Phosphotidylinositol 3 kinase 92E

PI3K92E and nutritional status

Studies in Drosophila have characterized insulin receptor/phosphoinositide 3-kinase (Inr/PI3K) signaling as a potent regulator of cell growth, but an understanding of its function during development has remained uncertain. Inhibiting Inr/PI3K signaling phenocopies the cellular and organismal effects of starvation, whereas activating this pathway bypasses the nutritional requirement for cell growth, causing starvation sensitivity at the organismal level. Consistent with these findings, studies using a pleckstrin homology domain-green fluorescent protein (PH-GFP) fusion as an indicator for PI3K activity show that PI3K is regulated by the availability of dietary protein in vivo. It is surmised that an essential function of insulin/PI3K signaling in Drosophila is to coordinate cellular metabolism with nutritional conditions (Britton, 2002).

To test whether PI3K is required for larval growth, its activity was inhibited by expressing p60, Deltap60, or Pten in large domains of the larva using the Gal4/UAS system. p60 is an adaptor that couples Inr to Pi3K92E (Dp110), and Deltap60 is a deletion variant lacking part of the Dp110 binding domain. These molecules and their mammalian homologs have dominant-negative effects on PI3K activity when overexpressed, presumably because they compete with endogenous Dp110/p60 complexes for binding sites on upstream activators such as insulin receptors and IRSs. When p60 is expressed under the control of Act-Gal4 (expressed ubiquitously) or Adh-Gal4 (expressed predominantly in fat body), larvae remain growth arrested in the first instar for as long as 2 weeks. Deltap60 and Pten had similar effects. Dissection of these animals revealed that all of their tissues and organs were proportionally reduced. This developmental arrest is indistinguishable from the effects of starvation or inhibition of protein synthesis (Britton, 2002).

To test whether PI3K is autonomously required for growth in the different larval cell types, the Flp/Gal4 technique was used to express p60, Deltap60, or Pten in scattered cells throughout the larva. This method employs the Act>CD2>Gal4 and hs-Flp transgenes to activate UAS-linked target genes, including the cell marker UAS-GFPnls, in cell clones. Heat shock-independent activation of Gal4 occurs prior to the onset of larval growth and DNA endoreplication in 1%-10% of cells (depending on organ) in the fat body, gut, salivary glands, renal (Malpigian) tubules, and epidermis. Cells overexpressing p60, Deltap60, or Pten in the salivary glands and fat body are greatly reduced in size and have much smaller nuclei with far less DNA than adjacent control cells. Similar effects are observed in other larval tissues. Despite their reduced growth, p60-, Deltap60-, and Pten-expressing cells are found at approximately the same frequencies as control GFP-marked cells. Apoptotic cells are not observed. Thus, reductions in PI3K activity are not incompatible with cell viability. Overt effects on cell morphology that might reflect changes in cell adhesion, motility, or identity are also not observed. It is concluded that reducing Inr/PI3K activity in the differentiated tissues of the larva has cell-autonomous effects that are limited to reducing cell growth and DNA replication (Britton, 2002).

These observations suggest that PI3K activity might be responsive to nutritional conditions. To directly test this possibility, a fusion protein was made for use as an in vivo reporter for PI3K activity. The pleckstrin homology (PH) domain of the Drosophila homolog of general receptor for phosphoinositides-1 (GRP1) was fused to green fluorescent protein (GFP), generating a protein called GPH (GFP-PH domain). PH domains from mammalian GRP1 genes bind specifically to phosphatidylinositol-3,4,5-P₃ (PIP₃), the second messenger generated by class I PI3-kinases. Since PIP₃ generally resides in lipid membranes, particularly the plasma membrane, GRP1 is recruited to membranes when PI3-kinase activity raises cellular levels of PIP₃. Fusion proteins containing the GRP1 PH domain are likewise recruited to plasma membranes by binding PIP₃, and thus serve as in situ reporters for PI3K activity (Britton, 2002).

For in vivo studies, the GPH gene was placed under control of the Drosophila ß-tubulin promotor, generating a gene called 'tGPH' (tubulin-GPH), and introduced into Drosophila by P element-mediated transformation. Membrane localization of tGPH was observed in the larval epidermis, fat body, salivary glands, malpighian tubules, and wing imaginal discs. Cytoplasmic and nuclear tGPH was also visible in these cell types. The degree of membrane localization depends upon the developmental stage. Epidermal cells show little membrane localization of tGPH in embryos or newly hatched first instar (L1) larvae, but have strong membrane localization in second (L2) and early third (L3) instar larvae. Later, in wandering stage L3 larvae, membrane-associated tGPH is again diminished. Similar trends are observed in the fat body. These variations might reflect changes in the levels of Inr, PI3K, Pten, or insulin-like peptides (dILPs) in the larva as it feeds and grows (Britton, 2002).

To test whether tGPH localization was responsive to PI3K activity in vivo, Inr or Dp110 was overexpressed using the Gal4 system. Either gene causes a striking redistribution of tGPH to plasma membranes in cells of the fat body, epidermis, Malpighian tubules, gut, and imaginal discs. To determine whether endogenous PI3K is responsible for the membrane localization of tGPH, PI3K activity was suppressed by expressing p60 with the heat-inducible driver hs-Gal4. In the larval epidermis, partial loss of membrane-bound tGPH is apparent 1.5 hr post-heat shock (phs), and by 4 hr, phs tGPH is nearly completely lost from cell membranes. Similar results were obtained in the fat body when either p60 or dPten were expressed mosaically using the Flp/Gal4 method (Britton, 2002).

To determine whether PI3K activity is nutritionally modulated, tGPH localization was monitored after starvation. Early L2 larvae (48-60 hr AED) were deprived of dietary protein by culture on either 20% sucrose or Sang's defined media lacking casein. These treatments arrest cell growth in all of the differentiated larval tissues. L2 larvae fed on either protein-free diet survive for up to 14 days, but a marked shrinkage of cells in the epidermis and fat body is observed. In the epidermis of early L2 larvae, culture on either protein-free diet causes membrane-bound tGPH to be diminished after 24 hr, and to be nearly undetectable after 48 hr. Levels of total tGPH also decrease after protein deprivation, but nuclear and cytoplasmic tGPH remain detectable for more than 6 days. Loss of membrane-associated tGPH also occurs in fat body cells of protein-deprived L2 larvae, with similar kinetics. Starvation causes shrinkage of the nuclei and nonlipid cytoplasm in fat body cells, leaving large tGPH-negative lipid droplets that occupy most of the cell volume. To test whether membrane association of tGPH is reversible, L2 larvae were cultured on 20% sucrose for 8 days and then returned to whole food. In this experiment, tGPH levels rose and the protein reassociated with plasma membranes in epidermal cells between 24 and 48 hr after feeding. These results indicate that cellular levels of PIP₃ drop as a consequence of starvation for dietary protein (Britton, 2002).

Terminally differentiated endoreplicating tissues (ERTs) constitute most of a Drosophila larva, and the growth of these tissues accounts for virtually all of the ~200-fold mass increase sustained by the animal during the larval stages. During larval life, the ERTs provide a physiologically nurturing environment for undifferentiated imaginal cells and neuroblasts, which generate much of the reproductive adult stage. Most of the biomass accumulated in the ERTs is eventually recycled into these progenitor cells as they form the adult. This study provides evidence that Inr/PI3K signaling coordinates nutritional status with ERT cell metabolism and growth. To determine whether Inr/PI3K signaling can maintain cell growth in the face of starvation, Flp/Gal4 was used to express Dp110 or Inr in scattered ERT cells, and then changes in cell size and DNA replication were assessed at time points during a protein starvation regime. At larval hatching, prior to starvation, cells expressing Dp110 or Inr in the gut, fat body, Malpighian tubules, and epidermis are only slightly larger than nonexpressing cells. After several days of starvation on 20% sucrose, Inr- or Dp110-expressing cells in these organs are much larger than adjacent control cells, and have visibly increased DNA content. BrdU incorporation indicated that gut and fat body cells expressing Dp110 or Inr continue to replicate their DNA for at least 2 to 3 days under starvation conditions. Normally, DNA endoreplication in these cells ceases within 1 to 2 days of starvation (Britton, 1998). A catalytically inactive PI3K, Dp110^D945A, does not promote cell growth or DNA endoreplication, indicating that lipid kinase activity was required. These experiments indicate that active Inr/PI3K signaling is sufficient to bypass the nutritional requirement for cellular growth and DNA replication in many larval cell types, and that this effect is cell autonomous (Britton, 2002).

To explore the means by which Inr/PI3K signaling induces cell growth, an examination was carried out of the morphology of fat body cells in which PI3K activity had been manipulated. Fat body cells accumulate large stores of protein, carbohydrate, and lipids during larval life, and also produce growth factors. During the third larval instar, these accumulations of nutrients cause fat body cells to become opaque. These nutrients are normally utilized during metamorphosis, but if a larva is starved, they are precociously mobilized into the haemolymph to support the animal during the ensuing dietary crisis. This causes the fat body cells to shrink and become clear as they lose organelles by autophagy and deplete stored metabolites (Britton, 2002).

Expression of Inr or PI3K in fat body cells increases the opacity of the cytoplasm, and thus promotes nutrient storage. A similar cytoplasmic effect is observed in intestinal cells from L1 animals. Close inspection has revealed that in both cell types, ectopic Inr or PI3K decreases the size of prominent vesicles in the cytoplasm. Induction of p60 in early L3 larvae has opposite effects, causing fat body cells to become more translucent. A loss of opacity was observed in fat bodies from Dp110 mutants after their growth arrest at L3. In further tests, fat body cells were stained for lipids with Nile red or for protein with Texas red X succinimidyl ester. This revealed that the cytoplasm of Inr-expressing cells contains many more, but much smaller, lipid droplets than neighboring control cells. In summary, activation of the Inr/PI3K pathway has profound effects on cytoplasmic composition. These effects mimic changes in the fat body that normally take place late in the L3 stage when nutrient storage by these cells is maximal. Suppression of Inr/PI3K activity has opposite effects on cytoplasmic composition, and these appear to mimic the mobilization of nutrients that normally accompanies starvation (Britton, 2002).

Considering the above results, it should be advantageous to larvae to downregulate insulin/PI3K signaling when nutrients are limited, since this would suppress nutrient storage and cell growth and allow nutrient mobilization by tissues such as the fat body. This idea was tested by hyperactivating Inr/PI3K signaling and then tracking development under different nutritional conditions. Several Gal4 drivers were used to induce expression in large numbers of cells, including Adh-Gal4 (expressed in the fat body, trachea, and a few cells in the gut), en-Gal4 (expressed in posterior epidermal cells, the hindgut, and some neural cells), Act-Gal4 (expressed ubiquitously), and hs-Flp/Act>Cd2>Gal4 (induced by heat shock in all cells). In several cases, overexpressed Dp110 and Inr were tolerated in feeding animals. For instance, animals expressing Dp110 under Adh-Gal4 or en-Gal4 control developed without delay and eclosed at the same frequency as controls, giving viable fertile adults. Inr was more deleterious, but some animals expressing Inr under Adh-Gal4 control developed to the L3 stage and a few viable adults eclosed. Ubiquitous expression of Dp110 or Inr using the Act-Gal4 driver, however, was 100% lethal at prelarval stages (Britton, 2002).

In contrast, hyperactivating Inr/PI3K signaling under starvation conditions was catastrophic. When Adh-Gal4 was used to drive Dp110 or Inr expression, for instance, L1 larvae raised on the sucrose/PBS diet all perished within 3 to 4 days of hatching, whereas control animals survived 8 to 9 days. Animals expressing Dp110 under en-Gal4 control also perished within 2 to 3 days, 4 to 5 days before controls, when deprived of dietary protein. Suppressing PI3K activity by expressing p60, Deltap60, Pten, or Dp110^D945A using Adh-Gal4, en-Gal4, or even Act-Gal4 had no effect on viability under starvation conditions. These results suggest that the starvation sensitivity caused by high Inr/PI3K activity is specifically related to nutrient uptake and storage, functions that appear to be unique to Inr and PI3K (Britton, 2002).

These results demonstrate that downregulation of Inr/PI3K activity is critical to maintaining metabolic homeostasis under starvation conditions. The remarkable ability of Inr/PI3K-expressing cells to continue stockpiling nutrients and grow, even in starved animals, may account for the starvation sensitivity observed at the organismal level. This idea was supported by observations made in starved tGPH larvae that overexpressed Dp110 in posterior compartment epidermal cells (genotype, en-Gal4 UAS-Dp110 tGPH). In these animals, high levels of membrane-bound tGPH persisted in Dp110-expressing epidermal cells until 2 days after nutrient withdrawal, at which point the animals died. In anterior (A) cells, which did not overexpress Dp110, tGPH was completely lost from plasma membranes within 18 hr after nutrient deprivation, and tGPH protein became undetectable by 36 hr. This is a much more rapid starvation response than observed in animals that did not contain Dp110-expressing cells. Anterior epidermal cells also shrank rapidly during starvation, whereas posterior, Dp110-expressing cells maintained their large size. These effects might result from the rapid depletion of nutrients by the PI3K-expressing cells, and a consequent drop in levels of hemolymph insulins (Britton, 2002).

In performing these experiments, it was noticed that larvae that overexpress Inr or Dp110 wander away from their food. To more carefully analyze this phenotype, animals expressing various PI3K signaling components under Adh-Gal4 control were cultured on agar plates with red-colored food (yeast paste) in the center for ~24 hr after hatching. These animals were then scored for the presence of red food in the gut as well as their proximity to the food source. Animals overexpressing Inr, Dp110, or Dp110^CAAX feed poorly (i.e., often have no food in the gut) and frequently wander away from their food. Similar aberrant behaviors were observed when Dp110 was expressed ubiquitously using Flp/Gal4, in which case nearly all animals wandered out of the food and pupated precociously. Thus, elevated levels of Inr/PI3K signaling alter larval feeding behavior, perhaps by affecting the animal's perceived level of hunger (Britton, 2002).

Is nutrition sensed in Drosophila at the cell level or by the animal as a whole? Although animal cells can sense amino acids directly, these studies suggest that cells in Drosophila larvae sense and respond to changes in dietary protein indirectly, using secondary humoral signals -- most likely insulins -- long before they become acutely starved for amino acids. In support of this idea, some cells in the larva can continue to grow and replicate their DNA long after the animal is deprived of dietary protein. Imaginal cells and neuroblasts do this, as do ERT cells in which Inr or PI3K have been artificially switched on. This attests to the fact that starvation for dietary protein does not completely deplete the larval hemolymph of amino acids or cause a global shutdown of protein synthesis. This is probably possible because nutrients stored in ERTs such as the fat body are mobilized during starvation to maintain levels of hemolymph nutrients. Nevertheless, starvation does cause a rapid, global shutdown of ERT cell growth, and thus some essential signal is lost (Britton, 2002).

One factor that all animal cells use for nutritional sensing is TOR (target of rapamycin), a protein kinase that mediates diverse effects on cell metabolism including protein synthesis, amino acid import, ribosome biogenesis, and autophagy (Raught, 2001; Schmelzle, 2000). What role might Drosophila TOR (dTOR) play in the nutrition response system addressed here? The mechanism by which TOR 'senses' nutrition remains uncertain (Kleijn and Proud, 2000), but cellular levels of amino acids, aminoacylated tRNAs, and ATP have been suggested as direct inputs (Dennis, 2001; Iiboshi, 1999). Drosophila dTOR mutations or the TOR-specific inhibitor rapamycin inactivate the TOR target S6-kinase and phenocopy starvation in fed larvae (Zhang, 2000; Oldham, 2000). Starvation, however, does not completely inactivate S6K, suggesting that dTOR retains some activity under starvation conditions (Oldham, 2000). Consistent with this interpretation, overexpressed PI3K is a potent promotor of cell growth in starved larvae, but PI3K cannot drive cell growth in dTOR mutant larvae (J. Lande and T. Neufeld, personal communication to Britton, 2002). This suggests that although dTOR may act as a cell-autonomous nutrient-dependent checkpoint for metabolism, the larva's physiology is so effective in buffering cells against absolute starvation that this checkpoint is rarely if ever fully engaged. TOR is found in fungi and plants and so seems to be a metabolic regulator that was used prior to the advent of multicellularity. Insulin signaling, which is absent from fungi and plants, probably evolved later when multicellular animals required a system to coordinate and fine-tune metabolism in communities of cells. The insulin system is clearly advantageous, since animals in which it is 'short-circuited' by hyperactivation of Inr or PI3K are unable to tolerate even brief periods of starvation (Britton, 2002).

The most direct evidence that insulin signaling is nutritionally controlled was obtained using a cellular indicator of PI3K activity, tGPH, which is recruited to plasma membranes by the second messenger product of PI3K, PIP₃. Subcellular tGPH distributions indicate that PIP₃ levels are high in many cell types in fed larvae, but low in larvae that have been starved for protein. Although these changes in PIP₃ levels might be due to altered expression of Inr, PI3K, or Pten, expression profiling experiments using cDNA microarrays indicate that levels of p60 and Dp110 mRNA are not depressed in L2 larvae that had been deprived of protein for 4 days. Perhaps the most attractive explanation for the apparent loss of PIP₃ upon starvation is that some of the seven Drosophila insulin-like peptides (dILPs) are produced in a nutrition-dependent fashion. Several of the dilp genes are expressed in the larval gut (Brogiolo, 2001) which, as the conduit of nutritional influx, might be expected to mediate metabolic responses to feeding throughout the animal. Other dilps are expressed in the salivary glands, imaginal discs, and small numbers of cells in the central nervous system (Brogiolo, 2001; Britton, 2002).

In mammals, insulin promotes the cellular uptake and storage of carbohydrates, proteins, and lipids, and is the strongest anabolic inducer known. Insulin-mediated responses are indirectly antagonized by the hormone glucagon, which stimulates catabolic reactions and the mobilization of stored nutrients. Insulin and glucagon are produced in the pancreas, and their relative levels are constantly adjusted to maintain proper blood sugar levels. The mammalian liver is also a key player in the regulation of metabolic homeostasis. In humans, most of the accessible glycogen, the principle form of stored carbohydrate, is found in the liver. This glycogen can be mobilized in response to exercise or starvation. In Drosophila larvae, hyperactivation of the Inr/PI3K pathway leads to increased accumulation of nutrients in the fat body, an organ that resembles the mammalian liver as the principal site of stored glycogen. Conversely, inhibition of PI3K activity depletes stored nutrients from the fat body, as does starvation. This suggests that like mammals, insects regulate storage of metabolites in response to changes in levels of Inr/PI3K signaling. Direct assays of the levels of carbohydrates, storage proteins, and lipids in the fat body after starvation or manipulation of Inr/PI3K activity should prove informative. While there are as yet no known Drosophila homologs of glucagons, there must be some mechanism by which stored resources can be mobilized during starvation or at the transition from feeding to metamorphosis (Britton, 2002).

A nutrient sensor mechanism controls Drosophila growth

Organisms modulate their growth according to nutrient availability. Although individual cells in a multicellular animal may respond directly to nutrient levels, growth of the entire organism needs to be coordinated. This study provides evidence that in Drosophila, coordination of organismal growth originates from the fat body, an insect organ that retains endocrine and storage functions of the vertebrate liver. A genetic screen for growth modifiers discovered slimfast, a gene that encodes an amino acid transporter. Remarkably, downregulation of slimfast specifically within the fat body causes a global growth defect similar to that seen in Drosophila raised under poor nutritional conditions. This involves TSC/TOR signaling in the fat body, and a remote inhibition of organismal growth via local repression of PI3-kinase signaling in peripheral tissues. These results demonstrate that the fat body functions as a nutrient sensor that restricts global growth through a humoral mechanism (Colombani, 2003).

In multicellular organisms, the control of growth depends on the integration of various genetic and environmental cues. Nutrient availability is one of the major environmental signals influencing growth and, as such, has dictated adaptative responses during evolution toward multicellularity. In particular, complex humoral responses ensure that growth and development are properly coordinated with nutritional conditions (Colombani, 2003).

In isolated cells, amino acid withdrawal leads to an immediate suppression of protein synthesis, suggesting that cells are protected by active sensing mechanims that block translation prior to depletion of internal amino acid stores. In many mammalian cell types, changes in amino acid diet affect the binding of the translation repressor 4EBP1 to initiation factor eIF4E and the activity of ribosomal protein S6 kinase (S6K). These two signaling events require the activity of TOR (target of rapamycin), a conserved kinase recently shown to participate in a nutrient-sensitive complex both in mammalian cells and in yeast. Mutations in the Drosophila TOR homolog (dTOR) results in cellular and physiological responses characteristic of amino acid deprivation and establish that TOR is cell autonomously required for growth in a multicellular organism. Furthermore, the TSC (tuberous sclerosis complex) tumor suppressor, consisting of a TSC1 and TSC2 heterodimer (TSC1/2), as well as the small GTPase Rheb participate to the regulation of TOR function. Overall, these data suggest that TSC, Rheb, TOR, and S6K participate in a conserved pathway that coordinates growth with nutrition in a cell-intrinsic manner (Colombani, 2003).

In multicellular organisms, humoral controls are believed to buffer variations in nutrient levels. However, little is known about how growth of individual cells is coordinated. In vertebrates, growth-promoting action of the growth hormone (GH) is mostly relayed to peripheral tissues through the production of IGF-I. Binding of IGF-I to its cognate receptor tyrosine kinase (IGF-IR) induces phosphorylation of insulin receptor substrates (IRS), which in turn activate a cascade of downstream effectors. These include phospho-inositide 3-kinase (PI3K), which generates the second messenger phosphatidylinositol-3,4,5-P₃ (PIP₃), and thereby activates the AKT/PKB kinase. Genetic manipulation of IGF-I, IGF-IR, PI3K, and AKT in mice modulates tissue growth in vivo thus demonstrating a requirement of the IGF pathway for growth. In Drosophila, both loss- and gain-of function studies have also exemplified the role of a conserved insulin/IGF signaling pathway in the control of growth. Ligands for the unique insulin receptor (Inr) constitute a family of seven peptides related to insulin, the Drosophila insulin-like peptides (Dilps). Remarkably, three dilp genes (dilp2, dilp3, and dilp5) are expressed in a cluster of seven median neurosecretory cells (m-NSCs) in the larval brain, suggesting that they have an endocrine function. Indeed, ablation of the seven dilp-expressing mNSCs in larvae induces a systemic growth defect (Colombani, 2003).

Both in flies and mice, mutations in IRS provoke growth retardation as well as female sterility similar to what is observed in starved animals. Moreover, PI3K activity in Drosophila larvae depends on the availability of proteins in the food. Overall, this supports the notion that the insulin/IGF pathway might coordinate tissue growth with nutritional conditions. However, upon amino acid withdrawal, neither PI3K nor AKT/PKB activities are downregulated in mammalian or insect cells in culture, suggesting that this pathway does not directly respond to nutrient shortage. Hence, an intermediate sensor mechanism must link nutrient availability to insulin/IGF signaling (Colombani, 2003).

An intriguing possibility is that specific organs could function as nutrient sensors and induce a nonautonomous modulation of insulin/IGF growth signaling in response to changes in nutrient levels. This study used a genetic approach in Drosophila to assess both the cellular and humoral responses to amino acid deprivation in the context of a developing organism. The insect fat body (FB) has important storage and humoral functions associated with nutrition, comparable to vertebrate liver and adipose tissue. During larval stages, the FB accumulates large stores of proteins, lipids, and carbohydrates, which are normally degraded by autophagy during metamorphosis in order to supply the developing tissues but can also be remobilized during larval life to compensate transitory nutrient shortage. In addition to its storage function, the FB also has endocrine activity and supports growth of imaginal disc explants and DNA replication of larval brains in coculture. This study demonstrates that the FB operates as a sensor for variations in nutrient levels and coordinates growth of peripheral tissues accordingly via a humoral mechanism (Colombani, 2003).

In the course of a P[UAS]-based overexpression screen for growth modifiers, a P[UAS]-insertion line (UY681) was found to cause growth retardation upon ectopic activation. Sequence analysis revealed that P(UY)681 is inserted in a predicted gene (CG11128) that encodes a putative protein showing strong homology with amino acid permeases of the cationic amino acid transporter (CAT) family. The P[UAS] element is inserted in the first intron of the CG11128 gene, potentially driving transcription of an antisense RNA in a GAL4-dependent manner. To assess the function of this transporter, ³H-arginine uptake was measured in S2 cells. Results indicate that amino acid uptake is either enhanced by transfection of a CG11128 cDNA or suppressed by RNAi, indicating that the encoded protein presents CAT activity. In situ hybridization revealed basal levels of CG11128 expression in most larval tissues but much higher levels in the FB and the gut, two tissues involved in amino acid processing (Colombani, 2003).

By P element remobilization, an imprecise excision was obtained that deletes the sequences encoding the N-terminal half of the protein. 87% of homozygous mutant animals die during larval stages. The few viable adults emerged after a 2 day delay and were smaller and markedly slimmer than control animals. The associated gene was named slimfast (slif) and the excision allele slif¹. Weight measurement indicated that homozygous slif¹ adult males displayed a 16% mass reduction compared to control. Accordingly, adult wing size was reduced by 8% due to a reduction of both cell size and cell number. When the slif¹ allele was in trans to Df(3L)Δ1AK, a deficiency covering the locus, larval lethality was slightly enhanced, suggesting that slif¹ corresponds to a strong hypomorphic allele. The amino acid transporter function of slif, as well as the phenotypes observed upon reduction of slif function suggest that slif mutant animals might suffer amino acid deprivation. A major consequence of amino acid deprivation in larvae is the remobilization of nutrient stores in the FB, which typically results in aggregation of storage vesicles. Consistently, fusion of storage vesicles was observed in the FB of slif¹ larvae and was indistinguishable from that observed in animals fed on protein-free media (Colombani, 2003).

GAL4 induction of P(UY)681 resulted in a growth-deficient phenotype similar to that of slif¹ loss of function. The antisense orientation of P(UY)681 suggested that the growth defect following GAL4 induction was due to an RNAi effect. Indeed, Northern blot analysis revealed that ubiquitous GAL4-dependent activation of P(UY)681 using the daughterless-GAL4 (da-GAL4) driver strongly reduced slif mRNA levels. Only two of the three alternative first exons are potentially affected by the antisense RNA, possibly explaining the residual accumulation of slif mRNAs in da-GAL4; P(UY)681 animals. Most of these animals died at larval stage, similar to what was observed for slif¹ mutants. Specific induction of P(UY)681in the wing disc using the MS1096-GAL4 driver provoked a reduction of the adult wing size, which could be either rescued by coactivation of a UAS-slif transgene or enhanced by reducing slif gene dosage with the heterozygous Df(3L)Δ1AK deficiency. Thus, GAL4-dependent activation of P(UY)681 reduces slif function and defines a conditional loss-of-function allele hereafter termed slif^Anti (Colombani, 2003).

