InteractiveFly: GeneBrief

raptor: Biological Overview | References

BIOLOGICAL OVERVIEW

Nutrient restriction (NR) decreases the incidence and growth of many types of tumors, yet the underlying mechanisms are not fully understood. This study identified Headcase (Hdc) and Unkempt (Unk) as two NR-specific tumor suppressor proteins that form a complex to restrict cell cycle progression and tissue growth in response to NR in Drosophila. Loss of Hdc or Unk does not confer apparent growth advantage under normal nutrient conditions but leads to accelerated cell cycle progression and tissue overgrowth under NR. Hdc and Unk bind to the TORC1 component Raptor and preferentially regulate S6 phosphorylation in a TORC1-dependent manner. It was further shown that HECA and UNK, the human counterparts of Drosophila Hdc and Unk, respectively, have a conserved function in regulating S6 phosphorylation and tissue growth. The identification of Hdc and Unk as two NR-specific tumor suppressors provides insight into molecular mechanisms underlying the anti-tumorigenic effects of NR (Li, 2019).

This study has identified Hdc and Unk as two nutrient-sensitive tumor suppressors that restrict cell cycle progression and hence tissue growth in response to NR. An interesting feature of Hdc and Unk is that their tumor-suppressing function is manifested only under nutrient-poor conditions. This feature distinguishes Hdc and Unk from the other nutrient-sensitive tumor suppressors such as PTEN and TSC1/2, which restrict tissue grow under both nutrient-rich and nutrient-poor conditions. This feature also provides a plausible explanation for the results of a previous study in which the role of Hdc and Unk in neuronal cell differentiation, but not in tissue growth, was revealed. Indeed, no growth advantage of hdc or unk mutant clones under nutrient-rich conditions was observed. However, when nutrients are limited, both hdc and unk mutant cells proliferate much faster than wild-type twin-spot control cells, resulting in overgrowth of mutant tissue. Thus, these results suggest that in addition to NR non-specific tumor suppressors like PTEN and TSC1/2, NR also involves NR-specific tumor suppressors to antagonize tumor growth. The identification of NR-specific tumor suppressors raises the possibility that mutation of these tumor suppressor genes may also contribute to the NR resistance of some types of tumors (Li, 2019).

The unk gene has been identified as a transcriptional target of TORC1 in multiple studies. It has been shown that upon TORC1 inhibition, the transcription factors Reptor and Reptor-BP enter the nucleus to upregulate unk expression (Tiebe, 2015). Consistent with the role of Unk in NR response, Reptor and Reptor-BP knockout flies are also very sensitive to starvation (Tiebe, 2015). Interestingly, Unk was co-purified with multiple TORC1 pathway components in a systematic protein interaction study, but its potential role in regulating TORC1 signaling has not been well investigated. This study uncovers the function of Hdc and Unk in regulating TORC1 signaling with evidence including (1) Hdc, Unk, and Raptor form a protein complex; (2) Hdc and Unk regulate S6 phosphorylation both in vivo and in cell culture, and (3) Raptor is required for Hdc and Unk to regulate S6 phosphorylation. In contrast to Tsc1/2, loss of Hdc or Unk affects the phosphorylation level of S6 but not S6K and 4E-BP. Thus, Hdc and Unk preferentially regulate S6 phosphorylation in a TORC1-dependent manner (Li, 2019).

An interesting finding from this study concerns the profound effects of Hdc and Unk on cell cycle progression. Consistent with the role of Hdc in cell cycle progression, the expression pattern of hdc has been found to correlate with the timing of imaginal cells entering into mitotic cell cycle. Previous studies in both mammals and Drosophila have uncovered an important role for mTOR in cell cycle progression. Interestingly, cell cycle marker analysis revealed that S6 phosphorylation, and therefore TORC1 activity, is selectively increased in S-phase cells in Drosophila imaginal discs. Thus, during Drosophila disc development, S6 phosphorylation is spatially regulated by TORC1 and correlates to proper cell cycle progression. However, both S6K and 4E-BP are not important for TORC1 to regulate cell cycle progression during Drosophila eye development, suggesting that there may be other substrates mediating TORC1 signaling in cell cycle regulation. Interestingly, this study found that Hdc and Unk regulate both cell cycle progression and S6 phosphorylation, but not S6K or 4E-BP phosphorylation, in a TORC1-dependent manner. Taken together, these results raise the possibility that the Hdc-Unk complex, through its physical interactions with Raptor, preferentially mediate the effects of TORC1 on cell cycle progression but not cell size. Future studies will be necessary to provide more evidence and understand the underlying molecular mechanisms (Li, 2019).

A question for further investigation concerns how Hdc and Unk regulate tissue growth specifically in response to NR. Based on the observed nutrient-sensitive interactions between Unk and Raptor, an attractive mechanism could be that the growth inhibition function of Unk is regulated by its physical interaction with Raptor. Under nutrient-rich conditions, Raptor interacts with Unk and suppresses its activity, which in turn suppresses the Unk-dependent growth inhibition function of Hdc. In contrast, upon nutrient starvation, Unk is released from Raptor and its growth inhibition function is activated. Activated Unk therefore functions with Hdc to suppress cell cycle progression and eventually tissue growth. Future studies are warranted to test this model (Li, 2019).

Given the conserved role of human HECA and UNK in regulating S6 phosphorylation, these results suggest that HECA and UNK may function as tumor suppressors in mammals. Consistent with this notion, silencing HECA expression in OSCC cells causes accelerated cell division, and overexpression of HECA slows down the proliferation of OSCC cells. Although the human ortholog of Unk has not been studied in the context of cell proliferation, it was shown that both HECA and UNK are able to inhibit tissue growth in vivo in the Drosophila model. Thus, it is worthwhile in the future to investigate the growth control function of HECA and UNK, especially their contribution to tumorigenesis with aberrant mTOR signaling (Li, 2019).

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 (see Drosophila RagATPases)-which form obligate heterodimers comprising RagA or RagB together with RagC or RagD. Amino acids cause Rag GTPases to switch to an active conformation, in which RagA/B is GTP-loaded and Rag C/D is GDP-loaded. The active Rag heterodimer physically interacts with raptor, recruiting mTORC1 to the lysosome where its activator Rheb resides. Extensive work has revealed several mechanisms implicated in the regulation of Rag activity that enables them to function as nutrient sensors. A common feature among these is the control of Rag nucleotide status, particularly through the activation of guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). These include the Ragulator (GEF for Rag A/B; Bar-Peled, 2012), the GATOR1 complex (GAP for RagA/B; Bar-Peled, 2013), folliculin (FLCN, GAP for RagC/D; Petit, 2013; Tsun, 2013), and leucyl-tRNA synthetase (LeuRS, GAP for RagD; Han, 2012). Ubiquitination has also recently emerged as a post-translational modification (PTM) capable of inhibiting Rag GTPase signaling by recruiting GATOR1 to RagA. Importantly, these pathways regulating Rag activity are all amino acid-dependent, and much less is known about the control of growth factor-mediated Rag GTPase signaling (Yang, 2018).

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

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

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

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

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

The InR/Akt/TORC1 growth-promoting signaling negatively regulates JAK/STAT activity and migratory cell fate during morphogenesis

Cell growth and cell differentiation are two distinct yet coupled developmental processes, but how they are coordinated is not well understood. During Drosophila oogenesis, this study found that the growth-promoting InR/Akt/TOR pathway was involved in suppressing the fate determination of the migratory border cells. The InR/Akt/TOR TOR and Raptor, components of TORC1, to downregulate the JAK/STAT pathway, which is necessary and sufficient for border cell fate determination. TORC1 promotes the protein stability of SOCS36E, the conserved negative regulator of JAK/STAT signaling, through physical interaction, suggesting that TORC1 acts as a key regulator coordinating both cell growth and cell differentiation (Kang, 2018).

