The Interactive Fly

Zygotically transcribed genes

Growth response - The TOR signaling pathway

The TOR signaling pathway is a portion of the Growth response - The Insulin receptor signaling pathway

  • Drosophila as a model for human diseases: Diabetes
  • Nutritional control of protein biosynthetic capacity by insulin via Myc in Drosophila
  • Dally proteoglycan mediates the autonomous and nonautonomous effects on tissue growth caused by activation of the PI3K and TOR pathways
  • Regulation of TORC1 by Rag GTPases in nutrient response
  • The Rag GTPases bind raptor and mediate amino acid signaling to mTORC1
  • Regulation of TORC1 in response to amino acid starvation via lysosomal recruitment of TSC2
  • eIF4A inactivates TORC1 in response to amino acid starvation
  • Somatic stem cell differentiation is regulated by PI3K/Tor signaling in response to local cues
  • mTORC1 activation during repeated regeneration impairs somatic stem cell maintenance
  • RagC phosphorylation autoregulates mTOR complex 1
  • Phosphatidic acid drives mTORC lysosomal translocation in the absence of amino acids
  • The GATOR complex regulates an essential response to meiotic double-stranded breaks in Drosophila
  • TOR signaling inhibition in intestinal stem and progenitor cells affects physiology and metabolism in Drosophila

    Regulation of TORC1 by Rag GTPases in nutrient response

    TORC1 (target of rapamycin complex 1) has a crucial role in the regulation of cell growth and size. A wide range of signals, including amino acids, is known to activate TORC1. This study reports the identification of Rag GTPases (RagA-B and RagC-D) as activators of TORC1 in response to amino acid signals. Knockdown of Rag gene expression suppressed the stimulatory effect of amino acids on TORC1 in Drosophila melanogaster S2 cells. Expression of constitutively active (GTP-bound) Rag in mammalian cells activated TORC1 in the absence of amino acids, whereas expression of dominant-negative Rag blocked the stimulatory effects of amino acids on TORC1. Genetic studies in Drosophila also show that Rag GTPases regulate cell growth, autophagy and animal viability during starvation. These studies establish a function of Rag GTPases in TORC1 activation in response to amino acid signals (Kim, 2008).

    The Rag GTPases bind raptor and mediate amino acid signaling to mTORC1

    The multiprotein mTORC1 protein kinase complex is the central component of a pathway that promotes growth in response to insulin, energy levels, and amino acids and is deregulated in common cancers. This study finds that the Rag proteins [a family of four related small guanosine triphosphatases (GTPases)] interact with mTORC1 in an amino acid-sensitive manner and are necessary for the activation of the mTORC1 pathway by amino acids. A Rag mutant that is constitutively bound to guanosine triphosphate interacted strongly with mTORC1, and its expression within cells made the mTORC1 pathway resistant to amino acid deprivation. Conversely, expression of a guanosine diphosphate-bound Rag mutant prevented stimulation of mTORC1 by amino acids. The Rag proteins do not directly stimulate the kinase activity of mTORC1, but, like amino acids, promote the intracellular localization of mTOR to a compartment that also contains its activator Rheb (Sancak, 2008).

    Regulation of TORC1 in response to amino acid starvation via lysosomal recruitment of TSC2

    TOR complex 1 (TORC1) is a potent anabolic regulator of cellular growth and metabolism. When cells have sufficient amino acids, TORC1 is active due to its lysosomal localization mediated via the Rag GTPases. Upon amino acid removal, the Rag GTPases release TORC1, causing it to become cytoplasmic and inactive. This study shows that, upon amino acid removal, the Rag GTPases also recruit TSC2 to the lysosome, where it can act on Rheb. Only when both the Rag GTPases and Rheb are inactive is TORC1 fully released from the lysosome. Upon amino acid withdrawal, cells lacking TSC2 fail to completely release TORC1 from the lysosome, fail to completely inactivate TORC1, and fail to adjust physiologically to amino acid starvation. These data suggest that regulation of TSC2 subcellular localization may be a general mechanism to control its activity and place TSC2 in the amino-acid-sensing pathway to TORC1 (Demetriades, 2014).

    eIF4A inactivates TORC1 in response to amino acid starvation

    Amino acids regulate TOR complex 1 (TORC1) via two counteracting mechanisms, one activating and one inactivating. The presence of amino acids causes TORC1 recruitment to lysosomes where TORC1 is activated by binding Rheb. How the absence of amino acids inactivates TORC1 is less well understood. Amino acid starvation recruits the TSC1/TSC2 complex to the vicinity of TORC1 to inhibit Rheb; however, the upstream mechanisms regulating TSC2 are not known. This study identified the the eIF4A-containing eIF4F translation initiation complex (composed of three subunits: eIF4E, eIF4A and eIF4G) as an upstream regulator of TSC2 in response to amino acid withdrawal in Drosophila. TORC1 and translation preinitiation complexes bind each other. Cells lacking eIF4F components retain elevated TORC1 activity upon amino acid removal. This effect is specific for eIF4F and not a general consequence of blocked translation. This study identifies specific components of the translation machinery as important mediators of TORC1 inactivation upon amino acid removal (Tsokanos, 2016).

    To maintain homeostasis, biological systems frequently use a combination of two distinct mechanisms that converge and counteract each other. For instance, the level of phosphorylation of a target protein depends not only on the rate of phosphorylation by the upstream kinase, but also on the rate of dephosphorylation by the phosphatase. Both the activating kinase and the inactivating phosphatase can be regulated separately. Likewise, the activity of TORC1 in response to amino acid levels appears to reflect a balance between activating and inactivating mechanisms that converge on Rheb. When amino acids are re-added to cells, TORC1 is activated via Rag or Arf1 GTPase-dependent recruitment to the lysosome where TORC1 binds Rheb (Kim, 2008; Sancak, 2008). In contrast, when amino acids are removed from cells, TORC1 activity drops in part by blocking this activation mechanism and in part via a distinct inactivation mechanism whereby TSC2 is recruited to the vicinity of TORC1 to act on Rheb (Demetriades, 2014). The existence of this distinct and counteracting mechanism is highlighted by the fact that in the absence of TSC2, both Drosophila and mammalian cells do not appropriately inactivate TORC1 in response to amino acid removal (Demetriades, 2014). The upstream mechanisms regulating TSC2 in response to amino acid withdrawal, however, are not known. This study has identified the translational machinery, and in particular components of the eIF4F complex, as one upstream regulatory mechanism working via TSC2 to inactivate TORC1 upon amino acid withdrawal (Tsokanos, 2016).

