PERK and GCN2 Contribute to eIF2α Phosphorylation and Cell Cycle Arrest after Activation of the Unfolded Protein Response Pathway

Abstract

Exposure of cells to endoplasmic reticulum (ER) stress leads to activation of PKR-like ER kinase (PERK), eukaryotic translation initiation factor 2␣ (eIF2␣) phosphorylation, repression of cyclin D1 translation, and subsequent cell cycle arrest in G 1 phase. However, whether PERK is solely responsible for regulating cyclin D1 accumulation after unfolded protein response pathway (UPR) activation has not been assessed. Herein, we demonstrate that repression of cyclin D1 translation after UPR activation occurs independently of PERK, but it remains dependent on eIF2␣ phosphorylation. Although phosphorylation of eIF2␣ in PERK؊/؊ fibroblasts is attenuated in comparison with wild-type fibroblasts, it is not eliminated. The residual eIF2␣ phosphorylation correlates with the kinetics of cyclin D1 loss, suggesting that another eIF2␣ kinase functions in the absence of PERK. In cells harboring targeted deletion of both PERK and GCN2, cyclin D1 loss is attenuated, suggesting GCN2 functions as the redundant kinase. Consistent with these results, cyclin D1 translation is also stabilized in cells expressing a nonphosphorylatable allele of eIF2␣; in contrast, repression of global protein translation still occurs in these cells, highlighting a high degree of specificity in transcripts targeted for translation inhibition by phosphorylated eIF2␣. Our results demonstrate that PERK and GCN2 function to cooperatively regulate eIF2␣ phosphorylation and cyclin D1 translation after UPR activation. INTRODUCTIONIntegral membrane proteins and secreted proteins are synthesized, folded, and posttranslationally modified in the endoplasmic reticulum (ER). When the integrity of protein folding in the ER is compromised and misfolded proteins accumulate, a signaling network referred to as the unfolded protein response pathway (UPR) is activated (Kaufman, 1999). Stresses that activate the UPR include disruption of proper protein glycosylation (glucose deprivation or treatment of cells with drugs that directly inhibit glycosylation such as tunicamycin), perturbations in ER calcium homeostasis (thapsigargin), perturbations in ER redox status (dithiothreitol [DTT]), and hypoxia (Kaufman, 1999;Koumenis et al., 2002).Activation of the UPR triggers a checkpoint that provides cells with the opportunity to adapt and survive, or under conditions of chronic stress, to commit to a program of cell death. The proximal effectors of the mammalian UPR include the three homologous, ER-resident transmembrane protein kinases Ire1 (␣ and ␤) and PKR-like ER kinase (PERK); the ER-resident transmembrane protease caspase 12; and the ER-resident transmembrane bZIP transcription factor ATF6 (Cox et al., 1993;Shi et al., 1998;Tirasophon et al., 1998;Wang et al., 1998;Harding et al., 1999;Haze et al., 1999;Nakagawa et al., 2000). Ire1 and PERK both have luminal, ER stress-sensing domains that regulate their dimerization and activation of their protein kinase activity. Activation of ATF6 occurs via a proteolytic cleavage that allows its translocation to the nucleus where it fun...

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