Cyclin-dependent kinase activity is required for progesterone receptor function: novel role for cyclin A/Cdk2 as a progesterone receptor coactivator - PubMed (original) (raw)

Cyclin-dependent kinase activity is required for progesterone receptor function: novel role for cyclin A/Cdk2 as a progesterone receptor coactivator

Ramesh Narayanan et al. Mol Cell Biol. 2005 Jan.

Abstract

Our studies examining the role of the cell cycle-regulated kinase cyclin A/Cdk2 in progesterone receptor (PR) action have demonstrated that cyclin-dependent kinase activity is required for PR function and that cyclin A/Cdk2 functions as a PR coactivator. Although Cdk2 can phosphorylate PR, elimination of these phosphorylation sites has little effect on the ability of cyclin A/Cdk2 to stimulate PR activity. PR interacts with cyclin A and recruits cyclin A/Cdk2 to progestin-responsive promoters, stimulating transcription. Inhibition of Cdk2 activity abolishes progesterone-dependent activation of PR target genes in part through inhibition of PR-dependent recruitment of steroid receptor coactivator 1 (SRC-1) and subsequent histone H4 acetylation at the target promoter. In vitro studies revealed that the interaction between SRC-1 and PR is dependent upon phosphorylation of SRC-1. This heretofore-unknown mechanism provides a potential means for integrating the regulation of PR activity with cell cycle progression. Moreover, the ability of PR to recruit cyclin A/Cdk2 to target promoters provides locally elevated levels of kinase, which can preferentially facilitate phosphorylation-dependent interactions and enzymatic activities of coactivators at the target promoter.

PubMed Disclaimer

Figures

FIG. 1.

Cyclin A increases the transcriptional activity of PR and AR but not VDR. HeLa cells (A to E, G, and H) or COS cells (F) were transfected as indicated in Materials and Methods with vector (0.5 μg of SRα296) or 0.5 μg of cyclin A, 10 ng of PR-B (A, D, E, and F), PR-A (B), or AR (G), and 0.25 μg of the reporter plasmids indicated in the panels (GRE, GRE2-_E1b_-luc; MMTV, MMTV-luc; VDRE, VDRE-tkLUC). The cells were treated or not 24 h after transfection with 10 nM concentrations of the indicated hormones. The cells were harvested 24 h after treatment, and luciferase activity was measured and normalized to β-Gal activity in panels A, B, and E to H and to the receptor level in panel D. (C) HeLa cells were cotransfected with 0.5 μg of cyclin A or vector and 0.05 μg of CMV β-Gal, and the β-Gal assay was performed on samples containing equal amounts of protein. The inset of panel A shows that cyclin A expression was increased in cells transfected with cyclin A. A representative Western blot of cyclin A over expression was performed with protein extracts from cells transfected with 0.5 μg of vector or cyclin A. Data shown are the mean and standard error of three independent experiments. RLU, relative light units; 1,25-D, 1,25-dihydroxyvitamin D3.

FIG. 2.

