Structure-activity relationship of leucyladenylate sulfamate analogues as leucyl-tRNA synthetase (LRS)-targeting inhibitors of Mammalian target of rapamycin complex 1 (mTORC1)

Leucyl-tRNA synthetase (LRS) plays an important role in amino acid-dependent mTORC1 signaling, which is known to be associated with cellular metabolism and proliferation. Therefore, LRS-targeting small molecules that can suppress mTORC1 activation may provide an alternative strategy to current anticancer therapy. In this work, we developed a library of leucyladenylate sulfate analogues by extensively modifying three different pharmacophoric regions comprising adenine, ribose and leucine. Several effective compounds were identified by cell-based mTORC1 activation assays and further tested for anticancer activity. The selected compounds mostly exhibited selective cytotoxicity toward five different cancer cell lines, supporting the hypothesis that the LRS-mediated mTORC1 pathway is a promising alternative target to current therapeutic approaches.     

Leucyl-tRNA synthetase is an intracellular leucine sensor for the mTORC1-signaling pathway

Amino acids are required for activation of the mammalian target of rapamycin (mTOR) kinase, which regulates protein translation, cell size, and autophagy. However, the amino acid sensor that directly couples intracellular amino acid-mediated signaling to mTORC1 is unknown. Here we show that leucyl-tRNA synthetase (LRS) plays a critical role in amino acid-induced mTORC1 activation by sensing intracellular leucine concentration and initiating molecular events leading to mTORC1 activation. Mutation of LRS amino acid residues important for leucine binding renders the mTORC1 pathway insensitive to intracellular levels of amino acids. We show that LRS directly binds to Rag GTPase, the mediator of amino acid signaling to mTORC1, in an amino acid-dependent manner and functions as a GTPase-activating protein (GAP) for Rag GTPase to activate mTORC1. This work demonstrates that LRS is a key mediator for amino acid signaling to mTORC1.     

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.     

Simultaneous inhibition of mTOR-containing complex 1 (mTORC1) and MNK induces apoptosis of cutaneous T-cell lymphoma (CTCL) cells

Background

mTOR kinase forms the mTORC1 complex by associating with raptor and other proteins and affects a number of key cell functions. mTORC1 activates p70S6kinase 1 (p70S6K1) and inhibits 4E-binding protein 1 (4E-BP1). In turn, p70S6K1 phosphorylates a S6 protein of the 40S ribosomal subunit (S6rp) and 4E-BP1, with the latter negatively regulating eukaryotic initiation factor 4E (eIF-4E). MNK1 and MNK2 kinases phosphorylate and augment activity of eIF4E. Rapamycin and its analogs are highly specific, potent, and relatively non-toxic inhibitors of mTORC1. Although mTORC1 activation is present in many types of malignancies, rapamycin-type inhibitors shows relatively limited clinical efficacy as single agents. Initially usually indolent, CTCL displays a tendency to progress to the aggressive forms with limited response to therapy and poor prognosis. Our previous study (M. Marzec et al. 2008) has demonstrated that CTCL cells display mTORC1 activation and short-term treatment of CTCL-derived cells with rapamycin suppressed their proliferation and had little effect on the cell survival.

Methods

Cells derived from CTCL were treated with mTORC1 inhibitor rapamycin and MNK inhibitor and evaluated for inhibition of the mTORC1 signaling pathway and cell growth and survival.

Results

Whereas the treatment with rapamycin persistently inhibited mTORC1 signaling, it suppressed only partially the cell growth. MNK kinase mediated the eIF4E phosphorylation and inhibition or depletion of MNK markedly suppressed proliferation of the CTCL cells when combined with the rapamycin-mediated inhibition of mTORC1. While MNK inhibition alone mildly suppressed the CTCL cell growth, the combined MNK and mTORC1 inhibition totally abrogated the growth. Similarly, MNK inhibitor alone displayed a minimal pro-apoptotic effect; in combination with rapamycin it triggered profound cell apoptosis.

Conclusions

These findings indicate that the combined inhibition of mTORC1 and MNK may prove beneficial in the treatment of CTCL and other malignancies.     

