BIOLOGICAL OVERVIEW

The Drosophila Insulin receptor (InR) regulates cell growth and proliferation through the PI3K/Akt pathway, which is conserved in metazoan organisms. The Drosophila forkhead-related transcription factor Foxo is a key component of the insulin signaling cascade. Foxo is phosphorylated by Akt upon insulin treatment, leading to cytoplasmic retention and inhibition of its transcriptional activity. Mutant Foxo lacking Akt phosphorylation sites no longer responds to insulin inhibition, remains in the nucleus, and is constitutively active. Foxo activation in S2 cells induces growth arrest and activates two key players of the InR/PI3K/Akt pathway: the translational regulator d4EBP/Thor (eukaryotic initiation factor 4E binding protein) and the InR itself. Induction of d4EBP likely leads to growth inhibition by Foxo, whereas activation of InR provides a novel transcriptionally induced feedback control mechanism. Targeted expression of Foxo in fly tissues regulates organ size by specifying cell number with no effect on cell size. These results establish Foxo as a key transcriptional regulator of the insulin pathway that modulates growth and proliferation (Puig, 2003).

During the development of multicellular organisms, growth is tightly regulated by controlling cell number and cell size so that each organ reaches its appropriate dimensions in relation to the size of the organism. Many studies indicate that growth and proliferation are coordinated but distinct processes and that cells progress through the cell cycle only when sufficient mass, size, and macromolecular biosynthesis have been reached. Organism growth is controlled by coordinating both cell cycle progression and survival, which is modulated by nutrient availability, growth factors, and temperature. Growth factors can stimulate cell division and survival by activating the insulin receptor, which in turn acts through two main signal transduction cascades: the Ras/MAP kinase and the PI3K/Akt kinase pathways. Insulin-mediated activation of PI3K increases production of 3'-phosphorylated phosphoinositide lipids (PIP₃) that serve as second messengers to recruit Akt to the plasma membrane. Once properly localized in the membrane, Akt becomes activated by phosphorylation and in turn phosphorylates a number of downstream targets that ultimately regulate cell growth. For example, Akt stimulates protein synthesis through activation of the Target of rapamycin (TOR) kinase, which subsequently phosphorylates and inactivates the translational repressor eukaryotic initiation factor 4E-binding protein (4EBP) (Puig, 2003 and references therein).

Mammalian Akt, in addition to modulating translation, regulates transcription through the forkhead-related FOXO family of transcription factors FOXO1, FOXO3a, and FOXO4 (Burgering, 2002) by phosphorylating these proteins at three conserved serine/threonine residues. This leads to retention of FOXO transcription factors in the cytoplasm, thereby down-regulating RNA synthesis of specific target genes (Burgering, 2002) that affect cell cycle progression (Kops, 1999; Alvarez, 2001) and apoptosis (Brunet, 1999; Dijkers, 2000) and that modulate metabolic genes (Ayala, 1999; Durham, 1999; Guo, 1999; Hall, 2000; Nasrin, 2000; Schmoll, 2000; Nakae, 2001; Nadal, 2002). Thus, FOXO transcription factors play a critical role in regulating cell growth and survival (Puig, 2003).

Recent genetic studies in Drosophila and Caenorhabditis elegans show that the InR/PI3K/Akt signaling pathway is largely conserved in metazoans. In invertebrates, this pathway apparently plays an essential role in regulating life span as well as body, organ, and cell size. C. elegans can enter the dauer state when food is limited; when conditions improve, growth is stimulated by activating the InR/PI3K/Akt signaling pathway. Worms with mutations in this pathway are small, their organs have fewer cells, and they live longer. Interestingly, mutations in the transcription factor DAF-16/FOXO suppress this phenotype (Lin, 1997; Ogg, 1997), suggesting that this key transcription factor is negatively regulated by the InR/PI3K/Akt pathway (Burgering, 2002). In Drosophila, the InR/PI3K/Akt pathway is also thought to regulate body size and life span. Flies with heteroallelic combinations of InR mutations are reduced in size because of fewer and smaller cells. Mutations in other components of the dInR/dPI3K/dAkt pathway produce similar phenotypes. Although the identity and number of specific gene targets of the insulin signaling pathway in Drosophila remain unclear, one important downstream effector of insulin signaling appears to be the translational inhibitor d4EBP (Miron, 2001; Puig, 2003).

