InteractiveFly: GeneBrief

Ras-related GTP binding A/B and Ras-related GTP binding C/D: Biological Overview | References

BIOLOGICAL OVERVIEW

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 GAP Activity Towards Rags complex 1 (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 mTORC1 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, 2020).

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, 2020).

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, 2020).

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, 2020).

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, 2020).

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, 2020).

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, 2020).

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, 2020).

The Rag GTPase regulates the dynamic behavior of TSC downstream of both amino acid and growth factor restriction

The dysregulation of the metabolic regulator TOR complex I (TORC1) contributes to a wide array of human pathologies. Tuberous sclerosis complex (TSC) is a potent inhibitor of TORC1. This study demonstrates that the Rag GTPase acts in both the amino-acid-sensing and growth factor signaling pathways to control TORC1 activity through the regulation of TSC dynamics in HeLa cells and Drosophila. TSC lysosomal-cytosolic exchange increases in response to both amino acid and growth factor restriction. Moreover, the rate of exchange mirrors TSC function, with depletions of the Rag GTPase blocking TSC lysosomal mobility and rescuing TORC1 activity. Finally, this study shows that the GATOR2 complex controls the phosphorylation of TSC2, which is essential for TSC exchange. These data support the model that the amino acid and growth factor signaling pathways converge on the Rag GTPase to inhibit TORC1 activity through the regulation of TSC dynamics (Yang, 2020).

Cells must sense and adapt to a constantly changing external environment. Accordingly, in both single-celled and multicellular organisms, the ability to switch between anabolism, which supports growth, and catabolism, which is activated in response to stress, is critical to the maintenance of metabolic homeostasis. The highly conserved target of rapamycin (TOR) complex 1 (TORC1) is central to the regulation of metabolism in eukaryotes. TORC1 serves as a hub to integrate multiple upstream signaling inputs and regulates the execution of downstream metabolic pathways that control growth, proliferation, and cell death. TORC1 consists of five subunits including mTOR, which functions as a serine-threonine kinase, as well as Deptor, Raptor, mLST8, and PRAS40. In the presence of positive upstream inputs, TORC1 promotes anabolic metabolism by phosphorylating downstream effectors such as 4E-BP1 and S6K to increase mRNA translation and protein synthesis. Concurrently, TORC1 inhibits catabolic pathways such as autophagy, by phosphorylating essential autophagic proteins including ULK1 and Atg13. Thus, through the regulation of mTORC1 activity, cells can rapidly react to a diverse array of positive and negative environmental cues (Yang, 2020).

An important aspect of TORC1 regulation is its recruitment to and activation of lysosomes. These two processes require different Ras-related small GTPases, Rag and Rheb, respectively. In the presence of amino acids, TORC1 is translocated from the cytosol to the lysosomal membrane by the Rags. Specifically, RagA or RagB dimerizes with RagC or RagD to form a heterodimeric complex that recruits TORC1 to the lysosome by directly interacting with the Raptor subunit in an amino-acid-dependent manner. Once on the lysosome, Rheb binds to the subunit mTOR resulting in a conformational change that exposes active site residues allowing TORC1 to bind and phosphorylate a wide array of substrates (Yang, 2020).

As small GTPases, the functions of Rags and Rheb are regulated by their guanine nucleotide-binding cycle, which is tightly controlled by upstream signals. The Rags switch their TORC1-recruiting function by changing their guanine-nucleotide-binding status. When cells lack access to appropriate levels of amino acids, the GTPase-activating protein toward Rags 1 (GATOR1) complex blocks the function of TORC1 by acting as a GTPase-activating protein (GAP) toward RagA/B. Thus, under amino acid starvation the guanine-nucleotide-binding status of RagA/B changes to GDP inhibiting the ability of the Rag heterodimer to recruit TORC1 onto lysosomes. Another GATOR subcomplex, GATOR2, functions to oppose GATOR1 activity and is composed of five highly conserved proteins (Mio, Wdr24 (absent from Drosophila), Wdr59, Seh1, and Sec13), several of which have been shown to localize to lysosomes. In the presence of sufficient amino acids, the GATOR2 complex activates TORC1 activity by opposing the activity of the GATOR1 complex. The mechanistic details of how the GATOR2 complex opposes the activity of the GATOR1 complex to activate TORC1 remain unclear (Yang, 2020).

The small GTPase Rheb is inhibited by a three-subunit complex, called the tuberous sclerosis complex (TSC), comprised the proteins TSC2, TSC1, and TBC1D7. In the absence of insulin, TSC2 possesses a GAP activity toward Rheb. TSC serves as a potent inhibitor of the TORC1 signaling pathway. Mutations or deletions in TSC subunits result in the growth of benign tumors, epilepsy, and developmental delay. The upstream signals controlling TSC have been extensively studied. Essentially, growth factors, including insulin, activate class I phosphatidylinositol-3-kinase (PI3K), which in turn stimulates the protein kinase AKT. AKT inhibits TSC by directly phosphorylating multiple serine residues in TSC2. Thus, growth factors and insulin positively regulate the TORC1 signaling pathway by preventing TSC from inhibiting the TORC1 activator Rheb (Yang, 2020).

While the model that TSC controls TORC1 activity through the inhibition of Rheb is well established, the detailed mechanism of how TSC is regulated remains poorly understood. It is widely accepted that TSC responds to insulin and other growth factors through a PI3K-AKT circuit. However, the role of TSC in response to changes in amino acid levels is unclear. One possibility is that TORC1 is regulated by two independent pathways; the PI3K-AKT pathway that regulates the activity of the TORC1 inhibitor TSC in response to growth factors and the Rag GTPase pathway that responds to amino acid levels. However, multiple laboratories have reported an essential role for TSC in the response to amino acid starvation. Additionally, it is unclear exactly how intracellular TSC dynamics impact the ability of TSC to inhibit TORC1. Several studies have shown that TSC shuttles between lysosomes and the cytoplasm in response to amino acid availability, whereas others have reported that the lysosomal localization of TSC is independent of amino acid status with the TSC complex being released from lysosomes in response to high levels of insulin. Notably, in some cell types, such as HeLa cells, TSC is constitutively present on lysosomes even in the presence of physiological concentrations of insulin. Thus, whether the recruitment of TSC from the cytosol to the lysosomes is a common feature of TSC regulation in all cell types remains unclear. Finally, the detailed mechanism of how the phosphorylation of TSC2 by AKT affects TSC's function is not well understood. Several studies proposed that TSC2 undergoes proteasomal degradation after phosphorylation by AKT. However, more recent work suggests that TSC is not regulated at the level of protein stability but that the entire TSC complex is released from lysosomes upon phosphorylation of the TSC2 subunit by AKT. Thus, a full understanding of TSC regulation requires further exploration (Yang, 2020).

This study demonstrates that the regulation of TSC lysosomal dynamics by the Rag GTPase is required for the full response to both amino acid and growth factor restriction. Moreover, using fluorescence recovery after photobleaching (FRAP) and a photoconvertible fluorescently tagged TSC2, this study demonstrates that in response to negative stimuli, the Rag GTPase drives rapid cycling of TSC on and off lysosomes in both HeLa cells and Drosophila. Importantly, this study finds that GATOR2 and the Rag GTPase impact the lysosomal cycling of TSC2 by regulating its inhibitory phosphorylation by AKT. These data support the model that the Rag GTPase works in concert with the PI3/AKT/TSC pathway to regulate TORC1 activity in response to both amino acid and growth factor restriction (Yang, 2020).

TSC is a critical inhibitor of TORC1 signaling. Currently, there are two working models for the role of TSC in the inhibition of TORC1 activity. The first model posits that TSC lies exclusively downstream of the PI3K-AKT growth factor signaling pathway, while the second model proposes that TSC is a critical downstream effector of both the growth factor signaling and amino-acid-sensing pathways. The current findings on the function of the GATOR2 complex are consistent with the second model, which implicate different nucleotide states of the Rag GTPase in the recruitment of TORC1 versus TSC to lysosomes in response to amino acid starvation. Moreover, this study tried to find that the Rag GTPase, which has previously been thought to exclusively function in amino acid sensing, regulates the recruitment of TSC to lysosomes in response to growth factor restriction. Thus, the data support a model in which both the amino-acid-sensing pathway and growth factor signaling pathway converge on the Rag GTPase to recruit TSC to lysosomes in response to inhibitory signals. Notably, this study found in both HeLa cells and Drosophila, the Rag GTPase promotes the rapid exchange of TSC between the lysosome and cytosol in response to negative inputs. Finally, demonstrating further integration of the amino-acid-sensing and growth factor signaling pathways, this study shows that the GATOR2 complex acts upstream of the Rag GTPase to promote both, the activating phosphorylation of AKT and the AKT-dependent inhibitory phosphorylation of TSC2 (Yang, 2020).

