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

43 Scopus citations

Abstract

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 (US)
Pages (from-to)C8-C21
JournalAmerican Journal of Physiology - Cell Physiology
Volume286
Issue number1 55-1
DOIs
StatePublished - Jan 2004

Keywords

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

ASJC Scopus subject areas

  • Clinical Biochemistry
  • Cell Biology
  • Physiology

Fingerprint Dive into the research topics of 'Rho-kinase-mediated Ca<sup>2+</sup>-independent contraction in rat embryo fibroblasts'. Together they form a unique fingerprint.

Cite this