These results support the following model. During mid-oogenesis (after stage 6), the InR/Akt/TORC1 pathway not only promotes the robust growth of follicle cells but also suppresses the fate determination and differentiation of migratory border cells specifically through TORC1, which promotes the protein stability of SOCS36E and thus attenuates the JAK/STAT signaling pathway (see TORC1 Physically Associated with SOCS36E and Prevented Its Degradation by Proteasome). This raises the question of why the important differentiation pathway of JAK/STAT would need to be antagonized by the ubiquitous growth-promoting signaling of InR/Akt/TORC1. There are two possible scenarios that can explain this. First, robustly growing follicle cells may not be suited for the specification and differentiation process that would turn them into highly motile cells. It is thus conceivable that those cells would need to downregulate the fate determination process until they are ready. This scenario implies that an active downregulation of the growth signaling pathway somehow occurs in the subset of anterior follicle cells that will become border cells, but there is no evidence yet to support this. Second, the InR/Akt/TORC1 pathway could be globally needed in all follicle cells to provide a uniform suppression of the migratory cell fate so that only a small population of 6-10 cells receiving the strongest cytokine signaling can overcome the suppression and turn into the border cells (Kang, 2018).

This study demonstrates that TORC1 genetically and physically interacts with SOCS36E, uncovering an interesting and novel connection between the InR/Akt/TORC1 and JAK/STAT signaling pathways during development. Furthermore, it is known that SOCS family members also serve as negative regulators of RTK (receptor tyrosine kinase) signaling, which like JAK/STAT is also commonly involved in various cell differentiation processes during development. So it will be interesting to test whether the growth signaling pathway could also cross-regulate the RTK pathway through its interaction with the SOCS family members. Recently, advances in immunology revealed that TORC1 signaling played critical roles in T cell fate decision, and it is well known that STAT family members are crucial for T cell function. It will be interesting to test whether SOCS5 or other SOCS members mediate TORC1's role in T cell fate determination (Kang, 2018).

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).

The role of TORC1 in muscle development in Drosophila

Myogenesis is an important process during both development and muscle repair. Previous studies suggest that mTORC1 plays a role in the formation of mature muscle from immature muscle precursor cells. This study shows that gene expression for several myogenic transcription factors including Myf5, Myog and Mef2c but not MyoD and myosin heavy chain isoforms decrease when C2C12 cells are treated with rapamycin, supporting a role for mTORC1 pathway during muscle development. To investigate the possibility that mTORC1 can regulate muscle in vivo, the essential dTORC1 subunit Raptor was ablated in Drosophila melanogaster, and it was found that muscle-specific knockdown of Raptor caused flies to be too weak to emerge from their pupal cases during eclosion. Using a series of GAL4 drivers it was also shown that muscle-specific Raptor knockdown also caused shortened lifespan, even when eclosure was unaffected. Together these results highlight an important role for TORC1 in muscle development, integrity and function in both Drosophila and mammalian cells (Hatfield, 2015).

LST8 regulates cell growth via target-of-rapamycin complex 2 (TORC2)

The evolutionarily conserved serine/threonine protein kinase target-of-rapamycin (TOR) controls cell growth as a core component of TOR complexes 1 (TORC1) and 2 (TORC2). Although TORC1 is the more central growth regulator, TORC2 has also been shown to affect cell growth. This study demonstrates that Drosophila LST8, the only conserved TOR-binding protein present in both TORC1 and TORC2, functions exclusively in TORC2 and is not required for TORC1 activity. In mutants lacking LST8, expression of TOR and RAPTOR, together with their upstream activator Rheb, was sufficient to provide TORC1 activity and stimulate cell and organ growth. Furthermore, using an lst8 knockout mutation, this study showed that TORC2 regulates cell growth cell autonomously. Surprisingly, however, TORC2 does not regulate cell growth via its best-characterized target, AKT. These findings support the possible application of TORC2-specific drugs in cancer therapy (Wang, 2012).

LST8 was originally identified genetically as a mutation in yeast that is synthetically lethal with sec13-1, a mutation that causes a sorting defect in the secretory pathway. Later, physical association studies showed that LST8 is a core component of TORC1 and TORC2 in both yeast and mammals. Structural data confirmed that LST8 stably interacts with the kinase domain of TOR without overlapping the Raptor binding site. While it has been clearly demonstrated that LST8 is essential for TORC2 activity, whether or not LST8 functioned in the TORC1 pathway had not been rigorously investigated. In mammalian cells, it was originally reported that the binding of LST8 to mTOR strongly stimulated the binding association between mTOR and Raptor and, as a result, elevated the kinase activity of mTORC1 toward S6K1 and 4E-BP1. In yeast, rapamycin, which is TORC1 specific, mimicked the Gap1p sorting defect of an lst8 mutant, and a temperature-sensitive lst8 mutant was hypersensitive to rapamycin, suggesting that LST8 functions in TORC1. However, the observations that the lst8 knockout did not affect the phosphorylation of S6K1 or 4E-BP1 and that lst8 knockout mice phenocopied rictor knockout mice but not mtor knockout mice suggested that LST8 might not function in TORC1, at least under normal conditions (Wang, 2012).

This report demonstrates that lst8 knockout flies are viable but small, similar to rictor mutants but dissimilar to files with tor or rheb mutations, which are lethal. Neither loss nor overexpression of LST8 affected the kinase activity of TORC1 toward S6K or autophagy, whereas the kinase activity of TORC2 toward AKT was completely lost in the lst8 mutants. Moreover, in the absence of LST8 the overexpression of Rheb still upregulated TORC1 activity and also promoted the growth of cells. Furthermore, the expression of TOR and Raptor was sufficient to drive phosphorylation of S6K and growth of cells when they were supplied with Rheb signaling, even when LST8 was entirely absent. Given these results, it is concluded that LST8 is an essential core component of TORC2 but is dispensable for the regulation and activity of TORC1 (Wang, 2012 and references therein).

Although TOR signaling functions primarily as a central regulator of cell growth, increasing evidence suggests that inappropriate activation of TOR also makes cells sensitive to death signals. For instance, tumor cells in TSC disease, which is caused by mutation of tsc1 or tsc2, exhibit morphological features of apoptosis such as activated caspase-8 and positive terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) results. In a mouse model of TSC disease, conditional knockout of tsc1 induced cell death in the hippocampus and neocortex. In a recent study in flies, it has been shown that activation of TORC1 by either overexpression of Rheb or mutation of Tsc1 led to neurodegeneration. In the latter case the neurodegeneration was specifically attributed to TOR's ability to suppress autophagy (Wang, 2012).

Although overactivation of TORC1 can result in cell death, it is not always sufficient for the induction of cell death. For instance, in young flies, overexpression of Rheb alone promoted cell growth but did not drive cell death, whereas neuronal cell death was detected only in aged flies after long-term TOR hyperactivation. This slow neurodegeneration was also dependent on neuronal activity. The present study, however, found that when Rheb and TORC1 are overexpressed together (a condition which very strongly increases TORC1 activity), severe cell death rapidly occurred in all cell types, even in young animals, without any dependency on neuronal activity. Therefore, it is suggested that the regulation of TORC1 activity is crucial for the survival of all cell types and that the incidence of cell death within a tissue may be correlated with TORC1 activity levels. The cell death-promoting function of TORC1 might explain why mutations of tsc1 or tsc2 lead to benign, rather than malignant, tumors (Wang, 2012).

In both mammals and invertebrates, it has been demonstrated that TORC2 regulates growth and metabolism. However, whether TORC2 affects organismal growth cell autonomously or indirectly via systemic effects had not been thoroughly evaluated. In C. elegans, rictor mutants have been shown to be developmentally delayed, small in body size, and short-lived. Targeted expression of Rictor in the intestines of these mutants could rescue these growth defects, and therefore it was suggested that TORC2 might act directly in the intestine to regulate cell mass and growth of the whole animal, non-cell autonomously. This study presents evidence supporting a cell-autonomous role for TORC2 in the growth of several different cell types. In genetic mosaics, cells homozygous for the lst8¹ mutation were smaller than adjacent wild-type control cells in the retina, wing epithelium, and fat body. Moreover, expression of LST8 in posterior wing compartments specially rescued growth of the posterior cells without affecting the anterior (Wang, 2012).

