Myosin light chain kinase/actin interaction in phorbol dibutyrate-stimulated smooth muscle cells

J Pharmacol Sci. 2011;116(1):116-27. doi: 10.1254/jphs.10296fp. Epub 2011 May 3.

Abstract

Previous work has suggested that in addition to its kinase activity, myosin light chain kinase (MLCK) exhibits non-kinase properties within its N-terminus that could influence cytoskeletal organization of smooth muscle cells (A. Nakamura et al. Biochem Biophys Res Commun. 2008;369:135-143). Myosin ATPase activity measurements indicate that the 26-41 peptide of MLCK significantly decreases ATPase activity as the concentration of this peptide increases. Sliding velocity of actin-filaments on myosin and stress responses in skinned smooth muscle tissue are also inhibited. Peptide-mediated uptake and the microinjection technique in cells indicate that the peptide was necessary for actin-filament stabilization. Fluorescence resonance energy transfer analysis indicated that in the presence of MLCK, α-actin but not β-actin remodeled during phorbol 12,13-dibutyrate (PDBu)-induced contractions. PDBu also induced podosomes in the cell. When MLCK expression was down-regulated by introduction of RNAi for MLCK by lentivirus vector into the cells, we failed to observe the podosome induction upon PDBu stimulation. Rescue experiments indicate that the non-kinase activity of MLCK plays an important role in maintaining actin stress fibers and in the PDBu-induced reorganization of actin-filaments in smooth muscle cells.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Actins / metabolism*
  • Animals
  • Cell Line
  • Cell Surface Extensions / drug effects
  • Cell Surface Extensions / metabolism
  • Cell Surface Extensions / ultrastructure
  • Chickens
  • Cytoskeleton / drug effects
  • Cytoskeleton / metabolism*
  • Enzyme Inhibitors / metabolism
  • Gene Silencing
  • Guinea Pigs
  • In Vitro Techniques
  • Kinetics
  • Microfilament Proteins / antagonists & inhibitors
  • Microfilament Proteins / genetics
  • Microfilament Proteins / metabolism
  • Muscle Contraction / drug effects*
  • Muscle, Smooth / drug effects
  • Muscle, Smooth / metabolism*
  • Muscle, Smooth / ultrastructure
  • Myocytes, Smooth Muscle / drug effects
  • Myocytes, Smooth Muscle / metabolism
  • Myocytes, Smooth Muscle / ultrastructure
  • Myosin-Light-Chain Kinase / antagonists & inhibitors
  • Myosin-Light-Chain Kinase / genetics
  • Myosin-Light-Chain Kinase / metabolism*
  • Myosins / antagonists & inhibitors
  • Peptide Fragments / metabolism
  • Phorbol 12,13-Dibutyrate / pharmacology*
  • Phosphorylation / drug effects
  • Protein Isoforms / antagonists & inhibitors
  • Protein Isoforms / genetics
  • Protein Isoforms / metabolism
  • Protein Processing, Post-Translational / drug effects
  • RNA, Small Interfering
  • Rats

Substances

  • Actins
  • Enzyme Inhibitors
  • Microfilament Proteins
  • Peptide Fragments
  • Protein Isoforms
  • RNA, Small Interfering
  • Phorbol 12,13-Dibutyrate
  • Myosin-Light-Chain Kinase
  • Myosins