Regulation of membrane-cytoskeletal interactions by tyrosine phosphorylation of erythrocyte band 3

Abstract

The cytoplasmic domain of band 3 serves as a center of erythrocyte membrane organization and constitutes the major substrate of erythrocyte tyrosine kinases. Tyrosine phosphorylation of band 3 is induced by several physiologic stimuli, including malaria parasite invasion, cell shrinkage, normal cell aging, and oxidant stress (thalassemias, sickle cell disease, glucose-6-phosphate dehydrogenase deficiency, etc). In an effort to characterize the biologic sequelae of band 3 tyrosine phosphorylation, we looked for changes in the polypeptide's function that accompany its phosphorylation. We report that tyrosine phosphorylation promotes dissociation of band 3 from the spectrin-actin skeleton as evidenced by: (1) a decrease in ankyrin affinity in direct binding studies, (2) an increase in detergent extractability of band 3 from ghosts, (3) a rise in band 3 cross-linkability by bis-sulfosuccinimidylsuberate, (4) significant changes in erythrocyte morphology, and (5) elevation of the rate of band 3 diffusion in intact cells. Because release of band 3 from its ankyrin and adducin linkages to the cytoskeleton can facilitate changes in multiple membrane properties, tyrosine phosphorylation of band 3 is argued to enable adaptive changes in erythrocyte biology that permit the cell to respond to the above stresses. (Blood. 2011; 117(22):5998-6006) IntroductionEarly 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 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 E 2 , 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][2][3][4][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 HCO 3 Ϫ for Cl Ϫ ) 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][14][15][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 a...

Effect of hypertonicity on the dynamic state of erythrocyte spectrin network as revealed by thermal dielectroscopy

“…The phosphorylated site is identified as Tyr-8, located at the extreme N-terminus of the cytoplasmic domain of band 3, responcible for the binding of band 3 to ankyrin. Consequently, the phosphorylation of the Tyr-8 residue of band 3 immediately disassociates the spectrin -band 3 bridge (19). As the phosphorylation reaction is reversible on restoring the initial volume of erythrocytes (18) the spectrin -band 3 bridge should reassociate in accordance with the result shown in Figure 5.…”

Thalassemic erythrocytes release microparticles loaded with hemichromes by redox activation of p72Syk kinase

“…20 In turn, phosphorylation of Tyr 8 and 21 residues located at the N-terminal of band 3 determines its uncoupling from the cytoskeleton and markedly increases its lateral mobility leading to an increased propensity to form clusters, to be easily extracted from the membrane and to induce membrane vesiculation. 19,46 A number of findings indicate that this mechanism may be working in the generation of MP in TI patients: (i) HMC are contained in MP found in patients' plasma or are released from TI-RBC in vitro; (ii) the degree of band 3 oxidation and phosphorylation in TI-RBC correlates with the amount of membrane-bound HMC; (iii) in TI-RBC and Redox activation of Syk leads to MP release in TI haematologica | 2014; 99 (3) 575 Table 1. Proteins identified in MP.…”

“…19 Moreover phosphorylation of band 3 has already been demonstrated to be increased in thalassemia, although its functional effects have not been investigated. In accordance with a previous report, 20 Figure 2A,B shows that oxidized and clustered band 3 is strongly hyper-phosphorylated in splenectomized TI patients in comparison with the situation in non-splenectomized patients.…”

“…20 In turn, the affinity of the phosphorylated band 3 molecules for ankyrin is drastically decreased, their lateral mobility is increased and they have a greater propensity to form large clusters inducing vesiculation. 19 In thalassemias, it has been previously demonstrated that HMC bind to band 3 causing free iron accumulation and free radical production, 7,21 but their role in inducing band 3 phosphorylation and membrane destabilization has never been investigated. In this study we investigated the role of HMC in the release of MP from TI-RBC.…”

“…11 The composition and pathogenic roles of MP have been extensively studied in various diseases, such as ischemia, diabetes and atherosclerosis, revealing complex pathogenic roles in modulating nitric oxide and prostacyclin production, stimulating cytokine release, inducing tissue factor expression, as well as monocyte chemotaxis and adherence to the endothelium. [12][13][14][15][16][17][18] Recent studies on the mechanisms of redox regulation of RBC membrane stability 19,20 indicate that oxidative stress induces a phosphorylative response that specifically involves two tyrosine residues located in the cytoplasmic domain of band 3. 20 Band 3 is the most abundant RBC membrane protein, and represents one of the major components of the junctional complexes that connect the lipid bilayer to the cytoskeleton.…”