Properties of a membrane-bound tyrosine kinase phosphorylating the cytosolic fragment of the red cell membrane band 3 protein.
Band 3 protein of human erythrocyte membrane is phosphorylated on a tyrosine residue located near the NH2 terminal by an endogenous tyrosine kinase activity (Dekowski, S., Rybicki, A. and Drickamer, K. (1983) J. Biol. Chem. 258, 2750-2753). A tyrosine kinase phosphorylating the band 3 protein in situ has been extracted from ghosts by non-ionic detergent and partially characterized (Phan-Dinh-Tuy, F., Henry, J. and Kahn, A. (1985) Biochem. Biophys. Res. Commun. 126, 304-312). We have studied the properties of the tyrosine kinase activity which remains bound to the ghosts after detergent extraction using the 43 kDa fragment of protein 3 as substrate. This activity, solubilized from the detergent-resistant material at 0.25 M NaCl and concentrated by phosphocellulose and tyrosine-agarose chromatographies, remains linked to high molecular weight complexes. It is specific for tyrosine. Assayed with the purified 43 kDa fragment it requires the presence of Mn2+ which cannot be replaced by Mg2+. Its affinity for 43 kDa fragment is very high with a Km of 3.3 microM. ATP acts as a phosphoryl donor with a Km of 0.55 microM. The tyrosine kinase activity was not modified by insulin, DMSO, phorbol ester and epidermal growth factor, vanadate and xanthine derivatives. Polyamines spermidine and the polylysine are inhibitors in the presence of Mn2+ but not in the presence of Mg2+. Heparin is a competitive inhibitor of ATP. 2,3-Diphosphoglycerate is an inhibitor at physiological concentrations (Ki = 2 mM). Purified red cell actin is not phosphorylated by the tyrosine kinase. These properties distinguish the red cell membrane-bound tyrosine kinase from other tyrosine kinases extracted from normal cells.
D019794:2,3-Diphosphoglycerate; D000199:Actins / Q000097:blood; D000255:Adenosine Triphosphate / Q000378:metabolism; D001457:Anion Exchange Protein 1, Erythrocyte / Q000378:metabolism*; D002845:Chromatography; D003600:Cytosol / Q000378:metabolism; D004163:Diphosphoglyceric Acids / Q000494:pharmacology; D004910:Erythrocyte Membrane / Q000201:enzymology*; D006493:Heparin / Q000494:pharmacology; D006801:Humans; D008345:Manganese / Q000494:pharmacology; D010446:Peptide Fragments / Q000378:metabolism; D010766:Phosphorylation; D011073:Polyamines / Q000494:pharmacology; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000097:blood*; D013379:Substrate Specificity; D014443:Tyrosine / Q000378:metabolism; D014638:Vanadates; D014639:Vanadium / Q000494:pharmacology
D000199:Actins; D001457:Anion Exchange Protein 1, Erythrocyte; D004163:Diphosphoglyceric Acids; D010446:Peptide Fragments; D011073:Polyamines; D014639:Vanadium; D019794:2,3-Diphosphoglycerate; D014638:Vanadates; D014443:Tyrosine; D008345:Manganese; D000255:Adenosine Triphosphate; D006493:Heparin; D011505:Protein-Tyrosine Kinases