REGULATION OF CALMODULIN-DEPENDENT PROTEIN KINASE II

钙调蛋白依赖性蛋白激酶 II 的调节

• 批准号: 2185403
• 负责人: ROGER J COLBRAN
• 金额: $ 10.79万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-07-01 至 1997-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2185403
• 关键词: Baculoviridae; SDS polyacrylamide gel electrophoresis; active sites; animal tissue; brain; calcium metabolism; calmodulin; calmodulin dependent protein kinase; enzyme activity; enzyme mechanism; enzyme substrate; laboratory rat; phosphatase inhibitor; phosphoprotein phosphatase; phosphorylation; radiotracer; site directed mutagenesis

The multifunctional Ca2+/calmodulin-dependent protein kinase II (CaMKII) plays a central role in many cellular responses to Ca2+-mobilization and is implicated in many aspects of health and disease. It is most abundant in brain where it regulates synaptic transmission in both the pre- and post-synaptic cells, and is implicated in learning and memory. The long-term aim is to understand mechanisms regulating phosphorylation of targeted substrates by CaMKII. Three related aspects of this aim are addressed: The mechanism by which Ca2+-independent autophosphorylation of CaMKII blocks interaction with calmodulin (CaM) and desensitizes the kinase to Ca2+/CaM will be investigated. A cDNA encoding the alpha subunit of mouse brain CaMKII will be mutated at positions corresponding to the known Ca2+-independent autophosphorylation sites (Thr3O5, Thr3O6 and Ser3l4) and expressed in insect cells using baculovirus. CaM-binding and autophosphorylation properties of mutant kinases will be characterized. These studies will be important in understanding the biochemical mechanisms involved in Ca2+-independent autophosphorylation as well as its physiological role. The phosphorylation state of CaMKII autophosphorylation sites depends on the rates of both phosphorylation and dephosphorylation. Therefore, protein phosphatases involved in dephosphorylating CAMKII will be identified. CaMKII will be [32P]-labeled at either Thr286, the Ca2+/CaM-dependent site responsible for the partially Ca2+-independent form of CaMKII, or at specific Ca2+-independent sites (Thr305/Thr306 or Ser3l4) and used as a substrate for neuronal protein phosphatases. Activities will be attributed to either known or novel enzymes by using a series of specific activators and inhibitors, and also by their chromatographic behavior. Characterization of the site specificity, identity and relative abundance of protein phosphatases acting on CaMKII is essential for a complete understanding of CaMKII regulation in vivo. Substrates phosphorylated in response to elevated Ca2+ in vivo may be targeted in part by co-localization of CaMKII with its substrates, in part on to regulation of kinase activity. The location of CaMKII may also affect its regulation by Ca2+ and phosphatases. The role of specific binding proteins in determining the subcellular localization of CaMKII will be investigated. CaMKII-binding proteins will be identified in rat tissue extracts and the possible regulation of binding by phosphorylation and other factors will be investigated.

多功能Ca2+/钙调蛋白依赖性蛋白激酶II（CAMKII） 在许多细胞对Ca2+杀伤中的反应中起着核心作用，并且 与健康和疾病的许多方面有关。 它最丰富 它调节前后的突触传播的大脑 突触后细胞，与学习和记忆有关。 这 长期目的是了解调节磷酸化的机制 CAMKII的目标底物。 这个目标的三个相关方面是 地址： CAMKII的Ca2+非依赖性自磷酸化的机制 阻止与钙调蛋白（CAM）相互作用，并使激酶脱敏 CA2+/CAM将进行研究。 编码小鼠α亚基的cDNA Brain Camkii将在与已知的位置进行突变 CA2+非依赖性自磷酸化位点（Thr3O5，Thr3O6和Ser3L4），并且 使用杆状病毒在昆虫细胞中表达。 凸轮结合和 突变激酶的自磷酸化特性将被表征。 这些研究对于理解生化机制至关重要 参与CA2+非依赖性自磷酸化及其 生理角色。 CAMKII自磷酸化位点的磷酸化状态取决于 磷酸化和去磷酸化的速率。 所以， 参与去磷酸化的CAMKII的蛋白质磷酸酶将是 确定。 camkii将是[32p]在THR286上标记的 CA2+/CAM依赖性位点负责部分Ca2+非依赖性形式 CAMKII，或在特定的Ca2+非依赖性位点（THR305/THR306或SER3L4） 并用作神经元蛋白磷酸酶的底物。 活动将 通过使用一系列的酶归因于已知或新型酶 特定的激活剂和抑制剂，也通过色谱法 行为。 场地特异性，身份和相对的表征 作用于CAMKII的蛋白质磷酸酶的丰度对于A是必不可少的 对体内CAMKII调节的完全了解。 响应升高的Ca2+体内磷酸化的底物可能是 部分是由CaMKII及其底物共定位的部分目标 对激酶活性的调节。 Camkii的位置也可能 影响其对Ca2+和磷酸酶的调节。 特定的作用 结合蛋白在确定CAMKII的亚细胞定位时将 被调查。 CAMKII结合蛋白将在大鼠组织中鉴定 提取物以及可能通过磷酸化和 其他因素将被研究。