Ribosomal protein S6 kinase


The S6k gene gives rise to two major transcripts that are expressed throughout development, suggesting a continuous requirement for this enzyme. Two major transcripts of 3.0 and 5.0 kb are found at abundant steady-state levels in whole animal poly(A)+ or total RNA. Two smaller transcripts (<3.0 kb) are expressed at lower levels at all tested developmental stages. All of these transcripts hybridize with probes that include the catalytic domain, C-terminal domain and the 3' UTR. Only the two major transcripts are detected with 5'-UTR probes common to class 1 and class 2 S6k cDNAs that also share a complete ORF. These 5'-UTR probes did not detect the smaller transcripts, suggesting that they have different N-terminal sequences. The smaller S6k transcripts are the predominant forms found in cultured air8 blood cells, which are derived from RpS6air8 mutant larvae suggesting that the S6k gene gives rise to tissue-specific expression patterns. An alternative explanation is that the S6k transcripts expressed in air8 cultured cells are influenced by the RpS6air8 mutation (Watson, 1996).

TSC1/2 tumour suppressor complex maintains Drosophila germline stem cells by preventing differentiation

Tuberous sclerosis complex human disease gene products TSC1 and TSC2 form a functional complex that negatively regulates target of rapamycin (TOR), an evolutionarily conserved kinase that plays a central role in cell growth and metabolism. This study describes a novel role of TSC1/2 in controlling stem cell maintenance. In the Drosophila ovary, disruption of either the Tsc1 or Tsc2 gene in germline stem cells (GSCs) leads to precocious GSC differentiation and loss. The GSC loss can be rescued by treatment with TORC1 inhibitor rapamycin, or by eliminating S6K, a TORC1 downstream effecter, suggesting that precocious differentiation of Tsc1/2 mutant GSC is due to hyperactivation of TORC1. One well-studied mechanism for GSC maintenance is that BMP signals from the niche directly repress the expression of a differentiation-promoting gene bag of marbles (bam) in GSCs. In Tsc1/2 mutant GSCs, BMP signalling activity is downregulated, but bam expression is still repressed. Moreover, Tsc1 bam double mutant GSCs could differentiate into early cystocytes, suggesting that TSC1/2 controls GSC differentiation via both BMP-Bam-dependent and -independent pathways. Taken together, these results suggest that TSC prevents precocious GSC differentiation by inhibiting TORC1 activity and subsequently differentiation-promoting programs. As TSC1/2-TORC1 signalling is highly conserved from Drosophila to mammals, it could have a similar role in controlling stem cell behaviour in mammals, including humans (Sun 2010).

TSC1/2 is known to regulate cell growth via inhibition on TORC1. This study demonstrates that it also functions by inhibiting the activity of TORC1 to maintain GSCs. Treatment with rapamycin, a TORC1-specific inhibitor, can completely rescue GSC loss in Tsc1 mutants. In addition, eliminating S6K, which functions downstream of TORC1 in regulating protein translation, could also completely rescue GSC loss in Tsc2 mutants. Interestingly, the daughters of Tor mutant GSCs can differentiate into germline cyst properly, indicating that TOR is normally not required for differentiation, but its hyperactivation in Tsc1/2 mutants drives precocious GSC differentiation. The simplest explanation of the delayed cystoblast differentiation in rapamycin-treated females might be a non-specific effect of drug treatment. However, it is also possible that TORC1 inhibition by rapamycin might cause repression of some, but not all, aspects of TOR function, which leads to uncoordinated development and/or differentiation of cystoblasts in response to GSC division. Consistently, accumulated cystoblasts where also observed when overexpressing both Tsc1 and Tsc2 in the germline. Together with the observation that TSC1/2-TORC1 signaling controls cell growth of germline cysts, this study suggests that TSC1/2-TORC1 may serve as a signaling integration point that orchestrates germline division, differentiation and development in order to control egg production in response to the local micro-environment and the system environment of the animals (Sun 2010).

In the Drosophila ovary, BMP signaling from the niche directly suppresses bam expression in GSCs to prevent differentiation. This signaling is crucial for GSC maintenance. As revealed by pMad expression, BMP signaling activity is significantly downregulated in Tsc1 mutant GSCs. This study also demonstrated that downregulation of pMad in Tsc1 mutant GSCs is mediated by TORC1 hyperactivation, as rapamycin treatment is able to restore the downregulated pMad level. However, TOR is not required for proper BMP signaling activity because pMad expression is not altered in rapamycin-treated germaria. Therefore, only TORC1 hyperactivation could inhibit BMP signaling in GSCs through unknown mechanisms, and this inhibitory effect occurs specifically in GSCs, as BMP signaling activity is not altered in Tsc1 mutant imaginal disc cells (Sun 2010).

Logically, bam expression could be derepressed in Tsc1 mutant GSCs as a consequence of BMP pathway downregulation. Surprisingly, no significant upregulation of bam-GFP expression could be detected in mutant GSCs, although in other GSCs that were compromised by BMP signaling, such as tkv mutant and mad mutant GSCs, bam transcription is significantly upregulated. Nevertheless, there might still be residual BMP signaling activities in Tsc1/2 mutant GSCs that are sufficient to suppress bam expression. Consistent with this notion is the observation that bam-GFP is not obviously upregulated in aged GSCs, even if BMP signaling activity has been significantly reduced. Together with the observation that bam mutation could not rescue the differentiation of Tsc1 mutant germ cells, it is suggested that the compromised BMP signaling activity may not be primarily responsible for Tsc1/2 mutant GSC loss. It is not clear why the effect of TSC1/2 on BMP signaling occurs specifically in GSCs. Possibly, Tsc1/2 mutant GSCs, once induced, have already primed for differentiation through a Bam-independent mechanism, which may trigger a positive feedback signal to inhibit BMP signaling activity, in order to facilitate differentiation (Sun 2010).

This study also reveals a BMP-Bam-independent mechanism that probably underlies the major role of TSC1/2-TORC1 signaling in GSC maintenance. The phenotype of Tsc1 bam double mutant germ cells differs from the bam alone mutant germ cells, as the double mutant GSCs can still become lost from the niche over time and undergo further differentiation into early cystocytes. Interestingly, the phenotype of Tsc1/2 mutant GSCs is similar to that of pelota (pelo) mutants. Pelo encodes a translational release factor-like protein and may regulate GSC maintenance at the translational level. In pelo mutant GSCs, there is also a downregulation of BMP signaling but no obvious upregulation of bam expression, and bam pelo double mutant germ cells are able to undergo similar limited differentiation into cystocytes, suggesting that TSC1/2 and Pelo might function in the same or parallel pathway to control GSC differentiation. It is proposed that similar to Pelo, TSC1/2 might function in a parallel pathway with the BMP-Bam pathway to control GSC differentiation, possibly by regulating the translation of differentiation-related mRNAs (Sun 2010).

Pum and Nos, which are known to function together to repress translation of the target mRNAs in embryos, are also essential for GSC maintenance. Recent genetic and biochemical studies suggest that Bam/Bgcn may directly inhibit the function of Pum/Nos to allow cystoblast differentiation. However, BMP signaling activation is able to prevent differentiation of nos mutant primordial germ cells, indicating that Pum/Nos could also function in parallel with the BMP-Bam pathway to control germ cell differentiation. In the future, it would be important to determine the functional relationships between the TSC1/2-TORC1 pathway, Pelo and Pum/Nos in regulating GSCs, and whether these factors, together with the microRNA pathway, target similar mRNAs to control GSC differentiation (Sun 2010).

This study has identified a novel role of TSC1/2 in controlling GSC maintenance and differentiation in the Drosophila ovary. Increasing evidence also suggests similar roles for TSC1/2-TOR signaling in regulating adult stem cell differentiation in mammals. For example, TSC1/2-mTOR signaling is also required for maintaining the quiescence of haematopoietic stem cells (HSCs), as Tsc1 deletion drives HSCs from quiescence to rapid cycling, which compromises HSC self-renewal. Thus, TSC1/2-TOR signaling could have an evolutionarily conserved role in regulating stem cell maintenance and differentiation from Drosophila to mammals (Sun 2010).

Anaplastic lymphoma kinase spares organ growth during nutrient restriction in Drosophila

Developing animals survive periods of starvation by protecting the growth of critical organs at the expense of other tissues. This study used Drosophila to explore the as yet unknown mechanisms regulating this privileged tissue growth. As in mammals, it was observed in Drosophila that the CNS is more highly spared than other tissues during nutrient restriction (NR). Anaplastic lymphoma kinase (Alk) efficiently protects neural progenitor (neuroblast) growth against reductions in amino acids and insulin-like peptides during NR via two mechanisms. First, Alk suppresses the growth requirement for amino acid sensing via Slimfast/Rheb/TOR complex 1. And second, Alk, rather than insulin-like receptor, primarily activates PI3-kinase. Alk maintains PI3-kinase signaling during NR as its ligand, Jelly belly (Jeb), is constitutively expressed from a glial cell niche surrounding neuroblasts. Together, these findings identify a brain-sparing mechanism that shares some regulatory features with the starvation-resistant growth programs of mammalian tumors (Cheng, L. Y. 2011).

This study found that CNS progenitors are able to continue growing at their normal rate under fasting conditions severe enough to shut down all net body growth. Jeb/Alk signaling was identified as a central regulator of this brain sparing, promoting tissue-specific modifications in TOR/PI3K signaling that protect growth against reduced amino acid and Ilp concentrations. These findings highlight that a 'one size fits all' wiring diagram of the TOR/PI3K network should not be extrapolated between different cell types without experimental evidence. The two molecular mechanisms by which Jeb/Alk signaling confers brain sparing is discussed, and how they may be integrated into an overall model for starvation-resistant CNS growth (Cheng, L. Y. 2011).

One mechanism by which Alk spares the CNS is by suppressing the growth requirement for amino acid sensing via Slif, Rheb, and TORC1 components in neuroblast lineages. An important finding of this study is that in the presence of Alk signaling Tor has no detectable growth input (evidence from Tor clones), but in its absence (evidence from UAS-AlkDN; Tor clones) Tor reverts to its typical role as a positive regulator of both growth and proliferation. The growth requirement for Slif/TORC1 is clearly much less in the CNS than in other tissues such as the wing disc but a low-level input cannot be ruled out due to possible perdurance inherent in any clonal analysis. Although Slif, Rheb, Tor, and Raptor mutant neuroblast clones attain normal volume, this reflects increased cell numbers offset by reduced average cell size. Atypical suppression of proliferation by TORC1 has also been observed in wing discs, where partial inhibition with rapamycin advances G2/M progression (Cheng, L. Y. 2011).

Alk signaling in neuroblast lineages does not override the growth requirements for all TOR pathway components. The downstream effectors S6k and 4E-BP retain functions as positive and negative growth regulators, respectively. 4E-BP appears to be particularly critical in the CNS as mutant animals have normal mass, but mutant neuroblast clones are twice their normal volume. In many tissues, 4E-BP is phosphorylated by nutrient-dependent TORC1 activity. In CNS progenitors, however, 4E-BP phosphorylation is regulated in an NR-resistant manner by Alk, not by TORC1. Hence, although the pathway linking Alk to 4E-BP is not yet clear, it makes an important contribution toward protecting CNS growth during fasting (Cheng, L. Y. 2011).

A second mechanism by which Alk spares CNS growth is by maintaining PI3K signaling during NR. Alk suppresses or overrides the genetic requirement for InR in PI3K signaling, which may or may not involve the direct binding of intracellular domains as reported for human ALK and IGF-IR (Shi, 2009). Either way, in the CNS, glial Jeb expression stimulates Alk-dependent PI3K signaling and thus neural growth at similar levels during feeding and NR. In contrast, in tissues such as the salivary gland, where PI3K signaling is primarily dependent upon InR, falling insulin-like peptides concentrations during NR strongly reduce growth (Cheng, L. Y. 2011).

The finding that Alk signals via PI3K during CNS growth differs from the Ras/MAPK transduction pathway described in Drosophila visceral muscle. However, a link between ALK and PI3K/Akt/Foxo signaling during growth is well documented in humans, both in glioblastomas and in non-Hodgkin lymphoma. Similarities with mammals are less obvious with regard to Alk ligands, as there is no clear Jeb ortholog and human ALK can be activated, directly or indirectly, by the secreted factors Pleiotrophin and Midkine (Cheng, L. Y. 2011).

A comparison of these results with those of previous studies indicates that CNS super sparing only becomes fully active at late larval stages. Earlier in larval life, dietary amino acids are essential for neuroblasts to re-enter the cell cycle after a period of quiescence. This nutrient-dependent reactivation involves a relay whereby Slif-dependent amino acid sensing in the fat body stimulates Ilp production from a glial cell niche (Sousa-Nunes, 2011). In turn, glial-derived Ilps activate InR and PI3K/TOR signaling in neuroblasts thus stimulating cell cycle re-entry. Hence, the relative importance of Ilps versus Jeb from the glial cell niche may change in line with the developmental transition of neuroblast growth from high to low nutrient sensitivity (Cheng, L. Y. 2011).

The results of this study suggest a working model for super sparing in the late-larval CNS. Central to the model is that Jeb/Alk signaling suppresses Slif/ RagA/Rheb/TORC1, InR, and 4E-BP functions and maintains S6k and PI3K activation, thus freeing CNS growth from the high dependence upon amino acid sensing and Ilps that exists in other organs. The CNS also contrasts with other spared diploid tissues such as the wing disc, in which PI3K-dependent growth requires a positive Tor input but is kept in check by negative feedback from TORC1 and S6K. Alk is both necessary (in the CNS) and sufficient (in the salivary gland) to promote organ growth during fasting. However, both Alk manipulations produce organ-sparing percentages intermediate between the 2% salivary gland and the 96% neuroblast values, arguing that other processes may also contribute. For example, some Drosophila tissues synthesize local sources of Ilps that could be more NR resistant than the systemic supply from the IPCs. In mammals, this type of mechanism may contribute to brain sparing as it has been observed that IGF-I messenger RNA (mRNA) levels in the postnatal CNS are highly buffered against NR. It will also be worthwhile exploring whether mammalian neural growth and brain sparing involve Alk and/or atypical TOR signaling. In this regard, it is intriguing that several studies show that activating mutations within the kinase domain of human ALK are associated with childhood neuroblastomas. In addition, fetal growth of the mouse brain was recently reported to be resistant to loss of function of TORC1. Finally, a comparison between the current findings and those of a cancer study, highlights that insulin/IGF independence and constitutive PI3K activity are features of NR-resistant growth in contexts as diverse as insect CNS development and human tumorigenesis (Cheng, L. Y. 2011).

Regulation of cuticle pigmentation in Drosophila by the nutrient sensing insulin and TOR signaling pathways

Insect pigmentation is a phenotypically plastic trait that plays a role in thermoregulation, desiccation tolerance, mimicry, and sexual selection. The extent and pattern of pigmentation of the abdomen and thorax in Drosophila melanogaster is affected by environmental factors such a growth temperature and access to the substrates necessary for melanin biosynthesis. This study aimed to determine the effect of nutritional status during development on adult pigmentation and test whether nutrient sensing through the Insulin/IGF and target of rapamycin (TOR) pathways regulates the melanization of adult cuticle in Drosophila. Flies reared on low quality food were shown to exhibit decreased pigmentation, which can be phenocopied by inhibiting expression of the Insulin receptor (InR) throughout the entire fly during mid to late pupation. The loss of Insulin signaling through PI3K/Akt and FOXO in the epidermis underlying the developing adult cuticle causes a similar decrease in adult pigmentation, suggesting that Insulin signaling acts in a cell autonomous manner to regulate cuticle melanization. In addition, TOR signaling increases pigmentation in a cell autonomous manner, most likely through increased S6K activity. These results suggest that nutrient sensing through the Insulin/IGF and TOR pathways couples cuticle pigmentation of both male and female Drosophila with their nutritional status during metamorphosis (Shakhamtsir, 2013).

An investigation of nutrient-dependent mRNA translation in Drosophila larvae

The larval period of the Drosophila life cycle is characterized by immense growth. In nutrient rich conditions, larvae increase in mass approximately two hundred-fold in five days. However, upon nutrient deprivation, growth is arrested. The prevailing view is that dietary amino acids drive this larval growth by activating the conserved insulin/PI3 kinase and Target of rapamycin (TOR) pathways and promoting anabolic metabolism. One key anabolic process is protein synthesis. However, few studies have attempted to measure mRNA translation during larval development or examine the signaling requirements for nutrient-dependent regulation. This work addresses this issue. Using polysome analyses, it was observed that starvation rapidly (within thirty minutes) decreased larval mRNA translation, with a maximal decrease at 6-18 hours. By analyzing individual genes, it was observed that nutrient-deprivation led to a general reduction in mRNA translation, regardless of any starvation-mediated changes (increase or decrease) in total transcript levels. Although sugars and amino acids are key regulators of translation in animal cells and are the major macronutrients in the larval diet, this study found that they alone were not sufficient to maintain mRNA translation in larvae. The insulin/PI3 kinase and TOR pathways are widely proposed as the main link between nutrients and mRNA translation in animal cells. However, this study found that genetic activation of PI3K and TOR signaling, or regulation of two effectors - 4EBP and S6K - could not prevent the starvation-mediated translation inhibition. Similarly, it was shown that the nutrient stress-activated eIF2α kinases, GCN2 and PERK, were not required for starvation-induced inhibition of translation in larvae. These findings indicate that nutrient control of mRNA translation in larvae is more complex than simply amino acid activation of insulin and TOR signaling (Nagarajan, 2014: PubMed).