As expected, loss of slif function using the slif^Anti allele also mimicked amino acid deprivation. Accordingly, ubiquitous slif^Anti induction in growing larvae resulted in storage vesicle aggregation and strong reduction of global S6 kinase activity, similar to what was reported in animals raised on protein-free diet. Additionally, an increase in PEPCK1 gene transcription was observed, similar to the effect of amino acid withdrawal. In summary, this study has identified two loss-of-function alleles of the slif gene whose defects mimic physiological aspects of amino acid deprivation. Importantly, the conditional slif^Anti allele provides a unique tool to mimic an amino acid deprivation in a tissue-specific manner (Colombani, 2003).

This study established that the FB is a sensor tissue for amino acid levels, as downregulation of the Slif amino acid transporter within the FB is sufficient to induce a general reduction in the rate of larval growth. In contrast, specific disruption of slif in imaginal discs, larval gut, or salivary glands did not induce a nonautonomous growth response, suggesting that these tissues do not participate in the systemic control of growth. The dilp-expressing median neurosecretory cells (m-NSCs) also affect growth control, since selective ablation of these cells in the larval brain induces an overall reduction of animal size. In response to complete sugar and protein starvation, the m-NSCs stop expressing dilp3 and dilp5 genes, suggesting that these neurons also sense nutrient levels. This study shows that the selective reduction of slif function in these cells has no obvious effect on tissue growth and animal development. This indicates that the seven dilp-expressing m-NSCs do not constitute a general amino acid sensor. In contrast, the role of m-NSCs in carbohydrate homeostasis and the observation that they stop expressing certain dilp genes when larvae are deprived of sugar rather suggests that these cells have a role in sensing carbohydrate levels (Colombani, 2003 and references therein).

This analysis also provides a framework in which to understand the phenotype of minidisc, a mutation in an amino acid transporter gene that exhibits nonautonomous growth defects in imaginal discs (Colombani, 2003).

In a number of model systems, both PI3K and TOR have been implicated in linking growth to nutritional status and, until recently, were considered as intermediates of a common regulatory pathway. In yeast, the TOR kinase is part of a cell-autonomous nutrient sensor, which controls protein synthesis, ribosome biogenesis, nutrient import, and autophagy. Genetic analysis in Drosophila indicates that dTOR is required for cell-intrinsic growth control. The results obtained using the slif^Anti allele in the wing disc indicate that individual tissues have indeed the potential to respond to amino acid deprivation in a cell-autonomous manner. Nonetheless, this study also demonstrates that the TOR nutritional checkpoint participates in a systemic control of larval growth emanating from the FB. Within a developing organism, each cell may integrate these two distinct inputs regarding nutritional status, one originating from a systemically-acting FB sensor, and the other from TOR-dependent signaling in individual cells. One can further speculate that depending on the strength and duration of starvation, different in vivo nutritional checkpoints will be hierarchically recruited to protect the animal and that the systemic control might, in most physiological situations, override the cell-autonomous control. Indeed, as the data demonstrate, the FB sensor is sufficient to induce a general and coordinated response to starvation without calling individual cell-autonomous mechanisms into play (Colombani, 2003).

Several lines of evidence indicate that the PI3K pathway is not part of the sensor mechanism in FB cells. First, a sensor for PI3K activity in the FB is only marginally affected by amino acid deprivation in that tissue, indicating that the cell-autonomous response to amino acid starvation does not directly influence PI3K signaling. This is reminiscent of previous observations in mammalian cultured cells, showing that PI3K activity does not respond to variations in amino acid levels. Moreover, inhibition of PI3K signaling by dPTEN expression in the FB is not sufficient to trigger the sensing mechanism. Although, dPTEN overexpression causes a complete disappearance of the PI3K sensor accompanied by growth suppression of FB cells, the FB maintains a critical mass that allows for normal larval growth. In contrast, the regulatory subunit p60 whose overexpression potently inhibits PI3-kinase in flies has been shown to induce a systemic effect on larval growth when overexpressed in the FB using an Adh-Gal4 driver. This study found that a pumpless ppl-GAL4-directed expression of p60 also provokes a strong suppression of larval growth and a dramatic inhibition of FB development in young larvae. Thus, the systemic effect on growth observed upon p60 overexpression most likely results from a drastic reduction of FB mass, which then fails to support normal larval growth (Colombani, 2003).

These results further indicate that PI3K signaling is a remote target of the humoral message that originates from the FB in response to amino acid deprivation. This is in agreement with previous data showing that PI3K activity is downregulated by dietary amino acid deprivation and explains why global PI3-kinase inhibition mimics cellular and organismal effects of starvation. The existence of a humoral relay reconciles these in vivo studies with the absence of direct PI3K responsiveness to amino acid levels (Colombani, 2003).

The relative resistance of imaginal disc growth to the systemic control exerted by the FB correlates with maintenance of PI3K activity in these tissues. This is in agreement with previous observations that cells in the larval brain and in imaginal discs maintain a slow rate of proliferation under protein starvation, while larval endoreduplicating tissues (ERTs) arrest. This difference might be attributed to the basal levels of dilp2 expression observed in imaginal discs, allowing a moderate growth rate of these tissues through an autocrine/paracrine mechanism. It was recently shown that clonal induction of PI3K potently induces cell-autonomous growth response even in fasting larvae, indicating that some nutrients are still accessible to support cell growth within a fasted larva. The main function of a general sensor could be to preserve these limited nutrients for use by high priority tissues. In this context, local PI3K activation through an autocrine loop in imaginal tissues could favor the growth of prospective adult structures in adverse food conditions. Thus, the FB would have an active role in controlling the allocation of resources depending on nutritional status. In this respect, it is noteworthy that FB cells are relatively resistant to the FB-derived humoral signal, since the PI3K sensor is not drastically affected in the FB of ppl>slif^Anti animals. Thereby, essential regulatory functions of the FB could be preserved even in severely restricted nutritional conditions (Colombani, 2003).

How does the FB signal to other tissues? This study suggests that a humoral signal relays information from the FB amino acid sensor and systemically inhibits PI3K signaling. In addition, this downregulation is not due to a direct inhibition of dilp expression by neurosecretory cells in the brain. Nevertheless, it cannot be ruled out that the secretion of these molecules is subjected to regulation in the mNSCs. Both in vivo and in insect cell culture, several imaginal discs growth factors (IDGF) secreted by the FB have been proposed to function synergistically with Dilp signaling to promote growth. However, this study did not find any modification of IDGF expression in the FB of larvae raised on water- or sugar-only diet, or upon FB induction of slif^anti. In vertebrates, the different functions of the circulating IGF-I are modulated through its association with IGF-BPs and acid labile subunit (ALS). In particular, the formation of a ternary complex with ALS leads to a considerable extension of IGF-I half-life. The finding that a Drosophila ALS ortholog is expressed within the FB in an amino acid-dependent manner provides a new avenue to study the molecular mechanisms of nonautonomous growth control mediated by the FB (Colombani, 2003).

This study highlights the contribution that genetics can provide to unravel the mechanisms of physiological control. Using a genetic tool to mimic amino acid deprivation, it was demonstrated that nutrition systemically controls body size through an amino acid sensor operating in the FB. It is proposed that (1) in metazoans, a systemic nutritional sensor modulates the conserved TOR-signaling pathway, and (2) the response to sensor activation is relayed by a hormonal mechanism, which triggers an Inr/PI3K-dependent response in peripheral tissues (Colombani, 2003).

Programmed autophagy in the Drosophila fat body is induced by ecdysone through regulation of the PI3K pathway

Eukaryotic cells catabolize their own cytoplasm by autophagy in response to amino acid starvation and inductive signals during programmed tissue remodeling and cell death. The Tor and PI3K signaling pathways have been shown to negatively control autophagy in eukaryotes, but the mechanisms that link these effectors to overall animal development and nutritional status in multicellular organisms remain poorly understood. This study reveals a complex regulation of programmed and starvation-induced autophagy in the Drosophila fat body. Gain-of-function genetic analysis indicates that Ecdysone receptor signaling induces programmed autophagy whereas PI3K signaling represses programmed autophagy. Genetic interaction studies show that ecdysone signaling downregulates PI3K signaling and that this represents the effector mechanism for induction of programmed autophagy. Hence, these studies link hormonal induction of autophagy to the regulatory function of the PI3K signaling pathway in vivo (Rusten, 2004).

The fat body is a primary nutrient-responsive tissue that emulates the functions of the liver and adipose tissue of vertebrates. Fat body cells undergo programmed autophagy during the last larval stage (L3) preceding pupariation. Findings in Mamestra brassicae have demonstrated that autophagy can be induced by ecdysone. In Drosophila, autophagy is developmentally upregulated from mid-L3 stage in fat body cells. Although previously the levels of ecdysone during the L3 stage of development in Drosophila have been detected only at low levels at the wandering-L3 stage and increase markedly before puparium formation in most studies, a small hormonal peak before the initiation of wandering has been reported. Expression of dominant-negative Ecdysone receptor from the mid-L3 stage under Lsp-Gal4 control results in a dramatic reduction of autophagy. The autophagic area is severely reduced, and fewer acidic structures are observed. Since complete inhibiton of autophagy was not observed, the timing of Lsp-Gal4 expression was closely followed and it was found that expression initiated reporter gene expression 20-30 min after programmed autophagy had been started. Most likely, this latency of Lsp-Gal4 expression accounts for the incomplete penetrance of the phenotypes. In fact, driving expression of the dominant-negative EcR using a constitutive fat body Gal4 driver (cg-Gal4), or placing a temperature-sensitive mutant of ecdysoneless (ecd¹) to restrictive temperature at the start of the L3 stage, led to a complete inhibition of autophagy. This suggests that ecdysone has a regulatory role on programmed autophagy already at the early L3 stage and that the ecdysone titer at this stage of development is at the threshold of detection. The issue of ecdysone levels during larval development has recently been revisited: radioimmunoassay measurements detected low but continuously increasing levels of ecdysone during mid-L3 stage. Taken together, these results suggest that programmed autophagy is due to this low but rising level of ecdysone during the L3 stage of development (Rusten, 2004).

A simple explanation of ecdysone-induced programmed autophagy could be the initiation of wandering and therefore starvation-induced autophagy. This is not likely, however, for the following reasons. Developmental autophagy is initiated in fat bodies of late feeding animals at least 6 hr before the animals stop feeding and leave the food, and 12 hr before ingested food starts to disappear from the anterior part of the midgut. In addition, the autophagic response in fat body cells is uncoupled from the change in feeding behavior since expression of a dominant-negative ecdysone receptor cell autonomously inhibited programmed autophagy in late wandering L3 animals that ceased feeding 8 hr earlier (Rusten, 2004).

PI3K signaling is able to regulate autophagy and is unlikely to be a part of the amino acid sensing mechanism during an acute starvation response; amino acids and insulin have been shown to control autophagic proteolysis through different signaling pathways in rat hepatocytes. This is supported by the fact that the presence of the PIP₃ binding probe, GFP-PH, at the cell membrane is not affected by amino acid deprivation in the fat body. The loss of PI3K signaling was only observed after 24 hr of starvation, long after the acute starvation response of autophagy. This concurs with observations in cultured mammalian cells in which insulin signaling and PI3K activity do not respond to variations in nutrient levels (Rusten, 2004).

What is then the physiological significance of PI3K signaling regulating autophagy? PI3K signaling was modulated in the fat body to see if it could influence the autophagic response to ecdysone. Elevation of PI3K signaling during the period of programmed autophagy prevented the biogenesis of autolysosomes. This epistatic regulation of PI3K signaling over ecdysone-induced programmed autophagy suggests that PI3K signaling is a part of the same pathway or a dominant repressor. A strong reduction and ultimately loss of PI3K signaling was observed in the fat body during the induction of programmed autophagy, suggesting that ecdysone downregulates PI3K signaling. In addition, a reduction in PI3K signaling failed to increase the autophagic activity during programmed autophagy, in line with the idea that it is in the same pathway and is already inhibited completely. In contrast, reducing Tor signaling in the fat body could further increase autophagic activity during developmental autophagy, suggesting that Tor is not inhibited completely, or not involved in programmed autophagy. Elucidating the role of Tor signaling during this process requires further studies (Rusten, 2004).

Several lines of evidence support a role for PI3K signaling in ecdysone-induced programmed autophagy in the fat body. (1) Inhibition of Ecdysone receptor activity or an increase in PI3K signaling produced very similar phenotypes, indicating that these pathways perform opposite regulatory roles on programmed autophagy. (2) Administration of the ecdysone analog RH5849 to feeding larvae promoted attenuation of PI3K signaling. (3) Clonal inactivation of ecdysone receptor signaling led to a failure of this attenuation. (4) Simultaneous downregulation of PI3K signaling and inhibiton of ecdysone receptor activity restored programmed autophagy to wild-type levels. Thus, ecdysone signaling is both necessary and sufficient for downregulation of the PI3K pathway during programmed autophagy. Taken together, these results suggest a model in which ecdysone receptor signaling has the ability to promote autophagy through the downregulation of PI3K signaling (Rusten, 2004).

Growth arrest and autophagy are required for salivary gland cell degradation in Drosophila

Autophagy is a catabolic process that is negatively regulated by growth and has been implicated in cell death. This study finds that autophagy is induced following growth arrest, and precedes developmental autophagic cell death of Drosophila salivary glands. Maintaining growth by expression of either activated Ras or positive regulators of the class I phosphoinositide 3-kinase (PI3K) pathway inhibits autophagy and blocks salivary gland cell degradation. Developmental degradation of salivary glands is also inhibited in autophagy gene (atg) mutants. Caspases are active in PI3K-expressing and atg mutant salivary glands, and combined inhibition of both autophagy and caspases increases suppression of gland degradation. Further, induction of autophagy is sufficient to induce premature cell death in a caspase-independent manner. These results provide in vivo evidence that growth arrest, autophagy, and atg genes are required for physiological autophagic cell death, and that multiple degradation pathways cooperate in the efficient clearance of cells during development (Berry, 2007).

These studies indicate that arrest of PI3K-dependent growth is an important determinant of autophagic cell death of salivary glands during Drosophila development. Maintenance of growth by expression of either activated Ras, Dp110, or Akt in salivary glands is sufficient to inhibit salivary gland degradation. It is possible that the larger Dp110-, Akt- and Ras^V12-expressing salivary glands simply have more material to degrade, and this is why they persist. It is suspected that this is not the case, however, since Dp110-expressing glands are larger than Ras^V12-expressing glands, yet Ras^V12-expressing glands are less degraded. Although PI3K-dependent growth inhibits autophagy, growth could influence other downstream targets. However, the Atg1-induced suppression of the Dp110 persistent salivary gland phenotype and the persistence of vacuolated salivary gland cell fragments in atg loss-of-function mutants support the conclusion that growth arrest and autophagy are required for proper salivary gland degradation (Berry, 2007).

Ras and class I PI3K signaling are complex, and cross-talk occurs between these pathways. Although the data indicate that both activated Ras and PI3K have similar effects on salivary gland cell growth and inhibition of autophagy, it is observed that Ras-expressing cells were more intact 24 hours apf. Caspase activity is detected in Dp110-and Akt-expressing glands, and it is speculated that part of the degradation observed in Dp110 and Akt glands was due to caspases. Indeed, combining Dp110 expression with caspase inhibition resulted in intact salivary glands. This additive phenotype indicates that multiple degradation pathways are involved in autophagic cell death in vivo. Caspases were also active in Ras-expressing glands that were predominantly intact; thus activated Ras likely influences factors separate from caspases and the PI3K pathway. Ras regulates PI3K-independent pathways including MAPK and the cell cycle. Proliferating cells usually double in size prior to division, and because of this, cell growth and division are often considered synonymous. These studies demonstrate that although expression of either Myc or CyclinD with Cdk4 is sufficient to induce nuclear size, they do not inhibit salivary gland degradation. These data support the conclusion that growth arrest, but not cell cycle arrest, is an important determinant of salivary gland autophagic cell death. While many studies have defined relationships between cell cycle arrest and cell death, this study defines a unique relationship between cell growth arrest and cell death (Berry, 2007).

Given autophagy’s well established function in cell survival, a role for autophagy in cell death seems paradoxical. The discovery that caspases function in cells dying with a Type II autophagic morphology led to speculation that all programmed cell death is regulated by apoptosis factors. Further, the preponderance of in vitro evidence shows a role for autophagy in cell death when caspases or apoptosis factors are inhibited. This study found that reduced function of any one of seven atg genes inhibits salivary gland degradation. The incomplete degradation of salivary glands in multiple atg loss-of-function mutants provides the first in vivo evidence that autophagy and atg genes are required for proper degradation of cells during developmental cell death. Caspase activity and caspase-dependent DNA fragmentation occurs in these atg mutants, indicating that autophagy is a caspase-independent degradation pathway required for complete cell degradation in autophagic cell death during development. Further, induction of autophagy by Atg1 expression leads to premature caspase-independent salivary gland degradation. The data do not exclude a role for caspases in autophagic cell death. Either inhibition of caspases by p35 or reduced atg gene function result in delayed and incomplete degradation of salivary glands, and the combined inhibition of caspases with reduced atg function results in increased persistence of this tissue. These data suggest that autophagy and caspases function in parallel pathways during salivary gland cell death, and that both independently contribute to cell destruction. Further, the presence of both autophagy and caspases appears to be more typical of autophagic cell death that occurs under physiological conditions. Autophagic cell death models of mammary lumen formation and embryonic cavitation, as well as amphibian developmental cell death, all involve both processed caspase-3 and autophagy (Berry, 2007).

The designations of type I apoptotic death and type II autophagic death are based on morphological criteria. The current studies indicate that cell morphology likely reflects difference in the factors that are used to activate cell death and degrade the dying cell. The degradation of salivary glands in caspase mutants indicates that caspase-independent factors are involved in autophagic cell death. The presence of autophagosomes in dying salivary glands led to an investigation of cell death in atg gene mutants; stronger defects were observed in salivary gland degradation with perturbed atg gene function than with drice, ark, and dronc mutants. These data indicate that cell morphology is informative, given that it suggested autophagy is involved in the death of salivary glands. However, it is important to note that cell death classification that is based on morphology can be misleading, since salivary glands clearly use both caspases and autophagy, degradation mechanisms that had been speculated to be strictly associated with a single morphological form of cell death (Berry, 2007).

Now that it is clear that autophagy participates in cell death under some circumstances, it will be critical to determine how autophagy participates in cell killing and removal. A recent study showed that autophagy is required to generate the energy needed to promote phagocytosis signaling in an in vitro model of embryonic cavitation (Qu, 2007). This is not believed to be the same as the role of autophagy in salivary glands, since no phagocytosis is observed during salivary gland death. Alternatively, autophagy may be used to recruit and degrade factors that promote cell survival, such as the degradation of cytoplasmic catalase in mouse L929 cells. Finally, extreme levels of autophagy may be sufficient to cause a metabolic catastrophe by degrading substrates and mitochondria that are needed for energy. The latter possibility does exist in salivary glands, as expression of the Atg1 kinase is sufficient to induce the death of fat (Scott, 2007) and salivary gland cells. Unlike fat cells, elevated autophagy does not induce caspase-dependent DNA fragmentation in salivary gland cells, and expression of p35 does not inhibit Atg1-induced death (Berry, 2007).

The prevalence of apoptosis and the potent killing potential of caspases raise the question of why autophagy participates in developmental cell death. In the context of Drosophila and other insects, larval cells have a modified endoreplication cell cycle that results in the production of gigantic cells. The number and size of cells may prohibit engulfment and digestion by phagocytes, and autophagy may be necessary for self-degradation. Further, the life history of the organism may lead to understanding of why autophagy participates in the destruction of tissues. Drosophila do not feed during the 3 day period of metamorphosis. Thus, the differentiation and morphogenesis of the entire adult occurs in the absence of food, and the resources to build the adult fly must come from reserves that are set aside during larval development. One important source of these resources is the fat that exhibits elevated levels of autophagy at the onset of metamorphosis. Several other large larval tissues are destroyed by autophagic cell death during metamorphosis including the midgut and salivary glands. It is speculated that like fat, catabolism of these tissues by autophagy provides resources that are needed to construct the adult. Similarly, it is speculated that the large number of autophagosomes observed in dying amphibian cells may serve to recycle nutrients during metamorphosis when these animals do not feed (Berry, 2007).

These studies have indicated that it is necessary to be cautious when considering autophagy to be either a cell survival or cell death process. Perhaps it is useful to consider autophagy for what it is; a catabolic process that contributes to many cellular and biological processes. This is not that different from the caspase proteases that are widely considered to be apoptosis proteases, as it is now clear that caspases also function in cell differentiation. Future studies are likely to show that autophagy functions in many cell types, and that its contribution to cell survival and cell death are dependent on the type and physiological context of the cell (Berry, 2007).

PI3K and Dpp signaling: regulation of cell growth and proliferation in the wing

Cell proliferation and patterning must be coordinated for the development of properly proportioned organs. If the same molecules were to control both processes, such coordination would be ensured. This possibility has been investigated in the Drosophila wing using the Dpp signaling pathway. Previous studies have shown that Dpp forms a gradient along the AP axis that patterns the wing, that Dpp receptors are autonomously required for wing cell proliferation, and that ectopic expression of either Dpp or an activated Dpp receptor, Tkv^Q253D, causes overgrowth. These findings are extended with a detailed analysis of the effects of Dpp signaling on wing cell growth and proliferation. Increasing Dpp signaling by expressing Tkv^Q253D accelerates wing cell growth and cell cycle progression in a coordinate and cell-autonomous manner. Conversely, autonomously inhibiting Dpp signaling using a pathway specific inhibitor, Dad, or a mutation in tkv, slows wing cell growth and division, also in a coordinate fashion. Stimulation of cell cycle progression by Tkv^Q253D is blocked by the cell cycle inhibitor RBF, and requires normal activity of the growth effector, PI3K. Among the known Dpp targets, vestigial was the only one tested that was required for Tkv^Q253D-induced growth. The growth response to altering Dpp signaling varies regionally and temporally in the wing disc, indicating that other patterned factors modify the response (Martín-Castellanos, 2002).

If growth and cell cycle progression are independently regulated by Tkv, one would expect to detect the proliferative effect of Tkv^Q253D even in growth-impaired cells. Alternatively, if Tkv^Q253D were to promote cell cycle progression indirectly via stimulating cellular growth, the proliferative effect of Tkv^Q253D should be inhibited when cell growth is impaired (Martín-Castellanos, 2002).

To suppress cell growth a truncated version of p60, Deltap60, was expressed. This is an adaptor molecule for the class I Phosphoinositide 3-Kinase (PI3K/Dp110 in Drosophila. Dp110 signaling is a potent growth inducer. Adaptor molecules, such as p60, bind to the Dp110 kinase and recruit it to the Insulin Receptor, allowing full activation of the enzyme. Deltap60 binds the Insulin Receptor but cannot bind Dp110, and thus inhibits Dp110 signaling in a dominant-negative manner. When expressed in wing cells, Deltap60 reduces cell size and strongly delays G1 progression. Flp/Gal4 clones expressing Deltap60 contain very few cells compared with controls. Overexpressed Deltap60 also dominantly blocks the growth and proliferation effects of Tkv^Q253D. Clones of cells that co-express Deltap60 and Tkv^Q253D contain as few cells as those expressing Deltap60 alone, and these cells are just slightly larger than those expressing Deltap60 alone. Thus, loss of growth resulting from loss of PI3K activity cannot be rescued by hyperactivating Dpp signaling, and cell proliferation induced by Dpp probably requires Dp110 activity. These results are consistent with the model in which Dpp-driven cell growth indirectly promotes cell cycle progression (Martín-Castellanos, 2002).

Although clonal growth is blocked by co-expressing Deltap60 and Tkv^Q253D, cells that co-express Deltap60 and Tkv^Q253D do not show the G1 delay characteristic of cells expressing Deltap60 alone. Thus, Tkv^Q253D appears to be able to promote G1/S progression even in the presence of Deltap60. This suggests that some aspects of cell cycle progression induced by Tkv^Q253D may be Dp110 independent. However, the slight increase in size observed in cells co-expressing Deltap60 and Tkv^Q253D makes it difficult to rule out the possibility that this effect on G1/S progression also occurs indirectly, as a consequence of increased growth (Martín-Castellanos, 2002).

Signaling from Akt to FRAP/TOR targets both 4E-BP and S6K in Drosophila melanogaster

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).

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).

Ras activity in the Drosophila prothoracic gland regulates body size and developmental rate via ecdysone release

In Drosophila, each of the three larval instars ends with a molt, triggered by release of steroid molting hormone ecdysone from the prothoracic gland (PG). Because all growth occurs during the larval stages, final body size depends on both the larval growth rate and the duration of each larval stage, which in turn might be regulated by the timing of ecdysone release. This study shows that the expression of activated Ras, PI3 kinase (PI3K), or Raf specifically in the PG reduces body size, whereas activated Ras or PI3K, but not Raf, increases PG cell size. In contrast, expression of either dominant-negative (dn) Ras, Raf, or PI3K increases body size and prolongs the larval stages, leading to delayed pupariation, whereas expression of dn-PI3K, but not of dn-Raf or dn-Ras, reduces PG cell size. To test the possibility that altered ecdysone release is responsible for these phenotypes, larval ecdysone levels were measured indirectly, via the transcriptional activation of two ecdysone targets, E74A and E74B. It was found that the activation of Ras within the PG induces precocious ecdysone release, whereas expression of either dn-PI3K or dn-Raf in the PG greatly attenuates the [ecdysone] increase that causes growth cessation and pupariation onset. It is concluded that Ras activity in the PG regulates body size and the duration of each larval stage by regulating ecdysone release. It is also suggested that ecdysone release is regulated in two ways: a PI3K-dependent growth-promoting effect on PG cells, and a Raf-dependent step that may involve the transcriptional regulation of ecdysone biosynthetic genes (Caldwell, 2005; full text of article).

