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Regulatory Mechanisms of Smooth Muscle Contraction

平滑肌收缩的调节机制

基本信息

批准号：
08044269
负责人：
NAKANO Takeshi
金额：
$ 1.41万
依托单位：
Mie University
依托单位国家：
日本
项目类别：
Grant-in-Aid for international Scientific Research
财政年份：
1996
资助国家：
日本
起止时间：
1996 至 1997
项目状态：
已结题

项目摘要

The regulatory mechanisms of myosin phosphatase (MP), the domain structure of the large subunit of MP (MYPT1) and human isoform of MYPT1 have been investigated.1.The activity of myosin phosphatase is inhibited by the phosphorylation of the large regulatory subunit (MYPT1). The kinases involved are the endogenous kinase and Pho-kinase. Introduciton of Rho-kinase into the permeabilized smooth muscle provoked a contraction. These results suggest that Rho-Kinase modulates smooth muscle contraction through the regulation of MP.2.MYPT1 binds to PP1c and also to myosin and thereby effects an activation of phosphatase activity. To investigate the sequence involved in these interaction various mutants of MYPT1 were expressed and assayd. Two regions of MYPT1 are important for binding of PP1c, i.e.the ankyrin repeats and the N-terminal sequence 1 to 38. The C-terminal ankyrin repeats appear dominant in binding to PP1c but there is an interaction that N-terminal repeats may also be involved. The N-terminal peptide has two apparent functions ; the binding to PP1c via the consensus binding sequence and activation of PP1c by the sequence to 16.3.Two cDNA clones encoding the human large subunit of myosin phosphatase were isolated and termed MYPT1 and MYPT2. Sequence analysis revealed that MYPT1 and MYPT2 contained 1030 and 982 amino acids, respectively. The amino acid sequence of MYPT1 was more than 89% identical to that of the rat MYPT1, and MYPT2 contained 61% identity with human MYPT1. Conserved sequences for the two isoforms were in the ankyrin repeats, the leucine zipper motif, and the central 50 residues containing the putative regulatory phosphorylation site. MYPT1 was present mainly in smooth muscle, while MYPT2 is dominant in heart. FISH analysis placed human MYPT1 and MYPT2 genes over chromosome 12q5-21.2 and 1q32-1, respectively.

本研究对肌球蛋白磷酸酶（myosin phosphatase，MP）、MP大亚基（myosin large subunit，MYPT 1）的结构域结构以及MYPT 1的人亚型的调控机制进行了探讨。所涉及的激酶是内源性激酶和Pho-激酶。将Rho激酶导入透化平滑肌引起收缩。这些结果表明Rho激酶通过调节MP.2调节平滑肌收缩。MYPT 1与PP 1c结合，也与肌球蛋白结合，从而影响磷酸酶活性的激活。为了研究参与这些相互作用的序列，表达并测定了MYPT 1的各种突变体。MYPT 1的两个区域对于PP 1c的结合是重要的，即锚蛋白重复序列和N-末端序列1至38。C-末端锚蛋白重复序列在与PP 1c的结合中占主导地位，但N-末端重复序列也可能参与相互作用。N-末端肽具有两种明显的功能：通过共有结合序列与PP 1c结合和通过序列16.3激活PP 1c。分离了两个编码人肌球蛋白磷酸酶大亚基的cDNA克隆，命名为MYPT 1和MYPT 2。序列分析表明，MYPT 1和MYPT 2分别含有1030和982个氨基酸。MYPT 1与大鼠MYPT 1的氨基酸序列同源性在89%以上，MYPT 2与人MYPT 1的氨基酸序列同源性为61%。两种亚型的保守序列位于锚蛋白重复序列、亮氨酸拉链基序和含有推定的调节磷酸化位点的中心50个残基中。MYPT 1主要存在于平滑肌中，而MYPT 2在心脏中占优势。FISH分析将人MYPT 1和MYPT 2基因分别定位于染色体12 q5 -21.2和1 q32 -1。