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A Functional DNA Binding Domain Is Required for Growth Hormone-induced Nuclear Accumulation of Stat5B

1999; Elsevier BV; Volume: 274; Issue: 8 Linguagem: Inglês

10.1074/jbc.274.8.5138

1083-351X

James Herrington, Liangyou Rui, Guoyang Luo, Li‐Yuan Yu‐Lee, Christin Carter‐Su,

Growth Hormone and Insulin-like Growth Factors

The mechanisms regulating the cellular distribution of STAT family transcription factors remain poorly understood. To identify regions of Stat5B required for ligand-induced nuclear accumulation, we constructed a cDNA encoding green fluorescent protein (GFP) fused to the N terminus of Stat5B and performed site-directed mutagenesis. When co-expressed with growth hormone (GH) receptor in COS-7 cells, GFP-Stat5B is tyrosyl-phosphorylated, forms dimers, and binds DNA in response to GH in a manner indistinguishable from untagged Stat5B. In multiple cell types, laser scanning confocal imaging of GFP-Stat5B co-expressed with GH receptor shows that GFP-Stat5B undergoes a rapid, dramatic accumulation in the nucleus upon GH stimulation. We introduced alanine substitutions in several regions of Stat5B and assayed for GH-dependent nuclear localization. Only the mutation that prevented binding to DNA (466VVVI469) abrogated GH-stimulated nuclear localization. This mutant fusion protein is tyrosyl-phosphorylated and dimerizes in response to GH. These results suggest that either high affinity binding to DNA contributes to nuclear accumulation of Stat5B or that this region is crucial for two functions, namely accumulation of Stat5B in the nucleus and DNA binding. Thus, we have identified a mutant Stat5 defective in nuclear localization despite its ability to be tyrosyl-phosphorylated and to dimerize. The mechanisms regulating the cellular distribution of STAT family transcription factors remain poorly understood. To identify regions of Stat5B required for ligand-induced nuclear accumulation, we constructed a cDNA encoding green fluorescent protein (GFP) fused to the N terminus of Stat5B and performed site-directed mutagenesis. When co-expressed with growth hormone (GH) receptor in COS-7 cells, GFP-Stat5B is tyrosyl-phosphorylated, forms dimers, and binds DNA in response to GH in a manner indistinguishable from untagged Stat5B. In multiple cell types, laser scanning confocal imaging of GFP-Stat5B co-expressed with GH receptor shows that GFP-Stat5B undergoes a rapid, dramatic accumulation in the nucleus upon GH stimulation. We introduced alanine substitutions in several regions of Stat5B and assayed for GH-dependent nuclear localization. Only the mutation that prevented binding to DNA (466VVVI469) abrogated GH-stimulated nuclear localization. This mutant fusion protein is tyrosyl-phosphorylated and dimerizes in response to GH. These results suggest that either high affinity binding to DNA contributes to nuclear accumulation of Stat5B or that this region is crucial for two functions, namely accumulation of Stat5B in the nucleus and DNA binding. Thus, we have identified a mutant Stat5 defective in nuclear localization despite its ability to be tyrosyl-phosphorylated and to dimerize. The Signal Transducers andActivators of Transcription (STAT) 1The abbreviations STATsignal transducers and activators of transcriptionSH2 domainSrc homology-2 domainGASinterferon-γ activation sequenceGHgrowth hormoneGHRgrowth hormone receptorPRLprolactinGFPgreen fluorescent proteinEMSAelectrophoretic mobility shift assayIRF-1interferon regulatory factor-1GFPgreen fluorescent proteinAPalkaline phosphataseαPYanti-phosphotyrosine antibodyN/Cnuclear-to-cytosol family of transcription factors provide a crucial signaling link between complexes of cytokine/hematopoietin receptors and Janus (JAK) family tyrosine kinases at the plasma membrane and gene transcription in the nucleus (1Darnell Jr., J.E. Science. 