Although the major associations of the red cell membrane skeletal proteins are well known, the factors which regulate these associations are almost entirely unknown. Protein phosphorylation is the most prevalent mechanism for the regulation of protein function, and each of the major red cell membrane skeletal proteins (with the exception of actin) is phosphorylated, in some cases by several distinct protein kinases. The principal hypothesis behind this application is that phosphyrylation is a key route by which membrane skeletal organization is governed, and that defective membrane skeletal phosphorylation any be at the root of certain hereditary hemolytic anemias. We will focus our attention on two membrane skeletal phosphoproteins, band 4.1 and ankyrin, proteins which serve crucial linkage functions within the skeleton, via multiple relatively well defined interactions. We will purify four kinases from red cell membranes and cytosol: protein kinase C, cAMP- dependent kinase, Ca-calmodulin kinase and cAMP independent kinase. We will define the target sites of each enzyme in band 4.1 and ankyrin using two-dimensional gels and peptide mapping. We will examine each of the major associations of these proteins as a function of its state of phosphorylation after treatment with each kinase. We will also purify phosphatases from red cells and seek to define individual or groups of phosphatases which may act on specific phosphorylated loci within band 4.1 and ankyrin. Our studies will also involve 1) Purification and characterization of a novel Ca-calmodulin-dependent red cell kinase which we have recently described and which acts principally on band 4.1. 2) Elucidation of the molecular mechanism behind the transmembrane regulation of protein kinase C by agents which affect only the extracellular portions of band 3 and glycophorin. 3) Measurement of the effects of membrane skeletal phosphorylation on the mobility of the integral membrane proteins band 3 and glycophorin using the fluorescence photobleaching recovery technique. 4) Screening red cells from individuals with inherited hemolytic anemias for deficiency or lack of specific kinases, and 5) Defining the molecular basis for an apparent lack of Ca-calmodulin kinase activity which we have uncovered in two individuals having hereditary sphereocytosis. The long-range goal of these studies is to provide insight into the molecular mechanisms which regulate the shape and flexibility on red blood cells, and how these may be defective in disease states.
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