Ayuso María I, Martinez-Alonso Emma, Salvador Nelida, Bonova Petra, Regidor Ignacio, Alcázar Alberto
Department of Investigation, Hospital Ramón y Cajal, IRYCIS, Madrid, Spain.
Institute of Neurobiology, Slovak Academy of Sciences, Košice, Slovakia.
PLoS One. 2015 Mar 30;10(3):e0121958. doi: 10.1371/journal.pone.0121958. eCollection 2015.
Eukaryotic initiation factor (eIF) 4E-binding proteins (4E-BPs) are translational repressors that bind specifically to eIF4E and are critical in the control of protein translation. 4E-BP2 is the predominant 4E-BP expressed in the brain, but their role is not well known. Here, we characterized four forms of 4E-BP2 detected by two-dimensional gel electrophoresis (2-DGE) in brain. The form with highest electrophoretic mobility was the main form susceptible to phosphorylation at Thr37/Thr46 sites, phosphorylation that was detected in acidic spots. Cerebral ischemia and subsequent reperfusion induced dephosphorylation and phosphorylation of 4E-BP2 at Thr37/Thr46, respectively. The induced phosphorylation was in parallel with the release of 4E-BP2 from eIF4E, although two of the phosphorylated 4E-BP2 forms were bound to eIF4E. Upon long-term reperfusion, there was a decrease in the binding of 4E-BP2 to eIF4E in cerebral cortex, demonstrated by cap binding assays and 4E-BP2-immunoprecipitation experiments. The release of 4E-BP2 from eIF4E was without changes in 4E-BP2 phosphorylation or other post-translational modification recognized by 2-DGE. These findings demonstrated specific changes in 4E-BP2/eIF4E association dependent and independent of 4E-BP2 phosphorylation. The last result supports the notion that phosphorylation may not be the uniquely regulation for the binding of 4E-BP2 to eIF4E under ischemic stress.
真核生物起始因子（eIF）4E结合蛋白（4E - BPs）是特异性结合eIF4E的翻译抑制因子，在蛋白质翻译控制中起关键作用。4E - BP2是大脑中表达的主要4E - BP，但它们的作用尚不清楚。在此，我们对通过二维凝胶电泳（2 - DGE）在大脑中检测到的四种4E - BP2形式进行了表征。电泳迁移率最高的形式是在Thr37 / Thr46位点易发生磷酸化的主要形式，这种磷酸化在酸性斑点中被检测到。脑缺血及随后的再灌注分别诱导了4E - BP2在Thr37 / Thr46位点的去磷酸化和磷酸化。诱导的磷酸化与4E - BP2从eIF4E的释放平行，尽管两种磷酸化的4E - BP2形式与eIF4E结合。长期再灌注后，通过帽结合试验和4E - BP2免疫沉淀实验证明，大脑皮层中4E - BP2与eIF4E的结合减少。4E - BP2从eIF4E的释放并未伴随4E - BP2磷酸化或2 - DGE识别的其他翻译后修饰的变化。这些发现证明了4E - BP2 / eIF4E结合的特异性变化，这些变化依赖和不依赖于4E - BP2磷酸化。最后一个结果支持了这样一种观点，即在缺血应激下，磷酸化可能不是4E - BP2与eIF4E结合的唯一调节方式。