Hyperactivation of Mammalian Target of Rapamycin Complex 1 (mTORC1) Promotes Breast Cancer Progression through Enhancing Glucose Starvation-induced Autophagy and Akt Signaling

The mammalian target of rapamycin complex 1 (mTORC1) is a master regulator of cell growth and proliferation. Recent studies have suggested that constitutive activation of mTORC1 in normal cells could lead to malignant tumor development in several tissues. However, the mechanisms of mTORC1 hyperactivation to promote the growth and metastasis of breast or other cancers are still not well characterized. Here, using a new inducible deletion system, we show that deletion of Tsc1 in mouse primary mammary tumor cells, either before or after their transplantation, significantly increased their growth in vivo. The increase in tumor growth was completely rescued by rapamycin treatment, suggesting a major contribution from mTORC1 hyperactivation. Interestingly, glucose starvation-induced autophagy, but not amino acid starvation-induced autophagy, was increased significantly in Tsc1-null tumor cells. Further analysis of these cells also showed an increased Akt activation but no significant changes in Erk signaling. Together, these results provide insights into the mechanism by which hyperactivation of mTORC1 promotes breast cancer progression through increasing autophagy and Akt activation in vivo.     

Improvement of mTORC1-driven overproduction of apoB-containing triacylglyceride-rich lipoproteins by short-chain fatty acids, 4-phenylbutyric acid and (R)-α-lipoic acid,in human hepatocellular carcinoma cells

The activation of hepatic kinase mechanistic target of rapamycin complex 1 (mTORC1) is implicated in the development of obesity-related metabolic disorders. This study investigated the metabolic sequelae of mTORC1 hyperactivation in human hepatoma cells and the lipid-regulating mechanisms of two short-chain fatty acids: 4-phenylbutyric acid (PBA) and (R)-α-lipoic acid (LA). We created three stable cell lines that exhibit low, normal, or high mTORC1 activity. mTORC1 hyperactivation induced the expression of lipogenic (DGAT1 and DGAT2) and lipoprotein assembly (MTP and APOB) genes, thereby raising cellular triacylglyceride (TG) and exacerbating secretion of apoB-containing TG-rich lipoproteins. LYS6K2, a specific inhibitor of the p70 S6 kinase branch of mTORC1 signaling, reversed these effects. PBA and LA decreased secreted TG through distinct mechanisms. PBA repressed apoB expression (both mRNA and protein) and lowered secreted TG without mitigation of mTORC1 hyperactivity or activation of AMPK. LA decreased cellular and secreted TG by attenuating mTORC1 signaling in an AMPK-independent manner. LA did not regulate apoB expression but led to the secretion of apoB-containing TG-poor lipoproteins by repressing the expression of lipogenic genes, FASN, DGAT1, and DGAT2. Our studies provide new mechanistic insight into the hypolipidemic activity of PBA and LA in the context of mTORC1 hyperactivation and suggest that the short-chain fatty acids may aid in the prevention and treatment of hypertriglyceridemia.     

Oncogenic mutations in adenomatous polyposis coli (Apc) activate mechanistic target of rapamycin complex 1 (mTORC1) in mice and zebrafish

Truncating mutations in adenomatous polyposis coli (APC) are strongly linked to colorectal cancers. APC is a negative regulator of the Wnt pathway and constitutive Wnt activation mediated by enhanced Wnt–β-catenin target gene activation is believed to be the predominant mechanism responsible for APC mutant phenotypes. However, recent evidence suggests that additional downstream effectors contribute to APC mutant phenotypes. We previously identified a mechanism in cultured human cells by which APC, acting through glycogen synthase kinase-3 (GSK-3), suppresses mTORC1, a nutrient sensor that regulates cell growth and proliferation. We hypothesized that truncating Apc mutations should activate mTORC1 in vivo and that mTORC1 plays an important role in Apc mutant phenotypes. We find that mTORC1 is strongly activated in apc mutant zebrafish and in intestinal polyps in Apc mutant mice. Furthermore, mTORC1 activation is essential downstream of APC as mTORC1 inhibition partially rescues Apc mutant phenotypes including early lethality, reduced circulation and liver hyperplasia. Importantly, combining mTORC1 and Wnt inhibition rescues defects in morphogenesis of the anterior-posterior axis that are not rescued by inhibition of either pathway alone. These data establish mTORC1 as a crucial, β-catenin independent effector of oncogenic Apc mutations and highlight the importance of mTORC1 regulation by APC during embryonic development. Our findings also suggest a new model of colorectal cancer pathogenesis in which mTORC1 is activated in parallel with Wnt/β-catenin signaling.KEY WORDS: APC, Wnt, mTOR, mTORC1, Zebrafish, Colon cancer, Polyposis, GSK-3     

The synergistic inhibition of breast cancer proliferation by combined treatment with 4EGI-1 and MK2206

