mTORC1 Kinase Activity Research Articles

Abstract 427: Targeted inhibition of the mTOR complex components, RAPTOR and RICTOR, attenuates progression of colorectal cancer

Abstract Colorectal cancer (CRC) is the second leading cause of cancer deaths in the USA. The mammalian target of rapamycin (mTOR) acts downstream of PI3K to regulate cell growth, proliferation and survival. The mTOR kinase nucleates two distinct complexes, mTORC1 and mTORC2. The mTOR-RAPTOR complex (mTORC1) regulates translation initiation through the effectors S6K and 4E-BP1, while the mTOR-RICTOR complex (mTORC2) regulates the actin cytoskeleton and activates Akt, a key regulator of cell survival. Rapamycin inhibits the kinase activity of mTORC1; prolonged rapamycin treatment also inhibits mTORC2 assembly and Akt activation in certain cells. The purpose of the present study was to determine the effect of targeting mTORC1 and mTORC2 upon progression of CRCs. METHODS. HCT116 and SW480 human colon cancer cells were treated with rapamycin. Cells were also transfected with shRNA directed against RAPTOR or RICTOR. Effects on migration (measured by wound healing assay) and invasion (measured by transwell matrigel assay) were analyzed. Changes in actin cytoskeleton and cell-cell contacts were determined by immunofluorescence microscopy. RESULTS. (i) Immunohistochemical analysis showed that the mTORC1 and mTORC2 components, mTOR, RAPTOR and RICTOR, are overexpressed in primary CRCs and matched liver metastases compared to normal colonic tissue. (ii) Treatment with rapamycin significantly decreased the migration and invasion of HCT116 and SW480 cells. (iii) Stable shRNA-mediated knockdown of RAPTOR and RICTOR significantly decreased the migration and invasion of HCT116 and SW480 cells. (iv) Stable shRNA-mediated knockdown of RAPTOR and RICTOR leads to mesenchymal-to-epithelial transition, as evidenced by increased E-cadherin and decreased vimentin levels. (v) Stable shRNA-mediated knockdown of RAPTOR and RICTOR leads to characteristic rearrangements in actin cytoskeleton as well as increased cell-cell contact. CONCLUSIONS. The mTORC1 and mTORC2 components, mTOR, RAPTOR and RICTOR, are overexpressed in both primary CRCs as well as matched liver metastases. Although the mTOR signaling axis is primarily associated with growth of normal and cancer cells, we demonstrate that stable inhibition of both mTORC1 and mTORC2 decreases migration and invasion capabilities of CRC cells as well as causing a mesenchymal-to-epithelial change in phenotype. The mechanism for this decrease in migration and invasion may be via modulation of actin cytoskeletal rearrangement and cell-cell contact. Thus, targeted inhibition of mTOR signaling represents a novel therapeutic strategy to attenuate the progression and metastasis of CRCs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 427.

MTORC1-Activated S6K1 Phosphorylates Rictor on Threonine 1135 and Regulates mTORC2 Signaling

The mammalian target of rapamycin (mTOR) is a conserved Ser/Thr kinase that forms two functionally distinct complexes important for nutrient and growth factor signaling. While mTOR complex 1 (mTORC1) regulates mRNA translation and ribosome biogenesis, mTORC2 plays an important role in the phosphorylation and subsequent activation of Akt. Interestingly, mTORC1 negatively regulates Akt activation, but whether mTORC1 signaling directly targets mTORC2 remains unknown. Here we show that growth factors promote the phosphorylation of Rictor (rapamycin-insensitive companion of mTOR), an essential subunit of mTORC2. We found that Rictor phosphorylation requires mTORC1 activity and, more specifically, the p70 ribosomal S6 kinase 1 (S6K1). We identified several phosphorylation sites in Rictor and found that Thr1135 is directly phosphorylated by S6K1 in vitro and in vivo, in a rapamycin-sensitive manner. Phosphorylation of Rictor on Thr1135 did not affect mTORC2 assembly, kinase activity, or cellular localization. However, cells expressing a Rictor T1135A mutant were found to have increased mTORC2-dependent phosphorylation of Akt. In addition, phosphorylation of the Akt substrates FoxO1/3a and glycogen synthase kinase 3 alpha/beta (GSK3 alpha/beta) was found to be increased in these cells, indicating that S6K1-mediated phosphorylation of Rictor inhibits mTORC2 and Akt signaling. Together, our results uncover a new regulatory link between the two mTOR complexes, whereby Rictor integrates mTORC1-dependent signaling.

