Increased tyrosine phosphorylation of band 3 in hemoglobinopathies

In order to investigate the tyrosine phosphorylation of band 3, we performed immunoblotting of intact red cells using anti-phosphotyrosine antibody of 21 patients with sickle cell disorders (11 SS, 5 Sbeta, 5 SC), 7 patients with beta thalassemias (5 beta thal intermedia, 2 deltabeta thal), 10 normal controls, and 1 patient with hereditary spherocytosis. They had not received transfusion for the last 4 months and all were clinically stable. Our results showed an increased tyrosine phosphorylation of two proteins, in the 100 and 80 kD regions, in sickle cell and beta-thalassemic red cells when compared to the normal controls and to the patient with hereditary spherocytosis. Immunoprecipitation of the lysed red cells with anti-band 3 antibody and immunoblotting with anti-phosphotyrosine antibody confirmed that the 100 kD tyrosine phosphorylated protein was band 3. In the sickle cell disease group, the band 3 tyrosine phosphorylation varied from 2- to 10-fold increase compared to control (x +/- SD; SS = 7.8- +/- 2.7-fold; SC = 3.8- +/- 1.3-fold; Sbeta = 5.2- +/- 2.0-fold). It was also higher in the beta-thalassemic group (beta-thal = 4.3- +/- 3.7-fold). There was no significant difference in tyrosine phosphorylation among the various groups tested, except when we compared the phosphorylation in intact red cells of patients with sickle cell anemia and hemoglobinopathy SC (U = 6, P < 0.02). The tyrosine phosphorylation of band 3 was increased in hemoglobinopathies even in the absence of high reticulocyte count. At least two mechanisms might be involved in the increased tyrosine phosphorylation of band 3 in these hemoglobin disorders, probably related to the endogenous reactive oxygen intermediates generated by the abnormal erythrocyte: an inhibition of protein tyrosine phosphatase activity or an activation of the protein tyrosine kinase p72syk.