CALMODULIN-MYOSIN LIGHT CHAIN KINASE INTERACTIONS

钙调蛋白-肌球蛋白轻链激酶相互作用

• 批准号: 2231004
• 负责人: HAROLD W DAVIS
• 金额: $ 10.02万
• 依托单位: UNIVERSITY OF CINCINNATI
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-02-14 至 1998-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2231004
• 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

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.

血管活性剂，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。 单层屏障 功能障碍至少部分是由于EC收缩，其通过 最终形成肌球蛋白轻链（MLC 20）的信号传导事件 磷酸化肌球蛋白轻链激酶对MLC 20的磷酸化作用 （MLCK）是平滑肌收缩的必要步骤， 非肌肉细胞关于规范的事件的信息有限 MLCK，一种Ca 2 +/钙调蛋白（CaM）依赖性酶。然而，已经表明， 凝血酶和组胺诱导MARCKS磷酸化， （豆蔻酰化富含丙氨酸的C激酶底物），一种钙调蛋白结合蛋白。 在目前的建议中，假设受体激动剂诱导的 培养EC中MLCK的激活受MARCKS磷酸化的调节 和CaM。为了检验这一假设，以下具体目标（SA）是 提出：SA#1）为了表征激动剂诱导的MARCKS磷酸化， 培养EC中MLCK活性。 在被蛋白激酶C磷酸化后， MARCKS释放CaM，然后可以用作MLCK的辅因子。Marcks 磷酸化将被确定并与MLCK激活相关（如 通过MLC 20磷酸化评估），以评估磷酸化是否 的MARCKS可以参与激动剂诱导的MLCK激活。SA#2）至 确定激动剂诱导的EC中CaM的磷酸化。 凸轮 被胰岛素受体或酪蛋白激酶II磷酸化， 更长的增强，但抑制体外MLCK活性，这表明一种新的 MLCK调节机制。最近，人们发现钙调素 也可以被MLCK磷酸化， 时间上跟随MLC 20磷酸化。CaM磷酸化将是 评估并与MLCK激活相关。SA#3）表征CaM 通过MLCK的磷酸化。CaM在体外会被MLCK磷酸化， 该反应的特征将由生物化学决定， 手段SA#4）为了确定CaM磷酸化对CaM的影响， MLCK相互作用。磷酸化钙调素对MLCK激活的影响 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磷酸化与MLCK激活之间存在相关性， 为EC收缩和屏障的机制提供重要见解 功能障碍