Binding of myosin light chain kinase to cellular actin-myosin filaments.
Myosin light chain kinase binds to the actomyosin-containing filaments in smooth and nonmuscle cells. However, the region of the kinase necessary for this high affinity binding in vivo is not known, although it has been proposed that the N and C termini bind to actin and myosin in vitro, respectively. Truncated myosin light chain kinases containing the catalytic core and calmodulin-binding domain but lacking N (amino acids 1-655) and/or C (amino acids 1004-1147) termini were expressed in the baculovirus system and purified. All enzymes were catalytically active and Ca2+/calmodulin-dependent. The C-terminal truncated myosin light chain kinase bound to detergent-washed smooth muscle contractile proteins similar to recombinant full-length myosin light chain kinase or enzyme purified from smooth muscle. The apparent affinity of the full-length kinase was greater for the actomyosin-containing filaments with associated proteins than for purified smooth muscle F-actin or actomyosin filaments from skeletal muscle. In contrast, truncations at the N terminus alone or at both N and C termini resulted in no significant binding. Similar effects were observed by two other assays: binding of fluorescently labeled myosin light chain kinases to actin-containing stress fibers in detergent-treated fibroblasts and localization of fluorescently labeled kinases after microinjection into primary smooth muscle cells in culture. The full-length and the C-terminal truncated myosin light chain kinases, but not myosin light chain kinases truncated at the N terminus or both N and C termini, associated with filaments in cells. Thus, the N terminus and not the C terminus of myosin light chain kinase is necessary for high affinity binding to actomyosin-containing filaments in smooth and nonmuscle cells.
Lin
P j
Pj
Department of Physiology, University of Texas Southwestern Medical Center, Dallas, Texas 75235, USA.
Luby-Phelps
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Stull
J T
JT
D008841:Actin Cytoskeleton / Q000378:metabolism*; D000199:Actins / Q000378:metabolism* / Q000648:ultrastructure; D000818:Animals; D009130:Muscle, Smooth / Q000378:metabolism* / Q000648:ultrastructure; D009219:Myosin-Light-Chain Kinase / Q000378:metabolism*; D009218:Myosins / Q000378:metabolism* / Q000648:ultrastructure; D011817:Rabbits
D000199:Actins; D009219:Myosin-Light-Chain Kinase; D009218:Myosins