by

Rheb Activation Of Mtor

  1. ‡ and

Of insulin/nutrient stimulation of mTOR complex 1 signalling, in addition to Rheb-GTP activation of the mTOR catalytic function, also involves a stable modification of the configuration of mTORC1 (mTOR complex 1) that increases access of substrates to their binding site on the raptor polypeptide. The mechanism. The regulation mTOR activity by growth factors is mediated by the PI3K/Akt signaling pathway leading to phosphorylation and inhibition of TSC2 by Akt and to the subsequent activation of Rheb, which activates mTOR by an as yet unknown mechanism. Activation of mTORC1. RHEB localizes at the lysosome to activate mTORC1 and Rag7 proteins localize mTORC1 to the lysosome, allowing RHEB to activate the protein. RHEB acts as an activator for mTORC1 in its GTP-bound form, therefore GTP -bound RHEB activates cell growth and proliferation within the cell. RheB has been shown to interact with C-Raf, Mammalian target of rapamycin (mTOR), TSC2, Ataxia telangiectasia mutated (ATM), KIAA1303 and Ataxia telangiectasia and Rad3 related. Biochemical studies have shown that activation of Rheb and, as a consequence, the mTOR pathway leads to the phosphorylation of S6K1 and 4E‐BP1, as well as the activation of S6K1 phosphotransferase activity (Brown 1995, Hara 1998).

Recent studies suggest that Rheb is involved in the activation of mTOR, a serine/threonine kinase that belongs to the family of PI3-kinase–related kinases. This family of kinases shares common features that include the presence of the HEAT domain, FAT domain, kinase domain, and FATC.

  1. Program in Signal Transduction Research, The Burnham Institute, La Jolla, California 92037
  1. ↵‡ Supported by a Kirschstein-NRSA Fellowship F32 CA099354 from the NCI, National Institutes of Health. To whom correspondence should be addressed: Program in Signal Transduction Research, The Burnham Institute, 10901 N. Torrey Pines Road, La Jolla, CA 92037. Tel.: 858-646-3100 (ext. 3943); Fax: 858-713-6274; E-mail: gchiang{at}burnham.org.

Abstract

The mammalian target of rapamycin (mTOR) coordinates cell growth with the growth factor and nutrient/energy status of the cell. The phosphatidylinositol 3-kinase-AKT pathway is centrally involved in the transmission of mitogenic signals to mTOR. Previous studies have shown that mTOR is a direct substrate for the AKT kinase and identified Ser-2448 as the AKT target site in mTOR. In this study, we demonstrate that rapamycin, a specific inhibitor of mTOR function, blocks serum-stimulated Ser-2448 phosphorylation and that this drug effect is not explained by the inhibition of AKT. Furthermore, the phosphorylation of Ser-2448 was dependent on mTOR kinase activity, suggesting that mTOR itself or a protein kinase downstream from mTOR was responsible for the modification of Ser-2448. Here we show that p70S6 kinase phosphorylates mTOR at Ser-2448 in vitro and that ectopic expression of rapamycin-resistant p70S6 kinase restores Ser-2448 phosphorylation in rapamycin-treated cells. In addition, we show that cellular amino acid status, which modulates p70S6 kinase (S6K1) activity via the TSC/Rheb pathway, regulates Ser-2448 phosphorylation. Finally, small interfering RNA-mediated depletion of p70S6 kinase reduces Ser-2448 phosphorylation in cells. Taken together, these results suggest that p70S6 kinase is a major effector of mTOR phosphorylation at Ser-2448 in response to both mitogen- and nutrient-derived stimuli.

The mammalian target of rapamycin (mTOR)1 is a member of the phosphatidylinositol 3 (PI-3)-kinase-related kinase family (PIKK), which includes ATM, ATR, hSMG-1, and DNA-PK (1–3). These large Ser/Thr protein kinases play essential roles in cellular responses to growth factors and stress. In particular, mTOR plays a critical role in coordinating cell growth with growth factor inputs as well as cellular nutrient and energy status.

The bacterially derived macrolide ester, rapamycin, is clinically approved as an immunosuppressant and shows promising anti-tumor activity. Rapamycin, when complexed with FKBP-12 (FK506-binding protein, 12 kDa), binds specifically to mTOR at a conserved stretch of ∼100 amino acids termed the FKBP-12·rapamycin binding domain (4). Mutation of a critical serine residue (Ser-2035) in the FKBP-12·rapamycin binding domain to a more bulky amino acid, such as isoleucine, abrogates FKBP-12·rapamycin binding to mTOR, and generates a rapamycin-resistant form of mTOR (4, 5). The mechanism by which rapamycin inhibits mTOR function remains poorly understood.

