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（由申请方提供）：拟定项目的目标是确定肌球蛋白轻链磷酸酶磷酸酶（MYPT 1磷酸酶）的分子特性，并阐明这种研究不足的关键组分的调节作用，为理解平滑肌收缩的生理学和病理生理学奠定分子和细胞基础。 平滑肌收缩除了由众所周知的Ca 2+依赖性途径调节外，还由Ca 2+非依赖性途径调节。钙非依赖性途径的关键组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（MYPT 1））的磷酸化调节。过去的研究集中在RhoA/ROCK通路，一种磷酸化MYPT 1的蛋白激酶。然而，最近的研究表明，MLC磷酸化的Ca 2+非依赖性调节不能单独用RhoA/ROCK来解释。我们认为MYPT 1磷酸酶是缺失的调节成分，解释了理解平滑肌收缩调节的未解决的研究问题。对这一重要的监管组成部分一无所知。我们最近的结果表明，MYPT 1磷酸酶在平滑肌的收缩-舒张周期中受到调节（中村等人，2007年）。此外，MYPT 1磷酸酶不受CPI 17的抑制，CPI 17可有效抑制MLCP活性，表明MYPT 1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。外界刺激改变MYPT 1磷酸酶活性，引起MYPT 1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MYPT 1磷酸酶，并使用质谱技术确定MYPT 1亚基的部分氨基酸序列。基于序列信息，我们将鉴定编码MYPT 1磷酸酶全酶的基因并功能性表达该酶（Aim 1）。我们将在分子水平上研究MYPT 1磷酸酶的特性和调控。一个关键问题是MYPT 1磷酸酶活性是如何调节的。我们假设MYPT 1磷酸酶的非催化亚基在调节中起关键作用，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（目的2）。在特定目标3中，我们将检测消除已鉴别MYPT 1磷酸酶对平滑肌中MLCP活性和MLC磷酸化的影响，以确认已鉴别MYPT 1磷酸酶的重要性。最后，我们将研究MYPT 1磷酸酶在平滑肌的调节由外部刺激。预期所获得的MYPT 1磷酸酶的信息将为了解含有平滑肌的器官的生理和病理生理提供线索。 公共卫生相关性：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，并且维持或改变器官的尺寸以抵抗施加的负荷。因此，平滑肌在维持脉管系统中的血流和气道中的气流中起关键作用，并且平滑肌的功能障碍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的，但研究不足的监管组成部分，一种酶，去磷酸化肌球蛋白轻链磷酸酶。预计所获得的信息将提供分子基础，以了解这些器官系统中的平滑肌功能障碍。