PHOSPHORYLATION OF BRUSH-BORDER MYOSIN-I BY PROTEIN-KINASE-C IS REGULATED BY CA2+-STIMULATED BINDING OF MYOSIN-I TO PHOSPHATIDYLSERINE CONCERTED WITH CALMODULIN DISSOCIATION

Brush border myosin I from chicken intestine is phosphorylated in vitro by chicken intestinal epithelial cell protein kinase C. Phosphorylation on serine and threonine to a maximum of 0.93 mol of P/mol of myosin I occurs within an approximately 20 kDa region at the end of the COOH-terminal tail of the 119-kDa heavy chain. The effects of Ca2+ on myosin I phosphorylation by protein kinase C are complex, with up to 4-fold stimulation occurring at 0.5-3-mu-M Ca2+, and up to 80% inhibition occurring at 3-320-mu-M Ca2+. Phosphorylation required that brush border myosin I be in its phosphatidylserine vesicle-bound state. Previously unknown Ca2+ stimulation of brush border myosin I binding to phosphatidylserine vesicles was found to coincide with Ca2+ stimulation of phosphorylation. A myosin I proteolytic fragment lacking approximately 20 kDa of its tail retained Ca2+-stimulated binding, but showed reduced Ca2+-independent binding. Ca2+-dependent phosphatidylserine binding is apparently due to the concomitant phosphatidylserine-promoted, Ca2+-induced dissociation of up to three of the four calmodulin light chains from myosin I. Four highly basic putative calmodulin-binding sites in the Ca2+-dependent phosphatidylserine binding region of the heavy chain were identified based on the similarity in their sequence to the calmodulin- and phosphatidylserine-binding site of neuromodulin. Calmodulin dissociation is now shown to occur in the low micromolar Ca2+ concentration range and may regulate the association of brush border myosin I with membranes and its phosphorylation by protein kinase C.