CLEAVAGE AND ACTIVATION OF PAL-ACTIVATED PROTEIN-KINASE GAMMA-PAK BY CPP32 (CASPASE-3) - EFFECTS OF AUTOPHOSPHORYLATION ON ACTIVITY

p21-activated protein kinase gamma-PAK (Pak2, PAK I) is cleaved by CPP 32 (caspase 3) during apoptosis and plays a key role in regulation of cell death, In vitro, CPP32 cleaves recombinant gamma-PAK into two pep tides; 1-212 contains the majority of the regulatory domain whereas 21 3-524 contains 34 amino acids of the regulatory domain plus the entire catalytic domain. Following cleavage, both peptides become autophosph orylated with [gamma-P-32]ATP. Peptide 1-212 migrates at 27,000 dalton s (p27) upon SDS-polyacrylamide gel electrophoresis and at 32,000 dalt ons following autophosphorylation on serine (p27P); the catalytic subu nit migrates at 34,000 daltons (p34) before and after autophosphorylat ion on threonine, Following caspase cleavage, a significant lag (simil ar to 5 min) is observed before autophosphorylation and activity are d etected. When gamma-PAK is autophosphorylated with ATP(Mg) alone and t hen cleaved, only p27 contains phosphate, and the enzyme is inactive w ith exogenous substrate. After autophosphorylation of gamma-PAK in the presence of Cdc42(GTP gamma S) or histone 4, both cleavage products c ontain phosphate and gamma-PAK is catalytically active. Mutation of th e conserved Thr-402 to alanine greatly reduces autophosphorylation and protein kinase activity following cleavage. Thus activation of gamma- PAK via cleavage by CPP32 is a two-step mechanism wherein autophosphor ylation of the regulatory domain is a priming step, and activation coi ncides with autophosphorylation of the catalytic domain.