Properties of Acanthamoeba myosin I heavy chain kinase bound to phospholipid vesicles.
The actin-activated Mg(2+)-ATPase and in vitro motility activities of the 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, which is enhanced by acidic phospholipids, or simply by binding to purified plasma membranes in the absence of significant autophosphorylation. In this paper, we show that the rate of phosphorylation of myosin I by unphosphorylated kinase is approximately 20-fold faster when both the myosin I and the kinase are bound to acidic phospholipid vesicles than when both are soluble. This activation is not due to an increase in the local concentrations of vesicle-bound kinase and myosin I. Thus, acidic phospholipids, like membranes, can activate myosin I heavy chain kinase in the absence of significant autophosphorylation, i.e. membrane proteins are not required. Kinetic studies show that both binding of kinase to phospholipid vesicles and autophosphorylation of kinase in the absence of phospholipid increase the Vmax relative to soluble, unphosphorylated kinase with either an increase in the apparent Km (when myosin I is the substrate) or no significant change in Km (when a synthetic peptide is the substrate). Kinetic data showed that autophosphorylation of phospholipid-bound kinase is both intermolecular and intervesicular, 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 results to the activation of myosin I heavy chain kinase and phosphorylation of myosin I isozymes in situ are discussed.
D000048:Acanthamoeba / Q000201:enzymology*; D000818:Animals; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000302:isolation & purification / Q000378:metabolism*; D002462:Cell Membrane / Q000201:enzymology; D002784:Cholesterol; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008051:Lipid Bilayers; D009218:Myosins / Q000378:metabolism; D010743:Phospholipids / Q000494:pharmacology; D010766:Phosphorylation; D011485:Protein Binding; D015800:Protozoan Proteins; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D008051:Lipid Bilayers; D010743:Phospholipids; D015800:Protozoan Proteins; D002784:Cholesterol; D017871:Calcium-Calmodulin-Dependent Protein Kinases; C033142:myosin-heavy-chain kinase; D009218:Myosins