2 HEADS ARE REQUIRED FOR PHOSPHORYLATION-DEPENDENT REGULATION OF SMOOTH-MUSCLE MYOSIN

Recent structural evidence (Rayment, I., Holden, H. M., Whittaker, M., Yohn, C. B., Lorenz, M., Holmes, K. C., and Milligan, R. A. (1993) Sc ience 261, 58-65) suggests that the two heads of skeletal muscle myosi n interact when the protein is bound to filamentous actin, Direct chem ical cross-linking experiments show that the two heads of smooth muscl e myosin interact in the presence of filamentous actin and the absence of ATP (Onishi, H., Maita, T., Matsuda, G., and Fujiwara, K. (1992) B iochemistry 31, 1201-1210). Head-head interactions may be important in the mechanism of phosphorylation-dependent regulation of smooth muscl e myosin. To explore the structural elements essential for phosphoryla tion-dependent regulation, we purified a proteolytic fragment of chick en gizzard myosin containing only one head attached to an intact tail. This molecule contained a partially digested regulatory light chain, which was replaced with exogenously added intact light chain in either the thiophosphorylated or the unphosphorylated state, Control experim ents showed that this replacement was nearly quantitative and did not alter the actin-activated ATPase of this myosin. Electron micrographs confirmed that the single-headed preparation contained an intact form of single-headed myosin. The unphosphorylated single-headed myosin hyd rolyzed ATP rapidly and moved actin filaments in an in vitro motility assay, Phosphorylation had minimal effects upon these properties, Ther efore, we conclude that phosphorylation-dependent regulation in this m yosin requires two heads. These findings may have important implicatio ns in studies of other regulated motor proteins that contain two motor domains.