Tuberous sclerosis complex gene products,Tuberin and Hamartin,control mTOR signaling by acting as a GTPase-activating protein complex toward Rheb

BACKGROUND: Tuberous Sclerosis Complex (TSC) is a genetic disorder that occurs through the loss of heterozygosity of either TSC1 or TSC2, which encode Hamartin or Tuberin, respectively. Tuberin and Hamartin form a tumor suppressor heterodimer that inhibits the mammalian target of rapamycin (mTOR) nutrient signaling input, but how this occurs is unclear. RESULTS: We show that the small G protein Rheb (Ras homolog enriched in brain) is a molecular target of TSC1/TSC2 that regulates mTOR signaling. Overexpression of Rheb activates 40S ribosomal protein S6 kinase 1 (S6K1) but not p90 ribosomal S6 kinase 1 (RSK1) or Akt. Furthermore, Rheb induces phosphorylation of eukaryotic initiation factor 4E binding protein 1 (4E-BP1) and causes 4E-BP1 to dissociate from eIF4E. This dissociation is completely sensitive to rapamycin (an mTOR inhibitor) but not wortmannin (a phosphoinositide 3-kinase [PI3K] inhibitor). Rheb also activates S6K1 during amino acid insufficiency via a rapamycin-sensitive mechanism, suggesting that Rheb participates in nutrient signaling through mTOR. Moreover, Rheb does not activate a S6K1 mutant that is unresponsive to mTOR-mediated signals, confirming that Rheb functions upstream of mTOR. Overexpression of the Tuberin-Hamartin heterodimer inhibits Rheb-mediated S6K1 activation, suggesting that Tuberin functions as a Rheb GTPase activating protein (GAP). Supporting this notion, TSC patient-derived Tuberin GAP domain mutants were unable to inactivate Rheb in vivo. Moreover, in vitro studies reveal that Tuberin, when associated with Hamartin, acts as a Rheb GTPase-activating protein. Finally, we show that membrane localization of Rheb is important for its biological activity because a farnesylation-defective mutant of Rheb stimulated S6K1 activation less efficiently. CONCLUSIONS: We show that Rheb acts as a novel mediator of the nutrient signaling input to mTOR and is the molecular target of TSC1 and TSC2 within mammalian cells.     

Analysis of mTOR signaling by the small G-proteins, Rheb and RhebL1

The small G protein Rheb (Ras homologue enriched in brain) is known to promote mammalian target of rapamycin (mTOR) signaling. In this study, we show that Rheb like-1 protein (RhebL1) rescues mTOR signaling during nutrient withdrawal and that tuberous sclerosis complex-1 (TSC) and TSC2 impairs RhebL1-mediated signaling through mTOR. We identify critical residues within the switch I region (N41) and 'constitutive' effector (Ec) region (Y/F54 and L56) of Rheb and RhebL1, which are required for their efficient activation of mTOR signaling. Mutation of Rheb and RhebL1 at N41 impaired their interaction with mTOR, which identifies mTOR as a common downstream target of both Rheb and RhebL1.     

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.     

Novel role of the small GTPase Rheb: its implication in endocytic pathway independent of the activation of mammalian target of rapamycin

The Ras-homologous GTPase Rheb that is conserved from yeast to human appears to be involved not only in cell growth but also in nutrient uptake. Recent biochemical analysis revealed that tuberous sclerosis complex (TSC), a GTPase-activating protein (GAP), deactivates Rheb and that phosphatidylinositol 3'-kinase (PI3k)-Akt/PKB kinase pathway activates Rheb through inhibition of the GAP-mediated deactivation. Although mammalian target of rapamycin (mTOR) kinase is implicated in the downstream target of Rheb, the direct effector(s) and exact functions of Rheb have not been fully elucidated. Here we identified that Rheb expression in cultured cells induces the formation of large cytoplasmic vacuoles, which are characterized as late endocytic (late endosome- and lysosome-like) components. The vacuole formation required the GTP form of Rheb, but not the activation of the downstream mTOR kinase. These results suggest that Rheb regulates endocytic trafficking pathway independent of the previously identified mTOR pathway. The physiological roles of the two Rheb-dependent signaling pathways are discussed in terms of nutrient uptake and cell growth or cell cycle progression.     

