Phosphorylation of the α-Subunit of the Eukaryotic Initiation Factor-2 (eIF2α) Reduces Protein Synthesis and Enhances Apoptosis in Response to Proteasome Inhibition

Abstract

Protein ubiquitination and subsequent degradation by the proteasome are important mechanisms regulating cell cycle, growth and differentiation, and apoptosis. Recent studies in cancer therapy suggest that drugs that disrupt the ubiquitin/proteasome pathway induce apoptosis and sensitize malignant cells and tumors to conventional chemotherapy. In this study we addressed the role of phosphorylation of the ␣-subunit eukaryotic initiation factor-2 (eIF2), and its attendant regulation of gene expression, in the cellular stress response to proteasome inhibition. Phosphorylation of eIF2␣ in mouse embryo fibroblast (MEF) cells subjected to proteasome inhibition leads to a significant reduction in protein synthesis, concomitant with induced expression of the bZIP transcription regulator, ATF4, and its target gene CHOP/GADD153. The primary eIF2␣ kinase activated by exposure of these fibroblast cells to proteasome inhibition is GCN2 (EIF2AK4), which has a central role in the recognition of cytoplasmic stress signals. Endoplasmic reticulum (ER) stress is not effectively induced in MEF cells subjected to proteasome inhibition, with minimal activation of the ER stress sensory proteins, eIF2␣ kinase PEK (PERK/EIF2AK3), IRE1 protein kinase and the transcription regulator ATF6 following up to 6 h of proteasome inhibitor treatment. Loss of eIF2␣ phosphorylation thwarts caspase activation and delays apoptosis. Central to this pro-apoptotic function of eIF2␣ kinases during proteasome inhibition is the transcriptional regulator CHOP, as deletion of CHOP in MEF cells impedes apoptosis. We conclude that eIF2␣ kinases are integral to cellular stress pathways induced by proteasome inhibitors, and may be central to the efficacy of anticancer drugs that target the ubiquitin/proteasome pathway.

A Bifunctional Intronic Element Regulates the Expression of the Arginine/Lysine Transporter Cat-1 via Mechanisms Involving the Purine-rich Element Binding Protein A (Purα)

“…C6 rat glioma cells were cultured in media supplemented with 5% heat-inactivated fetal bovine serum and 5% calf serum. Mouse embryonic fibroblasts (MEFs) with or without homozygous deletions of the Pur␣ gene (15) or the CHOP gene (16) were grown in media supplemented with 10% fetal bovine serum. FuGENE 6 HD (Roche Applied Science) was used to transfect cultured cells according to the manufacturer's instructions.…”

Rôle de l’heme regulated inhibitor(HRI) dans la résistance à l’apoptose

“…De plus, la phosphorylation d'eIF2 est connue, soit pour activer des signaux pro-apoptotiques (activation de la voie de mort cellulaire ATF4-CHOP [14,47], induction de l'expression du récepteur Fas de mort cellulaire [48]), soit pour empêcher la traduction de protéines de survie telles Bcl-x, inhibant ainsi les voies anti-apoptotiques et favorisant la mort cellulaire [16]. Ce rôle pro-apoptotique de la phosphorylation d'eIF2α suppose que son induction par les kinases eIF2α puisse contrer le développement tumoral en induisant la mort des cellules cancéreuses.…”

PKR , a p53 target gene, plays a crucial role in the tumor-suppressor function of p53

“…These data suggest that even though PKR plays a role in genotoxin-mediated inhibition of translation and apoptosis, the genotoxin-mediated apoptosis is, to some extent, functionally associated with but temporally dissociated from PKR-mediated inhibition of translation. The phosphorylation of eIF2␣ at Ser-51 leads to a significant reduction in protein synthesis, concomitant with induced expression of the basic leucine zipper (bZIP) regulator, activating transcription factor 4 (ATF4), and its target gene CCAAT/enhancer binding protein (C/EBP) homologous protein (CHOP), resulting in caspase activation and cell apoptosis (27). In accordance with our recent report (28), enhancements of ATF4/CHOP, followed by caspase-mediated cleavage of poly(ADP-ribose) polymerase (PARP) (29) were detected readily in p53 ϩ/ϩ PKR ϩ/ϩ cells but were barely detectable in the p53 ϩ/ϩ PKR KD (sh-PKR) cells under conditions of DNA damage (Fig.…”