CYCLIC NUCLEOTIDE INDEPENDENT PHOSPHORYLATION

p21激活的蛋白激酶γ-PAK在高渗应激、低水平电离辐射、DNA损伤药物和鞘氨醇的条件下被激活。 此外，γ-PAK在细胞凋亡早期通过半胱天冬酶3（CPP 32）切割成两个片段而被组成性激活。γ-PAK似乎通过许多不同底物的磷酸化发挥作用。 如通过将活性γ-PAK注射到青蛙卵母细胞中所示，γ-PAK诱导细胞停滞，并且γ-PAK在哺乳动物细胞中的瞬时转染和表达降低细胞活力。 应激条件抑制细胞分裂，减少和改变转录和翻译的特异性，并引起代谢的变化。 这些研究的重点如下。1)将在哺乳动物细胞培养物中通过鉴定不同应激条件下的γ-PAK上游活化剂来检查γ-PAK的细胞抑制特性，并确定其在蛋白激酶活化中的作用。 将通过在哺乳动物细胞中表达定点突变体来检查γ-PAK易位和激活以实现细胞抑制的要求以及蛋白质：蛋白质相互作用在靶向蛋白激酶中的作用。 2)将在体内和体外分析通过自身磷酸化和通过其他蛋白激酶磷酸化对γ-PAK活性的调节。 将鉴定与内源性活性γ-PAK相关的蛋白质，并确定γ-PAK对蛋白质磷酸化的影响。 3)将使用肽底物确定α-PAK和γ-PAK之间底物特异性的比较。 将鉴定γ-PAK的蛋白质底物，确定目标底物中的磷酸化位点，并分析磷酸化对底物活性的影响。 4)准备蛋白质，以通过X射线晶体学获得调节结构域（p27）和催化结构域（p34）以及γ-PAK全酶的结构。

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.