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Physiological role of myosin light chain kinase in regulating smooth muscle contraction : An approach by gene targeting followed by rescue.

肌球蛋白轻链激酶在调节平滑肌收缩中的生理作用：一种基因靶向随后救援的方法。

基本信息

批准号：
06454156
负责人：
KOHAMA Kazuhiro
金额：
$ 4.42万
依托单位：
Gunma University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1994
资助国家：
日本
起止时间：
1994 至 1995
项目状态：
已结题

项目摘要

Smooth muscle contraction is induced by an ATE-dependent interaction between actin and myosin, on which Ca^<2+> exerts regulatory activity, The site of action of Ca^<2+> is calmodulin (CaM). The role of CaM is to activate myosin light chain kinase (MLCK), which is able to phosphorylate the 20 kDa regulatory light chain of myosin. The myosin thus phosphorylated is in an active form that is able to interact with actin ATP-dependently. But the effect of Ca^<2+> on the actual contraction of smooth muscle is much more complex. There are regulatory ways by Ca^<2+> which are not subject to phosphorylation.To approach such a problem, we are interested in the actin binding activity of MLCK, a property that has been known for many years. We examined the effect of the actin-binding activity, and found that the activity regulates the interaction in association with CaM.Thus, MLCK regulates the interaction by its kinase activity as well as its actin-binding activity.We transfected plasmid containing antisence DNA of MLCK into smooth muscle cells and observed a reduction in the amount of MLCK.We also produced MLCK fragments by transfecting expression plasmieds containing various length of sence DNA of MLCIK into E.Coli. We obtained a few fragments that regulate the actin-myosin interaction. These results enable the study to examine physiological role of MLCK by introducing the recombinant fragments into the cells where endogenouse levels of MLCK is lowered.

平滑肌收缩是由肌动蛋白和肌球蛋白之间的ATE依赖性相互作用引起的，Ca^2+对这种相互作用具有调节作用，Ca^2+的作用部位是钙调蛋白（CaM）。CaM的作用是激活肌球蛋白轻链激酶（MLCK），其能够磷酸化肌球蛋白的20 kDa调节轻链。这样磷酸化的肌球蛋白处于能够与肌动蛋白ATP依赖性相互作用的活性形式。但Ca^<2+>对平滑肌实际收缩的影响要复杂得多。Ca^<2+>的调节方式不受磷酸化的影响，为了解决这一问题，我们对MLCK的肌动蛋白结合活性很感兴趣，这是一个多年前就已知道的性质。我们检查了肌动蛋白结合活性的影响，发现该活性调节与CaM的相互作用。因此，MLCK通过其激酶活性以及其肌动蛋白-我们将含有MLCK反义DNA的质粒转染到平滑肌细胞中，观察到MLCK的量减少。将MLCIK的DNA插入大肠杆菌中。我们获得了一些片段，调节肌动蛋白-肌球蛋白的相互作用。这些结果使得该研究能够通过将重组片段引入MLCK内源水平降低的细胞中来检查MLCK的生理作用。