Rheb binds and regulates the mTOR kinase

BACKGROUND: The target of rapamycin (TOR), in complex with the proteins raptor and LST8 (TOR complex 1), phosphorylates the p70S6K and 4E-BP1 to promote mRNA translation. Genetic evidence establishes that TOR complex activity in vivo requires the small GTPase Rheb, and overexpression of Rheb can rescue TOR from inactivation in vivo by amino-acid withdrawal. The Tuberous Sclerosis heterodimer (TSC1/TSC2) functions as a Rheb GTPase activator and inhibits TOR signaling in vivo. RESULTS: Here, we show that Rheb binds to the TOR complex specifically, independently of its ability to bind TSC2, through separate interactions with the mTOR catalytic domain and with LST8. Rheb binding to the TOR complex in vivo and in vitro does not require Rheb guanyl nucleotide charging but is modulated by GTP and impaired by certain mutations (Ile39Lys) in the switch 1 loop. Nucleotide-deficient Rheb mutants, although capable of binding mTOR in vivo and in vitro, are inhibitory in vivo, and the mTOR polypeptides that associate with nucleotide-deficient Rheb in vivo lack kinase activity in vitro. Reciprocally, mTOR polypeptides bound to Rheb(Gln64Leu), a mutant that is nearly 90% GTP charged, exhibit substantially higher protein kinase specific activity than mTOR bound to wild-type Rheb. CONCLUSIONS: The TOR complex 1 is a direct target of Rheb-GTP, whose binding enables activation of the TOR kinase.     

Biochemical and functional characterizations of small GTPase Rheb and TSC2 GAP activity

Tuberous sclerosis complex (TSC) is a genetic disease caused by a mutation in either the tsc1 or tsc2 tumor suppressor gene. Recent studies have demonstrated that TSC2 displays GAP (GTPase-activating protein) activity specifically towards the small G protein Rheb and inhibits its ability to stimulate the mTOR signaling pathway. Rheb and TSC2 comprise a unique pair of GTPase and GAP, because Rheb has high basal GTP levels and TSC2 does not have the catalytic arginine finger found in Ras-GAP. To investigate the function of TSC2 and Rheb in mTOR signaling, we analyzed the TSC2-stimulated Rheb GTPase activity. We found that Arg15, a residue equivalent to Gly12 in Ras, is important for Rheb to function as a substrate for TSC2 GAP. In addition, we identified asparagine residues essential for TSC2 GAP activity. We demonstrated a novel catalytic mechanism of the TSC2 GAP and Rheb that TSC2 uses a catalytic "asparagine thumb" instead of the arginine finger found in Ras-GAP. Furthermore, we discovered that farnesylation and membrane localization of Rheb is not essential for Rheb to stimulate S6 kinase (S6K) phosphorylation. Analysis of TSC1 binding defective mutants of TSC2 shows that TSC1 is not required for the TSC2 GAP activity but may function as a regulatory component in the TSC1/TSC2 complex. Our data further demonstrate that GAP activity is essential for the cellular function of TSC2 to inhibit S6K phosphorylation.     

The tuberous sclerosis protein TSC2 is not required for the regulation of the mammalian target of rapamycin by amino acids and certain cellular stresses

Amino acids positively regulate signaling through the mammalian target of rapamycin (mTOR). Recent work demonstrated the importance of the tuberous sclerosis protein TSC2 for regulation of mTOR by insulin. TSC2 contains a GTPase-activator domain that promotes hydrolysis of GTP bound to Rheb, which positively regulates mTOR signaling. Some studies have suggested that TSC2 also mediates the control of mTOR by amino acids. In cells lacking TSC2, amino acid withdrawal still results in dephosphorylation of S6K1, ribosomal protein S6, the eukaryotic initiation factor 4E-binding protein, and elongation factor-2 kinase. The effects of amino acid withdrawal are diminished by inhibiting protein synthesis or adding back amino acids. These studies demonstrate that amino acid signaling to mTOR occurs independently of TSC2 and involves additional unidentified inputs. Although TSC2 is not required for amino acid control of mTOR, amino acid withdrawal does decrease the proportion of Rheb in the active GTP-bound state. Here we also show that Rheb and mTOR form stable complexes, which are not, however, disrupted by amino acid withdrawal. Mutants of Rheb that cannot bind GTP or GDP can interact with mTOR complexes. We also show that the effects of hydrogen peroxide and sorbitol, cell stresses that impair mTOR signaling, are independent of TSC2. Finally, we show that the ability of energy depletion (which impairs mTOR signaling in TSC2+/+ cells) to increase the phosphorylation of eukaryotic elongation factor 2 is also independent of TSC2. This likely involves the phosphorylation of the elongation factor-2 kinase by the AMP-activated protein kinase.     

