Regulated Translation Initiation Controls Stress-Induced Gene Expression in Mammalian Cells

Abstract

Protein kinases that phosphorylate the alpha subunit of eukaryotic initiation factor 2 (eIF2alpha) are activated in stressed cells and negatively regulate protein synthesis. Phenotypic analysis of targeted mutations in murine cells reveals a novel role for eIF2alpha kinases in regulating gene expression in the unfolded protein response (UPR) and in amino acid starved cells. When activated by their cognate upstream stress signals, the mammalian eIF2 kinases PERK and GCN2 repress translation of most mRNAs but selectively increase translation of Activating Transcription Factor 4 (ATF4), resulting in the induction of the downstream gene CHOP (GADD153). This is the first example of a mammalian signaling pathway homologous to the well studied yeast general control response in which eIF2alpha phosphorylation activates genes involved in amino acid biosynthesis. Mammalian cells thus utilize an ancient pathway to regulate gene expression in response to diverse stress signals.

Upregulation of tryptophanyl-tRNA synthethase adapts human cancer cells to nutritional stress caused by tryptophan degradation

“…To further elucidate the downstream effects of GCN2 activation we next set out to establish whether Trp starvation leads to phosphorylation of the translation initiation factor eIF2α as GCN2 is known to inhibit eIF2α by phosphorylation 27 . Indeed, Trp starvation enhanced eIF2α phosphorylation at serine 51 (Figure 4J).…”

“…Trp shortage mediates WARS upregulation by signaling via the GCN2-peIF2a-ATF4 axis (Figure 4), as low levels of Trp result in the accumulation of uncharged tRNAs, which activate the stress kinase GCN2 10 . GCN2 then phosphorylates its substrate, eIF2a leading to reduced global protein synthesis, but increased translation of specific mRNAs such as ATF4 27,28 . Our results reveal that expression of GCN2 and ATF4 as well as phosphorylation of eIF2a are each required for the induction of WARS upon Trp degradation (Figure 4).…”

Approaches to imaging unfolded secretory protein stress in living cells

“…Activation of other mammalian UPR targets can also be followed in living cells. For example, the protein CHOP is upregulated during ER stress [26]. CHOP is a transcription factor that activates expression of genes involved in protection of cells from stress [67] and induction of apoptosis [5].…”

“…CHOP is a transcription factor that activates expression of genes involved in protection of cells from stress [67] and induction of apoptosis [5]. CHOP expression is regulated by the UPR via increased translation of ATF4 [26]. The fluorescence intensity of a GFP reporter, consisting of an FP under the control of the CHOP promoter, parallels the expression of the endogenous CHOP and can be successfully detected in cells upon UPR activation [68].…”

“…Alternative activation pathways have been reported in which no peptide binding by Ire1 is necessary [25]. PERK phosphorylates eIF2α to attenuate global translation and also dramatically enhances translation of ATF4, which then upregulates transcription of ER chaperones (Figure 1) [26, 27]. IRE1 cleaves XBP1 mRNA as part of a splicing reaction to generate an in frame form to generate a transcription factor that upregulates chaperones, ERAD components, and XBP1 (Figure 1) [15, 18].…”