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

1 Departments of Pathology, Saint Louis University School of Medicine, St. Louis 63104; 2 Department of Biochemistry & Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri 63110; and 3 Department of Physiology, Indiana University School of Medicine, Indianapolis, Indiana 46202 Submitted 16 September 2002 ; accepted in final form 22 August 2003 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 Rhokinase 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 PO 4 /mol RLC, respectively, within 2.5 min. Ca 2+ 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. myosin light chain kinase; RhoA; myosin II regulatory light chain phosphorylation Address for reprint requests and other correspondence: R. B. Wysolmerski, Dept. of Pathology, Saint Louis Univ. School of Medicine, 1402 S. Grand Blvd., St. Louis, MO 63104 (E-mail: wysolmer{at}slucare1.sluh.edu ).