Androgen Receptor Phosphorylation

Abstract

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 crosstalk between growth factor signaling and androgen in prostate development, physiology, and cancer.The steroid hormone receptors are ligand-activated transcription factors. In addition to regulation by steroids, they are also regulated by post-translational modifications generated by signal transduction pathways. Thus, they function not only as transcription factors but also as nodes that integrate multiple extracellular signals. The human progesterone receptor (PR) 1 is phosphorylated on multiple residues; the basal phosphorylation sites, including serines 81, 102, and 162, are rapidly induced in the presence of steroid (1). However, phosphorylation of serines 102, 294, and 345 in response to hormone is temporally delayed, reaching a maximum at 2 h (2). Phosphorylation of PR on Ser-676 in the hinge region has recently been identified (3); the analogous site in the chicken PR is also phosphorylated. When this site is mutated to alanine, subsaturating levels of hormone show severalfold less transcriptional activity compared with wild type (4). At least seven phosphorylation sites have been identified on the glucocorticoid receptor, and the relative level of phosphorylation of these sites appears to be cell cycle-regulated (5-7). Recent evidence indicates that the phosphorylation status of the glucocorticoid receptor plays a prominent role in receptor protein turnover (8). Growth factors are known to stimulate the ligand-independent activity of the estrogen receptor through the activation of the mitogen-activated protein kinase (MAPK) cascade and the direct phosphorylation of estrogen receptor by MAPK at Ser-118 (9). Hormone binding also regulates the phosphorylation ...

Androgen receptor activity is inhibited in response to genotoxic agents in a p53-independent manner

“…Although the knowledge of the functional significance of these sites is currently limited, it is well established that phosphorylation of serine 81 (Ser81) is hormone dependent (Gioeli et al, 2002;Black et al, 2004). Accordingly, we observed an increase in phosphorylation of Ser81 when hormone-depleted cells were stimulated with DHT ( Figure 6).…”

“…Although AR phosphorylation at residue Ser81 is a DHT-stimulated event (Gioeli et al, 2002;Black et al, 2004), the cellular function of this modification is not known. As the half-life of the AR is increased in response to androgens (Gregory et al, 2001), it is possible that phosphorylation of Ser81 is involved in this stabilization of the AR.…”

“…The AR is a 112 kDa protein and is reported to contain at least 10 different phosphorylation sites (Lin et al, 2001;Gioeli et al, 2002;Wong et al, 2004). Although the knowledge of the functional significance of these sites is currently limited, it is well established that phosphorylation of serine 81 (Ser81) is hormone dependent (Gioeli et al, 2002;Black et al, 2004).…”

PTEN and GSK3β: key regulators of progression to androgen-independent prostate cancer

“…Akt phosphorylation of AR has been reported to yield both activation (Wen et al, 2000) and inhibition of AR transactivation (Lin et al, 2001(Lin et al, , 2003. Additional controversy arises from mass spectrometry analysis of AR enrichments from LNCaP PrCa cells, which failed to detect putative Akt phosphorylation sites (Gioeli et al, 2002). In fact, mutational alteration of Akt phosphorylation sites show little effect upon AR transcription (Gioeli et al, 2002).…”

“…Additional controversy arises from mass spectrometry analysis of AR enrichments from LNCaP PrCa cells, which failed to detect putative Akt phosphorylation sites (Gioeli et al, 2002). In fact, mutational alteration of Akt phosphorylation sites show little effect upon AR transcription (Gioeli et al, 2002). Conflicting information regarding the direct post-translation modification that Akt confers upon AR (either directly or via GSK3b) could be attributed to use of varying androgen concentrations, systems of study or passage of cell lines.…”