Phosphotidylinositol 3 kinase 92E
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, Dp110D945A, 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 Dp110D945A 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 Dp110CAAX 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).
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).
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, TkvQ253D, 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
TkvQ253D 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 TkvQ253D 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 TkvQ253D-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 TkvQ253D even in growth-impaired cells. Alternatively, if TkvQ253D were to promote cell cycle progression indirectly via stimulating cellular growth, the proliferative effect of TkvQ253D 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 TkvQ253D. Clones of cells that co-express Deltap60 and TkvQ253D 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 TkvQ253D, cells that co-express Deltap60 and TkvQ253D do not show the G1 delay characteristic of cells expressing Deltap60 alone. Thus, TkvQ253D 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 TkvQ253D may be Dp110 independent. However, the slight increase in size observed in cells co-expressing Deltap60 and TkvQ253D 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).
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).
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).
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, Daktmyr, were expressed in late-prepupal salivary glands. Daktmyr 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 Daktmyr 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).
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 Drosphila, 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 RacN17 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 Drosphila 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).
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).
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.
RukL 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 RukL 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 RukL 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).
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 Cdc25stg 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 (Dp110D954A) 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 Dp110D954A 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).
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 (Pten2L117) and a hypomorphic (Pten2L100) 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 (Pten2L117) 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 Pten2L117and InR327, however, are almost the size of heads singly mutant for Pten2L117. 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 (Pten2L117/Pten2L100). 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 Pten2L117/Pten2L100. 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 Pten2L117 allele and a null mutation for PI 3-kinase p110 or its p60 adaptor. Loss of PTEN function (Pten2L117) 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 Pten2L117 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 Pten2L117/Pten2L100 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).
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 (RasV12) promotes these processes in the developing wing, but the effector pathways involved are unclear. Evidence is presented indicating that RasV12 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 RasV12-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 (RasV12) is capable of increasing
dMyc protein levels as well as levels of PI3K signaling, suggesting
that RasV12