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. 2008 May 15;22(8):1754–1766. doi: 10.1210/me.2007-0219

Glucocorticoid Receptor Phosphorylation Differentially Affects Target Gene Expression

Weiwei Chen 1,a, Thoa Dang 1,a, Raymond D Blind 1,a, Zhen Wang 1, Claudio N Cavasotto 1, Adam B Hittelman 1, Inez Rogatsky 1, Susan K Logan 1, Michael J Garabedian 1
PMCID: PMC2725771  PMID: 18483179

Abstract

The glucocorticoid receptor (GR) is phosphorylated at multiple sites within its N terminus (S203, S211, S226), yet the role of phosphorylation in receptor function is not understood. Using a range of agonists and GR phosphorylation site-specific antibodies, we demonstrated that GR transcriptional activation is greatest when the relative phosphorylation of S211 exceeds that of S226. Consistent with this finding, a replacement of S226 with an alanine enhances GR transcriptional response. Using a battery of compounds that perturb different signaling pathways, we found that BAPTA-AM, a chelator of intracellular divalent cations, and curcumin, a natural product with antiinflammatory properties, reduced hormone-dependent phosphorylation at S211. This change in GR phosphorylation was associated with its decreased nuclear retention and transcriptional activation. Molecular modeling suggests that GR S211 phosphorylation promotes a conformational change, which exposes a novel surface potentially facilitating cofactor interaction. Indeed, S211 phosphorylation enhances GR interaction with MED14 (vitamin D receptor interacting protein 150). Interestingly, in U2OS cells expressing a nonphosphorylated GR mutant S211A, the expression of IGF-binding protein 1 and interferon regulatory factor 8, both MED14-dependent GR target genes, was reduced relative to cells expressing wild-type receptor across a broad range of hormone concentrations. In contrast, the induction of glucocorticoid-induced leucine zipper, a MED14-independent GR target, was similar in S211A- and wild-type GR-expressing cells at high hormone levels, but was reduced in S211A cells at low hormone concentrations, suggesting a link between GR phosphorylation, MED14 involvement, and receptor occupancy. Phosphorylation also affected the magnitude of repression by GR in a gene-selective manner. Thus, GR phosphorylation at S211 and S226 determines GR transcriptional response by modifying cofactor interaction. Furthermore, the effect of GR S211 phosphorylation is gene specific and, in some cases, dependent upon the amount of activated receptor.

THE GLUCOCORTICOID RECEPTOR (GR) is a transcription factor responsible for managing developmental and metabolic processes in response to glucocorticoids (1,2,3,4). Before ligand binding, the GR is predominantly cytoplasmic and is bound to the heat shock protein 90-p23 chaperone complex (5,6,7), which is thought to inactivate GR DNA binding and transcriptional regulatory functions, while maintaining the receptor competent for a high-affinity ligand binding (8,9). Upon ligand binding, the hormone-receptor complex translocates to the nucleus where it binds specific DNA sequences and modulates transcription. Although GR is expressed in virtually every mammalian cell type, it regulates the expression of distinct sets of genes in a promoter- and cell type-specific manner (3,10). Even though the determinants of this promoter selectivity are not completely understood, our recent findings suggest that components of the Mediator complex, such as MED14, associate with GR and contribute to gene-specific regulation (11).

Like many other regulators, GR is a phosphoprotein (12,13). The ligand-free receptor is phosphorylated, with additional phosphorylation events occurring in conjunction with ligand binding (14). GR isolated from cultured mammalian cells or ectopically expressed in yeast is phosphorylated on multiple sites (15). Three sites cluster to the N-terminal transcriptional regulatory domain and include serine 203 (S203), serine 211 (S211), and serine 226 (S226) in the human (h) GR numbering scheme (Fig. 1A) (16). We have demonstrated previously that cyclin E/cyclin-dependent kinase 2 (Cdk2) phosphorylates GR at S203, whereas cyclin A/Cdk2 phosphorylates both S203 and S211 (15). Mutations at these sites, or of particular Cdk genes in yeast, reduce GR-dependent transcriptional activation, suggesting that phosphorylation of S203 and S211 is required for full GR transcriptional enhancement. Furthermore, mammalian cells lacking a Cdk inhibitor p27KIP1 display a concomitant increase in cyclin/Cdk2 activity and GR phosphorylation at the Cdk sites, and enhanced receptor transcriptional activity (17). In addition, GR S211 also appears to be a substrate for p38 MAPK (18). In contrast, phosphorylation of S226 by c-Jun N-terminal kinase (JNK), another member of the MAPK family, inhibits GR transcriptional activation (19). Phosphorylation of S226 by JNK has also been shown to regulate GR export from the nucleus upon hormone withdrawal (20).

Figure 1.

Figure 1

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GR Structure, Specificity, and Kinetics of S226 Phosphorylation

A, Functional domains and phosphorylated residues of the hGR. Shown is a schematic representation of hGR with major phosphorylation sites and the sequence of the hGR phosphopeptide used to produce the phospho-S226-specific antibody. B, Immunoblotting of hGR with phospho-S226 antibody is shown. Whole-cell extracts prepared from U2OS cells expressing an HA-tagged hGR (U2OS-hGR), either WT or phosphorylation site mutants S226A or S211A, or A549 cells expressing endogenous GR, untreated or treated with 100 nm Dex for 1 h, were analyzed by immunoblotting with phospho-S226 (top panel), or a phosphorylation state-independent GR antibody (bottom panel) as a measure of total GR. C, Kinetics of S226, S211, and S203 phosphorylation in response to Dex treatment. U2OS-hGR or A549 cells were treated with ethanol (−) or Dex (100 nm) for the time indicated. Whole-cell lysates were prepared, normalized for total protein concentration, and analyzed by immunoblotting with phospho-S226, phospho-S203, phospho-S211, phosphorylation state-independent GR (total GR), and actin antibodies. Quantitative analysis of immunoblot results in panel C normalized to actin. DBD. DNA-binding domain; LBD, ligand-binding domain.

GR dephosphorylation at S203 and S226 is regulated by protein phosphatase 5 (21), whereas S211 phosphatase is as yet unidentified. The fact that protein phosphatase 5 differentially affects GR target gene expression further suggests a link between GR phosphorylation and transcriptional regulation. We have recently shown that GR phospho-isoforms selectively occupy receptor target genes (22). Yet mechanistically how GR phosphorylation regulates gene expression remains enigmatic.

In this study we demonstrate gene-specific differences in the requirement for GR phosphorylation. Our data further show that the phosphorylation state of S211 is a key regulator of receptor transcriptional activation and repression.

RESULTS

Characterization of hGR S226 Phosphorylation Site-Specific Antibody

The polyclonal antibody GR phospho-S226 was raised against the phosphopeptide LLIDENLLS(P)PLAG corresponding to residues 218–230 of the hGR (Fig. 1A). The antibodies were tested for their ability to detect GR by immunoblotting extracts from U2OS cells stably expressing an hemagglutinin (HA)-tagged hGR (U2OS-hGR) either untreated or treated for 1 h with dexamethasone (Dex). The phospho-S226 antibody showed substantial immunoreactivity toward GR from Dex-treated, but not untreated, cells, even though equal amounts of GR were present as determined by immunoblotting for total GR (Fig. 1B). No immunoreactivity toward GR is observed with preimmune sera (data not shown). As a control for specificity, we tested the ability of the antibody to detect phosphorylation-deficient GR S226A, stably integrated in U2OS cells (U2OS-hGRS226A). The phospho-S226 antibody did not recognize GR S226A in either the absence or presence of Dex (Fig. 1B). Thus, the GR phospho-S226 antibody recognized its phosphorylation site specifically.

