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. 2019 Nov 21;17(1):153.
doi: 10.1186/s12964-019-0457-9.

SUMO3 Modification by PIAS1 Modulates Androgen Receptor Cellular Distribution and Stability

Affiliations
Free PMC article

SUMO3 Modification by PIAS1 Modulates Androgen Receptor Cellular Distribution and Stability

Nanyang Yang et al. Cell Commun Signal. .
Free PMC article

Abstract

Background: Abnormal reactivation of androgen receptor (AR) signaling in castration-resistant prostate cancer (CRPC) mainly results from overexpression and down-regulation of AR. Sumoylation of AR can influence its function. However, regulation of AR sumoylation by SUMO E3 ligases PIASs to modify AR distribution and stability are not well understood.

Methods: We assessed the potential effect of SUMO3 modification on AR intracellular localization by immunostaining in AR-negative prostate cancer DU145 cells, and detected the effect of PIAS1/SUMO3 overexpression on AR sumoylation related degradation. Then we characterized AR sumoylation sites involved modified by SUMO3, and the key residue of PIAS1 involved in itself sumoylation and further mediated AR sumoylation (sumo3-conjugated), translocation and degradation. Finally we detected the recognition of PIAS1 (sumoylation ligase) to MDM2, a ubiquin ligase mediated AR degradation.

Results: We demonstrate that SUMO E3 ligase PIAS1, along with SUMO3, mediates AR cytosolic translocation and subsequent degradation via a ubiquitin-proteasome pathway. Although AR sumoylation occurs prior to ubiquitination, the SUMO-acceptor lysine 386 on AR, together with ubiquitin-acceptor lysine 845, contribute to PIAS1/SUMO3-induced AR nuclear export, ubiquitination and subsequent degradation. Moreover, PIAS1 itself is modified by SUMO3 overexpression, and mutation of SUMO-acceptor lysine 117 on PIAS1 can impair AR cytoplasmic distribution, demonstrating the essential role of sumoylated PIAS1 in AR translocation. We further determine that sumoylated PIAS1 interacts with AR lysine 386 and 845 to form a binary complex. Consistent with the effect on AR distribution, SUMO3 modification of PIAS1 is also required for AR ubiquitination and degradation by recruiting ubiquitin E3 ligase MDM2.

Conclusion: Taken together, SUMO3 modification of PIAS1 modulates AR cellular distribution and stability. Our study provided the evidence the crosstalk between AR sumoylation and ubquitination mediated by PIAS1 and SUMO3.

Keywords: Androgen receptor; SUMO3; Sumoylation, PIAS1.

Conflict of interest statement

The authors declare that they have no competing of interest.

