The p21-activated protein kinase gamma-PAK is activated under conditions of hyperosmotic stress, by low levels of ionizing radiation, by DNA-damaging drugs, and by sphingosine. In addition, gamma-PAK is constitutively activated in early apoptosis via cleavage into two fragments by caspase 3 (CPP32). Gamma-PAK appears to function through phosphorylation of a number of different substrates. Gamma-PAK induces cytostasis as shown by injection of active gamma-PAK into frog oocytes, and transient transfection and expression of gamma-PAK in mammalian cells reduces cell viability. Conditions of stress inhibit cell division, reduce and alter the specificity of transcription and translation, and cause changes in metabolism. The focus of these studies are as follows. 1) The cytostatic properties of gamma-PAK will be examined in mammalian cell culture by identification of the upstream activators of gamma-PAK under different stress conditions and their role in activation of the protein kinase will be determined. The requirements for translocation and activation of gamma-PAK to achieve cytostasis and the role of protein:protein interactions in targeting the protein kinase will be examined by expression of site-directed mutants in mammalian cells. 2) Regulation of gamma-PAK activity by autophosphorylation and by phosphorylation by other protein kinases will be analyzed in vivo and in vitro. Proteins associated with endogenously active gamma-PAK will be identified and the effects of phosphorylation of the proteins by gamma-PAK will be determined. 3) A comparison of the substrate specificity between alpha-PAK and gamma-PAK will be determined using peptide substrates. Protein substrates for gamma-PAK will be identified, the sites of phosphorylation in substrates of interest will be determined, and the effects of phosphorylation on substrate activity will be analyzed. 4) Prepare protein for a collaborative effort to obtain structures of the regulatory (p27) and catalytic domain (p34) and of the gamma-PAK holoenzyme by x-ray crystallography.
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