells both in vitro and in vivo (61Trentin G.A. He Y. Wu D.C. Tang D. Rozakis-Adcock M. FEBS Lett. 2004; 578: 323-330Crossref PubMed Scopus (21) Google Scholar, 62Chen C.Y. Chiou S.H. Huang C.Y. Jan C.I. Lin S.C. Hu W.Y. Chou S.H. Liu C.J. Lo J.F. J. Pathol. 2009; 219: 347-355Crossref PubMed Scopus (38) Google Scholar). The mechanisms involved in hTid1-mediated growth suppression remain elusive because hTid1 modulates the activity of many proteins involved in signal transduction and hence in the survival/apoptosis and/or cell proliferation. Indeed, hTid1 regulates the activity and/or stability of the tyrosine kinase JAK2, the IKK-IKβ complex, and therefore the activation of NFκB as well as the HIF1α transcription factor (42Cheng H. Cenciarelli C. Nelkin G. Tsan R. Fan D. Cheng-Mayer C. Fidler I.J. Mol. Cell Biol. 2005; 25: 44-59Crossref PubMed Scopus (37) Google Scholar, 43Bae M.K. Jeong J.W. Kim S.H. Kim S.Y. Kang H.J. Kim D.M. Bae S.K. Yun I. Trentin G.A. Rozakis-Adcock M. Kim K.W. Cancer Res. 2005; 65: 2520-2525Crossref PubMed Scopus (37) Google Scholar, 63Sarkar S. Pollack B.P. Lin K.T. Kotenko S.V. Cook J.R. Lewis A. Pestka S. J. Biol. Chem. 2001; 276: 49034-49042Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). In addition, hTid1 interacts with the tumor suppressor APC (adenomatous polyposis coli) and regulates the activity of p53 by affecting its subcellular localization (64Qian J. Perchiniak E.M. Sun K. Groden J. Gastroenterology. 2010; 138: 1418-1428Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar, 65Ahn B.Y. Trinh D.L. Zajchowski L.D. Lee B. Elwi A.N. Kim S.W. Oncogene. 2010; 29: 1155-1166Crossref PubMed Scopus (64) Google Scholar). Our data suggest that hTid1 inhibits the growth of hematopoietic cell lines by interfering with the STAT5-dependent expression of genes involved in cell proliferation and survival, such as bcl-xL. Interestingly, several reports have shown that STAT5b but not STAT5a plays an important role in the proliferation and/or tumor invasion of glioblastoma multiforme cells and that constitutive activation of STAT5b contributes to squamous cell tumorigenesis in vitro and in vivo (25Xi S. Zhang Q. Gooding W.E. Smithgall T.E. Grandis J.R. Cancer Res. 2003; 63: 6763-6771PubMed Google Scholar, 26Liang Q.C. Xiong H. Zhao Z.W. Jia D. Li W.X. Qin H.Z. Deng J.P. Gao L. Zhang H. Gao G.D. Cancer Lett. 2009; 273: 164-171Crossref PubMed Scopus (67) Google Scholar). The apparent opposite effects of hTid1 in these two types of tumors suggest that hTid1 interferes with STAT5b activity in these neoplastic cells. In conclusion, our findings identified hTid1 as a new partner of STAT5b that has the ability to inhibit its transcriptional activity and its effects on hematopoietic cell growth. Whether hTid1 could suppress STAT5b-mediated cell transformation needs to be investigated. The finding that the 83 amino acids containing the DnaJ cysteine-rich region of hTid1 constitute the unique STAT5b-interacting domain opens interesting perspectives in the development of short peptides that could block the oncogenic activity of STAT5b. IntroductionSTAT transcription factors play a central role in cytokine-dependent survival, proliferation, and differentiation of a large spectrum of cells. Following cytokine addition, STAT proteins become tyrosine-phosphorylated and subsequently dimerize, forming homo- or heterodimers, and translocate into the nucleus, where they bind to specific elements in the promoter of target genes and activate transcription (1Leonard W.J. Int. J. Hematol. 