Interactive Fly, Drosophila

REGULATION
Rheb functions downstream of PTEN

Cytoplasmic activated protein kinase Akt regulates lipid-droplet accumulation in Drosophila nurse cells

The insulin/insulin-like growth factor signalling (IIS) cascade performs a broad range of evolutionarily conserved functions, including the regulation of growth, developmental timing and lifespan, and the control of sugar, protein and lipid metabolism. Recently, these functions have been genetically dissected in Drosophila, revealing a crucial role for cell-surface activation of the downstream effector kinase Akt in many of these processes. However, the mechanisms regulating lipid metabolism and the storage of lipid during development are less well characterized. The nutrient-storing nurse cells of the fly ovary were used to study the cellular effects of intracellular IIS components on lipid accumulation. These cells normally store lipid in a perinuclear pool of small neutral triglyceride-containing droplets. Loss of the IIS signalling antagonist PTEN, which stimulates cell growth in most developing tissues, produces a very different phenotype in nurse cells, inducing formation of highly enlarged lipid droplets. Furthermore, the accumulation of activated Akt in the cytoplasm is responsible for this phenotype and leads to a much higher expression of LSD2, the fly homologue of the vertebrate lipid-storage protein perilipin. This work therefore reveals a signalling mechanism by which the effect of insulin on lipid metabolism could be regulated independently of some of its other functions during development and adulthood. It is speculated that this mechanism could be important in explaining the well-established link between obesity and insulin resistance that is observed in Type 2 diabetes (Vereshchagina, 2006).

These data suggest that the effect of cytoplasmic P-Akt on lipid storage may be cell type-specific. Increasing IIS throughout the whole organism in viable Pten mutants surprisingly reduces total lipid content, whereas decreasing IIS elevates lipid levels both in flies and mice. It has not been possible to show biochemically whether triglycerides are increased rather than merely redistributed in Pten mutant ovaries, because only a small minority of the nurse cells is mutant. However, in some late-stage clones, in which cytoplasmic volumes are decreasing, lipid levels often appear elevated in mutant cells. Insulin-stimulated increases in lipid-droplet number and size have been observed in mammals, and, interestingly, when tumours are induced in mouse liver by raising IIS, lipid-storage mechanisms are also activated. It is proposed that these cell type-specific responses are due to selective accumulation of an IIS-modulated, cytoplasmic activated Akt pool that must, to some extent, be controlled independently of cell-surface P-Akt. Indeed, a Drosophila phosphatase has been identified that regulates levels of cytoplasmic P-Akt in nurse cells, and it has been shown that mutations in this gene produce a very similar enlarged lipid-droplet phenotype, confirming this hypothesis (Vereshchagina, 2006).

How could elevated cytoplasmic P-Akt induce such a dramatic lipid-droplet phenotype in nurse cells? In mammals, Akt can promote the transcription of genes involved in lipid biosynthesis and storage pathways. The data indicate LSD2/perilipin is one of these targets. IIS also post-translationally upregulates the activity of mammalian perilipin. Interestingly, ovaries mutant for Lsd2 show altered lipid accumulation, but droplets are still formed. Therefore, LSD2 is almost certainly one, but not the only, target for IIS in the control of lipid-droplet accumulation in nurse cells. In this context, it is interesting to note that, in addition to its proposed role in coating lipid droplets, LSD2 has recently been shown to regulate microtubule-dependent trafficking of these organelles. Since cytoplasmic P-Akt could still be associated with intracellular membranes or the droplet surface, it may be well positioned to modulate this transport process (Vereshchagina, 2006).

Obesity is a well-established predisposing factor in the acquisition of cellular insulin resistance and Type 2 diabetes. Increased levels of circulating free fatty acids (FFAs) associated with obesity appear to be important in this link. However, it is unclear whether other mechanisms are also involved or how reduced insulin sensitivity ultimately impacts on lipid storage. Molecules downstream of Akt are known to regulate cell-surface IIS through at least two negative-feedback loops involving downstream S6 kinase and the transcription factor FOXO. This work therefore raises the possibility that any predisposition towards increased cytoplasmic P-Akt could specifically promote lipid storage and also selectively suppress insulin-dependent events at the cell surface. It will be interesting to investigate further the molecules involved in controlling this P-Akt pool and whether the feedback mechanisms have any role to play in linking obesity and insulin resistance in Type 2 diabetes (Vereshchagina, 2006).

The role of the prothoracic gland in determining critical weight for metamorphosis in Drosophila melanogaster

The timely onset of metamorphosis in holometabolous insects depends on their reaching the appropriate size known as critical weight. Once critical weight is reached, juvenile hormone (JH) titers decline, resulting in the release of prothoracicotropic hormone (PTTH) at the next photoperiod gate and thereby inducing metamorphosis. How individuals determine when they have reached critical weight is unknown. Evidence is presented that in Drosophila, a component of the ring gland, the prothoracic gland (PG), assesses growth to determine when critical weight has been achieved. The GAL4/UAS system was used to suppress or enhance growth by overexpressing PTEN or Dp110 (Pi3K92E), respectively, in various components of the ring gland. Suppression of the growth of the PG and CA, but not of the CA alone, produced larger-than-normal larvae and adults. Suppression of only PG growth resulted in nonviable larvae, but larvae with enlarged PGs produced significantly smaller larvae and adults. Rearing larvae with enlarged PGs under constant light enhanced these effects, suggesting a role for photoperiod-gated PTTH secretion. These larvae are smaller, in part as a result of their repressed growth rates, a phenotype that could be rescued through nutritional supplementation (yeast paste). Most importantly, larvae with enlarged PGs overestimated size so that they initiated metamorphosis before surpassing the minimal viable weight necessary to survive pupation. It is concluded that the PG acts as a size-assessing tissue by using insulin-dependent PG cell growth to determine when critical weight has been reached (Mirth, 2005; full text of article).

These manipulations of insulin-dependent PG growth showed that this growth is inversely related to larval growth. Suppressing the growth of the PG (P0206>PTEN - ectopically driven PTEN) produced larvae that spent more time in each instar and were larger than normal. These effects are presumably due to a combination of reduced ecdysteroid biosynthesis, which is known to delay development, and increased growth rate. Conversely, larvae with enlarged PGs (phm>Dp110; phm is a phantom GAL4 line which was used to drive expression of Dp110) showed accelerated development in the L3. Their growth rate was dependent on nutritional conditions. Whereas phm>Dp110 larvae reared on suboptimal food grew slowly, well-fed phm>Dp110 larvae grew at the same rate as controls. Together, these data indicate that the growth of the PG negatively regulates the growth rate of the whole animal and that this regulation is modulated by nutrition (Mirth, 2005).

In addition, decreasing PG size in P0206>PTEN larvae resulted in premature metamorphosis and the formation of L2 puparia. Similar L2 puparia have been described in larvae with mutations that affect the regulation of ecdysteroid biosynthesis or signaling and in larvae where the Broad isoform Z3 was overexpressed in the ring gland, resulting in its apoptosis. L2 puparia are seen in situations where ecdysone synthesis is compromised because larvae cross the threshold weight for metamorphosis prior to the production of sufficient ecdysone to initiate a larval molt, redirecting their development to the metamorphic pathway (Mirth, 2005).

Reducing PG size resulted in reduced ecdysteroid biosynthesis; P0206>PTEN larvae showed reduced ecdysteroid titers at 44 hr AEL3, and phm>PTEN larvae only molted to L2 when fed 20E. Under conditions of low ecdysteroid synthesis, fast-growing larvae could surpass the threshold for metamorphosis before the ecdysteroid titer was sufficient to induce a molt, resulting in L2 prepupae. Slower-growing larvae would be unable to reach this threshold weight before the rise in ecdysteroid titer induced the molt to L3. Indeed, undernourished, and presumably slow-growing, P0206>PTEN L2 larvae all molted to L3, whereas only 33% of the well-fed P0206>PTEN larvae molted to L3 (Mirth, 2005).

