Integrated stress response of vertebrates is regulated by four eIF2α kinases

The integrated stress response (ISR) is a cytoprotective pathway initiated upon phosphorylation of the eukaryotic translation initiation factor 2 (eIF2 alpha) residue designated serine-51, which is critical for translational control in response to various stress conditions. Four eIF2 alpha kinases, namely heme-regulated inhibitor (HRI), protein kinase R (PKR), PKR-like endoplasmic reticulum kinase, (PERK) and general control non-depressible 2 (GCN2), have been identified thus far, and they are known to be activated by heme depletion, viral infection, endoplasmic reticulum stress, and amino acid starvation, respectively. Because eIF2 alpha is phosphorylated under various stress conditions, the existence of an additional eIF2 alpha kinase has been suggested. To validate the existence of the unidentified eIF2 alpha kinase, we constructed an eIF2 alpha kinase quadruple knockout cells (4KO cells) in which the four known eIF2 alpha kinase genes were deleted using the CRISPR/Cas9-mediated genome editing. Phosphorylation of eIF2 alpha was completely abolished in the 4KO cells by various stress stimulations. Our data suggests that the four known eIF2 alpha kinases are sufficient for ISR and that there are no additional eIF2 alpha kinases in vertebrates.