Erythrocyte membrane skeleton phosphoproteins: identification of two unrelated phosphoproteins in band 4.9

Abstract

Human erythrocyte membrane band 4.9 is phosphorylated by several erythrocyte protein kinases. Chromatography of erythrocyte membrane skeleton proteins on DEAE-Sephacel produces two proteins with relative mobilities, on gel electrophoresis, similar to that of band 4.9. The first, with a molecular mass of 49 kDa, is quite basic (p I > 8) while the second, 50.5 kDa, is slightly acidic (p I = 6.2). Comparative two-dimensional peptide mapping reveals that both proteins are present in band 4.9 on one-dimensional gels of total erythrocyte membrane proteins and membrane skeleton proteins. The 49 kDa protein, but not the 50.5 kDa protein, binds to actin filaments in a sedimentation assay. In intact erythrocytes metabolically labelled with [ 32P]orthophosphate, the 49 kDa protein is phosphorylated by protein kinase C, cAMP-dependent protein kinase, and protein kinases which are active in the absence of exogenous kinase activators. In contrast, the 50.5 kDa protein is phosphorylated by protein kinase C but not by the other protein kinases examined. Finally, two-dimensional peptide mapping was employed to compare the 49 kDa protein and a 57 kDa protein which copurifies with, and has many characteristics of, the 49 kDa protein. Significant similarities were found in both 125I-labeled chymotryptic peptide maps and 32P-labeled tryptic peptide maps, suggesting that the 49 kDa and 57 kDa proteins are closely related.