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
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Research Support, N.I.H., Extramural
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Research Support, Non-U.S. Gov't
MeSH terms
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Actins / metabolism*
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Animals
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Cell Line
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Cell Surface Extensions / drug effects
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Cell Surface Extensions / metabolism
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Cell Surface Extensions / ultrastructure
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Chickens
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Cytoskeleton / drug effects
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Cytoskeleton / metabolism*
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Enzyme Inhibitors / metabolism
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Gene Silencing
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Guinea Pigs
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In Vitro Techniques
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Kinetics
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Microfilament Proteins / antagonists & inhibitors
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Microfilament Proteins / genetics
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Microfilament Proteins / metabolism
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Muscle Contraction / drug effects*
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Muscle, Smooth / drug effects
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Muscle, Smooth / metabolism*
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Muscle, Smooth / ultrastructure
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Myocytes, Smooth Muscle / drug effects
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Myocytes, Smooth Muscle / metabolism
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Myocytes, Smooth Muscle / ultrastructure
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Myosin-Light-Chain Kinase / antagonists & inhibitors
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Myosin-Light-Chain Kinase / genetics
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Myosin-Light-Chain Kinase / metabolism*
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Myosins / antagonists & inhibitors
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Peptide Fragments / metabolism
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Phorbol 12,13-Dibutyrate / pharmacology*
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Phosphorylation / drug effects
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Protein Isoforms / antagonists & inhibitors
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Protein Isoforms / genetics
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Protein Isoforms / metabolism
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Protein Processing, Post-Translational / drug effects
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RNA, Small Interfering
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Rats
Substances
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Actins
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Enzyme Inhibitors
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Microfilament Proteins
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Peptide Fragments
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Protein Isoforms
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RNA, Small Interfering
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Phorbol 12,13-Dibutyrate
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Myosin-Light-Chain Kinase
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Myosins