REGULATION OF CA PUMP BY THE PROTEIN KINASE PATHWAY

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DESCRIPTION: This is a study of the protein kinase pathway by which extracellular agonists regulate the plasma membrane Ca2+ pump. The Ca2+ pump exists in the plasma membrane of heart, smooth muscle, kidney, brain, intestinal epithelium, erythrocyte and many other cell types. It is involved in control of intracellular Ca2+ levels in cells of all kinds and in movement of Ca2+ across epithelial cell layers such as the intestinal and kidney tubule epithelia. Studies on the phosphorylation of plasma membrane Ca2+ pump by protein kinases will be combined with studies on the effect of such phosphorylation on the activity of the plasma membrane Ca2+ pump. Information about the effect of protein kinases on this pump will provide an essential link in determining how agonists influence intracellular function by means of the Ca2+ signal. The pump consists of a single polypeptide chain of molecular weight about 135,000 (the exact molecular weight depends on the isoform). We have succeeded in expressing this pump in COS cells and have developed an assay system for determining activity of the expressed enzyme. We propose studies designed to determine the exact locus and nature of the control of the pump s activities by protein kinases. We will use truncated mutants and point mutants of the pump to determine the exact site of phosphorylation and will carry out activity assays under conditions almost identical to those of phosphorylation to determine how the phosphorylation at a given point activates the enzyme. We will raise antibodies against the phosphoenzymes, which should be very useful in determining the role of phosphorylation after stimulation of specific kinds of cells.