InteractiveFly: GeneBrief

Gcn2: Biological Overview | References


Gene name - Gcn2

Synonyms -

Cytological map position - 100C3-100C3

Function - protein kinase

Keywords - promotes avoidance of the essential amino acid-deficient diet, blocks translation initiation through eIF2a phosphorylation, required for infection-induced host translational blockage

Symbol - Gcn2

FlyBase ID: FBgn0019990

Genetic map position - chr3R:27,221,380-27,227,735

Classification - Serine/Threonine protein kinases, catalytic domain

Cellular location - cytoplasmic



NCBI links: Precomputed BLAST | EntrezProtein
BIOLOGICAL OVERVIEW

The brain is the central organizer of food intake, matching the quality and quantity of the food sources with organismal needs. To ensure appropriate amino acid balance, many species reject a diet lacking one or several essential amino acids (EAAs) and seek out a better food source. This study shows that, in Drosophila larvae, this behavior relies on innate sensing of amino acids in dopaminergic (DA) neurons of the brain. The amino acid sensor GCN2 acts upstream of GABA signaling in DA neurons to promote avoidance of the EAA-deficient diet. Using real-time calcium imaging in larval brains, this study shows that amino acid imbalance induces a rapid and reversible activation of three DA neurons that are necessary and sufficient for food rejection. Taken together, these data identify a central amino-acid-sensing mechanism operating in specific DA neurons and controlling food intake (Bjordal, 2014).

All organisms need to sense and adapt to changes in nutrient levels and nutrient demand. In vertebrates, this is achieved through close monitoring of available nutrients by sentinel tissues such as the gut, adipose tissue, and the pancreas, which, in turn, signals the nutritional status to the brain, ultimately leading to changes in metabolism and food intake. In the brain, nutrient-sensing neurons also respond directly to fuel-related stimuli like glucose, fatty acids, or amino acids, engaging neurophysiological responses that control energy intake. However, the neurochemical identity of these neurons and the molecular sensors used are in many instances unknown (Bjordal, 2014).

The complexity of the vertebrate brain presents a challenge to understand the integration of nutrient signals and the molecular and cellular mechanisms of neuronal nutrient sensing. A possible alternative is to use genetically tractable organisms with simpler brain structures like the fruit fly Drosophila. Indeed, Drosophila recapitulates many of the hallmarks of peripheral and central nutrient sensing seen in mammals. At the periphery, a main nutrient sensor located in fat cells signals to the brain and controls the release of Drosophila insulin-like peptides (Dilps). In conditions of nutrient restriction, the drop in general insulin signaling affects growth of peripheral tissues as well as the function of specific neuropeptides such as the Drosophila orthologs of neuropeptide Y, called NPF and sNPF, leading to changes in feeding behavior (Bjordal, 2014).

Recent reports also point to the presence of central nutrient sensors regulating food intake. Experiments made on tasteless animals have revealed that mice and flies are able to evaluate the caloric content of carbohydrates independently of sweet tasting. Interestingly, the Drosophila fructose receptor Gr43a is expressed in specific neurons of the adult brain and controls feeding according to circulating hemolymph fructose. Therefore, central fructose-sensing neurons could represent a new type of sensor for carbohydrates, although the cellular and molecular mechanisms by which GR43a acts to regulate food intake remain elusive (Bjordal, 2014).

Besides sugar, adult flies sense changes in dietary amino acid levels, and a deprivation in amino acid induces a change in their feeding preference toward amino acids (Ribeiro, 2010; Vargas, 2010; Toshima, 2012). The downstream effector of the target of rapamycin (TOR) pathway, S6-kinase, and the neurotransmitter serotonin are involved in this regulation. However, the detailed molecular mechanisms and the cellular identity of such amino acid sensor are unknown (Bjordal, 2014).

One aspect of amino acid sensing concerns the necessity to provide essential amino acids (EAAs) that cannot be synthesized or stored. Earlier experiments in rodents have demonstrated that animals rapidly evaluate the lack of one essential amino acid in the food and initiate a series of drastic changes in behavioral strategies, starting with food avoidance. Injection of imbalanced amino acid mixes in defined areas of the rodent brain is sufficient to trigger a reduction in food intake, suggesting that the sensor for EAA deficiency (EAAD) is located in the brain. Additionally, mice with a mutation in the gene encoding the conserved GC nonderepressing 2 (GCN2) kinase do not reject the imbalanced diet (Hao, 2005; Maurin, 2005), indicating a role for this cell-based amino acid sensor in triggering the EAAD response (Wek, 1989; Dong, 2000). The neural circuitry involved in this behavior remains uncharacterized (Bjordal, 2014).

This study has identified a neural circuitry involved in amino acid sensing and the control of feeding behavior in the Drosophila larval brain. Drosophila larvae were shown to reduce food intake when encountering an EAAD, and it was demonstrated that amino acid sensing takes place in a limited number of dopaminergic (DA) neurons. Calcium imaging in live brain show that DA cells are rapidly and reversibly activated by EAAD in a GCN2-dependent manner. Finally, using tissue-targeted genetic loss and gain-of-function tools, it was demonstrated that EAAD-induced food avoidance involves a GCN2-dependent inhibition of GABA signaling in dopaminergic neurons. This demonstrates the existence of a dopaminergic circuitry providing homeostatic control on feeding through a central amino acid sensing mechanism (Bjordal, 2014).