    The subcellular localization of TORC1 plays an important role in its regulation. A significant body of evidence shows that TORC1 needs to translocate to the lysosome or Golgi to become reactivated following amino acid starvation and re-addition. Whether active TORC1 then remains on the lysosome, or whether it can move elsewhere in the cell to phosphorylate target proteins, is less clear. Several findings in the literature, as well as the data presented in this study, indicate that active TORC1 can leave the lysosome, yet remain active: (1) Upon amino acid re-addition in starved cells, the Rag GTPases are necessary for mTORC1 lysosomal localization and reactivation. In contrast, Rag depletion in cells growing under basal conditions, replete of serum and amino acids, does not cause a strong drop in mTORC1 activity, although it causes a similar delocalization of mTORC1 away from lysosomes. Hence, under these conditions, mTORC1 is non-lysosomal, but still active to a large extent. (2) Similarly, particular stresses such as arsenite treatment can cause TORC1 to localize away from the lysosome, yet remain active. (3) The Rag GTPases tether TORC1 to the LAMTOR complex present on the lysosome. Amino acid restimulation, which activates TORC1, actually decreases binding between Rag GTPases and LAMTOR, suggesting that active Rag-bound TORC1 complexes can leave the lysosome and reside elsewhere in the cell. Additional mechanisms also contribute to the delocalization of the Rag GTPases away from lysosomes (4) Active TORC1 phosphorylates target proteins such as 4E-BP and S6K, which are physically associated with translation preinitiation complexes. Indeed, this study reports physical interactions between the TORC1 complex and translation preinitiation complexes, in agreement with what has also been observed by others. Therefore, either translation preinitiation complexes need to translocate to lysosomes to meet TORC1, or TORC1 needs to come off the lysosome to meet translation preinitiation complexes in the cytoplasm. (5) Using proximity ligation assay, an interaction was observed between Raptor and eIF4A, which does not colocalize with either lysosomes or endoplasmic reticulum, suggesting that it takes place in the cytoplasm. (6) In agreement with these PLA data, antibody staining of cells in the presence of amino acids with anti-TOR antibody reveals an accumulation of TOR on lysosomes, as well as a more diffuse, non-lysosomal TORC1 localization throughout the cytoplasm. (7) A recent report employing a FRET-based probe detects mTORC1 activity at lysosomes as well as in the cytoplasm and nucleus. Taken together, these data suggest that although TORC1 is activated on the lysosome, it then in part translocates to other sites in the cell including the cytoplasm to phosphorylate target proteins (Tsokanos, 2016).

    Upon amino acid withdrawal, both cytoplasmic and lysosomal fractions of active TORC1 need to be inactivated. The data presented in this study suggest that upon amino acid removal, inactivation of TORC1 happens in part via an eIF4A-dependent mechanism acting on TSC2 to inactivate Rheb in the cytosol. In agreement with this, TORC1 inactivation upon amino acid removal can be rescued by supplying cells with dominantly active, but not wild-type Rheb. It has been previously reported that a pool of TSC2 is also recruited to lysosomes upon amino acid removal (Demetriades, 2014). This study shows in Drosophila cells, upon amino acid removal, some TSC2 accumulates in lysosomes, whereas some remains in the cytosol. Therefore, TSC2 is likely recruited to all subcellular sites where active TORC1 is located to inactivate it. Indeed, Rheb and TSC2 have been observed at several subcellular compartments. Since Rheb localizes to many endomembranes in the cell, Rheb that is not bound to TORC1 could potentially remain active, to provide a pool for subsequent TORC1 reactivation (Tsokanos, 2016).

    Upon inactivation, the data indicate that TORC1 remains bound to preinitiation complexes, in agreement with previous reports. This finding is reminiscent of the fact that Raptor is also recruited to stress granules, which are essentially stalled preinitiation complexes, in response to another stress-oxidative stress. Whether the Rag GTPases also remain bound to preinitiation complexes upon amino acid removal is unclear because some experiments showed a decrease in binding between Rag GTPases and initiation factors, and some did not (Tsokanos, 2016).

    How could eIF4A affect TORC1 activity? The data indicate that the effects of eIF4A knockdown cannot be explained as a consequence of generally impaired translation, since other means of blocking translation do not have the same effects on TORC1 activity upon amino acid starvation. Instead, knockdown of any of the three members of the eIF4F complex gives this elevated TORC1 phenotype, indicating that it is specific for the eIF4F complex. The data are consistent with two interpretations: One option is that the eIF4F complex is specifically required to translate a protein that promotes TSC2 function. An alternate option is that the eIF4F complex acts directly on TSC2, regulating its activity. The latter is supported by the fact that eIF4A and TSC2 proteins are seen interacting with each other. Interestingly, eIF4A has been reported to have additional functions that are not translation-related (Tsokanos, 2016).

    Some differences were noted between Drosophila cells and mammalian cells. The first is that overexpression of wild-type Rheb is sufficient to activate TORC1 upon amino acid removal in mammalian cells, whereas this is not the case in Drosophila cells. This could be due to a difference in the biology of the two cell types, or simply to a technical difference having to do with levels of Rheb overexpression. A second difference is that cycloheximide treatment is sufficient to maintain elevated TORC1 levels in HeLa or HEK293 cells upon amino acid removal, whereas this is not the case in Drosophila cells. This could be due to differences in rates of amino acid efflux and levels of autophagy in mammalian compared to S2 and Kc167 cells, causing intracellular amino acid levels to remain elevated in mammalian cells when both amino acid import from the medium and amino acid expenditure via translation are simultaneously blocked (Tsokanos, 2016).

    A number of studies have looked at the involvement of Rheb in the cellular response to amino acids, with some disagreement on whether amino acids affect Rheb GTP-loading or Rheb-mTOR binding. The current data fit with previous reports that Rheb GTP-loading is affected by amino acids and with the conclusion that amino acids affect TORC1 activity via both a Rheb-dependent and a Rheb-independent mechanism (Tsokanos, 2016).

