The Androgen Receptor (AR) is essential for the normal development of the prostate gland as well as for the growth and survival of prostate cancers. Most androgen-independent prostate tumors continue to express the AR as well as androgen responsive genes, despite the near absence of circulating androgen in these patients. Although functionally androgen-independent, late-stage prostate cancer is still dependent on the AR. Several studies have demonstrated that 1) inhibiting AR expression reduced the growth of androgen-independent prostate cancer, 2) AR expression levels increase with prostate cancer progression to androgen independence and 3) the AR accumulates mutations that broaden the range of activating ligands. Thus, the AR is a critical regulator of prostate cancer progression. Moreover, it is increasingly clear that the AR is regulated not only by its cognate steroid hormone, but also by interactions with a constellation of co-regulatory and signaling molecules, many of which are elevated as prostate cancer progresses. The ability of the AR to function in the absence of physiologic levels of androgen is clearly a consequence of these alternative regulatory events. Our guiding hypothesis is that AR phosphorylation plays a critical role in regulating AR function. We further hypothesize that one or more of these phosphorylations play a role in prostate cancer, affecting gene expression, cell growth, and/or survival. While much is known about which sites of the AR undergo post-translational modification, little is known about how these post-translational modifications regulate AR function. By determining how AR phosphorylation choreographs AR function, we may uncover critical information for the development of novel prostate cancer therapeutics. Previously, we identified the major serine phosphorylation sites on the AR and observed regulation of AR phosphorylation by multiple agonists. Most recently, we have determined that stress kinase signaling regulates Ser650 phosphorylation and that this phosphorylation is required for optimal nuclear export. Since our last submission, we have determined two new candidate AR kinases and uncovered evidence that these kinases and AR phosphorylations may regulate AR transcriptional activity. These data have allowed us to enhance the focus of this revised application, and provide a solid foundation for examining the functional consequences and regulators of AR phosphorylation. Thus, this work will provide important insights into the function of the AR, a major regulator of prostate cancer progression. The following specific aims are proposed: 1) Specific Aim 1: Examine the effect of AR Ser81 phosphorylation by CDK9 on AR transcriptional activity; 2) Specific Aim 2: Determine the effect of the cell cycle on AR phosphorylation and transcription; 3) Specific Aim 3: Determine the role of AR phosphorylation in gene expression, growth and survival in prostate cancer. PUBLIC HEALTH RELEVANCE: There are no effective treatments for castration-resistant metastatic prostate cancer. In order to develop these therapies we must understand the molecular events that contribute to the progression of prostate cancer to a disease that is functionally androgen independent but that still requires the androgen receptor (AR). This proposal focuses on identifying the functional consequences and regulators of AR phosphorylation. This work should yield significant insights into how AR function is integrated with other cell regulatory activities, and ways to more effectively target the AR and regulators of AR activity in advanced and androgen independent prostate cancer.