IDENTIFICATION OF THE MAJOR CASEIN KINASE-I PHOSPHORYLATION SITES ON ERYTHROCYTE BAND-3

Human erythrocyte band 3 is a major substrate of two red blood cell pr otein kinases, casein kinase I and p72(syk) protein tyrosine kinase. A lthough the phosphorylation sites and physiologic consequences of p72( syk) phosphorylation have been characterized, little is known regardin g casein kinase I phosphorylation, In this report, we identify the maj or phosphorylation site of casein kinase I. Using isolated components, casein kinase I was found to phosphorylate the cytoplasmic domain of band 3 (CDB3), primarily on Thr residues. Classical peptide mapping na rrowed the major phosphorylation site to a peptide encompassing residu es 24-91. Computer-assisted evaluation of this sequence not only showe d two consensus casein kinase I phosphorylation sites, but also provid ed information on how to proteolytically separate and isolate the cand idate sites. Following the suggested protocols, a heptapeptide contain ing the major phosphorylation site was isolated, subjected to amino ac id sequencing, and found to be phosphorylated on Thr 42. A minor phosp horylation site was similarly identified as Ser 303. Because Thr 42 is situated near the binding sites on CDB3 of ankyrin, protein 4.1, prot ein 4.2, and the glycolytic enzymes, phosphorylation of CDB3 by casein kinase I could conceivably impact erythrocyte structure and/or functi on. (C) 1997 by The American Society of Hematology.