Figure 1
Activated Rac inhibits thrombin signaling to Rho. (A) PAE cells transfected with pCEFL-GFP as a control or with the activated RacV12 mutant and pCEFL-GFP together (RacV12) were starved overnight on poly-D-lysine coated glass cover-slips, treated with thrombin 5 U/ml for indicated times, fixed, and stained with TRITC-phalloidin. Arrows point to transfected cells as judged by co-transfection with pCEFL-GFP. (B) HEK293T cells were transfected with pCEFL-GFP as a control or with the activated RacV12 mutant and pCEFL-GFP together with RacV12 (RacV12), cultured overnight, starved for 4 h, and treated with thrombin for indicated times. 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.
Figure 2
Activated Rac requires Pak1 binding for inhibition of thrombin signaling to Rho. (A) PAE cells transfected with pCEFL-GFP as a control or with RacV12, RacV12C40, RacV12Δins mutants together with pCEFL-GFP as indicated were starved overnight on poly-D-lysine coated glass cover-slips, placed in serum-free media (control) or treated with thrombin (5 U/ml) 30 min, fixed and stained with TRITC-labelled phalloidin. Transfected cells were detected by GFP flourescence. (B) HEK293T cells were transfected with pCEFL-GFP as a control or with the RacV12 mutants shown in (A) together with pCEFL-GFP, cultured overnight, starved 4 h, treated with thrombin (5 U/ml) for 1 min, and lysed. 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.
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.
Figure 4
Pak1 binds the DH-PH domain of P115RhoGEF. (A) Constructs used in these experiments. (B) HEK293T cells were transfected with expression plasmids encoding the AU1-tagged constructs shown in (A) 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-AU1 antibody. (C) HEK293T cells were transfected with expression plasmids encoding AU1-tagged DH-PH domains of P115RhoGEF (P115), PDZ-RhoGEF (PRG), and LARG (LARG) in the presence or absence of wild-type myc tagged Pak1 and/or the activated Rac mutant RacQL (Rac). The P115RhoGEF ΔN construct is included as a control. Lysates were immunoprecipitated with anti-myc antibody, and immunoprecipitates tested for GEF content by western blotting with anti-AU1 antibody.
Figure 5
Pak1 inhibitory domain (PID) reverts Rac inhibition of Rho by Rac. (A) HEK293T cells were transfected with plasmids expressing a GFP or GFP-PID fusion protein (PID) (1 μg/plate) and incubated for two days, serum-starved 4 h, and treated with thrombin (5 U/ml) 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. GFP western blot from total cell lysate was used to detect the GFP-PID fusion protein. (A) Cells were co-transfected with plasmids encoding the active RacV12 (Rac) mutant in the presence of control pCEFL-GFP or pCEFL-GFP-PID (2 μg/plate). After 2 days incubation cells were serum-starved 4 h, stimulated with thrombin (5 U/ml, 2 min), and assayed for Rho-GTP levels with pulldown assays. Data are expressed as fold increase ± SEM in Rho-GTP with respect to unstimulated control cells.