Tyrosine phosphorylation of band 3 protein in Ca2+/A23187-treated human erythrocytes.
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.
Minetti
G
G
Dipartimento di Biochimica A. Castellani, Università di Pavia, Italy.
Piccinini
G
G
Balduini
C
C
Seppi
C
C
Brovelli
A
A
D001457:Anion Exchange Protein 1, Erythrocyte / Q000187:drug effects / Q000378:metabolism*; D001030:Apamin / Q000494:pharmacology; D000001:Calcimycin / Q000494:pharmacology*; D002118:Calcium / Q000494:pharmacology*; D018999:Charybdotoxin / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004910:Erythrocyte Membrane / Q000187:drug effects / Q000378:metabolism*; D004912:Erythrocytes / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D010750:Phosphoproteins / Q000097:blood* / Q000302:isolation & purification; D010766:Phosphorylation; D019000:Phosphotyrosine; D026902:Potassium Channel Blockers; D015221:Potassium Channels / Q000502:physiology; D011803:Quinine / Q000494:pharmacology
D001457:Anion Exchange Protein 1, Erythrocyte; D010750:Phosphoproteins; D026902:Potassium Channel Blockers; D015221:Potassium Channels; D018999:Charybdotoxin; D019000:Phosphotyrosine; D001030:Apamin; D000001:Calcimycin; D011803:Quinine; D002118:Calcium