Phosphorylation of progesterone receptor serine 400 mediates ligand-independent transcriptional activity in response to activation of cyclin-dependent protein kinase 2

Abstract

Human progesterone receptors (PR) are phosphorylated by cyclin-dependent protein kinase 2 (CDK2) at multiple sites, including Ser400. Herein, we have addressed the significance of phosphorylation of this residue. PR phospho-Ser400-specific antibodies revealed regulated phosphorylation of Ser400 in response to progestins and mitogens, and this correlated with increased CDK2 levels and activity. Expression of cyclin E elevated CDK2 activity and downregulated PR independently of ligand. Similarly, overexpression of activated mutant CDK2 increased PR transcriptional activity in the absence and presence of progestin. Mutation of PR Ser400 to alanine (S400A) blocked CDK2-induced PR activity in the absence, but not in the presence, of progestin. PR was unresponsive to activated CDK2 in breast cancer cells with elevated p27, and RNA interference knock-down of p27 partially restored CDK2-induced ligand-independent PR activation. Similarly, in p27(-/-) mouse embryonic fibroblasts, elevated CDK2 activity increased wild-type (wt) but not S400A PR transcriptional activity in the absence of progestin. CDK2 induced nuclear localization of unliganded wt but not S400A PR; liganded S400A PR exhibited delayed nuclear accumulation. These studies demonstrate that CDK2 regulates PR in the absence of progestins via phosphorylation of Ser400, thus revealing a novel mechanism for upregulated PR transcriptional activity in human breast cancer cells expressing altered cell cycle regulatory molecules.

FIG. 1.

Regulation of PR Ser400 phosphorylation. (A) Characterization of PR phospho-Ser400-specific antibodies. T47Dco cells expressing endogenous PR-A and PR-B isoforms or PR-null T47D cells were treated with R5020 (+) or EtOH vehicle control (−) for 1 h. Equal amounts of cell lysate (100 μg) were electrophoresed on SDS-7.5% PAGE gels, transferred to nitrocellulose, and blotted with antibodies specific for either total PR protein or PR phospho-Ser400(P400). As a control for the specificity of PR phospho-Ser400 antibodies, blots were incubated with antibodies in the presence or absence of a blocking peptide representing the peptide used to make the phospho-specific antibodies. (B) Time course of PR Ser400 phosphorylation. T47Dco, T47D-YA, and T47D-YB cells were treated with R5020 for 0 to 24 h, and Western blotting was performed as for panel A to detect either total PR or phospho-Ser400 PR. (C) CDK2 activity in R5020-treated T47D-YB cells. T47D-YB cells were treated for 0 to 24 h with either R5020 (+) or EtOH vehicle control (−), and CDK2 immune complexes were purified and subjected to in vitro kinase assays with histone H1 (HH1) as a substrate. Phosphorylation of HH1 was detected by autoradiography and quantified by phosphorimaging, and data are presented as a bar graph. Western blotting was performed to detect total levels of CDK2 in each cell lysate. (D) Mitogen-induced PR Ser400 phosphorylation. T47D-YB cells were treated with various mitogens (R5020, 10 nM; EGF, 30 ng/ml; heregulin [HRG], 10 ng/ml; PMA, 10 nM; IGF, 30 ng/ml; FBS, 10%) or vehicle control for 5 to 60 min, and cell lysates were subjected to SDS-PAGE and immunoblotting with antibodies specific for either total PR or phospho-Ser400 PR. Results are representative of two to three independent experiments.

FIG. 2.

