An important question with regard to the possibilities of Thor induction being involved in cell growth regulation is whether or not eIF4E also is induced by infection. In mammalian cells, overexpression of eIF4E is associated with malignant transformation, and concomitant overexpression of 4E-BP has been shown to negate this overgrowth (for review see Clemens, 1999). In the Drosophila immune response, large quantities of antimicrobial proteins are produced and one possibility is that eIF4E could be up-regulated to increase translation. In this scenario, Thor up-regulation would have the homeostatic function of producing more 4E-BP to keep growth regulation in balance. The hypothesis that eIF4E also is up-regulated by bacterial infection was tested. This is not the case; Thor is up-regulated upon infection while the levels of eIF4E mRNA remain the same. Therefore, if these two components are coordinately regulated, it is not at the level of transcription (Bernal, 2000).
All of the promoter regions studied for Drosophila antimicrobial genes induced by infection have been found to have elements similar to those of immune inducible genes in mammals. Sequence analysis of the 5'-flanking region has determined that Thor has an array of these types of elements. The Thor promoter has the canonical NFkappaB recognition sequence that has been shown to be essential for immune induction. Furthermore, Thor has the GATA sequence associated with NFkappaB elements that has been also shown to be important for immune induction and conserved in other Drosophila species. Additional sequences found upstream of Drosophila immune response genes have been also identified, in particular those involved in vertebrate cytokine regulation and liver specific expression. TRANSFAC analysis has identified more sequences related to liver-specific regulation, such as hepatocyte nuclear factor/forkhead, and also interferon-related regulatory sequences (Bernal, 2000).
Foxo regulates cell cycle arrest possibly by transcriptionally activating genes implicated in cell division or in cell growth. As an initial attempt to identify target genes of Foxo, DNA microarrays were used to assess gene expression profiles in S2 cells stably transfected with mutant Foxo and grown in the presence of insulin. Cells expressing wild-type Foxo or untransfected S2 cells subjected to the same treatment were assayed as controls (Puig, 2003).
Two-hundered and seventy-seven genes were found to be up-regulated in Foxoa3-expressing cells when compared with Foxo-expressing cells or untransfected S2 cells. Interestingly, two genes that were consistently and specifically up-regulated in these conditions were the Drosophlia InR gene (13.5-fold) and the Drosophila 4EBP gene (25-fold). Both genes have been implicated in the regulation of cell growth by insulin. To confirm that InR and 4EBP are bona fide transcriptional targets of Foxo, the same experiment described above was performed but in the presence of cycloheximide to inhibit translation. As expected, both InR and 4EBP continue to be transcriptionally activated (2.5- and 3.1-fold, respectively) by FOXOA3 but not Foxo in the insulin-repressed state. This result suggests that Foxo, when released from control by the insulin/dAkt cascade, is involved in transcription from the InR and 4EBP promoters (Puig, 2003).
To confirm these microarray results and to independently quantitate the increase in mRNA transcription, RNase protection assays were performed with mRNAs extracted from cells stably transfected with either Foxo or FoxoA3. Indeed, FoxoA3 stimulates transcription of Drosophila 4EBP and InR by 16.3- and 11-fold, respectively. A time-course experiment confirmed that Drosophila InR mRNA increases rapidly upon FoxoA3 expression: 3 h after CuSO4 addition, there is already an 8-fold increase, reaching 20-fold after 9 h of CuSO4 induction. Similar results were obtained for Drosophila 4EBP. These experiments suggest that Foxo expression specifically activates both Drosophila InR and 4EBP transcription, thus unmasking an important feedback control mechanism in this pathway involving Foxo and InR (Puig, 2003).
Having obtained evidence that exogenously transfected Foxo responds to insulin and regulates both the downstream target gene 4EBP and the feedback control target InR, it was of interest to know if endogenous Foxo would also activate transcription of these genes. The PI3K inhibitor LY294002 was used to activate endogenous Foxo or insulin to deactivate it. S2 cells grown in the absence of serum for 48 h were treated either with LY294002 or insulin. Total RNA was extracted and RNase protection was performed to detect Drosophila InR and 4EBP mRNAs. Both mRNA levels are significantly increased after LY294002 treatment (5.3-fold for dInR and 4-fold for d4EBP) when compared with insulin treatment. This result provides further evidence indicating that the PI3K–Akt pathway regulates InR and 4EBP transcription via Foxo (Puig, 2003).
It was of interest to determine whether Foxo directly binds to the promoters of Drosophila 4EBP and InR. To identify the DNA region recognized by Foxo in these two promoters, a 1708-bp fragment of the 4EBP promoter and a 1562-bp fragment of the InR promoter were inserted into a luciferase reporter vector. When transfected into S2 cells, these fragments responded to Foxo activation (3-fold for 4EBP, >200-fold for InR. A series of deletions lacking upstream sequences still responded to Foxo activation, albeit more weakly, suggesting that Foxo can bind the DNA in a region close to the start of transcription (485 bp for the d4EBP promoter and 194 bp for the dInR promoter). In contrast, Foxo completely fails to activate a reporter construct in which upstream activating sequences (UAS) for the transcription factor GAL4 are fused to the luciferase gene, confirming that transcription activation is specific for both 4EBP and InR promoters (Puig, 2003).
Interestingly, 125 bp upstream of the transcription start site of the d4EBP promoter there are three tandem copies of a putative FOXO4 recognition element (FRE). These elements are reminiscent of the ones present in the human glucose-6-phosphatase promoter, previously shown to bind FOXO4 (Yang, 2002). This was reassuring because Foxo and FOXO4 share 85% identity in the core of the forkhead DNA-binding domain. Similarly, several putative FRE sequences appear in the InR promoter in the region comprising nucleotides -1434 to -70 (Puig, 2003).
To determine whether Foxo binds these putative FREs, band shift experiments were performed with a 113-bp DNA probe encompassing the 4EBP FRE motifs and with 12 separate DNA probes (ranging from 100 to 150 bp) spanning a region of 1.4 kb from the InR promoter. Purified recombinant Foxo expressed in Escherichia coli efficiently binds the 113-bp FRE-containing fragment from the 4EBP promoter compared with control DNA fragments. Furthermore, Foxo binding to the 4EBP promoter fragment can be efficiently competed with an unlabeled 113-bp 4EBP promoter fragment but not with nonspecific DNA. Similarly, purified recombinant Foxo binds efficiently to 5 out of 12 of the DNA fragments located within the InR promoter. As expected, each of the five DNA fragments bound by Foxo contains putative FREs. Thus, Foxo can specifically bind to both promoters in vitro. To determine whether Foxo also binds these same DNA regions in vivo, chromatin immunoprecipitation (ChIP) experiments were performed with S2 cells expressing either Foxo or dFoxoA3. Cells were incubated with serum, and Foxo expression was induced with the addition of CuSO4. After 6 h, cells were cross-linked with formaldehyde, and extracts were prepared and immunoprecipitated. After reversal of cross-links, DNA was recovered, and PCR was performed with primers encompassing regions containing putative FREs in both promoters. The results indicate that Foxo can directly bind to both the 4EBP and InR promoters in vivo. These results establish that Foxo can specifically bind the 4EBP and InR promoters both in vitro and in vivo (Puig, 2003).
To demonstrate that Foxo can directly activate transcription of these promoters in vitro, the constructs were used that contain 485 bp of the 4EBP promoter region and 514 bp of the InR promoter region, respectively. Addition of purified recombinant Foxo to in vitro reactions activates transcription of these promoters by at least 3-fold (4EBP) and 5.5-fold (InR), which is comparable to the activation observed in vivo. Under in vitro transcription conditions, activation of the 4EBP promoter by Foxo becomes rapidly saturated with increasing amounts of Foxo. As expected, Foxo also activates (up to sixfold) a synthetic promoter bearing four FOXO4-binding sites placed upstream of the alcohol dehydrogenase distal promoter. Together these results show that transcription of 4EBP and InR can be directly activated by Foxo in vitro (Puig, 2003).
Drosophila embryonic Kc167 cells respond to insulin stimulation with upregulated activities of PKB and S6K. mRNA profiling experiments were performed using the Affymetrix GeneChip system to measure on a genome-wide scale the transcriptional changes induced by insulin in these cells. On the basis of the currently held model that FOXO transcription factors are transcriptional activators that are negatively regulated by insulin, potential Foxo target genes were expected to be repressed in Kc167 cells upon insulin stimulation. Foxo target gene candidates were selected that are transcriptionally downregulated by a factor of two or more upon insulin stimulation and whose promoter regions contain one or more conserved forkhead-response elements (FHREs) with the consensus sequence (G/A)TAAACAA. Three of these candidate gene products are each involved in one of two biological processes known to be negatively regulated by insulin, namely gluconeogenesis (PEPCK) and lipid catabolism (CPTI and long-chain-fatty-acid-CoA-ligase). The remaining candidates are involved in stress responses (cytochrome P450 enzymes), DNA repair (DNA polymerase iota), transcription and translation control (4E-BP and CDK8), and cell-cycle control (centaurin gamma and CG3799). Several of the insulin-repressed genes have been reported to be transcriptionally induced in Drosophila larvae under conditions of complete starvation (4E-BP and PEPCK) or sugar-only diet (CPTI and long-chain-fatty-acid-CoA-ligase) (Jünger, 2003).
