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

By G. Totsukawa, Y. Yamakita, S. Yamashiro, D. Hartshorne, Y. Sasaki, F. Matsumura. Journal of Cell Biology, 2000

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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.

Subfields: Cell Biology, Molecular Biology

View Paper: https://www.semanticscholar.org/paper/2828c120402496232c1c100ce93849dacc4dcf84

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Citations in the corpus

Rho GTPases and cell migration. (2001, background): “It is likely that ROCKs and MLCK act in concert to regulate different aspects of cell contractility, because ROCKs appear to be required for MLC phosphorylation associated with actin filaments in the cell body, whereas MLCK is required at the cell periphery (Totsukawa et al., 2000).”
Functional diversity of protein phosphatase-1, a cellular economizer and reset button. (2004, background): “For instance, Rho-activated Rho-kinase phosphorylates myosin in central stress fibers, whereas Ca(2) /calmodulin-activated MLCK or Cdc42-activated MRCK act on peripheral stress fibers (112, 197, 366).”
RHO GTPASES in neuronal morphogenesis (2000, background): “Both lead to increased phosphorylation of a critical residue in the myosin light chain, which then promotes the ATPase and motor activity of myosin II (ref”
Random versus directionally persistent cell migration (2009, background): “RHOA activates ROCK1, which phosphorylates myosin phosphatase and the regulatory light chain on myosin II to increase actin–myosin contractilit”
Rac and Rho GTPases in cancer cell motility control (2010, background): “It is likely that ROCKs and MLCK act in concert to regulate different aspects of cell contractility, because ROCK appears to be required for MLC phosphorylation associated with actin filaments in the cell body, whereas MLCK is required at the cell periphery [49].”
Viscoelastic retraction of single living stress fibers and its impact on cell shape, cytoskeletal organization, and extracellular matrix mechanics. (2006, background): “The factors that determine the relative contributions of these two pathways to myosin activity are poorly understood, and it has recently been proposed that these pathways are spatially regulated as well (i.e., the contractility of fibers at different locations in the same cell may be under the infl”
Focal Contacts as Mechanosensors (2001, background): “…II–driven contractility in smooth muscle and nonmuscle cells by phosphorylating, and thereby inactivating, the myosin light chain phosphatase (Kimura et al., 1996; Kawano et al., 1999) and possibly by direct phosphorylation of the myosin light chain (Kureishi et al., 1997; Totsukawa et al., 2000).”
The microenvironment of the tumour–host interface (2001, background): “Many signalling molecules are involved in regulating cell motility, including myosin light chain kinase (MLCK”