Conversion of Threonine 757 to Valine Enhances Stat5a Transactivation Potential*

P. Mangala GowriDagger , Tanmoy C. GangulyDagger §, Jingsong CaoDagger , Madhav N. DevalarajaDagger , Bernd Groner||, and Mary VoreDagger **

From the Dagger  Graduate Center for Toxicology, University of Kentucky, Lexington, Kentucky 40536-0305 and the || Georg Speyer Haus, Paul Ehrlich Strasse 42-44, 60596 Frankfurt, Germany

Received for publication, August 7, 2000, and in revised form, November 30, 2000


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The growth hormone family of cytokines transduces intracellular signals through the Jak2-Stat5 pathway to activate the transcription of target genes. Amino acids within the C termini of Stats constitute the transactivation domain but also regulate the time course of tyrosine phosphorylation and extent of DNA binding. We mutated Thr757 in the C-terminal of Stat5a (Thr-Stat5) to Val (Val-Stat5) and Asp (Asp-Stat5) and examined the effect on nuclear translocation, DNA binding, and prolactin-induced transcriptional activation of a Stat5-responsive luciferase reporter gene. Val-Stat5 produced a 5-fold higher increase in transcriptional activity relative to Thr-Stat5; Asp-Stat5 produced a similar response to Thr-Stat5. The increased transactivation was ligand induced and was not due to differences in basal expression of Val-Stat5 or to a constitutively activated Stat5 protein. Similar rates of loss of DNA binding ability and phosphorylation of Val- and Thr-Stat5 were observed following a single pulse of prolactin, indicating that the dephosphorylation pathways were unaltered. The serine-threonine kinase inhibitor H7 inhibited the transactivation potential of Thr-, Val-, and Asp-Stat5 to a similar extent, eliminating phosphorylation of Thr757 as a regulatory mechanism. The results suggest that Thr757 modulates the transactivation potential of Stat5 by a mechanism(s) that is dependent on the formation of Stat5 dimers and/or their nuclear translocation.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Stat (signal transducers and activators of transcription) proteins constitute a family of latent cytoplasmic transcription factors that are activated by a large number of cytokines and growth factors (1-3). Ligand binding by the cytokines to their cognate cell surface receptors leads to a conformational change in the receptor, which in turn results in activation of the receptor-associated Janus kinases (Jak).1 The activated Jak phosphorylate the receptor at specific tyrosine residues within the cytoplasmic domain, thus recruiting the Stat proteins, which bind via their Src homology 2 (SH2) domains (2). Jak-mediated tyrosine phosphorylation of the receptor-bound Stats leads to their dissociation from the receptor, dimerization by means of reciprocal SH2-phosphotyrosine interactions and translocation to the nucleus, where the Stat dimers bind to specific DNA sequences and induce gene transcription. Seven mammalian members of the Stat family have been reported and show a high degree of similarity. The highest degree of similarity occurs in the SH2 domain, whereas the C-terminal domain is the least conserved region (4). C-terminal deletion mutants and alternatively spliced variants lacking this region have provided evidence that this region serves as the transactivation domain (5). The transactivation domain of Stat5 has been mapped to a region between amino acids 750-772 (6). Deletion of the C terminus of Stat5 resulted in a dominant negative phenotype with sustained tyrosine phosphorylation and DNA binding relative to the wild type but with no transcriptional activation.