Recent studies have provided significant insights into the growth factor and nutrient signaling pathway(s) upstream of mTOR. Stimulation of many growth factor receptors leads to activation of the PI-3 kinase-AKT pathway, and it appears that this pathway is centrally involved in the coupling of mitogenic stimuli to mTOR. Moreover, loss of the tumor suppressor PTEN provides a powerful signal for tumor progression and simultaneously confers increased sensitivity to the anti-proliferative effect of rapamycin (6, 7). Nutrient/energy status also regulates mTOR signaling. Although it has been suggested that mTOR directly senses ATP levels in the cell (8), a more plausible mechanism of mTOR regulation involves the ATP-regulated LKB1/AMPK pathway (9, 10). Signaling through both the PI-3 kinase/AKT and LKB1/AMPK pathways as well as nutrient cues all converge at the level of the TSC1/2 complex, which genetic and biochemical evidence establish as a negative regulator of mTOR (11–14). TSC2 is a direct target of both AKT and AMPK. Phosphorylation of TSC2 by AKT results in the inactivation of the TSC1/2 complex (15–17), whereas phosphorylation of TSC2 by AMPK results in enhanced activity (9).

In addition to the regulation of mTOR by the TSC/Rheb pathway, our laboratory and others have provided evidence that mTOR may be a direct substrate for AKT (18, 19). The proposed AKT phosphorylation site (Ser-2448) in mTOR lies within a C-terminal regulatory region, which, when deleted, results in elevated mTOR activity in vitro and in cells (18, 20).

In this study, we demonstrate that mTOR phosphorylation at Ser-2448 is blocked by rapamycin, and this effect is independent of the AKT activation status, which suggests that Ser-2448 phosphorylation is catalyzed by a protein kinase other than AKT. We show that Ser-2448 is directly phosphorylated by p70S6 kinase in vitro and that expression of a rapamycin-resistant p70S6 kinase in cells maintains Ser-2448 phosphorylation in the presence of rapamycin. Furthermore, changes in amino acid availability, which affects p70S6 kinase activity, also modulates Ser-2448 phosphorylation. Finally, Ser-2448 phosphorylation is reduced in cells by siRNA-mediated depletion of p70S6 kinase. Taken together, these data suggest that p70S6 kinase is the major protein kinase responsible for Ser-2448 phosphorylation in mammalian cells.

EXPERIMENTAL PROCEDURES

Antibodies and Reagents—Rapamycin was obtained from the NCI, National Institutes of Health (Bethesda, MD). Wortmannin, the anti (α)-FLAG M2 antibody, α-FLAG M2-agarose, and the FLAG peptide were obtained from Sigma. The monoclonal α-AU1 and α-HA (12CA5) antibodies were purchased from Covance (Berkeley, CA). α-AKT and α-phospho-AKT (Ser-473) antibodies were purchased from Cell Signaling Technology (Beverly, MA), and α-p70S6 kinase antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The α-mTOR and α-phospho-Ser-2448 mTOR (α-mTORp2) antibodies have been described previously (18, 21).

Cell Culture, DNA Constructs, siRNA, and Transfections—HEK 293 cells, MCF-7 cells, and HeLa cells were maintained in high glucose Dulbecco's modified essential medium (DMEM) (Irvine Scientific, Santa Ana, CA) supplemented with 10% FCS (Hyclone, Logan, UT). HEK 293 cells stably expressing AU1 epitope-tagged, rapamycin-resistant (SI) mTOR have been described (22). For experiments involving serum starvation, cells were washed twice with phosphate-buffered saline and then serum-starved for 16–18 h in DMEM supplemented with 0.1% FCS. For serum stimulation, cells were incubated for 15 min with 10% FCS prior to cell harvest. For amino acid deprivation, cells were washed twice with phosphate-buffered saline and incubated for 50 min in high glucose DMEM minus amino acids. Cells were restimulated for 10 min with serum-free, high glucose DMEM prior to cell harvest. Where indicated, cells were pretreated for 30 min with rapamycin or wortmannin before stimulation with amino acids or serum.

The wild type (WT), SI phosphorylation site mutants (Ser-2448 → Ala and Ser-2448 → Glu), and rapamycin-resistant, kinase-inactive mTOR cDNAs cloned into the expression vector pcDNA3 (Invitrogen) have been described (18, 22). The HA-tagged wild-type p70S6 kinase and the FLAG-tagged p70S6 kinase were provided by Dr. Naohiro Terada (University of Florida, Gainesville, FL). The HA-tagged, kinase-inactive (K100R) p70S6K mutant in the expression vector pRK7 and the HA-tagged, rapamycin-resistant ΔN/C p70S6K in the expression vector pBJ were provided by Dr. John Blenis (Harvard Medical School, Boston, MA).

The luciferase siRNA control and the p70S6K siRNA SMARTpool were purchased from Dharmacon (Lafayette, CO). siRNAs were transfected into HeLa cells with Oligofectamine (Invitrogen) according to the manufacturer's protocol. Cells were harvested 48 h posttransfection.

HEK 293 cells were transfected with FuGENE 6 (Roche Applied Science) according to the manufacturer's protocol. Cells were used in experiments at 36–48 h posttransfection.