Abstract B15: Targeted inhibition of mTOR signaling attenuates the growth and progression of colorectal cancer

Abstract Introduction: Colorectal cancer (CRC) is the second leading cause of cancer deaths in the USA. The mammalian target of rapamycin (mTOR) acts downstream of PI3K to regulate cell growth, proliferation and survival. The mTOR kinase nucleates two distinct complexes, mTORC1 and mTORC2. The mTOR-RAPTOR complex (mTORC1) regulates translation initiation through the effectors S6K and 4E-BP1, while the mTOR-RICTOR-mSin1 complex (mTORC2) regulates the actin cytoskeleton and activates Akt, a key regulator of cell survival. Rapamycin inhibits the kinase activity of mTORC1; prolonged rapamycin treatment also inhibits mTORC2 assembly and Akt activation in certain cells. Furthermore, rapamycin mediated mTORC1 inhibition leads to loss of mTOR-S6K-IRS1 dependent feedback inhibition, thereby resulting in increased Akt activation. The purpose of the present study was to determine: (i) sensitivity of CRC cells to rapamycin treatment, and (ii) effect of targeting mTORC1 and mTORC2 upon CRC proliferation, apoptosis, cell cycle progression, migration, invasion and xenograft growth. Methods: HCT116, KM20, Caco-2 and SW480 human colon cancer cells were treated with rapamycin. Cells were also transfected with siRNA/shRNA directed against mTOR, RAPTOR or RICTOR. Effects on cell proliferation (measured by Coulter counter cell counts), apoptosis (measured by detecting the level of histone-associated DNA fragments), cell cycle progression (measured by fluorescence-activated cell sorting), migration (measured by wound healing assay), invasion (measured by transwell matrigel assay) and subcutaneous growth were analyzed. Results: (i) Immunohistochemical analysis showed that the mTORC1 and mTORC2 components, mTOR, RAPTOR and RICTOR are overexpressed in primary CRCs and matched liver metastases compared to normal colonic tissue. (ii) Treatment with rapamycin significantly decreased the proliferation of HCT116 and KM20 cells (rapamycin sensitive); however, rapamycin did not alter SW480 or Caco-2 proliferation (rapamycin resistant). (iii) Treatment with rapamycin significantly decreased the migration and invasion of HCT116 and SW480 cells. (iv) Transient siRNA-mediated knockdown of RAPTOR decreased proliferation of KM20 cells only, while knockdown of RICTOR decreased proliferation of all four cell lines. (v) Stable shRNA-mediated knockdown of mTORC1 and mTORC2 components leads to decreased proliferation, increased apoptosis and inhibition of G1-S phase cell cycle progression in HCT116 cells. (vi) Stable shRNA-mediated knockdown of mTORC1 and mTORC2 leads to decreased proliferation, but only mTORC2 knockdown leads to increased apoptosis in SW480 cells. (vii) Targeted inhibition of mTORC1 and mTORC2 attenuates growth of rapamycin-sensitive and — resistant CRCs in xenografted nude mice. Conclusions: The mTORC1 and mTORC2 components, mTOR, RAPTOR and RICTOR are overexpressed in CRCs. Transient inhibition of mTORC2, but not mTORC1, decreased the proliferation of rapamycin-sensitive and —resistant CRC cells. Stable inhibition of mTORC2 decreased proliferation and induced apoptosis in rapamycin-sensitive and —resistant CRC cells. Finally, targeted inhibition of both mTORC1 and mTORC2 attenuates subcutaneous growth of CRCs in vivo. Thus, inhibition of mTORC2 activity represents a novel therapeutic strategy for treatment of rapamycin-sensitive and, more importantly, rapamycin-resistant CRCs. Citation Information: Cancer Res 2009;69(23 Suppl):B15.