Functional and physical interactions between cyclin A and PR. (A) HeLa cells were cotransfected with 10 ng of full-length PR-B or PR-B constructs coding for the N-terminal DNA binding domain (DBD) hinge (left panel) or DBD hinge LBD (right panel), 0.5 μg of vector or cyclin A, 0.25 μg of GRE2-_E1b_-LUC, and 0.05 μg of β-Gal. The cells were treated or not with 10 nM R5020, and luciferase activity was normalized to β-Gal levels. White bars are vector-transfected samples, and black bars are cyclin A-transfected samples. Also shown are the schematic representations of the PR-B constructs used. (B) HeLa cells were transfected with 0.25 μg of GRE2-_E1b_-LUC, 10 ng of wild-type PR-A, or a 10 Ala mutant of PR-A (all of the serines and threonines in Ser/Thr-Pro motifs were mutated to alanines), and 0.5 μg of cyclin A or vector. The cells were treated with 10 nM R5020, and luciferase activity was measured and normalized to β-Gal levels. The top panel shows the sites that were mutated to alanine. The hormone-treated vector-transfected samples were set to 1 to facilitate a comparison of fold activation. The actual luciferase and β-Gal values for the hormone-treated wild-type and the 10 Ala mutant-transfected samples were 12.09 and 7.24, respectively. (C and D) HeLa cells were cotransfected with 0.25 μg of PR-B (C) or PR-A (D) and with 2.5 μg of myc-tagged cyclin A or pCMV vector. Twenty-four hours after transfection, the cells were treated with 10 nM R5020 for 60 min and harvested, and 100 μg of extract was incubated with myc 9E10 antibody as indicated in Materials and Methods. The immunoprecipitated protein extracts were run on an SDS-PAGE, and PR was detected with the 1294 antibody. (E) Cyclin A interacts with the N terminus of PR-B. HeLa cells were cotransfected with 0.25 μg of A/BCD PR-B and with 2.5 μg of myc-tagged cyclin A or backbone pCMV; immunoprecipitation and blotting were performed as described for panel C. (F) T47D cells were treated with or without 10 nM R5020 for 60 min, and the cell extracts were immunoprecipitated with cyclin A antibody or a nonspecific rabbit anti-mouse IgG (RAM) as described in Materials and Methods. The immunoprecipitated protein extract was run on an SDS-PAGE gel, and PR was detected using the PR antibody. The films were quantified densitometrically, and the fold change in PR-B immunoprecipitated by cyclin A antibody compared to the RAM-immunoprecipitated samples is given under each lane. The inputs in panels C to E were 10% of the total protein extract used for immunoprecipitation. (G) Cyclin A interacts with PR-B in vitro. One microgram of baculovirus-expressed GST-tagged cyclin A or GST was bound to glutathione-Sepharose beads, and then in vitro-translated [35S]PR-B was added. The beads were washed and eluted, the eluate was run with 20% input on an SDS-PAGE, and radiolabeled PR-B was detected by autoradiography. Myc cyc A, myc-tagged cyclin A; IP, immunoprecipitation; S, serine; T, threonine; DBD, DNA binding domain; Wt, wild type.

FIG. 3.

Cyclin A is rapidly and stably recruited to the MMTV promoter. (A) T47D cells stably transfected with MMTV-CAT (the region of the MMTV promoter probed is shown in the top panel) were treated with 10 nM R5020 for 60 min, protein DNA complexes were cross-linked and then immunoprecipitated with PR or cyclin A antibodies, and ChIP assays were performed as described in Materials and Methods. PCR was also performed with primers generated to the CAT region of the construct. Left panels show the ratios of the signals obtained by PCR amplification of immunoprecipitated MMTV or the CAT region divided by the corresponding input values, and the top right panel shows the fold change in recruitment, with that for the vehicle assigned a value of 1. (B) Cyclin A is recruited to the PR promoter rapidly, and the sites remain occupied for at least 60 min. T47D cells stably transfected with MMTV-CAT were treated with 10 nM R5020 for the indicated times, cross-linked complexes were immunoprecipitated with PR (panel 1), SRC-1 (panel 2), or cyclin A (panel 3) antibodies, and ChIP assays were performed as described in Materials and Methods. Data shown are the mean and standard error of three independent experiments and are shown as the fold change in recruitment, with the time zero value divided by input assigned a value of 1.

FIG. 4.

Cyclin A requires Cdk2 to stimulate PR activity but not for interaction with PR. (A) Myc-tagged cyclin A wild type or the Cdk2 binding mutant (2.5 μg) was cotransfected with Cdk2 (2.5 μg) into HeLa cells, and the cell extracts were immunoprecipitated with myc 9E10 antibody and blotted for Cdk2. (B) The Cdk2 binding mutant of myc-tagged cyclin A was cotransfected (2.5 μg) with 250 ng of PR-B, immunoprecipitated with myc 9E10 antibody, and blotted for PR. (C) Wild-type, Cdk2 binding mutant of cyclin A or vector backbone was cotransfected (0.5 μg) with 0.25 μg of GRE2-_E1b_-LUC and 10 ng of PR-B, luciferase activity was measured, and the values were normalized to β-Gal levels. The Western assay in the lower panel shows the expression of the myc cyclin A and the Cdk2 binding mutant of myc cyclin A. (D) HeLa cells were transfected as described in Materials and Methods with 0.25 μg of GRE2-_E1b_-LUC, 0.5 μg of cyclin A, Cdk2, or combinations of both, and 10 ng of PR-A or the phosphorylation-deficient mutant (10 Ala PR-A). The cells were treated with 10 nM R5020 for 24 h and harvested, and luciferase activity was measured and normalized to the β-Gal levels. The values of hormone-treated vector-transfected samples were set to 1. (E) Cdk2 interacts with PR-B, and the interaction is increased by transfection of cyclin A. HeLa cells were transfected with 0.25 μg of PR-B, 2.5 μg of Cdk2 or myc cyclin A, or a combination of both. After 24 h, the cells were treated with R5020 for 60 min. Protein extracts were immunoprecipitated with PR antibody and then blotted with Cdk2 antibody. I.P., immunoprecipitation; myc cyc A, myc-tagged cyclin A.

