The messenger RNA 5' cap-binding protein eIF4E is regulated by its binding protein (4E-BP), a downstream target of phosphatidylinositol-3-OH kinase [PI(3)K] signaling. 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. 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 (Tettweiler, 2005).

4E-BP has been studied extensively in cell culture; however, to date the biological role of 4E-BP in developing organisms is unclear. Since TOR (see Drosophila Tor) has been shown to control tissue growth during animal development, 4E-BP has also been assumed to serve as a growth regulator. The relevance of 4E-BP function for organismal development has been studied, and evidence is presented for an alternate view of 4E-BP function. 4E-BP strongly affects fat metabolism in Drosophila. It is suggested 4E-BP works as a metabolic brake that is activated under conditions of environmental stress to control fat metabolism. 4E-BP mutants lack this regulation, reducing their ability to survive under unfavorable conditions (Teleman, 2005).

A rapid response is a crucial early line of defense in preventing cellular death in situations of stress. Translational regulation allows an organism to generate quick responses to environmental cues by controlling the expression of protein from existing cellular mRNAs. Translation initiation of most eukaryotic mRNAs requires binding of eIF4F, a protein complex made up of eIF4A, eIF4G, and eIF4E, to the 5' cap structure. eIF4E activity is highly regulated both by Mnk1/Mnk2-dependent phosphorylation and by repressor proteins termed eIF4E-binding proteins (4E-BPs), which compete with eIF4G for the same binding site on eIF4E (Haghighat, 1995; Mader, 1995). 4E-BPs themselves are negatively regulated by phosphorylation, and are downstream effectors of the PI3K/TOR pathway (Miron, 2001). Under nutritionally favorable conditions, the evolutionarily conserved TOR pathway is active and results in 4E-BP phosphorylation. This prevents 4E-BP binding to eIF4E, thus upregulating translation (Schmelzle, 2000; Hay, 2004). Conversely, poor nutrition causes inhibition of the TOR pathway, such that unphosphorylated 4E-BP represses translation through eIF4E binding (Tettweiler, 2005).

Activation of the PI3K pathway stimulates Akt activity. Akt directly phosphorylates the forkhead transcription factor dFOXO, which is a transcriptional activator of d4E-BP in flies, resulting in reduced d4E-BP transcription (Jünger, 2003; Puig, 2003). Akt also activates TOR through tuberous sclerosis complex 2 (TSC2/Gigas), which functions as a GTPase-activating protein (GAP) for the GTPase protein Rheb that activates TOR. Thus, activation of the PI3K pathway in Drosophila represses both the expression of the d4E-BP gene and the activity of d4E-BP protein (Tettweiler, 2005).

Whether d4E-BP is essential under starvation and oxidative stress conditions was investigated, because dFOXO activates the transcription of d4E-BP and d4E-BP mRNA levels increase upon starvation (Zinke, 2002). Evidence is provided that d4E-BP activity is linked to life span, since overexpression of dFOXO is linked to increased longevity. This work establishes that d4E-BP is the critical effector of the dFOXO-induced stress-sensitive phenotype (Tettweiler, 2005).

To investigate whether 4E-BP activity influences life span in Drosophila, the median life span of d4E-BP^null flies was determined in comparison to revertant control flies that were produced by a precise excision of the P{lacW} insertion of Thor¹ (which disrupts the gene encoding d4E-BP) but which otherwise have the identical genetic background (Rodriguez, 1996; Bernal, 2000; Bernal, 2004). These will be referred to as revertant flies. A null mutation in d4E-BP caused a significant decrease in longevity. The median life span of mutant males was 19.8 d, ~25% shorter than that of control males, median life span of 26.6 d). The life span of females was longer than for males, but a comparable relative effect of the null mutation in d4E-BP on life span was observed in both sexes. These results show that d4E-BP, a target of the conserved PI3K/TOR signaling pathway that has been strongly implicated in longevity, has a significant impact itself on life span (Tettweiler, 2005).

In Drosophila larvae, protein-rich nutrition is critical for survival, while adults can survive up to 3 wk without protein. Strikingly, it was observed that larval d4E-BP protein levels rise rapidly during nutritional stress. A dramatic increase of ~10-fold is observed after 8 h of starvation. To determine whether this increase in d4E-BP level is important for survival following starvation, eggs were collected from Oregon-R, d4E-BP^null, and revertant flies, transferred to starvation medium: mortality was scored at 12-h intervals. Drosophila 4E-BP^null larvae die substantially faster than their control counterparts under starvation, (median life span of 20.8 h for starved d4E-BP^null larvae, 26.4 h for revertant, and 26.2 h for Oregon-R larvae). To examine whether this effect can be overcome by induced expression of a d4E-BP(wt) transgene in a d4E-BP^null background, the UAS-GAL4 system was used with the heat-shock inducible Hsp70 promoter. Induction of d4E-BP expression in this manner fully rescues the increased sensitivity of d4E-BP^null larvae to starvation (median life span of 27 h). Importantly, transgenic flies expressing a mutant version of d4E-BP [d4E-BP(Y54A,M59A)] that does not bind to eIF4E (Miron, 2001) are susceptible to starvation similar to the d4E-BP^null animals (median life-span 22.6 h). While the d4E-BP(Y54A,M59A) larvae resist nutritional stress somewhat better in the first 12 h, after 36 h of complete starvation their survival was as poor as that of d4E-BP^null larvae (survival rates of 8.5% and 9.6%, respectively) and far lower than that of control animals, or animals expressing d4E-BP from a transgene [survival rates of 26.8% for revertant larvae, 27.6% for Oregon-R larvae, and 30.3% for transgenic d4E-BP(wt) larvae]. These results demonstrate clearly that the protective role of d4E-BP during starvation requires binding to eIF4E (Tettweiler, 2005).