Members of the growth hormone family of cytokines such as prolactin (PRL) and growth hormone transduce intracellular signals via activation of the Jak2-Stat5 signal transduction pathway to regulate the transcription of a variety of genes (7-13). The specificity of transcriptional regulation by Stat5 is believed to result from its association with coactivators and corepressors of transcription and binding of additional transcription factors to the promoter regions of target genes (14). We have demonstrated that PRL acts via the long form of the PRL receptor (PRLRL) to facilitate the binding of Stat5 multimers to two interferon-gamma -activated sequences (GAS)-like elements in the promoter of the rat liver ntcp (Na+/taurocholate cotransporting polypeptide) gene to increase its transcription (15). Obligatory tyrosine phosphorylation at Tyr694 and subsequent serine/threonine phosphorylation has been demonstrated for hepatic Stat5 activation following growth hormone stimulation (9, 10, 16). Stat5a and Stat5b are highly homologous genes that share about 96% amino acid sequence homology (17-20). The amino acids within the C-terminal region in Stat5a and Stat5b isoforms from various species are conserved and contain a serine/threonine at position 757, which could serve as a potential phosphorylation site. Two serine residues (Ser779 and Ser725) in the C-terminal domain of Stat5a have been shown to be phosphorylation sites. Although mutation of Ser779 to Ala had no effect on PRL-induced transcriptional activation of a beta -casein reporter construct, mutation of Ser725 to Ala resulted in prolonged DNA binding activity following PRL treatment (21). Thus, serine phosphorylation may serve to modulate Stat5a transactivation. The present studies were designed to evaluate the role of the conserved Thr757 in transcriptional regulation of ntcp and used site-directed mutagenesis to alter Thr757 to Val and Asp and compare the transactivation properties of these mutants to those of wild type Stat5. We also studied the effect of the serine/threonine kinase inhibitor, H7, on the transactivation potential of wild type Stat5 and its mutants. The present studies show that the mutation of Thr757 to Val markedly increased the transactivation potential of Stat5, whereas mutation to Asp did not significantly alter this potential.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Reagents and Plasmids-- All reagents were of molecular biology or cell culture grade. Polynucleotide kinase was obtained from Life Technologies, Inc. Ovine PRL was kindly provided by Dr. A. F. Parlow of the National Institute of Diabetes and Digestive and Kidney Diseases, National Hormone and Pituitary Program. Stat5 monoclonal antibodies raised against ovine Stat5 amino acids 451-469 were obtained from Transduction Laboratories (Lexington, KY). Polyclonal antiserum directed against Stat5a was from Zymed Laboratories Inc. (San Francisco, CA), and anti-phosphotyrosine antibody (PY20) was from Santa Cruz Biotechnology (Santa Cruz, CA). The cDNA of PRLRL was a gift from Dr. Paul Kelly (INSERM, Paris, France). Plasmid pRSV-beta -galactosidase was kindly provided by Dr. Noonan of the University of Kentucky. The luciferase reporter plasmid 4X0.2SpGL3 was constructed as described (15) by linking four GAS elements (TTCTTGGAA) to the ntcp minimal promoter (-158 to +47) and ligating this fragment in the HindIII site of pGL3 basic vector (Promega, Madison, WI). Plasmid 0.5pT109luc was constructed by inserting the 500-base pair Hindlll fragment of the ntcp promoter (-1237 to -758) upstream of the herpes simplex virus thymidine kinase minimal promoter (HSVtk) in pT109Luc (American Type Culture Collection, Manassas, VA) (15). Plasmid DNA was extracted and purified using the Qiagen midicolumns or by CsCl gradient centrifugation twice. The inhibitor, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine·2HCl (H7) was obtained from Alexis Biochemicals (San Diego, CA).

Construction of Mutants-- Val-Stat5 was generated using the "megaprimer" polymerase chain reaction method (22) using 5'-GGGAGTACTACACTCCTGTGCTTGCCAAA-3' as the forward primer, 5'-GGGACTAGTCAACATTCAGGAGAGCGAGCCT-3' as the reverse primer, 5'-GGCCACATCCATGACCTCGTCCAGGTC-3' as the mutagenesis primer, and the 2.4-kilobase NotI-SalI fragment of Thr-Stat5 as a template. Asp-Stat5 was constructed using 5'-GGCCACATCCATGTCCTCGTCCAGGTC-3' as the mutagenesis primer. The 0.4-kilobase polymerase chain reaction product was digested with SpeI and ScaI and ligated with the 2-kilobase SalI-ScaI fragment of ovine Stat5 and subsequently ligated to SalI-SpeI digested pXM vector. Integrity of all plasmids was ascertained by sequencing.

Cell Culture and Transfection-- HepG2 cells were cultured in Dulbecco's modified Eagle's medium/Ham's F-12 medium with 10% fetal bovine serum as described (15). Cells were transfected using the calcium phosphate:DNA coprecipitation method (23). A 10-cm plate of cells was transfected with 5 µg of luciferase reporter constructs 4X0.2pGL3 or 0.5pT109luc, 5 µg of expression vectors for Thr-Stat5 (pXM-Thr-Stat5)/Val-Stat5 (pXM-Val-Stat5)/Asp-Stat5 (pXM-Asp-Stat5), 1 µg of expression vector for the PRLRL (pL3-PRLRL), and pUC19 as carrier, to a total of 20 µg. pRSV-beta -galactosidase (5 µg) was included in each transfection to monitor transfection efficiency. Single transfections were conducted in triplicate, and the mean was calculated for each data point. The data are the means (± S.E.) for three independent transfections.

Luciferase and beta -Galactosidase Assays-- Cells were treated with varying concentrations of PRL 6-8 h post-transfection. Luciferase and beta -galactosidase assays were performed 40-44 h post-transfection, and the normalized luciferase response was determined as relative light units divided by beta -galactosidase activity (A415 nm/min). The hormone-dependent fold induction was calculated relative to normalized luciferase response obtained in the absence of PRL treatment.