FOXO-independent suppression of programmed cell death by the PI3K/Akt signaling pathway in Drosophila

Signaling through the PI3K/Akt/FOXO pathway plays an important role in vertebrates in protecting cells from programmed cell death. PI3K and Akt have been similarly shown to be involved in survival signaling in Drosophila. However, it is not known whether PI3K and Akt execute this function by controlling a pro-apoptotic activity of Drosophila FOXO. This study shows that elevated signaling through PI3K and Akt can prevent developmentally controlled death in the salivary glands of the fruit fly. Drosophila FOXO is not required for normal salivary gland death and the rescue of salivary gland death by PI3K occurs independent of FOXO. These results give support to the notion that FOXOs have acquired pro-apoptotic functions after separation of the vertebrate and invertebrate lineages (Liu, 2006).

To determine whether elevated signaling through PI3K can rescue normal salivary gland death, the catalytic subunit of PI3K, Dp110, was expressed in late-prepupal glands using P{UAS-Dp110} and a heat-shock GAL4 driver. Most of the pupae expressing the subunit still possessed intact salivary glands 20 h APF, i.e., ~6 h after the glands are normally destroyed. This result shows that a high level of PI3K activity can overcome the stimuli that normally lead to the destruction of the salivary glands in early pupae. Moreover, it suggests that the PI3K pathway is normally inactive or, at least, strongly downregulated in dying salivary glands (Liu, 2006).

If the effect of PI3K is mediated by the canonical PI3K/Akt pathway, an elevated activity of Akt should have the same or a similar effect as an elevated PI3K activity. To test this prediction, both wild-type Akt and a constitutively active form of Drosophila Akt, Dakt^myr, were expressed in late-prepupal salivary glands. Dakt^myr carries a myristoylated amino terminus that targets the protein to the cell membrane. Under normal conditions, Akt is recruited to the membrane by the PI3K product PIP₃ and subsequently activated by phosphorylation through PDK1. The expression of both UAS-Dakt ^myr and UAS-akt was driven by heat-shock GAL4. The constitutively active Dakt^myr led to a complete rescue of salivary gland death, whereas unmodified Akt had no effect. It is concluded that only membrane-associated Akt can rescue salivary gland death. This is consistent with the normal mechanism of Akt activation that requires PI3K-induced recruitment of Akt to the cell membrane. The inhibition of death by active Akt, but not by inactive Akt, confirms that PI3K activity is limited in late-prepupal salivary glands and underscores the specificity of the observed effect (Liu, 2006).

In summary, the results show that the survival function of PI3K/Akt does not depend on the inactivation of dFOXO and that dFOXO has no apparent role in the activation of PCD in the salivary glands. Moreover, they indicate that an intact PI3K/Akt signaling pathway is not required for salivary gland survival. However, downregulation of the pathway may be required for salivary gland death, because elevated signaling through the pathway can rescue the salivary glands. Importantly, this study on the role of PI3K/Akt/dFOXO signaling in salivary gland death did not reveal a pro-apoptotic role of dFOXO, further strengthening the assumption that the functions of FOXOs in apoptosis are a late evolutionary acquisition in the vertebrate lineage (Liu, 2006).

Phosphatidylinositol 3-kinase and Akt nonautonomously promote perineurial glial growth in Drosophila peripheral nerves

Drosophila peripheral nerves, structured similarly to their mammalian counterparts, comprise a layer of motor and sensory axons wrapped by an inner peripheral glia (analogous to the mammalian Schwann cell) and an outer perineurial glia (analogous to the mammalian perineurium). Growth and proliferation within mammalian peripheral nerves are increased by Ras pathway activation: loss-of-function mutations in Nf1, which encodes the Ras inhibitor neurofibromin, cause the human genetic disorder neurofibromatosis, which is characterized by formation of neurofibromas (tumors of peripheral nerves). However, the signaling pathways that control nerve growth downstream of Ras remain incompletely characterized. This study shows that expression specifically within the Drosophila peripheral glia of the constitutively active Ras^V12 increases perineurial glial thickness. Using chromosomal loss-of-function mutations and transgenes encoding dominant-negative and constitutively active proteins, it was shown that this nonautonomous effect of Ras^V12 is mediated by the Ras effector phosphatidylinositol 3-kinase (PI3K) and its downstream kinase Akt. The nonautonomous, growth-promoting effects of activated PI3K are suppressed by coexpression within the peripheral glia of FOXO, a transcription factor inhibited by Akt-dependent phosphorylation. It is suggested that Ras-PI3K-Akt activity in the peripheral glia promotes growth of the perineurial glia by inhibiting FOXO. In mammalian peripheral nerves, the Schwann cell releases several growth factors that affect the proliferative properties of neighbors. Some of these factors are oversecreted in Nf1 mutants. These results raise the possibility that neurofibroma formation in individuals with neurofibromatosis might result in part from a Ras-PI3K-Akt-dependent inhibition of FOXO within Schwann cells (Lavery, 2007).

Activating Ras specifically within the peripheral glia was sufficient to increase growth of the perineurial glia. In addition, activating the Ras effector PI3K, but not Raf or Ral, within the peripheral glia was sufficient to increase perineurial glial growth, and inhibiting PI3K activity in the peripheral glia, but not perineurial glia, suppressed the growth-promoting effects of activated Ras. It was also found that activity within the peripheral glia of the PI3K-activated kinase Akt was both necessary and sufficient to mediate the growth-promoting effects of PI3K. Finally, it was found that overexpression within the peripheral glia of FOXO, the forkhead-box transcription factor that is phosphorylated and inactivated by Akt-dependent phosphorylation, was sufficient to suppress the growth-promoting effects of PI3K. Together, these results suggest that Ras activity in the peripheral glia activates nonautonomous growth via the PI3K and Akt-dependent inhibition of FOXO. This observation is consistent with the previous observations that Nf1^– mouse Schwann cells oversecrete growth factor(s) that cause increased recruitment of mast cells into the peripheral nerve and is consistent in part with the observation that the proliferation defects of Nf1^– mutant mouse or human cells requires hyperactivation of Tor in a PI3K- and Akt-dependent manner (Lavery, 2007).

Perineurial glial growth in Drosophila peripheral nerves is regulated by several genes. These genes include Nf1, which is the Drosophila ortholog of human Nf1, push, which is thought to encode an E3 ubiquitin ligase and two genes implicated in neurotransmitter signaling: amnesiac, which is thought to encode a neuropeptide similar to vertebrate pituitary adenylate cyclase-activating polypeptide, and inebriated (ine), which encodes a member of the Na⁺/Cl^–-dependent neurotransmitter transporter family. Some of these genes might regulate perineurial glial growth via the activity of Ras or PI3K within peripheral glia. For example, mutations in push, but not ine, enhance the perineurial glial growth phenotype of Ras^V12 expressed in peripheral glia. These observations are consistent with the possibility that the activity of ine regulates Ras-GTP levels within peripheral glia. In contrast, push might regulate PI3K in a Ras-independent manner or act in the perineurial glia to regulate sensitivity to peripheral glial growth factors. Additional experiments will be required to distinguish between these possibilities (Lavery, 2007).

There are several lines of evidence from mice and humans suggesting that cell nonautonomous growth regulation, as a consequence of intercellular signaling, underlies neurofibroma formation. First, although neurofibromas arise in individuals heterozygous for Nf1^– after loss of Nf1⁺ from cell(s) within peripheral nerves, neurofibromas are heterogeneous and contain cells that are not clonally related, such as Schwann cells, perineurial cells, and fibroblasts. These observations suggest that neurofibromas arise when a core of Nf1^– cells cause overproliferation of their heterozygous neighbors via nonautonomous means. Second, neurofibroma formation in mice and humans requires a homozygous Nf1 mutant genotype in Schwann cells but not other cells within the tumor. Third, Ras-GTP levels in Schwann cells from the mouse Nf1 knock-out mutant are uniformly elevated. In contrast, only a subset of Schwann cells from human neurofibromas exhibit elevated Ras- GTP levels; the possibility has been raised that this subset, but not other Schwann cells from the tumor, is homozygous for Nf1^–. In this view, these Nf1^– cells recruited neighboring Schwann cells that were heterozygous for Nf1^– into the tumor by nonautonomous means, such as by the excessive release of one or more growth factors. Fourth, it has been demonstrated that Nf1^– Schwann cells oversecrete the ligand for the c-Kit receptor. This oversecretion increased migration of mast cells into peripheral nerves and might be an essential step in neurofibroma formation. These Schwann cells also oversecrete additional factors whose physiological role remains unclear. The molecular mechanisms by which neurofibromin regulates the synthesis or release of these molecules remain incompletely understood. The current observations that Ras activity in the peripheral glia promotes growth nonautonomously via the PI3K- and Akt-dependent inhibition of FOXO might provide insights into the mechanisms by which peripheral nerve growth is regulated nonautonomously by the mammalian Schwann cell (Lavery, 2007).

By hyperactivating Ras, Nf1 mutations could in principle cause tumors via any of several Ras effector pathways. In addition, the diverse types of tumors observed in individuals with neurofibromatosis could result from hyperactivation of distinct Ras effector pathways. The Raf pathway has been viewed as a more relevant effector pathway than the PI3K pathway, mostly because the importance of Ras in the activation of PI3K under physiological conditions remains controversial. In particular, although it is clear that the oncogenic Ras^V12 mutant is sufficient to activate PI3K, it has sometimes been difficult to demonstrate that wild-type Ras is necessary for PI3K activation. Presumably, this difficulty reflects the fact that PI3K can be activated by Ras-independent as well as Ras-dependent mechanisms, such as direct activation by activated receptor tyrosine kinases or by PIKE-L (phosphatidylinositol kinase enhancer). However, more recently, it has been demonstrated that PI3K and Akt are hyperactivated in several Nf1 mutant cell types and that this hyperactivation is Ras dependent. Furthermore, PI3K activation plays an essential functional role in Nf1^–-mediated growth defects, as is demonstrated by the observation that PI3K- and Akt-dependent Tor activation is necessary for the proliferation defects of Nf1 mutants to occur: application of rapamycin, a Tor inhibitor, attenuates the ability of Nf1 mutant cells to proliferate. These observations demonstrate that PI3K and Akt play key roles in at least some aspects of Nf1^–-induced tumor growth (Lavery, 2007).

The results are consistent with these observations. By comparing the effects on perineurial glial growth of peripheral–glial expression of activated Raf, PI3K, or Ral, it was possible to demonstrate that activation of PI3K, not Raf or Ral, is sufficient to promote perineurial glial growth and that PI3K activity in the peripheral glia is necessary to observe the nonautonomous effect of activated Ras on perineurial glial growth. It was similarly shown that Akt activity os necessary and sufficient to mediate the growth-promoting effects of PI3K. However, previous studies have observed that Tor activation is critical for the PI3K- and Akt-dependent growth regulation of Nf1 mutant cells, this study observed a critical role for the PI3K- and Akt-dependent inhibition of the transcription factor FOXO. It is possible that the phenotype observed in previous studies reflects the well characterized ability of PI3K–Tor to activate growth cell autonomously, whereas the phenotype reported in this study reflects nonautonomous growth regulation. In this view, PI3K and Akt regulate autonomous and nonautonomous growth via the Tor and FOXO pathways, respectively (Lavery, 2007).

FOXO presumably inhibits the growth-promoting effects of PI3K and Akt by transcriptional regulation of target genes. Candidate FOXO target genes include those encoding the molecules oversecreted by Nf1^– Schwann cells, whereas other targets might be represented in the distinct transcript profiles exhibited by Nf1^– Schwann cells or malignant peripheral nerve sheath tumors compared with wild-type Schwann cells. For example, Schwann cells from neurofibromas, but not normal Schwann cells, express the epidermal growth factor (EGF) receptor. Other potential targets include genes encoding growth factors, although ectopic expression within the peripheral glia of two candidate genes, Hedgehog and the EGF ligands spitz and gurken, failed to induce perineurial glial growth. Additional experiments will be required to identify the FOXO targets that regulate nonautonomous growth in peripheral nerves (Lavery, 2007).

Distinct mechanisms regulate hemocyte chemotaxis during development and wound healing in Drosophila melanogaster

Drosophila hemocytes are highly motile macrophage-like cells that undergo a stereotypic pattern of migration to populate the whole embryo by late embryogenesis. The migratory patterns of hemocytes at the embryonic ventral midline are orchestrated by chemotactic signals from the PDGF/VEGF ligands Pvf2 and Pvf3; these directed migrations occur independently of phosphoinositide 3-kinase (PI3K) signaling. In contrast, using both laser ablation and a novel wounding assay that allows localized treatment with inhibitory drugs, PI3K is shown to be essential for hemocyte chemotaxis toward wounds and Pvf signals and PDGF/VEGF receptor expression are not required for this rapid chemotactic response. These results demonstrate that at least two separate mechanisms operate in Drosophila embryos to direct hemocyte migration and show that although PI3K is crucial for hemocytes to sense a chemotactic gradient from a wound, it is not required to sense the growth factor signals that coordinate their developmental migrations along the ventral midline during embryogenesis (Wood, 2007).

During Drosophila embryogenesis, hemocytes derive exclusively from head mesoderm at around 2 h after gastrulation. From this point of origin, these cells migrate along stereotypical routes to populate the whole embryo by stage 17. It has been shown that the developmental migration of these cells is dependent on the expression of the VEGF/PDGF ligands Pvf1, Pvf2, and Pvf3. The PDGF/VEGF receptor (PVR) is expressed in hemocytes, and pvr mutant embryos fail to exhibit normal hemocyte migrations, resulting in an accumulation of these cells at their head end. A recent study has demonstrated a role of PVR in controlling anti-apoptotic cell survival of embryonic hemocytes and suggests that the defect in hemocyte distribution observed in the mutant is largely due to high numbers of hemocytes undergoing apoptosis and becoming engulfed by their neighbors. However, this study also showed that Pvr expression within hemocytes is required for the directed migration of a subset of these cells that enter the extended germ during normal development, suggesting that this population of hemocytes may well be using Pvf signals as a chemoattractant to guide their migrations. Additionally, ectopic expression of Pvf2 within the embryo has been shown to be sufficient to induce a chemotactic response from embryonic hemocytes (Wood, 2007).

In addition to migrating along developmental pathways, embryonic hemocytes have been shown to migrate toward a laser-induced wound in a process that resembles the vertebrate inflammatory response. For a hemocyte to chemotax toward a chemotactic source, be it a wound or a guidance cue expressed along developmental migration routes, it has to be able to sense a chemotactic gradient and polarize in alignment with that gradient. Studies using Dictyostelium discoideum and mammalian neutrophils have demonstrated that the phosphoinositides PtdIns(3,4,5)P₃ (PIP₃) and PtdIns(3,4)P₂ (PIP₂) are key signaling molecules that become rapidly and highly polarized in cells that are exposed to a gradient of chemoattractant. In these actively chemotaxing cells, phosphoinositide 3-kinases (PI3Ks) rapidly translocate from the cytosol to the membrane at the leading edge of the cell, whereas phosphatase and tensin homologue (PTEN) dissociates from the leading edge and becomes restricted to the sides and the rear. The difference in localization of these two enzymes leads to localized PIP₃ production at the leading edge of the cell. Down- or up-regulation of PIP₃ by deletion of PI3Ks or of PTEN, respectively, results in severely reduced efficiency of chemotaxis. Though PI3K has been shown to be important for cell motility using these model systems, its role for single-cell chemotaxis in vivo in a multicellular organism has yet to be clarified. D. melanogaster has one class I PI3K, Dp110, whose role in cell growth control and cell survival has been well characterized; however, no role in cell migration and chemotaxis in Drosophila for this protein has been shown (Wood, 2007).

This study analyzed the developmental migrations of hemocytes and characterized in detail their migration patterns along the ventral midline. Quantitative analysis shows that ventral midline hemocytes undergo a rapid lateral migration, during which they are highly polarized. Pvf2 and -3 expression in the central nervous system (CNS), and Pvf2 alone in the dorsal vessel, are essential for directing the migration of hemocytes along these structures, and a decrease in expression of these ligands in the CNS is essential for the normal lateral migration of hemocytes in this region. The function of PI3K was analyzed in hemocytes. Using both dominant-negative PI3K-expressing hemocytes and the specific PI3K inhibitory drug LY294002, PI3K is shown not to be required for the Pvf-dependent normal dispersal of hemocytes during development but is essential for chemotaxis toward wounds. Additionally, hemocyte chemotaxis toward wounds is shown to be dependent on actin polymerization but that PI3K is not required for lamellipodial formation and instead appears to be required to sense a chemotactic gradient from a wound and polarize the hemocyte accordingly. These results demonstrate that at least two separate mechanisms operate in Drosophila embryos to direct hemocyte migration and show that although PI3K is crucial for hemocytes to sense a chemotactic gradient from a wound, it is not required to sense the Pvf growth factor signals that coordinate their developmental migrations along the ventral midline and dorsal vessel during embryogenesis (Wood, 2007).

Many obvious parallels exist between the migration of hemocytes along the ventral midline CNS and another developmentally regulated migration in Drosophila, that of border cell migration. Border cells take ~6 h to migrate a distance of 100 µm, a speed consistent with that describe for hemocyte migration along the CNS. Successful border cell migration, like hemocyte migration, requires the expression of the Pvr in the migrating cells and, just as is see for hemocytes, the chemotactic signals detected by the PVR in the border cells are not transduced through PI3K. Successful migration of border cells does, however, require Rac signaling and the Rac activator myoblast city (mbc), the D. melanogaster homologue of Dock 180. It has been shown that hemocyte-specific expression of dominant-negative Rac^N17 disrupts all hemocyte developmental migrations, demonstrating that Rac is required for the successful migration of ventral midline hemocytes along the CNS. Given that Pvr couples to the Dock 180 signaling pathway during border cell migration and that Dock 180 has been shown to be involved in the migration of lymphocytes, Mbc/Dock 180 is a potentially important protein for hemocyte migration. Despite the fact that mbc mutant embryos display a grossly normal pattern of hemocyte dispersal, it would be interesting to look in detail at the migration of these mutant cells along the ventral nerve cord. More work is needed to investigate what other similarities may exist between border cell migration and ventral midline hemocyte migration. During development, only a subset of the hemocytes present in the embryo respond to the midline Pvf expression and migrate along the CNS accordingly. Other cells follow other migratory pathways. What specifies these cells to migrate along the midline? Important studies in border cell migration have shown that the JAK-Stat signaling pathway signaling through the Domeless receptor (Dome) is necessary and sufficient to transform nonmotile epithelial cells into invasive ones. Whether a similar signaling mechanism is operating to specify future ventral midline hemoctyes and initiate their migration remains to be seen (Wood, 2007).

From stage 14 onwards, once hemocytes occupy the entire ventral midline, individual cells begin to rapidly leave the midline and occupy more lateral positions. At this stage of development, hemocytes appear to be highly polarized, exhibiting large lamellipodia at their leading edges and migrating at a speed more than three times faster than their earlier midline migration. This lateral movement requires a down-regulation in the attractive signal provided by Pvf2 in the midline, but is this the only driving force for the lateral movement? One possibility is that a different source of chemoattractant exists in the more lateral positions and that once Pvf2 expression is sufficiently down-regulated, this chemoattractant source operates to pull hemocytes laterally. Alternatively, hemocytes may be actively repelled from the midline or from one another, and the lateral migration observed by a subset of these hemocytes is a consequence of these cells attempting to maximize the distance between one another while maintaining contact with the CNS. It remains to be seen which, if any, of these hypotheses is true, but what is certain is that the guidance of hemocytes along the ventral midline of the embryo is not as simple as was first thought, and more studies are required to determine the exact relationship between this subpopulation of hemocytes and the different structures within the CNS as well as the overlying ectodermal cells, any of which could provide either chemoattractants or repellents for the migrating hemocytes to respond to (Wood, 2007).

This study has demonstrated a requirement of PI3K for the polarization and active chemotaxis of hemocytes toward an epithelial wound. This is the first demonstration of the role of PI3K for single-cell chemotaxis in Drosophila and shows a striking correlation with the mechanism of cell chemotaxis used by D. discoideum and mammalian neutrophils. In these model systems, class I PI3Ks are activated upon stimulation of G protein-coupled chemoattractant receptors and, once activated, PI3Ks catalyze the production of the phosphoinositides PIP₃ and PIP₂ at the leading edge of the cell. The accumulation of PIP₃/PIP₂ leads to a rapid and transient recruitment of pleckstrin homology domain-containing proteins, including the serine/threonine kinase Akt/PKB. Akt/PKB itself becomes activated upon recruitment to the membrane and, in D. discoideum, activates the serine/threonine kinase p21-activated kinase a, which eventually leads to the phosphorylation of Myosin II and subsequent polarization of the cytoskeleton. Evidence also exists to support a role for the PI3K antagonist PTEN in helping to establish and maintain the intercellular PIP₃ gradient required for successful chemotaxis by down-regulating the PIP₃ pathway at the rear of the migrating cell. How much of this signaling pathway is operating in chemotaxing hemocytes remains to be seen. The current study demonstrates the involvement of PI3K, and previous work has shown that the small GTPase Rac is required for efficient hemocyte chemotaxis toward wounds. In neutrophils, PIP₃ production has been shown to be autocatalytic and to require Rac but not Cdc42. In the proposed positive feedback loop, it is thought that PIP₃ may stimulate Rac through activation of a specific Rac GEF, which in turn activates PI3K, as well as effectors that mediate lamellipodial protrusion. Because Rac is absolutely required for hemocyte chemotaxis and lamellipodia formation, it is tempting to speculate that a similar feedback loop may be operating in Drosophila hemocytes. Further work is required to determine the complex relationships operating among PI3K, Rho family small GTPases, and the actin cytoskeleton that coordinate chemotactic migration in these highly motile cells (Wood, 2007).

The PI3K-dependent mechanism of polarization required for hemocyte chemotaxis toward a wound is extremely fast and perfectly suited for mature, highly motile hemocytes that need to rapidly react to a source of attractive signal, be it a wound, an invading organism, or an apoptotic cell. In contrast, the mechanics to developmentally disperse need not be so rapid, since the aim during development is simply to ensure that hemocytes migrate toward and arrive at their target tissue in a given amount of time and does not require the rapid response to constantly changing environments required for mature hemocytes. The mechanism controlling the developmental migration of hemocytes along the ventral midline is consequently much slower and is dependent on slow-diffusing growth factors of the Pvf family providing short-range guidance information signaling through the receptor tyrosine kinase PVR. These two mechanisms may not be the only ways in which hemocytes are able to chemotax toward an attractive source; indeed, the observation that hemocytes travel different migratory routes in the embryo suggests that they may not all be using the same machinery to polarize and migrate. What does seem to be consistent for both chemotaxis toward developmental signals and toward wounds, like motility in many cell types, is a requirement for Rac signaling and the formation of actin protrusions (Wood, 2007).

The fact that hemocyte migrations within the embryo are strictly regulated and adhere to a stereotyped pattern is important in a developmental context. Throughout embryogenesis, hemocytes carry out important developmental functions within the embryo, such as the engulfment and removal of apoptotic cells and the laying down of many extracellular matrix molecules, including collagen IV and laminin, that compose the basement membrane surrounding internal organs. The failure of hemocytes to travel along their normal migratory routes therefore has serious consequences. Such defects have been described in pvr mutants, where a lack of hemocyte migration along the ventral nerve cord results in a failure in CNS condensation, as well as a disruption in axon patterning. It is therefore vital for the embryo to ensure that hemocytes arrive at their correct target tissues during development. For this to occur, it is not sufficient to allow these cells to passively disperse throughout the embryo by random migrations; instead, a directed and tightly controlled migration is required (Wood, 2007). In this study, drugs were directly applied to Drosophila embryos using bead implantation. The application of drugs has been a powerful tool in cell culture and in vitro cell motility studies but remains largely unused in Drosophila. Using a bead assay, it will be possible to take advantage of the many useful drugs available to block both specific signaling pathways as well as important cytoskeletal processes. Combined with the powerful genetics available in Drosophila and the relative ease of live imaging in this system, the study of Drosophila hemocytes provides a powerful model to address the process of cell motility and chemotaxis and will undoubtedly provide a clearer understanding of the regulation and mechanics of single-cell migration in the complex setting of a multicellular organism (Wood, 2007).

Warts is required for PI3K-regulated growth arrest, autophagy, and autophagic cell death in Drosophila

Cell growth arrest and autophagy are required for autophagic cell death in Drosophila. Maintenance of growth by expression of either activated Ras, Dp110, or Akt is sufficient to inhibit autophagy and cell death in Drosophila salivary glands, but the mechanism that controls growth arrest is unknown. Although the Warts (Wts) tumor suppressor is a critical regulator of tissue growth in animals, it is not clear how this signaling pathway controls cell growth. This study shows that genes in the Wts pathway are required for salivary gland degradation and that wts mutants have defects in cell growth arrest, caspase activity, and autophagy. Expression of Atg1, a regulator of autophagy, in salivary glands is sufficient to rescue wts mutant salivary gland destruction. Surprisingly, expression of Yorkie (Yki) and Scalloped (Sd) in salivary glands fails to phenocopy wts mutants. By contrast, misexpression of the Yki target bantam is able to inhibit salivary gland cell death, even though mutations in bantam fail to suppress the wts mutant salivary gland-persistence phenotype. Significantly, wts mutant salivary glands possess altered phosphoinositide signaling, and decreased function of the class I PI3K-pathway genes chico and TOR suppressed wts defects in cell death. Although it has been shown that salivary gland degradation requires genes in the Wts pathway, this study provides the first evidence that Wts influences autophagy. These data indicate that the Wts-pathway components Yki, Sd, and bantam fail to function in salivary glands and that Wts regulates salivary gland cell death in a PI3K-dependent manner (Dutta, 2008).

Wts was identified as a protein that is expressed during autophagic cell death of Drosophila larval salivary glands with a high-throughput proteomics approach. This was surprising, given that wts RNA was not detected with DNA microarrays. Therefore, this study investigated whether Wts is present in salivary glands, and it was determined to be constitutively expressed at stages before and after the rise in ecdysone that triggers autophagic cell death. Animals that are homozygous for the hypomorphic wts^P2 allele, which is caused by a P element insertion, are defective in salivary gland cell death (Martin, 2007). Significantly two forms of Hpo are expressed during stages preceding salivary gland cell death, suggesting that phosphorylated Hpo is present in these cells and that this signaling pathway is activated (Dutta, 2008).

These studies indicate that Wts and other core components of this tumor-suppressor pathway are required for autophagic cell death of Drosophila salivary glands. wts is required for cell growth arrest and for proper regulation of caspases and autophagy, which contribute to the destruction of salivary glands. Although it is well known that cell division, cell growth, and cell death are important regulators of tissue and tumor size, it has been unclear whether a mechanistic relationship exists between cell growth and control of cell death (Dutta, 2008).

