Deoxygenation of sickle cells stimulates Syk tyrosine kinase and inhibits a membrane tyrosine phosphatase

Merciris, Patrick; Hardy‐Dessources, Marie‐Dominique; Giraud, Françoise

doi:10.1182/blood.v98.10.3121

Cited by 35 publications

(30 citation statements)

References 49 publications

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“…Protein tyrosine kinase activity proved to be insensitive to deoxygenation in normal human RBCs [71], whereas in sickle cells deoxygenation leads to inhibition of protein tyrosine phosphatase and activation of Syk tyrosine kinase [72]. On the other hand, in normal human erythrocytes, deoxygenation was shown to increase tyrosine phosphorylation of band 3, apparently via stimulation of tyrosine kinase activity [13].…”

Section: Hb Oxygenation and Signalling Pathwaysmentioning

confidence: 99%

Erythrocyte Signal Transduction Pathways, their Oxygenation Dependence and Functional Significance

Barvitenko

Adragna

Weber³

2005

Cell Physiol Biochem

135

104

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Erythrocytes play a key role in human and vertebrate metabolism. Tissue O₂ supply is regulated by both hemoglobin (Hb)-O₂ affinity and erythrocyte rheology, a key determinant of tissue perfusion. Oxygenation-deoxygenation transitions of Hb may lead to re-organization of the cytoskeleton and signalling pathways activation/deactivation in an O₂-dependent manner. Deoxygenated Hb binds to the cytoplasmic domain of the anion exchanger band 3, which is anchored to the cytoskeleton, and is considered a major mechanism underlying the oxygenation-dependence of several erythrocyte functions. This work discusses the multiple modes of Hb-cytoskeleton interactions. In addition, it reviews the effects of Mg²⁺, 2,3-diphosphoglycerate, NO, shear stress and Ca²⁺, all factors accompanying the oxygenation-deoxygenation cycle in circulating red cells. Due to the extensive literature on the subject, the data discussed here, pertain mainly to human erythrocytes whose O₂ affinity is modulated by 2,3-diphosphoglycerate, ectothermic vertebrate erythrocytes that use ATP, and to bird erythrocytes that use inositol pentaphosphate.

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Section: Hb Oxygenation and Signalling Pathwaysmentioning

confidence: 99%

Erythrocyte Signal Transduction Pathways, their Oxygenation Dependence and Functional Significance

Barvitenko

Adragna

Weber³

2005

Cell Physiol Biochem

135

104

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“…The SS cell fraction between densities 1.076 and 1.106 (20-30% reticulocytes and 70-80% discocytes) was collected, washed 3 times, resuspended in solution A (in mM): 15 NaCl,125 KCl, 20 HEPES/TRIS (pH 7.7 at 4 C), 295 mOsM, and stored overnight at 4 C. The concentration of total and ionized magnesium in this SS cell fraction is reportedly 1.7-2.2 and 0.37-0.49 mM respectively, close to the values in normal red cells [39]. All the preceding steps were performed at 4 C. Syk and Lyn activities were determined by an autophosphorylation assay, a method reported previously to correlate well with their activity towards an exogenous substrate [36]. IC assays were started by adding 2 l 250 M [g-32 P]-ATP (37 kBq) and incubated for 15 min at 37 C. Reactions were stopped by addition of 25 l SDS sample buffer.…”

Section: Cell Preparationmentioning

confidence: 72%

“…Furthermore, the activity of Lyn was not modified significantly by deoxygenation, whether or not [Mg 2+ ] i was clamped at its level in oxygenated cells, indicating that the deoxygenation-induced rise in [Mg 2+ ] i from 0.4 to 0.7 mM in the SS cell fraction used [39], was not sufficient to affect Lyn activity. After 60 min deoxygenation, activation of Syk and lack of change in Lyn activity were still observed (data not shown, see [36]). …”

Section: Effect Of Deoxygenation On Syk and Lyn Activity In Mg 2+ -Clmentioning

confidence: 89%

“…Deoxygenation of SS cells increases PTK activity and band-3 tyrosine phosphorylation [35] and stimulates Syk without changing Lyn activity [36]. In addition, the PTK inhibitors methyl-2,5-dihydroxycinnamate (DHC) and tyrphostin 47 (T 47) reduce deoxygenation-induced K + efflux in SS cells in the absence of external Ca 2+ and without affecting SIP, suggesting the involvement of PTK in the activation of KCC [35].…”

Section: Introductionmentioning

confidence: 99%

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Regulation of K-Cl cotransport by Syk and Src protein tyrosine kinases in deoxygenated sickle cells