    The data indicate a close physical relationship between TORC1 and the translational machinery. This is in part mediated by a direct interaction between the major scaffolding subunit of the initiation complex, eIF4G, and RagC and in part likely mediated by additional interactions between TORC1 and preinitiation supercomplexes as previously reported. Interestingly, TORC2 is also physically associated with the ribosome and requires ribosomes, but not translation, for its activation. Hence, both TORC1 and TORC2 have close physical connections to the translational machinery (Tsokanos, 2016).

    Some side observations in this study are interesting and could constitute a starting point for further studies. For instance, eIF4A-knockdown cells inactivate TORC1 more robustly than control cells upon serum removal. Also, eIF2b knockdown causes S6K phosphorylation to decrease significantly in S2 cells. It is not known why this occurs. The latter might suggest that there are additional points of cross-talk between TORC1 and the translation machinery (Tsokanos, 2016).

    How cells sense the presence or the absence of amino acids has been an open question in the field. The data presented in this study indicate that the translational machinery itself might sense the absence of amino acids. Indeed, the relevant parameter for a cell is likely not the absolute levels of intracellular amino acids, but rather whether the available amino acid levels are sufficient to support the amount of translation that a cell requires. Hence, the translation machinery itself might be best poised to make this assessment. Binding is observed between eIF4A and NAT1 that is strong in the presence of amino acids, and is reduced upon amino acid withdrawal, independently of TORC1 signaling. These epistasis experiments are consistent with NAT1 acting as the upstream mediator of the amino acid signal, binding and inhibiting eIF4A in the presence of amino acids, but not in the absence of amino acids. Hence, NAT1 might play a role in this sensing process (Tsokanos, 2016).

    In sum, these data identify the eIF4F complex as an important upstream regulator of TORC1, which acts via TSC2 to inactivate TORC1 upon withdrawal of amino acids (Tsokanos, 2016).

    Somatic stem cell differentiation is regulated by PI3K/Tor signaling in response to local cues

    Stem cells reside in niches that provide signals to maintain self-renewal, and differentiation is viewed as a passive process that depends on losing access to these signals. This study demonstrates that differentiation of somatic cyst stem cells (CySCs) in the Drosophila testis is actively promoted by PI3K/Tor signaling, as CySCs lacking PI3K/Tor activity cannot properly differentiate. An insulin peptide produced by somatic cells immediately outside of the stem cell niche was found to act locally to promote somatic differentiation through Insulin receptor (InR) activation. These results indicate that there is a local 'differentiation' niche which upregulates PI3K/Tor signaling in the early daughters of CySCs. Finally, it was demonstrated that CySCs secrete the Dilp-binding protein ImpL2, the Drosophila homolog of IGFBP7, into the stem cell niche, which blocks InR activation in CySCs. Thus, this study shows that somatic cell differentiation is controlled by PI3K/Tor signaling downstream of InR and that local production of positive and negative InR signals regulate the differentiation niche. These results support a model in which leaving the stem cell niche and initiating differentiation is actively induced by signaling (Amoyel, 2016).

    This study shows that PI3K/Tor activity is required for the differentiation of somatic stem cells in the Drosophila testis. Additionally, a 'differentiation' niche was identified immediately adjacent to the stem cell niche that, through the local production of Dilps, leads to the upregulation of PI3K/Tor activity in early CySC daughters and to their commitment to differentiation. The secretion of ImpL2 by CySCs antagonizes the initiation of differentiation in CySCs by blocking available Dilps in the stem cell niche. As a result, CySCs receive little free Dilp ligands. However, as their daughters move away from the hub, they encounter increasing levels of Dilps and decreasing levels of ImpL2, which leads to the upregulation of PI3K/Tor signaling and proper somatic cell differentiation. The fact that ImpL2 is upregulated by the main self-renewal signal (i.e., JAK/STAT) in CySCs leads to a model accounting for the spatial separation of the stem cell niche and the differentiation niche (Amoyel, 2016).

    The results are consistent with a model in which autocrine or paracrine production of Dilp6by early cyst cells serves as a differentiation niche in the testis, defining where in the tissue upregulation PI3K/Tor signaling - a prerequisite for differentiation - occurs. This differentiation niche is critical for somatic development because stem cell markers like Zfh1 are maintained in the absence of signals like PI3K/Tor. Notably, JAK/STAT activity is not expanded outside of the niche upon somatic loss of PI3K/Tor signaling, suggesting that differentiation signals play a critical role in downregulating stem cell factors. Intriguingly, recent studies in the Drosophila ovary have identified a differentiation niche in this tissue: autocrine Wnt ligands produced by somatic support escort cells regulate escort cell function, proliferation and viability. Taken together, these studies reveal that at least in Drosophila gonads, there is a defined region immediate adjacent to the stem cell niche where autocrine production of secreted factors induces the differentiation of somatic cells, which in turn promote development of the germ line (Amoyel, 2016).

    Several studies have examined the role of insulin signaling in gonadal stem cells. In both testes and ovaries, systemic Dilps have been shown to affect stem cell behavior. In both tissues, nutrition through regulation of systemic insulin controls the proliferation rate of GSCs. The current data showing that Akt1, Dp110 or Tor mutant CySC clones proliferate poorly are consistent with these findings and indicate that basal levels of insulin signaling are required for the proliferation and/or survival of both stem cell pools in the testis. This work also demonstrates that production of a secreted Insulin binding protein ImpL2 by CySCs reduces available Dilps in the stem cell niche, and ImpL2 in the niche milieu should reduce insulin signaling in GSCs and CySCs. While these data seemingly contradict the results that insulin is required for GSC maintenance, a model is suggested in which low constitutive levels of insulin signaling are required for stem cell proliferation and that higher levels are required to induce stem cell differentiation. (Amoyel, 2016).

