Bi-directional regulation between RNF6 and AKT: Potential mediators of prostate cancer progression

Abstract

Currently androgen deprivation therapy (ADT) is the initial treatment for men with metastatic prostate cancer. Despite the initial response to ADT, nearly all men develop advanced castrate-resistant prostate cancer (CRPC). Due to the lack of effective therapy for CRPC patients, the biology of such tumors has been under intense investigation. Previously, our laboratory demonstrated that protein expression of ring finger protein 6 (RNF6) ubiquitin ligase is increased in advanced human prostate tumors and that RNF6 was required for the growth of CRPC. The purpose of this study was to identify mechanisms by which RNF6 promote growth of CRPC cells. Using mass spectrometry, several RNF6 associated proteins were identified, one of which was AKT kinase (also called protein kinase B). A growing body of data suggests that AKT kinase promotes development and progression of CRPC. This study identifies novel roles the RNF6 ubiquitin ligase and AKT kinase in the growth of CRPC through mechanisms involving cross-regulation between the two proteins. We demonstrate that RNF6 is required for basal and growth factor mediated AKT kinase activity. RNF6 induces AKT polyubiquitination at lysine 183 within the kinase domain and this ubiquitination is required for the kinase activity. Additionally AKT ubiquitination at lysine 183 is required for proper nuclear/cytosol distribution and disruption of association with PH domain and leucine rich repeat protein phosphatase (PHLPP), thereby maintaining AKT phosphorylation required for the kinase activity. Functional assays reveal that RNF6 induced AKT ubiquitination at lysine 183 is required for prostate cancer cells growth under androgen depleted conditions. Mass spectrometry analysis also reveals that AKT phosphorylates RNF6 at serine 219. AKT induced RNF6 phosphorylation at serine 219 is required for RNF6 association with the androgen receptor (AR) and regulation of its activity. This is a novel mechanism of AR regulation by AKT through an effector protein, RNF6. In conclusion, RNF6 regulates AKT kinase activity through ubiquitination. On the other hand, AKT mediated RNF6 phosphorylation is required for RNF6 induced upregulation of AR activity. These are novel roles of AKT and RNF6 in driving castrate resistant growth of prostate cancer cells.