4E-BP was chosen for further investigation, because it has previously been reported to be insulin-regulated at the level of protein phosphorylation, but not at the level of gene expression. The 4E-BP gene encodes a translational repressor and was initially identified as the immune-compromised Thor mutant in a genetic screen for genes involved in the innate immune response to bacterial infection. There are several FHREs in the genomic region around the 4E-BP locus. The 4E-BP protein is negatively regulated by insulin through LY294002- and rapamycin-sensitive phosphorylation, suggesting involvement of the Dp110 and TOR signaling pathways. Phosphorylation of 4E-BP leads to the dissociation of 4E-BP from its binding partner, the translation initiation factor eIF4E, which then participates in the formation of a functional initiation complex. Positive transcriptional regulation of 4E-BP by Foxo, which corresponds to negative transcriptional regulation by insulin, would be a complementary mechanism of regulation (Jünger, 2003).
Whether overexpression of endogenous foxo can induce transcriptional upregulation of the 4E-BP gene was investigated. On the basis of overexpression results, the Dp110DN-Foxo coexpression was used to efficiently activate Foxo. Eye imaginal discs from Dp110DN-expressing third instar larvae display a low level of basal 4E-BP transcription throughout the disc, which is not induced by the driver construct alone. Coexpression of foxo elicits a dramatic upregulation of 4E-BP transcription posterior to the morphogenetic furrow. Consistent with this observation, it was possible to induce expression of the 4E-BP enhancer trap line Thor1 with human FOXO3a-TM . It remains unclear, however, whether regulation of d4E-BP expression by Foxo is of physiological relevance (Jünger, 2003).
Overexpression of 4E-BP partially suppresses the PKB overexpression phenotype, but since ectopic expression experiments have to be interpreted with some caution, whether loss of 4E-BP function suppresses the cell-number reduction in insulin-signaling mutants as does loss of Foxo function was investigated. Double-mutant flies were generated for PKB and 4E-BP and it was observed that the Thor1 mutation slightly but significantly suppressed the reduced cell-number phenotype in a dose-dependent manner. The Thor1 mutation itself had no effect on ommatidial number compared to wild-type flies, so additive effects of d4E-BP and dPKB can be ruled out. These observations strongly argue that under conditions of reduced insulin-signaling activity, the Foxo-dependent reduction in cell number is in part mediated by the transcriptional upregulation of its target 4E-BP. Microarray studies in both mammalian and Drosophila cells imply that FOXO transcription factors exert their physiological functions by modulating expression of large sets of target genes (Jünger, 2003).
All animals coordinate growth and maturation to reach their final size and shape. In insects, insulin family molecules control growth and metabolism, whereas pulses of the steroid 20-hydroxyecdysone (20E) initiate major developmental transitions. 20E signaling also negatively controls animal growth rates by impeding general insulin signaling involving localization of the transcription factor dFOXO and transcription of the translation inhibitor 4E-BP. The larval fat body, equivalent to the vertebrate liver, is a key relay element for ecdysone-dependent growth inhibition. Hence, ecdysone counteracts the growth-promoting action of insulins, thus forming a humoral regulatory loop that determines organismal size (Colombani, 2005).
FOXO is thought to function as a repressor of growth that is, in turn, inhibited by insulin signaling. However, inactivating mutations in Drosophila melanogaster FOXO result in viable flies of normal size, which raises a question over the involvement of FOXO in growth regulation. Previously, a growth-suppressive role for FOXO under conditions of increased target of rapamycin (TOR) pathway activity was described. This study further characterizes this phenomenon. Tuberous sclerosis complex 1 mutations cause increased FOXO levels, resulting in elevated expression of FOXO-regulated genes, some of which are known to antagonize growth-promoting pathways. Analogous transcriptional changes are observed in mammalian cells, which implies that FOXO attenuates TOR-driven growth in diverse species (Harvey, 2008).
To investigate mechanisms by which the TOR pathway controls tissue growth, transcriptional profiles were analyzed of tissue lacking Tsc1, which leads to hyperactivation of the TOR pathway and excessive growth. Eye-antennal imaginal discs from third instar Drosophila larvae were generated that were composed almost entirely of tissue derived from one of two different genotypes: Tsc1 or wild-type isogenic control. Three biologically independent first strand cDNA samples from each genotype were hybridized to microarray chips. Expression levels of 157 genes were elevated 1.5-fold or more, whereas 211 genes were repressed 1.5-fold or more when compared with control tissue. These genes have been implicated in diverse cellular functions including metabolism, membrane transport, stress response, cell growth, and cell structure (Harvey, 2008).
Observed transcriptional changes were validated for several genes using Drosophila gene-enhancer trap lines. The UAS-Gal4 system was used to activate the TOR pathway in a specific tissue domain by driving expression of Rheb under the control of the glass multiple reporter (GMR) promoter. Induction of astray (aay) and 4E-BP (both of which were found to be elevated in Tsc1 tissue by microarray analysis) were observed in the GMR expression domain (posterior to the morphogenetic furrow) when Rheb was misexpressed but were not induced when the negative control Gal4 gene was misexpressed. QPCR was also used to confirm expression changes observed in Tsc1 tissue for charybdis (chrb), scylla (scy), phosphoenolpyruvate carboxy kinase, 4E-BP, and aay (Harvey, 2008).
Intriguingly, several gene products whose expression was elevated in Tsc1 tissue have been implicated in tissue growth controlled by the insulin and TOR pathways, including 4E-BP, Chrb, and Scy. 4E-BP is a repressor of cap-dependent translation. Upon phosphorylation by TOR, 4E-BP dissociates from eIF4e, allowing assembly of the initiation complex at the mRNA cap structure, ribosome recruitment, and subsequent translation. Scy and Chrb, and their mammalian orthologues REDD1 and REDD2, inhibit insulin and TOR signaling in response to hypoxia and energy stress and restrict growth during Drosophila development. The finding that inhibitors of growth are highly expressed in Tsc1 tissue led to the hypothesis that such genes are transcriptionally induced as part of a feedback loop that restricts tissue growth under conditions of excessive TOR activity. Feedback loops are an important activity-modulating feature of many signaling pathways, including the TOR and insulin pathways (Harvey, 2008).
To examine the mechanism whereby transcription of growth inhibitors is induced in response to TOR hyperactivation, attempts were made to determine which transcription factors were responsible for their expression. One obvious candidate was FOXO, a member of the forkhead transcription factor family, which has a well-established role as an effector of insulin signaling. If FOXO has a role in inducing expression of negative regulators of growth in Tsc tissue, then expression of some of those genes should be elevated under conditions of increased FOXO activity. To investigate this hypothesis, the expression profiles were examined of Tsc1 LOF tissue and Drosophila S2 cells expressing FOXOA3, a mutant version of FOXO that is insensitive to phosphorylation-dependent inhibition by Akt. This analysis revealed that 25 genes were up-regulated 1.5-fold or greater in both Tsc1 LOF and FOXO GOF expression profiles, which represents a highly significant degree of overlap as determined by calculation of the hypergeometric distribution. A highly statistically significant P value strongly suggests that there is a functional overlap between these two datasets that cannot be explained by random variation (Harvey, 2008).
Interestingly, two genes previously implicated in tissue growth regulated by the insulin and TOR pathways 4E-BP and scy were elevated in both microarray experiments, whereas the chrb growth-inhibiting gene was not. Thus, a subset of genes elevated in Tsc1 tissue appears to respond to FOXO activity and was investigated further (Harvey, 2008).
4E-BP is a well-characterized FOXO target gene. To determine whether FOXO could directly activate transcription of genes that were elevated in Tsc1 tissue other than 4E-BP, focus was placed on scy and the phosphoserine phosphatase aay (one of the most highly elevated transcripts in each microarray experiment). scy and aay both possess consensus FOXO recognition elements (FREs) in their promoters comparable to those found in dInR and 4E-BP promoters. Therefore, whether these genes are bona fide FOXO targets was examined by measuring their expression in Drosophila S2 cells misexpressing FOXOA3 in the presence of insulin. aay and scy mRNAs were up-regulated 19.4- and 4.3-fold, respectively, relative to a control gene, actin, as determined by QPCR (Harvey, 2008).
Luciferase reporter assays in S2 cells were used to determine whether the aay promoter region containing putative FREs was sensitive to FOXO activity. Luciferase activity dependent on the aay promoter was strongly induced by FOXOA3. In addition, using in vitro band shift assays, it was demonstrated that FOXO directly binds to the aay promoter, indicating that FOXO likely activates expression of aay by directly binding to the FRE. Surprisingly, in parallel luciferase reporter assays, activation of the scy promoter by FOXO could not be demonstrated, despite the fact that strong binding of FOXO to the putative scy FRE was observed using in vitro band-shift assays. A possible explanation is that the scy-promoter construct lacked the minimal promoter elements required for transcription of luciferase (Harvey, 2008).
TOR pathway hyperactivation caused by Tsc deficiency has been shown to strongly repress activity of Akt. FOXO is normally inactivated by Akt-dependent phosphorylation, which restricts nuclear entry of FOXO and leads to its ubiquitin-dependent destruction. Therefore, in response to TOR pathway hyperactivation, it was predicted that reduced Akt activity would cause FOXO protein to accumulate. To examine this hypothesis, expression of FOXO protein was analyzed in mosaic Tsc1 imaginal discs. It was found that FOXO protein was markedly increased in Tsc1 clones when compared with neighboring wild-type tissue (Harvey, 2008).