TORC1 consists of TOR, Raptor, LST8, and PRAS40 (proline-rich AKT substrate, 40 kDa), a negative regulator. mTORC1 purified with PRAS40 at substoichiometric levels has been shown to have kinase activity toward S6K1 and 4E-BP1 in vitro. Raptor-free mTORC1 appears unable to support 4E-BP1 phosphorylation but might be capable of phosphorylating full-length S6K1. Interestingly, the phosphorylation of S6K1 by Raptor-free mTORC1 could still be inhibited by FKBP12-rapamycin. Nevertheless, overexpression of TOR and Raptor together has been shown to significantly increase in vitro TORC1 kinase activity compared to overexpressed TOR alone. Overexpressed TOR has been found in some cancer patients, but the significance of this relative to disease progression or TORC1 activity levels has not been assessed. By overexpression of TORC1 components in flies, this study demonstrated that expression of all three positive components of TORC1 (TOR, Raptor, and LST8) cannot drive increased TORC1 activity or cell overgrowth. However, in the presence of the upstream activator, Rheb, overexpression of TOR and Raptor either with or without LST8 increased TORC1 activity toward S6K and dramatically stimulated cell growth. These results further confirm that TOR and Raptor but not LST8 are bone fide components of TORC1. In contrast to results from in vitro studies, expression of TORC1 components did not function in the absence of activating signals, perhaps because cellular translocation of TORC1 is a key event in TORC1 activation. Since expression of TOR alone did not generate functional TORC1, the significance of overexpression of TOR in cancer cells might be reconsidered (Wang, 2012).

The fact that the two TOR complexes display distinct cellular functions and phosphorylate different downstream substrates suggests that they might respond to different upstream signals. It has been well established that TORC1 can be regulated by upstream signals such as amino acids and growth factors, but little is known about whether TORC2 can be activated by such signals. When purified from insulin- or IGF1-stimulated cells and assayed in vitro, TORC2 kinase activity is elevated, suggesting that growth factors can activate TORC2 through PI3K signaling. Data from tissue culture cells suggest that the TSC complex physically associates with TORC2 via Rictor, but surprisingly the regulation of TORC2 by the TSC1/2 complex appeared to be independent of its GTPase-activating protein activity toward Rheb. This study shows that AKT phosphorylation is downregulated when Rheb is overexpressed, suggesting a role for the Rheb GTPase in regulating TORC2. The recent finding that Rictor is directly phosphorylated by the mTORC1-dependent kinase S6K1 might help explain the regulation of TSC2 by Rheb. However, it is also possible that through negative feedback from TORC1, which phosphorylates and deactivates insulin receptor substrate 1, TORC2 is negatively regulated by Rheb signaling (Wang, 2012).

Nutrient availability is one of the major environmental signals influencing growth. Conflicting data have been proffered as to whether amino acids might regulate TORC2 activity. Although amino acids originally were not considered regulation signals for TORC2, it was recently reported that amino acids could increase AKT phosphorylation by TORC2. In addition, leucine addition to starved cells promotes cell migration in a TORC2-dependent manner. The fat body is an insect organ that retains the endocrine and storage functions of the vertebrate liver and adipose tissues and serves as a nutrient sensor that restricts global growth. Thus, fat body cells are very sensitive to nutritional conditions, and since TORC1 is controlled by nutrition, its activity has a profound influence in fat body cells. For instance, downregulation of TORC1 by expression of TSC1/2 strongly decreases fat body cell size relative to other tissues, where it has modest effects. In addition, fat body cells with upregulated TORC1 are capable of massive growth under starvation conditions, essentially bypassing the normal cessation of growth caused by starvation, whereas deregulated TORC1 has mild effects in fat body cells in fed animals. As this study shows, the disruption of TORC2 by mutation of lst8 affected the growth of every tissue to a similar extent. Moreover, starvation did not change the size reduction in lst8¹ cells in the fat body, suggesting that TORC2-regulated cell growth might not be nutrition sensitive (Wang, 2012).

TORC2 is believed to control cell survival, cell growth, and cytoskeletal organization by phosphorylating several AGC kinases including SGK, cPKCα, and AKT. Among these TORC2 substrates, AKT is especially important because of its general role in growth and survival. Therefore, it has been proposed that TORC2 might serve as a drug target in tumors that have lost the expression of PTEN, a tumor suppressor that inhibits AKT activity. However, recent evidence suggests that TORC2-mediated phosphorylation of AKT does not determine its absolute activity but, instead, determines substrate specificity. AKT variants immune to TORC2-mediated phosphorylation retain the ability to control glycogen synthase kinase 3 (GSK3) and TSC2 but show decreased activity toward FOXO1/3a. These observations align with those of this study in supporting the view that TORC2 might only help to inactivate the FOXO branch of the AKT signaling pathway without affecting the TORC1 branch (Wang, 2012).

The results of this study suggest that TORC2-mediated AKT phosphorylation does not regulate the growth of cells and argue against the importance of AKT as an important downstream target of TORC2 in cell growth control. Expression of nonphosphorylatable mutant forms of AKT did not change cellular growth status either with or without TORC2, and a constitutively active phospho-mimetic form of AKT was unable to suppress the growth defect of lst8 mutants. Moreover, mutation of foxo did not compensate for the reduction in cell growth caused by disruption of TORC2. In fact, similar results have been reported in C. elegans, where the reduced size of rictor mutants was proposed not to be a consequence of reduced AKT activity. Meanwhile, it was suggested that TORC2 controls cell growth by affecting SGK activity. However, the lack of an SGK homolog in flies casts some doubt on this mechanism. The unexpected finding of this study that AKT does not mediate TORC2-regulated cell growth emphasizes the importance of finding TORC2's critical substrates and also raises questions about the efficacy of TORC2 inhibitors as drugs for cancer therapy (Wang, 2012).

The TSC1/2 complex controls Drosophila pigmentation through TORC1-dependent regulation of catecholamine biosynthesis

In Drosophila, the pattern of adult pigmentation is initiated during late pupal stages by the production of catecholamines DOPA and dopamine, which are converted to melanin. The pattern and degree of melanin deposition is controlled by the expression of genes such as ebony and yellow as well as by the enzymes involved in catecholamine biosynthesis. This study shows that the conserved TSC/TORC1 cell growth pathway controls catecholamine biosynthesis in Drosophila during pigmentation. High levels of Rheb, an activator of the TORC1 complex, promote premature pigmentation in the mechanosensory bristles during pupal stages, and alter pigmentation in the cuticle of the adult fly. Disrupting either melanin synthesis by RNAi knockdown of melanogenic enzymes such as tyrosine hydroxylase (TH), or downregulating TORC1 activity by Raptor knockdown, suppresses the Rheb-dependent pigmentation phenotype in vivo. Increased Rheb activity drives pigmentation by increasing levels of TH in epidermal cells. These findings indicate that control of pigmentation is linked to the cellular nutrient-sensing pathway by regulating levels of a critical enzyme in melanogenesis, providing further evidence that inappropriate activation of TORC1, a hallmark of the human tuberous sclerosis complex tumor syndrome disorder, can alter metabolic and differentiation pathways in unexpected ways (Zitserman, 2012).

