CYCLIC NUCLEOTIDE INDEPENDENT PHOSPHORYLATION

磷酸化是最常见的共价修饰， 真核生物新陈代谢的调节。这笔赠款支持我们的研究， 蛋白激酶的结构和功能特性，它们的 调节、底物鉴定和功能性后果 磷酸化对活性的影响。具体目标包括： 下面的. I.我们将继续研究酪蛋白激酶（CK）的调控模式 II通过检查体内酶的磷酸化反应， 激素和体外对各种效应化合物的反应。阿尔法 CK Ⅱ β亚基在E.大肠杆菌，并将继续 单独表征和作为重构全酶表征， 将检查位点特异性突变对活性的影响。 二.将完成编码酪蛋白激酶（CK）I的DNA测序 并与其它蛋白激酶的序列进行了比较。我们将 继续CK I的物理、化学和功能表征。 还将研究培养细胞中的磷酸化和活性调节。 考察 三.我们将研究蛋白酶自磷酸化的影响 活化激酶（PAK）I活性，确定蛋白激酶 被其他蛋白激酶修饰，检查 蛋白激酶活性和酶的连续表征。我们 还将检查培养细胞中PAK I的激活模式。 四.我们将研究蛋白质复杂结合的条件 底物与其他蛋白质/肽的结合，以促进蛋白质/肽的磷酸化。 底物和改变活性，使用钙调蛋白作为 模型系统 诉蛋白质的位点特异性和多重磷酸化的影响 将继续审查感兴趣的问题， 和多磷酸化事件对酶活性、核酸 结合和蛋白质/蛋白质相互作用。

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.