In addition, FOXO protein appeared to be mostly nuclear in Tsc1 tissue and cytoplasmic in wild-type tissue. Consistent with this observation, nuclear localization of the mouse FOXO orthologue FOXO1 is observed in endothelial cells of Tsc2 mutant hemangiomas, whereas FOXO1 is mostly cytoplasmic in normal cells. FOXO mRNA levels are unchanged in Tsc1 tissue as determined by microarray analysis, which suggests that changes in translation or stability of FOXO protein account for its accumulation in Tsc1 tissue. The presence of increased FOXO protein in the nuclei of Tsc1 cells is consistent with the hypothesis that FOXO is responsible for increased expression of some of the growth inhibitors that are up-regulated in Tsc1 cells (Harvey, 2008).
To determine whether FOXO was necessary for transcriptional induction of genes that were elevated in Tsc1 tissue, QPCR analysis was used to measure 4E-BP, aay, and scy expression in Tsc1 and Tsc1-FOXO double mutant eye-antennal imaginal discs. Consistent with microarray analysis, increased expression of 4E-BP, aay, and scy was observed in Tsc1 tissue. In Tsc1-FOXO tissue, however, 4E-BP was expressed at approximately equivalent amounts as in wild-type tissue, whereas aay and scy expression was only partially reduced. This demonstrates that elevated expression of 4E-BP in Tsc1 tissue is dependent on the FOXO transcription factor and provides evidence that FOXO activity increases when the TOR pathway is hyperactivated. Expression of aay and scy appear to be partially dependent on FOXO but are likely stimulated by additional transcription factors in Tsc1 tissue (Harvey, 2008).
Next, attempts were made to determine whether FOXO is required to limit growth of tissues with increased TOR pathway activity. In addition, a potential role was examined for another transcription factor, HIF-1, for retardation of TOR-driven growth. HIF-1 is a dual-subunit transcription factor consisting of α and β subunits that functions in response to insulin/TOR signaling and drives transcription of the growth-inhibiting genes scy and chrb, both of which are elevated in Tsc1 tissue (Harvey, 2008).
Drosophila possesses several HIF-1α subunits and a sole HIF-1β subunit, tango (tgo), which partners with each HIF-1α subunit. If FOXO and/or HIF-1 are required to induce expression of genes that limit tissue growth when the TOR pathway is hyperactivated, one might predict that Tsc1-FOXO and/or Tsc1-tgo double mutant tissue would possess a greater capacity to grow than Tsc1 tissue alone. To test this hypothesis, the size was examined of Drosophila eyes comprised almost entirely of the following genotypes: control, tgo, FOXO, Tsc1, Tsc1-tgo, and Tsc1-FOXO. Mutant eyes were created by driving mitotic recombination of chromosomes bearing flipase recognition target (FRT) sites and the appropriate gene mutations, specifically in developing Drosophila eye-antennal imaginal discs. Eyes lacking either tgo or FOXO were approximately the same size as control eyes, whereas Tsc1 eyes were considerably larger. Tsc1-tgo double mutant eyes did not exhibit a further increase in size, which suggests that HIF-1 is not required to inhibit tissue growth in response to Tsc1 loss. In contrast, Tsc1-FOXO double mutant eyes were substantially larger than Tsc1 eyes. This finding is particularly significant in light of the finding that eyes lacking FOXO were indistinguishable in size from wild-type eyes. Thus, it appears that FOXO is normally dispensable for control of eye size, but when growth control is altered by virtue of increased TOR activity, FOXO partially offsets the increased tissue growth. These findings are consistent with observations that FOXO protein accumulates in Tsc1 tissue and that transcriptional profiles of FOXO GOF and Tsc1 LOF cells overlap significantly (Harvey, 2008).
Because individual components of the insulin and TOR pathways are highly conserved among eukaryotes, important regulatory mechanisms that control tissue growth via these pathways are also likely to be conserved. To investigate this idea, transcriptional control was analyzed of mouse orthologues of genes that were elevated in D. melanogaster Tsc1 tissue. Initially, Northern blotting analysis was performed on Tsc2 primary mouse embryonic fibroblasts (MEFs; derived on a p53 background to overcome premature senescence induced by Tsc2 loss). It is reasonable to predict that transcriptional changes that occur because of loss of either Tsc1 or Tsc2 should be very similar because TSC1 and TSC2 function together in an obligate fashion, and mutation of either gene leads to almost indistinguishable phenotypes. It was found that several gene expression changes observed in Drosophila Tsc1 tissue are conserved in Tsc2 MEFs (Harvey, 2008).
The homologues of aay, heat shock protein (hsp) 23, scy, and chrb (PSPH, hsp 27, REDD1, and REDD2, respectively) were all significantly up-regulated in Tsc2 MEFs when compared with control MEFs and expression of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) control. To demonstrate that these expression changes were a specific consequence of Tsc2 loss, Tsc2 expression was reconstituted in Tsc2 null cells, which substantially suppressed mammalian TOR activity and expression of these genes. Interestingly, expression of phosphoenolpyruvate carboxy kinase and 4E-BP1/2 was not altered between wild-type and Tsc2 cells, which might reflect tissue- or species-specific differences in the transcriptome of Drosophila epithelial cells and MEFs (Harvey, 2008).
To determine whether the mode of transcription of these genes was also conserved in mammals, expression was analyzed of the scy homologue REDD1. Like scy, mammalian REDD1 orthologues possess a putative consensus FRE within their proximal promoters. Cotransfection of a version of FOXO that is insensitive to phosphorylation-dependent inhibition by Akt (TM-FKHRL-1) induced robust activation of a mouse REDD1 reporter construct in primary MEFs. To determine whether induction was mediated through the identified FRE, a mutant reporter was created lacking this sequence. Deletion of the REDD1 FRE consistently reduced FOXO-mediated induction of the REDD1 promoter. Finally, to directly assess whether FOXO-dependent transcription was activated in mammalian cells lacking Tsc2, activity of the REDD1 promoter reporter or the corresponding mutant FRE reporter was examined in wild-type and Tsc2 MEFs. As predicted, the wild-type REDD1 promoter exhibited robust activation in Tsc2 cells compared with wild-type cells, and this activation was substantially reduced by deletion of the FRE. Together, these findings provide evidence that transcriptional changes resulting from Tsc1/Tsc2 deficiency are conserved in diverse species (Harvey, 2008).
This study has identified of an evolutionary conserved transcriptional program important for restricting tissue overgrowth driven by excessive activation of the TOR pathway. The FOXO transcription factor plays a key role in this transcriptional response, likely by stimulating expression of several growth inhibitory genes. Thus, although the requirement for FOXO in restricting growth under normal development conditions appears dispensable, this is no longer the case under conditions of excessive TOR activation. These findings have important implications for cancer syndromes that arise because of inappropriate TOR pathway activation, such as the human hamartomatous syndrome, tuberous sclerosis. TOR-dependent feedback inhibition is thought to contribute to the benign nature of Tsc1 and Tsc2 tumors (Ma, 2005; Manning, 2005). Conceivably, inactivating mutations in FOXO family transcription factors and/or FOXO target genes that possess growth-inhibiting properties could promote further growth in normally benign Tsc1 and Tsc2 tumors (Harvey, 2008).
It is generally accepted that the growth rate of an organism is modulated by the availability of nutrients. One common mechanism to control cellular growth is through the global down-regulation of cap-dependent translation by eIF4E-binding proteins (4E-BPs). Evidence is reported for a novel mechanism that allows eukaryotes to coordinate and selectively couple transcription and translation of target genes in response to a nutrient and growth signaling cascade. The Drosophila insulin-like receptor (dINR) pathway incorporates 4E-BP resistant cellular internal ribosome entry site (IRES) containing mRNAs, to functionally couple transcriptional activation with differential translational control in a cell that is otherwise translationally repressed by 4E-BP. Although examples of cellular IRESs have been previously reported, their critical role mediating a key physiological response has not been well documented. These studies reveal an integrated transcriptional and translational response mechanism specifically dependent on a cellular IRES that coordinates an essential physiological signal responsible for monitoring nutrient and cell growth conditions (Marr, 2007).
Coupled transcription and protein synthesis is a hallmark of prokaryotic gene expression. The advantages of such a linked system are well recognized as it provides smooth coordination to ensure that cells respond appropriately to signals such as nutrient availability. A rapid response to such environmental signals also allows for multiple points of regulation and a fine-tuning mechanism for controlling gene expression. In eukaryotic organisms, the compartmentalization of the cell nucleus makes the direct coupling of transcription and translation problematic. Nevertheless, like prokaryotes, the metazoan cell must respond to many external as well as internal signals, and a coupled response would be highly advantageous. However, there is currently little evidence for such a direct linkage, either physical or functional, in metazoans. In attempts to dissect the transcriptional regulatory circuitry of the insulin-like signaling cascade in Drosophila, a potentially new mechanism that functionally links transcription and translation has been identified (Marr, 2007).
