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

Liliana A Ponguta,Christopher W Gregory,Frank S French,Elizabeth M Wilson

doi:10.1074/jbc.m802392200

Abstract

The androgen receptor (AR) is required for prostate cancer development and contributes to tumor progression after remission in response to androgen deprivation therapy. Epidermal growth factor (EGF) increases AR transcriptional activity at low levels of androgen in the CWR-R1 prostate cancer cell line derived from the castration-recurrent CWR22 prostate cancer xenograft. Here we report that knockdown of AR decreases EGF stimulation of prostate cancer cell growth and demonstrate a mechanistic link between EGF and AR signaling. The EGF-induced increase in AR transcriptional activity is dependent on phosphorylation at mitogen-activated protein kinase consensus site Ser-515 in the AR NH(2)-terminal region and at protein kinase C consensus site Ser-578 in the AR DNA binding domain. Phosphorylation at these sites alters the nuclear-cytoplasmic shuttling of AR and AR interaction with the Ku-70/80 regulatory subunits of DNA-dependent protein kinase. Abolishing AR Ser-578 phosphorylation by introducing an S578A mutation eliminates the AR transcriptional response to EGF and increases both AR binding of Ku-70/80 and nuclear retention of AR in association with hyperphosphorylation of AR Ser-515. The results support a model in which AR transcriptional activity increases castration-recurrent prostate cancer cell growth in response to EGF by site-specific serine phosphorylation that regulates nuclear-cytoplasmic shuttling through interactions with the Ku-70/80 regulatory complex.

Highlights

androgen receptor (AR) has a modular structure characteristic of steroid hormone receptors, with an NH2-terminal transcriptional activation domain, DNA binding domain, hinge region, and carboxyl-terminal ligand binding domain [1, 2]
AR-dependent CWR-R1 Prostate Cancer Cell Proliferation— We investigated the requirement for AR in castration-recurrent prostate cancer cell growth using the CWR-R1 cell line that was derived from the castration-recurrent CWR22 xenograft of human prostate cancer [6]
The reduction in AR levels determined by immunoblot of EGFtreated CWR-R1 cells 2 and 5 days after AR-siRNA-Self-complementary Adeno-associated Virus (scAAV) infection (Fig. 2A, right panels) provided evidence that Epidermal growth factor (EGF) stimulation of CWR-R1 cell growth was mediated in part by AR

Summary

Introduction

AR has a modular structure characteristic of steroid hormone receptors, with an NH2-terminal transcriptional activation domain, DNA binding domain, hinge region, and carboxyl-terminal ligand binding domain [1, 2]. Androgen-independent AR Transactivation—To pursue evidence that AR functions in castration-recurrent prostate cancer in the absence of androgen, we tested whether EGF can increase endogenous AR transcriptional activity in CWR-R1 cells using an MMTV-Luc reporter vector in the absence and presence of DHT.

Results

Conclusion