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 nece ssary 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 i n 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 contractil e proteins similar to recombinant full-length myosin light chain kinas e 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 a ctomyosin filaments from skeletal muscle, In contrast, truncations at the N terminus alone or at both N and C termini resulted in no signifi cant binding, Similar effects were observed by two other assays: bindi ng of fluorescently labeled myosin light chain kinases to actin-contai ning stress fibers in detergent-treated fibroblasts and localization o f fluorescently labeled kinases after microinjection into primary smoo th muscle cells in culture. The full-length and the C-terminal truncat ed myosin light chain kinases, but not myosin light chain kinases trun cated at the N terminus or both N and C termini, associated with filam ents in cells, Thus, the N terminus and not the C terminus of myosin l ight chain kinase is necessary for high affinity binding to actomyosin -containing filaments in smooth and non-muscle cells.