    Prior reports have found that both male and female flies with reduced Insulin or Tor activity are sterile, and the results presented in this study suggest that this is due at least in part to a lack of somatic cell differentiation. The results indicate that Dilp6, the IGF homolog, plays a local role in CySC differentiation, but acts redundantly with other presumably systemic factors, suggesting that both constitutive and nutrient-responsive inputs control CySC differentiation. Indeed, this study shows that in addition to controlling the proliferation of stem cells, systemic insulin is required for their differentiation, as the poorly proliferative Akt1, Dp110 or Tor mutant CySC clones do not differentiate and eventually die by apoptosis. This combination of reduced proliferation and increased apoptosis may explain why other studies suggest that Tor is required for self- renewal in GSCs; indeed prior reports indicate that while Tor mutant GSCs are lost, hyper-activation of Tor leads to faster loss of GSCs through differentiation and recent work indicates that lineage-wide Tor loss blocks the differentiation of GSCs. The use of hypomorphic alleles enabled a genetic separation of the proliferative effects and differentiation requirements of PI3K and Tor in CySCs. Finally, there is evidence that PI3K/Tor activity promotes differentiation of stem cells in gonads in mammals, suggesting that these findings may reflect a conserved role of Tor activity in promoting germ cell differentiation, both through autonomous and non- autonomous mechanisms involving somatic support cells. Moreover, it seems likely that Tor activity may be a more general requirement for the differentiation of many stem cell types, as increased PI3K or Tor has been shown to induce differentiation in many instances. In particular, mouse long term hematopoietic stem cells are lost to differentiation when the PI3K inhibitor Pten is mutated, while Drosophila intestinal stem cells differentiate when Tor is hyperactive due to Tsc1/2 complex inactivation. Moreover, inhibition of Tor activity by Rapamycin promotes cellular reprogramming to pluripotency, while cells with increased Tor activity cannot be reprogrammed, suggesting a conserved role for Tor signaling in promoting differentiated states (Amoyel, 2016).

    mTORC1 activation during repeated regeneration impairs somatic stem cell maintenance

    The balance between self-renewal and differentiation ensures long-term maintenance of stem cell (SC) pools in regenerating epithelial tissues. This balance is challenged during periods of high regenerative pressure and is often compromised in aged animals. This study shows that target of rapamycin (TOR) signaling is a key regulator of SC loss during repeated regenerative episodes. In response to regenerative stimuli, SCs in the intestinal epithelium of the fly and in the tracheal epithelium of mice exhibit transient activation of TOR signaling. Although this activation is required for SCs to rapidly proliferate in response to damage, repeated rounds of damage lead to SC loss. Consistently, age-related SC loss in the mouse trachea and in muscle can be prevented by pharmacologic or genetic inhibition, respectively, of mammalian target of rapamycin complex 1 (mTORC1) signaling. These findings highlight an evolutionarily conserved role of TOR signaling in SC function and identify repeated rounds of mTORC1 activation as a driver of age-related SC decline (Haller, 2017).

    RagC phosphorylation autoregulates mTOR complex 1

    The mechanistic (or mammalian) target of rapamycin complex 1 (mTORC1) controls cell growth, proliferation, and metabolism in response to diverse stimuli. Two major parallel pathways are implicated in mTORC1 regulation including a growth factor-responsive pathway mediated via TSC2/Rheb and an amino acid-responsive pathway mediated via the Rag GTPases. This study identified and characterize three highly conserved growth factor-responsive phosphorylation sites on RagC, a component of the Rag heterodimer, implicating cross talk between amino acid and growth factor-mediated regulation of mTORC1. RagC phosphorylation is associated with destabilization of mTORC1 and is essential for both growth factor and amino acid-induced mTORC1 activation. Functionally, RagC phosphorylation suppresses starvation-induced autophagy, and genetic studies in Drosophila reveal that RagC phosphorylation plays an essential role in regulation of cell growth. Finally, mTORC1 was identified as the upstream kinase of RagC on S21. These data highlight the importance of RagC phosphorylation in its function and identify a previously unappreciated auto-regulatory mechanism of mTORC1 activity (Yang, 2018).

    mTOR is an evolutionarily conserved atypical serine/threonine kinase belonging to the phosphoinositide 3 kinase (PI3K)-related kinase family. mTOR is found in two structurally and functionally distinct complexes-mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2)-defined by their unique components, in particular raptor (mTORC1) and rictor (mTORC2). Through the coordinated phosphorylation of its downstream effectors, mTORC1 integrates extra- and intra-cellular signal inputs such as amino acids, growth factors (GF), stress, and energy status, to regulate major cellular processes including growth, proliferation, and survival. Underlining its crucial role in cellular and organismal homeostasis, mTORC1 dysregulation occurs in numerous human diseases including cancer, metabolic disorders, and neurodegeneration. Growth factors and amino acids both acutely enhance mTORC1 activity, and two different types of small GTPases-Ras-homolog enriched in brain (Rheb) and the Rag GTPases-cooperatively regulate mTORC1 activity via these two parallel activation mechanisms. Rheb is activated under conditions of high cellular ATP and upstream growth factor signals. Once activated, Rheb interacts with and activates mTORC1 and is required for mTORC1 activation by all signals, including amino acids. Rag GTPases are considered amino acid-specific regulators of the mTORC1 pathway. Mammals have four Rag proteins-RagA to RagD-which form obligate heterodimers comprising RagA or RagB together with RagC or RagD. Amino acids cause Rag GTPases to switch to an active conformation, in which RagA/B is GTP-loaded and Rag C/D is GDP-loaded. The active Rag heterodimer physically interacts with raptor, recruiting mTORC1 to the lysosome where its activator Rheb resides. Extensive work has revealed several mechanisms implicated in the regulation of Rag activity that enables them to function as nutrient sensors. A common feature among these is the control of Rag nucleotide status, particularly through the activation of guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). These include the Ragulator (GEF for Rag A/B; Bar-Peled, 2012), the GATOR1 complex (GAP for RagA/B; Bar-Peled, 2013), folliculin (FLCN, GAP for RagC/D; Petit, 2013; Tsun, 2013), and leucyl-tRNA synthetase (LeuRS, GAP for RagD; Han, 2012). Ubiquitination has also recently emerged as a post-translational modification (PTM) capable of inhibiting Rag GTPase signaling by recruiting GATOR1 to RagA. Importantly, these pathways regulating Rag activity are all amino acid-dependent, and much less is known about the control of growth factor-mediated Rag GTPase signaling (Yang, 2018).

    In a recent global mass spectrometry-based phosphoproteomics study in adipocytes, insulin-dependent phosphorylation was observed of several highly conserved residues on RagC including S2, S21, and T394. These data highlight a possible role for the Rag GTPases in mTORC1 growth factor sensing. This study demonstrates that both growth factors and amino acids trigger RagC phosphorylation and that phosphorylated RagC potentiates mTORC1 activity and affects mTORC1-dependent cell growth and autophagy. Moreover, the phosphorylation of RagC at S21 (and likely T394) was shown to be catalyzed directly by mTORC1, revealing a novel auto-regulatory feedback loop within the mTORC1 signaling pathway (Yang, 2018).

