Characterization of the hypertonically induced tyrosine phosphorylation of erythrocyte band 3.
Human erythrocyte band 3 becomes rapidly phosphorylated on tyrosine residues after exposure of erythrocytes to hypertonic conditions. The driving force for this phosphorylation reaction seems to be a decrease in cell volume, because (1) changes in band 3 phosphotyrosine content accurately track repeated changes in erythrocyte volume through several cycles of swelling and shrinking; (2) the level of band 3 phosphorylation is independent of the osmolyte employed but strongly sensitive to the magnitude of cell shrinkage; and (3) exposure of erythrocytes to hypertonic buffers under conditions in which intracellular osmolarity increases but volume does not change (nystatin-treated cells) does not promote an increase in tyrosine phosphorylation. We hypothesize that shrinkage-induced tyrosine phosphorylation results either from an excluded-volume effect, stemming from an increase in intracellular crowding, or from changes in membrane curvature that accompany the decrease in cell volume. Although the net phosphorylation state of band 3 is shown to be due to a delicate balance between a constitutively active tyrosine phosphatase and constitutively active tyrosine kinase, the increase in phosphorylation during cell shrinkage was demonstrated to derive specifically from an activation of the latter. Further, a peculiar inhibition pattern of the volume-sensitive erythrocyte tyrosine kinase that matched that of p72syk, a tyrosine kinase already known to associate with band 3 in vivo, suggested the involvement of this kinase in the volume-dependent response.
Dipartimento di Biochimica 'A. Castellani', Università di Pavia, Via Bassi 21, I-27100 Pavia, Italy.
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Minetti
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Seppi
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Ciana
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Balduini
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Brovelli
D000818:Animals; D001457:Anion Exchange Protein 1, Erythrocyte / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D048429:Cell Size / Q000187:drug effects; D003600:Cytosol; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004792:Enzyme Precursors / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D004912:Erythrocytes / Q000187:drug effects / Q000502:physiology*; D006801:Humans; D006982:Hypertonic Solutions / Q000494:pharmacology*; D047908:Intracellular Signaling Peptides and Proteins; D009994:Osmolar Concentration; D010744:Phosphoric Monoester Hydrolases / Q000037:antagonists & inhibitors / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D019311:Staurosporine / Q000494:pharmacology; D000072377:Syk Kinase; D013997:Time Factors; D014443:Tyrosine / Q000378:metabolism*
D001457:Anion Exchange Protein 1, Erythrocyte; D004791:Enzyme Inhibitors; D004792:Enzyme Precursors; D006982:Hypertonic Solutions; D047908:Intracellular Signaling Peptides and Proteins; D011994:Recombinant Proteins; D014443:Tyrosine; D011505:Protein-Tyrosine Kinases; C000606896:SYK protein, human; D000072377:Syk Kinase; D010744:Phosphoric Monoester Hydrolases; D019311:Staurosporine