[Targeting of myosin light chain kinase in smooth muscle cell]

We constructed a plasmid vector to have a 1.4 kb 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 (VSMCs), 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 chemotaf1p4++ motility to the platelet derived growth factor (PDGF), which was supported by the membrane ruffling. However, the transfectants showed neither chemotaxic motility nor developed membrane ruffling, indicating the essential role of MLCK in the motility. The specificity for the targeting was assessed by demonstrating that Rho-kinase activity, which also phosphorylates the myosin light chain (MLC), was well preserved in both SM3 and the transfectants. In spite of this importance of MLCK, PDGF failed to induce MLC 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 myosin-binding property of MLCK (Ye et al. Proc Natl. Acad. Sci. USA 96 6666-6671, 1999).