Regulation of mTORC1 by the Rab and Arf GTPases

The mammalian target of rapamycin (mTOR) is a key cell growth regulator, which forms two distinct functional complexes (mTORC1 and mTORC2). mTORC1, which is directly inhibited by rapamycin, promotes cell growth by stimulating protein synthesis and inhibiting autophagy. mTORC1 is regulated by a wide range of extra- and intracellular signals, including growth factors, nutrients, and energy levels. Precise regulation of mTORC1 is important for normal cellular physiology and development, and dysregulation of mTORC1 contributes to hypertrophy and tumorigenesis. In this study, we screened Drosophila small GTPases for their function in TORC1 regulation and found that TORC1 activity is regulated by members of the Rab and Arf family GTPases, which are key regulators of intracellular vesicle trafficking. In mammalian cells, uncontrolled activation of Rab5 and Arf1 strongly inhibit mTORC1 activity. Interestingly, the effect of Rab5 and Arf1 on mTORC1 is specific to amino acid stimulation, whereas glucose-induced mTORC1 activation is not blocked by Rab5 or Arf1. Similarly, active Rab5 selectively inhibits mTORC1 activation by Rag GTPases, which are involved in amino acid signaling, but does not inhibit the effect of Rheb, which directly binds and activates mTORC1. Our data demonstrate a key role of Rab and Arf family small GTPases and intracellular trafficking in mTORC1 activation, particularly in response to amino acids.     

Hyperactivation of mammalian target of rapamycin (mTOR) signaling by a gain-of-function mutant of the Rheb GTPase

Gain-of-function mutants of Ras and Rho family small GTPases have proven to be important tools in analyzing signaling downstream of these small GTPases. The Ras-related GTPase Rheb has emerged as a key player downstream of TSC1-2 in activating signaling to mammalian target of rapamycin (mTOR) effectors of cell growth such as S6K and 4E-BP1. The TSC1-2 tumor suppressor complex has been shown to act as a RhebGAP, converting Rheb from a GTP-bound to a GDP-bound form. Here we report the identification of a mutant Rheb (S16HRheb) that exhibits gain-of-function properties. At endogenous levels of expression S16HRheb exhibits increased GTP loading in vivo and is resistant to TSC1-2 GAP in vitro. Compared with wild-type Rheb, S16HRheb is more active at promoting the phosphorylation of the mTOR effectors S6K1 and 4E-BP1. Thus S16HRheb will help to identify proximal signaling events downstream of Rheb and allow potential Rheb-independent functions downstream of TSC1-2 to be investigated.     

Amino acid regulation of TOR complex 1

TOR complex 1 (TORC1), an oligomer of the mTOR (mammalian target of rapamycin) protein kinase, its substrate binding subunit raptor, and the polypeptide Lst8/GbetaL, controls cell growth in all eukaryotes in response to nutrient availability and in metazoans to insulin and growth factors, energy status, and stress conditions. This review focuses on the biochemical mechanisms that regulate mTORC1 kinase activity, with special emphasis on mTORC1 regulation by amino acids. The dominant positive regulator of mTORC1 is the GTP-charged form of the ras-like GTPase Rheb. Insulin, growth factors, and a variety of cellular stressors regulate mTORC1 by controlling Rheb GTP charging through modulating the activity of the tuberous sclerosis complex, the Rheb GTPase activating protein. In contrast, amino acids, especially leucine, regulate mTORC1 by controlling the ability of Rheb-GTP to activate mTORC1. Rheb binds directly to mTOR, an interaction that appears to be essential for mTORC1 activation. In addition, Rheb-GTP stimulates phospholipase D1 to generate phosphatidic acid, a positive effector of mTORC1 activation, and binds to the mTOR inhibitor FKBP38, to displace it from mTOR. The contribution of Rheb's regulation of PL-D1 and FKBP38 to mTORC1 activation, relative to Rheb's direct binding to mTOR, remains to be fully defined. The rag GTPases, functioning as obligatory heterodimers, are also required for amino acid regulation of mTORC1. As with amino acid deficiency, however, the inhibitory effect of rag depletion on mTORC1 can be overcome by Rheb overexpression, whereas Rheb depletion obviates rag's ability to activate mTORC1. The rag heterodimer interacts directly with mTORC1 and may direct mTORC1 to the Rheb-containing vesicular compartment in response to amino acid sufficiency, enabling Rheb-GTP activation of mTORC1. The type III phosphatidylinositol kinase also participates in amino acid-dependent mTORC1 activation, although the site of action of its product, 3'OH-phosphatidylinositol, in this process is unclear.     