Diet and energy-sensing inputs affect TorC1-mediated axon misrouting but not TorC2-directed synapse growth in a Drosophila model of tuberous sclerosis

The Target of Rapamycin (TOR) growth regulatory system is influenced by a number of different inputs, including growth factor signaling, nutrient availability, and cellular energy levels. While the effects of TOR on cell and organismal growth have been well characterized, this pathway also has profound effects on neural development and behavior. Hyperactivation of the TOR pathway by mutations in the upstream TOR inhibitors TSC1 (tuberous sclerosis complex 1) or TSC2 promotes benign tumors and neurological and behavioral deficits, a syndrome known as tuberous sclerosis (TS). In Drosophila, neuron-specific overexpression of Rheb, the direct downstream target inhibited by Tsc1/Tsc2, produced significant synapse overgrowth, axon misrouting, and phototaxis deficits. To understand how misregulation of Tor signaling affects neural and behavioral development, this study examined the influence of growth factor, nutrient, and energy sensing inputs on these neurodevelopmental phenotypes. Neural expression of Pi3K, a principal mediator of growth factor inputs to Tor, caused synapse overgrowth similar to Rheb, but did not disrupt axon guidance or phototaxis. Dietary restriction rescued Rheb-mediated behavioral and axon guidance deficits, as did overexpression of AMPK, a component of the cellular energy sensing pathway, but neither was able to rescue synapse overgrowth. While axon guidance and behavioral phenotypes were affected by altering the function of a Tor complex 1 (TorC1) component, Raptor, or a TORC1 downstream element (S6k), synapse overgrowth was only suppressed by reducing the function of Tor complex 2 (TorC2) components (Rictor, Sin1). These findings demonstrate that different inputs to Tor signaling have distinct activities in nervous system development, and that Tor provides an important connection between nutrient-energy sensing systems and patterning of the nervous system (Dimitroff, 2012).

Remote control of insulin secretion by fat cells in Drosophila

Insulin-like peptides (ILPs) couple growth, metabolism, longevity, and fertility with changes in nutritional availability. In Drosophila, several ILPs called Dilps are produced by the brain insulin-producing cells (IPCs), from which they are released into the hemolymph and act systemically. In response to nutrient deprivation, brain Dilps are no longer secreted and accumulate in the IPCs. The larval fat body, a functional homolog of vertebrate liver and white fat, couples the level of circulating Dilps with dietary amino acid levels by remotely controlling Dilp release through a TOR/RAPTOR-dependent mechanism. Ex vivo tissue coculture was used to demonstrate that a humoral signal emitted by the fat body transits through the hemolymph and activates Dilp secretion in the IPCs. Thus, the availability of nutrients is remotely sensed in fat body cells and conveyed to the brain IPCs by a humoral signal controlling ILP release (Géminard, 2009).

Due to the lack of immunoassay, the study of the regulation of Dilp levels in Drosophila has been limited so far to the analysis of their expression level in response to nutritional conditions. This study presents evidence that the secretion of Dilp2 and Dilp5 as well as a GFP linked to a signal peptide (secGFP) is controlled by the nutritional status of the larva. The data also indicate that the IPCs have the specific ability to couple secretion with nutritional input. This suggests that all Dilps produced in the IPCs could be subjected to a common control on their secretion that could therefore override differences in their transcriptional regulation. It was further shown that the regulation of Dilp secretion plays a key role in controlling Dilp circulating levels and biological functions, since blocking neurosecretion in the IPCs led to growth and metabolic defects, and conversely, expression of Dilp2 in nonregulated neurosecretory cells is lethal upon starvation. Interestingly, previous reports suggest that Dilp release could also be controlled in the adult IPCs, raising the possibility that this type of regulation contributes to controlling metabolic homeostasis, reproduction, and aging during adult life (Géminard, 2009).

Dilp release is not activated by high-carbohydrate or high-fat diets, but rather depends on the level of amino acids and in particular on the presence of branched-chain amino acids like leucine and isoleucine. This finding is consistent with the described mechanism of TOR activation by leucine in mammalian cells (Avruch, 2009; Nicklin, 2009). In particular, it was recently shown that Rag GTPases can physically interact with mTORC1 and regulate its subcellular localization in response to L-leucine (Sancak, 2008). Interestingly, the present work indicates that amino acids do not directly signal to the IPCs, but rather they act on fat-body cells to control Dilp release. TOR signaling has been previously shown to relay the nutritional input in fat-body cells. Tor signaling is required for the remote control of Dilp secretion, since inhibition of Raptor-dependent TOR activity in fat cells provokes Dilp retention. Surprisingly, activation of TOR signaling in fat cells of underfed larvae is sufficient to induce Dilp release, indicating that TOR signaling is the major pathway relaying the nutrition signal from the fat body to the brain IPCs. In contrast, inhibition of PI3K activity in fat cells does not appear to influence Dilp secretion in the brain. This result is in line with previous in vivo data showing that reduction of PI3K levels in fat cells does not induce systemic growth defects. Altogether, this suggests that the nutritional signal is read by a TOR-dependent mechanism in fat cells, leading to the production of a secretion signal that is conveyed to the brain by the hemolymph (Géminard, 2009).

Ex vivo brain culture experiments demonstrate that hemolymph or dissected fat bodies from fed larvae constitute an efficient source for the Dilp secretion factor. This signal is absent in underfed animals, suggesting that it could be identified by comparative analysis of fed and underfed states. The nature of the secretion signal is unknown. It is produced and released in the hemolymph by fat cells, and its production relies on TORC1 function. Given the role of TORC1 in protein translation, one could envisage that the secretion factor is a protein or a peptide for which translation is limited by TORC1 activity and relies on amino acid input in fat-body cells. In mammals, fatty acids and other lipid molecules have the capacity to amplify glucose-stimulated insulin secretion in pancreatic β cells. The fly fat body carries important functions related to lipid metabolism, and a recent link has been established between TOR signaling and lipid metabolism in flies (Porstmann, 2008), leaving open the possibility that a TOR-dependent lipid-based signal could also operate in this regulation. Interestingly, carbohydrates do not appear to contribute to the regulation of insulin secretion by brain cells in flies. This finding is reminiscent of the absence of expression of the Sur1 ortholog in the IPCs and suggests that global carbohydrate levels are controlled by the glucagon-like AKH produced by the corpora cardiaca cells (Géminard, 2009).

These experiments demonstrate that Dilp secretion is linked to the polarization state of the IPC membrane, suggestive of a calcium-dependent granule exocytosis, like the one observed for insulin and many other neuropeptides. The nature of the upstream signal controlling membrane depolarization is not known. Recent data concerning the function of the nucleostemin gene ns3 in Drosophila suggest that a subset of serotonergic neurons in the larval brain act on the IPCs to control insulin secretion (Kaplan, 2008). Therefore, it remains to be known whether the IPCs or upstream serotonergic neurons constitute a direct target for the secretion signal. So far, no link has been established between the serotonergic stimulation of IPC function and the nutritional input (Géminard, 2009).

In 1998, J. Britton and B. Edgar presented experiments where starved brain and fed fat bodies were cocultured, allowing arrested brain neuroblasts to resume proliferation in the presence of nutrients (Britton and Edgar, 1998). From these experiments, the authors proposed that quiescent neuroblasts were induced to re-enter the cell cycle by a mitogenic factor emanating from the fed fat bodies. The present data extend these pioneer findings and suggest the possibility that the factor sent by the fed fat bodies is the secretion factor that triggers Dilp release from the IPCs, allowing neuroblasts to continue their growth and proliferation program through paracrine Dilp-dependent activation (Géminard, 2009).

In conclusion, this work combines genetic and physiology approaches on a model organism to decipher key physiological regulations and opens the route for a genetic study of the molecular mechanisms controlling insulin secretion in Drosophila (Géminard, 2009).

Discrete functions of rictor and raptor in cell growth regulation in Drosophila

The TOR signaling pathway regulates cell growth and metabolism in response to various nutrient signals by forming complexes with either rictor or raptor. To distinguish the physiological roles of the complexes formed by the two different TOR partners in vivo functions of rictor and raptor were compared in Drosophila. In rictor-null mutants, Akt-induced tissue hyperplasia was reduced and Akt-Ser-505 phosphorylation was decreased. Furthermore, FOXO-dependent apoptosis, which is inhibited by Akt, was augmented in the rictor-null background, indicating that rictor is essential for the Akt-FOXO signaling module. Neither S6K-dependent cell growth nor S6K-Thr-398 phosphorylation was affected in rictor-null mutants. However, the knockdown of another TOR binding partner, raptor, decreased S6K-Thr-398 phosphorylation and inhibited S6K-induced cell overgrowth. Collectively, these findings strongly support that the association of TOR with rictor or raptor plays pivotal roles in TOR-mediated cell apoptosis and growth control by differentially regulating Akt- and S6K-dependent signaling pathways, respectively (Lee, 2007).