Cap-dependent translation is a potential cancer-related target (oncotarget) due to its critical role in cancer initiation and progression. 4EGI-1, an inhibitor of eIF4E/eIF4G interaction, was discovered by screening chemical libraries of small molecules. 4EGI-1 inhibits cap-dependent translation initiation by impairing the assembly of the eIF4E/eIF4G complex, and therefore is a potential anti-cancer agent. Here, we report that 4EGI-1 also inhibits mTORC1 signaling independent of its inhibitory role on cap-dependent translation initiation. The inhibition of mTORC1 signaling by 4EGI-1 activates Akt due to both abrogation of the negative feedback loops from mTORC1 to PI3K and activation of mTORC2. We further validated that mTORC2 activity is required for 4EGI-1-mediated Akt activation. The activated Akt counteracted the anticancer effects of 4EGI-1. In support of this model, inhibition of Akt potentiates the antitumor activity of 4EGI-1 both in vitro and in a xenograft mouse model in vivo. Our results suggest that a combination of 4EGI-1and Akt inhibitor is a rational approach for the treatment of cancer.     

The synergistic inhibition of breast cancer proliferation by combined treatment with 4EGI-1 and MK2206

Cap-dependent translation is a potential cancer-related target (oncotarget) due to its critical role in cancer initiation and progression. 4EGI-1, an inhibitor of eIF4E/eIF4G interaction, was discovered by screening chemical libraries of small molecules. 4EGI-1 inhibits cap-dependent translation initiation by impairing the assembly of the eIF4E/eIF4G complex, and therefore is a potential anti-cancer agent. Here, we report that 4EGI-1 also inhibits mTORC1 signaling independent of its inhibitory role on cap-dependent translation initiation. The inhibition of mTORC1 signaling by 4EGI-1 activates Akt due to both abrogation of the negative feedback loops from mTORC1 to PI3K and activation of mTORC2. We further validated that mTORC2 activity is required for 4EGI-1-mediated Akt activation. The activated Akt counteracted the anticancer effects of 4EGI-1. In support of this model, inhibition of Akt potentiates the antitumor activity of 4EGI-1 both in vitro and in a xenograft mouse model in vivo. Our results suggest that a combination of 4EGI-1and Akt inhibitor is a rational approach for the treatment of cancer.     

mTORC1 Down-Regulates Cyclin-Dependent Kinase 8 (CDK8) and Cyclin C (CycC)

In non-alcoholic fatty liver disease (NAFLD) and insulin resistance, hepatic de novo lipogenesis is often elevated, but the underlying mechanisms remain poorly understood. Recently, we show that CDK8 functions to suppress de novo lipogenesis. Here, we identify the mammalian target of rapamycin complex 1 (mTORC1) as a critical regulator of CDK8 and its activating partner CycC. Using pharmacologic and genetic approaches, we show that increased mTORC1 activation causes the reduction of the CDK8-CycC complex in vitro and in mouse liver in vivo. In addition, mTORC1 is more active in three mouse models of NAFLD, correlated with the lower abundance of the CDK8-CycC complex. Consistent with the inhibitory role of CDK8 on de novo lipogenesis, nuclear SREBP-1c proteins and lipogenic enzymes are accumulated in NAFLD models. Thus, our results suggest that mTORC1 activation in NAFLD and insulin resistance results in down-regulation of the CDK8-CycC complex and elevation of lipogenic protein expression.     

Mammalian Target of Rapamycin Complex 1 (mTORC1) Plays a Role in Pasteurella multocida Toxin (PMT)-induced Protein Synthesis and Proliferation in Swiss 3T3 Cells

