WW domain binding protein-2 (WBP-2) was cloned as an E6-associated protein interacting protein, and its role in steroid hormone receptors functions was investigated. We show that WBP-2 specifically enhanced the transactivation functions of progesterone receptor (PR) and estrogen receptor (ER), whereas it did not have any significant effect on the androgen receptor, glucocorticoid receptor, or the activation functions of p53 and VP-16. Depletion of endogenous WBP-2 with small interfering RNAs indicated that WBP-2 was required for the proper functioning of PR and ER. We also demonstrated that WBP-2 contains an intrinsic activation domain. Moreover, chromatin immunoprecipitation assays demonstrate the hormone-dependent recruitment of WBP-2 onto an estrogen-responsive promoter. Mutational analysis suggests that one of three polyproline (PY) motifs of WBP-2 is essential for its coactivation and intrinsic activation functions. We show that WBP-2 and E6-associated protein each enhance PR function, and their effect on PR action are additive when coexpressed, suggesting a common signaling pathway. In this study, we also demonstrate that the WBP-2 binding protein, Yes kinase-associated protein (YAP) enhances PR transactivation, but YAP's coactivation function is absolutely dependent on WBP-2. Taken together, our data establish the role of WBP-2 and YAP as coactivators for ER and PR transactivation pathways.

In this study we report that deletion of E6-associated protein (E6-AP) in mice results in a smaller prostate gland compared with that in normal wild-type animals. To investigate the mechanism(s) by which E6-AP affects prostate gland growth and development, we carried out both in vitro and in vivo experiments. In this study we show that E6-AP interacts with androgen receptor (AR) in a hormone-dependent manner and enhances the transactivation function of AR. Our in vivo data from E6-AP-null prostate glands show that the level of AR protein is elevated while the level of the AR target protein, probasin, is decreased. In contrast, the level of AR protein is decreased, and its target protein is increased in an E6-AP-overexpressing stable cell line, suggesting that E6-AP modulates both the protein level and the activity of AR. In addition, we show that the levels of phosphatidylinositol 3-kinase, total Akt, and phosphorylated Akt are decreased in E6-AP-null prostate, suggesting that E6-AP deletion down-regulates the signaling of the phosphatidylinositol 3-kinase-Akt pathway. We also show that RhoA negatively regulates AR function, and RhoA levels are increased in E6-AP-null prostate. Furthermore, expression levels of p53, Bax, active caspases, and apoptotic index are increased in E6-AP-null prostate. Collectively, our data suggest that E6-AP deletion attenuates the growth and development of the prostate gland by interfering with AR function as well as by stimulating p53-mediated apoptosis.

Expression of RGS2, but not other RGS proteins, abolished androgen-independent AR activity in androgenindependent LNCaP cells and CWR22Rv1 cells. In LNCaP cells, RGS2 inhibited G q -coupled GPCR signaling. Expression of exogenous wild-type RGS2, but not its GAP-deficient mutant, significantly reduced AR activation by constitutively activated G q Q209L mutant whereas silencing endogenous RGS2 by siRNA enhanced G q Q209L-stimulated AR activity. RGS2 had no effect on RGS-insensitive G q Q209L/G188S-induced AR activation. Furthermore, extracellular signal-regulated kinase 1/2 (ERK1/2) was found to be involved in RGS2-mediated regulation of androgen-independent AR activity. In addition, RGS2 functioned as a growth suppressor for androgen-independent LNCaP cells whereas androgensensitive LNCaP cells with RGS2 silencing had a growth advantage under steroid-reduced conditions. Finally, RGS2 expression level was significantly decreased in human prostate tumor specimens. Taken together, our results suggest RGS2 as a novel regulator of AR signaling and its repression may be an important step during prostate tumorigenesis and progression.

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