Enlarging the PG of larvae reared under constant light caused larvae to initiate metamorphosis earlier and at smaller sizes. Nevertheless, even though larvae starved early after the L3 molt were able to pupariate, they were unable to survive to pupation unless they had fed for at least 11.5 hr. This suggests that phm>Dp110 larvae starved prior to 11.5 hr AL3E initiated metamorphosis before surpassing the minimal viable weight. Furthermore, although in control larvae, critical weight and minimal viable weight are apparently attained at the same time, they are uncoupled in phm>Dp110 larvae. Therefore, the assessment of critical weight is dependent on PG growth, whereas the minimal viable weight is not (Mirth, 2005).

In Drosophila, the PGs are responsible for a size-assessment event, early in the L3, that induces the onset of metamorphosis once critical weight is surpassed. Enhancing PG growth resulted in an overestimation of body size, thereby causing the larva to initiate metamorphosis early, at a subnormal size. Under LL, the effects of enlarging the PG were enhanced, producing individuals that pupariated even earlier at even smaller sizes, suggesting that when PTTH release was unconstrained by circadian gating, the PTTH delay period was reduced. These data provide the first indication in Drosophila that the post-critical-weight PTTH release may be under photoperiod control, as it is in Manduca (Mirth, 2005).

There has been some discussion in the literature as to whether critical weight as described in Drosophila is the same as critical weight as defined in Manduca. This discussion has arisen because the definition for Manduca states that critical weight is the minimal size at which starvation can no longer delay the onset of metamorphosis, but when Drosophila larvae are starved before critical weight is reached, they die. The current data suggest that this is due to a tight relationship between minimal viable weight and critical weight in Drosophila. Effects more similar to those observed in Manduca can be obtained when pre-critical-weight Drosophila larvae are starved for an interval and then re-fed. Under these conditions, they delay metamorphosis for a period greater than the period of starvation. Much of the confusion surrounding critical weight in Drosophila has arisen because in wild-type larvae, minimal viable weight and critical weight are achieved at the same time (Mirth, 2005).

After critical weight has been surpassed, the metamorphic pathway appears to be partially suppressed by continued feeding in Drosophila. Hence, the nutrition pathway appears to promote growth and suppress metamorphosis, whereas insulin-dependent PG growth suppresses larval growth and promotes differentiation (Mirth, 2005).

The effects of increased growth in the PG are not simply due to increasing cell size, but rather are specific to the nutrition-dependent InR signaling pathway. Studies have indicated that when either dMYC or cyclinD/cdk4 are used to enlarge the PG cells, there is no reduction in overall body size. Overexpression of dMYC, of cyclinD/cdk4, and of Dp110 all enhance cell growth, but they do so in fundamentally different manners by using separate cascades. Whether the size-assessment mechanism operates via increased intracellular PIP3 levels in the PG cells or the accumulation of some other downstream component of the InR cascade in these cells is unknown (Mirth, 2005).

Although no difference in was detected ecdysteroid titers in larvae with enlarged PGs, there is evidence that increased InR signaling in the PG cells can produce mild increases in ecdysteroidogenesis and ecdysone signaling, increases that are below the level of detection of ecdysteroid-titer assays. Larvae with enlarged PGs showed both a mild increase in the transcription of phantom during feeding stages and an increase in the transcription of the early ecdysone response gene E74B. These subtle differences in ecdysteroid titers may be important for determining growth rates and for size assessment. A gradual rise in ecdysteroid titers is coincident with the time that critical weight is reached in Drosophila. Also, subtle shifts in 20E concentrations are important for growth. Basal concentrations of 20E in combination with bombyxin enhance the growth of wing imaginal tissues in vitro; slightly higher concentrations of 20E suppress growth (Mirth, 2005).

Mutations that cause imaginal disc and larval overgrowth often cause delayed pupariation and, in some cases, show low L3 ecdysteroid titers. In the case of the mutant lethal (2) giant larvae, the ring glands are smaller than normal and have the ultrastructural appearance of glands that have low rates of ecdysteroid biosynthesis. Delayed pupariation in these larvae can be rescued by implanting wild-type ring glands. Lastly, hypomorphic mutations in DHR4, a repressor of ecdysone-induced early genes, cause reductions in critical weight and early-pupariation phenotypes similar to those described in this study. Thus, the size-assessment mechanism is likely to involve surpassing a threshold ecdysteroid titer above which the activation of the ecdysone cascade occurs (Mirth, 2005).

These data allow construction of the following model for size assessment in Drosophila. As PG cells grow in response to increased InR signaling, they increase their basal level of ecdysteroid biosynthesis. Critical weight is then reached when systemic ecdysteroid concentrations surpass a threshold that sets into motion the endocrine events that will end the growth phase of larval development and allow the larva to begin metamorphosis (Mirth, 2005).

Studies in the mid-1970s defined a size-assessment event during the final instar of the moth Manduca sexta; termed critical weight, it is the minimal size required for the timely initiation of metamorphosis. How insect larvae determine when they have reached critical weight has long been a mystery. It is hypothesized that a size-assessing tissue determines when critical weight had been reached. Suppressing growth in this size-assessing tissue would cause an underestimation of body size, resulting in metamorphosis at larger than normal sizes, whereas enlarging this tissue would result in subnormal sizes. Studies in Drosophila have shown that manipulation of the growth of the PG via the InR pathway produced these types of effects. Furthermore, larvae with enlarged PGs metamorphosed at even smaller sizes when reared under LL, suggesting a role for PTTH circadian gating in this response. Smaller size arose both as a result of a reduction in growth rate, an effect that could be rescued via nutritional supplementation, and the early onset of metamorphosis. Most importantly, larvae with enlarged PGs had a remarkably reduced critical weight, suggesting that they are severely overestimating their own body size. These results offer a very new perspective on the problem of size control in insects, uniting the recent data exploring the role of nutrition and the insulin-receptor pathway on growth with the classical physiological experiments that defined critical weight (Mirth, 2005).

Effects of Mutation or Overexpression

To gain insight into the functions of Pten during development, an examination was made of clones of cells in the Drosophila eye that lack Pten. Pten mutant ommatidia in the mosaic eyes appear larger in size when compared to their neighboring wild-type ommatidia. Sections of these mosaic eyes reveal that the mutant ommatidia have normal photoreceptor cell composition and orientation. However, the sizes of the individual mutant cells are much larger than their neighboring wild-type cells (the average size of a mutant ommatidium is approximately 2.5 times that in a wild-type cell). Careful examination of the chimeric ommatidia that contain both mutant and wild-type cells reveals that the Pten mutant phenotype is cell autonomous, since every mutant cell has an enlarged cell body while its immediate wild-type neighbors have normal sized cell bodies. Unlike the Drosophila tumor suppressor genes such as lats, no obvious overproliferation defect is seen in Pten mutant clones in the eye. This could be because Pten may not normally function in the eye in regulating cell proliferation, or alternatively, there may be redundancy for Pten-like molecules in the eye (Huang, 1999).