Rheb1 is required for mTORC1 and myelination in postnatal brain development

mTor kinase is involved in cell growth, proliferation, and differentiation. The roles of mTor activators, Rheb1 and Rheb2, have not been established in?vivo. Here, we report that Rheb1, but not Rheb2, is critical for embryonic survival and mTORC1 signaling. Embryonic deletion of Rheb1 in neural progenitor cells?abolishes mTORC1 signaling in developing brain and increases mTORC2 signaling. Remarkably, embryonic and early postnatal brain development appears grossly normal in these Rheb1f/f,Nes-cre mice with the notable exception of deficits of myelination. Conditional expression of Rheb1 transgene in neural progenitors increases mTORC1 activity and promotes myelination in the brain. In addition the Rheb1 transgene rescues mTORC1 signaling and hypomyelination in the Rheb1f/f,Nes-cre mice. Our study demonstrates that Rheb1 is essential for mTORC1 signaling and myelination in the brain, and suggests that mTORC1 signaling plays a role in selective cellular adaptations, rather than general cellular viability.     

Key mediators of intracellular amino acids signaling to mTORC1 activation

Mammalian target of rapamycin complex 1 (mTORC1) is activated by amino acids to promote cell growth via protein synthesis. Specifically, Ras-related guanosine triphosphatases (Rag GTPases) are activated by amino acids, and then translocate mTORC1 to the surface of late endosomes and lysosomes. Ras homolog enriched in brain (Rheb) resides on this surface and directly activates mTORC1. Apart from the presence of intracellular amino acids, Rag GTPases and Rheb, other mediators involved in intracellular amino acid signaling to mTORC1 activation include human vacuolar sorting protein-34 (hVps34) and mitogen-activating protein kinase kinase kinase kinase-3 (MAP4K3). Those molecular links between mTORC1 and its mediators form a complicate signaling network that controls cellular growth, proliferation, and metabolism. Moreover, it is speculated that amino acid signaling to mTORC1 may start from the lysosomal lumen. In this review, we discussed the function of these mediators in mTORC1 pathway and how these mediators are regulated by amino acids in details.

     

Re-evaluating the roles of proposed modulators of mammalian target of rapamycin complex 1 (mTORC1) signaling

Signaling through mammalian target of rapamycin complex 1 (mTORC1) is stimulated by amino acids and insulin. Insulin inactivates TSC1/2, the GTPase-activator complex for Rheb, and Rheb.GTP activates mTORC1. It is not clear how amino acids regulate mTORC1. FKBP38 (immunophilin FK506-binding protein, 38 kDa), was recently reported to exert a negative effect on mTORC1 function that is relieved by its binding to Rheb.GTP. We confirm that Rheb binds wild type FKBP38, but inactive Rheb mutants showed contrasting abilities to bind FKBP38. We were unable to observe any regulation of FKBP38/mTOR binding by amino acids or insulin. Furthermore, FKBP38 did not inhibit mTORC1 signaling. The translationally controlled tumor protein (TCTP) in Drosophila was recently reported to act as the guanine nucleotide-exchange factor for Rheb. We have studied the role of TCTP in mammalian TORC1 signaling and its control by amino acids. Reducing TCTP levels did not reproducibly affect mTORC1 signaling in amino acid-replete/insulin-stimulated cells. Moreover, overexpressing TCTP did not rescue mTORC1 signaling in amino acid-starved cells. In addition, we were unable to see any stable interaction between TCTP and Rheb or mTORC1. Accumulation of uncharged tRNA has been previously proposed to be involved in the inhibition of mTORC1 signaling during amino acid starvation. To test this hypothesis, we used a Chinese hamster ovary cell line containing a temperature-sensitive mutation in leucyl-tRNA synthetase. Leucine deprivation markedly inhibited mTORC1 signaling in these cells, but shifting the cells to the nonpermissive temperature for the synthetase did not. These data indicate that uncharged tRNA(Leu) does not switch off mTORC1 signaling and suggest that mTORC1 is controlled by a distinct pathway that senses the availability of amino acids. Our data also indicate that, in the mammalian cell lines tested here, neither TCTP nor FKBP38 regulates mTORC1 signaling.     