    This study identified a new auto-regulatory branch of mTORC1 signaling, involving phosphorylation of the Rag GTPase RagC. This is the first report that Rag GTPase phosphorylation can regulate mTORC1 activity. More importantly, the results confirm that Rag GTPases are not only involved in the amino acid-sensing mTORC1 pathway, but could also participate in growth factor sensing in the mTORC1 pathway. Although previous studies show that in Rag heterodimers, the GTP/GDP loading of Rag heterodimers plays a dominant role in the interaction between Rag heterodimers and mTORC1, the data indicate that RagC is also a positive regulator of mTORC1 through post-translational modification. Interestingly, phosphoproteomics data suggest that most phosphorylation is concentrated on RagC compared with other Rag GTPases, and S21 is not conserved between RagC and RagD, suggesting that RagC is not functionally redundant and potentially has distinct biological functions to RagD (Yang, 2018).

    One of the RagC phosphorylation sites, S21, was established as a novel rapamycin-insensitive mTORC1 substrate in vitro and in cells, and the T394 is phosphorylated by mTOR in vitro. The S2 and T394 sites may also be mTORC1 substrates in vivo, because the kinetics of their phosphorylation resembles that of other bona fide mTORC1 substrates and they also have surrounding sequence features matching the preferred sequence motif of mTORC1. These findings indicate the presence of a positive feedback loop between mTORC1 and RagC, which may contribute to the fine-tuning of mTORC1 activity (Yang, 2018).

    There is evidence that the stability of the raptor-mTOR complex is related to mTORC activity, and the current data implicate RagC phosphorylation in the destabilization of mTORC1. This is likely to be a direct effect of RagC phosphorylation, because RagC 3E still destabilized mTOR-raptor complex under serum starvation. This is consistent with the observation that RagC 3E causes hyper-phosphorylation of ULK1 and inhibits autophagy under serum starvation. The next major question is what is the underlying cause of this instability. One possibility is that RagC phosphorylation influences the interaction with other regulators, resulting in 'locking' or 'opening' of the mTOR-raptor complex. Interestingly, it was observed that RagC 3A binds more FLCN, which is a GAP for RagC/D, and RagC 3E can bind more raptor under both steady and amino acid starvation/re-fed condition. One possibility is that RagC phosphorylation regulates its nucleotide binding status by modulating the interaction with FLCN. However, no substantial difference was observed in FLCN binding between wild-type RagC and RagC 3E, or raptor binding between wild-type RagC and 3A. The temporal change in mTOR/raptor stability upon amino acid re-feeding is similar to that of FLCN/RagC stability, but not with raptor/RagC interaction. A possible explanation is that FLCN has two functions: serving as a GAP for RagC/D and a 'lock' for mTORC1. This model could help explain why FLCN releases from Rag GTPases in the presence of amino acids if it is a GAP for RagC/D, which is a positive regulator for mTORC1 activity: After activating RagC/D, FLCN needs to be disassociated from the lysosome to unlock mTORC1, and RagC phosphorylation may affect this process. Further studies will be needed to investigate these possibilities (Yang, 2018).

    Other explanations cannot be ruled out for the impact of RagC phosphorylation on impaired mTORC1 activity. For example, it is well established that raptor recruits substrate proteins such as S6K and 4E-BP1 to mTORC1 so that they can be phosphorylated by mTOR. Therefore, RagC phosphorylation may affect the recruitment of mTORC1 substrates by raptor. Recently, two elegant studies showed that under amino acid or growth factor starvation, the Rag heterodimer binds and recruits TSC2 to lysosomes to inhibit Rheb, resulting in mTORC1 inactivation. Therefore, a final possibility is that RagC phosphorylation may mediate its effects by acting through TSC2. Future studies into the underlying mechanics of how RagC phosphorylation exerts its effects on mTORC1 signaling are therefore likely to shed light on this newly identified mechanism that sits at the intersection between amino acid sensing and growth factor signaling (Yang, 2018).

    Phosphatidic acid drives mTORC lysosomal translocation in the absence of amino acids

    mTOR Complex (mTORC1) promotes cell growth and proliferation in response to nutrients and growth factors. Amino acids induce lysosomal translocation of mTORC via the Rag GTPases. Growth factors activate Ras homolog enriched in brain (Rheb), which in turn, activates mTORC at the lysosome. Amino acids and growth factors also induce the phospholipase D (PLD)-phosphatidic acid (PA) pathway, required for mTORC signaling through mechanisms that are not fully understood. Using human and murine cell lines, along with immunofluorescence, confocal microscopy, endocytosis, PLD activity, and cell viability assays, this study shows that exogenously supplied PA vesicles deliver mTORC to the lysosome in the absence of amino acids, Rag GTPases, growth factors, and Rheb. Of note, pharmacological or genetic inhibition of endogenous PLD prevented mTORC lysosomal translocation. This study observed that precancerous cells with constitutive Rheb activation through loss of TSC complex subunit (TSC2) exploit the PLD-PA pathway and thereby sustain mTORC activation at the lysosome in the absence of amino acids. These findings indicate that sequential inputs from amino acids and growth factors trigger PA production required for mTORC translocation and activation at the lysosome (Frias, 2019).

    mTORC18 is a conserved serine/threonine catalytic complex that integrates signals from nutrients and growth factors to regulate cell growth, proliferation, survival, and metabolism. Activation of mTORC1 is a two-step process whereby amino acids induce Rag-dependent translocation of mTORC1 from the cytoplasm to the lysosome, followed by mTOR kinase activation by the lysosomal small GTPase Rheb upon growth factor stimulation (Frias, 2019).

    Phospholipase D (PLD) and its product, the signaling lipid phosphatidic acid (PA) play a role in mTORC1 activation in response to amino acids and growth factors. Amino acids induce lysosomal translocation of PLD1. Once on the lysosome, PLD1 binds to Rheb, which activates PLD1 in response to growth factors. PLD1 is widely expressed in mammals and converts the most abundant membrane phospholipid phosphatidylcholine to choline and PA. Conserved basic amino acids in the FKBP12-rapamycin binding (FRB) domain of mTOR lead to proton dissociation to generate PA with two negative charges. This locks mTOR onto deprotonated PA, promoting mTORC1 assembly and stability (Frias, 2019).

