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Endocrinology Vol. 138, No. 8 3426-3434
Copyright © 1997 by The Endocrine Society

ARTICLES

Growth Hormone-Induced Tyrosyl Phosphorylation and Deoxyribonucleic Acid Binding Activity of Stat5A and Stat5B1

Lisa S. Smit2, Joyce A. VanderKuur2, Aeisha Stimage, Yulong Han, Guoyang Luo, Li-yuan Yu-Lee, Jessica Schwartz and Christin Carter-Su

Department of Physiology (L.S.S., J.A.V., A.S., J.S., C.C.-S.), University of Michigan Medical School, Ann Arbor, Michigan 49109-0622; Department of Neuroscience (Y.H.), The Cleveland Clinic Foundation, Cleveland, Ohio 44195; and Departments of Medicine and Microbiology and Immunology (G.L., L.-Y.Y.-L.), Baylor College of Medicine, Houston, Texas 77030

Address all correspondence and requests for reprints to: Christin Carter-Su, Department of Physiology, University of Michigan Medical School, Ann Arbor, Michigan 49109-0622.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
GH is known to activate JAK2 tyrosine kinase and members of the Stat family of transcription factors, including Stats 1, 3, and 5. The recent observation that at least two Stat5 proteins (Stat5A and Stat5B) exist in mouse and human, raises the question of whether GH activates both Stat5A and Stat5B and, if so, whether the requirements for activation are the same. An initial report investigating this issue demonstrated GH-dependent activation of Stat5A but not Stat5B. In this paper, we demonstrate (in COS cells expressing rat GH receptor (rGHR) and either Stat5A or Stat5B, 3T3-F442A fibroblasts, and CHO cells expressing rGHR) that GH induces tyrosyl phosphorylation of both Stat5A and Stat5B. Similar time courses of phosphorylation were observed for the two proteins. Interestingly, the pattern of observed bands differs for the two forms of Stat5. Two closely migrating Stat5A bands can be detected in cells treated with or without GH. Both of these bands become tyrosyl phosphorylated in response to GH. Three species of Stat5B are observed in untreated cells. An additional, more slowly migrating Stat5B band, appears upon treatment with GH. The three more slower migrating Stat5B bands observed in response to GH contain phosphorylated tyrosyl residues. We further demonstrate that GH induces binding of Stat5A and Stat5B, as well as Stat1, to the GAS-like element in the ß-casein promoter. We and others have demonstrated previously that specific regions of GHR are required for GH-dependent activation of what is here identified as Stat5B. To gain insight into the mechanism by which GH promotes tyrosyl phosphorylation of Stat5A, GH-dependent tyrosyl phosphorylation of Stat5A was examined in CHO cells expressing truncated and mutated rGHR. The results indicate that Stat5A and Stat5B require the same regions of rGHR for maximal activation by GH: the C-terminal half of the cytoplasmic domain; tyrosines 333 and/or 338 in the N-terminal half of the cytoplasmic domain; and the regions required for JAK2 activation. To dissect further the mechanism by which GH activates Stat5A and B, the requirement for JAK2 in GH-dependent Stat5 tyrosyl phosphorylation was assessed using JAK2-deficient cells expressing GHR ({gamma}2A-GHR) and the wild-type parental cell line expressing GHR (2C4-GHR). GH-induced tyrosyl phosphorylation of Stat5B in 2C4-GHR cells but not in the JAK2 deficient, {gamma}2A-GHR cells, indicating that JAK2 is required for GH-dependent tyrosyl phosphorylation of Stat5B. Western blotting revealed that Stat5A is not expressed in this cell type. Taken together, these findings suggest that: 1) GH activates both Stat5A and Stat5B in several cell types; 2) the pattern of bands observed differs for Stat5A and Stat5B; 3) GH-dependent tyrosyl phosphorylation of Stat5A requires specific regions of GHR, and these requirements are the same as for Stat5B; and 4) JAK2 kinase is required for GH-dependent tyrosyl phosphorylation of Stat5B and, most likely, Stat5A.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
GH PLAYS a critical role in a variety of physiological processes, including longitudinal-bone growth and body metabolism (1, 2). At the cellular level, GH regulates the expression of a variety of genes (3). The actions of GH are mediated by GH binding to and activating its receptor (GHR), which is a member of the cytokine receptor superfamily (3). GH, like a number of other ligands that bind to members of the cytokine receptor family, activates the tyrosine kinase JAK2 and other cellular proteins, including members of the Stat (signal transducers and activators of transcription) family of transcription factors. The current hypothesis for the activation of Stat proteins proposes that ligand binds to receptor and activates the receptor-associated tyrosine kinase, JAK2 (or other members of the JAK family of tyrosine kinases). JAK2 then phosphorylates tyrosyl residues in the receptor. These phosphorylated tyrosines or, in some instances, phosphorylated tyrosines in JAK2, then serve as docking sites for Stats. Stat proteins are then phosphorylated by JAK2 on tyrosyl residues, homo- or heterodimerize, and translocate to the nucleus, where they bind DNA and promote transcriptional activation of target genes. Seryl or threonyl phosphorylation also may be required for activation of Stats (4). The ability of GH to activate JAK2 and Stat proteins provides at least one pathway (GHR-JAK2-Stat) whereby GH mediates gene transcription.