Insulin delays the progression of Drosophila cells through G2/M by activating the dTOR/dRaptor complex

In Drosophila and mammals, insulin signalling can increase growth, progression through G1/S, cell size and tissue size. This study analysed the way insulin affects cell size and cell-cycle progression in two haemocyte-derived Drosophila cell lines. Surprisingly, it was found that although insulin increases cell size, it slows the rate at which these cells increase in number. By using BrdU pulse-chase to label S-phase cells and follow their progression through the cell cycle, insulin was shown to delay progression through G2/M, thereby slowing cell division. The ability of insulin to slow progression through G2/M is independent of its ability to stimulate progression through G1/S, so is not a consequence of feedback by the cell-cycle machinery to maintain cell-cycle length. Insulin's effects on progression through G2/M are mediated by dTOR/dRaptor signalling. Partially inhibiting dTOR/dRaptor signalling by dsRNAi or mild rapamycin treatment can increase cell number in cultured haemocytes and the Drosophila wing, respectively. Thus, insulin signalling can influence cell number depending on a balance between its ability to accelerate progression through G1/S and delay progression through G2/M (Wu, 2007).

Phosphorylation and regulation of Akt/PKB by the rictor-mTOR complex

Deregulation of Akt/protein kinase B (PKB) is implicated in the pathogenesis of cancer and diabetes. Akt/PKB activation requires the phosphorylation of Thr308 in the activation loop by the phosphoinositide-dependent kinase 1 (PDK1) and Ser473 within the carboxyl-terminal hydrophobic motif by an unknown kinase. This study shows in Drosophila and human cells the target of rapamycin (TOR) kinase and its associated protein rictor are necessary for Ser473 phosphorylation and that a reduction in rictor or mammalian TOR (mTOR) expression inhibits an Akt/PKB effector. The rictor-mTOR complex directly phosphorylated Akt/PKB on Ser473 in vitro and facilitated Thr308 phosphorylation by PDK1. Rictor-mTOR may serve as a drug target in tumors that have lost the expression of PTEN, a tumor suppressor that opposes Akt/PKB activation (Sarbassov, 2005a).

The Akt/PKB kinase is a well-characterized effector of phosphoinositide 3-kinase (PI3K), and its deregulation plays important roles in the pathogenesis of human cancers. PI3K is necessary for the activation of Akt/PKB, and current models suggest that phosphatidylinositol-3,4,5-triphosphates produced upon growth factor stimulation recruit Akt/PKB to the plasma membrane by binding to its N-terminal pleckstrin homology (PH) domain. At the membrane, Akt/PKB is phosphorylated on two key residues: Thr³⁰⁸ (T308) of the activation loop by PDK1 and Ser⁴⁷³ (S473) in the hydrophobic motif of the C-terminal tail by a kinase whose identity has been elusive. The role of S473 phosphorylation is controversial, but there is an emerging view that it precedes the phosphorylation of T308 and is important for the recognition and activation of Akt/PKB by PDK1 (Sarbassov, 2005a and references therein).

The molecular identity of the S473 kinase (S473K), at times referred to as 'PDK2' or the 'hydrophobic motif (HM) kinase,' has been hotly debated for many years. Several candidate S473Ks have been proposed, including PDK1, integrin-linked kinase (ILK), Akt/PKB itself, and, most recently, DNA-PK_cs. Many lines of evidence argue that neither PDK1, ILK, nor Akt/PKB is the physiological S473K, and for several reasons, DNA-PK_cs is also unlikely to have this function. There is no Drosophila ortholog of DNA-PK_cs, and, thus, if DNA-PK_cs is a physiological S473K in mammals, a distinct kinase must play that role in flies even though all other core components of the pathway (e.g., PI3K, Akt/PKB, PDK1, and PTEN) are well conserved. Moreover, it has not been shown that DNA-PK_cs phosphorylates full-length Akt/PKB, and DNA-PK_cs null mice do not suffer the growth retardation or insulin signaling defects associated with Akt1/PKB1 or Akt2/PKB2 (Sarbassov, 2005a).

Mammalian TOR (mTOR) is a large protein kinase that exists in two distinct complexes within cells: one that contains mTOR, GβL, and raptor and another containing mTOR, GβL, and rictor. The raptor-containing complex is sensitive to the drug rapamycin and regulates cell growth, in part by phosphorylating the hydrophobic motif of S6K1, a member of the same family of kinases to which Akt/PKB belongs. The rictor-containing complex does not appear to be rapamycin-sensitive, and its cellular function is just beginning to be understood. Despite its structural similarity to S6K1, Akt/PKB phosphorylation is not sensitive to acute rapamycin treatment, and thus mTOR has not previously been considered as the S473K (Sarbassov, 2005a).

This study used RNA interference (RNAi) in cultured Drosophila cells to determine the role of TOR pathway components in the phosphorylation of the hydrophobic motif sites of Drosophila Akt/PKB (dAKT/dPKB) and S6K (dS6K). In mammals and Drosophila, S6K suppresses signaling through the PI3K/Akt pathway so that inhibition of S6K boosts Akt/PKB phosphorylation. Knockdown of dS6K or Drosophila Raptor expression with double-stranded RNAs (dsRNAs) inhibited the phosphorylation and activity of dS6K and increased the phosphorylation of dAkt/dPKB. Despite reducing dS6K phosphorylation to the same extent as did dRaptor dsRNA, the dTOR dsRNA failed to increase dAkt/dPKB phosphorylation and, surprisingly, decreased it by a small amount. The contrasting effects on dAkt/dPKB phosphorylation by the dTOR and dRaptor dsRNAs suggest that dTOR has an unexpected positive role in dAkt/dPKB signaling that is not shared with dRaptor and that dTOR is required for the increase in dAkt/dPKB phosphorylation caused by dS6K inhibition. Consistent with the dRaptor-independent role for dTOR in dAkt/dPKB phosphorylation, a knockdown of dRictor reduced dAkt/dPKB phosphorylation (Sarbassov, 2005a).

Because basal dAkt/dPKB phosphorylation is low in Drosophila Kc₁₆₇ cells, the roles of dRictor and dTOR were verified in cells in which dAkt/dPKB phosphorylation was enhanced by decreasing the expression of dPTEN, the negative regulator of the PI3K/Akt pathway. Knockdown of dS6K or dRaptor expression in dPTEN-depleted cells further boosted dAkt/dPKB phosphorylation. In contrast, knockdown of dRictor expression almost completely prevented the dramatic increase in dAkt/dPKB phosphorylation caused by a dPTEN knockdown, whereas the knockdown of dTOR expression caused a slightly smaller suppression. Also, dRictor and dTOR were required for the increase in phosphorylation of dAkt/dPKB caused by a knockdown in the expression of dRaptor (Sarbassov, 2005a).

The results in Drosophila cells suggest that dTOR and dRictor have a shared positive role in the phosphorylation of the hydrophobic motif site of dAkt/dPKB. This finding was unexpected, because previously no decrease was observed in the phosphorylation of the hydrophobic motif site of Akt/PKB after reducing mTOR expression in human cells with small interfering RNAs (siRNAs). In retrospect, however, these experiments were undertaken when RNAi-mediated knockdowns of expression in mammalian cells were relatively inefficient. In this study, with the use of a lentiviral short hairpin RNA (shRNA) expression system that robustly suppresses gene expression, results in human cell lines were obtained analogous to those in Drosophila cells. In human HT-29 colon and A549 lung cancer cells, knockdown of rictor or mTOR expression using two different sets of shRNAs decreased phosphorylation of both S473 and T308 of Akt/PKB. Mammalian cells may try to compensate for the effects of the rictor and mTOR knockdowns by boosting Akt/PKB expression. The decrease in T308 phosphorylation is consistent with the importance of S473 phosphorylation for T308 phosphorylation and with the fact that the Ser⁴⁷³ --> Asp⁴⁷³ mutant of Akt/PKB is a better substrate than the wild-type protein for T308 phosphorylation by PDK1. Knockdown of raptor expression increased the phosphorylation of both S473 and T308 despite reducing Akt/PKB expression. Knockdown of rictor or mTOR expression also decreased S473 phosphorylation in HeLa and HEK-293T cells, two human cell lines that, like A549 and HT-29 cells, contain wild-type PTEN. In addition, the knockdowns also decreased S473 phosphorylation in the PTEN-null PC-3 prostate cancer cell line, a result reminiscent of that in Drosophila cells with reduced dPTEN expression. Furthermore, the knockdowns decreased S473 phosphorylation in M059J glioblastoma cells that are null for DNA-PK_cs, a proposed S473K candidate. Thus, in six distinct human cell lines, rictor and mTOR but not raptor are necessary for the phosphorylation of the hydrophobic motif of Akt/PKB (Sarbassov, 2005a).

