Tyrosine phosphorylation of band 3 protein in Ca2+/A23187-treated human erythrocytes

Abstract

Human erythrocytes were induced to release membrane vesicles by treatment with Ca2+ and ionophore A23187. In addition to the biochemical changes already known to accompany loading of human erythrocytes with Ca2+, the present study reveals that tyrosine phosphorylation of the anion exchanger band 3 protein also occurs. The relationship between tyrosine phosphorylation of band 3 and membrane vesiculation was analysed using quinine (a non-specific inhibitor of the Ca(2+)-activated K+ channel, and the only known inhibitor of Ca(2+)-induced vesiculation) and charybdotoxin, a specific inhibitor of the apamin-insensitive K(+)-channel. Both inhibitors suppressed tyrosine phosphorylation of band 3. In the presence of quinine, membrane vesiculation was also suppressed. In contrast, at the concentration of charybdotoxin required to suppress tyrosine phosphorylation of band 3, membrane vesiculation was only mildly inhibited (16-23% inhibition), suggesting that tyrosine phosphorylation of band 3 is not necessary for membrane vesiculation. Phosphorylation of band 3 was in fact observed when erythrocytes were induced to shrink in a Ca(2+)-independent manner, e.g. by treatment with the K+ ionophore valinomycin or with hypertonic solutions. These observations suggest that band 3 tyrosine phosphorylation occurs when cell volume regulation is required.

Band 3 is an anchor protein and a target for SHP-2 tyrosine phosphatase in human erythrocytes

“…Phosphorylation/dephosphorylation of protein tyrosine residues (Tyr) has been implicated in the regulation of several erythrocyte functions, including metabolic pathways, [1][2][3][4][5][6] membrane transport, 7,8 and cellular volume and shape. [8][9][10] The Tyr-phosphorylated state of cellular proteins reflects the balance between the competing activities of protein tyrosine kinases and protein tyrosine phosphatases (PTPs).…”

Human kidney anion exchanger 1 localisation in MDCK cells is controlled by the phosphorylation status of two critical tyrosines

“…Hypertonicity is known to induce tyrosine phosphorylation of AE1 in human red blood cells (Minetti et al, 1996;Minetti et al, 1998) and hypertonicity activates tyrosine kinases by cell shrinkage (Kapus et al, 1999;Krump et al, 1997). We do not know why only Y359 was phosphorylated during hypertonic or hyperosmotic stress.…”

“…In addition to the results seen with pervanadate (Harrison et al, 1994), human eAE1 is phosphorylated in cells exposed to hypertonic medium (Minetti et al, 1996;Minetti et al, 1998) or calcium (Zipser et al, 2002). We tested a range of potential stimuli on MDCKIkAE1 cells for their effects on kAE1 phosphorylation.…”

“…Erythroid AE1 is tyrosine phosphorylated under various conditions (Bordin et al, 2002;Brunati et al, 2000;Harrison et al, 1994;Minetti et al, 1996;Minetti et al, 1998) and a proportion of this phosphorylation is inhibitable by the Src-family-specific inhibitor PP1 (Brunati et al, 2000) and the related inhibitor PP2 (Bordin et al, 2002). Fig.…”

Macrophage Recognition of Cells with Elevated Calcium Is Mediated by Carbohydrate Chains of CD43

“…Indeed, A23187 are often used in the laboratory to increase intracellular calcium (Sasaki and HasegawaSasaki., 1985;Minetti et al, 1996). Additionally, when treated with Ca 2+ and A23187, human erythrocytes and lymphocytes respond by budding to release membrane vesicles (Allan and Michell, 1975;Allan et al, 1976a;PodszywalowBartnicka et al, 2007).…”