Walter B N, Huang Z, Jakobi R, Tuazon P T, Alnemri E S, Litwack G, Traugh J A
Department of Biochemistry, University of California, Riverside, Riverside, California 92521, USA.
J Biol Chem. 1998 Oct 30;273(44):28733-9. doi: 10.1074/jbc.273.44.28733.
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.
p21激活蛋白激酶γ-PAK（Pak2，PAK I）在细胞凋亡过程中被CPP32（半胱天冬酶3）切割，并在细胞死亡调控中起关键作用。在体外，CPP32将重组γ-PAK切割成两个肽段；1-212包含大部分调节结构域，而213-524包含34个氨基酸的调节结构域加上整个催化结构域。切割后，两个肽段都用[γ-32P]ATP进行自磷酸化。肽段1-212在SDS-聚丙烯酰胺凝胶电泳上迁移至27,000道尔顿（p27），在丝氨酸上自磷酸化后迁移至32,000道尔顿（p27P）；催化亚基在苏氨酸自磷酸化前后均迁移至34,000道尔顿（p34）。在半胱天冬酶切割后，在检测到自磷酸化和活性之前观察到明显的延迟（约5分钟）。当γ-PAK仅用ATP（Mg）进行自磷酸化然后切割时，只有p27含有磷酸，并且该酶对外源底物无活性。在Cdc42（GTPγS）或组蛋白4存在下γ-PAK自磷酸化后，两个切割产物都含有磷酸，并且γ-PAK具有催化活性。将保守的苏氨酸-402突变为丙氨酸会大大降低切割后的自磷酸化和蛋白激酶活性。因此，通过CPP32切割激活γ-PAK是一种两步机制，其中调节结构域的自磷酸化是引发步骤，激活与催化结构域的自磷酸化同时发生。