An important outstanding question in the field of TORC1 regulation concerns the role of the Rag GTPase and TSC. The data indicate that in both Drosophila and HeLa cells, the GATOR-Rag GTPase axis inhibits TORC1 activity through the regulation of the dynamic behavior of TSC. In cells without a functional GATOR2 complex, the Rag GTPase, locked in its RAGAGDP: RAGCGTP-bound form due to the activation of GATOR1 recruits TSC to lysosomes precluding the recruitment and activation of TORC1. This inhibited state is relieved by depleting RAGA or RAGC, resulting in the recruitment and activation of TORC1 on lysosomes. Mutations in GATOR2 components mimic amino acid starvation. Thus, the current results are consistent with reports from the Teleman laboratory indicating that amino acid starvation promotes the Rag GTPase-dependent recruitment of TSC to lysosomes (Demetriades, 2014). Moreover, the current data confirm that under conditions of metabolic homeostasis TORC1 can be recruited to lysosomes and activated by Rheb in the absence of the Rag GTPase (Yang, 2020).

Intriguingly, in budding yeast, which does not have TSC, the homolog of the Rag GTPase, GTRl1/GTRL2, has a similar dual role in the regulation of TORC1 activity with the overexpression of GTR1GTP promoting TORC1 activity while GTR1GDP is associated with low TORC1 activity. Thus, the Rag GTPase may have a conserved role in the inhibition of TORC1 activity that goes beyond the regulation of TSC dynamics (Yang, 2020).

Surprisingly, this study also demonstrated a central role of the Rag GTPase in the recruitment of TSC to lysosomes in response to growth factor restriction. Specifically, the low TORC1 activity and reduced TSC mobility associated with growth factor depletion were rescued by depleting the Rag GTPase component RagA. Taken together, the data argue that the Rag GTPase and TSC are critical components of both the amino-acid-sensing and growth factor signaling pathways (Yang, 2020).

The idea that TSC acts downstream of the GATOR-Rag GTPase pathway is consistent with previous observations in Drosophila showing that depleting components of TSC rescue the low TORC1 activity observed in GATOR2 mutant ovaries. Indeed, in Drosophila, TSC is epistatic to the GATOR2 complex with respect to TORC1 activity, in that, depleting TSC in GATOR2 mutant cells resulted in high TORC1 levels similar to those observed in TSC single mutants. As indicated above, these data are also consistent with several previous reports that TSC acts in the amino-acid-sensing pathway (Yang, 2020).

Recent reports indicate that the recruitment of TSC to lysosomes is a common response to cellular stress in many mammalian cell types. However, these studies have not examined if TSC remains static on lysosomes or actively exchanges with the cytoplasmic pool during periods of TORC1 inhibition. Using FRAP and a photoconvertible TSC2 to follow the intracellular dynamics of TSC components, this study determined that the rate of exchange of TSC between the lysosome and the cytosol increases in response to nutrient deprivation and growth factor restriction. This rapid increase in cycling on and off lysosomes requires the Rag GTPase in both HeLa cells and the Drosophila ovary. Notably, increased cycling of TSC correlates with its increased interaction with Rheb and a concomitant decrease in TORC1 activity. Depleting components of the Rag GTPase in cells grown in amino acid or growth factor restricted media dramatically reduced the lysosomal-cytosol TSC rate of exchange, decreased its binding with Rheb, and rescued TORC1 activity. Thus, the data support the model that the Rag GTPase increases the exchange rate of TSC between the lysosome and the cytoplasm in response to both amino acid starvation and growth factor restriction (Yang, 2020).

It has long been established that AKT controls TSC activity through the inhibitory phosphorylation of TSC2. More recent studies indicate that AKT controls TSC activity by regulating its association with Rheb on the surface of lysosomes. This study finds that in nutrient-replete conditions, the inhibitory phosphorylation of TSC2 by AKT slows the rate of lysosomal-cytosolic exchange of TSC and allows for TORC1 activation. When TSC2 is rendered resistant to AKT-dependent phosphorylation, TSC rapidly cycled on and off lysosomes independent of growth factor and amino acid status. Importantly, cells expressing AKT-resistant TSC have low TORC1 activity. This study determined that in WDR24-KO cells, in which the RAG GTPase is in the RAGAGDP: RAGCGTP state, the AKT-dependent phosphorylation of TSC2 is strongly diminished, resulting in the increased mobility of the TSC complex on lysosomes and decreased TORC1 activity. Depleting components of the Rag GTPase in WDR24-KO cells, rescued these phenotypes resulting in increased AKT-dependent phosphorylation of TSC, decreased TSC lysosomal motility, and increased TORC1 activity. Further examination revealed that levels of activated AKT are kept low in WDR24-KO cells by a Rag GTPase-dependent mechanism. These data are consistent with previous observations that RAGA-KO cells have increased levels of activated AKT while WDR24-KO cells fail to increase AKT activation after Sestrin2 overexpression. Taken together, the data indicate that the GATOR2 complex promotes the activation of AKT, which facilitates the AKT-dependent inhibitory phosphorylation of TSC, upstream of the Rag GTPase. Moreover, they demonstrate that the rate of TSC cycling on and off lysosomes reflects TSC activity (Yang, 2020).

Based on these data, the following model is proposed. Under conditions of amino acid and growth factor sufficiency, GATOR2 inhibits GATOR1 resulting in the Rag GTPase adopting the RAGAGTP: RAGCGDP configuration, which favors the recruitment and activation of TORC1 on lysosomes and results in the limited exchange of TSC between the lysosome and the cytoplasm. In contrast, under conditions of amino acid or growth factor depletion, or in GATOR2 mutant cells, GATOR1 is active, resulting in the Rag GTPase adopting the RAGAGDP: RAGCGTP configuration, which promotes the rapid exchange of TSC between the lysosome and the cytoplasm and decreases TORC1 activity. Under these restricted conditions (AA−, serum−, or WDR24-KO), knockdowns of RAGA or RAGC prevent the rapid cycling of TSC allowing for the recovery of TORC1 activity due to the inherent ability of TORC1 to bind Rheb directly (Demetriades, 2014). Currently, whether the rapid cycling of TSC on and off lysosomes in response to upstream signals directly promotes TSC activity, or serves a regulatory role, has not been definitively established. In summary, these data support a model in which both the amino-acid-sensing and growth factor signaling pathways utilize the Rag GTPase to inhibit TORC1 activity through the regulation of TSC lysosomal dynamics (Yang, 2020).

A recent study reported that RAGA rapidly cycles between the lysosome and the cytosol in response to nutrients. Notably, RAGAGTP cycles on and of lysosomes while RAGAGDP remains more tightly associated with the Ragulator at the lysosomal surface. Intriguingly, this study found that RAGAGDP promotes the rapid cycling of the TSC complex on and off the lysosome. Thus, in the future, it will be important to determine precisely how the dynamic behavior of RAGA and TSC are coordinated to control TORC1 activity (Yang, 2020).

Amino acid-dependent NPRL2 interaction with Raptor determines mTOR Complex 1 activation

This study assigns a new function to a tumor suppressor NPRL2 (see Drosophila Nprl2) that activates the mTOR complex 1 (mTORC1) activity. The positive regulation of mTORC1 activity by NPRL2 is mediated through NPRL2 interaction with Raptor. While NPRL2 interacts with Rag GTPases, RagD in particular, to interfere with mTORC1 activity in amino acid scarcity, NPRL2 interacts with Raptor in amino acid sufficiency to activate mTORC1. A reciprocal relationship exists between NPRL2 binding to Rag GTPases and Raptor. NPRL2 majorly locates in the lysosomal membranes and has a higher binding affinity to the dominant negative mutant heterodimer of RagA(GDP)/RagD(GTP) that inactivates mTORC1. However, the binding affinity of NPRL2 with Raptor is much less pronounced in cells expressing the dominant negative mutant heterodimer of RagA(GDP)/RagD(GTP) than in cells expressing the dominant positive mutant heterodimer, RagA(GTP)/RagD(GDP). The positive effect of NPRL2 on TORC1 pathway was also evidenced in Drosophila animal model. A 'seesaw' model is proposed in which the interactive behavior of NPRL2 with Raptor determines mTORC1 activation by amino acid signaling in animal cells (Kwak, 2016).

NPRL2 or NPRL3 has been described to inhibit TORC1 activity in Drosophila S2 cells. However, it was noticed that Nprl2 is also a positive TORC1 regulator in Drosophila animal model, in which the expression of Nprl2 was downregulated using double stranded Nprl2-targeted RNAi (Nprl2-Ri). In situ hybridization assay indicates that Nprl2 mRNA is effectively depleted in the wing imaginal discs of MS1096-Gal4 > Nprl2-Ri flies. Tests were performed to seecwhether or not TORC1 activity is affected by Nprl2 knockdown in the wing imaginal discs of MS1096-Gal4 > Nprl2-Ri and Nub-Gal4 $ #gt; Nprl2-Ri flies. Phosphorylated S6 has been also used as a marker of mTORC1, or TORC1 activity in both mice and Drosophila animal models. It was confirmed that the level of phosphorylated S6 in the wing imaginal discs of the transgenic flies is decreased by the knockdown of Nprl2. In addition, the severe impairments in size and curvature of the wings in the transgenic flies expressing Nprl2-Ri were notably rescued by the expression of the constitutively active S6 kinase (S6K-CA). Knockdown of Nprl2 also resulted in the reduction of adult eye size, which is rescued by the expression of S6K-CA. The knockdown of Nprl2 in the salivary glands also showed a significant reduction in cell size (Kwak, 2016).