We also examined phosphorylation of endogenous GR in A549 cells. In contrast to what was observed in U2OS cells ectopically expressing hGR, GR phosphorylation at S226 in A549 cells increases only slightly upon treatment with Dex (Fig. 1B).

To further explore GR phosphorylation in both cell types, we examined the kinetics of GR phosphorylation at S226, S203, and S211 using antibodies specific for these phosphoserine residues. An antibody that recognizes GR independent of its phosphorylation state was used to determine the total amount of GR, and actin was used as a control for loading. As shown in Fig. 1C, phosphorylation at S226 in U2OS cells increased rapidly within the first hour of Dex treatment and remained high for 4 h, after which the signal decreased progressively from 6–12 h. Phosphorylation of S211 followed a pattern similar to that of S226 throughout the hormone treatment, whereas S203 had a higher basal level of phosphorylation and a less dramatic hormone induction (Fig. 1C). Thus, in U2OS ectopically expressing hGR, basal phosphorylation of S226 is low, and Dex-dependent phosphorylation at S226 is high, which mirrors that of S211.

In contrast, GR phosphorylation at S226 in A549 cells increases only slightly upon treatment with Dex (Fig. 1C, right panel) whereas the dynamics of hormone-dependent phosphorylation at S211 and the high basal level of phosphorylation at S203 are similar to that in U2OS-hGR cells. Thus, phosphorylation at S226 appears to vary between the two cell types, which may reflect differences in the availability or activity of GR kinases and phosphatases.

Ligands Effect GR Phosphorylation Differentially

We next tested a battery of ligands [Dex, cortisol, deooxycorticosterone, progesterone, progestin (R5020), 17-α-hydroxyprogesterone, aldosterone, R1881, testosterone, and dihydrotestosterone] with different potencies for their ability to induce GR phosphorylation. As shown in Fig. 2A, Dex, cortisol, deooxycorticosterone, progesterone, and progestin but not aldosterone, R1881, testosterone, or dihydrotestosterone promote S226 phosphorylation. In addition, RU486 induced S226 phosphorylation to levels similar to Dex in U2OS cells (data not shown). For the S211 site, phosphorylation was induced only by Dex, cortisol, and to a lesser extent, deooxycorticosterone. We have previously shown that phosphorylation at S211 was not induced by RU486 (16). Surprisingly, S203 phosphorylation was augmented by virtually all of the ligands examined, suggesting that S203 phosphorylation may reflect a nonselective effect of steroid localization. These results suggest that GR phosphorylation is differentially affected by agonist potency.

Figure 2.

Figure 2

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Effects of Ligands on GR Phosphorylation and Transcriptional Activation

A, U2OS-hGR were treated with ethanol (−) or the ligands indicated (100 nm) for 1 h, and whole-cell extracts were prepared. Equal amounts of protein from each treatment were analyzed by immunoblotting with phospho-S226, phospho-S211, phospho-S203, total GR, or actin antibodies, reflective of the amount of total protein in each lane. The ratio of GR phosphorylation at S211 to S226 normalized to total GR is shown (S211P/S226P). B, U2OS-hGR WT or U2OS-hGR S226A (S226A) cells were transiently transfected with the MMTV-luciferase reporter construct along with pCMV-lacZ as an internal control. After 16 h, the cells were treated with ligands indicated (100 nm) for 1 h, conditions identical to those used for the immunoblot analysis, and luciferase activity was determined. DHT, Dihydrotestosterone; PROG, progesterone; RLU, relative light units.

To test the influence of S211 and S226 phosphorylation on GR transcriptional activation, we assessed the activity of mouse mammary tumor virus (MMTV)-luciferase reporter in U2OS cell lines expressing wild-type (WT) or S226A hGR in the presence of 100 nm Dex, an inducer of higher phosphorylation at S211 relative to S226, or progesterone, a weaker inducer of S211 phosphorylation compared with S226 (Fig. 2A). As expected, Dex induced reporter gene activation in the WT GR cells, whereas progesterone did not. Interestingly, Dex initiated a more robust induction in the S226A line (Fig. 2B), in which even the suboptimal agonist progesterone modestly stimulated reporter gene activity. Thus, the relative level of phosphorylation at S211 vs. S226 is an important determinant of receptor activity and is consistent with the idea that S226 phosphorylation is inhibitory to GR transcriptional activation.

GR Hormone-Dependent Phosphorylation at S211 Is Affected by BAPTA-AM and Curcumin

Cellular signaling pathways potentially involved in GR phosphorylation were investigated by using pharmacological inhibitors of kinases and other signaling pathways (see Table 1). U2OS-hGR cells were treated for 1 h with the compounds, and GR phosphorylation at S203, S211, and S226 was determined after a 1-h Dex treatment. BAPTA-AM, an intracellular calcium-chelating agent, dramatically decreased hormone-dependent phosphorylation at the S211 (Fig. 3A). Curcumin, the yellow coloring agent in curry powder known as turmeric, which suppresses the nuclear factor-κB (NFκB) pathway through an unknown mechanism, inhibited GR phosphorylation at S211 at the highest concentration tested (50 μm) and also reduced phosphorylation at S203 and S226 to the level observed in the absence of hormone. DRB, a potent inhibitor of cyclinH/CDK8 and cyclin T/CDK9, did not decrease GR phosphorylation (Fig. 3A). Surprisingly, none of the other CDK or MAPK inhibitors (Table 1), significantly reduced phosphorylation at any of the sites under the conditions of the assay (data not shown), despite previous findings that cyclinA/CDK2 and MAPK phosphorylate GR activation function 1 (AF-1) in vitro, and mutations in the kinases or their inhibitors affected GR transcriptional activation (15,17). This may reflect redundancy of the CDKs and MAPKs with respect to GR phosphorylation or alterations in the drugs or targets that render them insensitive to inhibition in U2OS cells. BAPTA-AM and curcumin also reduced GR S211 hormone-dependent phosphorylation in human A549 lung epithelial cells and in a rat hepatoma cell line (data not shown). Chelating extracellular calcium with EGTA had no effect on GR phosphorylation at any of the sites. Similarly, thapsigargin, which depletes intracellular calcium stores, had virtually no effect on GR phosphorylation, suggesting that the effect of BAPTA-AM on GR phosphorylation is independent of intracellular calcium levels.

Table 1.

Inhibitors Examined for Effects on GR Phosphorylation

Inhibitor (Concentration) Cellular Target
BAPTA-AM (40 μg/ml) Intracellular divalent cation chelator
Cyclosporine A (CSA) (1 μg/ml) Calcineurin inhibitor
Curcumin (50 μm) Multiple inhibitory functions
DRB (100 μm) CK2/CDK8/9 inhibitor
EGTA-AM (3 mm) Extracellular Ca2+ chelator
Genistein (300 μm) Tyrosine kinase inhibitor
Glybendamide (15 μm) Cl− channel blocker
HA1004 (45 μm) cAMP/cGMP kinase inhibitor
IC261 (10 μm) CK1 inhibitor
LY294002 (15 μm) PI3 kinase inhibitor
Rapamycin (10 μm) p70 S6 kinase inhibitor
Roscovine (7 μm) p34 cdc2/cyclinB kinase inhibitor
SB202190 (35 μm) p38 MAPK inhibitor
Staurosporine (0.5 μm) General kinase inhibitor
Thapsigargin (2 μm) ER Ca2+ pump inhibitor
Tunicamycin (2.5 μg/ml) N-linked glycosylation inhibitor
U0126 (0.7 μm) MEK1 and MEK2 inhibitor
Valinomycin (10 μm) Ca2+ ATPase pump blocker
Wortmanin (0.1 μm) PI3 kinase inhibitor
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Bold letteringindicates compounds that affected GR phosphorylation; the remaining compounds had little or no measurable effect on GR phosphorylation at the sites examined. MEK, MAPK kinase; PI3, phosphatidylinositol. 

