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. 2009 Mar;23(3):292-307.
doi: 10.1210/me.2008-0219. Epub 2008 Dec 30.

SUMO-specific protease 1 (SENP1) reverses the hormone-augmented SUMOylation of androgen receptor and modulates gene responses in prostate cancer cells

Sanna Kaikkonen  1 , Tiina Jääskeläinen, Ulla Karvonen, Miia M Rytinki, Harri Makkonen, Daniel Gioeli, Bryce M Paschal, Jorma J Palvimo
Affiliations

SUMO-specific protease 1 (SENP1) reverses the hormone-augmented SUMOylation of androgen receptor and modulates gene responses in prostate cancer cells

Sanna Kaikkonen et al. Mol Endocrinol. 2009 Mar.

Abstract

The acceptor sites for small ubiquitin-like modifier (SUMO) are conserved in the N-terminal domains of several nuclear receptors. Here, we show that androgens induce rapid and dynamic conjugation of SUMO-1 to androgen receptor (AR). Nuclear import of AR is not sufficient for SUMOylation, because constitutively nuclear apo-ARs or antagonist-bound ARs are only very weakly modified by SUMO-1 in comparison with agonist-bound ARs. Of the SUMO-specific proteases (SENP)-1, -2, -3, -5, and -6, only SENP1 and SENP2 are efficient in cleaving AR-SUMO-1 conjugates in intact cells and in vitro. Both SENP1 and -2 are nuclear and found at sites proximal to AR. Their expression promotes AR-dependent transcription, but in a promoter-selective fashion. SENP1 and -2 stimulated the activity of holo-AR on compound androgen response element-containing promoters. The effects of SENP1 and -2 on AR-dependent transcription were dependent on catalytic activity and required intact SUMO acceptor sites in AR, indicating that their coactivating effects are mainly due to their direct isopeptidase activity on holo-AR. In prostate cancer cells, ectopic expression of SENP1, but not that of SENP2, increased the transcription activity of endogenous AR. Silencing of SENP1 attenuated the expression of several AR target genes and blunted androgen-stimulated growth of LNCaP cells. Our results indicate that SENP1 reverses the ligand-induced SUMOylation of AR and helps fine tune the cellular responses to androgens in a target promoter-selective manner.

