Dataset Information

Global Phosphoproteomics Pinpoints Uncharted Gcn2-Mediated Modes of Translational Control

ABSTRACT: To identify new Gcn2 target proteins, we performed a set of Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC)-based quantitative phosphoproteomics experiments, in which we compared WT cells to gcn2∆ and sui2S52A cells (n=3), all treated, or not, with rapamycin, which has previously been shown to stimulate eIF2alpha phosphorylation by Gcn2.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Saccharomyces Cerevisiae (baker's Yeast)

SUBMITTER: Joern Dengjel

LAB HEAD: Joern Dengjel

PROVIDER: PXD021109 | Pride | 2021-03-22

REPOSITORIES: Pride

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			Action	DRS
	20190319_MS_PP19_07_FT_B01.raw	Raw
	20190319_MS_PP19_07_FT_B02.raw	Raw
	20190319_MS_PP19_07_FT_B03.raw	Raw
	20190319_MS_PP19_07_FT_B04.raw	Raw
	20190319_MS_PP19_07_FT_B05.raw	Raw

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Global phosphoproteomics pinpoints uncharted Gcn2-mediated mechanisms of translational control.

Dokládal Ladislav L Stumpe Michael M Pillet Benjamin B Hu Zehan Z Garcia Osuna Guillermo Miguel GM Kressler Dieter D Dengjel Jörn J De Virgilio Claudio C

Molecular cell 20210319 9

The conserved Gcn2 protein kinase mediates cellular adaptations to amino acid limitation through translational control of gene expression that is exclusively executed by phosphorylation of the α-subunit of the eukaryotic translation initiation factor 2 (eIF2α). Using quantitative phosphoproteomics, however, we discovered that Gcn2 targets auxiliary effectors to modulate translation. Accordingly, Gcn2 also phosphorylates the β-subunit of the trimeric eIF2 G protein complex to promote its associat ...[more]

PMID: 33743194

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Project description:In eukaryotes, regulation of mRNA translation enables a fast, localized and finely tuned expression of gene products. Within the translation process, the first stage of translation initiation is most rigorously modulated by the actions of eukaryotic initiation factors (eIFs) and their associated proteins. These 11 eIFs catalyze the joining of the tRNA, mRNA and rRNA into a functional translation complex. Their activity is influenced by a wide variety of extra- and intracellular signals, ranging from global, such as hormone signaling and unfolded proteins, to specific, such as single amino acid imbalance and iron deficiency. Their action is correspondingly comprehensive, in increasing or decreasing recruitment and translation of most cellular mRNAs, and specialized, in targeting translation of mRNAs with regulatory features such as a 5’ terminal oligopyrimidine tract (TOP), upstream open reading frames (uORFs), or an internal ribosomal entry site (IRES). In mammals, two major pathways are linked to targeted mRNA translation. The target of rapamycin (TOR) kinase induces translation of TOP and perhaps other subsets of mRNAs, whereas a family of eIF2 kinases does so with mRNAs containing uORFs or an IRES. TOR targets translation of mRNAs that code for proteins involved in translation, an action compatible with its widely accepted role in regulating cellular growth. The four members of the eIF2 kinase family increase translation of mRNAs coding for stress response proteins such as transcription factors and chaperones. Though all four kinases act on one main substrate, eIF2, published literature demonstrates both common and unique effects by each kinase in response to its specific activating stress. This suggests that the activated eIF2 kinases regulate the translation of both a global and a specific set of mRNAs. Up to now, few studies have attempted to test such a hypothesis; none has been done in mammals. We use array analysis to determine the global mRNA shift into polysomes following a stress response, and to compare the translational response following activation of GCN2 versus PERK, two of the four eIF2alpha kinases. Experiment Overall Design: Gcn2 wild-type or knockout mouse liver were perfused with complete amino acids media or media lacking methionine for RNA extraction and hybridization of Affymetrix microarrays. RNA was extracted from unfractionated liver samples and polysome fraction of samples separated on sucrose density gradient. To minimize biological variations, we pooled RNA from two perfused liver samples to use in each array analysis. The conditions were total and polysome fraction of Gcn2+/+, +Met or -Met; total and polysome fraction of Gcn2-/-, +Met or -Met. Each array analysis was done in duplicate.