Partial purification and characterization of cytosolic Tyr‐protein kinase(s) from human erythrocytes

Abstract

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...

Comparative characterization of membrane‐associated and cytosolic Tyr‐protein kinases in human erythrocytes

“…The white ghosts (100 ~ 250 pg protein) were phosphorylated by the protein kinase sample (varying amounts of the phosphocellulose peak) under the conditions described in a previous paper [5], except that the Pipes buffer pH 6.5 was replaced by 75 mM Hepes buffer, pH 7.5. After incubation, an aliquot (z 40 pg) of 32P-labelled membrane proteins was analyzed by SDS/PAGE as previously described in [ 5 ] .…”

“…After incubation, an aliquot (z 40 pg) of 32P-labelled membrane proteins was analyzed by SDS/PAGE as previously described in [ 5 ] .…”

“…Phosphoamino acid analysis of 32P-labelled membrane proteins Acid hydrolysis (6 M HCl at 1 1 0 T for 2 h) of 32P-labelled membrane proteins (300 pg protein) before and after alkali treatment (2 M NaOH at 55 "C for 1 h) and two-dimensional electrophoresis of the acid hydrolysate on cellulose thin-layer plates were carried out under the conditions previously described [5]. Phosphocellulose, heparin-Sepharose CL-6B and Ultrogel A& were from Sigma, Pharmacia and LKB, respectively.…”

“…The phosphocellulose peak samples of detergent-extracted and NaCIextracted Tyr-protein kinases employed in the assay were calibrated on the basis of having the same kinase activity (1.5 units) with angiotensin 11. After incubation, an aliquot ( z 40 pg) of 32P-labelled membrane proteins was analyzed by SDS/PAGE as described in [5]. Lane CB shows the Coomassie-blue-stained pattern.…”

“…In the past few years, two Tyr-protein kinases phosphorylating the transmembrane band-3 protein have been isolated and partially characterized from human erythrocyte membranes, i.e. one extracted from native ghosts by nonionic detergent [l] and the other from the detergent-insoluble residue by 0.25 M NaCl [2-41. More recently, a Tyr-protein kinase capable of phosphorylating band-3 protein has been isolated by our laboratory [5] from cytosol (hemolysate supernatant). The interrelationship between cytosolic and membranous Tyr-kinases remained to be established.…”