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

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    Date
    2013-01-03
    Authors
    Chiou, E
    Dubois, Laura
    Moseley, Martin Arthur III
    Schwartz, EA
    Soderblom, Erik James
    Thompson, JW
    Zennadi, Rahima
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    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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    Journal article
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    http://hdl.handle.net/10161/11163
    Published Version (Please cite this version)
    10.1186/1559-0275-10-1
    Publication Info
    Chiou, E; Dubois, Laura; Moseley, Martin Arthur III; Schwartz, EA; Soderblom, Erik James; Thompson, JW; & 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 http://hdl.handle.net/10161/11163.
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    Scholars@Duke

    Moseley

    Martin Arthur Moseley III

    Associate Professor in Medicine

    Erik James Soderblom

    Assistant Research Professor of Cell Biology
    Zennadi

    Rahima Zennadi

    Associate Professor in Medicine
    Sickle Cell Disease My research investigations in Hematology address the disorders associated with abnormalities affecting cell membrane proteins involved in cell-cell interactions and their role in sickle cell vasculopathy. In sickle cell disease (SCD), recurrent obstruction of the microvasculature leads to serious life-threatening complications such as acute pain crises, acute chest syndrome, kidney failure and cerebrovascular accidents triggered by ischemic injury
    Alphabetical list of authors with Scholars@Duke profiles.
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