2001; 73: 271-277Crossref PubMed Scopus (180) Google Scholar). The STAT protein family comprises seven members, including the two closely related STAT5a and STAT5b molecules (2Liu X. Robinson G.W. Gouilleux F. Groner B. Hennighausen L. Proc. Natl. Acad. Sci. U.S.A. 1995; 92: 8831-8835Crossref PubMed Scopus (455) Google Scholar, 3Buitenhuis M. Coffer P.J. Koenderman L. Int. J. Biochem. Cell Biol. 2004; 36: 2120-2124Crossref PubMed Scopus (66) Google Scholar). Mice in which stat5a and stat5b genes were deleted revealed redundant and specific functions of both proteins. stat5a−/− mice have a profound defect in mammary gland development and in prolactin response, whereas stat5b−/− mice display a defect in growth hormone response (4Liu X. Robinson G.W. Wagner K.U. Garrett L. Wynshaw-Boris A. Hennighausen L. Genes Dev. 1997; 11: 179-186Crossref PubMed Scopus (907) Google Scholar, 5Udy G.B. Towers R.P. Snell R.G. Wilkins R.J. Park S.H. Ram P.A. Waxman D.J. Davey H.W. Proc. Natl. Acad. Sci. U.S.A. 1997; 94: 7239-7244Crossref PubMed Scopus (822) Google Scholar). Simultaneous inactivation of stat5a/b genes demonstrated the requirement of both proteins in myeloid and lymphoid cell proliferation (6Teglund S. McKay C. Schuetz E. van Deursen J.M. Stravopodis D. Wang D. Brown M. Bodner S. Grosveld G. Ihle J.N. Cell. 1998; 93: 841-850Abstract Full Text Full Text PDF PubMed Scopus (1067) Google Scholar, 7Cui Y. Riedlinger G. Miyoshi K. Tang W. Li C. Deng C.X. Robinson G.W. Hennighausen L. Mol. Cell Biol. 2004; 24: 8037-8047Crossref PubMed Scopus (398) Google Scholar). Indeed, erythroblasts, myeloid cells, mast cells, peripheral T cells, NK cells, and B cells display impaired proliferation and/or survival in mice lacking expression of STAT5 proteins (8Shelburne C.P. McCoy M.E. Piekorz R. Sexl V. Roh K.H. Jacobs-Helber S.M. Gillespie S.R. Bailey D.P. Mirmonsef P. Mann M.N. Kashyap M. Wright H.V. Chong H.J. Bouton L.A. Barnstein B. Ramirez C.D. Bunting K.D. Sawyer S. Lantz C.S. Ryan J.J. Blood. 2003; 102: 1290-1297Crossref PubMed Scopus (113) Google Scholar, 9Socolovsky M. Fallon A.E. Wang S. Brugnara C. Lodish H.F. Cell. 1999; 98: 181-191Abstract Full Text Full Text PDF PubMed Scopus (613) Google Scholar, 10Yao Z. Cui Y. Watford W.T. Bream J.H. Yamaoka K. Hissong B.D. Li D. Durum S.K. Jiang Q. Bhandoola A. Hennighausen L. O'Shea J.J. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 1000-1005Crossref PubMed Scopus (286) Google Scholar, 11Kieslinger M. Woldman I. Moriggl R. Hofmann J. Marine J.C. Ihle J.N. Beug H. Decker T. Genes Dev. 2000; 14: 232-244PubMed Google Scholar). STAT5 promotes cell survival and/or proliferation by regulating the expression of genes involved in the control of cell cycle and survival like bcl-xL, cyclins D1 and D2, p21waf1, and the proto-oncogene pim-1 (12Dumon S. Santos S.C. Debierre-Grockiego F. Gouilleux-Gruart V. Cocault L. Boucheron C. Mollat P. Gisselbrecht S. Gouilleux F. Oncogene. 1999; 18: 4191-4199Crossref PubMed Scopus (134) Google Scholar, 13Moriggl R. Topham D.J. Teglund S. Sexl V. McKay C. Wang D. Hoffmeyer A. van Deursen J. Sangster M.Y. Bunting K.D. Grosveld G.C. Ihle J.N. Immunity. 1999; 10: 249-259Abstract Full Text Full Text PDF PubMed Scopus (475) Google Scholar, 14Nosaka T. Kawashima T. Misawa K. Ikuta K. Mui A.L. Kitamura T. EMBO J. 1999; 18: 4754-4765Crossref PubMed Scopus (434) Google Scholar). Besides the physiological role of STAT5 in hematopoietic cell development, there is increasing evidence suggesting that inappropriate activation of STAT5 may contribute to the development of leukemias and solid cancers (15Benekli M. Baer M.R. Baumann H. Wetzler M. Blood. 2003; 101: 2940-2954Crossref PubMed Scopus (283) Google Scholar, 16Tan S.H. Nevalainen M.T. Endocr. Relat. Cancer. 2008; 15: 367-390Crossref PubMed Scopus (93) Google Scholar). STAT5 is frequently hyperactivated in cancer and leukemias, most probably by alterations of tyrosine kinase activities. Importantly, STAT5 is a common and crucial target for different oncoproteins with tyrosine kinase activity, like Tel-Jak2, Bcr-Abl, the mutated forms of Flt3 and c-Kit, and the Jak2V617F mutant (17Lacronique V. Boureux A. Monni R. Dumon S. Mauchauffé M. Mayeux P. Gouilleux F. Berger R. Gisselbrecht S. Ghysdael J. Bernard O.A. Blood. 