MYPT1 phosphatase in smooth muscle

平滑肌中的 MYPT1 磷酸酶

• 批准号: 8031260
• 负责人: Mitsuo Ikebe
• 金额: $ 24.68万
• 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 2012-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8031260
• 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

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.

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（MYPT1磷酸酶）的分子身份，并阐明这一研究不足的关键成分的调节作用，为理解肌球蛋白轻链磷酸酶奠定分子和细胞基础。平滑肌收缩的生理学和病理生理学。 除了众所周知的 Ca2+ 依赖性途径外，平滑肌收缩还受到 Ca2+ 独立途径的调节。 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（称为肌球蛋白靶向亚基1（MYPT1））的磷酸化调节。过去的研究主要集中在 RhoA/ROCK 通路，即一种磷酸化 MYPT1 的蛋白激酶。然而，最近的研究表明MLC磷酸化的Ca2+独立调节不能仅仅用RhoA/ROCK来解释。我们认为 MYPT1 磷酸酶是缺失的调节成分，它解释了理解平滑肌收缩调节方面尚未解决的研究问题。关于这个重要的监管组成部分我们一无所知。我们最近的结果表明，MYPT1 磷酸酶在平滑肌的收缩-舒张周期中受到调节（Nakamura 等，2007）。此外，MYPT1磷酸酶不受CPI17抑制，CPI17可有效抑制MLCP活性，表明MYPT1磷酸酶是与MLCP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，从而引起MYPT1磷酸化水平的变化，从而与RhoA/ROCK通路的调节协同调节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 MYPT1 磷酸酶，并使用质谱技术确定 MYPT1 亚基的部分氨基酸序列。根据序列信息，我们将鉴定编码 MYPT1 磷酸酶全酶的基因并功能性表达该酶（目标 1）。我们将从分子水平上研究MYPT1磷酸酶的特性和调控。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发挥关键作用，我们将使用质谱分析研究非催化亚基的调节功能，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 磷酸酶对平滑肌中 MLCP 活性和 MLC 磷酸化的影响，以确认已识别的 MYPT1 磷酸酶的重要性。最后我们将检查外部刺激对平滑肌中 MYPT1 磷酸酶的调节。预计所获得的MYPT1磷酸酶信息将为了解含有平滑肌的器官的生理学和病理生理学提供线索。 公共健康相关性：平滑肌分布在许多器官中，例如脉管系统、气道、消化道、子宫和泌尿系统，并在施加的负荷下维持或改变器官的尺寸。因此，平滑肌在维持脉管系统的血流和气道的气流方面起着至关重要的作用，平滑肌的功能障碍会导致严重的健康问题，例如高血压和哮喘。拟议的项目将确定一个关键但尚未充分研究的调节成分，即一种使肌球蛋白轻链磷酸酶去磷酸化的酶。预计所获得的信息将为了解这些器官系统中平滑肌的功能障碍提供分子基础。