Constitutively active STAT5A and STAT5B in vitro and in vivo: mutation of STAT5 is not a frequent cause of leukemogenesis.
Koji YamadaKoya AriyoshiMisa OnishiAtsushi MiyajimaFumihiko HayakawaMasayuki TowatariHaruki SaitoY. OkaShigetaka AsanoTetsuya NosakaToshio Kitamura
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Abstract STAT5 is an essential transcription factor in hematopoiesis, which is activated through tyrosine phosphorylation in response to cytokine stimulation. Constitutive activation of STAT5 is a hallmark of myeloid and lymphoblastic leukemia. Using homology modeling and molecular dynamics simulations, a model of the STAT5 phosphotyrosine-SH2 domain interface was generated providing first structural information on the activated STAT5 dimer including a sequence, for which no structural information is available for any of the STAT proteins. We identified a novel intramolecular interaction mediated through F706, adjacent to the phosphotyrosine motif, and a unique hydrophobic interface on the surface of the SH2 domain. Analysis of corresponding STAT5 mutants revealed that this interaction is dispensable for Epo receptor-mediated phosphorylation of STAT5 but essential for dimer formation and subsequent nuclear accumulation. Moreover, the herein presented model clarifies molecular mechanisms of recently discovered leukemic STAT5 mutants and will help to guide future drug development.
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Abstract Cytokine-induced tyrosine phosphorylation of the transcription factor STAT5 is required for its transcriptional activity. In this article we show that the small dual-specificity phosphatase VHR selectively dephosphorylates IFN-α- and β-activated, tyrosine-phosphorylated STAT5, leading to the subsequent inhibition of STAT5 function. Phosphorylation of VHR at Tyr138 was required for its phosphatase activity toward STAT5. In addition, the Src homology 2 domain of STAT5 was required for the effective dephosphorylation of STAT5 by VHR. The tyrosine kinase Tyk2, which mediates the phosphorylation of STAT5, was also responsible for the phosphorylation of VHR at Tyr138.
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By searching a database of expressed sequences, we identified a member of the signal transducers and activators of transcription (Stat) family of proteins. Human and murine full-length cDNA clones were obtained and sequenced. The sequence of the human cDNA was identical to the recently published sequence for interleukin-4 (IL-4)-Stat (J. Hou, U. Schindler, W.J. Henzel, T.C. Ho, M. Brasseur, and S. L. McKnight, Science 265:1701-1706, 1994), while the murine Stat6 amino acid and nucleotide sequences were 83 and 84% identical to the human sequences, respectively. Using Stat6-specific antiserum, we demonstrated that Stat6 is rapidly tyrosine phosphorylated following stimulation of appropriate cell lines with IL-4 or IL-3 but is not detectably phosphorylated following stimulation with IL-2, IL-12, or erythropoietin. In contrast, IL-2, IL-3, and erythropoietin induce the tyrosine phosphorylation of Stat5 while IL-12 uniquely induces the tyrosine phosphorylation of Stat4. Inducible tyrosine phosphorylation of Stat6 requires the membrane-distal region of the IL-4 receptor alpha chain. This region of the receptor is not required for cell growth, demonstrating that Stat6 tyrosine phosphorylation does not contribute to mitogenesis.
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Stat5 is activated by a broad spectrum of cytokines, as well as non-receptor tyrosine kinases, such as Src. In this study, the DNA binding properties of the two closely related Stat5 proteins, Stat5a and Stat5b, induced either by prolactin (Prl) or by Src were analyzed by electrophoretic mobility shift assays using several different Stat5 binding sites. Src-induced Stat5b-DNA binding complexes consistently displayed a slightly faster mobility than those induced by Prl, as well as differences in their ability to be supershifted by anti-Stat5 antibodies. IP-Westerns performed using specific antibodies