Diacylglycerol lipase regulates lifespan and oxidative stress response by inversely modulating TOR signaling in Drosophila and C. elegans

Target of rapamycin (TOR) signaling is a nutrient-sensing pathway controlling metabolism and lifespan. Although TOR signaling can be activated by a metabolite of diacylglycerol (DAG), phosphatidic acid (PA), the precise genetic mechanism through which DAG metabolism influences lifespan remains unknown. DAG is metabolized to either PA via the action of DAG kinase or 2-arachidonoyl-sn-glycerol by diacylglycerol lipase (DAGL). This study reports that in Drosophila and Caenorhabditis elegans, overexpression of diacylglycerol lipase (DAGL/inaE/dagl-1) or knockdown of diacylglycerol kinase (DGK/rdgA/dgk-5) extends lifespan and enhances response to oxidative stress. Phosphorylated S6 kinase (p-S6K) levels are reduced following these manipulations, implying the involvement of TOR signaling. Conversely, DAGL/inaE/dagl-1 mutants exhibit shortened lifespan, reduced tolerance to oxidative stress, and elevated levels of p-S6K. Additional results from genetic interaction studies are consistent with the hypothesis that DAG metabolism interacts with TOR and S6K signaling to affect longevity and oxidative stress resistance. These findings highlight conserved metabolic and genetic pathways that regulate aging (Lin, 2014).

Effects of Mutation

The adaptation of growth in response to nutritional changes is essential for the proper development of all organisms. The identification of the Drosophila homolog of the target of rapamycin (TOR), a candidate effector for nutritional sensing, is described. Genetic and biochemical analyses indicate that dTOR impinges on the insulin signaling pathway by autonomously affecting growth through modulating the activity of Drosophila S6k. However, in contrast to other components in the insulin signaling pathway, partial loss of dTOR function preferentially reduces growth of the endoreplicating tissues. These results are consistent with dTOR residing on a parallel amino acid sensing pathway (Oldham, 2000).

A tissue-specific genetic screen has been carried out for recessive mutations affecting cell growth and proliferation in the Drosophila compound eye. In this screen, genetically mosaic flies are generated in which the eye and head capsule are homozygous for a randomly induced mutation, while the rest of the body and the germ line are heterozygous and, thus, phenotypically wild type. Remarkably, mosaic flies containing a mutation in a growth-promoting gene have eye and head structures that are strongly reduced in size relative to their wild-type sized heterozygous bodies and are termed pinhead flies. Two EMS-induced pinhead mutations (2L1 and 2L19) map to chromosomal position 34A, where the Drosophila homolog of TOR (dTOR) is located. These mutations fail to complement two lethal P-element insertions, EP(2)2353 and l(2)k17004, located 262 and 211 bp, respectively, upstream of the putative translation start site of dTOR. The dTOR genomic region spans ~10 kb and is composed of seven exons (7.4 kb cDNA) encoding a 2480-amino acid protein with a predicted Mw of 282 kD. The dTOR protein exhibits 35%, 38%, 35%, and 41% overall amino acid identity to yeast TOR1, TOR2, C. elegans TOR, and Arabidopsis thaliana TOR, respectively. The overall identity with mTOR is significantly higher (56%) and is especially conserved in the kinase and FRB domains (74% and 77%). Sequence analysis of DNA from flies heterozygous for the EMS-induced dTOR mutations reveal two nucleotide substitutions. The lesion of dTOR2L1 results in a change of a proline to a leucine at amino acid position 2303 (P2303L). The location of this mutation within a highly conserved region of the kinase domain implies that the kinase activity of dTOR is critical for its function, as has been shown for the yeast TORs. In contrast, the lesion in dTOR2L19 is an arginine changed to a nonsense mutation at amino acid residue 248 (R248Stop), giving rise to a stop codon. This mutation would be predicted to result in a short, truncated protein and should thus be a complete loss-of-function mutation (Oldham, 2000).

The phenotypes associated with the complete loss of dTOR function are remarkably similar to phenotypes associated with mutations in the Insulin-like receptor (Inr) pathway. (1) Strong dTOR mutants arrest development at a similar stage as do strong mutants in the Inr pathway or amino acid-starved larvae with little detectable imaginal tissue. (2) dTOR mutant clones have a significant proliferative disadvantage similar to Inr pathway mutant clones. Clones of dTOR null mutant cells, although severely affected, are not cell lethal. Similarly, in most mammalian cell types, rapamycin decreases but does not abolish cell growth, except for IL2-mediated T-cell proliferation. (3) The strict autonomous control of cell growth without disturbing the specification and differentiation is also seen with the dTOR mutants. Indeed, loss of dTOR function in clones of homozygous mutant cells in the adult eye show that only the mutant cells, as exemplified by the dark, circular rhabdomeres, are severely reduced in size. Analysis of imaginal wing disc cells at the end of the third larval instar by fluorescence-activated cell sorting (FACS), confirms that cells from the weak heteroallelic combination, dTOR2L1/dTORl(2)k17004, are smaller than those of wild type. The effect on cell size is more pronounced in G1 than G2, consistent with mTOR function having a predominante role on cell growth during G1 (Oldham, 2000).

Despite this observation, there is no apparent difference between the distribution of dTOR mutant and wild-type cells within each phase of the cell cycle. Although the similarities of the loss-of-function mutant phenotypes of dTOR and other components of the Inr pathway are consistent with a model in which dTOR acts downstream of dPI3K, the analysis of partial loss-of-function dTOR mutants and the biochemical analysis of Drosophila S6K activity indicates a more complex relationship between dTOR and the Inr pathway (Oldham, 2000).

Genetic interactions between dTOR mutants and other mutations in the insulin pathway were examined. Drosophila Pten encodes a negative effector of insulin signaling, and eyes and heads lacking Pten function are significantly larger than wild-type heads. Removal of dTOR function strongly reduces the size of Pten mutant heads, suggesting that dTOR is required for the increased growth generated by the loss of Pten function (Oldham, 2000).

In vertebrates, S6K activity is blocked by rapamycin, an inhibitor of TOR. Therefore, Drosophila S6K activity was examined in immunoprecipitates of extracts from larvae mutant for dTOR, S6K, chico, and larvae treated with rapamycin or deprived of amino acids. A severe reduction in the phosphorylation of ribosomal protein S6 was observed in extracts from strong dTOR2L1/dTOR2L19 mutant larvae. This was not caused by a reduction in Drosophila S6k protein as shown by Western blotting of these extracts. In addition, the S6k protein is up-regulated in the dTOR mutant larvae and amino acid-starved larvae. In all cases, Western blot analysis has shown equivalent amounts of initiation factor 4E (eIF-4E). S6k activity is not detected in S6kl-1 null mutants and is severely reduced when wild-type larvae are starved for amino acids or treated with rapamycin. Higher doses of rapamycin blocks development during early larval stages, leading to lethality. Analysis of the weak dTOR2L1/dTORl(2)k17004 or dTOR2L1/dTOREP(2)2353 heteroallelic combinations also reveal a reduction in S6k activity in the third larval instar and an up-regulation of the protein as compared with wild-type flies. The surprising fact that dTOR mutants and amino acid starvation result in an up-regulation of S6k levels suggests that dTOR and amino acids may negatively control the protein levels of S6k. Unexpectedly, S6k activity as well as protein levels are unaffected in chico mutants. It may be that Inr does not signal to S6k or that S6k resides on a parallel pathway that bifurcates upstream of Chico. In support of the latter possibility, Inr has been shown to genetically interact with PI3K independently of Chico, presumably through docking sites for the p60 adaptor of PI3K in the Inr C-terminal tail. This result suggests that there is a S6k independent pathway for growth control and that the reduced Inr-mediated PI3K signaling in a chico mutant is sufficient for S6k activation (Oldham, 2000).

The biochemical differences between the ability of Chico and dTOR to activate S6k argue for a more complex relationship between the Inr pathway and dTOR. Given the low number of pharate adults, the weights of dTOR, S6kl-1, and chico mutants were compared at an early pupal stage. The weight of the dTOR mutant pupae is more similar to S6k than to chico mutant pupae. Thus, in the absence of S6k function or the presence of reduced dTOR levels, cellular growth rates are diminished but larvae pupariate at a larger size as a result of a longer developmental delay. Importantly, S6k mutant flies have cells that are smaller but of the normal number. However, in chico mutants, pupariation is initiated at a much smaller size. The result is that chico mutants emerge after only a 2-d delay and are smaller than dTOR and S6k mutants because of fewer and smaller cells. Therefore, while insulin signaling controls cell size and cell number, S6k primarily controls cell size. It will be of interest to know whether dTOR is also limited to controlling only cell size (Oldham, 2000).

Larvae are composed of mitotic cells, largely represented by the imaginal discs, and of endoreplicating tissues, which form larval structures like the gut, fat body, and salivary glands. An increase in DNA ploidy of larval cells is required for the ~200-fold increase in mass obtained by the larvae during the 5-d period between the completion of embryogenesis and the beginning of pupation. During starvation, larvae sacrifice their endoreplicating tissue to maintain the growth and proliferation of the mitotic cells that are required to form the reproductive adult. Furthermore, S6k activity is reduced in starved larvae and dTOR mutants. These observations prompted an analysis of the mitotic and endoreplicating tissues of dTOR, S6k, and chico mutant larvae just before pupariation. Strong dTOR and PI3K mutants, as well as amino acid-starved larvae, are incapable of growth and have barely detectable imaginal and endoreplicative tissues. Surprisingly, the wing discs of the weak dTOR heteroallelic combination are of approximately equivalent size to that of wild-type larvae, whereas those of S6kl-1 mutants are reduced. However, the amount of endoreplicating tissue in the dTOR mutant as compared to wild-type larvae is severely decreased. This is clearly demonstrated by comparing the salivary glands of dTOR mutant and wild-type larvae. In contrast, the size of endoreplicating tissue and imaginal discs in S6k null mutants as well as chico null mutants is reduced to approximately the same extent. Staining of the salivary glands with DAPI and phalloidin reveals that the size of the nuclei and, thus, the degree of endoreplication is severely reduced in S6k, chico, and dTOR mutants. The difference in size between dTOR and S6k mutant salivary glands is largely caused by a very pronounced reduction in cytoplasmic volume in dTOR mutants. The nuclear to cytoplasmic ratio is higher in dTOR salivary glands than in y w, S6k, or chico mutant salivary glands. Thus, it appears that partial loss of dTOR function permits the growth of imaginal tissue to wild-type size, while endoreplicating tissue is disproportionally reduced, a phenotype distinct from S6k mutants. Consistent with this finding, the lethality of the different dTOR mutants could not be rescued by constitutive expression of a S6K1 variant, D3E-E389, which exhibits high basal activity in the absence of mitogens under the control of the alpha-tubulin promoter, which rescues all aspects of the S6kl-1 null phenotype. Therefore, S6k-independent processes must contribute to the weak dTOR phenotype (Oldham, 2000).

The effect of rapamycin and amino acids on translation in mammals is mediated through the S6Ks and the 4E-BPs. Unlike the other elements in the PI3K signaling pathway, absence of amino acids blocks both S6K activation and 4E-BP phosphorylation. Indeed, a mutant of S6K1, lacking a portion of both its amino and carboxyl termini, is resistant to rapamycin but still sensitive to the fungal metabolite wortmannin, an inhibitor of PI3K. This suggests that the PI3K-dependent signal to S6K activation does not involve TOR. This same mutant is also unaffected by amino acid withdrawal, consistent with the role of mTOR as an amino acid checkpoint in S6K activation. Although there is some controversy concerning the ability of mitogens to activate mTOR, the in vitro activity of mTOR from cultured cells toward either itself, S6K1, or 4E-BP1 is unaffected by mitogens. Thus, mTOR may act as a permissive signal that primes 4E-BP phosphorylation and S6K activation by the PI3K signaling pathway if amino acids, and possibly other nutrients, are at sufficient levels. Likewise, in Drosophila larvae, amino acids are necessary, but not sufficient, for imaginal disc and endoreplicating tissue proliferation, compatible with dTOR acting in a parallel pathway involved in amino acid sensing. The fact that chico mutant larvae have normal levels of S6k activity and that the dTOR larval phenotypes with respect to the imaginal discs and endoreplicating tissues are so distinct compared with other mutants in the Inr pathway, supports the possibility that dTOR is not responsive to insulin signaling (Oldham, 2000 and references therein).

It is well established in yeast that TOR is an important mediator of nutrient limitation, and it has been proposed that TOR acts as an amino acid effector to coordinate the response of yeast to different nutritional conditions (Barbet, 1996). Indeed, the similarities between dTOR mutant larvae and larvae deprived of amino acids are striking. Therefore, it is likely that dTOR also functions as an amino acid sensor in multicellular organisms. The fact that yeast and Arabidopsis do not have an insulin system suggests that TOR may be an ancestral and widespread nutritional sensor. To provide additional levels of control, it may have been integrated into the insulin system later to respond to different modes of nutrient deprivation with different developmental responses (Oldham, 2000 and references therein).

Insulin treatment of Drosophila Kc 167 cells induces the multiple phosphorylation of a Drosophila ribosomal protein, as judged by its decreased electrophoretic mobility on two-dimensional polyacrylamide gels. The extent to which insulin induces this response is potentiated by cycloheximide and blocked by pretreatment with rapamycin. Isolation and mass spectrometric analysis have revealed that the multiply phosphorylated protein is the larger of two Drosophila melanogaster orthologs of mammalian 40S ribosomal protein S6, termed here DS6A. Proteolytic cleavage of DS6A (derived from stimulated Kc 167 cells), with the endoproteinase Lys-C releases a number of peptides, one of which contains all the putative phosphorylation sites. Conversion of phosphoserines to dehydroalanines with Ba(OH)(2) shows that the sites of phosphorylation reside at the carboxy terminus of DS6A. The sites of phosphorylation have been identified by Edman degradation after conversion of the phosphoserine residues to S-ethylcysteine as Ser(233), Ser(235), Ser(239), Ser(242), and Ser(245). Phosphopeptide mapping of individual phosphoderivatives, isolated from two-dimensional polyacrylamide gels, indicate that DS6A phosphorylation, in analogy to mammalian S6 phosphorylation, appears to proceed in an ordered fashion (Oldham, 2000 and references therein).

Precise body and organ sizes in the adult animal are ensured by a range of signaling pathways. Rheb (Ras homolog enriched in brain), a novel, highly conserved member of the Ras superfamily of G-proteins, promotes cell growth. Overexpression of Rheb in the developing fly causes dramatic overgrowth of multiple tissues: in the wing, this is due to an increase in cell size; in cultured cells, Rheb overexpression results in accumulation of cells in S phase and an increase in cell size. Rheb is required in the whole organism for viability (growth) and for the growth of individual cells. Inhibition of Rheb activity in cultured cells results in their arrest in G1 and a reduction in size. These data demonstrate that Rheb is required for both cell growth (increase in mass) and cell cycle progression; one explanation for this dual role would be that Rheb promotes cell cycle progression by affecting cell growth. Consistent with this interpretation, flies with reduced Rheb activity are hypersensitive to rapamycin, an inhibitor of the growth regulator target of rapamycin (TOR), a kinase required for growth factor-dependent phosphorylation of ribosomal S6 kinase (S6K). In cultured cells, the effect of overexpressing Rheb was blocked by the addition of rapamycin. These results imply that Rheb is involved in TOR signaling (Patel, 2003). Additional studies show that Rheb functions downstream of the tumor suppressors Tsc1 (tuberous sclerosis 1)-Tsc2, with Tsc2 functioning as a GAP for Rheb (Saucedo, 2003; Zhang, 2003), and that a major effector of Rheb function in controlling growth is, in fact, ribosomal S6 kinase (Stocker, 2003). It is still not clear, however, how Rheb signals to TOR (Zhang, 2003).

Given the similarities between Rheb and mutants in the InR and TOR signalling pathways, it is conceivable that Rheb represents a novel component of one of these growth control pathways. To test this possibility, a detailed epistasis analysis was performed. Examined first was whether the negative regulators of InR and TOR signalling (PTEN and Tsc1-Tsc2, respectively) could counteract the effects of Rheb overexpression. All overexpression experiments were performed in the eye using the GMR-Gal4 driver line. Expression of either PTEN or Tsc1-Tsc2 alone results in a very similar size reduction of the ommatidia when compared with control ommatidia. However, whereas expression of PTEN has no influence on the increase in ommatidial size caused by Rheb overexpression, co-expression of Tsc1-Tsc2 results in ommatidia of approximately wild-type size, indicating that the activities of Rheb and Tsc1-Tsc2 can counteract each other. Next, the enlarged ommatidia phenotype of GMR-Rheb was assayed in a number of mutant backgrounds. Reducing the activity of Drosophila protein kinase B (PKB) has no effect on ommatidial size. Similar results were obtained with hypomorphic mutations in InR and Dp110, respectively. In contrast, ommatidial size is dominantly reduced by a mutation in TOR (TOR2L1), and a suppression to wild-type size is observed in a S6K mutant background. Thus, the Rheb overexpression phenotype is dependent on TOR and S6K function, but is independent of InR signal strength. Finally, the behaviors of Rheb PTEN and Rheb Tsc1 double mutants were examined. The phenotypic consequences were assayed in mosaic animals using the ey-Flp method. As expected, the Rheb PTEN double-mutant tissue clearly displays a Rheb phenotype. The Rheb Tsc1 mutant tissue also resembles Rheb single mutants, indicating that Rheb is epistatic over (functions downstream of) Tsc1 (Stocker, 2003).

