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Refulatory activity of myosin light chain kinase in smooth muscle cell

平滑肌细胞肌球蛋白轻链激酶的再激活活性

基本信息

批准号：
10470022
负责人：
KOHAMA Kazuhiro
金额：
$ 8.7万
依托单位：
Gunma University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1998
资助国家：
日本
起止时间：
1998 至 1999
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10470022/
关键词：
myosin light chain kinase myosin actin smooth muscle phosphorylation cell motility 遊走能

项目摘要

We constructed a plasmid vector having a 1.4-kilobase pair insert of myosin light chain kinase (MLCK) cDNA in an antisense direction to express antisense mRNA. The construct was then transfected to SM3, a cell line from vascular smooth muscle cells, producing a few stable transfectants. The down-regulation of MLCK expression in the transfectants was confirmed by both Northern and Western blots. The control SM3 showed chemotaxic motility to plastelet-derived growth factor-BB, which was supported by lamellipodia. However, the transfectants showed neither chemotaxic motility nor developed lamellipodia, indicating the essential role of MLCK in the motility. The specifity for the targeting was assessed by a few tests including the rescue experiment. Despite this importance of MLCK, platelet-derived growth factor-BB failed to induce MLC20 phosphorylation in not only the transfectants but also in SM3.MLCK of smooth muscle is multifunctional, being composed of N-terminal actin-binding domain, central kinase domain, and C-terminal myosin-binding domain. We also found that the C-terminal domain could stimulate the actin-activated ATPase activity of smooth muscle myosin without phosphorylating its light chain. We discussed that the C-terminal domain may develop the membrane ruffling.

我们构建了一个质粒载体，在反义方向上具有肌球蛋白轻链激酶（MLCK）cDNA的1.4-内切酶对插入片段，以表达反义mRNA。然后将该构建体转染到来自血管平滑肌细胞的细胞系SM 3中，产生一些稳定的转染子。通过北方和Western印迹证实了转染子中MLCK表达的下调。对照SM 3显示出对血小板衍生生长因子-BB的趋化性运动，其由板状伪足支持。然而，转染子既没有表现出趋化性运动，也没有发达的片状伪足，表明MLCK在运动中的重要作用。靶向的特异性通过包括拯救实验在内的一些测试来评估。尽管MLCK如此重要，但血小板源生长因子-BB不仅不能诱导转染细胞中MLC 20的磷酸化，也不能诱导SM 3细胞中MLC 20的磷酸化。平滑肌MLCK是多功能的，由N端肌动蛋白结合结构域、中央激酶结构域和C端肌球蛋白结合结构域组成。我们还发现，C端结构域可以刺激肌动蛋白激活的平滑肌肌球蛋白的ATP酶活性，而不磷酸化其轻链。我们讨论了C-末端结构域可能产生膜皱褶。