Metazoan organisms must strictly control both body and organ size during development. Thus, cell size and cell number are tightly controlled to determine the final size of an animal. One of the cues used in determining growth regulation is nutrient availability. The insulin receptor (INR) and insulin-like growth factor (IGF) receptor pathways have evolved as key sensors of nutrient availability and play an important role in both cell-autonomous and nonautonomous decisions controlling cellular proliferation, cell size determination, and the response to nutrient availability. In Drosophila, this pathway is critical for determining body and organ size as well as metabolic homeostasis and life span. Perhaps most notably, misregulation of this pathway in humans can lead to type 2 diabetes and all of its associated pathologies, which is becoming a rapidly escalating worldwide epidemic (Marr, 2007).
The INR/IGF pathway is highly conserved, with homologs of the key molecular players present in metazoan organisms from flies to humans. The downstream targets of this signaling cascade are thought to separately modulate both transcription and translation to potentiate signals for either growth or stasis. In the presence of insulin or insulin-like peptides, the signaling cascade activates the oncogenic protein kinase Akt. To control RNA synthesis, Akt phosphorylates the Forkhead-box-binding protein (dFOXO) family of transcription factors, sequestering them in the cytoplasm and thus effectively inactivating them. This in turn prevents activated transcription of the dFOXO target genes. In addition, Akt stimulates the modification of the target of rapamycin (TOR) protein, which in turn phosphorylates and inactivates the translation initiation inhibitor eIF4E-binding protein (d4E-BP). In its unphosphorylated and active state, d4E-BP binds to the 7-methyl-guanosine (m7G) cap-binding protein eIF4E. This prevents formation of the translation initiation complex eIF4F, thereby inhibiting cap-dependent translation. This combination of inactivated dFOXO and inactive d4E-BP efficiently drives the cell toward growth and proliferation. Conversely, active dFOXO and d4E-BP conspire to arrest cell growth until the cell receives favorable nutrient and physiological signals to continue proliferation (Marr, 2007).
Drosophila melanogaster has proven to be a valuable model organism for working out the molecular details of this conserved pathway. In the absence of insulin or insulin-like peptides, dFOXO activates the transcription of both the insulin-like receptor (dINR) gene and the gene for Drosophila 4E-BP, establishing a transcriptional signaling loop that sensitizes the cell to receive further nutrient-dependent signals while preventing the cell from proliferating. In order to investigate this intriguing transcriptional feedback control, the start site of transcription for the dINR gene was precisely mapped using a modification of the cap-trapping cDNA synthesis method. This method, which depends on an intact m7G cap for capture of the mRNA, when combined with rapid amplification of five prime (5') cDNA ends (5' RACE) maximizes the yield of full-length 5' untranslated regions (UTRs). The use of this methodology allowed detection of critical UTRs associated with the mRNA that had previously gone undocumented. The dINR gene is actually controlled by a complex set of three distinct promoters (P1, P2, and P3) spread over 38 kb of the Drosophila genome. These combined promoters and associated introns and exons encompass the entire region between the Drosophila E2F gene and the currently annotated dINR gene. This complex control region fills a gap in the genome annotation that contains no other annotated genes or gene predictions (Marr, 2007).
Each of the dINR promoters produces a transcript with a unique and unusually long 5'UTR spliced to a short common exon that is in turn spliced to the first coding exon. The UTR originating from P1 is 1118 bases, the UTR originating from P2 is 419 bases, and the UTR originating from P3 is 485 bases. In contrast, the average 5'UTR in Drosophila is only 256 bases. All three UTRs contain multiple AUG initiator codons upstream of the legitimate INR initiator codon. In the case of the transcript that originates from P1, there are 12 AUGs before the legitimate translational start signal (Marr, 2007).
The DNA sequences immediately upstream of the mapped transcript start sites contain easily recognizable sequences similar to the computationally and biochemically determined common core promoter elements. P1 contains a TATA box, an Initiator element, and a downstream promoter element (DPE). P2 contains a TATA-like box and a DPE but no recognizable Initiator. P3 contains a recognizable Initiator but no recognizable TATA box or DPE. Importantly, a constitutively active form of dFOXO (dFOXO-A3) activates all three promoters in Drosophila Schneider line 2 (S2) cells, and this increased RNA synthesis can produce dINR protein even in the presence of insulin. The transcript originating at P1 is by far the most abundant transcript under both unactivated and activated conditions. P2 is present at an intermediate level, and P3 is a low-abundance transcript. Interestingly, the level of transcription correlates with the number of recognized core promoter elements, illustrating the important role these different elements play in determining the total level of transcription from a gene in both activated and unactivated states (Marr, 2007).
In the animal, all three transcripts are detectable in multiple developmental stages. They are present in whole animal extracts in the same relative order of abundance that is detected in S2 cells (P1 >> P2 > P3). When compared with the Rp49 transcript, a common control transcript that changes little over the stages tested, all three transcripts fluctuate in abundance. Notably, all three transcripts diminish significantly in the L3 larva, a time when the animal is voraciously eating. In contrast, these dINR transcripts peak in the pupae, a time when the animal is fasting and expending much of the energy gained during the larval stage. This observation is consistent with a previous finding that dINR expression is linked to nutrient availability (Marr, 2007).
Strikingly, dINR is not only transcriptionally up-regulated but also robustly translated. Growing S2 cells in the absence of serum and insulin causes a marked decrease in the rate of incorporation of radiolabeled cysteine and methionine consistent with a global decrease in the rate of translation. Despite this slowing of overall translation, dINR protein accumulates in S2 cells. This is detectable by immunoblot of whole cell extracts with antisera raised against the dINR protein. The increase in dINR protein levels is at least partially due to the absence of insulin itself and not another component of serum because the accumulation of dINR protein is inhibited by addition of insulin to media containing insulin-depleted serum. In addition, the increased dINR protein level is most likely due to increased synthesis since serum starved cells contain more radiolabeled receptor that binds to insulin-agarose. This raises the intriguing question of how translation of dINR can proceed in the presence of a quantitatively dephosphorylated, potently active, and up-regulated inhibitor of protein synthesis, d4E-BP. This paradoxical finding that the dINR pathway transcriptionally up-regulates both dINR and d4E-BP combined with the newly discovered unusually long 5'UTRs of these transcripts suggest that perhaps the INR gene engages the translation machinery in an unconventional manner that bypasses the need for eIF4E. A potential d4E-BP resistant internal ribosome entry site (IRES) exists in these Drosophila genes that contain long UTRs, as has been seen in other instances. For example, both the Antennapedia and Ultrabithorax long 5'UTRs contain IRESs, although their physiological role has remained undetermined (Marr, 2007).
As a first test of whether the dINR 5'UTRs also contain an IRES activity, a bicistronic construct, commonly used to assess IRES activity, was generated. The various 5'UTRs of dINR were inserted in both the forward and reverse orientations between the Renilla and firefly luciferase genes. The reverse orientation was used as a spacer length control equivalent. The ratio of Renilla luciferase expression to firefly luciferase expression should provide an indication of the cap-independent translational potential of the various 5'UTRs. Since resistance to d4E-BP is most relevant to this pathway, these experiments were carried out in the presence and absence of a constitutively active form of d4E-BP. Because the Renilla luciferase ORF is the first in the mRNA, it should be uniquely sensitive to inhibition of cap-dependent translation, while the firefly gene expression, if any, should be dependent on internal ribosome entry. The data are expressed as a ratio of the activity in the presence of d4E-BP to the activity in the absence of d4E-BP. Therefore, a number close to 1 indicates that there is no resistance to d4E-BP. In these cell-based assays, the 5'UTR from both P1 and P2 showed significant resistance to d4E-BP (about fourfold better than the reverse orientation in both cases), but only when inserted in the forward direction. Curiously, the 5'UTR from P3 showed unusual resistance to d4E-BP in either orientation. Indeed, the P3 UTR showed a perplexing increase in expression of the firefly ORF in the presence of d4E-BP compared with no UTR in both orientations. This finding reveals a potential limitation of using the bicistronic assays since interfering effects from cryptic promoters, cryptic splicing, or secondary effects of expression of d4E-BP cannot be ruled out with this assay (Marr, 2007).
To circumvent some of the inherent idiosyncrasies of the bicistronic constructs, monocistronic constructs were used that more closely mimic the situation of the endogenous dINR gene. Potential IRES activity esd measured in two complementary ways. First, in a DNA-based transient transfection, either the constitutively active form of d4E-BP or a control protein, green fluorescent protein (GFP), was expressed and resistance to d4E-BP was measure as the ratio of luciferase activity (provided by a second plasmid) in the presence of d4E-BP to the activity in the presence GFP. In this set of experiments, the minimal Antennapedia IRES, a Drosophila 5'UTR known to support cap-independent initiation of translation, was included as a positive control. Under these cell-based assay conditions, the P1 and P2 UTRs again displayed robust resistance to d4E-BP, while P3 and the common exons showed little resistance. Notably, the P2 5'UTR is as efficient as the minimal Antennapedia IRES, and the P1 5'UTR is actually significantly more efficient than the control IRES. Taken together, these two cell-based assays suggest that the 5'UTRs of at least the P1 and P2 transcripts can direct substantial IRES activity, while the P3 UTR appears to have much less if any such activity in S2 cells. Second, to complement these plasmid-based assays and directly investigate the contribution of the UTRs to translation, an RNA-based transfection assay was used. The RNAs contained either a m7G cap or an ApppG cap mimic. Only the 7mG cap allows cap-dependent translation. The ApppG cap stabilizes the transcript but does not allow cap-dependent translation, so it is a direct measure of the contribution of IRES activity. In this assay, the UTRs again showed significant IRES activity. The P1 UTR confers the same activity with or without a m7G cap, indicating a strong IRES activity. The P2 and P3 UTRs also confer cap-independent translation activity, although the level of activity is not equal to UTR plus cap. In contrast, the common exon or nonspecific UTR retains only 20% of their translation potential without the m7G cap. Taken together, these cell-based assays provide encouraging evidence for IRES activity of the dINR 5'UTRs (Marr, 2007).
