Refulatory activity of myosin light chain kinase in smooth muscle cell

我们构建了一个1.4千碱基对的质粒载体，以反义方向插入肌球蛋白轻链激酶（myosin light chain kinase， MLCK） cDNA表达反义mRNA。然后将构建物转染到血管平滑肌细胞系SM3中，产生一些稳定的转染物。Northern和Western blot均证实了转染后MLCK表达的下调。对照SM3对质体源性生长因子- bb具有趋化活性，并有板足支持。然而，这些转基因植物既没有趋化运动，也没有形成板足，这表明MLCK在运动中发挥了重要作用。通过包括救援实验在内的一些试验评估了靶向的特异性。尽管MLCK具有如此重要的作用，但血小板衍生生长因子- bb不仅在转染物中未能诱导MLC20磷酸化，而且在SM3中也未能诱导MLC20磷酸化。平滑肌的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端结构域可能产生膜褶皱。

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.