Figure 5.

Src acts downstream of RhoA to mediate Y87-phosphorylation of PKD2. (A) HeLa cells (0.65 × 10⁶ cells/6 cm plate) were co-transfected with FLAG-PKD2 and vector or constitutively-active RhoA (RhoA.CA). Cells were stimulated with PP2 (2.5 μM, 16 hours) or DMSO, as indicated. PKD2 was immunoprecipitated (anti-FLAG) and samples were analyzed for Y87 phosphorylation using the anti-pY87-PKD2 antibody. Immunoprecipitates were re-probed for total PKD2 and cell lysates were evaluated by Western blot for RhoA.CA (anti-GST) expression. Uncropped blots are shown in Supplemental Figure S9. (B) HeLa cells, transfected with vector control or GST-tagged constitutively-active RhoA (RhoA.CA), were seeded on coverslips (70,000 cells/slip). 24 hours later, cells were analyzed by immunofluorescence for phosphorylation at Y87 (anti-pY95/87 and Alexa Flour 546). (C) HeLa cells, transfected with GST-tagged constitutively-active RhoA (RhoA.CA), were seeded on coverslips (70,000 cells/slip). 24 hours later they were analyzed by immunofluorescence for phosphorylation at Y87 (anti-pY95/87 labeled with Zenon Alexa Flour 488), for FAK (anti-FAK labeled with Zenon Alexa Flour 647) and for GST (anti-GST labeled with Zenon Alexa Flour 405). F-actin filaments were visualized using rhodamine-Phalloidin. (D) HeLa cells (6000/ channel) were seeded in ibidi channel μ-slides with 2 μg/ml Rho Inhibitor I, as indicated. After 16 hours, cells were subjected to immunofluorescence analysis to determine the localization of endogenous pY87-PKD2 (secondary antibody used: Alexa Fluor 488) and F-actin (phalloidin). Scale bar indicates 10 μm. (E) Shows quantitation for the number of pY87-PKD2-positive adhesions per cell with control (n = 65 cells) and Rho Inhibitor l-treated cells (n = 65 cells).Error bars represent standard error of the mean (SEM). *Indicates statistical significance (p < 0.05) as compared to control cells. All experiments have been performed at least three times with similar results.