L Smith

University of Texas Southwestern Medical Center, Dallas, Texas, United States

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Publications (3)19.06 Total impact

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    ABSTRACT: Ca2+-calmodulin-dependent phosphorylation of myosin regulatory light chains by the catalytic COOH-terminal half of myosin light chain kinase (MLCK) activates myosin II in smooth and nonmuscle cells. In addition, MLCK binds to thin filaments in situ and F-actin in vitro via a specific repeat motif in its NH2 terminus at a stoichiometry of one MLCK per three actin monomers. We have investigated the structural basis of MLCK-actin interactions by negative staining and helical reconstruction. F-actin was decorated with a peptide containing the NH2-terminal 147 residues of MLCK (MLCK-147) that binds to F-actin with high affinity. MLCK-147 caused formation of F-actin rafts, and single filaments within rafts were used for structural analysis. Three-dimensional reconstructions showed MLCK density on the extreme periphery of subdomain-1 of each actin monomer forming a bridge to the periphery of subdomain-4 of the azimuthally adjacent actin. Fitting the reconstruction to the atomic model of F-actin revealed interaction of MLCK-147 close to the COOH terminus of the first actin and near residues 228-232 of the second. This unique location enables MLCK to bind to actin without interfering with the binding of any other key actin-binding proteins, including myosin, tropomyosin, caldesmon, and calponin.
    The Journal of Cell Biology 09/2001; 154(3):611-7. · 10.82 Impact Factor
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    L Smith, J T Stull
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    ABSTRACT: Smooth muscle myosin light chain kinase (MLCK) plays important roles in contractile-motile processes of a variety of cells. Three DFRxxL motifs at the kinase N-terminus (residues 2-63) are critical for high-affinity binding to actin-containing filaments [Smith et al. (1999) J. Biol. Chem. 274, 29433-29438]. A GST fusion protein containing residues 1-75 of MLCK (GST75-MLCK) bound maximally to both smooth muscle myofilaments and F-actin at 0.28 and 0.31 mol GST75-MLCK/mol actin with respective K(D) values of 0.1 microM and 0.8 microM. High-affinity binding of MLCK to actin-containing filaments may be due to each DFRxxL motif binding to one actin monomer in filaments.
    FEBS Letters 10/2000; 480(2-3):298-300. · 3.58 Impact Factor
  • L Smith, X Su, P Lin, G Zhi, J T Stull
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    ABSTRACT: 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.
    Journal of Biological Chemistry 11/1999; 274(41):29433-8. · 4.65 Impact Factor

Publication Stats

81 Citations
19.06 Total Impact Points

Institutions

  • 1999–2001
    • University of Texas Southwestern Medical Center
      • Department of Physiology
      Dallas, Texas, United States