Identification of a novel actin binding motif in smooth muscle myosin light chain kinase.
Phosphorylation of the 20-kDa regulatory light chain of myosin catalyzed by a Ca(2+)/calmodulin-dependent myosin light chain kinase is important in the initiation of smooth muscle contraction and other contractile processes in non-muscle cells. It has been previously shown that residues 1-142 of smooth muscle myosin light chain kinase are necessary for high-affinity binding to actin-containing filaments in cells (1). To further localize the region of the kinase required for binding, a series of N-terminal deletion mutants as well as several N-terminal glutathione S-transferase fusion proteins were constructed. Cosedimentation assays showed that a peptide containing residues 1-75 binds to purified smooth muscle myofilaments. Furthermore, the N-terminal peptide was sufficient for high-affinity binding to actin stress fibers in smooth muscle cells in vivo. Alanine scanning mutagenesis in the fusion protein identified residues Asp-30, Phe-31, Arg-32, and Leu-35 as important for binding in vitro. There are two additional DFRXXL motifs located at residues 2-7 and 58-63. The DFR residues in these three motifs were individually replaced by alanine residues in the full-length kinase. Each of these mutations significantly decreased myosin light chain kinase binding to myofilaments in vitro, and each abolished high-affinity binding to actin-containing filaments in smooth muscle cells in vivo. These results identify a unique structural motif comprised of three repeat consensus sequences in the N terminus of myosin light chain kinase necessary for high-affinity binding to actin-containing filaments.
Smith
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Department of Physiology, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75235-9040, USA.
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Lin
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Zhi
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Stull
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D008841:Actin Cytoskeleton / Q000378:metabolism; D000199:Actins / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D009130:Muscle, Smooth / Q000201:enzymology*; D009154:Mutation; D009219:Myosin-Light-Chain Kinase / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D011817:Rabbits; D011993:Recombinant Fusion Proteins; D016415:Sequence Alignment; D017384:Sequence Deletion; D014162:Transfection
D000199:Actins; D011993:Recombinant Fusion Proteins; D009219:Myosin-Light-Chain Kinase