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Figures

Fig. 1.
Fig. 1.
AR is rapidly modified by SUMO-1 in response to agonist binding. A, AR is preferentially modified with SUMO-1 rather than with SUMO-2. COS-1 cells on six-well plates were transfected with expression vectors encoding AR (200 ng) and Myc-SUMO-1 or Myc-SUMO-2 (200 ng) as indicated. The total amount of DNA per well was balanced with empty pFlag-CMV-2. One day after transfection, cells were grown for 22 h with or without 100 nm testosterone. The cell extracts were analyzed by immunoblotting with AR antibody and anti-Myc antibody. Arrows depict SUMOylated AR forms. B, SUMO-1 is rapidly conjugated to AR in response to testosterone. COS-1 cells were transfected with AR and SUMO-1 as above and treated with testosterone for the indicated times. AR immunoreactivity was quantified with Odyssey infrared imaging system, and the amount of SUMO-1-modified AR bands was blotted in relation to total AR (unmodified plus modified forms, the mean ± sem; n = 3). C, Lack of SUMOylation of LBD-deficient AR is not rescued by overexpressed PIAS1 or Ubc9. COS-1 cells were transfected with pSG5-hAR or pSG5-hAR1-657 (200 ng) with and without pMyc-SUMO-1 (40 ng), pFlag-PIAS1 (200 ng), or pFlag-Ubc9 (200 ng) as indicated and treated with testosterone for 18 h, and cell extracts were analyzed by immunoblotting with AR antibody, anti-Flag, and anti-Myc antibodies. D, SUMOylation of AR does not simply occur as a result of the nuclear import of the receptor. COS-1 cells were transfected with expression vectors encoding wtAR, AR fused to SV40 large T antigen nuclear import signal (AR-NLS), or AR fused to c-Abl kinase NES (AR-NES) in the presence and absence of SUMO-1 as indicated. Cells were treated with testosterone for 18 h and analyzed as in B. The percentages of SUMO-modified AR forms of the total immunoreactive AR pool in the absence vs. presence of testosterone in this experiment were as follows: wtAR, 8 vs. 47%; AR-NLS, 17 vs. 44%; AR-NES, 6 vs. 33%. E, Comparison of agonistic and antagonistic ligands in promoting the SUMO-1 modification of AR. Cells were transfected with AR and SUMO-1 as in B and treated with increasing concentrations of testosterone (1, 10, and 100 nm), R1881 (0.25, 2.5, and 25 nm), cyproterone acetate (CPA; 15, 150, and 1500 nm), or CDX (60, 600, and 6000 nm) for 2 h and analyzed as described above. T, Testosterone.
Fig. 2.
Fig. 2.
Effect of phosphorylation and acetylation sites on AR modification by SUMO-1. A, SUMOylation of AR is not markedly affected by disruption of the receptor’s phosphorylation sites. COS-1 cells were transfected with expression vectors (100 ng) encoding wtAR, phosphorylation site-mutated ARS650A, or compound mutant AR5A in the presence and absence of SUMO-1 (100 ng) and p38 MAPK and MMK6E (150 ng of each) as indicated. Cells were grown with (+) or without (−) 100 nm testosterone (18 h) and analyzed by immunoblotting with AR antibody, p38 phosphorylation (P)-specific antibody, and anti-Myc antibody. Note that the forced MAPK expression clearly upshifts the main band of wtAR and that of ARS650A, but it has a negligible effect on the mobility of AR5A mutant. B, AR can be concomitantly SUMOylated at K386 and phosphorylated at S650. Cells were transfected with Flag-tagged AR forms and SUMO-1 and treated with T as indicated. Cell lysates were immunoprecipitated with anti-Flag M2 agarose, and the precipitates were analyzed by immunoblotting with anti-AR antibody (left) or with anti-AR-P-650-specific antibody (right). C, Substitution of acetylation sites by arginines do not influence AR SUMOylation, but deletion of the hinge amino acids 629–633 weakens AR modification by SUMO-1. Cells were transfected with pSG5-hARΔ629-633, pSG5-Flag-hARK630,632,633R, or corresponding wtAR vector and SUMO-1 and treated with testosterone as indicated. AR was analyzed by immunoblotting as described in Fig. 1. T, Testosterone.
Fig. 3.
Fig. 3.
Comparison of the isopeptidase activity of various SENPs toward overall SUMOylated cellular proteins and AR-SUMO-1 conjugates. A, Effect of SENP1, SENP2, SENP3, SENP5, and SENP6 on SUMO-1- and SUMO-2-modified cellular proteins. COS-1 cells on six-well plates were transfected with 200 ng expression vector encoding Myc-tagged SUMO-1 or SUMO-2 and 200–800 ng of various SENPs or their catalytically inactive mutants (SENP1M-6M) as indicated. As all SENPs were expressed as N-terminal Flag fusions, immunoblotting with anti-Flag antibody allowed adjustment of their expression levels, so that comparable expression levels were achieved (D). The total amount of DNA was kept constant (1000 ng) by adding empty pFlag-CMV-2 as needed. Cell extracts were separated on 4–12% Bis-Tris gradient gels and immunoblotted with anti-Myc antibody. B, Deconjugation of AR-SUMO-1 conjugates in COS-1 cells by different SENPs. COS-1 cells were cotransfected with pSG5-hAR (200 ng), pCMV-Myc-SUMO-1 (100 ng), and increasing amounts of different SENPs as indicated. The total amount of DNA was balanced by empty pFlag-CMV-2 vector. Cells were grown in the presence of testosterone (T) as in Fig. 2, and cell extracts were immunoblotted with anti-AR and anti-Flag antibodies. C, Deconjugation of SUMOylated AR by SENPs in vitro. [35S]Methionine-labeled AR that was modified with SUMO-1 in a recombinant protein-based system was used as a substrate for different SENPs affinity purified from COS-1 cells as described in Materials and Methods. Reaction products were analyzed by SDS-PAGE and fluorography.
