Analysis of band 3 cytoplasmic domain phosphorylation and association with ankyrin.
The phosphorylation of the cytoplasmic domain of band 3 by the human erythrocyte membrane kinase and casein kinase A has been investigated. The cytoplasmic domain of band 3 was released from erythrocyte vesicles by treatment with alpha-chymotrypsin and isolated as a 43,000-Da peptide. Both the membrane kinase and casein kinase A catalyzed the incorporation of about 1 mol of phosphate per mole of the band 3 fragment. The phosphorylation of the band 3 fragment by both kinases was not additive, suggesting that the two enzymes might recognize the same phosphorylation sites. Also in support of this notion was the observation that the phosphopeptide maps of the band 3 fragment phosphorylated by the two kinases were identical. Phosphoamino acid analysis of the band 3 fragment phosphorylated by casein kinase A revealed the presence of approximately equal amounts of phosphoserine and phosphothreonine and, to a lesser extent, phosphotyrosine. The interaction between the 43,000-Da peptide with ankyrin and the effect of phosphorylation on this interaction have been examined. The band 3 fragment was found to form two different types of complexes, termed C1 and C2, with ankyrin in a saturable manner. The C1 and C2 complexes contained about 1.7 and 0.43 mol of band 3 fragment per mole of ankyrin, respectively. Interestingly, these binding stoichiometries were found to be reduced by half by the phosphorylation of ankyrin but not by the phosphorylation of the band 3 fragment. The results suggest that the structure and dynamics of the erythrocyte membrane cytoskeletal network may be regulated by phosphorylation.
D000255:Adenosine Triphosphate / Q000097:blood; D000596:Amino Acids / Q000097:blood; D001457:Anion Exchange Protein 1, Erythrocyte / Q000378:metabolism*; D017487:Ankyrins; D047388:Casein Kinases; D003593:Cytoplasm / Q000378:metabolism*; D004910:Erythrocyte Membrane / Q000201:enzymology; D004912:Erythrocytes / Q000201:enzymology*; D006801:Humans; D007700:Kinetics; D008565:Membrane Proteins / Q000378:metabolism*; D010750:Phosphoproteins / Q000097:blood; D010766:Phosphorylation; D011494:Protein Kinases / Q000097:blood*; D013379:Substrate Specificity
D000596:Amino Acids; D001457:Anion Exchange Protein 1, Erythrocyte; D017487:Ankyrins; D008565:Membrane Proteins; D010750:Phosphoproteins; D000255:Adenosine Triphosphate; D011494:Protein Kinases; D047388:Casein Kinases