1997; 277: 1630-1635Crossref PubMed Scopus (3401) Google Scholar). Seven mammalian STAT genes have been identified and mouse genetics have revealed functions for most that are well supported by the in vitro experiments that led to their discovery (1Darnell Jr., J.E. Science. 1997; 277: 1630-1635Crossref PubMed Scopus (3401) Google Scholar, 2Ihle J.N. Cell. 1996; 84: 331-334Abstract Full Text Full Text PDF PubMed Scopus (1268) Google Scholar). A general model for cytokine activation of STATs has been proposed, based primarily on Stats 1–3 (2Ihle J.N. Cell. 1996; 84: 331-334Abstract Full Text Full Text PDF PubMed Scopus (1268) Google Scholar, 3Darnell Jr., J.E. Kerr I.M. Stark G.R. Science. 1994; 264: 1415-1421Crossref PubMed Scopus (5062) Google Scholar). In this model, STATs exist as monomers located in the cytoplasm prior to receptor activation, although evidence for preassociation prior to activation exists (4Stancato L.F. David M. Carter-Su C. Larner A.C. Pratt W.B. J. Biol. Chem. 1996; 271: 4134-4137Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). Upon cytokine stimulation, phosphotyrosine residues in the receptor recruit STATs through an SH2 domain interaction. Activated Janus kinases phosphorylate STATs on a carboxyl tyrosine, promoting STAT dimer formation by intermolecular SH2 domain interaction and dissociation from the receptor complex. Once dimerized, STATs translocate to the nucleus, bind DNA, and regulate gene transcription (2Ihle J.N. Cell. 1996; 84: 331-334Abstract Full Text Full Text PDF PubMed Scopus (1268) Google Scholar, 3Darnell Jr., J.E. Kerr I.M. Stark G.R. Science. 1994; 264: 1415-1421Crossref PubMed Scopus (5062) Google Scholar). Recent studies have shown that in some cells some STATs are present in the nucleus prior to activation (5Shuai K. Schindler C. Prezioso V.R. Darnell Jr., J.E. Science. 1992; 258: 1808-1812Crossref PubMed Scopus (659) Google Scholar, 6Shuai K. Stark G.R. Kerr I.M. Darnell Jr., J.E. Science. 1993; 261: 1744-1746Crossref PubMed Scopus (690) Google Scholar, 7Zhang X. Blenis J. Li H.C. Schindler C. Chen-Kiang S. Science. 1995; 267: 1990-1994Crossref PubMed Scopus (525) Google Scholar, 8Wang Y.F. Yu-Lee L.Y. Mol. Cell. Endocrinol. 1996; 121: 19-28Crossref PubMed Scopus (51) Google Scholar, 9Stout L.E. Svensson A.M. Sorenson R.L. Endocrinology. 1997; 138: 1592-1603Crossref PubMed Scopus (50) Google Scholar). Thus, it appears that beyond the general mechanism outlined above, there may be cell type-specific mechanisms that could further regulate some of the STATs. Finding STATs in the nucleus of some cells prior to activation suggests that constitutive nuclear import and export exist in these cells. signal transducers and activators of transcription Src homology-2 domain interferon-γ activation sequence growth hormone growth hormone receptor prolactin green fluorescent protein electrophoretic mobility shift assay interferon regulatory factor-1 green fluorescent protein alkaline phosphatase anti-phosphotyrosine antibody nuclear-to-cytosol The mechanism by which activated STATs accumulate in the nucleus is unknown. STATs have a mass above the upper limit for diffusion through the nuclear pore (∼45 kDa) (10Gorlich D. Matta I.W. Science. 1996; 271: 1513-1518Crossref PubMed Scopus (1067) Google Scholar, 11Jans D.A. Hubner S. Physiol. Rev. 1996; 76: 651-685Crossref PubMed Scopus (389) Google Scholar, 12Koepp D.M. Silver P.A. Cell. 1996; 87: 1-4Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar) and thus are assumed to be actively transported into the nucleus. The best characterized nuclear import pathway involves binding of the transported protein to the heterodimer protein complex importin α/β followed by energy-dependent transport through the nuclear pore complex requiring the GTPase activity of Ran/TC4 (10Gorlich D. Matta I.W. Science. 