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CALMODULIN-MYOSIN LIGHT CHAIN KINASE INTERACTIONS

钙调蛋白-肌球蛋白轻链激酶相互作用

基本信息

批准号：
2332531
负责人：
HAROLD W DAVIS
金额：
$ 11.22万
依托单位：
UNIVERSITY OF CINCINNATI
依托单位国家：
美国
项目类别：
财政年份：
1994
资助国家：
美国
起止时间：
1994-02-14 至 1999-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/2332531
关键词：
SDS polyacrylamide gel electrophoresis biological signal transduction calcium binding protein enzyme activity fluorimetry molecular biology muscle contraction myosin light chain kinase phosphorylation protein purification receptor binding stoichiometry tissue /cell culture vascular endothelium

项目摘要

Vasoactive agents, such as thrombin and histamine, induce endothelial cell (EC) monolayer barrier dysfunction which is involved in a number of disease processes, including atherosclerosis. Monolayer barrier dysfunction is, at least, partially due to EC contraction which occurs via signaling events that culminate in myosin light chain (MLC20) phosphorylation. The phosphorylation of MLC20 by myosin light chain kinase (MLCK), is an obligatory step in contraction by smooth muscle and nonmuscle cells. Information is limited regarding events which regulate MLCK, a Ca2+/calmodulin (CaM)-dependent enzyme. However, it has been shown that thrombin and histamine induce the phosphorylation of MARCKS (myristoylated alanine-rich C kinase substrate), a CaM-binding protein. In the current proposal it is hypothesized that receptor agonist-induced MLCK activation in cultured EC is regulated by phosphorylation of MARCKS and CaM. To test this hypothesis the following Specific Aims (SA) are proposed: SA#1)To characterize agonist-induced MARCKS phosphorylation and MLCK activity in cultured EC. Upon phosphorylation by protein kinase C, MARCKS releases CaM that can then be used as a cofactor for MLCK. MARCKS phosphorylation will be determined and correlated with MLCK activation (as assessed by MLC20 phosphorylation) to evaluate whether the phosphorylation of MARCKS can be involved in agonist-induced MLCK activation. SA#2) To determine agonist-induced phosphorylation of CaM in cultured EC. CaM phosphorylated by either the insulin receptor or casein kinase II no longer augments, but inhibits in vitro MLCK activity, suggesting a novel mechanism of MLCK regulation. Recently, it has been discovered that CaM can also be phosphorylated by MLCK and that this phosphorylation temporally follows MLC20 phosphorylation. CaM phosphorylation will be assessed and correlated with MLCK activation. SA#3) To characterize CaM phosphorylation by MLCK. CaM will be phosphorylated by MLCK in vitro and the characteristics of this reaction will be determined by biochemical means. SA#4) To determine the consequences of CaM phosphorylation on CaM- MLCK interactions. The effect of phosphorylated CaM on MLCK activation will be evaluated by fluorometric and molecular biological techniques. These studies will demonstrate whether a temporal and vectoral relationship between CaM phosphorylation and MLCK activation exists and provide important insights in the mechanisms of EC contraction and barrier dysfunction.

血管活性物质，如凝血酶和组胺，诱导内皮细胞 (EC)单层屏障功能障碍，涉及许多疾病过程，包括动脉粥样硬化。单层势垒功能障碍至少部分是由于EC收缩，这种收缩通过最终形成肌球蛋白轻链的信号事件(MLC20) 磷酸化。肌球蛋白轻链激酶对MLC20的磷酸化作用 (MLCK)，是平滑肌收缩的必经步骤，非肌肉细胞。有关规定的事件的信息有限 MLCK是一种依赖于钙/钙调素(CaM)的酶。然而，它已经被证明凝血酶和组胺诱导MARCKS的磷酸化 (肉豆蔻酰化的富含丙氨酸的C激酶底物)，一种CaM结合蛋白。在目前的建议中，假设受体激动剂诱导 MARCKS的磷酸化调控培养的EC中MLCK的激活还有卡姆。为了检验这一假设，以下具体目标(SA)是建议：SA#1)来表征激动剂诱导的Marcks磷酸化和培养内皮细胞的MLCK活性。在被蛋白激酶C磷酸化后， Marcks释放Cam，然后将其用作MLCK的辅因子。马克将测定磷酸化并将其与MLCK激活(AS)相关联通过MLC20磷酸化进行评估)，以评估磷酸化是否可能参与激动剂诱导的MLCK激活。SA#2)至测定激动剂诱导的培养内皮细胞CaM的磷酸化。凸轮被胰岛素受体或酪蛋白激酶II磷酸化在体外，较长的时间可以增强MLCK活性，但会抑制MLCK活性，这表明一种新的 MLCK的调控机制。最近，人们发现，卡姆也可以被MLCK磷酸化，这种磷酸化在时间上跟随MLC20的磷酸化。CaM的磷酸化将是评估MLCK活性并与之相关。SA#3)来表征凸轮 MLCK的磷酸化。在体外，MLCK将使CaM磷酸化这一反应的特性将由生物化学来决定。意思是。SA#4)，以确定CaM磷酸化对CaM- MLCK相互作用。磷酸化CaM对MLCK活性的影响将通过荧光和分子生物学技术进行评估。这些研究将证明时间和矢量 CaM磷酸化和MLCK激活之间存在关系对EC收缩和屏障的机制提供重要的见解功能障碍。