GH has been shown to activate Stats 1 and 3, and most recently, Stat 5 (5, 6, 7, 8, 9, 10, 11, 12). Stat5 was first cloned as a PRL-induced transcription factor from sheep mammary gland (13). Stat5 is known to be activated by erythropoietin; interleukins-2, -3, and -5; granulocyte macrophage colony-stimulating factor (GM-CSF); thrombopoietin; leptin; colony stimulating factor-1 (CSF-1); platelet-derived growth factor (PDGF); and epidermal growth factor; in addition to PRL and GH (10, 14, 15, 16, 17, 18, 19, 20, 21). In response to GH, Stat5 has been implicated as a transcription factor for the ß-casein, Spi2.1, P450–3A10/6ß-hydroxylase, and insulin-1 genes (9, 10, 22, 23). Whereas a single Stat5 gene was identified in sheep, two forms of Stat5 (Stat5A and Stat5B), encoded by two different genes, have now been identified in mouse, human, and rat cells (12, 14, 19, 24, 25, 26, 27). Stat5A and Stat5B are highly homologous (>93% identical at the amino acid level), differing mainly at the C-terminus. Stat5A is more similar to sheep Stat5 than is Stat5B. Stat5B is 7 amino acids shorter than Stat5A in all species. Stat5B lacks the C-terminal putative MAP kinase phosphorylation site that is present in Stat5A and sheep Stat5, as well as in Stat1 and Stat3. The evidence to date indicates that, at least for Stats1 and 3, the MAP kinase site is required for maximal transcriptional activity in vivo in response to interferon (4, 28). Both Stat5A and Stat5B are expressed in most tissues, with the exception of brain and muscle, where very little or no Stat5A is expressed (14, 24). The functional significance of the two forms is unclear. Studies have shown that interleukins-2 and -3, PRL, CSF-1, and PDGF activate both Stat5A and Stat5B (14, 18, 19; Luo, G., and L.-y. Yu-Lee, manuscript submitted for publication). However, the two forms may have different binding preferences. Stat5A, but not Stat5B, binds to the sis-inducible element of the c-fos promoter in T-56 cells in response to CSF-1 and PDGF, whereas CSF-1 and PDGF induce binding of both Stat5A and Stat5B to the PRL-inducible element in the ß-casein promoter (18). To date, most studies of GH-dependent Stat5 activation have focused primarily on proteins recognized by antisheep Stat5 antibody or antimouse Stat5B antibody (with the degree of cross-reactivity with Stat5A undefined) and have not sought to differentiate between Stat5A and Stat5B.

One recent study reports that GH is able to induce tyrosyl phosphorylation of Stat5A (modified with a FLAG epitope) and its association with GHR in L-cells transfected with GHR, but had no effect on a similarly tagged Stat5B (29). This study illustrates the need to examine the ability of GH to activate wild-type Stat5A and Stat5B. In this paper, we demonstrate that GH stimulates tyrosyl phosphorylation of both Stat5A and 5B in 3T3-F442A cells, CHO cells expressing rat (r) GHR and COS cells expressing rat GH receptor (rGHR) and either Stat5A or Stat5B. We also show that GH induces binding of Stat5A and Stat5B, as well as Stat1, to the GAS-like element in the ß-casein promoter. Maximal Stat5A tyrosyl phosphorylation is observed to require the C-terminal half of the cytoplasmic domain of rGHR, as well as tyrosines 333 and/or 338 and the regions of GHR required for JAK2 association and activation. These are the same regions of GHR that we have described previously as being required for activation of a Stat5 protein that we identify in this paper as Stat5B. In addition, we demonstrate that JAK2 is required for GH-dependent activation of Stat5B using JAK2-deficient cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
The stocks of 3T3-F442A cells, 2C4-GHR and {gamma}2A-GHR cells, rat Stat5A complementary DNA (cDNA), and recombinant human (h) GH were kindly provided by H. Green (Harvard University, Cambridge, MA), G. Stark (Cleveland Clinic, Cleveland, OH) and I. Kerr (Imperial Cancer Research Fund, London, UK), J. Rosen (Baylor College of Medicine), and Eli Lilly (Indianapolis, IN), respectively. Triton X-100 came from Pierce Chemical Company (Rockford, IL), recombinant protein A-agarose from Repligen (Cambridge, MA), and enhanced chemiluminescence detection system from Amersham Corporation (Arlington Heights, IL).

Antibodies
Polyclonal {alpha}Stat5A (100 µg/ml) and {alpha}Stat5AX (1000 µg/ml) antibodies, raised against a peptide corresponding to amino acids 774–793 of mouse Stat5A, and polyclonal {alpha}Stat5B(sc) (100 µg/ml), raised against a peptide corresponding to amino acids 711–727 of mouse Stat5B, were purchased from Santa Cruz (Santa Cruz, CA). Polyclonal rabbit antirat {alpha}Stat5B(yl) was raised against a peptide corresponding to the last 10 C-terminal amino acids (777–786) of rat Stat5B (Luo, G., and L.-y. Yu-Lee, manuscript submitted for publication). Monoclonal mouse antihuman {alpha}Stat1, raised against an epitope corresponding to the N-terminal 194 amino acids, was purchased from Transduction Laboratories (Lexington, KY). Anti-JAK2 serum ({alpha}JAK2), raised against a synthetic peptide corresponding to amino acids 758–776 of murine JAK2, was prepared in our laboratory in conjunction with Pel-Freez, as described previously (30). Mouse monoclonal antiphosphotyrosine antibody 4G10 ({alpha}PY) was purchased from Upstate Biotechnology Inc. (Lake Placid, NY).

Cell culture and transfections
Mouse 3T3-F442A fibroblasts, COS-7 cells, 2C4-GHR, and {gamma}2A-GHR cells were grown in DMEM supplemented with 1 mM L-glutamine, 100 units/ml penicillin, 100 µg/ml streptomycin, 0.25 µg/ml amphotericin B, and either 8% calf serum (3T3-F442A) or 10% FCS (COS-7, 2C4-GHR, and {gamma}2A-GHR). COS-7 cells were transfected with mammalian expression vectors containing cDNAs (10 µg) for rGHR (31) and either Stat5A (27) or Stat5B (Luo, G., and L.-y. Yu-Lee, manuscript submitted for publication). Cells were transfected by calcium phosphate precipitation (32). Cells were then cultured for 6–8 h and refed with growth medium. Cells were harvested 48 h after transfection. 2C4 and {gamma}2A cells had been transfected previously with a mammalian expression vector containing the cDNA encoding hGHR (33). 125I-GH binding in {gamma}2A-GHR cells was approximately 30% of that observed in 2C4-GHR cells (data not shown).