1996; 271: 1513-1518Crossref PubMed Scopus (1067) Google Scholar, 11Jans D.A. Hubner S. Physiol. Rev. 1996; 76: 651-685Crossref PubMed Scopus (389) Google Scholar, 12Koepp D.M. Silver P.A. Cell. 1996; 87: 1-4Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar). STATs appear to lack a conventional nuclear localization sequence, the single or dual stretch of basic amino acids that bind to importin α. Yet, Stat1 dimers have recently been shown to associate with the importin α-homologue NPI-1 (13Sekimoto T. Imamoto N. Nakajima K. Hirano T. Yoneda Y. EMBO J. 1997; 16: 7067-7077Crossref PubMed Scopus (306) Google Scholar). Furthermore, Ifn-γ-stimulated Stat1 nuclear accumulation requires NPI-1 (13Sekimoto T. Imamoto N. Nakajima K. Hirano T. Yoneda Y. EMBO J. 1997; 16: 7067-7077Crossref PubMed Scopus (306) Google Scholar) and the GTPase activity of Ran/TC4 (14Sekimoto T. Nakajima K. Tachibana T. Hirano T. Yoneda Y. J. Biol. Chem. 1996; 271: 31017-31020Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). Perhaps STAT dimers possess the structure required for binding to nuclear import proteins. In cells where STATs are found in the nucleus prior to activation, cytokine-induced nuclear accumulation might also arise from down-regulation of nuclear export. Insight into how STAT proteins interact with proteins needed for nuclear transport awaits identification of a mutant STAT that can dimerize but is unable to localize to the nucleus. Stat5 was first identified as a mammary gland factor required for prolactin (PRL)-stimulated gene transcription (15Schmitt-Ney M. Doppler W. Ball R. Groner B. Mol. Cell. Biol. 1991; 11: 3745-3755Crossref PubMed Google Scholar, 16Wakao H. Schmitt-Ney M. Groner B. J. Biol. Chem. 1992; 267: 16365-16370Abstract Full Text PDF PubMed Google Scholar) but was found to be activated by many hormones, growth factors, and cytokines (1Darnell Jr., J.E. Science. 1997; 277: 1630-1635Crossref PubMed Scopus (3401) Google Scholar, 17Groner B. Gouilleux F. Curr. Opin. Genet. & Dev. 1995; 5: 587-594Crossref PubMed Scopus (117) Google Scholar,18Mui A.L. Wakao H. AM O.F. Harada N. Miyajima A. EMBO J. 1995; 14: 1166-1175Crossref PubMed Scopus (541) Google Scholar). We now know that in humans and rodents Stat5 is actually two distinct proteins arising from different genes (Stat5a andStat5b) (18Mui A.L. Wakao H. AM O.F. Harada N. Miyajima A. EMBO J. 1995; 14: 1166-1175Crossref PubMed Scopus (541) Google Scholar, 19Liu X. Robinson G.W. Gouilleux F. Groner B. Hennighausen L. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8831-8835Crossref PubMed Scopus (462) Google Scholar, 20Ripperger J.A. Fritz S. Richter K. Hocke G.M. Lottspeich F. Fey G.H. J. Biol. Chem. 1995; 270: 29998-30006Crossref PubMed Scopus (144) Google Scholar, 21Lin J.X. Mietz J. Modi W.S. John S. Leonard W.J. J. Biol. Chem. 1996; 271: 10738-10744Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar, 22Silva C.M. Lu H. Day R.N. Mol. Endocrinol. 1996; 10: 508-518Crossref PubMed Scopus (62) Google Scholar). Stat5A and Stat5B are highly homologous, differing mainly in their C terminus, and are expressed in most tissues (18Mui A.L. Wakao H. AM O.F. Harada N. Miyajima A. EMBO J. 1995; 14: 1166-1175Crossref PubMed Scopus (541) Google Scholar, 19Liu X. Robinson G.W. Gouilleux F. Groner B. Hennighausen L. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8831-8835Crossref PubMed Scopus (462) Google Scholar). Genetic disruption of the Stat5a gene results in mice deficient in mammopoiesis and lactogenesis (23Liu X. Robinson G.W. Wagner K.U. Garrett L. Wynshaw-Boris A. Hennighausen L. Genes Dev. 1997; 11: 179-186Crossref PubMed Scopus (927) Google Scholar). Knock-out of theStat5b gene removes the sexual dimorphism of body growth and liver gene expression induced by growth hormone (GH) (24Udy 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 (838) Google Scholar, 25Teglund 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 (1080) Google Scholar). The simultaneous deletion of both genes results in the most severe growth and reproductive defects, revealing functional redundancy of the Stat5 proteins in physiological processes mediated by GH and PRL (25Teglund 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 (1080) Google Scholar). To probe the mechanisms regulating Stat5B localization within cells, we constructed a green fluorescent protein (GFP)-Stat5B fusion protein. We find that the fusion protein is tyrosyl-phosphorylated, dimerizes, accumulates in the nucleus, and binds DNA upon cytokine receptor stimulation in a manner indistinguishable from untagged Stat5B. We identify by site-directed mutagenesis residues required for DNA binding that are also needed for GH-dependent nuclear localization. These studies identify for the first time a region of Stat5 required for nuclear localization independent of those regions needed for dimerization. Human fibrosarcoma 2C4 and 2C4-GHR cells were provided courtesy of G. Stark, Y. Han (Cleveland Clinic, Cleveland, OH), and I. Kerr (Imperial Cancer Research Fund, London, UK). Rat Stat5A cDNA (26Kazansky A.V. Raught B. Lindsey S.M. Wang Y.-F. Rosen J.M. Mol. Endocrinol. 1995; 9: 1598-1609Crossref PubMed Google Scholar) was kindly provided by J. Rosen (Baylor College of Medicine, Houston, TX); rat GH receptor (GHR) cDNA (27Mathews L.S. Enberg B. Norstedt G. J. Biol. Chem. 1989; 264: 9905-9910Abstract Full Text PDF PubMed Google Scholar) was from G. Norstedt (Karolinska Institute, Stockholm, Sweden), and recombinant human GH was the gift of Lilly. Prestained molecular weight standards were from Life Technologies, Inc. All chemicals were reagent grade or better. pEGFP-C1 (CLONTECH), which encodes a red-shifted variant of GFP optimized for fluorescence intensity and high expression in mammalian cells, was used to construct a cDNA encoding a GFP-Stat5B fusion protein. The BglII-EcoRI fragment of rat Stat5B cDNA (28Luo G. Yu-Lee L. J. Biol. Chem. 1997; 272: 26841-26849Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar) was first subcloned into pEGFP-C1 (designated pEGFP-ΔStat5B). Polymerase chain reaction with Pfu DNA polymerase (Stratagene) and oligonucleotides 5′-GAAGATCTATGGCAATGTGGATACAG-3′ and 5′-GGAGCTGCGTGGCATAG-3′ as primers was used to engineer a BglII site and remove an in-frame stop codon upstream of the start codon of Stat5B. The polymerase chain reaction product was purified, digested with BglII, and inserted into pEGFP-ΔStat5B, yielding cDNA encoding GFP fused to the N terminus of full-length Stat5B by a five amino acid linker (SGLRS). DNA sequencing (Sequenase 2.0; U. S. Biochemical Corp.) was performed to verify the region created by polymerase chain reaction and all junctions. GFP-Stat5B mutants were generated using the QuickChangeTM site-directed mutagenesis kit (Stratagene) as described by the manufacturer. Amino acids Tyr568, Lys582-Lys583-Lys586, or466VVVI469 were replaced by alanine. The oligonucleotide primers were as follows (lowercase represents mutations): 5′-GCCAGGACGGAATgcCACTTTCTGGC-3′ and 5′-GCCAGAAAGTGgcATTCCGTCCTGGC-3′ (Tyr568); 5′-GGAGGTGTTAgcGgcACATCTCgcGCCTCACTGGAACG-3′and 5′-CGTTCCAGTGAGGCgcGAGATGTgcCgcTAACACCTCC-3′ (Lys582-Lys583-Lys586); and 5′-CCTTGTCGCTCCCGGcGGcGGcGgcCGTGCATGGCAGCC-3′ and 5′-GGCTGCCATGCACGgcCgCCgCCgCCGGGAGCGACAAGG-3′ (466VVVI469). All mutations were verified by DNA sequencing. For the 466VVVI469 mutant, two clones were analyzed for tyrosyl phosphorylation, dimerization, and cellular localization with similar results. 