directed at the N and C termini of Stat5b suggested that depending on the activating stimulus, Stat5b exhibited different conformations, which influenced antibody accessibility at its C terminus. These conformational differences may in part be due to differential effects of Prl and Src on Stat5b tyrosine phosphorylation, since Src induced several additional sites of tyrosine phosphorylation of Stat5b at residues other than Tyr-699, including Tyr-724 and Tyr-679. The latter Tyr-679 is conserved in all mammalian Stat5bs, but is not present in Stat5a. A Stat 5bY679F mutant induced by Src kinase exhibited an altered pattern of nuclear localization as compared with wild-type Stat5b. Furthermore, this mutation inhibited v-Src-induced cyclin D1-luciferase reporter activity in transient transfection assays performed in Stat5a/b-deficient MEFs, suggesting that Tyr-679 phosphorylation may play a role in v-Src induced proliferation. Thus, depending on the signal transduction pathway responsible for activation, different conformations of activated Stat5 may result in selective biological responses. Stat5 is activated by a broad spectrum of cytokines, as well as non-receptor tyrosine kinases, such as Src. In this study, the DNA binding properties of the two closely related Stat5 proteins, Stat5a and Stat5b, induced either by prolactin (Prl) or by Src were analyzed by electrophoretic mobility shift assays using several different Stat5 binding sites. Src-induced Stat5b-DNA binding complexes consistently displayed a slightly faster mobility than those induced by Prl, as well as differences in their ability to be supershifted by anti-Stat5 antibodies. IP-Westerns performed using specific antibodies directed at the N and C termini of Stat5b suggested that depending on the activating stimulus, Stat5b exhibited different conformations, which influenced antibody accessibility at its C terminus. These conformational differences may in part be due to differential effects of Prl and Src on Stat5b tyrosine phosphorylation, since Src induced several additional sites of tyrosine phosphorylation of Stat5b at residues other than Tyr-699, including Tyr-724 and Tyr-679. The latter Tyr-679 is conserved in all mammalian Stat5bs, but is not present in Stat5a. A Stat 5bY679F mutant induced by Src kinase exhibited an altered pattern of nuclear localization as compared with wild-type Stat5b. Furthermore, this mutation inhibited v-Src-induced cyclin D1-luciferase reporter activity in transient transfection assays performed in Stat5a/b-deficient MEFs, suggesting that Tyr-679 phosphorylation may play a role in v-Src induced proliferation. Thus, depending on the signal transduction pathway responsible for activation, different conformations of activated Stat5 may result in selective biological responses. signal transducers and activators of transcription IFN-γ-activated sequence interleukin epidermal growth factor prolactin electrophoretic mobility shift assay Src homology domain Janus kinase immunoprecipitation Signal transducers andactivators of transcription (STAT1 proteins) form a family of seven latent cytoplasmic transcription factors, which following activation in response to many different cytokines, dimerize and translocate into the nucleus to activate gene transcription (1Darnell Jr., J.E. Kerr I.M. Stark G.R. Science. 1994; 264: 1415-1421Crossref PubMed Scopus (5028) Google Scholar). Stats can be activated by a broad spectrum of cytokines interacting with specific cytokine receptors, leading to the activation of members of the Janus tyrosine kinase (JAK) family. The activated JAKs phosphorylate the recruited monomeric Stats on a specific tyrosine and induce their dimerization. The activated dimers then translocate into the nucleus, where they bind to specific DNA-response elements in the promoters of target genes. This linear signal transduction pathway is clearly an oversimplification and can be modulated by cross-talk with other signal transduction pathways (e.g. by steroid receptors, etc.) (2Stocklin E. Wissler M. Gouilleux F. Groner B. Nature. 