Figure 3

Pak1 binds and phosphorylates P115RhoGEF. (A) HEK293T cells were transfected with expression plasmids encoding GFP, GFP-PDZ-RhoGEF (PRG), GFP-P115RhoGEF (P115), or GFP-LARG (LARG) in the presence or absence of wild-type myc tagged Pak1 and/or the activated Rac mutant RacQL (Rac). Lysates were immunoprecipitated with anti-myc antibody, and immunoprecipitates tested for GEF content by western blotting with anti-GFP antibody. (B) HEK293T cells were transfected with expression plasmids encoding GFP, GFP-GFP-P115RhoGEF (P115) in the presence or absence of wild-type myc tagged Pak1 and/or the activated Rac mutant RacV12 (Rac). Lysates were incubated with anti-AU1 antibody, and immunoprecipitates tested for Pak1 content by western blotting with anti-Pak1 antibody. (C) HEK293T cells were transfected with AU1-tagged full length P115RhoGEF. Two days post-transfection, cells were harvested in lysis buffer. Lysates were incubated with anti-AU1 antibody and immunoprecipitates were placed in kinase buffer containing recombinant Pak1 and γ-³²P-ATP for times indicated. Kinase reactions were stopped with the addition of sample buffer and boiling. Samples were resolved by SDS-PAGE and acrylamide gels were transferred to PVDF membranes. Resulting blots were exposed to X-ray film 24 h to detect phosphorylated proteins. Total immunoprecipitated P115RhoGEF levels were assessed by coomassie staining. (D) HEK293T cells were transfected with control or P115RhoGEF-specific siRNA oligonucleotides, and treated with thrombin for times indicated. Fresh lysates were used for Rho-pulldown assays with GST-Rhotekin to detect relative amounts of Rho-GTP. Total Rho levels were detected by western blot in total cell lysates as a loading control.