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J Biol Chem, Vol. 275, Issue 2, 1414-1420, January 14, 2000
From the a Department of Pharmacology and b First
Department of Internal Medicine, Gunma University School of Medicine,
Gunma 371-8511, Japan, the c Department of Cell Biology, Cornell
University Medical College, New York, New York 10021, e Asahi
Chemical Industry Co. Ltd., Shizuoka 416-0934, Japan, f National
Institute of Health Sciences, Tokyo 158-8501, Japan, g National
Center of Neurology and Psychiatry, Tokyo 187-8551, Japan,
h First Department of Internal Medicine, Mie University School
of Medicine, Mie 514-8507, Japan, and the i Department of
Veterinary Pharmacology, Graduate School of Agriculture and Life
Science, University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan
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).
Stable Transfectants of Smooth Muscle Cell Line Lacking the
Expression of Myosin Light Chain Kinase and Their Characterization with
Respect to the Actomyosin System*
*
This work was supported by National Institutes of Health
Grants HL54128, HL56987, and HL62175 to (T. M.), grants (to K. K.) from the Mitsubishi Foundation, Smoking Research Foundation, and Ministry of Education, Science and Culture of Japan, and the Special Coordination Funds for Promoting Science and Technology of the Science
and Technology Agency of Japanese Government.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
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