RhoA modulates signaling through the mechanistic target of rapamycin complex 1 (mTORC1) in mammalian cells

The mechanistic target of rapamycin (mTOR) in complex 1 (mTORC1) pathway integrates signals generated by hormones and nutrients to control cell growth and metabolism. The activation state of mTORC1 is regulated by a variety of GTPases including Rheb and Rags. Recently, Rho1, the yeast ortholog of RhoA, was shown to interact directly with TORC1 and repress its activation state in yeast. Thus, the purpose of the present study was to test the hypothesis that the RhoA GTPase modulates signaling through mTORC1 in mammalian cells. In support of this hypothesis, exogenous overexpression of either wild type or constitutively active (ca)RhoA repressed mTORC1 signaling as assessed by phosphorylation of p70S6K1 (Thr389), 4E-BP1 (Ser65) and ULK1 (Ser757). Additionally, RhoA·GTP repressed phosphorylation of mTORC1-associated mTOR (Ser2481). The RhoA·GTP mediated repression of mTORC1 signaling occurred independent of insulin or leucine induced stimulation. In contrast to the action of Rho1 in yeast, no evidence was found to support a direct interaction of RhoA·GTP with mTORC1. Instead, expression of caRheb, but not caRags, was able to rescue the RhoA·GTP mediated repression of mTORC1 suggesting RhoA functions upstream of Rheb to repress mTORC1 activity. Consistent with this suggestion, RhoA·GTP repressed phosphorylation of TSC2 (Ser939), PRAS40 (Thr246), Akt (Ser473), and mTORC2-associated mTOR (Ser2481). Overall, the results support a model in which RhoA·GTP represses mTORC1 signaling upstream of Akt and mTORC2.     

Mammalian Target of Rapamycin Complex I (mTORC1) Activity in Ras Homologue Enriched in Brain (Rheb)-Deficient Mouse Embryonic Fibroblasts

The Ras-like GTPase Rheb has been identified as a crucial activator of mTORC1. Activation most likely requires a direct interaction between Rheb and mTOR, but the exact mechanism remains unclear. Using a panel of Rheb-deficient mouse embryonic fibroblasts (MEFs), we show that Rheb is indeed essential for the rapid increase of mTORC1 activity following stimulation with insulin or amino acids. However, mTORC1 activity is less severely reduced in Rheb-deficient MEFs in the continuous presence of serum or upon stimulation with serum. This remaining mTORC1 activity is blocked by depleting the cells for amino acids or imposing energy stress. In addition, MEK inhibitors and the RSK-inhibitor BI-D1870 interfere in mTORC1 activity, suggesting that RSK acts as a bypass for Rheb in activating mTORC1. Finally, we show that this rapamycin-sensitive, Rheb-independent mTORC1 activity is important for cell cycle progression. In conclusion, whereas rapid adaptation in mTORC1 activity requires Rheb, a second Rheb-independent activation mechanism exists that contributes to cell cycle progression.     

