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. Author manuscript; available in PMC: 2022 Oct 18.
Published in final edited form as: Methods Enzymol. 2016 Nov 9;587:405–428. doi: 10.1016/bs.mie.2016.09.068

Evaluating the mTOR Pathway in Physiological and Pharmacological Settings

S Hong *, K Inoki *,†,1
PMCID: PMC9578513  NIHMSID: NIHMS1555936  PMID: 28253969

Abstract

Mammalian/mechanistic target of rapamycin (mTOR) is an evolutionarily conserved genuine protein kinase, which phosphorylates serine/threonine in response to growth factors and nutrients. It functions as a catalytic core in two distinct multiprotein complexes: mTOR complex1 (mTORC1) and mTOR complex 2 (mTORC2). mTORC1 promotes cell growth and proliferation by positively regulating translation, transcription, and lipid biosynthesis in response to growth factors and amino acids, whereas it inhibits autophagy, an essential degradation and recycling pathway. mTORC2 regulates cell survival and cytoskeleton organization. Mechanistic insights into the function and regulation of mTOR complexes have been provided in various experimental settings and monitoring mTOR activity has been a most valuable way to judge whether levels of environmental cues such nutrients and growth factors can satisfy cellular needs for cell growth, proliferation, and autophagic response. Here, we describe useful methods to access mTOR activity in different experimental settings.

1. INTRODUCTION

Mammalian/mechanistic target of rapamycin (mTOR) is a master kinase that regulates autophagy, cell growth, proliferation, and survival in response to growth factors and nutrients such as amino acids. It forms two structurally and functionally distinct multiprotein kinase complexes named mTORC complex 1 (mTORC1) and mTORC complex 2 (mTORC2; Dibble & Cantley, 2015; Guertin & Sabatini, 2007; Wullschleger, Loewith, & Hall, 2006). While mTORC1 consists of mTOR, Raptor (regulatory-associated protein of mTOR), mLST8 (mammalian lethal with SEC Thirteen 8), PRAS40 (proline-rich Akt substrate of 40 kDa), and Deptor (DEP domain-containing mTOR-interacting protein), mTORC2 comprises of mTOR, Rictor (rapamycin-insensitive companion of mTOR), SIN1 (SAPK-interacting 1), mLST8, Protor (protein observed with rictor), and Deptor (Laplante & Sabatini, 2012). In mTORC1, Raptor functions as a scaffold for several specific mTORC1 substrates, including S6 kinase (S6K), eIF4E binding protein (4EBP), and ULK1 (Unc-51-like kinase 1), as well as for tethering mTORC1 to the endosomal membrane for its activation (Hara et al., 2002; Kim et al., 2002; Sancak et al., 2008). In contrast, PRAS40 and Deptor negatively regulate the activity of mTORC1 (Peterson et al., 2009; Sancak et al., 2007; Vander Haar, Lee, Bandhakavi, Griffin, & Kim, 2007). In mTORC2, Rictor, SIN1, and mLST8 play an essential role in the activity of mTORC2 to phosphorylate its substrates, including Akt, PKCα, and SGK1 (Jacinto et al., 2006; Su & Jacinto, 2011; Yang, Inoki, Ikenoue, & Guan, 2006).

Bacteria-produced rapamycin is a macrolide and its pharmaceutical derivatives are drugs approved by the FDA for organ transplantation, coronary artery stenosis, and several types of cancer (Cargnello, Tcherkezian, & Roux, 2015; Geissler, 2015). mTORC1 is defined as the rapamycin-sensitive complex, whereas mTORC2 is insensitive. Rapamycin forms a complex with FKBP12 to interact with the FKBP12–rapamycin-binding (FRB) domain of mTOR kinase in mTORC1 and allosterically suppresses the mTOR kinase activity by blocking the accessibility of substrates to the active site of mTOR kinase and ultimately disrupts the formation of mTORC1(Brown et al., 1994; Hara et al., 2002; Kim et al., 2002). It has been demonstrated that the FRB domain of mTOR in mTORC2 is hindered by Rictor once mTORC2 is established (Gaubitz et al., 2015). However, prolonged rapamycin treatment often decreases the expression of mTORC2 and inhibits its functions (Sarbassov et al., 2006). Thus, it is likely that the rapamycin–FKBP12 complex may gain access to newly synthesized mTOR and prevent mTOR from forming mTORC2. Recently, several specific mTOR kinase inhibitors have been synthesized and now are commercially available. These second-generation mTOR inhibitors function as an ATP-competitive inhibitor for mTOR kinase that potently inhibits the kinase activity of both mTORC1 and mTORC2 (Feldman et al., 2009; Hsieh et al., 2012; Thoreen et al., 2009).

Two important environmental cues have long been studied in the regulation of mTORC1 activation: growth factors such as insulin and nutrients such as amino acids. The activity of mTORC1 is stimulated by growth factors and nutrients through two distinct Ras-related small guanosine triphosphatases (GTPases): monomeric ras homolog enriched in the brain (Rheb) and the heterodimeric Rag complex, respectively, on the lysosomal membrane (Dibble & Manning, 2013; Jewell, Russell, & Guan, 2013; Sancak et al., 2010).

Growth factors activate Rheb by inhibiting the trimeric TSC1/TSC2/TBC1D7 complex (hereafter called the TSC complex), a well-known tumor suppressor and the specific GTPase-activating protein (GAP) for Rheb, through inhibitory phosphorylation of TSC2 by Akt (Dibble et al., 2012; Garami et al., 2003; Inoki, Li, Xu, & Guan, 2003; Inoki, Li, Zhu, Wu, & Guan, 2002; Manning, Tee, Logsdon, Blenis, & Cantley, 2002; Potter, Pedraza, & Xu, 2002; Zhang et al., 2003). Akt-dependent TSC2 phosphorylation induces the dissociation of the TSC complex from the lysosomal membrane, thereby maintaining lysosomal active Rheb, which directly activates mTORC1 (Demetriades, Doumpas, & Teleman, 2014; Menon et al., 2014).

