Nucleocytoplasmic Shuttling and Phosphorylation of the Androgen Receptor

The androgen receptor (AR) is an androgen-regulated transcription factor that governs epithelial cell growth in the prostate gland. Its involvement in the progression of prostate cancer to an incurable state makes it an important therapeutic target. Androgenbound AR facilitates the formation of preinitiation complexes on target-promoters for gene expression. Even though the steady-state distribution of androgen-bound AR is nuclear, AR continuously shuttles between the nucleus and the cytoplasm. The regulatory contributions of nucleocytoplasmic shuttling to AR function are examined here. The consequences of shuttling on transcription, ligand-binding, and phosphorylation of the AR protein were evaluated by forcing the localization of AR to the nucleus or the cytoplasm. While ligand-binding was unaffected, transiently nuclear AR was sufficient to induce transcription of several genes. Cytoplasmic AR was enriched for androgenindependent phosphorylation at Ser94, whereas nuclear localization of AR was required for androgen-induced phosphorylation at Ser81, Ser256, and Ser308. That differential phosphorylation of AR occurs based on nucleocytoplasmic localization suggests that one of the purposes of shuttling is to connect signal transduction in the cytoplasm to AR actions in the nucleus. Within both the cytoplasm and nucleus, AR is known to interact with numerous binding partners, including several modulators of the G 1 phase of the cell cycle. Pharmacological inhibition of the cyclin-dependent kinase(CDK)-4 decreased phosphorylation of AR at Ser81, Ser308, and Ser424 and altered AR-mediated transcription of several endogenous genes. In vitro kinase assays revealed that CDK4 can directly phosphorylate AR at Ser308. Co-immunoprecipitation assays provided evidence of an androgen-independent AR complex with CDK4, where cyclin-binding to CDK4 ii was not required. Furthermore, CDK4 protein inhibitor p16 INK4a associated with the AR complex only in the absence of androgen, suggesting a possible mechanism for androgendependent activation of an AR kinase. This dynamic, multi-component complex indicates that AR may function as a node to integrate the CDK4/CyclinD/INK4/Rb cell cycle signaling pathway to gene expression in prostate cells. Together, these data establish nucleocytoplasmic shuttling and CDK4-mediated signaling as contributors to the regulation of AR phosphorylation and function.

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