| Androgen receptor (AR) re-activation under low androgen environment is the hallmark of castration resistant or hormone refractory prostate cancer (HRPC). Non-receptor tyrosine kinases phosphorylate AR in an androgen independent manner, and are thereby involved in HRPC. Herein, we investigated the role of Ack1 target phosphorylation sites (Tyr-267 and Tyr-363) on growth factor-regulated AR phosphorylation as well as AR transcriptional and functional activity. Both Tyr-267 and Tyr-363 have a critical role for ligand-dependent and --independent control of activity of AR. Treatment of LNCaP cells overexpressing full length or truncated AR (missing the ligand binding domain (LBD) with epidermal growth factor (EGF), heregulin, or Gas6 (ligand binding to Mer receptor tyrosine kinase and activating Ack1 downstream) induced AR phosphorylation at Tyr-267 and that phosphorylation was lost in the AR-Y267F mutant protein. The full length (FL) AR overexpressing cells proliferated strongly without androgen treatment and they reached optimal growth at a lower dose of DHT treatment (0.1 nM DHT). However, cells expressing full- or -truncated AR-Y267F mutant did not show androgen-independent proliferation and did not respond to androgen treatment. Overexpression of the Y267F mutant within full length- or truncated-AR showed significant reduction in soft agar colony formation, compared to the AR-WT. The extent of reduction in colony formation of Y363F AR expressing cells was moderate, but not as much as Y267F. The analysis of AR subcellular localization by both immunoblotting and immunofluoresence assays suggested that mutating the Tyr-267 site impaired both androgen-dependent and --independent nuclear translocation, compared to the AR-WT. Global gene expression profiling analysis demonstrated that there were no common genes regulated by both full length AR and AR-Y267F, suggesting that mutating Tyr-267 has a significant impact on not only AR dependent target gene expression but also global gene expression of LNCaP cells. Taken together, the results of our study demonstrate that tyrosine kinase target phosphorylation sites are important for both ligand-dependent and ligand-independent activity of AR protein. Targeting upstream tyrosine kinases and the N terminal domain of AR in truncated AR missing LBD may expand the repertoire of therapeutics for combating prostate cancer. |
