Signature Research Programmes in Neuroscience and Behavioural Disorders, and Cardiovascular and Metabolic Disorders, Duke-NUS Medical School, Singapore, Singapore.
School of Biological Sciences, Nanyang Technological University, Singapore, Singapore.
Cell Death Differ. 2018 Feb;25(2):255-267. doi: 10.1038/cdd.2017.152. Epub 2017 Oct 6.
Phosphorylation of the eukaryotic translation initiation factor, eIF2α, by stress-activated protein kinases and dephosphorylation by the growth arrest and DNA damage-inducible protein (GADD34)-containing phosphatase is a central node in the integrated stress response. Mass spectrometry demonstrated GADD34 acetylation at multiple lysines. Substituting K and K with alanines or glutamines did not impair GADD34's ability to recruit protein phosphatase 1α (PP1α) or eIF2α, suggesting that GADD34 acetylation did not modulate eIF2α phosphatase activity. Arsenite (Ars)-induced oxidative stress increased cellular GADD34 levels and enhanced Sirtuin 1 (SIRT1) recruitment to assemble a cytoplasmic complex containing GADD34, PP1α, eIF2α and SIRT1. Induction of GADD34 in WT MEFs paralleled the dephosphorylation of eIF2α (phosphoserine-51) and SIRT1 (phosphoserine-47). By comparison, eIF2α and SIRT1 were persistently phosphorylated in Ars-treated GADD34-/- MEFs. Expressing WT GADD34, but not a mutant unable to bind PP1α in GADD34-/- MEFs restored both eIF2α and SIRT1 dephosphorylation. SIRT1 dephosphorylation increased its deacetylase activity, measured in vitro and in cells. Loss of function of GADD34 or SIRT1 enhanced cellular p-eIF2α levels and attenuated cell death following Ars exposure. These results highlighted a novel role for the GADD34/PP1α complex in coordinating the dephosphorylation and reactivation of eIF2α and SIRT1 to determine cell fate following oxidative stress.
真核翻译起始因子 eIF2α 的磷酸化由应激激活蛋白激酶完成，去磷酸化由包含生长停滞和 DNA 损伤诱导蛋白（GADD34）的磷酸酶完成，这是整合应激反应的一个核心节点。质谱分析表明 GADD34 在多个赖氨酸上发生乙酰化。用丙氨酸或谷氨酰胺取代 K 和 K 并没有损害 GADD34 招募蛋白磷酸酶 1α（PP1α）或 eIF2α 的能力，这表明 GADD34 乙酰化并没有调节 eIF2α 磷酸酶活性。亚砷酸盐（Ars）诱导的氧化应激增加了细胞内 GADD34 的水平，并增强了 Sirtuin 1（SIRT1）的募集，以组装一个包含 GADD34、PP1α、eIF2α 和 SIRT1 的细胞质复合物。WT MEFs 中的 GADD34 诱导与 eIF2α（磷酸丝氨酸-51）和 SIRT1（磷酸丝氨酸-47）的去磷酸化平行。相比之下，在 Ars 处理的 GADD34-/- MEFs 中，eIF2α 和 SIRT1 持续磷酸化。在 GADD34-/- MEFs 中表达 WT GADD34，但不能表达不能与 PP1α 结合的突变体，恢复了 eIF2α 和 SIRT1 的去磷酸化。SIRT1 的去磷酸化增加了其在体外和细胞内的去乙酰化酶活性。GADD34 或 SIRT1 的功能丧失增加了细胞内 p-eIF2α 的水平，并减轻了 Ars 暴露后的细胞死亡。这些结果突出了 GADD34/PP1α 复合物在协调 eIF2α 和 SIRT1 的去磷酸化和再激活以决定氧化应激后细胞命运方面的新作用。