2C4, 2C4-GHR, COS-7, and NIH-3T3 cells were cultured at 37 °C in Dulbecco's modified Eagle's medium supplemented with 1 mml-glutamine, 100 units/ml penicillin, 100 μg/ml streptomycin, 0.25 μg/ml amphotericin B, and either 10% fetal bovine serum (2C4, 2C4-GHR, COS-7) or 9% calf serum (NIH-3T3). Medium for all lines except COS-7 was further supplemented with 5 mmsodium pyruvate. COS-7, 2C4, and 2C4-GHR cells were transiently transfected by calcium phosphate precipitation (29Chen C. Okayama H. Mol. Cell. Biol. 1987; 7: 2745-2752Crossref PubMed Scopus (4824) Google Scholar) by incubating subconfluent cultures with DNA precipitates for 6–16 h. For transfection of 2C4 and 2C4-GHR cells, 20 μg/ml CalPhos maximizer (CLONTECH) was used. The cultures were then rinsed twice with Dulbecco's modified Eagle's medium and fed with culture medium. To equalize the amount of DNA transfected in non-imaging experiments, empty pEGFP-C1 was used. NIH-3T3 cells were transfected with Lipofectin (Life Technologies, Inc.) as described by the manufacturer. Cells were harvested or used for imaging between 48 and 72 h post-transfection. 2C4 cells stably transfected with a mammalian expression vector containing the cDNA for human GHR (2C4-GHR) have been described previously (30Han Y. Leaman D.W. Watling D. Rogers N.C. Groner B. Kerr I.M. Wood W.I. Stark G.R. J. Biol. Chem. 1996; 271: 5947-5952Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). 10-cm plates of cells were incubated in serum-free medium containing 1% bovine serum albumin for 6–16 h and then treated with 500 ng/ml human GH at 37 °C. Cells were rinsed three times with ice-cold phosphate-buffered saline containing 1 mmNa3VO4, lysed in ice-cold lysis buffer (50 mm Tris-HCl, pH 7.5, 150 mm NaCl, 2 mm EGTA, 0.1% Triton X-100, 1 mmphenylmethylsulfonyl fluoride, 10 μg/ml aprotinin, 10 μg/ml leupeptin, 1 mm Na3VO4), and scraped from the plates on ice. Cellular proteins were precipitated from the supernatant (2 h on ice) with αStat5B (C-17, Santa Cruz Biotechnology; 1:100) or αStat5A (L-20, Santa Cruz Biotechnology; 1:100), immobilized on protein A-coated agarose beads (Repligen; 1.5 h at 8 °C), eluted by boiling, and separated by SDS-polyacrylamide gel electrophoresis (5–12% gradient or 7.5% gels). Proteins were transferred to nitrocellulose membrane (Amersham Pharmacia Biotech) and detected by Western blotting using enhanced chemiluminescence (ECL) (Amersham Pharmacia Biotech) with Stat5B-specific, affinity purified polyclonal rabbit anti-rat αStat5B (1:5000) (28Luo G. Yu-Lee L. J. Biol. Chem. 1997; 272: 26841-26849Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar), Stat5A-specific αStat5A (Santa Cruz Biotechnology; 1:2000), α-phosphotyrosine (4G10, Upstate Biotechnology; 1:7500), or α-GFP (8362-1, CLONTECH; 1:500). For the experiment shown in Fig. 9, cytosol and nuclear extracts were prepared (31Sadowski H.B. Shuai K. Darnell Jr., J.E. Gilman M.Z. Science. 