However, given the well-recognized limitations inherent with using cell-based assays to establish IRES activity, a Drosophila embryo-derived cap-dependant in vitro translation system was used to test more directly the putative IRES activity and more specifically the potential d4E-BP resistance of the INR UTRs. The translation extracts were treated with micrococcal nuclease to destroy the bulk of competing endogenous transcripts so that translation would be largely dependent on exogenously added RNA. As expected, addition of normal capped transcripts results in robust translation from all of the UTR-containing RNAs as well as the common UTR and a short nonspecific UTR control RNA. To test the dependence of translation on eIF4E, exogenous m7G cap analog was added as a competitor. This excess free cap efficiently binds and sequesters the available eIF4E, preventing this essential initiation factor from binding capped RNA, thus effectively blocking the nucleation of the eIF4F complex and cap-dependent initiation. Remarkably, only the transcripts containing the P1, P2, and P3 UTRs are resistant to exogenously added competitor cap analog, whereas the common UTR fragment and the short nonspecific leader are effectively inhibited. This finding strongly suggests that the various dINR-specific UTRs, indeed, provide a cap-independent mechanism of translation initiation. To directly test the resistance of these transcripts to d4E-BP-mediated translation inhibition, recombinant d4E-BP was added to the reactions. Whereas the common exon and control RNAs are efficiently inhibited by this blocker of eIF4E-mediated translation initiation, the P1, P2, and P3 UTR-containing transcripts are highly resistant to d4E-BP. These findings taken together with cell-based assays suggest that, indeed, dINR protein synthesis can proceed via an IRES-mediated eIF4E-independent mechanism of initiation both in vitro and in vivo (Marr, 2007).
What purpose might a cap-independent translation activity serve beyond simple resistance to the active d4E-BP in the absence of insulin? Perhaps by functionally coupling transcription and translation, such a mechanism could serve to amplify the signal received from the insulin receptor pathway. To test this idea, in vitro translation experiments were used. In the absence of miccrococal nuclease treatment, the endogenous transcripts present in the translation extract should effectively compete with the experimental dINR transcripts for limiting amounts of the translation machinery. Advantage was taken of this inevitable competition for translation machinery to test the response of the various UTRs in a situation that may more closely reflect the cellular environment, where multiple variable abundant transcripts must compete for a limited supply of the translational apparatus. Under these competitive conditions, addition of either m7G or d4E-BP actually results in an even more robust increase in translation of the dINR UTR-containing RNAs relative to the unchallenged state. This finding suggests that these RNAs that contain dINR UTRs, and presumably IRES activity, are highly effective at out-competing other transcripts for access to the translational machinery when m7G cap-dependent initiation is inhibited. While the molecular mechanism of 4E-BP resistance of the dINR transcripts have not been unequivocally defined, it is clear that the UTRs allow significant translation in conditions when cap-dependent translation is inhibited (Marr, 2007).
These data allowed formulation of a new model to explain the effects of nutrients and insulin levels on dINR feedback regulation. In times of high nutrients and therefore high insulin-like peptides, both dFOXO and d4E-BP are phosphorylated and inactive. Under these 'rich' conditions, dFOXO is sequestered in the cytoplasm and phosphorylated d4E-BP is unable to interact with eIF4E. This situation allows efficient translation of most cellular transcripts regardless of the mechanism of initiation (cap-dependent vs. cap-independent). In contrast, in low nutrient conditions or in the absence of insulin or insulin-like peptides, both dFOXO and d4E-BP become dephosphorylated and active. Activated dFOXO directs a robust increase in the transcription of both dINR and d4E-BP (among other genes). Additionally, the active and up-regulated d4E-BP effectively inhibits cap-dependent translation, freeing up the protein synthesis machinery to selectively translate IRES-containing transcripts like dINR. These two coordinated mechanisms consequently orchestrate the integration of a specific transcriptional response and simultaneously a translational response that greatly amplifies the signal and sensitizes the cell for detection of small changes in nutrient availability as well as, possibly, developmental and environmental cues (Marr, 2007).
Interestingly, the dFOXO-responsive dINR promoters produce three distinct transcripts. Why such a complex regulatory network? A hint may be that the P3 UTR does not seem to have detectable IRES activity in the S2 cells but shows substantial activity in vitro with extracts derived from whole Drosophila embryos. It is likely that the three transcripts are produced in a tissue- or temporal-specific manner during development, and it is speculated that each may depend on cell-specific IRES trans-acting factors (ITAFs) that are required for activity. This would direct tissues to respond differentially to dINR signaling. In tissues lacking specific ITAFs, the IRES activity would be diminished and the tissue may produce only a moderate level of dINR protein (Marr, 2007).
An interesting parallel was found between mechanisms for reprogramming the gene expression machinery in a cell to respond to physiological cues and the more commonly observed viral takeover of the cellular macromolecular synthesis machinery. When some viruses, such as polio, infect a cell, they target the translation initiation machinery (either eIF4G or 4E-BP) so that there is a switch from cap-dependent synthesis to IRES-dependent synthesis. This leads to a robust and specific stimulation of viral protein synthesis at the expense of most cellular protein synthesis. By the evolution of cellular mechanisms that activate 4E-BP and simultaneously produce transcripts containing cellular IRESs, a critical physiological signaling cascade can evidently adopt a similar mechanism to effectively usurp the macromolecular synthesis machinery to drive cellular physiology in a very specific direction. Indeed, viruses may have merely co-opted the mechanism from cells in the eternal battle between host and virus (Marr, 2007).
Although the initial characterization of the INR transcriptional feedback loop was carried out in Drosophila, a similar regulatory circuit has been found in vertebrates. It is interesting to note that the transcripts for human insulin receptor and IGF-2 receptor remain associated with polysomes when cap-dependant translation is inhibited by poliovirus infection. Although the level of INR mRNA up-regulation by FOXO in mouse muscle cells is only twofold, the levels of INR protein increase much more dramatically (six- to eight-fold), consistent with a coupled transcription/translation mechanism of the signal in vertebrates. It seems likely, given the findings report in this study, that the same type of coupling between the transcriptional program of FOXO proteins and translational control by IRES activity is also occurring in vertebrate systems. Understanding this novel mechanism that couples transcription and translation may provide new insight into disease states such as insulin-resistant type 2 diabetes (Marr, 2007).
The eIF4E-binding proteins (4E-BPs) interact with translation initiation factor 4E to inhibit translation. Their binding to eIF4E is reversed by phosphorylation of several key Ser/Thr residues. In Drosophila, S6 kinase (dS6K) and a single 4E-BP (d4E-BP) are phosphorylated via the insulin and target of rapamycin (TOR) signaling pathways. Although S6K phosphorylation is independent of phosphoinositide 3-OH kinase (PI3K) and serine/threonine protein kinase Akt, that of 4E-BP is dependent on PI3K and Akt. This difference prompted an examination of the regulation of d4E-BP in greater detail. Analysis of d4E-BP phosphorylation using site-directed mutagenesis and isoelectric focusing-sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the regulatory interplay between Thr37 and Thr46 of d4E-BP is conserved in flies and that phosphorylation of Thr46 is the major phosphorylation event that regulates d4E-BP activity. RNA interference (RNAi) was used to target components of the PI3K, Akt, and TOR pathways. RNAi experiments directed at components of the insulin and TOR signaling cascades show that d4E-BP is phosphorylated in a PI3K- and Akt-dependent manner. Surprisingly, RNAi of dAkt also affects insulin-stimulated phosphorylation of dS6K, indicating that dAkt may also play a role in dS6K phosphorylation (Miron, 2003).
Insulin treatment caused a strong increase in the immunoreactivity of d4E-BP to antibodies directed against human phospho-4E-BP1(Thr37/46). Since the residues in the region of Thr46, but not Thr37, are perfectly conserved between 4E-BP1 and d4E-BP, it is probably Thr46 that is hyperphosphorylated in d4E-BP after insulin treatment. Regulation of d4E-BP phosphorylation thus appears to differ from that of mammalian 4E-BP1. Phosphorylation of Thr37 and Thr46 of 4E-BP1 is only modestly induced after serum stimulation in serum-starved HEK293 cells (Gingras, 1999). In serum-starved HEK293 cells treated with rapamycin, the phosphorylation of 4E-BP1 at Thr37 and Thr46 is reduced, but upon serum addition, it is restored to its original state, whereas phosphorylation of Ser65 and Thr70 remains blocked (Gingras, 1999; Gingras, 2001). Thus, Ser65 and Thr70 are the rapamycin-sensitive sites of 4E-BP1. In contrast, in S2 cells, phosphorylation of d4E-BP at Thr46 is robustly induced after insulin treatment, and rapamycin completely blocks its phosphorylation. Therefore, unlike 4E-BP1, phosphorylation at Thr46 is a major insulin-stimulated and rapamycin-sensitive event involved in d4E-BP regulation. A dependency between Thr37 and Thr46 that is analogous to that of 4E-BP1 is important for d4E-BP phosphorylation. For 4E-BP1, phosphorylations at Thr37 and Thr46 are intimately linked; these two phosphorylation events are regulated coordinately by mTOR (Gingras, 1999). The link between Thr37 and Thr46 in d4E-BP is conserved, but it is not known if Thr37 acts as a priming event for the subsequent phosphorylation of Thr46 or if Thr37 and Thr46 are regulated coordinately, similar to 4E-BP1. Hence, phosphorylation of d4E-BP may be explained by three possible models: (1) In the primed model, d4E-BP is already phosphorylated at Thr37 and is subsequently phosphorylated on one additional site, Thr46, after insulin stimulation. (2) In the sequential model, d4E-BP is phosphorylated on Thr37 and then Thr46 (or vice versa). (3) In the coordinated model, d4E-BP is phosphorylated coordinately on Thr37 and Thr46 (Miron, 2003).