CHO cells had been stably transfected previously with a mammalian expression vector containing the cDNA encoding full-length rat liver GHR (GHR1–638), the same cDNA with termination codons replacing lysine codons at positions 455 (GHR1–454), 319 (GHR1–318) or 295 (GHR1–294), or GHR1–454 cDNA with phenylalanine replacing tyrosines at positions 333 and 338 (GHR1–454Y333,338F) or the full-length cDNA with amino acids 297–311 deleted (GHR{Delta}P) (31, 34). CHO cells were grown in Ham’s F-12 medium supplemented with 10% FCS, 0.5 mg/ml G418, 1 mM L-glutamine, 100 units/ml penicillin, 100 µg/ml streptomycin, and 0.25 µg/ml amphotericin. Cells expressed the appropriate sized receptor, as assessed by cross-linking to 125I-hGH, as previously described (35). The relative affinities of the different GHR for 125I-hGH do not differ, as described previously (31, 34).

GH binding
hGH labeled with 125I to an estimated specific activity of 75 or 95 µCi/µg, using chloramine-T, was prepared by The University of Michigan Reproductive Sciences Training Program Grant Core Facility. For binding studies, cells in 100-mm or 6-well dishes were incubated in serum-free medium with 1% BSA for approximately 18 h, washed with Krebs-Ringer phosphate buffer (KRP) (128 mM NaCl, 7 mM KCl, 1 mM CaCl2, 1.2 mM MgSO4, 1 mM NaHPO4, 1 mM glucose, pH 7.4) with 1% BSA and then incubated at 25 C for 1 h with 1.6 x 105 cpm/ml 125I-hGH in KRP-1% BSA, in the absence (total) or presence (nonspecific) of 4 µg/ml unlabeled hGH in KRP. Cells were washed four times in ice-cold KRP and lysed with 1 N NaOH. 125I-GH binding (total, nonspecific) was quantified using {gamma}-counting, and results were normalized to protein content.

Immunoprecipitation and Western blotting
Cells were incubated in serum-free medium with 1% BSA for approximately 18 h before treatment with GH. Cells were incubated for 15 min (or varying time points; see Fig. 3BGo) with 500 ng/ml hGH at 37 C. Cells were then rinsed three times with ice-cold PBSV and scraped in lysis buffer, as described above. Cell lysates were centrifuged at 12,000 x g for 10 min, and the supernatants were incubated on ice for 2 h with antibody to either Stat5A, Stat5B, or JAK2. Immune complexes were collected on protein A-agarose during a 1-h incubation at 8 C and washed three times with wash buffer (50 mM Tris, 0.1% Triton X-100, 137 mM NaCl, 2 mM EGTA, pH 7.5). Proteins were eluted by boiling for 5 min in 100 µl 20% SDS-PAGE sample buffer in lysis buffer. The immunoprecipitates were subjected to SDS-PAGE, followed by Western blot analysis with {alpha}PY, {alpha}Stat5A, or {alpha}Stat5B, using the enhanced chemiluminescence detection system. To reprobe blots, the membranes were submerged in stripping buffer (100 mM ß-mercaptoethanol, 2% SDS, 62.5 mM Tris-HCl, pH 6.7) and incubated at 50 C for 30 min while shaking. The membranes were then probed with the desired antibody. In some cases, the blots were not stripped but reprobed immediately with the appropriate antibody. For quantification of the amount of tyrosyl phosphorylation of Stat5A in the CHO cells, autoradiographs were scanned (ArcusII desktop scanner and FotoLook software; AGFA, Mortsel, Belgium) and the signal quantified using Molecular Analyst Image Analysis software (Bio-Rad, Hercules, CA).



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  		Figure 3. GH promotes tyrosyl phosphorylation of Stat5A and Stat5B in 3T3-F442A fibroblasts and CHO-GHR1–638 cells. A, 3T3-F442A fibroblasts were incubated without hormone (-) or with 500 ng/ml hGH (+) for 15 min. Stat5 proteins were immunoprecipitated with {alpha}Stat5A (5A) (1:100), {alpha}Stat5B(sc) [5B(sc)] (1:100), or {alpha}Stat5B(yl) [5B(yl)] (1:500). Immunoprecipitates were subjected to Western blot analysis using {alpha}PY (1:7500), {alpha}Stat5A (1:2500), or {alpha}Stat5B(yl) (1:5000). The migration of prestained molecular weight markers is indicated on the right. B, 3T3-F442A fibroblasts were incubated without hormone (-) or with 500 ng/ml hGH (+) for the times indicated. Whole cell lysates were immunoprecipitated with {alpha}Stat5A (1:100) or {alpha}Stat5B(sc) (1:100). Immunoprecipitates were subjected to Western blot analysis using {alpha}PY (1:7500). Two different exposures are shown. C, CHO-GHR1–638 cells were incubated with (+) or without (-) 500 ng/ml hGH for 15 min. Stat5 proteins were immunoprecipitated with {alpha}Stat5A (1:100) or {alpha}Stat5B(sc) (1:100). Immunoprecipitates were subjected to Western blot analysis using {alpha}PY (1:7500) and then reprobed with either {alpha}Stat5A (1:2500) or {alpha}Stat5B(sc) (1:2500). Lanes A–B, C and D–E are from separate experiments.