Amino acids, especially leucine and arginine, activate mTORC1 through the activation of Rag GTPases (Kim, Goraksha-Hicks, Li, Neufeld, & Guan, 2008; Sancak et al., 2008). There are four mammalian Rag proteins, which form obligate heterodimers. RagA and RagB are functionally redundant and form heterodimeric complexes with either RagC or RagD (Nakashima, Noguchi, & Nishimoto, 1999; Sekiguchi, Hirose, Nakashima, Ii, & Nishimoto, 2001). Intriguingly, when Rag complexes are active, RagA and RagB are GTP-bound forms, whereas RagC and RagD are GDP-bound forms (Kim et al., 2008; Sancak et al., 2008). Another unique feature of Rags is their lack of a lipid moiety, even though they reside on lysosomes. The lysosomal expression of Rags is dependent on the lysosome-anchored Ragulator (Bar-Peled, Schweitzer, Zoncu, & Sabatini, 2012; Sancak et al., 2010). The Ragulator is a pentameric protein complex consisting of five subunits, p18 (LAMTOR1), p14, MP1, HBXIP, and C7orf59. Importantly, Ragulator functions as not only a scaffold but also a guanine nucleotide exchange factor (GEF) for RagA and RagB (Bar-Peled et al., 2012). In response to amino acids, Ragulator is activated through v-ATPase on the lysosomal membrane, thereby stimulating the activity of Rags. In contrast, GATOR1, a trimetric protein complex consisting of DEPDC5, NPRL2, and NPRL3, functions as a GAP for both RagA and RagB. GATOR1 is inhibited by another pentameric protein complex, GATOR2, which interacts with Sestrins and CASTORs (Bar-Peled et al., 2013; Chantranupong et al., 2014, 2016; Kim et al., 2015; Parmigiani et al., 2014). Importantly, recent studies have revealed that Sestrin2 and CASTOR1 directly interact with leucine and arginine, respectively (Chantranupong et al., 2016; Wolfson et al., 2016). Both leucinebinding to Sestrin2 and arginine-binding to CASTOR1 are required for leucine and arginine to activate mTORC1 through GATOR2 activation. Although it has been proposed that lysosomal v-ATPase transmits luminal amino acid signal to the Rag complexes through Ragulator (Wolfson et al., 2016), newly identified cytosolic amino acid sensors such as Sestrins and CASTORs are not expressed on the lysosomal membrane (Chantranupong et al., 2016; Wolfson et al., 2016). Therefore, these observations suggest that essential amino acids are sensed at the lysosome and cytosol for mTORC1 activation.

In addition to the role of amino acids in recruiting mTORC1 to lysosomal membranes, recent studies have revealed other roles of amino acids in the regulation of the TSC complex. Under amino acid starvation conditions, TSC2 interacts with the inactive form of RagA on lysosomes, and this interaction is required for complete inactivation of mTORC1 upon amino acid starvation (Demetriades et al., 2014). Furthermore, amino acids, especially arginine, disrupt the interaction between TSC2 and Rheb, which enhances the accessibility of mTORC1 to active Rheb on the lyososomal membrane (Carroll et al., 2016). Presumably, this is why the activity of mTORC1 is relatively insensitive to amino acid depletion in cells lacking a functional TSC complex.

Compared to mTORC1, molecular mechanisms of mTORC2 activation have not been clearly shown, although a recent study proposed that ribosomes are required for mTORC2 activation (Zinzalla, Stracka, Oppliger, & Hall, 2011). mTORC2 interacts with ribosomal proteins (including RpL26) in a manner dependent on the activity of PI3K, and reduction of ribosomal proteins mitigates cellular mTORC2 activity.

Given that mTOR, especially mTORC1, plays a critical role in suppressing the induction of autophagy, monitoring cellular mTOR activity is a valuable tool to determine the status of cellular autophagic activity. Here, we summarize established methods for monitoring the activity of mTOR, its subcellular localization, and the activity of Rheb to determine cellular mTOR activity.

2. FUNCTIONAL READOUTS AND INHIBITORS FOR THE mTOR PATHWAY

There are a plethora of substrates that have been shown to be phosphorylated by the mTOR complexes. These substrates include S6K1, 4EBP1, PRAS40, and ULK1 (UNC-51-like kinase 1) as mTORC1 substrates, and serum- and glucocorticoid-induced kinase 1 (SGK1) and Akt (Laplante & Sabatini, 2012). For monitoring the activity of mTORC1 in vivo and in vitro, levels of S6K1 phosphorylation on Thr389 (hydrophobic motif ) and 4EBP1 phosphorylation on Thr37/Thr46 and Ser65 have been widely used. These substrates are known to play essential roles in mTORC1-dependent mRNA translation (Fig. 1) (Moschetta, Reale, Marasco, Vacca, & Carratu, 2014). In addition, ULK1 phosphorylation on Ser757 can be monitored to determine cellular mTORC1 activity in the regulation of autophagy (Kim, Kundu, Viollet, & Guan, 2011). Along with the abovementioned biochemical approaches, monitoring lysosomal localization of mTOR has begun to be accepted as a new biological method for mTORC1 activation.

Fig. 1.

Fig. 1

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mTORC1 and mTORC2 and mTOR pathway inhibitors. Schematic illustration shows the key components of mTORC1 and mTORC2, essential cellular cues activating these complexes, inhibitors for the mTOR pathway; rapamycin for mTORC1, Torin1, PP242, and INK128 for mTORC1 and mTORC2, PF-470861 for S6K1, and MK-2206 for Akt. Major phosphorylation sites of mTORC1 and mTORC2 substrates are indicated.

Akt phosphorylation on Ser473 (hydrophobic motif ) has been widely used for monitoring mTORC2 activity both in vivo and in vitro (Fig. 1) (Sarbassov, Guertin, Ali, & Sabatini, 2005).

To assess functions of mTOR in a variety of experimental settings, two types of mTOR inhibitors have been well used; allosteric inhibitors such as rapalogs, and ATP-competitive inhibitors, including Torin1, PP242, and INK128. Since ATP-competitive inhibitors directly inhibit the kinase activity of mTOR, a catalytic core in both mTORC1 and mTORC2, these inhibitors completely block the activity of both mTORC1 and mTORC2 (Figs. 1 and 3). While rapamycin and its derivatives, rapalogs are well-known mTORC1 inhibitors, and the effect of these allosteric inhibitors on mTORC1 inhibition is widely acknowledged, they do not completely suppress the phosphorylation of some mTORC1 substrates, such as 4EBP1 and ULK1. In addition, S6K1 and Akt, which are downstream kinases and substrates of mTORC1 and mTORC2, respectively, can be inhibited by PF-4708671 (S6K1 inhibitor) and MK-2206 (Akt inhibitor) (Figs. 1 and 3).

Fig. 3.

Fig. 3

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Pharmacological and physiological inhibition of mTOR and downstream effector kinases. Regulation of the mTOR pathway by amino acids and growth factors. HEK293T cells were starved in HBSS (with calcium and magnesium) for 60 min, followed by amino acids and serum stimulation for another 30 min in the absence or presence of various inhibitors [rapamycin (mTORC1 inhibitor) 100 nM, Torin 1 (mTORC1 and mTORC2 inhibitor) 250 nM, MK2206 (Akt inhibitor) 2 μM, or PF-4708671 (S6K1 inhibitor) 20 μM]. The indicated proteins were analyzed in Western blotting using the indicated antibodies. α, β, and γ denote non/hypo-, less-, and hyperphosphorylated forms of 4E-BP1, respectively.

mTORC1 receives at least two essential signals from growth factors and amino acids for its activation. These two signals impinge on the lysosomal membrane for mTORC1 activation through Rheb and Rag small GTPases. Thus, monitoring the active status of these two small GTPases provides important information for the molecular mechanisms by which mTORC1 regulators stimulate mTORC1 activity (Fig. 2).

Fig. 2.

Fig. 2

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Diagnostic posttranslational modifications in the mTORC1 pathway. Diagnostic phosphorylation events and critical regulators such as small GTPases and their GAPs and GEF are shown. The phosphorylation sites depicted in red have a positive role, whereas those in green play a negative role for the phosphorylated proteins.

