Global transformation of erythrocyte properties via engagement of an SH2-like sequence in band 3

Abstract

Src homology 2 (SH2) domains are composed of weakly conserved sequences of ∼100 aa that bind phosphotyrosines in signaling proteins and thereby mediate intra-and intermolecular protein-protein interactions. In exploring the mechanism whereby tyrosine phosphorylation of the erythrocyte anion transporter, band 3, triggers membrane destabilization, vesiculation, and fragmentation, we discovered a SH2 signature motif positioned between membrane-spanning helices 4 and 5. Evidence that this exposed cytoplasmic sequence contributes to a functional SH2-like domain is provided by observations that: (i) it contains the most conserved sequence of SH2 domains, GSFLVR; (ii) it binds the tyrosine phosphorylated cytoplasmic domain of band 3 (cdb3-PO 4 ) with K d = 14 nM; (iii) binding of cdb3-PO 4 to erythrocyte membranes is inhibited both by antibodies against the SH2 signature sequence and dephosphorylation of cdb3-PO 4 ; (iv) label transfer experiments demonstrate the covalent transfer of photoactivatable biotin from isolated cdb3-PO 4 (but not cdb3) to band 3 in erythrocyte membranes; and (v) phosphorylation-induced binding of cdb3-PO 4 to the membrane-spanning domain of band 3 in intact cells causes global changes in membrane properties, including (i) displacement of a glycolytic enzyme complex from the membrane, (ii) inhibition of anion transport, and (iii) rupture of the band 3-ankyrin bridge connecting the spectrin-based cytoskeleton to the membrane. Because SH2-like motifs are not retrieved by normal homology searches for SH2 domains, but can be found in many tyrosine kinase-regulated transport proteins using modified search programs, we suggest that related cases of membrane transport proteins containing similar motifs are widespread in nature where they participate in regulation of cell properties.SH2 domain motif | tyrosine phosphorylation | erythrocyte glycolysis | anion exchanger 1 | regulation of transport proteins P rotein tyrosine phosphorylation is involved in the regulation of most cellular process, including proliferation, survival, differentiation, responses to stress, and control of cell shape/motility (1). Whereas phosphotyrosine binding (PTB) domains may mediate association with tyrosine-containing sequences regardless of their phosphorylation state, Src homology 2 (SH2) domains promote protein association only when critical tyrosine residues become phosphorylated. This strict dependence on tyrosine phosphorylation creates a molecular switch that can be sensitively controlled by upstream tyrosine kinases (1). Whereas some SH2 domains facilitate association between heterologous proteins in a signaling pathway, others mediate intrapolypeptide interactions that change protein conformation and thereby alter signaling function (2). In all cases, association between the SH2 domain and the interacting phosphotyrosine involves formation of weak interactions between a highly conserved G(S/T)FLVR sequence of the SH2 domain and the phosphate present on the phosphorylated tyrosine. To date, all 120 known SH2 domain...

Identification of tyrosine kinase inhibitors that halt Plasmodium falciparum parasitemia

“…Oxidation of active site cysteines in both major RBC tyrosine phosphatases then inhibits their activity [ 22 , 57 , 60 – 62 ], leading to stable band 3 tyrosine phosphorylation by constitutively active Syk. This tyrosine phosphorylation then promotes an intramolecular interaction between the phosphotyrosines on band 3 and an SH2-like domain within the membrane-spanning domain of band 3 [ 20 ] that in turn triggers dissociation of ankyrin and the spectrin-based RBC cytoskeleton from band 3 [ 21 , 63 ]. The resulting disjunction of the cytoskeleton from the membrane then causes the predicted weakening of the RBC membrane that allows parasite egress from the weakened RBC [ 24 , 37 ].…”

“…During previous studies of human erythrocyte membranes, we observed that tyrosine phosphorylation of the erythrocyte transmembrane protein, band 3, promotes dissociation of the spectrin-based membrane cytoskeleton from the lipid bilayer via a mechanism that involves an intramolecular association of the phosphorylated cytoplasmic domain of band 3 (cdb3) with an SH2-like (MESH) sequence within the membrane-spanning domain of band 3 [20,21]. Because this phosphorylation-induced cytoskeleton dissociation was found to cause membrane vesiculation and fragmentation [20][21][22], and since band 3 was observed to become increasingly tyrosine phosphorylated during maturation of Plasmodium falciparum within infected erythrocytes [23,24], we hypothesized that egress of the parasite from its red blood cell (RBC) host might require the parasite-stimulated tyrosine phosphorylation of band 3 in order to weaken the RBC membrane in preparation for parasite escape. A subsequent search for possible P. falciparum tyrosine kinases that might perform this membrane-weakening function, however, yielded no obvious tyrosine kinase gene candidate in the P. falciparum genome [25,26], suggesting that the band 3 tyrosine phosphorylation might be performed by a red blood cell encoded tyrosine kinase.…”

Vesiculation of Red Blood Cells in the Blood Bank: A Multi-Omics Approach towards Identification of Causes and Consequences

“…These changes may reflect alterations in enzyme and/or membrane transport activity. Glycolytic enzymes are organized in multi-protein complexes that bind to band 3 and/or various other membrane proteins, and changes in band 3 conformation regulate their activity [44,45,[61][62][63]. Metabolomic data of misshapen RBCs support the hypothesis that alterations in membrane protein conformation affect various metabolic pathways [2,[64][65][66].…”

Inhibition of Band 3 tyrosine phosphorylation: a new mechanism for treatment of sickle cell disease

“…It has been frequently reported that oxidative stress leads to inactivation of erythrocyte tyrosine phosphatases, 30,31,49 which in turn allow unimpeded tyrosine phosphorylation of Band 3 by constitutively active tyrosine kinases 30,35,50 . Because this tyrosine phosphorylation induces an intramolecular interaction in Band 3, which causes dissociation of the spectrin‐actin cortical cytoskeleton from the membrane, 34,35 we hypothesised that oxidative stress deriving from premature HbS denaturation 25,28,29 might initiate a phosphorylation cascade, which would lead to dissociation of the spectrin‐based cytoskeleton from the membrane, causing membrane destabilisation and fragmentation. To test this hypothesis, we compared tyrosine phosphorylation of Band 3 in sickle cells and healthy controls.…”

“…Increased oxidative stress can then cause inhibition of RBC tyrosine phosphatases which normally prevent constitutive Band 3 tyrosine phosphorylation 30–33 . Upon inhibition of these phosphatases, over‐phosphorylation of Band 3 then induces global destabilisation of the erythrocyte membrane, 34,35 accelerating intravascular haemolysis and MP release. The increased plasma haemoglobin and heme can ‘activate’ the vascular endothelium, causing expression of adhesion receptors (e.g., p‐selectin, E‐selectin and von Willebrand factor), 10–12 as well as sequestration of the vasodilator (NO), 11,36–38 while the release of MPs can trigger intravascular thrombosis via activation of prothrombin 12,14,15,17 .…”