The general control nonderepressible-2 kinase mediates stress response and longevity induced by target of rapamycin inactivation in Caenorhabditis elegans - PubMed (original) (raw)
The general control nonderepressible-2 kinase mediates stress response and longevity induced by target of rapamycin inactivation in Caenorhabditis elegans
Aris Rousakis et al. Aging Cell. 2013 Oct.
Abstract
The general control nonderepressible 2 (GCN2) kinase is a nutrient-sensing pathway that responds to amino acids deficiency and induces a genetic program to effectively maintain cellular homeostasis. Here we established the conserved role of Caenorhabditis elegans GCN-2 under amino acid limitation as a translation initiation factor 2 (eIF2) kinase. Using a combination of genetic and molecular approaches, we showed that GCN-2 kinase activity plays a central role in survival under nutrient stress and mediates lifespan extension conferred by dietary restriction (DR) or inhibition of the major nutrient-sensing pathway, the target of rapamycin (TOR). We also demonstrated that the GCN-2 and TOR signaling pathways converge on the PHA-4/FoxA transcription factor and its downstream target genes to ensure survival of the whole organism under a multitude of stress conditions, such as nutrient scarcity or environmental stresses. This is one step forward in the understanding of evolutionary conserved mechanisms that confer longevity and healthspan.
Keywords: Caenorhabditis elegans; PHA-4; aging; general control nonderepressible 2; target of rapamycin.
© 2013 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.
Figures
Figure 1
Conservation in gene structure and function of Caenorhabditis elegans gcn-2. (A) Gene structure and predicted protein domains of GCN-2, designed using the Prosite MyDomains (
http://prosite.expasy.org/mydomains/
): Black boxes represent exons linked by lines corresponding to introns, and white boxes indicate the 5′ and 3′ UTR found in a second alternative isoform (
). The graphic was created using the Exon-Intron Graphic Maker (
). Branches point to the sequences deleted in the two alleles ok871 and ok886. Black arrowheads indicate the position of primers used in this study (Table S2). (B) Reverse transcription (RT)–PCR analysis with primers G1/G2 (shown in A pane) and qRT-PCR analysis with primers G4/G5 (shown in A pane). (C) Western blot analysis showing the levels of phosphorylated (P-eIF2α) normalized by the total amount of eIF2α, in whole worm extracts. Basal levels of P-eIF2α under well-fed conditions in untreated or _gcn-2(RNAi)_-treated N2 and gcn-2 mutants (left pane). Induced levels of P-eIF2α under amino acid limitation in krs-1(RNAi) treated worms (right pane).
Figure 2
Translational control of atf-5 gene expression. (A) Schema of uORFs within the atf-5 5′-UTR. The amino acid length of the predicted translated uORF and the coding sequence of atf-5 gene are shown. (B) Confocal and bright field images of 1-day-old transgenic worms expressing the translational fusion of the intact atf-5 (left box) or the uORF-less atf-5 transgene (right box) under normal feeding conditions. A larger magnification of the area in the red box is shown on the top left. White arrows indicate fluorescent nuclei, and white arrowheads show regions of autofluorescence. All images were taken at 20× magnification under the same microscopy settings (scale bar: 50 μm). The levels of atf-5 mRNA in N2, BRF140, and BRF152 worms, normalized to ama-1(mRNA), were quantified using qRT–PCR.
Figure 3
General control nonderepressible-2-dependent translational control of atf-5 under amino acid limitation. Confocal images of N2 or gcn-2(ok871) worms, both carrying a translational fusion of atf-5::gfp, fed Control or krs-1(RNAi) expressing bacteria. The images show 1-day adults fed with each RNAi from eggs (P0) or their L3-arrested progeny (F1) in krs-1(RNAi) worms. White arrows indicate fluorescent nuclei; white arrowheads show regions of autofluorescence. All images were taken at 20× magnification under the same microscopy settings (scale bar: 50 μm).
Figure 4
Loss of GCN-2 function affects lifespan only under nutrient stress. Survival curves of (A) N2, gcn-2, and atf-5 mutants fed OP50 bacteria (B) N2 worms treated with gcn-2(RNAi) or atf-5(RNAi) from L4s (C) eat-2(ad465) and eat-2(ad465);gcn-2(ok871) fed OP50 bacteria (D) N2 and gcn-2(ok871) treated with let-363(RNAi) from their first day of adulthood (E) N2 and gcn-2(ok871) treated with rsks-1(RNAi) from L4s (F) N2 and ife-2(ok306) treated with gcn-2(RNAi) from L4s.
Figure 5
General control nonderepressible-2 influences the induction of pha-4 and its downstream targets in response to TOR inactivation. qRT–PCR of pha-4(mRNA) in (A) 1-day adults of N2 and gcn-2(ok871) treated with let-363(RNAi) from L3 stage; N2, eat-2, and eat-2;gcn-2 young adults raised on OP-50 bacteria at 20 °C; and in the F1 progenies (L2–L3 stage) of N2 and gcn-2(ok871) treated with rrt-1(RNAi) (B) qRT–PCR of sod-1, sod-2, sod-3, sod-4, mtl-1, and lgg-1 on 1-day adults of N2 and gcn-2(ok871) treated with let-363(RNAi) from L3 stage. Quantification of each mRNA level, relative to ama-1 mRNA and the mean ± SD of biological triplicates are shown. The asterisks represent statistical significant difference from N2/Control or gcn-2/Control (*P < 0.05, ** P < 0.01, ***P < 0.001 in unpaired _t_-test).
Figure 6
GCN-2 affects pha-4 induction and survival of TOR-deficient and eat-2 worms under oxidative stress. Survival to sodium arsenite (SA) of 1-day adults of (A) N2 and gcn-2(ok871) fed with let-363(RNAi) from L3 stage (B) eat-2(ad465) and eat-2(ad465);gcn-2(ok871) (C) N2 and gcn-2(ok871) fed with pha-4(RNAi) from eggs (D) Quantification in arbitrary units (AU) of GFP signal in the intestine of 1-day adults expressing a membrane-bound GFP under the pha-4 promoter, fed either Control or gcn-2(RNAi) expressing bacteria and treated or not with SA (15 m
m
for 3 h before observation). Fluorescence intensity was measured from several confocal images using ImageJ. The total number (n) of areas counted and the mean ± SD are shown (E) qRT–PCR of pha-4(mRNA) in 1-day N2 or gcn-2 worms treated or not with SA (15 m
m
for 1.5 h). Quantification of each mRNA level, relative to ama-1 mRNA, and the mean ± SD of biological triplicates are shown (*P < 0.05, **P < 0.01, ***P < 0.001 in unpaired _t_-test) (F) A model illustrating the function of GCN-2 in response to nutrient and other stresses.
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