FIG. 5.

Cdk2 activity is important for PR function. (A) HeLa cells were transfected with 10 ng of PR-B and 0.25 μg of GRE-_E1b-_LUC and then treated 24 h after transfection with vehicle, 30 μM roscovitine, 10 nM R5020, or a combination of roscovitine and R5020 for 4 h. The cells were harvested, and luciferase activity was measured and normalized to the β-Gal levels. (B) HeLa cells were transfected with increasing concentrations of constitutively active luciferase (CMV LUC) and 0.05 μg of β-Gal and then treated or not immediately after transfection with 30 μM roscovitine for 4 h; luciferase activity was measured and normalized to β-Gal levels. (C) T47D cells stably transfected with MMTV-CAT were treated with vehicle, 30 μM roscovitine, 10 nM R5020, or a combination of roscovitine and R5020 for 4 h. The cells were harvested, and CAT activity was measured and normalized to total cellular protein levels. (D) T47D cells were treated with vehicle, 10 nM R5020, 5 μM CdCl2, or a combination of roscovitine and R5020 or CdCl2 for 6 h. Total cellular RNA was extracted, and then a reverse transcriptase real-time PCR with TaqMan primers and probe was performed for the MT2A gene as described in Materials and Methods. MT2A gene transcription was normalized to the 18S rRNA, and the value for vehicle-treated cells was set at 1. *, significantly different from vehicle-treated samples. (E) HeLa cells were transfected with the indicated amounts of Cdk2 siRNA plasmid, and the concentration of DNA was matched with an siRNA plasmid for lamin. The cells were treated 48 h after transfection with ethanol or 10 nM R5020, harvested 24 h later, and luciferase activity was measured and normalized to β-Gal levels. The activity is expressed relative to activity obtained with 1 μg of lamin siRNA plus R5020. (F) Cdk2 in the samples from panel E was detected by Western blotting using a Cdk2 antibody. Ros, roscovitine; Const. Active Luc, constitutively active luciferase.

FIG. 6.

Roscovitine inhibits recruitment of SRC-1 to the MMTV promoter and the acetylation of histone H4 at lysine 5. T47D cells stably transfected with MMTV-CAT were treated with 10 nM R5020 for 30 min or pretreated with 30 μM roscovitine for 3 h and then treated with R5020 for 30 min, and ChIP assays performed using antibodies to PR (A), cyclin A (B), SRC-1 (C), histone H4 acetyl lysine 5 (D), or TRAP-220 (E). In panels A through E the ratio of signal to input was determined and fold enhancement relative to an assigned value of 1 for vehicle-treated samples is plotted. (F) SRC-1 levels were measured by Western blotting of extracts from cells that were treated or not with roscovitine for 4 h. Also shown in the lower panel is the actin loading control. TRAP-220, thyroid receptor-associated protein; acetyl Lys 5, histone H3 acetyl lysine 5.

FIG.7.