TORC1 regulators Iml1/GATOR1 and GATOR2 control meiotic entry and oocyte development in Drosophila

In single-cell eukaryotes the pathways that monitor nutrient availability are central to initiating the meiotic program and gametogenesis. In Saccharomyces cerevisiae an essential step in the transition to the meiotic cycle is the down-regulation of the nutrient-sensitive target of rapamycin complex 1 (TORC1; see Drosophila Tor pathway) by the increased minichromosome loss 1/ GTPase-activating proteins toward Rags 1 (Iml1/GATOR1) complex in response to amino acid starvation. How metabolic inputs influence early meiotic progression and gametogenesis remains poorly understood in metazoans. This study defined opposing functions for the TORC1 regulatory complexes Iml1/GATOR1 and GAP Activity Towards Rags complex 2 (GATOR2) during Drosophila oogenesis. As is observed in yeast, the Iml1/GATOR1 complex inhibits TORC1 activity to slow cellular metabolism and drive the mitotic/meiotic transition in developing ovarian cysts. In iml1 germline depletions, ovarian cysts undergo an extra mitotic division before meiotic entry. The TORC1 inhibitor rapamycin can suppress this extra mitotic division. Thus, high TORC1 activity delays the mitotic/meiotic transition. Conversely, mutations in Tor, which encodes the catalytic subunit of the TORC1 complex, result in premature meiotic entry. Later in oogenesis, the GATOR2 components Missing oocyte (Mio) and Seh1 are required to oppose Iml1/GATOR1 activity to prevent the constitutive inhibition of TORC1 and a block to oocyte growth and development. These studies represent the first examination of the regulatory relationship between the Iml1/GATOR1 and GATOR2 complexes within the context of a multicellular organism. The data imply that the central role of the Iml1/GATOR1 complex in the regulation of TORC1 activity in the early meiotic cycle has been conserved from single cell to multicellular organisms (Wei, 2014b).

In yeast, the inhibition of the nutrient-sensitive target of rapamycin complex 1 (TORC1) in response to amino acid limitation is essential for cells to transit from the mitotic cycle to the meiotic cycle. In response to amino acid starvation, the Iml1 complex, comprising the Iml1, Nitrogen permease regulator-like 2 (Npr2), and Nitrogen permease regulator-like 3 (Npr3) proteins in yeast and the respective orthologs DEPDC5, Nprl2, and Nprl3 in mammals, inhibits TORC1 activity. The Iml1 complex, which has been renamed the 'GTPase-activating proteins toward Rags 1' (GATOR1) complex in higher eukaryotes, functions as a GTPase-activating protein complex that inactivates RagsA/B or Gtr1 in mammals and yeast, respectively, thus preventing the activation of TORC1. In the yeast Saccharomyces cerevisiae, mutations in the Iml1 complex components Npr2 and Npr3 result in a failure to down-regulate TORC1 activity in response to amino acid starvation and block meiosis and sporulation. As is observed in yeast, in Drosophila, Nprl2 and Nprl3 mediate a critical response to amino acid starvation (Wei, 2014a). However, their roles in meiosis and gametogenesis remain unexplored (Wei, 2014b).

Recent reports indicate that the Iml1, Npr2, and Npr3 proteins are components of a large multiprotein complex originally named the 'Seh1-associated' (SEA) complex in budding yeast and the 'GATOR' complex in higher eukaryotes. The SEA/GATOR complex contains eight highly conserved proteins. The three proteins described above, Iml1/DEPDC5, Npr2/Nprl2, and Npr3/Nprl3, form the Iml1/GATOR1 complex and inhibit TORC1. The five remaining proteins in the complex, Seh1, Sec13, Sea4/Mio, Sea2/WDR24, and Sea3/WDR59, which have been designated the 'GATOR2' complex in multicellular organisms, oppose the activity of Iml1/GATOR1 and thus promote TORC1 activity (Wei, 2014b).

Little is known about the physiological and/or developmental requirements for the GATOR2 complex in multicellular organisms. However, in Drosophila the GATOR2 components Mio and Seh1 interact physically and genetically and exhibit strikingly similar ovarian phenotypes, with null mutations in both genes resulting in female sterility (Senger, 2011; Wei, 2014a). In Drosophila females, oocyte development takes place within the context of an interconnected germline syncytium, also referred to as an 'ovarian cyst'. Ovarian cyst formation begins at the tip of the germarium when a cystoblast, the daughter of a germline stem cell, undergoes four synchronous divisions with incomplete cytokinesis to produce 16 interconnected cells. Actin-stabilized cleavage furrows, called 'ring canals', connect cells within the cyst. Each 16-cell cyst develops with a single oocyte and 15 polyploid nurse cells which ultimately are encapsulated by a somatically derived layer of follicle cells to produce an egg chamber. Each ovary is comprised of ~15 ovarioles that consist of a single germarium followed by a string of egg chambers in successively older stages of development. In mio- and seh1-mutant egg chambers, the oocyte enters the meiotic cycle, but as oogenesis proceeds, the oocyte fate and the meiotic cycle are not maintained stably (Senger, 2011; Wei, 2014a). Ultimately, a large fraction of mio and seh1 oocytes enter the endocycle and develop as polyploid nurse cells. A mechanistic understanding of how mio and seh1 influence meiotic progression and oocyte fate has remained elusive (Wei, 2014b).

This study demonstrates that the Iml1/GATOR1 complex down-regulates TORC1 activity to promote the mitotic/meiotic transition in Drosophila ovarian cysts. Depleting iml1 in the female germ line delays the mitotic/meiotic transition, so that ovarian cysts undergo an extra mitotic division. Conversely, mutations in Tor result in premature meiotic entry before the completion of the four mitotic divisions. Moreover, it was demonstrated that in the female germ line, the GATOR2 components Mio and Seh1 are required to oppose the TORC1 inhibitory activity of the Iml1/GATOR1 complex to prevent the constitutive down-regulation of TORC1 activity in later stages of oogenesis. These studies represent the first examination of the regulatory relationship between Iml1/GATOR1 and GATOR2 components within the context of a multicellular animal. Finally, these data reveal a surprising tissue-specific requirement for the GATOR2 complex in multicellular organisms and suggest a conserved role for the SEA/GATOR complex in the regulation of TORC1 activity during gametogenesis (Wei, 2014b).

Previous work demonstrated that in Drosophila the Iml1/GATOR1 complex mediates an adaptive response to amino acid starvation. This study tested the hypothesis that the Iml1/GATOR1 complex also has retained a role in the regulation of the early events of gametogenesis. Consistent with this model, this study found that in germline knockdowns of iml1, ovarian cysts delay meiotic entry and undergo a fifth mitotic division. This meiotic delay can be suppressed with the TORC1 inhibitor rapamycin. Thus, during Drosophila oogenesis the Iml1/GATOR1 complex promotes the transition from the mitotic cycle to the meiotic cycle through the down-regulation of the metabolic regulator TORC1. Increasing TORC1 activity by disabling its inhibitor delays meiotic progression, whereas germline clones of a Tor-null allele enter meiosis prematurely. Taken together, these data indicate that the level of TORC1 activity contributes to the timing of the mitotic/meiotic switch in Drosophila females and suggest that low TORC1 activity may be a conserved feature of early meiosis in many eukaryotes (Wei, 2014b).

However, in Drosophila, meiotic entry is not contingent on amino acid limitation at the organismal level. Indeed, the energy-intensive process of Drosophila oogenesis is curtailed dramatically when females do not have access to a protein source. Thus, to promote meiotic entry, Drosophila females must activate the Iml1/GATOR1 complex in a tissue-specific manner, using a mechanism that is independent of the overall nutrient status of the animal. At least two models can explain how Drosophila females might activate the Iml1/GATOR1 complex specifically in the germ line. In the first model, ovarian cysts locally experience low levels of amino acids during the mitotic cyst divisions and/or at the point of meiotic entry. These low levels of amino acids could reflect a non-cell-autonomous effect: The somatically derived escort cells that surround dividing ovarian cysts may function to create a low amino acid environment that triggers the activation of the Iml1/GATOR1 complex within developing ovarian cysts. Alternatively, the effect may be cell autonomous: The germ cells within dividing ovarian cysts may have a reduced ability to sense and/or import amino acids. In a second model, a developmental signaling pathway that is completely independent of local or whole-animal amino acid status directly activates the Iml1/GATOR1 complex. The identification of the upstream requirements for Iml1/GATOR1 activation in the female germ line will help distinguish between these two models (Wei, 2014b).