Regulation of lifespan in Drosophila by modulation of genes in the TOR signaling pathway

In many species, reducing nutrient intake without causing malnutrition extends lifespan. Like DR (dietary restriction), modulation of genes in the insulin-signaling pathway, known to alter nutrient sensing, has been shown to extend lifespan in various species. In Drosophila, the target of rapamycin (TOR) and the insulin pathways have emerged as major regulators of growth and size. Hence, the role of TOR pathway genes in regulating lifespan has been examined by using Drosophila. Inhibition of TOR signaling pathway by alteration of the expression of genes in this nutrient-sensing pathway, which is conserved from yeast to human, extends lifespan in a manner that may overlap with known effects of dietary restriction on longevity. In Drosophila, TSC1 and TSC2/Gigas (tuberous sclerosis complex genes 1 and 2) act together to inhibit TOR (target of rapamycin), which mediates a signaling pathway that couples amino acid availability to S6 kinase, translation initiation, and growth. Overexpression of dTsc1, dTsc2, or dominant-negative forms of dTOR or dS6K all cause lifespan extension. Modulation of expression in the fat is sufficient for the lifespan-extension effects. The lifespan extensions are dependent on nutritional condition, suggesting a possible link between the TOR pathway and dietary restriction (Kapahi, 2004).

The Drosophila homologs of human Tsc1 (Hamartin) and Tsc2 (tuberin) function in vivo as a complex that controls growth and size in a cell-autonomous manner. To examine their role in regulating lifespan, dTsc1 and dTsc2 were overexpressed through the ubiquitously expressed driver, daughterless (da-GAL4). Overexpression in transgenic flies carrying UAS constructs containing dTsc1 or dTsc2 extends mean lifespan at 29°C by 14% and 12%, respectively. Since GAL4 enhancer traps generally yield stronger effects at 29°C, most of the experiments were performed at that temperature (Kapahi, 2004).

dTsc1 and dTsc2 physically interact with dTOR, which is conserved from yeast to human as a nutrient sensor. Loss of dTsc1 in Drosophila eye leads to an increase in cell size, provided that dTOR is present. Surprisingly, however, dTOR overexpression causes a reduction in cell size, a phenotype similar to dTOR loss-of-function mutations, perhaps due to titration of cofactors required for TOR signaling. The effect of dTOR on lifespan was examined by using three UAS. One carries the full-length wild-type TOR gene. The second carries FRB, the 11 kDa FKBP12-rapamycin binding domain, which has been shown to prevent S phase entry when injected into human osteosarcoma cells. The third carries TED (toxic effector domain), containing the 754 amino acid central region, which inhibits cell growth and arrests cells in G1 when overexpressed in yeast (Hennig, 2002). Ubiquitous overexpression with the da-GAL4 driver of UAS-dTORFRB led to a mean lifespan increase at 29°C of 24%. However, overexpression of UAS -dTORWT or UAS-dTORTED prevented eclosion to adulthood (Kapahi, 2004).

S6 kinase activation upon phosphorylation has been implicated in mediating the downstream effects of TOR on translation initiation in flies and mammals. S6 kinase phosphorylation of ribosomal protein S6 is accompanied by upregulation of a class of mRNAs containing an oligopyrimidine tract at their transcriptional start site termed 5'TOP (Thomas, 2002). Some 200 genes, most of which encode components of the translational apparatus including ribosomal proteins and elongation factors, have this sequence and can account for about 20% of total cellular mRNA. Flies carrying homozygous mutations in dS6K show a developmental delay and a reduction in body size. The stimulation of dS6K phosphorylation by dTOR is abrogated when dTsc1 and dTsc2 are overexpressed. Furthermore, flies with reduced dTSC1 show increased dS6 kinase activation, and genetic reduction of S6 kinase level can rescue the lethality caused by loss of function of dTsc1 (Kapahi, 2004).

The role of S6 kinase in regulating lifespan was examined by using dominant-negative and constitutively active constructs. The dominant-negative effect was achieved by replacing the conserved lysine in the ATP binding site by glutamine (UAS-dS6KKQ), which causes cell-size reduction. The constitutively active form was generated by replacing the phosphorylation sites of S6 kinase by acidic amino acids (UAS-dS6KSTDETE), causing an autonomous cell size increase. By using da-GAL4 to drive ubiquitous overexpression of the dominant-negative form, a mean lifespan increase of 22% at 29°C was observed. Conversely, overexpression of the constutively active form of S6 kinase caused a mean lifespan decrease of 34% at 29°C. Overexpression of dTsc2 and dTORFRB was also tested at 25°C and led to a 20% and 26% increase in mean lifespan increase, respectively (Kapahi, 2004).

To determine which tissues are responsible for the lifespan extension, various GAL4 drivers with specific GAL4 expression pattern were employed to overexpress dTsc2 via a UAS promoter. Overexpression in the eye by using the driver gmr-GAL4 or in the nervous system by using appl-GAL4 did not extend lifespan. In contrast, by using the drivers 24B-GAL4 and PO188-GAL4, enhancer traps that are predominantly expressed in the muscle and fat, results in mean lifespan extensions of 27% and 37%, respectively, at 29°C. The fat-specific drivers DJ634-GAL4 and PO163-GAL4, when used to overexpress dTsc2, also led to a mean lifespan extension of 22% and 31%, respectively, at 29°C. Using DJ634-GAL4 to overexpress the dominant-negative form of TOR (UAS-dTORFRB) or of S6 kinase (UAS-UAS-dS6KKQ) also led to mean lifespan increases of 30% and 29%, respectively, at 29°C. These results indicate that manipulation of the TSC, TOR, and S6 kinase genes in the fat tissue is sufficient for their lifespan extension effects in Drosophila (Kapahi, 2004).

Amino acids have been shown to activate dS6k via TOR, an effect that can be abrogated in the presence of increased levels of dTsc1 and dTsc2. Since nutrients in the diet can modulate lifespan and because the TOR pathway is a critical mediator of nutrient signaling, it was asked whether the observed lifespan-extension effects are dependent on nutrient conditions. This was tested with overexpression of dTsc2 by using the ubiquitously expressing da-GAL4 driver. Flies were allowed to develop to adulthood under standard laboratory food and then maintained on specially prepared food containing various concentrations of yeast extract. At high concentrations of yeast extract, which may be regarded as the opposite of dietary restriction, the lifespan of control flies (da-GAL4/+) is severely reduced. However, overexpression of dTsc2 protects the fly from the deleterious effects of rich food, as if mimicking the effect of dietary restriction. Similar results were observed by overexpression of the dominant-negative form of S6 kinase (Kapahi, 2004).

Recent evidence from Drosophila suggests that signaling through TSC is both parallel to and interacting with the insulin pathway. This is supported by the finding that heterozygosity of dTsc1 or dTsc2 is sufficient to rescue the lethality of loss-of-function dInR mutants. However, the finding that loss-of-function mutations of dTsc1 and dPTEN, a phosphatase that negatively regulates the insulin-signaling pathway, cause cell autonomous and additive increases in cell size suggests that they may be in parallel pathways. Furthermore, in Drosophila, dPTEN loss of function, which leads to an increase in cell size, is only slightly suppressible by loss of function of dFOXO, a fly homolog of C. elegans daf-16. However, the increase in cell size resulting from dTsc1 is enhanced by dFOXO loss of function. Interestingly, unlike long-lived daf-2 mutants, the lifespan extension due to TOR deficiency in C. elegans is not suppressible by a daf-16 mutation. However, the TOR mutant animals do not further extend lifespan in a daf-2 background, leading to the possibility that TOR may be acting downstream or separately from daf-16 to exert its lifespan effects (Kapahi, 2004).

Lifespan extension has been linked with other phenotypes, including stress resistance, metabolic rate, lipid level, reproductive capacity, and body size. The long-lived strains described above with their respective controls for resistance to starvation were compared but no significant differences were found. Similarly, no significant differences were observed for weight and lipid content among these strains. It may be that lifespan extension can be produced by mild modulation of these genes, whereas effects on other phenotypes require severe perturbations. While lifespan extension is observed by using the da-GAL4 driver to overexpress dTsc1 or dTsc2 alone, simultaneous overexpression of dTsc1 and dTsc2 prevented eclosion to adulthood. Similarly, no change in size is observed if dTsc1 or dTsc2 alone are overexpressed in the eye, but a cell-autonomous decrease in size is seen when both are overexpressed simultaneously. Lifespan extension by chico is semidominant, but its effect on body size is recessive. Dominant effects on lifespan are observed with the genes Inr, EcR, Indy, and Rpd3, but their effects on lifespan can be uncoupled from other phenotypes such as fecundity, stress resistance, or lipid accumulation (Kapahi, 2004).

In humans, mutations in TSC1 and TSC2 lead to tuberous sclerosis, a common disorder characterized by the presence of benign tumors in various tissues, with some having large cells. DR in mice has been shown to protect against age-related tumorigenesis. These results suggest a link between lifespan extension by DR and the activities of genes in the TOR pathway. Hence, it is conceivable that the protective effects of DR on tumorigenesis and age-related decline might come from inhibition of such nutrient-responsive pathways (Kapahi, 2004).

These results show that upregulation of dTsc2 in the fat is sufficient for lifespan extension effects in Drosophila. Reduction of daf-2 levels in the C. elegans nervous system has been shown to be sufficient for lifespan extension. However, the lifespan extensions due to mutations in the insulin pathway or germline ablation in C. elegans are dependent on daf-16 activity in the intestine, the fat storage tissue in C. elegans. In Drosophila, the fat body has been proposed to modulate insulin signaling in peripheral tissues by secretion of dALS (acid-labile subunit), which, in mammals, forms a ternary complex with insulin-like growth factor, leading to an extension of the half-life of its ligand. Recently, mice with FIRKO (fat-specific insulin receptor knockout) have been shown to live 18% longer than controls . Hence, it is possible that secondary endocrine signals downstream of the insulin and TOR signaling pathways are released from the fat, and these affect the rate of aging in other tissues. Juvenile hormone and ecdysone are two such endocrine signals that have been implicated in regulating lifespan in conjunction with the insulin pathway in Drosophila (Kapahi, 2004).

Role and regulation of starvation-induced autophagy in the Drosophila fat body

In response to starvation, eukaryotic cells recover nutrients through autophagy, a lysosomal-mediated process of cytoplasmic degradation. Autophagy is known to be inhibited by TOR signaling, but the mechanisms of autophagy regulation and its role in TOR-mediated cell growth are unclear. Signaling through TOR and its upstream regulators PI3K and Rheb is necessary and sufficient to suppress starvation-induced autophagy in the Drosophila fat body. In contrast, TOR's downstream effector S6K promotes rather than suppresses autophagy, suggesting S6K downregulation may limit autophagy during extended starvation. Despite the catabolic potential of autophagy, disruption of conserved components of the autophagic machinery, including ATG1 and ATG5, does not restore growth to TOR mutant cells. Instead, inhibition of autophagy enhances TOR mutant phenotypes, including reduced cell size, growth rate, and survival. Thus, in cells lacking TOR, autophagy plays a protective role that is dominant over its potential role as a growth suppressor (Scott, 2004).

Autophagy likely evolved in single-cell eukaryotes to provide an energy and nutrient source allowing temporary survival of starvation. In yeast, Tor1 and Tor2 act as direct links between nutrient conditions and cell metabolism. These proteins sense nutritional status by an unknown mechanism, and effect a variety of starvation responses including changes in transcriptional and translational programs, nutrient import, protein and mRNA stability, cell cycle arrest, and induction of autophagy. Autophagy thus occurs in the context of a comprehensive reorganization of cellular activities aimed at surviving low nutrient levels (Scott, 2004).

In multicellular organisms, TOR is thought to have retained its role as a nutrient sensor but has also adopted new functions in regulating and responding to growth factor signaling pathways and developmental programs. Thus in a variety of signaling, developmental, and disease contexts, TOR activity can be regulated independently of nutritional conditions. In these cases, autophagy may be induced in response to downregulation of TOR despite the presence of abundant nutrients and may potentially play an important role in suppressing cell growth rather than promoting survival. Identification of the tumor suppressors PTEN, and TSC1 and TSC2 as positive regulators of autophagy provides correlative evidence supporting such a role for autophagy in growth control. Alternatively, since TOR activity is required for proper expression and localization of a number of nutrient transporters, inactivation of TOR may lead to reduced intracellular nutrient levels, and autophagy may therefore be required under these conditions to provide the nutrients and energy necessary for normal cell metabolism and survival (Scott, 2004).

The results presented here provide genetic evidence that under conditions of low TOR signaling, autophagy functions primarily to promote normal cell function and survival, rather than to suppress cell growth. This conclusion is based on the finding that genetic disruption of autophagy does not restore growth to cells lacking TOR, but instead exacerbates multiple TOR mutant phenotypes. It is important to note that mutations in TOR do not disrupt larval feeding, and thus disruption of autophagy is detrimental in TOR mutants despite the presence of ample extracellular nutrients. The finding that autophagy is critical in cells lacking TOR further supports earlier studies suggesting that inactivation of TOR causes defects in nutrient import, resulting in an intracellular state of pseudo-starvation (Scott, 2004).

Can the further reduction in growth of TOR mutant cells upon disruption of autophagy be reconciled with the potential catabolic effects of autophagy? TOR regulates the bidirectional flow of nutrients between protein synthesis and degradation through effects on nutrient import, autophagy, and ribosome biogenesis. When TOR is inactivated, rates of nutrient import and protein synthesis decrease, resulting in a commensurate reduction in mass accumulation and cell growth. In addition, autophagy is induced to maintain intracellular nutrient and energy levels sufficient for normal cell metabolism. When autophagy is experimentally inhibited in cells lacking TOR, this reserve source of nutrients is blocked, leading to a further decrease in energy levels, protein synthesis, and growth. It is noted that autophagy may have additional functions in cells with depressed TOR signaling, including recycling of organelles damaged by the absence of TOR activity, or selective degradation of cell growth regulators, analogous to the regulatory roles of ubiquitin-mediated degradation (Scott, 2004).

Autophagy is required for normal developmental responses to inactivation of insulin/PI3K signaling in the nematode C. elegans. In response to starvation or disruption of insulin/PI3K signaling, C. elegans larvae enter a dormant state called the dauer. Autophagy has been observed in C. elegans larvae undergoing dauer formation: disruption of a number of ATG homologs interfers with normal dauer morphogenesis. Importantly, simultaneous disruption of insulin/PI3K signaling and autophagy genes results in lethality, similar to the results presented in this study. Thus despite significant differences in developmental strategies for surviving nutrient deprivation, autophagy plays an essential role in the starvation responses of yeast, flies, and worms (Scott, 2004).

The prevailing view that S6K acts to suppress autophagy was founded on correlations between induction of autophagy and dephosphorylation of rpS6 in response to amino acid deprivation or rapamycin treatment. However, the genetic data presented in this study argue strongly against a role for S6K in suppressing autophagy: unlike other positive components of the TOR pathway, null mutations in S6K do not induce autophagy in fed animals. It is suggested that the observed correlation between S6K activity and suppression of autophagy is due to common but independent regulation of S6K and autophagy by TOR. Thus, autophagy suppression and S6K-dependent functions such as ribosome biogenesis represent distinct outputs of TOR signaling (Scott, 2004).

How might TOR signal to the autophagic machinery, if not through S6K? In yeast, this is accomplished in part through regulation of Atg1 kinase activity and ATG8 gene expression (Kamada, 2000 and Kirisako, 1999). The demonstration of a role for Drosophila ATG1 and ATG8 homologs [see TG8a (CG32672) and ATG8b (CG12334)] in starvation-induced autophagy, and the genetic interaction observed between ATG1 and TOR, are consistent with a related mode of regulation in higher eukaryotes. However, it is noted that other components of the yeast Atg1 complex such as Atg17 and Atg13, whose phosphorylation state is rapamycin sensitive, do not have clear homologs in metazoans, indicating that differences in regulation of autophagy by TOR are likely (Scott, 2004).

In addition to excluding a role for S6K in suppression of autophagy, these results reveal a positive role for S6K in induction of autophagy. S6K may promote autophagy directly, through activation of the autophagy machinery, or indirectly through its effects on protein synthesis. The latter possibility is consistent with previous reports that protein synthesis is required for expansion and maturation of autophagosomes. Interestingly, despite being required for autophagy, S6K is downregulated under conditions that induce it, including chronic starvation and TOR inactivation. Consistent with this, it was found that lysotracker staining is significantly weaker in chronically starved animals or in TOR mutants than in wild-type animals starved 3-4 hr. Furthermore, expression of constitutively activated S6K has no effect in wild-type, but restores lysotracker staining in TOR mutants to levels similar to those of acutely starved wild-type animals. It is suggested that downregulation of S6K may limit rates of autophagy under conditions of extended starvation or TOR inactivation and that this may protect cells from the potentially damaging effects of unrestrained autophagy (Scott, 2004).

Co-culture and conditioned media experiments have shown that the Drosophila fat body is a source of diffusible mitogens. The fat body has also been shown to act as a nutrient sensor through a TOR-dependent mechanism and to regulate organismal growth through effects on insulin/PI3K signaling. The results in this study extend these findings by showing that this endocrine response is accompanied by the regulated release of nutrients through autophagic degradation of fat body cytoplasm. Preventing this reallocation of resources, either through constitutive activation of PI3K or through inactivation of ATG genes, results in profound nutrient sensitivity. Thus, in response to nutrient limitation, the fat body is capable of simultaneously restricting growth of peripheral tissues through downregulation of insulin/PI3K signaling and providing these tissues with a buffering source of nutrients necessary for survival through autophagy (Scott, 2004).

