Fig. 2. Phosphorylation-dependent AR degradation by Akt. (A) COS-1 cells were transfected with wtAR or mtAR in combination with vector or cAkt for 16 h in 10% CDS medium, treated with 10 nM DHT for 16 h, and then harvested for a western blot assay. (B) COS-1 cells were transfected with wtAR or mtAR in combination with pCDNA3, cAkt or dAkt in 10% CDS medium for 16 h and treated with ethanol or 10 nM DHT for another 16 h. Cells were fixed and stained with AR antibodies, followed by examination with a confocal microscope. The red and blue colors represent AR and nuclei staining, respectively. (C) COS-1 cells were transfected with wtAR or mtAR for 16 h in 10% CDS medium, treated with 20 µg/ml cycloheximide (CYH) in the presence of 10 nM DHT, and then harvested for a western blot assay at different times as indicated.

Fig. 4. Mdm2 forms a complex with Akt and AR in vivo and interacts with AR in vitro. (A) LNCaP cells were cultured in 10% FBS, harvested and lysed for the co-immunoprecipitation assay. (B) COS-1 cells were transfected with plasmids, as indicated, and harvested for co-immunoprecipitation. (C) GST–Mdm2 or GST–C464A was incubated with AR for 2 h and subjected to SDS–PAGE, followed by autoradiography. (D) GST–Mdm2 was incubated with wtAR or mtAR for 2 h (left panel). GST–Mdm2 was incubated with AR in the presence or absence of alkaline phosphatase (AP) for 2 h (right panel). (E) GST–Mdm2 was incubated with full-length AR or AR deletion mutants for 2 h. (F) GST–Mdm2 or GST–Mdm2 deletion mutants were incubated with AR for 2 h. (G) LNCaP cells were transfected with vector, Flag-AR-D (amino acids 483–651) or Flag-M3 (amino acids 341–491) for 36 h, followed by harvesting for the co-immunoprecipitation assay.

Fig. 6. Mdm2 is an E3 ligase for AR and induces AR ubiquitylation and degradation. (A) Mdm2-deficient MEFs were transfected with AR in combination with vector, Mdm2, C464A or S166A/S186A in 10% CDS medium for 16 h, treated with 10 nM DHT for 16 h and harvested for the ubiquityl ation assay. (B) COS-1 cells were transfected with AR in combination with vector or Mdm2 for 16 h, pulsed with [³⁵S]methionine for 45 min and harvested at different chase times as indicated in the presence of 10 nM DHT. (C) COS-1 cells were transfected with AR in combination with different amounts of Mdm2 for 16 h, treated with 10 nM DHT in the presence or absence of 5 µM MG132 for 8 h and harvested for a western blot assay. (D) Mdm2-null MEF cells were transfected with AR in combination with vector, Mdm2, C464A or S166A/S186A in 10% CDS medium for 16 h, treated with 10 nM DHT for 16 h and then harvested for a western blot assay. (E) COS-1 cells were transfected with wtAR or mtAR in combination with vector or Mdm2 and then harvested for a western blot assay.

Fig. 8. The model for AR degradation. IGF-1 or IL-6 activates PI3K, resulting in activation of Akt. Akt phosphorylates AR and Mdm2 and increases the binding between AR and Mdm2, leading to AR ubiquitylation by Mdm2. The 26S proteasome recognizes the polyubiquitylated AR, leading to AR degradation.

Fig. 1. Akt promotes AR degradation via a proteasome-dependent pathway. (A) COS-1 cells were transfected with plasmids as indicated for 16 h in 10% CDS medium, followed by treatment with ethanol or 10 nM DHT for another 16 h. The cells were harvested for a western blot assay. (B) COS-1 cells were transfected with AR along with various amounts of cAkt for 16 h in 10% CDS medium, treated with dimethylsulfoxide (DMSO) or 5 µM MG132 prior to 10 nM DHT treatment for 6 h, and then harvested for a western blot assay. (C) LNCaP cells were transfected with vector or cAkt for 16 h in 10% CDS medium, treated with 10 nM DHT for 16 h, and then harvested for a western blot assay. (D) LNCaP cells were treated with ethanol or 20 µM LY294002 15 min prior to 50 ng/ml IGF-1 treatment in 10% CDS medium for 16 h and then were harvested for a western blot assay.

Fig. 3. Akt and Mdm2 promote AR ubiquitylation dependent on AR phosphorylation. COS-1 cells were transfected with AR in combination with vector, cAkt or Mdm2 in the presence or absence of HA-Ub in 10% CDS medium for 16 h, followed by treatment with 10 nM DHT for 16 h. The cells were then harvested for ubiquitylation assay. (Ub)n-AR, polyubiquitylated AR.

Fig. 5. Mdm2 is involved in Akt-mediated AR ubiquitylation and degradation. (A) Mdm2-intact MEFs or Mdm2-deficient MEFs were transfected with AR in combination with cAkt in 10% CDS medium for 16 h, treated with 10 nM DHT for 16 h and then harvested for a western blot assay. (B) Mdm2-intact MEFs or Mdm2-null MEFs were tranfected with plasmids as indicated in 10% CDS medium for 16 h, and treated with 10 nM DHT for 16 h. The cells were then harvested for the ubiquitylation assay. (C) COS-1 cells were transfected with plasmids as indicated in 10% CDS medium for 16 h, treated with 10 nM DHT for 16 h and then harvested for a western blot assay.

Fig. 7. Mdm2-mediated AR degradation reduces AR transcriptional activity. (A) PC-3 prostate cancer cells were transfected with AR in combination with vector or Mdm2 in the presence or absence of cAkt in 10% CDS medium for 16 h, treated with ethanol or 10 nM DHT for 16 h and harvested for a luciferase assay. At least three independent experiments were performed and the data are presented as means ± SD. (B) LNCaP cells were transfected with vector or Mdm2 in the presence or absence of cAkt in 10% CDS medium for 16 h, treated with ethanol or 10 nM DHT for 24 h and then harvested for a northern blot assay. (C) PC-3 cells were transfected with AR in combination with plasmids as indicated in 10% CDS medium for 16 h, treated with ethanol or 10 nM DHT for 16 h and harvested for the luciferase assay. (D) PC-3(AR)6 cells were transfected with plasmids for 16 h as indicated and treated or not with 10 nM DHT. After 2 weeks of culture, the cells were stained with crystal violet, and colony formation was counted by microscope. (E) PC-3(AR)6 cells were transfected with plasmids for 16 h as indicated, treated with 10 nM DHT for 16 h and fixed for immunocytochemistry.