Phosphotyrosine phosphatase associated with band 3 protein in the human erythrocyte membrane

Abstract

The anion-exchange band 3 protein is the main erythrocyte protein that is phosphorylated by tyrosine kinase. To study the regulation of band 3 phosphorylation, we examined phosphotyrisine phosphatase (PTP) activity in the human erythrocyte. We show that the human erythrocyte membrane contains a band 3-associated neutral PTP which is activated by Mg2+ and inhibited by Mn2+ and vanadate. The PTP is active in the intact cell and in the isolated membrane. A major fraction of the PTP is tightly bound to the membrane and can be extracted from it by Triton X-100; a minor part is associated with Triton X-100-insoluble cytoskeleton. The behaviour of the PTP parallels that of band 3, the major fraction of which is extractable by detergents with a minor fraction being anchored to the cytoskeleton. Moreover, band 3 is co-precipitated when the PTP is immunoprecipitated from solubilized membranes, and PTP is co-precipitated when band 3 is immunoprecipitated. The PTP appears to be related to PTP1B (identified using an antibody to an epitope in its catalytic domain and by molecular mass). The system described here has a unique advantage for PTP research, since it allows the study of the interaction of a PTP with an endogenous physiological substrate that is present in substantial amounts in the cell membrane. The membrane-bound, band 3-associated, PTP may play a role in band 3 function in the erythrocyte and in other cells which have proteins analogous to band 3.

Metabolic Effects of Endogenous and Exogenous Heterotropic Hemoglobin Modulators on Anion Transport: The Case of Pig Erythrocytes

“…RV binds preferentially to oxyhemoglobin and causes a significant shift of the oxygen dissociation curve (ODC) towards higher oxygen affinity indicating a stabilization of the R state of the Hb molecule (on the basis of the simple two-states allosteric model). However, RV causes also an upregulation of B3 tyrosine phosphorylation through the activation of lyn, a kinase of the src family [44]; this Tyr phosphorylation could well be the onset of oxidative stress [45] which might be followed by caspase 3 activation [46] [47] starting a "vicious cycle" that would deprive B3 of its important modulatory site (cdb3) [27] [48]. In pig, the impact on the B3 activity due to phosphorylation may be reduced by the contrasting effect of 2,3-DPG, which causes the release of kinase from the erythrocyte membrane [20] inhibiting the phosphorylation of B3.…”

Insulin activation of insulin receptor kinase in erythrocytes is not altered in non-insulin-dependent diabetes and not influenced by hyperglycemia

“…Another proposed mechanism for the glucose-mediated reduction in insulin receptor kinase activation in rat embryo fibroblasts with overexpressed insulin receptors involves alterations in phosphotyrosine phosphatase activity (Maegawa et al 1995). Phosphotyrosine phosphatase-1B (PTP-1B) appears to have a major role in the dephosphorylation of the insulin receptor and IRS-1 (Goldstein et al 2000), and erythrocytes contain a phosphotyrosine phosphatase that immunoprecipitates with monoclonal antibodies against this phosphatase (Zipser & Kosower 1996), and therefore most probably represents PTP-1B. It is nevertheless possible that erythrocytes lack certain components that are present in the cultured cell systems and that are necessary for the effect of glucose on receptor kinase activity or its reversal.…”

Protein Kinases and Protein Phosphatases as Participants in Signal Transduction of Erythrocytes

“…The final effect of peroxynitrite is the ampliphication of tyrosine dependent signaling, a finding of general interest in nitrite oxide related pathophysiology (Mallozzi et al 2005) Т 1В is localized at the erythrocyte membrane associated with band 3. It is activated by Mg 2+ and inhibited by Mn 2+ and vanadate ions (VO 3 -), (Zipser and Kosower, 1996). PTP1B, unlike the other enzymes examined, was quantitatively conserved during erythrocyte aging (Minetti et al 2004) and erythrocytes may undergo in vivo activation of the Ca 2+ -dependent calpain system that proteolytically regulates PTP1B activity (Ciana et al 2004).…”

“…(Harrison et al 1991). 72 syk and p56/53 lyn tyrosine kinases are also involved in band 3 phosphorylation (Hubert et al 2000), and phosphorylation/dephosphorylation cycle is maintained by protein phosphatases Т 1В and SHP-2 (Bordin et al 2002;Zipser and Kosower, 1996;Minetti et al 2004).…”

“…Band 3 is a target for tyrosine phosphatases SH -1 and SHP-2 (Bordin et al 2002) and PTP1B activities (Zipser and Kosower, 1996). Ca 2+ promotes erythrocyte band 3 phosphorylation via dissociation of phosphotyrosine phosphatase from band 3 (Ziper et al 2002) SHP-2 is an essential soluble protein tyrosine phosphatase (PTP) containing two spectrin homologous domains -SH2.…”