MYPT1 phosphatase in smooth muscle

描述（由申请人提供）：拟议项目的目标是确定肌球蛋白轻链磷酸酶磷酸酶（MYPT1磷酸酶）的分子特性，并阐明这一研究较少的关键成分的调节作用，为理解平滑肌收缩的生理和病理生理建立分子和细胞基础。除了众所周知的Ca2+依赖途径外，平滑肌收缩还受Ca2+独立途径的调节。Ca2+独立通路的关键成分是肌球蛋白轻链磷酸酶（MLCP），其活性受MLCP调控亚基的磷酸化调节，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，这是一种磷酸化MYPT1的蛋白激酶。然而，最近的研究表明，Ca2+对MLC磷酸化的独立调控不能仅仅用RhoA/ROCK来解释。我们认为MYPT1磷酸酶是缺失的调控成分，解释了理解平滑肌收缩调控的未解决的研究问题。对于这一重要的监管组成部分，人们一无所知。我们最近的研究结果表明，MYPT1磷酸酶在平滑肌的收缩-松弛周期中受到调节（Nakamura等，2007）。此外，MYPT1磷酸酶不受CPI17的抑制，而CPI17能有效抑制MLCP活性，这表明MYPT1磷酸酶与MLCP是不同的分子。基于这些发现，我们提出以下假设。外部刺激改变MYPT1磷酸酶活性，引起MYPT1磷酸化水平的改变，从而与RhoA/ROCK通路的调控协同调节MLCP活性。拟议的项目将解决这一假设。首先，我们将从平滑肌中分离出MYPT1磷酸酶，并使用质谱技术确定MYPT1亚基的部分氨基酸序列。基于序列信息，我们将鉴定编码MYPT1磷酸酶全酶的基因并功能性表达该酶（目的1）。然后我们将在分子水平上研究MYPT1磷酸酶的特性和调控。一个关键问题是如何调节MYPT1磷酸酶活性。我们假设MYPT1磷酸酶的非催化亚基在调控中发挥关键作用，我们将使用质谱分析研究非催化亚基的调控功能，包括磷酸化的影响（Aim 2）。在Specific Aim 3中，我们将测试消除已鉴定的MYPT1磷酸酶对平滑肌MLCP活性和MLC磷酸化的影响，以确认已鉴定的MYPT1磷酸酶的重要性。最后，我们将研究外部刺激对平滑肌中MYPT1磷酸酶的调节。期望获得的MYPT1磷酸酶的信息将为了解含平滑肌器官的生理和病理生理提供线索。

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: Smooth muscle is distributed in many organs such as vasculature, airway, digestive tract, uterus, and urinary system, and maintains or alters the dimensions of an organ against imposed loads. Hence, smooth muscle plays a critical role in maintaining blood flow in vasculature and airflow in airway and the malfunction of smooth muscle causes severe health problems such as high blood pressure and asthma. The proposed project will identify a critical, but under-investigated regulatory component, an enzyme that dephosphorylates myosin light chain phosphatase. It is anticipated that the obtained information will provide a molecular basis to understand the malfunction of smooth muscle in these organ systems.