From the Department of Microbiology and the Kaplan Comprehensive Cancer Center, New York University School of Medicine, New York, New York 10016
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Transcriptional activation by the glucocorticoid receptor (GR) is regulated by both glucocorticoid binding and phosphorylation. The rat GR N-terminal transcriptional regulatory domain contains four major phosphorylation sites: threonine 171 (Thr171), serine 224 (Ser224), serine 232 (Ser232), and serine 246 (Ser246). We have previously demonstrated that Ser224 and Ser232 are phosphorylated by cyclin-dependent kinases, while Ser246 is phosphorylated by the c-Jun N-terminal kinase. We report here that the remaining GR phosphorylation site, Thr171, is a target for glycogen synthase kinase-3 (GSK-3) in vitro and in cultured mammalian cells. Increasing GSK-3 activity through its overexpression in cultured cells inhibits GR transcriptional enhancement, an effect dependent upon Thr171. Correspondingly, overexpression of a constitutively active form of the GSK-3 inhibitor, protein kinase B/Akt, increases GR transcriptional enhancement. Overexpression of GSK-3 had no effect on GR-mediated transcriptional repression of AP1-dependent gene expression. Importantly, transcriptional activation by the human GR (hGR), which contains an alanine (Ala150) at the position equivalent to Thr171 in rat GR, is not affected by GSK-3 overexpression. Introduction of a threonine residue at this position (A150T) establishes GSK-3-mediated inhibition of hGR transcriptional activation. These findings demonstrate species-specific differences in GR signaling, as revealed through GSK-3 phosphorylation, which suggests that GR function in rodents may not fully recapitulate receptor action in humans and that hGR is capable of adopting the GSK-3 signaling pathway through a somatic mutation.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Glucocorticoid hormones control cellular proliferation and metabolism through their association with the glucocorticoid receptor (GR),1 a member of the intracellular receptor superfamily of transcriptional regulatory proteins (1). Upon glucocorticoid binding, GR enters the nucleus, associates with specific DNA sequences termed glucocorticoid response elements (GREs), and increases transcriptional initiation from nearby promoters. GR can also repress transcription mediated by the heterodimeric AP1 transcription factor complex (c-Jun and c-Fos) (2). Although glucocorticoids act as the primary signal in activating GR's transcriptional regulatory functions, GR-mediated transcriptional activation is also modulated by phosphorylation (3-5).
Rat GR isolated from cultured mammalian cells or ectopically expressed in yeast (Saccharomyces cerevisiae) is phosphorylated on four major residues (6). These sites cluster to the N-terminal transcriptional regulatory domain and include threonine 171 (Thr171), serine 224 (Ser224), serine 232 (Ser232), and serine 246 (Ser246) (Fig. 1A). Each of these residues is followed by a proline, thereby forming a motif phosphorylated by a family of serine/threonine-proline-directed kinases that includes the cyclin-dependent kinases (Cdk), the mitogen-activated protein kinases, and glycogen synthase kinase-3 (GSK-3). Differential phosphorylation at these sites both positively and negatively regulate GR transcriptional activation. Positive regulation is accomplished by cyclin-Cdk complexes: cyclin E-Cdk2 phosphorylates Ser224, while cyclin A-Cdk2 phosphorylates both Ser224 and Ser232. Mutations at these sites, or of particular Cdk genes in yeast, reduce GR-dependent transcriptional activation, suggesting that phosphorylation of Ser224 and Ser232 is required for full GR transcriptional enhancement (7). In contrast, phosphorylation of Ser246 by c-Jun N-terminal kinase, a member of the mitogen-activated protein kinases family, inhibits GR transcriptional activation (8).
The remaining GR phosphorylation site, Thr171, also resides in a motif recognized by serine/threonine-proline-directed kinases. However, our previous studies indicate that neither the Cdks, nor c-Jun N-terminal kinase efficiently phosphorylate Thr171 in vitro. Furthermore, phosphorylation of Thr171 is evident in both serum-deprived quiescent and serum-stimulated proliferating cells (8), suggesting that Cdks and c-Jun N-terminal kinase are unlikely to phosphorylate Thr171 in vivo, since these kinases are largely inactive in serum-starved, nonproliferating cells. GSK-3, on the other hand, is active throughout the cell cycle, as well as in serum-deprived cells (9). Thus, GSK-3 may represent the GR kinase that phosphorylates Thr171.
