Cellular and molecular characterization of the mammalian integrated stress response (ISR)
Amino acid deprivation, viral infection, heme depletion, and endoplasmic reticulum (ER) stress converge at the level of phosphorylation of eukaryotic translation initiation factor eIF2α by the activation of stress-specific eIF2α kinases in the Integrated Stress Response (ISR). Phosphorylated eIF2α (eIF2α-P) attenuates protein synthesis by inhibiting the recycling of ternary complexes required for translation initiation. However, eIF2α phosphorylation also leads to the paradoxical increase in translation of the transcription factor ATF4, and the induction of a gene expression program, which is important for promoting cell survival, as mutations in the upstream kinases or downstream effectors render cells and animals hypersensitive to stress.
To determine whether eIF2α phosphorylation pre-conditions cells against stress, I generated cells expressing a ligand-activatable eIF2α kinase, Fv2E-PERK, that is activated independently of stress. Fv2E-PERK activation induced eIF2α phosphorylation and ISR targets, and protected cells from oxidative and ER stress-induced cell death. Expression profiling indicated that Fv2E-PERK activates many of the same genes upregulated by the eIF2α kinase PERK during ER stress. Furthermore, transgenic mice expressing Fv2E-PERK in cardiomyocytes were generated and are being used in an ongoing collaborative study to determine whether pre-emptive activation of the ISR is protective during myocardial ischemia/reperfusion injury.
As ATF4 activation is key to the ISR, I analyzed the mechanism of its translational upregulation by eIF2α phosphorylation, which occurs when protein synthesis is inhibited. Mutational analysis performed on an ATF4 translational reporter, containing its two conserved upstream open reading frames (uORFs), suggested that although both uORFs are translated efficiently by scanning ribosomes, uORF1 functions as a positive regulatory element whereas uORF2 is inhibitory. In unstressed cells, low levels of eIF2α-P and high levels of ternary complex allow ribosomes that have translated uORF1 to reinitiate translation at uORF2, precluding translation of ATF4. In stressed cells, high levels of eIF2α-P and decreased levels of ternary complex favor reinitiation at ATF4 rather than at uORF2. These features are reminiscent of yeast in which translation of the transcription factor GCN4 increases during amino acid starvation. Thus, the mammalian ISR has been conserved from the yeast general control response both in its target genes and mechanism of activation.
0307: Molecular biology