1996; 383: 726-728Crossref PubMed Scopus (575) Google Scholar). Most Stats bind to similar GAS (IFN-γ-activated sequence) motifs (TTCNnGAA) with slightly different affinities for optimal binding; n = 4 for Stat6 and n = 3 for all other Stats (3Horvath C.M. Wen Z. Darnell Jr., J.E. Genes Dev. 1995; 9: 984-994Crossref PubMed Scopus (452) Google Scholar, 4Seidel H.M. Milocco L.H. Lamb P. Darnell Jr., J.E. Stein R.B. Rosen J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3041-3045Crossref PubMed Scopus (381) Google Scholar, 5Xu X. Sun Y.L. Hoey T. Science. 1996; 273: 794-797Crossref PubMed Scopus (407) Google Scholar). Non-receptor tyrosine kinases such as Src or Abl can also directly phosphorylate Stat proteins without involving JAKs (6Ilaria Jr., R.L. Van Etten R.A. J. Biol. Chem. 1996; 271: 31704-31710Abstract Full Text Full Text PDF PubMed Scopus (435) Google Scholar, 7Frank D.A. Varticovski L. Leukemia. 1996; 10: 1724-1730PubMed Google Scholar, 8Olayioye M.A. Beuvink I. Horsch K. Daly J.M. Hynes N.E. J. Biol. Chem. 1999; 274: 17209-17218Abstract Full Text Full Text PDF PubMed Scopus (312) Google Scholar, 9Kazansky A.V. Kabotyanski E.B. Wyszomierski S.L. Mancini M.A. Rosen J.M. J. Biol. Chem. 1999; 274: 22484-22492Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). In addition to tyrosine phosphorylation, Stat proteins can be phosphorylated on serine residues (10Beuvink 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, 11Yamashita H. Xu J. Erwin R.A. Farrar W.L. Kirken R.A. Rui H. J. Biol. Chem. 1998; 273: 30218-30224Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar, 12Wen Z. Darnell Jr., J.E. Nucleic Acids Res. 1997; 25: 2062-2067Crossref PubMed Scopus (267) Google Scholar, 13Wen Z. Zhong Z. Darnell Jr., J.E. Cell. 1995; 82: 241-250Abstract Full Text PDF PubMed Scopus (1744) Google Scholar, 14Kirken R.A. Malabarba M.G. Xu J. Liu X. Farrar W.L. Hennighausen L. Larner A.C. Grimley P.M. Rui H. J. Biol. Chem. 1997; 272: 14098-14103Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). While regulation of Stats by serine phosphorylation is poorly understood, it has been shown that this phosphorylation can modulate both their transcriptional activity and DNA binding (13Wen Z. Zhong Z. Darnell Jr., J.E. Cell. 1995; 82: 241-250Abstract Full Text PDF PubMed Scopus (1744) Google Scholar, 15Beadling C. Ng J. Babbage J.W. Cantrell D.A. EMBO J. 1996; 15: 1902-1913Crossref PubMed Scopus (161) Google Scholar). The DNA binding region of Stat proteins is located between amino acids 350 and 500, and the activation domain is located in the C-terminal region (16Moriggl R. Gouilleux-Gruart V. Jahne R. Berchtold S. Gartmann C. Liu X. Hennighausen L. Sotiropoulos A. Groner B. Gouilleux F. Mol. Cell. Biol. 1996; 16: 5691-5700Crossref PubMed Scopus (247) Google Scholar, 17Schaefer T.S. Sanders L.K. Park O.K. Nathans D. Mol. Cell. Biol. 1997; 17: 5307-5316Crossref PubMed Google Scholar, 18Vinkemeier U. Moarefi I. Darnell Jr., J.E. Kuriyan J. Science. 1998; 279: 1048-1052Crossref PubMed Scopus (213) Google Scholar). C-terminal-truncated Stat proteins still retain DNA binding activity, but may in some cases suppress transcriptional activation of responsive genes in a dominant-negative manner (19Muller M. Laxton C. Briscoe J. Schindler C. Improta T. Darnell Jr., J.E. Stark G.R. Kerr I.M. EMBO J. 1993; 12: 4221-4228Crossref PubMed Scopus (373) Google Scholar, 20Caldenhoven E. van Dijk T.B. Solari R. Armstrong J. Raaijmakers J.A.M. Lammers J.W.J. Koenderman L. de Groot R.P. J. Biol. Chem. 1996; 271: 13221-13227Abstract Full Text Full Text PDF PubMed Scopus (322) Google Scholar, 21Wang D. Stravopodis D. Teglund S. Kitazawa J. Ihle J.N. Mol. Cell. Biol. 1996; 16: 6141-6148Crossref PubMed Scopus (225) Google Scholar). There is some evidence that these naturally occurring dominant-negative Stat5 isoforms may be functionally correlated with biological responses (22Bovolenta C. Testolin L. Benussi L. Lievens P.M. Liboi E. J. Biol. Chem. 1998; 273: 20779-20784Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar), but the potential physiological significance