Covalent modification of the androgen receptor by small ubiquitin-like modifier 1 (SUMO-1)

Abstract

Modification by SUMO-1 is proposed to play a role in protein targeting and/or stability. The SUMO-1-conjugating enzyme Ubc9 interacts with androgen receptor (AR), a ligand-activated transcription factor belonging to the steroid receptor superfamily. We show here that AR is covalently modified by SUMO-1 (sumoylated) in an androgen-enhanced fashion and identify the principal acceptor site in the N-terminal domain of AR. Substitutions of sumoylated Lys residues enhanced transcriptional activity of AR without influencing its transrepressing activity. Interestingly, the same Lys residues form the cores of the recently described transcriptional synergy control motifs in AR [Iñiguez-Lluhi, J. A. & Pearce, D. (2000) Mol. Cell. Biol. 20, 6040-6050]. These motifs, which match perfectly with the sumoylation consensus sequence, are also present in the N-terminal domains of glucocorticoid, mineralocorticoid, and progesterone receptor. Taken together, our data suggest that reversible sumoylation is a mechanism for regulation of steroid receptor function.

Figure 1

AR is modified by SUMO-1. COS-1 cells grown on 6-cm dishes were transfected with vectors encoding Flag-tagged wt AR (wt) or AR lacking most of the DBD and the hinge region (ΔDBD), and GFP-tagged SUMO-1 (SUMO) or SUMO-1GA (SUMOGA) expression vector. The cells received 100 nM testosterone (T) or vehicle 12 h before harvesting as indicated. Five percent of the cell extracts were immunoblotted with AR antibody (A), and the rest were subjected to immunoprecipitation with anti-Flag antibody. The immunoprecipitates were immunoblotted with anti-GFP antibody (B) or anti-SUMO-1 antibody (C). Arrowheads depict the slowly migrating sumoylated forms of AR.

Figure 2

K386 is the major sumoylation site in AR. COS-1 cells were transfected with pGFP-SUMO-1 or empty expression vector as indicated along with wt Flag-hAR, Flag-K386R, Flag-K520R, or Flag-K386R/K520R. The cells received 100 nM testosterone 12 h before harvesting. Immunoblots and immunoprecipitations were performed as described in Fig. 1. (A Upper) Immunoblotting (WB) of cell extracts with anti-AR antibody (α-AR); (A Lower) immunoprecipitation with anti-Flag antibody and subsequent immunoblotting with anti-GFP antibody (α-GFP). (B Upper) COS-1 cells were transfected with expression vectors encoding wt AR or the K386R/K520R mutant, and the cell extracts were analyzed by immunoblotting with anti-AR antibody (α-AR); (B Lower) immunoprecipitation with anti-Flag antibody and subsequent immunoblotting with anti-SUMO-1 antibody (α-SUMO). Arrowheads depict the slowly migrating SUMO-1-conjugated ARs.

Figure 3

Ubc9 catalyzes attachment of SUMO-1 to AR and GR in vitro. (A) In vitro-translated ³⁵S-Met-labeled wt and mutated AR constructs were incubated with GST-SUMO-1 and HeLa cell fraction containing SUMO-1-activating enzyme in the presence (+) or absence (−) of GST-Ubc9. The samples were resolved on 7.5% SDS/PAGE and subjected to fluorography. Arrowheads depict the slowly migrating sumoylated forms of AR; Star, unmodified AR. (B) In vitro SUMO-1 conjugation reactions with GR, ERα, ERβ, and TRα. The reactions were performed in the presence (+) or absence (−) of GST-Ubc9 as described in A.

Figure 4

Substitutions of the sumoylation sites activate AR. (A) Transactivation by 10 ng of wt Flag-AR and the mutants K386R, K520R, and K386R/K520R was studied on minimal pARE₂-TATA-LUC reporter (150 ng) in COS-1 cells. The cells received 100 nM testosterone (+T) or vehicle (−T) 18 h after transfection. LUC activities in cell extracts were adjusted to the transfection efficiency by using β-galactosidase activity. The activity of AR in the presence of testosterone is set as 100, and the means ± SD from at least six independent experiments are shown. (B) As in A, except that cells were transfected with 1–100 ng of AR expression plasmids as indicated. The amount of transfected DNA was balanced by the addition of empty expression vector. (Inset) Expression levels of wt AR and K386R/K520R mutant in an experiment corresponding to data shown in B. AR was immunoblotted with AR antibody from the same lysates (pooled from triplicate wells) that were used for reporter gene assays. (C) Effect of coexpressed Ubc9 on transactivation by AR and K386R/K520R. The conditions were as in A, except that indicated amounts of pFLAG-Ubc9 (in μg) were cotransfected and the total amount of DNA was balanced by adding empty pFLAG-CMV2. Black bars depict wt AR and gray ones K386R/K520R mutant.

Figure 5

Effect of K386R/K520R mutation on transrepressing activity of AR and DNA binding in intact cells. (A) Repression of RelA-dependent transactivation by wt AR and K386R/K520R mutant. COS-1 cells were transfected with pκB₆tk-LUC (150 ng), pCMV-RelA (30 ng), and AR expression vectors (150 ng). The relative LUC activity in the absence of cotransfected AR is set as 100. Values are means ± SD from six independent experiments. (B) Binding of the wt and mutant AR to androgen response elements in intact cells as determined by a promoter interference assay (30). COS-1 cells were transfected with pCMV-ARE₂-LUC reporter (100 ng) and increasing amounts (10, 50, and 100 ng) of wt Flag-AR and K386R/K520R mutant expression vectors. The total amount of DNA was kept constant by adding empty pcDNA3.1(+) when necessary. Reporter gene activity in the absence of AR expression vector is set as 100, and the means ± SD from three independent experiments are shown. Black and gray bars depict wt AR and K386R/K520R mutant, respectively.