Microtubule-associated Protein/Microtubule Affinity-regulating Kinase 4 (MARK4) Is a Negative Regulator of the Mammalian Target of Rapamycin Complex 1 (mTORC1)

The mammalian target of rapamycin (mTOR) is a central cell growth regulator. It resides in two protein complexes, which in mammals are referred to as mTORC1 and mTORC2. mTORC1, which is directly inhibited by rapamycin, promotes cell growth by stimulating protein synthesis and inhibiting autophagy. A wide range of extra and intracellular signals, including growth factors, nutrients, energy levels, and various stress conditions, regulates mTORC1. Dysregulation of mTORC1 contributes to many human diseases, including cancer, cardiovascular disease, autoimmunity, and metabolic disorder. In this study, we identified MARK4, an AMP-activated kinase-related kinase, as a negative regulator of mTORC1. In Drosophila S2 cells and mammalian cells, knockdown of MARK family member increased mTORC1 activity, whereas overexpression of MARK4 in mammalian cells significantly inhibited mTORC1 activity. Interestingly, MARK4 selectively inhibits mTORC1 activation by Rag GTPases, which are involved in amino acid signaling, but does not inhibit the effect of Rheb, which directly binds to and activates mTORC1. In addition, we found that MARK4 phosphorylates Raptor, a key component of mTORC1, and this phosphorylation may interfere with Raptor-Rag interaction. Our data demonstrate MARK4 as a new negative regulator of mTORC1.     

Glycolytic Flux Signals to mTOR through Glyceraldehyde-3-Phosphate Dehydrogenase-Mediated Regulation of Rheb

The mammalian target of rapamycin (mTOR) interacts with raptor to form the protein complex mTORC1 (mTOR complex 1), which plays a central role in the regulation of cell growth in response to environmental cues. Given that glucose is a primary fuel source and a biosynthetic precursor, how mTORC1 signaling is coordinated with glucose metabolism has been an important question. Here, we found that the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) binds Rheb and inhibits mTORC1 signaling. Under low-glucose conditions, GAPDH prevents Rheb from binding to mTOR and thereby inhibits mTORC1 signaling. High glycolytic flux suppresses the interaction between GAPDH and Rheb and thus allows Rheb to activate mTORC1. Silencing of GAPDH or blocking of the Rheb-GAPDH interaction desensitizes mTORC1 signaling to changes in the level of glucose. The GAPDH-dependent regulation of mTORC1 in response to glucose availability occurred even in TSC1-deficient cells and AMPK-silenced cells, supporting the idea that the GAPDH-Rheb pathway functions independently of the AMPK axis. Furthermore, we show that glyceraldehyde-3-phosphate, a glycolytic intermediate that binds GAPDH, destabilizes the Rheb-GAPDH interaction even under low-glucose conditions, explaining how high-glucose flux suppresses the interaction and activates mTORC1 signaling. Taken together, our results suggest that the glycolytic flux regulates mTOR''s access to Rheb by regulating the Rheb-GAPDH interaction, thereby allowing mTORC1 to coordinate cell growth with glucose availability.The mTOR complex 1 (mTORC1) signal transduction pathway acts as a central controller of cell growth in mammals (20, 23, 29). mTORC1 integrates a wide range of intracellular and extracellular signals, including insulin, availability of nutrients (glucose and amino acids), cellular energy status, and hypoxia, to regulate protein synthesis and cell growth (11, 12, 17, 36, 46). Many of these environmental cues are integrated into tuberous sclerosis complex (TSC1-TSC2), the major upstream regulator of mTORC1. In response to the absence of insulin and to the low-energy status of cells, the TSC1-TSC2 complex stimulates the GTPase function of Rheb, a small GTPase that acts as a proximal key activator of mTORC1, which leads to the inhibition of Rheb-mediated mTORC1 activation. In contrast, inactivation of the TSC1-TSC2 complex results in the accumulation of GTP-bound Rheb and thus activation of mTORC1 (3, 13, 21, 27, 32, 39). For this reason, both the loss of TSC proteins and the overexpression of Rheb cause hyperactivation of mTORC1 signaling, which is frequently observed in many common human cancers (2, 5, 19, 25, 33). Therefore, a tight regulation of Rheb activity is critical for the proper operation of the mTORC1 pathway in response to environmental cues.Rheb is an atypical member of the Ras superfamily of GTPases (1, 10, 47). As with other small GTPases, the activity of Rheb is regulated by its guanine nucleotide binding status. However, the negative control of GTP-bound Rheb by the TSC1-TSC2 complex has only recently been investigated, and the regulation of the nucleotide binding status of Rheb is not fully understood. A recent study proposed that translationally controlled tumor protein may function as a guanine nucleotide exchange factor for Rheb that causes the accumulation of GTP-bound Rheb (18). GTP-bound Rheb is essential for activating mTOR kinase (21, 28, 38). However, the interaction between Rheb and mTOR does not depend on the GTP binding status of Rheb (30), raising questions regarding the mechanism by which Rheb activates mTORC1. Recently, FKBP38 (immunophilin FK506-binding protein, 38 kDa) was found to be a direct binding partner of Rheb and an inhibitor of mTORC1 (4). GTP-bound Rheb binds FKBP38 and releases FKBP38 from mTORC1, resulting in activation of the mTORC1 pathway. However, there have been conflicting results regarding the effects of nutrient availability on Rheb activity (31, 37, 42, 50) and the effect of these newly identified regulators of Rheb function (44, 45). Thus, the precise molecular mechanisms underlying Rheb regulation and Rheb-mediated mTORC1 activation have remained unclear.In this study, we identified glyceraldehyde-3-phosphate (Gly-3-P) dehydrogenase (GAPDH) as a novel Rheb binding protein and a negative regulator of Rheb. We found that the interaction between GAPDH and Rheb is induced when the glycolytic flux is suppressed under low-glucose conditions to inhibit mTORC1. Here, we provide a molecular mechanism underlying the cross talk between the glycolytic flux and the mTORC1 signaling.     

