FIGURE 7.

PKC-mediated phosphorylation at AR Ser-578 and EGF-dependent CWR-R1 cell growth. A, in vitro kinase assays were performed using GST-AR-(1–660) (2.5 μg, lane 1) and GST-AR-(1–660)-S578A (2.5 μg, lane 2) expressed in E. coli and purified by adsorption to glutathione beads. Histone H1 served as a PKC substrate control (2.5 μg, lane 3). Assays were performed as described under “Experimental Procedures” using the PKC catalytic subunit in the presence of 10 μCi of [γ-³²P]adenosine triphosphate (upper panel). Parallel immunoblots were probed with AR52 antibody (lower panel). Samples were analyzed by autoradiography and band intensities measured by densitometry. NS designates a nonspecific phosphorylated band. B, data from four independent experiments described in A were averaged. C, inhibition of CWR-R1 cell proliferation by calphostin. CWR-R1 cells were plated and serum-starved the next day for 24 h and treated as described under “Experimental Procedures” in serum-free media with and without 10 ng/ml EGF alone or with 50 nm calphostin, a PKC inhibitor (day 0). Media and additives were replenished every other day over 7 days. Cell proliferation indexes were measured using WST-8 reagent on days 3, 5, and 7 after seeding.