Cell-specific Regulation of Androgen Receptor Phosphorylation in Vivo

Abstract

The biological ramifications of phosphorylation of the androgen receptor (AR) are largely unknown. To examine the phosphorylation of AR at serine 213, a putative substrate for Akt, a phosphorylation site-specific antibody was generated. The use of this antibody indicated that AR Ser-213 is phosphorylated in vivo and that phosphorylation is tightly regulated in a cell type-specific manner. Furthermore, Ser-213 phosphorylation took place with rapid kinetics and was inhibited by the phosphatidylinositol 3-kinase inhibitor LY294002. Phosphorylation occurred in response to R1881 and dihydrotestosterone but weakly if at all in response to testosterone. It did not occur in response to AR antagonists or growth factor stimulation in the absence of an AR agonist. Transcription assays using an AR-responsive reporter gene construct showed that activated phosphatidylinositol 3-kinase inhibited transcription mediated by wild type AR but not that of a mutant AR variant (S213A), which could not be phosphorylated at Ser-213. By immunohistochemistry, the AR Ser(P)-213 antigen was detected in prostate epithelial but not stromal cells despite the fact that an antibody recognizing both phosphorylated and non-phosphorylated forms of AR demonstrates that AR is present in both cell types as expected. In fetal tissue the AR-Ser(P)-213 antigen was present in epithelial cells of the urogenital sinus when endogenous androgen levels were high and activated Akt was prevalent, but absent at a later stage of development when endogenous androgen levels were low and Akt activation was minimal. Immunoreactivity was evident in differentiated cells lining the lumen of the urogenital sinus but not in rapidly dividing, Ki67 positive cells within the developing prostate or stromal tissue, suggesting that site-specific phosphorylation of AR Ser-213 by cellular kinases occurs in a non-proliferating cellular milieu. Like other steroid hormone receptors, the androgen receptor (AR)2 is a phosphoprotein (1). Phosphorylated amino acids within the AR include serines 16, 81, 94, 256, 308, 424, and 650 (2-4). All of these sites show increased phosphorylation in the presence of androgen, with the exception of Ser-94, which is constitutively phosphorylated. Although the biological function of AR phosphorylation is relatively obscure, analysis of AR receptor mutants that are defective in nuclear export shows that they accumulate in subnuclear foci and are hyperphosphorylated at Ser-81 (5). Interestingly, phosphorylation of Ser-650 is enhanced by treatment with forskolin, epidermal growth factor (EGF), and phorbol-12-myristate-13-acetate (4), suggesting that AR phosphorylation may be intricately linked to signal transduction processes regulating tumor promotion and cell growth. In addition, it has been reported that Akt, a cytoplasmic protein kinase activated by the lipid products of PI3K, phosphorylates AR at serines 213 and 791 in vitro (6, 7) and that overexpression of activated Akt destabilizes AR (8).Multiple laboratories have investigated the functional link ...

Progression of prostate cancer by synergy of AKT with genotropic and nongenotropic actions of the androgen receptor

“…Previous work using cell-culture models has generated alternative views about the potential crosstalk between AKT and AR signaling by using reporter assays that measure AR transcriptional activity. AKT has been claimed either to activate or to suppress AR transcriptional activity, depending on the cell types and passage number of the cells used in the assays (14,17,18). In this paper we demonstrate a direct synergy between AKT and AR signaling that can transform naïve prostatic epithelium into androgen-insensitive, but AR-dependent, carcinoma.…”

“…Additionally, in a nongenotropic signaling pathway, AR is also capable of binding and activating c-Src kinase through a polyproline region to stimulate prostate cancer cell proliferation and bone growth (24)(25)(26). The stability of AR is also reported to be modulated by AKT through phosphorylation (13,14,17,18). In the AR S213A/S791A mutant, the two putative AKT phosphorylation serines (Ser-213 and Ser-791) are mutated to alanines (12,13,18).…”

“…The stability of AR is also reported to be modulated by AKT through phosphorylation (13,14,17,18). In the AR S213A/S791A mutant, the two putative AKT phosphorylation serines (Ser-213 and Ser-791) are mutated to alanines (12,13,18). This mutant acts similarly to wild-type AR in an in vitro transcriptional reporter assay (12).…”

Site-specific Androgen Receptor Serine Phosphorylation Linked to Epidermal Growth Factor-dependent Growth of Castration-recurrent Prostate Cancer

“…Mass spectrometry confirmed AR phosphorylation at Ser-81, -94, and -650 and identified Ser-16, -256, -308, and -424 as phosphorylation sites in the AR NH 2 -terminal region (14). AR NH 2 -terminal Ser-213 was phosphorylated in nonproliferating prostate epithelial cells (36), and Akt-mediated phosphorylation at AR Ser-213 and Ser-791 was linked to anti-apoptotic and proliferative effects in prostate cancer cells (37,38). However, the synergistic effects of Akt and AR on neoplastic proliferation of murine prostatic epithelium were independent of phosphorylation at these sites (39).…”

Regulation of the androgen receptor by post-translational modifications

“…Caution should also be taken when investigating AR-P in the literature as some of the phosphorylation sites are given different numbers, which can confuse the reader. For example, Ser210 is described as phosphorylated (Lin et al 2001b, McCall et al 2008, Shank et al 2008, Bryant et al 2011; however, when referring to the standard AR protein sequence (ANDR HUMAN, P10275), an arginine is found at this site, while in other studies, the same serine in question is referred to as Ser213 (Wen et al 2000, Nan et al 2003, Burgdorf et al 2004, Taneja et al 2005, Kim & Lee 2009, Lee 2009). In this review, the nomenclature used will correspond to the standard sequence to avoid such confusion.…”