To explore other potential roles for Pten during eye development, the UAS/GAL4 system was used to overexpress both human and fly PTEN in Drosophila. Interestingly, the phenotypes caused by overexpression of human PTEN and Drosophila Pten are indistinguishable, suggesting that the functions of the two homologs are conserved. For simplicity, the name PTEN will be used here. To verify whether the effects that are observed with expression of PTEN are related to its enzymatic activity, a construct, UAS-PTENC124S, was used in which a critical residue in the phosphatase domain has been mutated. This change also leads to elimination of catalytic activity in vitro. While ectopic expression of wild-type PTEN causes specific phenotypes, expression of PTENC124S under the same conditions has no effect. Together with the PTEN^c494 mutant, these experiments demonstrate that phosphatase activity is necessary for PTEN to exert its functions during development. Since ubiquitous expression of PTEN directed by heat-shock induction of GAL4 causes lethality during embryonic and larval stages, PTEN is expressed specifically in the developing eye using the eyeless GAL4 line (EYE-GAL4). The eyeless enhancer directs gene expression in the young developing eye disc where cells are actively proliferating. Overexpression of PTEN in proliferating cells of the eye disc results in dramatic reduction of eye size in a dosage-dependent manner. In fact, multiple copies of UAS-PTEN can completely eliminate the eye. This small adult eye phenotype could be caused either by the inhibition of cell proliferation by PTEN, which would result in eye discs of smaller than normal size, or by the failure of ommatidium differentiation in eye discs of normal size. The sizes of eye discs dissected from third instar larvae carrying EYE-GAL4/UAS-PTEN are dramatically reduced, suggesting a defect in cell proliferation. Consistent with this idea, sections of these small adult eyes show that all types of photoreceptor cells are present and the pigment lattice is essentially regular, indicating that cell differentiation and pattern formation are not disrupted. Moreover, although these eye discs are much smaller, neural differentiation in the posterior region occurs normally as indicated by neuronal-specific anti-Elav staining (Huang, 1999).

Suppression of cell proliferation in mammalian tissue culture cells by PTEN has been reported to act through blocking cell cycle progression in the G1 phase. To determine whether PTEN blocks G1/S transition in the developing eye, Drosophila eye discs were examined with GMR-GAL4 driven PTEN expression. The GMR promoter directs gene expression in the region posterior to the morphogenetic furrow (MF) in the eye imaginal disc. While most cells in this region are differentiating, a stripe of cells posterior to MF undergoes a synchronized, last round of cell division. GMR-directed expression of p21^C1P1/WAF1, a human cyclin-dependent kinase inhibitor, blocks this last round of division by preventing these cells from entering S phase. Unlike the GMR-directed p21 expression, BrdU labeling shows that these PTEN-expressing discs have the characteristic stripe of staining similar to that of the wild-type discs, suggesting that expression of PTEN does not block G1/S transition. In contrast, propidium iodide staining of the EYE-GAL4/UAS-PTEN eye discs reveals that the discs accumulate many cells with brighter nuclear staining consistent with a DNA content of 4C. FACS analysis of dissociated cells from these discs also shows an increased percentage of cells in G2. These results suggest that overexpression of PTEN during eye development causes cell cycle arrest at the G2 or G2/M phase (Huang, 1999).

Overexpression of PTEN under the direction of GMR results in adult eyes that are not only smaller but also rough. Sections of these adult eyes reveal a complex phenotype with varied degrees of severity. While the apical lenses of the ommatidia are smaller, the sizes of photoreceptor cells in the cross sections are larger than normal. Furthermore, these GMR/PTEN adult eyes have a flattened appearance with a narrowed retina, suggesting the mutant phenotype could result from changes in both cell shape and size. In addition, and different from the phenotype caused by inactivation of PTEN, the rhabdomeres of the PTEN-overexpressed photoreceptors are reduced in size. Moreover, these sections show that some ommatidia have missing pigment or photoreceptor cells. Third instar eye discs were therefore stained with the anti-Elav antibody to monitor retinal neuron differentiation. Elav staining reveals a neuronal differentiation pattern comparable to wild type, indicating that photoreceptor differentiation is not affected. To pinpoint the cause of the phenotype, an assessment was made as to whether cell death might have contributed to the GMR-GAL4/UAS-PTEN eye phenotype. These eye discs were stained with acridine orange. Unlike wild-type third instar eye discs, which have few dying cells, the GMR-GAL4/UAS-PTEN eye discs have a substantially increased amount of cell death in the posterior region of the eye disc, where GMR-GAL4 is expressed. To further verify the contribution of cell death to the eye phenotype, GMR-GAL4 was used to coexpress PTEN with the p35 baculovirus gene, which can block apoptotic cell death. The eye phenotype induced by GMR/PTEN is largely rescued by coexpression of p35 (Huang, 1999).

These experiments suggest the possibility that cell death might also contribute to the EYE/PTEN small eye phenotype. However, EYE/PTEN eye discs do not have increased acridine orange staining and the EYE/PTEN small eye phenotype is not suppressed by coexpression of p35. Thus, cell death appears not to be a major factor in PTEN-mediated inhibition of cell proliferation. Instead, these results suggest that cell death is a consequence of cells at different developmental stages in response to the PTEN product. It is concluded that overexpression of PTEN arrests cell-cycle progression in proliferating cells while promoting apoptosis in differentiating cells during eye development. These results suggest that PTEN may suppress tumorigenesis by preventing damaged cells from dividing and/or promoting a response to apoptotic signals in a cell context-dependent manner (Huang, 1999).

Pten, a Drosophila homolog of the mammalian PTEN tumor suppressor gene, plays an essential role in the control of cell size, cell number, and organ size. In mosaic animals, Pten minus cells proliferate faster than their heterozygous siblings, show an autonomous increase in cell size, and form organs of increased size, whereas overexpression of Pten results in opposite phenotypes. The loss-of-function phenotypes of Pten are suppressed by mutations in the PI3K target Dakt1 and the translational initiation factor eif4A, suggesting that Pten acts through the PI3K signaling pathway to regulate translation. Although activation of PI3K and Akt has been reported to increase rates of cellular growth but not proliferation, loss of Pten stimulates both of these processes, suggesting that PTEN regulates overall growth through PI3K/Akt-dependent and -independent pathways. Furthermore, Pten does not play a major role in cell survival during Drosophila development. These results provide a potential explanation for the high frequency of PTEN mutation in human cancer (Gao, 2000).

The observation that clones of Pten mutant cells have a proliferative advantage over their wild-type twin spots indicates that Pten controls progression through the cell division cycle. To determine which phase(s) of the cell cycle Pten regulates, FACS analysis was used to examine the DNA content of dissociated wing imaginal disc cells containing clones of dPTEN mutant cells. Loss of Pten results in a decrease in the percentage of cells in the G1 phase of the cell cycle and a relative increase in the S and G2 population. Clonal overexpression of Pten has a complementary effect, causing a slight decrease in the number of S-phase cells. FACS analysis also reveals complementary changes in cell size in response to Pten levels: loss of Pten causes an increase in average cell size, while Pten overexpression decreases average cell size. Similar effects were observed throughout all phases of the cell cycle and at multiple developmental stages. The cell size and cell cycle FACS profile of Pten mutant cells is remarkably similar to that of cells overexpressing Drosophila PI3K. In both cases, the percentage of cells in G1 is reduced, indicating an acceleration of this phase of the cell cycle. Since overexpression of PI3K does not increase proliferation rates, this shortened G1 appears to be balanced by a commensurate lengthening in the duration of S and/or G2 phases. A similar phenomenon has been described for cells whose G1 phase is accelerated by overexpression of Cyclin E, dMyc, or activated Ras. In contrast, the rapid proliferation rate of Pten mutant cells indicates that S and G2 phases do not lengthen in response to the abbreviated G1 and thus suggest that Pten regulates multiple phases of the cell cycle (Gao, 2000).

