Analysis of band 3 cytoplasmic domain phosphorylation and association with ankyrin |
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| Authors: | C J Soong P W Lu M Tao |
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| Affiliation: | 1. Large Lakes Observatory, University of Minnesota, Duluth, MN, USA;2. King County Environmental Lab, 322 W Ewing St, Seattle, WA, USA;1. School of Civil Engineering, Guangzhou University, Guangzhou, China;2. School of Engineering, Swinburne University of Technology, VIC 3122, Australia;1. Neuropharmacology Division, Department of Pharmacology, ISF College of Pharmacy, Moga-142001, Punjab, India;2. Department of Pharmaceutical Chemistry, ISF College of Pharmacy, Moga-142001, Punjab, India;3. Molecular Modeling and Pharmacoinformatics, Department of Pharmaceutical Chemistry, ISF College of Pharmacy Moga, Punjab 142001, India;1. Naomi Berrie Diabetes Center, Department of Medicine, Columbia University, New York, NY 10032, USA;2. Department of Genetics and Integrated Program in Cellular, Molecular, and Biomedical Studies, Columbia University, New York, NY 10032, USA;3. Division of Nephrology, The Helen L. and Martin S. Kimmel Center for Biology and Medicine at the Skirball Institute for Biomolecular Medicine, New York University Langone Medical Center, New York, NY 10016, USA;4. Molecular Nutrition Unit and Montreal Diabetes Research Center at the CRCHUM and Departments of Nutrition and Biochemistry, and Molecular Medicine, Université de Montréal, Montréal, QC H2X 0A9, Canada |
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| Abstract: | 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. |
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