Despite the importance of the InR/PI3K/Akt pathway in regulating cell growth and proliferation in Drosophila, little is known about how signaling is controlled downstream of Akt. In C. elegans and mammals, a critical member of this pathway downstream of Akt is the transcription factor DAF-16/FOXO, which counteracts insulin signaling. However, the Drosophila equivalent of DAF-16/FOXO has thus far not been described. In addition, the mechanisms, if any, that are used to provide feedback regulation of the InR pathway are unknown. Drosophila Foxo proves to be a key transcriptional regulator that controls both downstream target genes responsible for growth as well as upstream feedback targets in the insulin signaling pathway (Puig, 2003).

Foxo functions in feedback regulation of the insulin signaling pathway. Surprisingly, Foxo transcriptionally activates downstream as well as upstream targets of the InR/PI3K/Akt signaling cascade, providing the first evidence for a transcriptional feedback mechanism in the InR pathway that regulates cell growth and proliferation. Furthermore, it was found that Foxo modulates the InR signaling pathway by transcriptionally activating two key elements of this signaling cascade: the downstream effector 4EBP and InR itself. Activation of InR provides an interesting way to modulate the InR/PI3K/Akt pathway via a feedback regulatory loop that may have important implications during development (Puig, 2003).

It has been shown that regulation of growth during development depends on the availability of nutrients and that food limitation decreases the Drosophila insulin-like peptide (DILP) levels. An activated InR pathway promotes growth, whereas mutations in this pathway impair normal development. For instance, flies mutant for chico, which encodes the Drosophila homolog of IRS1-4, are developmentally delayed, have severely reduced body size and increased fat accumulation. Likewise, mutations in several other components of the InR pathway produce related phenotypes. The finding that Foxo is involved with feedback activation of InR provides a novel mechanism for the cells to regulate growth by responding rapidly to changes in nutrient conditions. When nutrients are abundant, elevated levels of DILPs are secreted to activate the InR pathway, and the resulting downstream signaling promotes growth, in part by inhibiting Foxo. These favorable nutrient conditions allow growth and development. However, in a situation in which nutrients are limiting, DILPs are secreted at a reduced rate, the InR pathway is activated, and Foxo remains dephosphorylated, nuclear, and active. As a result, growth is inhibited, in part by Foxo activation of 4EBP. However, because Foxo is active when nutrients are limited, InR becomes up-regulated and primed to signal when triggered by changes in DILPs levels. In this way, when nutrient conditions change, the cells are highly sensitized and able to respond rapidly by turning on the mechanisms that stimulate growth, including shutting down Foxo via Akt phosphorylation. In addition, Foxo-mediated InR transcriptional activation presents a highly sensitive way to regulate the InR/PI3K/Akt signaling pathway in response to subtle developmental cues that modulate DILP levels. High levels of DILPs activate InR, which lead to the inhibition of its own transcription, turning off the pathway. Conversely, reduced levels of DILPs activate dInR transcription. This sensitizes the pathway and provides a mechanism to amplify growth factor signals by allowing detection of lower levels of DILPs. Once this pathway is activated, feedback regulation via Foxo dampens InR expression and signaling. Growth and development through the InR pathway is thus exquisitely balanced and regulated. Interestingly, overexpression of Foxo under the control of strong promoters (GMR, tubulin) in flies results in severe morphological defects. These results suggest that abnormally high levels of Foxo may produce growth arrest in developing organs. Surprisingly, flies with loss-of-function mutations in Foxo appear to develop normally, indicating that Foxo is not essential during fly development. These results suggest the existence of additional mechanisms that modulate insulin signaling and underscore the complexity of such developmental pathways (Puig, 2003).

