STRUCTURE/FUNCTION OF GENETIC ENGINEERED CALMODULINS

基因工程钙调蛋白的结构/功能

• 批准号: 6180152
• 负责人: ANTHONY R MEANS
• 金额: $ 33.49万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1984
• 资助国家: 美国
• 起止时间: 1984-07-01 至 2002-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6180152
• 关键词: Aspergillus; Caenorhabditis elegans; alternatives to animals in research; antisense nucleic acid; calcium flux; calmodulin; calmodulin dependent protein kinase; cell cycle; cell line; cell nucleus; cytoplasm; enzyme activity; enzyme mechanism; enzyme structure; enzyme substrate; gene expression; genetic library; messenger RNA; molecular cloning; phosphorylation; protein binding; protein engineering; protein localization; protein sequence; protein structure function; recombinant proteins; reporter genes; site directed mutagenesis; tissue /cell culture

DESCRIPTION: The overall goal is to determine how the ubiquitous intracellular Ca2+ binding calmodulin (CaM) can regulate a wide variety of target proteins in a specific manner. The objective is to elucidate mechanisms that govern regulation of a proposed CaM kinase (CaMK) cascade that was discovered in part, as a outcome of the previous work. The first aim is to use the novel reagents, and current knowledge of mammalian CaMKI and IV to determine the mechanisms involved in their regulation by activating CaMK kinases (CaMKK) and examine the physiological relevance of this cascade in mammalian cells. The investigators have cloned and expressed several members of the CaMK family from 4 species (humans, rats, A. nidulans and C. elegans). In mammals the investigators have CaMKI and IV, CaMKK alpha and beta. Whereas the KKs both phosphorylate a single Thr residue located in the activation loops of CaMKI an IV, the regulation of CaMKIV is more complicated than CaMKI. In addition CaMKI is a nuclear enzyme whereas CaMKI is not. The investigators will address the sequence of events required to activate CaMKIV and to enter the nucleus as well as the mechanism of activation and subcellular localization of the 2CaMKKs. These experiments will be carried out using recombinant proteins as well as in Jurkat T cells. They have developed an assay in which CaMKIV is used to drive Ga14-CREB system via phosphorylation of CREB on SER-133 in Jurkact cells. This assay requires an increase in intracellular Ca2+ and phosphorylation of Thr-20 on CamKIV by a CaMKK. They will examine which steps occur in cytoplasm versus nucleus. CaMKK beta contains putative SH3 binding sites in the N-terminus and bipartite nuclear localization sequence/14.3.3 binding site that overlaps the C-terminus of the putative CaM binding site. The investigators will determine the relevance of these domains in vitro and in Jurkat cells. The second aim is to evaluate mechanisms involved in activation of enzymes that are homologs to CaMKI/IV and CaMKK in A. nidulans and C. elegans and how these cascades might influence well cycle progression, development or differentiation in these genetically tractable organisms. They will examine the properties of these kinases, determine if they are essential in A. nidulans and, if so, what biological pathway they control. Similar genetic studies in C. elegans will reveal in what cells the CaMKK is expressed and how expression changes during development. Finally, antisense RNA will be used to deplete the CaMKK mRNA to test the functional significance of the enzyme. These complementary approaches will yield exciting new insights into structure/function relationships between CaM and physiologically important CaMKs in these organisms.

描述：总体目标是确定无处不在的 细胞内Ca2+结合钙调蛋白（CAM）可以调节多种多样 以特定方式靶向蛋白质。目的是阐明 控制拟议的CAM激酶（CAMK）级联的机制 这部分是作为先前工作的结果。第一个 目的是使用新颖的试剂和当前对哺乳动物CAMKI的知识 和iv确定其调节所涉及的机制 激活CAMK激酶（CAMKK）并检查 该级联在哺乳动物细胞中。调查人员克隆了 表达了来自4种的CAMK家族的几个成员（人类，大鼠， A. Nidulans和C. exegrans）。在哺乳动物中，调查人员有CAMKI和 IV，Camkk Alpha和Beta。而KKS都磷酸化单个THR 残留物位于CaMki A和IV的激活环中，调节 Camkiv比Camki更复杂。此外，CaMki是核 酶，而camki则没有。调查人员将解决序列 激活camkiv并进入核所需的事件以及 2CAMKKS的激活和亚细胞定位机理。 这些实验也将使用重组蛋白进行 如在Jurkat T细胞中。他们开发了一种使用Camkiv的测定法 通过在Ser-133上磷酸化驱动GA14-CREB系统 Jurkact细胞。该测定需要增加细胞内Ca2+和 CAMKK在CAMKIV上的THR-20磷酸化。他们将检查哪个 在细胞质与核中发生了步骤。 camkk beta包含推定的SH3 N末端和两分核定位中的结合位点 序列/14.3.3结合位点与推定的C端重叠 凸轮绑定位点。调查人员将确定这些相关性 体外和Jurkat细胞的结构域。第二个目的是评估 与CAMKI/IV同源的酶激活有关的机制 和A. nidulans和C. elegans的Camkk以及这些级联 影响井周期的进展，发展或分化 遗传上可牵引的生物。他们将检查这些特性 激酶，确定它们是否在A. nidulans中是必不可少的，如果是的，则是什么 他们控制的生物途径。秀丽隐杆线虫中的类似遗传研究 将在CAMKK表达的细胞中揭示以及如何表达 开发过程中的变化。最后，反义RNA将用于耗尽 CAMKK mRNA测试酶的功能意义。这些 互补方法将为您带来令人兴奋的新见解 CAM与生理上重要的结构/功能关系 这些生物中的凸轮。