Proteomic analysis of ERK1/2-mediated human sickle red blood cell membrane protein phosphorylation.
Abstract
UNLABELLED: BACKGROUND: 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). 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. RESULTS: To 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.
CONCLUSIONS: These 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.
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https://hdl.handle.net/10161/11163Published Version (Please cite this version)
10.1186/1559-0275-10-1Publication Info
Soderblom, Erik J; Thompson, J Will; Schwartz, Evan A; Chiou, Edward; Dubois, Laura
G; Moseley, M Arthur; & Zennadi, Rahima (2013). Proteomic analysis of ERK1/2-mediated human sickle red blood cell membrane protein
phosphorylation. Clin Proteomics, 10(1). pp. 1. 10.1186/1559-0275-10-1. Retrieved from https://hdl.handle.net/10161/11163.This is constructed from limited available data and may be imprecise. To cite this
article, please review & use the official citation provided by the journal.
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Show full item recordScholars@Duke
Martin Arthur Moseley III
Adjunct Professor in the Department of Cell Biology
Erik James Soderblom
Associate Research Professor of Cell Biology
Director, Proteomics and Metabolomics Core Facility
J. Will Thompson
Adjunct Assistant Professor in the Department of Pharmacology & Cancer Biology
Dr. Thompson's research focuses on the development and deployment of proteomics and
metabolomics mass spectrometry techniques for the analysis of biological systems.
He served as the Assistant Director of the Proteomics and Metabolomics Shared Resource
in the Duke School of Medicine from 2007-2021. He currently maintains collaborations
in metabolomics and proteomics research at Duke, and develops new tools for chemical
analysis as a Princi
Rahima Zennadi
Associate Professor in Medicine
My research in Hematology addresses three areas of investigation: disorders associated
with sickle cell disease pathophysiology, venous thrombosis/thromboembolism (VT/E)
associated with aging, and cerebrovascular injury.
In sickle cell disease, vaso-occlusion leads to serious life-threatening complications,
including acute pain crises and irreversible organ damage. Vaso-occlusion is caused
largely by sickle red blood cell adhesion to the vascular endothelium. Prevention
of
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