GSK-3 was originally isolated as the kinase that phosphorylates
glycogen synthase, the rate-limiting enzyme of glycogen synthesis (10).
Two mammalian GSK-3 isoforms have been identified (GSK-3 and
GSK-3
) that are 85% homologous at the level of primary amino acid
sequence, and share substrate specificity (11). GSK-3 is conserved
throughout evolution, with homologues present in yeast (S. cerevisiae and Schizosaccharomyces pombe) (12, 13),
Dictyostelium discoideum (14), Drosophila
melanogaster (15, 16), and Xenopus laevis (17-19).
Recent studies in Dictyostelium, Xenopus, and
Drosophila have implicated GSK-3 in pathways other than
glycogen metabolism. GSK-3 has been implicated in cell fate
determination and differentiation through its ability to phosphorylate
and regulate factors involved in cellular proliferation including CREB,
c-Myc, c-Jun, and -catenin (10, 20-23). Although GSK-3 has no known
activators, its activity in cultured cells can be increased through
overexpression. GSK-3 enzymatic activity is, however, negatively
regulated by protein kinase B/Akt, an enzyme that phosphorylates and
inhibits GSK-3 (24). Akt is, in turn, activated through an association
with lipid products generated by phosphatidylinositol-3 kinase at the cell membrane and through phosphorylation (25-27). The
phosphatidylinositol-3 kinase-Akt pathway is induced in response to
insulin, insulin-like growth factor, epidermal growth factor, and other
mitogens (28, 29). Recently, the phosphatidylinositol-3 kinase-Akt
pathway has been implicated in cell survival, with a constitutively
activated form of Akt leading to a reduction in apoptosis in neuronal
cells (30, 31). GSK-3 activity is also inhibited by the Wnt signaling pathway through an unknown mechanism, involving the Dishevelled protein
(32, 33). Here we examine whether rat GR is a substrate for GSK-3
in vitro and investigate the consequences of GSK-3
activation and inhibition on GR transcriptional regulation in cultured
mammalian cells.
![]() |
EXPERIMENTAL PROCEDURES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Purification of Receptor Derivatives and in Vitro Kinase Assays-- A wild type rat GR derivative containing amino acids 106-318 or receptor mutant with a single amino acid substitution T171A, and a wild type human ER derivative containing estrogen receptor (ER) N-terminal amino acids 1-121, were expressed in Escherichia coli as glutathione S-transferase (GST)-fusion proteins (GST-GR106-318 and GST-ER1-121) exactly as described previously (7). The most concentrated fractions (1 mg/ml) were used as substrates for the in vitro kinase assays.
GST-GR106-318 substrate (2 µg) was bound to 100 µl of a 50% slurry of glutathione beads for 20 min on ice and washed twice with 1 ml of DK buffer (50 mM potassium phosphate, pH 7.15, 10 mM MgCl2, 5 mM NaF, 5 mM dithiothreitol, supplemented with protease inhibitors, 1 mM phenylmethylsulfonyl fluoride, and 1 µg/ml each of aprotinin, pepstatin A, and leupeptin). Approximately 50 milliunits of rabbit purified GSK-3 (Upstate Biotechnology) were added to the immobilized receptor along with 25 µM ATP, 10 mM MgCl2, 1 mM dithiothreitol, and [Phosphopeptide Mapping and Phosphoamino Acid Analysis-- For phosphopeptide mapping, polyacrylamide gels containing the labeled receptor were washed three times for 10 min each in 500 ml of water and dried between cellophane sheets. Following autoradiography, the GR band was excised and the rehydrated gel slice was placed into a microcentrifuge tube in 50 mM ammonium acetate (pH 4.0), 1 mM dithiothreitol, and 50 µg of V8 protease (endoproteinase Glu-C, Boehringer Mannheim). After 10 h at room temperature, an additional 50 µg of V8 protease was added and incubation was continued for 5 h at room temperature. Samples was centrifuged for 5 min at 12,000 × g and the supernatant containing the digested peptides was evaporated to dryness. Peptides were resuspended in 0.5 ml of water, dried, washed again, and dissolved in 10 µl of electrophoresis buffer I, pH 1.9 (15% acetic acid, 5% formic acid). Peptides were electrophoresed in the same buffer on a thin layer chromatography plate (microcrystalline cellulose adsorbent without fluorescent indicator; Kodak) at 1000 V for 50 min. Plates were then dried, subjected to ascending chromatography in the second dimension for 3.5 h with 37.5% butanol, 25% pyridine, and 7.5% acetic acid, air-dried, and exposed to film (34).