Drosophila larvae reduce their food intake on EAAD diet. This behavior does not rely on smell or taste because it can be specifically mimicked or suppressed by interfering with amino acid sensing in the brain. In addition, ex vivo brain imaging demonstrates that DA cells directly and rapidly activate in response to EAAD. The fast kinetics of the response observed in DA cells suggests that uncharged tRNA levels are instantly linked to variations in intracellular amino acid concentrations and translated into changes in GCN2 activity. GCN2 activation leads to several cellular responses, including a block in translation initiation through eIF2a phosphorylation and the consequent activation of a specific transcription program in which the ATF4 transcription factor plays a key role (Chaveroux, 2010). This study demonstrates that dATF4 in DA cells is required for the rejection of EAAD food. Given the very fast kinetics of neuronal activation, it is unlikely that transcription participates in acute EAAD avoidance. Using genetic interactions and ex vivo calcium imaging, it was shown that EAAD-induced feeding inhibition requires the repression of GABA signaling by dGCN2 activation in DA cells. In addition, bioluminescence resonance energy transfer (BRET) analysis demonstrates that ATF4 and GABA(B)R1 directly interact in living cells. These data are supported by observations made in rodents indicating that suppression of GABAergic inhibition contributes to EAAD-induced food avoidance (Truong, 2002). A model is proposed whereby, in response to EAAD, activation of dGCN2 induces dATF4-mediated GABA signaling inhibition, dopamine release, and food rejection (Bjordal, 2014).

TOR signaling couples amino acid availability with the systemic control of growth in fat body cells and ecdysone production in the larval ring gland (Colombani, 2003; Layalle, 2008). Interestingly, TOR inhibition in DA cells does not attenuate EAAD-induced food avoidance. Similarly, rapamycin injection in the antero-piriform complex of rodent brain does not alter EAAD-induced feeding inhibition (Hao, 2010), supporting the notion that GCN2, but not TOR signaling, is the sensor for EAAD response. How independent these pathways are is still an open question. Work in yeast suggests that TOR acts upstream of GCN2 (Cherkasova, 2003; Kubota, 2003; Staschke, 2010; Valbuena, 2012). Such functional epistasis has not been established in metazoan cells, and the present data suggest that the two pathways operate independently in vivo (Bjordal, 2014).

Not all DA neurons are activated by EAAD. Using live imaging, this study repeatedly observed that the DM1 and DL1 cluster, but not the DL2 cluster, are activated by EAAD. Interestingly, this cluster was recently implicated in olfactory reward-driven feeding (Wang, 2013), indicating subfunctionality among different dopamine circuits. Using subtraction analysis, it was possible to show that only three neurons in the DL1 cluster are responsible for EAAD-induced food avoidance. Nevertheless, additional DA cells are activated by EAAD, suggesting that they could contribute to other EAAD-induced behaviors. Indeed, EAAD induces long-term effects such as the development of a learned aversion to a deficient or imbalanced food and memory for the place associated with EAAD food. Hence, activation of other DA cells by EAAD may contribute to these additional behaviors (Bjordal, 2014).

This work demonstrates a direct role of DA in nutrient sensing and food rejection in flies; however, its role in aversive learning is well established. The activity of the PPL1 cluster of DA neurons in the adult fly brain can produce aversive memory when paired with an odor (Claridge-Chang, 2009; Aso, 2010). Distinct DA neurons in the PPL1 cluster provide motivational control over memory expression, suggesting that these neurons constitute a dopaminergic circuitry that regulates the internal motivational state of hunger and satiety (Krashes, 2009). Direct lineage tracing remains to be done, but the D0 and C1 Gal4 drivers targeting the subdomains of the larval DL1 cluster also target the PPL1 cluster in the adult brain (Liu, 2012), suggesting that DL1 and PPL1 cells may be related. Therefore, DA signaling in the DL1/PPL1 cluster could act as a general satiation signal, reducing food intake and abolishing appetitive performance (Bjordal, 2014).

The dopaminergic circuitry is known for its role in the motivational control of feeding. This study has shown that it also plays a key role in the homeostatic regulation of food intake. In light of recent studies showing that metabolic hormones also exert their effect on the dopamine reward circuit, the emerging picture is that dopamine is a central player in the regulation of food intake through the integration of nutrient sensing and motivational drives (Bjordal, 2014).

4E-BP is a target of the GCN2-ATF4 pathway during Drosophila development and aging

Reduced amino acid availability attenuates mRNA translation in cells and helps to extend lifespan in model organisms. The amino acid deprivation-activated kinase GCN2 mediates this response in part by phosphorylating eIF2α. In addition, the cap-dependent translational inhibitor 4E-BP (Thor) is transcriptionally induced to extend lifespan in Drosophila melanogaster, but through an unclear mechanism. This study shows that GCN2 and its downstream transcription factor, ATF4 (Cryptocephal), mediate 4E-BP induction, and GCN2 is required for lifespan extension in response to dietary restriction of amino acids. The 4E-BP intron contains ATF4-binding sites that not only respond to stress but also show inherent ATF4 activity during normal development. Analysis of the newly synthesized proteome through metabolic labeling combined with click chemistry shows that certain stress-responsive proteins are resistant to inhibition by 4E-BP, and gcn2 mutant flies have reduced levels of stress-responsive protein synthesis. These results indicate that GCN2 and ATF4 are important regulators of 4E-BP transcription during normal development and aging (Kang, 2016).

Previous studies had established the importance of 4E-BP transcription by FOXO in several distinct biological contexts, including the regulation of cell number, metabolism, response to oxidative stress, and cardiac function. Alternative transcriptional regulatory mechanisms for 4E-BP and their biological significance have remained poorly characterized. This study shows evidence that another pathway, mediated by GCN2 and ATF4, mediates the induction of 4E-BP transcription in response to the restriction of amino acids in the diet and during the development of specific tissues. The specific data presented in this study include examination of 4E-BP protein through Western blot from starved larval extracts and examination of transcripts through quantitative PCR in cultured S2 cells, larvae, and adult tissues. A new 4E-BP intron reporter, which responds to ATF4 activation, is widely expressed in Drosophila, indicating that ATF4 is a major mediator of 4E-BP induction during normal development as well as in response to dietary restriction of amino acids (Kang, 2016).

