Abstract
In erythrocytes, the regulation of the redox sensitive Tyr phosphorylation of band 3 and its functions are still partially defined. A role of band 3 oxidation in regulating its own phosphorylation has been previously suggested. The current study provides evidences to support this hypothesis: (i) in intact erythrocytes, at 2 mM concentration of GSH, band 3 oxidation, and phosphorylation, Syk translocation to the membrane and Syk phosphorylation responded to the same micromolar concentrations of oxidants showing identical temporal variations; (ii) the Cys residues located in the band 3 cytoplasmic domain are 20-fold more reactive than GSH; (iii) disulfide linked band 3 cytoplasmic domain docks Syk kinase; (iv) protein Tyr phosphatases are poorly inhibited at oxidant concentrations leading to massive band 3 oxidation and phosphorylation. We also observed that hemichromes binding to band 3 determined its irreversible oxidation and phosphorylation, progressive hemolysis, and serine hyperphosphorylation of different cytoskeleton proteins. Syk inhibitor suppressed the phosphorylation of band 3 also preventing serine phosphorylation changes and hemolysis. Our data suggest that band 3 acts as redox sensor regulating its own phosphorylation and that hemichromes leading to the protracted phosphorylation of band 3 may trigger a cascade of events finally leading to hemolysis.
Highlights
Due to their function in carrying oxygen and their high iron content, red blood cells (RBCs) are constantly exposed to oxidative stress [1]
RBCs may transiently experience oxidative stress when they are exposed to Reactive oxygen species GSH (ROS) crossing inflammatory tissues or interacting with oxidant contained in drugs or foods [2,3,4]
A number of hemolytic disorders are known to damage the RBC membrane increasing the production of free radicals originating from denatured hemoglobin species, invariably present in thalassemia, sickle cell disease [5,6,7] or with decreased ability of RBCs to deal with extracellular oxidants as in Glucose 6 phosphate dehydrogenase PTKs (G6PD) deficiency [8]
Summary
Due to their function in carrying oxygen and their high iron content, red blood cells (RBCs) are constantly exposed to oxidative stress [1]. A number of hemolytic disorders are known to damage the RBC membrane increasing the production of free radicals originating from denatured hemoglobin species (hemichromes), invariably present in thalassemia, sickle cell disease [5,6,7] or with decreased ability of RBCs to deal with extracellular oxidants as in G6PD deficiency [8]. In RBCs, hyperphosphorylation of band 3 has been constantly reported in all the prooxidant hemolytic disorders [13,14,15] and in malaria [16, 17], but the mechanisms
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