Although low TORC1 activity is required during early ovarian cyst development to promote the mitotic/meiotic switch, the dramatic growth of egg chambers later in oogenesis is a metabolically expensive process that is predicted to require high TORC1 activity. The current data indicate that the GATOR2 components Mio and Seh1 function to oppose the TORC1-inhibitory activity of the GATOR1 complex in the female germ line. In mio and seh1 mutants, TORC1 activity is constitutively repressed in the germ line of developing egg chambers, resulting in the activation of catabolic metabolism and the blocking of meiotic progression and oocyte development and growth (Wei, 2014b).

Previous data indicate that Mio and Seh1 act very early in oogenesis soon after the formation of the 16-cell cyst. The mio and seh1 ovarian phenotypes can be rescued by depleting the GATOR1 components nprl2, nprl3, or iml1 in the female germ line or by raising baseline levels of TORC1 activity by disabling an alternative TORC1 inhibitory complex, TSC1/2. These data are consistent with the model that the failure to maintain the meiotic cycle and the oocyte fate in mio and seh1 mutants is a direct result of inappropriately low TORC1 activity in the female germ line brought on by the deregulation of the Iml1/GATOR1 complex (Wei, 2014b).

Notably, null alleles of both mio and seh1 are viable, with many somatic tissues exhibiting no apparent developmental abnormalities and only limited reductions in cell growth. Thus, although Mio and Seh1 are critical for the activation of TORC1 and the development of the female gamete, these proteins play a relatively small role in the development and growth of many somatic tissues under nutrient-replete conditions. Whether this small role reflects the fact that components of the Iml1/GATOR1 complex are expressed at low levels in some somatic cell types or that the complex is present but needs to be activated by a signal, such as nutrient stress or a developmental signaling pathway, remains to be elucidated (Wei, 2014b).

In the future it will be important to gain a fuller understanding of the potential environmental and developmental inputs that regulate the activity of the Iml1/GATOR1 and GATOR2 complexes in multicellular organisms. These studies will provide much-needed insight into the basic mechanisms by which both environmental and developmental signaling pathways interface with the metabolic machinery to influence cell growth and differentiation (Wei, 2014b).

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).

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

Small GTPases act as molecular switches that alternate between GTP-bound and GDP-bound states, thereby regulating a vast array of cellular parameters, including mitochondrial activity, cell growth, cell metabolism, and cell morphology. Small GTPases are activated or inactivated by guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs), respectively, which regulate the GDP/GTP load of the GTPase. Hence, understanding how GEFs and GAPs are regulated is an important aspect of understanding GTPase function. Compared to the regulation of GEFs, relatively little is known about how the activity of GAPs is regulated. GAPs acting on members of the Rho and Arf GTPase superfamilies are activated via membrane recruitment, causing rearrangements in the structure of the GAPs upon membrane binding. Regulation of GAPs acting on members of the Ras superfamily of GTPases is less well understood. One such GAP is composed of the TSC1/TSC2/TBC1D7 trimeric tumor suppressor complex, which acts on the small GTPase Rheb. Although it is known that activity of this complex is regulated by phosphorylation on multiple sites, it is not yet clear how these phosphorylations affect TSC1/2 activity at the molecular level (Demetriades, 2014).

The kinase TOR complex 1 (TORC1) is a potent anabolic regulator of cellular growth and metabolism that is often hyperactivated in human cancers. To be active, TORC1 needs to bind a molecule of Rheb in the active, GTP-bound state. By inactivating Rheb, the TSC1/2 complex is therefore a critical upstream inhibitor of TORC1. The TSC1/2 complex acts as a central point of integration of almost all known inputs regulating TORC1, including cellular stresses such as low oxygen or low ATP, and various growth-promoting signals, such as PI3K, Ras, TNF, and Wnt signaling. The importance of the tuberous sclerosis complex (TSC) on TORC1 signaling and growth is highlighted by the fact that TSC2-inactivating mutations have been found in various human growth-related diseases. One other important input regulating TORC1 activity is the availability of amino acids. Whether TSC2 is also involved in regulating TORC1 in response to amino acids, however, is unclear because various studies have come to differing conclusions (Demetriades, 2014).

Unlike all the other inputs that regulate TORC1 via TSC1/2 and Rheb, amino acids regulate TORC1 via a separate set of small GTPases, the Rag GTPases (Kim, 2008, Sancak, 2008). The Rag GTPases form heterodimeric complexes consisting of RagA or RagB bound to RagC or RagD. These complexes are stably anchored to lysosomal membranes via the LAMTOR/Ragulator complex (Sancak, 2010). In the presence of amino acids, the Rag dimers are in an 'active' conformation with RagA or RagB bound to GTP and RagC or RagD bound to GDP. The active Rag dimers recruit TORC1 to the lysosomal surface, where it binds Rheb to form an active holoenzyme. In the absence of amino acids, the Rag GAP complex termed GATOR1 causes the Rag dimers to switch into an inactive conformation containing GDP-bound RagA/B, thereby releasing TORC1 from the lysosomal surface (Bar-Peled, 2013, Panchaud, 2013). This causes TORC1 to become inactive, presumably because it no longer binds active Rheb on the lysosomal surface. Hence, TORC1 activation can currently be viewed as consisting of two aspects-the activation of Rheb in response to a plethora of regulatory inputs and the localization of TORC1 to lysosomal membranes in response to amino acids, which allows it to meet Rheb (Demetriades, 2014).

This study uncover subcellular localization as a mechanism regulating activity of the TSC1/2 GAP complex. Upon amino acid removal, TSC1/2 is recruited to lysosomes via binding to the Rag proteins, thereby bringing TSC1/2 in close proximity to its target, Rheb. This suggests that relocalization of GAPs to the vicinity of their substrates is one mechanism for their regulation. Unexpectedly, it was found that regulation of Rheb by TSC1/2 upon amino acid starvation is required for TORC1 to be released from lysosomal membranes. This suggests a 'dual anchoring' mechanism of TORC1 at the lysosome, perhaps with the Rag proteins playing a crucial role in recruiting TORC1 to the lysosomal membrane and Rheb helping to retain it there. Xells lacking TSC2 were found to be impaired in their response to amino acid starvation, failing to efficiently turn off TORC1. As a result, cells lacking TSC2 are very sensitive to amino acid starvation and die under conditions that control cells can cope with. In sum, these data indicate that the TSC1/2 complex is responsive to amino acid starvation and participates in amino acid signaling to TORC1. Hence, the TSC1/2 complex appears to play a role in regulating TORC1 in response to all regulatory inputs known to date (Demetriades, 2014).

The data presented in this study suggest a model whereby, in the presence of amino acids, mTORC1 accumulates on lysosomes due to a dual anchoring activity composed primarily of the Rag proteins but supported by Rheb. While amino acids are present, binding between the Rag proteins and TSC1/2 is low, causing the TSC1/2 complex to remain cytoplasmic. Upon amino acid removal, the Rag proteins cause mTORC1 to be released from lysosomes via two independent activities, both of which result from the Rag proteins shifting to an inactive conformation. First, the Rag proteins reduce their binding for mTORC1, thereby releasing one of the two activities tethering mTORC1 at the lysosome. Second, the Rag proteins actively recruit TSC2 to the lysosome. This allows TSC2 to act on Rheb, thereby releasing the second tethering activity keeping mTORC1 on the lysosome. In the absence of TSC2, this second activity is unaffected, causing mTORC1 to remain lysosomally localized (Demetriades, 2014).

The LAMTOR complex (composed of the p18, p14, and MP1 proteins) has been shown to serve as a docking point for mTORC1 (Sancak, 2010) and MEK/ERK complexes, regulating their recruitment to late endosomes/lysosomes and their activation status. These data demonstrate that integrity of the LAMTOR complex is critical for proper TSC2 subcellular localization upon amino acid withdrawal, therefore highlighting the importance of this scaffold complex for endomembrane-mediated activation/inactivation of signaling pathways (Demetriades, 2014).

The data presented in this study show that the TSC1/2 complex is part of the molecular machinery required for mTORC1 to respond properly to the absence of amino acids. The TSC1/2 complex responds to amino acid starvation by changing its subcellular localization and TSC2 is required for mTORC1 to be fully released from lysosomes and fully inactivated upon amino acid removal. That said, however, in cells lacking TSC2, there is nonetheless a clear initial drop in TORC1 activity upon amino acid removal. The remaining activity is then sustained indefinitely. Hence, mTORC1 appears to consist of two pools or two degrees of activation, one of which requires TSC2 to become inactive upon amino acid withdrawal and one of which responds independently of TSC2. This might explain why previous studies arrived at differing interpretations of their data because there is some response of TORC1 to amino acid removal in TSC2 null cells; however, the response is severely blunted compared to controls. Further work will hopefully shed light on these two pools of activity. Although the impairment in mTORC1 response to amino acids in TSC2 null cells is partial, it is nonetheless of critical physiological relevance because TSC2 null MEFs die upon amino acid removal in sharp contrast to control MEFs (Demetriades, 2014).

