TI - Identification of the major casein kinase I phosphorylation sites on erythrocyte band 3 . AB - Human erythrocyte band 3 is a major substrate of two red blood cell protein kinases , casein kinase I and p72syk protein tyrosine kinase . Although the phosphorylation sites and physiologic consequences of p72syk phosphorylation have been characterized , little is known regarding casein kinase I phosphorylation . In this report , we identify the major 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 narrowed the major phosphorylation site to a peptide encompassing residues 24-91 . Computer-assisted evaluation of this sequence not only showed two consensus casein kinase I phosphorylation sites , but also provided information on how to proteolytically separate and isolate the candidate sites . Following the suggested protocols , a heptapeptide containing the major phosphorylation site was isolated , subjected to amino acid sequencing , and found to be phosphorylated on Thr 42 . A minor phosphorylation site was similarly identified as Ser 303 . Because Thr 42 is situated near the binding sites on CDB3 of ankyrin , protein 4.1 , protein 4.2 , and the glycolytic enzymes , phosphorylation of CDB3 by casein kinase I could conceivably impact erythrocyte structure and/or function .