Because the rictor and mTOR knockdowns inhibit phosphorylation events critical for Akt/PKB activity, they should affect Akt/PKB-regulated effectors. In HeLa cells, a reduction in the expression of rictor or mTOR but not raptor decreased phosphorylation of AFX (Foxo4a), a forkhead family transcription factor that is a direct substrate of Akt/PKB. Because the raptor-mTOR complex directly phosphorylates the hydrophobic motif site of S6K1, whether rictor-mTOR has an analogous function for Akt/PKB was determined. Rictor-mTOR complexes isolated from HEK-293T or HeLa phosphorylated S473 but not T308 of full-length, wild-type Akt/PKB in vitro. Immunoprecipitates of raptor, the ataxia telagiectasia mutated (ATM) protein, or protein kinase C α (PKCα) did not phosphorylate either site, and Akt/PKB did not autophosphorylate S473. Importantly, the raptor immunoprecipitates also contain mTOR but did not phosphorylate Akt/PKB, suggesting that for mTOR to phosphorylate Akt/PKB, it must be bound to rictor and that raptor cannot substitute. This lack of phosphorylation holds even in the raptor immunoprecipitates isolated from HEK-293T cells that contain as much mTOR as the rictor immunoprecipitates. Consistent with a key role for rictor, mTOR immunoprecipitates prepared from the rictor knockdown cells did not phosphorylate Akt/PKB despite containing a similar amount of mTOR as the controls. To verify that mTOR is the S473K in the rictor immunoprecipitates, immunoprecipitates were prepared from control cells and from two different lines of mTOR knockdown cells. Although rictor levels were equivalent in all the immunoprecipitates, only those prepared from cells expressing mTOR phosphorylated Akt/PKB in vitro. Both the LY294002 and wortmannin mTOR kinase inhibitors blocked the in vitro phosphorylation of Akt/PKB by rictor-mTOR, and LY294002 acted at concentrations that inhibit S473 phosphorylation in cells. Staurosporine, an inhibitor of Akt/PKB kinase activity, did not decrease the phosphorylation of Akt/PKB by rictor-mTOR. Thus, in vitro the rictor-mTOR complex phosphorylates S473 of Akt/PKB in a rictor- and mTOR-dependent fashion and with a drug sensitivity profile consistent with mTOR being the phosphorylating kinase (Sarbassov, 2005a).

To determine whether the phosphorylation of Akt/PKB on S473 by rictor-mTOR activates Akt/PKB activity, rictor-mTOR was used to phosphorylate Akt/PKB on S473, and then PDK1 was added to the assay to phosphorylate T308. Prior phosphorylation of Akt/PKB on S473 boosted subsequent phosphorylation by PDK1 of T308, consistent with the importance of S473 phosphorylation for T308 phosphorylation and with the inhibitory effects of the rictor and mTOR knockdowns on T308 phosphorylation. After phosphorylation with rictor-mTOR and PDK1, Akt1/PKB1 had about four- to fivefold more activity than after phosphorylation with PDK1 alone, confirming the important role of S473 in fully activating Akt/PKB. Because growth factors control the phosphorylation of Akt/PKB on S473, it was determined whether the concentration of serum in the cell media regulated the in vitro kinase activity of rictor-mTOR toward Akt/PKB. Rictor-mTOR had decreased activity in HeLa cells deprived of serum and was reactivated by serum stimulation for 30 min, indicating that modulation of the intrinsic kinase activity of rictor-mTOR may be a mechanism for regulating S473 phosphorylation (Sarbassov, 2005a).

These results indicate that the rictor-mTOR complex is a hydrophobic motif kinase for Akt/PKB. Rictor-TOR has essential roles in Akt/PKB hydrophobic motif site phosphorylation in Drosophila and human cells and in vitro phosphorylates full-length, wild-type Akt/PKB in a serum-sensitive fashion. No other proposed hydrophobic motif kinase has been shown to fulfill all these criteria. With hindsight, clues are seen in the literature to the important role of mTOR in Akt/PKB activation. Prolonged but not acute treatment of certain human cells with rapamycin partially inhibits Akt/PKB phosphorylation, and the current findings provide a possible rationale to explain these results. Although rapamycin does not bind to a preformed rictor-mTOR complex, during long-term rapamycin treatment the drug should eventually sequester many of the newly synthesized mTOR molecules within cells. Thus, as the rictor-mTOR complex turns over, rapamycin may interfere with its reassembly or over time become part of the new complexes. It is reasonable to expect then that prolonged rapamycin treatment may partially inhibit rictor-mTOR activity, which would explain why rapamycin is particularly effective at suppressing the proliferation of tumor cells with hyperactive Akt/PKB. The PI3K/Akt pathway is frequently deregulated in human cancers that have lost the expression of the PTEN tumor suppressor gene, and the current findings suggest that direct inhibitors of mTOR-rictor should strongly suppress Akt/PKB activity. Thus, the rictor-mTOR complex, like its raptor-mTOR sibling, may be a valuable drug target (Sarbassov, 2005a).

Phosphorylation and regulation of Akt/PKB by the rictor-mTOR complex

Deregulation of Akt/protein kinase B (PKB) is implicated in the pathogenesis of cancer and diabetes. Akt/PKB activation requires the phosphorylation of Thr308 in the activation loop by the phosphoinositide-dependent kinase 1 (PDK1) and Ser473 within the carboxyl-terminal hydrophobic motif by an unknown kinase. This study shows that in Drosophila and human cells the target of rapamycin (TOR) kinase and its associated protein rictor are necessary for Ser473 phosphorylation and that a reduction in rictor or mammalian TOR (mTOR) expression inhibited an Akt/PKB effector. The rictor-mTOR complex directly phosphorylated Akt/PKB on Ser473 in vitro and facilitated Thr308 phosphorylation by PDK1. Rictor-mTOR may serve as a drug target in tumors that have lost the expression of PTEN, a tumor suppressor that opposes Akt/PKB activation (Sarbassov, 2005b).

The raptor-mTOR protein complex is a key component of a nutrient-sensitive signaling pathway that regulates cell size by controlling the accumulation of cellular mass. How nutrients regulate signaling through the raptor-mTOR complex is not well known. This study shows that a redox-sensitive mechanism regulates the phosphorylation of the raptor-mTOR effector S6K1, the interaction between raptor and mTOR, and the kinase activity of the raptor-mTOR complex. In cells treated with the oxidizing agents diamide or phenylarsine oxide, S6K1 phosphorylation increased and became insensitive to nutrient deprivation. Conversely, the reducing reagent BAL (British anti-Lewisite, also known as 2,3-dimercapto-1-propanol) inhibits S6K1 phosphorylation and stabilizes the interaction of mTOR and raptor to mimic the state of the complex under nutrient-deprived conditions. These findings suggest that a redox-based signaling mechanism may participate in regulating the nutrient-sensitive raptor-mTOR complex and pathway (Sarbassov, 2005b).

