The objectives of this proposal are to study the role of protein kinases in membrane phosphorylation and in the regulation of red cell shape and metabolism. Our major effort will be to purify and characterize the membrane-bound and soluble cyclic AMP-independent protein kinases from human erythrocytes. The role of these kinases in the phosphorylation of various membrane proteins including spectrin and band 3 will be examined. Since each mole of spectrin is known to contain at least four moles of phosphates, attempts will be made to determine whether one or more kinases are responsible for these phosphates found in the spectrin molecule. These studies will be conducted utilizing completely dephosphorylated spectrin. The dephosphorylation of spectrin can be achieved by treatment with E. coli alkaline phosphatase. In addition, the possibility that these kinases may phosphorylate and regulate the activities of metabolic enzymes of erythrocytes will be investigated. Finally, we will also investigate the possibility that these kinases may themselves be regulated by protein factors or red cell metabolites, such as 2,3-DPG.
