Proteomic analysis of ERK1/2-mediated human sickle red blood cell membrane protein phosphorylation

Erik J Soderblom,M Arthur Moseley,Edward Chiou,J Will Thompson,Laura G Dubois,Rahima Zennadi,Evan A Schwartz

doi:10.1186/1559-0275-10-1

Erik J Soderblom, M Arthur Moseley + Show 5 more

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https://doi.org/10.1186/1559-0275-10-1

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Abstract

BackgroundIn sickle cell disease (SCD), the mitogen-activated protein kinase (MAPK) ERK1/2 is constitutively active and can be inducible by agonist-stimulation only in sickle but not in normal human red blood cells (RBCs). ERK1/2 is involved in activation of ICAM-4-mediated sickle RBC adhesion to the endothelium. However, other effects of the ERK1/2 activation in sickle RBCs leading to the complex SCD pathophysiology, such as alteration of RBC hemorheology are unknown.ResultsTo further characterize global ERK1/2-induced changes in membrane protein phosphorylation within human RBCs, a label-free quantitative phosphoproteomic analysis was applied to sickle and normal RBC membrane ghosts pre-treated with U0126, a specific inhibitor of MEK1/2, the upstream kinase of ERK1/2, in the presence or absence of recombinant active ERK2. Across eight unique treatment groups, 375 phosphopeptides from 155 phosphoproteins were quantified with an average technical coefficient of variation in peak intensity of 19.8%. Sickle RBC treatment with U0126 decreased thirty-six phosphopeptides from twenty-one phosphoproteins involved in regulation of not only RBC shape, flexibility, cell morphology maintenance and adhesion, but also glucose and glutamate transport, cAMP production, degradation of misfolded proteins and receptor ubiquitination. Glycophorin A was the most affected protein in sickle RBCs by this ERK1/2 pathway, which contained 12 unique phosphorylated peptides, suggesting that in addition to its effect on sickle RBC adhesion, increased glycophorin A phosphorylation via the ERK1/2 pathway may also affect glycophorin A interactions with band 3, which could result in decreases in both anion transport by band 3 and band 3 trafficking. The abundance of twelve of the thirty-six phosphopeptides were subsequently increased in normal RBCs co-incubated with recombinant ERK2 and therefore represent specific MEK1/2 phospho-inhibitory targets mediated via ERK2.ConclusionsThese findings expand upon the current model for the involvement of ERK1/2 signaling in RBCs. These findings also identify additional protein targets of this pathway other than the RBC adhesion molecule ICAM-4 and enhance the understanding of the mechanism of small molecule inhibitors of MEK/1/2/ERK1/2, which could be effective in ameliorating RBC hemorheology and adhesion, the hallmarks of SCD.

Highlights

In sickle cell disease (SCD), the mitogen-activated protein kinase (MAPK) ERK1/2 is constitutively active and can be inducible by agonist-stimulation only in sickle but not in normal human red blood cells (RBCs)
Sickle red blood cells homozygous for HbS (SS RBCs) are characterized by a panoply of abnormalities, including polymerization of deoxygenated HbS [1,2], persistent oxidative membrane damage associated with HbS cyclic polymerization [3], abnormal activation of membrane cation transports, cell dehydration [4], cytoskeletal dysfunction [5], and increased adhesion [6]
These stresses are thought to be propagated through alterations in normal protein phosphorylation events within complex intracellular signaling pathways which may subsequently affect protein structural stability [11,12], formation of protein–protein complexes [13,14], activation of ion transport leading to cell dehydration [15,16,17] and RBC adhesive function [18,19]

Summary

Introduction

In sickle cell disease (SCD), the mitogen-activated protein kinase (MAPK) ERK1/2 is constitutively active and can be inducible by agonist-stimulation only in sickle but not in normal human red blood cells (RBCs). Sickle red blood cells homozygous for HbS (SS RBCs) are characterized by a panoply of abnormalities, including polymerization of deoxygenated HbS [1,2], persistent oxidative membrane damage associated with HbS cyclic polymerization [3], abnormal activation of membrane cation transports, cell dehydration [4], cytoskeletal dysfunction [5], and increased adhesion [6]. These alterations in SS RBCs lead to the complex pathophysiology associated with SCD that includes vaso-occlusion, chronic hemolysis and ischemic tissue damage [7]. Several proteins involved in these pathways have been previously shown to be differentially tyrosine phosphorylated in SS RBCs compared to normal (AA) RBCs, including adducin, ankyrin 1, the actin binding protein dematin, and protein band 4.1, which stabilizes the spectrin-actin interaction [14,20]

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