Regulation Of The Transcriptional Activation Of The Androgen Receptor By The UXT Binding Protein VHL

VHL disease is a rare autosomal dominant genetic disease with an incidence of1/36,000live births and the average age is26.3-30.9years. VHL is a tumor suppressor; loss and/or inactivation of VHL cause various tumors. VHL forms a CRL ubiquitin E3ligase complex together with Cul-2, Rbx1, Elongin B and Elongin C, which can promote the ubiquitin-mediated proteosome degradation of target substrate proteins such as HIFla. The loss or mutation of VHL induces tumorigenesis, because HIF1? can not be degraded, instead, aggregates in the cytoplasm and then translocated into the nucleus and forms hetero-dimers with HIF1? to bind several promoters of downstream genes, including GLUT-1, PDGF, CA-IX, VEGF and TGFa.As a subunit of E3ubiquitin ligase, VHL promotes ubiquitination and degradation of several proteins including HIF1a, SUMO-conjugated HIF2a, hsRPB7, ?-catenin, Dv12, STRA13and ER-a. VHL is widely expressed and plays key roles in tumourigenesis, and although these targets of VHL and their regulation have been characterised, other targets or partners of VHL remain to be identified.This study focuses on identification of new targets of VHL by an improved yeast two hybrid system. Main results are as follows:1. An improved yeast two hybrid approachThe systematic procedure of yeast two hybrid were improved, including cDNA library amplification, the processes of yeast transformation and screening. The yeast two hybrid system can overcome several drawbacks of traditional yeast two hybrid method. This approach can be used for identification of low abundance protein-protein interaction and obtain a high proportion of positive colonies.2. Interacted proteins with VHL by screening with the improved yeast two hybrid systemWe have identified25candidate prey proteins using the mouse VHL as a "bait" to screen in the human embryo brain cDNA library by the improved yeast two hybrid system. Through re transformation verification experiment, four preys showed interaction with VHL:Dv12, UXT, PFDN5and RANBP9.3. VHL promoted the K48-linked ubiqutination of Dvl2 Our research showed that VHL can promote K48-linked polyubiquitin chain to bind with the K301site in Dv12and resulted in the ubiquitination-dependent proteosome degradation of Dv12.4. The interaction between VHL and UXT can regulate the transcriptional activation of androgen receptor (AR)Another VHL interacting protein UXT that we identified in the yeast two hybrid system is an AR co-activator. GST pull-down assays and co-immunoprecipitation experiments as well as the separation of cell fractions showed that UXT and VHL could interact with each other in the nucleus. In addition, domain mapping and ubiquitination analysis showed that VHL associated with the DBD and Hinge domains of AR and induced AR ubiquitination mainly by binding with an atypical ubiquitin-linked chain. Moreover, the interaction of VHL and AR activated AR transactivation upon DHT treatment. Knockdown of VHL by RNAi impeded AR ubiquitination and decreased its transcriptional activation. Our results suggested that VHL-UXT interaction and VHL-mediated AR ubiquitination could regulate the transcriptional activation of AR.