The Rheb family of GTP-binding proteins

Rheb proteins represent a novel and unique family of the Ras superfamily GTP-binding proteins that is conserved from yeast to human. Biochemical studies establish that they bind and hydrolyze GTP. Molecular modeling studies reveal a few structural differences between Rheb and Ras, which may suggest that residues involved in biochemical activities differ between the two G-proteins. The function of Rheb has been studied in a number of organisms that point to the involvement of Rheb in cell growth and cell cycle progression. In addition, studies in fungi suggest that Rheb is involved in arginine uptake. Further studies in Drosophila and mammalian cells have shown that the effects of Rheb on growth and cell cycle progression are mediated by the effect on the insulin/TOR/S6K signaling pathway. These studies have also shown that a complex consisting of the tuberous sclerosis gene products, Tsc1/Tsc2, serves as a GTPase activating protein (GAP) for Rheb, implying Rheb's role in this genetic disorder. Finally, Rheb proteins have been shown to be farnesylated and small molecule inhibitors of protein farnesyltransferase can block the ability of Rheb to activate the TOR/S6K signaling.     

Specific Activation of mTORC1 by Rheb G-protein in Vitro Involves Enhanced Recruitment of Its Substrate Protein

Rheb G-protein plays critical roles in the TSC/Rheb/mTOR signaling pathway by activating mTORC1. The activation of mTORC1 by Rheb can be faithfully reproduced in vitro by using mTORC1 immunoprecipitated by the use of anti-raptor antibody from mammalian cells starved for nutrients. The low in vitro kinase activity against 4E-BP1 of this mTORC1 preparation is dramatically increased by the addition of recombinant Rheb. On the other hand, the addition of Rheb does not activate mTORC2 immunoprecipitated from mammalian cells by the use of anti-rictor antibody. The activation of mTORC1 is specific to Rheb, because other G-proteins such as KRas, RalA/B, and Cdc42 did not activate mTORC1. Both Rheb1 and Rheb2 activate mTORC1. In addition, the activation is dependent on the presence of bound GTP. We also find that the effector domain of Rheb is required for the mTORC1 activation. FKBP38, a recently proposed mediator of Rheb action, appears not to be involved in the Rheb-dependent activation of mTORC1 in vitro, because the preparation of mTORC1 that is devoid of FKBP38 is still activated by Rheb. The addition of Rheb results in a significant increase of binding of the substrate protein 4E-BP1 to mTORC1. PRAS40, a TOR signaling (TOS) motif-containing protein that competes with the binding of 4EBP1 to mTORC1, inhibits Rheb-induced activation of mTORC1. A preparation of mTORC1 that is devoid of raptor is not activated by Rheb. Rheb does not induce autophosphorylation of mTOR. These results suggest that Rheb induces alteration in the binding of 4E-BP1 with mTORC1 to regulate mTORC1 activation.Rheb defines a unique member of the Ras superfamily G-proteins (1). We have shown that Rheb proteins are conserved and are found from yeast to human (2). Although yeast and fruit fly have one Rheb, mouse and human have two Rheb proteins termed Rheb1 (or simply Rheb) and Rheb2 (RhebL1) (2). Structurally, these proteins contain G1-G5 boxes, short stretches of amino acids that define the function of the Ras superfamily G-proteins including guanine nucleotide binding (1, 3, 4). Rheb proteins have a conserved arginine