Regulation of androgen receptor stability by the β1 Pix/STUB1 complex

Abstract

The androgen receptor (AR) is essential in the development and differentiation of testes and male genitalia. AR expression is tightly regulated at the translational and posttranslational levels. AR posttranscriptional regulation is a major determinant of AR availability since AR is a direct target of E3 ubiquitin ligase STUB1. Our work indicated that the Rac/Cdc42 guanosine triphosphatase guanine nucleotide exchange factor, β₁ Pix, enhanced AR levels after AR stimulation in HEK293 and HeLa cells. AR stimulation decreased AR ubiquitination which is accompanied by increased β₁ Pix binding to AR. Ectopic expression of β₁ Pix decreased AR ubiquitination in Tm4 and HEK293 cells. We demonstrated that the formation of a multimolecular complex comprised of AR/β₁ Pix/STUB1 responded in a time-dependent manner to AR stimulation. β₁ Pix binding dissociated STUB1 from AR and thus prevented STUB1 from catalyzing receptor ubiquitination. β₁ Pix enhanced AR transcriptional activity and increased AR target gene expression. Irrespective of treatment, immunofluorescence analysis showed a strong nuclear colocalization of endogenous AR and endogenous βPix in Tm4 cells. However, using Tm4 cell fractionation, AR stimulation decreased βPix/AR association in the cytosolic fraction and increased binding of AR to βPix in the nuclear fraction. To support the role of β₁ Pix in androgen regulation, we found that individuals lacking this gene have a significant increase in genitourinary malformations associated with androgen dysfunction. Our data indicate that β₁ Pix is an important modulator of AR stability and ligand-dependent AR transcriptional activity. We propose that β₁ Pix could serve as a promising therapeutic target to modulate AR signaling.

Figure 1.. Patients with microdeletions at 13q34 that encompass the ARHGEF7 gene exhibit penile and testicular anomalies.

All patients identified in the literature and Decipher database have microdeletions that are represented by red box. The ID, size of the deletion and phenotype of each patient is indicated in each box.

Figure 2.. βPIX is expressed in mouse testis and penis.

A) Detection of βPIX in the testis indicated expression mainly in the cytoplasm of the Sertoli cells. B) Detection of AR in the testis indicated expression in Sertoli and Leydig cells. C) Detection of βPIX in the penis indicated cytoplasmic expression mainly in urethral epithelium. D) Detection of AR in the penis indicated expression mainly in urethral epithelium.

Figure 3.. β₁Pix confers AR protein stabilization.

HEK293 (A) or HeLa-AR-GFP (B) cells were transfected with empty vector as a control or Myc β₁Pix for 24 h before stimulation with R1881 (10 nM). Total cell lysates were collected at 0, 1, 4 and 8 h after stimulation and examined by western blot. C) HEK293 cells were transfected with FlagAR along with HA-Ub and Mycβ₁Pix for 24 h before stimulation with 10 nM of R1881 for the indicated times. Total cell lysates were subjected to Co-IP using anti-Flag antibody followed by western blot analysis using the indicated antibodies. D) HEK293 cells were stimulated for the indicated times and the cell lysates were immunoprecipitated using anti-STUB1 antibody followed by Western blot using antibodies against βPix and STUB1. E) HEK293 cells were transfected with empty vector or Myc β₁Pix for 24 h before treatment with cycloheximide (CHX) for 0, 3 and 6 h. Total cell lysates were collected and analyzed by western blot.

Figure 4.. Effect of β₁Pix on AR ubiquitination.

A) HEK293 cells were co-transfected with FlagAR and HA-Ub for 24 h and treated with DMSO or MG132 (5 μM) for 16 h. Total cell lysates were collected and subjected to Western blot analysis using antibodies against AR, Flag and GAPDH. B) Portion of total cell lysates from (A) were subjected to immunoprecipitation using anti-AR antibody or normal IgG as a negative control followed by western blot analysis using antibodies against HA, Flag, βPix and AR. C) HEK293 cells were transfected with combination of FlagAR, HA-Ub and Myc β₁Pix and stimulated with vehicle or R1881 (10 nM). Total cell lysates were analyzed by Co-IP using anti-Flag antibody followed by Western blot with the indicated antibodies. D) HEK293 cells were transfected with HA-Ub, FlagSTUB1 and increasing amounts of Myc β₁Pix for 24 h. Total cell lysates were subjected to Co-IP using anti-AR antibody followed by Western blot analysis using antibodies against HA, AR, Myc and Flag.