For phosphoamino acid analysis, 32P-labeled receptor was transferred to Immobilon paper (Millipore Corp.), and the GR band was visualized by autoradiography, excised from the membrane, and hydrolyzed in 100 µl of 6 N HCl (Pierce) by heating to 110 °C for 60 min. Samples were washed twice in 0.5 ml of water, dried, and resuspended in 8 µl of electrophoresis buffer I. The hydrolysates were spotted onto a TLC plate, along with phosphoamino acid standards (1 µl of mixture of phosphoserine, phosphothreonine, and phosphotyrosine (Sigma), 1 mg/ml each), and resolved in the first dimension by electrophoresis at 1500 V for 20 min in electrophoresis buffer I, and in the second dimension by electrophoresis at 1300 V for 16 min in buffer II, pH 3.4 (5% acetic acid, 0.5% pyridine). After drying, plates were sprayed with 0.25% w/v ninhydrin in acetone and developed at 70 °C for 10 min to visualize the phosphoamino acid standards, and autoradiography was performed.Site-directed Mutagenesis-- Site-directed mutagenesis of the human GR alanine 150 to threonine was performed using Stratagene's Quick Change site-directed mutagenesis procedure and high fidelity Pfu DNA polymerase, according to the manufacturer's instructions, with the following oligos: 5'-GCTGTGTCTGCTACCCCCACAGAGAAG-3' and 5'-CTTCTCTGTGGGGGTAGCAGACACAGC-3'.
Plasmids--
pCMV-wt GR and pCMV-GR T171A expression plasmids
were used to produce rat GR, and XG46TL reporter plasmid,
containing two consensus GREs upstream of thymidine kinase promoter
(109) linked to a luciferase gene was used to assay GR
transcriptional activity. An XAP1TL reporter plasmid, containing a
single AP1 binding site upstream of the thymidine kinase promoter fused
to a luciferase gene, was used to assay transcriptional repression.
pcDNA3-hGR and pcDNA3-hGR A150T plasmids expressed the human
wild type and the alanine to threonine mutant GRs, respectively.
pCMV5-HA-GSK-3
expressed HA-tagged GSK-3 and pCMV6-HA-Akt plasmid
expressed a constitutively active myristylated HA-tagged form of Akt
(28). A pCMV5 empty vector was used to equalize the total amount of DNA
transfected in each experiment. pCMV-LacZ plasmid produced
-galactosidase.
Cell Lines and Treatments-- U-2 OS human osteosarcoma cells (ATCC HTB 96) and human HeLa cervical carcinoma cells (ATCC CCL 2) were maintained in Dulbecco's modified Eagle's medium (DMEM; Life Technologies, Inc.) supplemented with 10% fetal bovine serum (FBS; HyClone), 50 units/ml each of penicillin and streptomycin, and 2 mM L-glutamine (Life Technologies, Inc.). Rat pheochromocytoma PC-12 cells (ATCC CRL-1721) were maintained in DMEM supplemented with 10% FBS, 5% horse serum (Life Technologies, Inc.), 50 units/ml each of penicillin and streptomycin, and 2 mM L-glutamine. All hormone treatments were done in DMEM, 10% FBS containing either 100 nM glucocorticoid dexamethasone (resuspended in 100% ethanol) or an identical volume of 100% ethanol.
Transient Transfections and Reporter Activity Assays-- U-2 OS cells were plated on 60-mm dishes in DMEM, 10% FBS. One hour prior to transfection cells were refed with fresh medium and transfected with the indicated plasmids via the calcium phosphate precipitation method as described elsewhere (35). Eight hours later, cells were washed three times with prewarmed phosphate-buffered saline to remove calcium phosphate precipitates, allowed to recover overnight in DMEM, 10% FBS and incubated with fresh medium containing 100 nM dexamethasone where indicated, for an additional 8 h.
