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在凋亡早期通过caspase 3 （CPP32）裂解成两个片段而组成性激活。γ - pak似乎通过许多不同底物的磷酸化起作用。通过将活性γ - pak注射到青蛙卵母细胞中可以发现，γ - pak可以诱导细胞停滞，而在哺乳动物细胞中短暂转染和表达γ - pak会降低细胞活力。应激条件抑制细胞分裂，降低和改变转录和翻译的特异性，引起代谢的变化。这些研究的重点如下。1)在哺乳动物细胞培养中，通过鉴定不同应激条件下γ - pak上游激活因子来检测γ - pak的细胞抑制特性，并确定其在蛋白激酶激活中的作用。易位和激活γ - pak以实现细胞停滞的要求以及蛋白质的作用：蛋白质相互作用在靶向蛋白激酶中的作用将通过哺乳动物细胞中位点定向突变的表达来研究。2)将在体内和体外分析γ - pak活性通过自身磷酸化和其他蛋白激酶磷酸化的调节。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)将使用肽底物来比较α - pak和γ - pak的底物特异性。将鉴定γ - pak的蛋白质底物，确定感兴趣的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，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.