It is possible that wts and associated downstream growth-regulatory mechanisms could suppress cell death in other animals and cell types. Autophagic cell-death morphology has been reported in diverse taxa, but little is known about the mechanisms that control this form of cell death, and this lack of understanding is probably related to the limited investigation of physiologically relevant models of this process (Dutta, 2008).

This study used steroid-activated autophagic cell death of salivary glands as a system to study the relationship between cell growth and cell death. It is logical that cell growth influences cell death in salivary glands, given that autophagy is known to be regulated by class I PI3K signaling, which contributes to the death of these cells (Berry, 2007). It is unclear whether growth arrest is a determinant of autophagic cell death in other cell types and animals, and this question is important to resolve because of the importance of growth and autophagy in multiple disorders, including cancer. wts mutant salivary gland cells fail to arrest growth at the onset of puparium formation, and this suppresses the induction of autophagy. The inhibitor of apoptosis DIAP1 influences salivary gland cell death and is one of the best-characterized target genes of the Wts signaling pathway, but DIAP1 levels are not altered in wts mutant salivary glands. Significantly, the data provide the first evidence that Wts regulates autophagy and support previous studies indicating that caspases and autophagy function in an additive manner during autophagic cell death. Given the importance of both the Wts pathway and autophagy in human health, it is critical to determine whether this relationship exists in other cells (Dutta, 2008).

Cell growth and division are often considered to be synonymous, even though they are controlled by independent mechanisms. The Wts signaling pathway must influence cell growth, but most studies have emphasized the influence of this pathway on cell division and death. bantam is the only previously studied gene that is regulated by the Wts pathway and that is known to regulate cell growth. However, the mechanism of bantam action remains obscure. The current studies suggest the possibility that Wts may regulate growth via different mechanisms and that the nature of this regulation may depend on cell context. It is premature to conclude that bantam regulates a completely novel cell growth program, but the fact that misexpression of bantam stimulates cell growth in the absence of changes in a phosphoinositide marker and that chico and TOR fail to suppress the bantam-induced salivary gland-persistence phenotype minimally suggests that this microRNA regulates genes downstream of TOR. Significant progress has been made in the identification of microRNA targets, and future studies should resolve the mechanism underlying bantam regulation of cell growth (Dutta, 2008).

Recent studies of Wts signaling in Drosophila have identified a linear pathway that terminates with Yki and Sd regulation of effector genes that influence cell growth, cell division, and cell death. These studies indicate that the Wts pathway may not always regulate downstream effector genes via Yki and Sd, given that Yki expression was not able to phenocopy the wts mutant salivary gland destruction and expression of Sd induced premature degradation of salivary glands. Although bantam expression is sufficient to induce growth and inhibit cell death in salivary glands, bantam function is not required for the wts mutant phenotype. wts mutant salivary glands possess altered markers of PI3K signaling, and their defect in cell death is suppressed by chico and TOR. Combined, these results indicate that Wts regulates cell growth and cell death via a PI3K-dependent, and Yki- and Sd-independent, mechanism. Future studies will determine whether Wts regulates cell growth in a PI3K-dependent manner in other cells and animals (Dutta, 2008).

SREBP activity is regulated by mTORC1 and contributes to Akt-dependent cell growth

Cell growth (accumulation of mass) needs to be coordinated with metabolic processes that are required for the synthesis of macromolecules. The PI3-kinase/Akt signaling pathway induces cell growth via activation of complex 1 of the target of rapamycin (TORC1). This study shows that Akt-dependent lipogenesis requires mTORC1 activity. Furthermore, nuclear accumulation of the mature form of the sterol responsive element binding protein (SREBP1) and expression of SREBP target genes was blocked by the mTORC1 inhibitor rapamycin. Silencing of SREBP blocks Akt-dependent lipogenesis and attenuates the increase in cell size in response to Akt activation in vitro. Silencing of Drosophila SREBP (Helix loop helix protein 106) caused a reduction in cell and organ size and blocked the induction of cell growth by dPI3K. These results suggest that the PI3K/Akt/TOR pathway regulates protein and lipid biosynthesis in an orchestrated manner and that both processes are required for cell growth (Porstmann, 2008).

It was asked whether the PI3K/Akt pathway regulates the activity of SREBP in flies. Transient silencing of the catalytic subunit of PI3K, dp110, or dAkt reduced mRNA abundance of dFAS (Fatty acid synthase) and dSREBP in Kc167 cells. Conversely, silencing of dPTEN resulted in increased expression of both transcripts. Ubiquitous expression of dp110 using the da-GAL4 driver resulted in enhanced dSREBP and dFAS expression in second instar larvae compared to controls, indicating that the PI3K/Akt pathway activates dSREBP function (Porstmann, 2008).

Expression of mature dSREBP (m-dSREBP) using the MS1096-GAL4 driver resulted in significant lethality when flies were reared at 25°C (data not shown). When flies were reared at 18°C (resulting in a reduced activity of the GAL4/UAS system), severe deformation of the wing was observed. Expression of the full-length form of dSREBP (fl-dSREBP) in the wing caused less severely misshapen wings even when flies were reared at 25°C. However, coexpression of dp110 and full-length dSREBP resulted in severely deformed wings, a phenotype that is similar to that caused by expression of m-dSREBP. This result suggests that the activity of full-length dSREBP is enhanced by PI3K signaling (Porstmann, 2008).

Expression of dp110 causes an overgrowth phenotype in the wing, indicated by a 15%-20% increase in surface area of the wing. Silencing of dSREBP attenuated the increase in wing size induced by expression of dp110. Similar results were obtained using a nonoverlapping RNAi sequence targeting dSREBP or by heterozygous deletion of the dSREBP gene (Porstmann, 2008).

Expression of a kinase domain mutant of PI3K (dp110[KD]) decreases cell and organ size in Drosophila. Expression of dp110[KD] using the decapentaplegic (dpp)-GAL4 driver resulted in a 20%-30% reduction in the size of the dpp compartment. Expression of the dSREBP RNAi hairpin resulted in a small but significant decrease in wing area. However, coexpression of dSREBP RNAi with dp110KD did not further decrease the size of this compartment Taken together, these results suggest that dp110 and dSREBP are components of the same pathway in the regulation of cell growth in Drosophila (Porstmann, 2008).

Loss of PI3K blocks cell-cycle progression in a Drosophila tumor model

Tumorigenesis is a complex process, which requires alterations in several tumor suppressor or oncogenes. This study used a Drosophila tumor model to identify genes, which are specifically required for tumor growth. Reduction of phosphoinositide 3-kinase (PI3K) activity was found to result in very small tumors while only slightly affecting growth of wild-type tissue. The observed inhibition on tumor growth occurred at the level of cell-cycle progression. It is concluded that tumor cells become dependent on PI3K function and that reduction of PI3K activity synthetically interferes with tumor growth. The results of this study broaden insights into the intricate mechanisms underling tumorigenesis and illustrate the power of Drosophila genetics in revealing weak points of tumor progression (Willecke, 2011).

This study employed a genetic approach to identify genes required for the neoplastic growth phenotype of Ras^V12, Dlg^RNAi tumors. It was found that Ras^V12, Dlg^RNAi tumors are highly sensitive to reductions of the PI3K pathway and that changes in PI3K activity block cell-cycle progression (Willecke, 2011).

In agreement with previous reports, this study found that Ras^V12 induces the PI3K pathway in Drosophila. Yet curiously, Ras^V12, Dlg^RNAi tumors exhibit low levels of PI3K signaling. A possible reason for the paradoxical result may be derived from the cell polarity defects of Ras^V12, Dlg^RNAi tumors, which are not induced when Ras^V12 is expressed in otherwise wild-type cells or in combination with Upd. This interpretation implies that the activation of PI3K through Ras^V12 depends on proper cell polarity. In support of this explanation, it has been reported that Ras^V12 activates the PIP₃ reporter only at the apical side of cells. Additionally, studies have shown that PTEN directly binds to the polarity gene Bazooka and that the activity of the PI3K pathway is polarized in Drosophila oocyte cells. The activation of the PI3K pathway might, therefore, require the preservation of cell polarity, which could explain why Ras^V12 does not induce the PI3K pathway if Dlg is lost (Willecke, 2011).

Why are Ras^V12, Dlg^RNAi tumors sensitive to changes in PI3K signaling? Ras^V12, Dlg^RNAi tumor cells receive mitogenic signals from the JAK-STAT and MAPK pathways, which promote extensive tumor growth. Ras^V12, Dlg^RNAi tumors require a higher metabolic rate compared with wild-type cells, without gaining extra activation of PI3K through Ras^V12. As a result, PI3K signaling might be absolutely limiting for the growth of Ras^V12, Dlg^RNAi tumors. Expression of PI3K^RNAi then causes PI3K activity to drop below the threshold for such cells, triggering a block in cell-cycle progression. Tumors which express Ras^V12 together with Upd are not sensitive to changes in PI3K levels even though they overgrow as much as Ras^V12, Dlg^RNAi tumors. An pAkt western blot shows, however, that these tumors have high levels of PI3K signaling. Expression of PI3K^RNAi in this background might not cause PI3K activity to drop below a threshold for cell-cycle progression (Willecke, 2011).

Compounds that block PI3K pathway activity are known to be potent inhibitors of mammalian tumor growth and inhibitors that target PI3K, and other members of the pathway are currently being tested in clinical trials. Preclinical and clinical trials focus mainly on tumors that carry mutations in PI3K pathway components or that display abnormal levels of the biomarkers pAKT and PS6k1. The current results are, however, an example for a case where tumors with no genetic alterations in PI3K signaling components are also highly susceptible to reduction of PI3K levels. Understanding the molecular networks that create such PI3K dependency is a central topic in cancer research as it is highly relevant to identify PI3K-dependent tumors to predict the potential effectiveness of PI3K inhibitors. Genetic studies in Drosophila may therefore complement mammalian studies to more precisely determine which tumor-initiating pathways create PI3K sensitivity (Willecke, 2011).

In conclusion, this study has uncovered a synthetic interaction between the Drosophila PI3K signaling and Ras^V12, Dlg^RNAi tumor-initiating pathways. The results provide insights into the complex mechanisms underlying tumorigenesis and illustrate the power of Drosophila genetics in revealing the vulnerabilities of tumors. Identification of additional synthetic interactions through genetic screening in Drosophila may serve as a valuable resource for identifying potential drug targets in cancer therapy (Willecke, 2011).

Ligand activation of the metabotropic glutamate receptor (mGluR) activates the lipid kinase PI3K in both the mammalian central nervous system and Drosophila motor nerve terminal. In several subregions of the mammalian brain, mGluR-mediated PI3K activation is essential for a form of synaptic plasticity termed long-term depression (LTD), which is implicated in neurological diseases such as fragile X and autism. In Drosophila larval motor neurons, ligand activation of DmGluRA, the sole Drosophila mGluR, similarly mediates a PI3K-dependent downregulation of neuronal activity. The mechanism by which mGluR activates PI3K remains incompletely understood in either mammals or Drosophila. This study identified CaMKII and the nonreceptor tyrosine kinase DFak as critical intermediates in the DmGluRA-dependent activation of PI3K at Drosophila motor nerve terminals. Transgene-induced CaMKII inhibition or the DFak^CG1 null mutation each block the ability of glutamate application to activate PI3K in larval motor nerve terminals, whereas transgene-induced CaMKII activation increases PI3K activity in motor nerve terminals in a DFak-dependent manner, even in the absence of glutamate application. It was also found that CaMKII activation induces other PI3K-dependent effects, such as increased motor axon diameter and increased synapse number at the larval neuromuscular junction. CaMKII, but not PI3K, requires DFak activity for these increases. It is concluded that the activation of PI3K by DmGluRA is mediated by CaMKII and DFak (Lin, 2011).

Metabotropic glutamate receptors (mGluRs), G protein-coupled receptors for which glutamate is ligand, mediate aspects of synaptic plasticity in several systems. In several regions of the mammalian brain, including the hippocampus, the cerebellum, the prefrontal cortex, and others, ligand activation of group I mGluRs induces a long-term depression of synaptic activity, termed mGluR-mediated long-term depression (LTD). Induction of mGluR-mediated LTD both activates and requires the activation of the lipid kinase PI3 kinase (PI3K) and the downstream kinase Tor. Several genetic diseases of the nervous system are predicted to increase sensitivity to activation of mGluR-mediated LTD. For example, increased sensitivity to induction of mGluR-mediated LTD has been observed in the mouse model for fragile X. Furthermore, the genes affected in tuberous sclerosis (Tsc1 and Tsc2) and neurofibromatosis (Nf1) encode proteins that downregulate Tor activity. These observations raise the possibility that hyperactivation of mGluR-mediated LTD plays a causal role in the neurological phenotypes of fragile X, neurofibromatosis and tuberous sclerosis. Because these diseases are each associated with an extremely high incidence of autism spectrum disorders (ASDs), and because several lines of evidence suggest that elevated PI3K activity is associated with ASDs, it has been hypothesized that hyperactivation of this pathway might be responsible for ASDs as well. Thus it would be of interest to identify additional molecular components by which mGluR activation activates PI3K, and yet despite recent advances, this mechanism remains incompletely understood (Lin, 2011).

In Drosophila larval motor neurons, glutamate activation of the single mGluR, called DmGluRA, downregulates neuronal excitability; glutamate both activates PI3K and requires PI3K activity for this downregulation). Because glutamate is the excitatory neurotransmitter at the Drosophila neuromuscular junction (NMJ), it was hypothesized that this DmGluRA-mediated downregulation of neuronal excitability carried out a negative feedback on activity: glutamate released from motor nerve terminals would activate DmGluRA autoreceptors, which would then depress excitability (Lin, 2011).

This study identified additional molecular components that mediate the activation of PI3K by DmGluRA in Drosophila larval motor nerve terminals. It was found that activity of the calcium/calmodulin-dependent kinase II (CaMKII) is necessary for glutamate application to activate PI3K, and expression of the constitutively active CaMKII^T287D is sufficient both to activate PI3K even in the absence of glutamate and to confer several other neuronal phenotypes consistent with PI3K hyperactivation. It was also found that CaMKII^T287D requires the nonreceptor tyrosine kinase DFak for this PI3K activation: the DFak^CG1 null mutation blocks the ability of glutamate application to activate PI3K and prevents CaMKII^T287D from hyperactivating PI3K. Finally, CaMKII^T287D expression completely suppresses the hyperexcitability conferred by the DmGluRA null mutation DmGluRA^112b. It is concluded that ligand activation of DmGluRA activates PI3K via CaMKII and DFak (Lin, 2011).

In both mammalian central synapses and Drosophila larval motor neurons, activation by glutamate of the metabotropic glutamate receptor (mGluR) activates the lipid kinase PI3K, but the mechanism by which this activation occurs has not been elucidated. This study identified CaMKII as a critical intermediate in the ability of the single Drosophila mGluR (DmGluRA) to activate PI3K and shows that the ability of both activated DmGluRA and CaMKII to activate PI3K requires the nonreceptor tyrosine kinase, DFak (see A proposed mechanism for the DmGluRA-dependent activation of PI3K via CaMKII and DFak). These results provide novel insights into the mechanism by which DmGluRA activation triggers the observed downregulation of subsequent neuronal activity in Drosophila motor neurons. These results might also be relevant to the mechanism by which mGluR activates PI3K in mammalian central synapses, a process implicated in fragile X, ASDs, neurofibromatosis, and tuberous sclerosis (Lin, 2011).

How might CaMKII lead to the DFak-dependent activation of PI3K? Although the ability of CaMKII to activate PI3K has only recently been reported, it has been well established in mammals that CaMKII phosphorylates both Fak and Pyk2 on multiple serines on the C terminus. These phosphorylation events can activate Pyk2 by enabling subsequent tyrosine phosphorylations (particularly at Tyr402) via mechanisms that are incompletely understood. It has also been well established that Fak and Pyk2, when activated by tyrosine phosphorylation, are each able to activate PI3K: tyrosine-phosphorylated Fak binds p85, the PI3K regulatory subunit, via both the SH3 and SH2 domains. In addition, both tyrosine-phosphorylated Fak and Pyk2 are capable of activating Ras via the conserved Grb2-SoS pathway, which could in principle lead to the Ras-dependent, p85-independent activation of PI3K. These observations raise the possibility that Drosophila CaMKII might similarly activate PI3K by directly phosphorylating and activating DFak. Alternatively, DFak might function in a more indirect fashion, perhaps as a scaffold linking CaMKII and PI3K in a signaling complex. This alternative possibility would suggest that additional intermediates linking CaMKII and PI3K activation exist but are currently unidentified (Lin, 2011).

The observation that DmGluRA-mediated activation of PI3K requires CaMKII implies that DmGluRA activation increases intracellular Ca²⁺ levels in Drosophila motor nerve terminals as a necessary step in PI3K activation. The source of Ca²⁺ for this activation is not known. However in mammals, activation of group I mGluRs, which are responsible for mGluR-mediated LTD in the hippocampus and cerebellum, induce phospholipase C and IP3-mediated Ca²⁺ transients, which are essential intermediates in cerebellar mGluR-mediated LTD. Although the Drosophila DmGluRA is most similar to mammalian group II mGluRs, which are not known to activate Ca²⁺ transients, given that DmGluRA is the sole mGluR in Drosophila, it seems possible that DmGluRA might carry out many of the functions carried out by each of the three groups of mGluRs in mammals, as suggested previously. Alternatively, it is possible that DmGluRA activation might increase intracellular Ca²⁺ via the ryanodine receptor, which was previously shown to be an essential activator of CaMKII in Drosophila larval motor nerve terminals (Lin, 2011).

The ability of CaMKII to activate PI3K requires the nonreceptor tyrosine kinase DFak; the DFak^CG1 null mutation completely blocks the ability of glutamate applied to motor nerve terminals to activate PI3K, completely suppresses the increase in basal p-Akt levels conferred by CaMKII^T287D, and blocks the ability of CaMKII^T287D to confer two additional PI3K-dependent phenotypes: increased synapse number at the NMJ and increased motor axon diameter. These results identify DFak as an essential intermediate in PI3K activation by DmGluRA and CaMKII. However, DFak^CG1 mutants fail to exhibit other phenotypes conferred by decreased PI3K activity: in an otherwise wild-type background, DFak^CG1 larvae exhibit only minor effects on NMJ synapse number or motor axon diameters, which are each significantly decreased by decreased PI3K. These results raise the possibility that, whereas PI3K activation by DmGluRA and CaMKII is blocked in DFak^CG1, total PI3K activity is not strongly decreased because other significant routes to PI3K activation are DFak independent. Alternatively, DFak might participate in signaling pathways distinct from the CaMKII-DFak-PI3K pathway identified in this study that would oppose the effects of PI3K on synapse number and axon diameter. In this view, CaMKII would preferentially promote the ability of DFak to activate PI3K, rather than other DFak-dependent pathways (Lin, 2011).

In several subregions of the mammalian brain, ligand activation of group I mGluRs induces LTD, a type of synaptic plasticity. This induction both activates and requires the activity of PI3K as well as the PI3K-activated kinase Tor. Several lines of evidence have led to the proposal that increased sensitivity to mGluR-mediated LTD induction might underlie specific neurogenetic disorders. In particular, mice null for the gene affected in fragile X, which is associated with an extremely high incidence of autism as well as other cognitive deficits, exhibit increased sensitivity to mGluR-mediated LTD induction in both the hippocampus. Furthermore, the genes identified in two additional diseases associated with a high incidence of autism, neurofibromatosis (Nf1) and tuberous sclerosis (Tsc1 and Tsc2), each encode negative regulators of the PI3K pathway: Nf1 encodes a Ras GTPase activator, which inhibits the PI3K activator Ras, whereas the Tsc proteins are Tor inhibitors that are in turn inhibited by PI3K activity. Thus loss of Nf1 or Tsc might also increase sensitivity to mGluR-mediated LTD. Finally, several lines of direct evidence suggest that PI3K hyperactivation plays a causal role in autism. For example, DNA copy number variants observed in individuals with autism but not unaffected individuals identify at high frequency PI3K subunits or regulators, and each genetic change is predicted to elevate PI3K activity. In addition, a translocation that increases expression of the translation factor eIF-4E, which is known to be activated by the PI3K pathway, plays a direct, causal role in autism. The potential involvement of mGluR-mediated LTD in these neurogenetic disorders increases interest in identifying the molecular intermediates that participate in this pathway, but these intermediates are for the most part unidentified. Thus, the possibility that CaMKII and Fak might participate in mGluR-mediated PI3K activation in mammals as well as Drosophila might have significant medical interest (Lin, 2011).

Cell mixing induced by myc is required for competitive tissue invasion and destruction

Cell-cell intercalation is used in several developmental processes to shape the normal body plan. There is no clear evidence that intercalation is involved in pathologies. This study used the proto-oncogene myc to study a process analogous to early phase of tumour expansion: myc-induced cell competition. Cell competition is a conserved mechanism driving the elimination of slow-proliferating cells (so-called 'losers') by faster-proliferating neighbours (so-called 'winners') through apoptosis and is important in preventing developmental malformations and maintain tissue fitness. Using long-term live imaging of myc-driven competition in the Drosophila pupal notum and in the wing imaginal disc, this study showed that the probability of elimination of loser cells correlates with the surface of contact shared with winners. As such, modifying loser-winner interface morphology can modulate the strength of competition. Elimination of loser clones requires winner-loser cell mixing through cell-cell intercalation. Cell mixing is driven by differential growth and the high tension at winner-winner interfaces relative to winner-loser and loser-loser interfaces, which leads to a preferential stabilization of winner-loser contacts and reduction of clone compactness over time. Differences in tension are generated by a relative difference in F-actin levels between loser and winner junctions, induced by differential levels of the membrane lipid phosphatidylinositol (3,4,5)-trisphosphate. These results establish the first link between cell-cell intercalation induced by a proto-oncogene and how it promotes invasiveness and destruction of healthy tissues (Levayer, 2015).

To analyse quantitatively loser cell elimination, long-term live imaging was performed of clones showing a relative decrease of the proto-oncogene myc in the Drosophila pupal notum, a condition known to induce cell competition in the wing disc. Every loser cell delamination was counted over 10 h, and the probability of cell elimination was calculated for a given surface of contact shared with winner cells. A significant increase was observed of the proportion of delamination with winner-loser shared contact, whereas this proportion remained constant for control clones. The same correlation was observed in ex vivo culture of larval wing disc. Cell delamination in the notum was apoptosis dependent and expression of flower^lose (fwe^lose), a competition-specific marker for loser fate, was necessary and sufficient to drive contact-dependent delamination. Moreover it was confirmed that contact-dependent death is based on the computation of relative differences of fwe^lose between loser cells and their neighbours. Thus, cell delamination in the notum recapitulates features of cell competition (Levayer, 2015).

This suggests that winner-loser interface morphology could modulate the probability of eliminating loser clones. Using the wing imaginal disc, winner-loser contact was reduced by inducing adhesion- or tension-dependent cell sorting and observed a significant reduction of loser clone elimination. This rescue was not driven by a cell-autonomous effect of E-cadherin (E-cad) or active myosin II regulatory light chain (MRLC) on growth, death or cell fitness but rather by a general diminution of winner-loser contact. Competition is ineffective across the antero-posterior compartment boundary, a frontier that prevents cell mixing through high line tension. Accordingly, there was no increase in death at the antero-posterior boundary in wing discs overexpressing fwe^loseA in the anterior compartment. However, reducing tension by reducing levels of myosin II heavy chains was sufficient to increase the shared surface of contact between cells of the anterior and posterior compartments, and induced fwe^lose death at the boundary. Altogether, it is concluded that the reduction in surface contact between winners and losers is sufficient to block competition, which explains how compartment boundaries prevent competition (Levayer, 2015).

Loser clones have been reported to fragment more often than controls, whereas winner clones show convoluted morphology, suggesting that winner-loser mixing is increased during competition. This could affect the outcome of cell competition by increasing the surface shared between losers and winners. Clone splitting was used as a readout for loser–winner mixing. Two non-exclusive mechanisms can drive clone splitting: cell death followed by junction rearrangement, or junction remodelling and cell–cell intercalation independent of death. To assess the contribution of each phenomenon, the proportion of clones fragmented 48 h after clone induction (ACI) was systematically counted. A twofold increase was observed in the frequency of split clones in losers (wild type (WT) in tub-dmyc) versus WT in WT controls. Overexpressing E-cad or active myosin II was sufficient to prevent loser clone splitting, whereas blocking apoptosis or blocking loser fate by silencing fwe^lose did not reduce splitting. Finally, the proportion of split clones was also increased for winner clones either during myc-driven competition or during Minute-dependent competition. Altogether, this suggested that winner–loser mixing is increased independently of loser cell death or clone size by a factor upstream of fwe, and could be driven by cell–cell intercalation. Accordingly, junction remodelling events leading to disappearance of a loser–loser junction were three times more frequent at loser clone boundaries than control clone boundaries in the pupal notum. The rate of junction remodelling was higher in loser–loser junctions and in winner–winner junctions than in winner–loser junctions. The preferential stabilization of winner–loser interfaces should increase the surface of contact between winner and loser cells over time. Accordingly, loser clone compactness in the notum decreased over time whereas it remains constant on average for WT clones in WT background. Similarly, the compactness of clones in the notum also decreased over time for conditions showing high frequency of clone splitting in the wing disc, whereas clone compactness remained constant for conditions rescuing clone splitting. Altogether, it is concluded that both Minute- and myc-dependent competition increase loser–winner mixing through cell–cell intercalation (Levayer, 2015).

It was then asked what could modulate the rate of junction remodelling during competition. The rate of junction remodelling can be cell-autonomously increased by myc. Interestingly, downregulation of the tumour suppressor PTEN is also sufficient to increase the rate of junction remodelling through the upregulation of phosphatidylinositol (3,4,5)-trisphosphate (PIP3). It was reasoned that differences in PIP3 levels could also modulate junction remodelling during competition. Using a live reporter of PIP3 that could detect modulations of PIP3 in the notum, a significant increase of PIP3 was observed in the apico-lateral membrane of tub-dmyc–tub-dmyc interfaces compared with WT–WT and WT–tub-dmyc interfaces (Fig. 3a, b). Moreover, increasing/reducing Myc levels in a full compartment of the wing disc was sufficient to increase/decrease the levels of phospho-Akt (a downstream target of PIP3, whereas fwe^loseA overexpression had no effect. Similarly, levels of phospho-Akt were relatively higher in WT clones than in the surrounding M^-/+ cells. Thus differences in PIP3 levels might be responsible for winner–loser mixing. Accordingly, reducing PIP3 levels by overexpressing a PI3 kinase dominant negative (PI3K-DN) or increasing PIP3 levels by knocking down PTEN (UAS-pten RNAi) were both sufficient to induce a high proportion of fragmented clones and to reduce clone compactness over time in the notum , whereas increasing PIP3 in loser clones was sufficient to prevent cell mixing. Moreover, abolishing winner–loser PIP3 differences through larval starvation prevented loser clone fragmentation, the reduction of clone compactness over time in the notum and could rescue WT clone elimination in tub-dmyc background. It is therefore concluded that differences in PIP3 levels are necessary and sufficient for loser–winner mixing and required for loser cell elimination (Levayer, 2015).