Figure 5.. AR stimulation induces AR/STUB1/β₁Pix complex dissociation.

HEK293 cells were transfected with combination of AR, HA-Ub, FlagSTUB1 and Mycβ₁Pix or β₁PixΔ(602-611) for 24 h before stimulation with R1881 (10 nM) for 4 h. The cell lysates were collected and subjected to Co-IP using anti-AR antibody (A) or anti-Flag antibody (B) and the resulting immunocomplexes were analyzed by Western blot using the indicated antibodies. (C), Tm4 cells were transfected with combination of AR, HA-Ub, FlagSTUB1 and Mycβ₁Pix or β₁PixΔ(602-611) for 24 h before stimulation with R1881 (10 nM) for 4 h. The cell lysates were collected and subjected to Co-IP using anti-AR antibody and the resulting immunocomplexes were analyzed by Western blot using the indicated antibodies.

Figure 6.. Inhibition of Proteasomal activity results in increased AR/β₁Pix/STUB1 complex formation.

HEK293 cells were transfected with a combination of HA-Ub, FlagSTUB1, Mycβ₁Pix and Mycβ₁PixΔ(602-611) for 24 h before addition of vehicle or MG132 (5 μM) for 16 h. Total cell lysates were subjected to immunoprecipitation using anti-AR (A) or anti-Flag (B) antibodies followed by Western blot analysis with antibodies against AR, Myc and Flag.

Figure 7.. β₁Pix modulates AR transcriptional activity and AR targets gene expression in HEK293.

(A) HEK293 were transfected with Renilla, AR Luc plasmid and AR along with β₁Pix or its mutant β₁PixΔ(602-611) for 24 h and the media was replaced by DMEM containing 5% charcoal stripped FBS for 24 h. After 24 h stimulation with R1881 (10 nM), cells were harvested, and the luciferase activity was measured using the luciferase reporter assay (Promega) according to the manufacturer’s instructions. Data shown are representative of three independent experiments performed in triplicates. (B) qPCR analysis of four AR dependent selected genes (FOXJ1, ATF2, RAC3, and FKB) is indicated. All four genes were upregulated after transfection of HEK293 cells with βPix. Statistical analyses were conducted using the REST Software from Qiagen that indicated significance (*p<0.005).

Figure 8.. AR and βPix co-localization.

(A) Tm4 cells were serum starved for 24 h followed by stimulation with testosterone (10 nM) for 18 h. Cells were stained for AR and βPix endogenous expression as described under “Material and Methods”. Immunofluorescence staining was carried out using mouse monoclonal anti-βPix (red) and rabbit polyclonal anti-AR (green). C, Control; T, Testosterone. Representative images of three independent experiments are shown. (B) Sertoli Tm4 cells were transfected with HA-AR construct for 24 h in DMEM containing charcoal stripped FBS, then stimulated with R1881 (10 nM) for 2 h. cytosolic (C) and nuclear (N) fractions were immunoprecipitated with anti-βPix antibody followed by immunoblotting with anti-HA antibody. GAPDH was used as a cytosolic marker and histone H3 as a nuclear marker.

Figure 9.. Proposed pathway of AR/βPix/STUB1 complex in the modulation of AR stability.

(A) STUB1 binding to AR results in STUB1-mediated AR ubiquitination and degradation. (B) β₁Pix physical association with AR results in AR/STUB1 complex dissociation. β₁Pix-mediated AR physical sequestration prevents STUB1 binding to AR resulting in AR stabilization and AR/βPix nuclear translocation and activation of AR target gene expression.