Glucocorticoid receptor phosphorylation differentially affects target gene expression

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.

Figure 1

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.

Figure 2

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.

Figure 3

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.

Figure 4

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. MED14_1214–1434 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.

Figure 5

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.

Figure 6

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.

Figure 7

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.