Figures

Fig. 1
Fig. 1
Subcellular localization of AR in the presence of PIAS and SUMO. (a/b) DU145 cells in a 12-well plate were cotransfected with indicated plasmids for 48 h. Cells were then fixed and stained with anti-AR rabbit polyclonal antibody, then PI-conjugated anti-rabbit IgG antibody (red). Nuclei were visualized by DAPI staining. Representative images of transfected cells were acquired using immunofluorescence microscope
Fig. 2
Fig. 2
PIAS1 together with SUMO3 facilitates ubiquitin-proteasome mediated AR degradation. (a) DU145 cells in a 12-well plate were transiently transfected with empty vector, AR or AR along with PIAS1 and GFP-SUMO3. Cells were then fixed at different transfection periods (24 h, 48 h, 72 h and 96 h), and stained for AR (red). Representative images of transfected cells were shown. (b) DU145 cells were transfected with plasmids as described in A. The AR mRNA or beta-actin mRNA levels were analyzed by reverse-transcriptional PCR at indicated time course after transfection (24 h, 48 h, 72 h and 96 h). (c) DU145 cells were transfected with plasmids as described in A. Whole cell lysates at indicated time course after transfection were generated together and immunoprepapited with anti-AR antibody. The Immunoprecipitate was detected by anti-AR (IP, top panel), anti-ubiquitin (IB, second panel), and anti-SUMO3 immunoblotting (IB, third panel). Whole-cell lysates (Input) were immunoblotted with anti-AR (fourth panel) or anti- actin (bottom panel) antibodies. (d) DU145 cells were cotransfected with empty vectors, AR or AR together with GFP-SUMO3 and PIAS1. Cells were then treated with or without MG132 (5 μM) for 16 h before cells were collected at 72 h or 96 h as indicated after transfection. Whole-cell lysates were immunoblotted with anti-AR or anti-actin antibodies
Fig. 3
Fig. 3
Lysines 386 and 845 of AR are critical for PIAS1/SUMO3 overexpression induced nuclear export and degradation of AR. (a) DU145 cells cotransfected with myc-PIAS1, GFP-SUMO3 and wildtype AR or various point mutants of AR expression constructs as indicated for 48 h were subjected to immunoprecipitation with anti-AR antibody (IP), and this was followed by immunoblotting with anti-AR (IP) or anti-SUMO3 (IB) antibodies. (b) DU145 cells cotransfected with plasmids as described in A were fixed and stained with anti-flag mouse monoclonal antibody, then PI-conjugated anti-mouse IgG antibody (red). Nuclei were visualized by DAPI staining. Representative images of transfected cells were acquired using immunofluorescence microscope. (c) DU145 cells were cotransfected with plasmids as described in A for 72 h and then subjected to immunoprecipitation with anti-AR antibody, and followed by immunoblotting with anti-AR (IP) or anti-ubiquitin (IB) antibodies
Fig. 4
Fig. 4
PIAS1 itself was modified by SUMO3 at 117th lysine residue. (a) DU145 cells were cotransfected with plasmids as indicated for 48 h. Whole-cell lysates were immunoprepapited with anti-AR antibody and then analyzed by immunoblot analysis using the indicated antibodies against AR (IP, top panel) and PIAS1 (IB, second panel). Whole-cell lysates (Input) were also immunoblotted with anti-AR (third panel), anti-PIAS1 (fourth panel) or anti-actin (bottom panel) antibodies. (b) DU145 cells were co-transfected with plasmids as indicated for different transfection periods (24 h, 48 h, 72 h and 96 h). Whole cell lysates generated together were immunoprepapited with anti-myc antibody. The myc-immunoprepapite was then detected by anti-myc (IP, top panel) and anti-SUMO3 immunoblotting (IB, second panel). Whole-cell lysates (Input) were also immunoblotted with anti-myc (third panel) or anti-actin (bottom panel) antibodies. (c) Diagrammatic representation of putative typical sumoylation site on human PIAS1 sequense predicted by using the GPS-SUMO software. Analysis of human PIAS1 sequence indicated the presence of only one putative typical sumoylation site, Lys-117, which located near to the PINIT domain of PIAS1. (d) DU145 cells were co-transfected with plasmids as indicated for 48 h and Whole cell lysates were immunoprepapited with anti-myc antibody. The myc-immunoprepapite was then detected by anti-myc (IP, top panel) and anti-SUMO3 immunoblotting (IB, second panel)
Fig. 5
Fig. 5
