Tyrosine phosphorylation of band 3 inhibits peripheral protein binding.
The cytoplasmic domain of band 3 (cdb3) of the human erythrocyte membrane is a good substrate of endogenous and exogenous protein-tyrosine kinases. Because one site of tyrosine phosphorylation is within the glycolytic enzyme/hemoglobin-binding region at the N terminus of the polypeptide, we have investigated whether tyrosine phosphorylation of cdb3 might influence its interaction with the above peripheral proteins. Using p40, a protein-tyrosine kinase isolated from bovine thymus, we demonstrate that aldolase binding to cdb3 linked to Affi-Gel 15 is significantly inhibited by phosphorylation of the immobilized band 3. Importantly, upon dephosphorylation of the gel with acid phosphatase, aldolase binding returns to prephosphorylated values. Similarly, cdb3 phosphorylation was found to inhibit glyceraldehyde-3-phosphate dehydrogenase, phosphofructokinase, and hemoglobin binding to immobilized cdb3. In the converse experiment, untreated soluble cdb3 was shown to bind to immobilized aldolase, whereas phosphorylated cdb3 (approximately equal to 1.8 mol of Pi/mol of cdb3) did not. Furthermore, phosphorylated cdb3 was unable to inhibit aldolase catalysis, whereas untreated cdb3, as shown previously by others, was a potent inhibitor. Taken together, these results demonstrate that phosphorylation of cdb3 on tyrosine residues inhibits peripheral protein binding at the polypeptide's N terminus. In view of the known effect of glycolytic enzyme binding to band 3 on catalytic activity, tyrosine phosphorylation of band 3 may modulate glycolysis in vivo.
D001457:Anion Exchange Protein 1, Erythrocyte / Q000378:metabolism*; D004910:Erythrocyte Membrane / Q000378:metabolism; D005634:Fructose-Bisphosphate Aldolase / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D010766:Phosphorylation; D011485:Protein Binding; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism*
D001457:Anion Exchange Protein 1, Erythrocyte; D014443:Tyrosine; D011505:Protein-Tyrosine Kinases; D005634:Fructose-Bisphosphate Aldolase