Figure 3.

Figure 3

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Signaling Pathways Involved in GR Phosphorylation

U2OS-hGR cells were pretreated for 1 h with indicated compounds at the concentrations found in Table 1, and GR phosphorylation at S203, S211, and S226 was determined after a 1-h Dex treatment. Equal amounts of protein from each treatment were analyzed by immunoblotting with phospho-S226, phospho-S211, phospho-S203, or total GR antibodies. B, Subcellular distribution of GR upon treatment with BAPTA-AM, curcumin, and DRB. U2OS-hGR cells, treated exactly as above, were fixed, and the subcellular location of GR was examined by indirect immunofluorescence using a total GR antibody. The data shown are from a single experiment that is representative of at least three independent experiments. DAPI, 4′,6-Diamidino-2-phenylindole.

We also examined whether BAPTA-AM, curcumin, and DRB alter Dex-dependent nuclear localization of GR by immunofluorescence (Fig. 3B). BAPTA-AM and curcumin, but not DRB, resulted in retention of GR in the cytoplasm and also reduced the activity of an MMTV-luciferase reporter (data not shown). This is consistent with a previous report demonstrating that BAPTA-AM inhibited hormone-dependent GR nuclear import (23). This suggests that reducing GR hormone-dependent phosphorylation decreases localization of GR to the nucleus even in the presence of Dex. Alternatively, reduced GR phosphorylation may be secondary to the failure to localize to the nucleus. Additional experiments will be required to distinguish between these possibilities.

Molecular Modeling of GR Polypeptides Encompassing the S211 Phosphorylation Site

To gain insight into the potential local structural changes in GR upon phosphorylation at S211, we performed extensive global energy minimization simulations with two peptides, bearing a phosphorylated vs. nonphosphorylated S211 (Q195DLEFSSGSPGKETNES211PWRSDLLI220) (Fig. 4). The predicted conformation of the unmodified peptide exhibits a largely random conformation (Fig. 4A), consistent with biophysical measurements of the GR AF-1 structure (24). Interestingly, phosphorylation of S211 induces a marked trend toward a more structured conformation with the peptide adopting a helical structure on both sides of the phosphorylation site (Fig. 4B). This suggests that phosphorylation of S211 can confer structure to the GR AF-1 domain. It is also worth noting that many of the conformations in the phosphopeptide exhibited a hydrogen bond interaction between the phosphate moiety on the S211 and an adjacent arginine at residue 214 (R214), which in turn makes contact with glutamic acid 207 (E207) (Fig. 4C). Interestingly, E207 and R214 are conserved among receptor from diverse species, and R214 is also part of the consensus sequence for CDK phosphorylation [S-(P)-P-X-R/K]. This implies that flanking residues in the kinase consensus site might have evolved to contribute to the stabilization of local structure upon phosphorylation.

Figure 4.

Figure 4

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GR Phosphorylation at S211 Results in Structural Changes and Modulates Interaction with MED14 (Vitamin D Receptor Interacting Protein 150)

Representative low-energy conformations of the (A) non-phosphorylated (WT) and (B) phosphorylated (P-S211) peptide spanning the S211 site. Peptide is colored blue (N-term) to red (C-term) and S211 is depicted. Note that the phosphorylated peptide displays a more structured conformation around the phosphorylated residue, with the peptide adopting a helical structure on both sides of the phosphorylation site. C, Interaction of P-S211with R214. Residues P-S211, R214, and E207 are shown forming a hydrogen bond network that is displayed as dots between donor and acceptor atoms. D, Location of E198, F220, and W213, which flank the S211 phosphorylation site and have been shown to be essential for transcriptional activation at certain GREs. E, Phosphorylation-dependent interaction of MED14 with GR AF-1 derivatives in yeast two-hybrid assay. MED141214–1434 expressed in yeast as a fusion protein to the LexA DNA-binding domain (pEG202) was analyzed for its ability to interact with the WT GR AF-1 (WT), GR 30IIB (30IIB), which harbors three point mutations in AF-1, and S211A and S211D fused to the HA epitope and B42 activation domain in a galactose-inducible expression vector (pJG4/5). Strength of interaction is determined by quantitative liquid β-galactosidase assays after a 12-h incubation at 30 C in galactose containing media. Data represent the average β-galactosidase activity of three independent clones, and error bars are the sd. Western blots of extracts from strains expressing the indicated HA-GR fusions were performed using a HA-specific monoclonal antibody, a polyclonal antibody to LexA to detect the LexA-Med14 derivatives, and actin as loading control. Shown is protein expression from the three independent clones used in the assay. B-Gal, β-Galactosidase; VO, vector only.

In close proximity to the S211 phosphorylation site are three amino acid residues within the GR AF-1 (E198, F220, and W213) the simultaneous disruption of which (30IIB mutant) reduces receptor transcriptional activation (25). We therefore examined the location of these amino acids on the predicted structure. Interestingly, these residues localize within the helical portions of the GR polypeptide formed upon phosphorylation (Fig. 4D), indicating that the regions surrounding S211 are of critical importance to GR transcriptional activation perhaps because they contribute to the formation of the putative protein-protein interaction surfaces.

GR S211 Phosphorylation Modulates Interaction with MED14 (Vitamin D Receptor Interacting Protein 150)

Previous studies from our laboratory demonstrated that MED14 associates with the GR AF-1 (26). To determine whether GR phosphorylation at S211 affects its interaction with MED14, we examined the effect of S211 substitution with an alanine (S211A) and an aspartic acid (S211D), which potentially mimics the phosphorylated state of GR, on MED14 binding in a yeast two-hybrid assay. GR ectopically expressed in yeast is phosphorylated at the same sites observed in mammalian cells (15). The GR phosphorylation site derivatives were expressed in yeast, along with MED14 (Fig. 4E, lower panels) and a β-galactosidase reporter, and their interaction was determined.

As expected, the GR N-terminal activation domain interacted with MED14, whereas the mutation (30IIB) significantly reduced the interaction (26). A similar, albeit smaller, decrease was observed with the GR S211A mutant (Fig. 4E). In contrast, the phosphomimetic S211D substitution increased GR interaction with MED14. This suggests that GR phosphorylation at S211 modulates its interaction with MED14.

GR Phosphorylation Site Mutations Differentially Affect GR Target Gene Expression

To better understand the effect of phosphorylation on GR-dependent gene expression, we measured the mRNA accumulation of established GR target genes glucocorticoid-induced leucine zipper (GILZ), ladinin 1 (LAD1), IGF-binding protein 1 (IGFBP1), and interferon regulatory factor 8 (IRF8) in U2OS cells that stably express GR derivatives bearing serine to alanine substitutions at S226 (U2OS-hGRS226A) and S211 (U2OS-hGRS211A) at levels similar to WT GR in U2OS-hGR cells (11,21,27). U2OS-hGRS226A is a pool of clones stably expressing S226A, whereas the U2OS-hGRS211A line is a single clone. We have verified our findings with a pool stably expressing S211A (data not shown). After a 2-h Dex treatment at the indicated doses, total RNA was isolated and subjected to qPCR using gene-specific primer pairs (11). We first examined the expression of GR target genes in U2OS cells harboring the S226A mutant relative to cells with WT GR, and found it to be enhanced for all genes tested (Fig. 5A). For example, the induction of LAD1 by GR S226A at 10 nm Dex is 6-fold greater than that by the WT, and the induction of IGFBP1, IRF8, and GILZ is 2-fold over WT. This establishes that S226 phosphorylation inhibits glucocorticoid induction of the four endogenous GR targets examined.