The results suggest a simpler mode of regulation of d4E-BP by phosphorylation compared with the hierarchical phosphorylation of mammalian 4E-BP1. The phosphorylation of 4E-BP1 is the best understood, but not all mammalian 4E-BPs are regulated in a similar manner. 4E-BP2 is phosphorylated on fewer residues (Lin, 1996) and is dephosphorylated more slowly than 4E-BP1. Also, 4E-BP3 is weakly stimulated by insulin treatment, causing poor release from eIF4E. This has been attributed to the lack of the four-residue RAIP motif found in the N terminus of 4E-BP1 and -2 but not in 4E-BP3 Tee, 2003). The RAIP motif seems to be required for the efficient overall phosphorylation of 4E-BP1, and intriguingly, this motif is also lacking in d4E-BP. Another important motif for 4E-BP1 regulation is the TOR signaling (TOS) motif (Schalm, 2002), which is conserved (FQLDL, at the C terminus) in d4E-BP. Hence, a consequence of the lack of the RAIP motif may be the simpler regulation of d4E-BP to improve its release from eIF4E when d4E-BP is recruited to the dTOR/dRaptor complex through the TOS motif (Nojima, 2003, Schalm, 2002). Moreover, because of a divergent eIF4E-binding site, d4E-BP does not interact as strongly with deIF4E as 4E-BP1 (Miron, 2001). It is conceivable that because of the poorer interaction of d4E-BP with deIF4E, phosphorylation at Thr37 and Thr46 is sufficient to bring about its release from eIF4E. IEF-SDS-PAGE results indicate that in serum-starved S2 cells, some d4E-BP is already phosphorylated. Although these additional sites do not prevent the interaction between deIF4E and d4E-BP, it is possible that they contribute to the release from deIF4E once Thr46 becomes phosphorylated (Miron, 2003).
A large body of work has established the paramount importance of the InR-IRS-PI3K-PTEN signaling module in the control of cell growth. This module coordinates cellular metabolism with the nutritional state. The primary outcome of its activation is the modulation of the PI3K/PTEN cycle and consequent PIP3 production. The increase in PIP3 facilitates the recruitment of pleckstrin homology domain-containing proteins, such as dAkt and dPDK1, to the plasma membrane. dPTEN mutant flies die because of increased membrane translocation and activation of dAkt. Phosphorylation of the Tsc2 subunit of TSC by Akt results in inhibition of the complex by causing its dissociation or by blocking its interaction with other proteins. TSC inhibits S6K and 4E-BP1 by repressing the GTPase Rheb, preventing it from activating mTOR through an unknown mechanism. Thus, mTOR, is clearly a critical regulator of S6K and 4E-BP1 in mammals and Drosophila (Miron, 2003 and references therein).
Is d4E-BP regulated by a PI3K/Akt-independent pathway similar to that described for dS6K? Analysis of signaling to d4E-BP using RNAi indicates that it is not. It is more likely that d4E-BP is a direct downstream target of the dInR-dPI3K-dPTEN-dAkt-dTSC-dTOR signaling cascade. Thus, a linear pathway from InR to Akt that is important for 4E-BP regulation is conserved between Drosophila and mammals (Miron, 2003)
dPDK1 is critical for regulating growth by phosphorylating dAkt and dS6K. RNAi of dPDK1 does not significantly affect insulin-induced phosphorylation of d4E-BP. However, consistent with the direct phosphorylation of dS6K by dPDK1, the phosphorylation of dS6K at Thr398 is completely blocked by RNAi of PDK1. Thus, the results favor a model in which d4E-BP regulation is effected through dAkt, even when dPDK1 levels are dramatically reduced, whereas dS6K requires both dAkt and dPDK1. The differential effects of dPDK1 RNAi on d4E-BP and dS6K phosphorylation can be explained as follows: dPDK1 levels may be reduced below a threshold that is required to phosphorylate dS6K but is still adequate to activate dAkt, allowing d4E-BP phosphorylation. Since dS6K requires direct phosphorylation by dPDK1, it may be more susceptible to variations in its levels. In contrast, d4E-BP, which relies on a signal relayed by dAkt, may be less affected by variations in dPDK1. In mammalian PDK1-hypomorphic mutants, a kinase activity that is 10-fold lower than normal still results in normal Akt and S6K1 activation, yet these animals are greatly reduced in size. This observation supports the notion that reduced PDK1 activity may differentially activate downstream targets (Miron, 2003).
In Drosophila, coexpression of dS6K with dPI3K does not cause additive cellular overgrowth, unlike coexpression of dAkt and dPI3K. RNAi of dPTEN in Kc 167 cells and overexpression of dPTEN in Drosophila larvae had little effect on dS6K activity. Moreover, removal of both dS6K and dPTEN in cell clones does not prevent the dPTEN-dependent overgrowth phenotype. Together, these results and the results of dPI3K and dPTEN RNAi experiments would seemingly support the notion that dS6K-dependent cell growth is not influenced by dPI3K and dPTEN. However, a different effect of dPTEN RNAi on dS6K has been reported in another study: increase in dS6K phosphorylation following RNAi of dPTEN. Consistent with this observation RNAi directed against dPI3K and dPTEN has been shown to modulate dS6K phosphorylation. A reasonable explanation for these discrepancies is that the knockdown of dPI3K and dPTEN achieved in the current experiments was not sufficient to completely deplete these proteins and affect dS6K phosphorylation (Miron, 2003 and references therein).
The role of dAkt in regulating dS6K is subject to debate. In Drosophila, Akt plays a predominant role in mediating the effects of increased PIP3 levels, and all Akt-mediated growth signals are thought to be transduced via Tsc1/2. Tsc2 is directly phosphorylated by Akt, implying that S6K is downstream of Akt in the PI3K signaling pathway. The observation that RNAi of dAkt reduces dS6K phosphorylation at Thr398 supports a direct link among dAkt, dTSC, and dS6K but contradicts the finding that TSC modulates dS6K activity in a dAkt-independent manner. Recent data also support the conclusion of a link between dAkt and dS6K. Clones of cells doubly mutant for dPTEN and dTsc1 display an additive overgrowth phenotype, suggesting that the tumor suppressors act on two independent pathways, from dPTEN to dAkt and from dTSC to dS6K. The findings demonstrate clear effects of dPTEN, dAkt, and dTSC on d4E-BP, which does not preclude the possibility that two pathways regulate d4E-BP; however, a simpler interpretation is that a single pathway is important for its regulation. A possibility is that d4E-BP requires higher dAkt activity than dS6K in order to be phosphorylated. In circumstances of low PI3K activation, low levels of PIP3 are produced, resulting in weaker dAkt activity that is sufficient for dS6K activation but not for d4E-BP phosphorylation. A differential threshold of activation could be the source of the discrepancies between the current results and those of others. This model is strongly supported by recent data showing that in cells lacking both Akt1 and Akt2 isoforms, the low level of Akt activity remaining is sufficient for robust S6K1 phosphorylation, but phosphorylation of 4E-BP1 is dramatically reduced (Miron, 2003 and references therein).
Alternatively, the results could also be explained by the existence of a negative feedback loop between dPI3K and dS6K that dampens insulin signaling by suppressing dAkt activity. This negative feedback loop has been described. Similar observations were made in mammals; insulin-induced activation of Akt is inhibited in Tsc2-deficient mouse embryonic fibroblasts. Thus, depletion of dAkt may trigger this negative feedback loop, which diminishes dS6K phosphorylation and activation. Interestingly, engagement of this feedback mechanism can also provide an explanation for the reduction in total d4E-BP levels observed in dPDK1 RNAi-treated cells. Under these conditions, the reduction of dS6K signaling is accompanied by a concomitant reduction in growth signaling on the dPI3K-dAkt branch of the pathway. Thus, a reduced level of d4E-BP is required to accommodate the reduced need for deIF4E inhibition (Miron, 2003).
