Band 3 (AE1), the most prominent polypeptide of the human erythrocyte membrane, becomes heavily tyrosine phosphorylated following treatment of intact cells with protein tyrosine phosphatase inhibitors such as diamide, pervanadate, vanadate, or N-ethylmaleimide (NEM). The mechanism underlying this tyrosine phosphorylation is thought to involve the sequential action of two protein tyrosine kinases, Syk (p72syk) and Lyn (p53/56lyn). While Lyn catalysed phosphorylation appears to be strictly dependent on prior phosphorylation of Tyr8 and 21 of band 3 by Syk, little is known about the mechanism of induction of Syk phosphorylation. Data presented here show that both the fraction of Syk that associates with the membrane and the extent of phosphorylation of band 3 differ in response to the above inhibitors. While diamide and NEM stimulate syk translocation to the membrane during their induction of band 3 tyrosine phosphorylation, pervanadate and vanadate induce no change in kinase distribution. Moreover, diamide and NEM-induced Syk recruitment to the membrane are phosphotyrosine independent and involve their preferential association with Triton X-100-insoluble membrane skeletons. Together these data reveal a complex process controlling the association and catalytic activity of protein tyrosine kinases syk and lyn with the human erythrocyte membrane.

Haemolysis is usually episodic in glucose-6-phosphate dehydrogenase (G6PD) deficiency, often triggered by a period of oxidative stress. In the present work, we investigate a possible biochemical mechanism underlying the enhanced susceptibility of G6PD deficient red blood cells (RBC) to oxidative stress. We analysed eight male subjects with Mediterranean glucose-6P-dehydrogenase deficiency (G6PDd), class II, for their ability in phosphorylating erythrocyte membrane band 3 following oxidative and osmotic stress. Our findings show that this sensitivity is connected to an early membrane band 3 Tyr-phosphorylation in the presence of diamide. However, since both Syk, and Lyn kinases, and SHP-2 phosphatase, mostly implicated in the band 3 P-Tyr level regulation, are alike in content and activity in normal and patient erythrocytes, an alteration in the membrane organization is likely the cause of the anomalous response to the oxidant. We report, in fact, that hypertonic-induced morphological change in G6PDd erythrocyte induces a higher membrane band 3 Tyr-phosphorylation, suggesting a pre-existing membrane alteration, likely due to the chronic lowering of the redox systems in patients. We also report that 1-chloro-2,4-dinitrobenzene-pre-treatment of normal red cells can alter the normal protein-protein and protein-membrane interaction under hypertonic rather than oxidative stress, thus partially resembling the response in patients, and that RBC may utilize a wider range of redox defence, under oxidative conditions, including, but not exclusively, NADPH and glutathione. On the whole, these results would encourage a different approach to the evaluation of the effects of pharmacological administration to patients, giving more attention to the possible drug-induced membrane alteration evidenced by the abnormal band 3 Tyr-phosphorylation.

No abstract

SHP-1 is a SH2-domain containing protein Tyr-phosphatase expressed in hematopoietic cell lines, which is hypothesized to play a negative role in signal transduction. In human erythrocytes, the phospho-Tyr level of proteins, mainly transmembrane band 3, is closely controlled by the antithetic activity of Tyr-protein kinases and phosphatases, resulting in a dephosphorylated state. Only after particular stimuli, as with oxidizing agents, diamide or pervanadate, or thiol alkylating compound, N-ethyl maleimide (NEM), Tyr-phosphorylation of band 3 can be triggered, inhibiting Tyr-phosphatase action and inducing erythrocyte membrane reorganization. We demonstrate that, in human erythrocytes, SHP-1 is present in membranes from resting cells, but in 5% of the protein amount. Interestingly, this amount increases up to threefold following NEM treatment of intact cells, whereas diamide and pervanadate do not alter the normal protein location. In addition, SHP-1 translocation from cytosol to membrane is not affected by band 3 P-Tyr level, because it is not mediated by the SH2-P-Tyr recruitment mechanism, and localizes into the cytoskeletal compartment. Band 3 is the target of SHP-1, which dephosphorylates Tyr 8, 21, and 904. These findings support the idea that, in human erythrocytes, the normal level of Tyr-phosphorylation of membrane protein, mainly band 3, must be downregulated. We hypothesize that the presence of both SHP-2 and SHP-1 ensures band 3 dephosphorylation in different conditions: SHP-2, through interaction of its SH2 domain/s to P-Tyr protein, is regulated by the band 3 Tyr-phosphorylation level; SHP-1 may be involved by simple membrane rearrangement.