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).
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).
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