Evidence for regulation of lamellipodial and tail contraction of glycerinated chicken embryonic fibroblasts by myosin light chain kinase.

Permeabilized cell models of muscle and nonmuscle cells have proven useful for examining the regulation of actin, myosin, and other cytoskeletal proteins during cell contraction. Upon addition of Ca2+ and ATP, glycerinated chick embryonic skin fibroblasts retract their tails and lamellipodia. Ca2+-independent contractions are obtained by preincubation of cell models in Ca2+ ATP gamma S, followed by EGTA and ATP addition, or by addition of trypsin-treated myosin light chain kinase that no longer requires Ca2+ for reactivation. By pretreating cells before glycerination with colchicine, it is possible to study lamellipodial contraction independent of tail contraction. Similar responses to ATP gamma S pretreatment and unregulated myosin light chain kinase are observed in cells that only contain lamellipodia. SDS-PAGE electrophoresis of glycerinated fibroblasts incubated in ATP gamma 35S and Ca2+ shows that only two major proteins are thiophosphorylated, and that one of them, a band that comigrates with the 20K MW light chain of myosin, is thiophosphorylated in a Ca2+-dependent manner. Since the rate of tail contraction is several-fold faster after Ca2+ and ATP gamma S pretreatment or incubation in excess myosin light chain kinase, myosin light chain phosphorylation may be a rate-limiting step during contraction.