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The role of Mdm2 in androgen receptor regulation
The Role of Mdm2 in Androgen
Receptor
Regulation.
Luke Gaughan, David E. Neal* and Craig N. Robson. Prostate Research Group, of Newcastle Upon Tyne and Oncology Centre, Addenbrooke’s Hospital, Cambridge*.
University
Introduction. The androgen receptor (AR) is a member of the nuclear hormone receptor superfamily and plays a critical role in regulating expression of genes involved in prostate growth, homeostasis and androgen-dependent and -independent tumour formation. Work from our laboratory has highlighted an important role for co-activator and co-repressor proteins, such as Tip60 and HDAC respectively, in regulating AR activity though inducing changes to the acetylation status of the receptor. At present, several aspects of AR regulation remain largely ill defined, including mechanisms involved in controlling AR protein stability. However, the recent demonstration that the AR is a direct target for Mdm2-mediated ubiquitination and degradation suggests that Mdm2 and ubiquitination provide an additional facet for AR regulation. In this study, we sought to further examine the role of Mdm2 in AR regulation by firstly identifying the target lysine sites within the AR for Mdm2-mediated ubiquitination. We further investigated the effects of Mdm2 on both AR-mediated transcription and cellular localisation in an attempt to identify additional mechanisms of Mdm2-mediated AR control. Finally, using chromatin immunoprecipitation (ChlP) analysis, we examined the association of Mdm2 at an AR-responsive gene to establish a role for Mdm2 in regulation of AR-mediated transcription at the promoter level.
Materials
and Methods.
Using an in vitro ubiquitination assay, incorporating purified ubiquitylase enzymes and various AR mutants harbouring lysine substitutions, we attempted to determine the target ubiquitination sites within the AR. The effect of Mdm2 on AR-mediated transcription was assessed using transient transfection experiments in LNCaP cells, incorporating AR, wild-type and ubiquitylasedead Mdm2 together with an AR-responsive reporter. Using fluorescently-tagged AR and Mdm2 in LNCaP cells, the effect of Mdm2 on AR distribution was examined. To assess Mdm2 association with the AR-responsive PSA promoter, ChlP analysis incorporating an Mdm2 antibody was performed in LNCaP cells.
Results. We provide evidence that the AR is directly ubiquitinated by Mdm2 at several centrally positioned lysines, including the acetylation target sequence KLKK. In reporter assays, we show that AR-mediated transcription is abrogated by Mdm2 in an ubiquitylase-independent manner. Furthermore, fluorescence microscopy indicates that both wild-type and ubiquitylase-dead Mdm2 induces AR nuclear export, indicative of an additional mechanism for AR regulation. Finally, ChlP experiments confirm Mdm2 association at the AR-responsive PSA promoter and detail a multiprotein complex including Mdm2, AR, HDACI and TipGO.
Conclusion. In this work, we have furthered our understanding of AR-Mdm2 regulation and provided evidence of a multi-faceted mode of AR control by Mdm2. Intriguingly, the fact that Mdm2 is associated with the AR at both the promoter and during nuclear export is indicative of a very strict regulatory network and may provide new and novel targets for therapeutic intervention.
Receptor
Regulation.
Luke Gaughan, David E. Neal* and Craig N. Robson. Prostate Research Group, of Newcastle Upon Tyne and Oncology Centre, Addenbrooke’s Hospital, Cambridge*.
University
Introduction. The androgen receptor (AR) is a member of the nuclear hormone receptor superfamily and plays a critical role in regulating expression of genes involved in prostate growth, homeostasis and androgen-dependent and -independent tumour formation. Work from our laboratory has highlighted an important role for co-activator and co-repressor proteins, such as Tip60 and HDAC respectively, in regulating AR activity though inducing changes to the acetylation status of the receptor. At present, several aspects of AR regulation remain largely ill defined, including mechanisms involved in controlling AR protein stability. However, the recent demonstration that the AR is a direct target for Mdm2-mediated ubiquitination and degradation suggests that Mdm2 and ubiquitination provide an additional facet for AR regulation. In this study, we sought to further examine the role of Mdm2 in AR regulation by firstly identifying the target lysine sites within the AR for Mdm2-mediated ubiquitination. We further investigated the effects of Mdm2 on both AR-mediated transcription and cellular localisation in an attempt to identify additional mechanisms of Mdm2-mediated AR control. Finally, using chromatin immunoprecipitation (ChlP) analysis, we examined the association of Mdm2 at an AR-responsive gene to establish a role for Mdm2 in regulation of AR-mediated transcription at the promoter level.
Materials
and Methods.
Using an in vitro ubiquitination assay, incorporating purified ubiquitylase enzymes and various AR mutants harbouring lysine substitutions, we attempted to determine the target ubiquitination sites within the AR. The effect of Mdm2 on AR-mediated transcription was assessed using transient transfection experiments in LNCaP cells, incorporating AR, wild-type and ubiquitylasedead Mdm2 together with an AR-responsive reporter. Using fluorescently-tagged AR and Mdm2 in LNCaP cells, the effect of Mdm2 on AR distribution was examined. To assess Mdm2 association with the AR-responsive PSA promoter, ChlP analysis incorporating an Mdm2 antibody was performed in LNCaP cells.
Results. We provide evidence that the AR is directly ubiquitinated by Mdm2 at several centrally positioned lysines, including the acetylation target sequence KLKK. In reporter assays, we show that AR-mediated transcription is abrogated by Mdm2 in an ubiquitylase-independent manner. Furthermore, fluorescence microscopy indicates that both wild-type and ubiquitylase-dead Mdm2 induces AR nuclear export, indicative of an additional mechanism for AR regulation. Finally, ChlP experiments confirm Mdm2 association at the AR-responsive PSA promoter and detail a multiprotein complex including Mdm2, AR, HDACI and TipGO.
Conclusion. In this work, we have furthered our understanding of AR-Mdm2 regulation and provided evidence of a multi-faceted mode of AR control by Mdm2. Intriguingly, the fact that Mdm2 is associated with the AR at both the promoter and during nuclear export is indicative of a very strict regulatory network and may provide new and novel targets for therapeutic intervention.