Roobol Anne, Roobol Jo, Bastide Amandine, Knight John R P, Willis Anne E, Smales C Mark
*Centre for Molecular Processing and Protein Science Group, School of Biosciences, University of Kent, Canterbury, Kent, CT2 7NJ, U.K.
†MRC Toxicology Unit, Hodgkin Building, PO Box 138, Lancaster Road, Leicester LE1 9HN, U.K.
Biochem J. 2015 Jan 15;465(2):213-25. doi: 10.1042/BJ20140852.
One of the key cellular responses to stress is the attenuation of mRNA translation and protein synthesis via the phosphorylation of eIF2α (eukaryotic translation initiation factor 2α). This is mediated by four eIF2α kinases and it has been suggested that each kinase is specific to the cellular stress imposed. In the present study, we show that both PERK (PKR-like endoplasmic reticulum kinase/eIF2α kinase 3) and GCN2 (general control non-derepressible 2/eIF2α kinase 4) are required for the stress responses associated with conditions encountered by cells overexpressing secreted recombinant protein. Importantly, whereas GCN2 is the kinase that is activated following cold-shock/hypothermic culturing of mammalian cells, PERK and GCN2 have overlapping functions since knockdown of one of these at the mRNA level is compensated for by the cell by up-regulating levels of the other. The protein p58IPK {also known as DnaJ3C [DnaJ heat-shock protein (hsp) 40 homologue, subfamily C, member 3]} is known to inhibit the eIF2α kinases PKR (dsRNA-dependent protein kinase/eIF2α kinase 2) and PERK and hence prevent or delay eIF2α phosphorylation and consequent inhibition of translation. However, we show that p58IPK is a general inhibitor of the eIF2α kinases in that it also interacts with GCN2. Thus forced overexpression of cytoplasmic p58 delays eIF2α phosphorylation, suppresses GCN2 phosphorylation and prolongs protein synthesis under endoplasmic reticulum (ER), hypothermic and prolonged culture stress conditions. Taken together, our data suggest that there is considerable cross talk between the eIF2α kinases to ensure that protein synthesis is tightly regulated. Their activation is controlled by p58 and the expression levels and localization of this protein are crucial in the capacity the cells to respond to cellular stress via control of protein synthesis rates and subsequent folding in the ER.
细胞对应激的关键反应之一是通过真核翻译起始因子2α（eIF2α）的磷酸化来减弱mRNA翻译和蛋白质合成。这由四种eIF2α激酶介导，并且有人提出每种激酶对所施加的细胞应激具有特异性。在本研究中，我们表明，与过表达分泌重组蛋白的细胞所遇到的条件相关的应激反应需要PERK（PKR样内质网激酶/eIF2α激酶3）和GCN2（一般控制非去阻遏蛋白2/eIF2α激酶4）。重要的是，虽然GCN2是在哺乳动物细胞冷休克/低温培养后被激活的激酶，但PERK和GCN2具有重叠功能，因为在mRNA水平敲低其中之一会被细胞通过上调另一种的水平来补偿。已知蛋白p58IPK {也称为DnaJ3C [DnaJ热休克蛋白（hsp）40同源物，C亚家族，成员3]}可抑制eIF2α激酶PKR（双链RNA依赖性蛋白激酶/eIF2α激酶2）和PERK，从而预防或延迟eIF2α磷酸化以及随后对翻译的抑制。然而，我们表明p58IPK是eIF2α激酶的一般抑制剂，因为它也与GCN2相互作用。因此，细胞质p58的强制过表达会延迟eIF2α磷酸化，抑制GCN2磷酸化，并在内质网（ER）、低温和长时间培养应激条件下延长蛋白质合成。综上所述，我们的数据表明eIF2α激酶之间存在相当多的相互作用，以确保蛋白质合成受到严格调控。它们的激活由p58控制，并且该蛋白的表达水平和定位对于细胞通过控制蛋白质合成速率以及随后在内质网中的折叠来应对细胞应激的能力至关重要。