Studies with the Serine/Threonine Kinase Inhibitor-- To investigate the effect of the serine/threonine kinase inhibitor H7, HepG2 cells were trypsinized 6 h post-transfection, dispensed in 96-well plates, and incubated overnight. H7 (100 µM) was added to the cells, followed after 1 h by treatment with increasing doses of PRL. Luciferase activity was determined after 6 h of incubation with ligand.

Preparation of Nuclear Extracts and Electromobility Shift Assays (EMSA)-- Whole cell or nuclear extracts were prepared as described (24). Cells were transfected with recombinant plasmids and 6-8 h post-transfection were washed twice with phosphate-buffered saline (PBS). Fresh medium was added to the plate, and the cells were incubated for a further 36-40 h. PRL (0.5 µg/ml) was then added directly to the cells in the 10-cm culture dish and removed at the indicated times. For nuclear extracts, cells were washed with PBS, scraped and collected in cold PBS, pelleted, and resuspended in cold buffer A (10 mM HEPES, pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 1 mM NaF, 0.5 mM DTT, 0.2 mM PMSF, 1 µg/ml pepstatin, 5 µg/ml aprotinin, 2 µg/ml leupeptin, and 5 µg/ml antipain). The suspension was incubated on ice for 10 min and centrifuged for 10 s at high speed. The pellet was resuspended in 20-100 µl of cold buffer C (20 mM HEPES, pH 7.9, 25% glycerol, 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, pH 8.0, 1 mM Na3VO4, 10 mM NaF, 0.5 mM DTT, 0.2 mM PMSF, and the protease inhibitors at the above mentioned concentrations), incubated on ice for 20 min, and centrifuged at 4 °C for 2 min, and the extract was aliquoted and stored at -80 °C until further use. For whole cell extracts, cells were collected in ice-cold PBS. After a brief centrifugation, cells were suspended in ice-cold extraction buffer containing 400 mM NaCl, 50 mM KCl, 20 mM HEPES buffer, pH 7.9, 1 mM EDTA, pH 8.0, 20% glycerol, 1 mM Na3VO4, 1 mM NaF, 1 mM DTT, 0.2 mM PMSF, and protease inhibitors at the same concentrations as above. The cells were lysed with three freeze-thaw cycles (15 min at 37 °C, 15 min at -70 °C), followed by centrifugation at 20,800 × g at 4 °C for 15 min. The supernatant was removed and stored at -70 °C. The protein concentration in the nuclear and whole cell extracts was estimated by the procedure of Lowry et al. (25). The protease inhibitors, DTT and PMSF were added to the buffers just before use.

Double-stranded consensus Stat5 oligonucleotide (5'-agatTTCTAGGAAttcaatcc-3') was used as probe for EMSA. The probe was radiolabeled with [gamma -32P]ATP using polynucleotide kinase at the 5'-OH (blunt) ends. The labeled probe was gel eluted in 1× NET buffer (0.1 M NaCl, 1 mM EDTA, 1 mM Tris-HCl, pH 7.6) after polyacrylamide (12%) gel electrophoresis. Briefly, 10 µg of protein was incubated for 20 min at room temperature with 10 fmol of purified probe in a 15-µl buffer consisting of 5 mM Tris-HCl, pH 7.9, 15 mM HEPES-KOH, pH 7.9, 0.08 M KCl, 3.5 mM MgCl2, 5 mM EDTA, 5 mM DTT, 10% glycerol, 0.1% Tween 20, and 0.133 mg/ml (dI-dC):poly(dI-dC). Unbound probe was separated from protein-bound probe in a 4% polyacrylamide gel containing 2.5% glycerol and 0.25× TBE (25 mM Tris-HCl, 25 mM boric acid, and 0.25 mM EDTA, pH 8.0). The gel was dried and exposed to film at -80 °C. For competition assays, a 10-50-fold molar excess of unlabeled oligomer was added to the incubation mixture. For supershift studies, the medium was incubated with Stat5 monoclonal antibody for 15 min at 4 °C and 30 min at room temperature, before the addition of the labeled probe.

