Directed cell movements are important in many aspects of biology. In early development, primordial germ cells and neuro crest cells migrate long distances to appropriate developmental sites, while in the adult, macrophages move to sites of infection and exhibit phagocytic activities. In the transformed state, on the other hand, control of cell locomotion and attachment is disrupted. A central question of cell biology, therefore, concerns the mechanism by which extracellular signals influence the organization and activity of actin, myosin, and other contractile proteins. A good system for studying this type of regulation is the unicellular amoeba, Dictyostelium, which responds chemotactically to cAMP and folic acid. The heavy chain of Dictyostelium myosin is phosphorylated in vivo, and this phosphorylation causes the disassembly of myosin thick filaments. Since thick filaments are presumed to be essential for contraction, heavy chain phosphorylation appears to be an important mechanism for regulating directed cell movements. Specific heavy chain kinases have been partially purified from extracts of Dictyostelium. One of these kinases phosphorylates a threonine residue, which is unusual since in vivo phosphorylation is restricted to serine residues. To determine if threonine phosphorylation occurs in vegetative or developing cells, new techniques will be tried to purify myosin, and the effect of phosphotase inhibitors will be determined. The heavy chain kinases will be further purified by ion exchange and affinity chromatography techniques. Extensive characterization will be carried out, including peptide mapping studies to compare in vivo and in vitro sites of phosphorylation. To study the regulation of these enzymes, important cellular messengers such as cAMP, cGMP, pH, and Ca++ will be tested for their effects on the activity of the purified kinases. A heavy chain phosphatase has also been partially purified fom Dictyostelium and this enzyme will be further purified and characterized. An understanding how the heavy chain kinases and phosphatases are regulated should provide insights into the chain of events which links an extracellular signal to myosin assembly and ultimately to direct cell movements.