The drug rapamycin has important uses in oncology, cardiology, and transplantation medicine, but its clinically relevant molecular effects are not understood. When bound to FKBP12, rapamycin interacts with and inhibits the kinase activity of a multiprotein complex composed of mTOR, mLST8, and raptor (mTORC1). The distinct complex of mTOR, mLST8, and rictor (mTORC2) does not interact with FKBP12-rapamycin and is not thought to be rapamycin sensitive. mTORC2 phosphorylates and activates Akt/PKB, a key regulator of cell survival. This study shows that rapamycin inhibits the assembly of mTORC2 and that, in many cell types, prolonged rapamycin treatment reduces the levels of mTORC2 below those needed to maintain Akt/PKB signaling. The proapoptotic and antitumor effects of rapamycin are suppressed in cells expressing an Akt/PKB mutant that is rapamycin resistant. This work describes an unforeseen mechanism of action for rapamycin that suggests it can be used to inhibit Akt/PKB in certain cell types (Sarbassov, 2006b).

Tsc1-mTORC1 signaling controls striatal dopamine release and cognitive flexibility

Tuberous Sclerosis Complex (TSC) is a neurodevelopmental disorder caused by mutations in TSC1 (see Drosophila Tsc1) or TSC2 (see Drosophila Gigas), which encode proteins that negatively regulate mTOR complex 1 (mTORC1). TSC is associated with significant cognitive, psychiatric, and behavioral problems, collectively termed TSC-Associated Neuropsychiatric Disorders (TAND), and the cell types responsible for these manifestations are largely unknown. This study used cell type-specific Tsc1 deletion to test whether dopamine neurons, which modulate cognitive, motivational, and affective behaviors, are involved in TAND. Loss of Tsc1 and constitutive activation of mTORC1 in dopamine neurons causes somatodendritic hypertrophy, reduces intrinsic excitability, alters axon terminal structure, and impairs striatal dopamine release. These perturbations lead to a selective deficit in cognitive flexibility, preventable by genetic reduction of the mTOR-binding protein Raptor (see Drosophila Raptor). These results establish a critical role for Tsc1-mTORC1 signaling in setting the functional properties of dopamine neurons, and indicate that dopaminergic dysfunction may contribute to cognitive inflexibility in TSC (Kosillo, 2019).

Distinct roles for the mTOR pathway in postnatal morphogenesis, maturation and function of pancreatic islets

While much is known about the molecular pathways that regulate embryonic development and adult homeostasis of the endocrine pancreas, little is known about what regulates early postnatal development and maturation of islets. Given that birth marks the first exposure to enteral nutrition, this study investigated how nutrient-regulated signaling pathways influence postnatal islet development. To do this loss-of-function studies were performed of mechanistic target of rapamycin (mTOR; see Drosophila Tor), a highly conserved kinase within a nutrient-sensing pathway known to regulate cellular growth, morphogenesis and metabolism. Deletion of mTOR in pancreatic endocrine cells had no significant effect on their embryonic development. However, within the first 2 weeks after birth, mTOR-deficient islets became dysmorphic, beta-cell maturation and function was impaired, and animals lost islet mass. Moreover, it was discovered that these distinct functions of mTOR are mediated by separate downstream branches of the pathway, in that mTORC1 (Raptor; see Drosophila Raptor) is the main complex mediating maturation and function of islets, whereas mTORC2 (Rictor; see Drosophila Rictor) impacts islet mass and architecture. Taken together, these findings suggest that nutrient-sensing may be a trigger for postnatal beta cell maturation and islet development (Sinagoga, 2017).

Architecture of human mTOR complex 1

Target of rapamycin (TOR), a conserved protein kinase and central controller of cell growth, functions in two structurally and functionally distinct complexes: TORC1 and TORC2. Dysregulation of mammalian TOR (mTOR) signaling is implicated in pathologies that include diabetes, cancer, and neurodegeneration. This study resolved the architecture of human mTORC1 (mTOR with subunits Raptor and mLST8) bound to FK506 binding protein (FKBP)-rapamycin, by combining cryo-electron microscopy at 5.9 angstrom resolution with crystallographic studies of Chaetomium thermophilum Raptor at 4.3 angstrom resolution. The structure explains how FKBP-rapamycin and architectural elements of mTORC1 limit access to the recessed active site. Consistent with a role in substrate recognition and delivery, the conserved amino-terminal domain of Raptor is juxtaposed to the kinase active site of TOR (Aylett, 2016).

TDP-43 loss of function increases TFEB activity and blocks autophagosome-lysosome fusion

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that is characterized by selective loss of motor neurons in brain and spinal cord. TAR DNA-binding protein 43 (TDP-43) was identified as a major component of disease pathogenesis in ALS, frontotemporal lobar degeneration (FTLD), and other neurodegenerative disease. Despite the fact that TDP-43 is a multi-functional protein involved in RNA processing and a large number of TDP-43 RNA targets have been discovered, the initial toxic effect and the pathogenic mechanism underlying TDP-43-linked neurodegeneration remain elusive. This study found that loss of TDP-43 strongly induced a nuclear translocation of TFEB, the master regulator of lysosomal biogenesis and autophagy, through targeting the mTORC1 key component raptor. This regulation in turn enhanced global gene expressions in the autophagy-lysosome pathway (ALP) and increased autophagosomal and lysosomal biogenesis. However, loss of TDP-43 also impaired the fusion of autophagosomes with lysosomes through dynactin 1 downregulation, leading to accumulation of immature autophagic vesicles and overwhelmed ALP function. Importantly, inhibition of mTORC1 signaling by rapamycin treatment aggravated the neurodegenerative phenotype in a TDP-43-depleted Drosophila model, whereas activation of mTORC1 signaling by PA treatment ameliorated the neurodegenerative phenotype. Taken together, these data indicate that impaired mTORC1 signaling and influenced ALP may contribute to TDP-43-mediated neurodegeneration (Xia, 2016).

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

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

The TOR pathway interacts with the insulin signaling pathway to regulate C. elegans larval development, metabolism and life span

The highly conserved target-of-rapamycin (TOR) protein kinases control cell growth in response to nutrients and growth factors. In mammals, TOR has been shown to interact with raptor (regulatory associated protein of mTOR: potential Drosophila homolog CG4320) to relay nutrient signals to downstream translation machinery. Raptor associates in a near stoichiometric ratio with mTOR to form a complex that functions as the nutrient sensor. It was proposed that raptor acts as a scaffold to bridge TOR with its putative phosphorylation targets. In C. elegans, mutations in the genes encoding CeTOR and raptor result in dauer-like larval arrest, implying that CeTOR regulates dauer diapause. The daf-15 (raptor) and let-363 (CeTOR) mutants shift metabolism to accumulate fat, and raptor mutations extend adult life span. daf-15 transcription is regulated by DAF-16, a FOXO transcription factor that is in turn regulated by daf-2 insulin/IGF signaling. This is a new mechanism that regulates the TOR pathway. Thus, DAF-2 insulin/IGF signaling and nutrient signaling converge on DAF-15 (raptor) to regulate C. elegans larval development, metabolism and life span (Jia, 2004).

The mammalian target of rapamycin (mTOR) partner, raptor, binds the mTOR substrates p70 S6 kinase and 4E-BP1 through their TOR signaling (TOS) motif

The mammalian target of rapamycin (mTOR) controls multiple cellular functions in response to amino acids and growth factors, in part by regulating the phosphorylation of p70 S6 kinase (p70S6k) and eukaryotic initiation factor 4E-binding protein 1 (4E-BP1). Raptor (regulatory associated protein of mTOR) is a recently identified mTOR binding partner that also binds p70S6k and 4E-BP1 and is essential for TOR signaling in vivo. Raptor binds to p70S6k and 4E-BP1 through their respective TOS (conserved TOR signaling) motifs which are shown to be required for amino acid- and mTOR-dependent regulation of these mTOR substrates in vivo. A point mutation of the TOS motif also eliminates all in vitro mTOR-catalyzed 4E-BP1 phosphorylation and abolishes the raptor-dependent component of mTOR-catalyzed p70S6k phosphorylation in vitro. Raptor appears to serve as an mTOR scaffold protein, the binding of which to the TOS motif of mTOR substrates is necessary for effective mTOR-catalyzed phosphorylation in vivo and perhaps for conferring their sensitivity to rapamycin and amino acid sufficiency (Nojima, 2003).