Genetic interactions between Pten and PI3K, a component of the insulin signaling pathway in Drosophila, were examined. Overexpression of the PI3K catalytic subunit, Dp110, results in increased wing size, while overexpression of a dominant negative Dp110 construct (PI3KDN) results in the opposite phenotype. These phenotypes are due to changes in cell size. Coexpression of Pten with PI3KDN further reduces wing size. In addition, overexpression of Pten suppresses the increased wing size resulting from PI3K overexpression. To further examine the genetic interaction between Pten and PI3K, Pten mutant cells were examined in a genetic background of overexpressing PI3K or PI3KDN. When PI3K is overexpressed, Pten mutant cells are indistinguishable in cell size from their nonmutant siblings, suggesting that overexpression of PI3K results in increased PIP3 levels and increased signaling that cannot be further activated by removing Pten. Conversely, overexpression of PI3KDN partially suppresses the increased cell size of Pten mutant cells. Thus, PTEN and PI3K antagonize each other in regulating cell size (Gao, 2000).

Certain combinations of loss-of-function alleles of the Drosophila Insulin-like receptor (inr) result in flies with a decreased cell size. This provides an opportunity to examine the genetic epistasis between Pten and inr. The increased cell size of Pten mutant cells can not be reversed in inr mutant animals, and thus loss of Pten function is epistatic to (acts downstream from) mutations in inr (Gao, 2000).

Previous studies have shown that loss of PTEN function promotes cell survival in mammals through activation of Akt. In addition, PTEN acts through Akt in metabolic and longevity control in C. elegans. Dakt1, a Drosophila homolog of Akt, has been suggested to play a role in cell survival in embryogenesis (Staveley, 1998) and cell size control (Verdu, 1999). A hypomorphic allele of Dakt1 has been identified in the large-scale gene disruption project carried out by the Berkeley Drosophila Genome Project. This allele is semilethal, and homozygous survivors show reduced body size and cell size, consistent with a role of Dakt1 in growth control. To examine whether Pten controls cell size through regulating Akt activity, Pten mutant clones were generated in Dakt1 mutant animals. Dakt1 mutation completely suppressed the increase of cell size associated with the Pten mutation. This result provides strong in vivo evidence that Dakt1 functions downstream of (or in parallel to) Pten in the control of cell size. Taken together, these genetic interactions suggest that the role of Pten in opposing signaling through the PI3K/Akt pathway is conserved between flies and vertebrates (Gao, 2000).

eif4A is an ATP-dependent RNA helicase that is an essential component of the eif4F translation initiation complex. Previous studies have identified an allelic series of eif4A mutants that affects larval growth, DNA replication, and cell proliferation (Galloni, 1999). Since the defect in DNA synthesis in eif4A mutants can be bypassed by overexpressing the E2F transcription factor, it has been suggested that eif4A preferentially regulates a specific set of cell-cycle regulatory genes (Galloni, 1999). To examine whether the proliferative advantage of Pten mutant cells requires the same set of cell-cycle regulatory genes as those controlled by eif4A, Pten;eif4A double-mutant clones were examined. A hypomorphic allele and a stronger allele of eif4A were used. The weaker allele confers proliferation disadvantage to the cells, resulting in mutant clones that are smaller than the twin spots without affecting cell size (Galloni, 1999). Hypomorphilc eif4A can partially suppress the overproliferation of Pten mutant cells, but the increased cell size of Pten mutant cells is not suppressed. The stronger eif4A allele completely suppresses the proliferation of Pten mutant cells. Pten;eif4A double-mutant clones are undetectable, as has been observed in the eif4A strong single mutant (Galloni, 1999). It is suggested that modulation of translation initiation is an important aspect of Pten function in regulating cell proliferation (Gao, 2000).

It is proposed that Pten regulates cell proliferation by multiple mechanisms, both PI3K-dependent and -independent. One potential PI3K-independent mechanism is suggested by the domain in Pten related to tensin, an actin filament capping protein that localizes to focal adhesions. Overexpression of tensin can suppress anchorage-independent proliferation of Ras-transformed 3T3 cells, and therefore this domain may provide a growth-regulatory function in mammalian PTEN as well. Moreover, in addition to its role as a lipid phosphatase, PTEN also possesses a dual-specificity protein phosphatase activity. PTEN has been shown to bind and dephosphorylate the focal adhesion kinase FAK and to down-regulate the formation of focal adhesions. Such cell contacts play a critical role in regulating proliferation in Drosophila, and the gene products of several Drosophila tumor suppressors such as expanded, fat, and l(2) discs large all localize to adherens or septate junctions. The results for Pten are thus consistent with a model in which PTEN suppresses cell growth and G1/S progression by down-regulating the PI3K/Akt pathway and inhibiting the G2/M transition through an alternative mechanism, perhaps involving regulation of the cytoarchitecture. The ability to regulate both growth and cell division may explain why PTEN is such a common target in advanced tumors. This model is also consistent with the different mutant phenotypes between a null Pten allele and an allele that carries a point mutation (Huang, 1999). While the point mutation changes an invariant amino acid within the phosphatase active site and is likely to inactivate the lipid phosphatase activity, the other domains of Pten are still intact. Characterization of Pten mutants that are specifically defective in cell growth or proliferation may shed further light on its role in the control of overall growth (Gao, 2000).

Since loss of lilliputian function affects cell size and head size, tests were performed for genetic interactions between lilli and components of the PI3K/PKB pathway. Pten acts as a negative regulator in the PI3K/PKB pathway by dephosphorylating the second messenger phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P3; PIP3]. Tissues mutant for Pten show hyperplastic and hypertrophic growth: Pten mutant cells are larger and proliferate at a higher rate than wild-type cells. Removal of Pten function from the eye imaginal disc tissue using the ey-Flp system results in an increase in eye and head size. The increase in eye size is due to an increase in cell number and cell size as indicated by the increase in number and size of ommatidia. To test whether the Pten large-head phenotype is modified by the removal of Lilli function, Pten;lilli double mutant eyes were generated. Indeed, eyes double mutant for Pten and lilli are considerably smaller than Pten mutant eyes due to a reduction in cell number and cell size. Loss of Lilli function, however, does not completely suppress the Pten phenotype. Although these results suggest that Lilli and Pten cooperate in the control of cell and organ growth, the absence of a clear-cut epistasis between the two mutants indicates that Lilli does not act downstream of Pten in a simple linear pathway but it rather acts in a parallel pathway required for growth (Wittwer, 2001).

Retinal cells lacking lilli are significantly smaller than wild-type cells. Despite the small size of individual lilli mutant adult cells, relatively large clones of lilli mutant cells can be generated, suggesting that lilli may affect cell size without changing the overall rate of growth. This was tested by comparing the size of individual lilli mutant clones to their wild-type twinspots in third instar eye and wing imaginal discs. lilli clones induced either at 24-36 hours or 36-48 hours after egg deposition (AED) are indistinguishable in size and number of cells from their wild-type twinspots. Of 75 individual clones examined at 96 hours after induction, the average area of lilli mutant clones was 1440 pixels, compared to 1400 pixels for corresponding wild-type twinspots. Thus, lilli mutant cells grew at 1.03 times the rate of wild-type cells, indicating that lilli is not required for normal rates of cell growth (Tang, 2001).