HeLa and PC-12 cells were plated in 60-mm dishes, washed once with serum-free medium and transfected with the indicated plasmids using 20 µl of LipofectAMINE reagent (Life Technologies, Inc.) in a total volume of 2.5 ml of serum-free phenol red-free DMEM per 60-mm dish according to the manufacturer's instructions. Three hours post-transfection 2.5 ml of DMEM, 20% FBS was added to each dish and cells were incubated for another 12 h. The next day cells were refed with fresh DMEM, 10% FBS with 100 nM dexamethasone or identical volume of 100% ethanol and incubated for an additional 8 h. Transfected cells were washed twice in phosphate-buffered saline and harvested in 1X Reporter lysis buffer (Promega). Luciferase activity was quantified in a reaction mixture containing 25 mM glycylglycine, pH 7.8, 15 mM MgSO4, 1 mM ATP, 0.1 mg/ml bovine serum albumin, 1 mM dithiothreitol. Lumat LB 9507 luminometer (EG&G Berthold) was used with 1 mM D-luciferin (Analytical Luminescence Laboratory) as a substrate. Lysates were additionally assayed forWestern Blotting-- To make protein extracts from transfected cells, U-2 OS cells were washed twice with phosphate-buffered saline and lysed directly on the dishes in 200 µl of ice-cold lysis buffer (150 mM NaCl, 50 mM Hepes, pH 7.5, 1 mM EDTA, 1 mM EGTA, 10% glycerol, 1% Triton X-100, 1 mM NaF, 25 µM ZnCl2, supplemented with protease inhibitors (described above) and phosphatase inhibitor 1 mM sodium orthovanadate). The lysates were collected and precleared by centrifugation (10,000 × g for 10 min at 4 °C). The protein concentration in all samples was adjusted with the lysis buffer, and 200 µl of the whole cell extracts was boiled for 2 min with 50 µl of 5× SDS sample buffer. For immunoblotting, protein extracts were fractionated by 10% SDS-PAGE, transferred to Immobilon paper, and probed with mouse monoclonal antibodies against GSK-3 (Upstate Biotechnology), HA-tagged Akt (Boehringer Mannheim), or with anti-GR rabbit polyclonal antiserum (Santa Cruz Biotechnology, Inc.). The blots were developed using horseradish peroxidase-coupled sheep anti-mouse or donkey anti-rabbit antibodies and the enhanced chemiluminescence (ECL) substrate per the manufacturer's instructions (Amersham Corp.).
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
GSK-3 Phosphorylates the Rat Glucocorticoid Receptor in Vitro at
Threonine 171--
To examine whether GSK-3 can utilize the rat GR as
a substrate in vitro, we tested purified rabbit GSK-3 for
its ability to phosphorylate a GST-GR fusion protein containing the
receptor residues 106 through 318 (GST-GR106-318). We
compared GR phosphorylation by GSK-3 to that of the established GSK-3
substrate, the transcription factor c-Jun. Fig.
1B demonstrates that GSK-3 phosphorylates the GST-GR106-318 and
GST-c-Jun1-223 in vitro with similar
efficiency. In contrast, under the same experimental conditions GSK-3
failed to phosphorylate an ER derivative encompassing residues 1 through 121 (GST-ER1-121), which contains three
serine-proline phosphorylation sites at Ser104,
Ser106, and Ser118 and has been shown
previously to be substrate for cyclin A-Cdk2 complex (37) (Fig.
1B). Thus, it appears that the N-terminal transcriptional
regulatory domain of rat GR is a substrate for GSK-3 in
vitro.
|
|
Ectopic Expression of GSK-3 Inhibits GR Transcriptional Activation, but Not Repression, in Cultured Mammalian Cells-- Since GSK-3 is expressed ubiquitously and is constitutively active in virtually all mammalian cell lines, we examined whether GSK-3 overexpression alters GR-dependent transcriptional activation. We transiently co-expressed GSK-3, rat GR, and a GR-responsive reporter plasmid containing two consensus GREs upstream of the luciferase gene, in U-2 OS human osteosarcoma cells, which lack endogenous GR. Fig. 3A demonstrates that increasing amounts of transfected GSK-3 inhibit GR hormone-dependent transcriptional enhancement in a dose-dependent manner, with the maximal dose of GSK-3 used reducing GR transcriptional activity by over 50% compared with control cells transfected with the vector alone. This effect likely represents an underestimate of the total impact of GSK-3 on GR activity since the results are obtained in a cell line expressing endogenous GSK-3. The reduction in GR activity was not due to the inhibition of GR protein expression, since the steady-state level of GR was not affected by the GSK-3 overexpression (Fig. 3B). Inhibition of GR activity by ectopically expressed GSK-3 was also observed in rat PC-12 cells (Fig. 3C), which express GR endogenously, suggesting that the effect of GSK-3 on GR transcriptional activity extends to multiple cell types.