 
Electrophoretic mobility shift assays (EMSAs)
EMSAs were performed as described previously (36). Briefly, cells were deprived of serum overnight and treated with 500 ng/ml hGH for 30 min at 37 C, and nuclear extracts were prepared. The extracts were preincubated with 1 µl {alpha}Stat5AX, {alpha}Stat5A, {alpha}Stat5B(yl), or {alpha}Stat1, incubated with a probe corresponding to the PRL response element in the ß-casein promoter (5'AGATTTCTAGGAATTCAA3'; 40,000 cpm, 5 x 10-15 mol) (13), analyzed on a nondenaturing polyacrylamide gel, and subjected to autoradiography.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Specificity of Stat5A and Stat5B antibodies
To examine the ability of GH to activate Stat5A and Stat5B, we first determined the specificity of {alpha}Stat5A, {alpha}Stat5B(sc), and {alpha}Stat5B(yl) antibodies for immunoblotting and immunoprecipitation. COS cells were mock transfected or transiently transfected with either rat Stat5A or Stat5B cDNA expression vectors. To assess the specificity of these antibodies in blotting, whole cell lysates were blotted with {alpha}Stat5A, {alpha}Stat5B(sc), or {alpha}Stat5B(yl). A Stat5A band was observed when lysates of COS cells, transiently expressing Stat5A, were blotted with {alpha}Stat5A (Fig. 1AGo, lane B). A similarly migrating band of much weaker intensity (by densitometry, approximately 15% of that observed in cells expressing Stat5A) was observed in both the mock-transfected cells and cells transfected with a Stat5B expression vector (lanes A and C). These results suggest that {alpha}Stat5A recognizes exogenous Stat5A and an endogenous protein in COS cells (thought to be COS Stat5A) but does not immunoblot detectable levels of exogenous Stat5B. A Stat5B band, migrating slightly faster than Stat5A, was observed when lysates of COS cells, transiently expressing Stat5B, were probed with {alpha}Stat5B(sc) (lane F) or {alpha}Stat5B(yl) (lane I). In lysates of COS cells mock, transfected or transfected with Stat5A and blotted with {alpha}Stat5B(sc), bands with the same migration, but of lesser intensity (approximately 10% of that observed in cells expressing Stat5B), were observed (lanes D and E). Similar bands were not observed when these lysates were probed with {alpha}Stat5B(yl) (lanes G and H) unless the blot was overexposed (data not shown). These results suggest that {alpha}Stat5B(sc) and {alpha}Stat5B(yl) do not cross-react with Stat5A at detectable levels in an immunoblot, but {alpha}Stat5B(sc), and to a lesser extent, {alpha}Stat5B(yl), do recognize an endogenous COS protein (thought to be COS Stat5B).



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  		Figure 1. Stat5A and Stat5B antibodies are specific for their respective form of Stat5. A, COS-7 cells were mock transfected (-) or transfected with cDNA expression vectors for Stat5A (A) or Stat5B (B). Whole cell lysates were probed with {alpha}Stat5A (1:2500), {alpha}Stat5B(sc) (1:2500), or {alpha}Stat5B(yl) (1:5000). B, COS-7 cells were transfected with cDNA expression vectors for Stat5A or Stat5B. Lysates were immunoprecipitated with {alpha}Stat5A (1:100), {alpha}Stat5B(sc) (1:100), or {alpha}Stat5B(yl) (1:500) and probed with {alpha}Stat5A, {alpha}Stat5B(sc), or {alpha}Stat5B(yl).

 
To assess the specificity of the Stat5 antibodies in immunoprecipitation, whole cell lysates of the transfected cells were immunoprecipitated and blotted with {alpha}Stat5A, {alpha}Stat5B(sc), or {alpha}Stat5B(yl). A prominant band was observed when {alpha}Stat5B(sc or yl) immunoprecipitates of COS cells expressing Stat5B were probed with {alpha}Stat5B(sc) (Fig. 1BGo, lanes E and F) or {alpha}Stat5B(yl) (lanes H and I). No band was detected when {alpha}Stat5A immunoprecipitates of COS cells expressing Stat5B were probed with either of the Stat5B antibodies (lanes D and G), demonstrating that {alpha}Stat5A does not immunoprecipitate a detectable amount of Stat5B. A band was observed in {alpha}Stat5A, but not {alpha}Stat5B(yl), immunoprecipitates of COS cells expressing Stat5A probed with {alpha}Stat5A (lanes A and C), demonstrating that {alpha}Stat5B(yl) does not immunoprecipitate Stat5A. A faint band with a migration appropriate for Stat5A was observed when lysates of COS cells expressing Stat5A were immunoprecipitated with {alpha}Stat5B(sc) and immunoblotted with {alpha}Stat5A (lane B) [the signal was less than 1% that observed when the same lysates were immunoprecipitated with {alpha}Stat5A (compare lane B with A)], suggesting that {alpha}Stat5B(sc) immunoprecipitates Stat5A but only to a very small extent. Taken together, these results demonstrate that all three Stat5 antibodies are highly specific for the form of Stat5 they were raised against, with {alpha}Stat5B(sc) immunoprecipitating a trivial amount of Stat5A. Although Stat5A and Stat5B appeared as single bands in the experiment shown in Fig. 1Go, two Stat5A bands and three Stat5B bands were detected in immunoprecipitates from untreated cells in other experiments when optimal resolution was achieved (See Fig. 2Go).



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  		Figure 2. GH promotes tyrosyl phosphorylation of Stat5A and Stat5B in transfected COS-7 cells. COS-7 cells were mock transfected or transfected with cDNA expression vectors for GHR, Stat5A, or Stat5B alone or in combination, as indicated. Cells were incubated without (-) or with (+) 500 ng/ml hGH for 15 min. Whole cell lysates were immunoprecipitated with {alpha}Stat5A (1:100) (lanes A–H) or {alpha}Stat5B(sc) (1:100) (lanes I–R), blotted with {alpha}PY (1:7500), and then reprobed with either {alpha}Stat5A (1:2500) (lanes A–H) or {alpha}Stat5B(sc) (1:2500) (lanes I–R). Lanes A–P and Q–R are from different experiments.

 
GHR mediates GH-induced tyrosyl phosphorylation of Stat5A and Stat5B in COS cells
To determine whether GH is able to activate both Stat5A and Stat5B, we examined the ability of GH to stimulate tyrosyl phosphorylation of Stat5A and Stat5B in COS cells transiently expressing rGHR in combination with either rat Stat5A or rat Stat5B. Cells were incubated with or without 500 ng/ml hGH for 15 min, Stat5A or Stat5B was immunoprecipitated with antibodies to the respective Stat5, and phosphorylated tyrosines were detected by Western blotting with antiphosphotyrosine antibody ({alpha}PY). GH promotes the tyrosyl phosphorylation of proteins precipitated by {alpha}Stat5A or {alpha}Stat5B(sc) from lysates of GH-treated COS cells expressing GHR and Stat5A or Stat5B, respectively (Fig. 2Go; lanes G, H, and O-R), suggesting that GH induces tyrosyl phosphorylation of both Stat5A and Stat5B. Greatly reduced levels of tyrosyl-phosphorylated Stat5A and Stat5B are also detected in response to GH in cells transfected with Stat5A or Stat5B alone (lanes F and N), presumably caused by the presence of a small amount of endogenous GHR. Consistent with these tyrosyl-phosphorylated proteins being exogenous Stat5A and Stat5B, respectively, they are not observed in mock-transfected cells. Faint bands corresponding to Stat5A and Stat5B are detectable in cells transfected with GHR cDNA alone but only after long exposures (data not shown). These bands are presumably caused by the small amount of endogenous Stat5A and Stat5B (see Fig. 1AGo).