Regulation of hunger-driven behaviors by neural ribosomal S6 kinase in Drosophila

Hunger elicits diverse, yet coordinated, adaptive responses across species, but the underlying signaling mechanism remains poorly understood. This study reports on the function and mechanism of the Drosophila insulin-like system in the central regulation of different hunger-driven behaviors. Overexpression of Drosophila insulin-like peptides (DILPs) in the nervous system of fasted larvae suppresses the hunger-driven increase of ingestion rate and intake of nonpreferred foods (e.g., a less accessible solid food). Moreover, up-regulation of Drosophila p70/S6 kinase activity in DILP neurons leads to attenuated hunger response by fasted larvae, whereas its down-regulation triggered fed larvae to display motivated foraging and feeding. Finally, evidence is provided that neural regulation of food preference but not ingestion rate may involve direct signaling by DILPs to neurons expressing neuropeptide F receptor 1, a receptor for neuropeptide Y-like neuropeptide F. This study reveals a prominent role of neural Drosophila p70/S6 kinase in the modulation of hunger response by insulin-like and neuropeptide Y-like signaling pathways (Wu, 2006).

The relatively simple Drosophila larva offers a genetically tractable model to define and characterize different neuronal signaling pathways that constitute a complete central feeding apparatus. Younger third-instar larvae forage actively and use their mouth hooks for food intake. Larvae normally feed on liquid food, and their food ingestion can be quantified by measuring the contraction rate of the mouth hooks. This study examined how food deprivation affects larval feeding response to a liquid (e.g., 10% glucose-agar paste) and less accessible solid food (e.g., 10% glucose agar blocks). To extract embedded glucose from the solid food, larvae have to pulverize the food by scraping agar surface with mouth hooks. Unless stated otherwise, synchronized third-instar larvae (74 h after egg laying) were used for the assays (Wu, 2006).

When fed ad libitum, normal larvae (w1118) display significant feeding activity in the liquid food with an average mouth-hook contraction frequency of ~30 times in a 30-s test period; in contrast, these larvae declined the solid food. However, larvae withheld from food (on a wet tissue) for 40 or 120 min display increased intake of both liquid and solid foods. For example, larvae fasted for 120 min show a 100% and >500% increase in mouth-hook contraction rate in liquid and solid food, respectively. Thus, deprivation not only enhances feeding rate in a graded fashion, but also triggers motivated foraging on the less accessible food normally rejected by fed larvae. In addition, larvae display virtually identical feeding responses to liquid and solid foods containing 10% glucose, apple juice, or 10% glucose/yeast under deprived and nondeprived conditions. Therefore, these paradigms appear to provide a general assessment of larval feeding response (Wu, 2006).

dS6K is a cell-autonomous effector of nutrient-sensing pathways. This study investigated a possible role of neural dS6K in coupling peripheral physiological hunger signals and neuronal activities critical for hunger-driven behaviors. The transcripts of dilp1, dilp2, dilp3, and dilp5 are predominantly expressed in two small clusters of medial neurosecretory cells that project to the ring gland, the fly heart, and the brain lobes. A gal4 driver containing a 2-kb fragment from the dilp2 promoter (dilp2-gal4) was generated that directs the specific expression of a GFP reporter in those cells. Using dilp2-gal4, two transgenes, UAS-dS6KDN, encoding a dominant negative, and UAS-dS6KACT, a constitutively active form of dS6K, were expressed. When fed ad libitum, control larvae (w x UAS-dS6KDN or UAS-dS6KACT) behave like w larvae. However, dilp2-gal4 x UAS-dS6KDN larvae displayed a 50% increase in the rate of liquid-food intake and significant feeding of the solid food. Conversely, fasted larvae overexpressing dS6K activity (dilp2-gal4 x UAS-dS6KACT) showed attenuated feeding response to both liquid and solid foods. These findings reveal that dS6K in DILP neurons mediates hunger regulation of approaching/consumptive behaviors, controlling both quality and quantity of food for ingestion. The body size and the developmental rate of all four groups of larvae were measured, and no significant differences were detected (Wu, 2006).

DILPs act as neurohormones in Drosophila larvae. Down-regulation of dS6K activity in DILP neurons may reduce DILP release, thereby promoting increased food intake that is normally triggered only by hunger. A corollary of this interpretation is that overproduction of DILPs in the nervous system should interfere with hunger response by deprived animals. To test this idea, a neural-specific elav-gal4 driver was used to direct dilp expression in the larval nervous system. Three UAS-dilp lines (UAS-dilp2, UAS-dilp3, and UAS-dilp4) were chosen for the analysis. The elav-gal4 x UAS-dilp2 and UAS-dilp4 larvae displayed normal feeding response when fed ad libitum. However, the same larvae fasted for 120 min displayed significantly attenuated feeding rates, similar to those of dilp2-gal4 x UAS-dS6KACT larvae. For example, the comparative analysis of the elav-gal4 x UAS-GFP control and elav-gal4 x UAS-dilp2 and UAS-dilp4 experimental larvae showed that the latter were ~30% and 33–45% lower in the ingestion rate of the liquid and solid food, respectively; surprisingly, elav-gal4 x UAS-dilp3 and UAS-GFP larvae showed virtually identical feeding responses. Therefore, DILP2 and DILP4 negatively regulate hunger-driven feeding activities. Taken together, these results suggest that a high level of dS6K activity in DILP neurons may suppress hunger response by reducing DILP release (Wu, 2006).

Attempts were made to delineate the signaling mechanism that couples the dS6K activity in DILP neurons with its broad impact on hunger-driven feeding activities. A previous study showed that fasted larvae ablated of NPF or its receptor (NPFR1) neurons are deficient in motivated feeding of the less-preferred solid food but normal in feeding of richer liquid food. It was of interest to enquire whether the NPF/NPFR1 neuronal pathway might be one of the downstream effectors of the DILP pathway. To test this hypothesis, the function of three components of the dInR signaling pathway were analyzed in NPFR1 neurons: dInR, phosphatidylinositol 3,4,5-trisphosphate 3-phosphatase (dPTEN), and phosphatidylinositol 3-kinase (dPI3K). Five different transgenes were used: UAS-dInRACT and UAS-dInRDN encode a constitutively active and a dominant-negative form of dInR, respectively; UAS-Dp110 and UAS-dPI3KDN encode a catalytic subunit and a dominant-negative form of dPI3K, respectively; and UAS-dPTEN encodes a functional enzyme. When fed ad libitum, npfr1-gal4 x UAS-dInRDN, UAS-dPTEN, or UAS-dPI3KDN larvae display hyperactive feeding of the solid food, similar to w larvae deprived for 40 min. In contrast, fasted larvae overexpressing dInR or dPI3K (npfr1-gal4 x UAS-dInRACT or UAS-Dp110) display attenuated feeding response to the solid food. Importantly, larvae with up- or down-regulated dInR signaling in NPFR1 neurons do not exhibit significant changes in the intake rate of the richer liquid food relative to the paired controls. Taken together, these findings suggest that the dInR pathway negatively regulates the activity of NPFR1 neuron and mediates the DILP-regulated change in food preference but not ingestion rate. Furthermore, the results suggest that NPFR1 neurons are the direct targets of DILPs (Wu, 2006).

A possible role of dS6K in hunger regulation of the functioning of NPFR1 neurons was evaluated, by expressing UAS-dS6KDN and UAS-dS6KACT using npfr1-gal4. When fed ad libitum, npfr1-gal4 x UAS-dS6KDN larvae display hyperactive feeding of the solid food, similar to npfr1-gal4 x UAS-dInRDN larvae. However, these larvae, unlike dilp2-gal4 x UAS-dS6KDN animals, display no increases in the ingestion rate of the richer liquid food. Conversely, fasted larvae overexpressing dS6K (npfr1-gal4 x UAS-dS6KACT) display attenuated feeding response to the solid food. These findings suggest that dS6K also negatively regulates the activity of NPFR1 neurons in food preference, but does not mediate the regulation of feeding rate by DILP signaling (Wu, 2006).

The food response was evaluated of the solid and liquid food by larvae overexpressing an npfr1 cDNA under the control of an npfr1-gal4 driver. In the presence of the liquid food, both experimental (npfr1-gal4 x UAS-npfr1) and control larvae (e.g., npfr1-gal4 x UAS-ANF-GFP), fed or fasted, show similar intake rates and comparable increases in feeding response to hunger. However, when forced to feed on the solid food, fed experimental larvae exhibit significant intake of the solid food (30 times per 30 s), whereas fed controls rejected the same food. Thus, NPFR1 overexpression selectively promotes change in food preference without increasing ingestion rate. It was also observed that the feeding responses of NPFR1-overexpressing larvae and controls fasted for 120 min were indistinguishable. Thus, the effect of NPFR1 overexpression on food preference is detectable only in fed or mildly fasted larvae, suggesting hunger-activated NPFR1 signaling approaches a plateau in severely fasted animals (Wu, 2006).

npfr1 activity was selectively knocked down by expressing npfr1 dsRNA in the nervous system. The UAS-npfr1dsRNA lines were previously used to functionally disrupt npfr1 activity. It was found that 120-min fasted larvae expressing npfr1 dsRNA in NPFR1 or the nervous system (npfr1-gal4, elav-gal4, or appl-gal4 x UAS-npfr1dsRNA) were deficient in motivated feeding of the solid but not liquid food. In contrast, all control larvae, including those expressing npfr1dsRNA in muscle cells (MHC82-gal4 x UAS-npfr1dsRNA), showed normal feeding responses. These results indicate that neural NPFR1 mediates hunger regulation of food selection (Wu, 2006).

A potential problem of the previous transgenic studies is that NPF/NPFR1 signaling is likely to be disrupted in a relatively early stage of larval development. Conceivably, the NPF/NPFR1 neuronal pathway could be essential for ad libitum or hunger-driven feeding of richer liquid foods, but such an activity might be masked by some yet-unidentified compensatory mechanism triggered by its early loss. To test this idea, attempts were made to disrupt NPF/NPFR1 neuronal signaling in a temporally controlled manner by expressing a temperature-sensitive allele of shibire (shits1) driven by npf-gal4 or npfr1-gal4. The shits1 allele encodes a semidominant-negative form of dynamin that blocks neurotransmitter release at a restrictive temperature (>29°C). At the permissive temperature of 23°C, 120-min-fasted experimental larvae (npf-gal4 and npfr1-gal4 x UAS-shits1) and paired controls (y w x UAS-shits1 and npf-gal4 and npfr1-gal4 x w1118) displayed normal feeding responses to both liquid and solid foods. However, if larvae were incubated at 30°C for 15 min, controls still displayed normal feeding activities, whereas the experimental larvae showed attenuated feeding response to the solid but not liquid food. Therefore, there was no detectable developmental or physiological compensation for the loss of NPF signaling in Drosophila larvae. These results also suggest that the NPF/NPFR1 neuronal pathway is acutely required to initiate and maintain larval hunger response. The foraging activity of the experimental larvae was completely restored when the assay temperature was reduced to 23°C, suggesting that the NPF system can modulate the intensity and duration of feeding response (Wu, 2006).

This study has shown that dS6K regulates different, yet coordinated, behaviors controlling quantitative and qualitative aspects of hunger-adaptive food response. Evidence is provided that dS6K mediates hunger regulation of two opposing insulin- and NPY-like signaling activities, dynamically modifying larval food preference and feeding rate based on the nutritional state. For example, hunger stimuli may cause a reduction of dS6K activity in DILP neurons, resulting in the suppression of DILP signaling that negatively regulates a downstream NPF/NPFR1-dependent and another NPF-independent neuronal pathway. The DILP/NPFR1 neuronal pathway selectively mediates hunger-adaptive change in food preference, possibly by overriding the high threshold of food acceptance set by a separate default pathway, enabling hungry animals to be receptive to less preferred foods. The NPF/NPFR1-independent pathway promotes a general increase in the ingestion rate of preferred/less preferred foods, enabling animals to compete effectively for limited food sources. This study also implicates the presence of a separate default pathway for mediating the selective intake of preferred foods (baseline feeding) in larvae fed ad libitum. This default pathway may be largely insensitive to DILP or NPF signaling, because overexpression of dS6K, DILPs, or NPFR1 in nondeprived larvae does not affect ad libitum feeding in the liquid food. It is suggested that the conserved S6K pathway may be critical for regulating behavioral adaptation to hunger in diverse organisms, including humans, and its components are potential drug targets for appetite control (Wu, 2006).

The functional differences of DILP1–7 have not been reported previously. In this study, dilp2, dilp3, and dilp4 were shown to be functionally distinct. DILP2 and DILP3 both are produced in the same medial neurosecretory cells. However, unlike DILP2, DILP3 is apparently not involved in suppressing deprivation-motivated feeding. It is still unclear whether the differential activities of DILP2 and DILP3 reflect their structural divergence or are caused by the presence of yet-unidentified dInR isoforms. DILP4 is not expressed within the two medial clusters of DILP neurons. Under acute deprivation, the level of dilp4 transcripts showed a 5-fold reduction in the larval CNS. Thus, it is possible that DILP4 may play a localized role in promoting feeding response inside the CNS (Wu, 2006).

Feeding is a reward-seeking behavior, and deprivation strengthens the reinforcing effect (reward value) of food. These studies suggest a previously uncharacterized role of the DILP/dInR signaling pathway in regulating an animal's perception of food quality. The DILP/NPF neural network may regulate an animal's incentive to acquire lower-quality foods by modifying the reward circuit. This hypothesis is interesting in light of the findings that foods and abused substances may act on the same reward circuit, and highly palatable foods can reduce drug-seeking behaviors. It is also possible that the DILP/NPF system might represent a specialized neural circuit that positively alters the reward value of lesser-quality foods. Conceivably, a better understanding of the action of this signaling system may provide fresh insights into neural mechanisms for controlling eating and drug-seeking behaviors (Wu, 2006).

Given its prominent role in behavioral adaptation to hunger, the insulin/NPY-like neural network is likely of primary importance to animal evolution. In addition, insulin and NPY family molecules have been found in a wide range of animals from humans to worms. Therefore, the insulin/NPY-like network may be a useful model for studying comparatively how diverse animals have evolved distinct ways of adapting an ancestral neural system to suit their respective lifestyles (Wu, 2006).

Drosophila TCTP is essential for growth and proliferation through regulation of dRheb GTPase

Cellular growth and proliferation are coordinated during organogenesis. Misregulation of these processes leads to pathological conditions such as cancer. Tuberous sclerosis (TSC) is a benign tumour syndrome caused by mutations in either TSC1 or TSC2 tumour suppressor genes. Studies in Drosophila and other organisms have identified TSC signalling as a conserved pathway for growth control. Activation of the TSC pathway is mediated by Rheb (Ras homologue enriched in brain), a Ras superfamily GTPase. Rheb is a direct target of TSC2 Gigas in Drosophila) and is negatively regulated by its GTPase-activating protein activity. However, molecules required for positive regulation of Rheb have not been identified. This study shows that a conserved protein, translationally controlled tumour protein (TCTP), is an essential new component of the TSC-Rheb pathway. Reducing Drosophila TCTP (dTCTP) levels reduces cell size, cell number and organ size, which mimics Drosophila Rheb (dRheb) mutant phenotypes. dTCTP is genetically epistatic to Tsc1 and dRheb, but acts upstream of dS6k, a downstream target of dRheb. dTCTP directly associates with dRheb and displays guanine nucleotide exchange activity with it in vivo and in vitro. Human TCTP (hTCTP) shows similar biochemical properties compared to dTCTP and can rescue dTCTP mutant phenotypes, suggesting that the function of TCTP in the TSC pathway is evolutionarily conserved. These studies identify TCTP as a direct regulator of Rheb and a potential therapeutic target for TSC disease (Hsu, 2007).

S6k is identified in a screen for genes that function in leg disc regeneration in Drosophila

Many diverse animal species regenerate parts of an organ or tissue after injury. However, the molecules responsible for the regenerative growth remain largely unknown. The screen reported in this study aimed to identify genes that function in regeneration and the transdetermination events closely associated with imaginal disc regeneration using Drosophila melanogaster. A collection of 97 recessive lethal P-lacZ enhancer trap lines were screened for two primary criteria: first, the ability to dominantly modify wg-induced leg-to-wing transdetermination and second, for the activation or repression of the lacZ reporter gene in the blastema during disc regeneration. Of the 97 P-lacZ lines, six genes (Krüppelhomolog- 1, rpd3, jing, combgap, Aly and S6 kinase) were identified that met both criteria. Five of these genes suppress, while one enhances, leg-to-wing transdetermination and therefore affects disc regeneration. Two of the genes, jing and rpd3, function in concert with chromatin remodeling proteins of the Polycomb Group (PcG) and trithorax Group (trxG) genes during Drosophila development, thus linking chromatin remodeling with the process of regeneration (McClure, 2008).