Western Blots-- Proteins from whole cell extracts or nuclear extracts from unstimulated or PRL-stimulated (0.5 µg/ml) cells were immunoprecipitated with polyclonal antiserum directed against Stat5a or PY20, prior to immunoblotting with either PY20 or a Stat5a (1:3,000 dilution) antibody. An anti-rabbit or anti-mouse IgG horseradish peroxidase antibody (1:5,000) was used as a secondary antibody, and the proteins were visualized by the ECL detection system (Amersham Pharmacia Biotech). Bands were digitized and quantified by computer-assisted imaging using MCID/M4 software supplied by Imaging Research Inc. (St. Catharines, Ontario, Canada).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Val-Stat5 Exhibits a Higher Transactivation Potential than Thr-Stat5 or Asp-Stat5-- Within the C terminus of Stat5 is a stretch of 22 amino acids (750) indispensable for PRL-induced transactivation (6, 26). The conserved sequence, FDL(D/E)(E/D)V(T/S)DVARHVEELLRRPMD, in this transactivation domain in the Stat5a and Stat5b isoforms of various species contains a single threonine/serine as a potential phosphorylation site (6). We mutated the Thr757 in Stat5a to a Val or an Asp to determine the effect of loss of this putative phosphorylation site and replacement with an acidic amino acid residue, respectively, on the transactivation potential of Thr-Stat5. When cells were transfected with Val-Stat5 and stimulated with PRL, a dose-dependent increase in the transcription of the reporter construct 4X0.2pGL3 was observed. This induction was 5-5.5 times greater than that in cells transfected with Thr-Stat5 (Fig. 1A). The response of cells transfected with Asp-Stat5 and the 4X0.2pGL3 construct was similar to that of cells transfected with Thr-Stat5. The basal luciferase activity remained unaltered (Fig. 1B), indicating that neither of the mutants was constitutively active. In transfection experiments with the reporter construct of the ntcp promoter containing the two native GAS elements coupled to the thymidine kinase promoter, Val-Stat5 also showed a significantly increased transactivation potential relative to that of Thr-Stat5 and Asp-Stat5 (Fig. 1C).


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Fig. 1.   Comparison of the transactivation potentials of mutants versus wild type Stat5. HepG2 cells were transfected with 4X0.2pGL3 (A and B) or 0.5pT109luc (C), expression vectors for Thr-Stat5 (black-square), Val-Stat5 (), or Asp-Stat5 (black-triangle), PRLRL, and beta -galactosidase. Cells were treated with increasing concentrations of PRL and luciferase activity assayed after 40-44 h. A, Val-Stat5 exhibited greater transactivation potential than Thr-Stat5 or Asp-Stat5. B, estimation of basal luciferase activity showed that the mutants were not constitutively active. C, Val-Stat5 also showed a higher transactivation potential in cells transfected with the ntcp promoter containing the two native GAS elements.

Effect of the Serine/Threonine Kinase Inhibitor H7 on the Transactivation Potential of Thr-, Val-, and Asp-Stat5-- Protein kinase inhibitors of the H series are the most commonly used inhibitors of serine/threonine phosphorylation (27). H7 is one of the three widely used inhibitors and inhibits protein kinase C, in addition to cAMP- and cGMP-dependent protein kinase (cAPK and cGPK, respectively). To determine whether phosphorylation of Thr757 of Stat5 plays a role in signal transduction, HepG2 cells pretreated with vehicle or H7 for 1 h were treated for 6 h with increasing doses of PRL before determining the luciferase activity. H7 decreased the PRL-induced transcription of the reporter gene by ~90% at 1 µg/ml PRL. This observation is consistent with previous reports that serine/threonine phosphorylation of Stat molecules is required for the activation of its DNA binding and subsequent transactivation potential (5, 16, 28). If the phosphorylation of Thr757 were essential for Stat5 activation, then the Val-Stat5 and Asp-Stat5 mutants should not be sensitive to the inhibitor. However, the effect of H7 was similar in the wild type and both Val-Stat5 and Asp-Stat5 transfected cells (Fig. 2).


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Fig. 2.   Effect of a serine/threonine kinase inhibitor on the transactivation potential of Thr-, Val-, and Asp-Stat5. HepG2 cells were transfected with 4X0.2pGL3, expression vectors for Thr-Stat5 (open bars), Val-Stat5 (black bars), and Asp-Stat5 (hatched bars), PRLRL, and beta -galactosidase. Cells were pretreated with 100 µM H7 and then exposed to increasing concentrations of PRL for 6 h before quantitation of luciferase activity.

Increased Nuclear Localization of Val-Stat5 as Compared with Thr-Stat5-- We carried out further studies comparing Thr-Stat5 and Val-Stat5 to investigate the mechanism of the enhanced response to Val-Stat5. The increase in luciferase activity in cells transfected with Val-Stat5 versus Thr-Stat5 was not due to differences in their expression, because similar levels of immunoreactive Val- and Thr-Stat5 protein were present in unstimulated whole cell extracts (Fig. 3A, lanes 1 and 2). In contrast, increased levels of immunoreactive Stat5 were observed in nuclear extracts from Val-Stat5-transfected cells relative to those obtained from Thr-Stat5-transfected cells following stimulation with PRL for 1 or 6 h (Fig. 3A, compare lanes 4 and 6 with lanes 3 and 5). The increased Stat5 levels in the nuclear extracts from Val-Stat5 transfected cells were observed as early as 5 min after PRL induction (Fig. 3B).