Interestingly, despite an approximately 50% reduction in the size of lilli photoreceptor cells and wing margin bristles in the adult, other cell types in the adult eye and wing are unaffected. For example, the surface of eyes containing lilli mutant clones appears normal by SEM analysis, suggesting that loss of lilli does not reduce the size of cone cells. The size of lilli cells in developing wing and eye imaginal discs appears normal as well. This was confirmed by FACS analysis of dissociated wing discs, which has revealed no significant difference in size between lilli and wild-type cells. In addition, unlike mutations in components of the PI3K pathway, lilli mutant cells display a normal cell cycle profile. To test whether lilli is required for PI3K-mediated growth, cells doubly mutant for lilli and Pten, an inhibitor of this pathway, were examined. Mutations in Pten increase cell size and advance G1/S progression; these effects are prevented by mutations in downstream components such as torso (tor). In contrast, loss of lilli does not prevent the cell enlargement or cell cycle changes caused by Pten mutation, indicating that lilli is not an essential element of the PI3K pathway. Interestingly, it was noticed that ommatidia containing the enlarged lilli;Pten photoreceptor cells are severely disorganized, and contain malformed rhabdomeres characteristic of cytoskeletal defects. Together, these results indicate that lilli affects the cell size through a growth-independent and PI3K-independent mechanism. It is suggested that mutations in lilli may affect final cell size by disrupting the morphological changes that cells such as rhabdomeres and bristles, which are the specializations of photoreceptor and trochogen cells respectively, undergo during pupal development (Tang, 2001).

Mutations have been characterized in the Drosophila Tsc1 and Tsc2/gigas genes. Inactivating mutations in either gene cause an identical phenotype characterized by enhanced growth and increased cell size with no change in ploidy. Overall, mutant cells spend less time in G1. Coexpression of both Tsc1 and Tsc2 restricts tissue growth and reduces cell size and cell proliferation. This phenotype is modulated by manipulations in cyclin levels. In postmitotic mutant cells, levels of Cyclin E and Cyclin A are elevated. This correlates with a tendency for these cells to reenter the cell cycle inappropriately as is observed in the human lesions (Tapon, 2001).

The enhanced growth observed in the Tsc1 or Tsc2 mutants most resembles the results of inactivating PTEN or increasing Ras1 or dmyc activity. In each of these situations, there is a reduction in the length of the G1 phase. In contrast, increased growth driven by Cyclin D/cdk4 does not alter the distribution of cells in different phases of the cell cycle. The effects of the combined overexpression of Tsc1 and Tsc2 displays genetic interactions with multiple pathways. The phenotype is influenced by alterations in the levels of dS6K, PTEN, Ras1, dmyc, cyclin D, and cdk4. Thus, Tsc1 and Tsc2 may function downstream of the point of convergence of these pathways. Alternatively, Tsc1 and Tsc2 may primarily antagonize one of these pathways, but this effect could be overcome by increasing the activity of one of the others (Tapon, 2001).

A mutation has been isolated in the Drosophila homolog of TSC1 (Tsc1). Cells mutant for Tsc1 are dramatically increased in size yet differentiate normally. Organ size is also increased in tissues that contain a majority of mutant cells. Clones of Tsc1 mutant cells in the imaginal discs undergo additional divisions but retain normal ploidy. Flow cytometry analysis indicates that the increase in cell size is not due to endoreplication. Tsc1 protein is shown to bind to Drosophila Tsc2 in vitro. Overexpression of Tsc1 or Tsc2 alone in the wing and eye has no effect, but co-overexpression leads to a decrease in cell size, cell number, and organ size. Genetic epistasis data are consistent with a model that Tsc1 and Tsc2 function together in the insulin signaling pathway (Potter, 2001).

Recent work has demonstrated that the insulin signaling pathway plays an important role in the regulation of cell size, cell number, and organ size. Mutations of Drosophila PTEN (dPTEN), which functions as a negative regulator of insulin signaling, result in phenotypes that resemble the effects of Tsc1 and Tsc2 mutations. Therefore, genetic epistasis experiments were performed to test whether Tsc1 or Tsc2 might also function to negatively regulate insulin signaling. Overexpression of Drosophila insulin receptor (dinr) using the eyGAL4 driver leads to lethality at 25°C and a dramatic increase in ommatidia number in escapers at room temperature. Co-overexpression of Tsc1 and Tsc2 (but not either Tsc1 or Tsc2 alone) rescues both the lethality and the extra ommatidia phenotype caused by dinr overexpression. Furthermore, overexpression of dinr using the pGMR-GAL4 driver leads to an increase in ommatidium size, which is also suppressed by co-overexpression of Tsc1 and Tsc2. Clones of dinr mutant ommatidia are smaller in size than wild-type. Ommatidia that are mutant for both dinr and Tsc1, however, exhibit the Tsc1 mutant phenotype of increased ommatidium size (Potter, 2001).

Overexpression of dPTEN using the pGMR-GAL4 driver leads to eyes with a decreased ommatidium size. However, overexpression of dPTEN is unable to suppress the clonal Tsc1 mutant phenotype. Similar to dinr, clones of dAkt mutant ommatidia are smaller in size. Ommatidia mutant for both dAkt and Tsc1 display the Tsc1 phenotype. Similarly, ommatidia that contained Tsc2 mutant clones in a dAkt mutant background exhibit the Tsc2 mutant phenotype. These results suggest that in the eye, Tsc1 and Tsc2 function genetically epistatic to (downstream of) dinr, dPTEN, and dAkt (Potter, 2001).

Genetic analyses suggest that the TSC genes act in a parallel pathway that converges on the insulin pathway downstream from Akt. The most convincing evidence for a functional link between the TSC genes and insulin signaling comes from the observation that heterozygosity of TSC1 or TSC2 is sufficient to rescue the lethality of loss-of-function InR mutants. This argues that the TSC genes are intimately linked to insulin signaling, rather than functioning in a totally independent cell-growth pathway. These results suggest that the TSC tumor suppressor genes are novel negative regulators of insulin signaling, and modulating the activities of the TSC genes might provide a potential way to correct insulin signaling defects in certain diseases such as diabetes and obesity (Gao, 2001).

To distinguish between these two possibilities, cells were generated that were doubly mutant for null alleles of PTEN and TSC1. PTEN is a negative regulator of the InR-PI3K-Akt pathway, and loss of PTEN results in increased Akt activity and cellular growth. It was reasoned that if TSC1 acts downstream from Akt within the InR-PI3K-Akt pathway, it might be expected that PTEN;TSC1 double-mutant cells would show a similar cell-size phenotype to either single mutant. However, if TSC1 acts parallel to the InR-PI3K-Akt the pathway, it might be expected that PTEN;TSC1 double-mutant cells would show additive effects on cell size as compared with each single mutant. PTEN;TSC1 double-mutant photoreceptors are 2.9 times the size of wild-type cells, as compared with 1.9 for PTEN^- and 1.8 for TSC1^-. This result strongly suggests that the TSC genes function in a parallel pathway that converges on the insulin pathway at a point downstream from Akt (Gao, 2001).

The insulin/insulin-like growth factor-1 signaling pathway promotes growth in invertebrates and vertebrates by increasing the levels of phosphatidylinositol 3,4,5-triphosphate through the activation of p110 phosphatidylinositol 3-kinase. Two key effectors of this pathway are the phosphoinositide-dependent protein kinase 1 (PDK1) and Akt/PKB. Although genetic analysis in C. elegans has implicated Akt as the only relevant PDK1 substrate, cell culture studies have suggested that PDK1 has additional targets. In Drosophila, dPDK1 (FlyBase name: Protein kinase 61C) controls cellular and organism growth by activating Akt1 and S6 kinase, dS6K (FlyBase name: RPS6-p70-protein kinase). Furthermore, dPDK1 genetically interacts with dRSK but not with dPKN (FlyBase name: Protein kinase related to protein kinase N), encoding two substrates of PDK1 in vitro. Thus, the results suggest that dPDK1 is required for dRSK but not dPKN activation and that it regulates insulin-mediated growth through two main effector branches, dAkt and dS6K (Rintelen, 2001).

