FIGURE 6.

AR Ser-578 required for EGF-induced AR transactivation in CWR-R1 and Ishikawa cells. A, CWR-R1 cells were transiently transfected with 0.1 μg of PSA-Enh-Luc and 10 ng wt pCMV-AR and the S578A, S515A, S515A-S578A, S650A, and S578A-S650A mutants and incubated for 24 h with and without 0.1 nm DHT and 10 ng/ml EGF as indicated, and luciferase activity was determined (upper panel). COS cells were transfected using DEAE dextran as described under “Experimental Procedures” with 2 μg of pCMV5 empty vector (-), wild-type pCMV-AR (wt), and the S578A and S515A mutants (lower panel). COS cells were incubated in the absence and presence of 10 nm DHT and 10 ng/ml EGF as indicated, and immunoblots were performed to confirm similar expression levels. B, CWR-R1 cells were transfected with 0.1 μg of PSA-Enh-Luc and 50 ng of pCMV-AR-(1–660) wt and S578A mutant and incubated for 24 h with and without 10 ng/ml EGF, and luciferase activity was determined (upper panel). Similar expression levels were determined by transfecting COS cells with 2 μg of wt pCMV-AR-(1–660) and the S578A mutant and incubating cells for 24 h in the absence and presence of 10 ng/ml EGF (lower panel). C, Ishikawa cells were transfected with 0.1 μg of PSA-Enh-Luc and 25 ng of wild-type pCMV-AR (wt) or S578A mutant and incubated for 24 h with increasing concentrations of DHT with and without 10 ng/ml EGF as indicated, and luciferase activity was determined.