The results also show that Drosophila gcn2 mutants have a shorter lifespan than wild-type controls when reared in food with low yeast content. These results are similar to what had been observed with mutants of C. elegans gcn2 and yeast GCN4, an ATF4 equivalent gene in that organism. The Drosophila gcn2 mutant phenotype is also similar to the reported phenotype of 4E-BP mutant flies. However, this study did not examine through double-mutant analysis whether the two genes have a strictly linear genetic relationship in regulating lifespan. Based on current understanding, the two genes do not have a strictly linear relationship: GCN2-ATF4 has other transcriptional targets that also contribute to their phenotypes, and ATF4-independent regulatory inputs into 4E-BP exist, such as those mediated by FOXO and TOR. Thus, it is speculated that the similar reported phenotypes of gcn2 and 4E-BP mutants on lifespan may be due to a broad effect of 4E-BP on other GCN2-ATF4 target gene expression, as 4E-BP's target, eIF-4E, is thought to be involved in the expression of most eukaryotic genes (Kang, 2016).

Emerging evidence indicates that 4E-BP is not indiscriminate in the inhibition of general translation. For example, ribosome profiling studies in mammalian cultured cells have found that 4E-BP1's effect on translation is highly selective, with some transcripts being highly sensitive to 4E-BP1 and others indifferent. Accordingly, it appears that 4E-BP activation would have cells shift their overall protein synthesis profile. The data in this study are consistent with that view. Specifically, it was found that BiP and other stress-responsive transcripts score positive in the Internal Ribosomal Entry Site (IRES) assay and are resistant to suppression by 4E-BP. It is noted that mammalian BiP also reportedly has an IRES element in its 5' UTR. The finding that 4E-BP is a target of the UPR helps make sense of such an observation; IRES would help transcripts evade suppression by 4E-BP, whose expression level is high in stressed cells, allowing BiP to be expressed and help resolve stress. As 4E-BP activation results in a specific biological phenotype of enhanced stress resistance and lifespan extension, it appears that the proteome shift brought on by 4E-BP favors stress-responsive gene expression (Kang, 2016).

In Drosophila, 4E-BP is widely understood as a transcriptional target of FOXO. However, the role of FOXO in mediating the effects of dietary restriction of amino acids has been disputed. The experiments presented in this paper show that the loss of foxo does not impair 4E-BP transcription, at least under conditions of amino acid restriction. Notably, a foxo mutant allele was used that is different from those used in the earlier studies on 4E-BP. Although the earlier studies had used foxo21/25 alleles with premature stop codons, recent studies indicate that full-length FOXO protein is still expressed in the foxo25 mutants. Thus, it is possible that these alleles have neomorphic properties that may have led to results different from the current work. On the other hand, the foxo mutant allele used in this study has been validated to be a null allele. The negative result with the foxo mutant is mostly related to the amino acid deprivation response and does not contradict FOXO's known role in the induction of 4E-BP in other contexts (Kang, 2016).

In regards to the cellular response to amino acid deprivation, much focus had been placed on the TOR signaling pathway. It is interesting that the other amino acid-response pathway mediated by GCN2 leads to the transcriptional regulation of this TOR phosphorylation substrate. The current observation suggests that the two amino acid-responsive pathways work cooperatively (Kang, 2016).

Adipocyte amino acid sensing controls adult germline stem cell number via the amino acid response pathway and independently of Target of Rapamycin signaling in Drosophila

How adipocytes contribute to the physiological control of stem cells is a critical question towards understanding the link between obesity and multiple diseases, including cancers. Previous studies have revealed that adult stem cells are influenced by whole-body physiology through multiple diet-dependent factors. For example, nutrient-dependent pathways acting within the Drosophila ovary control the number and proliferation of germline stem cells (GSCs). The potential role of nutrient sensing by adipocytes in modulating stem cells in other organs, however, remains largely unexplored. This study report that amino acid sensing by adult adipocytes specifically modulates the maintenance of GSCs through a Target of Rapamycin-independent mechanism. Instead, reduced amino acid levels and the consequent increase in uncoupled tRNAs trigger activation of the GCN2-dependent amino acid response pathway within adipocytes, causing increased rates of GSC loss. These studies reveal a new step in adipocyte-stem cell crosstalk (Armstrong, 2014).

Drosophila is an ideal model for molecular physiology studies, owing to the ease of cell/tissue-specific manipulations, which are essential to dissect how individual systemic signaling events contribute to complex physiological networks. Indeed, recent years have seen an explosion in metabolism and physiology studies using Drosophila. Particularly useful throughout these studies is the UAS/Gal4 system, which allows tissue- and/or cell-type-specific genetic manipulations; however, a crucial consideration when designing such studies is the specificity of Gal4 expression to avoid misinterpretation of phenotypes. Indeed, most of the published fat body drivers tested were not expressed exclusively in adipocytes in adult females. By contrast, the robust and highly specific expression of 3.1Lsp2-Gal4 in adipocytes makes it a valuable tool for exclusive genetic manipulation of adipocytes to test how they impact not only GSCs, but also other adult stem cell types (Armstrong, 2014).