Although Stat5A and 5B are both phosphorylated on tyrosines in response to GH, the pattern of observed bands differs for the two proteins. A broad Stat5A band was observed when lysates from cells transfected with GHR and Stat5A and treated with GH are immunoprecipitated with {alpha}Stat5A and blotted with {alpha}PY or {alpha}Stat5A (Fig. 2Go, lane H). However, in {alpha}PY blots of GH-treated cells transfected with Stat5A alone, a doublet was visible (lane F). This suggests that the broad Stat5A band in lane H ({alpha}PY and {alpha}Stat5A) might actually be a tight doublet that could not be resolved because of the amount of protein present. Multiple Stat5B bands were observed in both {alpha}PY and {alpha}Stat5B(sc) blots of GH-treated COS cells expressing GHR and Stat5B (lanes P and R). In the best resolved gels, three Stat5B bands were observed in {alpha}Stat5B(sc) blots of {alpha}Stat5B(sc) immunoprecipitates from untreated (no GH) cells (Fig. 2Go, lane Q; the two fastest migrating bands are running very close together). A fourth, slower migrating band appears upon stimulation with GH (Fig. 2Go, lane R, the upper band is relatively faint). When the {alpha}Stat5B(sc) immunoprecipitates from the GH-treated cells are blotted with {alpha}PY, three bands are observed that comigrate with the upper three bands detected by {alpha}Stat5B(sc) (Fig. 2Go, lane R; a longer exposure of lane P also reveals a third, slightly faster migrating band). The existence of multiple bands suggests that Stat5A and Stat5B are phosphorylated on serines and/or threonines, even in the absence of ligand.

GH induces tyrosyl phosphorylation of Stat5A and Stat5B in 3T3-F442A fibroblasts and CHO-GHR1–638 cells
We next compared the ability of GH to promote tyrosyl phosphorylation of endogenous Stat5A and Stat5B in mouse 3T3-F442A fibroblasts and CHO cells stably expressing rGHR (CHO-GHR1–638). Lysates from GH-treated 3T3-F442A cells were immunoprecipitated with {alpha}Stat5A, {alpha}Stat5B(sc), or {alpha}Stat5B(yl). The immunoprecipitates were loaded on a gel in triplicate and then blotted with {alpha}PY, {alpha}Stat5A, or {alpha}Stat5B(yl). Fig. 3AGo (lanes A–F) demonstrates that GH induces tyrosyl phosphorylation of proteins recognized by {alpha}Stat5A, {alpha}Stat5B(sc), and {alpha}Stat5B (yl), suggesting that GH induces tyrosyl phosphorylation of both Stat5A and Stat5B in 3T3-F442A fibroblasts. As with the COS cells, a broad band of approximately 94 kDa is observed in lysates from cells treated with or without GH, immunoprecipitated and blotted with {alpha}Stat5A (lanes I and J). Two faster migrating bands (approximately 72 kDa and 60 kDa) also are observed. The 94-kDa band comigrates with the band observed in the {alpha}PY blot (lane D). (In some experiments, we have resolved this latter band as a very tight doublet, data not shown). In contrast, and as in COS cells, three bands (approximately 86, 89, and 92 kDa) are observed in lysates from untreated (no GH) cells immunoprecipitated with either Stat5B antibody and blotted with {alpha}Stat5B(yl) (lanes M and Q). An additional, slower migrating band (approximately 94 kDa) is observed in lysates from cells treated with GH (lanes N and R). The three slower migrating bands in the immunoprecipitates from GH-treated cells comigrate with the three bands observed in the {alpha}PY blot. In the latter, the two slower migrating bands (approximately 92 and 94 kDa) are of relatively equal intensity, and the third, fastest migrating band (approximately 89 kDa) is very faint. A small amount of protein is observed when lysates are immunoprecipitated with {alpha}Stat5B(sc or yl) and blotted with {alpha}Stat5A (lanes G, H, K, and L) and vice versa (lane P). This is thought to indicate the presence of Stat5A/Stat5B heterodimers, rather than a small degree of cross-reactivity of the immunoprecipitating antibody, because in transfected COS cells, {alpha}Stat5A and {alpha}Stat5B(yl) did not immunoprecipitate any detectable Stat5B or Stat5A, respectively (Fig. 1BGo). The detection of coprecipitating Stat5A and Stat5B in unstimulated cells (lanes G and K) suggests that Stat5A/5B dimers exist, even in the absence of ligand, with ligand binding enhancing dimer formation (lanes H and L), as observed previously for Stat1 and Stat2 (37).

A time course (Fig. 3BGo) revealed that GH-dependent tyrosyl phosphorylation of Stat5A and Stat5B in 3T3-F442A cells follows similar time courses, although the Stat5B signal seems to be sustained at maximal levels longer than the Stat5A signal. Tyrosyl phosphorylation of both Stat5A and the slowest migrating Stat5B band is observed as early as one min (Fig. 3BGo, short exposure) and peaks by 5 min. Tyrosyl phosphorylation of Stat5A is decreasing by 30 min and is further decreased, but still visible, at 60 min. The slowest migrating Stat5B band remains at approximately maximal tyrosyl phosphorylation out to 30 min (see short exposure) but is reduced by 60 min compared with earlier time points. Tyrosyl phosphorylation of the two faster migrating tyrosyl phosphorylated Stat5B bands peaks at one min, is still visible at 30 min (Fig. 3BGo, long exposure) and disappears by 60 min. Some variability in the ratio of the different Stat5B bands was observed. In this experiment, the top band was most prevalent at 15 min, whereas in other experiments, the two faster migrating bands were more prevalent (see Fig. 3AGo, lane F).

