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)的分子特性，并阐明这一研究较少的关键成分的调节作用，以创建一个分子和细胞基础，以了解平滑肌收缩的生理学和病理生理学。除了众所周知的钙依赖途径外，平滑肌收缩还受钙非依赖途径的调节。肌球蛋白轻链磷酸酶(MLCP)是钙非依赖性途径的关键组成部分，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)。过去的研究集中在RhoA/ROCK通路上，这是一种磷酸化MYPT1的蛋白激酶。然而，最近的研究表明，RhoA/ROCK不能完全解释钙离子对MLC磷酸化的非依赖性调节。我们认为，MYPT1磷酸酶是缺失的调节成分，解释了尚未解决的研究问题，以了解平滑肌收缩调节。对于这一重要的监管组成部分，人们一无所知。我们最近的结果表明，MYPT1磷酸酶在平滑肌的收缩-松弛周期中受到调节(Nakamura等人，2007年)。此外，CPI17不抑制MYPT1磷酸酶，而CPI17能有效地抑制MLCP活性，这表明MYPT1磷酸酶是不同于MLCP的分子。基于这些发现，我们提出了以下假设。外界刺激改变MYPT1磷酸酶活性，从而引起MYPT1磷酸化水平的改变，从而通过调节RhoA/ROCK通路来调节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，并用质谱学技术测定MYPT1亚基的部分氨基酸序列。根据序列信息，我们将确定编码MYPT1磷酸酶全酶的基因，并在功能上表达该酶(目标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关键作用，我们将利用质谱仪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，我们将测试已鉴定的MYPT1磷酸酶的消除对平滑肌MLCP活性和MLC磷酸化的影响，以证实已鉴定的MYPT1磷酸酶的重要性。最后，我们将研究外界刺激对平滑肌MYPT1磷酸酶的调节作用。MYPT1磷酸酶的获得将为理解含有平滑肌的器官的生理学和病理生理学提供线索。 公共卫生相关性：平滑肌分布在许多器官中，如血管、呼吸道、消化道、子宫和泌尿系统，并在施加负荷时维持或改变器官的尺寸。因此，平滑肌在维持血管中的血液流动和呼吸道中的气流方面起着至关重要的作用，而平滑肌功能障碍会导致严重的健康问题，如高血压和哮喘。拟议的项目将确定一个关键但未被研究的调节成分，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的平滑肌功能障碍提供分子基础。