    This study reports that PA with an unsaturated fatty acid stimulates lysosomal translocation and activation of mTORC1 in the absence of amino acids, Rag GTPases, growth factors, or Rheb. This work provides a unifying model showing that PA is critical for translocation and full activation of mTORC1 at the lysosome in response to sequential signals provided by amino acids and growth factors (Frias, 2019).

    The data support a model where PLD1, similar to mTORC1, acts like a coincidence detector and effector of both amino acids and growth factors. Amino acids induce PLD activity and production of PA. Exogenously supplied PA vesicles enter the cell through endocytosis and drive mTOR to the lysosome, suggesting that amino acids induce production of PA-containing endosomes that carry mTOR to the lysosome. In agreement, inhibition of endogenous PLD prevented mTOR translocation to the lysosome in response to amino acids. Amino acid-induced RagA/B-GTP RagC/D-GDP heterodimers provide a parallel pathway that locks mTOR on the lysosome. Amino acids also induce the translocation of PLD1 from cytoplasmic puncta to the lysosome. Once on the lysosome, PLD1 binds to Rheb. Growth factors activate Rheb, which then activates PLD1. Lysosomal PA production promotes further binding of PA to mTOR to allow complex stability and activation (Frias, 2019).

    Previous studies showed that exogenously supplied PA induced mTORC1 activation in the presence but not in the absence of amino acids. This study was able to induce mTORC1 translocation to the lysosome and mTORC1 activity with exogenously supplied PA-18:1 vesicles in the absence of amino acids. The main difference between the two studies is that this study performed amino acid and serum deprivation (to prevent contamination of amino acids present in serum) for 1 h, followed by amino acid stimulation for 10 min. In contrast, the previous study performed amino acid starvation for 2 h after overnight serum starvation, followed by amino acid stimulation for 30 min (Frias, 2019).

    Previous findings suggest that mTORC1 assembles before reaching the lysosome because binding of mTOR to the Rag GTPases requires the mTORC1 component raptor. PA promotes mTORC1 assembly and stability. PA-containing endosomes carrying mTORC1 to the lysosome is therefore an attractive model in which PA would allow mTORC1 formation and stability. This study showed that exogenously supplied PA can drive mTOR to the lysosome and induce mTORC1 activity in TSC2-null MEFs where RagC and D were genetically ablated. Therefore, it is proposed that delivery of mTORC1 to the lysosome does not require the Rags. However, this study found that residual retention of mTOR at the lysosome in TSC2-null MEFs was lost upon RagC and D knockdown. This suggests that the Rags operate in parallel to PA to lock mTORC1 on the lysosome, after mTORC1 delivery by PA. This study showed that genetic ablation of PLD1 induced lysosomal scattering. This favors the idea that PA-containing endosomes carry mTORC1 to the lysosome, fuse with the lysosome, and increase its size (Frias, 2019).

    Exogenously supplied PA vesicles induce mTORC1 translocation and activation in the absence of Rheb, indicating that the key step in Rheb activation of mTORC1 is increased PLD1 activity and PA production. Consistent with this observation, the Rheb association with mTOR is independent of GTP loading, whereas the Rheb association with PLD1 depends on GTP loading. Additionally, this study found that PLD1/2 inhibitors, in combination, abolished mTORC1 activity in RagAGTP/GTP MEFs, indicating that PA production is required for complex assembly and stability. If mTORC1 is not intact, then constitutive Rag activation is lost. Thus, the effect of PA on mTORC1 is downstream of Rheb and parallel to Rag GTPases. Genetic deletion of mTOR in mice is embryonic lethal. Unlike mTOR, mice with genetic deletion of PLD1, PLD2, or both are viable, suggesting that PLD and PA may not be required for steady state but rather acute activation of mTOR. PLD inhibitors preferentially killed TSC-null MEFs, whereas rapamycin did not. This suggests an advantage in terms of cancer therapeutics because targeting PLD may selectively target cancer cells, with minimal side effects. PLD inhibitors were developed from halopemide, a psychotropic drug extensively used in humans without toxicities. Thus, PLD inhibition might be a viable alternative to current therapies in cancers with mTORC1 hyperactivation that requires PLD-generated PA (Frias, 2019).

    The GATOR complex regulates an essential response to meiotic double-stranded breaks in Drosophila

    The TORC regulator GATOR1/SEACIT controls meiotic entry and early meiotic events in yeast. However, how metabolic pathways influence meiotic progression in metazoans remains poorly understood. This study examined the role of the TORC regulators GATOR1 and GATOR in the response to meiotic double-stranded breaks (DSB) during Drosophila oogenesis. In mutants of the GATOR component mio, meiotic DSBs trigger the constitutive downregulation of TORC activity and a permanent arrest in oocyte growth. Conversely, in GATOR mutants, high TORC activity results in the delayed repair of meiotic DSBs and the hyperactivation of p53. Unexpectedly, it was found that GATOR inhibits retrotransposon expression in the presence of meiotic DSBs in a pathway that functions in parallel to p53. Thus, these studies have revealed a link between oocyte metabolism, the repair of meiotic DSBs and retrotransposon expression (Wei, 2019).

    Metabolism impacts meiotic progression during oogenesis. Target of Rapamycin Complex 1 (TORC1) is a multi-protein complex that functions as a master regulator of metabolism. In the presence of adequate nutrients and positive upstream growth signals, TORC1, which contains the serine/threonine kinase Target of Rapamycin, becomes active and functions to stimulate growth and inhibit catabolic metabolism through the phosphorylation of down-stream effector proteins. The Seh1 Associated Complex Inhibits TORC1 (SEACIT), originally identified in yeast, inhibits TORC1 activity in response to amino acid limitation. SEACIT, known as the GAP Activity Towards Rags complex 1 (GATOR1) in metazoans, is comprised of three highly conserved proteins Npr2/Nprl2, Npr3/Nprl3 and Iml1/Depdc5. In Drosophila and mammals, depleting any of the three GATOR1 components results in increased TORC1 activity and growth, as well as a reduced response to amino acid starvation. Thus, the role of the SEACIT/GATOR1 complex in the regulation of TORC1 activity is highly conserved in eukaryotes (Wei, 2019).

