MYPT1 phosphatase in smooth muscle

摘要：该项目的目标是确定肌球蛋白轻链的分子同一性。 磷酸酶磷酸酶(MYPT1磷酸酶)，并阐明这一调节作用的研究很少 创建分子和细胞基础的关键组件，以了解生理和 平滑肌收缩的病理生理学。 除了众所周知的钙离子非依赖途径外，平滑肌收缩还受到钙离子非依赖性途径的调节 钙依赖途径。钙非依赖性途径的关键成分是肌球蛋白轻链 磷酸酶(MLCP)，其活性受MLCP调节亚基的磷酸化调节， 称为肌球蛋白靶向亚基1(MYPT1)。过去的研究主要集中在RhoA/ROCK途径上， 一种使MYPT1磷酸化的蛋白激酶。然而，最近的研究表明，钙离子不依赖于 MLC磷酸化的调控不能完全用RhoA/ROCK来解释。我们建议MYPT1 磷酸酶是缺失的调节成分，解释了尚未解决的研究问题 了解平滑肌收缩的调节。关于这一重要的监管规定，我们一无所知 组件。我们最近的结果表明，MYPT1磷酸酶在 平滑肌的收缩-松弛循环(Nakamura等人，2007年)。此外，MYPT1磷酸酶是 不被CPI17抑制，CPI17可以有效地抑制MLCP活性，这表明MYPT1磷酸酶是不同的 来自MLCP的分子。基于这些发现，我们提出了以下假设。外部刺激会改变 MYPT1磷酸酶活性导致MYPT1磷酸化水平的变化，从而调节 MLCP活性与RhoA/ROCK途径的调控相一致。拟议的项目将解决 这个假说。首先，我们将从平滑肌中分离MYPT1磷酸酶，并测定其部分氨基酸 用质谱法测定MYPT1亚基的酸序列。根据序列 信息，我们将确定编码MYPT1磷酸酶全酶的基因并进行功能表达 该酶(目标1)。然后，我们将研究MYPT1磷酸酶的特性和调控 分子水平。一个关键问题是MYPT1磷酸酶活性是如何调节的。我们假设 MYPT1磷酸酶的非催化亚基在调节中起着关键作用，我们将研究 包括磷酸化效应在内的非催化亚基功能的质谱分析 (目标2)。在特定目标3中，我们将测试已鉴定的MYPT1磷酸酶的消除对MLCP的影响 MYPT1在平滑肌中的活性和MLC的磷酸化以确认其重要性 磷酸酶。最后，我们将研究外加外源激素对平滑肌MYPT1磷酸酶的调节作用。 刺激物。预计获得的MYPT1磷酸酶信息将为理解 含有平滑肌的器官的生理学和病理生理学。

ABSTRACT: The goal of proposed project is to determine the molecular identity of myosin light chain phosphatase phosphatase (MYPT1 phosphatase) and clarify the regulatory role of this poorly investigated critical component to create a molecular and cellular basis for understanding of the physiology and pathophysiology of smooth muscle contraction. Smooth muscle contraction is regulated by the Ca2+ independent pathway in addition to the well known Ca2+ dependent pathway. The key component of the Ca2+ independent pathway is myosin light chain phosphatase (MLCP), whose activity is regulated by the phosphorylation of the regulatory subunit of MLCP, called myosin targeting subunit 1(MYPT1). The research in the past has centered on the RhoA/ROCK pathway, a protein kinase phosphorylating MYPT1. However, recent studies have suggested that the Ca2+ independent regulation of MLC phosphorylation cannot solely be explained by RhoA/ROCK. We propose that MYPT1 phosphatase is the missing regulatory component that explains the unsolved research problem for understanding smooth muscle contractile regulation. Nothing is known about this important regulatory component. Our recent results have suggested that MYPT1 phosphatase is regulated during the contraction-relaxation cycle in smooth muscle (Nakamura et al., 2007). Furthermore, MYPT1 phosphatase is not inhibited by CPI17,which potently inhibits MLCP activity, suggesting that MYPT1 phosphatase is a different molecule from MLCP. Based upon these findings, we propose the following hypothesis. External stimuli alters the MYPT1 phosphatase activity, which causes the change in the MYPT1 phosphorylation level, thus regulates MLCP activity concertedly with the regulation of the RhoA/ROCK pathway. The proposed project will address this hypothesis. First we will isolate MYPT1 phosphatase from smooth muscle and determine the partial amino acid sequence of the subunits of MYPT1 using a Mass Spectrometry technique. Based upon the sequence information, we will identify the genes encoding the MYPT1 phosphatase holoenzyme and functionally express this enzyme (Aim 1). We will then study the characteristics and the regulation of MYPT1 phosphatase at the molecular level. A key question is how MYPT1 phosphatase activity is regulated. We hypothesize that the non-catalytic subunits of MYPT1 phosphatase play a key role in the regulation, and we will study the regulatory function of the non-catalytic subunits including the effect of phosphorylation using Mass Spectrometry analysis (Aim 2). In Specific Aim 3, we will test the effect of elimination of the identified MYPT1 phosphatase on MLCP activity and MLC phosphorylation in smooth muscle to confirm the importance of the identified MYPT1 phosphatase. Finally we will examine the regulation of MYPT1 phosphatase in smooth muscle by external stimuli. It is anticipated that the obtained information of MYPT1 phosphatase will provide a clue to understand the physiology and pathophysiology of organs containing smooth muscle.