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The article presents a study related to the protein synthesis and dephosphorylation of translation initiation factor 2a in eukaryotic cells. Topics include substrate selectivity, crude cell lysate or purified G-actin, and computational docking of the N-terminal lobe of eIF2a. The study infers that avidity for the substrate plays an important role in impartingspecificity on the PPP1R15B-PP1G-actin ternary complex.

Loss-of-function mutations in EIF2AK3, encoding the pancreatic endoplasmic reticulum (ER) kinase, PERK, are associated with dysfunction of the endocrine pancreas and diabetes. However, to date it has not been possible to uncouple the long term developmental effects of PERK deficiency from sensitization to physiological levels of ER unfolded protein stress upon interruption of PERK modulation of protein synthesis rates. Here, we report that a selective PERK inhibitor acutely deregulates protein synthesis in freshly isolated islets of Langerhans, across a range of glucose concentrations. Acute loss of the PERK-mediated strand of the unfolded protein response leads to rapid accumulation of misfolded pro-insulin in cultured beta cells and is associated with a kinetic defect in pro-insulin processing. These in vitro observations uncouple the latent role of PERK in beta cell development from the regulation of unfolded protein flux through the ER and attest to the importance of the latter in beta cell proteostasis. [ABSTRACT FROM AUTHOR]

Interferon-γ (IFN-γ) is believed to contribute to immune- mediated demyelinating disorders by targeting the myelin-producing oligodendrocyte, a cell known to be highly sensitive to the disruption of protein synthesis and to the perturbation of the secretory pathway. We found that apoptosis induced by IFN-γ in cultured rat oligodendrocytes was associated with endoplasmic reticulum (ER) stress. ER stress also accompanied oligodendrocyte apoptosis and hypomyelination in transgenic mice that inappropriately expressed IFN-γ in the central nervous system (CNS). Compared with a wild-type genetic background, the enforced expression of IFN-γ in mice that were heterozygous for a loss of function mutation in pancreatic ER kinase (PERK) dramatically reduced animal survival, promoted CNS hypomyelination, and enhanced oligodendrocyte loss. PERK encodes an ER stress-inducible kinase that phosphorylates eukaryotic translation initiation factor 2α and specifically maintains client protein homeostasis in the stressed ER. Therefore, the hypersensitivity of PERK+/- mice to IFN-γ implicates ER stress in demyelinating disorders that are induced by CNS inflammation. [ABSTRACT FROM AUTHOR]

Hypoxic stress results in a rapid and sustained inhibition of protein synthesis that is at least partially mediated by eukaryotic initiation factor 2α (eIF2α) phosphorylation by the endoplasmic reticulum (ER) kinase PERK. Here we show through microarray analysis of polysome-bound RNA in aerobic and hypoxic HeLa cells that a subset of transcripts are preferentially translated during hypoxia, including activating transcription factor 4 (ATF4), an important mediator of the unfolded protein response. Changes in mRNA translation during the unfolded protein response are mediated by PERK phosphorylation of the translation initiation factor eIF2α at Ser-51. Similarly, PERK is activated and is responsible for translational regulation under hypoxic conditions, while inducing the translation of ATF4. The overexpression of a C-terminal fragment of GADD34 that constitutively dephosphorylates eIF2α was able to attenuate the phosphorylation of eIF2α and severely inhibit the induction of ATF4 in response to hypoxic stress. These studies demonstrate the essential role of ATF4 in the response to hypoxic stress, define the pathway for its induction, and reveal that GADD34, a target of ATF4 activation, negatively regulates the eIF2α-mediated inhibition of translation. Taken with the concomitant induction of additional ER-resident proteins identified by our microarray analysis, this study suggests an important integrated response between ER signaling and the cellular adaptation to hypoxic stress. [ABSTRACT FROM AUTHOR]

The endoplasmic reticulum (ER)-resident protein kinase PERK attenuates protein synthesis in response to ER stress through the phosphorylation of translation initiation factor eIF2α at serine 51. ER stress induces PERK autophosphorylation at several serine/threonine residues, a process that is required for kinase activation and phosphorylation of eIF2α. Herein, we demonstrate that PERK also possesses tyrosine kinase activity. Specifically, we show that PERK is capable of autophosphorylating on tyrosine residues in vitro and in vivo. We further show that tyrosine 615, which is embedded in a highly conserved region of the kinase domain of PERK, is essential for autocatalytic activity. That is, mutation of Tyr-615 to phenylalanine compromises the autophosphorylation capacity of PERK and the phosphorylation of eIF2α in vitro and in vivo. The Y615F mutation also impairs the ability of PERK to induce translation of ATF4. Immunoblot analyses with a phosphospecific antibody confirm the phosphorylation of PERK at Tyr-615 both in vitro and in vivo. Thus, our data classify PERK as a dual specificity kinase whose regulation by tyrosine phosphorylation contributes to its optimal activation in response to ER stress. [ABSTRACT FROM AUTHOR]

