Limits...
The general control nonderepressible-2 kinase mediates stress response and longevity induced by target of rapamycin inactivation in Caenorhabditis elegans.

Rousakis A, Vlassis A, Vlanti A, Patera S, Thireos G, Syntichaki P - Aging Cell (2013)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Biomedical Research Foundation of the Academy of Athens, Center of Basic Research II, Athens, 11527, Greece; School of Medicine, University of Athens, Athens, 11527, Greece.

Show MeSH

Related in: MedlinePlus

Conservation in gene structure and function of Caenorhabditis elegansgcn-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 (http://www.wormbase.org). The graphic was created using the Exon-Intron Graphic Maker (http://wormweb.org/exonintron). 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).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4225475&req=5

fig01: Conservation in gene structure and function of Caenorhabditis elegansgcn-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 (http://www.wormbase.org). The graphic was created using the Exon-Intron Graphic Maker (http://wormweb.org/exonintron). 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).

Mentions: In C. elegans, the sole homolog of yeast/mammalian GCN2 is encoded by the gene Y81G3A.3 (gcn-2) and phosphorylates the eIF2α subunit at the putative phosphorylation site Ser49 (Nukazuka et al., 2008). Lately, it was shown that GCN-2 is required for the induced phospho-eIF2α levels during mitochondrial or osmotic stress (Baker et al., 2012; Lee & Strange, 2012), but its function during nutrient deprivation or other stresses has not been established. The predicted GCN-2 protein of 1696 amino acids shares 24.2% identity (40.7% similarity) with human HsGCN2 and 21% identity (35.3% similarity) with yeast ScGCN2 (EMBOSS Align-EMBL/EBI), having all the functional domains that characterize the kinase across species (Fig. 1A). To investigate the function of GCN-2, we used the existing gcn-2 mutants (ok871 and ok886), both of which have an in-frame deletion (Wormbase WS230), lacking part of the internal coding sequence (Fig. 1A). In both gcn-2 alleles, we detected a truncated mRNA transcript (lanes 1 and 2 in Fig. 1B, left pane) expressed at the same levels as the wild-type (N2) transcript (Fig. 1B, right pane). By measuring the basal levels of eIF2α phosphorylation in whole protein extracts of N2 worms subjected to gcn-2(RNAi) (lane 2 in Fig. 1C) or of each gcn-2 mutant (lanes 3–4 in Fig. 1C), compared to untreated animals (lane 1 in Fig. 1C), we verified that both ok871 and ok886 are loss-of-function alleles of gcn-2.


The general control nonderepressible-2 kinase mediates stress response and longevity induced by target of rapamycin inactivation in Caenorhabditis elegans.

Rousakis A, Vlassis A, Vlanti A, Patera S, Thireos G, Syntichaki P - Aging Cell (2013)

Conservation in gene structure and function of Caenorhabditis elegansgcn-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 (http://www.wormbase.org). The graphic was created using the Exon-Intron Graphic Maker (http://wormweb.org/exonintron). 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).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4225475&req=5

fig01: Conservation in gene structure and function of Caenorhabditis elegansgcn-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 (http://www.wormbase.org). The graphic was created using the Exon-Intron Graphic Maker (http://wormweb.org/exonintron). 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).
Mentions: In C. elegans, the sole homolog of yeast/mammalian GCN2 is encoded by the gene Y81G3A.3 (gcn-2) and phosphorylates the eIF2α subunit at the putative phosphorylation site Ser49 (Nukazuka et al., 2008). Lately, it was shown that GCN-2 is required for the induced phospho-eIF2α levels during mitochondrial or osmotic stress (Baker et al., 2012; Lee & Strange, 2012), but its function during nutrient deprivation or other stresses has not been established. The predicted GCN-2 protein of 1696 amino acids shares 24.2% identity (40.7% similarity) with human HsGCN2 and 21% identity (35.3% similarity) with yeast ScGCN2 (EMBOSS Align-EMBL/EBI), having all the functional domains that characterize the kinase across species (Fig. 1A). To investigate the function of GCN-2, we used the existing gcn-2 mutants (ok871 and ok886), both of which have an in-frame deletion (Wormbase WS230), lacking part of the internal coding sequence (Fig. 1A). In both gcn-2 alleles, we detected a truncated mRNA transcript (lanes 1 and 2 in Fig. 1B, left pane) expressed at the same levels as the wild-type (N2) transcript (Fig. 1B, right pane). By measuring the basal levels of eIF2α phosphorylation in whole protein extracts of N2 worms subjected to gcn-2(RNAi) (lane 2 in Fig. 1C) or of each gcn-2 mutant (lanes 3–4 in Fig. 1C), compared to untreated animals (lane 1 in Fig. 1C), we verified that both ok871 and ok886 are loss-of-function alleles of gcn-2.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Biomedical Research Foundation of the Academy of Athens, Center of Basic Research II, Athens, 11527, Greece; School of Medicine, University of Athens, Athens, 11527, Greece.

Show MeSH
Related in: MedlinePlus