3. METHODS

3.1. Cell Culture and Treatments

The signal transductions from growth factors such as insulin and amino acids to mTOR are well conserved in mammalian cells. Representative mammalian cells widely used in the research of mTOR signaling include HEK293T (human embryonic kidney 293T), MEFs (mouse embryonic fibroblasts), and some cancer cell lines such as HeLa cells. These cells can be cultured in standard culture media such as DMEM (Dulbecco’s modified eagle medium) with fetal bovine serum (FBS). However, it is noteworthy that the mTOR pathway in HEK293T and some cancer cells is less sensitive to growth factor stimulation or depletion, in part due to a lack of the activity of phosphatase and tensin homolog (PTEN), a key lipid phosphatase that removes the phosphate in the D3 position of inositol rings from a variety of phophstidylinositols. In addition, HeLa cells that lack the expression of serine/threonine-protein kinase STK11 (LKB1), a master kinase for the T-loop of AMPK family of proteins, show less sensitive to glucose stimulation or depletion in the regulation of the mTOR pathway (Lizcano et al., 2004; Shaw et al., 2004).

In order to inhibit mTOR activity by suppressing upstream inputs, cells need to be starved with growth factor, amino acids, or both by culturing growth factor-free DMEM, PBS containing dialyzed FBS, and PBS containing calcium, magnesium, and glucose (DPBS, ThermoFisher, cat# 14040216), respectively. By depleting growth factors in medium, the activity of both mTORC1 and mTORC2 is inhibited. By depleting amino acids in medium, the activity of mTORC1 but not mTORC2 is greatly inhibited. Media lacking a specific amino acid such as leucine or glutamine are also commercially available. To ensure complete removal of growth factors or amino acids, cells should be washed at least once with growth factor- or amino acid-free media before culturing with the starvation media. To inhibit mTORC1 activity by suppressing Akt activity, specific and potent pan-Akt inhibitors such as MK-2206 (IC50 = 8/12/65 nM for Akt1/2/3, respectively), AZD5363 (IC50 = 3/8/8 nM), and GSK690693 (IC50 = 2/13/9) are commercially available.

In order to inhibit mTORC1 activity directly, cells are treated with rapamycin (sirolimus) or RAD001 (everolimus). To inhibit both mTORC1 and mTORC2, ATP-competitive inhibitors such as Torin 1 (IC50 = 2–10 nM, 1000-fold selectivity for mTOR than PI3K), KU-006379 (IC50 = ~10 nM) AZD8055 (IC50 = ~1 nM, 1000-fold selectivity), INK128 (IC50 = 1 nM, 200-fold selectivity), and Torkinib (PP242) (IC50 = 8 nM, 10- to 100-fold selectivity) can be used (Figs. 1 and 3).

3.2. Transfection

Treatments with physiological cues including mitogens, growth factors, and nutrients, or pharmacological compounds such as inhibitors generally produce their effects in all of cultured cells. Therefore, it is able to examine the mTOR signaling by analyzing endogenous proteins. However, in order to determine the role of an exogenous protein in the regulation of mTORC1 or mTORC2, coexpression of the exogenous protein with mTORC1 substrate (e.g., S6K1) or mTORC2 substrate (i.e., Akt) as a reporter helps to analyze its effects on the mTOR pathway in cells that have low transfection efficiency.

Liposome-mediated transfection is a common and efficient method for introducing negatively charged nucleic acid molecules, including cDNA and RNA. Lipofectamine (Invitrogen) and fugene (Promega) are two representative commercially available transfection reagents with less cytotoxicity and high efficiency of cDNA transfection. However, due to the cost of these transfection reagents, calcium phosphate transfection is a more attractive method, especially for large-scale transfections such as shRNA-expressing virus production. In addition, PEI (polyethylenamine) can be used as a transfection reagent.

3.2.1. Calcium Phosphate Transfection

All the solutions should be warmed at room temperature before transfection.

  1. Grow cells to 70% confluent in 10-cm plates: less confluent cells can die due to cytotoxicity of transfection mixtures.

  2. Change with fresh growth media 3 h before transfection.

  3. Add 10–50 μg of DNA into a 50-mL disposable tube and add autoclaved water to 1095 μL.

  4. Add 155 μL of 0.22-μm-filtered 2 M calcium chloride (stored at 4°C) and mix by gentle swirling.

  5. Add 1250 μL of 2 × HBS dropwise within 1 min to evenly form calcium phosphate particles: 2 × HBS: For 500 mL, 8 g NaCl, 0.2 g Na2HPO4, 6.5 g HEPES, pH 7.0 stored at −80°C.

  6. After 12 h, remove the media, wash once with media to remove calcium phosphates and add fresh growing media.

  7. Cells can be harvested and expression can be assessed 36–48 h after transfection.

3.2.2. Transfection Using Liposome (Lipofectamine)

  1. Grow cells to 60% confluent in one well of a six-well plate.

  2. Before transfection, wash and change media with 0.85 mL of Opti-MEM (ThermoFisher, cat# 31985062).

  3. In a microcentrifuge tube, add 150 μL of Opti-MEM and DNA constructs.

  4. Mix by gentle vortex.

  5. Add 5 μL of lipofectamine into the same tube. (This step is different from the manufacturer’s instructions.)

  6. Mix by gentle vortex and incubate at room temperature for 30 min.

  7. Add the transfection mixture from step 6 dropwise and do not disturb attached cells.

  8. Incubate cells in the CO2-humidified incubator for 4–12 h.

  9. Change with growing media.

  10. Cells can be harvested and levels of an exogenous protein can be assessed 36–48 h after transfection.

3.2.3. Transfection Using PEI

  1. Grow cells to reach 70–90% confluent in a 10-cm plate.

  2. Change media with 10 mL of serum-free DMEM (without antibiotics and FBS).

  3. In a microcentrifuge tube, add 800 μL of serum-free DMEM, and DNA constructs (less than 50 μg) and mix by vortex.

  4. In another microcentrifuge tube, add 800 μL of serum-free DMEM and 60 μL of PEI (DNA: PEI ratio is 1:3) and vortex; 2 mg/mL PEI pH 7.0 stored at −80°C.

  5. Mix the abovementioned two tubes and incubate at room temperature for 20 min.

  6. Add the PEI transfection mixture dropwise and incubate for 6–8 h.

  7. Change media with growing media.

  8. Cells can be harvested and levels of an exogenous protein expression can be assessed 36–48 h after transfection.

3.3. Protein Extraction for Western Blotting

Since mTORC1 senses intracellular nutrient levels, we recommend that cells be lysed promptly with lysis buffer without washing with nutrient-free solution such as PBS buffer.

  1. For six-well plates, remove media completely by aspiration and immediately add 300 μL of cold NP-40 lysis buffer (10 mM Tris–Cl pH 7.5, 2 mM EDTA, 150 mM NaCl, 1% NP-40, 10 mM pyrophosphate, 10 mM glycerophosphate, 50 mM NaF, 1.5 mM Na3VO4, 1% Triton X-100, and EDTA-free protease inhibitors [Roche]). For a 10-cm plate, add 1 mL of lysis buffer.