1993; 261: 1739-1744Crossref PubMed Scopus (642) Google Scholar) and divided for immunoprecipitation or electrophoretic mobility shift assay (see below). Extracts were diluted and supplemented where appropriate to make final volumes (500 μl) and concentrations of NaCl (150 mm), glycerol (7%), and Triton X-100 (0.2%) equal for immunoprecipitation from the cytosolic and nuclear fractions. The Western blot data presented in the figures are representative of at least three separate experiments except for Fig. 9, which was performed twice with similar results. Cell lysates were immunoprecipitated with αStat5B and incubated at 37 °C for 60 min in 100 μl of dephosphorylation buffer (50 mm Tris-HCl, pH 8.5, 0.1 mm EDTA, 0.2 mm MgCl2, 0.02 mm ZnCl2) containing 40 units of calf intestinal alkaline phosphatase (AP; Boehringer Mannheim). As controls, 10 mm Na3VO4 was added to the dephosphorylation buffer or AP was omitted. The reaction was terminated, and proteins were eluted by boiling in a 4:1 mixture of lysis buffer and SDS-polyacrylamide gel electrophoresis sample buffer. The resultant dephosphorylated proteins were separated by SDS-polyacrylamide gel electrophoresis and immunoblotted with αStat5B as described above. EMSAs were performed using nuclear extracts of COS-7 cells transfected with cDNAs encoding rat GHR and either Stat5B or GFP-Stat5B (wild-type or VVVI mutant). Forty-eight hours after transfection and 16 h after serum deprivation, cells were treated with 500 ng/ml GH for 5–60 min and nuclear extracts prepared (31Sadowski H.B. Shuai K. Darnell Jr., J.E. Gilman M.Z. Science. 1993; 261: 1739-1744Crossref PubMed Scopus (642) Google Scholar). The extracts were incubated with or without 1 μg of αStat5B (C-17 10x, Santa Cruz Biotechnology) and then with a probe corresponding to the PRL response element of the β-casein promoter (5′-AGATTTCTAGGAATTCAA-3′; 40,000 cpm, 5 × 10−15mol) (32Wakao H. Gouilleux F. Groner B. EMBO J. 1994; 13: 2182-2191Crossref PubMed Scopus (717) Google Scholar). Samples were analyzed on a non-denaturing polyacrylamide gel and subjected to autoradiography. The EMSA experiments were performed twice with similar results. Confocal imaging was performed with a Noran OZ laser scanning confocal microscope equipped with a 60× Nikon objective. GFP was excited at 488 nm by a krypton-argon laser, and fluorescence above 500 nm was captured. Cells were grown on glass coverslips attached to the bottom of a 60-mm culture dish, transfected with cDNA, incubated in serum-free medium for 6–16 h, and then imaged at room temperature in Krebs-Ringer phosphate buffer (128 mm NaCl, 7 mm KCl, 1 mm CaCl2, 1.2 mm MgSO4, 1 mm NaHPO4, 10 mm glucose, pH 7.4) containing 0.1% bovine serum albumin. The contribution of cellular autofluorescence was judged to be less than 1%. Preliminary experiments revealed that repeated laser exposure inhibited GH-dependent migration of GFP-Stat5B into the nucleus, presumably because of phototoxicity. Thus, the following protocol was adopted. Once control images were obtained, cell location was recorded by capturing a low-power image, and the cells were stimulated with GH in a 37 °C incubator. Following the times indicated in the figures, the same cells were found and imaged a second time. The presented images are representative of at least three separate experiments during which at least 20 cells were imaged. For quantitative analysis of fluorescence distribution, cells were fixed (3.7% paraformaldehyde in phosphate-buffered saline for 10 min) following stimulation. The mean intensities of neighboring cytosolic and nuclear regions (approximately 5 μm2 each) were calculated using Adobe PhotoshopTM, corrected for background, and expressed as nuclear-to-cytosol fluorescence ratios. As a tool for the study of the cellular localization of Stat5B, we constructed a GFP-Stat5B fusion protein. We first examined whether this protein, when expressed in COS-7 cells, is regulated by GH in a manner similar to untagged Stat5B. To examine whether GH stimulates tyrosyl phosphorylation of GFP-Stat5B, cells transfected with cDNAs encoding rat GHR and either Stat5B or GFP-Stat5B were treated with or without GH for 15 min. Proteins were