9] Isolation and characterization of calmodulin-dependent myosin heavy chain kinase from intestinal brush border

Abstract

This chapter describes methods to identify myosin heavy chain kinase activity in crude extracts of brush borders from chicken intestinal epithelial cells. A procedure for isolation of the myosin heavy chain kinase is described, and it shows that it is completely dependent on calcium and calmodulin for activity. No mechanism for regulation of myosin heavy chain kinase was previously reported in any cell type. Phosphorylation of myosin on its heavy chains, in addition to the well-established light-chain-phosphorylation mechanism, may regulate actomyosin-based contractile events in nonmuscle cells. Myosin heavy chain phosphorylation is shown to regulate actomyosin ATPase activity and myosin filament assembly in vitro in several invertebrates (Acanthamoeba, Dictyostelium, and Physarum). Heavy chain phosphorylation is demonstrated in several vertebrate cell types as well (fibroblasts, macrophages, lymphocytes, leukemic myeloblasts, and brain). Isolated myosin heavy chain kinase is completely dependent on calcium and calmodulin for its activity. The total dependence of the myosin heavy chain kinase activity in the brush border extract on calcium indicates that no calcium-independent heavy chain kinase is present in brush borders. The brush border kinase phosphorylates a threonine residue located very near the tip of the tail of myosin. This region of the molecule is phosphorylated in myosins from other cell types.