The pronounced effect of loss of dPDK1 function on head size suggests that it is a dominant constituent in the dInr pathway. To test this possibility, the ability of complete and partial loss-of-function alleles of dPDK1 to reverse phenotypes caused by either overexpression of dInr or by mutations in dPTEN, the 3-phosphatidylinositide phosphatase, was evaluated. Overexpression of a wild-type dInr cDNA under the control of GMR-Gal4 leads to a marked increase in eye size and a slightly rough eye surface, an effect dominantly suppressed by removing one copy of dPDK1. Further reduction of dPDK1 function by the dPDK1^1/4 heteroallelic combination reduces the eye to almost wild-type size, suggesting that the amount of dPDK1 protein is rate-limiting for the dInr overgrowth phenotype. Null mutations in dPTEN cause lethality, and removal of dPTEN function in clones stimulates cell autonomous growth, suggesting that increased levels of PIP₃ promote growth and are the likely cause of lethality. Thus, if dPDK1 is an essential target of PIP₃, mutations in dPDK1 may suppress the dPTEN phenotype. Surprisingly, some dPTEN/dPDK1 double mutant flies survive to adulthood, indicating that the presumed PIP₃-induced lethality is primarily caused by the hyperactivation of dPDK1 or of one of its targets (Rintelen, 2001).

These results show that dPDK1 is an essential component in the insulin signaling pathway in the control of cell growth and body size through its two substrates, dAkt and dS6K. These results are distinct from the genetic evidence in C. elegans where Akt is the primary target of PDK1 in dauer formation. Because mutations in the insulin signaling pathway do not show an autonomous alteration of cell size in C. elegans, the regulation of the rate of protein synthesis through S6K does not seem to be a primary target of this pathway. However, the fact that dPDK1 may yet have additional substrates is suggested by the genetic interaction with dRSK gain-of-function mutations and because viable dPDK1 males are almost completely sterile. Although mutations in components of the insulin signaling pathway such as dInr, chico, Dp110/PI(3)K, and dAkt cause female sterility, male sterility is not observed. Further genetic dissection of dPDK1 function is required to determine the role of dPDK1 in male fertility. These findings in Drosophila are consistent with the absence of insulin growth factor-1-induced activation of S6K, Akt, and RSK in mammalian PDK1^-/- embryonic stem cells, and therefore provide evidence for the functional conservation of branch points in kinase networks during evolution (Rintelen, 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 test whether increasing PtdInsP₃ levels in an InR or PI 3-kinase p110 mutant background is sufficient to restore growth, the function of a negative regulator of the insulin pathway was eliminated. The 3'-phosphoinositol-specific lipid phosphatase, PTEN acts as a negative regulator of the PI 3-kinase pathway by converting PtdInsP₃ generated by PI 3-kinase into PtdInsP₂. Used were a null (Pten^2L117) and a hypomorphic (Pten^2L100) allele of Pten, identified in a screen for genes involved in growth control. As shown by HPLC analysis of the phospholipids in extracts of Pten mutant larvae, the loss of PTEN function results in a 2-fold increase in PtdInsP₃ levels. This is consistent with the increase in PtdInsP₃ seen in Pten-deleted murine fibroblasts. One prominent biological effect of these increased PtdInsP₃ levels in Drosophila is a substantial increase in size in both larvae and pupae. To test whether loss of PTEN function, and consequently increased PtdInsP₃ levels, is sufficient to restore growth or viability in InR null mutants, InR and Pten double mutants were generated by creating mosaic animals using the eyeless-Flipase (eyFlp) tissue-specific recombination system. In such animals, the head consists of homozygous mutant tissue, whereas the rest of the body is heterozygous for the same mutation. While loss of PTEN function (Pten^2L117) in the head results in a fly with a disproportionately larger head (with more and larger cells), loss of InR function (InR³²⁷) results in flies with smaller heads (pinhead) compared to the wild type. Heads doubly mutant for Pten^2L117and InR³²⁷, however, are almost the size of heads singly mutant for Pten^2L117. Also, two different lethal heteroallelic InR combinations (InR³⁰⁴/InR³²⁷ or InR³⁰⁴/InR²⁵), which arrest at the second larval instar stage, develop to the pupal stage (15%-17% of 33% expected) and even to pharate adults in the presence of reduced PTEN levels (Pten^2L117/Pten^2L100). These results demonstrate that complete loss of PTEN function can largely substitute for InR-mediated growth and proliferation in the absence of InR function and that the Ras/MAPK pathway plays little or no role in the InR mediated control of cell growth. This notion is further supported by the observation that complete loss of InR function in the compound eye does not result in a loss of photoreceptors, a hallmark of loss of Ras pathway function (Oldham, 2002).

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

Because loss of Chico function results in increased lipid levels as well as a dramatic decrease in body size, attempts were made to determine whether PTEN might also have a function in metabolic and body size control like Chico. Partial loss of PTEN function results in flies that are considerably bigger than controls. They weigh approximately 50 percent more than their heterozygous siblings without showing any apparent effect on cell differentiation. The increase in size is due to an increase in both cell size and number as determined by a morphometric analysis of the wing and eye. When the levels of lipids and glycogen were measured, a decrease per mass in lipid and glycogen was observed compared to Pten mutant flies rescued by a genomic Pten transgene. One biological outcome of this difference is a twofold increase in the rate of mortality under water-only starvation conditions compared to a 2-fold decrease in chico mutant flies. Thus lipid and glycogen levels strongly correlate with the survival time under starvation conditions. Since Chico and PTEN activity regulates growth during development and the accumulation of energy stores in the adult, the effects of InR-mediated growth and lipid/glycogen metabolism must diverge downstream of Pten (Oldham, 2002).

Thus, growth deficency associated with the loss of InR function is fully compensated by loss of PTEN function. This suggests that the levels of PtdInsP₃ in the cell control the amount of cellular growth. Also, partial loss of PTEN function increases body size and decreases lipid and glycogen stores in the adult, suggesting that the levels of PtdInsP₃ also control metabolism in the adult (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).

Loss of PTEN function results in a metabolically similar phenotype as loss of murine PTP1B (Ptpn1), an IR-specific tyrosine phosphatase, in that hyperactivation of the IR pathway causes resistance to high-fat-diet-induced obesity because of increased basal metabolism. These metabolic lipid effects have likely been conserved during evolution because the increased lipid levels in chico mutants are reminiscent of the enhanced lipid content in Irs2 deleted and NIRKO mice (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).

Insulin-IGF receptor (InR) signaling has a conserved role in regulating lifespan, but little is known about the genetic control of declining organ function. This study describes progressive changes of heart function in aging fruit flies: from one to seven weeks of a fly's age, the resting heart rate decreases and the rate of stress-induced heart failure increases. These age-related changes are minimized or absent in long-lived flies when systemic levels of insulin-like peptides are reduced and by mutations of the only receptor, InR, or its substrate, Chico. Moreover, interfering with InR signaling exclusively in the heart, by overexpressing the phosphatase PTEN or the forkhead transcription factor FOXO, prevents the decline in cardiac performance with age. Thus, insulin-IGF signaling influences age-dependent organ physiology and senescence directly and autonomously, in addition to its systemic effect on lifespan. The aging fly heart is a model for studying the genetics of age-sensitive organ-specific pathology (Wessells, 2004).