GH also is able to induce tyrosyl phosphorylation of Stat5A and Stat5B in CHO cells expressing wild-type rGHR (CHO-GHR1–638) (Fig. 3CGo). The pattern of bands is similar to that observed in 3T3-F442A fibroblasts and transfected COS cells. One (lane B) or two (lane C) bands are observed in lysates from cells treated with GH, immunoprecipitated with {alpha}Stat5A, and blotted with either {alpha}PY or {alpha}Stat5A. Three bands are present in lysates from untreated cells that are immunoprecipitated and blotted with {alpha}Stat5B (sc) (lane D). A fourth, slower migrating protein appears upon GH treatment (lane E). The three slowest migrating bands in lane E, {alpha}Stat5B blot, comigrate with the three bands in the {alpha}PY blot.

GH induces DNA binding activity of Stat5A and Stat5B
The ability of GH to induce binding of Stat5A and Stat5B to DNA was assessed by EMSA (Fig. 4Go). When nuclear extracts from GH-treated COS cells expressing GHR and Stat5A were incubated with a probe corresponding to the GAS-like element in the ß-casein promoter, a band shift was observed (Fig. 4AGo, lanes D and J). The Stat5A band could be completely supershifted by pretreatment of the nuclear extracts with {alpha}Stat5AX (lane K) but was not shifted by pretreatment with {alpha}Stat5B(yl) (lane L). When nuclear extracts from GH-treated COS cells expressing GHR and Stat5B were incubated with the same probe, a band shift with a slightly faster migration was observed (Fig. 4AGo, lanes F and M). The Stat5B band was supershifted with {alpha}Stat5B(yl) (lane O) but not {alpha}Stat5AX (lane N). No band shift was detected in mock-transfected cells (lane B). These results indicate that GH induces binding of both Stat5A and Stat5B to the ß-casein promoter. These results also demonstrate that {alpha}Stat5AX and {alpha}Stat5B(yl) are highly specific for Stat5A and Stat5B, respectively, when used in an EMSA. {alpha}Stat5B(sc) cross-reacted significantly with Stat5A in this system (unpublished observation). Two complexes, designated I and II, were observed when nuclear extracts from GH-treated 3T3-F442A cells are incubated with the ß-casein promoter probe (Fig. 4AGo, lanes H and I; and Fig. 4BGo, lanes B and H). The upper portion of the 3T3-F442A complex II comigrates with the COS Stat5A band (Fig. 4AGo, compare lanes I and J), and the lower portion of the 3T3-F442A complex II comigrates with the COS Stat5B band (compare lanes I and M), suggesting that complex II contains Stat5A and Stat5B. Consistent with this hypothesis, the upper portion of the 3T3-F442A complex II is supershifted by both {alpha}Stat5A and the more concentrated {alpha}Stat5AX (Fig. 4BGo, lanes C and D). Multiple supershifted complexes were observed, which are thought to represent Stat5 homo- or heterodimer binding complexes with different numbers of antibodies bound. The lower portion of the 3T3-F442A complex II is supershifted by {alpha}Stat5B(yl) (lane E). Preincubating nuclear extracts of GH-treated 3T3-F442A cells with both {alpha}Stat5AX and {alpha}Stat5B(yl) resulted in a complete shift of complex II (lane F). Complex I is supershifted by {alpha}Stat1 (lane I), but not by antibody, to either form of Stat5 (lanes C and D) or {alpha}Stat3 (data not shown). These results suggest that Stat5A, Stat5B, and Stat1 bind to the GAS-like element in the ß-casein promoter in response to GH.



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  		Figure 4. GH promotes DNA binding activity of Stat5A and Stat5B. Nuclear extracts were prepared from transiently transfected COS cells (A) or 3T3-F442A fibroblasts (B), incubated without (-) or with (+) 500 ng/ml hGH for 30 min. EMSAs were performed using a GAS-like PRL response element from the ß-casein promoter (13). Nuclear extracts were preincubated with 1 µl {alpha}Stat5A, {alpha}Stat5AX, {alpha}Stat5B(yl), or Stat1 for 20 min where indicated. The migrations of complexes I and II are indicated.

 
Regions of GHR required for GH-induced phosphorylation of Stat 5A
We have demonstrated previously, using mutated and truncated rat liver GHR stably expressed in CHO cells (Fig. 5AGo), that the C-terminal half of the cytoplasmic domain of GHR, as well as two amino-terminal tyrosines (333 and 338), are required for maximal GH-dependent activation of protein recognized by {alpha}Stat5B(sc) [(35); see summary of results shown in Fig. 5BGo (open bars) for reference]. Our present observation that {alpha}Stat5B(sc) is quite specific for Stat5B suggests that our previous study focused on Stat5B rather than Stat5A. Here we compare the regions of GHR required for GH-dependent tyrosyl phosphorylation of Stat5A with those required for Stat5B tyrosyl phosphorylation. The ability of GH to stimulate tyrosyl phosphorylation of Stat5A in CHO cell lines expressing truncated and mutated GHR was assayed, and the results were quantified, normalized for 125I-GH binding to account for the variable levels of GHR expression in the different cell lines, and expressed as a percent of the signal observed in GH-treated CHO cells expressing the full-length receptor, GHR1–638 (Fig. 5BGo). GH promotes the tyrosyl phosphorylation of proteins precipitated by {alpha}Stat5A from lysates of GH-treated CHO cells expressing GHR1–638 (wild-type) and GHR1–454. Tyrosyl phosphorylation of Stat5A, in response to GH, was reduced in cells expressing GHR1–454 compared with cells expressing GHR1–638 and absent in cells expressing GHR1–318, GHR1–294, or GHR{Delta}P (GHRs that do not bind JAK2) (38, and data not shown; Fig. 5BGo). Reprobing the blots with {alpha}Stat5A indicated that the observed differences in the abilities of the various CHO cells to show GH-mediated tyrosyl phosphorylation of Stat5A could not be attributed to clonal differences in the expression of Stat5A (data not shown). These results indicate that, as for GH-dependent tyrosyl phosphorylation of Stat5B, three regions are required for maximal GH-dependent phosphorylation of Stat5A: amino acids 455–638, 318–455, and 297–311 ({Delta}P). The latter two regions are required for JAK2 binding and activation (38).