CDK2 activity decreases PR protein levels. (A) CDK2 inhibitors block ligand-dependent PR Ser 400 phosphorylation and PR downregulation. T47D-YB cells were preincubated with either vehiclecontrol or a CDK2-specific inhibitor (CDK2 inhibitor II) for 1 h andthen incubated with or without R5020 for an additional 11 h. Cell lysates were blotted for either total PR or phospho-Ser400 PR (P400). (B) CDK2 inhibitors block mitogen-induced phosphorylation of PR Ser400. T47D-YB cells were pretreated with vector control (dimethyl sulfoxide [DMSO]) or the CDK inhibitor roscovatine (700 nM) for 30 min and then treated with R5020 or EGF for 1 h. Cell lysates were blotted for total PR or phospho-Ser400 PR (P400). (C) Adenoviral expression of cyclin E stimulates CDK2 activity. HeLa cells stably expressing PR-B were infected with Ad-cyclin E (Ad-E) or a control adenovirus (Ad-control) for 48 h. CDK2 activity in immunoglobulin G (IgG-control) or CDK2 immune complexes was measured as described above (Fig. 1 legend) with histone H1 (HH1) as a substrate. Purified active cyclin E/CDK2 complexes (1 to 3 ng) were added to the reaction mixture as a positive control for CDK2 activity. (D) Ad-cyclin E induces loss of PR protein. Western blotting for total PR, phospho-Ser400 PR, cyclin E, and β-actin was performed on cell lysates from HeLa cells stably expressing PR-B and transduced with either control adenovirus (Ad-control) or Ad-cyclin E (Ad-E) in the presence and absence of R5020 for 1 h. (E) Inhibition of CDK2 activity stabilizes PR protein. T47D-YB cells were treated with a CDK2-specific inhibitor (CDK2 inhibitor II) or vehicle control (DMSO) for 12 h. Western blotting assays for total PR and actin were performed on cell lysates. Data from three separate experiments are shown.

FIG. 3.

CDK2 increases PR transcriptional activity in the presence and absence of progestin. (A) Expression of S400A PR-B and time course of ligand-dependent wt and S400A PR downregulation. HeLa cells were transiently transfected with either wt or S400A mutant PR-B (1 μg each) and treated with R5020 (+) or ETOH vehicle control (−) for 1 h (upper panel) or for 1 to 9 h (lower panels). Equal amounts of cell lysates (100 μg) were probed for total PR, phospho-Ser400 PR, or β-actin by Western blotting. (B) CDK2-induced transcriptional activity of wt and S400A PR. HeLa cells were transiently cotransfected withPRE-driven luciferase and constitutive Renilla reporter plasmids, either wt or S400A PR-B (100 ng each), and either mutant activated CDK2 (CDK2-TY) or its parental control vector. Following treatment of cell cultures with R5020 for 18 h, PRE-luciferase activity was measured and normalized to that in the Renilla controls. Cell lysates were also immunoblotted for total PR levels at the same time point. (C) CDK2 dose dependence of PR transcriptional activity. HeLa cells were transiently cotransfected with wt or S400A PR-B (100 ng each) and PRE-luciferase and Renilla reporter plasmids, along with increasing amounts of mutant CDK2-TY cDNA. PRE-luciferase activity was measured and normalized to that of Renilla controls. Control cultures (vector) received 1.0 μg of parental vector control (for CDK2-TY). Results are representative of three independent experiments. The data are presented as means ± standard deviations of three replicates for each data point.

FIG. 4.

Activated CDK2 induces ligand-independent PR transcriptional activity in PR-null MDA-MB-435 (A) but not T47D-Y (B) breast cancer cells. (A) PR-B activity in MDA-MD-435 cells. MDA-MB-435 cells were transiently transfected with wt or S400A PR-B along with PRE-luciferase and Renilla reporter plasmids and increasing concentrations of mutant CDK2-TY or its empty parental vector as a control (vector). PRE-luciferase activity was measured and is reported in arbitrary units as the luciferase/Renilla ratio. (B) PR-B activity in T47D-Y cells. T47D-Y PR-null cells were transiently transfected with either wt or S400A PR along with PRE-luciferase and Renilla reporter plasmids and increasing concentrations of CDK2-TY or its empty parental vector as a control (vector). PRE-luciferase activity was measured and is reported in arbitrary units as the luciferase/Renilla ratio. The data are presented as means ± standard deviations of three replicates for each data point. Results are representative of two to three independent experiments.

