INCREASED TYROSINE PHOSPHORYLATION OF BAND-3 IN HEMOGLOBINOPATHIES

In order to investigate the tyrosine phosphorylation of band 3, we per formed immunoblotting of intact red cells using anti-phosphotyrosine a ntibody of 21 patients with sickle cell disorders (11 SS, 5 S beta, 5 SC), 7 patients with beta thalassemias (5 beta that intermedia, 2 delt a beta thal), 10 normal controls, and 1 patient with hereditary sphero cytosis, They had not received transfusion for the last 4 months and a ll were clinically stable. Our results showed an increased tyrosine ph osphorylation of two proteins, in the 100 and 80 kD regions, in sickle cell and beta-thalassemic red cells when compared to the normal contr ols and to the patient with hereditary spherocytosis, Immunoprecipitat ion of the lysed red cells with anti-band 3 antibody and immunoblottin g with anti-phosphotyrosine antibody confirmed that the 100 kD tyrosin e phosphorylated protein was band 3, In the sickle cell disease group, the band 3 tyrosine phosphorylation varied from 2- to 10-fold increas e compared to control (x +/- SD; SS = 7.8- +/- 2.7-fold; SC = 3,8- +/- 1.3-fold; S beta = 5.2- +/- 2.0-fold), It was also higher in the beta -thalassemic group (beta-thal = 4,3- +/- 3.7-fold). There was no signi ficant difference in tyrosine phosphorylation among the various groups tested, except when we compared the phosphorylation in intact red cel ls of patients with sickle cell anemia and hemoglobinopathy SC (U = 6, P< 0.02). The tyrosine phosphorylation of band 3 was increased in hem oglobinopathies even in the absence of high reticulocyte count, At lea st two mechanisms might be involved in the increased tyrosine phosphor ylation of band 3 in these hemoglobin disorders, probably related to t he 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, Am, J. Hematol, 58:224-230, 1998. (C) 1998 Wiley-Liss, Inc.