2000; 95: 2076-2083Crossref PubMed Google Scholar, 18Nieborowska-Skorska M. Wasik M.A. Slupianek A. Salomoni P. Kitamura T. Calabretta B. Skorski T. J. Exp. Med. 1999; 189: 1229-1242Crossref PubMed Scopus (241) Google Scholar, 19Mizuki M. Fenski R. Halfter H. Matsumura I. Schmidt R. Müller C. Grüning W. Kratz-Albers K. Serve S. Steur C. Büchner T. Kienast J. Kanakura Y. Berdel W.E. Serve H. Blood. 2000; 96: 3907-3914Crossref PubMed Google Scholar, 20Harir N. Boudot C. Friedbichler K. Sonneck K. Kondo R. Martin-Lannerée S. Kenner L. Kerenyi M. Yahiaoui S. Gouilleux-Gruart V. Gondry J. Bénit L. Dusanter-Fourt I. Lassoued K. Valent P. Moriggl R. Gouilleux F. Blood. 2008; 112: 2463-2473Crossref PubMed Scopus (87) Google Scholar, 21Levine R.L. Wadleigh M. Cools J. Ebert B.L. Wernig G. Huntly B.J. Boggon T.J. Wlodarska I. Clark J.J. Moore S. Adelsperger J. Koo S. Lee J.C. Gabriel S. Mercher T. D'Andrea A. Fröhling S. Döhner K. Marynen P. Vandenberghe P. Mesa R.A. Tefferi A. Griffin J.D. Eck M.J. Sellers W.R. Meyerson M. Golub T.R. Lee S.J. Gilliland D.G. Cancer Cell. 2005; 7: 387-397Abstract Full Text Full Text PDF PubMed Scopus (2425) Google Scholar). Furthermore, it has been shown that STAT5 plays a critical role in Bcr-Abl- and Tel-Jak2-induced myeloproliferative disease (22Hoelbl A. Kovacic B. Kerenyi M.A. Simma O. Warsch W. Cui Y. Beug H. Hennighausen L. Moriggl R. Sexl V. Blood. 2006; 107: 4898-4906Crossref PubMed Scopus (172) Google Scholar, 23Schwaller J. Parganas E. Wang D. Cain D. Aster J.C. Williams I.R. Lee C.K. Gerthner R. Kitamura T. Frantsve J. Anastasiadou E. Loh M.L. Levy D.E. Ihle J.N. Gilliland D.G. Mol. Cell. 2000; 6: 693-704Abstract Full Text Full Text PDF PubMed Scopus (269) Google Scholar). The most direct evidence that constitutive activation of STAT5 is an important causative event in cell transformation came from the analysis of the STAT5 mutants, STAT5a1*6 and STAT5b1*6, and cS5F. These proteins with mutations at residues His299 → Arg and Ser711/716 → Phe (STAT5a1*6 or STAT5b1*6) or with the single mutation Ser711 → Phe (cS5F) possess constitutive tyrosine phosphorylation and are capable of inducing leukemias in mice (23Schwaller J. Parganas E. Wang D. Cain D. Aster J.C. Williams I.R. Lee C.K. Gerthner R. Kitamura T. Frantsve J. Anastasiadou E. Loh M.L. Levy D.E. Ihle J.N. Gilliland D.G. Mol. Cell. 2000; 6: 693-704Abstract Full Text Full Text PDF PubMed Scopus (269) Google Scholar, 24Moriggl R. Sexl V. Kenner L. Duntsch C. Stangl K. Gingras S. Hoffmeyer A. Bauer A. Piekorz R. Wang D. Bunting K.D. Wagner E.F. Sonneck K. Valent P. Ihle J.N. Beug H. Cancer Cell. 2005; 7: 87-99Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar). In addition, STAT5b plays an important role in the proliferation and/or survival of tumor cells from head and neck cancer, glioblastomas, and prostate cancer (16Tan S.H. Nevalainen M.T. Endocr. Relat. Cancer. 2008; 15: 367-390Crossref PubMed Scopus (93) Google Scholar, 25Xi S. Zhang Q. Gooding W.E. Smithgall T.E. Grandis J.R. Cancer Res. 2003; 63: 6763-6771PubMed Google Scholar, 26Liang Q.C. Xiong H. Zhao Z.W. Jia D. Li W.X. Qin H.Z. Deng J.P. Gao L. Zhang H. Gao G.D. Cancer Lett. 2009; 273: 164-171Crossref PubMed Scopus (67) Google Scholar, 27Gu L. Dagvadorj A. Lutz J. Leiby B. Bonuccelli G. Lisanti M.P. Addya S. Fortina P. Dasgupta A. Hyslop T. Bubendorf L. Nevalainen M.T. Am. J. Pathol. 