SRC-1 activity and its interaction with PR is regulated by Cdk2 kinase activity. (A) Cyclin A and SRC-1 synergistically increase PR-B transactivation. HeLa cells were transfected with 0.25 μg of GRE2-_E1b_-LUC, 0.5 μg of cyclin A, 0.5 μg of SRC-1, vectors for cyclin A or SRC-1, and 10 ng of PR-B. The cells were treated 24 h after transfection with 10 nM R5020 and harvested 48 h after transfection, and luciferase activity was measured and normalized to the β-Gal levels. (B) Cyclin A/Cdk2 increases the intrinsic activity of SRC-1 but not CBP. HeLa cells were cotransfected with 0.25 μg of pBind, pBind SRC-1, or pBind CBP, increasing concentrations of cyclin A/Cdk2, and with the 17-mer LUC reporter. The cells were harvested 48 h after transfection, and luciferase activity was measured and normalized to β-Gal levels. (C) Cyclin A/Cdk2 increases the interaction between PR LBD and SRC-1. HeLa cells were transfected with 0.25 μg of the PR LBD fused to Gal4 DNA binding domain or SRC-1 fused to the VP16 activation domain along with 0.5 μg of vector or 0.25 μg of cyclin A and 0.25 μg of Cdk2. The cells were treated 24 h after transfection and harvested 48 h after transfection, and luciferase activity was measured and normalized to β-Gal levels. As a control, cells were also transfected with Gal VP16 (Gal DNA binding domain fused to the VP16 activation domain) to distinguish between enhanced VP16 activity and authentic enhancement of interactions between PR and SRC-1. (D) Cyclin A/Cdk2 phosphorylates SRC-1 in vitro. Baculovirus-expressed SRC-1 was immunoprecipitated with an SRC-1 antibody or an unrelated control (AR) antibody. The beads were washed and incubated with 5 μl of baculovirus-expressed and purified cyclin A/Cdk2 in an in vitro phosphorylation assay mixture containing [γ32P]ATP. SRC-1 was extracted with 4× Laemmli buffer and run on an SDS-PAGE, and phosphorylated SRC-1 visualized by autoradiography. (E) Interaction between PR and SRC-1 is dependent on phosphorylation of SRC-1. In vitro-translated 35S-labeled SRC-1 was treated with λ phosphatase, and the reaction was stopped with the phosphatase inhibitor sodium vanadate. Samples which received sodium vanadate prior to the addition of phosphatase are indicated as containing sodium vanadate in the figure. Samples containing cyclin A/Cdk2 were incubated with the kinase after the phosphatase treatment. The treated SRC-1 fractions were then incubated with baculovirus-expressed and purified PR-B attached to protein A-Sepharose beads through a PR antibody, and bound SRC-1 was extracted with 2× Laemmli buffer, run on an SDS-PAGE, transferred to nitrocellulose, and detected by autoradiography. SRC-1 incubated with protein A-Sepharose and PR antibody without PR was used as a negative control. The right panel shows the densitometric evaluation of the SRC-1 bands detected by autoradiography. N-Term PRB, N terminus of PR-B; Unrelated Ab, unrelated antibody control.

References

1. Agoulnik, I. U., W. C. Krause, W. E. Bingman III, H. T. Rahman, M. Amrikachi, G. E. Ayala, and N. L. Weigel. 2003. Repressors of androgen and progesterone receptor action. J. Biol. Chem. 278**:**31136-31148. - PubMed
1. Ait-Si-Ali, S., S. Ramirez, F. X. Barre, F. Dkhissi, L. Magnaghi-Jaulin, J. A. Girault, P. Robin, M. Knibiehler, L. L. Pritchard, B. Ducommun, D. Trouche, and A. Harel-Bellan. 1998. Histone acetyltransferase activity of CBP is controlled by cycle-dependent kinases and oncoprotein E1A. Nature 396**:**184-186. - PubMed
1. Archer, T. K., P. Lefebvre, R. G. Wolford, and G. L. Hager. 1992. Transcription factor loading on the MMTV promoter: a bimodal mechanism for promoter activation. Science 255**:**1573-1576. - PubMed
1. Bai, W., and N. L. Weigel. 1996. Phosphorylation of Ser211 in the chicken progesterone receptor modulates its transcriptional activity. J. Biol. Chem. 271**:**12801-12806. - PubMed
1. Beck, C. A., N. L. Weigel, and D. P. Edwards. 1992. Effects of hormone and cellular modulators of protein phosphorylation on transcriptional activity, DNA binding, and phosphorylation of human progesterone receptors. Mol. Endocrinol. 6**:**607-620. - PubMed

Cyclin-dependent kinase activity is required for progesterone receptor function: novel role for cyclin A/Cdk2 as a progesterone receptor coactivator - PubMed (original) (raw)