Starvation and oxidative stress resistance in Drosophila are mediated through the eIF4E-binding protein, d4E-BP

Abstract

eIF4E, the mRNA 5' cap-binding protein, is regulated by its binding protein (4E-BP), a downstream target of phosphatidylinositol-3-OH kinase [PI(3)K] signaling. We show that Drosophila 4E-BP (d4E-BP) activity becomes critical for survival under dietary restriction and oxidative stress, and is linked to life span. The Drosophila forkhead transcription factor (dFOXO) activates d4E-BP transcription. We show that ectopic expression of d4E-BP in dFOXO-null flies restores oxidative stress resistance to control levels. Thus, d4E-BP is an important downstream effector of a dFOXO phenotype, and regulation of translation by eIF4E is vital during environmental stress.

Figure 1.

Life span of D. melanogaster is affected by the eIF4E inhibitory protein, 4E-BP. (A) The median life span of d4E-BP^null males (n = 97) is ∼25% reduced compared with revertant males (n = 157). (—•—) d4E-BP^null flies; (—▵—) revertant control. Statistical comparison (log-rank test): revertant control vs. d4E-BP^null, p < 0.0001. (B) A null mutation in d4E-BP has a comparable effect on the life span of female animals. Statistical comparison (log-rank test): revertant control (n = 165) vs. d4E-BP^null (n = 86), p < 0.0001.

Figure 2.

Time course of increasing expression of d4E-BP in response to nutrient stress: 40-h-old larvae of revertant animals were subjected to complete starvation. Identical amounts of total protein (30 μg) were analyzed by Western blot with 1868 antibody to d4E-BP. Loading control, α-tubulin.

Figure 3.

d4E-BP binding to eIF4E is required to rescue nutritional stress resistance. Eggs were subjected to complete starvation, and surviving larvae were counted every 12 h. d4E-BP^null larvae (—•—) died significantly faster than revertant control larvae (—▵—). This effect can be rescued by overexpression of d4E-BP (—□—), but not by overexpression of d4E-BP(Y54A,M59A) (—▪—), a mutant form that cannot bind to eIF4E. Statistical comparison (log-rank test): revertant control (n = 157) vs. d4E-BP^null (n = 200), and revertant control vs. d4E-BP^null; Hsp70-GAL4/UAS-d4E-BP(Y54A,M59A) (n = 270), p < 0.0001. Revertant control vs. d4E-BP^null; Hsp70-Gal4/UAS-d4E-BP(wt) (n = 102), p = 0.345 (not significant). Each data point represents the aggregate of results from three independent experiments.

Figure 4.

d4E-BP mediates the oxidative stress resistance of dFOXO. Flies were exposed to 5% H₂O₂, and survival was determined at 12-h intervals. Oregon-R flies were used as control. For d4E-BP^null (—•—) and dFOXO-null flies (—♦—), mortality at 60 h was 100% and 98%, respectively, compared with 33.9% for Oregon-R (—▴—). Ectopic expression of d4E-BP in a d4E-BP^null background rescued this effect (—□—, 64% mortality at 60 h), but not ectopic expression of d4E-BP(Y54A,M59A) (—▪—, 99.6% mortality at 60 h). Overexpression of d4E-BP in a dFOXO-null background (—⋄—, 60.3% mortality after 60 h) rescued the stress-sensitive phenotype of dFOXO-null flies. Survival data from three to six independent experiments were combined to clarify statistical analysis. Statistical comparison (log-rank test): Oregon-R (n = 118) vs. dFOXO-null (n = 224), Oregon-R vs. d4E-BP^null (n = 120), Oregon-R vs. d4E-BP^null;Hsp70-Gal4/UAS-d4E-BP(wt) (n = 150), Oregon-R vs. d4E-BP^null; Hsp70GAL4/UAS-d4E-BP(Y54A,M59A) (n = 274), all p < 0.0001. Oregon-R vs. Act5CGAL4/UAS-d4E-BP(wt);dFOXO-null (n = 156), p = 0.109 (not significant). Supplementary Figure S2 provides evidence that ectopic expression of d4E-BP can also rescue starvation sensitivity of dFOXO-null larvae.

Figure 5.

d4E-BP is an important downstream effector of the dFOXO phenotype. Under oxidative stress, dFOXO becomes dephosphorylated, and enters the nucleus to activate d4E-BP transcription. Amino acid starvation inhibits the dTOR pathway in the larval fat body, which results in suppression of the Inr/PI3K pathway in peripheral tissues. This affects d4E-BP regulation in two ways. First, suppression of Inr/PI3K signaling leads to inactivation of dAkt. Consequently, dAkt cannot phosphorylate dFOXO, resulting in enhanced transport of dFOXO to the nucleus and transcriptional activation of d4E-BP. Also, the d4E-BP kinase dTOR is a downstream target of the PI3K pathway. Under starvation, dTOR is inactive, such that d4E-BP does not become phosphorylated, and can interact with eIF4E. Thus, amino acid starvation increases both the protein level and activity of d4E-BP, which results in repression of cap-dependent translation.