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 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 platelet-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 specificity 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. 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 phosphorylating its light chain (Ye, L.-H., Kishi, H., Nakamura, A., Okagaki, T., Tanaka, T., Oiwa, K., and Kohama, K. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 6666-6671).
Kishi
H
H
Department of Pharmacology, Gunma University School of Medicine, Gunma 371-8511, Japan.
Mikawa
T
T
Seto
M
M
Sasaki
Y
Y
Kanayasu-Toyoda
T
T
Yamaguchi
T
T
Imamura
M
M
Ito
M
M
Karaki
H
H
Bao
J
J
Nakamura
A
A
Ishikawa
R
R
Kohama
K
K
D000205:Actomyosin / Q000378:metabolism*; D000818:Animals; D001011:Aorta; D000077214:Becaplermin; D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects / Q000648:ultrastructure*; D002633:Chemotaxis; D003001:Cloning, Molecular; D005822:Genetic Vectors; D006801:Humans; D009131:Muscle, Smooth, Vascular / Q000166:cytology* / Q000502:physiology; D009219:Myosin-Light-Chain Kinase / Q000235:genetics* / Q000378:metabolism*; D010982:Platelet-Derived Growth Factor / Q000494:pharmacology; D020574:Proto-Oncogene Proteins c-sis; D011817:Rabbits; D017479:Receptors, Platelet-Derived Growth Factor / Q000502:physiology; D011994:Recombinant Proteins / Q000378:metabolism / Q000494:pharmacology; D015183:Restriction Mapping; D014158:Transcription, Genetic; D014162:Transfection
D010982:Platelet-Derived Growth Factor; D020574:Proto-Oncogene Proteins c-sis; D011994:Recombinant Proteins; D000077214:Becaplermin; D000205:Actomyosin; D017479:Receptors, Platelet-Derived Growth Factor; D009219:Myosin-Light-Chain Kinase