The Drosophila genome-sequencing project has revealed a total of seven genes encoding eight eukaryotic initiation factor 4E (eIF4E) isoforms. Four of them (eIF4E-1,2, eIF4E-3, eIF4E-4 and eIF4E-5) share exon/intron structure in their carboxy-terminal part and form a cluster in the genome. All eIF4E isoforms bind to the cap (m7GpppN) structure. All of them, except eIF4E-6 and eIF4E-8 are able to interact with Drosophila eIF4G or eIF4E-binding protein (4E-BP). eIF4E-1, eIF4E-2, eIF4E-3, eIF4E-4 and eIF4E-7 rescue a yeast eIF4E-deficient mutant in vivo. Only eIF4E-1 mRNAs and, at a significantly lower level, eIF4E3 and eIF4E-8 are expressed in embryos and throughout the life cycle of the fly. The transcripts of the remaining isoforms are detected from the third instar larvae onwards. This indicates the cap-binding activity relies mostly on eIF4E-1 during embryogenesis. This agrees with the proteomic analysis of the eIF4F complex purified from embryos and with the rescue of l(3)67Af, an embryonic lethal mutant for the eIF4E-1,2 gene, by transgenic expression of eIF4E-1. Overexpression of eIF4E-1 in wild-type embryos and eye imaginal discs results in phenotypic defects in a dose-dependent manner (Hernandez, 2005).
Dominant mutations in leucine-rich repeat kinase 2 (LRRK2) are the most frequent molecular lesions so far found in Parkinson's disease (PD), an age-dependent neurodegenerative disorder affecting dopaminergic (DA) neuron. The molecular mechanisms by which mutations in LRRK2 cause DA degeneration in PD are not understood. This study shows that both human LRRK2 and the Drosophila orthologue of LRRK2 phosphorylate eukaryotic initiation factor 4E (eIF4E)-binding protein (4E-BP), a negative regulator of eIF4E-mediated protein translation and a key mediator of various stress responses. Although modulation of the eIF4E/4E-BP pathway by LRRK2 stimulates eIF4E-mediated protein translation both in vivo and in vitro, it attenuates resistance to oxidative stress and survival of DA neuron in Drosophila. These results suggest that chronic inactivation of 4E-BP by LRRK2 with pathogenic mutations deregulates protein translation, eventually resulting in age-dependent loss of DA neurons (Imai, 2008).
This study used Drosophila as a model system to understand the normal physiological function of LRRK2 and how its dysfunction leads to DA neurodegeneration. Genetic and biochemical evidence is provided that dLRRK modulates the maintenance of DA neuron by regulating protein synthesis. LRRK2 primes phosphorylation of 4E-BP, and this event has an important function in mediating the pathogenic effects of mutant dLRRK. These results link deregulation of the eIF4E/4E-BP pathway of protein translation with DA degeneration in PD (Imai, 2008).
eIF4E is a key component of the eIF4F complex that initiates cap-dependent protein synthesis. It has long been recognized that a key mechanism regulating eIF4E function is through phosphorylation-induced release of 4E-BP from eIF4E. A number of candidate kinases, including mTOR, have been implicated on the basis of in vitro or cell culture studies, but the physiological kinases remain to be identified. This study shows that LRRK2 is one of the physiological kinases for 4E-BP. LRRK2 exerts an effect on 4E-BP primarily at the T37/T46 sites. Phosphorylation at T37/T46 by LRRK2 likely facilitates subsequent phosphorylation at T70 and S65 in vivo by other kinase or LRRK2 itself. 4E-BP phosphorylation by LRRK2, therefore, could serve as an initiating event in an ordered, multisite phosphorylation process to generate hyperphosphorylated 4E-BP, similar to the phosphorylation of the Alzheimer's disease-associated tau. These results show that LRRK2 is not the only kinase that phosphorylates 4E-BP T37/T46 sites. Similarly, 4E-BP is unlikely the only substrate of LRRK2. A recent study showed that human LRRK2 phosphorylates moesin, the physiological relevance of which remains to be determined (Imai, 2008).
The role of 4E-BP in regulating eIF4E function has been well established in vitro. Recent studies in Drosophila, however, have revealed the complexity of the in vivo function of 4E-BP. Loss of the only d4E-BP gene does not affect cell size or animal viability, suggesting that it is dispensable for cell growth or survival under normal conditions. However, d4E-BP mutant flies are defective in responses to various stress stimuli. d4E-BP has also been proposed to exert an effect as a metabolic brake for fat metabolism under stress condition. Whether this role of 4E-BP is relevant to dLRRK function in stress resistance and DA neuron maintenance remains to be tested. eIF4E, the target of 4E-BP, functions primarily in regulating general protein translation in vitro. It has been suggested that overactivation of eIF4E is linked to the aging process and lifespan regulation. This study observed that overexpression of eIF4E as well as dLRRK leads to an aging-related phenotype in DA neurons, which strongly suggested that chronic attenuation of 4E-BP activity promotes oxidative stress and consequent aging in DA neurons. This is consistent with the finding of similar patterns of gene expression under oxidative stress and aging conditions, and the fact that PD caused by LRRK2 mutations is of late onset, with aging being a major risk factor (Imai, 2008).
This study analysed effects of removing dLRRK activity using a transposon insertion allele (dLRRK−), a chromosomal deletion allele (dLRRK Df) and gene knockdown (dLRRK RNAi). dLRRK(−/−), dLRRK (Df/−) and dLRRK RNAi flies are all resistant to oxidative stress treatments and show reduced endogenous ROS damages. In the paraquat treatment assay, dLRRK (Df/−) appeared more resistant than dLRRK(−/−). It is possible that dLRRK(−/−), which contains a transposon insertion in the COR domain of dLRRK, is not a null allele, although it has not been possible to detect a truncated protein product using an antibody against the N-terminus of the protein. Alternatively, the chromosomal deletion in dLRRK (Df) may include other gene(s) relevant to stress sensitivity. One candidate is the gene for PI3K Dp110 subunit. A recent study reported that dLRRK(−/−) animals are slightly sensitive to hydrogen peroxide but are comparable to control animals in response to paraquat. It is possible that the different genetic backgrounds and the nutrient conditions may account for the divergent results. In the current studies, the mutant chromosome was backcrossed to w− WT background for six generations in an effort to eliminate potential background mutations. A consistent finding from this study and two other studies of dLRRK(−/−) animals is that dLRRK is dispensable for the maintenance of DA neurons, although in one study it was reported that dLRRK(−/−) animals show reduced TH immunoreactivity and shrunken morphology of DA neurons. In contrast, overexpression of hLRRK2 containing a pathogenic G2019S mutation, or overexpression of mutant dLRRK as reported in this study, caused DA neuron degeneration, supporting the fact that the pathogenic mutations cause disease by a GOF mechanism (Imai, 2008).
The pathogenesis caused by mutations in LRRK2 could be partially explained by their higher kinase activity. Indeed, some pathogenic mutants of both hLRRK2 and dLRRK show elevated kinase activity towards 4E-BP. However, other mutants (e.g., hLRRK2 Y1699C and dLRRK Y1383C) did not show elevated kinase activity in vivo. Therefore, these pathogenic hLRRK2 mutations might confer cellular toxicity through mechanisms other than protein translation. For example, some hLRRK2 mutants are prone to aggregation in cultured cells. Consistently, dLRRK Y1383C mutant appeared as more prominent vesicular aggregates in fly DA neurons. Nevertheless, the facts that overexpression of eIF4E is sufficient to confer hypersensitivity to oxidative stress and DA neuron loss and that co-expression of 4E-BP suppresses the dopaminergic toxicity caused by more than one pathogenic dLRRK mutants provide compelling evidence that the eIF4E-4E-BP axis has an important function in mediating the pathogenic effects of overactivated LRRK2. The more downstream events that lead to DA neurotoxicity remain to be elucidated. So far, no clear evidence has been found of altered autophagy, caspase activation or DNA fragmentation (Imai, 2008).
There are several possibilities of how elevated protein translation could contribute to PD pathogenesis. First, given that protein synthesis is a highly energy-demanding process, stimulation of protein translation by LRRK2 could perturb cellular energy and redox homoeostasis. This could be especially detrimental in aged cells or stressed post-mitotic cells such as DA neurons. Second, increased protein synthesis could lead to the accumulation of misfolded or aberrant proteins, overwhelming the already compromised ubiquitin proteasome and molecular chaperone systems in aged or stressed cells. Third, altered LRRK2 kinase activity may affect synapse structure and function, which is known to involve local protein synthesis. Deregulation of this process could lead to synaptic dysfunction and eventual neurodegeneration (Imai, 2008).
Mutations in leucine-rich repeat kinase 2 (LRRK2) are linked to familial as well as sporadic forms of Parkinson's disease (PD), a neurodegenerative disease characterized by dysfunction and degeneration of dopaminergic and other types of neurons. The molecular and cellular mechanisms underlying LRRK2 action remain poorly defined. This study shows that LRRK2 controls synaptic morphogenesis at the Drosophila neuromuscular junction. Loss of Drosophila LRRK2 results in synaptic overgrowth, whereas overexpression of Drosophila LRRK or human LRRK2 has opposite effects. Alteration of LRRK2 activity also affects neurotransmission. LRRK2 exerts its effects on synaptic morphology by interacting with distinct downstream effectors at the presynaptic and postsynaptic compartments. At the postsynapse, LRRK2 interacts with the previously characterized substrate 4E-BP (Imai, 2008), an inhibitor of protein synthesis. At the presynapse, LRRK2 phosphorylates and negatively regulates the microtubule (MT)-binding protein Futsch. These results implicate synaptic dysfunction caused by deregulated protein synthesis and aberrant MT dynamics in LRRK2 pathogenesis and offer a new paradigm for understanding and ultimately treating PD (Lee, 2010).