It was then asked which downstream effectors of PIP3 could affect junction stability. A relative growth decrease can generate mechanical stress that can be released by cell-cell intercalation. Accordingly, growth reduction through Akt downregulation is sufficient to increase clone splitting and could contribute to loser clone splitting. However, Akt is not sufficient to explain winner-loser mixing because, unlike PIP3, increasing Akt had no effect on clone splitting. PIP3 could also modulate junction remodelling through its effect on cytoskeleton and the modulation of intercellular adhesion or tension. No obvious modifications of E-cad, MRLC or Dachs (another regulator of tension) was detected in loser cells. However, a significant reduction of F-actin levels and a reduction of actin turnover/polymerization rate were observed in loser-loser and loser-winner junctions in the notum. Similarly, modifying Myc levels in a full wing disc compartment was sufficient to modify actin levels, and F-actin levels were higher in WT clones than M-/+ cells. This prompted a test of the role of actin organization in winner-loser mixing. Downregulating the formin Diaphanous (Dia, a filamentous actin polymerization factor) by RNA interference (RNAi) or by using a hypomorphic mutant was sufficient to obtain a high proportion of fragmented clones and to reduce clone compactness over time, whereas overexpressing Dia in loser clones prevented clone splitting (UAS-dia::GFP) and compactness reduction. This effect was specific to Dia as modulating Arp2/3 complex (a regulator of dendritic actin network) had no effect on clone splitting. Thus, impaired filamentous actin organization was necessary and sufficient to drive loser-winner mixing. These actin defects were driven by the differences in PIP3 levels between losers and winners. Thus Dia could be an important regulator of competition through its effect on cell mixing. Overexpression of Dia was indeed sufficient to reduce loser clone elimination significantly (Levayer, 2015).

Filamentous actin has been associated with tension regulation. It was therefore asked whether junction tension was modified in winner and loser junctions. The maximum speed of relaxation of junction after laser nanoablation (which is proportional to tension) was significantly reduced in loser-loser and winner-loser junctions compared with winner-winner junctions. This distribution of tension has been proposed to promote cell mixing. Accordingly, decreasing PIP3 in clones reduced tension both in low-PIP3-low-PIP3 and low-PIP3-normal-PIP3 junctions, whereas overexpressing Dia in loser clones or starvation were both sufficient to abolish differences in tension, in agreement with their effect on winner-loser mixing and the distribution of F-actin. Thus the lower tension at winner-loser and loser-loser junctions is responsible for winner-loser mixing. Altogether, it is concluded that the relative PIP3 decrease in losers increases winner-loser mixing through Akt-dependent differential growth and the modulation of tension through F-actin downregulation in winner-loser and loser-loser junctions (Levayer, 2015).

Several modes of tissue invasion by cancer cells have been described, most of them relying on the departure of the tumour cells from the epithelial layer. This study suggests that some oncogenes may also drive tissue destruction and invasion by inducing ectopic cell intercalation between cancerous and healthy cells, and subsequent healthy cell elimination. myc-dependent invasion could be enhanced by other mutations further promoting intercalation (such as PTEN). Stiffness is increased in many tumours, suggesting that healthy cell-cancer cell mixing by intercalation might be a general process (Levayer, 2015).

Protein Interactions

Regulatory subunits of PI3K

The mammalian phosphoinositide 3-kinases (PI3Ks) p110alpha, beta, and delta form heterodimers with Src homology 2 (SH2) domain-containing adaptors such as p85alpha or p55(PIK). The two SH2 domains of these adaptors bind to phosphotyrosine residues (pY) found within the consensus sequence pYXXM. A heterodimer of Drosophila PI3K, Dp110, with an adaptor, p60, can be purified from S2 cells with a pYXXM phosphopeptide affinity matrix. Using amino acid sequence from the gel-purified protein, the gene encoding p60 was cloned and mapped to the genomic region 21B8-C1, and the exon/intron structure was determined. p60 contains two SH2 domains and an inter-SH2 domain but lacks the SH3 and breakpoint cluster region homology (BH) domains found in mammalian p85alpha and beta. Analysis of the sequence of p60 shows that the amino acids responsible for the SH2 domain binding specificity in mammalian p85alpha are conserved and predicts that the inter-SH2 domain has a coiled-coil structure. The Dp110.p60 complex was immunoprecipitated with p60-specific antisera and shown to possess both lipid and protein kinase activity. The complex was found in larvae, pupae, and adults, consistent with p60 functioning as the adaptor for Dp110 throughout the Drosophila life cycle (Weinkove, 1997).

Mammalian phosphatidylinositol 3-kinase (PI 3-kinase) plays an important role in the regulation of various cellular and receptor tyrosine kinase-mediated processes, such as mitogenesis and transformation. PI 3-kinase is composed of a 110-kDa catalytic subunit and a regulatory subunit of 85 kDa or 55 kDa. A regulatory subunit from Drosophila melanogaster, named droPIK57, has been cloned from head-specific cDNA libraries. The droPIK57 gene encodes a protein containing two SH2 domains with significant sequence homology to those in p85 and p55. Like the p55 subunits, DroPIK57 is missing the SH3 domain and the bcr homology region of the p85 subunit. The short N-terminus as well as the C-terminus of the DroPIK57 protein show no identity to the known PI 3-kinase subunits, suggesting that it is a new member in the family of regulatory subunits. In-situ hybridization and Northern blot analysis indicate a widespread function of this gene during embryogenesis and in the CNS (Albert 1997).

Susi, a negative regulator of Drosophila PI3-kinase: Susi regulates PI3K activity by directly binding to P60, the regulatory subunit of PI3K

The Phosphatidylinositol-3 kinase/Protein Kinase B (PI3K/PKB) signaling pathway controls growth, metabolism, and lifespan in animals, and deregulation of its activity is associated with diabetes and cancer in humans. Susi (also known as B4), a coiled-coil domain protein acts as a negative regulator of insulin signaling in Drosophila. Whereas loss of Susi function increases body size, overexpression of Susi reduces growth. Genetic evidence is provided that Susi negatively regulates dPI3K activity. Susi directly binds to dP60, the regulatory subunit of dPI3K. Since Susi has no overt similarity to known inhibitors of PI3K/PKB signaling, it defines a novel mechanism by which this signaling cascade is kept in check. The fact that Susi is expressed in a circadian rhythm, with highest levels during the night, suggests that Susi attenuates insulin signaling during the fasting period (Wittwer, 2005).

To identify negative regulators of dINR signaling, a misexpression screen was carried out for genes that suppress the overgrowth phenotype caused by overexpression of wild-type dINR in the developing eye using the UAS/Gal4 system. 5,400 fly lines were tested containing random insertions of an enhancer-promoter (EP) element that permits the transcription of genes flanking the insertion in response to Gal4. Several independent lines that strongly suppress the dINR-induced eye phenotype (among them EP(7-66)) contain EP insertions upstream of the first coding exon of the B4 gene. The suppression is caused by the overexpression of the B4 gene, since overexpression of B4 from a UAS transgene had the same effect. Therefore, the B4 locus was renamed Suppressor of signaling by insulin (Susi). Susi encodes a novel protein with a predicted coiled-coil (CC) domain (amino acids 916-942. Proteins that are obvious orthologs of Susi exist in other insect species such as Drosophila pseudoobscura (75% identical amino acids) and Anopheles gambiae. Owing to the low sequence conservation and the large size of the family of CC domain-containing proteins, it was not possible to resolve whether one of the CC domain proteins from higher organisms is a Susi ortholog (Wittwer, 2005).

Susi overexpression suppresses dINR function in other developmental processes also. For example, embryonic lethality associated with the expression of dINR by en-Gal4 was suppressed by the concomitant expression of Susi. Importantly, the effects of Susi overexpression appear to be specific for dINR/dPI3K signaling. The complete set of EP lines was tested in parallel for effects on other signaling pathways, including the growth promotion by dMyc, and the EP insertions in the Susi locus were not found in screens other than the dINR screen (Wittwer, 2005).

Several lines of evidence suggest that Susi is a novel negative regulator in insulin signaling that acts between dINR and dPI3K. (1) Gain and loss of Susi function mimic the loss-of-function phenotypes of positive and negative regulators of the insulin pathway, respectively. Susi regulates cell growth by controlling cell number and cell size, but does not affect programmed cell death. (2) Susi, like dPTEN, releases PKB-dependent inhibition of dFOXO, thus enabling the expression of the dFOXO target gene d4EBP. (3) Susi, like dPTEN, attenuates PIP₃ levels induced by increased dINR activity, but, unlike dPTEN, fails to reduce PIP₃ levels induced by a membrane-tethered form of dPI3K (Wittwer, 2005).

Although complete loss of Susi function results in qualitatively similar phenotypes as loss of dPTEN function, the phenotypes are generally weaker. Whereas homozygous Susi flies are viable and increased in size, complete loss of dPTEN function results in lethality. Consistently, removal of dPTEN function using the ey-FLP system also results in a stronger increase in head size than removal of Susi function. Thus, the negative regulatory function of Susi on PI3K/PKB signaling is less pronounced than that of dPTEN (Wittwer, 2005).

Genetic analysis indicates that Susi acts between dINR and dPI3K, making dPI3K a possible target for Susi function. The protein sequence of Susi includes a putative coiled-coil (CC) protein interaction domain. In vitro binding studies show that Susi binds to the regulatory subunit dP60 of dPI3K. This result suggests that Susi inhibits PI3K/PKB signaling by binding and thereby inhibiting PI3K (Wittwer, 2005).

How does binding of Susi to dPI3K inhibit PI3K/PKB signaling? Susi may cause the degradation of dP60 and dP110. Alternatively, Susi may interfere with any step required for the activation of dPI3K, such as the formation of the dP60/dP110 heterodimer, recruitment of dPI3K to the membrane, or the conformational changes required for the activation of dPI3K. Overexpression of Susi in flies does not reduce dP110 protein levels and does not suppress the dP60 overexpression phenotype, making it unlikely that Susi causes the degradation of either dP110 or dP60 (Wittwer, 2005).

Co-overexpression of Susi with dP110 and dP60 in a cell culture system does not interfere with the formation of the dP110/P60 heterodimer. Therefore, it is unlikely that Susi interferes with the assembly of the dPI3K holoenzyme. In fact, binding of Susi to dP60 is independent of the binding of dP60 to the dP110 catalytic subunit. Since Susi regulates the activity of the wild-type but not of the membrane-tethered form of dPI3K, it may regulate the membrane recruitment and/or activation of dPI3K. Upon overexpression, a significant fraction of Susi protein is located at the membrane. It is therefore unlikely that Susi functions by retaining the dP60/dP110 complex in the cytoplasm. Since binding studies suggest that Susi forms a trimolecular complex with dP60 and dP110, it is possible that Susi interferes with PI3K activity by suppressing a conformational change required for dPI3K activation (Wittwer, 2005).

Is Susi function conserved in mammals? Based on sequence comparison, no clear ortholog of Susi has been identified outside insect species. Furthermore, Susi is unable to negatively regulate insulin signaling in mammalian cells. Under conditions where GST-Susi interacts with dP60, no interaction with the mammalian homologs of dP60, P85α and P85β, could be observed. Moreover, overexpression of Susi in COS-7 cells is unable to counteract the increased phospho-PKB levels caused by the overexpression of the human insulin receptor. The only domain recognizable in Susi is the CC domain, which is involved in protein-protein interactions and is present in a large number of different proteins. Interestingly, the domain is also present in another negative regulator of PI3K activity, the mammalian regulator of ubiquitous kinase (Ruk) protein. Ruk, also known as CIN85 or SETA, is an adaptor-type protein belonging to the CD2AP/CMS family and exists in three isoforms. Ruk_L consists of three SH3 domains, a proline-rich domain, and a C-terminal CC domain. Its interaction with the P85 subunit of PI3K requires the proline-rich domain of Ruk_L and the SH3 domain of P85. The role of the C-terminal CC domain has not been tested. Susi lacks SH3 and proline-rich domains. Thus, Susi and Ruk_L appear to interact in different ways with the corresponding PI3K adaptors dP60 and P85, respectively. Susi may therefore define a novel, possibly insect-specific, type of negative regulation of PI3K activity (Wittwer, 2005).

The relatively weak Susi loss-of-function phenotype suggests that Susi is involved in fine-tuning the cellular response to insulin. Interestingly, Susi expression in adult flies is modulated in a circadian rhythm. According to three independent studies, expression levels of Susi are higher during the night than during the day, reaching a maximum during the second part of the night. In mammals, insulin sensitivity shows diurnal changes. In humans and rats, insulin sensitivity increases toward the onset of the activity period (day for humans and night for rats). The mechanisms underlying this phenomenon are unknown. Insulin regulates carbohydrate metabolism in adult flies in a similar way as in mammals, and flies show a sleep-like behavior during the night. Susi may cause diurnal changes of insulin sensitivity in Drosophila similar to those in mammals. High levels of Susi during the night may contribute to a reduction in cell growth and metabolism in anticipation of the lack of feeding during this time. It should be pointed out, however, that the circadian expression of Susi has been described in adult flies. This study has characterized the role of Susi in larval growth regulation. The fact that Susi mutant flies are viable provides an ideal basis for addressing the potential role of Susi in circadian regulation of metabolism and physiology in the adult (Wittwer, 2005).

Signaling downstream of PI3K

The decision between survival and death is an important aspect of cellular regulation during development and malignancy. Central to this regulation is the process of apoptosis, which is conserved in multicellular organisms. A variety of signaling cascades have been implicated in modulation of apoptosis, including the phosphatidylinositol (Pl) 3-kinase pathway. Activation of Pl 3-kinase is protective, and inhibition of this lipid kinase enhances cell death under several conditions, including deregulated expression of c-Myc, neurotrophin withdrawal and anoikis. Recently, the protective effects of Pl 3-kinase have been linked to its activation of the pleckstrin homology (PH)-domain-containing protein kinase B (PKB or AKT). PKB/AKT was identified from an oncogene, v-akt, found in a rodent T-cell lymphoma. To initiate a genetic analysis of PKB, a Drosophila Akt1 mutant was isolated and characterized. It exhibits ectopic apoptosis during embryogenesis as judged by induction of membrane blebbing, DNA fragmentation and macrophage infiltration. Apoptosis caused by loss of Dakt function is rescued by caspase suppression but is distinct from previously described reaper/grim/hid functions. These data implicate Dakt1 as a cell survival gene in Drosophila, consistent with cell protection studies in mammals (Staveley, 1998).

Organism size is determined by a tightly regulated mechanism that coordinates cell growth, cell proliferation and cell death. The Drosophila insulin receptor/Chico/Dp110 pathway regulates cell and organism size. Genetic manipulation of the phosphoinositide-3-OH-kinase-dependent serine/threonine protein kinase Akt (protein kinase B) during development of the Drosophila imaginal disc affects cell and organ size in an autonomous manner. Ectopic expression of Akt does not affect cell-fate determination, apoptosis or proliferation rates in imaginal discs. Thus, Akt appears to stimulate intracellular pathways that specifically regulate cell and compartment size independent of cell proliferation in vivo (Verdu, 1999).

To determine whether Drosophila Akt1 participates in insulin-receptor signal transduction, Akt1 activity was measured in Schneider (S2) cells. Insulin stimulates Akt1 activity sevenfold in S2 cells overexpressing a wild-type Akt1 transgene. Furthermore, membrane localization of Akt1 by addition of an src myristoylation sequence to its amino terminus is sufficient to confer constitutive kinase activity. In contrast, kinase-deficient Akt1 shows activity neither in the basal state nor after insulin stimulation, thus indicating that the measured phosphotransferase activity is not due to a contaminating kinase. These observations confirm that Akt1 is regulated in a way similar to that of its mammalian homolog. Consistent with this proposal, pretreatment with the PI(3)K inhibitor wortmannin blocks Akt1 activation by insulin. These data indicate that, as in mammalian cells, Drosophila PI(3)K is a component required for mediating activation of Akt1 (Verdu, 1999).

To determine whether Akt1 transduces growth-related signals, Akt1-deficient somatic clones were generated in the developing eye by mitotic recombination. Adult eyes exhibit a reduction in size in Akt1-deficient rhabdomeres, which, in some instances, co-exist with normal-sized heterozygous cells in the same ommatidium. Akt1 mutant clones are rare and small and are obtained only after heat-shock during the third instar larval stage. These observations indicate that the lack of Akt1 clones in the adult retina following induction at early larval stages may have resulted from cell competition, by which the Akt1-deficient cells would be eliminated and replaced by the surrounding wild-type sister cells. The phenotype of the Akt1-deficient rhabdomeres may have resulted from perturbations of cell growth or proliferation. The smaller size of these rhabdomeres shows that Akt1 is essential for normal cell growth, but dispensable for cell-fate determination. Moreover, the co-existence of Akt1 mutant rhabdomeres with wild-type twin-spot rhabdomeres in the same ommatidium suggests a cell-autonomous control of cell growth by Akt1 (Verdu, 1999).

The small size of the clones of Akt1-deficient cells could be the result of an impairment in the proliferation, survival, or both, of homozygous null cells. To evaluate more specifically the effects of Akt1 on proliferation or growth in vivo, upstream activation sequence (UAS)-Akt1 lines were generated with which to investigate the effects of altering the amount of Akt1 during Drosophila eye and wing imaginal disc development. The gmrGAL4 transgene targets expression of Akt1 to cells posterior to the morphogenetic furrow, producing flies exhibiting enlarged and bulging eyes with a mild disruption of the regular, external lattice. Similar, but less pronounced, effects are observed with a sevGAL4 transgene. Quantitative analysis reveals that the Akt1-induced increase in the size of the eye is caused by an increase in the size but not in the number of ommatidia. To determine the extent of this phenotype, tangential sections of these eyes were examined. In spite of the rough appearance of the adult compound eye, Akt1 expression does not affect the normal process of photoreceptor cell-fate determination in these larger ommatidia (Verdu, 1999).

Akt plays a central part in promoting the survival of a wide range of cell types in mammalian systems and in Drosophila embryos. However, overexpression of Akt1 does not alter the normal rate of apoptosis in the eye, as shown by equivalent acridine orange staining in control and gmrGAL4/UAS-Akt1 eye imaginal discs. Hence, overexpression of Akt1 affects neither the normal processes of cell-fate determination nor apoptosis in the developing retina (Verdu, 1999).

To determine whether ectopic expression of Akt1 increases the size of tissues other than the eye, Akt1 was targeted to the wing using a 71BGAL4 line. This results in a marked enlargement of the wing imaginal disc and an expansion of the surface of the adult wing blade as well as an increase in vein thickness. This increase in size is often accompanied by a mild disruption of the proximo-distal alignment characteristic of the hairs present on the wing-blade surface. Morphometric analysis of 71BGAL4/UAS-Akt1 wings reveals a 29% increase in wing surface area. Furthermore, ectopic expression of Akt1 along the anteroposterior boundary of the wing imaginal disc results in enlargement of only the corresponding region of the adult wing. In spite of the increased size of the wing in 71BGAL4/UAS-Akt1 flies, there is no change in the number of cells, resulting in a cell density in 71BGAL4/UAS-Akt1 flies that is 15% lower than that in 71BGAL4/+ controls. Together, these observations show that ectopic expression of Akt1 increases the size of the wing imaginal disc, leading to enlargement of the adult wing. The question of whether the effect of Akt1 on compartment growth in the wing is cell autonomous was addressed further. Targeting of Akt1 to the posterior compartment of the wing imaginal disc with an engrailed-GAL4 line results in a marked expansion of this region, whereas the anterior compartment remains unaffected (Verdu, 1999).

To evaluate the Akt1-selective increase in cell size more quantitatively, Akt1 was expressed in the posterior compartment of wing imaginal discs; measured were compartment areas, cell size and cell number, the latter two by flow cytometry. Expression or inactivation of cell-cycle regulators, such as E2F, RBF and Cdc2, in the posterior compartment affects cell size and number without altering compartment size. Akt1 expression increases the area occupied by the posterior compartment concomitant with a marked enlargement of its cells as measured by forward light scatter. Strikingly, no changes in the number of cells in the posterior compartment are detected. Thus, overexpression of Akt1 affects compartment size by altering cell growth without a concomitant increase in the final number of cells within the compartment. Studies of mammalian cells have indicated that Akt may positively regulate cell-cycle progression. However, in the wing imaginal disc, no differences were found in cell proliferation between control cells in the anterior compartment and cells expressing Akt1 in the posterior compartment, as judged by the pattern or frequency of bromodeoxyuridine incorporation (Verdu, 1999).

Akt overrides G1 arrest induced by PTEN (see Drosophila Pten) and by interleukin-2 deprivation in cell-culture models. To determine whether ectopic Akt1 could bypass cell-cycle arrest in imaginal tissues, a population of physiologically arrested cells in the wing imaginal disc, the zone of non-proliferating cells (ZNC), was studied. Expression of positive regulators of the cell cycle, such as the phosphatase Cdc25^stg and cyclin E, bypasses both G1 and G2 arrests in the ZNC. Interestingly, Akt1 expression in the posterior compartment does not rescue the cells of the ZNC from their G1 arrest. As a more quantitative assay of Akt1 effects on cell-cycle progression, wing imaginal discs expressing Akt1 ubiquitously were dissected and cellular DNA content was measured by flow cytometry. The proportions of cells in G1, S and G2 phase remain indistinguishable in cells expressing Akt1 and wild-type cells, despite the differences in compartment size (Verdu, 1999).

A compartment functions as an independent unit of growth and size control. Ectopic expression of Akt1 overrides the intrinsic control mechanisms regulating the final size of the compartment. To circumvent potential compartment controls on cell number, clones of cells overexpressing Akt1 were generated in the wing imaginal disc. Clone size was assessed 48 h after induction by heat-shock. Akt1 markedly increases clonal size through an enlargement of the cells rather than an increase in the cell number. As a more sensitive assay of cell number, clones of cells expressing Akt1 were induced in the wing disc 72 h after egg deposition and cell number was assessed 48 h later. Analysis reveals that the increase in clonal size induced by ectopic Akt1 expression is due to a selective increase in cell size but not cell number. Thus, it is concluded that Akt1 affects compartment size by increasing cell growth (that is, cell size) without altering cell proliferation (Verdu, 1999).

Several lines of evidence indicate a requirement for components of the protein-synthetic regulatory apparatus for cell growth. The large-cell and small-cell phenotypes resulting from increasing or removing Akt activity, respectively, are consistent with concomitant alterations in the translational machinery. In mammals, Akt appears to influence the rate of protein synthesis through mTOR (for mammalian target of rapamycin)-mediated activation of p70S6kinase (see Drosophila RPS6-p70-protein kinase) and inhibition of the 4E-binding protein-1 (4E-BP1 or PHAS-1), a repressor of translation initiation. These results implicate Akt as an activator of messenger RNA translation and indicate that regulation of this pathway could be relevant to the ability of Akt to promote cell growth in vivo. A critical question is whether increases in protein synthesis are merely permissive for expansion of cell size, implying the existence of a distinct growth-regulatory mechanism, or whether Akt-dependent enhancement of protein translation is in itself sufficient to cause an increase in organ size. Alternatively, the augmentation in cell growth produced by Akt could be the result of activation of a concerted anabolic program, of which protein synthesis would be a vital component (Verdu, 1999 and references therein).

An important question arising from this and other papers is how signaling from the insulin receptor regulates compartment size. From the data presented here it can be concluded that manipulation of Akt levels affects compartment size by increasing cell growth without significant changes in cell number. Similar findings have been obtained from study of wing discs with reduced levels of S6 kinase (Montagne, 1999). The insulin receptor, Chico and Dp110 appear to influence both cell size and number in the Drosophila wing. Thus, a plausible scenario is that the pathway bifurcates directly upstream of Akt, which is required for cell growth (through a Drosophila TOR, S6 kinase and 4E-BP1), while a second branch mediates cell proliferation through a parallel pathway. However, it is not yet clear that activation of the insulin-receptor signaling pathway promotes cell proliferation in Drosophila. Reduction in levels of the insulin receptor, Chico or Dp110 negatively affects cell growth and cell number. Nonetheless, it remains unclear whether this is a direct result of modulation of the cell-cycle machinery, or secondary to an impairment in cell growth. Inadequate cell growth may well function as a mitotic checkpoint, or render the cell more susceptible to apoptosis as cell division proceeds unabated. Either mechanism would result in a decrease in cell number. Interestingly, ectopic expression of Dp110 in clones of cells in the wing imaginal disc results in a dramatic increase in cell and clone size, with no effects in cell number. In any case, clearly the phenotypes resulting from ectopic expression of cell-cycle regulators in the wing disc do not resemble those reported for ectopic expression of Dp110, Akt and S6 kinase. Thus, the effects of the insulin-receptor pathway on cell growth are unlikely to be secondary to alterations in cell cycle, but probably represent the major biological output for Chico, Dp110 and Akt in Drosophila. Other regulatory pathways probably function as primary determinants of proliferation (Verdu, 1999 and references therein).

The initiation factor 4E for eukaryotic translation (eIF4E) binds the messenger RNA 5'-cap structure and is important in the regulation of protein synthesis. Mammalian eIF4E activity is inhibited when the initiation factor binds to the translational repressors, the 4E-binding proteins (4E-BPS). The Drosophila 4E-BP (d4E-BP) is a downstream target of the phosphatidylinositol-3-OH kinase [PI(3)K] signal-transduction cascade, which affects the interaction of d4E-BP with eIF4E. Ectopic expression of a highly active d4E-BP mutant in wing-imaginal discs causes a reduction of wing size, brought about by a decrease in cell size and number. A marked reduction in cell size is also observed in post-mitotic cells. Expression of d4E-BP in the eye and wing together with PI(3)K or dAkt1, the serine/threonine kinase downstream of PI(3)K, results in suppression of the growth phenotype elicited by these kinases. These results support a role for d4E-BP as an effector of cell growth (Miron, 2001).

