Mammalian eukaryotic initiation factor 2 alpha kinases functionally substitute for GCN2 protein kinase in the GCN4 translational control mechanism of yeast.

Dever, Thomas E.; Chen, Jane-Jane; Barber, Glen N.; Cigan, A. Mark; Feng, Lan; Donahue, Thomas F.; London, Irving M.; Katze, Michael G.; Hinnebusch, A G

doi:10.1073/pnas.90.10.4616

Cited by 215 publications

(186 citation statements)

References 16 publications

(26 reference statements)

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“…These results support the idea that GADD34 recruits PP1 to dephosphorylate eIF2α without affecting GCN2 kinase activity. Previously, it was shown that overexpression of human PKR is lethal in yeast because of the high levels of eIF2α phosphorylation and inhibition of translation (27,28). To test whether expression of GADD34 would suppress the toxicity associated with high-level expression of PKR in yeast, vectors encoding different variants of GADD34 were introduced into a yeast strain containing one copy of the human PKR gene.…”

Section: Gadd34 Promotesmentioning

confidence: 99%

An eIF2α-binding motif in protein phosphatase 1 subunit GADD34 and its viral orthologs is required to promote dephosphorylation of eIF2α

Rojas

Vasconcelos

Dever

2015

Proc. Natl. Acad. Sci. U.S.A.

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Transient protein synthesis inhibition, mediated by phosphorylation of the α subunit of eukaryotic translation initiation factor 2 (eIF2α), is an important protective mechanism cells use during stress conditions. Following relief of the stress, the growth arrest and DNA damage-inducible protein GADD34 associates with the broadly acting serine/threonine protein phosphatase 1 (PP1) to dephosphorylate eIF2α. Whereas the PP1-binding motif on GADD34 has been defined, it remains to be determined how GADD34 directs PP1 to specifically dephosphorylate eIF2α. In this report, we map a novel eIF2α-binding motif to the C terminus of GADD34 in a region distinct from where PP1 binds to GADD34. This motif is characterized by the consensus sequence Rx [Gnl], where capital letters are preferred and x is any residue. Point mutations altering the eIF2α-binding motif impair the ability of GADD34 to interact with eIF2α, promote eIF2α dephosphorylation, and suppress PKR toxicity in yeast. Interestingly, this eIF2α-docking motif is conserved among viral orthologs of GADD34, and is necessary for the proteins produced by African swine fever virus, Canarypox virus, and Herpes simplex virus to promote eIF2α dephosphorylation. Taken together, these data indicate that GADD34 and its viral orthologs direct specific dephosphorylation of eIF2α by interacting with both PP1 and eIF2α through independent binding motifs.

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Section: Gadd34 Promotesmentioning

confidence: 99%

An eIF2α-binding motif in protein phosphatase 1 subunit GADD34 and its viral orthologs is required to promote dephosphorylation of eIF2α

Rojas

Vasconcelos

Dever

2015

Proc. Natl. Acad. Sci. U.S.A.

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“…Because PERK is a member of the eIF2␣ kinase family, we considered it likely that another family member might cooperate and thereby promote eIF2␣ phosphorylation and attenuation of cyclin D1 synthesis (Dever et al, 1993). Two candidates for a redundant eIF2␣ kinase include PKR and GCN2, which are activated by double-stranded RNA and nutrient deprivation, respectively.…”

Section: Cyclin D1 Loss Requires Phosphorylation Of Eif2␣mentioning

confidence: 99%

“…Indeed, cyclin D1 protein levels were maintained in eIF2␣ S51A knockin fibroblasts after ER stress, demonstrating that cyclin D1 loss was absolutely dependent upon eIF2␣ phosphorylation. Furthermore, eIF2␣ S51A fibroblasts failed to arrest in G 1 phase during ER stress, providing further support for the requirement for eIF2␣ phosphorylation in cyclin D1 regulation.Because PERK is a member of the eIF2␣ kinase family, we considered it likely that another family member might cooperate and thereby promote eIF2␣ phosphorylation and attenuation of cyclin D1 synthesis (Dever et al, 1993). Two candidates for a redundant eIF2␣ kinase include PKR and GCN2, which are activated by double-stranded RNA and nutrient deprivation, respectively.…”

mentioning

confidence: 99%

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

Hamanaka

Bennett

Cullinan

et al. 2005

MBoC

231

180

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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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“…High-level expression of human PKR is toxic in yeast because of phosphorylation of eIF2␣ and the attendant inhibition of translation initiation (13,14). Substitution of Ser-51 in eIF2␣ by Ala suppresses PKR toxicity as do mutations that block kinase activity (9,14,15).…”

mentioning

confidence: 99%

Protein kinase PKR mutants resistant to the poxvirus pseudosubstrate K3L protein

Seo

Liu

Kawagishi-Kobayashi

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

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As part of the mammalian cell innate immune response, the doublestranded RNA activated protein kinase PKR phosphorylates the translation initiation factor eIF2␣ to inhibit protein synthesis and thus block viral replication. Poxviruses including vaccinia and smallpox viruses express PKR inhibitors such as the vaccinia virus K3L protein that resembles the N-terminal substrate-targeting domain of eIF2␣. Whereas high-level expression of human PKR was toxic in yeast, this growth inhibition was suppressed by coexpression of the K3L protein.We used this yeast assay to screen for PKR mutants that are resistant to K3L inhibition, and we identified 12 mutations mapping to the C-terminal lobe of the PKR kinase domain. The PKR mutations specifically conferred resistance to the K3L protein both in yeast and in vitro. Consistently, the PKR-D486V mutation led to nearly a 15-fold decrease in K3L binding affinity yet did not impair eIF2␣ phosphorylation. Our results support the identification of the eIF2␣-binding site on an extensive face of the C-terminal lobe of the kinase domain, and they indicate that subtle changes to the PKR kinase domain can drastically impact pseudosubstrate inhibition while leaving substrate phosphorylation intact. We propose that these paradoxical effects of the PKR mutations on pseudosubstrate vs. substrate interactions reflect differences between the rigid K3L protein and the plastic nature of eIF2␣ around the Ser-51 phosphorylation site.eIF2 ͉ translational control

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Mammalian eukaryotic initiation factor 2 alpha kinases functionally substitute for GCN2 protein kinase in the GCN4 translational control mechanism of yeast.

Cited by 215 publications

References 16 publications

An eIF2α-binding motif in protein phosphatase 1 subunit GADD34 and its viral orthologs is required to promote dephosphorylation of eIF2α

An eIF2α-binding motif in protein phosphatase 1 subunit GADD34 and its viral orthologs is required to promote dephosphorylation of eIF2α

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

Protein kinase PKR mutants resistant to the poxvirus pseudosubstrate K3L protein

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