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  		Figure 5. GH-dependent tyrosyl phosphorylation of Stat5A and Stat5B in CHO cells expressing mutant GHR. A, Full-length and mutant GHRs expressed in CHO cells. The lengths of the GHRs are indicated by the numbers. The hatched area indicates the transmembrane domains, separating the extracellular and cytoplasmic domains. Y denotes intracellular tyrosine residues and F denotes tyrosines mutated to phenylalanines. Box 1 is shown. Results of 125I-GH binding experiments in cells expressing the various GHRs are shown as a percent of 125I-GH binding, observed in cells expressing GHR1–638. The ability of JAK2 to associate with these receptors is indicated (34, 38). B, CHO cells expressing wild-type or mutant GHR, as indicated, were incubated without hormone or with 500 ng/ml hGH for 15 min. Proteins in whole cell lysates were immunoprecipitated with {alpha}Stat5A (1:100). Immunoprecipitates were subjected to Western blot analysis using {alpha}PY (1:7500) and reprobed with {alpha}Stat5A (1:2500). The amount of tyrosyl phosphorylation of Stat5A was quantified and the results are normalized for 125I-GH binding and expressed as a percent of Stat5A tyrosyl phosphorylation in cells expressing GHR1–638. The means of GH-dependent Stat5A tyrosyl phosphorylation ± SE (or range for n = 2) are shown. n = 16 for GHR1–454, 4 for GHR1–294 and GHR1–454Y333,338F, 3 for GHR{Delta}P, and 2 for GHR1–318. Tyrosyl phosphorylated Stat5A was not detected in cells treated without GH. The

 
Because maximal GH-dependent activation of Stat5B requires tyrosines 333 and/or 338 (35), we examined the ability of GH to stimulate tyrosyl phosphorylation of Stat5A in CHO cells expressing GHR1–454 in which tyrosines 333 and 338 are mutated to phenylalanines (GHR1–454Y333,338F). Tyrosines 333 and/or 338 are hypothesized to be the only tyrosines in GHR1–454 that are phosphorylated in response to GH (34). Statistically significant tyrosyl phosphorylated Stat5A was not detectable in GH-treated CHO cells expressing GHR1–454Y333,338F (Fig. 5BGo). This indicates that tyrosines 333 and/or 338 contribute to GH-dependent Stat5A tyrosyl phosphorylation, most likely by serving as Stat5A binding sites. The analysis of the GHR truncations and tyrosine mutations suggests that, in addition to the JAK2 binding site, multiple tyrosines (or other sequences) in GHR are required for Stat5A activation: tyrosines 333 and/or 338, as well as a tyrosine(s) (or another sequence) between amino acids 455 and 638.

JAK2 is required for GH-dependent tyrosyl phosphorylation of Stat5B
To verify that JAK2 is required for GH-dependent tyrosyl phosphorylation of Stats 5A and 5B, the ability of GH to stimulate tyrosyl phosphorylation of Stats 5A and 5B in JAK2-deficient, {gamma}2A cells and the parental cell line, 2C4, was assessed. Both cell lines lack endogenous GHR and, therefore, were stably transfected with a hGHR cDNA (2C4-GHR and {gamma}2A-GHR) (33). GH induced tyrosyl phosphorylation of Stat5B in 2C4-GHR cells (Fig. 6Go). However, no tyrosyl-phosphorylated Stat5B was observed in {gamma}2A-GHR cells in response to GH treatment, indicating that JAK2 is required for GH-dependent phosphorylation of Stat5B. Immunoprecipitation with {alpha}JAK2 and blotting with {alpha}PY revealed that JAK2 is expressed and tyrosyl phosphorylated in response to GH in 2C4-GHR, but not in {gamma}2A-GHR cells. No tyrosyl-phosphorylated Stat5A was observed in 2C4-GHR or {gamma}2A-GHR cells (data not shown). Western blots of 2C4-GHR lysates revealed that the cells do not contain detectable quantities of Stat5A (data not shown). Thus, GH-dependent Stat5A activation may require JAK2, as suggested by the data obtained in CHO cells; however, the 2C4-GHR cells do not permit a more direct analysis. The presence of Stat5B and not Stat5A in 2C4-GHR cells suggests that differential expression of the Stat5 proteins may provide a mechanism for achieving cell type-specific responses to GH.



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  		Figure 6. JAK2 is required for GH-dependent tyrosyl phosphorylation of Stat5B. 2C4-GHR cells and {gamma}2A-GHR cells were incubated without hormone (-) or with 500 ng/ml hGH (+) for 15 min. Cellular proteins were immunoprecipitated with {alpha}Stat5B(sc) (1:100) or {alpha}JAK2 (1:500) and Western blotted with {alpha}PY (1:7500).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this paper, we demonstrate that GH is capable of stimulating tyrosyl phosphorylation of both Stat5A and Stat5B in 3T3-F442A fibroblasts, CHO-GHR1–638 cells and COS-7 cells transfected with GHR cDNA, and the respective Stat5 cDNA. We further show that GH induces the DNA binding activity of Stat5A and Stat5B in transfected COS cells and 3T3-F442A fibroblasts. We and others have shown previously that GH activates Stats 1 and 3 in 3T3-F442A and CHO-GHR1–638 cells (5, 7, 35, 39). Thus, GH activates multiple Stats (1, 3, 5A, and 5B) in the same cell type, providing a means by which GH may potentially stimulate transcription of multiple genes. The observation that Stats 1, 5A, and 5B bind to the ß-casein promoter in response to GH, whereas Stats 1 and 3 bind to the SIE of the c-fos promoter (7), suggests that different combinations of Stats may serve as transcriptional activators for different genes. Cell-type specific expression of Stats, resulting in a different array of Stats being activated in response to GH in different cells, may provide a means by which tissue-specific responses to GH are achieved. For example, because of the lack of Stat5A, Stat5B (but not Stat5A) may serve as a signaling molecule for GH in 2C4-GHR cells.

