Stimulation of the AMP-activated protein kinase leads to activation of eukaryotic elongation factor 2 kinase and to its phosphorylation at a novel site, Serine 398
Stimulation of the AMP-activated protein kinase leads to activation of eukaryotic elongation factor 2 kinase and to its phosphorylation at a novel site, Serine 398
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DOI：
10.1074/jbc.m309773200
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发表时间：
2004-03-26
影响因子：
4.8
通讯作者：
Proud, CG
中科院分区：
文献类型：
--
作者：
Browne, GJ;Finn, SG;Proud, CG
蛋白质合成消耗了哺乳动物细胞代谢能量的很高比例，其中大部分被肽链延长所用。真核细胞中一个重要的能量供需调节因子是AMP激活的蛋白激酶(AMPK)。肽链延长的速度可以通过真核延长因子(EEF)2的磷酸化来调节，真核延长因子2抑制其活性，并由一种称为eEF2的特异性钙/钙调蛋白依赖的蛋白激酶催化。在这里，我们发现AMPK直接磷酸化eEF2激酶，我们确定磷酸化的主要部位是eEF2激酶调节域中的Ser-398。在体外，AMPK还使eEF2激酶的另外两个位点(Ser-78和Ser-366)磷酸化。我们开发了合适的磷酸化特异性抗血清，并表明在激活AMPK和增加eEF2磷酸化的一系列条件下，完整细胞中eEF2激酶中Ser-398的磷酸化水平得到增强。Ser-78和Ser-366在细胞内似乎不被AMPK磷酸化。虽然心肌细胞似乎含有一种不同的eEF2激酶亚型，但它也包含一个与Ser-398相对应的位点，该位点在体外被AMPK磷酸化。在细胞内激活AMPK和增加eEF2磷酸化的刺激会增加eEF2激酶的活性。因此，AMPK和eEF2激酶可能在细胞能量状态和蛋白质合成抑制之间提供了关键联系，蛋白质合成是代谢能量的主要消耗者。
Protein synthesis consumes a high proportion of the metabolic energy of mammalian cells, and most of this is used by peptide chain elongation. An important regulator of energy supply and demand in eukaryotic cells is the AMP-activated protein kinase (AMPK). The rate of peptide chain elongation can be modulated through the phosphorylation of eukaryotic elongation factor (eEF) 2, which inhibits its activity and is catalyzed by a specific calcium/calmodulin-dependent protein kinase termed eEF2 kinase. Here we show that AMPK directly phosphorylates eEF2 kinase, and we identify the major site of phosphorylation as Ser-398 in a regulatory domain of eEF2 kinase. AMPK also phosphorylates two other sites (Ser-78 and Ser-366) in eEF2 kinase in vitro. We develop appropriate phosphospecific antisera and show that phosphorylation of Ser-398 in eEF2 kinase is enhanced in intact cells under a range of conditions that activate AMPK and increase the phosphorylation of eEF2. Ser-78 and Ser-366 do not appear to be phosphorylated by AMPK within cells. Although cardiomyocytes appear to contain a distinct isoform of eEF2 kinase, it also contains a site corresponding to Ser-398 that is phosphorylated by AMPK in vitro. Stimuli that activate AMPK and increase eEF2 phosphorylation within cells increase the activity of eEF2 kinase. Thus, AMPK and eEF2 kinase may provide a key link between cellular energy status and the inhibition of protein synthesis, a major consumer of metabolic energy.