Regulation of Myosin Light Chain Kinase by Phosphorylat*

描述(由申请人提供) 肌球蛋白轻链激酶(MLCK)是调节肌球蛋白轻链的关键元件 肌肉收缩。特别是MLCK的磷酸化，已经成为一种 可能将收缩与其他信号通路联系在一起的事件。在这 应用程序我们的目标是填补我们在如何针对特定站点的知识方面的空白 磷酸化会影响MLCK的活性。我们的长期目标是了解 血管内皮细胞的信号转导过程及其机制 它的规定。具体地说，我们建议(I)开发特定于磷的 用抗体监测细胞内MLCK的磷酸化水平并评估 肌动蛋白MLCK的磷酸化与收缩特性的关系 对平滑肌/非肌肉细胞的研究；(Ii)检验以下假设 MAPK激活的MLCK对酶活性的影响及其机制探讨 研究MLCK磷酸化对其构象的影响；以及(Iii) 确定MAPK和PKA的磷酸化如何影响MLCK与 阐明平滑肌和非肌肉收缩机械的组成 原位定位。拟议的研究是对 美国国立卫生研究院正在进行的项目资助(P01-AR41637)，标题为 “平滑肌调节的分子机制。”建议的协作 不仅将加强申请者(C-L·阿尔伯特·王， 博士，美国首席研究员)，但也将使科学 外国合作者亚历山大·沃罗特尼科夫博士的利益，并强烈 影响在国外实验室进行的研究。本文件中描述的工作 提案将提供信息，以扩大正在进行的 对MLCK的磷酸化调控进行了研究，并提供了一种较好的 对平滑肌调节的理解。

DESCRIPTION (provided by applicant) Myosin light chain kinase (MLCK) is a key element in the regulation of smooth muscle contraction. Phosphorylation of MLCK, in particular, has emerged as an event that may link contractility to other signaling pathways. In this application we aim to fill the gaps in our knowledge of how site-specific phosphorylation affects the MLCK activity. Our long term goal is to understand the signal transduction process in smooth muscle cells and the mechanisms for its regulation. Specifically, we propose (i) to develop phospho-specific antibodies to monitor the phosphorylation level of MLCK in cells and to assess the relationship between phosphorylation of MLCK and the contractile properties of smooth/non-muscle cells; (ii) to test the hypothesis that phosphorylation of MLCK by MAPK affects the activity of the enzyme and to explore the mechanism by investigating effects of MLCK phosphorylation on its conformation; and (iii) to determine how phosphorylation by MAPK and PKA affects interaction of MLCK with components of smooth muscle and non-muscle contractile machinery by elucidating the in situ localization. The proposed research is a logical extension of the ongoing Program Project Grant funded by NIH (P01-AR41637) under the title of "Molecular Mechanisms of Smooth Muscle Regulation." The proposed collaboration will not only enhance the research program of the applicant (C.-L. Albert Wang, Ph.D., the US Principal Investigator), but will also benefit the scientific interests of the Foreign Collaborator, Dr. Alexander Vorotnikov, and intensely impact the research conducted in the Foreign Laboratory. Work described in this proposal will provide information to extend the applicability of the ongoing studies to the regulation of MLCK by phosphorylation, and afford a better understanding of smooth muscle regulation.