TOS motif-mediated raptor binding regulates 4E-BP1 multisite phosphorylation and function

The mammalian target of rapamycin, mTOR, is a serine/threonine kinase that controls cell growth and proliferation via the translation regulators eukaryotic initiation factor 4E (eIF4E) binding protein 1 (4E-BP1) and ribosomal protein S6 kinase 1 (S6K1). A TOR signaling (TOS) motif has been identified in the N terminus of S6K1 and the C terminus of 4E-BP1; in S6K1, the TOS motif is necessary to facilitate mTOR signaling to phosphorylate and activate S6K1. However, it is unclear how the TOS motif in S6K1 and 4E-BP1 mediates mTOR signaling. This study shows that a functional TOS motif is required for 4E-BP1 to bind to raptor (a recently identified mTOR-interacting protein), for 4E-BP1 to be efficiently phosphorylated in vitro by the mTOR/raptor complex, and for 4E-BP1 to be phosphorylated in vivo at all identified mTOR-regulated sites. mTOR/raptor-regulated phosphorylation is necessary for 4E-BP's efficient release from the translational initiation factor eIF4E. Consistently, overexpression of a mutant of 4E-BP1 containing a single amino acid change in the TOS motif (F114A) reduces cell size, demonstrating that mTOR-dependent regulation of cell growth by 4E-BP1 is dependent on a functional TOS motif. These data demonstrate that the TOS motif functions as a docking site for the mTOR/raptor complex, which is required for multisite phosphorylation of 4E-BP1, eIF4E release from 4E-BP1, and cell growth (Schalm, 2003).

Search PubMed for articles about Drosophila Raptor

Avruch, J., Long, X., Ortiz-Vega, S., Rapley, J., Papageorgiou, A. and Dai, N. (2009). Amino acid regulation of TOR complex 1. Am J Physiol Endocrinol Metab 296(4): E592-602. PubMed ID: Aylett, C. H., Sauer, E., Imseng, S., Boehringer, D., Hall, M. N., Ban, N. and Maier, T. (2016). Architecture of human mTOR complex 1. Science 351(6268): 48-52. PubMed ID: 26678875

Dimitroff, B., Howe, K., Watson, A., Campion, B., Lee, H. G., Zhao, N., O'Connor, M. B., Neufeld, T. P. and Selleck, S. B. (2012). Diet and energy-sensing inputs affect TorC1-mediated axon misrouting but not TorC2-directed synapse growth in a Drosophila model of tuberous sclerosis. PLoS One 7(2): e30722. PubMed ID: 22319582

Géminard, C., Rulifson, E. J. and Léopold, P. (2009). Remote control of insulin secretion by fat cells in Drosophila. Cell Metab. 10(3): 199-207. PubMed Citation: 19723496

Hatfield, I., Harvey, I., Yates, E.R., Redd, J.R., Reiter, L.T. and Bridges, D. (2015). The role of TORC1 in muscle development in Drosophila. Sci Rep 5: 9676. PubMed ID: 25866192

Jia, K. Chen, D. and Riddle, D. L. (2004). The TOR pathway interacts with the insulin signaling pathway to regulate C. elegans larval development, metabolism and life span. Development 131: 3897-3906. 15253933

Kang, D., Wang, D., Xu, J., Quan, C., Guo, X., Wang, H., Luo, J., Yang, Z., Chen, S. and Chen, J. (2018). The InR/Akt/TORC1 growth-promoting signaling negatively regulates JAK/STAT activity and migratory cell fate during morphogenesis. Dev Cell 44(4): 524-531. PubMed ID: 29456138

Kosillo, P., Doig, N. M., Ahmed, K. M., Agopyan-Miu, A., Wong, C. D., Conyers, L., Threlfell, S., Magill, P. J. and Bateup, H. S. (2019). Tsc1-mTORC1 signaling controls striatal dopamine release and cognitive flexibility. Nat Commun 10(1): 5426. PubMed ID: 31780742

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

Lee, G. and Chung, J. (2007). Discrete functions of rictor and raptor in cell growth regulation in Drosophila. Biochem Biophys Res Commun 357(4): 1154-1159. PubMed ID: 17462592

Li, N., Liu, Q., Xiong, Y. and Yu, J. (2019). Headcase and Unkempt regulate tissue growth and cell cycle progression in response to nutrient restriction. Cell Rep 26(3): 733-747. PubMed ID: 30650363

Nicklin, P., Bergman, P., Zhang, B., Triantafellow, E., Wang, H., Nyfeler, B., Yang, H., Hild, M., Kung, C., Wilson, C., Myer, V. E., MacKeigan, J. P., Porter, J. A., Wang, Y. K., Cantley, L. C., Finan, P. M. and Murphy, L. O. (2009). Bidirectional transport of amino acids regulates mTOR and autophagy. Cell 136(3): 521-534. PubMed ID: 19203585

Nojima, H., Tokunaga, C., Eguchi, S., Oshiro, N., Hidayat, S., Yoshino, K., Hara, K., Tanaka, N., Avruch, J. and Yonezawa, K. (2003). The mammalian target of rapamycin (mTOR) partner, raptor, binds the mTOR substrates p70 S6 kinase and 4E-BP1 through their TOR signaling (TOS) motif. J Biol Chem 278(18): 15461-15464. PubMed ID: 12604610

Porstmann, T., Santos, C. R., Griffiths, B., Cully, M., Wu, M., Leevers, S., Griffiths, J. R., Chung, Y. L. and Schulze, A. (2008). SREBP activity is regulated by mTORC1 and contributes to Akt-dependent cell growth. Cell Metab 8(3): 224-236. PubMed ID: 18762023

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(5882): 1496-1501. PubMed ID: 18497260

Sarbassov, D., Guertin, D. A., Ali, S. M. and Sabatini, D. M. (2005a). Phosphorylation and regulation of Akt/PKB by the rictor-mTOR complex. Science 307: 1098-1101. Medline abstract: 15718470

Sarbassov, D. D. and Sabatini, D. M. (2005b). Redox regulation of the nutrient-sensitive raptor-mTOR pathway and complex. J. Biol. Chem. 280(47): 39505-9. Medline abstract: 16183647

Schalm, S. S., Fingar, D. C., Sabatini, D. M. and Blenis, J. (2003). TOS motif-mediated raptor binding regulates 4E-BP1 multisite phosphorylation and function. Curr Biol 13(10): 797-806. PubMed ID: 12747827

Sinagoga, K. L., Stone, W. J., Schiesser, J. V., Schweitzer, J. I., Sampson, L., Zheng, Y. and Wells, J. M. (2017). Distinct roles for the mTOR pathway in postnatal morphogenesis, maturation and function of pancreatic islets. Development 144(13):2402-2414. PubMed ID: 28576773

Wang, T., Blumhagen, R., Lao, U., Kuo, Y. and Edgar, B. A. (2012). LST8 regulates cell growth via target-of-rapamycin complex 2 (TORC2). Mol Cell Biol 32: 2203-2213. PubMed ID: 22493059

Wu, M. Y., Cully, M., Andersen, D. and Leevers, S. J. (2007). Insulin delays the progression of Drosophila cells through G2/M by activating the dTOR/dRaptor complex. EMBO J 26(2): 371-379. PubMed ID: 17183368

Xia, Q., Wang, H., Hao, Z., Fu, C., Hu, Q., Gao, F., Ren, H., Chen, D., Han, J., Ying, Z. and Wang, G. (2016). TDP-43 loss of function increases TFEB activity and blocks autophagosome-lysosome fusion. EMBO J 35: 121-142. PubMed ID: 26702100

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

Zitserman, D., Gupta, S., Kruger, W. D., Karbowniczek, M. and Roegiers, F. (2012). The TSC1/2 complex controls Drosophila pigmentation through TORC1-dependent regulation of catecholamine biosynthesis. PLoS One 7(11): e48720. PubMed ID: 23144943

date revised: 10 November 2021

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