IGF-1 stimulates protein synthesis by enhanced signaling through mTORC1 in bovine mammary epithelial cells

Using the MAC-T cell line as a model, the effects of insulin-like growth factor (IGF)-1 on the regulation of protein synthesis through the mammalian target of rapamycin complex 1 (mTORC1) signaling in bovine mammary epithelial cells were evaluated. Global rates of protein synthesis increased by 47% within 30 min of IGF-1 treatment. The effect of IGF-1 on protein synthesis was associated with enhanced association of the eukaryotic initiation factor (eIF) 4E with eIF4G and a concomitant reduction of eIF4E association with eIF4E-binding protein-1 (4E-BP1). There was a progressive increase in the phosphorylation state of ribosomal protein S6 kinase-1, a downstream target of mTORC1 in response to IGF-1. In addition, IGF-1 stimulated mTORC1 kinase activity toward 4E-BP1 in vitro. Phosphorylation on Ser473 of Akt was induced by IGF-1 within 5 min and remained elevated throughout a 30-min time course. The effect of IGF-1 on Akt phosphorylation was also concentration dependent. Activation of Akt by IGF-1 led to increased phosphorylation of tuberous sclerosis complex 2 on Thr1426, without any change in its association with tuberous sclerosis complex 1. Phosphorylation of proline-rich Akt substrate of 40-kDa (PRAS40) at Thr246 was stimulated by IGF-1. The amount of PRAS40 associated with mTORC1 decreased in response to IGF-1, and PRAS40 binding to mTORC1 was inversely related to its phosphorylation level. Overall, these results suggest that activation of the PI3K-Akt pathway by IGF-1 stimulated global protein synthesis in bovine mammary epithelial cells through changes in the phosphorylation and association state of components of the mTORC1 signaling pathway.

Rictor is a novel target of p70 S6 kinase-1

The rapamycin-insensitive companion of mammalian target of rapamycin (mTOR) (Rictor) is a key member of mTOR complex-2 (mTORC2), which phosphorylates the AGC kinases Akt/PKB, PKC and SGK1 at a C-terminal hydrophobic motif. We identified several novel sites on Rictor that are phosphorylated, including Thr1135, which is conserved across all vertebrates. Phosphorylation of this site on Rictor is stimulated by amino acids and growth factors through a rapamycin-sensitive signaling cascade. We demonstrate here that Rictor is a direct target of the ribosomal protein S6 kinase-1 (S6K1). Rictor phosphorylation at Thr1135 does not lead to major changes in mTORC2-kinase activity. However, phosphorylation of this site turns over rapidly and mediates 14-3-3 binding to Rictor and mTORC2, providing possibility for altered interactions of the complex. These findings reveal an unexpected signaling input into mTORC2, which is regulated by amino acids, growth factors and rapamycin.

Insulin resistance without elevated mammalian target of rapamycin complex 1 activity in muscles of mice fed a high-fat diet

The mammalian target of rapamycin complex 1 (mTORC1) appears to mediate the development of insulin resistance in cultured cells. We studied in vivo insulin action and mTORC1 signaling in skeletal muscles of mice fed a normal chow [control (CON)] diet or a high-fat diet (HFD) for 16 wk. We assessed in vivo insulin action by measuring glucose tolerance (GT), insulin tolerance (IT), and insulin-assisted GT (IAGT). Although GT was not altered, the HFD significantly reduced IT and IAGT. Acute treatment with rapamycin, a highly specific inhibitor of mTORC1, did not improve GT, IT, or IAGT in mice fed the CON diet or the HFD. Phosphorylation of S6 kinase (S6K) on Thr(389), a surrogate measure of mTORC1 kinase activity, was assessed in skeletal muscles of mice 15 min after an intraperitoneal injection of insulin or saline. In the basal state and after insulin stimulation, phosphorylation of S6K on Thr(389) was similar in muscles of mice fed the HFD and mice fed the CON diet, indicating that mTORC1 activity is not elevated. Furthermore, phosphorylation of insulin receptor substrate 1 on Ser(636), a site phosphorylated by mTORC1, was similar in muscles of mice fed the HFD and mice fed the CON diet. Taken together, these findings indicate that in vivo insulin resistance can occur without an increase in mTORC1 activity in skeletal muscle and that inhibition of mTORC1 with rapamycin does not improve insulin action.