Figure 5.

Figure 5

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GR Target Genes Are Differentially Responsive to Mutations in the GR Phosphorylation Sites, S226 and S211

A, GR constructs S211A and S226A stably expressed in U2OS cells were analyzed by immunoblotting with antibodies to GR and actin as an internal control for loading. B and C, U2OS cells stably expressing GR WT, GR S226A, or S211A were treated with ethanol vehicle (0) or the indicated amount of Dex for 2 h, and total RNA was analyzed by real-time PCR as described in Materials and Methods. The values indicate expression of target genes normalized to Rpl19 RNA and presented as fold induction relative to vehicle-treated cells. The data shown are from three independent experiments. The error bar indicates the sd.

Quantitative analysis revealed two patterns of dose-dependent accumulation of mRNA in U2OS-hGRS211A as compared with cells expressing the WT GR (Fig. 4B). Dex-dependent induction of IGFBP1 and IRF8 mRNAs by S211A was reduced relative to WT at all hormone concentrations tested, which shifts the dose-response curve to the right. This change may suggest that the S211A mutant is less efficient at engaging in the associations with cofactors, such as MED14, necessary for transcriptional activation of IGFBP1 and IRF8. This is consistent with our previous findings that IRF8 and IGFBP1 required MED14 for activation by GR (11). In contrast, induction of GILZ and LAD1 by S211A is lower than that by WT at Dex concentrations below 10 nm, and similar to or even higher at hormone concentrations above 10 nm. This suggests that distinct mechanisms are employed by GR in the induction of GILZ and LAD1 at low vs. high Dex concentrations, which is affected by receptor phosphorylation. Interestingly, our previous analysis indicates that GILZ, for example, is largely MED14 independent, at least at 100 nm Dex (11). This effect was confirmed in three independent clones as well as a pooled line (data not shown). These findings indicate that the effect of GR S211 phosphorylation is gene specific and, in some cases, may also be dependent upon the amount of activated receptor.

We also examined whether GR phosphorylation site mutations affected the ability of GR to repress gene expression using the U2OS cell system. Seven GR target genes were examined that were repressed by GR in U20S cells (I. Rogatsky, unpublished data). These include cMyc, ephrin, Fra, cJun, JunB, Smad7, and PAC1. As with activation, the lack of GR phosphorylation at S211 results in gene-specific effects on repression by GR. For example, the ability of the S211A mutant to repress JunB was severely compromised compared with wild-type GR, whereas cMyc and Smad7 was only moderately affected, and repression of ephrin, Fra, and cJun was largely insensitive to the S211A mutation (Fig. 6, B and C). Unexpectedly, a majority of these genes were only minimally affected by the S226A mutation (Fig. 6, A and B). Interestingly, Pac 1, which is modestly repressed by the wild-type GR, in fact was induced by the S211A and to a lesser extent by the S226A mutants compared with WT GR (Fig. 6C). Basal levels of the repressed target genes were virtually unchanged between phosphorylation site mutants and wild-type lines. Thus, phosphorylation appears to affect the magnitude of repression by GR in a gene-selective manner, and in the case of Pac1, can convert a repressed gene to one that is now activated by GR. This may suggest that common surfaces within GR affected by phosphorylation may be responsible for both positive and negative regulation.

Figure 6.

Figure 6

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Repression by GR Is Phosphorylation Sensitive

U2OS cells stably expressing GR WT, GR S211A, or S266A were treated with ethanol vehicle or 100 nm Dex for 2 h, and total RNA was analyzed by real-time PCR. The values indicate expression of target genes normalized to RPL19 mRNA levels and presented relative to the expression of vehicle-treated cells, which was set as 100%. A, The level of hormone-dependent repression over the untreated control in each clone is shown. B, The percent repression of each gene by GR S211A and S226A relative to that of WT. C, The level of hormone-dependent repression (left) and the percent repression of PAC1 by the GR mutants relative to WT is shown. The error bar represents the sd from a single experiment performed in triplicate. This experiment was repeated twice, and the same patterns were observed. SMAD.

DISCUSSION

It has been proposed that hormone-dependent phosphorylation of steroid receptors is a mechanism to modulate receptor transcriptional responses (14,28,29,30). Here we provide evidence that GR phosphorylation at S211 and S226 can indeed affect receptor transcriptional activation in a gene-specific manner. Furthermore, this regulation can be both positive and negative, and the relative level of phosphorylation at S211 vs. S226 is an important determinant of receptor activity. Thus, comparatively higher phosphorylation at S211 relative to S226 correlates with GR nuclear localization and greater transcriptional activation and vice versa. This is consistent with previous findings that phosphorylation of GR S226 by JNK specifies receptor nuclear export under conditions of hormone withdrawal, a condition that favors low S211 and high S226 phosphorylation levels (19,20). Given that the glucocorticoid induction of all GR target genes examined (GILZ, IGFBP1, IRF8, and LAD1) was increased in the GR S226A-expressing cells (Fig. 5A), we conclude that phosphorylation at S226 reduces GR transcriptional activation through some general mechanism such as the enhanced nuclear export. A careful kinetic examination of nuclear import and export of the GR phosphorylation site mutations will help to address mechanism. It should be noted that our previous work has shown that S226A phosphorylation enhances hormone-dependent phosphorylation at S211 (21), which could also contribute to increased transcriptional activation of GR S226A.

In contrast, the impact of S211 phosphorylation on GR target gene expression is gene specific (Fig. 5B) and paralleled the requirements for MED14 in gene activation by GR (11). Structure simulations reveal that phosphorylation of GR at S211 may induce the receptor to acquire a helical conformation around the site (Fig. 4, A and B). In addition, S211 phosphorylation enhanced receptor interaction with MED14 (Fig. 4E), further reinforcing the idea that phospho-S211 may comprise a part of a cofactor-binding surface. Indeed, disruption of this interaction by the S211A mutation results in a shift of the ligand dose-response curve in the case of IGFBP1 and IRF8 (Fig. 4), genes the glucocorticoid induction of which is MED14 dependent. Modulation of the dose-response curve by changing GR-MED14 interaction would be predicted by important work from the Simons laboratory (31). In addition, elegant studies by Kumar et al. (24) have shown that acquisition of structure in the GR AF-1 can lead to enhanced interaction with the TATA-binding protein (TBP). It will be interesting to test whether this interaction is also modulated by receptor phosphorylation.

Phosphorylation-dependent coregulator recruitment is not limited to GR. Classic studies with CREB have shown that phosphorylation by protein kinase A results in binding of CBP (32). Hammer et al. (33) revealed that phosphorylation of SF-1 by MAPK enhanced coregulator recruitment. Likewise, estrogen receptor (ER)α phosphorylation of S118 facilitates interactions with (SF) 3ap120, a splicing factor (34), with steroid receptor coactivator 3 (35), as well as with stromelysin-1 platelet-derived growth factor-responsive element-binding protein that represses ER activity (36,37).