The amino acid sensitive TOR pathway from yeast to mammals

The target of rapamycin (TOR) is an ancient effector of cell growth that integrates signals from growth factors and nutrients. Two downstream effectors of mammalian TOR, the translational components S6K1 and 4EBP1, are commonly used as reporters of mTOR activity. The conical signaling cascade initiated by growth factors is mediated by PI3K, PKB, TSC1/2 and Rheb. However, the process through which nutrients, i.e., amino acids, activate mTOR remains largely unknown. Evidence exists for both an intracellular and/or a membrane bound sensor for amino acid mediated mTOR activation. Research in eukaryotic models, has implicated amino acid transporters as nutrient sensors. This review describes recent advances in nutrient signaling that impinge on mTOR and its targets including hVps34, class III PI3K, a transducer of nutrient availability to mTOR.     

Regulation of mTOR and cell growth in response to energy stress by REDD1

The tuberous sclerosis tumor suppressors TSC1 and TSC2 regulate the mTOR pathway to control translation and cell growth in response to nutrient and growth factor stimuli. We have recently identified the stress response REDD1 gene as a mediator of tuberous sclerosis complex (TSC)-dependent mTOR regulation by hypoxia. Here, we demonstrate that REDD1 inhibits mTOR function to control cell growth in response to energy stress. Endogenous REDD1 is induced following energy stress, and REDD1-/- cells are highly defective in dephosphorylation of the key mTOR substrates S6K and 4E-BP1 following either ATP depletion or direct activation of the AMP-activated protein kinase (AMPK). REDD1 likely acts on the TSC1/2 complex, as regulation of mTOR substrate phosphorylation by REDD1 requires TSC2 and is blocked by overexpression of the TSC1/2 downstream target Rheb but is not blocked by inhibition of AMPK. Tetracycline-inducible expression of REDD1 triggers rapid dephosphorylation of S6K and 4E-BP1 and significantly decreases cellular size. Conversely, inhibition of endogenous REDD1 by short interfering RNA increases cell size in a rapamycin-sensitive manner, and REDD1-/- cells are defective in cell growth regulation following ATP depletion. These results define REDD1 as a critical transducer of the cellular response to energy depletion through the TSC-mTOR pathway.     

Inactivation of Rheb by PRAK-mediated phosphorylation is essential for energy-depletion-induced suppression of mTORC1

Cell growth can be suppressed by stressful environments, but the role of stress pathways in this process is largely unknown. Here we show that a cascade of p38β mitogen-activated protein kinase (MAPK) and p38-regulated/activated kinase (PRAK) plays a role in energy-starvation-induced suppression of mammalian target of rapamycin (mTOR), and that energy starvation activates the p38β-PRAK cascade. Depletion of p38β or PRAK diminishes the suppression of mTOR complex 1 (mTORC1) and reduction of cell size induced by energy starvation. We show that p38β-PRAK operates independently of the known mTORC1 inactivation pathways--phosphorylation of tuberous sclerosis protein 2 (TSC2) and Raptor by AMP-activated protein kinase (AMPK)--and surprisingly, that PRAK directly regulates Ras homologue enriched in brain (Rheb), a key component of the mTORC1 pathway, by phosphorylation. Phosphorylation of Rheb at Ser 130 by PRAK impairs the nucleotide-binding ability of Rheb and inhibits Rheb-mediated mTORC1 activation. The direct regulation of Rheb by PRAK integrates a stress pathway with the mTORC1 pathway in response to energy depletion.     

Insulin activation of Rheb,a mediator of mTOR/S6K/4E-BP signaling,is inhibited by TSC1 and 2

Tumor suppressor genes evolved as negative effectors of mitogen and nutrient signaling pathways, such that mutations in these genes can lead to pathological states of growth. Tuberous sclerosis (TSC) is a potentially devastating disease associated with mutations in two tumor suppressor genes, TSC1 and 2, that function as a complex to suppress signaling in the mTOR/S6K/4E-BP pathway. However, the inhibitory target of TSC1/2 and the mechanism by which it acts are unknown. Here we provide evidence that TSC1/2 is a GAP for the small GTPase Rheb and that insulin-mediated Rheb activation is PI3K dependent. Moreover, Rheb overexpression induces S6K1 phosphorylation and inhibits PKB phosphorylation, as do loss-of-function mutations in TSC1/2, but contrary to earlier reports Rheb has no effect on MAPK phosphorylation. Finally, coexpression of a human TSC2 cDNA harboring a disease-associated point mutation in the GAP domain, failed to stimulate Rheb GTPase activity or block Rheb activation of S6K1.     