at residue 15 that corresponds to residue 12 of Ras (1). The effector domain required for the binding with downstream effectors encompasses the G2 box and its adjacent sequences (1, 5). Structural analysis by x-ray crystallography further shows that the effector domain is exposed to solvent, is located close to the phosphates of GTP especially at residues 35–38, and undergoes conformational change during GTP/GDP exchange (6). In addition, all Rheb proteins end with the CAAX (C is cysteine, A is an aliphatic amino acid, and X is the C-terminal amino acid) motif that signals farnesylation. In fact, we as well as others have shown that these proteins are farnesylated (7–9).Rheb plays critical roles in the TSC/Rheb/mTOR signaling, a signaling pathway that plays central roles in regulating protein synthesis and growth in response to nutrient, energy, and growth conditions (10–14). Rheb is down-regulated by a TSC1·TSC2 complex that acts as a GTPase-activating protein for Rheb (15–19). Recent studies established that the GAP domain of TSC2 defines the functional domain for the down-regulation of Rheb (20). Mutations in the Tsc1 or Tsc2 gene lead to tuberous sclerosis whose symptoms include the appearance of benign tumors called hamartomas at different parts of the body as well as neurological symptoms (21, 22). Overexpression of Rheb results in constitutive activation of mTOR even in the absence of nutrients (15, 16). Two mTOR complexes, mTORC1 and mTORC2, have been identified (23, 24). Whereas mTORC1 is involved in protein synthesis activation mediated by S6K and 4EBP1, mTORC2 is involved in the phosphorylation of Akt in response to insulin. It has been suggested that Rheb is involved in the activation of mTORC1 but not mTORC2 (25).Although Rheb is clearly involved in the activation of mTOR, the mechanism of activation has not been established. We as well as others have suggested a model that involves the interaction of Rheb with the TOR complex (26–28). Rheb activation of mTOR kinase activity using immunoprecipitated mTORC1 was reported (29). Rheb has been shown to interact with mTOR (27, 30), and this may involve direct interaction of Rheb with the kinase domain of mTOR (27). However, this Rheb/mTOR interaction is a weak interaction and is not dependent on the presence of GTP bound to Rheb (27, 28). Recently, a different model proposing that FKBP38 (FK506-binding protein 38) mediates the activation of mTORC1 by Rheb was proposed (31, 32). In this model, FKBP38 binds mTOR and negatively regulates mTOR activity, and this negative regulation is blocked by the binding of Rheb to FKBP38. However, recent reports dispute this idea (33).To further characterize Rheb activation of mTOR, we have utilized an in vitro system that reproduces activation of mTORC1 by the addition of recombinant Rheb. We used mTORC1 immunoprecipitated from nutrient-starved cells using anti-raptor antibody and have shown that its kinase activity against 4E-BP1 is dramatically increased by the addition of recombinant Rheb. Importantly, the activation of mTORC1 is specific to Rheb and is dependent on the presence of bound GTP as well as an intact effector domain. FKBP38 is not detected in our preparation and further investigation suggests that FKBP38 is not an essential component for the activation of mTORC1 by Rheb. Our study revealed that Rheb enhances the binding of a substrate 4E-BP1 with mTORC1 rather than increasing the kinase activity of mTOR.     