Immunoprecipitations, in Vitro Kinase Reactions, and Immunoblotting—For mTOR immunoprecipitations, cells were suspended in lysis buffer (50 mm Tris-HCl, pH 7.4, 100 mm NaCl, 50 mm β-glycerophosphate, 10% glycerol (w/v), 1% Triton X-100, 1 mm EDTA, 10 μg/ml aprotinin, 1 μg/ml pepstatin A, 10 μg/ml leupeptin, 2 mm phenylmethylsulfonyl fluoride, 20 μm microcystin-LR, 25 mm NaF). Cleared extracts were immunoprecipitated with rabbit α-mTOR antibody or α-AU1 antibody. Immune complexes were collected on protein A-Sepharose (Sigma) or α-mouse IgG-agarose (Sigma), washed three times with lysis buffer, and resuspended in SDS-PAGE sample buffer.

For p70S6 kinase reactions, transfected HEK 293 cells were lysed as described above. Soluble proteins were immunoprecipitated with monoclonal α-HA antibody. Immune complexes were collected on α-mouse IgG-agarose, washed three times in lysis buffer, followed by one wash in p70S6 kinase buffer (50 mm Tris-Cl, pH 7.4, 10 mm NaCl, 10 mm MgCl2, 10% glycerol, 1 mm dithiothreitol). Immunoprecipitates were resuspended in 40 μl of p70S6 kinase buffer, and kinase reactions were initiated by addition of 1 μg of GST-mTOR RD fragment (amino acids 2405–2517) and 100 μm ATP. Reactions were incubated for 20 min at 30 °C and terminated with 2× SDS-PAGE sample buffer.

For phosphorylation of full-length mTOR by soluble p70S6 kinase, HEK 293 cells transfected with a FLAG-tagged p70S6 kinase expression vector were lysed as described above. Soluble proteins were immunoprecipitated with α-FLAG-agarose, and bound p70S6 kinase was eluted with p70S6 kinase buffer containing 0.15 mg/ml FLAG peptide. Anti-mTOR immunoprecipitates prepared from serum-starved HEK 293 cells were resuspended in 30 μl of p70S6 kinase buffer, and kinase reactions were initiated by the addition of 10 μl of soluble Flag-p70S6 kinase and 100 μm ATP. Reactions were incubated for 20 min at 30 °C and terminated with 2× SDS-PAGE sample buffer.

Samples were resolved by SDS-PAGE and transferred to polyvinylidene difluoride membranes (PerkinElmer Life Sciences). Western blotting was carried out essentially as described (21). Bound antibodies were detected with horseradish peroxidase-conjugated protein A (Amersham Biosciences) for polyclonal antibodies or horseradish peroxidase-conjugated goat anti-mouse antibody (Amersham Biosciences) for monoclonal antibodies followed by enhanced chemiluminescence using Western Lightning reagent (PerkinElmer Life Sciences).

RESULTS

Rapamycin Blocks Serum-stimulated Ser-2448 Phosphorylation—Previous reports demonstrated that mTOR was phosphorylated in vitro by AKT at a serine residue (Ser-2448) located in a C-terminal “repressor domain” of mTOR (18, 19). In later studies, we unexpectedly observed that phosphorylation at the Ser-2448 site was consistently suppressed in cells treated with rapamycin. As short term exposure to rapamycin does not inhibit AKT, these results suggested that Ser-2448 phosphorylation was mediated by a rapamycin-sensitive protein kinase that shared an overlapping substrate preference with AKT. To test this hypothesis, we cultured MCF-7 cells overnight in low serum (0.1% FCS)-containing medium. The cells were restimulated with 10% FCS in the absence or presence of 100 nm rapamycin or 100 nm wortmannin (Fig. 1A). As previously shown, serum stimulation increased Ser-2448 phosphorylation, with a concomitant increase in AKT phosphorylation at Ser-473. Treatment of the cells with 100 nm wortmannin, a concentration that inhibits PI-3 kinase, but not mTOR or other PI-3 kinase-related kinase family members (23), blocked the serum-dependent phosphorylation of mTOR, as well as the modification of AKT, consistent with the requirement for PI-3 kinase in AKT activation (24). As noted previously, Ser-2448 phosphorylation was also strongly suppressed by rapamycin; however, in contrast to wortmannin, rapamycin had no effect on the phosphorylation of AKT at Ser-473. Thus, changes in the phosphorylation of mTOR at Ser-2448 were not tightly correlated with the activation state of AKT in these cells.

We next asked whether binding of FKBP-12·rapamycin to mTOR was required for the observed decrease in Ser-2448 phosphorylation. To test this, we used HEK 293 cells that stably express a mTOR polypeptide bearing a Ser-2035 → Ile mutation in the FKBP-12·rapamycin binding domain (SI mTOR). The SI mTOR protein exhibits markedly reduced binding affinity for FKBP-12·rapamycin and confers rapamycin resistance when transfected into mammalian cells (5). We repeated the experiments described above, this time with serum-starved HEK 293 SI mTOR cells (Fig. 1B). In these cells, serum stimulation provoked a clear increase in the phosphorylation of the ectopically expressed SI mTOR protein at Ser-2448. The increase in Ser-2448 phosphorylation was inhibited by wortmannin; however, rapamycin had little or no effect on the phosphorylation of the SI mTOR protein. These results indicate that, as is the case for endogenous wild-type mTOR (see Fig. 1A), phosphorylation of the Ser-2448 site in SI mTOR remains dependent on PI 3-kinase. However, in contrast to the endogenous protein, phosphorylation of SI mTOR at Ser-2448 is resistant to rapamycin. This result confirms that the inhibitory effect of rapamycin on Ser-2448 phosphorylation is dependent on the inhibition of mTOR-dependent signaling by this drug.

