TY - JOUR
T1 - Identification of a novel actin binding motif in smooth muscle myosin light chain kinase
AU - Smith, Lula
AU - Su, Xujun
AU - Lin, Pei Ju
AU - Zhi, Gang
AU - Stull, James T.
PY - 1999/10/8
Y1 - 1999/10/8
N2 - 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 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.
AB - 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 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.
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U2 - 10.1074/jbc.274.41.29433
DO - 10.1074/jbc.274.41.29433
M3 - Article
C2 - 10506206
AN - SCOPUS:0032883786
SN - 0021-9258
VL - 274
SP - 29433
EP - 29438
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 41
ER -