Deficiency of Src family kinases Fgr and Hck results in activation of erythrocyte K/Cl cotransport.

Franceschi, Lucia De; Fumagalli, Laura; Olivieri, Oliviero; Corrocher, Roberto; Lowell, Clifford A.; Berton, Giorgio

doi:10.1172/jci119150

Cited by 112 publications

(96 citation statements)

References 46 publications

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“…26,28,29 Tyrosine phosphorylation modulates the system in complex ways, with some tyrosine kinase inhibitors activating KCC 33 and others inhibiting. 34,35 Some of these effects may be mediated by modulation of protein phosphatase activity by tyrosine phosphorylation 36 and may explain the complex kinetics that have prompted more complicated models of KCC activation. 1,37 Nevertheless, a 2-stage model involving activation by protein phosphatase(s) and inactivation by a volume-sensitive kinase explains most of the kinetics of KCC activation (for more detail, see Document S1, available on the Blood website; see the Supplemental Materials link at the top of the online article).…”

Section: Discussionmentioning

confidence: 99%

Urea stimulation of KCl cotransport induces abnormal volume reduction in sickle reticulocytes

Joiner

Rettig

Jiang

et al. 2006

Blood

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KCl cotransport (KCC) activity contributes to pathologic dehydration in sickle (SS) red blood cells (RBCs). KCC activation by urea was measured in SS and IntroductionThe KCl cotransporter (KCC) mediates electroneutral coupled transport of K ϩ and Cl Ϫ in a variety of cell types. 1,2 KCC activation results in net efflux of KCl from cells with high potassium content, with accompanying water loss and volume reduction. KCC is active in reticulocytes and diminishes with red cell maturation. 3,4 It is thought to function physiologically to reduce red blood cell (RBC) volume and establish the high cellular hemoglobin concentration (CHC) characteristic of mature cells. KCC is activated in vitro by cell swelling, acid pH, sulfhydryl oxidation/alkylation, and exposure to urea, but the relative importance of these stimuli in vivo is unknown.In RBCs containing sickle hemoglobin (Hb S), KCC activity is high, in part because of the high percentage of reticulocytes. 4 However, Hb S or Hb C in trait RBCs (AS or AC) with normal mean cell age, or after incorporation into normal RBC ghosts, appears to increase KCC activity. 5,6 Expression of Hb S or C in transgenic mice also increases red cell KCC activity. 7 Recently, we demonstrated an abnormal response to acid pH in SS RBCs that was partially corrected by incubation with the sulfhydryl reducing agent, dithiothreitol. 8 These findings suggested that regulation of KCC is abnormal in SS RBCs.As a volume regulator, KCC activity is responsive to CHC. 9,10 KCC is activated on cell swelling, and, as KCl is lost, cell volume falls, cellular hemoglobin concentration increases, and transport activity diminishes. The resultant CHC thus reflects the physiologic "volume set point" of the system. The inactivation of KCC as the cell shrinks toward its new steady state is most likely a key factor in defining the volume regulatory function of the transporter, which in turn is crucial for establishing the steady state CHC for RBCs in vivo.Volume reduction mediated by KCC has been demonstrated with RBC density measurements. 7,11,12 However, these studies, like flux studies, are complicated by varying numbers of reticulocytes (or young cells) in unfractionated SS blood, which preclude comparisons of KCC-mediated volume reduction in SS and AA cells. We recently compared the RVD mediated by KCC and stimulated by cell swelling and acid pH in SS and AA reticulocytes by tracking the changes in reticulocyte CHC by means of density gradient analysis. 8 Acid-stimulated volume reduction was exaggerated in SS reticulocytes and was diminished by sulfhydryl reduction, parallel to the behavior of KCC flux activity. However, despite the normal response of KCC flux activation to cell swelling in SS RBCs, swelling-stimulated volume reduction was markedly abnormal in SS reticulocytes and resulted in higher final CHC than in AA reticulocytes. Thus, volume reduction and KCC fluxes appear to reflect different functional aspects of the KCC system in RBCs and may be subject to different pathologic influences in SS RBCs...

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Section: Discussionmentioning

confidence: 99%

Urea stimulation of KCl cotransport induces abnormal volume reduction in sickle reticulocytes

Joiner

Rettig

Jiang

et al. 2006

Blood

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“…14,[19][20][21] Red cell membrane (ghost) and cytosol fractions were obtained as previously reported. [22][23][24] Immunoblot 23, 25 and immunoprecipitation (IP) 14,[26][27][28] were carried out as previously reported (see supplemental Materials and methods for details). When required, the IP assays were carried out as required in the absence or presence of the GST-Lyn/SH3 domain or of the inhibitors PP2 or geldanamycin (GA).…”

Section: Red Cell Membrane Cytosol Preparation Immunoblot Analysismentioning

confidence: 99%

A new molecular link between defective autophagy and erythroid abnormalities in chorea-acanthocytosis