|
|
Akt, an Inhibitor of GSK-3, Increases GR Transcriptional Enhancement-- We next asked whether a decrease in GSK-3 activity would increase GR transcriptional activation. Since no GSK-3-deficient cell line is available, we chose to inhibit GSK-3 activity by using Akt, a protein kinase that phosphorylates GSK-3 and inhibits its catalytic activity. We overexpressed a constitutively active membrane-targeted myristylated form of Akt (28) in U-2 OS cells and measured GR-dependent transcriptional activation. GR transcriptional enhancement in the presence of ectopically expressed Akt was increased to nearly 300% compared with that of control cells receiving an empty expression vector (Fig. 5A). Akt also increased transcriptional activation of endogenous GR in rat PC-12 cells, and the rat GR introduced into GR-negative SAOS2 human osteosarcoma cells (data not shown), suggesting that this effect is not confined to a single cell type. Thus, activation of Akt increases GR transcriptional activation.
|
Inhibition of GR Transcriptional Activity by GSK-3 Is Species-restricted-- The analysis of GRs isolated and sequenced from different species demonstrates that the majority of GR phosphorylation sites are evolutionarily conserved. For example, serine residues 224, 232, and 246 in the rat GR are conserved among human, primates, mouse, guinea pig and Xenopus receptors (40-44). In contrast, Thr171 is conserved between rat, mouse, and guinea pig GR, but corresponds to an alanine (Ala150) residue in the hGR, suggesting that hGR may be insensitive to signaling by GSK-3. To examine this possibility, we tested the effects of GSK-3 overexpression on the transcriptional activation of endogenous hGR in HeLa cells, and in U-2 OS cells, where hGR was introduced ectopically. Transient overexpression of GSK-3 failed to inhibit hGR transcriptional activation in HeLa cells (Fig. 6A). Since our transfection studies with the rat GR were performed in U-2 OS cells, we had to eliminate the possibility that these cells contain a specific co-factor necessary for GSK-3 action on GR. Fig. 6B demonstrates that in U-2 OS cells, no decrease in the hGR transcriptional activity was observed upon GSK-3 overexpression. Thus, under conditions identical to those used for the experiments with the rat GR (Fig. 3A), GSK-3 did not inhibit transcriptional enhancement by hGR, revealing species-specific differences in GR signaling.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We have demonstrated that GSK-3 phosphorylates the rat GR at Thr171 in vitro. In cultured mammalian cells, overexpression of GSK-3 inhibits GR transcriptional activation, while decreasing GSK-3 activity, through expression of the GSK-3 inhibitor, Akt, increases GR transcriptional enhancement. GR-mediated repression of AP1-dependent transcriptional activity, however, was not affected by GSK-3 overexpression. A threonine to alanine mutation at Thr171, the site of rat GR phosphorylation by GSK-3 in vitro, eliminates the effect of GSK-3 on rat GR transcriptional enhancement. Although the effect of GSK-3 overexpression on GR-mediated transcriptional activation was relatively modest (~50%), this likely represents an underestimate of the impact of GSK-3 on GR, since the studies were performed in cell lines containing active endogenous GSK-3. Our in vitro phosphorylation and mapping studies, coupled with activity assays using GR mutants, strongly suggest that GSK-3 phosphorylates rat GR at Thr171, and as a consequence, reduces GR transcriptional activation.
