Properties of filament-bound myosin light chain kinase

Myosin light chain kinase binds to actin-containing filaments from cells wi th a greater affinity than to F-actin, However, it is not known if this bin ding in cells is regulated by Ca2+/calmodulin as it is with F-actin. Theref ore, the binding properties of the kinase to stress fibers were examined in smooth muscle-derived A7r5 cells, Full-length myosin light chain kinase or a truncation mutant lacking residues 2-142 was expressed as chimeras conta ining green fluorescent protein at the C terminus. In intact cells, the ful l-length kinase bound to stress fibers, whereas the truncated kinase showed diffuse fluorescence in the cytoplasm. After permeabilization with saponin , the fluorescence from the truncated kinase disappeared, whereas the fluor escence of the full-length kinase was retained on stress fibers. Measuremen ts of fluorescence intensities and fluorescence recovery after photobleachi ng of the full-length myosin light chain kinase in saponin-permeable cells showed that Ca2+/calmodulin did not dissociate the kinase from these filame nts. However, the filament-bound kinase was sufficient for Ca2+ dependent p hosphorylation of myosin regulatory light chain and contraction of stress f ibers. Thus, dissociation of myosin light chain kinase from actin-containin g thin filaments is not necessary for phosphorylation of myosin light chain in thick filaments. We note that the distance between the N terminus and t he catalytic core of the kinase is sufficient to span the distance between thin and thick filaments.