In addition to adipocytes, nutrient sensing by other tissues also affects GSCs. For example, insulin-like peptides secreted from the brain act directly on the germline to modulate GSC proliferation, cyst growth and vitellogenesis, and also indirectly affect GSC maintenance through effects on the nich. Other adult stem cell types are also modulated by insulin signaling, including male GSCs and intestinal stem cells. Much remains unknown, however, about how other tissues influence stem cells, despite evidence suggesting endocrine roles for muscle, intestines and the brain (Armstrong, 2014).

The findings of this study that amino acid sensing by adipocytes controls GSC maintenance through the amino acid response (AAR) pathway and ovulation through TOR clearly illustrate the high degree of specificity of adipocyte-to-ovary communication. The results also imply that relatively small fluctuations in amino acid levels (e.g. those resulting from partial knockdown of single amino acid transporters) within adipocytes can be effectively transmitted to the ovary to modulate stem cell number. These same slight reductions in amino acid levels resulted in less significant effects on ovulation, consistent with the distinct amino acid sensing mechanisms involved. It will be very interesting to identify and study the effectors downstream of AAR and TOR signaling that mediate these distinct effects on the GSC lineage (Armstrong, 2014).

Not surprisingly, inhibition of TOR signaling impacted ovulation more severely than manipulation of single amino acid transporters, in agreement with its role downstream of transporters and as an integrator of multiple inputs, including nutrients, energy status and growth factors. It is likely that additional stimuli upstream of TOR within adipocytes also regulate ovulation (Armstrong, 2014).

The AAR pathway is evolutionarily conserved from yeast to humans; however, its downstream targets are context dependent. In yeast, for example, phosphorylation of eIF2α by activated GCN2 causes selective upregulation of translation of the transcriptional factor GCN4, which in turn induces genes involved in amino acid transport as well as in amino acid biosynthesis. Translational derepression of ATF4 (the GCN4 equivalent in Drosophila and humans), by contrast, leads to expression of oxidative stress genes in mouse embryonic fibroblasts. The targets of the AAR pathway in the context of intact multicellular organisms remain largely unidentified. Nevertheless, it is reasonable to speculate that the sets of targets regulated by the AAR pathway in different tissues and cell types may be quite different, given the diversity of processes being modulated. For example, the AAR pathway acts in the brains of Drosophila larvae, mice and rats to reduce intake of food sources that lack essential amino acids. This study demonstrates a starkly different role of the AAR pathway in adipocytes in the control of GSC numbers. A fascinating challenge for future studies will be to identify the subsets of targets activated in a cell-type, context-dependent manner, and to investigate how the specificity of this pathway is achieved from budding yeast to Drosophila adipocytes to Drosophila and rodent neurons to achieve such differing cellular outcomes. These studies raise the possibility that specific targets downstream of ATF4 induced in adipocytes signal to the ovary to control GSC number. Additional studies in different tissues and conditions will elucidate how much overlap exists of targets induced by the AAR pathway. Finally, it is also possible that activation of the AAR pathway in adipocytes in response to increased levels of unloaded tRNAs could alter signals from adipocytes to GSCs downstream of either global reduction in translation or of increased levels of ATF4 and its targets (Armstrong, 2014).

Obesity and high calorie intake are associated with increased risk of multiple cancer types, including breast, colon and prostate cancer. Similar to GSCs and other stem cells, cancers are highly responsive to nutrient-sensing pathways, and components of the insulin and TOR pathways are often misregulated in cancers. Given the parallels between cancer cells and stem cells, investigations of the role of adipocytes in adult stem cell regulation will likely provide valuable insights into the link between obesity and cancer risk. Based on these results, it is speculated that aberrant communication of the nutrient-sensing status of fat cells could modulate the activity of cancer cells and might explain the link between diet, adiposity and cancer (Armstrong, 2014).

Coordinate regulation of eIF2alpha phosphorylation by PPP1R15 and GCN2 is required during Drosophila development

Phosphorylation of eukaryotic translation initiation factor 2 alpha (eIF2alpha) by the kinase GCN2 attenuates protein synthesis during amino acid starvation in yeast, whereas in mammals a family of related eIF2alpha kinases regulate translation in response to a variety of stresses. Unlike single-celled eukaryotes, mammals also possess two specific eIF2alpha phosphatases, PPP1R15a and PPP1R15b, whose combined deletion leads to a poorly understood early embryonic lethality. This paper reports the characterisation of the first non-mammalian eIF2alpha phosphatase and the use of Drosophila to dissect its role during development. The Drosophila protein demonstrates features of both mammalian proteins, including limited sequence homology and association with the endoplasmic reticulum. Of note, although this protein is not transcriptionally regulated, its expression is controlled by the presence of upstream open reading frames in its 5'UTR, enabling induction in response to eIF2alpha phosphorylation. Moreover, its expression was shown to be necessary for embryonic and larval development, and this serves to oppose the inhibitory effects of GCN2 on anabolic growth (Malzer, 2013).

Dephosphorylation of eIF2α by PPP1R15 in mammals is important in the response to many forms of cellular stress, in haematopoiesis and in mammalian embryonic development. However, studies of its role in development have been hampered by functional redundancy between the two mammalian isoforms and by the severity of the double knockout phenotype. Moreover, until now, no invertebrate PPP1R15 had been identified to enable its study during development and, by extension, a better understanding of its evolution. This study has determined that dPPP1R15 (formerly CG3825) shares sequence homology with both mammalian PPP1R15 proteins, has a similar localisation at the ER and functionally antagonises the eIF2α kinases both in vivo and in cultured cells. Its tight ER association and lack of significant transcriptional regulation both suggest that it shares more functional homology with the PPP1R15b isoform than PPP1R15a (Malzer, 2013).