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Fig. 3.   Western blots of basal and PRL-induced levels of Thr-Stat5 and Val-Stat5. A, whole cell extracts (200 µg of protein) from unstimulated Thr-Stat5 and Val-Stat5 transfected cells (lanes 1 and 2, respectively) or nuclear extracts (200 µg of protein) prepared from Thr-Stat5-transfected (lanes 3 and 5) and Val-Stat5-transfected (lanes 4 and 6) cells 1 and 6 h after stimulation with 0.5 µg/ml PRL, respectively, were immunoprecipitated with 5 µg of polyclonal Stat5a antibody. Western blots were performed on immunoprecipitated proteins using polyclonal Stat5a antibody. B, nuclear extracts prepared from Thr-Stat5 or Val-Stat5 transfected cells without (lanes 1 and 2) or with exposure to 0.5 µg/ml PRL for 2 min (lanes 3 and 4), 5 min (lanes 5 and 6), and 15 min (lanes 7 and 8) were immunoprecipitated with 2 µg of anti-phosphptyrosine antibody (PY-20), and Western blots were performed using mouse monoclonal Stat5 antibody. Samples in lanes 1, 3, 5, and 7 are from Thr-Stat5-transfected cells, and those in lanes 2, 4, 6, and 8 are from Val-Stat5-transfected cells.

Increased Transactivation Potential of Val-Stat5 Correlates with Increased Nuclear Translocation and DNA Binding Ability-- To investigate the mechanism(s) for the enhanced transactivation potential of Val-Stat, nuclear extracts from transfected cells were probed for DNA binding ability. In the absence of transfected Stat5, no specific DNA binding ability was observed in HepG2 cells transfected with PRLRL and treated with PRL (Fig. 4A, lane 2 versus lane 4), indicating nondetectable levels of endogenous Stat5. These results are consistent with our studies that showed no PRL-mediated induction of luciferase activity in HepG2 cells transfected with PRLRL and an ntcp-promoter luciferase construct under these same conditions (15). Consistent with observations of low basal levels of luciferase activity, no specific DNA binding ability was observed in unstimulated cells (Fig. 4B, lanes 1 and 2). Specific DNA-protein complex formation was observed in both Thr-Stat5- and Val-Stat5-transfected cells as early as 15 min following stimulation with PRL, with DNA-binding of Val-Stat5 being higher than that of Thr-Stat5 at all time points up to 24 h (Fig. 4, B and C). The observed bands were confirmed as Stat5 by incubation with the anti-Stat5 antibody that supershifted the Stat5 complex (Fig. 4C, lanes 11 and 12). The specificity of the Stat5 bands was also confirmed by coincubation with 100-fold excess unlabeled Stat5 oligomer in the EMSA reaction, which markedly reduced the signal (Fig. 4C, lanes 13 and 14).


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Fig. 4.   Increased nuclear translocation and DNA binding ability of Val-Stat5 versus Thr-Stat5. HepG2 cells were transfected with expression vectors for Thr-Stat5 or Val-Stat5 and/or PRLRL and were either untreated or treated with 0.5 µg/ml PRL for varying time periods. A, 32P-labeled Stat5 consensus oligonucleotide was incubated with nuclear extracts from untransfected cells (lane 1) or cells transfected with PRLRL (lane 2) or Thr-Stat5 (lane 3) or both PRLRL and Thr-Stat5 (lane 4). B, 32P-labeled Stat5 consensus oligonucleotide was incubated with nuclear extracts from Thr-Stat5-transfected (lanes 1, 3, 5, and 7) or Val-Stat5-transfected (lanes 2, 4, 6, and 8) cells that were either untreated (lanes 1 and 2) or treated for 15 min (lanes 3 and 4), 30 min (lanes 5 and 6), or 60 min (lanes 7 and 8) with PRL, respectively. C, 32P-labeled Stat5 consensus oligonucleotide was incubated with nuclear extracts from Thr-Stat5- or Val-Stat5-transfected cells that were either untreated (lanes 1 and 2) or treated for 1 h (lanes 3 and 4), 6 h (lanes 5 and 6), 12 h (lanes 7 and 8), and 24 h (lanes 9 and 10), respectively, with PRL (0.5 µg/ml). Nuclear extracts prepared from Thr-Stat5-transfected (lanes 11 and 13) or Val-Stat5-transfected (lanes 12 and 14) cells 1 h after PRL treatment were treated with 1 µg of polyclonal rabbit Stat5a antibody to induce a supershift (SS) complex (lanes 11 and 12) or exposed to a 100-fold molar excess of unlabeled Stat5 consensus oligonucleotide (lanes 13 and 14).

