Androgen receptor phosphorylation. Regulation and identification of the phosphorylation sites

Activation of signal transduction kinase cascades has been shown to alter androgen receptor (AR) activity. Although it has been suggested that changes in AR phosphorylation might be directly responsible, the basal and regulated phosphorylations of the AR have not been fully determined. We have identified the major sites of AR phosphorylation on ARs expressed in COS-1 cells using a combination of peptide mapping, Edman degradation, and mass spectrometry. We describe the identification of seven AR phosphorylation sites, show that the phosphopeptides seen with exogenously expressed ARs are highly similar to those seen with endogenous ARs in LNCaP cells and show that specific agonists differentially regulate the phosphorylation state of endogenous ARs in LNCaP prostate cancer cells. Treatment of LNCaP cells with the synthetic androgen, R1881, elevates phosphorylation of serines 16, 81, 256, 308, 424, and 650. Ser-94 appears constitutively phosphorylated. Forskolin, epidermal growth factor, and phorbol 12-myristate 13-acetate increase the phosphorylation of Ser-650. The kinetics of phosphorylation of most sites in response to hormone or forskolin is temporally delayed, reaching a maximum at 2 h post-stimulation. The exception is Ser-81, which continues to display increasing phosphorylation at 6 h. These data provide a basis for analyzing mechanisms of cross-talk between growth factor signaling and androgen in prostate development, physiology, and cancer.     

Ligand-dependent corepressor acts as a novel androgen receptor corepressor, inhibits prostate cancer growth, and is functionally inactivated by the Src protein kinase

The activated androgen receptor (AR) promotes prostate cancer (PCa) growth. AR antagonists repress the AR by recruitment of corepressors. Not much is known about the inactivation of AR by corepressors in the presence of agonists (androgens). Here we show that the corepressor LCoR acts as an androgen-dependent corepressor that represses human PCa growth in vivo. In line with this, progressive decrease of ligand-dependent corepressor expression was observed in the PCa TRAMP mouse model with increasing age. LCoR interacts with AR and is recruited to chromatin in an androgen-induced manner. Unexpectedly, the LXXLL motif of LCoR is dispensable for interaction with the AR. Rather, the data indicate that LCoR interacts with the AR DNA binding domain on DNA. Interestingly, the interaction of LCoR with AR is inhibited by signaling pathways that are associated with androgen-independent PCa. Here we also show that the Src kinase inactivates the corepressive function of LCoR. Interfering with endogenous Src function by a dominant negative Src mutant, the growth inhibitory activity of LCoR is enhanced in vivo in a xenograft mouse model system. Thus, our studies indicate a role of LCoR as an AR corepressor and a tumor suppressor. Further, the decreased expression or inactivation of LCoR is as an important step toward PCa carcinogenesis in vivo.