The presence of regulatory uORFs upstream of the dPPP1R15 start codon is reminiscent of both PPP1R15a and PPP1R15b. It is noteworthy that the 5′UTR found within the mRNA of both mouse and human PPP1R15a contain two uORFs (Lee, 2009). In each case, uORF2 is necessary for translational regulation of the true coding sequence, while uORF1 appears to play a less important role. This contrasts with the mRNA of mammalian ATF4 in which uORF1 is required for the regulatory function since its translation renders the scanning ribosome depleted of active eIF2, thus reducing the likelihood of translating the inhibitory uORF2. The current findings suggest that the regulation of dPPP1R15 shares a uORF2-dominant mechanism with mammalian PPP1R15a. Loss of uORF1 from the 5′UTR of dPPP1R15 had little effect on translation of the true coding sequence following treatment with tunicamycin, while loss of uORF2 markedly reduced translation during ER stress. The mRNA of human PPP1R15b also contains two non-overlapping uORFs but it remains unclear whether these play a regulatory function (Malzer, 2013).

Mice lacking both Ppp1r15 genes and Drosophila depleted of dPPP1R15 by RNAi driven by tubulin-Gal4 or e22c-Gal4 (this study) show markedly impaired embryonic development. Use of the weaker ubiquitous driver daughterless-Gal4 allowed for a less dramatic depletion of dPPP1R15 enabling a study of the role of eIF2α phosphorylation at later stages of development, which revealed a genetic interaction between dPPP1R15 and the nutrient sensing kinase GCN2. The biphasic effects of genetic manipulation of eIF2α phosphorylation, with toxicity caused by the loss of the kinase GCN2 or the phosphatase dPPP1R15 is striking. While lack of dPPP1R15 may lead to excessive phosphorylation of eIF2α and thus a global impairment of protein synthesis, it is less clear why loss of the kinase GCN2 under conditions of adequate nutrient availability should impair development. A link between dysregulated eIF2α phosphorylation and impaired larval growth has been shown previously by the overexpression of a mutant form of eIF2α in which the phosphorylated serine was mutated to alanine (phosphorylation resistant) or aspartic acid (phosphomimetic). The phosphorylation resistant animals grew faster and larger. In contrast, the phosphomimetic mutants experienced developmental delay. This is consistent with the observation of impaired growth of animals depleted of PPP1R15 by RNAi in which eIF2α is likely to remain phosphorylated for longer periods. The defects of growth and molting have also been seen with mutations of the DmATF4 gene, cryptocephal. This transcription factor is regulated in many organisms by a translational mechanism similar to that observed for dPPP1R15. Since loss of GCN2 will be accompanied by reduction of eIF2α phosphorylation it will also have lower ATF4 levels mimicking loss of cryptocephal. Thus, either excessive or deficient phosphorylation of eIF2α is likely to result in impaired larval development (Malzer, 2013).

A previous genetic screen for modifiers of growth in Drosophila identified slimfast, an amino acid transporter. Remarkably, when slimfast was depleted selectively in the fatbody, animals suffered a global defect in growth. It is known that the TOR pathway is involved in the balancing protein synthesis with nutrient supply via ribosomal protein S6 kinase and eIF4E-BP and much of the effect of amino acid starvation has been attributed to these pathways. However, this study suggests that GCN2 may also participate. Moreover, there is evidence for cross talk between the TOR and GCN2 pathways (Cherkasova, 2003). Further work is required to determine if such cross talk might play a role in nutrient sensing by the fat body (Malzer, 2013).

While the interactions between dPPP1R15 and its cognate kinases during larval development are readily explained by a simple antagonistic relationship, the apparent synthetic lethal interaction between dPPP1R15 and dGCN2 knockdown during embryogenesis is curious. One might speculate that during embryogenesis the phosphorylation of eIF2α by dGCN2 may serve to promote translation of dPPP1R15. If partial knockdown of dPPP1R15 (by daughterless-Gal4 driven RNAi) is compensated for during embryogenesis by enhanced translation of the residual dPPP1R15 mRNA in response to phosphorylation of eIF2α by GCN2, then reduction in the level of dGCN2 may paradoxically reduce the level of dPPP1R15 still further. If dPPP1R15 protein is required at a late stage of embryogenesis, for example to limit the effects of dPERK signaling triggered by the development of secretory tissues, then the earlier failure to translate dPPP1R15 might lead to failure to eclose (Malzer, 2013).

This study suggests that coordinate regulation of eIF2α phosphorylation during larval development is important in determining the rate of anabolic growth and, in turn, successful completion of pupariation. This study has characterised the first example of an invertebrate eIF2α phosphatase and demonstrated that during development it primarily antagonises the function of GCN2. Further studies will address the mechanism by which alteration of eIF2α phosphorylation selectively within the fat body can impair global tissue growth (Malzer, 2013).

Infection-induced host translational blockage inhibits immune responses and epithelial renewal in the Drosophila gut

Typically, immune responses control the pathogen, while repair and stress pathways limit damage caused by pathogenesis. The relative contribution of damage to the outcome of pathogenesis and the mechanistic links between the immune and repair pathways are poorly understood. This study analyzed how the entomopathogenic bacterium Pseudomonas entomophila induces irreversible damage to the Drosophila gut. P. entomophila ingestion was found to induce a global translational blockage that impairs both immune and repair programs in the fly gut. P. entomophila-induced translational inhibition is dependent on bacterial pore forming toxins and reactive oxygen species produced by the host in response to infection. Translational arrest is mediated through activation of the GCN2 kinase and inhibition of the TOR pathway as a consequence of host damage. Together, this study study draws a model of pathogenesis in which bacterial inhibition of translation by excessive activation of stress responsive pathways inhibits both immune and regenerative epithelial responses (Charkrabarti, 2012).