Regulated phosphorylation of the α subunit of eukaryotic translation initiation factor 2 (eIF2α) by the endoplasmic reticulum (ER) stress-activated protein kinase PERK modulates protein synthesis and couples the production of ER client proteins with the organelle's capacity to fold and process them. PERK activation by ER stress is known to involve transautophosphorylation, which decorates its unusually long kinase insert loop with multiple phosphoserine and phosphothreonine residues. We report that PERK activation and phosphorylation selectively enhance its affinity for the nonphosphorylated eIF2 complex. This switch correlates with a marked change to the protease sensitivity pattern, which is indicative of a major conformational change in the PERK kinase domain upon activation. Although it is dispensable for catalytic activity, PERK's kinase insert loop is required for substrata binding and for eIF2α phosphorylation in viva. Our findings suggest a novel mechanism for eIF2 recruitment by activated PERK and for unidirectional substrate flow in the phosphorylation reaction. [ABSTRACT FROM AUTHOR]

Unfolded and malfolded client proteins impose a stress on the endoplasmic reticulum (ER), which contributes to cell death in pathophysiological conditions. The transcription factor C/EBP homologous protein (CHOP) is activated by ER stress, and CHOP deletion protects against its lethal consequences. We find that CHOP directly activates GADD34, which promotes ER client protein biosynthesis by dephosphorylating phospho-Ser 51 of the α-subunit of translation initiation factor 2 (eIF2α) in stressed cells. Thus, impaired GADD34 expression reduces client protein load and ER stress in CHOP-/- cells exposed to perturbations that impair ER function. CHOP-/- and GADD34 mutant cells accumulate less high molecular weight protein complexes in their stressed ER than wild-type cells. Furthermore, mice lacking GADD34-directed eIF2α dephosphorylation, like CHOP-/- mice, are resistant to renal toxicity of the ER stress-inducing drug tunicamycin. CHOP also activates ERO1α, which encodes an ER oxidase. Consequently, the ER of stressed CHOP-/- cells is relatively hypo-oxidizing. Pharmacological and genetic manipulations that promote a hypo-oxidizing ER reduce abnormal high molecular weight protein complexes in the stressed ER and protect from the lethal consequences of ER stress. CHOP deletion thus protects cells from ER stress by decreasing ER client protein load and changing redox conditions within the organelle. [ABSTRACT FROM AUTHOR]

The small molecule ISRIB antagonizes the activation of the integrated stress response (ISR) by phosphorylated translation initiation factor 2, eIF2(αP). ISRIB and eIF2(αP) bind distinct sites in their common target, eIF2B, a guanine nucleotide exchange factor for eIF2. We have found that ISRIB-mediated acceleration of eIF2B's nucleotide exchange activity in vitro is observed preferentially in the presence of eIF2(αP) and is attenuated by mutations that desensitize eIF2B to the inhibitory effect of eIF2(αP). ISRIB's efficacy as an ISR inhibitor in cells also depends on presence of eIF2(αP). Cryoelectron microscopy (cryo-EM) showed that engagement of both eIF2B regulatory sites by two eIF2(αP) molecules remodels both the ISRIB-binding pocket and the pockets that would engage eIF2α during active nucleotide exchange, thereby discouraging both binding events. In vitro , eIF2(αP) and ISRIB reciprocally opposed each other's binding to eIF2B. These findings point to antagonistic allostery in ISRIB action on eIF2B, culminating in inhibition of the ISR. • Mutually antagonistic binding of phosphorylated eIF2 and ISRIB to eIF2B • Opposing structural rearrangement of eIF2B by binding ISRIB or phosphorylated eIF2 • Phosphorylated eIF2 exposes ISRIB's ability to enhance eIF2B activity in vitro • Weak ISR-inhibitory activity of ISRIB in cells lacking phosphorylated eIF2 ISRIB is a drug-like compound that restores memory and cognitive function in mouse models of certain human diseases. Its beneficial properties derive from interference with a stress-response pathway that hinges on regulating rates of protein synthesis. Here, Zyryanova, Kashiwagi et al. dissect the molecular basis of ISRIB action in cells. [ABSTRACT FROM AUTHOR]