The formation of AR and sumoylated PIAS1 complex. (a/b) DU145 cells were cotransfected with plasmids as indicated for 48 h and the total amount of plasmids per well was normalized by empty vectors. Whole-cell lysates were immunoprepapited with anti-AR or anti-myc antibodies. The immunoprepapite was then detected by indicated antibodies against AR (IP, top panel of a) and anti-PIAS1 (IB, second panel of a), or against myc (IP, top panel of b) and anti-SUMO3 (IB, second panel of b). Whole-cell lysates (Input) were also immunoblotted with anti-AR (third panel of a), anti-myc (fourth panel of a) and anti-actin (bottom panel of a) antibodies, or with anti-myc (third panel of b), anti-AR (fourth panel of b) and anti-actin (bottom panel of b) antibodies. (c) The mammalian two-hybrid assay was performed in DU145 cells. Cells were transiently transfected in 48-well with 100 ng 5 × GAL4-luc, 25 ng Renilla luciferase reporter,30 ng SUMO3, 30 ng VP16-AR and 30 ng GAL4-PIAS1 or GAL4-PIAS1 (K117 L) as indicated. The total amount of plasmids per well was normalized in all transfections by the addition of empty vectors. Transfected cells were grown for 48 h and then harvested for luciferase assay. Values represent mean ± S.D. *P < 0.01. (d) DU145 were transiently transfected in 48-well with 100 ng 5 × GAL4-luc, 25 ng Renilla luciferase reporter,30 ng SUMO3, 30 ng GAL4-PIAS1, and 30 ng VP16-AR or various points mutants of VP16-AR constructs as indicated for 48 h, and then harvested for luciferase assay. Values represent mean ± S.D. *P < 0.05,**P < 0.01
Fig. 6
Fig. 6
Inability of PIAS1(K117 L) to promote cytoplasmic translocation of AR from nucleus. (a) DU145 cells were transiently co-transfected with flag-AR, GFP-SUMO3 and myc-PIAS1 or myc-PIAS1 (K117 L) for 48 h or 72 h. Cells were fixed and stained with anti-flag mouse monoclonal antibody(red). Nuclei were visualized by DAPI staining. Representative images of transfected cells were shown. (b) DU145 cells were transiently co-transfected with flag-AR, GFP-SUMO3 and myc-PIAS1 or myc-PIAS1 (K117 L) for 48 h. Cells were fixed and stained with anti-myc mouse monoclonal antibody (red). Nuclei were visualized by DAPI staining. Representative images of transfected cells were shown
Fig. 7
Fig. 7
MDM2 is recruited by SUMO3 modificd PIAS1 and required for degradation of AR. (a) DU145 cells were transiently co-transfected with flag-AR, GFP-SUMO3 and myc-PIAS1 or myc-PIAS1 (K117 L) for 72 h. Whole-cell lysates were immunoblotted with anti-AR, anti-myc and anti-actin antibodies. (b) DU145 cells were transiently co-transfected as described in A for 48 h. Whole-cell lysates were immunoprepapited with anti-myc antibodies. The immunoprepapite was then detected by indicated antibodies against myc, MDM2 and ChIP. Whole-cell lysates (Input) were also immunoblotted with anti-myc, anti-MDM2, anti-ChIP and anti-actin antibodies. (c) DU145 cells were transiently co-transfected with PIAS1, empty vector or GFP-SUMO3 or GFP-SUMO3 and myc-PIAS1. Whole-cell lysates were immunoprepapited with anti-myc antibody. The immunoprepapite was then detected by indicated antibodies against myc, SUMO3, MDM2 and ChIP. Whole-cell lysates (Input) were also immunoblotted with anti-myc, anti-MDM2, anti-ChIP and anti-actin antibodies. (d) DU145 cells were transiently co-transfected with flag-AR, myc-PIAS1 and empty vector or GFP-SUMO3 or GFP-SUMO3 and MDM2 shRNA for 72 h. Whole-cell lysates were immunoblotted with anti-AR, anti-myc and anti-actin antibodies
Fig. 8
Fig. 8
MDM2 is not required for SUMO3 modified PIAS1 induced AR nuclear export. DU145 cells were transiently co-transfected with flag-AR, SUMO3, myc-PIAS1 and control shRNA or MDM2 shRNA for 48 h. Cells were then fixed and stained with anti-MDM2 (green) and anti-flag (red) or anti-myc mouse monoclonal antibody (red). Nuclei were visualized by DAPI staining. Representative images of transfected cells were shown
Fig. 9
Fig. 9
Model for the regulation of AR subcellular localization and turnover by sumoylation and ubiquitination systems. In castration-resistant prostate cancer cells, the binding of androgen contained in the serum makes AR to be released from the cytoplasmic associated heat shock proteins (HSP) and translocate to the nucleus; likewise, the overexpressed PIAS1 and SUMO3 are also gathered in nucleus. SUMO3 can be conjugated to the 117th lysine of PIAS1 which is a SUMO E3 ligase itself (a), and then the sumoylated PIAS1 recruit the MDM2 protein(b) and also interact with AR through its 386th and 845th lysines, which may block the AR dimer formation (c), further resulting in the nuclear export of AR and its binding partners. The MDM2 cooperating with ubiquitin E1 and E2 promotes the polyubiquitination of AR and its subsequent proteasome-mediated degradation. The SUMO3 modification of partial AR is also accompanied in this process (d)

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