FIGURE 4.

EGF-dependent phosphorylation at AR Ser-515. A, schematic representation of 15 predicted phosphorylation sites in part of the AR NH₂-terminal region (N-term), DNA binding domain (DBD), and hinge region that were mutated in FLAG-AR-(507–660) and tested for band shift on immunoblots. B, COS cells were transfected with 2 μg of FLAG-AR-(507–660), serum-depleted for 24 h, and treated for 5 h with and without 10 ng/ml EGF as indicated. Cells were harvested, and lysates were incubated with and without 2.5 units of λ-phosphatase for 30 min at 30 °C in the absence (lanes 1–4) and presence of phosphatase inhibitors (lanes 5–8). C, COS cells (upper panel) and CWR-R1 cells (lower panel) were transfected with 2 μg of wild-type (wt) FLAG-AR-(507–660) or the S515A and S578A mutants. Cells were serum-depleted for 24 h, treated with and without 10 ng/ml EGF for 5 h, collected, and lysed in the presence of phosphatase inhibitors for immunoprecipitation using FLAG affinity resin. AR52 antibody was used to detect wt and mutant FLAG-AR-(507–660) and an associated slower migrating band indicative of phosphorylation. Also indicated is the nonspecific IgG band. D, reduced AR Ser-515 phosphorylation by mitogen-activated protein kinase inhibitor, U0126. COS cells were treated in the absence (lanes 1–2) and presence of increasing concentrations of U0126 (lanes 3–8) for 1 h before transfection with 2 μg of FLAG-AR-(507–660). The next day cells were serum-starved for 24 h in the absence and presence of U0126. Cells were treated again for 5 h with and without U0126 in the absence (lanes 1, 3, 5, and 7) and presence of 10 ng/ml EGF (lanes 2, 4, 6, and 8). FLAG-AR-(507–660) was detected using AR52 antibody, and β-actin served as the loading control.