Pten negatively regulates PKB

Tuberous sclerosis complex (TSC) is a genetic disorder caused by mutations in one of two tumor suppressor genes, TSC1 and TSC2. Absence of Drosophila Tsc1 and/or Tsc2 (Gigas) leads to constitutive S6k activation and inhibition of PKB, the latter effect being relieved by loss of S6K. In contrast, the Pten tumor suppressor, a negative effector of PI3K, has little effect on S6k, but negatively regulates PKB (Akt1). More importantly, reducing S6k signaling rescues early larval lethality associated with loss of Tsc1/2 function, arguing that the S6k pathway is a promising target for the treatment of TSC (Radimerski, 2002b).

To determine whether loss of Tsc1/2 or Pten directly affected S6k activity, each was depleted in Drosophila Kc167 cells by dsRNAi. Quantitative Real Time PCR showed that such treatment strongly reduced levels of both transcripts. Compared with control cells, depletion of Tsc1 increases S6k activity and T398 phosphorylation, consistent with the reduced electrophoretic mobility of S6k. These results are in agreement with recent findings in TSC1 null mammalian cells (Kwiatkowski, 2002). Insulin treatment of either control cells or Tsc1-depleted cells did not significantly increase these responses beyond that of Tsc1 depletion alone, indicating that loss of Tsc function leads to full S6k activation. RAD001, a rapamycin derivative, blocks S6k activity in both control and Tsc1-depleted cells treated with insulin. However, it was consistently noted that the RAD001 block of insulin-induced S6k activation is not as strong in Tsc1-depleted cells, suggesting that not all the effects of Tsc on S6k are dependent on Tor, the Drosophila target of rapamycin. Similar results to those described here were obtained by Tsc2 depletion. In addition, the effects appear specific, since Tsc1 depletion has no effect on the basal activity of other AGC-kinase family members, such as PKB or Drosophila atypical PKC. However, insulin-induced PKB activation and S505 phosphorylation are repressed in Tsc1-depleted cells as compared with control cells, consistent with S6k acting in a negative feedback loop to dampen PKB signaling. In contrast to loss of Tsc1, depletion of Pten has little effect on S6k activity and T398 phosphorylation, whereas it leads to elevated levels of both basal and insulin-stimulated PKB activity and S505 phosphorylation. Thus, loss of Tsc1/2, but not Pten, leads to constitutive S6k activation (Radimerski, 2002b).

To determine whether the findings above could be corroborated in the animal, S6k activity was measured in extracts of Tsc1, Pten, and S6k null larvae. The results show that S6k activity in extracts derived from Tsc1 null larvae is strongly increased over that of wild-type larvae, whereas it is slightly increased in larvae lacking Pten. The opposite was found for PKB activity, which is strongly repressed in Tsc1 null larvae, and up-regulated in Pten-deficient larvae. Hence, it cannot be excluded that reduced PKB activity contributes to larval lethality of Tsc mutants. Given that loss of Tsc function leads to increased S6k activity, it was reasoned that ectopic expression of Tsc1/2, but not Pten, would inhibit S6k activity. To test this hypothesis, both tumor suppressors were expressed ubiquitously in larvae using the GAL4/UAS system, such that the GAL4 promoter chosen in each case led to developmental arrest at late larval second instar. Extracts from larvae overexpressing Tsc1/2 display strongly reduce S6k activity, whereas those from Pten overexpressing larvae have normal levels of S6k activity. In contrast, PKB activity is strongly suppressed in Pten overexpressing larvae and little affected in extracts from larvae overexpressing Tsc1/2. These data corroborate previous findings that S6k and PKB act in parallel signal transduction pathways (Radimerski, 2002a), and provide compelling evidence that they are negatively controlled by distinct tumor suppressor genes (Radimerski, 2002b).

Despite the fact that S6k and PKB act in parallel signaling pathways, loss of Tsc1/2 function leads to inhibition of PKB activity, suggesting cross-talk between the two pathways. Compatible with such a model, recent studies have shown that rapamycin treatment of adipocytes inhibits a negative feedback loop, which normally functions to dampen insulin-induced PKB activation. Since RAD001 inhibits S6k activity (Radimerski, 2002a) and increases PKB activity (Radimerski, 2002a), it raised the possibility that the effects of Tsc mutants on PKB are mediated through S6k. Consistent with this hypothesis, inhibition of PKB activity due to loss of Tsc function was relieved in the absence of S6k. Similar results were obtained by using dsRNAi in cell culture. Thus, the suppression of PKB by loss of Tsc function requires S6k (Radimerski, 2002b).

To genetically test the specificity of Tsc1/2 and Pten tumor suppressor function, either Tsc1 or Pten were removed in cells giving rise to the adult eye structure, by inducing mitotic recombination with the FLP/FRT system under the control of the eyeless promoter. In a wild-type genetic background, loss of either Tsc1 or Pten within the developing eye causes strong overgrowth of the head. Eye overgrowth by removal of Tsc1 is strongly suppressed in a genetic background null for S6k, as is ommatidia size, in agreement with a previous report analyzing double mutant clones of Tsc2 and S6k in the eye (Potter, 2001). In contrast, removal of Pten in the eyes of S6k null flies still induces overgrowth of clones with enlarged ommatidia. These findings are supported by results showing that eye overgrowth by removal of Tsc1 is still observed in clones devoid of PKB function (Potter, 2001) and overgrowth by removal of Pten is suppressed in a viable PKB mutant genetic background (Stocker, 2002). Thus, Tsc1/2 appears to be specific for the S6k-signaling pathway, whereas Pten antagonizes PI3K signaling to counteract PKB activation by decreasing PIP3 levels (Radimerski, 2002b).

Taken together, these results demonstrate that the tumor suppressor Tsc1/2 is a critical component in controlling S6k activation. Interestingly, this effect may be Tor independent, as insulin-induced S6k activation is more elevated in Tsc1/2-depleted cells pretreated with RAD001 than in control cells, and in preliminary studies, clonal overgrowth in the eye induced by loss of Tsc1 is not suppressed in a semiviable, heterorallelic Tor mutant background. Overexpression of Tsc1/2 selectively suppresses the S6k-signaling pathway, whereas Pten operates on the dPI3K-signaling pathway. Double mutations for Pten and Tsc1 are additive for clonal overgrowth, compatible with S6k and PKB independently mediating growth. Nevertheless, inhibition of PKB by loss of Tsc function shows that there is negative cross-talk between the two signaling pathways. Given this negative cross-talk, the observation that in double mutant clones growth is additive, suggests that in the absence of Pten, inhibition of PKB by loss of Tsc is circumvented. However, despite the observation that double mutations for Pten and Tsc1 are additive for clonal overgrowth, overgrowth induced by absence of Pten is suppressed in clones mutant for Tor. Since S6k does not prevent such overgrowth, it is possible that this suppression actually represents an intermediate phenotype, or that Pten negatively acts on a Tor target distinct from S6k. At this point, it is important to gain a deeper knowledge of the molecular mechanisms by which Tsc1/2 acts to suppress S6k function and how the signaling components of these two pathways cross-talk with one another (Radimerski, 2002b).

