FIGURE 6.

PAK1 regulates PAK3 activity through heterodimerization. A, control of PAK expression. HA-tagged PAK3a-WT and PAK3b-WT plasmids were transfected in HeLa cells, and cell lysates were HA-immunoprecipitated (IP HA), then the amount of PAK3 proteins was determined using HA antibodies. B, control of co-immunoprecipitation of PAK3s proteins with PAK1. FLAG-PAK1-KD plasmid was transfected in HeLa cells alone or in the presence of HA-PAK3a-WT or HA-PAK3b-WT. Cell lysates were FLAG-immunoprecipitated (IP FLAG) then the amount of PAK1 proteins was determined using FLAG antibodies (second panel). The co-immunoprecipitated PAK3a-WT and PAK3b-WT proteins were revealed by HA blotting (WB HA, first panel). TCLs were controlled using the HA antibody (third panel). C, PAK3a displays an inducible kinase activity whereas PAK3b possesses a constitutive activity. Catalytic activity of HA-PAK3a-WT; HA-PAK3b-WT and FLAG-PAK1-KD previously obtained by immunoprecipitation was tested in the presence or in the absence of recombinant Cdc42-V12. D and E, catalytic activity of complexed or dissociated PAK3 proteins. HA-PAK3a-WT (D) or HA-PAK3b-WT (E) obtained by co- immunoprecipitation (B, first panel) was analyzed after incubation with or without recombinant Cdc42-V12 protein. Images shown are representative of three experiments