In mammals, it has been reported that Akt promotes protein synthesis through TOR-mediated phosphorylation and subsequent inactivation of the translational inhibitor 4EBP. Hypophosphorylated 4EBP interacts strongly with eIF4E, providing a mechanism for Akt to regulate EBP via Tor. In flies, a similar mechanism has been reported. In contrast, the role of Foxo in transcriptionally modulating d4EBP has not been previously described. The finding that Foxo directly regulates 4EBP transcription provides an alternative and parallel mechanism for Akt to inhibit 4EBP function. Under conditions in which the insulin pathway is active, Akt sequesters Foxo in the cytoplasm, and 4EBP transcription is turned off. When the InR/PI3K/Akt pathway is inactive, Foxo is free to stimulate transcription of 4EBP and inhibit protein synthesis. Overexpression of 4EBP has been shown to slow growth in mammalian cells, and this study shows that Foxo overexpression leads to growth arrest in Drosophila S2 cells. It is therefore likely that up-regulation of 4EBP by Foxo contributes to the observed growth arrest. However, additional mechanisms (i.e., induction of apoptosis by Foxo), which could also contribute to the observed phenotype, cannot be ruled out (Puig, 2003).

It has been well documented that the InR/PI3K/Akt pathway regulates cell number and cell size in Drosophila. However, the precise mechanisms by which the insulin pathway controls these parameters remain unknown. Mutations in some members of this pathway affect cell size as well as cell number, whereas mutations in other members appear to affect only cell size. For example, mutations in both InR and PI3K produce smaller flies with reduced numbers of cells and smaller cell size. Mutations in the negative regulator PTEN produce bigger cells and increased proliferation. In contrast, mutations in Akt produce smaller organs without affecting cell number, only cell size. Overexpression of mutant 4EBP with increased binding affinity for eIF4E produces flies with smaller and fewer cells. Thus, until now, none of the components of the Drosophila InR pathway has been found to regulate only cell number without influencing cell size. It was therefore intriguing to find that overexpression of Foxo produces a reduction in cell number without any measurable effect on cell size. This is reminiscent of the transcription factor c-myc, which in mammals regulates cell number without altering cell size but in Drosophila affects both cell size and number. Taken together, the results of this study reveal the species specific complexity of the mechanisms that regulate cell growth and proliferation. Indeed, the results suggest that the InR/PI3K/Akt pathway is far from being a simple linear cascade. Instead, Akt appears to regulate numerous targets, each one with its own set of downstream effectors. In addition, it is conceivable that Foxo may be regulated by kinases other than Akt. In mammals, FOXO4 has been shown to be regulated by the Ras/MAP kinase pathway (Kops, 1999), and a similar mechanism may exist in flies. Interestingly, microarray experiments identified >200 genes in addition to 4EBP that are up-regulated by Foxo, which increases the complexity of transcriptional regulation affected by Foxo. Additional studies will be necessary to determine the multiple mechanisms by which the insulin signaling cascade dictates cell number and size during development of the metazoan body plan (Puig, 2003).

Genetic studies in C. elegans and Drosophila have led to two models regarding the output of the insulin pathway. (1) The complete epistasis of daf-16, coding for the Foxo homolog, over the insulin pathway mutants daf-2, age-1, akt-1 and akt-2 suggests that the primary function of PKB/AKT is to inactivate FOXO transcription factors (Paradis, 1998). (2) It has been proposed that the TSC tumor suppressor complex is the major target of PKB in the regulation of cell growth in Drosophila. Analysis of Drosophila Foxo indicates that it is indeed a critical PKB target, but that it mediates only one aspect of PKB function. Several lines of evidence support this model. (1) The effects of ectopic overexpression of Foxo and hFOXO3a in the developing Drosophila eye are altered by Dp110 and PKB signaling as well as by nutrient levels. Under conditions of lowered insulin signaling, the phenotypes resulting from expression of foxo and hFOXO3a are dramatically enhanced. This situation was mimicked by expressing a PKB-insensitive phosphorylation mutant, suggesting that endogenous PKB signaling is required to mitigate the effects of ectopically expressed Foxo and hFOXO3a. (2) The physiological relevance of Foxo in PKB signaling is most vividly demonstrated by the observation that the larval lethality associated with the complete loss of PKB is rescued by foxo mutations to the extent that some flies develop to pharate adults. The lethality associated with loss of PKB function is therefore to a large extent due to the hyperactivation of Foxo. (3) Loss of Foxo function suppresses the effects of insulin-signaling mutations only partially; Foxo mediates a reduction in cell number but not in cell size in response to reduced insulin signaling (Jünger, 2003).