One established pathway leading to arrest of cap-dependent protein synthesis is the phosphorylation of the eukaryotic initiation factor 2α (eIF2α). Under resting conditions, eIF2α is not phosphorylated and is part of the complex that recruits the initiator Methionine-tRNA (transfer RNA)to the start codon. When phosphorylated, however, it acts as an inhibitor of general translation. To elucidate the mechanisms underlying translation inhibition by P. entomophila, this study analyzed the status of eIF2α phosphorylation in guts of flies after ingestion of Ecc15 (a bacterial species that induces a systemic immune response in Drosophila) or P. entomophila. Consistent with a general inhibition of translation, western blot analysis showed that eIF2α is phosphorylated in gut extracts collected after P. entomophila, but not after Ecc15 infection. In mammals, a family of kinases (PKR, GCN2, PERK, HRI) that respond to starvation or stresses induce eIF2α phosphorylation. Two of them, GCN2 (general control nonrepressed 2) and PERK (PKR-like endoplasmic reticulum kinase) are conserved in Drosophila. GCN2 is mainly activated by the accumulation of uncharged tRNAs after nutrient starvation, while PERK is activated when unfolded proteins accumulate in the endoplasmic reticulum (Wek, 2007). Using the Dpt-lacZ enzymatic activity/Dpt-lacZ transcript ratio as readout of P. entomophila translation inhibition, the implication of these two kinases in P. entomophila-mediated blockage of translation was tested. The Dpt-lacZ activity/transcript ratio upon P. entomophila infection was similar in guts of flies deficient for PERK or the wild-type. In contrast, inactivation of GCN2 in the gut by RNA interference (RNAi) restored the levels of Dpt-lacZ activity. Similarly, the level of global translation as measured by AHA incorporation was higher in GCN2 RNAi guts compared to the wild-type, upon P. entomophila infection. It is concluded that phosphorylation of eIF2α by GCN2 is involved in the bulk arrest of protein synthesis upon P. entomophila infection (Charkrabarti, 2012).

To further elucidate the mechanism underlying gut translation inhibition by P. entomophila, the ability of the bacteria to modulate the activity of the translational repressor 4E-BP1, another key regulator of translation, was examined. 4E-BP1 is a target of the TOR kinase that alleviates its inhibitory activity through its phosphorylation. Under positive-growth conditions, TOR is active and maintains 4E-BP1 in its phosphorylated state, rendering 4E-BP1 incapable of inhibiting translation. However, under nutritional and environmental stress conditions, TOR is inactive, and 4E-BP1 becomes hypophosphorylated and inhibits cap-dependent translation. At 16 hr postinfection, P. entomophila caused a strong reduction in 4E-BP1 phosphorylation, while the total amount of 4E-BP1 remained unaffected. This suggested that P. entomophila infection also inhibits translation through 4E-BP1. Therefore, it was hypothesized that P. entomophila infection could inhibit TOR activity and thereby reduce protein synthesis. In animals, the Tuberous sclerosis protein complex (Tsc1/2) is a negative regulator of TOR kinase activity. Interestingly, knockdown by RNAi of the Tsc2 gene restored the Dpt-lacZ activity in guts infected by P. entomophila. Consistent with this observation, knockdown of TSC by RNAi also increased the amount of phosphorylated 4E-BP1. This increase was not only detected in guts infected with P. entomophila, but also in both unchallenged and Ecc15-infected intestines. Conversely, the knockdown of TOR by RNAi was sufficient to block translation of the Dpt-lacZ reporter upon infection with the nonlethal bacterium Ecc15 (Charkrabarti, 2012).

The inhibition of the TOR pathway by the TSC complex is determined by several inputs, the main two being the activation of the AMP kinase (AMPK) that senses low intracellular ATP levels and the decrease of insulin receptor signaling in response to a decrease in systemic growth signals. This dual regulation ensures an optimal coordination between translation and nutrient/energy availability. Therefore, which of the two branches mediates the TSC inhibition of TOR upon P. entomophila infection was tested. No inhibition of translation by Ecc15 was observed in flies deficient for chico that encodes an insulin receptor (InR) adaptor protein, or in flies expressing a dominant negative form of InR in the gut. Additionally, P. entomophila was still able to block translation in the gut of flies expressing a constitutively active form of insulin receptor. Finally, no change in expression of insulin-like peptide genes was detected in flies that ingested P. entomophila. These experiments indicate that the insulin receptor pathway is not involved in P. entomophila repression of host translation. Conversely, inhibition of translation by P. entomophila was less marked in flies lacking one copy of ampkα. Silencing of the ampkα gene by RNAi in the midgut also partially restored Dpt-lacZ activity in P. entomophila infected flies. Collectively, these results show that at least two mechanisms, eIF2α phosphorylation through GCN2 activation and 4E-BP hypophosphorylation through AMPK-TSC inhibition of TOR activity, repress host translation after P. entomophila infection (Charkrabarti, 2012).