Unlike IRF8 and IGFBP1, the glucocorticoid induction of which was lessened by the S211A mutation across the hormone dose curve, activation of GILZ and LAD1 was impaired at a low concentration of Dex (<10 nm), but equal to or greater than that of WT at greater than 10 nm Dex (Fig. 5). GILZ harbors multiple glucocorticoid response elements (GREs) that contribute to activation by GR (38), whereas IGFBP1 (39) and IRF8 (11) have noncanonical GREs that display low GR occupancy relative to GILZ. We previously suggested that weak GR binding to DNA might bias genes toward MED14-dependent RNA polymerase II recruitment, whereas high GR recruitment bypasses this requirement (11). Consequently, under low hormone concentrations GR occupancy at GILZ is low, and its expression is more dependent on S211 phosphorylation and MED14 recruitment (Fig. 5B), whereas at higher hormone concentration GILZ induction becomes independent of phospho-S211 and MED14. In contrast, genes like IRF8, which are regulated through weak GREs, would operate entirely through the S211 phosphorylation- and MED14-dependent pathway (Fig. 7). We conclude that the role of GR phosphorylation at S211 is largely to facilitate the GR-MED14 interaction and transcriptional enhancement.

Figure 7.

Figure 7

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Interplay among GR Phosphorylation, GRE Occupancy, and Cofactor Recruitment Regulates GR Target Gene Response

A, Models for GR S211 phosphorylation-dependent and -independent gene expression. For IGFBP1 and IRF8, GR S211 phosphorylation (encircled P) facilitates recruitment of the Mediator complex via MED14, which, in turn, recruits the RNA polymerase II (Pol II) and the basal transcriptional machinery. The S211A mutation would reduce this interaction and transcriptional activation. A similar scenario is envisioned at GILZ under low hormone concentrations when only a fraction of the GREs are occupied by GR. At high hormone concentrations, GR will occupy all of the GILZ GREs and bypass the need for the MED14 and S211 phosphorylation by recruiting Pol II through another cofactor (hatched). B, GR S211 phosphorylation- and MED14-dependent genes are those with an inherently modest GR occupancy due to low affinity or number of GREs. Genes independent of S211 phosphorylation and MED14 contain multiple and/or high-affinity GREs capable of high GR occupancy under conditions of high hormone and/or receptor levels. Note that genes independent of S211 may display the behavioral characteristics of S211 phosphorylation-dependent genes under conditions of fractional GR occupancy as a result of low hormone and/or receptor levels.

From our analysis, we can begin to classify GR target genes into phosphorylation-dependent and -independent behaviors (Fig. 6B). The S211 phosphorylation-dependent genes are expected to be MED14 dependent and would include those at which GR occupancy is modest by virtue of a low GRE number or affinity (e.g. IGFBP1), or under suboptimal conditions, such as low hormone levels (e.g. GILZ). In contrast, S211 phosphorylation-independent genes are largely MED14 independent and would include those with multiple high-affinity GREs, as exemplified by GILZ under conditions of high hormone and receptor levels. Notably, phosphorylation-independent genes may behave like phosphorylation-dependent genes under conditions of fractional GR occupancy as a result of low hormone and/or receptor levels.

Because LAD1 induction by GR S211A mirrors that of GILZ, one would expect that LAD1 would harbor multiple high-affinity GREs. However, we have been unable to locate the GR binding sites in LAD1 by chromatin immunoprecipitation scanning of approximately 5 kb of DNA upstream of the start site of transcription. In light of the recent analysis of ER binding throughout the genome, this may not be surprising given that a majority of regulatory regions bound by the receptor are neither upstream nor nearby the start site of transcription (40). Additional studies will have to be performed to determine the location and nature of the LAD1 GREs.

These studies reveal one potential mechanism of how GR phosphorylation may modulate gene expression. They suggest that some GR-responsive genes will be hard wired and inherently GR phosphorylation dependent because of the restricted number and affinity of their GREs (e.g. IGFBP1). GR occupancy could also be influenced by adjacent transcription factor-binding sites, such as, for example, binding of FoxA1 for ER recruitment (41). In contrast, other genes (e.g. GILZ) may differentially respond to GR phosphorylation, due to multiple high-affinity GREs capable of wide-ranging GR occupancy states. This model may explain the often-conflicting data on the role of phosphorylation in GR function, which relied heavily on cell-based transient assays using synthetic reporter genes containing multiple high-affinity GREs and saturating receptor and hormone levels, all of which would favor a phosphorylation-independent response (42,43,44). This model is also consistent with early findings from Bai and Weigel (45) that a mutation of a phosphorylation site in the chicken progesterone receptor reduced transcriptional activity and that the magnitude of this effect was dependent upon the level of receptor expression. Elegant work from the laboratory of Alarid and associates (46) has shown that high nuclear concentrations of ERα in breast cancer cells can bypass the ligand requirement for activated transcription, and that this is independent of ER phosphorylation. Hence, increased receptor occupancy at a target gene, which in this case is driven by increased receptor expression, overrides the need for receptor phosphorylation. Likewise, important studies from the laboratory of Stallcup and co-workers (47) have shown that synergy among coactivators (e.g. GRIP1, CARM1, and PRMT1) occurs only when receptor levels are low, suggesting that different mechanisms of gene activation occur at high and low receptor concentrations.

Such an occupancy model for phosphorylation-dependent gene expression may hold true for other nuclear receptors and transcription factors, especially those that display cell type-specific patterns of phosphorylation (48). For example, signal transducer and activator of transcription 1 phosphorylation by IκB kinase ε in response to interferon β treatment has recently been shown to influence gene expression and signal transducer and activator of transcription 1 occupancy of select interferon-stimulated genes (49). Multiple androgen-responsive elements control prostate-specific antigen (PSA) activation by the androgen receptor (AR) (50). Depending on the level of AR expression or ligand concentration, PSA induction could be either dependent or independent of AR phosphorylation. This could explain the complex pattern of PSA expression (51,52) as a function of AR and ligand concentration observed, for example, in androgen-independent prostate cancer where AR levels are often elevated (53,54). Recently, we have found that liver X receptor (LXR)α phosphorylation has a greater impact on LXR target genes with noncanonical LXR binding sites, rather than those with strong consensus LXR-response elements (55). Thus, it appears that receptor levels and hormone strength, along with intracellular cofactor concentration, can set the stage for gene regulation through receptor phosphorylation. We also provide the evidence that GR phosphorylation plays a gene-selective role in transcriptional repression (Fig. 6). Whether receptor levels and cofactors such as MED14 play roles in repression remains to be elucidated. The interplay among GR phosphorylation, cofactor recruitment, and GRE type and/or abundance has likely evolved to adjust GR-dependent gene regulation to changing cellular needs.

MATERIALS AND METHODS

Antibody Production

The phosphopeptide, which corresponds to the following sequence in hGR: 218LLIDENLLSPO4PLAG230, was synthesized by Anaspec, Inc. (San Jose, CA). A cysteine residue was added to the N terminus of the peptide to facilitate chemical cross-linking. The peptide was coupled to KLH and used to immunize rabbits (Covance Research Products, Inc., Denver, PA). Sera from immunized rabbits were tested for antibody titer and specificity for the phosphorylated peptide was tested by ELISA. High-titer antibodies were further tested on hGR expressed in U2OS by immunoblotting.