mTOR Kinase Activity Is Required for Ser-2448 Phosphorylation—To test whether mTOR kinase activity was required for Ser-2448 phosphorylation, we transiently transfected HEK 293 cells with AU1 epitope-tagged, WT, SI mTOR, or a rapamycin-resistant, kinase-inactive mTOR double mutant. After 48 h, the transfected cells were treated with 100 nm rapamycin, and the phosphorylation of Ser-2448 was comparatively examined in the various AU1-tagged mTOR proteins (Fig. 1C). Rapamycin treatment strongly reduced the phosphorylation of both the WT and rapamycin-resistant, kinase-inactive mTOR polypeptides, whereas phosphorylation of the rapamycin-resistant SI mTOR was unaffected by the drug. Like SI mTOR, the rapamycin-resistant, kinase-inactive mTOR double mutant does not bind FKBP-12·rapamycin; however, this kinase-inactive mTOR mutant cannot confer rapamycin resistance when transfected into mammalian cells (18). Thus, the phosphorylation of the Ser-2448 site depends on the expression of catalytically active mTOR. Because Ser-2448 is not a mTOR autophosphorylation site (18), this finding raises the possibility that a downstream target of mTOR directly or indirectly mediates the phosphorylation of Ser-2448 in intact cells.

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Fig. 1.

Inhibition of Ser-2448 phosphorylation by rapamycin and wortmannin.A, MCF-7 cells were starved in DMEM + 0.1% FCS for 16 h. Cells were incubated for 30 min with 100 nm rapamycin (Rap) or 100 nm wortmannin (Wm) as indicated prior to stimulation for 15 min with 10% FCS. Anti-mTOR immunoprecipitates were immunoblotted with phospho-Ser-2448-specific mTOR antibodies (α-mTORp2), and the same membrane was reprobed with α-mTOR antibodies. Whole cell extracts from the same samples were immunoblotted with α-phospho-AKT (Ser-473) antibodies followed by α-AKT antibodies. B, HEK 293 cells stably expressing AU1 epitope-tagged SI mTOR were serum-starved for 16 h in DMEM + 0.1% FBS. Cells were stimulated with 10% FBS in the presence of the indicated drug and were harvested after 15 min. Anti-AU1 immunoprecipitates were immunoblotted with α-mTORp2, and the same membrane was stripped and reprobed with α-mTOR antibodies. C, HEK 293 cells were transfected with AU1 epitope-tagged WT, SI, or rapamycin-resistant, kinase-inactive (SIDA) mTOR cDNAs. At 48 h posttransfection, cells were treated with 100 nm rapamycin for 30 min where indicated and harvested. Anti-AU1 immunoprecipitates were immunoblotted with α-mTORp2, and the same membrane was stripped and reprobed with α-mTOR antibodies. The data shown are representative of three independent experiments.

Phosphorylation of Ser-2448 by p70S6 Kinase in Vitro—Of the known mTOR target proteins, p70S6 kinase represents a prime candidate for the putative mTOR-regulated Ser-2448 kinase. To test whether p70S6 kinase phosphorylates mTOR at Ser-2448 in vitro, we transfected HEK 293 cells with an expression plasmid encoding a HA-tagged version of wild-type p70S6 kinase (WT) or a catalytically inactive p70S6 kinase mutant bearing a Lys to Arg substitution in the catalytic domain (KR) (25). Immune complex kinase reactions were performed with α-HA immunoprecipitates as a source of enzyme and a GST fusion protein containing mTOR amino acids 2405–2517 (GST-mTOR RD) as substrate. Phosphate incorporation into the substrate was visualized with the α-phospho-Ser-2448 antibody, α-mTORp2 (Fig. 2A). Immunoprecipitates containing the recombinant WT p70S6 kinase, but not the kinase-inactive KR mutant, mediated substantial phosphorylation of GST-mTOR RD at the Ser-2448 site. As a control for the specificity of the α-mTORp2 antibody, we repeated the immune complex kinase assays with a GST-mTOR RD fusion protein containing a Ser-2448 → Ala substitution as the substrate. Incubation of this substrate with WT p70S6 kinase-containing immunoprecipitates failed to increase the immunoreactivity of the mutated mTOR RD fragment with the α-mTORp2 antibody (data not shown).

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Fig. 2.

