CYCLIC NUCLEOTIDE INDEPENDENT PHOSPHORYLATION

这项研究旨在研究磷酸化在 调节新陈代谢。 这些研究将探讨四个作用 多能蛋白激酶，酪蛋白激酶I和II以及蛋白酶 活化激酶（PAK）I和II。 重点将放在以下方面的作用： 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、表皮生长 EGF和佛波醇酯。 在未来的资助期内，我们 建议识别PAK II响应EGF的激活模式， 在分子水平上的佛波酯。 总酶和活性量 和非活性PAK II将在正常和转化中进行检查和比较。 细胞的激素状态和生长阶段。 水平 还将检查酶在细胞周期中的位置， 在获得性耐药性时基因扩增。 PAK II和PAK I 将被纯化为同质的多克隆和单克隆抗体 产生的，和物理和化学特性的酶。 通过PAK II在体外磷酸化的纯化蛋白将被检查为 底物在体内;磷酸化对酶/蛋白的影响 将对活动进行分析。 调节酪蛋白激酶I，酪蛋白 激酶II和PAK I将根据细胞类型、在 细胞周期和激素状态，以及它们之间的相互关系 将检测这些酶和PAK II。 纯化蛋白，已鉴别 作为这三种多能蛋白激酶的底物， 分析和功能后果的个人和多个 将测定磷酸化事件。

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.