Cell Culture and Preparation of Cell Extracts

The human lung carcinoma cell line A549 (CCL-185) containing endogenous GR and the human osteosarcoma cell line U2OS (HTB 96) lacking endogenous GR were obtained from the American Type Culture Collection (Manassas, VA) and were cultured in DMEM (Cellgro; Mediatech, Inc., Manassas, VA), supplemented with either 5% or 10% fetal bovine serum (HyClone Laboratories, Inc., Logan, UT), respectively, 2 mm l-glutamine, 50 μg/ml penicillin, and 50 μg/ml streptomycin (Cellgro). Generation of stable U2OS cell lines ectopically expressing hGR (wild-type and phosphorylation site mutants, S203A, S211A, and S226A) was performed as previously described. Individual neomycin-resistant clones and pools of clones were isolated and assayed for hGR expression by indirect immunofluorescence and immunoblotting with HA- and GR-specific antibodies. Clones homogeneously expressing HA-hGR were maintained at 500 μg/ml Geneticin.

Extracts for immunoblotting were prepared from a subconfluent 10-cm plate of A549 and U2OS-hGR cells treated with 100 nm Dex or equal volume of the ethanol vehicle 1 h before lysis. Cells were placed on ice, washed twice in PBS, lysed in 0.5 ml of buffer containing 50 mm HEPES (pH 7.5), 150 mm NaCl, 1 mm EDTA, 1 mm EGTA, 1 mm NaF, 1% Triton X-100, 10% glycerol, and additional protease and phosphatase inhibitors: 1 mm phenylmethylsulfonyl fluoride, 20 mm β-glycerophosphate, 8 mm sodium pyrophosphate, 1 μg/ml leupeptin, pepstatin A, and aprotinin (Roche, Indianapolis, IN). Lysates were centrifuged at 12,000 rpm for 15 min at 4 C. The soluble supernatants were normalized for total protein concentration using the Bio-Rad protein assay (Bio-Rad Laboratories, Hercules, CA), and the samples were boiled for 3 min in 2× sodium dodecyl sulfate sample buffer and stored at −20 C.

Immunoblotting

Cell extracts or immunoprecipitates containing GR were separated by 10% SDS-PAGE and were transferred to Immobilon paper (Millipore Corp., Bedford, MA) at 110 V for 80 min in Tris-glycine transfer buffer. The membranes were blocked overnight in 5% BSA in Tris-buffered saline (TBS, pH 7.4; blocking solution) at 4 C, and then were incubated in the blocking buffer with primary antibody at room temperature (RT) for 2–4 h using 1:1,000 dilution of serum for phospho-S226 (Ab459) and phospho-S211 (Ab353), and 1:10,000 dilution of serum for phospho-S203 (Ab221). Endogenous GR was detected using the N499 polyclonal antibody, raised against residues 1–499 of the hGR. The membranes were washed three times for 10 min in TBS/0.1% Triton X-100 and twice in TBS and incubated for 1 h at RT with 0.2 μg/ml protein A conjugated to horseradish peroxidase (Kirkegaard & Perry Laboratories, Gaithersburg, MD). For blots using the anti-HA-tag monoclonal antibody (α-HA; Roche), an horseradish peroxidase-conjugated goat-antimouse IgG secondary antibody was used. Blots were then washed three times for 10 min in TBS-0.1% Triton X-100 and twice in TBS and developed using enhanced chemiluminescence according to manufacturer’s instructions (GE Health Sciences, Piscataway, NJ). Quantitative analysis of immunoblots was performed using the National Institutes of Health Image software package (version 1.62).

Immunofluorescence

U2OS-hGR cells were cultured in phenol red-free DMEM containing 10% charcoal-stripped fetal bovine serum on coverslips coated with poly-d-lysine and were treated with Dex for 1 h. Cells were fixed in cold acetone (−20 C) for 15 min, air-dried, and incubated in PBS containing 2.5% BSA for 1 h to block nonspecific protein binding. Cells were incubated with primary antibodies in blocking solution for 1 h at RT, washed five times in PBS-0.1% Triton X-100, followed by incubation with goat antimouse or goat antirabbit fluorescein-conjugated secondary antibody (Vector Laboratories, Inc., Burlingame, CA) diluted in blocking solution, for 1 h at RT. Secondary antibody was removed by washing the cells five times in PBS-0.1% Triton X-100 and three times in PBS. Coverslips were mounted onto Citifluor (Ted Pella, Redding, CA), and the fluorescein signal was visualized and photographed using a Zeiss Axioplan 2 microscope (Carl Zeiss, Inc., Thornwood, NY).

Peptide Simulations

Modeling of peptides was performed using ICM software (MolSoft, La Jolla, CA) as previously described (56). Global energy minimization simulations were performed with two peptides, one with S211 phosphorylated and one without: Q195DLEFSSGSPGKETNES211PWRSDLLI220. For each peptide, 40 parallel independent Biased Probability Monte Carlo global energy optimizations were performed starting from randomized conformations. The simulations of each peptide were terminated after 200 million energy evaluations. During stimulations, low-energy conformations were collected, merged, and sorted by energy, and redundant conformations were eliminated.

Real-Time PCR

Total RNA from U20S-hGR cells was extracted, and cDNA was synthesized and amplified on a LightCycler (Roche) with primers for the GILZ, LAD1, IGFBP1, and IRF8 as described elsewhere (11). The following primer pairs were used to profile the GR-repressed genes: cMyc, forward (F), 5′-TGAACCAGAGTTTCATCTGCG-3′; reverse (R), 5′-TTCTCTGAGACGAGCTTGGC-3′; Fra1, F, 5′-CATCGCAAGAGTAGCAGCAG-3′; R, 5′-AGGAGACATTGGCTAGGGTG-3′; Ephrin1, F, 5′-CTTCACACCTTTCACCCTGG-3′; R, 5′-CAGTCACCTTCAACCTCAAGC-3′: cJun, F, 5′-TGCAAAGATGGAAACGACC-3′; R, 5′-TGCTCTGTTTCAGGATCTTGG-3′: JunB, F, 5′-AACATGGAAGACCAAGAGCG-3′; R, 5′-TCTTCACCTTGTCCTCCAGG-3′; SMAD7, F, 5′-TGCTGTGAATCTTACGGGAAG-3′; R, 5′-AATCCATCGGGTATCTGGAG-3′: PAC1, F, 5′-ATACCTCATGCAGATCGCC-3′; R, 5′-ACAGCACCTGGGTCTCAAAC-3′.

Acknowledgments

We thank Evelyn Kono and Sara Schoenfeld for help with the inhibitor studies, Ines Pindea Torra for assistance with qPCR, and Susan Ha and Jerome Nwachukwu for evaluating the manuscript.

Footnotes

This work was supported by grants from the Concern Foundation (to S.K.L.), The American Cancer Society (to S.K.L.), and National Institutes of Health Grant DK54836 (to M.J.G.).

Present address for C.N.C.: School of Health Information Sciences, University of Texas Health Science Center at Houston, Houston, Texas 77030.

Disclosure Statement: The authors have nothing to disclose.

First Published Online May 15, 2008

Abbreviations: AF-1, Activation function 1; AR, androgen receptor; Cdk2, cyclin-dependent kinase 2; Dex, dexamthasone; ER, estrogen receptor; GILZ, glucocorticoid-induced leucine zipper; GR, glucocorticoid receptor; GRE, glucocorticoid response element; HA, hemagglutinin; IGFBP1, IGF-binding protein 1; IRF8, interferon regulatory factor 8; JNK, c-Jun N-terminal kinase; LAD1, ladinin 1; LXR, liver X receptor; MMTV, mouse mammary tumor virus; PSA, prostate-specific antigen; RT, room temperature; TBS, Tris-buffered saline; WT, wild type.

