BIOCHEMISTRY OF CONTRACTILE PROTEINS

平滑肌是大多数中空器官的收缩元件，是 对包括血管在内的许多生理功能都是必不可少的 组织、呼吸道和胃肠道。一个主要的监管机构 控制平滑肌收缩活动的机制是 肌球蛋白的磷酸化。肌球蛋白的磷酸化水平反映了 肌球蛋白轻链激酶和肌球蛋白两种关键酶的活性 磷酸酶(MP)。最近有研究表明，在某些条件下，例如 在几种生理激动剂的刺激下，MP的活性是 抑制，这导致肌球蛋白磷酸化显著增加 在次最大钙离子浓度下。此应用程序的目标是 为这一新的调控机制建立分子基础。国会议员 由3个亚基组成：一个催化亚基和两个假定的调节亚基 130kD和20kD亚基。另一个重要的发现是 130kD亚基的磷酸化抑制MP的活性。然而， 未鉴定出激活酶。具体目的是为了调查 MP的功能及其调控的三个方面。第一个遗嘱 研究不同亚基之间的相互作用并建立 重要的结合部位或结构域，其上有一个分子框架 可以构建MP全酶。20kD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、构建和表达及多种检测方法 磷酸酶活性和蛋白质相互作用。第二个目标是 检测130kD亚基磷酸化的影响。这个 将确定磷酸化的关键位点(S)。根据计划 为MP全酶建立的，那些改变的相互作用 在磷酸化之后，这些基因参与了 抑制作用将被识别出来。最终目标是将 所涉及的激酶和程序概述，以确定其身份和 也是为了筛选其他可能具有生理作用的底物。 130kD激酶的候选者是Rho激活的激酶，这将 被调查。最终，这个激酶的作用(S)必须是 与信号转导通路(S)整合在一起的平滑肌 在细胞膜上启动，并以收缩装置为目标。

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.