Date Published: December 9, 2013
Publisher: Impact Journals LLC
Author(s): Kamindla Rajesh, Andreas I. Papadakis, Urszula Kazimierczak, Philippos Peidis, Shuo Wang, Gerardo Ferbeyre, Randal J. Kaufman, Antonis E. Koromilas.
Eukaryotic cells respond to various forms of stress by blocking mRNA translation initiation via the phosphorylation of the alpha (α) subunit of eIF2 at serine 51 (S51) (eIFαP). An important role of eIF2αP is the regulation of redox homeostasis and adaptation of cells to oxidative stress. Herein, we demonstrate that eIF2αP guards cells from intracellular reactive oxygen species (ROS) via the inhibition of senescence. Specifically, genetic inactivation of either eIF2αP or eIF2α kinase PERK in primary mouse or human fibroblasts leads to proliferative defects associated with increased DNA damage, G2/M accumulation and induction of premature senescence. Impaired proliferation of either PERK or eIF2αP-deficient primary cells is caused by increased ROS and restored by anti-oxidant treatment. Contrary to primary cells, immortalized mouse fibroblasts or human tumor cells become tolerant to elevated intracellular ROS levels caused by impaired eIF2αP. However, eIF2αP-deficient human tumor cells are highly susceptible to extrinsic ROS generated by the pro-oxidant drug doxorubicin by undergoing premature senescence. Our work demonstrates that eIF2αP determines cell destiny through its capacity to control senescence in response to oxidative stress. Also, inhibition of eIF2αP may be a suitable means to increase the anti-tumor effects of pro-oxidant drugs through the induction of senescence.
Metazoans respond to various forms of stress by phosphorylating the α subunit of the eukaryotic initiation factor 2 (eIF2) at the serine 51 (herein referred to as eIF2αP), a modification that causes a general inhibition of protein synthesis [1,2]. In mammalian cells, eIF2αP is mediated by a family of four kinases each of which responds to distinct stimuli . The family consists of the heme-regulated inhibitor (HRI), the general control non-derepressible-2 (GCN2), the endoplasmic reticulum (ER)-resident protein kinase PERK and the RNA-dependent protein kinase PKR [1,2]. These enzymes exhibit significant sequence similarities, particularly in the kinase domain (KD), which explains their specificity towards eIF2α [1,2]. Despite the general shutdown of protein synthesis, certain mRNAs like those encoding the activating transcription factor 4 (ATF4) in mammals and the general control non-derepressible-4 (GCN4) in yeast are efficiently translated under conditions of increased eIF2αP. This is because the 5′ untranslated regions (5′ UTRs) of these mRNAs consist of upstream open reading frames (uORFs), which impede translation of the downstream ORFs [1,2]. Increased eIF2αP decreases the formation of the eIF2-GTP-Met-tRNAi ternary complex, an event that bypasses the inhibitory effects of the uORFs leading to efficient re-initiation at the downstream ORFs [1,2].
The anti-oxidant function of eIF2αP depends on its translational properties and requires efficient ATF4 synthesis, which in turn induces transcription of genes involved in the import of thiol-containing amino acids and glutathione biosynthesis as a means to counteract oxidative insults . In mammalian cells, ATF4 has additional transcriptional roles by acting alone or in combination with other transcription factors to induce the expression of anti-oxidant genes like heme oxygenase-1 and sequestosome1/A170 . In a pathway different from eIF2αP, PERK can phosphorylate nuclear factor (erythroid-derived 2)-like 2 (NFE2L2), also known as NRF2, and promote its dissociation from Keap1 resulting in increased translocation of active NRF2 to the nucleus . Also, attenuation of general protein synthesis caused by increased eIF2αP is another mechanism that contains intracellular ROS levels and contributes to adaptation of cells to oxidative stress. This is because client protein load in the ER decreases, which in turn prevents illegitimate disulfide bond formation in the under chaperoned ER. As such, cells are supplied with a sufficient amount of reducing equivalents to alleviate themselves from oxidative stress . Due to close proximity of the ER to mitochondria, activation of the PERK and increased eIF2αP have also been proposed to be involved in the regulation of mitochondrial ROS production. Specifically, attenuation of protein synthesis prevents cells from ATP depletion, which takes place when the ER is overwhelmed with misfolded client proteins . Depletion of ATP stimulates mitochondrial oxidative phosphorylation and ROS production, which can be further induced by increased efflux of Ca2+ from ER to cytosol as a result of ER stress . In line with this notion, a previous study provided evidence that mitochondrial function contributes to ROS production in eIF2αP-deficient pancreatic β cells .