The cytoplasmic domain name of music group 3 serves as a center of erythrocyte membrane organization and constitutes the major substrate of erythrocyte tyrosine kinases. by: (1) a decrease in ankyrin affinity in direct binding studies (2) an increase in detergent extractability of band three or more from ghosts (3) a rise in music group 3 cross-linkability by bis-sulfosuccinimidyl-suberate (4) significant changes in Ziyuglycoside II erythrocyte morphology and (5) elevation of the price of music group 3 diffusion in intact cells. Because release of band three or more from its ankyrin and adducin linkages to the cytoskeleton can facilitate changes in multiple membrane properties tyrosine phosphorylation of band three or more is argued to enable adaptive changes in erythrocyte biology that permit the cell to respond to the above stresses. Introduction Early views of the human erythrocyte argued that the cell was inert to external stimuli and that its complement of protein kinases phospholipases G proteins phosphatases and hormone receptors simply constituted nonfunctional vestiges of signaling pathways that were once operational in erythroid precursor cells. More recent evidence however has revealed that the human erythrocyte is highly responsive to its Ziyuglycoside II environment and that the cell’s rich ensemble of signaling proteins likely comprise critical components in the cell’s communication with its extracellular milieu. Classic hormones/signaling molecules such as prostaglandin E2 insulin epinephrine endothelin ADP and NO are now known to modulate erythrocyte properties in an adaptive manner and the functional activities of many intracellular signaling intermediates have been demonstrated to regulate erythrocyte behavior. 1–5 One of the major targets of erythrocyte signaling appears to be the predominant membrane-spanning protein band 3. Band 3 (AE1) catalyzes the exchange of anions (primarily HCO3? for Cl? ) across the erythrocyte membrane 6 anchors the spectrin/actin cytoskeleton to the lipid bilayer 7 organizes and regulates a complex of glycolytic enzymes 8 9 participates in control of erythrocyte lifespan 10 11 nucleates several important membrane-spanning proteins 12 and serves as a docking site for multiple peripheral membrane proteins including protein 4. 1 protein 4. 2 and several kinases and phosphatases. 13–16 Not surprisingly mutations in band 3 are frequently associated with various hemolytic diseases. 17 Perhaps because of its many important functions band 3 is also a prominent substrate of Ser/Thr kinases 18 19 and is the major substrate of the cell’s protein tyrosine kinases. 20 21 In response to physiologic stimuli such as hypertonic conditions or oxidative stress and in severe hematologic disorders such as thalassemias sickle cell anemia and glucose-6-phosphate dehydrogenase deficiency 22 23 phosphorylation of band 3 on tyrosine residues can increase by several orders of magnitude. 20 22 Ziyuglycoside II Although the protein tyrosine kinase Lyn has been shown to participate in this phosphorylation 21 the protein tyrosine kinase Ziyuglycoside II Syk can be argued to play SC35 the more prominent role because it has been reported to phosphorylate tyrosines 8 and 21 of band 3 20 which in turn generates a binding site for other protein tyrosine kinases. 21 Of more direct relevance to erythrocyte signal transduction Syk may also mediate the effects of oxidant stress on band 3 tyrosine Ziyuglycoside II phosphorylation because it strongly prefers to phosphorylate a reversibly oxidized conformation of AE1. 20 Except for displacement of glycolytic enzymes from their binding sites on band 3 27 28 the functional consequences of its tyrosine phosphorylation remain largely unexplored. However because the sites of band 3 tyrosine phosphorylation are distributed throughout the protein (ie at least tyrosines 8 21 359 and 90419 29 it can be hypothesized that the biologic impact of tyrosine phosphorylation should extend beyond its regulation of glycolysis. The only suggestion that other functions of band 3 might also be affected by tyrosine phosphorylation lay in the observation that tyrosine phosphorylation of the polypeptide’s cytoplasmic domain is somehow inhibited by ankyrin binding30; however no connection between this observation and the regulation of any membrane-cytoskeletal interaction has ever been examined. Therefore the purpose of this study was to evaluate whether tyrosine phosphorylation of band 3 might alter its interaction with the spectrin/actin skeleton. We report here that tyrosine phosphorylation of band 3.