MYPT1 phosphatase in smooth muscle

平滑肌中的 MYPT1 磷酸酶

• 批准号: 8207884
• 负责人: Mitsuo Ikebe
• 金额: $ 20.56万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-01-03 至 2013-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8207884
• 关键词: Address; Affinity Chromatography; Agonist; Amino Acid Sequence; Amino Acids; Ants; Asses; Asthma; Blood flow; Cyclic Nucleotides; Dimensions; Enzymes; Functional disorder; Gastrointestinal tract structure; Gene Silencing; Genes; Goals; Health; Holoenzymes; Hypertension; Mass Spectrum Analysis; Molecular; Muscle Contraction; Myosin ATPase; Myosin Regulatory Light Chains; Organ; Pathway interactions; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphotransferases; Physiology; Play; Property; Protein Dephosphorylation; Protein Kinase; Regulation; Relaxation; Research; Rho-associated kinase; Role; Series; Site; Smooth Muscle; Specificity; Stimulus; Techniques; Testing; Up-Regulation; Urinary system; Uterus; base; body system; inhibitor/antagonist; membrane-associated placental tissue protein 1; myosin phosphatase; rho; study characteristics

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.

摘要：拟议项目的目标是确定肌球蛋白轻链的分子身份 磷酸酶（MYPT1 磷酸酶）并阐明这种研究不足的调节作用 为理解生理学和理解奠定分子和细胞基础的关键组成部分 平滑肌收缩的病理生理学。 除了众所周知的途径外，平滑肌收缩还受到 Ca2+ 独立途径的调节 Ca2+ 依赖性途径。 Ca2+ 独立途径的关键组成部分是肌球蛋白轻链 磷酸酶（MLCP），其活性受MLCP调节亚基磷酸化的调节， 称为肌球蛋白靶向亚基 1 (MYPT1)。过去的研究主要集中在RhoA/ROCK通路上， 一种磷酸化 MYPT1 的蛋白激酶。然而，最近的研究表明，Ca2+ 独立 MLC 磷酸化的调节不能仅用 RhoA/ROCK 来解释。我们建议 MYPT1 磷酸酶是缺失的调节成分，它解释了未解决的研究问题 了解平滑肌收缩调节。对于这项重要的监管规定一无所知 成分。我们最近的结果表明 MYPT1 磷酸酶在 平滑肌的收缩-舒张循环（Nakamura et al., 2007）。此外，MYPT1 磷酸酶是 不受 CPI17 抑制，而 CPI17 有效抑制 MLCP 活性，表明 MYPT1 磷酸酶是一种不同的磷酸酶 MLCP 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磷酸化水平的变化，从而调节 MLCP 活性与 RhoA/ROCK 通路的调节一致。拟议的项目将解决 这个假设。首先我们从平滑肌中分离出MYPT1磷酸酶并测定部分氨基 使用质谱技术分析 MYPT1 亚基的酸序列。根据顺序 信息，我们将鉴定编码 MYPT1 磷酸酶全酶的基因并进行功能表达 这种酶（目标 1）。接下来我们将研究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控中发挥着关键作用，我们将研究其调控机制 使用质谱分析非催化亚基的功能，包括磷酸化的影响 （目标 2）。在具体目标 3 中，我们将测试消除已识别的 MYPT1 磷酸酶对 MLCP 的影响 平滑肌中的活性和 MLC 磷酸化，以确认已识别的 MYPT1 的重要性 磷酸酶。最后我们将检查外部环境对平滑肌中 MYPT1 磷酸酶的调节。 刺激。预计所获得的MYPT1磷酸酶信息将为理解提供线索。 含有平滑肌的器官的生理学和病理生理学。