Drosophila 4E-BP (d4E-BP) was isolated by interaction cloning from a complementary DNA expression library using ³²P-labelled deIF4EI. d4E-BP is identical to Drosophila Thor (Bernal, 2000) and homologous to 4E-BPs from other species. Phosphorylation sites in mammalian 4E-BP1 are conserved in d4E-BP, but the predicted eIF4E-binding motif in d4E-BP (YERAFMK) diverges from the canonical consensus sequence (Miron, 2001).

To examine the binding of d4E-BP to deIF4E, residues within the consensus eIF4E-binding site were mutated. Recombinant proteins were expressed in Escherichia coli, and far Western blotting was performed using ³²P-labelled deIF4EI. Mutation of Tyr 54 to Ala (Y54A) or Phe (Y54F), and Met 59 to Ala (M59A) abrogates the interaction of d4E-BP with deIF4E. Mutation of Lys 60 to Ala (K60A) decreases deIF4E binding by 87%, indicating that Lys 60 contributes to deIF4E binding. However, when either Met 59 or Lys 60 are mutated to the consensus Leu, the interaction of d4E-BP with deIF4EI is 2.5-fold higher than with the wild type, and when both Met 59 and Lys 60 are so changed, deIF4E binding increases by 3.4-fold. These results indicate that d4E-BP interacts with deIF4E, albeit more weakly than previously characterized 4E-BPs, owing to its divergent eIF4E-binding motif (Miron, 2001).

4E-BP1 is hyperphosphorylated in response to insulin in many cell types. To test whether this response operates in Drosophila, Schneider-2 (S2) cells were deprived of serum and treated with insulin. Increasing levels of a slower migrating form of d4E-BP (d4E-BP) were observed consequent to insulin treatment. To determine whether the ß-form corresponds to phosphorylated d4E-BP, extracts from insulin-stimulated S2 cells were treated with either calf intestine alkaline phosphatase (CIP) or protein phosphatase 2A (PP2A). Untreated extracts (or extracts kept on ice) contain both the faster migrating alpha- and the slower migrating ß-forms. In contrast, phosphatase-treated extracts contained only the alpha-form (Miron, 2001).

LY294002 and rapamycin inhibit PI(3)K and target of rapamycin (TOR) activity, respectively, and block the insulin-induced hyperphosphorylation of 4E-BP1. Similarly, exposure of serum-deprived S2 cells to either drug before treatment with insulin, results in a dose-dependent decrease in d4E-BP phosphorylation. To determine whether phosphorylation of d4E-BP prevents its binding to deIF4E, m⁷GDP-agarose precipitation was performed. The alpha form is present primarily in the bound fraction, whereas the ß-form is found exclusively in the unbound fraction. These results show that d4E-BP is a downstream target of the PI(3)K pathway, and that the binding of d4E-BP to deIF4E is modulated by its phosphorylation state (Miron, 2001).

Assembly of eIF4F is essential for translational control, and overexpression of eIF4E in mammalian cells results in malignant transformation. To investigate whether eIF4F is also linked to growth control, eIF4F assembly was perturbed in Drosophila. UAS transgenic fly lines were generated that express wild-type d4E-BP or the mutant d4E-BP that binds deIF4E most strongly, d4E-BP(LL). Expression of d4E-BP was targeted to the wing-imaginal disc using MS1096-GAL4. The size and cell number of wings from males were measured. Expression of wild-type d4E-BP has no effect on wing size or pattern. However, expression of d4E-BP(LL) from one line [d4E-BP(LL)^w] causes a marked reduction of wing size without affecting cell number. Another line, [d4E-BP(LL)^s], which expresses d4E-BP(LL) more strongly, causes a larger reduction, which is partly due to a decrease in cell number. Since direct inhibition of cellular proliferation increases, rather than decreases, cell size, it is possible that d4E-BP(LL) also affects cell size directly, and cell proliferation as a consequence. This is supported by analysis of the effects of d4E-BP(LL) expression in larval-wing discs. Although discs from the d4E-BP(wt) and d4E-BP(LL)^w lines are indistinguishable from control discs, d4E-BP(LL)^s discs are 52% smaller. d4E-BP(LL)^s males also required 1-2 days longer to eclose, which would account for the smaller decrease in adult wings (Miron, 2001).

Acridine-orange staining shows that d4E-BP(LL)^s discs contain many apoptotic cells. Co-expression of p35, the baculovirus inhibitor of apoptosis, with d4E-BP(LL)^s partially rescues the size of adult wings. To distinguish between apoptosis and decreased proliferation, cell clones expressing d4E-BP(LL), with or without p35, and co-expressing green fluorescent protein (GFP), were induced 72 h after egg deposition in developing wing discs using the flip-out technique. Clones expressing d4E-BP(LL)^w contain fewer cells than wild-type clones, but co-expression of p35 with d4E-BP(LL)^w does not affect the number of cells per clone, indicating that decreased proliferation, but not increased apoptosis, is the cause of reduction. Few clones expressing d4E-BP(LL)^s are recovered, and they usually contain 1-2 cells. Co-expression of p35 greatly increases the number of clones recovered, but only marginally increases the number of cells per clone (1-4 cells) (Miron, 2001).

Direct interference with cell proliferation using string mutants results in increased cell size. To help distinguish effects on size from effects on proliferation, cell size was evaluated by flow cytometry (FACS). Mean forward-light scatter values for GFP-positive cells that expressed d4E-BP(LL) were reduced by 6%-8%. Because cells that expressed d4E-BP(LL) are smaller and proliferate more slowly than their wild-type counterparts, it is conceivable that d4E-BP(LL) directly affects cell growth and consequently retards proliferation, which would lead to reduced viability and ultimately apoptosis. Similar results were observed in dTOR mutants, and interpreted as a primary defect in cellular growth coupled with a consequent decrease in cell proliferation. The possibility that growth and proliferation are affected independently by d4E-BP(LL) expression cannot be excluded (Miron, 2001).

To exclude proliferation effects, the growth and viability of d4E-BP(LL) cells were examined in a post-mitotic tissue. Polyploid fat-body cells undergo successive rounds of DNA synthesis without mitoses. Cells that express d4E-BP(LL)^s, induced 48 h after egg deposition in the fat body, are 45%-70% smaller than neighboring wild-type cells, but their frequency is much higher than in mitotically active tissues, such as the wing-imaginal disc. Thus, viability of cells that express d4E-BP(LL) is maintained in the absence of mitogenic signals, indicating that proliferation of wild-type neighboring cells is necessary to cause the disappearance of cells expressing d4E-BP(LL). In support of this notion is the finding that when d4E-BP(LL)^s clones are induced during development of eye-imaginal discs, only the clones that are generated posterior to the morphogenetic furrow survive; the clones generated anterior to the furrow (that is, in mitotically active cells), are eliminated (Miron, 2001).

To study the possible role of d4E-BP as an effector of cell growth through the PI(3)K signaling pathway, potential interactions between d4E-BP and relevant signaling genes of this pathway were examined. Expression of wild-type d4E-BP in eye-imaginal discs, using GMR-GAL4, does not engender any discernible phenotype, whereas expression of dAkt1 results in an enlarged eye. However, co-expression of wild-type d4E-BP and dAkt1 partially suppresses the enlarged-eye phenotype, and fully suppresses the roughness induced by expression of dAkt1. Since d4E-BP by itself has no effect on eye size but is able to suppress the dAkt1 phenotype, there is a genuine epistatic relationship between d4E-BP and dAkt1 (Miron, 2001).

Other components of the PI(3)K pathway were also examined for potential epistatic interactions with d4E-BP in the wing, using dpp-GAL4 and 4E-BP(LL)^s. Ectopic expression of Dp110 and dAkt1 causes an enlargement of the region encompassed by the third and fourth longitudinal veins, the anterior crossvein and wing margin. In contrast, expression of a dominant-negative mutant form of PI(3)K (Dp110^D954A) or d4E-BP(LL)^s results in reduction of the size of this region. Co-expression of d4E-BP(LL)^s with Dp110 or dAkt1 suppresses the growth enhancement engendered by expression of these kinases, whereas co-expression of d4E-BP(LL)^s with Dp110^D954A results in further size reduction. Flies that lacked a copy of the gene encoding the adaptor protein p60 [the Drosophila homolog of mammalian PI(3)K subunit p85] are also reduced in size when d4E-BP(LL)^s is co-expressed. These results provide genetic evidence that d4E-BP is a downstream effector of the PI(3)K pathway (Miron, 2001).

Null mutants of d4E-BP are viable and although their immune response is compromised (Bernal, 2000), they do not exhibit increased growth. Furthermore, ectopic expression of Drosophila eIF4E in a wild-type or d4E-BP null background fails to produce a growth-related phenotype. Therefore, an increase in eIF4E activity alone is not sufficient to promote cell growth in Drosophila imaginal discs. This is consistent with data in primary mouse-embryo fibroblasts, in which eIF4E overexpression leads only to oncogenic transformation when co-expressed with c-myc or E1A. Attempts were made to rescue the d4E-BP(LL)-induced growth defects in imaginal discs by co-expressing deIF4E. Unexpectedly, growth is further reduced. Thus, endogenous eIF4E expression levels are optimal for cell growth and proliferation, and in the absence of activation of the PI(3)K pathway, a further increase in eIF4E expression is either without effect or deleterious (Miron, 2001).

Many studies have shown that PI(3)K and TOR-mediated signaling is important for normal cell growth and proliferation. However, one downstream target of this pathway, S6K, regulates cell size but not proliferation. Constitutive expression of dS6K in dTOR mutants only partially suppresses the dTOR phenotype, indicating that S6K-independent targets operate downstream of dTOR. Regulation of eIF4E activity, independent of S6K, contributes to the control of cell size. In Drosophila, the activity of eIF4E is modulated through 4E-BP. Phosphorylation of eIF4E is correlated with increased translation rates. Mutation of the phosphorylation site in Drosophila eIF4E causes a cell size reduction. In summary, the results presented here show that d4E-BP acts as an important downstream effector of PI(3)K in the regulation of cell proliferation and growth, independent of S6K, and further underline the importance of translation initiation in the latter process (Miron, 2001).

Insulin signalling is a potent inhibitor of cell growth and has been proposed to function, at least in part, through the conserved protein kinase TOR (target of rapamycin). Recent studies suggest that the tuberous sclerosis complex Tsc1-Tsc2 may couple insulin signalling to Tor activity. However, the regulatory mechanism involved remains unclear, and additional components are most probably involved. In a screen for novel regulators of growth, Rheb (Ras homolog enriched in brain), a member of the Ras superfamily of GTP-binding proteins, was identified. Increased levels of Rheb in Drosophila promote cell growth and alter cell cycle kinetics in multiple tissues. In mitotic tissues, overexpression of Rheb accelerates passage through G1-S phase without affecting rates of cell division, whereas in endoreplicating tissues, Rheb increases DNA ploidy. Mutation of Rheb suspends larval growth and prevents progression from first to second instar. Genetic and biochemical tests indicate that Rheb functions in the insulin signalling pathway downstream of Tsc1-Tsc2 and upstream of TOR. Levels of rheb mRNA are rapidly induced in response to protein starvation, and overexpressed Rheb can drive cell growth in starved animals, suggesting a role for Rheb in the nutritional control of cell growth (Saucedo, 2003).

Because the growth and cell cycle phenotypes after Rheb overexpression are reminiscent of those caused by hyperactivation of insulin/phosphatidylinositol-3-OH kinase [PI(3)K] signalling, the potential role of Rheb in this network was tested. Using a pleckstrin homology (PH) domain-green fluorescent protein (GFP) fusion protein as a reporter of PI(3)K activity, it was found that Rheb dies not stimulate PI(3)K function, indicating that if Rheb has a role in insulin/PI(3)K signalling, it functions further downstream. The lipid phosphatase PTEN (phosphatase and tensin homolog deleted in chromosome 10) directly antagonizes the kinase function of PI(3)K and suppresses growth when overexpressed. Co-overexpression of Rheb bypasses PTEN-mediated growth inhibition in the adult eye, providing further evidence that Rheb functions downstream of PI(3)K activity. Whether PI(3)K signalling occurs in the absence of Rheb was tested. Animals overexpressing PI(3)K are sensitive to starvation, most probably because of inappropriate anabolic metabolism. Removal of one or both copies of rheb suppresses this hypersensitivity, suggesting that Rheb is required for PI(3)K signalling (Saucedo, 2003).

Drosophila Phosphoinositide-dependent kinase-1 acts downstream of PI3K

Phosphoinositide-dependent kinase-1 (PDK-1) is a central mediator of the cell signaling between phosphoinositide 3-kinase (PI3K) and various intracellular serine/threonine kinases including Akt/protein kinase B (PKB), p70 S6 kinases, and protein kinase C. Recent studies with cell transfection experiments have implied that PDK-1 may be involved in various cell functions including cell growth and apoptosis. However, despite its pivotal role in cellular signalings, the in vivo functions of PDK-1 in a multicellular system have rarely been investigated. Drosophila PDK-1 (dPDK-1) mutants have been isolated and the in vivo roles of their kinases have been characterized. Drosophila deficient in the dPDK-1 gene exhibit lethality and an apoptotic phenotype in the embryonic stage. Conversely, overexpression of dPDK-1 increases cell and organ size in a Drosophila PI3K-dependent manner. dPDK-1 not only can activate Drosophila Akt/PKB (Dakt1), but also substitutes the in vivo functions of its mammalian ortholog to activate Akt/PKB. This functional interaction between dPDK-1 and Dakt1 was further confirmed through genetic analyses in Drosophila. However, cAMP-dependent protein kinase, which has been proposed as a possible target of dPDK-1, did not interact with dPDK-1. In conclusion, these findings provide direct evidence that dPDK-1 regulates cell growth and apoptosis during Drosophila development via the PI3K-dependent signaling pathway and demonstrate this Drosophila system to be a powerful tool for elucidating the in vivo functions and targets of PDK-1 (Cho, 2001).

PDK-1 originally was identified as an upstream regulatory kinase of Akt/protein kinase B (PKB). Consequently, the in vivo roles of PDK-1 have been inferred mainly from those of Akt/PKB. Akt/PKB is a growth factor-regulated serine/threonine kinase that contains a pleckstrin homology domain, as does PDK-1. It acts downstream of phosphoinositide 3-kinase (PI3K) to regulate various cellular activities, including glucose metabolism, transcription, and protein translation. Akt/PKB also negatively regulates apoptosis in various ways. To exert its antiapoptotic effects, Akt/PKB either inhibits the activities of proapoptotic proteins, such as BAD and caspase-9, or induces antiapoptotic signals via the NF-kappaB- and forkhead transcription factor-dependent pathways. Recent transgenic studies in Drosophila have revealed an unexpected function of Akt/PKB and the PI3K signaling pathway: the pathway plays an essential role in the control of cell size. When the activities of one or multiple components of the pathway, including PI3K, Drosophila akt1 (Dakt1), and Drosophila p70 S6 kinase, are down-modulated, cell size as well as body size are dramatically reduced in a cell-autonomous manner (Cho, 2001 and references therein).

Recent studies also suggest that PDK-1 is involved in the activation of members of the AGC superfamily of serine/threonine protein kinases, through phosphorylation of their activation loop in response to extracellular stimulations induced by peptide growth factors and hormones. A number of important kinases in this family, including Akt/PKB, p70 S6 kinase, various protein kinase Cs, protein kinase C-related kinases, and cAMP-dependent protein kinase (PKA), have been proposed as either in vivo or in vitro targets of PDK-1. These results implicate that PDK-1 may play the role of a 'master kinase' in regulating these downstream kinases. However, further investigation is required to determine the actual in vivo targets of PDK-1, as it has been revealed that some of the AGC family kinases are not directly phosphorylated by PDK-1 in vivo, despite possessing a putative PDK-1 phosphorylation site at the activation loop and being phosphorylated by PDK-1 in vitro. In addition, although PDK-1 is regarded as a regulator of at least some of these important kinases, the physiological role of the kinase in a multicellular system has not yet been defined at all (Cho, 2001 and references therein).

The PDK-1 Drosophila homolog, dPDK-1 [ accepted FlyBase name Protein kinase 61C; referred to here as dPDK-1] is 54% identical to its human counterpart in the catalytic domain and is also highly homologous in the noncatalytic carboxyl terminus. Flies containing mutations in the dPDK-1 locus were isolated for genetic analyses. Three P-element insertion mutants, EP(3)837, EP(3)3553, and EP(3)3091, have been found containing P-element insertions in either the 5' or intron region of the dPDK-1 gene. In detail, the inserted positions of the P-element in EP(3)837 and EP(3)3553, which have been determined by inverse PCR, are located at 179 bp and 144 bp upstream of the dPDK-1 transcription start site, respectively. The insertion sites and directions of the P-elements are oriented to induce gene expression and imply that these mutants can be used to study the gain of function of dPDK-1. Another EP line, EP(3)3091, has a P-element in the fourth intron of dPDK-1. The insertion site of EP(3)3091 predicts that the transcription of dPDK-1 is disrupted by the insertion of the transposon. Indeed, EP(3)3091 displays a complete lethal phenotype. In addition, another PDK-1-deficient lethal line, DeltadPDK5, has been generated by an imprecise excision of P-element in EP(3)837. This line contains about a 10-kb deletion that includes the first exon of dPDK-1. This mutant fails to complement the lethality of EP(3)3091, suggesting that both lines are alleles of dPDK-1 mutants. Thus, EP(3)3091 and DeltadPDK5 are hereafter referred to as dPDK-1¹ and dPDK-1², respectively (Cho, 2001).

None of the homozygous dPDK-1¹ and dPDK-1² flies emerge as larva, and both display an embryonic lethality. To isolate dPDK-1¹ homozygous individuals, a GFP balancer chromosome was used. The GFP-negative embryos were selected as dPDK-1 homozygotes. All of the hatched larvae from dPDK-1¹ or dPDK-1²/TM3, GFP, Ser females show GFP expression. dPDK-1¹ homozygous embryos produce no ventral cuticles, and they do not develop into the larval stage. These results are similar to those seen in the mutation of Dakt1, whose mammalian homologs are well-known targets of PDK-1. Briefly, absence of maternal and zygotic Dakt1 activity also results in an embryonic lethality, along with defective cuticle formation (Cho, 2001).

Whether dPDK-1 also is involved in the cell survival-signaling pathway was tested. TUNEL assays were performed with dPDK-1¹ homozygous embryos to examine the involvement of the kinase in apoptosis. Apoptotic activity is dramatically induced in the dPDK-1 zygotic loss-of-function mutant. The induced apoptosis in dPDK-1¹ mutant embryos is extensively suppressed by expression of Dakt1 using the hs-GAL4-UAS system. Collectively, these results strongly suggest that PDK-1 plays an important role in Drosophila embryonic development and apoptosis (Cho, 2001).

A series of components in the PI3K pathway including Dakt1 and Drosophila p70 S6 kinase modulate cell size in a cell-autonomous manner. Thus, whether overexpression of dPDK-1 affects cell size was examined using the GAL4-UAS system. dPDK-1 was overexpressed under the control of gmr-GAL4, which directs expression of the gene in the developing eye. This ectopic overexpression of dPDK-1 causes an increase in ommatidia size, ~1.33-fold bigger than controls. In addition, the effect of overexpression of dPDK-1 was examined in a specific compartment of the wing disk. The wing disk is composed of two compartments (dorsal and ventral), which fold and generate the flattened wing blade. When dPDK-1 is ectopically overexpressed in the dorsal compartment with ap-GAL4 driver, the wing of EP(3)837 is convex toward the dorsal side. This is likely the result of an increase in the size of the cells in the dorsal compartment. Indeed, a similar situation is observed in the UAS-Drosophila p70 S6 kinase flies. These results suggest that dPDK-1 regulates cell and organ size (Cho, 2001).

Despite the fact that there is no clear evidence on how the intrinsic kinase activity of PDK-1 is regulated, the kinase has been found to act downstream of PI3K. Thus, whether dPDK-1 and PI3K can genetically interact was examined in fly lines in which dPDK-1 was coexpressed with PI3K or a dominant negative Dp110 (PI3KDN). Overexpression of the PI3K catalytic subunit, Dp110, increases cell size, whereas overexpression of a PI3KDN results in the opposite phenotype. This change in cell size results in the change of organ and body size. Overexpression of PI3KDN under ptc-GAL4 (the driver induces GAL4 expression throughout the anterior compartment with a stripe of maximal intensity along the border of anterior/posterior compartment extending into the posterior compartment) results in reduction of the distance between L3 and L4 veins. However, this phenotype is strongly suppressed by coexpression of dPDK-1 with PI3KDN, suggesting that dPDK-1 acts as a vital downstream effector of PI3K in cell and compartment size control. Conversely, overexpression of the PI3K wild-type causes an increase in the distance between L3 and L4 veins, and coexpression of dPDK-1 and PI3K further increases the distance in a synergistic manner. These results provide strong in vivo evidence that dPDK-1 functions downstream of Drosophila PI3K in the control of cell and compartment size (Cho, 2001).

To determine whether dPDK-1 functions in a manner similar to its mammalian counterpart, myc-tagged dPDK-1, myc-tagged human PDK-1 (hPDK-1), and/or HA-tagged human Akt/PKB were transiently expressed in COS cells. As expected, dPDK-1 strongly induces human Akt/PKB activity, to levels comparable to those induced by hPDK-1. Conversely, coexpression of a dominant negative hPDK-1 or a dominant negative dPDK-1 strongly inhibits the epidermal growth factor-induced activation of human Akt/PKB. These results indicate that the Drosophila ortholog of PDK-1 can properly function and substitute its mammalian counterpart to relay the growth factor-induced activation signals to a mammalian Akt/PKB (Cho, 2001).

Whether dPDK-1 can activate Dakt1 in Drosophila was examined. To test this, dPDK-1 and HA-tagged Dakt1 were coexpressed in the Drosophila eye using the gmr-GAL4 driver, and the phosphotransferase activities of Dakt1 were measured from the head extracts of gmr-GAL4, gmr-GAL4; UAS-HA-Dakt1, or gmr-GAL4; UAS-HA-Dakt1/EP(3)837. Dakt1 activity is strongly increased in the flies coexpressing dPDK-1. Consistent with this increased activity, an electrophoretically retarded Akt/PKB band, corresponding to a highly phosphorylated and activated form, is observed. This biochemical evidence strongly supports that Dakt1 is indeed a physiological target of dPDK-1 (Cho, 2001).

To further confirm the in vivo roles of dPDK-1, genetic interactions between Dakt1 and dPDK-1 were examined in flies. Overexpression of Dakt1 in the Drosophila eye increases eye size and generates a bulging eye with enlarged ommatidia. In addition to this change in size, the ommatidia array becomes irregular, and eye bristles are enlarged with a frequent loss of number. When dPDK-1 is coexpressed with Dakt1 in the eye, it displays a severely crumpled morphology. The eye bristles are enlarged even more severely, and the boundaries of all ommatidia and photoreceptor cells disappear. These dPDK-1/Dakt1 phenotypes are further enhanced by an increased dose of gmr-GAL4 driver. These findings, taken together, clearly demonstrate the functional and genetic interactions between dPDK-1 and Dakt1 (Cho, 2001).

The genetic interactions between dPDK-1 and Drosophila PKA were examined. Although PKA has been proposed to be a putative substrate of PDK-1, the in vivo relevance of this has not been clearly determined. When the catalytic subunit of Drosophila PKA (dPKAc) is overexpressed in the developing eye of Drosophila, the eye is discolored and swells up with wrinkles. Scanning electron microscopic views of the eye show that the boundaries of all ommatidia and photoreceptor cells disappear. However, unlike Akt/PKB, coexpression of dPDK-1 does not affect these phenotypes of dPKAc. Furthermore, the regulatory subunit of Drosophila PKA (dPKAr) also does not interact with dPDK-1. These results support that PKA is not regulated by PDK-1 in Drosophila, which is highly consistent with recent results that PKA is phosphorylated and activated normally in a PDK-1-deficient cell line. These results strongly support that the Drosophila system is a physiologically relevant tool for determining the actual in vivo targets of PDK-1 (Cho, 2001).

Insulin/IGF signaling during development controls growth and size, possibly by coordinating the activities of the Ras and PI 3-kinase signaling pathways. In vertebrates, the IR and IGFR act through IRS1-IRS4 proteins, which are multifunctional adaptors that link insulin and IGF signaling to the Ras/MAPK and phosphoinositide 3'-kinase (PI 3-kinase) signaling pathways. The pleckstrin homology domain (PH) and phosphotyrosine binding domain (PTB) of the IRS proteins are believed to mediate binding to phosphoinositol phosphates and the juxtamembrane NPXY motif of IR/IGFR, respectively. Grb2 (Drosophila homolog Drk) is an adaptor protein containing SH2 and SH3 domains. It has been suggested that Grb2 may, via its binding to IRS, link insulin/IGF to the Ras/MAPK pathway and thereby control proliferation. The Drosophila homolog of the SH2 domain containing p85 PI 3-kinase adaptor subunit, p60, binds Chico/IRS and thereby recruits the p110 catalytic subunit of PI 3-kinase [which converts phosphoinositol(4,5)P₂ (PtdIns(4,5)P₂) into phosphoinositol(3,4,5)P₃ (PtdIns(3,4,5)P₃)] to the plasma membrane. The p110 PI 3-kinase belongs to the class I PI 3-kinases implicated in the metabolic effects of insulin. The classical effectors that mediate the biological outcomes of insulin and IGF downstream of IRS have been divided into two functional branches: the Ras/MAPK proliferation pathway, and the PI 3-kinase metabolic, growth and survival pathway (Oldham, 2002).

To analyze the role of the different domains of Chico/IRS under physiological conditions, a panel of effector site mutants was created in a genomic rescue construct for chico that disrupts the PH or the PTB domains or the putative binding sites of Drk/Grb2 and p60. The constructs include the cis-regulatory sequences that permit expression of chico in its normal spatial and temporal pattern. The wild-type chico construct fully restores the defects of chico homozygous null mutants. In this manner, the effector site mutants were assayed for the ability to rescue the three different phenotypes associated with complete loss of Chico function: body size reduction, female sterility and lipid alterations. The Drk/Grb2 consensus binding site mutant is able to fully rescue the reduced weight to the same extent as the wild-type rescue construct. Therefore, the presence of a functional Drk binding site in Chico and thus the link to the activation of the Ras/MAPK kinase pathway is not required for its wild-type function. In contrast, the PH and PTB domain mutants and the double p60 PI 3-kinase binding site mutant were unable to rescue the reduced body weight. The latter result is surprising because InR contains additional functional PI 3-kinase binding sites in its C-terminal tail, an extension shared only with the C. elegans InR homolog, Daf-2, and not the mammalian IR or IGFR. This suggests that the presence of additional p60 binding sites in the InR C-terminal tail is not sufficient in vivo to mediate wild-type levels of growth and proliferation in the absence of the Chico p60 PI 3-kinase binding sites and that the InR C-terminal tail may contribute only low levels of PI 3-kinase signaling. Although the PTB domain mutant fails to restore normal body weight, it rescues the female sterility associated with the loss of Chico function. With the exception of the full rescue of the lipid accumulation observed in Drk/Grb2 mutant, all the other effectors only partially restore the change in lipid accumulation (Oldham, 2002).