of dominant-negative Stat isoforms is still presently unknown. The N-terminal region of Stats is required for cooperative binding of Stat dimers to form a tetrameric structure (23John S. Vinkemeier U. Soldaini E. Darnell Jr., J.E. Leonard W.J. Mol. Cell. Biol. 1999; 19: 1910-1918Crossref PubMed Scopus (165) Google Scholar, 24Meyer W.K. Reichenbach P. Schindler U. Soldaini E. Nabholz M. J. Biol. Chem. 1997; 272: 31821-31828Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). For example, Stat5 forms tetramers on the IL-2 response element in the human IL-2Ra promoter (23John S. Vinkemeier U. Soldaini E. Darnell Jr., J.E. Leonard W.J. Mol. Cell. Biol. 1999; 19: 1910-1918Crossref PubMed Scopus (165) Google Scholar, 24Meyer W.K. Reichenbach P. Schindler U. Soldaini E. Nabholz M. J. Biol. Chem. 1997; 272: 31821-31828Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 25Lecine P. Algarte M. Rameil P. Beadling C. Bucher P. Nabholz M. Imbert J. Mol. Cell. Biol. 1996; 16: 6829-6840Crossref PubMed Google Scholar), and the CIS gene (26Verdier F. Rabionet R. Gouilleux F. Beisenherz-Huss C. Varlet P. Muller O. Mayeux P. Lacombe C. Gisselbrecht S. Chretien S. Mol. Cell. Biol. 1998; 18: 5852-5860Crossref PubMed Scopus (131) Google Scholar), both of which contain two adjacent Stat binding sites. Stat5 plays a key role in mammary gland differentiation, as well as in the prolactin (Prl)- induced expression of milk protein genes, such as औ-casein (27Gouilleux F. Wakao H. Mundt M. Groner B. EMBO J. 1994; 13: 4361-4369Crossref PubMed Scopus (527) Google Scholar, 28Wakao H. Gouilleux F. Groner B. EMBO J. 1994; 13: 2182-2191Crossref PubMed Scopus (715) Google Scholar), αs1-casein (29Pierre S. Jolivet G. Devinoy E. Houdebine L.M. Mol. Endocrinol. 1994; 8: 1720-1730PubMed Google Scholar), औ-lactoglobulin (30Burdon T.G. Maitland K.A. Clark A.J. Wallace R. Watson C.J. Mol. Endocrinol. 1994; 8: 1528-1536PubMed Google Scholar), and whey acidic protein (31Li S. Rosen J.M. Mol. Cell. Biol. 1995; 15: 2063-2070Crossref PubMed Scopus (158) Google Scholar). However, the expression of Stat5 is not restricted to the mammary gland. Both isoforms of Stat5 (Stat5a and Stat5b, which share 937 identity at the amino acid level) can be activated by many cytokines in addition to Prl, such as IL-2 (32Gouilleux F. Moritz D. Humar M. Moriggl R. Berchtold S. Groner B. Endocrinology. 1995; 136: 5700-5708Crossref PubMed Scopus (67) Google Scholar, 33Lin J.X. Migone T.S. Tsang M. Friedmann M. Weatherbee J.A. Zhou L. Yamauchi A. Bloom E.T. Mietz J. John S. Immunity. 1995; 2: 331-339Abstract Full Text PDF PubMed Scopus (677) Google Scholar), IL-3 (34Azam M. Erdjument-Bromage H. Kreider B.L. Xia M. Quelle F. Basu R. Saris C. Tempst P. Ihle J.N. Schindler C. EMBO J. 1995; 14: 1402-1411Crossref PubMed Scopus (300) Google Scholar, 35Mui A.L. Wakao H. O'Farrell A.M. Harada N. Miyajima A. EMBO J. 1995; 14: 1166-1175Crossref PubMed Scopus (539) Google Scholar), IL-5 (35Mui A.L. Wakao H. O'Farrell A.M. Harada N. Miyajima A. EMBO J. 1995; 14: 1166-1175Crossref PubMed Scopus (539) Google Scholar), IL-7, IL-15 (33Lin J.X. Migone T.S. Tsang M. Friedmann M. Weatherbee J.A. Zhou L. Yamauchi A. Bloom E.T. Mietz J. John S. Immunity. 1995; 2: 331-339Abstract Full Text PDF PubMed Scopus (677) Google Scholar), thrombopoietin (36Pallard C. Gouilleux F. Benit L. Cocault L. Souyri M. Levy D. Groner B. Gisselbrecht S. Dusanter-Fourt I. EMBO J. 1995; 14: 2847-2856Crossref PubMed Scopus (200) Google Scholar), erythropoietin, GM-CSF (35Mui A.L. Wakao H. O'Farrell A.M. Harada N. Miyajima A. EMBO J. 1995; 14: 1166-1175Crossref PubMed Scopus (539) Google Scholar, 37Gouilleux F. Pallard C. Dusanter-Fourt I. Wakao H. Haldosen L.A. Norstedt G. Levy D. Groner B. EMBO J. 1995; 14: 2005-2013Crossref PubMed Scopus (333) Google Scholar), EGF (38Ruff-Jamison S. Chen K. Cohen S. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4215-4218Crossref PubMed Scopus (134) Google Scholar), and platelet-derived growth factor (39Paukku K. Valgeirsdottir S. Saharinen P. Bergman M. Heldin C.H. Silvennoinen O. Biochem. J. 2000; 345 (Pt 3): 759-766Crossref PubMed Scopus (38) Google Scholar) in various cells and tissues, as well as by non-receptor tyrosine kinases such as Src and Bcr-Abl (6Ilaria Jr., R.L. Van Etten R.A. J. Biol. Chem. 1996; 271: 31704-31710Abstract Full Text Full Text PDF PubMed Scopus (435) Google Scholar, 7Frank D.A. Varticovski L. Leukemia. 1996; 10: 1724-1730PubMed Google Scholar). Thus, Stat5 may play a variety of regulatory roles that control different functions including cell growth, survival, and differentiation. Recent studies have demonstrated for example, that activated Stat5 may regulate cyclin D1 promoter activity resulting in cell cycle progression (40Matsumura I. Kitamura T. Wakao H. Tanaka H. Hashimoto K. Albanese C. Downward J. Pestell R.G. Kanakura Y. EMBO J. 1999; 18: 1367-1377Crossref PubMed Scopus (293) Google Scholar, 41Wen X. Lin H.H. Shih H.M. Kung H.J. Ann D.K. J. Biol. Chem. 1999; 274: 38204-38210Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 42Schroeder M.D. Symowicz J. Schuler L.A. Mol. Endocrinol. 2002; 16: 45-57Crossref PubMed Scopus (83) Google Scholar). Targeted disruption of Stat5a or Stat5b genes in mice results in a distinctive tissue-specific phenotype.Stat5a knockout mice display, for example, defects in mammary gland development and lactation during pregnancy (43Liu X. Robinson G.W. Wagner K.U. Garrett L. Wynshaw-Boris A. Hennighausen L. Genes Dev. 1997; 11: 179-186Crossref PubMed Scopus (920) Google Scholar), whileStat5b knockout mice display sexually dimorphic liver gene expression (44Udy 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 (832) Google Scholar). There is also evidence suggesting that closely related Stat5a and Stat5b may be differently activated (45Meinke A. Barahmand-Pour F. Wohrl S. Stoiber D. Decker T. Mol. Cell. Biol. 1996; 16: 6937-6944Crossref PubMed Scopus (155) Google Scholar) and exhibit distinct biochemical differences (26Verdier F. Rabionet R. Gouilleux F. Beisenherz-Huss C. Varlet P. Muller O. Mayeux P. Lacombe C. Gisselbrecht S. Chretien S. Mol. Cell. Biol. 1998; 18: 5852-5860Crossref PubMed Scopus (131) Google Scholar) and distinct DNA binding specificities (46Boucheron C. Dumon S. Santos S.C. Moriggl R. Hennighausen L. Gisselbrecht S. Gouilleux F. J. Biol. Chem. 1998; 273: 33936-33941Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). However, the latter studies are controversial, because they were based on an incorrectly identified single amino acid difference between murine Stat5a and 5b. Instead, recent studies on the DNA binding properties of Stat5a and Stat5b suggest that baculovirus-expressed Stat5a and Stat5b homodimers recognize a similar optimal consensus sequence (47Ehret G.B. Reichenbach P. Schindler U. Horvath C.M. Fritz S Nabholz M. Bucher P. J. Biol. Chem. 2001; 276: 6675-6688Abstract Full Text Full Text PDF PubMed Scopus (311) Google Scholar). Significant differences, however, have been observed in the ability of Stat5a and Stat5b to form tetrameric complexes on adjacent consensus and nonconsensus GAS sites, thus in part explaining their ability to activate nonidentical sets of genes (48Soldaini E. John S. Moro S. Bollenbacher J. Schindler U. Leonard W. Mol. Cell. Biol. 2000; 20: 389-401Crossref PubMed Scopus (154) Google Scholar). Thus, the arrangement of weak affinity binding sites in pairs on target genes may help provide selectivity in response to Stat5a tetrameric complexes. Selectivity might also reflect the cooperation with other transcription factors that interact with Stat5, and their own adjacent weak affinity binding sites perhaps helping to stabilize coactivator complexes (49Wyszomierski S.L. Rosen J.M. Mol. Endocrinol. 