Various insights can be derived from these data. (1) Regulation of mTORC1 activation could previously be rationalized as consisting of two independent, parallel steps: first, regulation of Rheb via TSC1/2 in response to a plethora of signals including stresses and growth factor signaling, and second, regulation of mTORC1 subcellular localization to lysosomal membranes in response to amino acids. Only when mTORC1 is properly localized to meet active Rheb would an active holoenzyme form. The data presented in this study blur the distinction between these two steps because Rheb also affects mTORC1 localization, and amino acids also signal through TSC1/2. Instead, the two sets of regulatory inputs into mTORC1 appear to be more integrated. (2) Amino acid removal is 'dominant' over growth factor signaling, causing mTORC1 to shut off despite the presence of growth factors. This was previously explained by the fact that, in the absence of amino acids, mTORC1 could not localize near active Rheb to form an active complex. The dual tethering model is also consistent with this notion but for a slightly modified reason, which is that amino acid starvation acts to sever both the Rag and Rheb lysosomal tethering activities. (3) Seen from the perspective of the Rag proteins, they swap binding partners depending on the state of amino acid signaling, binding preferentially to mTORC1 in the presence of amino acids, and binding preferentially to the TSC1/2 complex in the absence of amino acids. Consequently, mTORC1 and TSC1/2 also swap subcellular localizations (Demetriades, 2014).

It was noticed that, knockdown of Rag proteins did not result in as strong a reduction in TORC1 activity in the presence of amino acids as has been previously reported. The simplest explanation is technical-that the Rag knockdowns are not strong enough to fully abrogate Rag recruitment of mTORC1 in the presence of amino acids but are sufficient to impair Rag recruitment of TSC2 in the absence of amino acids. In that case, optimizing the Rag knockdowns might lead to even stronger effects than the ones presented in this study. Two alternate biological explanations, however, might be worth investigating in the future. The first is that the data suggest a dual anchoring mechanism of mTORC1 at the lysosomal membrane-one by the Rag proteins and one by Rheb. It is possible that the relative contribution of lysosomal tethering of mTORC1 by the Rag proteins and by Rheb might depend on their relative levels of expression and activation in the system being studied. This balance will likely depend on the cell line and on cell culture conditions. A second possible explanation could be one of biological kinetics, influenced by treatment strategy. The outcome might be quantitatively different if one looks at acute amino acid removal from cells adapted to complete medium (which is done in this study) or if one looks at amino acid add-back to cells that have equilibrated their signaling to the absence of amino acids. Indeed, it was seen that, the Rag proteins were knocked down in HEK293FT cells, no dramatic reduction is seen in mTORC1 activity in the presence of amino acids. However, if amino acids are removed for 1 hr and then amino acids were re-added for 30 min, the same degree of Rag knockdown causes an obvious reduction in mTORC1 activity. Likewise, in Drosophila S2 cells, RagC knockdown only had a mild effect on TORC1 activity in untreated cells but severely blunted the ability of cells to respond to amino acid add-back. This difference between amino acid removal and amino acid add-back raises the interesting possibility that the Rag proteins are key in recruiting mTORC1 to the lysosome, a process that happens upon amino acid readdition, and that both the Rag proteins and Rheb work together to keep mTORC1 on the lysosome once it is there. Indeed, in agreement with this model, mTOR is able to be recruited to the lysosome upon amino acid readdition in TSC2 null MEFs in which Rheb is knocked down, indicating that, although Rheb tethers mTOR to the lysosome upon amino acid removal, it is not required for de novo recruitment of mTOR to the lysosome upon amino acid add-back (Demetriades, 2014).

Previous reports have shown that hyperactive mTORC1 signaling or dysregulated translation can lead to a metabolic mismatch in supply and demand, leading to cellular or organismal death. It is hypothesized that, if TSC2 is required for mTORC1 activity to respond to amino acid starvation, then TSC2 might also be necessary for cells to respond physiologically to this stress. Indeed, TSC2 knockout MEFs die upon removal of amino acids, whereas control cells do not. The fact that cells with elevated mTORC1 activity due to impaired nutrient sensing die when deprived of amino acids raises the interesting hypothesis that limiting nutrient supply to tumors of certain genotypes might have a beneficial effect on their treatment. Consistent with this effect being due to elevated mTORC1 activity, the death of TSC2 knockout MEFs is rescued by rapamycin treatment. This leads to the unexpected finding that rapamycin can actually promote cell survival under nutrient deprivation conditions, which might have therapeutic implications in mTOR-related malignancies (Demetriades, 2014).

This study identified the subcellular localization of TSC1/2 as one mechanism regulating this GAP holoenzyme. In an accompanying manuscript, Menon (2014) showed that TSC2 subcellular localization is also regulated by insulin signaling. They show that, in the absence of fetal bovine serum (FBS), TSC2 is lysosomally localized. This study shows that, in the absence of amino acids, TSC2 is lysosomally localized, even in the presence of growth factor signaling (FBS). Hence, the presence of both amino acids and growth factor signaling are required to keep TSC2 in the cytoplasm, and as long as one of the two is missing, TSC2 becomes lysosomally localized. Combined, these findings raise the possibility that TSC2 subcellular localization is a general mechanism for regulating this complex. It would be interesting to study whether the other inputs known to regulate TSC2 also affect its subcellular localization (Demetriades, 2014).

Sestrins function as guanine nucleotide dissociation inhibitors for Rag GTPases to control mTORC1 signaling

Mechanistic target of rapamycin complex 1 (mTORC1) (see Drosophila Tor) integrates diverse environmental signals to control cellular growth and organismal homeostasis. In response to nutrients, Rag GTPases (see Drosophila RagA-B) recruit mTORC1 to the lysosome to be activated, but how Rags are regulated remains incompletely understood. This study shows that Sestrins (see Drosophila Sestrin) bind to the heterodimeric RagA/B-RagC/D GTPases, and function as guanine nucleotide dissociation inhibitors (GDIs) for RagA/B. Sestrin overexpression inhibits amino-acid-induced Rag guanine nucleotide exchange and mTORC1 translocation to the lysosome. Mutation of the conserved GDI motif creates a dominant-negative form of Sestrin that renders mTORC1 activation insensitive to amino acid deprivation, whereas a cell-permeable peptide containing the GDI motif inhibits mTORC1 signaling. Mice deficient in all Sestrins exhibit reduced postnatal survival associated with defective mTORC1 inactivation in multiple organs during neonatal fasting. These findings reveal a nonredundant mechanism by which the Sestrin family of GDIs regulates the nutrient-sensing Rag GTPases to control mTORC1 signaling (Peng, 2014).

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 found 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).

The mTOR complex 1 (mTORC1) branch of the mammalian target of rapamycin (mTOR) pathway is a major driver of cell growth in mammals and is deregulated in many common tumors. It is also the target of the drug rapamycin, which has generated considerable interest as an anticancer therapy. Diverse signals regulate the mTORC1 pathway, including insulin, hypoxia, mitochondrial function, and glucose and amino acid availability. Many of these are integrated upstream of mTORC1 by the tuberous sclerosis complex (TSC1-TSC2) tumor suppressor, which acts as an important negative regulator of mTORC1 through its role as a guanosine triphosphatase (GTPase)-activating protein (GAP) for Rheb, a small guanosine triphosphate (GTP)-binding protein that potently activates the protein kinase activity of mTORC1. Loss of either TSC protein causes hyperactivation of mTORC1 signaling, even in the absence of many of the upstream signals that are normally required to maintain pathway activity. A notable exception is the amino acid supply, as the mTORC1 pathway remains sensitive to amino acid starvation in cells lacking either TSC1 or TSC2 (Sancak, 2008).

The mechanisms through which amino acids signal to mTORC1 remain mysterious. It is a reasonable expectation that proteins that signal the availability of amino acids to mTORC1 are also likely to interact with it, but, so far, no good candidates have been identified. Because most mTORC1 purifications rely on antibodies to isolate mTORC1, we wondered if in previous work antibody heavy chains obscured, during SDS-polyacrylamide electrophoresis (SDS-PAGE) analysis of purified material, mTORC1-interacting proteins of 45 to 55 kD. Indeed, using a purification strategy that avoids this complication, the 44-kD RagC protein was identified as copurifying with overexpressed raptor, the defining component of mTORC1 (Sancak, 2008).