References

  1. Buckingham JC 2006 Glucocorticoids: exemplars of multi-tasking. Br J Pharmacol 147(Suppl 1):S258–S268 [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Yudt MR, Cidlowski JA 2002 The glucocorticoid receptor: coding a diversity of proteins and responses through a single gene. Mol Endocrinol 16:1719–1726 [DOI] [PubMed] [Google Scholar]
  3. Yamamoto KR 1995 Multilayered control of intracellular receptor function. Harvey Lect 91:1–19 [PubMed] [Google Scholar]
  4. Gronemeyer H, Gustafsson JA, Laudet V 2004 Principles for modulation of the nuclear receptor superfamily. Nat Rev Drug Discov 3:950–964 [DOI] [PubMed] [Google Scholar]
  5. Segnitz B, Gehring U 1997 The function of steroid hormone receptors is inhibited by the hsp90-specific compound geldanamycin. J Biol Chem 272:18694–18701 [DOI] [PubMed] [Google Scholar]
  6. Scherrer LC, Hutchison KA, Sanchez ER, Randall SK, Pratt WB 1992 A heat shock protein complex isolated from rabbit reticulocyte lysate can reconstitute a functional glucocorticoid receptor-Hsp90 complex. Biochemistry 31:7325–7329 [DOI] [PubMed] [Google Scholar]
  7. Picard D 2006 Chaperoning steroid hormone action. Trends Endocrinol Metab 17:229–235 [DOI] [PubMed] [Google Scholar]
  8. Picard D, Khursheed B, Garabedian MJ, Fortin MG, Lindquist S, Yamamoto KR 1990 Reduced levels of hsp90 compromise steroid receptor action in vivo. Nature 348:166–168 [DOI] [PubMed] [Google Scholar]
  9. Hutchison KA, Czar MJ, Pratt WB 1992 Evidence that the hormone-binding domain of the mouse glucocorticoid receptor directly represses DNA binding activity in a major portion of receptors that are “misfolded” after removal of hsp90. J Biol Chem 267:3190–3195 [PubMed] [Google Scholar]
  10. Kumar R, Thompson EB 2005 Gene regulation by the glucocorticoid receptor: structure:function relationship. J Steroid Biochem Mol Biol 94:383–394 [DOI] [PubMed] [Google Scholar]
  11. Chen W, Rogatsky I, Garabedian MJ 2006 MED14 and MED1 differentially regulate target-specific gene activation by the glucocorticoid receptor. Mol Endocrinol 20:560–572 [DOI] [PubMed] [Google Scholar]
  12. Dalman FC, Sanchez ER, Lin AL, Perini F, Pratt WB 1988 Localization of phosphorylation sites with respect to the functional domains of the mouse L cell glucocorticoid receptor. J Biol Chem 263:12259–12267 [PubMed] [Google Scholar]
  13. Bodwell JE, Orti E, Coull JM, Pappin DJ, Smith LI, Swift F 1991 Identification of phosphorylated sites in the mouse glucocorticoid receptor. J Biol Chem 266:7549–7555 [PubMed] [Google Scholar]
  14. Ismaili N, Garabediana MJ 2004 Modulation of glucocorticoid receptor function via phosphorylation. Ann NY Acad Sci 1024:86–101 [DOI] [PubMed] [Google Scholar]
  15. Krstic MD, Rogatsky I, Yamamoto KR, Garabedian MJ 1997 Mitogen-activated and cyclin-dependent protein kinases selectively and differentially modulate transcriptional enhancement by the glucocorticoid receptor. Mol Cell Biol 17:3947–3954 [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Wang Z, Frederick J, Garabedian MJ 2002 Deciphering the phosphorylation “code” of the glucocorticoid receptor in vivo. J Biol Chem 277:26573–26580 [DOI] [PubMed] [Google Scholar]
  17. Wang Z, Garabedian MJ 2003 Modulation of glucocorticoid receptor transcriptional activation, phosphorylation, and growth inhibition by p27Kip1. J Biol Chem 278:50897–50901 [DOI] [PubMed] [Google Scholar]
  18. Miller AL, Webb MS, Copik AJ, Wang Y, Johnson BH, Kumar R, Thompson EB 2005 p38 Mitogen-activated protein kinase (MAPK) is a key mediator in glucocorticoid-induced apoptosis of lymphoid cells: correlation between p38 MAPK activation and site-specific phosphorylation of the human glucocorticoid receptor at serine 211. Mol Endocrinol 19:1569–1583 [DOI] [PubMed] [Google Scholar]
  19. Rogatsky I, Logan SK, Garabedian MJ 1998 Antagonism of glucocorticoid receptor transcriptional activation by the c-Jun N-terminal kinase. Proc Natl Acad Sci USA 95:2050–2055 [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Itoh M, Adachi M, Yasui H, Takekawa M, Tanaka H, Imai K 2002 Nuclear export of glucocorticoid receptor is enhanced by c-Jun N-terminal kinase-mediated phosphorylation. Mol Endocrinol 16:2382–2392 [DOI] [PubMed] [Google Scholar]
  21. Wang Z, Chen W, Kono E, Dang T, Garabedian MJ 2007 Modulation of glucocorticoid receptor phosphorylation and transcriptional activity by a C-terminal-associated protein phosphatase. Mol Endocrinol 21:625–634 [DOI] [PubMed] [Google Scholar]
  22. Blind RD, Garabedian MJ 2008 Differential recruitment of glucocorticoid receptor phospho-isoforms to glucocorticoid-induced genes. J Steroid Biochem Mol Biol 109:150–157 [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Strubing C, Clapham DE 1999 Active nuclear import and export is independent of lumenal Ca2+ stores in intact mammalian cells. J Gen Physiol 113:239–248 [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Kumar R, Lee JC, Bolen DW, Thompson EB 2001 The conformation of the glucocorticoid receptor af1/τ1 domain induced by osmolyte binds co-regulatory proteins. J Biol Chem 276:18146–18152 [DOI] [PubMed] [Google Scholar]
  25. Iniguez-Lluhi JA, Lou DY, Yamamoto KR 1997 Three amino acid substitutions selectively disrupt the activation but not the repression function of the glucocorticoid receptor N terminus. J Biol Chem 272:4149–4156 [DOI] [PubMed] [Google Scholar]
  26. Hittelman AB, Burakov D, Iniguez-Lluhi JA, Freedman LP, Garabedian MJ 1999 Differential regulation of glucocorticoid receptor transcriptional activation via AF-1-associated proteins. EMBO J 18:5380–5388 [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Rogatsky I, Wang JC, Derynck MK, Nonaka DF, Khodabakhsh DB, Ha CM, Darimont BD, Garabedian MJ, Yamamoto KR 2003 Target-specific utilization of transcriptional regulatory surfaces by the glucocorticoid receptor. Proc Natl Acad Sci USA 100:13845–13850 [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Weigel NL 1996 Steroid hormone receptors and their regulation by phosphorylation. Biochem J 319:657–667 [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Shao D, Lazar MA 1999 Modulating nuclear receptor function: may the phos be with you. J Clin Invest 103:1617–1618 [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Weigel NL, Moore NL 2007 Steroid receptor phosphorylation: a key modulator of multiple receptor functions. Mol Endocrinol 21:2311–2319 [DOI] [PubMed] [Google Scholar]
