Mechanisms underlying the control of progesterone receptor transcriptional activity by SUMOylation

Abstract

Posttranslational modification by small ubiquitin-like modifier (SUMO) is a major regulator of transcription. We previously showed that progesterone receptors (PR) have a single consensus psiKXE SUMO-conjugation motif centered at Lys-388 in the N-terminal domain of PR-B and a homologous site of PR-A. SUMOylation of the PR is hormone-dependent and has a suppressive effect on transcription of an exogenous promoter. Here we show that repression of PR activity by SUMOylation at Lys-388 is uncoupled from phosphorylation, involves synergy between tandem progesterone response elements, and is associated with lowered ligand sensitivity and slowed ligand-dependent down-regulation. However, paradoxically, cellular overexpression of SUMO-1 increases PR transcriptional activity even if Lys-388 is mutated, suggesting that the receptors are activated indirectly by other SUMOylated proteins. One of these is the coactivator SRC-1, whose binding to PR and enhancement of agonist-dependent N-/C-terminal interactions is augmented by the presence of SUMO-1. Increased transcription due to SRC-1 is independent of PR SUMOylation based on assays with the Lys-388 mutants and the pure antiprogestin ZK98299, which blocks N-/C-terminal interactions. In summary, SUMOylation tightly regulates the transcriptional activity of PR by repressing the receptors directly while activating them indirectly through augmented SRC-1 coactivation.

FIGURE 1.

SUMOylation slows ligand-dependent PR down-regulation and represses transcription. A, schematic of PR-A and PR-B and location of the Lys-388 SUMO conjugation site within an IKEE motif. BUS, PR-B-upstream segment; IF, inhibitory function. B and C, PR immunoblots were performed on extracts of PR-negative T47D-Y breast cancer cells stably expressing PR-A or PR-A K388R (B) or PR-B or PR-B K388R (C) and treated with 10 nm R5020 for 0–16 h. Cell extracts were probed with anti-PR1294 or anti-β-actin AC-74 monoclonal antibodies. D and E, T47D-Y cells were transiently transfected with expression vectors for each PR isoform or its SUMOylation-deficient mutant, the PRE₂-Luc reporter, and pSV40-Renilla as an internal control. Cells were treated for 24 h without (–) or with (+) 10 nm R5020. Luciferase activity is expressed in relative luciferase units (RLU). Data represent triplicates ± S.D. Similar results were obtained in several experiments.

FIGURE 2.

Modulation of PR transcriptional activity by SUMO-1 depends on the promoter context. HeLa cells were transfected with 2 μg of luciferase reporters containing zero or one (A) and two or three (B) tandem PREs together with 50 ng of a PR-A, PR-A K388R, PR-B, or PR-B K388R expression vector and Renilla-Luc as an internal control. The cells were treated for 24 h with 10 nm R5020 and then harvested and lysed. The extracts were assayed for luciferase activities. Transcription levels are expressed as the -fold induction over the no hormone control in duplicate samples. Note the differences in scale between A and B.

FIGURE 3.

Mutation of the SUMO attachment site raises PR sensitivity to hormone. A, HeLa cells were transfected with 10 or 100 ng of the PR-B expression vector, a PRE₂-Luc reporter, and the Renilla control plasmid and treated with ethanol or various concentrations of R5020 for 24 h. The average was plotted as a percentage of the maximal induction by 10^–8 m R5020 above no hormone levels. Curve fitting was performed by Prism Graph as described under “Experimental Procedures.” The S.D. of triplicate values is indicated by the error bars. B, the corresponding relative luciferase activities were plotted. C, HeLa cells were transfected with 10 ng of wild type PR-B or the PR-B K388R mutant and treated with increasing concentrations of R5020 for 24 h. D, the corresponding relative luciferase were plotted ± S.D. RLU, relative luciferase units.

FIGURE 4.

