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Deubiquitinating enzyme Usp12 regulates the interaction between the androgen receptor and the Akt pathway.

McClurg UL, Summerscales EE, Harle VJ, Gaughan L, Robson CN - Oncotarget (2014)

Bottom Line: We have recently reported that AR is deubiquitinated and stabilised by Usp12 resulting in increased transcriptional activity.In this report we demonstrate that Usp12, in complex with Uaf-1 and WDR20, directly deubiquitinates and stabilises the Akt phosphatases PHLPP and PHLPPL resulting in decreased levels of active pAkt.Decreased pAkt in turn down-regulates AR Ser213 phosphorylation resulting in enhanced receptor stability and transcriptional activity.

View Article: PubMed Central - PubMed

Affiliation: Solid Tumour Target Discovery Laboratory, Newcastle Cancer Centre, Northern Institute for Cancer Research, Medical School, Newcastle University, Newcastle upon Tyne NE2 4HH, UK.

ABSTRACT
The androgen receptor (AR) is a transcription factor involved in prostate cell growth, homeostasis and transformation regulated by post-translational modifications, including ubiquitination. We have recently reported that AR is deubiquitinated and stabilised by Usp12 resulting in increased transcriptional activity. In this study we have investigated the relationship between Usp12, PHLPP and PHLPPL tumour suppressors in the regulation of AR transcriptional activity in prostate cancer (PC). PHLPP and PHLPPL are pro-apoptotic phosphatases that dephosphorylate and subsequently deactivate Akt. Phosphorylated Akt is reported to deactivate AR in PC by phosphorylation at Ser213 and Ser791 leading to ligand dissociation and AR degradation. In contrast, PHLPP- and PHLPPL-mediated dephosphorylation and inactivation of Akt elevates the levels of active AR. In this report we demonstrate that Usp12, in complex with Uaf-1 and WDR20, directly deubiquitinates and stabilises the Akt phosphatases PHLPP and PHLPPL resulting in decreased levels of active pAkt. Decreased pAkt in turn down-regulates AR Ser213 phosphorylation resulting in enhanced receptor stability and transcriptional activity. Additionally, we observe that depleting Usp12 sensitises PC cells to therapies aimed at Akt inhibition irrespectively of their sensitivity to androgen ablation therapy. We propose that Usp12 inhibition could offer a therapeutic alternative for castration resistant prostate cancer.

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Usp12 deubiquitinates and stabilises PHLPP and PHLPPL and as a result controls the levels of pAkt(A) COS-7 cells were transfected with p-HA-PHLPP, p-HA-PHLPPL, p-His-Ub and p-Flag-Usp12 plasmids as indicated. 72h post transfection cells were treated with MG-132 and harvested 16h later. Lysates were denatured and subsequently immunoprecipitated (IP) with HA antibody (PHLPP and PHLPPL) followed by immunoblotting. (B-C) COS-7 cells were transfected with PHLPP (B) and PHLPPL (C) and with increasing amounts (75-300ng) of WT or C48A mutant Usp12 for 72h. Reactions were balanced with empty pCMV vector. Cells were lysed followed by immunoblotting. (D-E) LNCaP cells were treated with siRNA as indicated, at 96h cells were lysed followed by immunoblotting.
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Figure 2: Usp12 deubiquitinates and stabilises PHLPP and PHLPPL and as a result controls the levels of pAkt(A) COS-7 cells were transfected with p-HA-PHLPP, p-HA-PHLPPL, p-His-Ub and p-Flag-Usp12 plasmids as indicated. 72h post transfection cells were treated with MG-132 and harvested 16h later. Lysates were denatured and subsequently immunoprecipitated (IP) with HA antibody (PHLPP and PHLPPL) followed by immunoblotting. (B-C) COS-7 cells were transfected with PHLPP (B) and PHLPPL (C) and with increasing amounts (75-300ng) of WT or C48A mutant Usp12 for 72h. Reactions were balanced with empty pCMV vector. Cells were lysed followed by immunoblotting. (D-E) LNCaP cells were treated with siRNA as indicated, at 96h cells were lysed followed by immunoblotting.

Mentions: PHLPP protein stability was previously shown to be regulated by ubiquitination by SCF-b-TrcP complex [23], similar regulation probably occurs for PHLPPL. As Usp12 is a deubiquitinating enzyme we hypothesised that PHLPP and PHLPPL could be potential targets for ubiquitination reversal by Usp12. To assess this, we overexpressed ubiquitin, Usp12 and either PHLPP or PHLPPL in COS-7 cells prior to treatment with the proteosomal inhibitor MG-132, to maximise the levels of ubiquitinated phosphatase enzymes, followed by lysis under denaturing conditions that permits exclusive detection of ubiquitinated PHLPP or PHLPPL without contamination by interacting proteins. Lysates, were immunoprecipitated with anti-PHLPP or PHLPPL antibodies and the levels of ubiquitinated PHLPP and PHLPPL visualised by immunoblotting using an anti-ubiquitin antibody. In agreement with previous reports, both phosphatases were ubiquitinated in cells (Figure 2A, lanes 2 and 4). Importantly, overexpression of Usp12 deubiquitinated both PHLPP and PHLPPL (Figure 2A, lanes 1 and 3) and this elevated the steady-state levels of the enzymes, while overexpression of an enzymatically inactive Usp12C48A mutant failed to elevate PHLPP and PHLPPL levels suggesting the importance of Usp12 enzymatic activity for phosphatase regulation (Figure 2B and Figure 2C). Similarly, when we silenced Usp12, or its interacting partners Uaf-1 and WDR20, levels of PHLPP and PHLPPL were decreased in LNCaP PC cells (Figure 2D).