  31. Simons Jr SS 2006 How much is enough? Modulation of dose-response curve for steroid receptor-regulated gene expression by changing concentrations of transcription factor. Curr Top Med Chem 6:271–285 [DOI] [PubMed] [Google Scholar]
  32. Chrivia JC, Kwok RP, Lamb N, Hagiwara M, Montminy MR, Goodman RH 1993 Phosphorylated CREB binds specifically to the nuclear protein CBP. Nature 365:855–859 [DOI] [PubMed] [Google Scholar]
  33. Hammer GD, Krylova I, Zhang Y, Darimont BD, Simpson K, Weigel NL, Ingraham HA 1999 Phosphorylation of the nuclear receptor SF-1 modulates cofactor recruitment: integration of hormone signaling in reproduction and stress. Mol Cell 3:521–526 [DOI] [PubMed] [Google Scholar]
  34. Endoh H, Maruyama K, Masuhiro Y, Kobayashi Y, Goto M, Tai H, Yanagisawa J, Metzger D, Hashimoto S, Kato S 1999 Purification and identification of p68 RNA helicase acting as a transcriptional coactivator specific for the activation function 1 of human estrogen receptor α. Mol Cell Biol 19:5363–5372 [DOI] [PMC free article] [PubMed] [Google Scholar] [Retracted]
  35. Likhite VS, Stossi F, Kim K, Katzenellenbogen BS, Katzenellenbogen JA 2006 Kinase-specific phosphorylation of the estrogen receptor changes receptor interactions with ligand, deoxyribonucleic acid, and coregulators associated with alterations in estrogen and tamoxifen activity. Mol Endocrinol 20:3120–3132 [DOI] [PubMed] [Google Scholar]
  36. Gburcik V, Picard D 2006 The cell-specific activity of the estrogen receptor α may be fine-tuned by phosphorylation-induced structural gymnastics. Nucl Recept Signal 4:e005 [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Gburcik V, Bot N, Maggiolini M, Picard D 2005 SPBP is a phosphoserine-specific repressor of estrogen receptor α. Mol Cell Biol 25:3421–3430 [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Wang JC, Derynck MK, Nonaka DF, Khodabakhsh DB, Ha C, Yamamoto KR 2004 Chromatin immunoprecipitation (ChIP) scanning identifies primary glucocorticoid receptor target genes. Proc Natl Acad Sci USA 101:15603–15608 [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Suwanichkul A, DePaolis LA, Lee PD, Powell DR 1993 Identification of a promoter element which participates in cAMP-stimulated expression of human insulin-like growth factor-binding protein-1. J Biol Chem 268:9730–9736 [PubMed] [Google Scholar]
  40. Carroll JS, Meyer CA, Song J, Li W, Geistlinger TR, Eeckhoute J, Brodsky AS, Keeton EK, Fertuck KC, Hall GF, Wang Q, Bekiranov S, Sementchenko V, Fox EA, Silver PA, Gingeras TR, Liu XS, Brown M 2006 Genome-wide analysis of estrogen receptor binding sites. Nat Genet 38:1289–1297 [DOI] [PubMed] [Google Scholar]
  41. Carroll JS, Liu XS, Brodsky AS, Li W, Meyer CA, Szary AJ, Eeckhoute J, Shao W, Hestermann EV, Geistlinger TR, Fox EA, Silver PA, Brown M 2005 Chromosome-wide mapping of estrogen receptor binding reveals long-range regulation requiring the forkhead protein FoxA1. Cell 122:33–43 [DOI] [PubMed] [Google Scholar]
  42. Webster JC, Jewell CM, Bodwell JE, Munck A, Sar M, Cidlowski JA 1997 Mouse glucocorticoid receptor phosphorylation status influences multiple functions of the receptor protein. J Biol Chem 272:9287–9293 [DOI] [PubMed] [Google Scholar]
  43. Mason SA, Housley PR 1993 Site-directed mutagenesis of the phosphorylation sites in the mouse glucocorticoid receptor. J Biol Chem 268:21501–21504 [PubMed] [Google Scholar]
  44. Almlof T, Wright AP, Gustafsson JA 1995 Role of acidic and phosphorylated residues in gene activation by the glucocorticoid receptor. J Biol Chem 270:17535–17540 [DOI] [PubMed] [Google Scholar]
  45. Bai W, Weigel NL 1996 Phosphorylation of Ser211 in the chicken progesterone receptor modulates its transcriptional activity. J Biol Chem 271:12801–12806 [DOI] [PubMed] [Google Scholar]
  46. Fowler AM, Solodin NM, Valley CC, Alarid ET 2006 Altered target gene regulation controlled by estrogen receptor-α concentration. Mol Endocrinol 20:291–301 [DOI] [PubMed] [Google Scholar]
  47. Koh SS, Chen D, Lee YH, Stallcup MR 2001 Synergistic enhancement of nuclear receptor function by p160 coactivators and two coactivators with protein methyltransferase activities. J Biol Chem 276:1089–1098 [DOI] [PubMed] [Google Scholar]
  48. Taneja SS, Ha S, Swenson NK, Huang HY, Lee P, Melamed J, Shapiro E, Garabedian MJ, Logan SK 2005 Cell-specific regulation of androgen receptor phosphorylation in vivo. J Biol Chem 280:40916–40924 [DOI] [PubMed] [Google Scholar]
  49. Tenoever BR, Ng SL, Chua MA, McWhirter SM, Garcia-Sastre A, Maniatis T 2007 Multiple functions of the IKK-related kinase IKKε in interferon-mediated antiviral immunity. Science 315:1274–1278 [DOI] [PubMed] [Google Scholar]
  50. Wang Q, Carroll JS, Brown M 2005 Spatial and temporal recruitment of androgen receptor and its coactivators involves chromosomal looping and polymerase tracking. Mol Cell 19:631–642 [DOI] [PubMed] [Google Scholar]
  51. Yeung F, Li X, Ellett J, Trapman J, Kao C, Chung LW 2000 Regions of prostate-specific antigen (PSA) promoter confer androgen-independent expression of PSA in prostate cancer cells. J Biol Chem 275:40846–40855 [DOI] [PubMed] [Google Scholar]
  52. Jia L, Coetzee GA 2005 Androgen receptor-dependent PSA expression in androgen-independent prostate cancer cells does not involve androgen receptor occupancy of the PSA locus. Cancer Res 65:8003–8008 [DOI] [PubMed] [Google Scholar]
  53. Craft N, Shostak Y, Carey M, Sawyers CL 1999 A mechanism for hormone-independent prostate cancer through modulation of androgen receptor signaling by the HER-2/neu tyrosine kinase. Nat Med 5:280–285 [DOI] [PubMed] [Google Scholar]
  54. Wang LG, Ossowski L, Ferrari AC 2004 Androgen receptor level controlled by a suppressor complex lost in an androgen-independent prostate cancer cell line. Oncogene 23:5175–5184 [DOI] [PubMed] [Google Scholar]
  55. Torra IP, Ismaili N, Feig JE, Xu CF, Cavasotto C, Pancratov R, Rogatsky I, Neubert TA, Fisher EA, Garabedian MJ 2008 Phosphorylation of liver X receptor α selectively regulates target gene expression in macrophages. Mol Cell Biol 28:2626–2636 [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Li W, Cavasotto CN, Cardozo T, Ha S, Dang T, Taneja SS, Logan SK, Garabedian MJ 2005 Androgen receptor mutations identified in prostate cancer and androgen insensitivity syndrome display aberrant ART-27 coactivator function. Mol Endocrinol 19:2273–2282 [DOI] [PubMed] [Google Scholar]

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