Phosphorylation of Ser-2448 by p70S6 kinase.A, HEK 293 cells were transfected with empty vector, HA-tagged wild type (WT) or kinase-inactive (KR) p70S6 kinase. At 48 h posttransfection, cells were harvested, and HA-tagged p70S6 kinase was immunoprecipitated with α-HA antibodies. Top panel, immune complex kinase assay performed with α-HA immunoprecipitates and GST-mTOR RD (amino acids 2405–2517) as substrate. Substrate phosphorylation was visualized by immunoblotting with α-mTORp2. Bottom panel, the same membrane was immunoblotted with α-HA antibodies. B, mTOR was immunoprecipitated from serum-starved HEK 293 cells and used as the substrate for in vitro kinase assays with soluble FLAG-tagged p70S6K. Substrate phosphorylation was visualized by immunoblotting with α-mTORp2, and the membrane was stripped and reprobed with α-mTOR antibodies. FLAG-tagged p70S6K was visualized by immunoblotting with anti-FLAG antibodies. C, HEK 293 cells were co-transfected with AU1-tagged wild-type mTOR and either wild-type (WT) or rapamycin-resistant (ΔN/C) p70S6 kinase. At 48 h posttransfection, cells were treated for 30 min with 100 nm rapamycin where indicated and harvested. α-AU1 immunoprecipitates were immunoblotted with α-mTORp2, and the same membrane was stripped and reprobed with α-AU1 antibodies. The data shown are representative of three independent experiments.

We also examined whether p70S6 kinase phosphorylated full-length mTOR in vitro. To generate this substrate, we serum-starved HEK 293 cells to reduce Ser-2448 phosphorylation to basal levels and isolated mTOR by immunoprecipitation. We then assayed the ability of a soluble FLAG-tagged p70S6 kinase to phosphorylate the mTOR immunoprecipitates. As described above, we visualized phosphate incorporation into Ser-2448 with the α-mTORp2 antibody (Fig. 2B). Anti-mTOR immunoprecipitates incubated with ATP alone displayed a basal level of Ser-2448 phosphorylation. The background phosphorylation of mTOR at Ser-2448 was observed when the kinase reactions were performed in the absence of Mg2+-ATP, indicating that the basal Ser-2448 phosphorylation had occurred prior to isolation of mTOR from the cells (data not shown). In agreement with our previous result with our GST-RD fusion protein, Ser-2448 phosphorylation of full-length mTOR was substantially increased upon addition of ATP and Flag-p70S6 kinase. Taken together, these results indicate p70S6 kinase shares with AKT (18, 19) the ability to phosphorylate mTOR at Ser-2448, at least under in vitro assay conditions.

Rapamycin-resistant p70S6 Kinase Restores Ser-2448 Phosphorylation—If p70S6 kinase serves as an important Ser-2448 kinase in intact cells, then phosphorylation of mTOR at this residue should display resistance to rapamycin in cells transfected with a drug-resistant p70S6 kinase mutant. To test this hypothesis, we co-expressed AU1-epitope-tagged WT mTOR with either wild-type p70S6 kinase (WT) or a N- and C-terminally truncated p70S6 kinase mutant (ΔN/C) whose catalytic activity is insensitive to rapamycin (25). As expected, phosphorylation of mTOR at Ser-2448 remained sensitive to rapamycin in cells transfected with WT p70S6 kinase (Fig. 2C). In contrast, rapamycin failed to suppress Ser-2448 phosphorylation in the ΔN/C p70S6 kinase-expressing cells. These results strongly suggest that p70S6 kinase is a major effector of Ser-2448 phosphorylation in serum-stimulated cells.

Ser-2448 Phosphorylation Is Stimulated by Amino Acids— The activity of p70S6 kinase is regulated by extracellular amino acids, as well as polypeptide growth factors (26, 27). If p70S6 kinase is a major contributor to the phosphorylation of mTOR at Ser-2448 in intact cells, then we would expect that readdition of amino acids to starved cells would lead to concomitant increases in p70S6 kinase activity and Ser-2448 phosphorylation. To address this prediction, we deprived HEK 293 cells of amino acids and then restimulated the cells with amino acids in the absence or presence of rapamycin (Fig. 3). Amino acid starvation decreased Ser-2448 phosphorylation to a level comparable with that observed in rapamycin-treated cells cultured under amino-acid-replete conditions. Readdition of amino acids to these cells provoked a strong increase in Ser-2448 phosphorylation, which was paralleled by the appearance of electrophoretically retarded forms of p70S6 kinase, a well established marker of kinase activation. Rapamycin exposure blocked the phosphorylation of p70S6 kinase and mTOR induced by cellular stimulation with amino acids, indicating that both responses were dependent on mTOR signaling functions. Importantly, neither amino acid stimulation nor rapamycin treatment altered the activation state of AKT, as monitored by changes in the phosphorylation of this protein kinase at Ser-473. Thus, these results argue that p70S6 kinase, rather than AKT, plays the dominant role in the regulation of mTOR phosphorylation at Ser-2448 by amino acids.

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Fig. 3.

Amino acid stimulation increases Ser-2448 phosphorylation. HEK 293 cells were grown overnight in DMEM + 10% FCS. Cells were cultured in serum-free DMEM (+) or were starved for 50 min in serum-free DMEM minus amino acids (–). The indicated samples were stimulated for 10 min with serum-free DMEM plus amino acids (–/+). Cells were treated for 30 min with 100 nm rapamycin prior to harvest. Anti-mTOR immunoprecipitates were immunoblotted with α-mTORp2, and the same membrane was reprobed with α-mTOR antibodies. Whole cell extracts from the same samples were probed with α-p70S6 kinase antibodies, α-phospho-AKT (Ser-473) antibodies, and α-AKT antibodies. The data shown are representative of three independent experiments.