Signaling Events Downstream of Mammalian Target of Rapamycin Complex 2 Are Attenuated in Cells and Tumors Deficient for the Tuberous Sclerosis Complex Tumor Suppressors

Mutations in the TSC1 and TSC2 tumor suppressor genes give rise to the neoplastic disorders tuberous sclerosis complex (TSC) and lymphangioleiomyomatosis. Their gene products form a complex that is a critical negative regulator of mammalian target of rapamycin (mTOR) complex 1 (mTORC1) and cell growth. We recently found that the TSC1-TSC2 complex promotes the activity of mTOR complex 2 (mTORC2), an upstream activator of Akt, and this occurs independent of its inhibitory effects on mTORC1. Loss of mTORC2 activity in cells lacking the TSC1-TSC2 complex, coupled with mTORC1-mediated feedback mechanisms, leads to strong attenuation of the growth factor-stimulated phosphorylation of Akt on S473. In this study, we show that both phosphatidylinositol 3-kinase-dependent and phosphatidylinositol 3-kinase-independent mTORC2 substrates are affected by loss of the TSC1-TSC2 complex in cell culture models and kidney tumors from both Tsc2(+/-) mice (adenoma) and TSC patients (angiomyolipoma). These mTORC2 targets are all members of the AGC kinase family and include Akt, protein kinase Calpha, and serum and glucocorticoid-induced protein kinase 1. We also show that the TSC1-TSC2 complex can directly stimulate the in vitro kinase activity of mTORC2. The interaction between these two complexes is mediated primarily through regions on TSC2 and a core component of mTORC2 called Rictor. Hence, loss of the TSC tumor suppressors results in elevated mTORC1 signaling and attenuated mTORC2 signaling. These findings suggest that the TSC1-TSC2 complex plays opposing roles in tumor progression, both blocking and promoting specific oncogenic pathways through its effects on mTORC1 inhibition and mTORC2 activation, respectively.

Critical Role for Hypothalamic mTOR Activity in Energy Balance

The mammalian target of rapamycin (mTOR) promotes anabolic cellular processes in response to growth factors and metabolic cues. The TSC1 and TSC2 tumor suppressors are major upstream inhibitory regulators of mTOR signaling. Mice with Rip2/Cre-mediated deletion of Tsc1 (Rip-Tsc1cKO mice) developed hyperphagia and obesity, suggesting that hypothalamic disruption (for which Rip2/Cre is well known) of Tsc1 may dysregulate feeding circuits via mTOR activation. Indeed, Rip-Tsc1cKO mice displayed increased mTOR signaling and enlarged neuron cell size in a number of hypothalamic populations, including Pomc neurons. Furthermore, Tsc1 deletion with Pomc/Cre (Pomc-Tsc1cKO mice) resulted in dysregulation of Pomc neurons and hyperphagic obesity. Treatment with the mTOR inhibitor, rapamycin, ameliorated the hyperphagia, obesity, and the altered Pomc neuronal morphology in developing or adult Pomc-Tsc1cKO mice, and cessation of treatment reinstated these phenotypes. Thus, ongoing mTOR activation in Pomc neurons blocks the catabolic function of these neurons to promote nutrient intake and increased adiposity.

Phosphatidic acid signaling to mTOR: Signals for the survival of human cancer cells

