Division of Hematology and Duke Comprehensive Sickle Cell Center, Department of Medicine, Duke University Medical Center, Durham, NC, USA.
Clin Proteomics. 2013 Jan 3;10(1):1. doi: 10.1186/1559-0275-10-1.
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.
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.
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.
在镰状细胞病（SCD）中，丝裂原激活的蛋白激酶（MAPK）ERK1/2 持续激活，仅在镰状而非正常人类红细胞（RBC）中可被激动剂刺激诱导。ERK1/2 参与了 ICAM-4 介导的镰状 RBC 与内皮细胞的黏附。然而，ERK1/2 在镰状 RBC 中的其他激活效应导致了复杂的 SCD 病理生理学，例如 RBC 血液流变学的改变尚不清楚。
为了进一步描述 U0126（ERK1/2 的上游激酶 MEK1/2 的特异性抑制剂）预处理的镰状和正常 RBC 膜血影蛋白中 ERK1/2 诱导的膜蛋白磷酸化的全基因组 ERK1/2 诱导变化，应用无标记定量磷酸蛋白质组学分析方法。在存在或不存在重组活性 ERK2 的情况下，对 8 个独特的处理组进行了分析，从 155 个磷酸化蛋白中定量了 375 个磷酸肽，峰强度的平均技术变异系数为 19.8%。U0126 处理镰状 RBC 可减少 36 个磷酸肽，来自 21 个磷酸化蛋白，这些蛋白不仅参与 RBC 形状、柔韧性、细胞形态维持和黏附的调节，还参与葡萄糖和谷氨酸转运、cAMP 产生、错误折叠蛋白的降解和受体泛素化。糖蛋白 A 是受该 ERK1/2 途径影响最大的蛋白，其中包含 12 个独特的磷酸化肽，表明除了对镰状 RBC 黏附的影响外，ERK1/2 途径增加糖蛋白 A 的磷酸化也可能影响糖蛋白 A 与带 3 的相互作用，从而导致带 3 的阴离子转运和带 3 转运减少。在与重组 ERK2 共孵育的正常 RBC 中，随后增加了 36 个磷酸肽中的 12 个的丰度，因此代表了通过 ERK2 介导的特定 MEK1/2 磷酸抑制靶标。
这些发现扩展了 ERK1/2 信号在 RBC 中的作用模型。这些发现还确定了该途径的除 RBC 黏附分子 ICAM-4 以外的其他蛋白靶标，并增强了对 MEK/1/2/ERK1/2 小分子抑制剂机制的理解，这可能有效改善 RBC 血液流变学和黏附，这是 SCD 的特征。
