BIOCHEMISTRY OF CONTRACTILE PROTEINS

平滑肌是大多数中空器官中的收缩元件， 对于许多生理功能包括血管的功能是必需的。 组织、气道和胃肠道。 一个重要的监管 控制平滑肌收缩活动的机制是 肌球蛋白的磷酸化。 肌球蛋白磷酸化水平反映了 两种关键酶：肌球蛋白轻链激酶和肌球蛋白 磷酸酶（MP）。 最近的研究表明，在某些条件下，例如， 在几种生理激动剂的刺激下，MP活性是 这导致肌球蛋白磷酸化的显著增加 在次最大CA 2+浓度下。 此应用程序的目标是 为这种新的调节机制建立了分子基础。 的MP 由3个亚基组成：一个催化亚基和两个假定的调节亚基。 130 kD和20 kD亚基。 一个重要的额外发现是， 130 kD亚基的磷酸化抑制MP活性。 但 激酶未被鉴定。 具体目的是调查 MP功能及其调控的三个方面。 第一个将 研究不同亚基之间的相互作用， 重要的结合位点或结构域，其上的分子框架 可以构建MP全酶。 20 kD亚基的作用是 不知道。 将使用几种技术，包括酵母双杂交 几种突变体的系统、构建和表达以及各种测定 磷酸酶活性和蛋白质相互作用。 第二个目标将 检测130 kD亚基磷酸化的影响。 的 将鉴定磷酸化的关键位点。 基于所述计划 为MP全酶建立的那些相互作用， 在磷酸化之后， 抑制将被识别。 最后的目的是表征 激酶参与和程序概述，以建立其身份和 也可以筛选其他可能具有生理作用的底物。 130 kD激酶的候选者是rho活化激酶，这将 追究 最终，这种激酶的作用必须是 与平滑肌中的信号转导途径整合， 起始于细胞膜并以收缩器官为目标。

Smooth muscle is the contractile element in most hollow organs and is essential for many physiological functions including those of vascular tissue, airways, and the gastrointestinal tract. A major regulatory mechanism in controlling contractile activity of smooth muscle is phosphorylation of myosin. The level of myosin phosphorylation reflects the activites of two key enzymes: myosin light chain kinase and myosin phosphatase (MP). Recently it was shown that under some conditions, e.g. on stimulation by several physiological agonists, that MP activity is inhibited and this results in a marked increase of myosin phosphorylation at sub-maximal CA2+ concentrations. The goal of this application is to establish a molecular basis for this novel regulatory mechanism. The MP consists of 3 subunits: a catalytic subunit and two putative regulatory subunits of 130 kD and 20 kD. An important additional finding is that phosphylation of the 130 kD subunits inhibits MP activity. However, the kinase is not identified. The specific aims are designed to investigate three aspects of MP function and its regulation. The first will investigate interactions between different subunits and establish important binding sites, or domains, upon which a molecular framework of the MP holoenzyme can be constructed. The role of the 20 kD subunit is not known. Several techniques will be used including the yeast two-hybrid system, construction and expression of several mutants and various assays of phosphatase activity and protein interactions. The second aim will examine the effects of phosphorylation of the 130 kD subunit. The critical site(s) of phosphorylation will be identified. Based on the plan established for the MP holoenzyme, those interactions that change following phosphorylation and that are involved in the mechanism of inhibition will be identified. The final aim is to characterized the kinase involved and procedures are outlined to establish its identity and also to screen for other substrates that might have a physiological role. A candidate for the 130 kD kinase is a rho-activated kinase and this will be investigated. Ultimately, the action(s) of this kinase must be integrated with the signal transduction pathway(s) in smooth muscle that are initiated at the cell membrane and target the contractile apparatus.