2010; 176: 1959-1972Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). STAT5b acts downstream of epidermal growth factor receptor, which is frequently overexpressed or hyperactivated in these tumors (28Kloth M.T. Laughlin K.K. Biscardi J.S. Boerner J.L. Parsons S.J. Silva C.M. J. Biol. Chem. 2003; 278: 1671-1679Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). Furthermore, STAT5b is specifically activated in T-cell lymphomas transformed by the oncogenic fusion NPM1-ALK and contributes to the NPM1-ALK oncogenesis by promoting cell growth and survival, whereas STAT5a acts as a tumor suppressor in these malignant cells (29Zhang Q. Wang H.Y. Liu X. Wasik M.A. Nat. Med. 2007; 13: 1341-1348Crossref PubMed Scopus (100) Google Scholar). This suggests that STAT5a and STAT5b may have some non overlapping and opposite functions in the transformation of similar target cells.Like other STAT family members, STAT5a and STAT5b proteins contain in their carboxyl-terminal part a transactivation domain that is required for transcriptional activation (30Moriggl R. Gouilleux-Gruart V. Jähne R. Berchtold S. Gartmann C. Liu X. Hennighausen L. Sotiropoulos A. Groner B. Gouilleux F. Mol. Cell Biol. 1996; 16: 5691-5700Crossref PubMed Scopus (246) Google Scholar). In some early hematopoietic progenitors and in peripheral T cells, cleavage of full-length STAT5 proteins by proteases generates carboxyl-terminally truncated STAT5 proteins called STAT5γ that lack the transactivation domain and function as dominant negative proteins (3Buitenhuis M. Coffer P.J. Koenderman L. Int. J. Biochem. Cell Biol. 2004; 36: 2120-2124Crossref PubMed Scopus (66) Google Scholar). Mutagenesis analyses have shown that a small amphipathic α-helical region within this domain is required not only for transcriptional activation of STAT5 proteins but also for the rapid proteasome-dependent turnover of the molecules (31Wang D. Moriggl R. Stravopodis D. Carpino N. Marine J.C. Teglund S. Feng J. Ihle J.N. EMBO J. 2000; 19: 392-399Crossref PubMed Google Scholar). This region is also involved in the recruitment of the cofactors CBP/P300 and NCoA1/SRC-1 (32Litterst C.M. Kliem S. Marilley D. Pfitzner E. J. Biol. Chem. 2003; 278: 45340-45351Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 33Pfitzner E. Jähne R. Wissler M. Stoecklin E. Groner B. Mol. Endocrinol. 1998; 12: 1582-1593Crossref PubMed Google Scholar). Thus, transcriptional activation and down-regulation of STAT5 proteins are mediated via a similar region located in the transactivation domain. STAT5a and STAT5b share 96% homology at the amino acid level and differ mainly in the carboxyl-terminal region. Importantly, a serine residue at position 779 that is phosphorylated in STAT5a is absent at a similar position in STAT5b (34Beuvink I. Hess D. Flotow H. Hofsteenge J. Groner B. Hynes N.E. J. Biol. Chem. 2000; 275: 10247-10255Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). There is evidence that STAT5b is phosphorylated on tyrosine residues in the carboxyl terminus distinct from the residue Tyr699, which is necessary for STAT5b dimerization and activation (35Kloth M.T. Catling A.D. Silva C.M. J. Biol. Chem. 2002; 277: 8693-8701Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). Such phosphorylations may eventually affect STAT5b intracellular trafficking or interaction with cellular proteins (36Kazansky A.V. Rosen J.M. Cell Growth Differ. 2001; 12: 1-7PubMed Google Scholar). The carboxyl-terminal regions of STAT5a and STAT5b may therefore confer distinct functions to these two molecules that might be related in part to interactions with distinct molecular partners. In this work, we aimed to identify new STAT5 transactivation domain-interacting proteins that could differentially regulate STAT5a or STAT5b activity. For this purpose, we used a bacterial two-hybrid screening approach. We have identified hTid1, a human DnaJ protein, homolog of the Drosophila tumor suppressor Tid56, as a novel and specific negative regulator of STAT5b activity and expression in hematopoietic cell lines.
Referência(s)