The increase in specific DNA binding in cells transfected with Val-Stat5 did not seem to be due to increased affinity for the cognate DNA-recognition sequence but to increased nuclear concentrations. Incubation of the nuclear extracts of Thr-Stat5 and Val-Stat5 with increasing concentrations of unlabeled Stat5 consensus oligomer competed for specific Thr- and Val-Stat5 DNA binding with similar efficiency (Fig. 5A). There was no major change in the binding specificity, because incubation of the nuclear extracts with either the Stat3 oligomer (upper strand, 5'-gatccTTCTGGGAAtcctagatc-3') or an oligomer containing a GAS element essentially identical to that activated by interferon-gamma in the human interferon regulatory factor 1 gene promoter (31) (upper strand 5'-taattTTCCCCGAAgtaca-3') did not alter the Stat5 consensus oligomer binding to the proteins (Fig. 5B). In coexpression experiments, increasing the amounts of Thr-Stat5 plasmid partially decreased the transactivation produced by Val-Stat5, suggesting that the two proteins compete for the same DNA binding sequence (data not shown). A direct measurement of the "off time" of the preformed DNA-wild type or mutant protein complex using equilibrium displacement experiments was inconclusive because of the inherent low affinity of the Stat5 protein for the DNA recognition site and the rapid rate of dissociation. Thus, a 50-fold molar excess of competing unlabeled Stat5 oligomer displaced greater than 90% of the specifically bound complex within 1 min (data not shown).


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Fig. 5.   Thr-Stat5 and Val-Stat5 exhibit similar Stat5 DNA binding affinity. Nuclear extracts were prepared from HepG2 cells transfected with expression vectors for Thr-Stat5 or Val-Stat5 and PRLRL and treated for 1 h with PRL (0.5 µg/ml). A, nuclear extracts from Thr-Stat5-transfected (lanes 1, 3, 5, and 7) or Val-Stat5-transfected (lanes 2, 4, 6, and 8) cells were incubated with 1000-fold (lanes 1 and 2), 500-fold (lanes 3 and 4), 50-fold (lanes 5 and 6), and 10-fold (lanes 7 and 8) molar excess of unlabeled Stat5 oligomer before competition with 32P-labeled Stat5 consensus oligonucleotide. B, nuclear extracts from Thr-Stat5-transfected (lanes 1, 3, 5, and 7) or Val-Stat5-transfected (lanes 2, 4, 6, and 8) cells were incubated with a 50-fold molar excess of unlabeled Stat3 (lanes 1 and 2), IRF-1 (lanes 3 and 4), or Stat5 (lanes 5 and 6) consensus oligonucleotides before competition with 32P-labeled Stat5 consensus oligonucleotide. Lanes 7 and 8 represent experiments in which nuclear extracts were treated with 1 µg of polyclonal Stat5a antibody to induce a supershift (SS) complex.

Increased Nuclear Levels of Val-Stat5 Are Not a Consequence of Delayed Dephosphorylation-- Dephosphorylation of Stat5 Tyr694, rather than Stat5 protein degradation, has been shown to be the primary determinant of down-regulation of its DNA binding ability (6, 24, 32). The region between amino acids 750 and 794 exhibits a role in down-regulating the DNA binding activity of Stat5. We postulated that this region might regulate the interaction between Thr-Stat5 and a phosphatase, which by dephosphorylation of Tyr694, would lead to down-regulation of Stat5 DNA binding ability. Accordingly, mutation of Thr757 to Val could disrupt the interaction of the phosphatase with Stat5, leading to a delayed dephosphorylation of Tyr694 and/or other serine/threonine residues. This would increase the steady-state levels of the translocated Stat5 multimers and facilitate binding to the GAS elements, thus allowing for an increased transactivation potential. To test this possibility, cells transfected with either Thr-Stat5 or Val-Stat5 were treated with a single pulse of PRL for 30 min, after which the medium was removed and replaced with ligand-free medium. Nuclear extracts prepared at various times thereafter were then subjected to EMSA. These nuclear extracts were also immunoprecipitated with anti-phosphotyrosine antibody (PY-20) and probed with Stat5a antibody by Western analysis. Both Thr- and Val-Stat5 exhibited a rapid decrease in their DNA binding ability, such that by 2 h, no DNA binding ability could be detected (Fig. 6A). The DNA-protein complexes formed were quantitated and plotted as a function of time. As shown in Fig. 6B, the DNA-protein complexes decayed with very similar kinetics. Similar rates of loss were also observed when the nuclear extracts were first immunoprecipitated with anti-phosphotyrosine antibody and the levels of phosphorylated Stat5 were visualized with a polyclonal Stat5a antibody by Western analysis (Fig. 6, C and D).