Genetic analysis of the control of body size in Drosophila has revealed two classes of mutations. Flies carrying mutations in chico or viable allelic combinations of Inr, Dp110, and PKB are reduced in body size by up to 50% owing to a reduction in both cell size and cell number. Conversely, flies mutant for S6K exhibit a more moderate reduction in body size, caused almost exclusively by a reduction in cell size. This suggests that the pathways controlling cell number and cell size bifurcate at or below PKB. Although foxo single mutants have no obvious size phenotype, loss of foxo substantially suppresses the cell-number reduction observed in insulin-signaling mutants. It appears that Foxo mediates the repression of proliferation in flies mutant for Inr, chico, Dp110, and PKB without being required for the reduction in cell size. Chico-Foxo double mutant flies even have slightly smaller cells than chico mutants, suggesting that removal of Foxo permits cell-cycle acceleration under conditions of impaired insulin signaling. The pathway controlling body size in response to insulin therefore bifurcates at the level of PKB: PKB controls cell number by inhibiting Foxo function and PKB controls cell size, at least under some conditions, by regulating S6K activity by phosphorylation of TSC2 (Jünger, 2003).

The signaling systems controlling cell size and cell number are tightly interconnected. Genetic and biochemical analyses have revealed five different links between the TSC-TOR-S6K pathway and the Inr-PKB-Foxo pathway. (1) Under conditions of unnaturally high insulin-signaling activity (that is, following the oncogenic activation of PKB) PKB phosphorylates and inactivates TSC2, resulting in increased activation of S6K. Under normal culture conditions this regulation does not seem critical, however, loss of dPKB function does not lower dS6K activity in larval extracts. (2) Under physiological conditions, PDK1 regulates PKB as well as S6K. (3) S6K itself downregulates dPKB activity in a negative feedback loop. (4) Under severe starvation conditions, nuclear Foxo presumably activates target genes that reduce cell proliferation. One of these target genes is 4E-BP, which encodes an inhibitor of translation initiation. When conditions improve, the insulin and TOR signaling pathways can stimulate translation by disrupting the 4E-BP/eIF4E complex via phosphorylation of 4E-BP, and in parallel by repressing FOXO-dependent 4E-BP expression. (5) Under even more severe starvation or stress conditions, full activation of Foxo upregulates expression of the insulin receptor itself, thus rendering the cell hypersensitive to low insulin levels. These multiple positive and negative interactions ensure a continuous fine adjustment of the growth rate to changing environmental conditions (Jünger, 2003).

Genetic dissection of signaling by insulin and its target DAF-16 has been pioneered in C. elegans and has helped to unravel the role of this pathway in dauer formation and longevity. The same pathway with the homologous nuclear targets operates in flies in the control of cell growth and proliferation, processes that do not involve insulin signaling in worms. Dauer formation and possibly longevity affect the entire organism and do not depend on cell-autonomous functions of the insulin signaling pathway. The cell-growth phenotype in Drosophila, however, depends on the cell-autonomous functioning of the insulin-signaling cascade. Insects enter diapause in response to diverse environmental cues (nutrients, day length or temperature) and arrest development or the aging process in a manner similar to dauer formation in worms. Ageing, and possibly diapause, is also under the control of the insulin pathway in Drosophila. It has recently been shown that heterozygous IGF-1R mutant mice also exhibit a prolonged lifespan. It therefore appears that the function of the insulin pathway, its components, and possibly at least some of its targets, have been conserved throughout evolution (Jünger, 2003).