2001; 15: 228-240Crossref PubMed Scopus (88) Google Scholar). Previous studies from our laboratory have suggested that Prl and Src signaling pathways may differentially regulate Stat5a and Stat5b nuclear translocation and retention (9Kazansky A.V. Kabotyanski E.B. Wyszomierski S.L. Mancini M.A. Rosen J.M. J. Biol. Chem. 1999; 274: 22484-22492Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). Thus, we postulated that the regulation of specific gene targets involved in proliferation, differentiation, or apoptosis by these two highly related isoforms of Stat5 might also reflect their activation by different signal transduction pathways. In this study, the DNA binding properties of Stat5a and Stat5b induced by Prl and Src to a variety of GAS-like sequences, such as औ-casein, APRE (α2-macroglobulin), APRE-2 (double APRE site), p21 (SIE2), and p21 (SIE3) were analyzed. As expected, Stat5a activated by Prl bound to the APRE-2 (double GAS site) as a tetramer, while Stat5b activated identically bound as a dimer. However, surprisingly, differences in the mobility of DNA binding complexes of Stat5b induced by Src versus Prl were observed. Based on immunoprecipitation studies and antibody supershift experiments, we propose that Stat5b induced by Src versusPrl may exhibit a different conformation at its C terminus, possibly due to additional tyrosine phosphorylation. In contrast to Prl, which induced only Tyr-699 phosphorylation of Stat5b, Src induced phosphorylation on Tyr-699 as well as on additional tyrosines. To identify the potential Src-induced tyrosine phosphorylation sites in the C-terminal region of Stat5b in addition to Tyr-699, a series of tyrosine to phenylalanine mutants were analyzed both individually and sequentially by IP-Western blots. Based on this analysis, Tyr-724 (conserved in both Stat5a and Stat5b) and Tyr-679 (a unique site present only in Stat5b, but absent in Stat5a) were shown to contribute to the Src-induced tyrosine phosphorylation of Stat5b. Furthermore, mutation of Tyr-679 to Phe in Stat5b altered its pattern of nuclear localization upon activation by Src kinase, and decreased v-Src-induced cyclin D1 promoter activity, suggesting that phosphorylation of this tyrosine may influence protein-protein interactions most likely of the Stat5b C-terminal transactivation domain and possibly may influence cell cycle progression. Thus, depending on the signal transduction pathway responsible for activation, different structural conformations of activated Stat5b may result in selective biological responses. The expression vectors for rat-Stat5a, rat-Stat5b, a C-terminal-truncated isoform, Stat5bΔ40, and for the long form of the PrlR have been described previously (50Kazansky A.V. Raught B. Lindsey S.M. Wang Y.F. Rosen J.M. Mol. Endocrinol. 1995; 9: 1598-1609Crossref PubMed Google Scholar, 51Ripperger J.A. Fritz S. Richter K. Hocke G.M. Lottspeich F. Fey G.H. J. Biol. Chem. 1995; 270: 29998-30006Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar, 52Luo G. Yu-Lee L. J. Biol. Chem. 1997; 272: 26841-26849Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar, 53Ali S. Edery M. Pellegrini I. Lesueur L. Paly J. Djiane J. Kelly P.A. Mol. Endocrinol. 1992; 6: 1242-1248Crossref PubMed Scopus (0) Google Scholar). c-Src kinase-active construct (54Courtneidge S.A. Fumagalli S. Koegl M. Superti-Furga G. Twamley-Stein G.M. Development. 1993; : 57-64PubMed Google Scholar) was kindly provided by Dr. Sara Courtneidge at Sugen Corporation (South San Francisco, CA). v-Src cDNA was kindly provided by Dr. Hiromitsu Hanafusa (The Rockefeller University, New York, NY) and cloned into pEFIRES vector in our laboratory by Dr. Alexander Kazansky. The −674CD1LUC plasmid containing the cyclin D1 promoter-driven luciferase reporter, as well as several other cyclin D1 constructs were kindly provided by Dr. Richard G. Pestell (Lombardi Cancer Center, Georgetown University, Washington, D. C.). Stat5aW37A and Stat5bW37A mutants were kindly provided by Dr. Warren J. Leonard (National Institutes of Health, Bethesda, MD). Stat5bS730A was provided by Dr. Robert Kirken (University of Texas Medical School, Houston, TX). Stat5b mutants were prepared using the QuickChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) as described by the manufacturer with oligonucleotide primers designed to alter tyrosine residues (TAC or TAT) to phenylalanines (TTC or TTT). The following mutants of rat Stat5b were generated by this method: Y699F, Y699F/Y742F, Y699F/Y739F, Y699F/Y724F, Y699F/Y683-82F, Y699F/Y679F, Y699F/Y668F, Y699F/Y742F/Y739F, Y699F/Y742F/Y739F/Y724F, Y699F/Y742F/Y739F/Y724F/Y683-82F, Y699F/Y742F/Y739F/Y724F/Y683-82F/Y679F, Y699F/Y742F/Y739F/Y724F/Y683-82F/Y679F/Y668F. For the dominant-negative C-terminal-truncated isoform Stat5bΔ40, the Y699F mutant was also generated. All mutations were confirmed by direct sequence analysis. HeLa cells were maintained in Opti-MEM media (Invitrogen), supplemented with 37 fetal bovine serum and gentamicin (50 ॖg/ml) in a 37 °C and 57 CO2 incubator. DNA constructs (10 ॖg) were transiently transfected into HeLa cells by using a calcium phosphate precipitation technique (5Prime-3Prime, Boulder, CO). For the co-transfection experiments, 4 ॖg of Stat5a or Stat5b expression vector and 6 ॖg of PrlR or Src kinase construct were utilized. Before Prl or Src induction, the cells were incubated overnight in medium containing 107 charcoal-stripped horse serum, insulin (5 ॖg/ml), gentamicin (50 ॖg/ml), and hydrocortisone (1 ॖg/ml) and then (for the Prl experiments only) stimulated with ovine Prl for 30 min (1 ॖg/ml, lot AFP-10677C, kindly provided by the National Hormone and Pituitary Program, NIDDK, National Institutes of Health, Bethesda, MD). Alternatively, for reporter assays, Stat5a/5b-deficient MEFs derived from Stat5a/5b knockout mice (66Teglund 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 (1077) Google Scholar) kindly provided by Dr. James Ihle, were transiently co-transfected with 0.5 ॖg of Stat5b or a Stat5bY679F mutant, 1 ॖg of v-Src and 1 ॖg of a −674CD1-Luc reporter construct. Experiments were performed in 6-well plates, and each transfection was done in triplicate. Lysates were prepared 24 h after transfection, and luciferase activity was measured using aPromega luciferase kit (Madison, WI). Luciferase values were normalized to protein amount by Western blot analysis for Stat 5b. The specific, affinity-purified rabbit polyclonal antiserum generated against peptides from the unique amino acid epitopes present in the C-terminal regions of Stat5a and Stat5b have been characterized previously (50Kazansky A.V. Raught B. Lindsey S.M. Wang Y.F. Rosen J.M. Mol. Endocrinol. 1995; 9: 1598-1609Crossref PubMed Google Scholar, 52Luo G. Yu-Lee L. J. Biol. Chem. 1997; 272: 26841-26849Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). The N-terminal Stat5 (N-20) antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Phosphotyrosine antibody (PY-20) was purchased from Transduction Laboratories (Lexington, KY). v-Src antibody was purchased from Calbiochem (La Jolla, CA). The site-specific antibody to Ser-730-phosphorylated Stat5b was kindly provided by Dr. Robert Kirken (University of Texas Medical School, Houston, TX). Cells were rinsed twice with ice-cold phosphate-buffered saline (9.1 mm dibasic sodium phosphate, 1.7 mm monobasic sodium phosphate, 150 mm sodium chloride, pH 7.4) and scraped into 2 volumes of Wu buffer (51Ripperger J.A. Fritz S. Richter K. Hocke G.M. Lottspeich F. Fey G.H. J. Biol. Chem. 1995; 270: 29998-30006Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar) containing 10 mm HEPES, pH 7.4, 1.5 mm magnesium chloride, 0.1 mm EGTA, 107 glycerol, 1 mm dithiothreitol, 10 ॖg/ml aprotinin, 10 ॖg/ml antipain, 10 ॖg/ml benzamidine, and 5 ॖg/ml leupeptin. After lysis in Wu buffer, cells were centrifuged for 15 min at 4 °C and 14,000 rpm. Supernatant was retained as the cytoplasmic extract. After washing of remaining pellets twice with 5 volumes of Wu buffer, they were resuspended in two volumes of the same buffer with 400 mm of sodium chloride. The samples were incubated on ice for 10 min at 4 °C and then centrifuged for 15 min at 4 °C and 14,000 rpm. The supernatants were dialyzed in Invitrogen microdialysis units for 2 h at 4 °C against 1 liter of Wu buffer with 100 mm sodium chloride and were retained as nuclear extracts. Protein concentrations were determined using a Bio-Rad protein assay. Five oligonucleotides were designed, annealed, and used for the EMSA experiments (Table I). औ-Casein and APRE-2 were end-labeled using the Klenow fragment