Lupo

Tibaldi

Mattè

et al. 2016

Blood

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Key Points• In chorea-acanthocytosis, spiculated red cells are characterized by heightened Lyn kinase activity and dysregulated autophagy.• Regulation of protein turnover by autophagy plays a key role in erythropoiesis and red cell integrity.Chorea-acanthocytosis is one of the hereditary neurodegenerative disorders known as the neuroacanthocytoses. Chorea-acanthocytosis is characterized by circulating acanthocytes deficient in chorein, a protein of unknown function. We report here for the first time that chorea-acanthocytosis red cells are characterized by impaired autophagy, with cytoplasmic accumulation of active Lyn and of autophagy-related proteins Ulk1 and Atg7. In chorea-acanthocytosis erythrocytes, active Lyn is sequestered by HSP90-70 to form high-molecular-weight complexes that stabilize and protect Lyn from its proteasomal degradation, contributing to toxic Lyn accumulation. An interplay between accumulation of active Lyn and autophagy was found in chorea-acanthocytosis based on Lyn coimmunoprecipitation with Ulk1 and Atg7 and on the presence of Ulk1 in Lyncontaining high-molecular-weight complexes. In addition, chorein associated with Atg7 in healthy but not in chorea-acanthocytosis erythrocytes. Electron microscopy detected multivesicular bodies and membrane remnants only in circulating chorea-acanthocytosis red cells. In addition, reticulocyte-enriched chorea-acanthocytosis red cell fractions exhibited delayed clearance of mitochondria and lysosomes, further supporting the impairment of authophagic flux. Because autophagy is also important in erythropoiesis, we studied in vitro CD341 -derived erythroid precursors. In chorea-acanthocytosis, we found (1) dyserythropoiesis; (2) increased active Lyn; (3) accumulation of a marker of autophagic flux and autolysososme degradation; (4) accumlation of Lamp1, a lysosmal membrane protein, and LAMP1-positive aggregates; and (5) reduced clearance of lysosomes and mitochondria. Our results uncover in choreaacanthocytosis erythroid cells an association between accumulation of active Lyn and impaired autophagy, suggesting a link between chorein and autophagic vesicle trafficking in erythroid maturation. (Blood. 2016;128(25):2976-2987

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“…In some experiments tyrosine-enriched proteins were obtained by immunoprecipitation with a specific anti-phosphotyrosine antibody (clone 4G10, UpState, NY, USA) as previously reported by De Franceschi et al 16,17 Briefly, red-cell ghosts were solubilized in a medium containing 50 mmol/L Tris-HCl, pH 7.4, 100 mmol/L NaCl, 5 mmol/L EDTA, 1% Triton X-100, 1 mmol/L Na-orthovanadate, 0.004% benzamidine, and 1 tablet of a protease inhibitor cocktail (Roche, Germany). After incubation for 60 min at 4°C, the protein extract was centrifuged at 15,000 g for 15 min and the supernatant was used for immunoprecipitation.…”

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confidence: 99%

A novel erythroid anion exchange variant (Gly796Arg) of hereditary stomatocytosis associated with dyserythropoiesis

Iolascon¹,

Falco²,

Borgèse³

et al. 2009

Haematologica

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Background \ud \ud Stomatocytoses are a group of inherited autosomal dominant hemolytic anemias and include overhydrated hereditary stomatocytosis, dehydrated hereditary stomatocytosis, hereditary cryohydrocytosis and familial pseudohyperkalemia. \ud \ud Design and Methods \ud \ud We report a novel variant of hereditary stomatocytosis clue to a de novo band 3 mutation (p. G796R-band3 CEINGE) associated with a dyserythropoietic phenotype. Band 3 genomic analysis, measurement at of hematologic parameters and red cell indices and morphological analysis of bone marrow were carried out. We then evaluated the red cell membrane permeability and ion transport systems by functional studies of the patients erythrocytes and Xenopus oocytes transfected with mutated band 3. We analyzed the red cell membrane tyrosine phosphorylation profile and the membrane association of the tyrosine kinases Syk and Lyn from the Src-family-kinase group, since the activity of the membrane cation transport pathways is related to cyclic phosphorylation-dephosphorylation events. \ud \ud Results \ud \ud The patient showed mild hemolytic anemia with circulating stomatocytes together with signs of dyserythropoiesis. Her red cells displayed increased Na(+) content with decreased K(+) content and abnormal membrane cation transport activities. Functional characterization of band 3 CEINGE in Xenopus oocytes showed that the mutated band 3 is converted from being an anion exchanger (Cl(-), HCO(3)(-)) to being a cation pathway for Na(+) and K(+). Increased tyrosine phosphorylation of some red cell membrane proteins was observed in diseased erythrocytes. Syk and Lyn membrane association was increased in the patient's red cells compared to in normal controls, indicating perturbation of phospho-signaling pathways involved in cell volume regulation events. \ud \ud Conclusions \ud \ud Band 3 CEINGE alters function from that of anion exchange to cation transport, affects the membrane tyrosine phosphorylation profile, in particular of band 3 and stomatin, and its presence during red cell development likely contributes to dyserythropiesis

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Deficiency of Src family kinases Fgr and Hck results in activation of erythrocyte K/Cl cotransport.

Cited by 112 publications

References 46 publications

Urea stimulation of KCl cotransport induces abnormal volume reduction in sickle reticulocytes

Urea stimulation of KCl cotransport induces abnormal volume reduction in sickle reticulocytes

A new molecular link between defective autophagy and erythroid abnormalities in chorea-acanthocytosis

A novel erythroid anion exchange variant (Gly796Arg) of hereditary stomatocytosis associated with dyserythropoiesis

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