dFOXO is a transcriptional activator of d4E-BP (Jünger, 2003; Puig, 2003; Wang, 2005). dFOXO loss-of-function mutants exhibit increased sensitivity to oxidative stress (Jünger, 2003). Since d4E-BP is a downstream target of dFOXO, the sensitivity of EP-dFOXO²¹/EP-dFOXO²⁵ flies and d4E-BP^null flies to oxidative stress was compared. EP-dFOXO²¹/EP-dFOXO²⁵ is null for dFOXO function (Jünger, 2003) and is subsequently referred to here as dFOXO-null for brevity. Indeed, on medium containing 5% hydrogen peroxide, a reduced median life span was observed of dFOXO-null and d4E-BP^null flies (median life spans of 34.6 h and 23.2 h, respectively). The survival rate at 60 h after exposure to oxidative stress was 0% for d4E-BP^null animals, and only 2% for dFOXO-null flies, compared with 66.1% for wild-type controls. Thus, an intriguing possibility is that d4E-BP acts as the downstream mediator of the dFOXO-null phenotype. As is the case for d4E-BP-mediated protection against starvation, this effect can be rescued by ectopic expression of d4E-BP (median life span of 55.4 h). The resistance to oxidative stress was also dependent on eIF4E binding, since d4E-BP(Y54A, M59A) was unable to rescue stress sensitivity (median life span of 24.1 h, 0.4% survival rate at 60 h). The heat shock itself did not significantly affect life span. To examine whether reduced levels of d4E-BP protein could account for the oxidative stress sensitivity of dFOXO-null mutants, d4E-BP was ectopically expressed in a dFOXO-independent manner in these animals. Remarkably, ectopic expression of d4E-BP completely rescues the sensitivity of dFOXO-null animals to oxidative stress (median life span of 56.8 h, 39.7% survival rate after 60 h exposure to 5% H₂O₂). Taken together, these results demonstrate that d4E-BP is a critical downstream mediator of dFOXO in oxidative stress resistance (Tettweiler, 2005).

Normal growth and development are suspended during stress in order to concentrate resources on an appropriate stress response, and repressing translation does, in fact, slow growth. d4E-BP is strongly up-regulated during starvation, and its absence leads to compromised survival under outright starvation. The importance of d4E-BP in nutrient stress response is underscored by the fact that it is one of a small set of ~14 genes whose expression is up-regulated as a consequence of starvation (Zinke, 2002; Tettweiler, 2005). The data support the current models to explain how oxidative stress activates transcription of d4E-BP. Oxidative stress promotes dephosphorylation of dFOXO, which causes its transport into the nucleus and activation of d4E-BP transcription (Jünger, 2003). Amino acid starvation activates the dTOR pathway in the larval fat body. This triggers a starvation signal that suppresses the Inr/PI3K pathway in peripheral tissues (Colombani, 2003). Suppression of this pathway results in inactivation of dAkt, which in turn can no longer phosphorylate and inactivate dFOXO, causing enhanced transcription of d4E-BP (Tettweiler, 2005).

Since the function of d4E-BP in stress resistance requires its eIF4E-binding activity, it is proposed that d4E-BP involves repression of cap-dependent translation, concomitant with the stimulation of cap-independent translation. The up-regulation of d4E-BP could result in preferential translation of mRNAs, which translate in a cap-independent manner, via an internal ribosome entry site (IRES) element. Such stimulation has been documented under stress conditions including irradiation, hypoxia, and nutrient deprivation. It is also possible that d4E-BP exerts its effects by inhibiting the translation of key mRNA targets as was documented in mammals (Gingras, 1999). Mammalian eIF4E preferentially stimulates the translation of mRNAs with a high degree of secondary structure in their mRNA 5 'UTRs (Gingras, 1999). These mRNAs, which are inefficiently translated, mostly encode proteins that play important roles in cell growth and proliferation (Tettweiler, 2005).

Recent studies show that overexpression of dFOXO in adult fat bodies can extend the Drosophila life span. Further, overexpression of dTSC1, dTSC2, or dominant-negative forms of dTOR or dS6K all extend the life span of Drosophila. Interestingly, TOR also regulates life span in Caenorhabditis elegans. The data support a role for d4E-BP in mediating life span, most likely as an effector of dFOXO. Taken together, these results add to the increasing body of evidence supporting a key role for 4E-BP-mediated regulation of eIF4E in controlling cell growth, proliferation, and survival (Miron, 2001; Fingar, 2002; Li, 2002; Avdulov, 2004; Bjornsti, 2004; Tettweiler, 2005 and references therein).

GENE STRUCTURE

cDNA clone length - 764

Bases in 5' UTR - 182

Exons - 2

Bases in 3' UTR - 228

PROTEIN STRUCTURE

Amino Acids - 117

Structural Domains

The Thor protein is similar to the mammalian translational regulators known as 4E-BPs. Sequences corresponding to 4E-BPs also have been identified in zebrafish, the slime mold Dictyostelium discoideum and the parasite Schistosoma mansoni. THOR is most similar to mouse PHAS-II and human 4E-BP2, with 39% and 38% identity respectively, and 67.5% similarity for both. Of importance is the high conservation in the central region, residues 34-59 for THOR: this includes the binding region of 4E-BPs to eIF4E. THOR has 66.5%-70% identity with all sequences in this region, except for D. discoideum, which has 55.5% identity (Bernal, 2000).

date revised: 10 November 2005

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