  2. Incubate on ice for 15 min with occasional tapping.

  3. Transfer suspension into 1.5-mL microcentrifuge tubes.

  4. Spin at maximum rpm for 15 min at 4°C.

  5. Transfer 150 μL of supernatant into a new tube.

  6. Add 50 μL of 4 × SDS sample buffer (200 mM Tris–Cl pH 6.8, 8% SDS, 40% glycerol, 20% β-mercaptoethanol, and 0.4% bromophenol blue) and vortex briefly.

  7. Denature proteins in a 95°C heating block for 5 min.

  8. Keep the samples at room temperature for 10 min.

  9. Use 10 μL of each sample to apply to a well of SDS-PAGE, followed by Western blot analysis. Samples can be stored at −20°C for several years.

3.4. Assessing Cellular mTOR Activity Using Phosphospecific Antibodies

Since mTORC1 is sensitive to nutrients and growth factors (Figs. 1 and 3), washing cells with buffers without them is not desirable, which may lower the activity of mTORC1 during washing or harvesting. If it is necessary to remove unwanted components in media from cell surfaces, cells should be rinsed quickly. As described earlier, the starvation of nutrients such as amino acids inhibits the activity of mTORC1 within 30 min in most cells. However, the inhibition of mTORC1 and mTORC2 caused by growth factor starvation varies among cells. For instance, the activity of mTORC1 and mTORC2 can be inhibited by serum depletion in MEFs within 60 min, whereas it takes much longer in HEK293T or certain cancer cells with diminished PTEN or TSC2 activity. Both the mTORC1 and mTORC2 activity suppressed by nutrient- and growth factor-starvation can be regained within 15 min by the replenishment of nutrients and growth factors (Fig. 3).

To determine the activity of mTORC1 in vivo, Western blot analysis with phosphospecific antibodies against mTORC1 substrates is the most accurate and straightforward method. In addition, monitoring mobility shift caused by protein phosphorylation in SDS-PAGE is an alternative way when the phosphospecific antibody is not available. However, the appropriate percentage of separating gel in SDS-PAGE needs to be empirically determined to obtain a clear mobility shift of target proteins by phosphorylation. For example, to detect the mobility shift of S6K1 or 4E-BP1, generally 8% or 13% SDS-PAGE, respectively, is considered as the ideal setting for obtaining clear mobility shift of these proteins. 4EBP1 is a representative protein that can be phosphorylated as multiple residues and detected as three major bands (α, β, and γ form) in Western blotting. The α, β, and γ 4EBP1 correspond to non/hypo-, less-, and hyperphosphorylated form, respectively (Fig. 3).

To monitor cellular mTORC2 activity, levels of Akt phosphorylation on serine 473 (hydrophobic site) can be determined by Western blotting with phospho-Ser473 Akt antibody. In this section, we describe methods to monitor the activity of mTORC1.

  1. Cells grow 70% confluent in a 10-cm plate.

  2. Wash with HBSS with calcium and magnesium twice.

  3. Add 10 mL of HBSS with calcium and magnesium and incubate in the humidified CO2 incubator for 1 h to inhibit mTORC1 activity completely.

  4. Remove HBSS by aspiration and add 10 mL of DMEM media containing amino acids and 10% FBS for maximum activation of mTORC1. For only amino acid stimulation, add DMEM without FBS.

  5. Incubate for additional 15–30 min.

  6. Extract proteins as mentioned earlier: For the Western blotting, NP-40 lysis buffer is preferred.

  7. In Western blotting, levels of phosphorylation of S6K1 and its total protein can be determined using phosphospecific S6K1 (phospho-T389) and S6K1 antibody, respectively. Levels of S6 phosphorylation and its total protein can also be monitored for determining cellular S6K activity using phospho-S6 (phospho-S240/244) and S6 antibodies. Note that Ser235/236 phosphorylation of S6 can be induced by not only mTORC1-S6K pathway, but also other kinases, including RSK. The activity of mTORC1 or S6K1 can be determined by the ratio of pS6K1/S6K or pS6/S6, respectively.

3.5. Accessing Levels of mTOR Complexes and Their Activities by Western Blotting

Although Western blotting is one of the most widely used techniques, the task of obtaining a clear band of high-molecular-weight proteins such as mTOR (288 kDa) and the components of mTOR complexes by Western blotting needs some extra effort. To detect mTOR, Raptor, and Rictor, approximately 8% SDS-PAGE can be used.

  1. During SDS-PAGE, briefly rinse a PVDF membrane with water.

  2. Activate a membrane with MeOH until it becomes transparent: this takes a couple of seconds.

  3. Remove MeOH and incubate with transfer buffer. The transfer buffer can be prepared a day before the experiment; keep it at 4°C to enhance transfer efficiency (for 4 L, Tris 12.1 g, glycine 57.6 g, MeOH 800 mL).

  4. Incubate the membrane in transfer buffer for at least 5 min at room temperature by shaking.

  5. Transfer proteins to the membrane at 4°C for 180 min at fixed 350 mA for mTOR, Raptor, S6K1, Rictor, and Akt in a 8% SDS-PAGE gel, and for 120 min at the same mA for S6 and 4EBP1 in a 13% SDS-PAGE gel.

  6. After transfer, briefly wash the membrane three times with TBST.

  7. Block the membrane with blocking buffer (5% nonfat dry milk in TBST) for 20–60 min.

  8. Wash the membrane as in step 6 to remove excess milk from the membrane.

  9. Incubate the membrane in antibody solution overnight at 4°C with gentle rocking: primary antibodies are diluted in 10 mL of TBST containing 5% BSA and 0.02% sodium azide.

  10. The next day, collect primary antibodies in polystyrene tubes to recycle these antibodies and keep them at −20°C.

  11. Wash the membrane three times with TBST for 30 min.

  12. Incubate the membrane with secondary antibodies conjugated with HRP (1:5000) in blocking solution and incubate for 2 h at room temperature with rocking.

  13. Wash the membrane with TBST four times for 40 min (longer than 40 min washing is also acceptable).

  14. Visualize target protein bands using ECL mixture onto X-ray film.

3.6. Membrane Stripping and Reprobing Membrane

  1. After visualizing proteins by ECL reagents, recover the membrane and wash with water several times to remove TBST and ECL: if membrane is dried, activate with MeOH and wash with water.

  2. Wash the membrane with stripping buffer (25 mM glycine, 1% SDS, pH 2) for 40 min (each 10 min × four times).

  3. Briefly wash the membrane with water several times to remove any trace of SDS in the stripping buffer and wash three times with TBST for 30 min.

  4. Repeat Western blotting from the blocking stage to probe other proteins.

3.7. Coimmunoprecipitation of mTORC1 and mTORC2

mTOR immunoprecipitation using mTOR antibodies can pull-down all the key components of mTORC1 and mTORC2, since mTOR is a common component in both mTORC1 and mTORC2. If specific isolation of mTORC1 or mTORC2 is desired, immunoprecipitation of Raptor for mTORC1 or Rictor for mTORC2 is necessary. For the coimmunoprecipitation (co-IP) of mTOR complexes, CHAPS lysis buffer must be used because other nonionic detergents such as NP-40 and TX-100 disrupt the integrity of these complexes.

