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J Biol Chem, Vol. 273, Issue 44, 28733-28739, October 30, 1998

Cleavage and Activation of p21-activated Protein Kinase -PAK by CPP32 (Caspase 3)
EFFECTS OF AUTOPHOSPHORYLATION ON ACTIVITY

Barbara N. Walter, Zhongdong Huang, Rolf Jakobi, Polygena T. Tuazon, Emad S. Alnemri^¶, Gerald Litwack, and Jolinda A. Traugh

From the  Department of Biochemistry, University of California, Riverside, Riverside, California 92521, and the ^¶ Department of Microbiology and Immunology and the  Department of Biochemistry and Molecular Pharmacology, Thomas Jefferson University, Philadelphia, Pennsylvania 19107

p21-activated protein kinase -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 -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 [-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 (~5 min) is observed before autophosphorylation and activity are detected. When -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 -PAK in the presence of Cdc42(GTPS) or histone 4, both cleavage products contain phosphate and -PAK is catalytically active. Mutation of the conserved Thr-402 to alanine greatly reduces autophosphorylation and protein kinase activity following cleavage. Thus activation of -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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Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.

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