of DNA polymerase with [α-32P]CTP, while all others were end-labeled by T4 polynucleotide kinase with [γ-32P]ATP. EMSA were performed as described previously (55Raught B. Khursheed B. Kazansky A. Rosen J. Mol. Cell. Biol. 1994; 14: 1752-1763Crossref PubMed Google Scholar). Briefly, nuclear extracts (10 ॖg for each sample) were preincubated in 10 ॖl of Wu-buffer containing 100 mm sodium chloride and 2 ॖg of poly(dI-dC) (Amersham Biosciences) for 15 min on ice. If antibody was used for supershift analysis, it was included in the 10 ॖl of Wu buffer. After preincubation to block nonspecific binding and allow the antibody to bind, 5 ॖl of binding mixture (50 ॖg/ml p(dN)6, 2.5 mg/ml bovine serum albumin, 47 Ficoll 400, and 107 glycerol) with labeled probe (20,000 cpm) was added to each sample and incubated on ice for 15 min. The reaction mixture was loaded onto a 47 polyacrylamide gel in 0.25× Tris borate-EDTA buffer, electrophoresed, dried, and subjected to autoradiography. Immunoprecipitation and Western blot analysis were performed as described previously (9Kazansky A.V. Kabotyanski E.B. Wyszomierski S.L. Mancini M.A. Rosen J.M. J. Biol. Chem. 1999; 274: 22484-22492Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar).Table ISequences of DNA probesStat5a consensus1-aRef. 44.N (A/g) (T/A)TTC(C/T) N (G/a)GAA(A/tc)(T/c) NStat5b consensus1-aRef. 44.N (A/tg)(T/A)TTC(C/T)(T/cag)(G/a)GAA(T/A)(T/ca)Nऔ-Casein1-bRef. 37.5′-AGATTTCTAGGAATTCAATCC-3′APRE1-bRef. 37.5′-GATCCTTCTGGGAATTCCTA-3′1-dBolded residues represent changes compared to the consensus sequences of Stat5a or Stat5b.APRE-25′-TGGATCATCCTTCTGGGAATTCTGATATCCTTCTGGGAATTCTG-3′p21-SIE21-cRef. 67.5′-GATCCTTTCTGAGAAATGG-3′p21-SIE31-cRef. 67.5′-GATCCCTCAGTCTTCTTGGAAATTC-3′1-a Ref. 44Udy 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 (832) Google Scholar.1-b Ref. 37Gouilleux F. Pallard C. Dusanter-Fourt I. Wakao H. Haldosen L.A. Norstedt G. Levy D. Groner B. EMBO J. 1995; 14: 2005-2013Crossref PubMed Scopus (333) Google Scholar.1-c Ref. 67Matsumura I. Ishikawa J. Nakajima K. Oritani K. Tomiyama Y. Miyagawa J.-I. Kato T. Miyazaki H. Matsuzawa Y. Kanakura Y. Mol. Cell. Biol. 1997; 17: 2933-2934Crossref PubMed Scopus (172) Google Scholar.1-d Bolded residues represent changes compared to the consensus sequences of Stat5a or Stat5b. Open table in a new tab For indirect immunofluorescence cells were cultured on glass coverslips coated with poly-d-lysine, fixed and stained as described in (9Kazansky A.V. Kabotyanski E.B. Wyszomierski S.L. Mancini M.A. Rosen J.M. J. Biol. Chem. 1999; 274: 22484-22492Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). Images were obtained using a DeltaVision deconvolution microscope and softWoRx imaging workstation from Applied Precision, Inc. (Issaquah, WA). To explore DNA binding specificities of rat Stat5a and Stat5b to different DNA sequences that contain the consensus Stat5 binding motif, e.g. औ-casein GAS site, as compared with those promoters with minor differences, e.g. p21-SIE2 and APRE GAS sites (Table I), HeLa cells were transfected with expression vectors for Stat5a or Stat5b and PrlR. After stimulation with Prl for 30 min, nuclear extracts were prepared, and EMSA was performed (Fig. 1,panel A). As expected, following Prl activation, both Stat 5a and Stat 5b formed complexes with the single GAS sites in the औ-casein, p21-SIE2, and to APRE probes with different intensities most likely reflecting slightly different affinities to the consensus and nonconsensus Stat5 binding sites (Fig. 1, panel A,lanes 4 and 6). A slight difference in the mobility of Prl-inducible DNA binding complexes for Stat5a and Stat5b was observed (compare lanes 4 and 6, Fig. 1,panel A), which also was expected based upon previous observations (9Kazansky A.V. Kabotyanski E.B. Wyszomierski S.L. Mancini M.A. Rosen J.M. J. Biol. Chem. 1999; 274: 22484-22492Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 46Boucheron C. Dumon S. Santos S.C. Moriggl R. Hennighausen L. Gisselbrecht S. Gouilleux F. J. Biol. Chem. 1998;
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