  1. Grow cells 70% confluent in 10-cm plates.

  2. Remove media completely by aspiration and add 1 mL of CHAPS lysis buffer [40 mM HEPES pH 7.5, 120 mM NaCl, 1 mM EDTA, 0.6% CHAPS 10 mM pyrophosphate, 10 mM glycerophosphate, 50 mM NaF, 1.5 mM Na3VO4, 1% Triton X-100, and EDTA-free protease inhibitors (Roche)] immediately.

  3. Incubate on ice for 15 min with occasional tapping.

  4. Collect suspension and spin at maximum rpm for 15 min at 4°C.

  5. Transfer 800 μL for immunoprecipitation and 150 μL for input (lysate).

  6. Add 1–2 μg of mTOR antibodies (Santa Cruz, cat# SC1549; Bethyl laboratory, cat# A300/A301) into 800 μL of extract and incubate for 2–3 h at 4°C with gentle rocking.

  7. Add 20 μL of protein G sepharose beads (50% slurry in CHAPS lysis buffer, GE health care, cat# 17–068-01) and incubate for another hour.

  8. Wash five times with CHAPS lysis buffer.

  9. Remove the lysis buffer completely and denature immunoprecipitated proteins with 50 μL of 1 × SDS sample buffer for 5 min at 95°C.

  10. After 10 min of incubation at room temperature, spin samples for 10 s and analyze those in 8% or lower SDS-PAGE, followed by Western blotting.

  11. In Western blotting, co-IPed mTORC1 and mTORC2 components can be detected by using specific antibodies (Cell Signaling Technology), such as mTOR (cat# 2983), Raptor (cat# 2280), mLST8 (cat# 3274), and Rictor (cat# 9476). All these antibodies are diluted 1/1000 in 5% BSA TBST and stored at −20°C. Frozen diluted antibodies are thawed at room temperature before use and incubated at 4°C for overnight.

3.8. In Vitro Kinase Assay

In order to measure the kinase activity of mTORC1 or mTORC2 directly, an in vitro kinase (IVK) assay can be performed using S6K1 or 4EBP1 as a substrate for mTORC1, and Akt for mTORC2 kinase assay.

3.8.1. Preparation of GST-S6K1 from Mammalian Cells

  1. Grow HEK293T cells 60% confluent in 5 × 15-cm plates.

  2. Transfect with 20 μg of mammalian expression GST-S6K1 using the calcium phosphate method.

  3. Next, 48 h after transfection, starve cells with HBSS for 2 h or treat cells with 250 nM of torin1 or 100 nM of rapamycin for 1 h to completely dephosphorylate GST-S6K1 within the cells.

  4. Rinse cells with ice-cold PBS one time

  5. Lyse cells with PBST buffer (PBS with 0.3% Tween-20, 1 mL per plate) with protease inhibitors.

  6. Incubate on ice for 15 min.

  7. Collect suspension into microcentrifuge tubes.

  8. Spin at 4°C for 15 min at maximum rpm.

  9. Transfer the supernatant into a new 15-mL tube.

  10. Add 50 μL of PBST-washed 50% slurry of glutathione sepharose beads and rock it at 4°C for 4 h.

  11. Wash three times with HNTG buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 10% glycerol, 0.1% Triton-X100).

  12. Wash twice with cold PBS.

  13. Elute with 10 mg/mL of GSH solution (reduced glutathione in 100 mM Tris pH 8) for 4 h.

  14. Dialyze the eluent with cold dialysis buffer (10 mM Tris pH 7.5, 50 mM NaCl, 0.5 mM EDTA, and 0.05% beta-mercaptoethanol).

  15. Determine the concentration by SDS-PAGE and snap-freeze in liquid nitrogen and store at −80°C until use.

Note that GST-4EBP1 or GST-Akt can be prepared using the same method. For GST-Akt purification, cells transfected with GST-Akt should be treated with mTOR kinase inhibitors such as Torin1 before harvesting. Low-molecular-weight substrates such as GST-4EBP1 can also be prepared from bacteria using a bacterial GST expression vector.

3.8.2. mTOR in vitro Kinase Assay Using Phosphospecific Antibody

For kinase assays, mTORC1 can be immunoprecipitated by Raptor antibodies, while mTORC2 is immunoprecipitated by Rictor antibodies. For in vitro kinase assays using exogenous mTORC1 and mTORC2, each component of the complexes needs to be transfected.

  1. Grow cells (e.g., HEK293T, MEF) at 80% confluency in 10-cm plates.

  2. Remove media and briefly wash cells with cold PBS.

  3. Lyse cells in CHAPS lysis buffer containing protease inhibitors and phosphatase inhibitors (1 mL per 10-cm plate) on ice for 15 min.

  4. Collect lysates and spin at 4°C for 15 min at maximum rpm.

  5. Transfer supernatant into a new tube.

  6. Add 1 μg of Raptor or Rictor antibodies and rock it for 3 h at 4°C.

  7. Add 20 μL of 50% slurry of protein G sepharose beads in CHAPS buffer.

  8. Rock it for 1 h at 4°C.

  9. Wash three times with CHAPS lysis buffer and wash once with HEPES washing buffer (25 mM HEPES–KOH, 20 mM KCl, pH 7.4).

  10. Wash once with 1 × kinase reaction buffer without ATP (20 mM Tris–HCl pH 7.5, 10 mM MgCl2).

  11. Add 25 μL of kinase reaction mixture: 5 μL of 5 × kinase reaction buffer, 120 ng of GST-S6K1 for mTORC1 and GST-AKT for mTORC2, 200 μM ATP.

  12. Incubate at 37°C for 20 min with gentle rocking.

  13. Terminate the reaction by adding 10 μL of 4 × SDS sample loading buffer and denature the samples at 95°C for 5 min.

  14. Analyze the samples by Western blotting.

For mTORC1 kinase assays, levels of GST-S6K1 phosphorylation can be detected by using phospho-Thr389 S6K1 antibodies. Similarly, phospho-Ser473 Akt antibodies can be used for mTORC2-dependent GST-Akt phosphorylation.

3.9. mTOR Immunofluorescence Staining

Lysosomal localization of mTORC1 is necessary for its activation and stimulated in a manner dependent on Rag GTPases activity. Thus, monitoring cellular mTORC1 localization can be used for an indirect measurement of Rag and mTORC1 activity. To determine mTOR localization on lysosomes, cells need to be starved with amino acid-free media such as HBSS or DPBS (with calcium and magnesium) for 50 min to dissipate mTORC1 from lysosomal membranes. Replenishment of amino acids (DMEM without FBS) sufficiently induces lysosomal mTOR localization within 5 min in MEF cells.

  1. Grow cells on a round-cover slide in a 12-well plate.

  2. Wash with PBS once to remove media.

  3. Fix cells with 1 mL of warmed PBS containing 4% paraformaldehyde at 37°C for 5 min.

  4. Wash with PBS twice and permeabilize with 1 mL of permeabilizing buffer (PBS containing 0.05% Triton X-100) at room temperature for 5 min.