RagC is a Ras-related small GTP-binding protein and one of four Rag proteins in mammals (RagA, RagB, RagC, and RagD). RagA and RagB are very similar to each other and are orthologs of budding yeast Gtr1p, whereas RagC and RagD are similar and are orthologs of yeast Gtr2p. In yeast and in human cells, the Rag and Gtr proteins function as heterodimers consisting of one Gtr1p-like (RagA or RagB) and one Gtr2p-like (RagC or RagD) component. The finding that RagC copurifies with raptor was intriguing because, in yeast, Gtr1p and Gtr2p regulate the intracellular sorting of the Gap1p amino acid permease and microautophagy, processes modulated by amino acid levels and the TOR pathway. The Gtr proteins have been proposed to act downstream or in parallel to TORC1 in yeast because their overexpression induces microautophagy even in the presence of rapamycin, which normally suppresses it (Sancak, 2008).

To verify identification of RagC as an mTORC1-interacting protein, raptor was expressed with different pairs of Rag proteins in human embryonic kidney (HEK)-293T cells. Consistent with the Rags functioning as heterodimers, raptor copurified with RagA-C or RagB-C, but not with RagA-B or the Rap2A control protein. Because the nucleotide loading state of most GTP-binding proteins regulates their functions, RagB, RagC, and RagD mutants predicted to be restricted to the GTP- or guanosine diphosphate (GDP)-bound conformations were generated (for simplicity, these mutants are called RagBGTP, RagBGDP, etc.). When expressed with mTORC1 components, Rag heterodimers containing RagBGTP immunoprecipitated with more raptor and mTOR than did complexes containing wild-type RagB or RagBGDP. The GDP-bound form of RagC increased the amount of copurifying mTORC1, so that RagBGTP-CGDP recovered the highest amount of endogenous mTORC1 of any heterodimer tested. Giving an indication of the strength of the mTORC1-RagBGTP-CGDP association, in this same assay, coimmunoprecipitation of mTORC1 with Rheb1, an established interactor and activator of mTORC1, could not be detected. When expressed alone, raptor, but not mTOR, associated with RagBGTP-DGDP, which suggests that raptor is the key mediator of the Rag-mTORC1 interaction. Consistent with this, rictor, an mTOR-interacting protein that is only part of mTORC2, did not copurify with any Rag heterodimer. Last, highly purified raptor interacted in vitro with RagB-D and, to a larger extent, with RagBGTP-DGDP, which indicates that the Rag-raptor interaction is most likely direct (Sancak, 2008).

Whether various Rag heterodimers affected the regulation of the mTORC1 pathway within human cells was tested. In HEK-293T cells, expression of the RagBGTP-DGDP heterodimer, which interacted strongly with mTORC1, not only activated the pathway, but also made it insensitive to deprivation for leucine or total amino acids, as judged by the phosphorylation state of the mTORC1 substrate T389 of S6K1. The wild-type RagB-C heterodimer had milder effects than RagBGTP-CGDP, making the mTORC1 pathway insensitive to leucine deprivation, but not to the stronger inhibition caused by total amino acid starvation. Expression of RagBGDP-DGTP, a heterodimer that did not interact with mTORC1, had dominant-negative effects, as it eliminated S6K1 phosphorylation in the presence, as well as absence, of leucine or amino acids. Expression of RagBGDP alone also suppressed S6K1 phosphorylation. These results suggest that the activity of the mTORC1 pathway under normal growth conditions depends on endogenous Rag function (Sancak, 2008).

To verify the actions of the Rags in a more physiological setting than that achieved by transient cDNA transfection, HEK-293T cell lines stably expressing Rheb1, RagB, or RagBGTP were generated (attempts to generate lines stably expressing RagBGDP failed). Under normal growth conditions, these cells were larger than control cells and had higher levels of mTORC1 pathway activity. Unlike transient Rheb1 overexpression, stable expression did not make the mTORC1 pathway insensitive to leucine or amino acid starvation, consistent with evidence that transiently overexpressed Rheb may have nonphysiological consequences on amino acid signaling to mTORC1. Stable expression of a Rheb1GTP mutant was also unable to make the mTORC1 pathway resistant to amino acid deprivation. In contrast, stable expression of RagBGTP eliminated the sensitivity of the mTORC1 pathway to leucine or total amino acid withdrawal, whereas that of wild-type RagB overcame sensitivity to leucine but not to amino acid starvation. Thus, transient or stable expression of the appropriate Rag mutants is sufficient to put the mTORC1 pathway into states that mimic the presence or absence of amino acids (Sancak, 2008).

To determine if the Rag mutants affect signaling to mTORC1 from inputs besides amino acids, tests were performed to see whether in RagBGTP-expressing cells the mTORC1 pathway was resistant to other perturbations known to inhibit it. This was not the case, as oxidative stress, mitochondrial inhibition, or energy deprivation still reduced S6K1 phosphorylation in these cells. Moreover, in HEK-293E cells, expression of RagBGTP-DGDP did not maintain mTORC1 pathway activity in the absence of insulin. Expression of the dominant-negative RagBGDP-DGTP heterodimer did, however, block insulin-stimulated phosphorylation of S6K1, as did amino acid starvation. Thus, although RagBGTP expression mimics amino acid sufficiency, it cannot substitute for other inputs that mTORC1 normally monitors (Sancak, 2008).

This evidence for a primary role of the Rag proteins in amino acid signaling to mTORC1 raised the question of where, within the pathway that links amino acids to mTORC1, the Rag proteins might function. The existence of the Rag-mTORC1 interaction, the effects on amino acid signaling of the Rag mutants, and the sensitivity to rapamycin of the S6K1 phosphorylation induced by RagBGTP, strongly suggested that the Rag proteins function downstream of amino acids and upstream of mTORC1. To verify this, advantage was taken of the established finding that cycloheximide reactivates mTORC1 signaling in cells starved for amino acids by blocking protein synthesis and thus boosting the levels of the intracellular amino acids sensed by mTORC1. Thus, if the Rag proteins act upstream of amino acids, cycloheximide should overcome the inhibitory effects of the RagBGDP-CGTP heterodimer on mTORC1 signaling, but if they are downstream, cycloheximide should not reactivate the pathway. The results were clear: cycloheximide treatment of cells reversed the inhibition of mTORC1 signaling caused by leucine deprivation, but not that caused by expression of RagBGDP-CGTP. Given the placement of the Rag proteins downstream of amino acids and upstream of mTORC1, whether amino acids regulate the Rag-mTORC1 interaction within cells was determined. Initial tests using transiently coexpressed Rag proteins and mTORC1 components did not reveal any regulation of the interaction. Because it was reasoned that pronounced overexpression might overcome the normal regulatory mechanisms that operate within the cell, an assay was developed, based on a reversible chemical cross-linker, that allows detection of the interaction of stably expressed FLAG-tagged Rag proteins with endogenous mTORC1. With this approach, it was readily found that amino acids, but not insulin, promote the Rag-mTORC1 interaction when either FLAG-tagged RagB or RagD were used to isolate mTORC1 from cells. As the GTP-loading state of the Rag proteins also regulates the Rag-mTORC1 interaction, it was determined whether amino acids modulate the amount of GTP bound to RagB. Indeed, amino acid stimulation of cells increased the GTP loading of RagB. Consistent with this, amino acids did not further augment the already high level of interaction between mTORC1 and the RagBGTP mutant (Sancak, 2008).

To determine whether the Rag proteins are necessary for amino acids to activate the mTORC1 pathway, combinations of lentivirally delivered short hairpin RNAs (shRNAs) were used to suppress RagA and RagB or RagC and RagD at the same time. Loss of RagA and RagB also led to the loss of RagC and RagD and vice versa, which suggests that, within cells, the Rag proteins are unstable when not in heterodimers. In cells with a reduction in the expression of all the Rag proteins, leucine-stimulated phosphorylation of S6K1 was strongly reduced. The role of the Rag proteins appears to be conserved in Drosophila cells as double-stranded RNA-mediated suppression of the Drosophila orthologs of RagB or RagC eliminated amino acid-induced phosphorylation of dS6K. Consistent with amino acids being necessary for activation of mTORC1 by insulin, a reduction in Rag expression also suppressed insulin-stimulated phosphorylation of S6K1. Thus, the Rag proteins appear to be both necessary and sufficient for mediating amino acid signaling to mTORC1 (Sancak, 2008).

Unlike Rheb, the Rag heterodimers did not directly stimulate the kinase activity of mTORC1 in vitro, so the possibility was considered that the Rag proteins regulate the intracellular localization of mTOR. mTOR is found on the endomembrane system of the cell, including the endoplasmic reticulum, Golgi apparatus, and endosomes. The intracellular localization of endogenous mTOR, as revealed with an antibody, recognizes mTOR in immunofluorescence assays, was strikingly different in cells deprived of amino acids than in cells starved and briefly restimulated with amino acids or growing in fresh complete media. In starved cells, mTOR was in tiny puncta throughout the cytoplasm, whereas in cells stimulated with amino acids for as little as 3 min, mTOR localized to the perinuclear region of the cell, to large vesicular structures, or to both. Rapamycin did not block the change in mTOR localization induced by amino acids, which indicated that it is not a consequence of mTORC1 activity but rather may be one of the mechanisms that underlies mTORC1 activation. The amino acid-induced change in mTOR localization required expression of the Rag proteins and of raptor, and amino acids also regulated the localization of raptor (Sancak, 2008).