Our data indicate that JAK2 is essential for GH-dependent tyrosyl phosphorylation of Stat5B. Similar results have been reported for GH-dependent activation of Stats 1 and 3 (33). We were unable to demonstrate a requirement for JAK2 in GH-dependent Stat5A tyrosyl phosphorylation using 2C4-GHR/{gamma}2A-GHR cells because of the absence of Stat5A in these cells. The absence of Stat5A may explain the apparent discrepancy between this work and an earlier work that was unable to demonstrate GH-dependent activation of Stat5 in a sister cell line using sheep Stat5 antibody (33). However, the observation that mutated GHRs that activate JAK2 expressed in CHO cells also stimulate tyrosyl phosphorylation of Stat5A, whereas mutated GHRs that fail to activate JAK2 also fail to stimulate tyrosyl phosphorylation of Stat5A, is consistent with JAK2 being required for GH-dependent tyrosyl phosphorylation of Stat5A, as well as for Stat5B.

We have demonstrated previously that, in addition to the regions of GHR required for JAK2 activation, the C-terminal half of the cytoplasmic domain of GHR and tyrosines 333 and/or 338 in the N-terminal half are required for maximal Stat5B tyrosyl phosphorylation and binding to a GAS element in the ß-casein promoter (35). The observation that Stat5A tyrosyl phosphorylation is decreased in CHO cells expressing GHR1–454 and further decreased in cells expressing GHR1–454Y333,338F suggests that, like Stat5B, maximal GH-dependent Stat5A tyrosyl phosphorylation requires sequences in the C-terminal half of rGHR and tyrosines 333 and/or 338. The requirement of the C-terminal half of GHR for maximal Stat5 activation is suggested also by the finding that specific tyrosines in the C-terminal half of the porcine GHR are required for GH-dependent transcription of the Spi2.1 promoter, a promoter known to bind Stat5 (40). Whether the requirement of tyrosines 333 and/or 338 for a maximal response is caused by Stat5 proteins binding to these tyrosines, or a result of a conformational change of the GHR as a result of substituting these tyrosines with phenylalanines, is not known.

Heterodimerization of Stat5A and Stat5B in murine mammary gland, in response to PRL, has been reported (41). The observations that Stat5A and Stat5B require the same regions of GHR for tyrosyl phosphorylation, and in 3T3-F442A fibroblasts {alpha}Stat5A immunoprecipitates some Stat5B and vice versa, raise the possibility that Stat5A and Stat5B may form heterodimers before being translocated to the nucleus. The role of Stat5 homo- vs. heterodimers in GH-dependent activation of transcription remains to be determined.

Although the requirements for GH-dependent tyrosyl phosphorylation of Stat5A and Stat5B seem to be similar or identical, the patterns of bands observed in {alpha}Stat5A and {alpha}Stat5B immunoprecipitations are different. A doublet or a single broad Stat5A was observed in 3T3-F442A and transfected COS and CHO cell lysates, which becomes tyrosyl phosphorylated upon treatment with GH. We believe that, because the two proteins in the doublet migrate so closely together, they are often visualized as a single band. In addition, two smaller, nontyrosyl phosphorylated proteins are observed (approximately 72 and 60 kDa). In contrast, three distinct Stat5B bands were detected in untreated 3T3-F442A and transfected COS and CHO cells, with an additional, slower migrating band observed with GH treatment. The three more slowly migrating Stat5B bands observed, in response to GH, contain tyrosyl phosphorylated proteins, as revealed by Western blotting with {alpha}PY. The multiple Stat5A and Stat5B bands cannot be explained by the alternatively spliced products reported by others (26, 27), because these smaller products lack the C-terminal epitope against which {alpha}Stat5A and {alpha}Stat5B(yl) were raised. The multiple Stat5A and Stat5B bands of approximately 90 kDa are likely to represent multiple phosphorylation states, with a greater number of phosphorylated states existing for Stat5B than for Stat5A. Multiple Stat5B bands have been detected also in rat liver, with two bands containing phosphorylated tyrosines (39). Experiments in rat liver have suggested that the slower migrating tyrosyl phosphorylated Stat5B protein also contains phosphorylated seryl and/or threonyl residues. If Stat5B is seryl and/or threonyl phosphorylated, it is likely to be by a kinase other than MAP kinase, because the conserved seryl residue found within the consensus site for phosphorylation by MAP kinase in other Stat family members is not present in Stat5B. Furthermore, the MAP kinase pathway is not required for lactogen-induced tyrosyl phosphorylation of Stat5A or Stat5B in mammary epithelial cells or transcriptional activation of a ß-casein reporter construct, as evidenced by the lack of an effect of the MAP kinase inhibitor, PD98059, on these events (42). This suggests that GH may induce seryl and/or threonyl phosphorylation of both Stat5A and Stat5B via a MAP kinase-independent pathway. Stat5A and Stat5B do contain multiple conserved putative protein kinase C and casein kinase II phosphorylation sites, suggesting the possible involvement of one or both of these kinases in Stat5 activation in response to GH. Protein kinase C{alpha} has been implicated in PRL activation of the ß-casein gene expression by Stat5 (43). A more complete identification of the Stat5A and Stat5B bands will require further study and will facilitate a better understanding of GH-initiated signal transduction via Stat5 proteins.


   Acknowledgments
 
We thank P. Du and P. Ho for technical assistance with experiments and cell culture; and Drs. H. Rui, J. Herrington, L. Argetsinger, G. Campbell, and D. Meyer for helpful discussions. We also thank Drs. G. Stark, I. Kerr, D. Watling, and N. Rogers for kindly providing the {gamma}2A-GHR and 2C4-GHR cells; Dr. Jeffrey Rosen for providing the Stat5A cDNA; and Drs. G. Norstedt and N. Billestrup for providing the transfected CHO cells.


   Footnotes
 
1 This work was supported by research grants from NIH (R01-DK-34171 to C.C.-S. and J.S., and R01-DK-44625 to L.-y.Y.-L.) and from NSF (IBN-9221667 to J.S.). Back

2 L.S.S. is a postdoctoral fellow of the Cancer Research Institute. J.A.V. is a postdoctoral fellow of the Arthritis Foundation. Both authors contributed equally. Back

Received January 8, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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A. S. Verma, R. N. Dhir, and B. H. Shapiro
Inadequacy of the Janus Kinase 2/Signal Transducer and Activator of Transcription Signal Transduction Pathway to Mediate Episodic Growth Hormone-Dependent Regulation of Hepatic CYP2C11
Mol. Pharmacol., March 1, 2005; 67(3): 891 - 901.
[Abstract] [Full Text]