The longevity phenotype of IGF-1R-deficient mice is associated with enhanced resistance to oxidative stress. It is likely that this phenomenon is due to hyperactivation of FOXO proteins, since several studies have shown that FOXO transcription factors play a role in the oxidative-stress response in mammalian cells as well as in C. elegans. The observation that foxo mutant flies are hypersensitive to oxidative stress confirms that, in addition to their role in insulin signaling, the role of FOXO proteins in protecting against cellular stress is highly conserved. The mechanism by which Foxo confers oxidative-stress resistance is not yet known. In a microarray experiment, several genes encoding cytochrome P450 enzymes were identified as Foxo target gene candidates. Since it has been shown that cytochrome P450 enzymes reduce the toxic effects of paraquat in mice, they might partially mediate the protective effect of Foxo. Furthermore, it remains to be established whether the regulation of Foxo by insulin is required for Foxo's protective properties. It is tempting to speculate that distinct stress-induced signaling pathways activate Foxo under conditions of cellular stress, in addition to the negative input from the insulin cascade, since several stress-induced phosphorylation sites are conserved between hFOXO3a and Foxo. This view is supported by the observation that overexpression of a FOXO variant that cannot be inactivated by PKB elicits cell death, a phenotype not observed in larval tissues lacking insulin-signaling components. This result argues that Foxo induces cellular responses that are independent of insulin (Jünger, 2003).

The emerging model postulates that positive and negative inputs converge on FOXO proteins in response to different environmental conditions, making them central and important integrators controlling cellular (cell-cycle progression) and organismal adaptations (dauer formation, diapause and longevity. Elucidating the positive inputs that converge on FOXO, by mutating conserved phosphorylation sites in the single Drosophila homolog of this class, should help arrive at a better understand Foxo's integrator function (Jünger, 2003).

GENE STRUCTURE

cDNA clone length - 3631 pb

Bases in 5' UTR - 522

Exons - 16

Bases in 3' UTR - 712

PROTEIN STRUCTURE

Amino Acids - 613

Structural Domains

Homology searches using FOXO4 or its DNA-binding domain reveal the presence of only one Drosophila gene with high similarity to FOXO transcription factors. Several cDNA clones were sequenced, and the longest open reading frame encodes a protein of 613 amino acids with a calculated molecular mass of 67,412.12 D. This protein has been designated 'Foxo'. The foxo gene contains 11 exons spanning 40 kb. The DNA-binding domain is located in the N-terminal region and has 45% identity with human FOXO4 (84% identity in the region that spans the three alpha-helices constituting the forkhead core domain. The homology with two other members of the FOXO family, FOXO1 and FOXO3a, is also significant in the DNA-binding domain. All four proteins share a characteristic five-amino-acid insertion (SNSSA) between alpha-helices 2 and 3 of the forkhead domain, which differentiates this subset from other forkhead-related family members. Importantly, the three residues phosphorylated by Akt in FOXO4 are conserved in Drosophila Foxo: T28, S193, and S258 in FOXO4 correspond to T44, S190, and S259 in Drosophila Foxo. The amino acids that define the motif recognized by Akt (RXRXXS/T) are also conserved. Drosophila Foxo has a C-terminal region with a high Ser and Gln content (11.7% and 13.4%, respectively), commonly found in transcription activation domains (Puig, 2003).

The Drosophila genome contains a single homolog of the DAF-16/FOXO family of transcription factors. This notion is supported by phylogenetic analysis calculated from a multiple sequence alignment. The foxo gene is more closely related to the mammalian FOXO subfamily and daf-16 than any other Drosophila forkhead gene. The amino-acid sequences of the predicted 613 amino-acid Foxo protein and hFOXO3a are 27% identical over the full protein length, and 82% identical within the forkhead DNA-binding domain. Furthermore, foxo is the only Drosophila forkhead gene encoding a putative protein containing conserved PKB phosphorylation sites. The orientation of the three PKB consensus sites relative to the forkhead domain is conserved among the mammalian FOXO proteins, DAF-16 and Foxo. There is a high degree of sequence conservation between Foxo and FOXO/DAF-16 proteins within the DNA-binding domain. Taken together, these observations strongly suggest that Foxo is the only Drosophila homolog of the mammalian FOXO transcription factors and C. elegans DAF-16 (Jünger, 2003).

