Faculty Bibliography

Protein phosphorylation is a versatile posttranslational modification that can regulate nuclear receptor function. Although the precise role of receptor phosphorylation is not fully understood, it appears that it functions to direct or refine receptor activity in response to particular physiological requirements. Identifying and characterizing specific nuclear receptor phosphorylation sites is an important step in elucidating the role(s) receptor phosphorylation plays in function. Although traditional methods of metabolic labeling and in vitro protein phosphorylation have been informative, receptor phosphorylation site-specific antibodies are simple and reliable tools to study receptor phosphorylation. This chapter will discuss how to develop nuclear receptor phosphorylation site-specific antibodies to elucidate function

Androgen receptor (AR) is expressed in both stromal and epithelial cells of the prostate. The majority of studies on AR expression and function in prostate cancer is focused on malignant epithelial cells rather than stromal cells. In this study, we examined the levels of stromal AR in androgen-dependent and -independent prostate cancer and the function of stromal AR in prostate cancer growth and invasion. We showed that stromal AR levels were decreased in the areas surrounding cancerous tissue, especially in androgen-independent cancer. Using two telomerase-immortalized human stromal cell lines, one AR-positive and the other AR-negative, we demonstrated that stromal cells lacking AR stimulated cell proliferation of co-cultured prostate cancer cells in vitro and enhanced tumour growth in vivo when co-injected with PC3 epithelial cells in nude mice. In contrast, stromal cells expressing AR suppressed prostate cancer growth in vitro and in vivo. In parallel with cancer growth, in vitro invasion assays revealed that stromal cells lacking AR increased the invasion ability of PC3 cell by one order of magnitude, while stromal cells expressing AR reduced this effect. These results indicate a negative regulation of prostate cancer growth and invasion by stromal AR. This provides potentially new mechanistic insights into the failure of androgen ablation therapy, and the reactivation of stromal AR could be a novel therapeutic approach for treating hormone refractory prostate cancer.

The glucocorticoid receptor (GR) is phosphorylated at multiple sites within its N terminus (S203, S211, S226), yet the role of phosphorylation in receptor function is not understood. Using a range of agonists and GR phosphorylation site-specific antibodies, we demonstrated that GR transcriptional activation is greatest when the relative phosphorylation of S211 exceeds that of S226. Consistent with this finding, a replacement of S226 with an alanine enhances GR transcriptional response. Using a battery of compounds that perturb different signaling pathways, we found that BAPTA-AM, a chelator of intracellular divalent cations, and curcumin, a natural product with antiinflammatory properties, reduced hormone-dependent phosphorylation at S211. This change in GR phosphorylation was associated with its decreased nuclear retention and transcriptional activation. Molecular modeling suggests that GR S211 phosphorylation promotes a conformational change, which exposes a novel surface potentially facilitating cofactor interaction. Indeed, S211 phosphorylation enhances GR interaction with MED14 (vitamin D receptor interacting protein 150). Interestingly, in U2OS cells expressing a nonphosphorylated GR mutant S211A, the expression of IGF-binding protein 1 and interferon regulatory factor 8, both MED14-dependent GR target genes, was reduced relative to cells expressing wild-type receptor across a broad range of hormone concentrations. In contrast, the induction of glucocorticoid-induced leucine zipper, a MED14-independent GR target, was similar in S211A- and wild-type GR-expressing cells at high hormone levels, but was reduced in S211A cells at low hormone concentrations, suggesting a link between GR phosphorylation, MED14 involvement, and receptor occupancy. Phosphorylation also affected the magnitude of repression by GR in a gene-selective manner. Thus, GR phosphorylation at S211 and S226 determines GR transcriptional response by modifying cofactor interaction. Furthermore, the effect of GR S211 phosphorylation is gene specific and, in some cases, dependent upon the amount of activated receptor

Overexpression of steroid receptor coactivator 3 (SRC-3) is associated with an increased incidence of breast cancer. A recent study shows that SRC-3 is protected from proteasomal degradation by atypical protein kinase C (aPKC)-mediated phosphorylation in an estrogen receptor alpha (ERalpha)-dependent manner. This finding provides a novel mechanism for coupling increased SRC-3 expression with enhanced estrogen-dependent cellular proliferation

Androgen receptor (AR) mediates transcriptional activation of diverse target genes through interactions with various coactivators that may alter its function and help mediate the switch between prostate cell proliferation and differentiation. We recently identified p44/MEP50 as an AR coactivator and further showed that it is expressed primarily in the nucleus and cytoplasm of benign prostate epithelial and prostate cancer cells, respectively. We also showed that haploinsufficiency in p44(+/-) mice causes prostate epithelial cell proliferation. To establish direct cause-and-effect relationships, we have used p44 fusion proteins that are selectively expressed in the nucleus or cytoplasm of prostate cancer cells (LNCaP), along with RNAi analyses, to examine effects of p44 both in vitro and in vivo (in tumor xenografts). We show that preferential expression of p44 in the nucleus inhibits proliferation of LNCaP cells in an AR-dependent manner, whereas preferential expression of p44 in the cytoplasm enhances cell proliferation. These effects appear to be mediated, at least in part, through the regulation of distinct cell-cycle regulatory genes that include p21 (up-regulated by nuclear p44) and cyclin D2 and CDK6 (up-regulated by cytoplasmic p44). Importantly, we also demonstrate that altered p44 expression is associated with androgen-independent prostate cancer. Our results indicate that nuclear p44 and cytoplasmic p44 have distinct and opposing functions in the regulation of prostate cancer cell proliferation