Abstract
This report presents an analysis of the phosphorylation of human and rabbit erythrocyte membrane proteins which migrate in NaDodSO~4~‐polyacrylamide gels in the area of the Coomassie Blue‐stained proteins generally known as band 3. The phosphorylation of these proteins is of interest as band 3 has been implicated in transport processes. This study shows that there are at least three distinct phosphoproteins associated with the band 3 region of human erythrocyte membranes. These are band 2.9, the major band 3, and PAS‐1. The phosphorylation of these proteins is differentially catalyzed by solubilized membrane and cytoplasmic cyclic AMP‐dependent and ‐independent erythrocyte protein kinases. Band 2.9 is present and phosphorylated in unfractionated human and rabbit erythrocyte ghosts but not in NaI‐ or dimethylmaleic anhydride (DMMA)‐extracted membranes. These latter membrane preparations are enriched in band 3 and in sialoglycoproteins. The NaI‐extracted ghosts contain residual protein kinase activity which can catalyze the autophosphorylation of band 3 whereas the DMMA‐extracted ghosts are usually devoid of any kinase activity. However, both NaI‐ and DMMA‐extracted ghosts, as well as Triton X‐100 extracts of the DMMA‐extracted ghosts, can be phosphorylated by various erythrocyte protein kinases. The kinases which preferentially phosphorylate the major band 3 protein are inactive towards PAS‐1 while the kinases active towards PAS‐1 are less active towards band 3. The band 3 protein in the DMMA‐extracted ghosts can be cross‐linked with the Cu^2+^ ‐σ‐phenanthroline complex. The cross‐linking of band 3 does not affect its capacity to serve as a phosphoryl acceptor nor does phosphorylation affect the capacity of band 3 to form cross‐links. In addition to band 2.9, the major band 3 and PAS‐1, another minor protein component appears to be present in the band 3 region in human erythrocyte membranes. This protein is specifically phosphorylated by the cyclic AMP‐dependent protein kinases isolated from the cytoplasm of rabbit erythrocytes. The rabbit erythrocyte membranes lack PAS‐1 and the cyclic AMP‐dependent protein kinase substrate.