There are three different mechanisms that organisms use to re-grow and replace lost or damaged body parts, and often, more than one mechanism can function within different tissues of the same organism. Muscle and bone, for example, repair themselves by activating a resident stem cell population, while the liver regenerates by compensatory proliferation of normally quiescent differentiated cells. Appendage/fin regeneration in lower vertebrates occurs by a process termed epimorphic regeneration, which proceeds in three distinct stages: (1) wound healing and migration of the surrounding epithelial cells to form the wound epidermis, (2) formation of the regeneration blastema -- a mass of undifferentiated and proliferating cells of mesenchymal origin and (3) regenerative outgrowth and pattern re-formation. Whether these diverse modes of regeneration share a common molecular and genetic basis is not known (McClure, 2008).

Regeneration in the Drosophila imaginal discs, the primordia of the adult fly appendages, closely parallels epimorphic limb/fin regeneration in lower vertebrates. Cells in the imaginal discs are rigidly determined to form specific adult structures (e.g., legs and wings) by the third larval instar. If the discs are fragmented at this time and cultured in vivo, they will regenerate. Disc regeneration begins 12 h after wounding, when transient heterotypic contacts are made between peripodial (squamous epithelium) and columnar cells (disc proper) near the cut edges of the wound. These initial contacts involve microvilli-like extensions and provide temporary wound closure. Then, approximately 24 h after wounding, homotypic cell contacts (between columnar or between squamous cells) are made involving the close apposition of cell membranes and cellular bridges, which eventually (48 h after wounding) restore the physical continuity of the disc. Before and during wound healing, cell division is randomly distributed throughout the disc. However, once completed (36-48 h after wounding), division is observed only in cells near the wound site. These cells are known as the regeneration blastema. Thus, like appendage regeneration in lower vertebrates, disc regeneration involves wound healing followed by blastema formation (McClure, 2008).

Blastema cells are responsible for the regeneration and repatterning of the entire missing disc fragment. Thus, these cells exhibit remarkable developmental plasticity. For example, in anterior- only leg disc fragments, some blastema cells will switch to posterior identity and establish a novel posterior compartment in the regenerate. This anterior/posterior conversion occurs during heterotypic wound healing, when hedgehog (hh)- expressing peripodial cells induce ectopic engrailed (en) expression in the apposing anterior columnar cells. In addition, the disc blastema, like its vertebrate counterpart, is able to form a normal regenerate (complete leg disc and adult leg) when isolated from the remaining disc fragment. Regenerative plasticity is also observed when a few blastema cells switch fate to that of another disc type (e.g., leg-to-wing), in a phenomenon known as transdetermination. Transdetermination events are closely associated with regenerative disc growth. Clonal analysis, for example, has shown that blastema cells first regenerate the missing disc structures, and only then, are they competent to transdetermine (McClure, 2008).

Little is known about how the regeneration blastema forms in the fragmented leg disc, although ectopic Wingless (Wg/Wnt1) expression is detected along the cut site, both prior to and during blastema formation. Wg is a developmental signal in many different tissues and animals; in flies Wg patterns all of the imaginal discs, functioning as both a morphogen and mitogen to regulate disc cell fate and growth. In lower vertebrates, Wnt ligands are key regulators of blastema formation during epimorphic regeneration. Thus, activation of Wg within the disc blastema is potentially important for regeneration. This idea is consistent with the observation that ubiquitous expression of wg during the second or third larval instars, in unfragmented leg discs, is sufficient to induce a regeneration blastema in the proximodorsal region of the disc, known as the weak point. Moreover, ubiquitous expression of wg mimics the pattern deviations associated with leg disc fragmentation and subsequent regeneration, including the duplication of ventral with concomitant loss of dorsal pattern elements and leg-to-wing transdetermination events. Thus, leg disc regeneration can be examined using two experimental protocols: fragmentation or ubiquitous wg expression. However, it is important to point out that only fragmentation-induced regeneration involves wound healing (McClure, 2008).

Precisely which molecules and signaling pathways are required for the process of regeneration remain poorly understood, partly because the organisms historically used to study regeneration (e.g., newts and salamanders) have been refractory to genetics and molecular manipulations. Recently, however, the use of new genetic techniques together with 'regeneration' model systems -- such as planarians, hydra and zebrafish have given researchers the opportunity to examine the mechanisms of regeneration and to identify the genes, proteins and signaling pathways that regulate different regenerative processes. For example, a large scale RNAi-based screen was performed to survey gene function in planarian tissue homeostasis and regeneration. Out of ~1000 genes examined, RNAi knock-down of 240 displayed regeneration-related phenotypes, including defects in wound healing, blastema formation and blastema cell differentiation. Despite these studies, however, it remains unclear whether regeneration requires only the modulation of genes expressed at the time of injury, the reactivation of earlier developmental genes and/or signaling pathways, or the activation of novel genes specific to the process of regeneration. Thus, a major interest in the field of regenerative biology is the identification of gene products that regulate blastema formation, blastema growth and regenerative cellular plasticity. A genetic screen, using wg-induced leg disc regeneration, aimed at identifying genes that regulate cellular plasticity and regeneration using Drosophila was carried out prothoracic leg discs. A collection of 97 recessive lethal P-element lacZ (PZ) insertion lines were screened for ectopic lacZ expression during wg-induced leg disc regeneration, and six genes were identified that function in wg-induced leg disc regeneration, including genes with functional ties to Wg signaling as well as chromatin remodeling proteins (McClure, 2008).

This study consisted of an enhancer trap screen designed to identify genes with changed gene expression during leg disc regeneration as well as required for regenerative proliferation and growth. The screen identified 19 genes that when heterozygous mutant (PZ/+), dominantly modify wg-induced leg-to-wing transdetermination, which serves as a functional assay for disc regeneration. Of the 19 genes, 37% are transcription factors or involved in transcriptional regulation (tai, Krh1, ken, jing, combgap (cg), rpd3 and Aly), 21% function in cell cycle regulation and growth (oho23B, S6k, polo and cycA), 10.5% play a role in protein secretion (Secβ61 and Syx13), and 31% are of other or unknown function [l(3)01629, CG30947, l(2)00248, l(3)05203, l(3)01344, Nup154]. The identification of transcription factors as the most frequent class of genes that modify wg-induced leg disc regeneration was similarly observed in a DNA microarray screen designed to identify genes enriched in leg disc cells that transdetermine to wing (Klebes, 2005). Together, these findings strongly suggest that transcription factors and their downstream targets play a prominent role in disc cell plasticity (McClure, 2008).

Using lacZ expression analyses, together with whole mount in situ hybridization experiments, the expression patterns of the 19 genes that modified wg-induced leg-to-wing transdetermination were verified. This analysis identified several different expression patterns upon wg-induced regeneration, including a loss of gene expression, ubiquitous expression and genes with expression limited to the regeneration blastema. Such observations indicate that a complex change of gene expression, both negative and positive, mediates the process of epimorphic regeneration. Six (jing, Alyi cg, rpd3, Kr-h1 and S6k) of the 19 modifiers displayed expression limited to the regeneration blastema, indicating that novel markers of regeneration and transdetermination have been identified. The blastema-specific expression patterns of jing, Aly, cg, Kr-h1, rpd3 and S6k raised the intriguing possibility that these genes may be functionally involved in the formation, cell proliferation or maintenance of the blastema during disc regeneration. Indeed, upon ubiquitous wg expression jing/+ animals rarely formed a regeneration blastema, indicating that two wild-type copies of jing are required for the initiation of the regenerative process. In contrast, Aly/+ and cg/+ animals formed a normal blastema, but only after a one-day delay. Therefore, two wild-type copies of the Aly and cg genes are required for the proper timing of regeneration. In addition, it was found that the frequency of blastema formation was reduced in rpd3/+ animals, implicating this gene in the process of regeneration. Interestingly, heterozygous mutations in all four of these genes (jing, Aly, cg and rpd3) strongly suppress wg-induced leg-to-wing transdetermination. It is speculated that the transdetermination frequency declines in these mutant animals because the initiation and/or timing of blastema formation is delayed. This idea is consistent with all previous work which has shown that blastema cells are only competent to transdetermine after they have regenerated the missing disc structures. Heterozygous mutations in Kr-h1 and S6k did not significantly alter the formation of the wg-induced regeneration blastema, however, these genes did affect regeneration-induced transdetermination. Such results suggest that Kr-h1 and S6k specifically function to modulate the cell fate changes that occur as a consequence of regeneration (McClure, 2008).

Investigations into the molecular basis of transdetermination have shown that inputs from the Wg, Decapentapelagic (Dpp) and Hedgehog (Hh) signaling pathways activate key selector genes out of their normal developmental context, such as ectopic Vg activation in the leg disc, which then drives cell-fate switches. Several of the genes identified in this screen have functional ties to Wg, Dpp and Hh signaling pathways. For example, Cg is a zinc-finger transcription factor that is required for proper transcriptional regulation of the Hh signaling effector gene Cubitus interruptus (Ci). In cg mutant wing and leg discs, Ci expression is lowered in the anterior compartment, resulting in the ectopic activation of wg and dpp and significant disc overgrowth. Another gene identified in this screen -- ken, functions in concert with Dpp to direct the development of the Drosophila terminalia. Further characterizations of whether these genes and other modifiers of transdetermination and regeneration affect Wg, Dpp and Hh expression and/or signaling may shed light on the regulation of regeneration and regeneration-induced proliferation and cell fate plasticity (McClure, 2008).

Regulation of neurogenesis and epidermal growth factor receptor signaling by the Insulin receptor/Target of rapamycin pathway in Drosophila

Determining how growth and differentiation are coordinated is key to understanding normal development, as well as disease states such as cancer, where that control is lost. Growth and neuronal differentiation are coordinated by the insulin receptor/target of rapamycin (TOR) kinase (InR/TOR) pathway. The control of growth and differentiation diverge downstream of TOR. TOR regulates growth by controlling the activity of S6 kinase (S6K) and eIF4E. Loss of s6k delays differentiation, and is epistatic to the loss of tsc2, indicating that S6K acts downstream or in parallel to TOR in differentiation as in growth. However, loss of eIF4E inhibits growth but does not affect the timing of differentiation. This study shows that there is crosstalk between the InR/TOR pathway and epidermal growth factor receptor (EGFR) signaling. InR/TOR signaling regulates the expression of several EGFR pathway components including pointedP2 (pntP2). In addition, reduction of EGFR signaling levels phenocopies inhibition of the InR/TOR pathway in the regulation of differentiation. Together these data suggest that InR/TOR signaling regulates the timing of differentiation through modulation of EGFR target genes in developing photoreceptors (McNeill, 2008).

Tight coordination of growth and differentiation is essential for normal development. InR/TOR signaling controls the timing of neuronal differentiation in the eye and leg in Drosophila. This study demonstrates that the InR/TOR pathway regulates neuronal differentiation in an S6K-dependent, but 4EBP/eIF4E-independent manner. It has previously been impossible to determine whether InR/TOR signaling was acting downstream or in parallel to the EGFR/MAPK pathway. Using argos and rho as reporters this study shows that the InR/TOR pathway is able to regulate EGFR/MAPK signaling downstream of MAPK. Moreover, pntP2 expression is up- and downregulated by activation or inhibition of InR/TOR signaling, respectively, and InR/TOR and EGFR pathways interact through pntP2. Taken together these data suggest that temporal control of differentiation by the InR/TOR pathway is achieved by modulation of EGFR pathway transcriptional targets in differentiating PRs (McNeill, 2008).

TOR is part of two multimeric complexes (TORC1 and TORC2) and is a core component of the InR pathway. TORC1 activity is regulated by nutrient and energy levels providing a conduit for hormonal and catabolic cellular inputs. Growth is regulated by two downstream targets of TORC1: S6K and 4EBP. The current data demonstrate that upstream of TORC1, differentiation and growth are regulated by the same factors. Downstream of TORC1, differentiation and growth differ significantly in that loss of s6k, but not eIF4E (or overexpression of 4EBP) affects differentiation. eIF4E regulates 7-methyl-guanosine cap-dependent translation and is the rate-limiting factor in translation initiation. The finding that eIF4E does not affect differentiation suggests that the temporal control of differentiation is not based on a translation initiation-dependent mechanism. Strikingly, loss of s6k blocks the precocious differentiation induced by loss of tsc2. Given the relatively weak effects of loss of s6k this may seem surprising. However, the degree of suppression is similar to the effect of loss of s6k on the overgrowth phenotype caused by loss of tsc2, namely, tsc2, s6k double-mutant cells are the same size as wild-type cells. Although loss of eIF4E has no affect on differentiation it may act redundantly with another factor, such as s6k. Testing this hypothesis though is technically challenging since the Drosophila genome contains eight different eIF4E isoforms. It will be interesting in future to test whether any of these isoforms regulate differentiation or alternatively whether eIF4E and s6k act redundantly. Although further work is required to determine the precise relationship between S6K and the InR/TOR pathway, the data point to a critical role of S6K in coordinating neuronal differentiation and growth (McNeill, 2008).

As in other neuronal systems, differentiation of PRs in the Drosophila eye occurs in a stereotyped manner. The advantage of the Drosophila retina as an experimental system is that the PRs differentiate spatiotemporally. Using this feature, as well as a series of cell-type-specific antibodies, this study has demonstrated that InR/TOR signaling is selective in the cell-types that it affects. The differentiation of PRs 2/5, 3/4, and 8 are unaffected by perturbations in InR/TOR signaling, whereas PRs 1, 6, and 7 and cone cells are dependent on this pathway for temporal control of differentiation. Interestingly the affected cells all differentiate after the second mitotic wave. However, regulators of the cell cycle do not affect the temporal control of differentiation. Why then are PRs 1, 6, and 7 and cone cells specifically affected? In cells with increased InR/TOR signaling, the expression of argos, rho, and pntP2 is precocious and increased throughout the clone, suggesting that the upregulation of EGFR signaling occurs in all cells. However, decreasing EGFR activity using a hypomorphic pntP2 allele specifically affects the differentiation of PRs 1, 6, and 7 and cone cells. Interestingly, pntP2 expression in differentiated cells is also restricted to PRs 1, 6, and 7 and cone cells. These observations suggest that differentiation of PRs 1, 6, and 7 and cone cells is critically dependent on EGFR levels signaling through pntP2. Therefore, although activation of InR/TOR signaling causes upregulation of EGFR transcriptional targets in all cells as they differentiate, the phenotypic effect is seen only in PRs 1, 6, and 7 and cone cells since these cells are highly sensitive to EGFR activity signaling through pntP2. This possibility is supported by the fact that precocious differentiation caused by overexpression of Dp110 can be suppressed by the simultaneous reduction of pntP2 levels. The complete suppression of the Dp110 differentiation phenotype by simultaneous reduction of pntP2 strongly suggests that pntP2 acts downstream of Dp110 and InR/TOR signaling in a pathway that regulates the temporal control of differentiation. It has been suggested that later differentiating PRs require higher levels of EGFR activity than their earlier differentiating neighbors. In particular, the activation of PR 7 requires both EGFR and Sevenless RTKs. In the case of InR/TOR pathway activation it may be that, through its regulation of EGFR downstream targets, the 'second burst' of RTK activity is enhanced causing PRs 1, 6, and 7 and cone cells to differentiate precociously. There may also be other as yet unidentified factors through which the InR/TOR pathway controls the expression of Aos and rho in PRs 2-5 and 8 (McNeill, 2008).

Activation of insulin and insulin-like growth factor receptors in mammalian systems is well known to elicit a response via the Ras/MAPK pathway. However, loss of the InR in the Drosophila eye does not result in a loss of PRs, a hallmark of the Ras pathway, nor does mutation of the putative Drk binding site in chico affect the function of the Drosophila IRS. In accordance with these data no change is seen in dpERK staining when the InR/TOR pathway is activated in the eye disc. Rather than a direct activation of Ras signaling by the InR, the data suggest that in the developing eye crosstalk between these pathways occurs at the level of regulation of the expression of EGFR transcriptional outputs. The most proximal component of the EGFR pathway that is regulated by InR/TOR signaling is pntP2. However, the data suggest that temporal control of PR differentiation requires concerted regulation of EGFR transcriptional outputs, since overexpression of pntP2 alone is not sufficient to cause precocious differentiation, whereas overexpression of activated EGFR is sufficient. Interestingly, microarray analyses of Drosophila and human cells have shown that the InR/TOR pathway regulates the expression of hundreds of genes. The mechanism by which this transcriptional control is exerted has yet to be elucidated. It will be interesting in future to determine the extent of transcriptional crosstalk between InR/TOR and EGFR pathways in developing neurons (McNeill, 2008).

Drosophila RSK negatively regulates bouton number at the neuromuscular junction

Ribosomal S6 kinases (RSKs) are growth factor-regulated serine-threonine kinases participating in the RAS-ERK signaling pathway. RSKs have been implicated in memory formation in mammals and flies. To characterize the function of RSK at the synapse level, the effect was investigated of mutations in the rsk gene on the neuromuscular junction (NMJ) in Drosophila larvae. Immunostaining revealed transgenic expressed RSK in presynaptic regions. In mutants with a full deletion or an N-terminal partial deletion of rsk, an increased bouton number was found. Restoring the wild-type rsk function in the null mutant with a genomic rescue construct reverted the synaptic phenotype, and overexpression of the rsk-cDNA in motoneurons reduced bouton numbers. Based on previous observations that RSK interacts with the Drosophila ERK homologue Rolled, genetic epistasis experiments were performed with loss- and gain-of-function mutations in Rolled. These experiments provided evidence that RSK mediates its negative effect on bouton formation at the Drosophila NMJ by inhibition of ERK signaling (Fischer, 2009).

