EAK-7 controls development and life span by regulating nuclear DAF-16/FoxO activity

Abstract

FoxO transcription factors control development and longevity in diverse species. Although FoxO regulation via changes in its subcellular localization is well established, little is known about how FoxO activity is regulated in the nucleus. Here, we show that the conserved C. elegans protein EAK-7 acts in parallel to the serine/threonine kinase AKT-1 to inhibit the FoxO transcription factor DAF-16. Loss of EAK-7 activity promotes diapause and longevity in a DAF-16/FoxO-dependent manner. Whereas akt-1 mutation activates DAF-16/FoxO by promoting its translocation from the cytoplasm to the nucleus, eak-7 mutation increases nuclear DAF-16/FoxO activity without influencing DAF-16/FoxO subcellular localization. Thus, EAK-7 and AKT-1 inhibit DAF-16/FoxO activity via distinct mechanisms. Our results implicate EAK-7 as a FoxO regulator and highlight the biological impact of a regulatory pathway that governs the activity of nuclear FoxO without altering its subcellular location.

Figure 1. EAK-7 acts in parallel to AKT-1 to inhibit dauer arrest via DAF-16/FoxO and DAF-12

A. Schematic of the DAF-2/InsR pathway. B. daf-16/FoxO null and C. daf-12 null mutations suppress the dauer arrest phenotype of an eak-7 null mutant at 27°C on NGM plates lacking supplemental cholesterol. D. Dauer arrest phenotypes of eak-7(mg338) at 25°C. The null allele daf-16(mgDf47) was used in this experiment. E. Dauer arrest phenotypes of the null allele eak-7(tm3188) at 25°C. F. and G. Genetic interactions of eak-7 null mutation with akt-1, akt-2, and sgk-1 null mutations at F. 25°C and G. 27°C on standard NGM plates containing supplemental cholesterol. H. A daf-12 null mutation suppresses the dauer arrest phenotype of eak-7;akt-1 double mutants at 25°C. All measurements in this figure represent the mean + S.D. See Table S1 for raw data and numbers of animals scored in each dauer assay.

Figure 2. Genetic interactions of eak-7 mutants in lifespan control

A. Lifespan phenotype of eak-7 null mutants at 25°C. B. daf-16/FoxO RNAi suppresses lifespan extension in eak-7 null mutants. C. eak-7 null mutation enhances the lifespan extension observed in akt-2 null mutants. D. sgk-1 activity is required for lifespan extension in eak-7 null mutants. E. eak-7 null mutation enhances lifespan extension of glp-1(e2141) mutants. F. Lifespan extension in eak-7 null mutants does not require eak-3 activity. G. akt-1;akt-2 double mutants are extremely long-lived compared to single mutants. H. sgk-1 activity is required for lifespan extension in akt-1 null mutants. See Table S2 for numbers of animals assayed and p-values for all lifespan experiments.

Figure 3. DAF-16/FoxO target gene expression in eak-7 null mutants

A. sod-3, B. mtl-1, and C. dod-3 mRNA quantification using qRT-PCR on total RNA from L2 larvae. Measurements represent the mean + S.D. D. sod-3 mRNA quantification in adult animals. E. sod-3p::GFP expression in various strains. The anterior of the animal is facing down in all images.

Figure 4. EAK-7 regulation of DAF-16/FoxO

A. An eak-7 null mutation does not affect the subcellular localization of DAF-16::GFP. White arrows denote nuclear fluorescence in akt-1 mutants. The anterior of the animal is facing right in all images. B. ftt-2 RNAi enhances dauer arrest at 25°C in an eak-7 null mutant but not in akt-1 or akt-2 null mutants. C. An eak-7 null mutation enhances dauer arrest at 25°C in animals harboring DAF-16^AM, a constitutively nuclear DAF-16/FoxO mutant GFP fusion protein lacking all canonical Akt/PKB phosphorylation sites. D. eak-7 null mutation does not affect endogenous daf-16/FoxO mRNA levels in L2 larvae. E. An eak-7 null mutation increases endogenous DAF-16/FoxO protein levels in an akt-1 null mutant. DAF-16/FoxO protein was quantified by anti-DAF-16/FoxO immunoblot of whole worm lysates. Anti-ACT-1 immunoblotting was used to confirm equal protein loading. Measurements in dauer assays represent the mean + S.D.

Figure 5. eak-7 gene structure, protein domain organization, and expression pattern

A. eak-7 gene structure. eak-7 is the downstream gene in an operon with hsb-1. The deletion in the eak-7(tm3188) allele begins 249 bp upstream of the eak-7 initiator methionine codon and deletes portions of exons 1 and 2 of eak-7. The mg338 allele is a C/T transition that results in a cysteine-to-tyrosine mutation at amino acid 315 (C315Y). B. EAK-7 domain organization. The N-myristoylation motif and TLDc domain are shown. C. hsb-1p::EAK-7::GFP expression pattern in L4 larvae. hsb-1p::EAK-7::GFP is strongly expressed in the pharynx and nerve ring (top panels), the vulva and ventral nerve cord (middle panels), and in intestinal cells and cells surrounding the anus (bottom panels). Images were taken at 400× magnification. The scale bar is 10 μm. D. Subcellular localization of wild-type EAK-7::GFP (top panels) and EAK-7::GFP harboring a mutation of glycine 2 to alanine (G2A; bottom panels) at 1000× magnification. The scale bar is 10 μm.

Figure 6. Rescue of eak-7 mutant phenotypes by tissue-specific EAK-7::GFP expression

Rescue of dauer arrest at 25°C in eak-7;akt-1 double mutants (A.-C.) and rescue of lifespan extension in an eak-7 null mutant (D.–F.) by EAK-7::GFP transgenes under the control of eak-4 (XXX-specific; A. and D.), ric-19 (neuronal; B. and E.), and ges-1 (intestinal; C. and F.) promoters. “EX” and “NTS” denote transgenic animals carrying the extrachromosomal array and their non-transgenic siblings, respectively. Measurements represent the mean + S.D. Data are from a single representative transgenic line. Data from additional lines are in Tables S3 and S4.

Figure 7. Genetic model of EAK-7 function during development and adulthood

A. During development, EAK-7 acts in parallel to AKT-1 to inhibit DAF-16/FoxO activity. SGK-1 plays a minor role in dauer regulation. B. In adults, EAK-7 acts in parallel to AKT-1, AKT-2, and the germline to inhibit DAF-16/FoxO activity. SGK-1 may be required for maximal DAF-16/FoxO activity.