Depletion of p70S6 Kinase Expression in Cells Reduces Ser-2448 Phosphorylation—To confirm whether p70S6 kinase was responsible for Ser-2448 phosphorylation in cells, we used siRNA to down-regulate p70S6 kinase expression (Fig. 4). Transfection of HeLa cells with p70S6 kinase-specific siRNA reduced p70S6 kinase expression with a corresponding decrease in Ser-2448 phosphorylation, whereas control siRNA-transfected cells were unaffected. In both control and p70S6 kinase siRNA-treated cells, AKT phosphorylation was unaffected. These data provide compelling evidence that p70S6 kinase is responsible for Ser-2448 phosphorylation in cells.

DISCUSSION

Phosphorylation of mTOR at Ser-2448 has become a popular biomarker for the activation state of the PI-3 kinase pathway as well as the activation status of mTOR (28–31). Based on the knowledge that AKT phosphorylates the Ser-2448 site, the operative assumption is that changes in Ser-2448 phosphorylation reflect alterations in PI-3 kinase activity induced by endogenous factors or by therapeutic agents. The present findings indicate that the regulation of Ser-2448 phosphorylation is more complex than originally proposed and that this phosphorylation event is not obligatorily linked to AKT. We have confirmed earlier findings that Ser-2448 phosphorylation is wortmannin-sensitive (18, 19). Although the wortmannin results are consistent with the earlier conclusion that AKT is an effector of Ser-2448 phosphorylation, an equally plausible conclusion is that Ser-2448 phosphorylation is sensitive to wortmannin because another PI-3 kinase-activated kinase, PDK-1 (32), is required for phosphorylation of the critical Thr-229 residue in the p70S6 kinase activation loop (33, 34). Thus, our results suggest that, at least in the cell types examined in this study, Ser-2448 phosphorylation primarily reflects a feedback signal to mTOR from its downstream target, p70S6 kinase. As mTOR activity is essential for the activation of p70S6 kinase, we conclude that Ser-2448 phosphorylation is a reasonable indicator of the level of mTOR signaling in cells or tissues.

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Fig. 4.

siRNA-mediated knock-down of p70S6K reduces Ser-2448 phosphorylation. HeLa cells were transfected with either a control luciferase siRNA or a p70S6K-specific siRNA SMARTpool. Cells were harvested at 48 h posttransfection. Anti-mTOR immunoprecipitates were immunoblotted with α-mTORp2, and the same membrane was reprobed with α-mTOR antibodies. Whole cell extracts from the same samples were probed with α-p70S6 kinase, α-phospho-AKT (Ser-473), α-AKT, and α-phospholipase Cγ (PLCγ) antibodies. The data shown are representative of three independent experiments.

Several lines of evidence support the notion that p70S6 kinase is the dominant kinase responsible for mTOR phosphorylation at Ser-2448 in cells. Previous studies demonstrated that Ser-2448 phosphorylation was stimulated by amino acids, which regulate p70S6 kinase activity, but not AKT (35, 36) Furthermore, perturbation of the TSC/Rheb pathway, which regulates p70S6 kinase, but not AKT, influences mTOR Ser-2448 phosphorylation. For example, overexpression of TSC1 and TSC2 decreased Ser-2448 phosphorylation and conversely, RNA interference-mediated depletion of TSC2 increased Ser-2448 phosphorylation (15). Overexpression of Rheb stimulates phosphorylation of p70S6 kinase and results in elevated Ser-2448 phosphorylation (12). Treatment of cells with 2-deoxyglucose, which mimics glucose deprivation by inhibiting hexokinase, activates the TSC pathway via AMPK, decreases p70S6 kinase activity, and results in a concomitant decrease in mTOR Ser-2448 phosphorylation (Ref. 9 and data not shown). Furthermore, the depletion of AKT-1 or AKT-2 isoforms by retrovirus-mediated RNA-interference did not affect mTOR Ser-2448 phosphorylation in macrophage colony-stimulating factor-stimulated bone marrow macrophages (37). It should be noted that other examples of feedback regulation by p70S6 kinase exist. In Drosophila, it has been recently described that phosphorylation of the IRS proteins by p70S6 kinase leads to their degradation (38). Similarly, hyperactivation of the mammalian p70S6 kinase pathway via loss of TSC1 or TSC2 results in the inactivation and degradation of IRS proteins, and leads to insulin resistance (39).