PR phosphorylation and SUMOylation are unconnected. HeLa cells were transiently transfected with expression vectors encoding either wild type (wt) PR-B or the PR-B S294A/S344A/S345A phosphorylation-deficient mutant together with a GFP-SUMO-1 expression vector (+) or an empty vector control (–). Cells were treated for 24 h without (–) or with (+) 10 nm R5020. Western blot analysis was performed on cell extracts with anti-PR1294, and β-actin detected with AC-74 was used as a protein loading control. Locations of molecular weight standards are shown on the left. MAb, monoclonal antibody.

FIGURE 5.

SUMO-1 expression enhances PR transcriptional activity. HeLa cells were transfected with the PRE₂-Luc reporter in the presence of pSV40-Renilla as internal control along with PR-B (A), PR-B K388R (B-K388R) (B), NT-B (C), or DBD-LBD (D) expression vectors and a GFP-SUMO-1 expression vector at doses of 20, 50, 100, and 200 ng of DNA or an empty vector control. Cells were treated without (–) or with (+) 10 nm R5020 for 24 h before being assayed for luciferase activity. The values are expressed as relative luciferase units normalized to Renilla controls. RLU, relative luciferase units.

FIGURE 6.

PR SUMOylation, N-/C-terminal interactions, and effects of progestin agonists and antagonists. A, schematic of the fusion constructs used in the mammalian two-hybrid assay; abbreviations are as in Fig. 1. Full-length PR or PR fragments were fused to the Gal4 DNA binding domain or the VP-16 activation domain. B, effect of progestins on N-/C-terminal interactions. HeLa cells were cotransfected with Gal4-LBD and VP16-NTB and the 5× Gal4-Luc reporter and treated with the agonist R5020 (10 nm), the partial antagonists RU486 and ZK 112993 (100 nm), or the pure antagonist ZK98299 (100 nm) alone or with 10 nm R5020 plus a 100 nm concentration of one of the antagonists. Interaction between the LBD and NTB is reported as -fold induction compared with hormone-untreated controls. C, R5020-dependent interaction between wild-type NTB or SUMOylation-deficient NTB mutant (m) and the PR C terminus. HeLa cells were cotransfected with Gal4-LBD and VP16 fusion constructs of wild type or K388R mutant (NTBm) NTB, along with a 5× Gal4-Luc reporter and treated without or with 10 nm R5020. The values are expressed as the -fold induction over the no hormone controls. D, SUMO-1 is covalently bound to PR in the presence of agonists and all antagonists. HeLa cells were transfected with PR and GFP-SUMO-1 expression vectors, and cells were treated with vehicle alone or with progestin or the antagonists shown. Western blots were performed with anti-PR 1294. β-Actin was used as a protein loading control.

FIGURE 7.

SUMO-1 enhances the interaction between SRC-1 and full-length PR-B or the SUMOylation-deficient PR-B mutant. A, both SRC-1 and SUMO-1 enhance PR N-/C-terminal interactions. HeLa cells were transfected as in Fig. 6 with Gal4-LBD and VP16-NTB, with and without SRC-1 or SUMO-1, in the absence or presence of R5020. Values are expressed as -fold induction over the no hormone controls. B, SUMO-1 enhances the interaction between SRC-1 and liganded PR LBD. HeLa cells were transfected with Gal4-LBD and VP16-SRC-1 in the presence or absence of SUMO-1 and the presence or absence of R5020. Values represent -fold induction over the no hormone controls. C, SUMO-1 inhibits the interaction between SRC-1 and the PR-B N terminus. HeLa cells were transfected with Gal4-SRC-1 and VP-16 NTB in the absence or presence of SUMO-1. Results are reported over the basal activity of Gal4-SRC-1. Note that in the absence of an LBD, no hormone is required. D, SUMO-1 enhances the interaction between SRC-1 and full-length PR-B even if the PR SUMOylation site is mutated. HeLa cells were cotransfected with Gal4-SRC-1 alone or together with VP16-PR-B or the VP16-PR-B K388R (VP-PR-Bm) mutant, in the absence or presence of SUMO-1, and treated without or with R5020. Results are reported as the -fold activity compared with no hormone controls.