In cells overexpressing RagB, Rheb1, or Rheb1GTP, mTOR behaved as in control cells, its localization changing upon amino acid stimulation from small puncta to the perinuclear region and vesicular structures. In contrast, in cells overexpressing the RagBGTP mutant that eliminates the amino acid sensitivity of the mTORC1 pathway, mTOR was already present on the perinuclear and vesicular structures in the absence of amino acids, and became even more localized to them upon the addition of amino acids. Thus, there is a correlation, under amino acid-starvation conditions, between the activity of the mTORC1 pathway and the subcellular localization of mTOR, which implies a role for Rag-mediated mTOR translocation in the activation of mTORC1 in response to amino acids (Sancak, 2008).

No established marker could be found of the endomembrane system that colocalized with mTOR in amino acid-starved cells. However, in cells stimulated with amino acids, mTOR in the perinuclear region and on the large vesicular structures overlapped with Rab7, which indicated that a substantial fraction of mTOR translocated to the late endosomal and lysosomal compartments in amino acid-replete cells. In cells expressing RagBGTP, mTOR was present on the Rab7-positive structures even in the absence of amino acids (Sancak, 2008).

The perinuclear region and vesicular structures on which mTOR appears after amino acid stimulation are similar to the Rab7-positive structures where green fluorescent protein (GFP)-tagged Rheb localizes in human cells. Unlike mTOR, however, amino acids did not appreciably affect the localization of Rheb, as GFP-Rheb1 colocalized with Discosoma red fluorescent protein (DsRed)-labeled Rab7 (DsRed-Rab7) in the presence or absence of amino acids. Unfortunately, it is currently not possible to compare, in the same cells, the localization of endogenous mTOR with that of Rheb, because the signal for GFP-Rheb or endogenous Rheb is lost after fixed cells are permeabilized to allow access to intracellular antigens. Nevertheless, given that both mTOR and Rheb are present in Rab7-positive structures after amino acid stimulation, it is proposed that amino acids might control the activity of the mTORC1 pathway by regulating, through the Rag proteins, the movement of mTORC1 to the same intracellular compartment that contains its activator Rheb. This would explain why activators of Rheb, like insulin, do not stimulate the mTORC1 pathway when cells are deprived of amino acids and why Rheb is necessary for amino acid-dependent mTORC1 activation. When Rheb is highly overexpressed, some might become mislocalized and inappropriately encounter and activate mTORC1, which could explain why Rheb overexpression, but not loss of TSC1 or TSC2, makes the mTORC1 pathway insensitive to amino acids (Sancak, 2008).

In conclusion, the Rag GTPases bind raptor, are necessary and sufficient to mediate amino acid signaling to mTORC1, and mediate the amino acid-induced relocalization of mTOR within the endomembrane system of the cell. Given the prevalence of cancer-linked mutations in the pathways that control mTORC1, it is possible that Rag function is also deregulated in human tumors (Sancak, 2008).

Ragulator and GATOR1 complexes promote fission yeast growth by attenuating TOR complex 1 through Rag GTPases

TOR complex 1 (TORC1) is an evolutionarily conserved protein kinase complex that promotes cellular macromolecular synthesis and suppresses autophagy. Amino-acid-induced activation of mammalian TORC1 is initiated by its recruitment to the RagA/B-RagC/D GTPase heterodimer, which is anchored to lysosomal membranes through the Ragulator complex. This study identified in the model organism Schizosaccharomyces pombe a Ragulator-like complex that tethers the Gtr1-Gtr2 Rag heterodimer to the membranes of vacuoles, the lysosome equivalent in yeasts. Unexpectedly, the Ragulator-Rag complex is not required for the vacuolar targeting of TORC1, but the complex plays a crucial role in attenuating TORC1 activity independently of the Tsc1-Tsc2 complex, a known negative regulator of TORC1 signaling. The GATOR1 complex, which functions as Gtr1 GAP, is essential for the TORC1 attenuation by the Ragulator-Rag complex, suggesting that Gtr1(GDP)-Gtr2 on vacuolar membranes moderates TORC1 signaling for optimal cellular response to nutrients (Chia, 2017).

Spatial control of the TSC complex integrates insulin and nutrient regulation of mTORC1 at the lysosome

mTORC1 promotes cell growth in response to nutrients and growth factors. Insulin activates mTORC1 through the PI3K-Akt pathway, which inhibits the TSC1-TSC2-TBC1D7 complex (the TSC complex) to turn on Rheb, an essential activator of mTORC1. However, the mechanistic basis of how this pathway integrates with nutrient-sensing pathways is unknown. This study demonstrates in cultured cells that insulin stimulates acute dissociation of the TSC complex from the lysosomal surface, where subpopulations of Rheb and mTORC1 reside. The TSC complex associates with the lysosome in a Rheb-dependent manner, and its dissociation in response to insulin requires Akt-mediated TSC2 phosphorylation. Loss of the PTEN tumor suppressor results in constitutive activation of mTORC1 through the Akt-dependent dissociation of the TSC complex from the lysosome. These findings provide a unifying mechanism by which independent pathways affecting the spatial recruitment of mTORC1 and the TSC complex to Rheb at the lysosomal surface serve to integrate diverse growth signals (Meon, 2014).

Rag GTPases are cardioprotective by regulating lysosomal function

The Rag family proteins are Ras-like small GTPases that have a critical role in amino-acid-stimulated mTORC1 activation by recruiting mTORC1 to lysosome. Despite progress in the mechanistic understanding of Rag GTPases in mTORC1 activation, little is known about the physiological function of Rag GTPases in vivo. This study shows that loss of RagA and RagB (RagA/B) in cardiomyocytes results in hypertrophic cardiomyopathy and phenocopies lysosomal storage diseases, although mTORC1 activity is not substantially impaired in vivo. Despite upregulation of lysosomal protein expression by constitutive activation of the transcription factor EB (TFEB) in RagA/B knockout mouse embryonic fibroblasts, lysosomal acidification is compromised owing to decreased v-ATPase level in the lysosome fraction. This study uncovers RagA/B GTPases as key regulators of lysosomal function and cardiac protection (Kim, 2014).

Amino acid deprivation inhibits TORC1 through a GTPase-activating protein complex for the Rag family GTPase Gtr1

The Rag family of guanosine triphosphatases (GTPases) regulates eukaryotic cell growth in response to amino acids by activating the target of rapamycin complex 1 (TORC1). In humans, this pathway is often deregulated in cancer. In yeast, amino acids promote binding of GTP (guanosine 5'-triphosphate) to the Rag family GTPase Gtr1, which, in combination with a GDP (guanosine diphosphate)-bound Gtr2, forms the active, TORC1-stimulating GTPase heterodimer. This study identified Iml1, which functioned in a complex with Npr2 and Npr3, as a GAP (GTPase-activating protein) for Gtr1. Upon amino acid deprivation, Iml1 transiently interacted with Gtr1 at the vacuolar membrane to stimulate its intrinsic GTPase activity and consequently decrease the activity of TORC1. These results delineate a potentially conserved mechanism by which the Iml1, Npr2, and Npr3 orthologous proteins in humans may suppress tumor formation (Panchaud, 2013).

A Tumor suppressor complex with GAP activity for the Rag GTPases that signal amino acid sufficiency to mTORC1

The mTOR complex 1 (mTORC1) pathway promotes cell growth in response to many cues, including amino acids, which act through the Rag guanosine triphosphatases (GTPases) to promote mTORC1 translocation to the lysosomal surface, its site of activation. Although progress has been made in identifying positive regulators of the Rags, it is unknown if negative factors also exist. This study identified GATOR as a complex that interacts with the Rags and is composed of two subcomplexes which were called GATOR1 and -2. Inhibition of GATOR1 subunits (DEPDC5, Nprl2, and Nprl3) makes mTORC1 signaling resistant to amino acid deprivation. In contrast, inhibition of GATOR2 subunits (Mios, WDR24, WDR59, Seh1L, and Sec13) suppresses mTORC1 signaling, and epistasis analysis shows that GATOR2 negatively regulates DEPDC5. GATOR1 has GTPase-activating protein (GAP) activity for RagA and RagB, and its components are mutated in human cancer. In cancer cells with inactivating mutations in GATOR1, mTORC1 is hyperactive and insensitive to amino acid starvation, and such cells are hypersensitive to rapamycin, an mTORC1 inhibitor. Thus, this study identified a key negative regulator of the Rag GTPases and reveal that, like other mTORC1 regulators, Rag function can be deregulated in cancer (Bar-Peled, 2013).