  5. Incubate cells with PBS containing 0.25% BSA at room temperature for 1 h for blocking.

  6. Add mTOR antibody (Cell Signaling Technology, cat# 2983, 1:100 dilution) into blocking solution and incubate at room temperature for 1 h or at 4°C for 16 h.

  7. Wash four times with PBS.

  8. Incubate with fluorescence-conjugated secondary antibodies (1:1000 dilution) at room temperature for 30 min in the dark.

  9. Wash four times with PBS and once with water.

  10. Mount slides and keep in a slide box at room temperature until analysis using a microscope.

3.10. GTP Loading Assay of Small GTPase

To investigate events upstream of mTORC1, it is important to measure the activity of Rheb or Rag to dissect the molecular mechanism underlying mTORC1 activation (Fig. 4). The activity of these small GTPases can be assessed by determining amounts GTP and GDP that bind to Rheb or Rag in vivo. Generally, an increased ratio of GTP/GDP indicates the activation of small GTPases. This assay can be done for both endogenous and exogenous small GTPases. Next, we introduce the assay for measuring the activity of exogenous Rheb (myc-Rheb) and discuss appropriate approaches to measure the activity of Rag small GTPases.

Fig. 4.

Fig. 4

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GTP loading assay for Rheb GTPase. HEK293T cells were transfected with the indicated cDNA constructs. 48 h after transfection, cells were labeled with radioactive 32P phosphate, and immunoprecipitated myc-Rheb was analyzed in the GTP loading assay. Rheb-bound radioactive GTP and GDP were visualized by a PhosphoImager and the amount of Rheb-bound GTP and GDP were quantified by an ImageQuant. The data were expressed as a ratio (GTP/GDP) (right panel). Coexpression of HA-TSC2 with myc-Rheb largely stimulated GTP hydrolysis of Rheb.

3.10.1. Accessing Guanine Nucleotide Loading Status on Rheb In Vivo

  1. On a six-well plate, transfect 50 ng of myc-Rheb construct using lipofectamine, as previously described.

  2. Wash cells once with phosphate-free DMEM (ThermoFisher, cat# 11971–025).

  3. Incubate cells with 0.8 mL/well of phosphate-free DMEM at the humidified CO2 incubator for 60 min.

  4. During the incubation, prepare a labeling master mix: 245 μL of phosphate-free DMEM, 105 μL of 32P orthophosphate (5 mCi/mL) per well of a six-well plate.

  5. Add 50 μL of the mixture per well.

  6. Incubate for 4 h.

  7. Prepare antibody-protein G sepharose bead conjugates: Add 1–2 μg of myc antibodies into 10 μL of the beads (50% slurry in the lysis buffer) per well and rock it at 4°C for 2 h.

  8. Remove labeling media and lyse the cells with 250 μL of lysis buffer (50 mM Tris pH 7.5, 0.5% NP-40, 100 mM NaCl, 10 mM MgCl2, 1 mM DTT, and protease inhibitors) per well.

  9. Gently rock the plate on ice for 30 s and transfer the lysate into a tube.

  10. Centrifuge for 15 min with maximal rpm at 4°C.

  11. Take 200 μL of supernatant and add 10 μL of the antibody-protein G bead conjugates and NaCl to a final concentration of 0.5 M to block any GAP activity in the immunoprecipitants.

  12. Rock the tube for 2 h at 4°C.

  13. Wash three times with washing buffer I (50 mM Tris–Cl pH 8.0, 500 mM MgCl2, 500 mM NaCl, 1 mM DTT, 0.5% TX-100).

  14. Wash three times with washing buffer II (50 mM Tris–Cl pH 8.0, 100 mM NaCl, 5 mM MgCl2, 1 mM DTT, 0.1% TX-100).

  15. Add 20 μL of elution buffer (2 mM EDTA, 0.2% SDS, 1 mM GDP, 1 mM GTP) and rock it at 68°C for 10 min.

  16. Spin shortly and recover supernatants for analysis.

  17. Apply 10 μL of each sample onto a PEI cellulose plate (approximately 3 cm above the bottom of the plate) and dry it completely with a regular hairdryer.

  18. Soak the plate in MeOH and dry it.

  19. Immerse the bottom portion of the plate (below where the samples are loaded) with MeOH.

  20. Stand the plate in the TLC chamber that is filled to a depth of 1 cm with TLC running buffer (1 M LiCl, 1 M formic acid).

  21. Close the chamber lid to keep humidified in the chamber and remove the plate from the chamber when the solvent ascends to the top of the plate.

  22. Dry the plate with a hairdryer.

  23. Expose the TLC plate on a PhosphorImager screen for 6 h and read the radioactive GTP and GDP in a PhosphorImager.

  24. Determine the amount of radioactive GTP and GDP by the using Imagequant software and calculate GTP/GDP ratio using the formula. GTP moles (=GTP signal/3), GDP moles (=GDP signal/2). Note that overexpression of Rheb sufficiently activates mTORC1 in HEK293T cells, indicating that excess expression of Rheb overcomes the activity of endogenous TSC2, a specific GAP for Rheb. Therefore, endogenous or lower levels of exogenous Rheb that do not affect basal mTORC1 activity need to be analyzed.

3.10.2. Accessing in vivo Guanine Nucleotide Loading Status on Rag

RagA or RagB forms an obligate heterodimer with RagC or RagD. In addition, in the most active state, RagA/B is GTP-charged, whereas RagC/D is the GDP-bound form. A Rag heterodimer is very stable; therefore, it is difficult to determine the amount of GTP and GDP that bind to one of the Rags within a heterodimer in vivo. Therefore, to analyze endogenous Rag activity in vivo, it may need to establish special cell lines. For instance, the RagC S75L mutant (Oshiro, Rapley, & Avruch, 2014), which is unable to bind guanyl nucleotides, can be stably expressing in cells lacking both RagC and RagD expression, and then endogenous RagA can be IPed and monitored levels of bound GTP and GDP.

4. CONCLUDING REMARKS

Over the past several decades, tremendous efforts to reveal molecular mechanisms underlying the regulation of mTOR signaling have been made, and hence numerous new members in the mTOR pathway have been discovered. Identification of new members in the mTORC1 signaling has shed light on the molecular mechanism by which mTORC1 is activated by amino acids. During the last decade, more than 20 new proteins have been identified in the regulation of amino acids-induced mTORC1 activation. Identifications of the molecular mechanisms by which mTORC1 receives signals from amino acids such as leucine and arginine are the most significant discoveries in the last few years. However, there are still unanswered questions. For example, the molecular mechanism by which glutamine induces mTORC1 activation remains elusive. It has been reported that glutamine is transported into cells through the SLC1A5 amino acid transporter; hence cellular glutamine in turn is used to import leucine via the antiporter SLC7A5-SLC3A2, thereby stimulating mTORC1 through Rag activation (Nicklin et al., 2009). In addition, α-ketoglutarate, a glutamine metabolite, can stimulate GTP loading of RagB. However, a recent study has shown that glutamine induces lysosomal mTORC1 localization and its activation in a manner independent of Rag small GTPases (Jewell et al., 2015). Secondarily, GEFs for Rheb and RagC/D have not been identified. It is possible that GEFs for Rheb or RagC/D may not be necessary for the regulation of these small GTPases. For instance, Rheb has little its own GTPase activity of its own, and high concentrations of cellular GTP may be spontaneously loaded to Rheb. Therefore, the regulation of the TSC complex may fulfill the sole mechanism of Rheb-induced mTORC1 activation. Finally, another interesting topic that has not been fully elucidated is where mTORC1 can physically interact with its distinct substrates, including S6K, 4EBPs, and ULK1. For instance, upon mTORC1 activation, the majority of these substrates can be sufficiently and fully phosphorylated. However, these substrates are not exclusively expressed at the surface or surrounding of the lysosomes where mTORC1 is activated. It remains unclear whether mTORC1 leaves lysosomes to find its substrates, and if it does, how the trafficking of mTORC1 from lysosomes is regulated.