During the past decade elevated phospholipase D (PLD) activity has been reported in virtually all cancers where it has been examined. PLD catalyzes the hydrolysis of phosphatidylcholine to generate the lipid second messenger phosphatidic acid (PA). While many targets of PA signaling have been identified, the most critical target of PA in cancer cells is likely to be mTOR — the mammalian target of rapamycin. mTOR has been widely implicated in signals that suppress apoptotic programs in cancer cells — frequently referred to as survival signals. mTOR exists as two multi-component complexes known as mTORC1 and mTORC2. Recent data has revealed that PA is required for the stability of both mTORC1 and mTORC2 complexes — and therefore also required for the kinase activity of both mTORC1 and mTORC2. PA interacts with mTOR in a manner that is competitive with rapamycin, and as a consequence, elevated PLD activity confers rapamycin resistance — a point that has been largely overlooked in clinical trials involving rapamycin-based strategies. The earliest genetic changes occurring in an emerging tumor are generally ones that suppress default apoptotic programs that likely represent the first line of defense of cancer. Targeting survival signals in human cancers represents a rational anti-cancer therapeutic strategy. Therefore, understanding the signals that regulate PA levels and how PA impacts upon mTOR could be important for developing strategies to de-repress the survival signals that suppress apoptosis. This review summarizes the role of PA in regulating the mTOR-mediated signals that promote cancer cell survival.

An ATP-competitive Mammalian Target of Rapamycin Inhibitor Reveals Rapamycin-resistant Functions of mTORC1

The mammalian target of rapamycin (mTOR) kinase is the catalytic subunit of two functionally distinct complexes, mTORC1 and mTORC2, that coordinately promote cell growth, proliferation, and survival. Rapamycin is a potent allosteric mTORC1 inhibitor with clinical applications as an immunosuppressant and anti-cancer agent. Here we find that Torin1, a highly potent and selective ATP-competitive mTOR inhibitor that directly inhibits both complexes, impairs cell growth and proliferation to a far greater degree than rapamycin. Surprisingly, these effects are independent of mTORC2 inhibition and are instead because of suppression of rapamycin-resistant functions of mTORC1 that are necessary for cap-dependent translation and suppression of autophagy. These effects are at least partly mediated by mTORC1-dependent and rapamycin-resistant phosphorylation of 4E-BP1. Our findings challenge the assumption that rapamycin completely inhibits mTORC1 and indicate that direct inhibitors of mTORC1 kinase activity may be more successful than rapamycin at inhibiting tumors that depend on mTORC1.

The TSC-mTOR Pathway Mediates Translational Activation of TOP mRNAs by Insulin Largely in a Raptor- or Rictor-Independent Manner

The stimulatory effect of insulin on protein synthesis is due to its ability to activate various translation factors. We now show that insulin can increase protein synthesis capacity also by translational activation of TOP mRNAs encoding various components of the translation machinery. This translational activation involves the tuberous sclerosis complex (TSC), as the knockout of TSC1 or TSC2 rescues TOP mRNAs from translational repression in mitotically arrested cells. Similar results were obtained upon overexpression of Rheb, an immediate TSC1-TSC2 target. The role of mTOR, a downstream effector of Rheb, in translational control of TOP mRNAs has been extensively studied, albeit with conflicting results. Even though rapamycin fully blocks mTOR complex 1 (mTORC1) kinase activity, the response of TOP mRNAs to this drug varies from complete resistance to high sensitivity. Here we show that mTOR knockdown blunts the translation efficiency of TOP mRNAs in insulin-treated cells, thus unequivocally establishing a role for mTOR in this mode of regulation. However, knockout of the raptor or rictor gene has only a slight effect on the translation efficiency of these mRNAs, implying that mTOR exerts its effect on TOP mRNAs through a novel pathway with a minor, if any, contribution of the canonical mTOR complexes mTORC1 and mTORC2. This conclusion is further supported by the observation that raptor knockout renders the translation of TOP mRNAs rapamycin hypersensitive.

