Figure 3.

hPAK65 activation by caspase cleavage. (A) hPAK65 constitutively activated by N-terminal truncation. Various PAK constructs as indicated were expressed in COS-7 cells, immunoprecipitated with anti-myc antibody, and incubated with control buffer (lanes 1 and 2) or recombinant caspase-3 (lanes 3 and 4) at 37°C for 30 min. Kinase assays were performed using histone H4 as substrate. Equal amounts of protein in each reaction were confirmed by immunoblotting by using anti-myc antibody (data not shown). (B) A 36-kDa kinase induced during Fas-induced apoptosis. The same Jurkat cell extracts prepared as in Fig. 1 were subjected to an in-gel kinase assay. The induced 36-kDa kinase is indicated. (C) Endogenous hPAK65 kinase activity induced during Fas-induced apoptosis. The Jurkat cell extracts as in Fig. 1 were immunoprecipitated with hPAK65-C antibody, followed by in vitro kinase reactions in kinase buffer containing substrate histone H4. The reaction mixtures were then separated by SDS/PAGE.