CYCLIC NUCLEOTIDE INDEPENDENT PHOSPHORYLATION

这项研究旨在研究磷酸化在细胞周期中的作用。 调节新陈代谢。这些研究将考察四个方面的作用 多潜能蛋白激酶、酪蛋白激酶I和II和蛋白水解酶 激活的激酶(PAK)I和II。将重点放在 PAK II在代谢调节中的作用，因为PAK II已被证明是 在血清饥饿的细胞中对胰岛素的反应被激活，表皮生长 因子(EGF)和佛波醇酯。在即将到来的授权期内，我们 建议确定PAK II的激活模式以响应EGF和 佛波醇酯在分子水平上。总酶和活性总量 将非激活的PAK II在正常和转化后进行检测和比较 与激素状态和生长阶段有关的细胞。的级别 还将检查酶在细胞周期中的位置和 基因扩增对获得性耐药的影响。PAK II和PAK I 将被提纯为均一、多克隆和单克隆抗体 生产，并对酶进行物理和化学表征。 在体外被PAK II磷酸化的纯化蛋白将被检测为 体内底物；磷酸化对酶/蛋白质的影响 将对活动进行分析。酪蛋白激酶I、酪蛋白水平的调节 将根据细胞类型、位置在 细胞周期、荷尔蒙状态以及二者之间的相互关系 这些酶和PAK II将被检测。纯化蛋白，经鉴定 作为这三种多潜能蛋白激酶的底物， 分析了个体和多重的功能后果 将确定磷酸化事件。

This research is designed to examine the role of phosphorylation in the regulation of metabolism. These studies will examine the role of four multipotential protein kinases, casein kinase I and II and protease activated kinase (PAK) I and II. Emphasis will be placed on the role of PAK II in the regulation of metabolism, since PAK II has been shown to be activated in serum-starved cells in response to insulin, epidermal growth factor (EGF), and phorbol esters. During the coming grant period, we propose to identify the mode of activation of PAK II in response to EGF and phorbol esters at a molecular level. Total enzyme and amounts of active and inactive PAK II will be examined and compared in normal and transformed cells with regard to hormonal status and growth phase. Levels of the enzyme will also be examined with regard to position in the cell cycle and with gene amplification upon acquired drug resistance. PAK II and PAK I will be purified to homogeneity, polyclonal and monoclonal antibodies produced, and the enzymes characterized physically and chemically. Purified proteins phosphorylated by PAK II in vitro will be examined as substrate in vivo; the effects of phosphorylation on enzymic/protein activity will be analyzed. Regulation of levels of casein kinase I, casein kinase II and PAK I will be examined with regard to cell type, position in the cell cycle, and hormonal status, and the interrelationship between these enzymes and PAK II will be examined. Purified proteins, identified as substrate for these three multipotential protein kinases, will be analyzed and the functional consequences of individual and multiple phosphorylation events will be determined.