The mechanism by which GSK-3 phosphorylation of Thr171
decreases GR transcriptional activity is unclear. GSK-3 phosphorylates and inactivates other regulatory factors including c-Myc, c-Jun, NF-ATc, and -catenin. Although the mechanism of c-Myc and c-Jun inactivation by GSK-3 phosphorylation is unknown, GSK-3 phosphorylation of
-catenin targets it for degradation (45, 46), while GSK-3 phosphorylation of NF-ATc promotes its export from the nucleus (47). It
is doubtful, however, that either of these established mechanisms
explain GSK-3 regulation of GR, since 1) neither GSK-3 nor Akt
overexpression alter steady state GR protein levels, and 2) increased
export of GR from the nucleus would also affect
GR-dependent transcriptional repression, which has not been
observed in our experiments. Alternatively, GSK-3-mediated
phosphorylation of rat GR at Thr171 may disrupt
protein-protein interactions that favor GR transcriptional enhancement, or recruit inhibitory proteins that antagonize
GR-dependent transcriptional activation, hypotheses that
are currently being tested.
Given the high degree of conservation between GRs from different species, it is particularly striking that the hGR does not contain a site of GSK-3-mediated phosphorylation, thereby making hGR insensitive to GSK-3 overexpression. However, when an alanine residue at the position homologous to Thr171 in rat GR is replaced with a threonine, transcriptional activation by the hGR A150T mutant becomes sensitive to GSK-3 overexpression. Sequence comparison between GRs isolated from different species shows that the primary amino acid sequence surrounding and including rat GR Thr171 (residues 164 through 173) is conserved among rodents, including rat, mouse, and guinea pig (40, 41, 43). The equivalent region from human, squirrel monkey, owl monkey, and cotton-top tamarin GR remains conserved among primates, but has diverged from rodents (42, 48, 49). Why this region of GR has diverged between primates and rodents, while the other major phosphorylation sites (Ser224, Ser232, and Ser246) are conserved remains unclear, but likely reflects alternative strategies adopted by each species to regulate GR action.
The differences in GR primary amino acid structure and signaling between rodents and humans may contribute to the greater sensitivity of murine lymphocytes to glucocorticoid-induced apoptosis relative to human cells. It is conceivable that GSK-3-mediated inhibition of GR transcriptional activation in rodents results in the reduced expression of a putative survival factor induced by GR. Recently, the cyclin-dependent kinase inhibitor p21Cip1 has been shown to be a GR-responsive gene (49-51) and forced expression of p21 can block apoptosis (52). Thus, p21 expression protects cells from apoptosis, and as such, can be considered a survival factor. It is tempting to speculate that inhibition of GR by GSK-3, and the subsequent lack of p21 induction, may facilitate apoptosis in murine but not human lymphocytes. It would be interesting to replace mouse GR with that of the human GR in vivo and examine whether the glucocorticoid-induced apoptosis of murine lymphocytes expressing hGR still occurs. We speculate further that a threonine at position 150 in hGR would result in greater glucocorticoid sensitivity compared with an alanine at this position.
Our findings demonstrate species-specific differences in human and rat GR signaling, which suggest that studies on GR function in mice and rats may not fully translate into hGR activity. In addition, our results indicate that hGR is capable of adopting the GSK-3 signaling pathway through a somatic mutation, which antagonizes hGR-dependent transcriptional activation. It would be informative to examine whether alanine to threonine substitutions at residue 150 in hGR are present in glucocorticoid-sensitive, but absent in glucocorticoid-resistant, malignancies.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank James Woodgett and Thomas Franke for the GSK-3 and Akt expression constructs, respectively. We also thank Roland Knoblauch, Adam Hittelman, Samir Taneja, Ian Mohr, and Angus Wilson for critically reading the manuscript.
![]() |
FOOTNOTES |
---|
* This work was supported in part by Army Breast Cancer Research Fund Grants DAMD17-94-J-4454 and DAMD17-96-1-6032 (to M. J. G.) and the Irma T. Hirschl Charitable Trust.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.
Supported by a National Institutes of Health Training Grant
5T32AI07180-17.
§ To whom correspondence should be addressed: Dept. of Microbiology and the Kaplan Comprehensive Cancer Center, New York University School of Medicine, 550 First Ave., New York, NY 10016. Tel.: 212-263-7662; Fax: 212-263-8276; E-mail: garabm01{at}mcrcr.med.nyu.edu.
1 The abbreviations used are: GR, glucocorticoid receptor; hGR, human GR; GRE, glucocorticoid response element; Cdk, cyclin-dependent kinases; GSK, glycogen synthase kinase; ER, estrogen receptor; GST, glutathione S-transferase; PAGE, polyacrylamide gel electrophoresis; HA, hemagglutinin; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; wt, wild type.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|