FIGURE 1.

PMA activates Rho and ROCK in LNCaP cells. A, LNCaP cells were treated with PMA (100 nm) for 1 h, and cell lysates were collected at different times. Rho-GTP levels were determined using a pull-down assay. B, effect of the PKC inhibitor GF 109203X (5 μm) on RhoA activation. The inhibitor was added 45 min before and during PMA or vehicle (ethanol) treatment. C (left), LNCaP cells were treated with PMA (100 nm) for different times, and MYPT1-Thr850 phosphorylation was determined in cell extracts by Western blot. A densitometric analysis is presented. Right, comparison of different prostate cancer cell lines. D, LNCaP cells were treated with PMA (100 nm), and at different times, ROCK activity was determined in immunoprecipitates. E, effect of the ROCK inhibitor Y-27632 (10 μm) on PMA-induced MYPT1-Thr⁸⁵⁰ phosphorylation in cell extracts. F, effect of the PKC inhibitor GF 109203X (5 μm) on PMA-induced MYPT1-Thr⁸⁵⁰ phosphorylation in cell extracts. For A–C, E, and F, densitometric analysis is presented in each case as mean ± S.D. (n = 3). G, LNCaP cells were treated with PMA (+PMA; 100 nm) or vehicle (−PMA) for 30 min and subjected to phalloidin staining. Cells were visualized by confocal microscopy. Experiments were carried out in the presence of GF 109203X (5 μm) or Y-27632 (10 μm). Similar results were observed in at least three experiments.