A complex interplay between Akt, TSC2 and the two mTOR complexes

Akt/PKB (protein kinase B) both regulates and is regulated by the TSC (tuberous sclerosis complex) 1-TSC2 complex. Downstream of PI3K (phosphoinositide 3-kinase), Akt phosphorylates TSC2 directly on multiple sites. Although the molecular mechanism is not well understood, these phosphorylation events relieve the inhibitory effects of the TSC1-TSC2 complex on Rheb and mTORC1 [mTOR (mammalian target of rapamycin) complex] 1, thereby activating mTORC1 in response to growth factors. Through negative-feedback mechanisms, mTORC1 activity inhibits growth factor stimulation of PI3K. This is particularly evident in cells and tumours lacking the TSC1-TSC2 complex, where Akt signalling is severely attenuated due, at least in part, to constitutive activation of mTORC1. An additional level of complexity in the relationship between Akt and the TSC1-TSC2 complex has recently been uncovered. The growth-factor-stimulated kinase activity of mTORC2 [also known as the mTOR-rictor (rapamycin-insensitive companion of mTOR) complex], which normally enhances Akt signalling by phosphorylating its hydrophobic motif (Ser(473)), was found to be defective in cells lacking the TSC1-TSC2 complex. This effect on mTORC2 can be separated from the inhibitory effects of the TSC1-TSC2 complex on Rheb and mTORC1. The present review discusses our current understanding of the increasingly complex functional interactions between Akt, the TSC1-TSC2 complex and mTOR, which are fundamentally important players in a large variety of human diseases.

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.

Regulation of Proline-rich Akt Substrate of 40 kDa (PRAS40) Function by Mammalian Target of Rapamycin Complex 1 (mTORC1)-mediated Phosphorylation

The rapamycin-sensitive mammalian target of rapamycin (mTOR) complex 1 (mTORC1) contains mTOR, raptor, mLST8, and PRAS40 (proline-rich Akt substrate of 40 kDa). PRAS40 functions as a negative regulator when bound to mTORC1, and it dissociates from mTORC1 in response to insulin. PRAS40 has been demonstrated to be a substrate of mTORC1, and one phosphorylation site, Ser-183, has been identified. In this study, we used two-dimensional phosphopeptide mapping in conjunction with mutational analysis to show that in addition to Ser-183, mTORC1 also phosphorylates Ser-212 and Ser-221 in PRAS40 when assayed in vitro. Mutation of all three residues to Ala markedly reduces mTORC1-mediated phosphorylation of PRAS40 in vitro. All three sites were confirmed to be phosphorylated in vivo by [(32)P]orthophosphate labeling and peptide mapping. Phosphorylation of Ser-221 and Ser-183 but not Ser-212 is sensitive to rapamycin treatment. Furthermore, we demonstrate that mutation of Ser-221 to Ala reduces the interaction with 14-3-3 to the same extent as mutation of Thr-246, the Akt/protein kinase B-phosphorylated site. We also find that mutation of Ser-221 to Ala increases the inhibitory activity of PRAS40 toward mTORC1. We propose that after mTORC1 kinase activation by upstream regulators, PRAS40 is phosphorylated directly by mTOR, thus contributing to the relief of PRAS40-mediated substrate competition.

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. We find 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.

PRAS40 and PRR5-Like Protein Are New mTOR Interactors that Regulate Apoptosis

TOR (Target of Rapamycin) is a highly conserved protein kinase and a central controller of cell growth. TOR is found in two functionally and structurally distinct multiprotein complexes termed TOR complex 1 (TORC1) and TOR complex 2 (TORC2). In the present study, we developed a two-dimensional liquid chromatography tandem mass spectrometry (2D LC-MS/MS) based proteomic strategy to identify new mammalian TOR (mTOR) binding proteins. We report the identification of Proline-rich Akt substrate (PRAS40) and the hypothetical protein Q6MZQ0/FLJ14213/CAE45978 as new mTOR binding proteins. PRAS40 binds mTORC1 via Raptor, and is an mTOR phosphorylation substrate. PRAS40 inhibits mTORC1 autophosphorylation and mTORC1 kinase activity toward eIF-4E binding protein (4E-BP) and PRAS40 itself. HeLa cells in which PRAS40 was knocked down were protected against induction of apoptosis by TNFα and cycloheximide. Rapamycin failed to mimic the pro-apoptotic effect of PRAS40, suggesting that PRAS40 mediates apoptosis independently of its inhibitory effect on mTORC1. Q6MZQ0 is structurally similar to proline rich protein 5 (PRR5) and was therefore named PRR5-Like (PRR5L). PRR5L binds specifically to mTORC2, via Rictor and/or SIN1. Unlike other mTORC2 members, PRR5L is not required for mTORC2 integrity or kinase activity, but dissociates from mTORC2 upon knock down of tuberous sclerosis complex 1 (TSC1) and TSC2. Hyperactivation of mTOR by TSC1/2 knock down enhanced apoptosis whereas PRR5L knock down reduced apoptosis. PRR5L knock down reduced apoptosis also in mTORC2 deficient cells. The above suggests that mTORC2-dissociated PRR5L may promote apoptosis when mTOR is hyperactive. Thus, PRAS40 and PRR5L are novel mTOR-associated proteins that control the balance between cell growth and cell death.