Parkinson's disease (PD) is one of the most common neurodegenerative diseases and is characterized by locomotor abnormalities as a result of the dysfunction and eventual loss of dopaminergic (DA) neurons. Most PD cases are sporadic with no known cause. Recent advances in PD genetics have led to the identification of familial PD (FPD) genes. It is anticipated that understanding the disease mechanisms of the FPD cases will provide insights into PD pathogenesis in general. Despite intensive studies of the FPD gene products at the biochemical and cell biological levels, understanding of their physiological function and the molecular and cellular pathways underlying disease pathogenesis is still fragmentary. Of all FPD genes, leucine-rich repeat kinase 2 (LRRK2) is the most frequently mutated. LRRK2 encodes a large serine/threonine kinase with multiple other domains. Some pathogenic mutations in LRRK2, such as the I2020T and G2019S substitutions in the kinase domain and R1441C substitution in the ROC domain, appear to augment kinase activity. In Drosophila and mouse models, pathogenic human (hLRRK2) or Drosophila (dLRRK) LRRK2 induce parkinsonian phenotypes in an age-dependent manner. A number of LRRK2-interacting proteins and substrates have been identified through in vitro studies, which implicate diverse biological functions for LRRK2 in translational control, vesicular trafficking, and cytoskeletal regulation. The physiological relevance of these interacting proteins and substrates remain to be established (Lee, 2010).
Actin and microtubule (MT) cytoskeleton dynamics play a crucial role in the formation of the nervous system, regulating such fundamental processes as axonal guidance and synaptogenesis. Dynamic modulation of synaptic structure and function is fundamental to neural network formation during development and is the molecular basis of learning and memory. Synaptic dysfunction is tightly linked to the pathogenesis of major neurodegenerative diseases such as Alzheimer's disease, and its role in PD is beginning to be appreciated. In Drosophila, the MT-associated protein 1B (MAP1B) homolog Futsch is required for axonal and dendritic growth during embryogenesis and for synaptic morphogenesis during larval neuromuscular junction (NMJ) development. This study shows that dLRRK phosphorylates and negatively regulates Futsch function at the presynapse. The previously characterized dLRRK substrate 4E-BP functionally interacts with LRRK2 at the postsynapse. These results implicate defects in presynaptic MT cytoskeleton dynamics and postsynaptic protein synthesis in LRRK2 pathogenesis (Lee, 2010).
Genetic mutations in LRRK2 are frequently found in familial and sporadic PD cases. Understanding the physiological function of LRRK2 will therefore offer insights into PD pathogenesis in general. This study reveals a new physiological function of LRRK2 and offers molecular mechanisms underlying such function. The key findings from this study are that LRRK2 plays an important role in regulating synaptic morphogenesis and that it does so through distinct substrate proteins at the presynaptic and postsynaptic compartments. The results also show that the precise level of LRRK2 activity is important for synaptic morphogenesis and neurotransmission, but the regulation of these two synaptic processes likely involve different mechanisms and players. Given the similarity of Drosophila NMJ synapse to mammalian excitatory glutamatergic synapses, it is possible that the findings reported here are relevant to mammalian systems (Lee, 2010).
Synaptic loss is a major neurobiological substrate of cognitive dysfunction in a number of neurological diseases. Extensive studies in patients and animal models have documented that synaptic failure is one of the earliest events in the pathogenesis of Alzheimer's disease. Interestingly, neurotransmission defects have been repeatedly observed in rodent FPD models, including the LRRK2 model, although no obvious signs of neurodegeneration accompany the electrophysiological defects. These results suggest that synaptic dysfunction is a primary effect of FPD gene mutations and that synaptic failure is intimately involved in PD pathogenesis. The molecular mechanisms underlying these synaptic transmission defects, however, remain elusive. This study of the LOF and GOF models of LRRK2 in Drosophila provides mechanistic insights into the possible cause of synaptic dysfunction in LRRK2-associated PD. It was found that LRRK2 regulates synaptic morphogenesis at the presynaptic and postsynaptic compartments through distinct substrates (Lee, 2010).
In the presynaptic side, LRRK2 forms a complex with tubulin and the MT-binding protein Futsch. Furthermore, LRRK2 phosphorylates Futsch and negatively regulates the presynaptic function of Futsch in controlling MT dynamics. MT cytoskeleton is critical for the generation and maintenance of neuronal axons and dendrites, transport of synaptic vesicles and organelles along the processes, and the initiation and maintenance of synaptic transmission. Disrupted MT dynamics in neuronal synapses has been implicated as an underlying cause for several neurological diseases, including hereditary spastic paraplegia and fragile X syndrome. LRRK2-associated PD may share this feature with the aforementioned diseases. Disrupted MT dynamics could be responsible for the presynaptic effects observed in LRRK2 LOF and GOF mutants, such as aberrant mitochondria distribution. The synaptic vesicle transport phenotypes seen in Caenorhabditis elegans LRK-1 mutant could also be attributable to altered MT dynamics. These could all contribute to synaptic dysfunction. Futsch/MAP1B proteins are large multidomain proteins that are phosphorylated by multiple kinases, including Sgg/GSK-3β, PAR-1/MARK, and Cdk5, which also phosphorylate tau and are implicated in tau pathology. Tau-related pathology has been observed in LRRK2 transgenic animals. It would be interesting to test for possible interplay between LRRK2 and these other kinases in regulating MT-binding proteins and MT dynamics. In mammalian hippocampal neurons, overexpression of pathogenic hLRRK2 led to reduced neurite length and branching, whereas deficiency of LRRK2 had opposite effects. Whether MT dynamics regulated by Futsch/MAP1B is contributing to this LRRK2 effect in mammals will await additional investigation (Lee, 2010).
This study also showed that LRRK2 interacts with 4E-BP at the postsynapse. 4E-BP acts as a negative regulator of the translational initiation factor eIF4E through direct binding and sequestration. Phosphorylation of 4E-BP by LRRK2 weakens 4E-BP binding to eIF4E (Imai, 2008), therefore releasing the inhibitory effect of 4E-BP on eIF4E. Previous studies have demonstrated an important postsynaptic role for eIF4E-mediated protein synthesis in activity-dependent synaptic growth at the Drosophila NMJ (Sigrist, 2000). Genetic interaction studies demonstrate a functional role for 4E-BP in mediating the synaptic effects of LRRK2. However, the exact roles of 4E-BP and LRRK2 in this process appear complex. For example, (1) despite the prominent effects of 4E-BP overexpression on NMJ development, its loss of function has no obvious effect. One would expect a gain of eIF4E function in the absence of 4E-BP and therefore a synaptic-overgrowth phenotype. (2) 4E-BP activity is predicted to be high in dLRRK mutant and low in LRRK2 overexpression condition, but this study observed synapse phenotypes opposite of what is predicted based on the presumed roles of eIF4E and 4E-BP on Drosophila NMJ morphogenesis. One possible explanation of these seemingly disparate results is that phospho-4E-BP, the product of LRRK2-mediated phosphorylation of 4E-BP, instead of being inactive and inert, may actually perform some new synaptic function at the NMJ. In this scenario, loss of 4E-BP function in d4E-BP mutant would not show the same phenotype as LRRK2 overexpression, which produces more phospho-4E-BP. Recent studies in Drosophila dopaminergic neurons suggest a functional role for phospho-4E-BP in vivo. Alternately, effectors other than 4E-BP may also mediate the effects of LRRK2 on NMJ development and neurotransmission. Although 4E-BP overexpression might have masked the effects of these other effectors, in dLRRK mutant background, the functional roles of these other effectors might manifest themselves. A similar situation was observed in pumillo mutant, in which a synapse-loss phenotype was observed despite the upregulation of eIF4E activity in this mutant attributable to the derepression of translational inhibition of eIF4E, which would have resulted in a predicted synapse-overgrowth phenotype. Involvement of other effectors in mediating the effects of LRRK2 on NMJ development and neurotransmission, and possibly different effectors for NMJ development versus neurotransmission, could also explain the complex electrophysiological phenotypes of dLRRK mutant and LRRK2 overexpression animals, as well as the uncoupling of the effects on synaptic differentiation and neurotransmission by the various genetic manipulations. Future studies will test these possibilities as well as the relevance of the NMJ studies to dopaminergic neuron synapses (Lee, 2010).
In addition to LRRK2, the TOR pathway also regulates 4E-BP function through phosphorylation. This pathway primarily regulates cell and organism growth through diverse outputs, including protein synthesis, cytoskeletal change, autophagy, and cell survival. This study found that treatment of flies with rapamycin, an inhibitor of TOR, has the same effects as 4E-BP overexpression in wild type as well as LRRK2 overexpression backgrounds. Although rapamycin has been extensively studied in the context of autophagy induction and neurodegeneration models, its effect on Drosophila NMJ development is unlikely attributable to autophagy, because the current results show that presynaptic or postsynaptic induction of autophagy through Atg1 overexpression had no obvious effect on synapse number. The similar effects of rapamycin and 4E-BP overexpression on NMJ development support that rapamycin acts via the 4E-BP translational control pathway to impact NMJ development. A recent report showed that either 4E-BP overexpression or 4E-BP activation by rapamycin could suppress the muscle and dopaminergic neurodegeneration phenotypes seen in Drosophila Pink1 and Parkin models of PD. These results suggest that deregulation of protein synthesis could be generally involved in PD pathogenesis and that rapamycin or its analogs could be developed into effective PD therapeutics (Lee, 2010).
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