Integrated stress response of vertebrates is regulated by four eIF2α kinases

Abstract

The integrated stress response (ISR) is a cytoprotective pathway initiated upon phosphorylation of the eukaryotic translation initiation factor 2 (eIF2α) residue designated serine-51, which is critical for translational control in response to various stress conditions. Four eIF2α 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 eIF2α is phosphorylated under various stress conditions, the existence of an additional eIF2α kinase has been suggested. To validate the existence of the unidentified eIF2α kinase, we constructed an eIF2α kinase quadruple knockout cells (4KO cells) in which the four known eIF2α kinase genes were deleted using the CRISPR/Cas9-mediated genome editing. Phosphorylation of eIF2α was completely abolished in the 4KO cells by various stress stimulations. Our data suggests that the four known eIF2α kinases are sufficient for ISR and that there are no additional eIF2α kinases in vertebrates.

Figure 1. Phylogenetic analysis of the eukaryotic translation initiation factor 2 (eIF2α) kinomes.

The amino acid sequences of kinases that are homologous to eIF2α kinases were aligned using CLUSTAL W 2.1 and the unrooted phylogenetic tree was created via the neighbour-joining method using MEGA 5.05. The scale bar represents 0.2 substitutions per site. The species abbreviations used in the Figure were as follows: Ha Hammondia hammondi (Hh), Toxoplasma gondii (Tg), Arabidopsis thaliana (At), Schizosaccharomyces pombe (Sp), Caenorhabditis elegans (Ce), Drosophila melanogaster, Danio rerio (Dr) and Mus musculus (Mm).

Figure 2. Eukaryotic translation initiation factor 2 (eIF2α) is phosphorylated by various stresses in mouse embryonic fibroblasts (MEFs).

(A) Representative immunoblots of phosphorylated eIF2α, total eIF2α and ribophorin (Rb) 1 h after treatment with the indicated stress stimulus in the wild-type MEFs. The ratio of autophosphorylated versus total eIF2α is indicated. (B) Representative immunoblots of phosphorylated PKR, PERK, GCN2 and Rb 1 h after treatment with the indicated stress stimulus in MEFs. The stress condition abbreviations used in the Figure were as follows: untreated (UT), 400 μM arsenate (Ars), 10 ng/μl polyinosinic-polycytidylic acid (pIC), 200 nM thapsigargin (Tg), 2 mM histidinol (His), 1 mM H₂O₂ (H₂O₂), 500 mM NaCl (NaCl), 254 nm 200 J/m² ultraviolet irradiation (UV), 42 °C heat shock (heat), cold shock (cold), low glucose (LG), serum starvation (SS) and anoxia (Ano).

Figure 3. Generation of quadruple eukaryotic translation initiation factor 2 (eIF2α) kinase knockout mouse embryonic fibroblasts (MEFs) (4KO) by CRISPR/Cas9-mediated genome editing.

(A) Schematic of each targeting locus of the four Eif2ak kinases with the target sequence underscored, PAM in bold type and exons in block boxes. Representative sequencing results showing the indel mutations generated in 4KO cells. (B) Representative immunoblots of protein kinase R (PKR), PKR-like ER kinase (PERK), general control non-depressible 2 (GCN2) and ribophorin (Rb) in the wild-type and 4KO MEFs. (C) RT-qPCR analysis (mean ± SD, n = 4, *p < 0.01) of the expression of heme-regulated inhibitor (Hri) mRNA in the wild-type and 4KO MEFs.

Figure 4. Eukaryotic translation initiation factor 2 (eIF2α) phosphorylation was completely abolished in quadruple eIF2α kinase knockout mouse embryonic fibroblasts (4KO MEFs) under the various stress conditions.

(A) Representative immunoblots of phosphorylated eIF2α, total eIF2α and ribophorin (Rb) 1 h after treatment with the indicated stress stimulus in the wild-type and 4KO MEFs. The ratio of autophosphorylated versus total eIF2α is indicated. The stress conditions abbreviations used in the Figure are as follows: untreated (UT), 400 μM arsenate (Ars), 10 ng/μl polyIC (pIC), 200 nM thapsigargin (Tg), 2 mM histidinol (His), 1 mM H₂O₂ (H₂O₂), 500 mM NaCl (NaCl), 254 nm 200 J/m² ultraviolet irradiation (UV), 42 °C heat shock (heat), cold shock (cold), low glucose (LG), serum starvation (SS) and anoxia (Ano). (B) Schematic representation of the structure and activation of the Fv2E-PERK fusion protein. AP20187(AP)-induced Fv2E-PERK homodimerization causes the activation of Fv2E-PERK, subsequently phosphorylating eIF2α. (C) Representative immunoblots of phosphorylated eIF2α, total eIF2α and Fv2E-PERK in the wild-type and 4KO MEFs overexpressing Fv2E-PERK after exposure to Mock (Ctrl) or 1 nM AP for 1 h.

Figure 5. Quadruple eukaryotic translation initiation factor 2 (eIF2α) kinase knockout mouse embryonic fibroblasts (4KO MEFs) overexpressing the indicated Flag-tagged eIF2α kinases are useful tools for validating the activation of each eIF2α kinase by various stressors.

(A) Representative immunoblots showing ectopically expression of the indicated Flag-tagged eIF2α kinases and endogenous ribophorin (Rb) in the six types of MEFs (wild-type, 4KO and 4KO overexpressing the indicated Flag-tagged eIF2α kinases). (B) Representative immunoblots of protein kinase R (PKR), PKR-like ER kinase (PERK), general control non-depressible 2 (GCN2) and Rb in the six types of MEFs (wild-type, 4KO and 4KO overexpressing the indicated Flag-tagged eIF2α kinases). (C) RT-qPCR analysis (mean ± SD, n = 4, *p < 0.01) of the mRNA expression of heme-regulated inhibitor (Hri) in the six types of MEFs (wild-type, 4KO and 4KO overexpressing the indicated Flag-tagged eIF2α kinases). (D) Representative immunoblots of phosphorylated eIF2α, total eIF2α and Rb 1 h after treatment with the indicated stress stimulus in the six types of MEFs (wild-type, 4KO and 4KO overexpressing the indicated Flag-tagged eIF2α kinases).