Rho-kinase-mediated Ca2+-independent contraction in rat embryo fibroblasts

Daniel A. Emmert, Judy A. Fee, Zoe M. Goeckeler, Jeremy M. Grojean, Tetsuro Wakatsuki, Elliot L. Elson, B. Paul Herring, Patricia J. Gallagher, Robert B. Wysolmerski

Research output: Contribution to journalArticlepeer-review

6 Scopus citations


Thus far, determining the relative contribution of Ca 2+/calmodulin-dependent myosin light chain kinase (MLCK) and Ca 2+-independent Rho-kinase pathways to myosin II activation and contraction has been difficult. In this study, we characterize the role of Rho-kinase in a rat embryo fibroblast cell line (REF-52), which contains no detectable MLCK. No endogenous MLCK could be detected in REF-52 cells by either Western or Northern blot analysis. In the presence or absence of Ca 2+, thrombin or lysophosphatidic acid (LPA) increased RhoA activity and Rho-kinase activity, correlating with isometric tension development and myosin II regulatory light chain (RLC) phosphorylation. Resting tension is associated with a basal phosphorylation of 0.31 ± 0.02 mol PO 4/mol RLC, whereas upon LPA or thrombin treatment myosin II RLC phosphorylation increases to 1.08 ± 0.05 and 0.82 ± 0.05 mol PO4/mol RLC, respectively, within 2.5 min. Ca2+ chelation has minimal effect on the kinetics and magnitude of isometric tension development and RLC phosphorylation. Treatment of REF-52 cells with the Rho-kinase-specific inhibitor Y-27632 abolished thrombin- and LPA-stimulated contraction and RLC phosphorylation. These results suggest that Rho-kinase is sufficient to activate myosin II motor activity and contraction in REF-52 cells.

Original languageEnglish
Pages (from-to)C8-C21
JournalAmerican Journal of Physiology - Cell Physiology
Issue number1 55-1
StatePublished - Jan 2004


  • Myosin II regulatory light chain phosphorylation
  • Myosin light chain kinase
  • RhoA


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