Our results help to explain the earlier finding that a mTOR Ser-2448 → Ala mutant retains the ability to signal to p70S6 kinase (18). With AKT as the proposed effector of Ser-2448 phosphorylation, it was difficult to explain why loss of the Ser-2448 site failed to disrupt signaling through the AKT → mTOR → p70S6 kinase pathway. The current results indicate that Ser-2448 phosphorylation is a consequence, rather than a cause of p70S6 kinase activation. Nonetheless, the functional significance of Ser-2448 phosphorylation with regard to mTOR signaling remains elusive. When assayed in vitro, we did not observe any apparent differences in mTOR kinase activity with either mTOR Ser-2448 → Ala or Ser-2448 → Glu (a “phosphomimetic” substitution) mutants when compared with wild-type mTOR (data not shown). Furthermore, ectopic expression of either mTOR Ser-2448 → Ala or Ser-2448 → Glu mutants that also contained the rapamycin-resistant SI mutation fully supported mTOR-dependent cell size and proliferative activity in rapamycin-treated HEK 293 cells (data not shown). However, we note several limitations of this experimental approach. First, rapamycin is now known to inhibit only a subset of mTOR-dependent signaling functions (22, 40, 41). A second, related caveat is that ectopic expression of any catalytically active, rapamycin-resistant mTOR mutant in the cells confers rapamycin resistance to p70S6 kinase and reconstitutes the phosphorylation of Ser-2448 on the endogenous mTOR polypeptide (data not shown). A definitive evaluation of the functional consequences of Ser-2448 mutations will require that the mTOR Ser-2448 → Ala and Ser-2448 → Glu alleles be expressed in cells that completely lack endogenous mTOR.

It is now apparent that the mTOR regulation through phosphorylation of the `repressor domain' is more complex than originally thought. In addition to Ser-2448 phosphorylation catalyzed by p70S6 kinase and AKT, Thr-2446 is phosphorylated by AMPK (42). Phosphorylation of Thr-2446 and Ser-2448 appears to be mutually exclusive and the significance of AMPK phosphorylation within this region of mTOR remains to be determined (42).

In summary, we conclude that mTOR phosphorylation at Ser-2448 is not controlled directly by AKT but rather occurs in a feedback fashion catalyzed by the downstream target of mTOR, p70S6 kinase. The presence of two juxtaposed, inter-regulated phosphorylation sites in the mTOR “repressor domain” (18, 42) strongly hints that these modifications are functionally significant, although their roles in mTOR signaling remain elusive. Further studies are required to determine whether feedback phosphorylation of mTOR by p70S6 kinase amplifies or dampens mTOR signaling in nutrient- or growth factor-stimulated cells.

Acknowledgments

We thank Dr. John Blenis and Dr. Naohiro Terada for supplying p70S6 kinase reagents. We also thank members of the Abraham laboratory for reagents and helpful discussions.

Footnotes

  • ↵1 The abbreviations used are: mTOR, mammalian target of rapamycin; PI, phosphatidylinositol; FKBP, FK506-binding protein; siRNA, small interfering RNA; HA, hemagglutinin; HEK, human embryonic kidney; DMEM, Dulbecco's modified essential medium; FCS, fetal calf serum; WT, wild type; SI, rapamycin-resistant; GST, glutathione S-transferase; AMPK, AMP-activated protein kinase; TSC, tuberous sclerosis complex.

  • ↵* This work was supported in part by Grants CA76193 and CA52995 (to R. T. A.) from the NCI, National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

  • Received February 15, 2005.
  • Revision received April 26, 2005.
  • The American Society for Biochemistry and Molecular Biology, Inc.

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Methods Enzymol. Author manuscript; available in PMC 2009 Jun 9.
Published in final edited form as:
doi: 10.1016/S0076-6879(07)38021-X
NIHMSID: NIHMS106485
The publisher's final edited version of this article is available at Methods Enzymol
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Abstract

Rheb (Ras homolog enriched in brain) is a GTPase conserved from yeast to human and belongs to a unique family within the Ras superfamily of GTPases. Rheb plays critical roles in the activation of mTOR, a serine/threonine kinase that is involved in the activation of protein synthesis and growth. mTOR forms two distinct complexes, mTORC1 and mTORC2. While mTORC1 is implicated in the regulation of cell growth, proliferation, and cell size in response to amino acids and growth factors, mTORC2 is involved in actin organization. However, the mechanism of activation is not fully understood. Therefore, studies to elucidate the Rheb-mTOR signaling pathway are of great importance. Here we describe methods to characterize this pathway and to evaluate constitutive active mutants of Rheb and mTOR that we recently identified. Constitutive activity of the mutants can be demonstrated by the phosphorylation of ribosomal protein S6 kinase 1 (S6K1) and eIF4E-binding protein 1 (4E-BP1) both in vivo and in vitro after starving cells for amino acids and growth factors. In addition, formation and activity of mTORC1 and mTORC2 can be measured by immunoprecipitating these complexes and carrying out in vitro kinase assays. We also describe a protocol for rapamycin treatment, which directly inhibits mTOR and can be used to investigate the mTOR signaling pathway in cell growth, cell size, etc.

1. Introduction

Small GTPases bind guanine nucleotides and serve as a molecular switch to regulate a number of physiological processes such as cell growth and morphology (). Rheb, a small GTPase that belongs to a unique family within the Ras superfamily of GTPases, controls cell growth and proliferation as well as cell size (; ; ; ). Unlike most small GTPases that are predominantly in an inactive GDP bound state, Rheb exists in a high activated state (), presumably due to a low intrinsic GTPase activity as well as to a limiting amount of Tsc1/Tsc2 GAP protein inside the cell.

