AR is a direct Akt target. (A) Akt-consensus phosphorylation sites (Ser-210 and Ser-790) of AR are responsible for AR phosphorylation. wtAR, mtAR S210A, or mtAR S790A was transfected into DU145. After transfection, whole-cell extract was immunoprecipitated with the anti-AR antibody, NH27. Half of the precipitated complex was treated with Akt and [γ-³²P]ATP for 2 h and analyzed by SDS/PAGE. To verify the equal expression levels of the wtAR and mtAR constructs, the remaining immunoprecipitates were subjected to Western blot analysis as shown (Lower). (B) Akt interacts with AR in LNCaP cells in vivo. LNCaP cell lysates were immunoprecipitated (IP) with anti-Akt or normal IgG (N-IgG). The immunoprecipitated complexes were immunoblotted (IB) with AR antibody (NH27) or anti-Akt antibody, respectively. (C) In vitro phosphorylation of AR by Akt, but not by PI(3)K. One microgram of N-DBD AR or 1 μg of DBD-LBD AR purified from E. coli was treated for 1 h with Akt or PI(3)K. Phosphorylation of the AR was detected by separation on 12.5% SDS/PAGE and autoradiography. The Akt and PI(3)K used in this experiment are active, as determined by phosphorylating H2B and PI, respectively, which is shown (C, Upper).

Phosphorylation of AR by Akt in vivo. (A) Activation of Akt by IGF-1. COS-1 cells were pretreated with ethanol or 20 μM LY294002 for 30 min, followed by treatment with 100 ng/ml IGF-1 for 30 min. The total cell lysates were immunoprecipitated by anti-Akt antibody (New England Biolabs). The immunocomplex was subjected to SDS/PAGE, followed by immunoblot with phospho-Akt (S473) antibody or Akt antibody. (B) IGF-1 phosphorylates AR in vivo via Akt. COS-1 cells were cultured in [³²P]PO₄-containing medium. AR immunocomplex was subjected to SDS/PAGE followed by autoradiography. Immunoblotting confirmed equivalent amounts of AR in immunocomplex. (C) cAkt, but not dAkt, phosphorylates wtAR, but not mtAR S210A and mtAR S790A, in vivo. COS-1 cells were transfected with wtAR, mtAR S210A, or mtAR S790A in combination with PCDNA3 vector, cAkt, or dAkt. After 24 h of transfection, cells were labeled with [³²P]PO₄ for 4 h. (D) Suppression of AR transactivation by Akt in a dose-dependent manner. The DU145 cells were transfected with various plasmids, as indicated, for 24 h, followed by DHT treatment for another 24 h. Transactivation was measured by CAT activity by using mouse mammary tumor virus–CAT as a reporter. (E) mtAR S210A is resistant to Akt suppressive effect on AR transactivation. DU145 cells were transfected with plasmids encoding wtAR, mtAR S210A, or mtAR S790A in presence of cAkt or dAkt for 24 h. Ligand treatment and AR transactivation were performed as previously described. The data are means ± SD from three independent experiments.

Inhibition of AR transactivation by PI(3)K/Akt pathway. (A) AR transactivation is enhanced through the inhibition of PI(3)K activity by Δp85 and LY294002. The DU145 cells were treated with LY294002 for 30 min before DHT treatment; the transactivation activity was determined after 24 h of transfection. (B) Suppression of AR transactivation by p110* in a dose-dependent manner. (C) Suppression of AR transactivation by PI(3)K via Akt but not via p70S6K. The DU145 cells were transfected with plasmids, as indicated, for 16 h. Cells were treated with 20 nM rapamycin or 20 μM LY294002 for 30 min before 1 nM DHT treatment. The data are means ± SD from three independent experiments.

Effect of PI(3)K/Akt pathway on the interaction between AR and ARA70. (A) Modulation of interaction between AR and ARA70 by cAkt, dAkt, or LY294002. The DU145 cells were transfected with 2.5 μg of GAL4-ARA70 and 2.5 μg of VP16-AR, followed by treatment with LY294002 or vehicle 30 min before DHT treatment. The interaction between AR and ARA70 was determined by CAT assay by using pG5-CAT as a reporter. (B) The enhanced AR transactivation by various AR coactivators, ARA70, ARA54, TIF2, and SRC-1, could be further promoted in the presence of LY294002 or Δp85. The data are means ± SD from three independent experiments.

PI(3)K/Akt pathway suppressed androgen/AR-induced apoptosis. (A) PC-3(AR)2 and PC-3(AR)6 expressed AR protein. PC-3 cells were stably transfected with AR, followed by selection with hygromycin B, and confirmed by Western blotting by using AR antibody NH27, whereas LNCaP was used as a positive control. (B) Androgen/AR-induced apoptosis in PC-3(AR)2, PC-3(AR)6, and SAR-91 were inhibited by PI(3)K/Akt pathway. SAR-91, S7MC, PC-3(AR)2, and PC-3(AR)6 were treated with LY294002 (20 μM) or HF (5 μM) for 30 min, followed by addition of IGF-1 (100 ng/ml) for another 30 min before DHT treatment. After 3 days, cell apoptosis was analyzed by TUNEL assay. The data are means ± SD from three independent experiments.

Androgen/AR-induced apoptosis and p21 expression were inhibited by Akt. (A) Akt suppressed androgen/AR-induced p21 promoter activity. PC-3 cells were transfected with different plasmids, as indicated, for 16 h, followed by DHT treatment for another 16 h. p21 promoter activity was determined by luciferase activity. (B) Androgen/AR-induced PC-3(AR)6 apoptosis and p21 protein expression was blocked by Akt. PC-3(AR)6 was transfected with pCDNA3, cAkt, or dAkt, as indicated, for 16 h. The cells then were treated with DHT for 3 days; cell apoptosis was then determined by TUNEL assay. p21 protein expression was detected by Western blotting by using p21 monoclonal antibody. (C) AR-induced p21 protein expression was enhanced by dAkt in LNCaP cells. LNCaP stable clones (pCDNA3 and dAkt) were treated with 10 nM DHT for 2 days; the p21 protein expression then was detected. LNCaP stable transfection with dAkt was confirmed by Western blot assay by using phospho-Akt (Ser-473) antibody. (D) DHT and PMA synergistically induced p21 expression and apoptosis in LNCaP cells. LNCaP cells were pretreated with HF or vehicle for 30 min followed by treatment with DHT for 24 h. PMA then was added for another 24 h, and cell apoptosis was determined by TUNEL assay. (E) Activation of PI(3)K/Akt pathway by IGF-1 suppresses DHT/PMA-induced apoptosis. LNCaP stable clones (pCDNA3 and dAkt) were treated with 10 nM DHT for 24 h followed by treatment with 20 μM LY294002 for 30 min. IGF-1 was added for another 30 min, followed by 10 nM PMA treatment for another 24 h. The apoptosis was determined by TUNEL assay. The data are means ± SD from three independent experiments.