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Residues required for phosphorylation of translation initiation factor eIF2? under diverse stress conditions are divergent between yeast and human.

ABSTRACT: PERK, PKR, HRI and GCN2 are the four mammalian kinases that phosphorylate the ? subunit of the eukaryotic translation initiation factor 2 (eIF2?) on Ser51. This phosphorylation event is conserved among many species and attenuates protein synthesis in response to diverse stress conditions. In contrast, Saccharmyces cerevisiae expresses only the GCN2 kinase. It was demonstrated previously in S. cerevisiae that single point mutations in eIF2?'s N-terminus severely impaired phosphorylation at Ser51. To assess whether similar recognition patterns are present in mammalian eIF2?, we expressed human eIF2?'s with these mutations in mouse embryonic fibroblasts and assessed their phosphorylation under diverse stress conditions. Some of the mutations prevented the stress-induced phosphorylation of eIF2? by all mammalian kinases, thus defining amino acid residues in eIF2? (Gly 30, Leu 50, and Asp 83) that are required for substrate recognition. We also identified residues that were less critical or not required for recognition by the mammalian kinases (Ala 31, Met 44, Lys 79, and Tyr 81), even though they were essential for recognition of the yeast eIF2? by GCN2. We propose that mammalian eIF2? kinases evolved to maximize their interactions with the evolutionarily conserved Ser51 residue of eIF2? in response to diverse stress conditions, thus adding to the complex signaling pathways that mammalian cells have over simpler organisms.

SUBMITTER: Majumder M

PROVIDER: S-EPMC4365977 | biostudies-literature | 2015 Feb

REPOSITORIES: biostudies-literature

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Residues required for phosphorylation of translation initiation factor eIF2α under diverse stress conditions are divergent between yeast and human.

Majumder Mithu M Mitchell Daniel D Merkulov Sergei S Wu Jing J Guan Bo-Jhih BJ Snider Martin D MD Krokowski Dawid D Yee Vivien C VC Hatzoglou Maria M

The international journal of biochemistry & cell biology 20141222

PERK, PKR, HRI and GCN2 are the four mammalian kinases that phosphorylate the α subunit of the eukaryotic translation initiation factor 2 (eIF2α) on Ser51. This phosphorylation event is conserved among many species and attenuates protein synthesis in response to diverse stress conditions. In contrast, Saccharmyces cerevisiae expresses only the GCN2 kinase. It was demonstrated previously in S. cerevisiae that single point mutations in eIF2α's N-terminus severely impaired phosphorylation at Ser51. ...[more]

PMID: 25541374

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Project description:Disruption of protein folding in the endoplasmic reticulum triggers the Unfolded Protein Response (UPR), a transcriptional and translational control network designed to restore protein homeostasis. Central to the UPR is PERK phosphorylation of the alpha subunit of eIF2 (eIF2~P), which represses global translation coincident with preferential translation of mRNAs, such as ATF4 and CHOP, that serve to implement the UPR transcriptional regulation. In this study, we used sucrose gradient ultracentrifugation and a genome-wide microarray approach to measure changes in mRNA translation during ER stress. Our analysis suggests that translational efficiencies vary across a broad range during ER stress, with the majority of transcripts being either repressed or resistant to eIF2~P, while a notable cohort of key regulators are subject to preferential translation. From this latter group, we identify IBTKa as being subject to both translation and transcriptional induction during eIF2~P in both cell lines and a mouse model of ER stress. Translational regulation of IBTKalpha mRNA involves the stress-induced relief of two inhibitory uORFs in the 5'-leader of the transcript. Depletion of IBTKalpha by shRNA reduced viability of cultured cells coincident with increased caspase 3/7 cleavage, suggesting that IBTKalpha is a key regulator in determining cell fate during the UPR. We used a genome-wide microarray approach to determine how individual mRNAs were differentially translated during endoplasmic reticulum stress. A microarray analysis from our laboratory identified gene transcripts suggested to be under translation control in mouse embryonic fibroblast (MEF) cells following a 6 hour treatment with thapsigargin, a potent inducer of ER stress, or no stress. The mRNAs were separated by sucrose gradient analyses to yield three fractions, those transcripts associated with large polysomes (?4 ribosomes per mRNA), those associated with monosome, disomes, or trisomes, and those fractionated at the top of the gradient with free ribosomes. RNA was extracted from sucrose gradients corresponding to these fractions and hybridized on Affymetrix microarrays. In parallel, we also measured total levels for each gene transcript in the presence or absence of thapsigargin treatment to address transcription regulation coincident with translational control. Please note that the treatment plus fractionation based on association with different numbers of ribosomes did yield different populations of mRNAs, which resulted in considerable variation in normalized data across the samples.

Dataset Information

Residues required for phosphorylation of translation initiation factor eIF2? under diverse stress conditions are divergent between yeast and human.

Publications

Residues required for phosphorylation of translation initiation factor eIF2α under diverse stress conditions are divergent between yeast and human.

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