FIGURE 3.

PAK6-mediated AR phosphorylation promotes its ubiquitin-mediated degradation. A, PAK6 reduced AR levels under DHT stimulation. HEK293 cells were transfected with pcDNA3.1-Myc-His-PAK6 and pcDNA 3.1-His-AR and treated with 10 nm DHT and 10 μm cycloheximide at the indicated times. PAK6 and AR protein levels were measured by Western blot analysis as indicated. B, PAK6 reduced wild-type AR under DHT stimulation, while AR S578A mutant was unaffected. HEK293 cells were co-transfected with pcDNA-FLAG-AR wild-type/S578A (505–919 amino acids) (including DNA binding domain and ligand binding domain), and pcDNA3.1-Myc-His-PAK6 was treated with 10 nm DHT and 10 μm cycloheximide. C, ubiquitination assay showed that PAK6 markedly enhanced AR WT but not AR S578A ubiquitination. HEK293 cells were transfected with Myc-ubiquitin and pcDNA-FLAG-AR WT/S578A (505–919 amino acids) in combination with pcDNA3.1-Myc-His PAK6/vector-treated or untreated with 5 μm MG132 for 6 h in the presence of 10 nm DHT. 30 μg of total lysates were set aside as input; equal amounts lysates of protein were then harvested for ubiquitination assay. IP, immunoprecipitation; IB, immunoblot. D, stable knockdown of PAK6 by shRNA in CWR22Rv1 cells showed impairment of the endogenous ubiquitination of AR when treated with MG132 and DHT (7th lane from left). CWR22Rv1 cells infected with lentiviruses harboring shRNA control/PAK6 were treated with or without 5 μm MG132 for 6 h in the presence/absence of 10 nm DHT. Equal amounts lysates of protein were then harvested for ubiquitination assay.