Pasteurella multocida toxin (PMT) is a potent mitogen known to activate several signaling pathways via deamidation of a conserved glutamine residue in the α subunit of heterotrimeric G-proteins. However, the detailed mechanism behind mitogenic properties of PMT is unknown. Herein, we show that PMT induces protein synthesis, cell migration, and proliferation in serum-starved Swiss 3T3 cells. Concomitantly PMT induces phosphorylation of ribosomal S6 kinase (S6K1) and its substrate, ribosomal S6 protein (rpS6), in quiescent 3T3 cells. The extent of the phosphorylation is time and PMT concentration dependent, and is inhibited by rapamycin and Torin1, the two specific inhibitors of the mammalian target of rapamycin complex 1 (mTORC1). Interestingly, PMT-mediated mTOR signaling activation was observed in MEF WT but not in Gα_q/11 knock-out cells. These observations are consistent with the data indicating that PMT-induced mTORC1 activation proceeds via the deamidation of Gα_q/11, which leads to the activation of PLCβ to generate diacylglycerol and inositol trisphosphate, two known activators of the PKC pathway. Exogenously added diacylglycerol or phorbol 12-myristate 13-acetate, known activators of PKC, leads to rpS6 phosphorylation in a rapamycin-dependent manner. Furthermore, PMT-induced rpS6 phosphorylation is inhibited by PKC inhibitor, Gö6976. Although PMT induces epidermal growth factor receptor activation, it exerts no effect on PMT-induced rpS6 phosphorylation. Together, our findings reveal for the first time that PMT activates mTORC1 through the Gα_q/11/PLCβ/PKC pathway. The fact that PMT-induced protein synthesis and cell migration is partially inhibited by rapamycin indicates that these processes are in part mediated by the mTORC1 pathway.     

Phosphorylation of Raptor by p38beta participates in arsenite-induced mammalian target of rapamycin complex 1 (mTORC1) activation

Cell growth is influenced by environmental stress. Mammalian target of rapamycin (mTOR), the central regulator of cell growth, can be positively or negatively regulated by various stresses through different mechanisms. The p38 MAP kinase pathway is essential in cellular stress responses. Activation of MK2, a downstream kinase of p38α, enhances mTOR complex 1 (mTORC1) activity by preventing TSC2 from inhibiting mTOR activation. The p38β-PRAK cascade targets Rheb to inhibit mTORC1 activity upon glucose depletion. Here we show the activation of p38β participates in activation of mTOR complex 1 (mTORC1) induced by arsenite but not insulin, nutrients, anisomycin, or H(2)O(2). Arsenite treatment of cells activates p38β and induces interaction between p38β and Raptor, a regulatory component of mTORC1, resulting in phosphorylation of Raptor on Ser(863) and Ser(771). The phosphorylation of Raptor on these sites enhances mTORC1 activity, and contributes largely to arsenite-induced mTORC1 activation. Our results shown here and in previous work demonstrate that the p38 pathway can regulate different components of the mTORC1 pathway, and that p38β can target different substrates to either positively or negatively regulate mTORC1 activation when a cell encounters different environmental stresses.     

Phosphatidic acid activates mammalian target of rapamycin complex 1 (mTORC1) kinase by displacing FK506 binding protein 38 (FKBP38) and exerting an allosteric effect

Phosphatidic acid (PA) is a critical mediator of mitogenic activation of mammalian target of rapamycin complex 1 (mTORC1) signaling, a master regulator of mammalian cell growth and proliferation. The mechanism by which PA activates mTORC1 signaling has remained unknown. Here, we report that PA selectively stimulates mTORC1 but not mTORC2 kinase activity in cells and in vitro. Furthermore, we show that PA competes with the mTORC1 inhibitor, FK506 binding protein 38 (FKBP38), for mTOR binding at a site encompassing the rapamycin-FKBP12 binding domain. This leads to PA antagonizing FKBP38 inhibition of mTORC1 kinase activity in vitro and rescuing mTORC1 signaling from FKBP38 in cells. Phospholipase D 1, a PA-generating enzyme that is an established upstream regulator of mTORC1, is found to negatively affect mTOR-FKBP38 interaction, confirming the role of endogenous PA in this regulation. Interestingly, removal of FKBP38 alone is insufficient to activate mTORC1 kinase and signaling, which require PA even when the FKBP38 level is drastically reduced by RNAi. In conclusion, we propose a dual mechanism for PA activation of mTORC1: PA displaces FKBP38 from mTOR and allosterically stimulates the catalytic activity of mTORC1.     

Kinase Suppressor of Ras 1 Is Not Required for the Generation of Regulatory and Memory T Cells

The mammalian target of rapamycin (mTOR) kinase is a critical regulator of the differentiation of helper and regulatory CD4+ T cells, as well as memory CD8+ T cells. In this study, we investigated the role of the ERK signaling pathway in regulating mTOR activation in T cells. We showed that activation of ERK following TCR engagement is required for sustained mTOR complex 1 (mTORC1) activation. Absence of kinase suppressor of Ras 1 (KSR1), a scaffold protein of the ERK signaling pathway, or inhibition of ERK resulted in decreased mTORC1 activity following T cell activation. However, KSR1-deficient mice displayed normal regulatory CD4+ T cell development, as well as normal memory CD8+ T cell responses to LCMV and Listeria monocytogenes infection. These data indicate that despite its role in mTORC1 activation, KSR1 is not required in vivo for mTOR-dependent T cell differentiation.     