Deubiquitinating enzyme Usp12 regulates the interaction between the androgen receptor and the Akt pathway.

McClurg UL, Summerscales EE, Harle VJ, Gaughan L, Robson CN - Oncotarget (2014)

Usp12 deubiquitinates and stabilises PHLPP and PHLPPL and as a result controls the levels of pAkt(A) COS-7 cells were transfected with p-HA-PHLPP, p-HA-PHLPPL, p-His-Ub and p-Flag-Usp12 plasmids as indicated. 72h post transfection cells were treated with MG-132 and harvested 16h later. Lysates were denatured and subsequently immunoprecipitated (IP) with HA antibody (PHLPP and PHLPPL) followed by immunoblotting. (B-C) COS-7 cells were transfected with PHLPP (B) and PHLPPL (C) and with increasing amounts (75-300ng) of WT or C48A mutant Usp12 for 72h. Reactions were balanced with empty pCMV vector. Cells were lysed followed by immunoblotting. (D-E) LNCaP cells were treated with siRNA as indicated, at 96h cells were lysed followed by immunoblotting.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4196185&req=5

Figure 2: Usp12 deubiquitinates and stabilises PHLPP and PHLPPL and as a result controls the levels of pAkt(A) COS-7 cells were transfected with p-HA-PHLPP, p-HA-PHLPPL, p-His-Ub and p-Flag-Usp12 plasmids as indicated. 72h post transfection cells were treated with MG-132 and harvested 16h later. Lysates were denatured and subsequently immunoprecipitated (IP) with HA antibody (PHLPP and PHLPPL) followed by immunoblotting. (B-C) COS-7 cells were transfected with PHLPP (B) and PHLPPL (C) and with increasing amounts (75-300ng) of WT or C48A mutant Usp12 for 72h. Reactions were balanced with empty pCMV vector. Cells were lysed followed by immunoblotting. (D-E) LNCaP cells were treated with siRNA as indicated, at 96h cells were lysed followed by immunoblotting.
Mentions: PHLPP protein stability was previously shown to be regulated by ubiquitination by SCF-b-TrcP complex [23], similar regulation probably occurs for PHLPPL. As Usp12 is a deubiquitinating enzyme we hypothesised that PHLPP and PHLPPL could be potential targets for ubiquitination reversal by Usp12. To assess this, we overexpressed ubiquitin, Usp12 and either PHLPP or PHLPPL in COS-7 cells prior to treatment with the proteosomal inhibitor MG-132, to maximise the levels of ubiquitinated phosphatase enzymes, followed by lysis under denaturing conditions that permits exclusive detection of ubiquitinated PHLPP or PHLPPL without contamination by interacting proteins. Lysates, were immunoprecipitated with anti-PHLPP or PHLPPL antibodies and the levels of ubiquitinated PHLPP and PHLPPL visualised by immunoblotting using an anti-ubiquitin antibody. In agreement with previous reports, both phosphatases were ubiquitinated in cells (Figure 2A, lanes 2 and 4). Importantly, overexpression of Usp12 deubiquitinated both PHLPP and PHLPPL (Figure 2A, lanes 1 and 3) and this elevated the steady-state levels of the enzymes, while overexpression of an enzymatically inactive Usp12C48A mutant failed to elevate PHLPP and PHLPPL levels suggesting the importance of Usp12 enzymatic activity for phosphatase regulation (Figure 2B and Figure 2C). Similarly, when we silenced Usp12, or its interacting partners Uaf-1 and WDR20, levels of PHLPP and PHLPPL were decreased in LNCaP PC cells (Figure 2D).

Bottom Line: We have recently reported that AR is deubiquitinated and stabilised by Usp12 resulting in increased transcriptional activity.In this report we demonstrate that Usp12, in complex with Uaf-1 and WDR20, directly deubiquitinates and stabilises the Akt phosphatases PHLPP and PHLPPL resulting in decreased levels of active pAkt.Decreased pAkt in turn down-regulates AR Ser213 phosphorylation resulting in enhanced receptor stability and transcriptional activity.

View Article: PubMed Central - PubMed

Affiliation: Solid Tumour Target Discovery Laboratory, Newcastle Cancer Centre, Northern Institute for Cancer Research, Medical School, Newcastle University, Newcastle upon Tyne NE2 4HH, UK.

ABSTRACT
The androgen receptor (AR) is a transcription factor involved in prostate cell growth, homeostasis and transformation regulated by post-translational modifications, including ubiquitination. We have recently reported that AR is deubiquitinated and stabilised by Usp12 resulting in increased transcriptional activity. In this study we have investigated the relationship between Usp12, PHLPP and PHLPPL tumour suppressors in the regulation of AR transcriptional activity in prostate cancer (PC). PHLPP and PHLPPL are pro-apoptotic phosphatases that dephosphorylate and subsequently deactivate Akt. Phosphorylated Akt is reported to deactivate AR in PC by phosphorylation at Ser213 and Ser791 leading to ligand dissociation and AR degradation. In contrast, PHLPP- and PHLPPL-mediated dephosphorylation and inactivation of Akt elevates the levels of active AR. In this report we demonstrate that Usp12, in complex with Uaf-1 and WDR20, directly deubiquitinates and stabilises the Akt phosphatases PHLPP and PHLPPL resulting in decreased levels of active pAkt. Decreased pAkt in turn down-regulates AR Ser213 phosphorylation resulting in enhanced receptor stability and transcriptional activity. Additionally, we observe that depleting Usp12 sensitises PC cells to therapies aimed at Akt inhibition irrespectively of their sensitivity to androgen ablation therapy. We propose that Usp12 inhibition could offer a therapeutic alternative for castration resistant prostate cancer.

Show MeSH
Related in: MedlinePlus