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Fig. 6.   Thr-Stat5 and Val-Stat5 exhibit similar rates of decay of DNA binding ability and dephosphorylation. HepG2 cells were transfected with expression vectors for Thr-Stat5 or Val-Stat5 and PRLRL. Cells were stimulated with a single 30-min pulse of PRL (0.5 µg/ml) after which the medium was replaced. A, 32P-labeled Stat5 consensus oligonucleotide was incubated with nuclear extracts from Thr-Stat5-transfected (lanes 1, 3, 5, and 7) or Val-Stat5-transfected (lanes 2, 4, 6, and 8) cells immediately following PRL stimulation (lanes 1 and 2), or 30 (lanes 3 and 4), 60 (lanes 5 and 6), and 120 min (lanes 7 and 8) following incubation in PRL-free medium. B, a plot of the optical density of the nuclear Stat5a complex versus time in Thr-Stat5-transfected () and Val-Stat5-transfected (open circle ) cells shown in A. C, nuclear extracts (100 µg) from Thr-Stat5- or Val-Stat5-transfected cells were immunoprecipitated with 5 µg of anti-phosphotyrosine antibody (PY-20) followed by Western analysis using polyclonal Stat5a antibody. The numbering of the lanes is the same as for A. D, a plot of the optical density of Stat5a versus time in Thr-Stat5-transfected () and Val-Stat5-transfected (open circle ) cells shown in C.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Signal transduction via the Stat5 pathway requires the recruitment of the latent cytoplasmic transcription factor to the phosphorylated tyrosine residue in the cytoplasmic domain of the receptor, phosphorylation of a key tyrosine (Tyr694) in Stat5 by the receptor-associated Jak, followed by the interaction of the SH2 domain, and formation of dimers by each of the phosphotyrosines in the two Stats in a reciprocal manner. The dimers are then translocated to the nucleus, where they bind to the GAS elements via their DNA-binding domain and direct an increase in specific transcriptional activity by the recruitment of the general transcription factors via their transactivation domain. A number of studies have demonstrated that amino acids 750-772 in the C termini of Stat5 are essential for the increase in transcriptional activity, and may also act to down-regulate activity by increasing the dephosphorylation of Tyr694. Other studies have demonstrated that although tyrosine phosphorylation is obligatory to Stat5 transcriptional activation, serine phosphorylation can modulate the binding to DNA (5). Thus, mutation of Ser725 to Ala in Stat5a resulted in sustained DNA binding activity relative to that of wild type Stat5a but with no change in transcriptional activation (21). The increased DNA binding observed in the present experiments is similar to the increased binding observed with C-terminal deletion mutants and naturally occurring splice variants of Stat5a and Stat5b (6, 30). However, unlike the C-terminal deletion variants of Stat5a and Stat5b that exert a dominant negative phenotype, mutation of Thr757 to Val resulted in a higher transactivation potential, whereas simulation of a phosphorylated site by mutation to Asp had no effect on activity. These data suggest that Thr757 is not an essential site for phosphorylation in the induction process mediated by the wild type version of Stat5. The observation that incubation with the serine/threonine kinase inhibitor H7 equally inhibited transactivation by wild type Stat5a and its mutants further supports the argument that Thr757 is not a site for phosphorylation of Stat5a. The similar inhibitory effect of H7 on Thr-, Val-, and Asp-Stat5-mediated transcription suggests that other phosphorylation sites that contribute to Stat5 function are targets for the serine/threonine kinase inhibitor. However, our results are consistent with reports that Stat5a/5b isoforms are phosphorylated at tyrosine and serine, but not threonine residues, following activation with PRL or interleukin-2 in a variety of cell lines (33-35).

Because dephosphorylation of Stat5 Tyr694 is the primary regulator of down-regulation of its DNA binding ability and amino acids 750-794 have been shown to down-regulate the DNA binding activity of Stat5, we postulated that Thr757 might interact with a phosphatase that mediated the dephoshorylation of Tyr694. However, when we compared the rates of loss of DNA binding ability and dephosphorylation of Thr- and Val-Stat5 following a pulse dose of oPRL, they were both very similar. These observations indicate that the increased transactivation potential of Val-Stat5 is not a consequence of delayed dephosphorylation of this mutant protein but rather because of an increased rate of formation and/or nuclear translocation of Stat5 dimers.

