Properties of filament-bound myosin light chain kinase

J Biol Chem. 1999 Feb 26;274(9):5987-94. doi: 10.1074/jbc.274.9.5987.

Abstract

Myosin light chain kinase binds to actin-containing filaments from cells with a greater affinity than to F-actin. However, it is not known if this binding in cells is regulated by Ca2+/calmodulin as it is with F-actin. Therefore, the binding properties of the kinase to stress fibers were examined in smooth muscle-derived A7r5 cells. Full-length myosin light chain kinase or a truncation mutant lacking residues 2-142 was expressed as chimeras containing green fluorescent protein at the C terminus. In intact cells, the full-length kinase bound to stress fibers, whereas the truncated kinase showed diffuse fluorescence in the cytoplasm. After permeabilization with saponin, the fluorescence from the truncated kinase disappeared, whereas the fluorescence of the full-length kinase was retained on stress fibers. Measurements of fluorescence intensities and fluorescence recovery after photobleaching of the full-length myosin light chain kinase in saponin-permeable cells showed that Ca2+/calmodulin did not dissociate the kinase from these filaments. However, the filament-bound kinase was sufficient for Ca2+-dependent phosphorylation of myosin regulatory light chain and contraction of stress fibers. Thus, dissociation of myosin light chain kinase from actin-containing thin filaments is not necessary for phosphorylation of myosin light chain in thick filaments. We note that the distance between the N terminus and the catalytic core of the kinase is sufficient to span the distance between thin and thick filaments.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Actin Cytoskeleton / enzymology*
  • Actins / metabolism
  • Amino Acid Sequence
  • Base Sequence
  • Calcium / metabolism
  • Calmodulin / metabolism
  • Cell Line
  • DNA Primers
  • Green Fluorescent Proteins
  • Luminescent Proteins / genetics
  • Molecular Sequence Data
  • Muscle, Smooth / cytology
  • Muscle, Smooth / enzymology
  • Myosin-Light-Chain Kinase / genetics
  • Myosin-Light-Chain Kinase / metabolism*
  • Phosphorylation
  • Protein Binding
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism

Substances

  • Actins
  • Calmodulin
  • DNA Primers
  • Luminescent Proteins
  • Recombinant Fusion Proteins
  • Green Fluorescent Proteins
  • Myosin-Light-Chain Kinase
  • Calcium