Androgen Receptor Serine 81 Phosphorylation Mediates Chromatin Binding and Transcriptional Activation

Shaoyong Chen,Sarah Gulla,Changmeng Cai,Steven P Balk

doi:10.1074/jbc.m111.325290

Shaoyong Chen, Sarah Gulla + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m111.325290

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Abstract

Our previous findings indicated that androgen receptor (AR) phosphorylation at serine 81 is stimulated by the mitotic cyclin-dependent kinase 1 (CDK1). In this report, we extended our previous study and confirmed that Ser-81 phosphorylation increases during mitosis, coincident with CDK1 activation. We further showed blocking cell cycle at G(1) or S phase did not disrupt androgen-induced Ser-81 phosphorylation and AR-dependent transcription, consistent with a recent report that AR was phosphorylated at Ser-81 and activated by the transcriptional CDK9. To assess the function of Ser-81 phosphorylation in prostate cancer (PCa) cells expressing endogenous AR, we developed a ligand switch strategy using a ligand-binding domain mutation (W741C) that renders AR responsive to the antagonist bicalutamide. An S81A/W741C double mutant AR stably expressed in PCa cells failed to transactivate the endogenous AR-regulated PSA or TMPRSS2 genes. ChIP showed that the S81A mutation prevented ligand-induced AR recruitment to these genes, and cellular fractionation revealed that the S81A mutation globally abrogated chromatin binding. Conversely, the AR fraction rapidly recruited to chromatin after androgen stimulation was highly enriched for Ser-81 phosphorylation. Finally, inhibition of CDK1 and CDK9 decreased AR Ser-81 phosphorylation, chromatin binding, and transcriptional activity. These findings indicate that Ser-81 phosphorylation by CDK9 stabilizes AR chromatin binding for transcription and suggest that CDK1-mediated Ser-81 phosphorylation during mitosis provides a pool of Ser-81 phosphorylation AR that can be readily recruited to chromatin for gene reactivation and may enhance AR activity in PCa.

Highlights

androgen receptor (AR) Ser-81 phosphorylation correlates with transcriptional activity and can be mediated by CDK9 and cyclin-dependent kinase 1 (CDK1), but its function is unknown
To address the functional significance of Ser-81 phosphorylation in AR-expressing prostate cancer (PCa) cells, we exploited a W741C mutation in the AR ligand-binding domain (LBD) that allows the LBD to fold into the agonistic conformation and AR to be activated by the antagonist bicalutamide [20]. Using this ligand switch strategy to stably express and selectively activate a Ser-81 by alanine (S81A) mutant in LNCaP PCa cells expressing endogenous AR, we found that the S81A/W741C double mutant versus the W741C single mutant AR was unable to stimulate endogenous AR-regulated genes in response to bicalutamide
Because CDK1 is activated during mitosis, in this study we first established that AR Ser-81 phosphorylation was increased during mitosis, coincident with CDK1 activation

Summary

Background

AR Ser-81 phosphorylation correlates with transcriptional activity and can be mediated by CDK9 and CDK1, but its function is unknown. In contrast to CDK1, which is activated in mitosis, CDK9 associates with cyclin T to form the P-TEFb complex that stimulates transcriptional elongation through phosphorylation of substrates, including Ser-2 in the RNA polymerase II C-terminal domain [19] This latter study showed that an S81A mutant AR was less effective at stimulating growth and, when expressed in AR-negative PCa cells, was decreased in its ability to transactivate a subset of AR-regulated genes [18]. We suggest that Ser-81 phosphorylation by CDK1 during mitosis provides a pool of Ser(P)-81-AR that can be rapidly recruited to AR-regulated genes when the cells enter G0/G1 Through this mechanism, increased CDK1 activity may contribute to AR activation in CRPC

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DISCUSSION