    The multi-protein GATOR2 complex, known as Seh1 Associated Complex Activates TORC1 (SEACAT) in yeast, inhibits the activity of GATOR1 and thus functions to activate TORC1 (see Mio prevents the constitutive downregulation of TORC1 activity in response to meiotic DSBs). In metazoans, the GATOR2 complex functions in multiple amino acid sensing pathways. In tissue culture cells, depleting GATOR2 components results in the constitutive activation of GATOR1 and the permanent downregulation of TORC1 activity. However, genetic studies of the role of individual GATOR2 components in Drosophila, indicate that the requirement for the GATOR2 complex is more nuanced when examined in the context of a multicellular animal. For example, mutations in the GATOR2 component mio, result in a block to oocyte growth and differentiation, due to the constitutive downregulation of TORC1 activity in the female germline. However, mio is not required to maintain TORC1 activity in most somatic tissues of Drosophila (Wei et al., 2016). Why there is a tissue specific requirement for mio in the female germline of Drosophila is currently unknown (Wei, 2019).

    In single celled eukaryotes, nutrient limitation often facilitates meiotic entry. In the yeast Saccharomyces cerevisiae, the down-regulation of TORC1 by SEACIT/GATOR1 in response to amino acid stress promotes both meiotic entry and early meiotic progression. Surprisingly, as is observed in yeast, during Drosophila oogenesis the GATOR1 complex promotes meiotic entry. These data raise the intriguing possibility that in Drosophila the GATOR1 complex and low TORC1 activity may be critical to the regulation of additional events of the early meiotic cycle (Wei, 2019).

    This study reports that the GATOR complex is critical to the response to meiotic DSB during Drosophila oogenesis. Restraining TORC1 activity via a pathway that involves both GATOR1 and the Tuberous sclerosis complex (TSC) promotes the timely repair of meiotic DSBs and prevents the hyperactivation of p53 in the female germline. Notably, the delayed repair of meiotic DSBs in GATOR1 mutants is due, at least in part, to the hyperactivation of the TORC1 target S6K. Conversely, the data indicate that the GATOR2 component Mio opposes the activity of GATOR1 in the female germline, thus preventing the constitutive downregulation of TORC1 activity and allowing for the growth and development of the oocyte in later stages of oogenesis. Thus, this study has identified a regulatory loop required to modulate TORC1 activity in response to meiotic DSBs during Drosophila oogenesis. Finally, during the course of these studies, it was observed that the GATOR1 complex prevents the derepression of retrotransposon expression in the presence of meiotic DSBs (Wei, 2019).

    Previous work has shown that in Drosophila, mutations in the GATOR2 component mio, result in the constitutive activation of the GATOR1 pathway in the female germline but not in somatic tissues. This study demonstrates that the tissue specific requirement for mio during oogenesis is due, at least in part, to the generation of meiotic DBSs during oogenesis. In Drosophila, only the female germline undergoes meiotic recombination and thus experiences the genotoxic stress associated with developmentally programmed DSBs. This study shows that in mio mutants, blocking the formation of meiotic DSBs prevents the constitutive downregulation of TORC1 activity thus allowing for the growth and development of the oocyte. These data are consistent with the model that meiotic DSBs trigger the activation of a TORC1 inhibitory pathway that must be opposed and/or attenuated by the GATOR2 component Mio (see A working model for the role of the GATOR complex in the response to meiotic DSBs) (Wei, 2019).

    While there are several possible models that might explain these data, it is believed the most parsimonious explanation for these results is that the TORC1 inhibitory pathway activated by meiotic DSBs, involves both GATOR1 and TSC. This model is consistent with the ability of both GATOR1 and TSC depletions to rescue the mio mutant phenotype. Additionally, recent reports indicate that GATOR1 and TSC act in a common pathway to downregulate TORC1 activity in response to multiple upstream inhibitory inputs. Previously, it was determined that in Drosophila, amino acid starvation induces a dramatic GATOR1/TSC dependent decrease in TORC1 activity in somatic tissues, that far exceeds any reduction in TORC1 activity observed in GATOR2 null mutants. This observation strongly suggests that, in addition to the removal of the GATOR2 inhibition of GATOR1, there is an activation step that is required to fully potentiate the GATOR1/TSC pathway (Wei, 2019).

    Thus, based on these data the following model is proposed. Meiotic DSBs activate, or are required to maintain, a GATOR1/TSC dependent pathway that downregulates TORC1 activity in the female germline. The GATOR2 component Mio is required to oppose or turnoff this pathway to prevent the constitutive downregulation of TORC1 activity in later stages of oogenesis. While it is believed that the data support the role of the GATOR1/TSC pathway, it is conceded that an alternative regulator of TORC1 activity may also be critical to the downregulation of TORC1 activity in response to meiotic DSBs (Wei, 2019).

    Hyperactivation of TORC1 has been linked to defects in the DNA damage response in single celled and multicellular organisms. The observation that meiotic DSBs likely promote the GATOR1 dependent downregulation of TORC1 activity during Drosophila oogenesis, suggested that limiting TORC1 activity may be important to the regulation of meiotic DSB repair. In previous work, it was found that GATOR1 mutant ovaries had TORC1 activity levels approximately three times higher than those observed in wild-type ovaries. This study demonstrates that GATOR1 mutant ovaries exhibit multiple phenotypes consistent with the misregulation of meiotic DSB repair including, an increase in the steady state number of Mei-W68/Spo-11 induced DSBs, the retention of meiotic DSBs into later stages of oogenesis and the hyperactivation of p53. Importantly, RNAi depletions of Tsc1 partially phenocopied the GATOR1 ovarian defects. Thus, the misregulation of meiotic DSBs observed in GATOR1 mutant oocytes are due to high TORC1 activity and not to a TORC1 independent function of the GATOR1 complex (Wei, 2019).

    Epistasis analysis between the GATOR1 component nprl3 and the Rad51 homolog spnA, strongly suggest that GATOR1 impacts the repair, rather than the generation, of meiotic DSBs. This study determined that double mutants of nprl2 and the Rad51 homolog spnA, which is required for the repair of meiotic DSBs, have approximately the same number of DSBs as spnA single mutants. These data are consistent with GATOR1 and spnA influencing the common process of DNA repair and are inconsistent with GATOR1 mutants producing supernumerary breaks (Wei, 2019).