mTORC2 Is Required for Rit-Mediated Oxidative Stress Resistance

Rit, a member of the Ras family of GTPases, has been shown to promote cell survival in response to oxidative stress, in part by directing an evolutionarily conserved p38 MAPK-Akt survival cascade. Aberrant Rit signaling has recently been implicated as a driver mutation in human cancer, adding importance to the characterization of critical Rit effector pathways. However, the mechanism by which Rit-p38 signaling regulated Akt activity was unknown. Here, we identify mTORC2 as a critical downstream mediator of Rit-dependent survival signaling in response to reactive oxygen species (ROS) stress. Rit interacts with Sin1 (MAPKAP1), and Rit loss compromises ROS-dependent mTORC2 complex activation, blunting mTORC2-mediated phosphorylation of Akt kinase. Taken together, our findings demonstrate that the p38/mTORC2/Akt signaling cascade mediates Rit-dependent oxidative stress survival. Inhibition of this previously unrecognized cascade should be explored as a potential therapy of Rit-dependent malignancies.     

Rac1 regulates the activity of mTORC1 and mTORC2 and controls cellular size

Mammalian target of rapamycin (mTOR) is a serine/threonine kinase that exists in two separate complexes, mTORC1 and mTORC2, that function to control cell size and growth in response to growth factors, nutrients, and cellular energy levels. Low molecular weight GTP-binding proteins of the Rheb and Rag families are key regulators of the mTORC1 complex, but regulation of mTORC2 is poorly understood. Here, we report that Rac1, a member of the Rho family of GTPases, is a critical regulator of both mTORC1 and mTORC2 in response to growth-factor stimulation. Deletion of Rac1 in primary cells using an inducible-Cre/Lox approach inhibits basal and growth-factor activation of both mTORC1 and mTORC2. Rac1 appears to bind directly to mTOR and to mediate mTORC1 and mTORC2 localization at specific membranes. Binding of Rac1 to mTOR does not depend on the GTP-bound state of Rac1, but on the integrity of its C-terminal domain. This function of Rac1 provides a means to regulate mTORC1 and mTORC2 simultaneously.     

Nuclear FAK: a new mode of gene regulation from cellular adhesions

Mammalian target of rapamycin complex 1 (mTORC1) is a master regulator of cell growth and autophagy. Its activity is regulated by the availability of amino acids and growth factors. The activation of mTORC1 by growth factors, such as insulin and insulin-like growth factor-1 (IGF-1), is mediated by tuberous sclerosis complex (TSC) 1 and 2 and Rheb GTPase. Relative to the growth factor-regulated mTORC1 pathway, the evolutionarily ancient amino acid-mTORC1 pathway remains not yet clearly defined. The amino acid-mTORC1 pathway is mediated by Rag GTPase heterodimers. Several binding proteins of Rag GTPases were discovered in recent studies. Here, we discuss the functions and mechanisms of the newly-identified binders of Rag GTPases. In particular, this review focuses on SH3 binding protein 4 (SH3BP4), the protein recently identifed as a negative regulator of Rag GTPases.     

RalA functions as an indispensable signal mediator for the nutrient-sensing system

Cells sense nutrients present in the extracellular environment and modulate the activities of intracellular signaling systems in response to nutrient availability. This study demonstrates that RalA and its activator RalGDS participate in nutrient sensing and are indispensable for activation of mammalian target of rapamycin complex 1 (mTORC1) induced by extracellular nutrients. Knockdown of RalA or RalGDS abolished amino acid- and glucose-induced mTORC1 activation, as judged by phosphorylation of S6 kinase and eukaryotic translation initiation factor 4E-binding protein 1. The amount of GTP-bound RalA increased in response to increased amino acid availability. In addition, RalA knockdown suppressed Rheb-induced S6 kinase phosphorylation, and the constitutively active form of RalA induced mTORC1 activation in the absence of Rheb. These results collectively suggest that RalGDS and RalA act downstream of Rheb and that RalA activation is a crucial step in nutrient-induced mTORC1 activation.     

