Fig. 5. PAK4 kinase activity is elevated by HGF. (A) MDCK cells were transfected with HA-PAK4wt and serum-starved for 4 hours prior to stimulation with HGF for the indicated times. PAK4 was immunoprecipitated from cell lysates with anti-HA mAb and its kinase activity assayed in the presence of Histone H1 and [-³²P]ATP. Phosphorylation of Histone is indicated. As a control (C) in each experiment (A-D), a kinase assay was also performed on protein G-Sepharose beads coupled to anti-HA mAb, which had been incubated with untransfected cell lysate. Autoradiographs and western blots were quantified using Kinetic Imaging Software and the level of kinase activity normalised to protein expression levels. (B,C) MDCK cells were transfected with HA-PAK4GBD or HA-PAK4wt and serum-starved for 4 hours prior to stimulation with HGF for 15 minutes. LY294002 was added to cultures after 3.5 hours of serum starvation. PAK4 was immunoprecipitated from cell lysates with anti-HA mAb and its kinase activity assayed in the presence of Histone H1 and [-³²P]ATP. Autoradiographs and western blots were quantified as above. Time in the presence of HGF in minutes is indicated; LY, LY294002. The results shown are mean±s.e.m. of three independent experiments. (D) MDCK cells were transfected with HA-PAK4wt and HA-PAK4M350 and starved for 4 hours. PAK4 proteins were immunoprecipitated from cell lysates with anti-HA mAb and their kinase activity assayed in the presence of Histone H1 and [-³²P]ATP.