This study investigated the effect of rsk loss of function mutations in Drosophila, and found higher numbers of synaptic boutons in these mutants. The effect could be rescued by transgenic rsk expression. Vice versa overexpression of RSK reduced bouton numbers. Furthermore, removal of one allele of the Drosophila erk/rl gene normalized the effect of rsk loss of function on bouton formation, indicating that RSK mediates its effect through ERK/RL. Indeed, RSK and ERK/RL proteins interact directly with each other, and this interaction is abolished in the rlSem mutant. Furthermore, rlSem mutants show enhanced bouton numbers, similarly as rsk mutants, indicating that RSK negatively regulates ERK/RL activity at the NMJ and thus modulates bouton formation (Fischer, 2009).

A role of vertebrate RSK2 in inhibition of the RAS/ERK pathway has been proposed in several studies, but different underlying mechanisms have been suggested. In isolated mouse motoneurons, RSK2 is a negative regulator of axon growth by inhibiting ERK phosphorylation. In skeletal muscles of RSK2 knock-out mice, increased ERK activation has been observed. This could be explained by lack of inhibition of the ERK pathway via RAS guanine exchange factor SOS. In Drosophila, this inhibition seems not to occur through SOS. Knockdown of RSK2 leads to increased ERK phosphorylation in PC12 cells and cortical neurons. Moreover basal and 5HT2A receptor-mediated ERK 1/2 phosphorylation is increased in RSK2 knock-out fibroblasts These data are consistent with the current results showing that RSK interacts with ERK/RL and that this interaction leads to inhibition of ERK/RL activity in bouton formation at the NMJ (Fischer, 2009).

Previous studies on RSK and RL in the developing eye and wing imaginal disc provided evidence that RSK inhibits translocation of ERK/RL from the cytoplasm to the nucleus and thereby controls RL dependent gene transcription. However, the NMJ constitutes a separate part of the cell, and it is also conceivable that the effects of RSK and RL are mediated locally and do not involve nuclear translocation of these proteins. RSK seems to be present in the presynapse, but its distribution is diffuse and not restricted to active zones. This corresponds to the known distribution of RL in axon terminals. Thus, it is possible that RSK determines the localization of RL within synaptic boutons. Interestingly, an antibody that only recognizes active, phosphorylated RL showed a restricted localization to spots most likely corresponding to active zones. Thus one could speculate that RSK binds ERK/RL in axon terminals, thus inhibiting its activation, and only ERK/RL that is unbound can be activated by phosphorylation and move to active zones (Fischer, 2009).

In conclusion, these data indicate that RSK negatively regulates bouton formation at the NMJ, and that negative regulation of RL signaling is involved in this effect. Thus, Drosophila RSK seems to have a similar function as the RSK2 isoform in vertebrates. Therefore, the memory defects observed in flies, mice, and human CLS patients with mutations in rsk could be caused by dysregulated synapse architecture, as observed in the Drosophila model (Fischer, 2009).

The nuclear receptor DHR3 modulates dS6 kinase-dependent growth in Drosophila

S6 kinases (S6Ks) act to integrate nutrient and insulin signaling pathways and, as such, function as positive effectors in cell growth and organismal development. However, they also have been shown to play a key role in limiting insulin signaling and in mediating the autophagic response. To identify novel regulators of S6K signaling, a Drosophila-based, sensitized, gain-of-function genetic screen was used. Unexpectedly, one of the strongest enhancers to emerge from this screen was the nuclear receptor (NR), Drosophila hormone receptor 3 (DHR3), a critical constituent in the coordination of Drosophila metamorphosis. This study demonstrates that DHR3, through dS6K, also acts to regulate cell-autonomous growth. Moreover, the ligand-binding domain (LBD) of DHR3 was shown to be essential for mediating this response. Consistent with these findings, an endogenous DHR3 isoform was identified that lacks the DBD. These results provide the first molecular link between the dS6K pathway, critical in controlling nutrient-dependent growth, and that of DHR3, a major mediator of ecdysone signaling, which, acting together, coordinate metamorphosis (Montagne, 2009).

By using Drosophila genetics and a gain-of-function strategy, the NR, DHR3, was identified as an enhancer of a dS6K-regulated growth phenotype. This effect can be mediated by an isoform of DHR3 lacking the DBD. Moreover, using a revertant screening strategy, LBD-specific DHR3 mutants were generated, and it was demonstrated that the LBD of DHR3 is necessary to maintain normal growth and dS6K activity. In contrast to the role DHR3 plays in transcriptional regulation affecting the onset of metamorphosis, these studies indicate that it also plays a role in regulating cell-autonomous growth. These effects are most likely mediated through dS6K, as the ability of ectopically expressed DHR3-RS to drive growth in the dorsal wing blade is blunted in Drosophila deficient for dS6K. Consistent with these findings, it has been demonstrated that dS6K also controls cell growth in a cell-autonomous manner. However, the effect on cell size is more pronounced in dS6K mutants than in the DHR3-mutant clones described in this study. This may reflect the fact that dS6K activity is blunted, but not abolished, in DHR3 LBD-mutant larvae. Compatible with this hypothesis, it was found that in a dS6K P-element-induced mutant (P{PZ}S6K[07084]) no dS6K protein could be detected; however, this mutation induced a much less severe phenotype as compared with the dS6Kl-1 null mutation. In homozygous DHR3 mutant eyes both the size and the number of ommatidia were decreased, whereas in dS6K mutant flies the size reduction of the eye was only due to a decrease in ommatidia size but not number. This difference might be attributed to the experimental settings. In the current study, DHR3 mutant eyes were generated by mitotic recombination in a heterozygous Minute background, whose developmental delay is less than two days. In contrast, the size and number of ommatidia in dS6K mutant eyes were measured in homozygous mutant flies that exhibit a five-day delay at eclosion. The longer time for the latter to emerge as adults allows additional cell divisions to proceed, leading to a higher number of ommatidia (Montagne, 2009).

Previous studies demonstrated that DHR3 participates in a hierarchal regulatory circuit in response to ecdysone signaling, but also acts in a negative feedback loop to repress ecdysone receptor-mediated signaling. Prothoracic gland production of ecdysone is mediated by the brain neuropeptide prothoracicotropic hormone (PTTH). Recent studies in Drosophila have shown that genetic ablation of PTTH-producing neurons induces a delay in larval development and results in larger adult flies as a direct consequence of reduced levels of ecdysone. Interestingly, in the tobacco hornworm, Manduca sexta, PTTH-induced ecdysone production is paralleled by the phosphorylation of the Manduca orthologue of Drosophila ribosomal protein S6. Moreover, this process is sensitive to rapamycin, and a burst of dS6K activity is observed at early pupation. As the body size of the adult fly appears to be determined by growth regulators, including dS6K, as well as by hormones that control the timing of developmental windows, such as PTTH, the results suggest that the DHR3/dS6K regulatory module acts to integrate these two processes (Montagne, 2009).

These studies supports the existence of a novel DHR3 polypeptide devoid of a DBD, DHR3-PS. Nonetheless, although DHR3-PS is sufficient to potentiate a dS6K-dependent growth phenotype, it is not possible to exclude that the other DBD-containing DHR3 isoforms also contribute to dS6K activation. In general, DHR3, like other NRs, is a transcription factor composed of four elements: a modulator domain, the DBD, the hinge region, and the LBD. The DBD of NRs typically consists of two zinc fingers, with the first being critical for conferring DNA-binding specificity. Like DHR3-PS, NRs lacking a DBD have been previously reported. Notably, in Drosophila, the NR E75B, a DHR3 partner, lacks one of the 2 zinc fingers that is required to form a functional DBD. However, E75B, through its ability to interact with DHR3, modulates DHR3 transcriptional activity in a gas-responsive manner (Reinking, 2005). Like the putative DHR3-PS, the NR short heterodimer partner (SHP) in mammals is also devoid of DBD, but, as with E75B, it interacts with other NRs to modulate their transcriptional activity. It is unlikely that DHR3-PS behaves as a dominant-interfering effector of full-length DHR3 as ectopic DHR3-PS expression induces growth, whereas DHR3-RNAi inhibits growth. However, DHR3 also heterodimerizes with two NRs: E75 and the ecdysone receptor. Thus, in the case of E75, ectopically expressed DHR3-PS may act to decrease the levels of free E75, leaving full-length DHR3 free to increase the transcription of target genes. In contrast, DHR3-PS binding to the ecdysone receptor could counteract the negative growth regulation mediated by ecdysone signaling. However, it should be noted that the negative effects of ecdysone are humoral and mediated by dFOXO-inactivation within the fat body, whereas, as this study has shown, DHR3 regulates growth in a cell-autonomous manner. Moreover, dFOXO subcellular distribution was not altered in DHR3 mutant clones in third instar wing imaginal discs, indicating that the DHR3 cell-autonomous effect on cell growth is not mediated by the PKB/dFOXO signaling (Montagne, 2009).

In contrast to acting as a dominant-interfering isoform, the results presented in this study also suggest that DHR3 activates dS6K through a non-genomic mechanism, an effect of NRs that does not require the DBD function. Such a model is supported by NR responses whose kinetics are too rapid to be explained by de novo transcription and translation of a gene product. Indeed, nongenomic effects typically occur within minutes following addition of the cognate ligand and are resistant to transcriptional inhibitors. In the case of DHR3, it is experimentally difficult to address this question as the ligand for DHR3 is unknown, and a genetic endpoint is being scored for, resulting from events induced much earlier in larval development. It has been demonstrated that vitamin D3 and all-trans-retinoic acid both induce activation of S6K1 within minutes of administration to cells. Moreover, in the case of vitamin D3, it was shown that these effects were mediated through protein phosphatases PP1 and PP2A in a vitamin D3 receptor (VDR)-dependent manner. VDR appears to directly interact with the catalytic subunits of PPI and PP2A, and vitamin D3 acts to disrupt this interaction and enhance an interaction between VDR and S6K1, stabilizing S6K1 in its phosphorylated active state. However, depleting DHR3 levels by RNA interference blunts both dS6K T398 and d4E-BP T37/T46 phosphorylation, suggesting that DHR3 acts upstream or at the level of dTORC1. Identification of potential partners for DHR3-PS may be useful in determining, at the molecular level, the mechanism by which DHR3 controls cell growth and dS6K activity (Montagne, 2009).

The data further support the notion that a ligand exists for DHR3, and that the ligand is required for many of the pleiotropic activities of DHR3. Those NRs that bind steroid hormones are, in general, high-affinity receptors, whereas the low-affinity NRs bind ligands that are present in high concentration, such as dietary nutrients. The observation that an NR, generated by fusing the DHR3 LBD with the DBD of Gal4, is transcriptionally active in a number of specific embryonic and larval tissues suggests that such a ligand is widely present. Given the role of dTOR/dS6K as a nutritional effector, it is interesting to note that the chimeric DHR3/Gal4 NR is active in organs that provide basal nutrients, in particular, in a group of cells of the larval midgut, which are essential for the transfer of nutrients to the hemolymph. Importantly, the mammalian orthologues to DHR3 and its partner E75 are retinoid-related orphan receptor (ROR)α and Rev-erb (NR1D)α, respectively. As in Drosophila, the NR1D subgroup functions as dominant transcriptional silencers by inhibiting transactivation mediated by RORα. Interestingly, it was recently reported that RORα-deficient mice, like S6K1-deficient mice, exhibit reduced fat-pad mass, smaller adipocytes, and resistance to diet-induced obesity. Moreover, in solving the X-ray structure of the RORα LBD, it was revealed that cholesterol was bound in the ligand-binding pocket. While the Drosophila NR, DHR96, has recently been shown to bind cholesterol thereby modulating cholesterol homeostasis, this does not exclude the possibility that DHR3 could also bind cholesterol. However, the predicted models of the structure of DHR3 indicate that the size of the ligand-binding pocket is smaller than those of either RORα or RORβ. Given the role of the mTOR/S6K1 nutrient-responsive pathway in mammals, it raises the possibility that DHR3 is a low-affinity receptor for an abundant nutrient ligand. Identification of this specific ligand constitutes the next issue to investigate (Montagne, 2009).

Rapamycin activation of 4E-BP prevents parkinsonian dopaminergic neuron loss

Mutations in PINK1 and parkin cause autosomal recessive parkinsonism, a neurodegenerative disorder characterized by the loss of dopaminergic neurons. To highlight potential therapeutic pathways, this study identified factors that genetically interact with parkin/PINK1. Overexpression of the translation inhibitor 4E-BP can suppress all pathologic phenotypes including degeneration of dopaminergic neurons in Drosophila. 4E-BP is activated in vivo by the TOR inhibitor rapamycin, which can potently suppress pathology in PINK1/parkin mutants. Rapamycin also ameliorates mitochondrial defects in cells from parkin-mutant patients. Recently, 4E-BP was shown to be inhibited by the most common cause of parkinsonism, dominant mutations in LRRK2. This study further shows that loss of the Drosophila LRRK2 homolog activates 4E-BP and is also able to suppress PINK1/parkin pathology. Thus, in conjunction with recent findings these results suggest that pharmacologic stimulation of 4E-BP activity may represent a viable therapeutic approach for multiple forms of parkinsonism (Tain, 2009).

This study used Drosophila as a model system to uncover genetic suppressors in order to understand the pathogenic mechanisms and to highlight putative therapeutic pathways for PD. Thor, the sole Drosophila homolog of mammalian 4E-BP1, has been identified as a genetic modifier of parkin. In the present study, the genetic interaction of Thor with parkin and PINK1 was investigated. While loss-of-function mutations in Thor dramatically decrease parkin and PINK1 mutant viability, overexpression of 4E-BP is able to suppress PINK1 and parkin mutant phenotypes, including degeneration of dopaminergic neurons. These results suggest that 4E-BP acts to mediate or promote a survival response implemented upon loss of parkin or PINK1 (Tain, 2009).

4E-BP1 is an inhibitor of 5' cap-dependent protein translation, which is known to play an important role in cellular response to changes in environmental conditions such as altered nutrient levels and various physiological stresses. It has been demonstrated that Drosophila 4E-BP is important for survival under a wide variety of stresses including starvation, oxidative stress, unfolded protein stress and immune challenge. Such a response pathway represents a likely target for possible manipulation by therapeutics. Fenetic evidence supports this idea, hence, this study attempted to validate whether this represented a viable therapeutic target (Tain, 2009).

4E-BP activity is regulated post-translationally by the TOR signaling pathway. Activated TOR hyper-phosphorylates 4E-BP inhibiting it leading to promotion of 5' cap-dependent translation. Rapamycin is a small molecule inhibitor of TOR signaling and has been shown to lead to 4E-BP hypo-phosphorylation. Genetic evidence suggested that administration of rapamycin to parkin/PINK1 mutants should relieve 4E-BP inhibition and confer a protective effect. Exposing mutant animals to rapamycin during development caused an increase in hypo-phosphorylated 4E-BP and, remarkably, was sufficient to suppress all pathologic phenotypes, including muscle degeneration, mitochondrial defects and locomotor ability. Continued administration of rapamycin during aging also completely suppressed progressive degeneration of dopaminergic neurons (Tain, 2009).

To validate this pathway as a viable target for therapy, the studies were extended to human tissue. There is growing evidence that mitochondrial dysfunction is a key pathologic event across the spectrum of parkinsonism. Mitochondrial defects have been demonstrated in a number of cell lines derived from patients with parkin mutations. This study shows that rapamycin is also capable of ameliorating mitochondrial bioenergetic and morphological defects in parkin-deficient PD patient cell lines. Thus, the results provide strong support for the proposition that modulating 4E-BP mediated translation by pharmaceuticals such as rapamycin can be efficacious in vivo and is relevant to human pathophysiology (Tain, 2009).

TOR signaling regulates a number of downstream effectors other than 4E-BP, for example, up-regulation of S6 kinase promoting protein synthesis and cell proliferation, and down-regulation of autophagy likely through inhibition of ATG1. The coordinated regulation of these pathways serves to optimize cellular activity in response to vital changes such as nutrient availability and environmental stresses. Stimulation of autophagy under nutrient-deprived conditions is a survival mechanism that recycles essential metabolic components, but this mechanism also promotes the degradation of aggregated or misfolded proteins. Thus, the potential therapeutic effects of rapamycin have been widely promoted as a strategy to combat a number of neurodegenerative diseases including PD primarily for its perceived role in promoting autophagic clearance of aggregated proteins. However, recent studies have provided compelling evidence that the pro-survival effects of rapamycin can be mediated in the absence of autophagy by reducing protein translation. This study has demonstrated that genetic ablation of 4E-BP is sufficient to completely abrogate any beneficial effects of rapamycin in vivo while inhibiting Atg5, a key mediator of autophagy, does not diminish the efficacy of rapamycin-mediated protection. Together, these results indicate that in this instance the major protective effects of rapamycin treatment are mediated through regulated protein translation, with little or no contribution from autophagy (Tain, 2009).

A switch from cap-dependent to cap-independent translation is likely to effect widespread changes in the proteome, particularly the induction of pro-survival factors including chaperones, anti-oxidants and detoxifying enzymes. In support of this, it was shown that transgenic or rapamycin-induced 4E-BP activation leads to increased protein levels of GstS1, a major detoxification enzyme in Drosophila. Interestingly, it was previously shown that transgenic overexpression of Drosophila GstS1 is able to suppress dopaminergic neuron loss in parkin mutants. Elucidating the global changes in response to 4E-BP activation will be crucial to understanding the exact molecular mechanisms of neuro-protection but currently remains unresolved (Tain, 2009).

