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© The Rockefeller University Press, 0021-9525/2001//569 $5.00
The Journal of Cell Biology, Volume 153, Number 3, , 2001 569-584

Rho-Kinase–Mediated Contraction of Isolated Stress Fibers

^a Department of Structural Analysis, National Cardiovascular Center Research Institute, Suita, Osaka 565-8565, Japan
^b Division of Signal Transduction, Nara Institute of Science and Technology, Ikoma, Nara 630-0101, Japan
^c Department of Cell Pharmacology, Nagoya University School of Medicine, Showa, Nagoya, Aichi 466-8550, Japan
^d Center for Cardiovascular Research, University of Rochester, Rochester, New York 14642
Department of Structural Analysis, National Cardiovascular Center Research Institute, 5 Fujishirodai, Suita, Osaka 565, Japan.81-6-6833-5012 ext

It is widely accepted that actin filaments and the conventional double-headed myosin interact to generate force for many types of nonmuscle cell motility, and that this interaction occurs when the myosin regulatory light chain (MLC) is phosphorylated by MLC kinase (MLCK) together with calmodulin and Ca²⁺. However, recent studies indicate that Rho-kinase is also involved in regulating the smooth muscle and nonmuscle cell contractility. We have recently isolated reactivatable stress fibers from cultured cells and established them as a model system for actomyosin-based contraction in nonmuscle cells. Here, using isolated stress fibers, we show that Rho-kinase mediates MLC phosphorylation and their contraction in the absence of Ca²⁺. More rapid and extensive stress fiber contraction was induced by MLCK than was by Rho-kinase. When the activity of Rho-kinase but not MLCK was inhibited, cells not only lost their stress fibers and focal adhesions but also appeared to lose cytoplasmic tension. Our study suggests that actomyosin-based nonmuscle contractility is regulated by two kinase systems: the Ca²⁺-dependent MLCK and the Rho-kinase systems. We propose that Ca²⁺ is used to generate rapid contraction, whereas Rho-kinase plays a major role in maintaining sustained contraction in cells.

Key Words: Rho • Rho-kinase • stress fiber • contraction • myosin regulatory light chain

© 2001 The Rockefeller University Press

Abbreviations used in this paper: CC, coiled coil; GFP, green fluorescent protein; GST, glutathione S-transferase; MBS, myosin-binding subunit; MLC, myosin regulatory light chain; MLCK, MLC kinase; RB, Rho-binding; Rho, Ras homology; TEA, triethanolamine (2, 2', 2''-nitrilotriethanol).

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