FIG. 5.

CDK2-induced PR transcriptional activity is inhibited by the CDK2 inhibitor p27. (A) p27 expression in cell line models. Cell lysates from T47D-Y, T47D-YB, MDA-MB-435, HeLa, and p27^−/− and p27^+/+ MEF cells were electrophoresed on 10% gels and blotted for p27; β-actin served as a loading control. (B) Reversal of CDK2-induced PR activity by p27. MDA-MB-435 cells were transiently cotransfected with wt PR (100 ng), mutant CDK2-TY in the presence and absence of either p27 or mutant p27^ck−, and the appropriate vector controls along with the PRE-luciferase and Renilla reporter plasmids. PRE-luciferase activity was measured following treatment with either EtOH vehicle control or R5020 for 18 h. (C) PR transcriptional activity is enhanced by ligand and EGF in MDA-MB-435 breast cancer cells. MDA-MB-435 cells were transiently transfected with a PRE-luciferase reporter, Renilla reporter, with wt or S400A PR (100 ng) with or without CDK2-TY. Following treatment with R5020 (10 nM) or EGF (30 ng/ml) for 18 h, PRE-luciferase activity was measured and normalized to that of Renilla controls. The data are presented as means ± standard deviations of three replicates for each data point. Results are representative of three independent experiments.

FIG. 6.

Ligand and mitogen-induced PR transcriptional activity is enhanced in cells with low levels of p27. (A) Absence of CDK2-induced PR activity in p27-rich T47D cells. T47D-YB cells were transiently transfected with a PRE-luciferase reporter along with increasing concentrations of CDK2-TY. PRE-luciferase activity was measured and normalized to that of Renilla controls. Vector control cultures (vector) received 1.0 μg of parental vector control (for CDK2-TY). (B) p27 RNAi restores CDK2-induced PR transcriptional activity in T47D-YB cells. T47D-YB cells were transiently transfected with PRE-luciferase and Renilla reporter plasmids, CDK2-TY (+) or its parental control vector (−), and pSHAG vectors expressing either control RNAi (−) or p27 RNAi (+). Following treatment with R5020 (10 nM) for 18 h, PRE-luciferase activity was measured and normalized to that of Renilla controls. (Inset) p27 RNAi knock-down of p27. P27 RNAi or control RNAi was cotransfected into T47D-YB cells, and cell lysates were blotted for p27 and β-actin. The data are presented as means ± standard deviations of three replicates for each data point. Results are representative of two to three independent experiments.

FIG. 7.

PR Ser400 is highly phosphorylated in p27^−/− relative to wt p27^+/+ MEF cells. (A) Endogenous CDK2 activity in MEF cells. Endogenous CDK2 in lysates from unstimulated wt p27^+/+ and knockout p27^−/− MEFs was purified by immunoprecipitation with CDK2-specific antibodies, and immune complexes were assayed in in vitro kinase assays with histone H1 (HH1) as a substrate. Controls included nonspecific immunoglobulin G immunoprecipitates (IgG control) and purified recombinant active cyclinE/CDK2 as a positive control for CDK2 activity. (B) Increased PR Ser400 phosphorylation in p27^−/− MEF cells. wt PR or S400A PR was transiently transfected into p27^+/+ or p27^−/− MEF cells. Cell lysates were electrophoresed on SDS-PAGE gels and subjected to Western blotting with either total PR or phospho-Ser400 PR antibodies (P400). The PR phospho-Ser400 antibody recognized a nonspecific protein (nsp), which migrated just under PR in MEF cells, that is not present in breast epithelial cells or HeLa cells.

FIG. 8.

