Stable transfectants of smooth muscle cell line lacking the expression of myosin light chain kinase and their characterization with respect to the actomyosin system

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 antise nse mRNA The construct was then transfected to SM3, a cell line from vascul ar smooth muscle cells, producing a few stable transfectants. The down-regu lation of MLCK expression in the transfectants was confirmed by both Northe rn and Western blots. The control SM3 showed chemotaxic motility to platele t-derived growth factor-BB, which was supported by lamellipodia. However, t he transfectants showed neither chemotaxic motility nor developed lamellipo dia, indicating the essential role of MLCK in the motility. The specificity for the targeting was assessed by a few tests including the rescue experim ent. Despite this importance of MLCK, platelet-derived growth factor-BB fai led to induce MLC20 phosphorylation in not only the transfectants but also in SM3. The mode in which MLCK was involved in the development of membrane ruffling is discussed with special reference to the novel property of MLCK that stimulates the ATPase activity of smooth muscle myosin without phospho rylating its light chain.