In terms of the mechanism of mTORC2 activation, it has been shown that ribosomes play an important role in the PI3K-dependent mTORC2 activation (Zinzalla et al., 2011). However, it has not been fully understood how the association of mTORC2 with ribosomes stimulates mTORC2. In this chapter, we described the basic but critical techniques and methods to examine cellular mTOR activity, and hopefully, these methods can help to elucidate these questions that are still in mystery.

ACKNOWLEDGMENTS

We thank members of the Inoki lab for helpful discussions and this work was supported by National Institutes of Health (NIH) Grants (DK083491 and GM110019).

REFERENCES

  1. Bar-Peled L, Chantranupong L, Cherniack AD, Chen WW, Ottina KA, Grabiner BC, et al. (2013). A tumor suppressor complex with GAP activity for the Rag GTPases that signal amino acid sufficiency to mTORC1. Science, 340, 1100–1106. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bar-Peled L, Schweitzer LD, Zoncu R, & Sabatini DM (2012). Ragulator is a GEF for the rag GTPases that signal amino acid levels to mTORC1. Cell, 150, 1196–1208. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Brown EJ, Albers MW, Shin TB, Ichikawa K, Keith CT, Lane WS, et al. (1994). A mammalian protein targeted by G1-arresting rapamycin-receptor complex. Nature, 369, 756–758. [DOI] [PubMed] [Google Scholar]
  4. Cargnello M, Tcherkezian J, & Roux PP (2015). The expanding role of mTOR in cancer cell growth and proliferation. Mutagenesis, 30, 169–176. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Carroll B, Maetzel D, Maddocks OD, Otten G, Ratcliff M, Smith GR, et al. (2016). Control of TSC2-Rheb signaling axis by arginine regulates mTORC1 activity. eLife, 5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chantranupong L, Scaria SM, Saxton RA, Gygi MP, Shen K, Wyant GA, et al. (2016). The CASTOR proteins are arginine sensors for the mTORC1 pathway. Cell, 165, 153–164. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Chantranupong L, Wolfson RL, Orozco JM, Saxton RA, Scaria SM, Bar-Peled L, et al. (2014). The Sestrins interact with GATOR2 to negatively regulate the aminoacid-sensing pathway upstream of mTORC1. Cell Reports, 9, 1–8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Demetriades C, Doumpas N, & Teleman AA (2014). Regulation of TORC1 in response to amino acid starvation via lysosomal recruitment of TSC2. Cell, 156, 786–799. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Dibble CC, & Cantley LC (2015). Regulation of mTORC1 by PI3K signaling. Trends in Cell Biology, 25, 545–555. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Dibble CC, Elis W, Menon S, Qin W, Klekota J, Asara JM, et al. (2012). TBC1D7 is a third subunit of the TSC1-TSC2 complex upstream of mTORC1. Molecular Cell, 47, 535–546. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Dibble CC, & Manning BD (2013). Signal integration by mTORC1 coordinates nutrient input with biosynthetic output. Nature Cell Biology, 15, 555–564. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Feldman ME, Apsel B, Uotila A, Loewith R, Knight ZA, Ruggero D, et al. (2009). Active-site inhibitors of mTOR target rapamycin-resistant outputs of mTORC1 and mTORC2. PLoS Biology, 7, e38. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Garami A, Zwartkruis FJ, Nobukuni T, Joaquin M, Roccio M, Stocker H, et al. (2003). Insulin activation of Rheb, a mediator of mTOR/S6K/4E-BP signaling, is inhibited by TSC1 and 2. Molecular Cell, 11, 1457–1466. [DOI] [PubMed] [Google Scholar]
  14. Gaubitz C, Oliveira TM, Prouteau M, Leitner A, Karuppasamy M, Konstantinidou G, et al. (2015). Molecular basis of the rapamycin insensitivity of target of rapamycin complex 2. Molecular Cell, 58, 977–988. [DOI] [PubMed] [Google Scholar]
  15. Geissler EK (2015). Post-transplantation malignancies: Here today, gone tomorrow? Nature Reviews. Clinical Oncology, 12, 705–717. [DOI] [PubMed] [Google Scholar]
  16. Guertin DA, & Sabatini DM (2007). Defining the role of mTOR in cancer. Cancer Cell, 12, 9–22. [DOI] [PubMed] [Google Scholar]
  17. Hara K, Maruki Y, Long X, Yoshino K, Oshiro N, Hidayat S, et al. (2002). Raptor, a binding partner of target of rapamycin (TOR), mediates TOR action. Cell, 110, 177–189. [DOI] [PubMed] [Google Scholar]
  18. Hsieh AC, Liu Y, Edlind MP, Ingolia NT, Janes MR, Sher A, et al. (2012). The translational landscape of mTOR signalling steers cancer initiation and metastasis. Nature, 485, 55–61. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Inoki K, Li Y, Xu T, & Guan KL (2003). Rheb GTPase is a direct target of TSC2 GAP activity and regulates mTOR signaling. Genes & Development, 17, 1829–1834. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Inoki K, Li Y, Zhu T, Wu J, & Guan KL (2002). TSC2 is phosphorylated and inhibited by Akt and suppresses mTOR signalling. Nature Cell Biology, 4, 648–657. [DOI] [PubMed] [Google Scholar]
  21. Jacinto E, Facchinetti V, Liu D, Soto N, Wei S, Jung SY, et al. (2006). SIN1/MIP1 maintains rictor-mTOR complex integrity and regulates Akt phosphorylation and substrate specificity. Cell, 127, 125–137. [DOI] [PubMed] [Google Scholar]
  22. Jewell JL, Kim YC, Russell RC, Yu FX, Park HW, Plouffe SW, et al. (2015). Metabolism. Differential regulation of mTORC1 by leucine and glutamine. Science, 347, 194–198. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Jewell JL, Russell RC, & Guan KL (2013). Amino acid signalling upstream of mTOR. Nature Reviews. Molecular Cell Biology, 14, 133–139. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Kim E, Goraksha-Hicks P, Li L, Neufeld TP, & Guan KL (2008). Regulation of TORC1 by Rag GTPases in nutrient response. Nature Cell Biology, 10, 935–945. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Kim J, Kundu M, Viollet B, & Guan KL (2011). AMPK and mTOR regulate autophagy through direct phosphorylation of Ulk1. Nature Cell Biology, 13, 132–141. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Kim JS, Ro SH, Kim M, Park HW, Semple IA, Park H, et al. (2015). Sestrin2 inhibits mTORC1 through modulation of GATOR complexes. Scientific Reports, 5, 9502. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Kim DH, Sarbassov DD, Ali SM, King JE, Latek RR, Erdjument-Bromage H, et al. (2002). mTOR interacts with raptor to form a nutrient-sensitive complex that signals to the cell growth machinery. Cell, 110, 163–175. [DOI] [PubMed] [Google Scholar]