A Highlights from MBoC Selection: The Late Endosome is Essential for mTORC1 Signaling

The multisubunit mTORC1 complex integrates signals from growth factors and nutrients to regulate protein synthesis, cell growth, and autophagy. To examine how endocytic trafficking might be involved in nutrient regulation of mTORC1, we perturbed specific endocytic trafficking pathways and measured mTORC1 activity using S6K1 as a readout. When early/late endosomal conversion was blocked by either overexpression of constitutively active Rab5 (Rab5CA) or knockdown of the Rab7 GEF hVps39, insulin- and amino acid–stimulated mTORC1/S6K1 activation were inhibited, and mTOR localized to hybrid early/late endosomes. Inhibition of other stages of endocytic trafficking had no effect on mTORC1. Overexpression of Rheb, which activates mTOR independently of mTOR localization, rescued mTORC1 signaling in cells expressing Rab5CA, whereas hyperactivation of endogenous Rheb in TSC2−/− MEFs did not. These data suggest that integrity of late endosomes is essential for amino acid– and insulin-stimulated mTORC1 signaling and that blocking the early/late endosomal conversion prevents mTOR from interacting with Rheb in the late endosomal compartment.     

Rag GTPases and AMPK/TSC2/Rheb mediate the differential regulation of mTORC1 signaling in response to alcohol and leucine

Leucine (Leu) and insulin both stimulate muscle protein synthesis, albeit at least in part via separate signaling pathways. While alcohol (EtOH) suppresses insulin-stimulated protein synthesis in cultured myocytes, its ability to disrupt Leu signaling and Rag GTPase activity has not been determined. Likewise, little is known regarding the interaction of EtOH and Leu on the AMPK/TSC2/Rheb pathway. Treatment of myocytes with EtOH (100 mM) decreased protein synthesis, whereas Leu (2 mM) increased synthesis. In combination, EtOH suppressed the anabolic effect of Leu. The effects of EtOH and Leu were associated with coordinate changes in the phosphorylation state of mTOR, raptor, and their downstream targets 4EBP1 and S6K1. As such, EtOH suppressed the ability of Leu to activate these signaling components. The Rag signaling pathway was activated by Leu but suppressed by EtOH, as evidenced by changes in the interaction of Rag proteins with mTOR and raptor. Overexpression of constitutively active (ca)RagA and caRagC increased mTORC1 activity, as determined by increased S6K1 phosphorylation. Furthermore, the caRagA-caRagC heterodimer blocked the inhibitory effect of EtOH. EtOH and Leu produced differential effects on AMPK signaling. EtOH enhanced AMPK activity, resulting in increased TSC2 (S1387) and eEF2 phosphorylation, whereas Leu had the opposite effect. EtOH also decreased the interaction of Rheb with mTOR, and this was prevented by Leu. Collectively, our results indicate that EtOH inhibits the anabolic effects that Leu has on protein synthesis and mTORC1 activity by modulating both Rag GTPase function and AMPK/TSC2/Rheb signaling.     

Inappropriate activation of the TSC/Rheb/mTOR/S6K cassette induces IRS1/2 depletion, insulin resistance, and cell survival deficiencies

Tuberous sclerosis is a largely benign tumor syndrome derived from the acquisition of somatic lesions in genes encoding the tumor suppressor products, TSC1 or TSC2. Loss of function of the TSC1-TSC2 complex, which acts as a Rheb GAP, yields constitutive, unrestrained signaling from the cell growth machinery comprised of Rheb, mTOR, and S6K. We demonstrate herein that constitutive activation of the Rheb/mTOR/S6K cassette, whether by genetic deletion of TSC1 or TSC2 or by ectopic expression of Rheb, is sufficient to induce insulin resistance. This is the result of downregulation of the insulin receptor substrates, IRS1 and IRS2, which become limiting for signal transmission from the insulin receptor to PI3K. Downstream of PI3K, the survival kinase, Akt, is completely refractory to activation by IRS-dependent growth factor pathways such as insulin or IGF-I in TSC1- or TSC2-deficient cells but not to activation by IRS-independent pathways such as those utilized by PDGF. The antiapoptotic program induced by IGF-I but not PDGF is severely compromised in TSC2 null cells. Our results suggest that inappropriate activation of the Rheb/mTOR/S6K pathway imposes a negative feedback program to attenuate IRS-dependent processes such as cell survival.     