The inability of the p60 binding site mutant to rescue the size defect indicates that the Chico PI 3-kinase docking sites are necessary for InR/Chico (insulin/IGF) action in size control. However, the issue of whether recruitment of PI 3-kinase to Chico is sufficient to mediate the attainment of wild-type body size is unresolved. It has been reported that overexpression of PI 3-kinase and Akt in Drosophila is sufficient for increased growth but not proliferation. Loss of zygotic InR function results in embryonic lethality with some small arrested larvae, but loss of zygotic Chico function results in viable small flies. Two parsimonious hypotheses could explain this difference. (1) InR activates not only the PI 3-kinase pathway but also another, Chico-independent, signal transduction pathway, or (2) InR signals predominantly through PI 3-kinase, but loss of Chico does not block PI 3-kinase activation completely because of direct interaction of p60 with the InR C-terminal tail. This provides residual PI 3-kinase activation sufficient to rescue viability, but not wild-type size. If the latter hypothesis were true, then increasing PtdInsP₃ levels should be sufficient to rescue loss of InR function (Oldham, 2002).

To test whether increasing PtdInsP₃ levels in an InR or PI 3-kinase p110 mutant background is sufficient to restore growth, the function of a negative regulator of the insulin pathway was eliminated. The 3'-phosphoinositol-specific lipid phosphatase, PTEN acts as a negative regulator of the PI 3-kinase pathway by converting PtdInsP₃ generated by PI 3-kinase into PtdInsP₂. Used were a null (Pten^2L117) and a hypomorphic (Pten^2L100) allele of Pten, identified in a screen for genes involved in growth control. As shown by HPLC analysis of the phospholipids in extracts of Pten mutant larvae, the loss of PTEN function results in a 2-fold increase in PtdInsP₃ levels. This is consistent with the increase in PtdInsP₃ seen in Pten-deleted murine fibroblasts. One prominent biological effect of these increased PtdInsP₃ levels in Drosophila is a substantial increase in size in both larvae and pupae. To test whether loss of PTEN function, and consequently increased PtdInsP₃ levels, is sufficient to restore growth or viability in InR null mutants, InR and Pten double mutants were generated by creating mosaic animals using the eyeless-Flipase (eyFlp) tissue-specific recombination system. In such animals, the head consists of homozygous mutant tissue, whereas the rest of the body is heterozygous for the same mutation. While loss of PTEN function (Pten^2L117) in the head results in a fly with a disproportionately larger head (with more and larger cells), loss of InR function (InR³²⁷) results in flies with smaller heads (pinhead) compared to the wild type. Heads doubly mutant for Pten^2L117and InR³²⁷, however, are almost the size of heads singly mutant for Pten^2L117. Also, two different lethal heteroallelic InR combinations (InR³⁰⁴/InR³²⁷ or InR³⁰⁴/InR²⁵), which arrest at the second larval instar stage, develop to the pupal stage (15%-17% of 33% expected) and even to pharate adults in the presence of reduced PTEN levels (Pten^2L117/Pten^2L100). These results demonstrate that complete loss of PTEN function can largely substitute for InR-mediated growth and proliferation in the absence of InR function and that the Ras/MAPK pathway plays little or no role in the InR mediated control of cell growth. This notion is further supported by the observation that complete loss of InR function in the compound eye does not result in a loss of photoreceptors, a hallmark of loss of Ras pathway function (Oldham, 2002).

Since increasing PtdInsP₃ levels can rescue loss of InR function, these results suggest that the level of PtdInsP₃ may be critical in determining the amount of growth. This possibility was explored by examining genetic interactions between Pten and PI 3-kinase p60 and p110. The lethality associated with the complete loss of PI 3-kinase p110 function, cannot be rescued by Pten^2L117/Pten^2L100. It is possible that without any PI 3-kinase p110 function, PTEN function becomes obsolete. In order to test this possibility, double mosaic clones were generated with the strong loss of function Pten^2L117 allele and a null mutation for PI 3-kinase p110 or its p60 adaptor. Loss of PTEN function (Pten^2L117) is unable to rescue the pinhead phenotype caused by loss of PI 3-kinase p110 function. However, clones that are doubly mutant for PI 3-kinase p60 and Pten^2L117 are of wild-type size. In the absence of PI 3-kinase p60 function, PI 3-kinase p110 might have residual activity as suggested by the weaker phenotype of the PI 3-kinase p60 null mutant. Indeed, flies doubly mutant for PI 3-kinase p60 and Pten^2L117/Pten^2L100 flies are viable. These data provide strong genetic support for the close relationship between PTEN and PI 3-kinase and indicate that the intracellular levels of PtdInsP₃ define the amount of cellular growth (Oldham, 2002).

The rescue of lethal, null InR mutant combinations to near viability by reducing PTEN activity strengthens the argument that a PtdInsP₃-dependent signaling pathway is the primary effector for InR-derived growth and proliferation. In support of this observation, PI 3-kinase and Akt have been isolated as retroviral oncogenes, suggesting that activation of PI 3-kinase and Akt is sufficient to mediate growth, proliferation, and oncogenesis in vertebrate systems. In Drosophila and mammals, overexpression of PI 3-kinase causes increased growth; but this is not sufficient for proliferation as is the removal of Pten. From this premise, it has been proposed that PI 3-kinase and PTEN regulate similar yet distinct pathways. Alternatively, it is possible that they do function uniquely in the same pathway and that the difference may be due to altered location and function because of overexpression, or to differential feedback of PI 3-kinase versus PTEN. For example, since PI 3-kinase has been shown to act as a serine/threonine protein kinase on IRS, this may have a negative feedback effect on the insulin pathway that might not be evident in Pten loss-of-function mutations. Nevertheless, PI 3-kinase is absolutely critical in controlling size because using an allelic series of PI 3-kinase mutants in combination with the ey-Flp sytem resulted in a range of different head sizes. Furthermore, expressing an activated and dominant-negative form of PI 3-kinase in Drosophila imaginal discs or the heart of the mouse also leads to a corresponding increase or decrease in cell and organ size. Thus, the PI 3-kinase/PTEN cycle can be considered a dedicated growth rheostat, and the InR pathway is an evolutionary conserved module for regulating the range of growth and size (Oldham, 2002).

Collectively, these data firmly establish Drosophila as a valid model organism for the study of metabolic diseases like diabetes and obesity as well as for the study of growth disorders like cancer. Pten mutant flies are larger in size due to increased cell size and number, but have a corresponding decrease in energy stores, a situation completely opposite that of mutations in positive components of the insulin signaling pathway like InR, chico, PI 3-kinase, and dAkt. These large viable Pten mutants show that a reduction of PTEN function is sufficient for increased organism size. This fact suggests that the four-fold size difference between viable InR and Pten mutants can simply be controlled by the amount of PtdInsP₃ and this phenomenon may possibly be extended to vertebrate size regulation. Thus, in Drosophila, the InR/PI 3-kinase/PTEN pathway combines both metabolism and growth control into one pathway that later diverged into two separate, yet interacting systems in mammals (Oldham, 2002).

Ras signaling and PI3K in the developing wing

The Ras GTPase links extracellular signals to intracellular mechanisms that control cell growth, the cell cycle, and cell identity. An activated form of Drosophila Ras (Ras^V12) promotes these processes in the developing wing, but the effector pathways involved are unclear. Evidence is presented indicating that Ras^V12 promotes cell growth and G1/S progression by increasing dMyc protein levels and activating PI3K signaling, and that it does so via separate effector pathways. Endogenous Ras is required to maintain normal levels of dMyc, but not PI3K signaling during wing development. Finally, induction of dMyc and regulation of cell identity are separable effects of Raf/MAPK signaling. These results suggest that Ras may only affect PI3K signaling when mutationally activated, such as in Ras^V12-transformed cells, and provide a basis for understanding the synergy between Ras and other growth-promoting oncogenes in cancer (Prober, 2002).

In the developing Drosophila wing, Ras, dMyc, and PI3K regulate rates of cellular growth (i.e., mass accumulation) and progression through the G₁/S transition of the cell cycle without affecting overall rates of cell division. These results concur with experiments in mice showing that Ras, Myc, and PI3K promote cell growth without affecting rates of cell division. This study shows that an activated form of Drosophila Ras (Ras^V12) is capable of increasing dMyc protein levels as well as levels of PI3K signaling, suggesting that Ras^V12 drives growth and G₁/S progression via both of these mechanisms. Ras^V12 effector loop mutants were used to show that Ras^V12 affects dMyc and PI3K signaling via separate pathways, and that overexpressed dMyc and PI3K do not cross-regulate each other. Thus, a hierarchy has been established for these growth-regulatory proteins (Prober, 2002).

Overexpressed Drosophila Ras^V12 recruits the tGPH (aPH-GFP fusion protein used as an indicator of dPI3K signaling) reporter to the cell membrane, suggesting that Ras^V12 activates PI3K signaling, and thereby increases PIP₃ levels, in the developing wing. It is inferred that Drosophila Ras^V12 directly activates PI3K, because mammalian studies have shown that Ras^V12 can directly bind and activate PI3K. Alternatively, Drosophila Ras^V12 may activate PI3K signaling via other mechanisms, such as by inhibiting the lipid phosphatase PTEN. This possibility seems less likely, however, since direct interactions between Ras and PTEN have not been described. Contradicting the generally accepted idea that PI3K is normally an effector of Ras signaling, this study found that localization of the PI3K reporter tGPH was not detectably affected in ras^-/- cells. Although the observations using the tGPH reporter were not quantitative, and small effects could have been missed, these results nevertheless indicate that Ras does not normally play a major role in regulating PI3K in the developing wing. Consistent with this hypothesis, expression of an activated form of Drosophila EGFR (EGFR^lambdatop) had no effect on PI3K signaling. Because the ability of dEGFR^lambdatop to activate downstream pathways is limited by the amount of endogenous Ras, this result suggests that higher levels of Ras activity than can be generated in wild-type cells are required to activate PI3K (Prober, 2002).

An alternative explanation for the discrepancy between the involvement of Drosophila and mammalian Ras in regulating PI3K signaling may relate to the evolution of ras genes. The Drosophila and C. elegans Ras homologs are more homologous to mammalian K-Ras than to H- or N-Ras, suggesting that K-Ras may have an older, more general function than the other mammalian ras genes. In support of this idea, H- and N-Ras are dispensable, whereas K-Ras is essential, for normal mouse development. It is also interesting to note that overexpressed K-Ras preferentially activates Raf over PI3K, whereas the opposite is true for H-Ras. Thus, K-Ras may play a more fundamental role in developmental processes dependent on Raf, but independent of PI3K, whereas H- and N-Ras may have evolved to perform less critical functions in which they regulate PI3K (Prober, 2002).

Using the tGPH reporter, it was found that levels of PI3K signaling are not patterned but rather are uniform throughout wing development. It is therefore unlikely that PI3K signaling is regulated by localized patterning signals such as the morphogens Vein, Dpp, and Wg, which are secreted from the notum, anterior-posterior boundary, and dorsal-ventral boundary of the wing, respectively, and are thought to pattern growth and cell proliferation of the wing. Furthermore, cell-autonomous activation of Dpp signaling using an activated form of its receptor (Tkv^Q253D), which is a potent growth driver in the wing, has no effect on tGPH localization. It may be that Dpp and Wg regulate cell growth rates by affecting the ability of cells to respond to ubiquitous PI3K-dependent growth signals. They may do so by regulating the expression or activity of signaling proteins or transcription factors required for transducing PI3K-dependent signals (Prober, 2002).

The tGPH reporter revealed that the polarized epithelial cells of Drosophila wing discs contain dense regions of tGPH colocalized with Armadillo at the apical region of the cell membrane, with lower tGPH levels present throughout the basolateral cell membrane. This does not simply reflect an apical accumulation of membrane microdomains enriched in PIP₃ in polarized cells, because inhibiting PI3K activity (by expressing Deltap60) dramatically reduces apical tGPH fluorescence. In contrast, tGPH is uniformly localized throughout the cell membranes of unpolarized Drosophila fat body cells in vivo and Drosophila S2 cells in culture. Similarly, mammalian PI3K is uniformly active throughout the cell membrane of cultured HEK 293 cells. Thus, the dynamics of PI3K signaling are dependent on the cellular context, which is likely disturbed when tissues are dissociated into single cells that are studied in culture. This process may allow signaling interactions not normally occurring in vivo. In support of this idea, overexpression of the Drosophila Insulin receptor homolog (Inr) does not activate MAPK in the developing wing or affect Ras-mediated cell fate specification in the developing eye, whereas addition of insulin to cultured Drosophila or mammalian cells does activate Ras/MAPK signaling. Alternatively, the failure to detect activation of Ras signaling in response to overexpressed Inr may reflect a cell-type specificity for this interaction or insufficient sensitivity of the assays. It will therefore be interesting to compare the subcellular localization of PI3K signaling complexes in cultured mammalian cells with the tissues from which they are derived (Prober, 2002).

PI3K signaling is thought to be regulated by a family of secreted Drosophila insulin-like peptides (dilps) that bind and activate Inr. dilp2 is ubiquitously expressed in imaginal tissues, whereas dilp2 and other dilp family members are expressed in a variety of larval tissues including the gut and neurosecretory cells in the brain. dilp2 that is expressed in imaginal tissues is likely secreted apically into the lumen between cells of the columnar epithelium and the overlying peripodial membrane. This would result in preferential binding of dIlp2 to Inr at the apical region of the cell, which could account for the high levels of apically localized tGPH that were observed. Alternatively, apical PI3K signaling may reflect a local concentration of PI3K-signaling complexes. Consistent with the latter possibility, Ras^V12 recruited tGPH to only apical regions of the cell membrane. This result suggests that Ras^V12 may require other apically localized factors to activate PI3K signaling. This possibility is supported by the finding that coexpressed Deltap60, which prevents PI3K from interacting with upstream activators, blocks Ras^V12-mediated activation of PI3K signaling, as it does in mammals. Because mammalian Ras can directly bind the catalytic subunit of PI3K, it is inferred that coexpressed Deltap60 should not affect the ability of Drosophila Ras to activate PI3K signaling unless Ras-dependent activation requires other apically localized factors that bind Deltap60. These factors may include the Insulin receptor substrate Chico, Inr itself or other receptor tyrosine kinases, G-protein coupled receptors, or components of signaling complexes that are recruited upon activation of these receptors. Several receptor tyrosine kinases, including EGFR, as well as phosphotyrosine-containing proteins, are concentrated at the apical cell surface, although Inr is distributed throughout the cell membrane. The Drosophila homolog of the heterotrimeric G-protein subunit Gαi, which presumably transduces signals from a large family of associated receptors, is also concentrated apically in wing disc cells. This is consistent with the possibility that heterotrimeric G-proteins may regulate PI3K signaling in Drosophila, as they do in mammals (Prober, 2002).

Much of the current understanding of Ras function, and that of most oncogenes, derives from studies in homogenous cell culture systems. These studies have focused primarily on cell-autonomous effects of oncogenes rather than upon the roles of interactions among cells within tissues in tumor development. Tissue homeostasis is maintained by a continuous exchange of signals between cells, the extracellular matrix, and the local environment. An important feature of tumor development is escape from this regulation, initially allowing the autonomous growth and proliferation of tumor cells, and eventually resulting in altered adhesion and migration of tumor cells away from their site of origin. The behavior of clones of cells with elevated Raf/MAPK signaling levels in developing Drosophila epithelia is strikingly similar to that of tumor cells within mammalian tissues. These cells have altered adhesive properties and cell identities, and as a result minimize contact with neighboring wild-type cells. In contrast, PI3K and dMyc do not regulate cell identity or adhesion. Studies in Drosophila and vertebrates have also suggested that even though both dMyc and PI3K stimulate growth, they appear to do so via different mechanisms. PI3K signaling promotes nutrient import and storage, whereas dMyc promotes nucleolar growth and protein synthesis. Thus, the ability of Ras^V12 to up-regulate both of these pathways may generate a more robust and balanced growth response than activation of either dMyc or PI3K alone. Furthermore, the ability of Ras^V12 to deregulate cell identity and adhesion may underlie the strong synergy between Ras and other growth-promoting oncogenes in vivo (Prober, 2002).

Conserved MicroRNA miR-8/miR-200 and its target USH/FOG2 control growth by regulating PI3K

How body size is determined is a long-standing question in biology, yet its regulatory mechanisms remain largely unknown. This study finds that a conserved microRNA miR-8 and its target, U-shaped (USH), regulate body size in Drosophila. miR-8 null flies are smaller in size and defective in insulin signaling in fat body that is the fly counterpart of liver and adipose tissue. Fat body-specific expression and clonal analyses reveal that miR-8 activates PI3K, thereby promoting fat cell growth cell-autonomously and enhancing organismal growth non-cell-autonomously. Comparative analyses identify USH and its human homolog, FOG2, as the targets of fly miR-8 and human miR-200, respectively. USH/FOG2 inhibits PI3K activity, suppressing cell growth in both flies and humans. FOG2 directly binds to p85α, the regulatory subunit of PI3K, and interferes with the formation of a PI3K complex. This study identifies two novel regulators of insulin signaling, miR-8/miR-200 and USH/FOG2, and suggests their roles in adolescent growth, aging, and cancer (Hyun, 2009).

Animal body size is a biological parameter subject to considerable stabilizing selection; animals of abnormal size are strongly selected against as less fit for survival. Thus, the way in which body size is determined and regulated is a fundamental biological question. Recent studies using insect model systems have begun to provide some clues by showing that insulin signaling plays an important part in modulating body growth. The binding of insulin (insulin-like peptides in Drosophila) to its receptor (InR) triggers a phosphorylation cascade involving the insulin receptor substrate (IRS; chico in Drosophila), phosphoinositide-3 kinase (PI3K), and Akt/PKB. An active PI3K complex consists of a catalytic subunit (p110; dp110 in Drosophila) and a regulatory subunit (p85α; dp60 in Drosophila). Phosphorylated Akt (p-Akt) phosphorylates many proteins -- including forkhead box O transcription factor (FOXO) -- which are involved in cell death, cell proliferation, metabolism, and life span control. Once activated, the kinase cascade enhances cell growth and proliferation (Hyun, 2009).

Organismal growth is achieved not only by cell-autonomous regulation but also by non-cell-autonomous control through circulating growth hormones. Recent studies in insects indicate that several endocrine organs, such as the prothoracic gland and fat body, govern organismal growth by coordinating developmental and nutritional conditions. However, detailed mechanisms of how body size is determined and modulated remain largely unknown (Hyun, 2009).

microRNAs (miRNAs) are noncoding RNAs of ~22 nt that act as posttranscriptional repressors by base-pairing to the 3' untranslated region (UTR) of their cognate mRNAs. The physiological functions of individual miRNAs remain largely unknown. Studies of miRNA function rely heavily on computational algorithms that predict target genes. In spite of their utility, however, these target prediction programs generate many false-positive results, because regulation in vivo depends on target message availability and complementary sequence accessibility. To overcome the difficulties in identifying real targets, various experimental approaches have been developed, including microarrays, proteomic analyses, and biochemical purification of the miRNA-mRNA complex. Genetic approaches using model organisms can also be useful tools for studying the biological roles of miRNAs at both the organismal and molecular levels. Despite these advances, however, it is still a daunting task to understand the biological function of a given miRNA and to identify its physiologically relevant targets (Hyun, 2009).

This study found using Drosophila as a model system that conserved miRNA miR-8 positively regulates body size by targeting a fly gene called u-shaped (ush) in fat body cells. It was further discovered that this function of miR-8 and USH is conserved in mammals and that the human homolog of USH, FOG2, acts by directly binding to the regulatory subunit of PI3K (Hyun, 2009).

Small body phenotype of the mir-8 mutant

: In a screen for miRNAs that modulate cell proliferation, it was observed that human miR-200 miRNAs (miR-200a, miR-200b, miR-200c, miR-141, and miR-429) promote cell growth when transfected into several human cell lines. Members of the miR-200 family are upregulated in certain cancers, including ovarian cancer, consistent with the observation of the proliferation promoting effects. Because the miR-200 family is highly conserved in bilaterian animals, with miR-8 being the sole homolog in Drosophila, D. melanogaster was used in order to uncover the biological function of the miR-200 family (Hyun, 2009).

The phenotype of the miR-8 null fly was first analyzed using mir-8^δ2. It has been shown that mir-8 mutation results in increased apoptosis in the brain and frequent occurrence of malformed legs and wings (in about one-third of the mutants). Interestingly, in addition to these phenotypes, it as found that miR-8 null flies are significantly smaller in size and mass than their wild-type counterparts (Hyun, 2009).

The determination of the final body size in insects during the larval stage is analogous to that which occurs during the human juvenile period. It is generally known that reduced body size in insects is caused by either slow larval growth, precocious early pupariation that shortens the larval growth period, or both. It was observed that, at 100 hr after egg laying (AEL), miR-8 null larvae exhibit a significantly smaller body volume than do wild-type larva. The onset of pupariation in miR-8 null flies was not significantly different from that in wild-type flies, and adult emergence was slightly delayed (~12 hr). Thus, the smaller body size of miR-8 null flies is likely to be caused by slower growth during the larval period rather than by precocious pupariation. Insufficient food intake has been reported to accompany either precocious or delayed pupariation, depending on the onset of reduced feeding. However, the levels of Drosophila insulin-like peptides (Dilps), which are known to be reduced in starvation conditions, were not downregulated in miR-8 null larvae. Given the unaffected onset time of pupariation and the levels of Dilps in this animal, the small body size of miR-8 null flies is unlikely due to reduced feeding (Hyun, 2009).

Next, it was asked whether the small body phenotype was caused by a reduction in cell size, cell number, or both. Cell size and number were measured and it was found that cell number was reduced in the wing in miR-8 null flies, whereas cell size was not significantly different from that of wild-type. Thus, assuming that similar regulation takes place in other body parts, the reduced growth in the peripheral tissues of the miR-8 null flies may be ascribed to decreased cell number rather than reduced cell size (Hyun, 2009).

To understand why miR-8 null animals grow slowly, the activities of the proteins involved in insulin signaling were examined in the miR-8 null flies. The level of activated Akt was measured by Western blotting using a p-Akt-specific antibody. The p-Akt level was reduced in the mutant flies, suggesting that Akt signaling is impaired in the absence of miR-8. Activated p-Akt is known to inactivate FOXO via phosphorylation. Phosphorylation prevents nuclear localization of FOXO, which, in turn, results in the reduction of transcription of FOXO target genes. Consistent with the reduced level of p-Akt, the FOXO target gene, 4EBP, was increased in mir-8 mutant larvae, indicating that insulin signaling is indeed significantly reduced in the miR-8 null animal (Hyun, 2009).

Fat body-specific expression of miR-8 rescues the small body phenotype

The spatial expression pattern of miR-8 was examined in larvae using a mir-8 enhancer trap GAL4 line (hereafter referred to as mir-8 gal4), which, when combined with UAS-GFP, expresses GFP under the control of the mir-8 enhancer. In addition to the signals in the brain, wing discs, and leg discs that were previously observed by Karres, strong signals were noticed in the fat body. The level of miR-8 was determined by Northern blotting using total RNA prepared from different larval organs, which showed that miR-8 is indeed highly abundant in the fat body (Hyun, 2009).

Recent studies suggested that Drosophila fat body may be an important organ in the control of energy metabolism and growth. Therefore, it was reasoned that if miR-8 in the larval fat body is critical for body size control, exclusive expression of miR-8 in the fat body alone should alleviate the whole body size defect observed in the mir-8 mutants. To test this idea, transgenic flies were generated to specifically reintroduce miR-8 into the fat bodies of mir-8 mutant larvae using a fat body-specific GAL4 driver, Cg gal4 (CgG4). Remarkably, miR-8 expression in the fat body alone rescued the phenotype to near wild-type levels in both body weight and body size, suggesting that miR-8 in the fat body is important for systemic body growth. Another interesting observation was that the miRNAs from the human miR-200c cluster, which includes miR-200c and miR-141, could also yield a comparable rescue effect. Human miR-200 family miRNAs, which are located in two chromosomal clusters, have extensive homology to miR-8. The fact that miRNAs of the human miR-200c cluster effectively compensate for the loss of miR-8 suggests that these human miRNAs can be processed by the Drosophila miRNA processing machinery and that they share a conserved biological function. Because CgG4 is expressed in the anterior lymph gland as well as in the fat body, an additional GAL4 driver, ppl gal4 (pplG4), was used that is active mainly in the fat body and slightly in the salivary gland (Zinke, 1999). Similar rescue effects were observed with pplG4, in support of the fat body-specific function of miR-8 (Hyun, 2009).

USH is a critical target of miR-8 in the regulation of body growth

To further understand the molecular function of miR-8, miR-8 target genes responsible for body size control were identified. Because miR-8 is a highly conserved miRNA and because the human homologs promote cell proliferation in human cells, it was assumed that the targets involved in the cell proliferation phenotype may be conserved throughout evolution. To discover such conserved targets,the candidate target genes of both fly miR-8 and human miR-200 miRNAs were listed using five different target prediction programs. The two groups of putative targets, one from flies and the other from humans, were then compared with each other to identify homologous gene pairs. The Drosophila gene u-shaped (ush) and its human homolog Friend of GATA 2 (FOG2, also known as ZFPM2) were the most frequently predicted gene pair conserved in both species. From the target candidate list, 15 genes, including ush/FOG2, were selected that are known as tumor suppressors or negative regulators of cell proliferation in at least one species. These target genes were validated with luciferase reporter plasmids. Briefly, each target's 3' UTR, which contains the miRNA target sites, was inserted downstream of a luciferase gene. The reporter activities were measured following cotransfection of miRNAs and the reporter plasmids into Drosophila S2 or human HeLa cells. After extensive reporter assays, seven gene pairs were identified that respond both to miR-8 and to miR-200 family miRNAs in fly and human cells, respectively (Hyun, 2009).