immunoprecipitated with αStat5B and analyzed by Western blotting using αStat5B, anti-phosphotyrosine antibody (αPY), or αGFP (Fig.1 A). Antibodies directed against the C terminus of Stat5B (αStat5B) and the N-terminal GFP tag (αGFP) both recognized a protein with an apparent molecular mass appropriate for GFP-Stat5B (∼120 kDa) in immunoprecipitates from cells transfected with GFP-Stat5B cDNA (Fig. 1 A,lanes E–H). Neither antibody detected a protein of smaller size (except for endogenous Stat5B in the case of αStat5B; Fig.1 A, lanes E–H, and data not shown) indicating that the vast majority of expressed protein was full-length fusion protein. In immunoprecipitates from cells transfected with Stat5B cDNA, αStat5B recognized an approximately 90-kDa protein (Fig.1 A, lanes A–D), appropriate for untagged Stat5B. GH stimulated to similar extents tyrosyl phosphorylation of GFP-Stat5B (Fig. 1 A, lane H) and untagged Stat5B (Fig.1 A, lane D). Hence, GFP-Stat5B expressed in COS-7 cells is full-length (not truncated at either the N or C terminus), is recognized by both αStat5B and αGFP, and like untagged Stat5B is tyrosyl-phosphorylated in response to GH. We next sought to determine whether GFP-Stat5B is capable of forming dimers in response to GH-induced tyrosyl phosphorylation. To analyze dimer formation, we utilized the ability of Stat5B to form dimers with Stat5A in response to many cytokines, including GH (33Smit L.S. VanderKuur J.A. Stimage A. Han Y. Luo G. Yu-lee L.-Y. Schwartz J. Carter-Su C. Endocrinology. 1997; 138: 3426-3434Crossref PubMed Scopus (64) Google Scholar). COS-7 cells were transiently transfected with cDNAs encoding GHR, Stat5A, and either GFP, Stat5B, or GFP-Stat5B. Stat5A was immunoprecipitated with Stat5A-specific antibody, and precipitated proteins were probed with Stat5B-specific antibody. GH induced co-immunoprecipitation of Stat5B (Fig. 1 B, lanes C and D) and GFP-Stat5B (Fig. 1 B, lanes E and F) with Stat5A. No proteins were detected when Stat5A was co-expressed with GFP alone (Fig. 1 B, lanes A andB), showing that αStat5B did not cross-react with Stat5A. The Stat5B which co-immunoprecipitated with Stat5A in response to GH migrates as three distinct bands (Fig. 1 B, lane D). Similarly, three GFP-Stat5B bands also co-immunoprecipitate with Stat5A in response to GH (Fig. 1 B, lane F), although the relative amounts differ when compared with Stat5B. The lower level of GFP-Stat5B compared with Stat5B bound to Stat5A in Fig.1 B is assumed to be due to a lower level of expression of GFP-Stat5B since in all experiments where it was examined, GFP-Stat5B was expressed at lower levels than untagged Stat5B. Interestingly, co-expression of Stat5A with either Stat5B or GFP-Stat5B but not with GFP leads to a low level of constitutive tyrosyl phosphorylation of Stat5A (Fig. 1 B, lanes A, C, and E). In sum, these experiments show that activated GFP-Stat5B, like untagged Stat5B, forms heterodimers with Stat5A. Based on studies of Stat5B regulation by GH in liver (34Ram P.A. Park S.H. Choi H.K. Waxman D.J. J. Biol. Chem. 1996; 271: 5929-5940Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar), we predicted that the multiple Stat5B and GFP-Stat5B bands seen in immunoprecipitates from GH-treated cells reflect differential phosphorylation of Stat5B on serines/threonines and tyrosines. To test for differential phosphorylation, we treated Stat5B immunoprecipitates with the general phosphatase alkaline phosphatase (AP). AP reduced the three Stat5B and GFP-Stat5B bands seen in immunoprecipitates from GH-treated cells to predominantly the fastest migrating band, with a variable amount of a slower migrating band (Fig.2, lanes B and C). The changes in band pattern produced by