Several mechanisms could be involved in the enhanced transactivation by Val-Stat5. Overcoming negative regulation could be one of the mechanisms by which Val-Stat5 elicits an increased response. The SOCS (suppressors of cytokine signaling) family of inhibitors either bind the activated Jaks or compete with Stat5 for binding to the receptor to bring about inhibition (reviewed in Ref. 36). Because the mutation is in Thr757 of Stat5 and not in the receptor or Jak2, such a mechanism is unlikely. Delayed dephosphorylation was experimentally ruled out as a possible reason for increased Val-Stat5 transactivation. The coactivators and corepressors of transcription like CBP/p300 are known to interact with the C-terminal of the activated Stat5 (37). However, in cotransfection experiments, we found no evidence of increased interaction of Val-Stat5 with p300, compared with Thr-Stat5 (data not shown). The most likely mechanism of enhanced transactivation by Val-Stat5, therefore, is either a more effective association between Val-Stat5 and Jak2 to facilitate phosphorylation by Jak2, increased stability and translocation of the Val-Stat5 dimers following activation, or both. Crystallographic studies have shown that in Stat dimers, the C termini are in very close proximity, whereas the N termini are far apart, forming a saddle-like structure around the DNA (38). The present data suggest that replacement of the OH group of Thr757 with the methyl group of Val results in increased hydrophobic bonding between the C termini of Val-Stat5, leading to more stable dimers. This hypothesis also explains the sustained high levels of phosphorylated Val-Stat5 in the nucleus. The faster nuclear translocation could result from faster dimerization as a result of better interaction between Jak2 and Val-Stat5, or alternatively, an enhanced rate of actual translocation into the nucleus. Mutation of Thr757 to a Val, however, does not appear to disrupt the interaction between Stat5 and the nuclear phosphatases that are involved in deactivation and resetting of the Jak-Stat pathway. Whether a specific PIAS (protein inhibitor of activated Stats) exists for Stat5, as has been shown for Stat1 and Stat3 (38), is not known. If identified, altered interaction with such a PIAS might also account for the increased transactivation potential of Val-Stat5.

Recently, Callus and Mathey-Prevot (39) further characterized the C termini of murine Stat5a and confirmed that deletion of the last 57 amino acids results in the loss of ability to transactivate. Within this region, deletion of a 12-amino acid block (749) also dramatically decreased the transactivation potential of a chimerae of the DNA-binding domain of Gal4 with the C-terminal transactivation domain of Stat5. However, when the full-length Stat5 containing this deletion was cotransfected into cells expressing the PRL receptor and a Stat5-responsive luciferase reporter gene, the luciferase activity in response to PRL was not affected. This was found to be due to enhanced DNA binding activity of the 12-amino acid deletion mutant, which these authors also postulated might reflect an enhanced rate of dimerization. Mutation of Ser756 (analogous to Thr757 in the present study) within this 12-amino acid block to Gly resulted in a 25% decrease in the transactivation following PRL treatment, even though neither the DNA binding nor phosphorylation of Tyr694 were altered. The basis for the modest decrease in transactivation in the S756G mutant was postulated to reflect subtle structural changes in the 12-amino acid region. Importantly, mutation of the hydrophobic Phe751 and Leu753 to Ala reduced transactivation about 70%, indicating the importance of hydrophobicity of this region to transactivation. These findings support the hypothesis that the increased hydrophobicity of Val-Stat5 is a key factor in its enhanced transactivation potential.

In summary, the present studies show that amino acid residues present in the C-terminal transactivation domain of wild type Stat5a, specifically Thr757, play a critical role in regulating its function as a transcription factor. The presence of the hydroxyl group in Thr757 appears to limit the rate of phosphorylation and/or nuclear translocation of Stat5, because substitution with a Val at this position markedly increased its ability to form transcriptionally active dimers. The increased hydrophobicity of Val-Stat5 appears to be a major contributor to its DNA binding and transactivation activity, consistent with the essential nature of other nearby hydrophobic amino acids.

    FOOTNOTES

* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ Present address: Millennium Pharmaceuticals Inc., 270 Albany St., Cambridge, MA 02139.

Present address: Dept. of Molecular Biology, 2800 Plymouth Rd., Parke-Davis Pharmaceutical Research, Ann Arbor, MI 48205.

** To whom correspondence should be addressed: 306 Health Sciences Research Bldg., Graduate Center for Toxicology, University of Kentucky, Lexington, KY 40536-0305. Tel.: 859-257-3760; Fax: 859-323-1059; E-mail: maryv@pop.uky.edu.

Published, JBC Papers in Press, December 22, 2000, DOI 10.1074/jbc.M007156200

    ABBREVIATIONS

The abbreviations used are: Jak, Janus kinases; SH, Src homology; PRL, prolactin; PRLRL, long form of the prolactin receptor; GAS, gamma -interferon activated sequences; H7, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine·2HCl; PBS, phosphate-buffered saline; DTT, dithiothreitol; PMSF, phenylmethylsulfonyl fluoride; EMSA, electromobility shift assays.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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