Identification of a Novel Actin Binding Motif in Smooth Muscle Myosin Light Chain Kinase
| Autor: | Pei Ju Lin, Xujun Su, Gang Zhi, Lula Smith, James T. Stull |
|---|---|
| Rok vydání: | 1999 |
| Předmět: |
Myofilament
Myosin light-chain kinase Calmodulin Recombinant Fusion Proteins Molecular Sequence Data macromolecular substances Transfection Biochemistry Myosin head Myosin Animals Amino Acid Sequence Myosin-Light-Chain Kinase Molecular Biology Rho-associated protein kinase Actin Sequence Deletion biology Muscle Smooth Cell Biology Smooth muscle contraction Actins Cell biology Actin Cytoskeleton Microscopy Fluorescence COS Cells Mutation biology.protein Rabbits Sequence Alignment Protein Binding |
| Zdroj: | Journal of Biological Chemistry. 274:29433-29438 |
| ISSN: | 0021-9258 |
| Popis: | Phosphorylation of the 20-kDa regulatory light chain of myosin catalyzed by a Ca2+/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 glutathioneS-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. |
| Databáze: | OpenAIRE |
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