Recently, a successful Phase I clinical trial was completed for a rapamycin analog in the treatment of solid tumors. The results of the trial demonstrated that the drug was efficacious at subtoxic doses, and suggested that specific tumor types may be more sensitive to inhibition by rapamycin than others. The question that arose from the trial is, which tumors would be susceptible to rapamycin treatment? Here, it has been demonstrated for the first time in vivo that a mild reduction in S6k signaling, which alone has no blatant phenotype, is sufficient to restore viability of flies devoid of Tsc function. Thus, these findings imply that rapamycin or its derivatives might be very promising pharmaceutical agents in the treatment of tumors arising from TSC (Radimerski, 2002b).

d4E-BP is a direct downstream target of the dInR-dPI3K-dPTEN-dAkt-dTSC-dTOR signaling cascade

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/Thor) 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).

insulin-PI3K/TOR pathway induces a HIF-dependent transcriptional response in Drosophila by promoting nuclear localization of HIF-alpha/Sima

The hypoxia-inducible factor (HIF) is a heterodimeric transcription factor composed of a constitutively expressed HIF-ß subunit and an oxygen-regulated HIF-alpha subunit. A hypoxia-inducible transcriptional response has been defined in Drosophila that is homologous to the mammalian HIF-dependent response. In Drosophila, the bHLH-PAS proteins Similar (Sima) and Tango (Tgo) are the functional homologues of the mammalian HIF-alpha and HIF-ß subunits, respectively. HIF-alpha/Sima is regulated by oxygen at several different levels that include protein stability and subcellular localization. Insulin can activate HIF-dependent transcription, both in Drosophila S2 cells and in living Drosophila embryos. Using a pharmacological approach as well as RNA interference, it has been determined that the effect of insulin on HIF-dependent transcriptional induction is mediated by PI3K-AKT and TOR pathways. Stimulation of the transcriptional response involves upregulation of Sima protein but not sima mRNA. Finally, the effect of the activation of the PI3K-AKT pathway on the subcellular localization of Sima protein was analyzed in vivo. Overexpression of dAKT and dPDK1 in normoxic embryos provokes a major increase in Sima nuclear localization, mimicking the effect of a hypoxic treatment. A similar increase in Sima nuclear localization was observed in dPTEN homozygous mutant embryos, confirming that activation of the PI3K-AKT pathway promotes nuclear accumulation of Sima protein. It is concluded that regulation of HIF-alpha/Sima by the PI3K-AKT-TOR pathway is a major conserved mode of regulation of the HIF-dependent transcriptional response in Drosophila (Dekanty, 2005).

Insulin stimulation or exposure to hypoxia can induce common target genes; such transcriptional response depends on the Drosophila HIFalpha and HIFß homologues Sima and Tango. Evidence is provided that insulin-stimulated HRE response is transduced by the PI3K-AKT pathway and, furthermore, that the effect depends on TOR and involves an increase in Sima protein levels, whereas mRNA levels are not affected. These results are in good agreement with the reported effect of the PI3K-AKT/TOR pathway on mammalian HIF, because in several cell lines activation of this pathway led to an increase in HIF protein levels or stabilization of the protein (Dekanty, 2005).

In addition, the subcellular localization of Sima depends on oxygen tension in a dose-dependent manner, and activation of the PI3K-AKT pathway also causes a major increase in Sima nuclear localization. This regulatory mechanism might represent another conserved aspect of HIF regulation, because one recent report suggests that HIF-alpha accumulates in the nucleus of retinal epithelial cells upon IGF-1alpha treatment. The molecular bases of HIFalpha nuclear accumulation upon hypoxia or PI3K activation are so far unclear. Nucleo-cytoplasmic localization of many transcription factors results from a steady-state equilibrium between nuclear import and nuclear export, and accumulation in one or the other compartment depends on the relative rate of import versus export. Whether HIF nuclear accumulation upon hypoxia or growth factor stimulation depends on regulated nuclear import or regulated nuclear export remains to be determined (Dekanty, 2005).

All major cellular features of oxygen-dependent regulation of HIF proteins are conserved in Drosophila and, thus, activation of the HRE response in Drosophila by the PI3K-AKT and TOR pathways extends even further the notion of a conserved HIF system in evolution. The functional significance of the regulation exerted by PI3K-AKT and TOR pathways over the HRE response was discussed in the context of cancer biology, because the loss of PTEN or the tuberous sclerosis complex 1 (TSC1) and TSC2 proteins is frequently associated with human tumors. What is the functional meaning of PI3K-AKT pathway regulation of the HRE response in normal cells? The PI3K-AKT and TOR pathways are regulated in part by growth factors and other endocrine signals and thus, endocrine control of the HRE response is conserved among animal species that have diverged 700 million years ago. It seems reasonable to postulate that the main physiological role of HRE induction by the PI3K-AKT pathway is the stimulation of glycolysis, but a function in the regulation of animal body size and growth control is another interesting possibility (Dekanty, 2005).

A cardinal function of the PI3K-AKT and TOR pathways throughout evolution is to regulate growth, and to determine the final size of developing organs and whole organisms. Genetic studies in Drosophila have shown that a reduction of the activity of the PI3K-AKT pathway results in flies with a reduced body size, bearing smaller cells. Likewise, a reduction in TOR signaling provokes growth decrease and, conversely, over-activation of TOR signaling due to loss-of-function of its negative regulators TSC1 and/or TSC2, leads to an increase in cell and body size. The effect of TOR on cell growth was reported to be mediated at least in part by S6K, a kinase that phosphorylates the ribosomal protein S6, leading to translational activation (Dekanty, 2005).

Besides its role in growth control, the insulin-PI3K-AKT pathway has been traditionally implicated in the regulation of circulating glucose levels and anabolic metabolism. It has been demonstrated that the cellular bases of glucose sensing and regulation of serum glucose are conserved between mammals and Drosophila, and it has been proposed that PI3K-AKT signaling in conjunction with the TOR pathway coordinates growth according to environmental conditions and the nutritional status of the organism. The mechanism involved in this coordination is still unclear. Oxygen tension is one environmental factor that has been shown to modulate growth in Drosophila, because hypoxic flies have a reduced body size (Frazier, 2001). A mechanistic explanation to this phenomenon has been provided by showing that overexpression of Sima protein causes a reduction in cell size in an autonomous manner (Centanin, 2005). Consistent with this, it has been shown that hypoxia provokes a reduction in Drosophila TOR pathway activity and that such reduction results from hyperactivation of the TSC1-TSC2 tumor suppressor complex. Similar results have been reported in mammalian cells, implying that TOR is regulated by hypoxia. Furthermore, hypoxia-dependent TSC1-TSC2 stimulation and growth inhibition are mediated by the product of a HIF/Sima-inducible gene called scylla in Drosophila and RTP801/REDD1 in mammals (Dekanty, 2005).

The results establish a direct link between pathways largely implicated in growth regulation (PI3K-AKT and TOR) and the hypoxia-responsive machinery (HIF/Sima). It is suggested that in hypoxia, HIF prolyl hydroxylase (Hph)/Fatiga activity is reduced, resulting in HIF/Sima stabilization and induction of an HRE response. One of the genes induced by hypoxia is scylla/RTP801/REDD1, which in turn activates TSC1-TSC2. Then, stimulation of the TSC complex provokes reduction of TOR activity and decreases S6K phosphorylation, resulting in growth inhibition. According to this model, PI3K-TOR activation of HIF-alpha/Sima might generate a negative feedback loop to limit or downregulate growth; in this scenario, low oxygen levels are expected to enhance Sima-dependent inhibition of growth (Dekanty, 2005).

It has been reported that mitochondrial dysfunction inhibits Hph/Fatiga activity, thereby triggering the transcriptional response to hypoxia, and also that it concomitantly provokes growth defects. It was proposed that Hph/Fatiga operates as an integration node between oxygen levels and growth regulation. The current results have shown that the effect of Hph/Fatiga on growth regulation is conveyed at least in part by Sima. Further studies will reveal whether Hph/Fatiga also plays Sima-independent roles in cell and organ size determination (Dekanty, 2005).

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