AP treatment are the result of dephosphorylation and not protein degradation since sodium orthovanadate inhibited the mobility changes (Fig. 2, lane D). The incomplete dephosphorylation of Stat5B seen in immunoprecipitates from GH-treated cells is thought to result from limited access of AP to one of the phosphorylated sites in the Stat5B dimer. In support of Stat5B being phosphorylated on serines/threonines as well as tyrosines, we have found that the serine/threonine-specific phosphatase PP2A condenses the three Stat5B bands from GH-treated cells to two, tyrosyl-phosphorylated bands (data not shown). Further support for the multiple GFP-Stat5B bands not being truncated forms of Stat5B is the finding that all bands are recognized by both antibody to the N-terminal GFP tag and antibody to the 10 amino acids at the C terminus of Stat5B which are unique to the B isoform (28Luo G. Yu-Lee L. J. Biol. Chem. 1997; 272: 26841-26849Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). Also, the bands do not arise from adventitious proteolysis during the immunoprecipitation since the same bands are seen in blots of the cell lysates (Fig. 2,lane A). Overall, these data indicate that GFP-Stat5B, like untagged Stat5B, is phosphorylated at multiple sites. To assess the ability of GFP-Stat5B to bind DNA, we performed EMSAs with a probe corresponding to the GAS-like element of theβ-casein promoter and nuclear extracts from COS-7 cells overexpressing GHR with or without either Stat5B or GFP-Stat5B (Fig.3). GH produced a similar time-dependent increase in the formation of a DNA-binding complex in nuclear extracts from cells expressing Stat5B (Fig. 3,lanes A–E) or GFP-Stat5B (Fig. 3, lanes G–K) but not in extracts from cells expressing GHR alone (Fig. 3,lanes M and N). This DNA-binding complex contains Stat5B or GFP-Stat5B since pretreatment of the nuclear extracts with αStat5B results in a supershifted complex (Fig. 3, lanes Fand L). We find that the DNA complex containing GFP-Stat5B migrates slightly slower than the complex containing untagged Stat5B, consistent with the addition of the GFP tag. Overall, Figs. 1 and 3illustrate that the critical cytokine-regulated events of tyrosyl phosphorylation, dimerization, and DNA binding are functionally intact for GFP-Stat5B. The EMSAs indicated that GFP-Stat5B was present in COS-7 cell nuclei following GH treatment (Fig. 3). We next sought to verify the presence of GFP-Stat5B in nuclei by directly visualizing GFP-Stat5B in single, living cells. For these experiments we employed human fibrosarcoma 2C4 cells because they possess a more uniform morphology than COS-7 cells and are easily transfected. Cells transiently expressing GHR and GFP, GFP-Stat5B alone, or GHR and GFP-Stat5B were imaged by confocal microscopy prior to and following stimulation with GH (Fig. 4). In cells expressing both GFP and GHR (Fig. 4, A and B), GFP was found throughout the cytoplasm and nucleus, and this distribution did not change upon GH treatment. Likewise, in cells expressing GFP-Stat5B but not GHR (Fig. 4, C andD), GFP-Stat5B was present both in the cytoplasm and nucleus prior to and following GH addition. A similar subcellular distribution of GFP-Stat5B was seen in cells expressing GFP-Stat5B and GHR in the absence of GH (Fig. 4 E). GFP-Stat5B was also present in both the cytosol and nucleus prior to activation when expressed in COS-7, NIH-3T3, and 2C4-GHR cells (Fig. 5,A, C, and E). The presence of some GFP-Stat5B in the nucleus as well as in the cytoplasm is unlikely to be the result of the GFP tag or overexpression since a similar distribution was seen for the following: 1) untagged Stat5B expressed in COS-7 cells and endogenous Stat5B in CHO-GHR cells (both de