PROPERTIES OF ACANTHAMOEBA MYOSIN-I HEAVY-CHAIN KINASE BOUND TO PHOSPHOLIPID-VESICLES

The actin activated Mg2+-ATPase and in vitro motility activities of th e three Acanthamoeba myosin I isozymes depend upon phosphorylation of their single heavy chains by myosin I heavy chain kinase, Previously, the kinase had been shown to be activated by autophosphorylation, whic h is enhanced by acidic phospholipids, or simply by binding to purifie d plasma membranes in the absence of significant autophosphorylation, Tn this paper, we show that the rate of phosphorylation of myosin I by unphosphorylated kinase is similar to 20-fold faster when both the my osin I and the kinase are bound to acidic phospholipid vesicles than w hen both are soluble, This activation is not due to an increase in the local concentrations of vesicle-bound kinase and myosin I, Thus, acid ic phospholipids, like membranes, can activate myosin I heavy chain ki nase in the absence of significant autophosphorylation, i.e. membrane proteins are not required, Kinetic studies show that both binding of k inase to phospholipid vesicles and autophosphorylation of kinase in th e absence of phospholipid increase the V-max relative to soluble, unph osphorylated kinase with either an increase in the apparent K-m (when myosin I is the substrate) or no significant change in K-m (when a syn thetic peptide is the substrate), Kinetic data showed that autophospho rylation of phospholipid bound kinase is both intermolecular and inter vesicular, and that phosphorylation of phospholipid-bound myosin I by phospholipid-bound kinase is also intervesicular even when the kinase and myosin are bound to the same vesicles. The relevance of these resu lts to the activation of myosin I heavy chain kinase and phosphorylati on of myosin I isozymes in situ are discussed.