Association of GCN1-GCN20 regulatory complex with the N-terminus of eIF2alpha kinase GCN2 is required for GCN2 activation

Stimulation of GCN4 mRNA translation due to phosphorylation of the alpha-subunit of initiation factor 2 (eIF2) by its specific kinase, GCN2, requires binding of uncharged tRNA to a histidyl-tRNA synthetase (HisRS)-like domain in GCN2. GCN2 function in vivo also requires GCN1 and GCN20, but it was unknown whether these latter proteins act directly to promote the stimulation of GCN2 by uncharged tRNA. This study found that the GCN1-GCN20 complex physically interacts with GCN2, binding to the N-terminus of the protein. Overexpression of N-terminal GCN2 segments had a dominant-negative phenotype that correlated with their ability to interact with GCN1-GCN20 and impede association between GCN1 and native GCN2. Consistently, this Gcn(-) phenotype was suppressed by overexpressing GCN2, GCN1-GCN20 or tRNA(His). The requirement for GCN1 was also reduced by overexpressing tRNA(His) in a gcn1Delta strain. It is concluded that binding of GCN1-GCN20 to GCN2 is required for its activation by uncharged tRNA. The homologous N-terminus of Drosophila GCN2 interacted with yeast GCN1-GCN20 and had a dominant Gcn(-) phenotype, suggesting evolutionary conservation of this interaction (Garcia-Barrio, 2000).

Isolation of the gene encoding the Drosophila melanogaster homolog of the Saccharomyces cerevisiae GCN2 eIF-2α kinase

Genomic and cDNA clones homologous to the yeast GCN2 eIF-2α kinase (yGCN2) were isolated from Drosophila melanogaster. The identity of the Drosophila GCN2 (dGCN2) gene is supported by the unique combination of sequence encoding a protein kinase catalytic domain and a domain homologous to histidyl-tRNA synthetase and by the ability of dGCN2 to complement a deletion mutant of the yeast GCN2 gene. Complementation of Δgcn2 in yeast by dGCN2 depends on the presence of the critical regulatory phosphorylation site (serine 51) of eIF-2α. dGCN2 is composed of 10 exons encoding a protein of 1589 amino acids. dGCN2 mRNA is expressed throughout Drosophila development and is particularly abundant at the earliest stages of embryogenesis. The dGCN2 gene was cytogenetically and physically mapped to the right arm of the third chromosome at 100C3 in STS Dm2514. The discovery of GCN2 in higher eukaryotes is somewhat unexpected given the marked differences between the amino acid biosynthetic pathways of yeast vs. Drosophila and other higher eukaryotes. Despite these differences, the presence of GCN2 in Drosophila suggests at least partial conservation from yeast to multicellular organisms of the mechanisms responding to amino acid deprivation (Olsen, 1998).

Cloning and characterization of a cDNA encoding a protein synthesis initiation factor-2α (eIF-2α) kinase from Drosophila melanogaster

Phosphorylation of the alpha subunit of the eukaryotic initiation factor 2 (eIF-2α) is one of the best-characterized mechanisms for downregulating protein synthesis in mammalian cells in response to various stress conditions. In Drosophila, such a regulatory mechanism has not been elucidated. This study reports the molecular cloning and characterization of DGCN2, a Drosophila eIF-2α kinase related to yeast GCN2 protein kinase. DGCN2 contains all of the 12 catalytic subdomains characteristic of eukaryotic Ser/Thr protein kinases and the conserved sequence of eIF-2α kinases in subdomain V. A large insert of 94 amino acids, which is characteristic of eIF-2α kinases, is also present between subdomains IV and V. It is particularly notable that DGCN2 possesses an amino acid sequence related to class II aminoacyl-tRNA synthetases, a unique feature of yeast GCN2 protein kinase. DGCN2 expression is developmentally regulated. During embryogenesis, DGCN2 mRNA is dynamically expressed in several tissues. Interestingly, at later stages this expression becomes restricted to a few cells of the central nervous system. Affinity-purified antibodies, raised against a synthetic peptide based on the predicted DGCN2 sequence, specifically immunoprecipitated an eIF-2α kinase activity and recognized an approximately 175 kDa phosphoprotein in Western blots of Drosophila embryo extracts (Santoyo, 1997).


REFERENCES

Search PubMed for articles about Drosophila Gcn2

Armstrong, A. R., Laws, K. M. and Drummond-Barbosa, D. (2014). Adipocyte amino acid sensing controls adult germline stem cell number via the amino acid response pathway and independently of Target of Rapamycin signaling in Drosophila. Development 141: 4479-4488. PubMed ID: 25359724

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Chaveroux, C., Lambert-Langlais, S., Cherasse, Y., Averous, J., Parry, L., Carraro, V., Jousse, C., Maurin, A. C., Bruhat, A. and Fafournoux, P. (2010). Molecular mechanisms involved in the adaptation to amino acid limitation in mammals. Biochimie 92: 736-745. PubMed ID: 20188139

Cherkasova, V. A. and Hinnebusch, A. G. (2003). Translational control by TOR and TAP42 through dephosphorylation of eIF2alpha kinase GCN2. Genes Dev 17: 859-872. PubMed ID: 12654728

Claridge-Chang, A., Roorda, R. D., Vrontou, E., Sjulson, L., Li, H., Hirsh, J. and Miesenbock, G. (2009). Writing memories with light-addressable reinforcement circuitry. Cell 139: 405-415. PubMed ID: 19837039

Colombani, J., Raisin, S., Pantalacci, S., Radimerski, T., Montagne, J. and Leopold, P. (2003). A nutrient sensor mechanism controls Drosophila growth. Cell 114: 739-749. PubMed ID: 14505573

Dong, J., Qiu, H., Garcia-Barrio, M., Anderson, J. and Hinnebusch, A. G. (2000). Uncharged tRNA activates GCN2 by displacing the protein kinase moiety from a bipartite tRNA-binding domain. Mol Cell 6: 269-279. PubMed ID: 10983975