PRAS40 Regulates mTORC1 Kinase Activity by Functioning as a Direct Inhibitor of Substrate Binding

Mammalian target of rapamycin (mTOR) functions in two distinct signaling complexes, mTORC1 and mTORC2. In response to insulin and nutrients, mTORC1, consisting of mTOR, raptor (regulatory-associated protein of mTOR), and mLST8, is activated and phosphorylates eukaryotic initiation factor 4E-binding protein (4EBP) and p70 S6 kinase to promote protein synthesis and cell size. Previously we found that activation of mTOR kinase in response to insulin was associated with increased 4EBP1 binding to raptor. Here we identify prolinerich Akt substrate 40 (PRAS40) as a binding partner for mTORC1. A putative TOR signaling motif, FVMDE, is identified in PRAS40 and shown to be required for interaction with raptor. Insulin stimulation markedly decreases the level of PRAS40 bound by mTORC1. Recombinant PRAS40 inhibits mTORC1 kinase activity in vivo and in vitro, and this inhibition depends on PRAS40 association with raptor. Furthermore, decreasing PRAS40 expression by short hairpin RNA enhances 4E-BP1 binding to raptor, and recombinant PRAS40 competes with 4E-BP1 binding to raptor. We, therefore, propose that PRAS40 regulates mTORC1 kinase activity by functioning as a direct inhibitor of substrate binding.

PRAS40 Is an Insulin-Regulated Inhibitor of the mTORC1 Protein Kinase

The heterotrimeric mTORC1 protein kinase nucleates a signaling network that promotes cell growth in response to insulin and becomes constitutively active in cells missing the TSC1 or TSC2 tumor suppressors. Insulin stimulates the phosphorylation of S6K1, an mTORC1 substrate, but it is not known how mTORC1 kinase activity is regulated. We identify PRAS40 as a raptor-interacting protein that binds to mTORC1 in insulin-deprived cells and whose in vitro interaction with mTORC1 is disrupted by high salt concentrations. PRAS40 inhibits cell growth, S6K1 phosphorylation, and rheb-induced activation of the mTORC1 pathway, and in vitro it prevents the great increase in mTORC1 kinase activity induced by rheb1-GTP. Insulin stimulates Akt/PKB-mediated phosphorylation of PRAS40, which prevents its inhibition of mTORC1 in cells and in vitro. We propose that the relative strengths of the rheb- and PRAS40-mediated inputs to mTORC1 set overall pathway activity and that insulin activates mTORC1 through the coordinated regulation of both.

Activation of Mammalian Target of Rapamycin (mTOR) by Insulin Is Associated with Stimulation of 4EBP1 Binding to Dimeric mTOR Complex 1

Insulin stimulates protein synthesis by promoting phosphorylation of the eIF4E-binding protein, 4EBP1. This effect is rapamycin-sensitive and mediated by mammalian target of rapamycin (mTOR) complex 1 (mTORC1), a signaling complex containing mTOR, raptor, and mLST8. Here we demonstrate that insulin produces a stable increase in the kinase activity of mTORC1 in 3T3-L1 adipocytes. The response was associated with a marked increase in 4EBP1 binding to raptor in mTORC1, and it was abolished by disrupting the TOR signaling motif in 4EBP1. The stimulatory effects of insulin on both 4EBP1 kinase activity and binding occurred rapidly and at physiological concentrations of insulin, and both effects required an intact mTORC1. Results of experiments involving size exclusion chromatography and coimmunoprecipitation of epitope-tagged subunits provide evidence that the major insulin-responsive form is dimeric mTORC1, a structure containing two heterotrimers of mTOR, raptor, and mLST8.