    These observations on the role of the GATOR1 complex during Drosophila oogenesis are particularly intriguing in light of similar meiotic defects observed in a npr3 mutants in Saccharomyces cerevisiae. In the sporulation proficient strain SK1, npr3 mutant cells enter meiosis and express the transcription factor and master regulator of gametogenesis IME1 with wild-type kinetics. Subsequently, npr3 mutants exhibit a mild delay in the generation of meiotic DNA breaks, but a substantial delay in the repair of meiotic DSBs. Thus, yeast and Drosophila SEACIT/GATOR1 mutants share a common meiotic phenotype, the delayed repair of meiotic DSBs. These results raise the intriguing possibility that low TORC1 activity may be a common feature of the early meiotic cycle in many organisms (Wei, 2019).

    Notably, the data indicate that the delay in the repair of meiotic DSBs in GATOR1 mutants is due to the hyperactivation of the TORC1 downstream target S6K. S6K is a critical downstream effector of TORC1 that impacts multiple essential cellular processes including, but not limited to cell growth, energy balance and aging. Intriguingly, in mammals, S6K has been implicated in the regulation of the DNA damage response with hyperactivation of the TORC1-S6K pathway resulting in the accumulation of unrepaired DSBs and genome instability. Thus, similar to what is reported in mammals, the data are consistent with the model that the hyperactivity of the TORC1/S6K axis delays the repair of DSBs in Drosophila (Wei, 2019).

    Finally, it was determined that GATOR1 mutants have a diminished response to DSBs outside the female germline in somatic tissues of Drosophila. Similar to what is observed in TSC mutant cells in humans that have increased levels of TORC1 activity, this study find that GATOR1 mutant embryos have a reduced ability to survive low levels of γ-irradiation. Moreover, in the somatic follicle cells of the ovary a delay was observed in the repair of DSBs after adult females are exposed to low levels of γ-irradiation. Thus, in Drosophila inappropriately high TORC1 activity delays the repair of DSBs in both the germline and somatic tissues (Wei, 2019).

    The initiation of homologous recombination through the programmed generation of DNA double-stranded breaks (DSBs) is a universal feature of meiosis. DSBs represent a dangerous form of DNA damage that can result in dramatic and permanent changes to the germline genome. To minimize this destructive potential, the generation and repair of meiotic DSBs is tightly controlled in space and time . The activation of transposable elements represents an additional threat to genome integrity in germ line cells. Genotoxic stress, resulting from DNA damage, has been implicated in the deregulation of transposons in multiple organisms. Thus, germ line cells may be at an increased risk for transposon derepression due to the genotoxic stress associated with meiotic recombination. Consistent with this hypothesis, germ line cells have evolved extensive surveillance systems to detect and silence transposons beyond the pathways present in most somatic tissues (Wei, 2019).

    Previous studies have shown that DNA damage promotes the deregulation of retrotransposon in multiple organisms, including Drosophila. In line with these studies, this study found that in GATOR1 mutants, the DSBs that initiate meiotic recombination trigger the deregulation of retrotransposon expression. Similarly, p53 mutant females derepress retrotransposon expression during oogenesis, but as observed in GATOR1 mutants, primarily in the presence of meiotic DSBs. Double mutants of nprl3, p53 exhibit a dramatic increase in retrotransposon expression relative to either p53 or nprl3 single mutants, implying that p53 and GATOR1 act through independent pathways to repress retrotransposon expression in the female germline. One possibility is that both GATOR1 and p53 independently impact genome stability. Thus, disabling both pathways may have an additive effect on both genome stability and retrotransposon expression. Consistent with the hypothesis that genome instability drives retrotransposon expression, this study found that mutants in spnA/Rad51, which fail to repair meiotic DSBs, also exhibit increased transcription of multiple retrotransposons. Intriguingly, the SpnA homolog Rad51, as well as other genes required for DNA repair, was recently identified in a high throughput screen for genes that suppress (Long Interspersed Element-1) LINE1 expression in mammalian tissue culture cells (Wei, 2019).

    However, the current data also suggest that the GATOR1 complex may influence retrotransposon expression independent of the regulation of TORC1 activity. While both GATOR1 and TSC are required for the efficient repair of meiotic DSBs, in contrast to GATOR1 mutant ovaries, little to no increase was observed in retrotransposon expression in the Tsc1 depleted ovaries. We believe this reflects the incomplete depletion of Tsc1 by RNAi resulting in a reduced retention of meiotic DSBs relative to GATOR1 mutants. However, a second possibility is that the GATOR1 complex inhibits retrotransposon expression independent of TORC1 inhibition. As is observed with spnA the depletion of GATOR1 components, but not TSC components result in the activation of LINE1 expression in HeLa cells. Taken together, these data hint that the GATOR1 complex may impact retrotransposon expression in the germline via two independent pathways: First by promoting the repair of meiotic DSBs through the downregulation of TORC1 activity and second via a pathway that functions independent of TORC1 inhibition (Wei, 2019).

    Genes encoding components of the GATOR1 complex are often deleted in cancers. As is observed in GATOR1 mutants, cancer cells frequently have increased TORC1 activity, increased genomic instability and increased retrotransposon expression. Thus, in the future it will be important to identify the molecular mechanism by which the GATOR1 complex influences both the response to genotoxic stress and the expression of retrotransposons under both normal and pathological conditions (Wei, 2019).

    TOR signaling inhibition in intestinal stem and progenitor cells affects physiology and metabolism in Drosophila

    In all eukaryotic organisms, the control of growth, metabolism, reproduction, and lifespan is realized by interactions of genetic and environmental signals. An important player in the regulatory network is the target of rapamycin (TOR) signaling pathway, which is triggered by nutritional cues. Given the pivotal role of TOR in regulating multiple processes in organisms, this study inhibited TOR by inducible expression of specific RNAi in Drosophila intestinal stem and progenitor cells or progenitor cells alone. TOR inhibition in stem and progenitor cells shortened the lifespan on both regular diet and under malnutrition. Moreover, flies became more short-lived under starvation or oxidative stress conditions if TOR was inhibited. TOR-RNAi expression resulted in a decrease in body glycogen and TAG levels. All these physiological and metabolic changes might be partially explained by significant changes in mRNA levels for genes encoding the Drosophila insulin-like peptides (dilp2, dilp3 and dilp5) with subsequent effects on insulin signaling to modulate gene expression in peripheral tissues (e.g. tobi and pepck transcripts). In the gut, a strong increase in transcript levels of cytokines upd2, upd3 and downstream target socs36e of the JAK/STAT signaling pathway in the gut indicate an important role for this signaling pathway when TOR is inhibited (Strilbytska, 2020).


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    Zygotically transcribed genes

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