The Drosophila foxo gene consists of 10 exons and is spread out over approximately 31 kb in polytene chromosome section 88A within the genomic scaffolding region, AE003703, of the Berkeley Drosophila Genome Project (BDGP). foxo encodes a theoretical protein of 463 amino acids. Analysis of the complete Drosophila genome for additional FOXO homologues revealed none. Alignment of Foxo with the human homologs of FOXO and Daf-16a1 using ClustalW reveals that although the overall identity of amino acids is not high, the identity in the forkhead box DNA binding domain is between 74 and 86 percent. The Akt phosphorylation sites are also well conserved in their relative position in the protein, and in sequence. The T1 site is located at T24 in Foxo, the S1 site at S160, and the S2 site at S239. These sites align well with the human FOXO homologs in the ClustalW alignment, however the Daf-16 S1, and S2 sites are slightly out of line. All three of the potential Akt phosphorylation sites in Foxo fit the Akt consensus target sequence (RxRxxS/T) (Kramer, 2003).

Other notable features found in FOXO homologs include a DYRK1a phosphorylation site, a 14-3-3 binding site, a nuclear localization signal (NLS), a nuclear export signal (NES), and Ral dependent phosphorylation sites. A DYRK1a phosphorylation site has confirmed experimentally in FOXO1 at S329. This serine residue is conserved in human FOXO3a (S324), FOXO4 (S267), Daf-16a1 (S317), and Foxo (S248). In addition, the sequence surrounding this site in Foxo (LS²⁴⁸PI) is identical to that in FOXO1. The high conservation of this sequence indicates that Foxo may be phosphorylated at this site by the Drosophila homolog of DYRK1a, minibrain (mnb) (Kramer, 2003).

Binding to 14-3-3 proteins is thought to be an important part of FOXO sequestration. 14-3-3 proteins normally bind to a consensus site containing a phosphoserine residue, either RSxS^PxP, or RxxxS^PxP. In the case of Drosophila Foxo, the sequence surrounding the T1 Akt phosphorylation site fits the former perfectly, aside from the substitution of a threonine for a serine. It has been shown experimentally that 14-3-3 does bind to this site in FOXO1, FOXO3a, and Daf-16, hence, it is likely that this region functions as a 14-3-3 binding site in Drosophila (Kramer, 2003).

The current model for FOXO deactivation suggests that a NES exists which causes constitutive localization of FOXO in the cytoplasm in the absence of a functional NLS. A non-conventional NLS was identified in human FOXO4 from amino acids 180-221. The corresponding sequence in Foxo (amino acids 147-194) is 38% identical and 66% similar in amino acid content. This similarity suggests that this region may act as an NLS in Foxo as well. A leucine rich NES has been identified in FOXO1 (368 MENLLDNLNL 377) and the conservation of this sequence is quite high FOXO3a, FOXO4, and Daf-16. The corresponding region in Foxo retains two of the important leucine residues (281 LTGTMADELTL 291). However, the remaining sequence is more divergent, and may or may not act as an NES in Drosophila (Kramer, 2003).

FOXO4 has previously been shown to be phosphorylated in a Ral-dependent manner at threonines 447 and 451. However, these sites do not appear to be conserved in the other human FOXO homologs, Daf-16, or Foxo, indicating that Ral dependent phosphorylation of FOXO may be specific to FOXO4 (Kramer, 2003).

Interestingly, the carboxy-terminal three amino acids are conserved between Foxo and FOXO1 (VSG). Also, FOXO3a contains a similar sequence in the final three amino acids (VPG). In view of this conservation, it is possible that this tail plays a functional role in Foxo regulation (Kramer, 2003).

date revised: 5 December 2003

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