Band-3 protein (approximately 95 kDa), the major and multifunctional transmembrane protein of human erythrocytes, has been shown to be phosphorylated by endogenous Tyr-protein kinases on different Tyr residues at its N and C cytoplasmic domains.Both the added ~3 6 "~ (catalytic domain of ~7 2 "~~) and Lyn kinases are able to phosphorylate the isolated cytoplasmic domain of band 3 (cdb3), yielded by chymotryptic digestion of band 3 in the isolated membranes (ghosts).However, the two Tyr-protein kinases exhibited different phosphorylation behaviours when added to the isolated erythrocyte membranes. More precisely, the added p36"jk markedly Tyr phosphorylates the band-3 protein, whereas the added Lyn phosphorylates it very poorly.It is of interest that Lyn can associate with membranes and markedly phosphorylate band 3 when this latter protein has been previously phosphorylated by p3eYk, i.e. the p36'vk-catalyzed phosphorylation is proposed to be a prerequisite for the association of Lyn with the membrane (likely to band 3) and for the Lyn-catalyzed phosphorylation of different band-3 Tyr sites.Keywords: p36"Yk-catalyzed Tyr phosphorylation; Lyn-catalyzed Tyr phosphorylation ; band 3 ; human erythrocytes.For a long time, the major multifunctional transmembrane band-3 protein (approximately 95 kDa) of human erythrocytes has been found to be endogenously phosphorylated at its Ncytoplasmic domain (approximately 43 kDa), protruding into the cytosol as an extended flexible finger, not only on the Ser/Thr residues [ l ] by CAMP-independent casein kinase I [2-61, but also on Tyr residues [7-181 by Tyr-kinases.Human erythrocyte Tyr-protein kinases, phosphorylating band 3 in the isolated ghosts or its isolated cytoplasmic domain of band 3 (cdb3), have been studied in several laboratories including our own [15, 161, where two Tyr-protein kinases have been isolated from human erythrocyte cytosol by DEAE-cellulose chromatography followed by Heparin-Sepharose chromatography.While the most abundant cytosolic Tyr-protein kinase (approximately 36 kDa) [15] belongs to the Syk family, being reported to be yielded quite likely by proteolytic breakdown of its larger parent ~7 2 ' '~ in the human erythrocyte membranes [19] and other cells [20, 211, the other minor cytosolic Tyr-protein kinase [15] has been shown (by immuno-blotting assay) to belong to the Lyn family, as previously reported in [19]. This paper deals with the different properties of the two Tyrprotein kinases and, in particular, with the mechanism by which the p36vk-catalyzed phosphorylation of band 3 in the isolated ghosts recruits band 3 for the Lyn-catalyzed phosphorylation of Tyr residue(s) not 32P labelled by ~3 6 "~.In particular, the results reported here show that Lyn, when added to isolated human erythrocyte ghosts, can associate with membranes and markedly phosphorylate the band-3 cytoplasmic domain, only once this latter had been previously disposed by the p36"yk-catalyzed phosphorylation in the presence of unlabeled ATP. MATERIALSAnti-Lyn was purchased from Sa...

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Tyrosine-protein kinase, phosphorylating tyrosine residues of transmembrane band 3 protein, has been partially purified from human erythrocyte cytosol by DEAE-Sepharose chromatography followed by heparinSepharose chromatography.Such a Tyr-protein kinase (36 kDa), as distinct from the Ser/Thre-protein kinases (casein kinase S and TS), appears to display a broader site specificity than does the previously described human erythrocyte P-Tyr-protein phosphatase, dephosphorylating band 3 protein. That is, it is able to phosphorylate not only the highly acidic copolymer poly(Glu-Tyr)4 but also angiotensin 11, lacking an acidic amino acid sequence around the target Tyr residue. Moreover, the phosphorylation of these two substrates exhibits a different pH dependence and a different response to NaCl and 2,3-bisphosphoglycerate.These results suggest that in intact erythrocytes the cytosolic Tyr-protein kinase might phosphorylate band 3 not only on Tyr-8, surrounded by several acidic side-chains (as demonstrated preferentially to occur in isolated ghosts), but also on other Tyr residues surrounded by other amino acid sequences.The multifunctional transmembrane band 3 protein (95 kDa) of human erythrocytes has been found to be endogenously phosphorylated not only on serine/threonine residues [l] but also on tyrosine residues [2].Up to now attention has been focused on the characterization of human erythrocyte Tyr-protein kinase(s) associated with membrane structures [3 -71.The present research has been undertaken to characterize the endogenous Tyr-protein kinase(s) located in the cytosol and presumably involved in the Tyr phosphorylation of band 3, taking into account the fact that the endogenous phosphorylation of this transmembrane protein has been found [2] to occur preferentially on its NH,-terminal region (23 kDa). Since this region protrudes from the membrane bilayer into the cytoplasm, it is expected to be accessible to the cytosolic protein kinases.On the other hand, human erythrocyte P-Tyr-protein phosphatase(s), dephosphorylating band 3 P-tyrosine residues, has been found to be located predominantly in the cytosol [8, 91.The results reported here indicate that cytosolic Tyr-protein kinase(s), and P-Tyr-protein phosphatase(s), involved in the Tyr phosphorylation state of band 3 protein, do not exhibit the same substrate specificity. That is, the Tyr-protein kinase(s) described here is able markedly to phosphorylate not only the very acidic random copolymer poly(Glu-Tyr)4: 1 but also angiotensin 11, lacking acidic amino acid sequences around the target Tyr residue. By contrast, the P-Tyr-protein MATERIALS AND METHODSHuman erythrocytes (9 ml packed cells), free of leucocytes and platelets, were prepared from venous blood (45 ml) (freshly collected from healthy donors), according to Beutler et al. [lo]. Thereafter they were washed once in isotonic phosphate buffer pH 8 and lysed in hypotonic ( 5 mM) phosphate buffer pH 8 according to Dodge et al. [Ill, except that the solutions contained 0.05 mM phenylmethylsulphonyl...

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