The potential importance of 4E-BP modulation as a therapeutic target is underscored by recent findings that report the most common genetic cause of PD, dominant mutations in LRRK2, inhibit 4E-BP function through direct phosphorylation. Expression of these mutations causes disruption of dopaminergic neurons in Drosophila and mouse, however, in striking similarity to the current results, overexpression of 4E-BP can circumvent the pathogenic effects of mutant LRRK2 and prevent neurodegeneration (Imai, 2008) in Drosophila. This study shows that loss of Drosophila LRRK leads to activation of 4E-BP and can suppress pathology in PINK1 and parkin mutants. These data further support a link between LRRK2 and 4E-BP activity and a common cause of PD. Thus, the results indicate that promoting 4E-BP activity may be beneficial in preventing neurodegeneration in multiple forms of parkinsonism. Since 4E-BP activity can be manipulated by small molecule inhibitors such as rapamycin, this pathway represents a viable therapeutic target. It will be particularly interesting to determine whether rapamycin is efficacious in ameliorating pathologic phenotypes in the recently reported LRRK2 transgenic mouse model, but further studies will be necessary to determine whether pharmacologic modulation of 4E-BP function is therapeutically relevant in all forms of parkinsonism including sporadic PD (Tain, 2009).

Reduction of protein translation and activation of autophagy protect against PINK1 pathogenesis in Drosophila melanogaster

Mutations in PINK1 and Parkin cause familial, early onset Parkinson's disease. In Drosophila, PINK1 and Parkin mutants show similar phenotypes, such as swollen and dysfunctional mitochondria, muscle degeneration, energy depletion, and dopaminergic (DA) neuron loss. PINK1 and Parkin have been shown to genetically interact with the mitochondrial fusion/fission pathway, and PINK1 and Parkin have been proposed to form a mitochondrial quality control system that involves mitophagy. However, the in vivo relationships among PINK1/Parkin function, mitochondrial fission/fusion, and autophagy remain unclear; and other cellular events critical for PINK1 pathogenesis remain to be identified. This study shows that PINK1 genetically interacte with the protein translation pathway. Enhanced translation through S6K activation significantly exacerbates PINK1 mutant phenotypes, whereas reduction of translation shows suppression. Induction of autophagy by Atg1 overexpression also rescues PINK1 mutant phenotypes, even in the presence of activated S6K. Downregulation of translation and activation of autophagy are already manifested in PINK1 mutant, suggesting that they represent compensatory cellular responses to mitochondrial dysfunction caused by PINK1 inactivation, presumably serving to conserve energy. Interestingly, the enhanced PINK1 mutant phenotype in the presence of activated S6K can be fully rescued by Parkin, apparently in an autophagy-independent manner. These results reveal complex cellular responses to PINK1 inactivation and suggest novel therapeutic strategies through manipulation of the compensatory responses (Liu, 2010; full text of article)

Previously, PINK1 and Parkin have been suggested to interact with mitochondrial fusion/fission machinery and the autophagy pathway. This study found that PINK1 also genetically interacts with the protein translation pathway. Increased global protein translation with S6K or eIF4E over expression (OE) exacerbates PINK1 mutant phenotypes, while decreased translation has the opposite effects. Overexpression of constitutively active S6Ks dramatically enhances muscle and DA neuron degeneration in PINK1 mutant flies, which can be mitigated by the co-expression of RpS6 RNAi or RpS9 RNAi, supporting that the TOR/S6K pathway modifies PINK1 mutant phenotypes through regulating global translation. Recently, it has been reported that pathogenic leucine-rich repeat kinase 2 (LRRK2), which represents the most frequent molecular lesions found in Parkinson's disease, promotes 4E-BP phosphorylation, resulting in increased eIF4E-mediated translation, enhanced sensitivity to oxidative stress, and DA neuron loss. Taken together, these results support the idea that deregulated protein translation is generally involved in the pathogenesis of Parkinson's disease (Liu, 2010).

Deregulated translation affects Parkinson's disease pathogenesis most likely at the level of energy metabolism, since protein translation is a very energy-consuming process, of which ribosomal biogenesis is the most costly, consuming approximately 80% of the energy in proliferating cells. This study shows that forced upregulation of ribosomal biogenesis in the fly muscle by the overexpression of constitutively active S6K is well tolerated in WT flies; however, such manipulation in PINK1 RNAi flies completely abolishes their flight ability, depletes ATP in the muscle and enhances muscle and DA neuron degeneration. The tolerance of increased protein translation by wild type flies is probably due to the existence of an intact mitochondrial quality control system containing PINK1 and Parkin, which can either eliminate damaged mitochondria generated during elevated energy production or minimize damages caused by increased ROS generated during energy production. However, in PINK1 or Parkin mutants that lack a functional mitochondrial quality control system, increased protein translation and the corresponding energy demand will translate into increased ROS generation, accumulation of dysfunctional mitochondria, and eventual energy depletion and tissue degeneration. Since downregulation of translation through knockdown of S6K, RpS6, or RpS9 is beneficial to PINK1 mutant flies, and S6K activity is already tuned down in PINK1 mutant flies, reduction of translation likely represents one of the cellular compensatory responses to the energy deficit caused by mitochondrial dysfunction in PINK1 mutants. Interestingly, partial reduction of S6K activity prolonges fly lifespan, whereas increased S6K activity has the opposite effects on longevity. The effects of S6K on animal lifespan and PINK1 mutant phenotypes can both be explained by the energy metabolism hypothesis and they offer a tantalizing link between aging and the pathogenesis of Parkinson's disease (Liu, 2010).

Supporting the energy metabolism model, it was shown that downregulation of protein translation by knocking down positive regulators of translation (S6K, RpS6, RpS9) or overexpressing a negative regulator (4E-BP) could rescue PINK1 mutant phenotypes. These manipulations presumably act by preserving cellular energy and reducing the workload and ROS production of mitochondria. Previously, 4E-BP OE was suggested to rescue PINK1 mutant phenotype by upregulating Cap-independent translation of stress related genes, including antioxidant genes, and boosting antioxidant gene activity has been suggested as a therapeutic strategy in the PINK1 and Parkin models of Parkinson's disease. This study found that although overexpression of antioxidant genes, such as Catalase, GTPx-1, SOD and GstS1, all showed some degree of rescue of PINK1 mutant phenotypes, their effects were in general weaker than that of Atg1 OE, Parkin OE, or Marf RNAi, particularly in the PINK1 RNAi/S6K-TE OE background. These data suggest that increasing autophagy and mitochondrial fission might be better choices to combat PINK1-related Parkinson's disease (Liu, 2010).

Autophagy is a conserved cellular process through which cytoplasmic content or defective intracellular organelles can be eliminated or recycled. Although autophagy is usually induced under adverse conditions to provide means for survival, basal level of autophagy in the cell is just as critical to the physiological health of the organism, since defects in autophagy are frequently associated with cancer, neurodegeneration, and aging. The induction of autophagy leads to the de novo formation of double membrane structure called isolation membrane, which expands to form a sealed compartment named autophagosome that will engulf materials destined for degradation. The large size of mitochondria likely poses a challenge for the autophagy machinery, as engulfment of an entire mitochondrion requires a significant amount of building materials for autophagosome formation. This is especially the case in PINK1 mutant where dysfunctional mitochondria becomes grossly swollen or aggregated. Previously, it has been shown that increased mitochondrial fission or Parkin OE could efficiently rescue the enlarged mitochondria phenotype in PINK1 mutants. The rescuing effect by increased mitochondrial fission could be due to the fact that it decreases mitochondrial size and makes it easier for the autophagosome to engulf the entire mitochondrion during mitophagy. In addition, increased mitochondrial fission could facilitate the segregation of the healthy part of a mitochondrion from the unhealthy part, thus enhancing the selective elimination of dysfunctional mitochondria through mitophagy. Supporting the mitophagy model, Parkin has been proposed to promote the efficient removal of damaged mitochondria by selectively ubiquitinating proteins on damaged mitochondria. A key prediction of the mitophagy model is that the protective effects of Parkin OE and increased mitochondrial fission as in the case of Marf RNAi will depend on the autophagy pathway. Surprisingly, this study found that blocking autophagy through Atg1 RNAi or Atg18 RNAi failed to block Parkin OE or Marf RNAi's rescuing abilities in PINK1 mutant, although Atg18 RNAi was effective in blocking the rescuing ability of Atg1 OE. This result suggests that the rescuing effect of Parkin OE or Marf RNAi is not entirely dependent on autophagy, and that other processes are likely involved. For example, Parkin has been suggested to promote mitochondrial biogenesis and regulate protein translation. Further studies are needed to elucidate the exact molecular functions of Parkin that are critically involved in mitochondrial function and tissue maintenance in vivo (Liu, 2010).

Given the well-established catabolic role of autophagy in degrading cytoplasmic contents, it helps recycle nutrients and provide energy source needed for survival under harsh conditions. In PINK1 mutants that suffer energy deficit due to mitochondrial dysfunction, induction of autophagy would present as a compensatory response to cope with the limited energy supply. Indeed, this study found that basal autophagy is induced in PINK1 mutant, and further increase of autophagy through Atg1 OE protects against PINK1 pathogenesis. Thus, decreased translation and increased autophagy both represent compensatory responses in PINK1 mutant flies, and further augmentation of these responses can effectively protect against the toxic effects of PINK1 inactivation. A previous study in cultured mammalian cells also indicated that autophagy is induced in response to PINK1 inactivation. Thus, the in vivo compensatory responses revealed in this study are likely relevant to PINK1 pathogenesis in mammals. Pharmacological interventions that promote these responses offer potential new treatment strategies for Parkinson's disease (Liu, 2010).

Insulin/IGF-Regulated Size Scaling of Neuroendocrine Cells Expressing the bHLH Transcription Factor Dimmed in Drosophila

Neurons and other cells display a large variation in size in an organism. Thus, a fundamental question is how growth of individual cells and their organelles is regulated. Is size scaling of individual neurons regulated post-mitotically, independent of growth of the entire CNS? Although the role of insulin/IGF-signaling (IIS) in growth of tissues and whole organisms is well established, it is not known whether it regulates the size of individual neurons. The role of IIS in the size scaling of neurons in the Drosophila CNS was studied. By targeted genetic manipulations of insulin receptor (dInR) expression in a variety of neuron types it was demonstrated that the cell size is affected only in neuroendocrine cells specified by the bHLH transcription factor DimmedD (Dimm). Several populations of Dimm-positive neurons tested displayed enlarged cell bodies after overexpression of the dInR, as well as PI3 kinase and Akt1 (protein kinase B), whereas Dimm-negative neurons did not respond to dInR manipulations. Knockdown of these components produce the opposite phenotype. Increased growth can also be induced by targeted overexpression of nutrient-dependent TOR (target of rapamycin) signaling components, such as Rheb (small GTPase), Tor and S6K (S6 kinase). After Dimm-knockdown in neuroendocrine cells manipulations of dInR expression have significantly less effects on cell size. It was also shown that dInR expression in neuroendocrine cells can be altered by up or down-regulation of Dimm. This novel dInR-regulated size scaling is seen during postembryonic development, continues in the aging adult and is diet dependent. The increase in cell size includes cell body, axon terminations, nucleus and Golgi apparatus. It is suggested that the dInR-mediated scaling of neuroendocrine cells is part of a plasticity that adapts the secretory capacity to changing physiological conditions and nutrient-dependent organismal growth (Luo, 2013).


Baar, K. and Esser, K. (1999). Phosphorylation of p70(S6k) correlates with increased skeletal muscle mass following resistance exercise. Am. J. Physiol. 276(1 Pt 1): C120-7. PubMed Citation: 9886927

Balendran, A., et al. (1999). Evidence that 3-phosphoinositide-dependent protein kinase-1 mediates phosphorylation of p70 S6 kinase in vivo at Thr-412 as well as Thr-252. J. Biol. Chem. 274(52): 37400-6. PubMed Citation: 10601311

Barbet, N. C., Schneider, U., Helliwell, S. B., Stansfield, I., Tuite, M. F. and Hall, M. N. (1996). TOR controls translation initiation and early G1 progression in yeast. Mol. Biol. Cell 7: 25-42. PubMed Citation: 8741837

Beck, T. and Hall, M. N. (1999). The TOR signalling pathway controls nuclear localization of nutrient-regulated transcription factors. Nature 402: 689-692. PubMed Citation: 10604478

Brennan, P., et al. (1999). p70(s6k) integrates phosphatidylinositol 3-kinase and rapamycin-regulated signals for E2F regulation in T lymphocytes. Mol. Cell Biol. 19(7): 4729-38. PubMed Citation: 10373522

Bryk, B., Hahn, K., Cohen, S. M. and Teleman, A. A. (2010). MAP4K3 regulates body size and metabolism in Drosophila. Dev. Biol. 344(1): 150-7. PubMed Citation: 20457147

Burnett, P. E., et al. (1998). RAFT1 phosphorylation of the translational regulators p70 S6 kinase and 4E-BP1. Proc. Natl. Acad. Sci. 95(4): 1432-7. PubMed Citation: 9465032

Cardenas, M. E., et al. (1999). The TOR signaling cascade regulates gene expression in response to nutrients. Genes Dev. 13: 3271-3279. PubMed Citation: 10617575

Chen, D., Li, P. W., Goldstein, B. A., Cai, W., Thomas, E. L., Chen, F., Hubbard, A. E., Melov, S. and Kapahi, P. (2013). Germline signaling mediates the synergistically prolonged longevity produced by double mutations in daf-2 and rsks-1 in C. elegans. Cell Rep 5: 1600-1610. PubMed ID: 24332851; Graphical Abstract

Cheng, L., Locke, C. and Davis, G. W. (2011). S6 kinase localizes to the presynaptic active zone and functions with PDK1 to control synapse development. J Cell Biol 194: 921-935. PubMed ID: 21930778

Cheng, L. Y., Bailey, A. P., Leevers, S. J., Ragan, T. J., Driscoll, P. C. and Gould, A. P. (2011). Anaplastic lymphoma kinase spares organ growth during nutrient restriction in Drosophila. Cell 146: 435-447. PubMed ID: 21816278

Choi, J. H., et al. (2000). TOR signaling regulates microtubule structure and function. Curr. Biol. 10: 861-864. PubMed Citation: 10899009

Chou, M. M. and Blenis, J. (1996). The 70 kDa S6 kinase complexes with and is activated by the Rho family G proteins Cdc42 and Rac1. Cell 85: 573-583. 8653792

Csibi, A., Lee, G., Yoon, S. O., Tong, H., Ilter, D., Elia, I., Fendt, S. M., Roberts, T. M. and Blenis, J. (2014). The mTORC1/S6K1 pathway regulates glutamine metabolism through the eIF4B-cependent control of c-Myc translation. Curr Biol 24: 2274-2280. PubMed ID: 25220053

Duan, C., Liimatta, M. B. and Bottum, O. L. (1999). Insulin-like growth factor (IGF)-I regulates IGF-binding protein-5 gene expression through the phosphatidylinositol 3-kinase, protein kinase B/Akt, and p70 S6 kinase signaling pathway. J. Biol. Chem. 274(52): 37147-53. PubMed Citation: 10601276

Dufner, A. and Thomas, G. (2000). Ribosomal S6 kinase signaling and the control of translation. Exp. Cell Res. 253(1): 100-9. PubMed Citation: 10579915

Fang, Y., et al. (2003). PLD1 regulates mTOR signaling and mediates Cdc42 activation of S6K1. Curr. Biol. 13: 2037-2044. 14653992

Findlay, G. M., Yan, L., Procter, J., Mieulet, V. and Lamb, R. F. (2007). A MAP4 kinase related to Ste20 is a nutrient-sensitive regulator of mTOR signalling. Biochem. J. 403(1): 13-20. PubMed Citation: 17253963

Fischer, M., Raabe, T., Heisenberg, M. and Sendtner, M. (2009). Drosophila RSK negatively regulates bouton number at the neuromuscular junction. Dev Neurobiol 69: 212-220. PubMed ID: 19160443

Garami, A., et al. (2003). Insulin activation of Rheb, a mediator of mTOR/S6K/4E-BP signaling, is inhibited by TSC1 and 2. Mol. Cell 11(6): 1457-66. 12820960

Hara, K., et al. (1998). Amino acid sufficiency and mTOR regulate p70 S6 kinase and eIF-4E BP1 through a common effector mechanism. J. Biol. Chem. 273: 14484-14494. PubMed Citation: 9603962

Hardwick, J. S., et al. (1999). Rapamycin-modulated transcription defines the subset of nutrient-sensitive signaling pathways directly controlled by the Tor proteins. Proc. Natl. Acad. Sci. 96(26): 14866-70. PubMed Citation: 10611304

Hari, K. L., et al. (1995). The mei-41 gene of D. melanogaster is a structural and functional homolog of the human ataxia telangiectasia gene. Cell 82: 815-821. PubMed Citation: 7671309

Hennig, K.M. and Neufeld, T.P. (2002). Inhibition of cellular growth and proliferation by dTOR overexpression in Drosophila. Genesis 34: 107-110. 12324961

Hsiu-Ling, Li., Davis, W. and Pure, E. (1999). Suboptimal cross-linking of antigen receptor induces Syk-dependent activation of p70S6 kinase through Protein kinase C and phosphoinositol 3-kinase. J. Biol. Chem. 274: 9812-9820. PubMed Citation: 10092671

