CYCLIC NUCLEOTIDE INDEPENDENT PHOSPHORYLATION

磷酸化是最常见的共价修饰，涉及 真核生物新陈代谢的调控。这笔助学金支持我们的研究 蛋白激酶的结构和功能特性，它们的模式 监管、底物的鉴定和功能后果 磷酸化对活性的影响。要解决的具体目标包括 以下是。 我们将继续研究酪蛋白激酶(CK)的调节方式 II通过检测体内该酶的磷酸化对 荷尔蒙和体外对各种效应化合物的反应。阿尔法 和CK II的β亚基在大肠杆菌中表达，并将继续 单独和作为重组全酶及其作用的表征 活性上的特定部位突变将被检查。 将完成酪蛋白激酶(CK)I编码DNA的测序 并将该序列与其他蛋白激酶的序列进行比较。我们会 继续CK I的物理、化学和功能表征。 培养细胞的磷酸化和活性调节也将是 检查过了。 III.我们将研究蛋白水解酶的自磷酸化效应 活化的蛋白激酶(PAK)I对活性的影响，决定蛋白激酶是否 被其他蛋白激酶修饰，检查潜在的效应因子 蛋白激酶活性，并继续对该酶进行表征。我们 还将检测培养细胞中PAK I的激活模式。 IV.我们将研究蛋白质复杂结合的要求 以其他蛋白质/多肽为底物来促进细胞的磷酸化 底物和改变活性，使用钙调蛋白作为 模型系统。 五、蛋白质位点特异性和多重磷酸化的影响 感兴趣的将继续审查和单一的职能作用 和多重磷酸化事件对酶活性、核酸 结合，以及蛋白质/蛋白质相互作用将被分析。

Phosphorylation is the most common covalent modification involved in regulation of metabolism in eukaryotes. This grant supports our studies on the structural and functional properties of protein kinases, their mode of regulation, identification of substrates, and the functional consequences of phosphorylation on activity. The specific aims to be addressed include the following. I. We will continue studies on the mode of regulation of casein kinase (CK) II by examining phosphorylation of the enzyme in vivo in response to hormones and in vitro in response to various effector compounds. The alpha and beta subunits of CK II are expressed in E. coli and will continue to be characterized alone and as the reconstituted holoenzyme and the effects of site-specific mutations on activity will be examined. II. Sequencing of the DNA coding for casein kinase (CK) I will be completed and the sequence compared with those of other protein kinases. We will continue the physical, chemical, and functional characterization of CK I. Phosphorylation and modulation of activity in cultured cells will also be examined. III. We will examine the effects of autophosphorylation of protease activated kinase (PAK) I on activity, determine whether the protein kinase is modified by other protein kinases, examine potential effectors of protein kinase activity and continue characterization of the enzyme. We will also examine the mode of activation of PAK I in cultured cells. IV. We will examine the requirements for complex association of protein substrates with other proteins/peptides to promote phosphorylation of the substrate and alter activity, using calmodulin as a model system. V. The effects of site-specific and multiple phosphorylation of proteins of interest will continue to be examined and the functional role of single and multiple phosphorylation events on enzyme activity, nucleic acid binding, and on protein/protein interactions will be analyzed.