Ragulator is a GEF for the rag GTPases that signal amino acid levels to mTORC1

The mTOR Complex 1 (mTORC1) pathway regulates cell growth in response to numerous cues, including amino acids, which promote mTORC1 translocation to the lysosomal surface, its site of activation. The heterodimeric RagA/B-RagC/D GTPases, the Ragulator complex that tethers the Rags to the lysosome, and the v-ATPase form a signaling system that is necessary for amino acid sensing by mTORC1. Amino acids stimulate the binding of guanosine triphosphate to RagA and RagB but the factors that regulate Rag nucleotide loading are unknown. This study identified HBXIP and C7orf59 as two additional Ragulator components that are required for mTORC1 activation by amino acids. The expanded Ragulator has nucleotide exchange activity toward RagA and RagB and interacts with the Rag heterodimers in an amino acid- and v-ATPase-dependent fashion. Thus, this study provides mechanistic insight into how mTORC1 senses amino acids by identifying Ragulator as a guanine nucleotide exchange factor (GEF) for the Rag GTPases (Bar-Peled, 2012).

Ragulator-Rag complex targets mTORC1 to the lysosomal surface and is necessary for its activation by amino acids

The mTORC1 kinase promotes growth in response to growth factors, energy levels, and amino acids, and its activity is often deregulated in disease. The Rag GTPases interact with mTORC1 and are proposed to activate it in response to amino acids by promoting mTORC1 translocation to a membrane-bound compartment that contains the mTORC1 activator, Rheb. This study shows that amino acids induce the movement of mTORC1 to lysosomal membranes, where the Rag proteins reside. A complex encoded by the MAPKSP1, ROBLD3, and c11orf59 genes, which was termed Ragulator, interacts with the Rag GTPases, recruits them to lysosomes, and is essential for mTORC1 activation. Constitutive targeting of mTORC1 to the lysosomal surface is sufficient to render the mTORC1 pathway amino acid insensitive and independent of Rag and Ragulator, but not Rheb, function. Thus, Rag-Ragulator-mediated translocation of mTORC1 to lysosomal membranes is the key event in amino acid signaling to mTORC1 (Sancak, 2010).

Search PubMed for articles about Drosophila RagA-B or Ragc-d

Bar-Peled, L., Schweitzer, L. D., Zoncu, R. and Sabatini, D. M. (2012). Ragulator is a GEF for the rag GTPases that signal amino acid levels to mTORC1. Cell 150(6): 1196-1208. PubMed ID: 22980980

Bar-Peled, L., Chantranupong, L., Cherniack, A. D., Chen, W. W., Ottina, K. A., Grabiner, B. C., Spear, E. D., Carter, S. L., Meyerson, M. and Sabatini, D. M. (2013). A Tumor suppressor complex with GAP activity for the Rag GTPases that signal amino acid sufficiency to mTORC1. Science 340(6136): 1100-1106. PubMed ID: 23723238

Chia, K. H., Fukuda, T., Sofyantoro, F., Matsuda, T., Amai, T. and Shiozaki, K. (2017). Ragulator and GATOR1 complexes promote fission yeast growth by attenuating TOR complex 1 through Rag GTPases. Elife 6. PubMed ID: 29199950

Demetriades, C., Doumpas, N. and Teleman, A. A. (2014). Regulation of TORC1 in response to amino acid starvation via lysosomal recruitment of TSC2. Cell 156: 786-799. PubMed ID: 24529380

Frias, M. A., Mukhopadhyay, S., Lehman, E., Walasek, A., Utter, M., Menon, D. and Foster, D. A. (2020). Phosphatidic acid drives mTORC1 lysosomal translocation in the absence of amino acids. J Biol Chem 295(1): 263-274. PubMed ID: 31767684

Han, J. M., Jeong, S. J., Park, M. C., Kim, G., Kwon, N. H., Kim, H. K., Ha, S. H., Ryu, S. H. and Kim, S. (2012). Leucyl-tRNA synthetase is an intracellular leucine sensor for the mTORC1-signaling pathway. Cell 149(2): 410-424. PubMed ID: 22424946

Kim, E., Goraksha-Hicks, P., Li, L., Neufeld, T. P. and Guan, K. L. (2008). Regulation of TORC1 by Rag GTPases in nutrient response. Nat Cell Biol 10: 935-945. PubMed ID: 18604198

Kim, Y. C., Park, H. W., Sciarretta, S., Mo, J. S., Jewell, J. L., Russell, R. C., Wu, X., Sadoshima, J. and Guan, K. L. (2014). Rag GTPases are cardioprotective by regulating lysosomal function. Nat Commun 5: 4241. PubMed ID: 24980141

Kwak, S. S., Kang, K. H., Kim, S., Lee, S., Lee, J. H., Kim, J. W., Byun, B., Meadows, G. G. and Joe, C. O. (2016). Amino acid-dependent NPRL2 interaction with Raptor determines mTOR Complex 1 activation. Cell Signal 28(2): 32-41. PubMed ID: 26582740

Menon, S., Dibble, C. C., Talbott, G., Hoxhaj, G., Valvezan, A. J., Takahashi, H., Cantley, L. C. and Manning, B. D. (2014). Spatial control of the TSC complex integrates insulin and nutrient regulation of mTORC1 at the lysosome. Cell 156(4): 771-785. PubMed ID: 24529379

Panchaud, N., Peli-Gulli, M. P. and De Virgilio, C. (2013). Amino acid deprivation inhibits TORC1 through a GTPase-activating protein complex for the Rag family GTPase Gtr1. Sci Signal 6(277): ra42. PubMed ID: 23716719

Peng, M., Yin, N. and Li, M. O. (2014). Sestrins function as guanine nucleotide dissociation inhibitors for Rag GTPases to control mTORC1 signaling. Cell 159: 122-133. PubMed ID: 25259925

Petit, C. S., Roczniak-Ferguson, A. and Ferguson, S. M. (2013). Recruitment of folliculin to lysosomes supports the amino acid-dependent activation of Rag GTPases. J Cell Biol 202(7): 1107-1122. PubMed ID: 24081491

Sancak, Y., Peterson, T. R., Shaul, Y. D., Lindquist, R. A., Thoreen, C. C., Bar-Peled, L. and Sabatini, D. M. (2008). The Rag GTPases bind raptor and mediate amino acid signaling to mTORC1. Science 320: 1496-1501. PubMed ID: 18497260

Sancak, Y., Bar-Peled, L., Zoncu, R., Markhard, A. L., Nada, S. and Sabatini, D. M. (2010). Ragulator-Rag complex targets mTORC1 to the lysosomal surface and is necessary for its activation by amino acids. Cell 141(2): 290-303. PubMed ID: 20381137

Senger, S., Csokmay, J., Akbar, T., Jones, T. I., Sengupta, P. and Lilly, M. A. (2011).The nucleoporin Seh1 forms a complex with Mio and serves an essential tissue-specific function in Drosophila oogenesis. Development 138(10): 2133-42. PubMed ID: 21521741

Tsokanos, F. F., Albert, M. A., Demetriades, C., Spirohn, K., Boutros, M. and Teleman, A. A. (2016). eIF4A inactivates TORC1 in response to amino acid starvation. EMBO J 35(10):1058-76. PubMed ID: 26988032

Tsun, Z. Y., Bar-Peled, L., Chantranupong, L., Zoncu, R., Wang, T., Kim, C., Spooner, E. and Sabatini, D. M. (2013). The folliculin tumor suppressor is a GAP for the RagC/D GTPases that signal amino acid levels to mTORC1. Mol Cell 52(4): 495-505. PubMed ID: 24095279

Wei, Y. and Lilly, M. A. (2014a). The TORC1 inhibitors Nprl2 and Nprl3 mediate an adaptive response to amino-acid starvation in Drosophila. Cell Death Differ 21: 1460-1468. PubMed ID: 24786828

Wei, Y., Reveal, B., Reich, J., Laursen, W. J., Senger, S., Akbar, T., Iida-Jones, T., Cai, W., Jarnik, M. and Lilly, M. A. (2014b). TORC1 regulators Iml1/GATOR1 and GATOR2 control meiotic entry and oocyte development in Drosophila. Proc Natl Acad Sci U S A 111(52):E5670-7. PubMed ID: 25512509

Yang, G., Humphrey, S. J., Murashige, D. S., Francis, D., Wang, Q. P., Cooke, K. C., Neely, G. and James, D. E. (2018). RagC phosphorylation autoregulates mTOR complex 1. EMBO J. PubMed ID: 30552228

Yang, S., Zhang, Y., Ting, C. Y., Bettedi, L., Kim, K., Ghaniam, E. and Lilly, M. A. (2020). The Rag GTPase regulates the dynamic behavior of TSC downstream of both amino acid and growth factor restriction. Dev Cell. PubMed ID: 32898476

date revised: 10 December 2020

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