Hsu, Y.-C., Chern, J. J., Cai, Y., Liu, M. and Choi, K.-W. (2007). Drosophila TCTP is essential for growth and proliferation through regulation of dRheb GTPase. Nature 445: 785-788. PubMed Citation: 17301792

Inoki, K., Li, Y., Xu, T. and Guan, K. L. (2003). Rheb GTPase is a direct target of TSC2 GAP activity and regulates mTOR signaling. Genes Dev. 17(15): 1829-34. 12869586

Isotani, S., et al. (2000). Immunopurified mammalian target of rapamycin phosphorylates and activates p70 S6 kinase alpha in vitro. J. Biol. Chem. 274(48): 34493-8. PubMed Citation: 10567431

Kamada, Y., Funakoshi, T., Shintani, T., Nagano, K., Ohsumi, M. and Ohsumi, Y. (2000). Tor-mediated induction of autophagy via an Apg1 protein kinase complex. J. Cell Biol. 150: 1507-1513. 10995454

Kapahi, K., et al. (2004). Regulation of lifespan in Drosophila by modulation of genes in the TOR signaling pathway. Curr. Biol. 14: 885-890. 15186745

Kawasome, H., et al. (1998). Targeted disruption of p70(s6k) defines its role in protein synthesis and rapamycin sensitivity. Proc. Natl. Acad. Sci. 95(9): 5033-8. PubMed ID: 9560223

Kelleher, R. J., et al. (2004). Translational control by MAPK signaling in long-term synaptic plasticity and memory. Cell 116: 467-479. 15016380

Kim, S., et al. (2000). Extracellular zinc activates p70 S6 kinase through the phosphatidylinositol 3-kinase signaling pathway. J. Biol. Chem. 275(34): 25979-84. PubMed ID: 10851233

Kim, D. H., et al. (2003). GbetaL, a positive regulator of the rapamycin-sensitive pathway required for the nutrient-sensitive interaction between raptor and mTOR. Mol. Cell 11(4): 895-904. 12718876

Kirisako, T., Baba, M., Ishihara, N., Miyazawa, K., Ohsumi, M., Yoshimori, T., Noda, T. and Ohsumi, Y. (1999). Formation process of autophagosome is traced with Apg8/Aut7p in yeast. J. Cell Biol. 147: 435-446. 10525546

Klebes, A., et al. (2005). Regulation of cellular plasticity in Drosophila imaginal disc cells by the Polycomb group, trithorax group and lama genes. Development 132: 3753-3765. PubMed citation: 16077094

Kumar, V., et al. (2000a). Functional interaction between RAFT1/FRAP/mTOR and protein kinase cdelta in the regulation of cap-dependent initiation of translation. EMBO J. 19: 1087-1097. PubMed Citation: 10698949

Kumar, V., et al. (2000b). Regulation of the rapamycin and FKBP-target 1/mammalian target of rapamycin and cap-dependent initiation of translation by the c-Abl protein-tyrosine kinase. J. Biol. Chem. 275(15): 10779-87. PubMed Citation: 10753870

Kwiatkowski, D., et al. (2002). A mouse model of TSC1 reveals sex-dependent lethality from liver hemangiomas, and up-regulation of p70S6 kinase activity in TSC1 null cells. Hum. Mol. Genet. 11: 525-534. 11875047

Lee, S. B., et al. (2007). ATG1, an autophagy regulator, inhibits cell growth by negatively regulating S6 kinase. EMBO Rep. 8(4): 360-5. PubMed Citation: 17347671

Lin, Y. H., Chen, Y. C., Kao, T. Y., Lin, Y. C., Hsu, T. E., Wu, Y. C., Ja, W. W., Brummel, T. J., Kapahi, P., Yuh, C. H., Yu, L. K., Lin, Z. H., You, R. J., Jhong, Y. T. and Wang, H. D. (2014). Diacylglycerol lipase regulates lifespan and oxidative stress response by inversely modulating TOR signaling in Drosophila and C. elegans. Aging Cell [Epub ahead of print]. PubMed ID: 24889782

Liu, H., Feng, X., Ennis, K. N., Behrmann, C. A., Sarma, P., Jiang, T. T., Kofuji, S., Niu, L., Stratton, Y., Thomas, H. E., Yoon, S. O., Sasaki, A. T. and Plas, D. R. (2017). Pharmacologic targeting of S6K1 in PTEN-deficient neoplasia. Cell Rep 18(9): 2088-2095. PubMed ID: 28249155

Liu, S. and Lu, B. (2010). Reduction of protein translation and activation of autophagy protect against PINK1 pathogenesis in Drosophila melanogaster. PLoS Genet. 6(12): e1001237. PubMed Citation: 21151574

Luo, J., Liu, Y. and Nassel, D. R. (2013). Insulin/IGF-Regulated Size Scaling of Neuroendocrine Cells Expressing the bHLH Transcription Factor Dimmed in Drosophila. PLoS Genet 9: e1004052. PubMed ID: 24385933

Lustig, Y., et al. (2011). Separation of the gluconeogenic and mitochondrial functions of PGC-1α through S6 kinase. Genes Dev. 25(12): 1232-44. PubMed Citation: 21646374

Manning, B. D., et al. (2002). Identification of the tuberous sclerosis complex-2 tumor suppressor gene product Tuberin as a target of the Phosphoinositide 3-kinase/Akt pathway. Molec. Cell 10: 151-162. 12150915

McClure, K. D. and Schubiger, G. (2008). A screen for genes that function in leg disc regeneration in Drosophila melanogaster. Mech. Dev. 125(1-2): 67-80. PubMed citation

McNeill, H., Craig, G. M. and Bateman, J. M. (2008). Regulation of neurogenesis and epidermal growth factor receptor signaling by the Insulin receptor/Target of rapamycin pathway in Drosophila. Genetics 179: 843-853. PubMed Citation: 18505882

Miron, M., Lasko, P. and Sonenberg, N. (2003). Signaling from Akt to FRAP/TOR targets both 4E-BP and S6K in Drosophila melanogaster. Mol. Cell. Biol. 23(24): 9117-26. 14645523

Montagne, J., et al. (1999). Drosophila S6 kinase: a regulator of cell size. Science. 285(5436): 2126-9. PubMed Citation: 10497130

Montagne, J., et al. (2009). The nuclear receptor DHR3 modulates dS6 kinase-dependent growth in Drosophila. PLoS Genet. 6: e1000937. PubMed Citation: 20463884

Mori, H., et al. (2000). 14-3-3tau associates with a translational control factor FKBP12-rapamycin-associated protein in T-cells after stimulation by pervanadate. FEBS Lett. 467: 61-64. PubMed ID: 10664457

Nagarajan, S. and Grewal, S. S. (2014). An investigation of nutrient-dependent mRNA translation in Drosophila larvae. Biol Open 3(11):1020-31. PubMed ID: 25305039

Oldham, S., et al. (2000). Genetic and biochemical characterization of dTOR, the Drosophila homolog of the target of rapamycin. Genes Dev. 14: 2689-2694. 0523807

Parekh, D., et al. (1999). Mammalian TOR controls one of two kinase pathways acting upon nPKCdelta and nPKCepsilon. J. Biol. Chem. 274: 34758-34764. PubMed ID: 10574945

Patel, P. H., et al. (2003). Drosophila Rheb GTPase is required for cell cycle progression and cell growth. J. Cell Sci. 116(Pt 17): 3601-10. 12893813

Pende, M., Um, S. H., Mieulet, V., Sticker, M., Goss, V. L., Mestan, J., Mueller, M., Fumagalli, S., Kozma, S. C. and Thomas, G. (2004). S6K1(-/-)/S6K2(-/-) mice exhibit perinatal lethality and rapamycin-sensitive 5?-terminal oligopyrimidine mRNA translation and reveal a mitogen-activated protein kinase-dependent S6 kinase pathway. Mol. Cell. Biol. 24: 3112-3124. 15060135

Peterson, R. T., et al. (1999). Protein phosphatase 2A interacts with the 70-kDa S6 kinase and is activated by inhibition of FKBP12-rapamycin associated protein. Proc. Natl. Acad. Sci. 96(8): 4438-42. PubMed ID: 10200280

Petritsch, C., et al. (2000). TGF-beta inhibits p70 S6 kinase via protein phosphatase 2A to induce G1 arrest. Genes Dev. 14: 3093-3101. PubMed ID: 11124802

Polymenis, M. and Schmidt, E. V. (1997). Coupling of cell division to cell growth by translational control of the G1 cyclin CLN3 in yeast. Genes Dev. 11: 2522-2531

Potter, C. J., Huang, H. and Xu, T. (2001). Drosophila Tsc1 functions with Tsc2 to antagonize insulin signaling in regulating cell growth, cell proliferation, and organ size. Cell 105: 357-368. 11348592

Powers, T. and Walter, P. (1999). Regulation of ribosome biogenesis by the rapamycin-sensitive TOR-signaling pathway in Saccharomyces cerevisiae. Mol. Biol. Cell. 10: 987-1000. PubMed ID: 10198052

Pullen, N., et al. (1998). Phosphorylation and activation of p70s6k by PDK1. Science 279(5351): 707-10

Radimerski, T., et al. (2000). Identification of insulin-induced sites of ribosomal protein S6 phosphorylation in Drosophila melanogaster. Biochemistry 39(19): 5766-74.

Radimerski, T., Montagne, J., Rintelen, F., Stocker, H., van Der Kaay, J., Downes, C. P., Hafen, E., and Thomas, G. (2002a). dS6K-regulated cell growth is dPKB/dPI(3)K-independent, but requires dPDK1. Nat. Cell. Biol. 4: 251-255. 11862217

Radimerski, T., et al. (2002b). Lethality of Drosophila lacking TSC tumor suppressor function rescued by reducing dS6K signaling. Genes Dev. 16: 2627-2632. 12381661

Reiling, J. H. and Hafen, E. (2004). The hypoxia-induced paralogs Scylla and Charybdis inhibit growth by down-regulating S6K activity upstream of TSC in Drosophila. Genes Dev. 18(23): 2879-92. 15545626

Reinking, J., et al. (2005). The Drosophila nuclear receptor e75 contains heme and is gas responsive. Cell 122: 195-207. PubMed Citation: 16051145

Richardson, C. J., et al. (2004). SKAR is a specific target of S6 kinase 1 in cell growth control. Curr. Biol. 14: 1540-1549. 15341740

Ruvinsky, I., et al. (2005). Ribosomal protein S6 phosphorylation is a determinant of cell size and glucose homeostasis. Genes Dev. 19: 2199-2211. 16166381

Saucedo, L. J., et al. (2003). Rheb promotes cell growth as a component of the insulin/TOR signalling network. Nat. Cell Biol. 5(6):566-71. 12766776

Schalm, S. S. and Blenis, J. (2002). Identification of a conserved motif required for mTOR signaling. Curr. Biol. 12(8): 632-9. 11967149

Scott, R. C., Schuldiner, O. and Neufeld, T. P. (2004). Role and regulation of starvation-induced autophagy in the Drosophila fat body. Dev Cell. 7: 167-178. 15296714

Shahbazian, D., et al. (2006). The mTOR/PI3K and MAPK pathways converge on eIF4B to control its phosphorylation and activity. EMBO J. 25(12): 2781-91. 16763566

Shakhmantsir, I., Massad, N. L. and Kennell, J. A. (2013). Regulation of cuticle pigmentation in Drosophila by the nutrient sensing insulin and TOR signaling pathways. Dev Dyn. [Epub ahead of print] PubMed ID: 24133012

Sheaffer, K. L., Updike, D. L. and Mango, S. E. (2008). The Target of Rapamycin pathway antagonizes pha-4/FoxA to control development and aging. Curr. Biol. 18(18): 1355-64. PubMed Citation: 18804378

Shi, P., Lai, R., Lin, Q., Iqbal, A. S., Young, L. C., Kwak, L. W., Ford, R. J. and Amin, H. M. (2009). IGF-IR tyrosine kinase interacts with NPM-ALK oncogene to induce survival of T-cell ALK+ anaplastic large-cell lymphoma cells. Blood 114: 360-370. PubMed ID: 19423729

Shima, H., et al. (1998). Disruption of the p70s6k/p85s6k gene reveals a small mouse phenotype and a new functional S6 kinase. EMBO J. 17: 6649-6659

Shioi, T., et al. (2000). The conserved phosphoinositide 3-kinase pathway determines heart size in mice. EMBO J. 19: 2537-2548

Sofer, A., Lei, K., Johannessen, C. M. and Ellisen, L. W. (2005). Regulation of mTOR and cell growth in response to energy stress by REDD1. Mol. Cell. Biol. 25(14): 5834-45. 15988001

Song, Q. and Gilbert, L. I. (1994). S6 phosphorylation results from prothoracicotropic hormone stimulation of insect prothoracic glands: a role for S6 kinase. Dev. Genet. 15(4): 332-8

Song, Q. and Gilbert, L. I. (1997). Molecular cloning, developmental expression, and phosphorylation of ribosomal protein S6 in the endocrine gland responsible for insect molting. J. Biol. Chem. 272(7): 4429-35

Sousa-Nunes, R., Yee, L. L. and Gould, A. P. (2011). Fat cells reactivate quiescent neuroblasts via TOR and glial insulin relays in Drosophila. Nature 471: 508-512. PubMed ID: 21346761

Stewart, M. J., et al. (1996). The Drosophila p70 s6k homolog exhibits conserved regulatory elements and rapamycin sensitivity. Proc. Natl. Acad. Sci. 93: 10791-10796

Stocker, H., Andjelkovic, M., Oldham, S., Laffargue, M., Wymann, M. P., Hemmings, B. A., and Hafen, E. (2002). Living with lethal PIP3 levels: Viability of flies lacking PTEN restored by a PH domain mutation in Akt/PKB. Science 295: 2088-2091. 11872800

Stocker, H., et al. (2003). Rheb is an essential regulator of S6K in controlling cell growth in Drosophila. Nat. Cell Biol. 5(6):559-65. 12766775

Sun, P., Quan, Z., Zhang, B., Wu, T. and Xi, R. (2010). TSC1/2 tumour suppressor complex maintains Drosophila germline stem cells by preventing differentiation. Development 137(15): 2461-9. PubMed Citation: 20573703

Sun, X., Wheeler, C. T., Yolitz, J., Laslo, M., Alberico, T., Sun, Y., Song, Q. and Zou, S. (2014). A mitochondrial ATP synthase subunit interacts with TOR signaling to modulate protein homeostasis and lifespan in Drosophila. Cell Rep 8: 1781-1792. PubMed ID: 25220459

Tabancay, A. P., et al. (2003). Identification of dominant negative mutants of Rheb GTPase and their use to implicate the involvement of human Rheb in the activation of p70S6K. J. Biol. Chem. 2003. 12869548

Thomas, G. (2002). The S6 kinase signaling pathway in the control of development and growth. Biol. Res. 35: 305-313. 12415748

Valentinis, B., et al. (2000). Insulin receptor substrate-1, p70S6K, and cell size in transformation and differentiation of hemopoietic cells. J. Biol. Chem. 275: 25451-25459

Vinals, F., Chambard, J. C. and Pouyssegur, J. (1999). p70 S6 kinase-mediated protein synthesis is a critical step for vascular endothelial cell proliferation. J. Biol. Chem. 274(38): 26776-82

Wassarman, D. A., Solomon, N. M. and Rubin, G. M. (1994). The Drosophila melanogaster ribosomal S6 kinase II-encoding sequence. Gene 144(2): 309-10

Watson, K. L., Konrad, K. D., Woods, D. F. and Bryant, P. J. (1992). Drosophila homolog of the human S6 ribosomal protein is required for tumor suppression in the hematopoietic system. Proc. Natl. Acad. Sci. 89(23): 11302-6.

Watson, K. L., Chou, M. M., Blenis, J., Gelbart, W. M, and Erikson, R. L. (1996). A Drosophila gene structurally and functionally homologous to the mammalian 70-kDa s6 kinase gene. Proc. Natl. Acad. Sci. 93(24): 13694-8

Weng, S. C. and Shiao, S. H. (2015). Frizzled 2 is a key component in the regulation of TOR signaling-mediated egg production in the mosquito Aedes aegypti. Insect Biochem Mol Biol. PubMed ID: 25890109

Wu, Q., Zhang, Y., Xu, J. and Shen, P. (2005). Regulation of hunger-driven behaviors by neural ribosomal S6 kinase in Drosophila. Proc. Natl. Acad. Sci. 102(37): 13289-94. 16150727

Yokogami, K., et al. (2000). Serine phosphorylation and maximal activation of STAT3 during CNTF signaling is mediated by the rapamycin target mTOR. Curr. Biol. 10: 47-50.

Zaragoza, D., et al. (1998). Rapamycin induces the G0 program of transcriptional repression in yeast by interfering with the TOR signaling pathway. Mol. Cell. Biol. 18: 4463-4470

Zhang, H., et al. (2000). Regulation of cellular growth by the drosophila target of rapamycin dTOR. Genes Dev. 14(21): 2712-24. PubMed ID: 11069888

Zhang, Y., et al. (2003). Rheb is a direct target of the tuberous sclerosis tumor suppressor proteins. Nat. Cell Biol. 5(6): 578-81. 12771962

Zheng, X. and Sehgal, A. (2010). AKT and TOR signaling set the pace of the circadian pacemaker. Curr. Biol. 20(13): 1203-8. PubMed Citation: 20619819

Ribosomal protein S6 kinase: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation

date revised: 30 June 2015

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