Johanns M, Pyr Dit Ruys S, Houddane A, Vertommen D, Herinckx G, Hue L, Proud C G, Rider M H
Université catholique de Louvain (UCL), de Duve Institute, Avenue Hippocrate 75 bte 74.02, 1200-Brussels, Belgium.
South Australian Health and Medical Research Institute (SAHMRI), University of Adelaide, North Terrace, Adelaide, SA 5000, Australia.
Cell Signal. 2017 Aug;36:212-221. doi: 10.1016/j.cellsig.2017.05.010. Epub 2017 May 11.
Eukaryotic elongation factor 2 (eEF2) kinase (eEF2K) is a key regulator of protein synthesis in mammalian cells. It phosphorylates and inhibits eEF2, the translation factor necessary for peptide translocation during the elongation phase of protein synthesis. When cellular energy demand outweighs energy supply, AMP-activated protein kinase (AMPK) and eEF2K become activated, leading to eEF2 phosphorylation, which reduces the rate of protein synthesis, a process that consumes a large proportion of cellular energy under optimal conditions.
The goal of the present study was to elucidate the mechanisms by which AMPK activation leads to increased eEF2 phosphorylation to decrease protein synthesis.
Using genetically modified mouse embryo fibroblasts (MEFs), effects of treatments with commonly used AMPK activators to increase eEF2 phosphorylation were compared with that of the novel compound 991. Bacterially expressed recombinant eEF2K was phosphorylated in vitro by recombinant activated AMPK for phosphorylation site-identification by mass spectrometry followed by site-directed mutagenesis of the identified sites to alanine residues to study effects on the kinetic properties of eEF2K. Wild-type eEF2K and a Ser491/Ser492 mutant were retrovirally re-introduced in eEF2K-deficient MEFs and effects of 991 treatment on eEF2 phosphorylation and protein synthesis rates were studied in these cells.
RESULTS & CONCLUSIONS: AMPK activation leads to increased eEF2 phosphorylation in MEFs mainly by direct activation of eEF2K and partly by inhibition of mammalian target of rapamycin complex 1 (mTORC1) signaling. Treatment of MEFs with AMPK activators can also lead to eEF2K activation independently of AMPK probably via a rise in intracellular Ca. AMPK activates eEF2K by multi-site phosphorylation and the newly identified Ser491/Ser492 is important for activation, leading to mTOR-independent inhibition of protein synthesis. Our study provides new insights into the control of eEF2K by AMPK, with implications for linking metabolic stress to decreased protein synthesis to conserve energy reserves, a pathway that is of major importance in cancer cell survival.
真核生物延伸因子2（eEF2）激酶（eEF2K）是哺乳动物细胞中蛋白质合成的关键调节因子。它使eEF2磷酸化并抑制eEF2，eEF2是蛋白质合成延伸阶段肽链转运所必需的翻译因子。当细胞能量需求超过能量供应时，AMP激活的蛋白激酶（AMPK）和eEF2K被激活，导致eEF2磷酸化，从而降低蛋白质合成速率，在最佳条件下蛋白质合成过程消耗细胞能量的很大一部分。
本研究的目的是阐明AMPK激活导致eEF2磷酸化增加从而降低蛋白质合成的机制。
使用基因改造的小鼠胚胎成纤维细胞（MEF），将常用的AMPK激活剂处理以增加eEF2磷酸化的效果与新型化合物991的效果进行比较。细菌表达的重组eEF2K在体外被重组激活的AMPK磷酸化，通过质谱鉴定磷酸化位点，然后将鉴定出的位点定点突变为丙氨酸残基，以研究对eEF2K动力学特性的影响。野生型eEF2K和Ser491/Ser492突变体通过逆转录病毒重新导入eEF2K缺陷的MEF中，并在这些细胞中研究991处理对eEF2磷酸化和蛋白质合成速率的影响。
AMPK激活主要通过直接激活eEF2K并部分通过抑制雷帕霉素复合物1（mTORC1）信号传导导致MEF中eEF2磷酸化增加。用AMPK激活剂处理MEF也可能通过细胞内Ca升高独立于AMPK导致eEF2K激活。AMPK通过多位点磷酸化激活eEF2K，新鉴定的Ser491/Ser492对激活很重要，导致不依赖mTOR的蛋白质合成抑制。我们的研究为AMPK对eEF2K的调控提供了新的见解，这对于将代谢应激与蛋白质合成减少联系起来以保存能量储备具有重要意义，这一途径在癌细胞存活中至关重要。