  28. Laplante M, & Sabatini DM (2012). mTOR signaling in growth control and disease. Cell, 149, 274–293. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Lizcano JM, Goransson O, Toth R, Deak M, Morrice NA, Boudeau J, et al. (2004). LKB1 is a master kinase that activates 13 kinases of the AMPK subfamily, including MARK/PAR-1. The EMBO Journal, 23, 833–843. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Manning BD, Tee AR, Logsdon MN, Blenis J, & Cantley LC (2002). Identification of the tuberous sclerosis complex-2 tumor suppressor gene product tuberin as a target of the phosphoinositide 3-kinase/akt pathway. Molecular Cell, 10, 151–162. [DOI] [PubMed] [Google Scholar]
  31. Menon S, Dibble CC, Talbott G, Hoxhaj G, Valvezan AJ, Takahashi H, et al. (2014). Spatial control of the TSC complex integrates insulin and nutrient regulation of mTORC1 at the lysosome. Cell, 156, 771–785. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Moschetta M, Reale A, Marasco C, Vacca A, & Carratu MR (2014). Therapeutic targeting of the mTOR-signalling pathway in cancer: Benefits and limitations. British Journal of Pharmacology, 171, 3801–3813. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Nakashima N, Noguchi E, & Nishimoto T (1999). Saccharomyces cerevisiae putative G protein, Gtr1p, which forms complexes with itself and a novel protein designated as Gtr2p, negatively regulates the Ran/Gsp1p G protein cycle through Gtr2p. Genetics, 152, 853–867. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Nicklin P, Bergman P, Zhang B, Triantafellow E, Wang H, Nyfeler B, et al. (2009). Bidirectional transport of amino acids regulates mTOR and autophagy. Cell, 136, 521–534. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Oshiro N, Rapley J, & Avruch J (2014). Amino acids activate mammalian target of rapamycin (mTOR) complex 1 without changing Rag GTPase guanyl nucleotide charging. The Journal of Biological Chemistry, 289, 2658–2674. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Parmigiani A, Nourbakhsh A, Ding B, Wang W, Kim YC, Akopiants K, et al. (2014). Sestrins inhibit mTORC1 kinase activation through the GATOR complex. Cell Reports, 9, 1281–1291. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Peterson TR, Laplante M, Thoreen CC, Sancak Y, Kang SA, Kuehl WM, et al. (2009). DEPTOR is an mTOR inhibitor frequently overexpressed in multiple myeloma cells and required for their survival. Cell, 137, 873–886. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Potter CJ, Pedraza LG, & Xu T (2002). Akt regulates growth by directly phosphorylating Tsc2. Nature Cell Biology, 4, 658–665. [DOI] [PubMed] [Google Scholar]
  39. Sancak Y, Bar-Peled L, Zoncu R, Markhard AL, Nada S, & Sabatini DM (2010). Ragulator-Rag complex targets mTORC1 to the lysosomal surface and is necessary for its activation by amino acids. Cell, 141, 290–303. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Sancak Y, Peterson TR, Shaul YD, Lindquist RA, Thoreen CC, Bar-Peled L, et al. (2008). The Rag GTPases bind raptor and mediate amino acid signaling to mTORC1. Science, 320, 1496–1501. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Sancak Y, Thoreen CC, Peterson TR, Lindquist RA, Kang SA, Spooner E, et al. (2007). PRAS40 is an insulin-regulated inhibitor of the mTORC1 protein kinase. Molecular Cell, 25, 903–915. [DOI] [PubMed] [Google Scholar]
  42. Sarbassov DD, Ali SM, Sengupta S, Sheen JH, Hsu PP, Bagley AF, et al. (2006). Prolonged rapamycin treatment inhibits mTORC2 assembly and Akt/PKB. Molecular Cell, 22, 159–168. [DOI] [PubMed] [Google Scholar]
  43. Sarbassov DD, Guertin DA, Ali SM, & Sabatini DM (2005). Phosphorylation and regulation of Akt/PKB by the rictor-mTOR complex. Science, 307, 1098–1101. [DOI] [PubMed] [Google Scholar]
  44. Sekiguchi T, Hirose E, Nakashima N, Ii M, & Nishimoto T (2001). Novel G proteins, Rag C and Rag D, interact with GTP-binding proteins, Rag A and Rag B. The Journal of Biological Chemistry, 276, 7246–7257. [DOI] [PubMed] [Google Scholar]
  45. Shaw RJ, Kosmatka M, Bardeesy N, Hurley RL, Witters LA, DePinho RA, et al. (2004). The tumor suppressor LKB1 kinase directly activates AMP-activated kinase and regulates apoptosis in response to energy stress. Proceedings of the National Academy of Sciences of the United States of America, 101, 3329–3335. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Su B, & Jacinto E (2011). Mammalian TOR signaling to the AGC kinases. Critical Reviews in Biochemistry and Molecular Biology, 46, 527–547. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Thoreen CC, Kang SA, Chang JW, Liu Q, Zhang J, Gao Y, et al. (2009). An ATP-competitive mammalian target of rapamycin inhibitor reveals rapamycin-resistant functions of mTORC1. The Journal of Biological Chemistry, 284, 8023–8032. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Vander Haar E, Lee SI, Bandhakavi S, Griffin TJ, & Kim DH (2007). Insulin signalling to mTOR mediated by the Akt/PKB substrate PRAS40. Nature Cell Biology, 9, 316–323. [DOI] [PubMed] [Google Scholar]
  49. Wolfson RL, Chantranupong L, Saxton RA, Shen K, Scaria SM, Cantor JR, et al. (2016). Sestrin2 is a leucine sensor for the mTORC1 pathway. Science, 351, 43–48. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Wullschleger S, Loewith R, & Hall MN (2006). TOR signaling in growth and metabolism. Cell, 124, 471–484. [DOI] [PubMed] [Google Scholar]
  51. Yang Q, Inoki K, Ikenoue T, & Guan KL (2006). Identification of Sin1 as an essential TORC2 component required for complex formation and kinase activity. Genes & Development, 20, 2820–2832. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Zhang Y, Gao X, Saucedo LJ, Ru B, Edgar BA, & Pan D (2003). Rheb is a direct target of the tuberous sclerosis tumour suppressor proteins. Nature Cell Biology, 5, 578–581. [DOI] [PubMed] [Google Scholar]
  53. Zinzalla V, Stracka D, Oppliger W, & Hall MN (2011). Activation of mTORC2 by association with the ribosome. Cell, 144, 757–768. [DOI] [PubMed] [Google Scholar]

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