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Published online 21 August 2000. doi:10.1083/jcb.150.4.797
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© The Rockefeller University Press, 0021-9525/2000//797 $5.00
The Journal of Cell Biology, Volume 150, Number 4, , 2000 797-806

Original Article

Distinct Roles of Rock (Rho-Kinase) and Mlck in Spatial Regulation of Mlc Phosphorylation for Assembly of Stress Fibers and Focal Adhesions in 3t3 Fibroblasts



Go Totsukawaa, Yoshihiko Yamakitaa, Shigeko Yamashiroa, David J. Hartshorneb, Yasuharu Sasakic, and Fumio Matsumuraa

a Department of Molecular Biology and Biochemistry, Rutgers University, Nelson Lab, Busch Campus, Piscataway, New Jersey 08855
b Muscle Biology Group, University of Arizona, Tucson, Arizona 85721
c Frontier 21 Project, Life Science Research Center, Asahi Chemical Industry Co. Ltd., Samejima, Fuji, Shizuoka 416-0934, Japan
Department of Molecular Biology and Biochemistry, Rutgers University, Nelson Lab, Busch Campus, Piscataway, NJ 08855.(732) 445-4213(732) 445-2838

matsumura{at}mbcl.rutgers.edu

ROCK (Rho-kinase), an effector molecule of RhoA, phosphorylates the myosin binding subunit (MBS) of myosin phosphatase and inhibits the phosphatase activity. This inhibition increases phosphorylation of myosin light chain (MLC) of myosin II, which is suggested to induce RhoA-mediated assembly of stress fibers and focal adhesions. ROCK is also known to directly phosphorylate MLC in vitro; however, the physiological significance of this MLC kinase activity is unknown. It is also not clear whether MLC phosphorylation alone is sufficient for the assembly of stress fibers and focal adhesions.

We have developed two reagents with opposing effects on myosin phosphatase. One is an antibody against MBS that is able to inhibit myosin phosphatase activity. The other is a truncation mutant of MBS that constitutively activates myosin phosphatase. Through microinjection of these two reagents followed by immunofluorescence with a specific antibody against phosphorylated MLC, we have found that MLC phosphorylation is both necessary and sufficient for the assembly of stress fibers and focal adhesions in 3T3 fibroblasts. The assembly of stress fibers in the center of cells requires ROCK activity in addition to the inhibition of myosin phosphatase, suggesting that ROCK not only inhibits myosin phosphatase but also phosphorylates MLC directly in the center of cells. At the cell periphery, on the other hand, MLCK but not ROCK appears to be the kinase responsible for phosphorylating MLC. These results suggest that ROCK and MLCK play distinct roles in spatial regulation of MLC phosphorylation.

Key Words: myosin phosphatase • myosin phosphorylation • stress fibers • focal adhesions • RhoA

© 2000 The Rockefeller University Press

Abbreviations used in this paper: MBS, myosin binding subunit of myosin phosphatase; MLC, regulatory light chain; MLCK, myosin light chain kinase; PP1c, catalytic subunit of type 1 protein phosphatase; ROCK, Rho-associated kinase.


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