Functional Link between Phosphorylation State of Membrane Proteins and Morphological Changes of Human Erythrocytes

Bordin, Luciana; Clari, Giulio; Moro, Isabella; Vecchia, Francesca Dalla; Moret, V.

doi:10.1006/bbrc.1995.2123

Cited by 36 publications

(30 citation statements)

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“…Tyrosine kinase assays of immunocomplexes were performed in 30 L incubation mixture containing 50 mmol/L Tris-HCl, pH 7.5; 10 mmol/L MnCl 2 , 50 mol/L ␥[ 32 P]ATP (specific activity 1000 cpm/pmol), 100 mol/L sodium orthovanadate (basal medium), and either 300 ng of the cytoplasmic domain of band 3 16 or 200 M cdc2 (6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20) peptide, 16 which served as substrates for Syk and Lyn, respectively. Following incubation for 10 minutes at 30°C, samples were subjected to SDS-PAGE (15% gels), and substrate phosphorylation was evaluated by an Instant Imager (Packard Instrument Company, Meriden, CT).…”

Section: Syk and Lyn Ip Kinase Assaysmentioning

confidence: 99%

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Band 3 is an anchor protein and a target for SHP-2 tyrosine phosphatase in human erythrocytes

Bordin

Brunati²,

Donella‐Deana³

et al. 2002

Blood

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Section: Syk and Lyn Ip Kinase Assaysmentioning

confidence: 99%

“…[8][9][10] The Tyr-phosphorylated state of cellular proteins reflects the balance between the competing activities of protein tyrosine kinases and protein tyrosine phosphatases (PTPs). Because the activity of PTPs is much higher than that of protein tyrosine kinases, basal levels of protein phosphotyrosine (P-Y) in cells are very low.…”

Section: Introductionmentioning

confidence: 99%

Band 3 is an anchor protein and a target for SHP-2 tyrosine phosphatase in human erythrocytes

Bordin

Brunati²,

Donella‐Deana³

et al. 2002

Blood

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“…It was recently reported that Tyr phosphorylation is implicated in the modification of several erythrocyte functions, including metabolic pathways (11), membrane transport (12), and cell volume and shape (13). Phosphorylation of proteins is a common characteristic of signal transduction systems.…”

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confidence: 99%

Hypoxia-Induced Post-Translational Changes in Red Blood Cell Protein Map of Newborns

et al. 2005

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Tyrosine (Tyr) phosphorylation is implicated in the modification of several erythrocyte functions, such as metabolic pathways and membrane transport, as well as in signal transduction systems. Here we describe the map of Tyr-phosphorylated soluble proteins of newborn red blood cells (RBC) using an in vitro model simulating RBC reoxygenation at birth after an intrauterine hypoxic event. We tested the hypothesis that a hypoxic environment and subsequent reoxygenation promote posttranslational changes in the RBC protein map of newborns, in addition to desferrioxamine (DFO)-chelatable iron (DCI) release and methemoglobin (MetHb) formation. Umbilical cord blood RBC were incubated under hypoxic conditions for 16 h at 37°C, and subsequently for 8 h under aerobic conditions. Control erythrocytes were incubated under aerobic conditions at 37°C for the period of the experiment, i.e. for 24 h. Tyr-phosphorylation proteins were assessed using advanced high-resolution twodimensional electrophoresis, 2-D immunoblot analysis with antiphosphotyrosine (anti-pTyr) antibodies, and computer-aided electrophoretogram analysis. Higher DCI release and MetHb formation were observed in newborn RBC incubated under hypoxic conditions than in those incubated aerobically. Different immunoreactivity patterns with anti-pTyr antibodies were also observed between newborn RBC incubated under hypoxic conditions and controls. A hypoxic environment is a factor promoting DCI release, a well-known condition of oxidative stress. This is the first map of Tyr-phosphorylated soluble proteins of newborn RBC obtained using an in vitro model simulating RBC reoxygenation at birth after an intrauterine hypoxic event. Our results suggest that hypoxia increases Tyr-phosphorylation of antioxidant proteins, protecting RBC against oxidative stress. RBC are exposed to oxidative stress more than other cells of the body as a result of their high membrane polyunsaturated fatty acid content and high cellular concentrations of oxygen and heme iron. Iron plays a central role in generating harmful ROS. Its redox cycling promotes the Fenton reaction, which produces the potent oxidant hydroxyl radical (1). To be redoxcycling active, iron must be released from its macromolecular complexes (mainly transport and storage proteins) (2). We previously showed that iron is released from Hb in a DFOchelatable form when erythrocytes are exposed to oxidative stress, such as when they are incubated with oxidizing agents or undergo prolonged aerobic incubation, two models of erythrocyte ageing (3). Iron release is accompanied by Met-Hb formation and oxidative damage to erythrocyte membrane proteins and lipids (3). In several studies, we used DCI in erythrocytes as an index of oxidative stress in various adult and neonatal pathophysiological conditions (4 -7). We found that RBC of hypoxic newborns have a higher DCI content (6,7) and greater susceptibility to iron release after aerobic incubation

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“…To test this hypothesis we performed a set of experiments in the presence of OV, a phosphatase inhibitor [22] well known to affect protein phosphorylation, including band 3 cytoplasmic domain (cdb3) [23] [24]. In terms of anion flux (Figure 4), the results show that OV induce a significant increase of the B3 activity both in human and pig RBCs; the analysis of the percentage of increase of the anion exchange in the presence of OV (about 30% in human and 20% in pig RBCs) indicates a difference between the two types of cells.…”

Section: Resultsmentioning

confidence: 99%

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

Tellone

Russo²,

Giardina³

et al. 2015

OALib

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This comparative study, focused on the anion kinetic of band 3 protein of pig erythrocytes, investigates in vitro the metabolic functionality of the red blood cells of this mammal which show particular characteristics of the cell membrane and significant stoichiometric variations of several cytosolic components. Pig red blood cells compared to human ones, are characterized by a higher anion flux strongly modulated by the conformational transition state (T-R) of hemoglobin; these findings are probably connected to the intracellular concentration of 2,3-disphosphoglycerate and to chloride activity. The experimental use, alone or in combination, of some exogenous heterotropic modulators of hemoglobin as orthovanadate, gemfibrozil and resveratrol, shows characteristic modulations of the anion flux which are related to the cytosolic metabolites physiologically present in pig erythrocytes, to the metabolism and oxidative stress. Our findings highlight new features about the pig red blood cells functionality and contribute to extending the current understanding of their life cycle, providing opportunities for new investigations in the field of comparative physiology.

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Functional Link between Phosphorylation State of Membrane Proteins and Morphological Changes of Human Erythrocytes

Cited by 36 publications

References 0 publications

Band 3 is an anchor protein and a target for SHP-2 tyrosine phosphatase in human erythrocytes

Band 3 is an anchor protein and a target for SHP-2 tyrosine phosphatase in human erythrocytes

Hypoxia-Induced Post-Translational Changes in Red Blood Cell Protein Map of Newborns

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

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