Glyceraldehyde-3-phosphate Dehydrogenase Enhances Transcriptional Activity of Androgen Receptor in Prostate Cancer Cells

Naoki Harada,Ryoko Yasunaga,Yasuki Higashimura,Ryoichi Yamaji,Katsumi Fujimoto,Joel Moss,Hiroshi Inui,Yoshihisa Nakano

doi:10.1074/jbc.m610724200

Abstract

Androgen receptor (AR) functions as a transcriptional factor for genes involved in proliferation and differentiation of normal and cancerous prostate cells. Coactivators that bind to AR are required for maximal androgen action. Here we report that increasing the expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in a prostate cancer cell line by as little as 1.8-fold enhances transcriptional activity of AR (but not the transcriptional activity of glucocorticoid receptor or estrogen receptor alpha) in a ligand-dependent manner and results in an increased expression of prostate-specific antigen. Small interference RNA-mediated knockdown of GAPDH significantly attenuated ligand-activated AR transactivation. Immunoprecipitation analysis revealed the presence of an endogenous protein complex containing GAPDH and AR in both the cytoplasm and nucleus. Addition of a nuclear localization signal (NLS) to GAPDH (GAPDH-NLS) completely abolished the ability of GAPDH to transactivate AR. Neither wild-type GAPDH nor GAPDH-NLS enhanced transcriptional activity of mutant AR (AR Delta C-Nuc) that is a constitutively active form of AR in the nucleus, even though GAPDH-NLS formed a complex with wild-type AR or AR Delta C-Nuc. AR transactivation was enhanced by a mutant GAPDH lacking dehydrogenase activity. GAPDH enhanced the transcriptional activity of AR(T875A) activated by an antagonist such as hydroxyflutamide or cyproterone acetate. These results indicate that GAPDH functions as a coactivator with high selectivity for AR and enhances AR transactivation independent of its glycolytic activity. Further, these data suggest that formation of a GAPDH.AR complex in the cytoplasm rather than nucleus is essential for GAPDH to enhance AR transactivation.

Highlights

Androgen receptor (AR) functions as a transcriptional factor for genes involved in proliferation and differentiation of normal and cancerous prostate cells
When similar experiments were performed in LNCaP cells or COS-7 cells, AR transactivation was enhanced by glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in both cell lines (Fig. 1C)
To determine whether GAPDH activates the expression of Prostate-specific antigen (PSA), GAPDH was overexpressed in LNCaP cells, and a cell lysate was subjected to Western blot analysis with anti-PSA antibodies

Summary

Introduction

Androgen receptor (AR) functions as a transcriptional factor for genes involved in proliferation and differentiation of normal and cancerous prostate cells. When the transcriptional activity of AR was determined at various concentrations of R1881 with PC-3 cells overexpressing GAPDH at the same level, the enhancing effect of GAPDH on AR transactivation was observed even in the presence of 0.05 nM R1881 (Fig. 1B).

Results

Conclusion