Cleavage and activation of p21-activated protein kinase gamma-PAK by CPP32 (caspase 3). Effects of autophosphorylation on activity |
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Authors: | BN Walter Z Huang R Jakobi PT Tuazon ES Alnemri G Litwack JA Traugh |
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Affiliation: | Department of Biochemistry, University of California, Riverside, Riverside, California 92521, USA. |
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Abstract: | p21-activated protein kinase gamma-PAK (Pak2, PAK I) is cleaved by CPP32 (caspase 3) during apoptosis and plays a key role in regulation of cell death. In vitro, CPP32 cleaves recombinant gamma-PAK into two peptides; 1-212 contains the majority of the regulatory domain whereas 213-524 contains 34 amino acids of the regulatory domain plus the entire catalytic domain. Following cleavage, both peptides become autophosphorylated with gamma-32P]ATP. Peptide 1-212 migrates at 27,000 daltons (p27) upon SDS-polyacrylamide gel electrophoresis and at 32,000 daltons following autophosphorylation on serine (p27P); the catalytic subunit migrates at 34,000 daltons (p34) before and after autophosphorylation on threonine. Following caspase cleavage, a significant lag (approximately 5 min) is observed before autophosphorylation and activity are detected. When gamma-PAK is autophosphorylated with ATP(Mg) alone and then cleaved, only p27 contains phosphate, and the enzyme is inactive with exogenous substrate. After autophosphorylation of gamma-PAK in the presence of Cdc42(GTPgammaS) or histone 4, both cleavage products contain phosphate and gamma-PAK is catalytically active. Mutation of the 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 autophosphorylation of the regulatory domain is a priming step, and activation coincides with autophosphorylation of the catalytic domain. |
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