Merciris

Claussen²,

Joiner³

et al. 2003

Pflugers Arch - Eur J Physiol

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Protein tyrosine kinases (PTK) of the Src family are thought to suppress K-Cl cotransport (KCC) activity via negative regulation of protein phosphatases. However, some PTK inhibitors reduce KCC activity, suggesting opposite regulation by different PTK families. We have reported previously that deoxygenation of sickle cells stimulates KCC and activates Syk (a Syk family PTK), but not Lyn (an Src family PTK). In this study the same results were obtained when PTK activities were measured under the conditions used to measure KCC activity and which prevent any change in intracellular [Mg(2+)]. Methyl-2,5-dihydroxycinnamate (DHC), a PTK inhibitor, was more selective for Syk than Lyn, while staurosporine (ST), a broad-specificity protein kinase inhibitor, inhibited Lyn more than Syk. Deoxygenation or 4-amino-5-(4-chlorophenyl)-7-( t-butyl)pyrazolo[3,4- d] pyrimidine (pp2, a specific Src inhibitor) stimulated KCC independently. These effects were not additive and were inhibited by DHC. In contrast, ST-induced KCC activation was resistant to DHC, suggesting a different pathway of activation. Overall, these data indicate that Syk activity is required for KCC activation, either induced by deoxygenation of sickle cells, or mediated by Src inhibition in oxygenated cells, and that Syk and Src PTKs exert opposing and interconnected regulatory effects on the activity of the transporter.

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“…These findings led us to suggest that Src family kinases down-regulated KCl cotransport activity by phosphorylating and inhibiting the K-Cl cotransport stimulator PP-1a [4,33]. Interestingly, other studies implicated erythroid Src and Syk kinases as modulators of K-Cl cotransport in human sickle red cells exposed to cyclic oxy-deoxygenation [34,35]. Similarities are evident in the mechanisms of red cell dehydration and K + loss observed in human sickle red cells and in erythrocytes from the SAD mouse, a transgenic mouse model for sickle cell disease [36].…”

Section: Introductionmentioning

confidence: 98%

Regulation of K–Cl cotransport by protein phosphatase 1α in mouse erythrocytes

Franceschi

Villa-Moruzzi

Biondani

et al. 2005

Pflugers Arch - Eur J Physiol

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The K-Cl cotransport (KCC) is an electroneutral-gradient-driven-membrane transport system, which is involved in regulation of red cell volume. Although the regulatory cascade of KCC is largely unknown, a signaling pathway involving phosphatases and kinases has been proposed. Here, we investigated the expression and the activity of protein phosphatase 1(PP-1) isoforms in mouse red cells, focusing on two models of abnormally activated KCC: mice genetically lacking the two Src-family tyrosine kinases, Hck and Fgr, (hck-/-fgr-/-) and the SAD transgenic sickle-cell-mice. The PP-1alpha, PP-1gamma, PP-1delta isoforms were expressed at similar levels in wild-type, hck-/-fgr-/- and SAD mouse erythrocytes and in each case were predominantly localized to cytoplasm. The PP-1alpha activity was significantly higher in both membrane and cytosol fractions of hck-/-fgr-/- and of SAD erythrocytes than in those of wild-type red cells, suggesting PP-1alpha as a target of the Hck and Fgr kinases. The PP2, a specific inhibitor of Src-family kinase, significantly increased KCC activity in wild-type mouse red cells, but failed to modify the already increased KCC activity in SAD erythrocytes. The lag-time for activation of KCC was considerably reduced in both hck-/-fgr-/- and SAD erythrocytes, suggesting that the rate limiting activation steps in both strains are freed from their tonic inhibition. Sulfhydryl reduction by dithiothreitol (DTT) lowered KCC activity only in SAD red cells, but did not affect the PP2-treated erythrocytes. These data suggest up-regulation of KCC in SAD red cells is mainly secondary to oxidative damage, which most likely reduces or removes the tonic KCC inhibition resulting from PP-1alpha activity controlled in turn by Src-family kinases.

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Deoxygenation of sickle cells stimulates Syk tyrosine kinase and inhibits a membrane tyrosine phosphatase

Cited by 35 publications

References 49 publications

Erythrocyte Signal Transduction Pathways, their Oxygenation Dependence and Functional Significance

Erythrocyte Signal Transduction Pathways, their Oxygenation Dependence and Functional Significance

Regulation of K-Cl cotransport by Syk and Src protein tyrosine kinases in deoxygenated sickle cells

Regulation of K–Cl cotransport by protein phosphatase 1α in mouse erythrocytes

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