To examine which targets among the candidates are physiologically relevant to the phenotype observed, the candidate genes were knocked down in the fat body of miR-8 null flies and it was asked whether the knockdown could rescue the small body phenotype. Using the UAS-RNA interference (RNAi) lines obtained from the Vienna RNAi Library Centre, dsRNAs of five candidate genes were expressed in the fat body of mir-8 mutants using CgG4. Lap1 knockdown was unsuccessful and, thus, did not rescue the mir-8 mutant phenotype. Among the RNAi lines tested, the one against ush rescued the dwarf phenotype most dramatically. RNAi of ush in wild-type background did not significantly increase body weight, ruling out the possibility that the effects of ush knockdown and mir-8 mutation are additive (Hyun, 2009).

Because a previous study showed that miR-8 targets atrophin (atro) to prevent neurodegeneration, whether atro is also involved in body size regulation was tested. Knockdown of atro in the fat body, however, failed to rescue the small body phenotype of miR-8 null flies. Thus, the reported function of miR-8 in the prevention of neurodegeneration may be separate from its function in body growth, not only spatially but also at the molecular level. To exclude possible off-target effects of ush RNAi, the ush¹⁵¹³ hypomorph, which expresses a reduced level of ush as the result of a mutation in the promoter region, was used. Consistent with the results of the ush RNAi, ush¹⁵¹³ heterozygotes have larger adult bodies than do the control flies. This result indicates that USH may indeed suppress body growth (Hyun, 2009).

Next, whether the level of USH was elevated in miR-8 null animals was examined. The endogenous ush mRNA level was determined by qRT-PCR analysis of the RNAs from whole larva or larval fat body. The ush mRNA is, indeed, significantly upregulated in the fat body of miR-8 null larvae (δ2.0 fold), suggesting that miR-8 suppresses ush in the fat body. Upregulation of ush mRNA in whole larval RNA was less prominent (~1.3 fold). Thus, ush may be more strongly suppressed in the fat body than in other body parts. Notably, USH protein levels are more dramatically affected than the mRNA levels, indicating that miR-8 represses USH production by both mRNA destabilization and translational inhibition. Furthermore, a point mutation of the miR-8 target site in the 3' UTR of ush abolished the suppression of the 3' UTR reporter, indicating that the suppression is mediated through the direct binding of miR-8 to the predicted target site. Putative target sites for miR-8 are found in all Drosophila species examined, including distant species such as D. virilis and D. grimshawi. Together, these results demonstrate that ush is an authentic target of miR-8 (Hyun, 2009).

miR-8 and USH regulate PI3K

Several reports have suggested that insulin signaling in the larval fat body controls organismal growth in a non-cell-autonomous manner. In support of this idea, it was observed that suppression of insulin signaling in the larval fat body yielded smaller flies and fewer cells in the wing. This phenotype is similar to that of miR-8 null flies. To further investigate whether insulin signaling is indeed defective in the fat body of miR-8 null animals, the tGPH reporter, a GFP protein fused to a PH domain, was used. Using this technique, the activity of PI3K can be measured by monitoring the membrane-associated GFP signal, because PH domains bind to the membrane-anchored phosphatidylinositol-3,4,5-triphosphate (PIP₃) produced by PI3K. It was found that PI3K activity was downregulated in mir-8 mutant fat bodies and that FOXO was more strongly localized to the nucleus of mutant fat bodies than the controls. Moreover, the level of p-Akt was significantly reduced in the fat body, whereas the phosphorylated JNK level did not change. Thus, insulin signaling is specifically disrupted in the fat bodies of miR-8 null larvae (Hyun, 2009).

To more precisely analyze miR-8's function in fat cells, flip-out GAL4 overexpressing clones of miR-8 were generated in the fat body of mir-8 heterozygote. In the mosaic fat cells overexpressing miR-8, the tGPH signals was augmented in the membrane, indicating that miR-8 promotes PI3K activity in a cell-autonomous manner. Cell size also increased with miR-8 overexpression (Hyun, 2009).

Next mitotic null clones were generated to observe the loss of function phenotype. Cells of the miR-8 null clone were smaller than the adjacent cells in the twin spot -- the cells harboring wild-type copies of miR-8. This suggests that miR-8 promotes fat cell growth in a cell-autonomous manner, as expected if miR-8 enhances insulin signaling in the fat body. Fewer (or no) null clone cells were often observed next to the twin spot cells when the mitotic clones were induced at embryonic stage or newly hatched larval stage. This suggests the frequent failure of proliferation and survival of miR-8 null cells during larval development. It is noted that null clones of miR-8 were generated in the wing or eye disc but little growth defect was found in these organs. Therefore, the effect of miR-8 on cell growth is dependent on tissue type, which may be explained by the fact that USH is present in the fat body but not in wing precursor cells or the eye disc (Hyun, 2009).

To determine whether USH negatively regulates insulin signaling, mosaic clones of fat cells overexpressing USH were generated. USH-overexpressing cells were smaller in size and showed significantly lower tGPH signals in the membrane and higher FOXO signals in the nucleus than did the neighboring wild-type cells. Also mosaic fat cells expressing dsRNA against ush were created to observe the knockdown phenotype. The tGPH signal was significantly enhanced in the mosaic cells depleted of USH. In mosaic ush mutant cells, the nuclear FOXO signals decreased. Together, these observations indicate that USH inhibits insulin signaling upstream of or in parallel with PI3K in a cell-autonomous manner (Hyun, 2009).

Whether reduced insulin signaling caused by the absence of miR-8 could be rescued by knockdown of USH was further examined. Excessive insulin signaling is known to reduce the levels of insulin receptor (Inr) and cytohesin Steppke (step) through negative feedback by FOXO. These two targets of FOXO were upregulated in the fat body of miR-8 null larvae, whereas reintroduction of miR-8 dramatically reduced their expression. Notably, ush RNAi also restores the mRNA levels of the FOXO target genes Inr and step in mir-8 mutant fat bodies. Thus, the defect of insulin signaling in the fat body of miR-8 null larvae is at least partially attributable to elevated ush levels (Hyun, 2009).

FOG2 directly binds to p85α to inhibit PI3K

Although FOG2 is thought to be a nuclear transcriptional coregulator, several studies have reported that FOG2 also localizes to the cytoplasm. To confirm this finding, subcellular fractionation and Western blotting were performed. FOG2 was, in fact, observed predominantly in the cytoplasm rather than in the nucleus in HeLa and PANC1 cells. Immunostaining also showed cytoplasmic localization of FOG2 in HepG2 cells, suggesting a cytoplasmic role of FOG2 (Hyun, 2009).

Given that FOG2 suppresses PI3K and colocalizes with p85α, it is suspected that FOG2 may interact with PI3K. Notably, a significant amount of p85α, the regulatory subunit of PI3K, was coprecipitated with anti-FOG2 antibody. Interaction between FOG2 and p85α was also observed when the FOG2 was ectopically expressed in a FLAG-tagged form (Hyun, 2009).

To map the interaction domain of FOG2, several truncated mutants of FOG2 were generated. The mutants containing a FLAG-tag in the N termini were coexpressed with V5-tagged p85α and were analyzed by immunoprecipitation using anti-FLAG antibody. The results indicate that the middle region of FOG2 (507-789 aa) mediates the interaction with p85α. It was then asked whether the middle region is sufficient to inhibit PI3K activity when it is ectopically expressed in HepG2 cells. The middle region suppressed PI3K, whereas neither the N-terminal part nor the C-terminal part had a significant effect on PI3K activity (Hyun, 2009).

To test whether FOG2 binds to p85α directly, the FOG2 protein was expressed and purified from bacteria and was used in an in vitro binding assay, along with purified recombinant p85α protein fused to GST. The recombinant FOG2 protein containing the middle region of FOG2 (413-789 aa) specifically bound to recombinant p85α (Hyun, 2009).

Finally, it was asked whether FOG2 can directly inhibit p85α by performing an in vitro PI3K assay using recombinant FOG2. Addition of the recombinant FOG2 protein containing the middle region (FOG2[413-789]) to the immunoprecipitated PI3K complex significantly inhibited the PI3K activity. This finding suggests that direct binding of FOG2 to p85α leads to the inhibition of PI3K activity. Notably, it was also found that Drosophila USH physically interacts with Drosophila p60 (dp60, the fly ortholog of p85α) when dp60 is coexpressed with USH in human HEK293T cells. Therefore, the action mechanism of USH/FOG2 may be conserved across the phyla (Hyun, 2009).

This study has revealed two novel regulatory components of insulin signaling: miR-8/miR-200 and USH/FOG2. miR-8/200 negatively regulates USH/FOG2 through direct base-pairing to the 3' UTR of the ush/FOG2 mRNA. USH/FOG2, in turn, inhibits the formation of an active PI3K complex via direct interaction with dp60/p85α, the regulatory subunit of PI3K. In fly fat bodies, miR-8 suppresses ush, which causes cell-autonomous increase of fat cell growth. The roles of miR-8 and USH are conserved in mammals; miR-200 miRNAs target FOG2 to upregulate insulin signaling and cell proliferation in human cells. Given that the PI3K-Akt-FOXO pathway plays central roles in many developmental processes and that defects of this pathway have been associated with cancer, diabetes, neuropathology, and aging, further investigation of the miR-8/200 family and USH/FOG2 may contribute to the understanding and amelioration of such human diseases (Hyun, 2009).

In Drosophila, miR-8 posttranscriptionally represses USH, thereby activating insulin signaling, which results in cell-autonomous growth of fat body cells. This process also causes nonautonomous organismal growth, likely through the induction of humoral factors. In human liver cells, miR-200 posttranscriptionally represses FOG2, which directly binds to p85α and blocks the formation of an active PI3K complex. As such, the repression of FOG2 by miR-200 stimulates insulin signaling and cell proliferation (Hyun, 2009).

The results support and extend the emerging theory that the fat body is a central organ coordinating metabolic condition and global growth of the organism. It is proposed that miR-8 regulates the growth of peripheral tissues in a non-cell-autonomous manner by modulating the secretion of the humoral factors that are under the control of insulin signaling (see A model for the functions of miR-8/miR-200 and USH/FOG2). Future investigation is needed to identify the humoral factors that mediate the communication between the fat body and other tissues. Because the larval fat body is considered the Drosophila counterpart of mammalian liver and adipose tissues, it will be interesting to study whether miR-200 and FOG2 play a similar role in liver and adipose tissues to control body growth during the human juvenile period (Hyun, 2009).

Previous studies suggest that USH/FOG2 may function as either transcriptional coactivators or corepressors by partnering with various GATA transcription factors. However, FOG2 is localized to the cytoplasm in some tissues. FOG1, the other human homolog of Drosophila USH, was also reported to remain in the cytoplasm of skin stem cells that lack GATA-3 and was shown to be sequestered in the cytoplasm by a cytoplasmic protein TACC3. USH/FOG2 have been studied mainly in hematopoiesis and heart development in both flies and mammals. However, it was recently shown that USH suppresses cell proliferation in Drosophila hemocytes. It is also noteworthy that FOG2 is frequently downregulated in human cancers of the thyroid, lung, and prostate, which suggests a role of FOG2 as a tumor suppressor. This study is the first report that FOG2 acts as a negative modulator of the PI3K-Akt pathway via direct binding to p85α. It remains to be determined whether the newly discovered molecular function of USH/FOG2 is related to the previously described phenotypes of ush/FOG2 (Hyun, 2009).

This study also offers a comprehensive way of discovering the physiological function of conserved miRNAs. By systematically mapping the protein homologs of miRNA targets and by validating them experimentally, seven gene pairs were identified as conserved targets of the miR-8/200 family. Also fly genetics and human cell biology were used to identify ush/FOG2 as the target gene that is responsible for one particular phenotype. Of note, six other genes (Lap1/ERBB2IP, CG8445/BAP1, dbo/KLHL20, Lar/PTPRD, Ced-12/ELMO2, and CG12333/WDR37) may also be authentic targets of miR-8/200, although they need to be further verified by additional methods. These six genes may function in different organs and/or at different developmental stages. It has been reported that miR-8 prevents neurodegeneration by targeting atro. This study observed that atro knockdown does not rescue the small body phenotype of mir-8 mutants and that ush knockdown cannot reverse the wing and leg defects attributed to atro. Thus, a single miRNA may have several distinct functions in different cell types, likely depending on the availability of specific targets or downstream effectors. In a recent study, miR-8 gain of function was shown to affect the WNT pathway, although this finding was not sufficiently supported by the phenotype resulting from miR-8 loss of function. The miR-200 family has also been shown to interfere with epithelial to mesenchymal transitions in humans to enhance cancer cell colonization in distant tissues and to regulate olfactory neurogenesis and osmotic stress in zebrafish. It remains to be determined whether these previously described functions of the miR-8/200 microRNAs are systemically interconnected in a single organism and how widely each of these functions is conserved among animals expressing miR-8/200 microRNAs (Hyun, 2009).

MAPK/ERK signaling regulates insulin sensitivity to control glucose metabolism in Drosophila

The insulin/IGF-activated AKT signaling pathway plays a crucial role in regulating tissue growth and metabolism in multicellular animals. Although core components of the pathway are well defined, less is known about mechanisms that adjust the sensitivity of the pathway to extracellular stimuli. In humans, disturbance in insulin sensitivity leads to impaired clearance of glucose from the blood stream, which is a hallmark of diabetes. This study presents the results of a genetic screen in Drosophila designed to identify regulators of insulin sensitivity in vivo. Components of the MAPK/ERK pathway were identified as modifiers of cellular insulin responsiveness. Insulin resistance was due to downregulation of insulin-like receptor gene expression following persistent MAPK/ERK inhibition. The MAPK/ERK pathway acts via the ETS-1 transcription factor Pointed. This mechanism permits physiological adjustment of insulin sensitivity and subsequent maintenance of circulating glucose at appropriate levels (Zhang, 2011).

The insulin signal transduction pathway is regulated by cross-talk from several other signaling pathways. This includes input from the amino-acid sensing TOR pathway into regulation of insulin pathway activity by way of S6 kinase regulating IRS. Signaling downstream of growth factor receptors has also been linked to regulation of insulin signaling. The active form of the small GTPase Ras can bind to the catalytic subunit of PI3K and promote its activity. Expression of a form of PI3K that cannot bind Ras allows insulin signaling, but at reduced levels. The work reported in this study provides evidence for a second mechanism through which growth factor receptor signaling through the MAPK/ERK pathway modulates insulin pathway activity. Transcriptional control of inr gene expression by EGFR signaling may provide a means to link developmental signaling to regulation of metabolism. In this context, a statistically significant correlation wass noted between EGFR target gene sprouty and inr gene expression at different stages during Drosophila development (Zhang, 2011).

Several steps of the insulin pathway can be regulated by phosphorylation. Given that the MAPK/ERK pathway is a kinase cascade, a priori, the possibility of phosphorylation-based interaction between these pathways would seem likely. However, this appears not to be the case. Acute pharmacological inhibition of the MAPK/ERK pathway proved to have no impact on insulin pathway activity. Thus short-term changes in MAPK/ERK pathway activity do not seem to be used for transient modulation of insulin pathway activity. Instead, the MAPK/ERK pathway acts through the ETS-1 type transcription factor Pointed to control expression of the inr gene. Transcriptional control of inr suggests a slower, less labile influence of the MAPK pathway. Taken together with the earlier studies, these findings suggest that growth factor signaling can regulate insulin sensitivity by both transient and long-lasting mechanisms (Zhang, 2011).

Why use both short-term and long-term mechanisms to modulate insulin responsiveness to growth factor signaling? The use of direct and indirect mechanisms that elicit a similar outcome is reminiscent of feed-forward network motifs. Although these motifs are often thought of in the context of transcriptional networks, the properties that they confer are also relevant in the context of more complex systems involving signal transduction pathways. In multicellular organisms, feed-forward motifs are often used to make cell fate decisions robust to environmental noise. The findings suggest a scenario in which a feed-forward motif is used in the context of metabolic control, linking growth factor signaling to insulin responsiveness. In this scenario, growth factor signaling acts directly via RAS to control PI3K activity and indirectly via transcription of the inr gene to elicit a common outcome: sensitization of the cell to insulin. This arrangement allows for a rapid onset of enhanced insulin sensitization, followed by a more stable long-lasting change in responsiveness. Thus a transient signal can both allow for an immediate as well as a sustained response. The transcriptional response also makes the system stable to transient decreases in steady-state growth factor activity. It is speculated that this combination of sensitivity and stability allows responsiveness while mitigating the effects of noise resulting from the intrinsically labile nature of RTK signaling. As illustrated by the data, failure of this regulation in the fat body leads to elevated circulating glucose levels, likely reflecting impaired clearance of dietary glucose from the circulation by the fat body. Maintaining circulating free glucose levels low is likely to be important due to the toxic effects of glucose. In contrast, circulating trehalose, glycogen or triglyceride levels showed no significant change in animals with reduced InR expression, suggesting that these aspects of energy metabolism can be maintained through compensatory mechanisms in conditions of moderately impaired insulin signaling (Zhang, 2011).

Earlier studies have shown that the transcription of the inr gene is under dynamic control. Activation of FOXO in the context of low insulin signaling leads to upregulation of inr transcription, thus constituting a feedback regulatory loop. Thus, InR expression appears to be under control of two receptor-activated cues, which have opposing activities: inr expression is positively regulated by the EGFR-MAPK/ERK module, but negatively regulated by its own activity on FOXO. In the setting of this study, the cross-regulatory input from the MAPK/ERK pathway was found to dominate over the autoregulatory FOXO-dependent mechanism. If conditions exist in which the FOXO-dependent mechanism was dominant, a limited potential for crossregulation by the MAPK/ERK pathway would be expected. Whether Pointed and FOXO display regulatory cooperativity at the inr promoter is an intriguing question for future study (Zhang, 2011).

Depletion of histone deacetylase 3 antagonizes PI3K-mediated overgrowth through the acetylation of histone H4 at lysine 16

Histone acetylation is one of the best-studied gene modifications and has been shown to be involved in numerous important biological processes. This study has demonstrated that the depletion of histone deacetylase 3 (Hdac3) in Drosophila melanogaster results in a reduction in body size. Further genetic studies showed that Hdac3 counteracts the overgrowth induced by InR, PI3K or S6K over-expression, and the growth regulation by Hdac3 is mediated through the deacetylation of histone H4 at lysine 16 (H4K16). Consistently, the alterations of H4K16 acetylation (H4K16ac) induced by the over-expression or depletion of males-absent-on-the-first (MOF), a histone acetyltransferase that specifically targets H4K16, results in changes in body size. Furthermore, H4K16ac was found to be modulated by PI3K signaling cascades. The activation of the PI3K pathway caused a reduction in H4K16ac, whereas the inactivation of the PI3K pathway results in an increase in H4K16ac. The increase in H4K16ac by the depletion of Hdac3 counteracts the PI3K-induced tissue overgrowth and PI3K-mediated alterations in the transcription profile. Overall, these studies indicated that Hdac3 serves as an important regulator of the PI3K pathway and reveals a novel link between histone acetylation and growth control (Lv, 2012).

Core histone modifications are known to play an essential role in the regulation of chromatin organization and transcription. These modifications include acetylation, methylation, phosphorylation, ubiquitination, sumoylation and poly(ADP-ribosyl)ation. Histone acetylation is one of the best-studied modifications and is thought to be involved in both the initiation and elongation steps of transcription. The acetylation of the core histone tails alters the folding dynamics of nucleosomal arrays and 30-nm chromatin fibers and recruits specific chromatin remodeling complexes that exert the specific function(s) of chromatin (Lv, 2012).

The acetylation of histones is regulated by two highly conserved classes of histone enzymes, histone acetyltransferases (HATs) and histone deacetylases (HDACs), which catalyze the addition and removal, respectively, of acetyl groups on histone lysine residues. Reversible histone acetylation and deacetylation are highly regulated processes that are crucial for chromatin reorganization and the regulation of gene transcription in response to extracellular conditions. The balance between the acetylation and deacetylation of histones serves as a key regulatory mechanism for gene expression and governs numerous developmental processes and disease states (Lv, 2012).

HDACs have been classified into four subfamilies based on their homologs and functional similarities. Hdac3 is a class HDAC that shares homology with yeast Rpd3. This protein is reportedly present in the nuclear, cytoplasmic and membrane fractions. The knockout of Hdac3 in mice leads to embryonic lethality before day 9.5. The inactivation of Hdac3 has been shown to delay cell cycle progression and result in cell cycle-dependent DNA damage, inefficient repair and increased apoptosis in mouse embryonic fibroblasts. Hdac3 has also been shown to be up-regulated in various tumor types. However, the precise function and underlying molecular mechanism of Hdac3 in these processes remain largely unknown (Lv, 2012).

The Drosophila ortholog to human Hdac3 is known to be Hdac3 or dHDAC3 (Johnson, 1998). This study used Drosophila to investigate the function of Hdac3 during development. Depletion of Hdac3 in Drosophila results in a reduction in both organ and body sizes. Hdac3 controls growth through the regulation of H4K16 deacetylation. Alterations in H4K16ac through the ectopic expression of MOF, a histone acetyltransferase that specifically targets H4K16, result in changes of cell/body size. It was also found that H4K16ac is modulated by PI3K signaling. Increasing the level of H4K16ac by depleting Hdac3 effectively reverses the PI3K-induced tissue overgrowth and alterations in the transcription profile (Lv, 2012).

Hdac3 is a component of the nuclear receptor co-repressor complex containing N-CoR (nuclear receptor corepressor) and SMRT (silencing mediator for retinoid and thyroid hormone receptors), both of which are recruited by nuclear hormone receptors to regulate gene transcription). Several substrates were found to be targets of Hdac3, including histones and non-histone proteins. Among the targets affected by Hdac3, this study found that H4K16ac is a critical epigenetic modification associated with animal growth, as demonstrated not only by the finding that alterations in H4K16ac were closely associated with Hdac3-induced organ/body growth but also by the finding that mutating H4K16 directly affected Hdac3-induced growth. Furthermore, transgenic lines in which MOF, the specific histone H4K16 HAT, was over-expressed or depleted exhibited similar changes in cell/body size, thus confirming that H4K16ac plays an essential role in animal growth. Histone H4K16 acetylation is known to function as a dual switch for higher-order chromatin and protein-histone interactions and has been shown to regulate embryonic stem cell self-renewal and cellular life span. Recent work in has suggested that H4K16ac in Drosophila not only is critical for the acetylation of H4K5, H4K8 and H3K9, which are hallmarks of active chromatin, but also exerts an effect on H3K9 methylation and the association of HP1 with chromatin, which are hallmarks of heterochromatin. It is therefore presumed that the changes in H4K16ac affect higher-order chromatin and alter the transcription of genes related to growth. However, the exact mechanism by which H4K16ac regulates the transcription of genes related to growth needs to be further investigated (Lv, 2012).

One of the main findings in this work is the genetic interaction between Hdac3/H4K16ac and the PI3K pathway. The PI3K pathway is a highly conserved signal transduction cascade from flies to humans. Previous studies have identified a number of the components of this signaling pathway. However, the mechanisms by which this pathway regulates nuclear events, such as gene transcription, remain largely unknown. This work shows that PI3K signaling modulates the acetylation of H4K16. This finding was supported by results showing that the activation of PI3K caused a corresponding reduction in H4K16ac, whereas the inactivation of the PI3K pathway resulted in an increase in H4K16ac. Furthermore, the introduction of the H4K16A mutant, in which H4K16 cannot be acetylated, further enlarged the PI3K-induced increase in ommatidial size, confirming the function of histone H4K16ac in PI3K signaling (Lv, 2012).

Although the exact mechanism by which PI3K regulates H4K16ac is still unknown, this study demonstrates that the loss of Hdac3 inhibited PI3K-mediated overgrowth, thus suggesting that PI3K targets the activity of Hdac3 and subsequently affects H4K16ac. This hypothesis is supported by the observations that Drosophila Hdac3 can form a complex with Akt and that the complex of human Hdac3 with the deacetylase activation domain (DAD), the human SMRT co-repressor and inositol tetraphosphate is required for the activation of Hdac3 enzymatic functionality. The observation that the depletion of Hdac3 decreased the level of phospho-Akt and affected the subcellular localization of GFP-PH also supported this possibility. However, the observation that Hdac3 depletion failed to counteract the PI3K-induced hyperphosphorylation of Akt while completely rescuing the decrease in H4K16ac and the tissue overgrowth induced by the PI3K over-expression indicated that Hdac3 likely counteracts the PI3K-induced tissue overgrowth by modulating the level of H4K16ac (Lv, 2012).

The hyperactivation of the PI3K pathway is known to be associated with many types of human cancer. A number of HDAC inhibitors have been developed and applied in clinical trials to inhibit tumor growth. However, the molecular mechanisms of these HDAC inhibitors in cancer prevention remain to be elucidated. The present study found that the over-expression of PI3K decreases H4K16ac in vivo. Further studies have shown that increasing the level of H4K16ac by depleting Hdac3 can antagonize the PI3K-induced tissue overgrowth. This finding, therefore, may provide further insight into the mechanisms by which the HDAC inhibitors inhibit tumor growth (Lv, 2012).

Ras-dependent and Ras-independent effects of PI3K in Drosophila motor neurons

The lipid kinase PI3K plays key roles in cellular responses to activation of receptor tyrosine kinases or G protein coupled receptors such as the metabotropic glutamate receptor (mGluR). Activation of the PI3K catalytic subunit p110 occurs when the PI3K regulatory subunit p85 binds to phosphotyrosine residues present in upstream activating proteins. In addition, Ras is uniquely capable of activating PI3K in a p85-independent manner by binding to p110 at amino acids distinct from those recognized by p85. Because Ras, like p85, is activated by phosphotyrosines in upstream activators, it can be difficult to determine if particular PI3K-dependent processes require p85 or Ras. This study asked if PI3K requires Ras activity for either of two different PI3K-regulated processes within Drosophila larval motor neurons. To address this question, the effects on each process were determined of transgenes and chromosomal mutations that decrease Ras activity, or mutations that eliminate the ability of PI3K to respond to activated Ras. It was found that PI3K requires Ras activity to decrease motor neuron excitability, an effect mediated by ligand activation of the single Drosophila mGluR DmGluRA. In contrast, the ability of PI3K to increase nerve terminal growth is Ras-independent. These results suggest that distinct regulatory mechanisms underlie the effects of PI3K on distinct phenotypic outputs (Johnson, 2012).

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