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.