Structure-guided Mutation of the Conserved G3-box Glycine in Rheb Generates a Constitutively Activated Regulator of Mammalian Target of Rapamycin (mTOR)

Constitutively activated variants of small GTPases, which provide valuable functional probes of their role in cellular signaling pathways, can often be generated by mutating the canonical catalytic residue (e.g. Ras Q61L) to impair GTP hydrolysis. However, this general approach is ineffective for a substantial fraction of the small GTPase family in which this residue is not conserved (e.g. Rap) or not catalytic (e.g. Rheb). Using a novel engineering approach, we have manipulated nucleotide binding through structure-guided substitutions of an ultraconserved glycine residue in the G3-box motif (DXXG). Substitution of Rheb Gly-63 with alanine impaired both intrinsic and TSC2 GTPase-activating protein (GAP)-mediated GTP hydrolysis by displacing the hydrolytic water molecule, whereas introduction of a bulkier valine side chain selectively blocked GTP binding by steric occlusion of the γ-phosphate. Rheb G63A stimulated phosphorylation of the mTORC1 substrate p70S6 kinase more strongly than wild-type, thus offering a new tool for mammalian target of rapamycin (mTOR) signaling.     

mTOR signaling: PLD takes center stage

The mammalian target of rapamycin (mTOR) assembles a signaling network that transduces nutrient signals and various other stimuli to regulate a wide range of cellular functions. Of the two distinct mTOR complexes, mTORC1 is under the control of the TSC-Rheb pathway, which serves as an integrator of multiple upstream signals. A lipid signaling cascade involving phospholipase D (PLD) and phosphatidic acid (PA) has also been known to mediate mitogenic signals upstream of mTORC1. A new study now reveals a direct connection between these two regulatory pathways and demonstrates that PLD1 is an effector of Rheb in the activation of mTORC1. A novel role of PLD as a nutrient sensor has also been suggested. In this extra-view, we discuss the emerging importance of PA and PLD in the mTORC1 signaling network and the biological processes it governs. We also consider the implications from several recent findings and propose mechanistic models of PLD-mTOR signaling to be tested in the near future.     

Phospholipase D mediates nutrient input to mammalian target of rapamycin complex 1 (mTORC1)

The mammalian target of rapamycin (mTOR) is a critical sensor of nutritional sufficiency. Although much is known about the regulation of mTOR in response to growth factors, much less is known about the regulation of mTOR in response to nutrients. Amino acids have no impact on the signals that regulate Rheb, a GTPase required for the activation of mTOR complex 1 (mTORC1). Phospholipase D (PLD) generates a metabolite, phosphatidic acid, that facilitates association between mTOR and the mTORC1 co-factor Raptor. We report here that elevated PLD activity in human cancer cells is dependent on both amino acids and glucose and that amino acid- and glucose-induced increases in mTORC1 activity are dependent on PLD. Amino acid- and glucose-induced PLD and mTORC1 activity were also dependent on the GTPases RalA and ARF6 and the type III phosphatidylinositol-3-kinase hVps34. Thus, a key stimulatory event for mTORC1 activation in response to nutrients is the generation of phosphatidic acid by PLD.     

To Cease or To Proliferate

Tor (Target of Rapamycin) pathway underlies a major signaling mechanism for controlling cell growth and proliferation1. Rheb (Ras homolog enriched in brain) is a small GTPase in the Tor pathway2-4. Similar to other small GTPases, Rheb cycles between a GTP-bound active state and a GDP-bound inactive state. TSC2 (Tuberous sclerosis complex 2), a gene mutated in an autosomal dominant disease Tuberous sclerosis, was shown to be the Rheb-GAP (GTPase activating protein)5, 6. However, a guanine nucleotide exchange factor (GEF) for Rheb had been missing. Human TCTP (Translationally controlled tumor protein) has been implicated in cancer, but its function in vivo has not been clearly elucidated. Recently we reported a molecular genetic characterization of TCTP function in Drosophila7. Drosophila TCTP (dTCTP) displays GEF activity to Rheb and is essential for Rheb activation in organ growth. Thus, our study provides a tight linkage of dTCTP to the Rheb-TOR pathway. In this addendum, we will briefly overview our findings and discuss our perspectives for future research on TCTP.