Fig. 4.
Fig. 4.
SENP1 can partially colocalize with holo-AR in the nucleus. EGFP-AR and Flag-tagged SENP1, SENP2, or SENP5 were expressed in COS-1 cells. A, In the absence of AR, SENP1 and SENP2 show nuclear and predominantly perinuclear localization, respectively, whereas SENP5 is localized to nucleoli. B, When expressed in the presence of AR and testosterone, a considerable amount of SENP1 colocalizes with holo-AR in the nucleoplasm. SENP2 and AR reside in close proximity to each other but do not colocalize. SENP5 resides separated from holo-AR. Anti-Flag antibody and Rhodamine-Red-X-labeled secondary antibody was used for detection of SENPs, and AR was detected as EGFP fluorescence. Cells were analyzed by confocal microscopy as described in Materials and Methods. Yellow in merged images indicates colocalization.
Fig. 5.
Fig. 5.
The stimulatory effect of SENP1 and SENP2 on the transcriptional activity of AR is dependent on SUMOylation sites and requires multiple AREs. A, COS-1 cells grown on 12-well plates were cotransfected with pARE2-TATA-luc driven by two canonical AREs and a TATA box and expression vectors encoding wtAR or SUMOylation-deficient ARK386,520R mutant and SENP1 and -2 or their catalytically inactive mutants (M) as indicated and described in Materials and Methods. Twenty-four hours after transfection, cells were cultured for 18 h in the absence (−) or presence (+) of 100 nm testosterone. The relative luciferase (LUC) activity of wtAR in the presence of hormone and empty pFlag-CMV-2 is set as 1 and other values are expressed in relation to that. The columns represent the mean ± sd values from at least three experiments. B, COS-1 cells were cotransfected with promoter constructs containing either one ARE (pARE1-TATA-luc) or two AREs (pARE2-TATA-luc) and wtAR or ARK386,520R and increasing amounts of SENP1. Cells were grown and treated with testosterone as in A. The relative luciferase activity of wtAR with promoter containing one ARE in the presence of hormone and absence of SENP1 is set as 1 and other values expressed in relation to that. The columns represent the mean ± sd values from three experiments. C, COS-1 cells on 12-well plates were cotransfected with pARE2-TATA-luc and expression vectors encoding wtAR or LBD truncated AR mutant AR1-657 with or without SENP1. Cells were cultured with or without testosterone as described above and luciferase activities were measured. (D) COS-1 cells grown on 12-well plates were cotransfected with pARE2-TATA-luc and expression vectors encoding wtAR or ARK386,520R and SENP1 or corresponding empty vector. One day after transfection, TSA (1000 nm) or vehicle (dimethylsulfoxide) was added 2 h before addition of testosterone or vehicle (ethanol). Cells were grown for 14 h before harvesting and luciferase measurements. T, Testosterone.
Fig. 6.
Fig. 6.
Overexpression of SENP1 stimulates endogenous AR activity in LNCaP prostate cancer cells. A–D, LNCaP cells on 12-well plates were transfected with pARE2-TATA-LUC (A), pFKBP51-I5E-TATA-LUC (B), pELK4(−543/+54)-LUC (C), or pPB(−285/+32)-LUC (D) with expression vectors encoding SENP1, SENP2, or SENP6 as indicated and described in Materials and Methods. Twenty-four hours after transfection, cells were grown for 18 h in the presence of R1881 (10 nm) or vehicle. The relative luciferase activity of AR in the presence of hormone and absence of SENP is set as 1 and other values expressed in relation to that. The columns represent the mean ± sd values from three experiments. E, Overexpressed SENP1 decreases the amount of AR-SUMO conjugates in LNCaP cells, whereas SENP2 or -6 does not have an evident effect on the conjugates. The protein lanes originate from the same experiment and analysis, but they have been regrouped as indicated by dividing lanes. F, Expression of transfected SENPs verified by immunoblotting with anti-Flag antibody (asterisk indicates a nonspecific band).
Fig. 7.
Fig. 7.
Silencing of SENP1 attenuates expression of AR target genes in LNCaP cells. A, Ablation of SENP1 mRNA and SENP2 mRNA by RNAi in LNCaP cells. B–D, Effect of silencing of SENP1 or SENP2 on the accumulation of FKBP51 mRNA, ELK4 mRNA, and TMPRSS2 mRNA. Cells were transfected with SENP1 siRNA, SENP2 siRNA, or scrambled (SCR) control siRNA as indicated and described in Materials and Methods. Forty-eight hours after the transfection, cells were treated with 10 nm R1881 (black bars) or vehicle (white bars) for 24 h before extracting total RNA for real-time RT-PCR-based quantification. The results are expressed as mean ± sd from four experiments. *, P ≤ 0.05 compared with cells transfected with siSCR and treated with R1881. E–G, Silencing of SENP1 does not decrease loading of AR onto FKBP51, ELK4, or TMPRSS2 regulatory regions. LNCaP cells were transfected with siSCR (white bars) or siSENP1 (gray bars) as above, and ChIPs were carried out with anti-AR antibody or IgG antibody. Precipitated DNA was used as a template in quantitative PCR analysis with respective primers. H, Silencing of SENP1 blunts the androgen-stimulated growth of LNCaP cells. Cell proliferation was measured using CellTiter96 AQueous cell proliferation assay reagent (Promega) at indicated times as described in Materials and Methods. ***, P ≤ 0.001 compared with cells transfected with siSCR and treated with R1881. I, Phase-contrast microscopic analysis of the LNCaP cells in the proliferation experiment.
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