The TSC/Rheb/TOR Signaling Pathway in Fission Yeast and Mammalian Cells: Temperature Sensitive and Constitutive Active Mutants of TOR

The TSC/Rheb/TOR signaling pathway plays important roles in growth and cell cycle regulation. The main player TOR belongs to the PI3K-related protein kinase family. Recent studies utilizing fission yeast Tor2 have led to the identification of a number of amino acid changes that lead to inactivation as well as activation of TOR kinase. Also, constitutive active mutations in its upstream regulator, Rheb, have been identified. Isolation and characterization of temperature sensitive Tor2 mutants have established that this kinase functions as a key switch that determines cell fate between growth and sexual development. Introduction of Tor2 activating mutations into mTOR conferred nutrient independent activation of mTOR. Interestingly, these studies point to regions of TOR kinase important for its function.     

Rheb binds tuberous sclerosis complex 2 (TSC2) and promotes S6 kinase activation in a rapamycin- and farnesylation-dependent manner

Recently the tuberous sclerosis complex 2 (TSC2) tumor suppressor gene product has been identified as a negative regulator of protein synthesis upstream of the mTOR and ribosomal S6 kinases. Because of the homology of TSC2 with GTPase-activating proteins for Rap1, we examined whether a Ras/Rap-related GTPase might be involved in this process. TSC2 was found to bind to Rheb-GTP in vitro and to reduce Rheb GTP levels in vivo. Over-expression of Rheb but not Rap1 promoted the activation of S6 kinase in a rapamycin-dependent manner, suggesting that Rheb acts upstream of mTOR. The ability of Rheb to induce S6 phosphorylation was also inhibited by a farnesyl transferase inhibitor, suggesting that Rheb may be responsible for the Ras-independent anti-neoplastic properties of this drug.     

Ras Homolog Enriched in Brain (Rheb) Enhances Apoptotic Signaling

Rheb is a homolog of Ras GTPase that regulates cell growth, proliferation, and regeneration via mammalian target of rapamycin (mTOR). Because of the well established potential of activated Ras to promote survival, we sought to investigate the ability of Rheb signaling to phenocopy Ras. We found that overexpression of lipid-anchored Rheb enhanced the apoptotic effects induced by UV light, TNFα, or tunicamycin in an mTOR complex 1 (mTORC1)-dependent manner. Knocking down endogenous Rheb or applying rapamycin led to partial protection, identifying Rheb as a mediator of cell death. Ras and c-Raf kinase opposed the apoptotic effects induced by UV light or TNFα but did not prevent Rheb-mediated apoptosis. To gain structural insight into the signaling mechanisms, we determined the structure of Rheb-GDP by NMR. The complex adopts the typical canonical fold of RasGTPases and displays the characteristic GDP-dependent picosecond to nanosecond backbone dynamics of the switch I and switch II regions. NMR revealed Ras effector-like binding of activated Rheb to the c-Raf-Ras-binding domain (RBD), but the affinity was 1000-fold lower than the Ras/RBD interaction, suggesting a lack of functional interaction. shRNA-mediated knockdown of apoptosis signal-regulating kinase 1 (ASK-1) strongly reduced UV or TNFα-induced apoptosis and suppressed enhancement by Rheb overexpression. In conclusion, Rheb-mTOR activation not only promotes normal cell growth but also enhances apoptosis in response to diverse toxic stimuli via an ASK-1-mediated mechanism. Pharmacological regulation of the Rheb/mTORC1 pathway using rapamycin should take the presence of cellular stress into consideration, as this may have clinical implications.     

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.     

Simultaneous inhibition of mTORC1 and mTORC2 by mTOR kinase inhibitor AZD8055 induces autophagy and cell death in cancer cells

mTOR is a major biological switch, coordinating an adequate response to changes in energy uptake (amino acids, glucose), growth signals (hormones, growth factors) and environmental stress. mTOR kinase is highly conserved through evolution from yeast to man and in both cases, controls autophagy and cellular translation in response to nutrient stress. mTOR kinase is the catalytic component of two distinct multiprotein complexes called mTORC1 and mTORC2. In addition to mTOR, mTORC1 contains Raptor, mLST8 and PRAS40. mTORC2 contains mTOR, Rictor, mSIN1 and Protor-1. mTORC1 activates p70S6K, which in turn phosphorylates the ribosomal protein S6 and 4E-BP1, both involved in protein translation. mTORC2 activates AKT directly by phosphorylating Serine 473. pAKT(S473) phosphorylates TSC2 (tuberin) and inactivates it, preventing its association with TSC1 (hamartin) and the inhibition of Rheb, an activator of mTOR. pAKT also phosphorylates PRAS40, releasing it from the mTORC1 complex, increasing its kinase activity. Finally, AKT regulates FOXO3 phosphorylation, sequestering it in the cytosol in an inactive state.