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.     

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.     

Effects of mTORC1 inhibition on proteasome activity and levels

The mechanistic target of rapamycin (mTOR) regulates numerous extracellular and intracellular signals involved in the maintenan-ce of cellular homeostasis and cell growth. mTOR also functions as an endogenous inhibitor of autophagy. Under nutrient-rich conditions, mTOR complex 1 (mTORC1) phosphorylates the ULK1 complex, preventing its activation and subsequent autophagosome formation, while inhibition of mTORC1 using either rapamycin or nutrient deprivation induces autophagy. Autophagy and proteasomal proteolysis provide amino acids necessary for protein translation. Although the connection between mTORC1 and autophagy is well characterized, the association of mTORC1 inhibition with proteasome biogenesis and activity has not been fully elucidated yet. Proteasomes are long-lived cellular organelles. Their spatiotemporal rather than homeostatic regulation could be another adaptive cellular mechanism to respond to starvation. Here, we reviewed several published reports and the latest research from our group to examine the connection between mTORC1 and proteasome. We have also investigated and described the effect of mTORC1 inhibition on proteasome activity using purified proteasomes. Since mTORC1 inhibitors are currently evaluated as treatments for several human diseases, a better understanding of the link between mTORC1 activity and proteasome function is of utmost importance.     

Targeting AMP-activated protein kinase (AMPK) for treatment of autosomal dominant polycystic kidney disease

Autosomal dominant polycystic kidney disease (ADPKD) is the most common monogenetic kidney disease worldwide and an important cause of chronic kidney disease. Multiple experimental studies have highlighted the role of increased mammalian target of rapamycin complex 1 (mTORC1) and reduced AMP-activated protein kinase (AMPK) signaling in modulating cyst growth in ADPKD. Notably, mTORC1 and AMPK are two diametrically opposing sensors of energy metabolism which regulate cell growth and proliferation. Although pharmacological mTORC1 inhibition was highly effective in experimental studies of ADPKD, clinical trials of mTORC1 inhibitors showed a lack of efficacy with low-dose treatment and poor tolerability with high-dose treatment. Therapeutic AMPK activation has been shown to attenuate cystic kidney disease severity in Pkd1 mutant animal models by improving mitochondrial biogenesis and reducing tissue inflammation. This review summarizes the current knowledge on the function of AMPK as a regulator of cellular energy metabolism and how AMPK activation by pharmacological and non-pharmacological means can potentially be exploited to treat ADPKD in the clinical settings.     

Amino Acids Activate Mammalian Target of Rapamycin (mTOR) Complex 1 without Changing Rag GTPase Guanyl Nucleotide Charging

Activation of mammalian target of rapamycin complex 1 (mTORC1) by amino acids is mediated in part by the Rag GTPases, which bind the raptor subunit of mTORC1 in an amino acid-stimulated manner and promote mTORC1 interaction with Rheb-GTP, the immediate activator. Here we examine whether the ability of amino acids to regulate mTORC1 binding to Rag and mTORC1 activation is due to the regulation of Rag guanyl nucleotide charging. Rag heterodimers in vitro exhibit a very rapid, spontaneous exchange of guanyl nucleotides and an inability to hydrolyze GTP. Mutation of the Rag P-loop corresponding to Ras^Ser-17 abolishes guanyl nucleotide binding. Such a mutation in RagA or RagB inhibits, whereas in RagC or RagD it enhances, Rag heterodimer binding to mTORC1. The binding of wild-type and mutant Rag heterodimers to mTORC1 in vitro parallels that seen with transient expression, but binding to mTORC1 in vitro is entirely independent of Rag guanyl nucleotide charging. HeLa cells stably overexpressing wild-type or P-loop mutant RagC exhibit unaltered amino acid regulation of mTORC1. Despite amino acid-independent raptor binding to Rag, mTORC1 is inhibited by amino acid withdrawal as in parental cells. Rag heterodimers extracted from ³²P-labeled whole cells, or just from the pool associated with the lysosomal membrane, exhibit constitutive [³²P]GTP charging that is unaltered by amino acid withdrawal. Thus, amino acids promote mTORC1 activation without altering Rag GTP charging. Raptor binding to Rag, although necessary, is not sufficient for mTORC1 activation. Additional amino acid-dependent steps couple Rag-mTORC1 to Rheb-GTP.