PR Ser400 is required for ligand-independent PR transcriptional activity in p27^−/− MEF cells. (A) Increased PR transcriptional activity in p27^−/− MEF cells. P27^+/+ or p27^−/− MEF cells were transiently transfected with PRE-luciferase and Renilla reporter plasmids and either wt or S400A PR-B (100 ng) and, 48 h after transfection, cell cultures were treated without or with R5020 for 18 h. PRE-luciferase activity in cell lysates was measured and normalized to that of Renilla controls. (B) PR dose dependence of PR transcriptionalactivity in p27^−/− MEF cells. p27^−/− cells were transiently transfected with increasing concentrations (10 to 250 ng) of vectors expressing either wt or S400A PR-B, control empty vectors to normalize total cDNA concentrations, and a constant amount of PRE-luciferase reporter plasmid. PRE-luciferase activity in lysates from unstimulated cells was measured and is reported as relative luminometer units (RLUs). (C) Absence of ligand-independent PR transcriptional activity in p27^+/+ cells. P27^+/+ MEF cells were transiently transfected with increasing concentrations of either wt or S400A PR-B (10 to 1,000 ng), control empty vector as appropriate, and a constant amount of PRE-luciferase reporter plasmid as for panel B, and PRE-luciferase activity in lysates from unstimulated cells was measured. (Note the difference in scales between Fig. 7B and C.) (D) RU486 blocks ligand-independent PR transcriptional activity in p27^−/− cells. p27^−/− cells were transiently transfected with a PRE-luc reporter along with either 100 or 250 ng wt PR. 48 h following transfection, cultures were treated with 10⁻⁷ M RU486 or EtOH vehicle control, and PRE-luciferase activity was measured and normalized to that of Renilla controls. The data are presented as means ± standard deviations of three replicates for each data point. Results are representative of three to four independent experiments.

FIG. 9.

Confocal microscopy showing the subcellular localization of wt and S400A PR. (A) Delayed ligand-induced nuclear translocation of S400A PR. HeLa cells growing on coverslips in six-well dishes were transiently transfected with wt or S400A PR-B and treated with EtOH vehicle control or R5020 for either 1 or 3 h. The cells were fixed and subjected to IHC with total PR monoclonal antibodies followed by fluorescein isothiocyanate (FITC)-conjugated secondary antibodies, and PR-stained cells were visualized by confocal microscopy. Controls for the primary andsecondary antibodies demonstrated the specificity of PR staining (data not shown). (B) Percentage of transfected cells containing exclusively nuclear PR. Transfected HeLa cells containing wt or S400A PR were prepared as for panel A and counted (100 each), and the data are expressed graphically as the percentage of transfected cells with PR exclusively in the nucleus relative to PR staining in both compartments. (C) Wild-type but not S400A PR nuclear association induced by activated CDK2. HeLa cells growing on coverslips in six-well dishes were transiently cotransfected with either wt or S400A PR-B along with mutant CDK2-TY or empty vector control. PR in transfected cells was immunostained as above with total PR monoclonal antibodies followed by FITC-conjugated secondary antibodies. PR-transfected cells were visualized by confocal microscopy. (D) Percentage of cells with exclusively nuclear PR. HeLa cells transfected with either wt or S400A PR were prepared as for panel C and counted (100 each), and the data are expressed graphically as the percentage of cells with PR exclusively in the nucleus relative to those with PR in both cytoplasmic and nuclear compartments. (E) p27 and CDK2-dependent cytoplasmic retention of liganded PR. HeLa cells growing on coverslips in six-well dishes were transiently transfected with wt PR-B and either empty vector control or vectors encoding wt p27 or a p27^ck− mutant unable to bind CDK2 and, 48 h later, transfected cells were treated with R5020 for 1 h. Cells were stained with PR-specific monoclonal antibodies as described above and visualized by confocal microscopy. These experiments were repeated two times in HeLa cells and one time in MDA-MB-435 cells with similar results.