Hao, S., Sharp, J. W., Ross-Inta, C. M., McDaniel, B. J., Anthony, T. G., Wek, R. C., Cavener, D. R., McGrath, B. C., Rudell, J. B., Koehnle, T. J. and Gietzen, D. W. (2005). Uncharged tRNA and sensing of amino acid deficiency in mammalian piriform cortex. Science 307: 1776-1778. PubMed ID: 15774759

Hao, S., Ross-Inta, C. M. and Gietzen, D. W. (2010). The sensing of essential amino acid deficiency in the anterior piriform cortex, that requires the uncharged tRNA/GCN2 pathway, is sensitive to wortmannin but not rapamycin. Pharmacol Biochem Behav 94: 333-340. PubMed ID: 19800362

Garcia-Barrio, M., Dong, J., Ufano, S. and Hinnebusch, A. G. (2000). Association of GCN1-GCN20 regulatory complex with the N-terminus of eIF2alpha kinase GCN2 is required for GCN2 activation. EMBO J 19: 1887-1899. PubMed ID: 10775272

Kang, M. J., Vasudevan, D., Kang, K., Kim, K., Park, J. E., Zhang, N., Zeng, X., Neubert, T. A., Marr, M. T., and Don Ryoo, H. (2016). 4E-BP is a target of the GCN2-ATF4 pathway during Drosophila development and aging. J Cell Biol 216(1):115-129. PubMed ID: 27979906

Krashes, M. J., DasGupta, S., Vreede, A., White, B., Armstrong, J. D. and Waddell, S. (2009). A neural circuit mechanism integrating motivational state with memory expression in Drosophila. Cell 139: 416-427. PubMed ID: 19837040

Kubota, H., Obata, T., Ota, K., Sasaki, T. and Ito, T. (2003). Rapamycin-induced translational derepression of GCN4 mRNA involves a novel mechanism for activation of the eIF2 alpha kinase GCN2. J Biol Chem 278: 20457-20460. PubMed ID: 12676950

Layalle, S., Arquier, N. and Leopold, P. (2008). The TOR pathway couples nutrition and developmental timing in Drosophila. Dev Cell 15: 568-577. PubMed ID: 18854141

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Liu, Q., Liu, S., Kodama, L., Driscoll, M. R. and Wu, M. N. (2012). Two dopaminergic neurons signal to the dorsal fan-shaped body to promote wakefulness in Drosophila. Curr Biol 22: 2114-2123. PubMed ID: 23022067

Malzer, E., Szajewska-Skuta, M., Dalton, L. E., Thomas, S. E., Hu, N., Skaer, H., Lomas, D. A., Crowther, D. C. and Marciniak, S. J. (2013). Coordinate regulation of eIF2alpha phosphorylation by PPP1R15 and GCN2 is required during Drosophila development. J Cell Sci 126: 1406-1415. PubMed ID: 23418347

Maurin, A. C., Jousse, C., Averous, J., Parry, L., Bruhat, A., Cherasse, Y., Zeng, H., Zhang, Y., Harding, H. P., Ron, D. and Fafournoux, P. (2005). The GCN2 kinase biases feeding behavior to maintain amino acid homeostasis in omnivores. Cell Metab 1: 273-277. PubMed ID: 16054071

Olsen, D. S., Jordan, B., Chen, D., Wek, R. C. and Cavener, D. R. (1998). Isolation of the gene encoding the Drosophila melanogaster homolog of the Saccharomyces cerevisiae GCN2 eIF-2α kinase. Genetics 149: 1495-1509. PubMed ID: 9649537

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Santoyo, J., Alcalde, J., Mendez, R., Pulido, D. and de Haro, C. (1997). Cloning and characterization of a cDNA encoding a protein synthesis initiation factor-2α (eIF-2α) kinase from Drosophila melanogaster. Homology To yeast GCN2 protein kinase. J Biol Chem 272: 12544-12550. PubMed ID: 9139706

Staschke, K. A., Dey, S., Zaborske, J. M., Palam, L. R., McClintick, J. N., Pan, T., Edenberg, H. J. and Wek, R. C. (2010). Integration of general amino acid control and target of rapamycin (TOR) regulatory pathways in nitrogen assimilation in yeast. J Biol Chem 285: 16893-16911. PubMed ID: 20233714

Toshima, N. and Tanimura, T. (2012). Taste preference for amino acids is dependent on internal nutritional state in Drosophila melanogaster. J Exp Biol 215: 2827-2832. PubMed ID: 22837455

Truong, B. G., Magrum, L. J. and Gietzen, D. W. (2002). GABA(A) and GABA(B) receptors in the anterior piriform cortex modulate feeding in rats. Brain Res 924: 1-9. PubMed ID: 11743989

Valbuena, N., Rozalen, A. E. and Moreno, S. (2012). Fission yeast TORC1 prevents eIF2alpha phosphorylation in response to nitrogen and amino acids via Gcn2 kinase. J Cell Sci 125: 5955-5959. PubMed ID: 23108671

Vargas, M. A., Luo, N., Yamaguchi, A. and Kapahi, P. (2010). A role for S6 kinase and serotonin in postmating dietary switch and balance of nutrients in D. melanogaster. Curr Biol 20: 1006-1011. PubMed ID: 20471266

Wang, Y., Pu, Y. and Shen, P. (2013). Neuropeptide-gated perception of appetitive olfactory inputs in Drosophila larvae. Cell Rep 3: 820-830. PubMed ID: 23453968

Wek, R. C., Jackson, B. M. and Hinnebusch, A. G. (1989). Juxtaposition of domains homologous to protein kinases and histidyl-tRNA synthetases in GCN2 protein suggests a mechanism for coupling GCN4 expression to amino acid availability. Proc Natl Acad Sci U S A 86: 4579-4583. PubMed ID: 2660141

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