Regulation of Rheb is catalyzed by tuberous sclerosis 2 (Tsc2), which acts as a GTPase activating protein (GAP) that enhances the hydrolysis of GTP to GDP in Rheb (; ; ; ; ). Tsc2 forms a complex with tuberous sclerosis 1 (Tsc1) and directly inhibits the Rheb activation. It has been found that the negative regulation of Rheb by the Tsc1/2 complex is controlled by insulin. Insulin binding to its receptor triggers the activation of the class I PI3-kinase/Akt pathway. The activated Akt then increases Tsc2 phosphorylation at serine 939 and 981 (), leading to the dissociation of the Tsc1/2 complex.

Recent studies suggest that Rheb is involved in the activation of mTOR, a serine/threonine kinase that belongs to the family of PI3-kinase–related kinases. This family of kinases shares common features that include the presence of the HEAT domain, FAT domain, kinase domain, and FATC domain (). In addition, TOR kinases contain the FRB domain where FKBP/rapamycin complex binds. mTOR acts as a central protein that controls cell growth and proliferation through transcriptional and translational mechanisms in response to amino acids and growth factors such as insulin. However, amino acids and insulin use two distinct pathways to activate mTOR. Vps34, a class III PI3-kinase, but not class I PI3-kinase, is activated by amino acid stimulation (; ). On the other hand, growth factors activate the class I PI3-kinase/Akt signaling pathway, which then inactivates Rheb GAP, Tsc1/2, as described previously (; ). It has been reported that mTOR forms two distinct complexes, which respond to amino acids or insulin (; ). mTOR complex 1 (mTORC1) is rapamycin sensitive and contains Raptor, GβL/mlST8, and PRAS40 (; ). This complex phosphorylates S6K1 and 4E-BP1, and plays an essential role in the regulation of cell growth and proliferation (, ). Within the complex, Raptor acts as a scaffold protein that connects mTOR to its substrates. PRAS40 is a negative regulator of mTOR that is affected by insulin (; ). GβL is also involved in mTORC1 activity, but its mechanism remains to be elucidated (). On the other hand, mTOR complex 2 (mTORC2) is relatively rapamycin insensitive and contains Rictor, Sin1, and GβL (; ; ; ). mTORC2 is involved in actin organization and cell survival, and mediates insulin signal to Akt by the phosphorylation at serine 473 (; ; ; ; ). Interestingly, inhibition of mTORC2 decreases the phosphorylation level of Akt substrates, forkhead transcription factor, FOXO1/3 proteins, but not other Akt substrates such as Tsc2 and GSK3β, suggesting that mTORC2 preferentially affects downstream events mediated by Akt (; ).

Our genetic analysis of the Tsc/Rheb/TOR signaling pathway in fission yeast led to the identification of novel Rheb and TOR mutants. In the case of Rheb, we first developed screening assays to identify active Rheb mutants in yeast. Screening of a random mutant library of Rheb identified a number of yeast Rheb mutants that showed phenotypes similar to those exhibited by the cells lacking the Tsc1/2 complex, which negatively regulates Rheb (). Comparison of Rheb sequences from different organisms led to the identification of other active mutants of human Rheb (). These mutants will be valuable in elucidating Rheb function and the activation mechanism for the Rheb-mTOR signaling. Constitutive active mutants of Tor2p have also been identified from the analysis of fission yeast signaling (). Altogether, 22 single amino acid changes have been identified in Tor2p. Introduction of some of these mutations to mTOR conferred nutrient-independent activation of mTOR.

In this chapter, we present methods to characterize the constitutive active mutants of Rheb and mTOR. We also describe methods to detect activation of mTOR and to characterize mTOR complexes. Finally, rapamycin sensitivity will be examined.

2. Methods

2.1. Detection of mTOR activation by examining phospho-S6K1 or phospho-4E-BP1

Activation of mTOR is detected by examining phosphorylation of downstream proteins. We usually examine phosphorylation of S6K1 at Ser 389 and/or phosphorylation of 4E-BP1 at Thr 37/46 or Thr 70 for this experiment. To enhance the sensitivity of detection, genes encoding these proteins are transfected. Figure 21.1 shows an example of detecting mTOR activation after amino acid addition. Briefly, cells are transfected with FLAG-tagged S6K1 and then starved for both serum and amino acids. Then amino acid mixture containing glucose is added and incubated for 30 min. Cells are collected and the level of phospho-S6K1 is examined by using an antibody specific for phosphorylated S6K1. The total level of S6K1 is examined by using anti-FLAG antibody. Phosphorylation of S6K1 is shut down after serum and amino acid starvation (PBS lane). However, the addition of amino acids and glucose leads to the appearance of phospho-S6K1 band (+glucose +AA lane). We cannot detect the increased phosphorylation of S6K1 in HEK293 cells when treated with glucose (+glucose lane) or amino acid mixture only (not shown).

Activation of mTOR by the addition of amino acids. HEK293 cells were transfected with FLAG-S6K1 and starved for serum for 24 h (DMEM). These cells were then cultured in D-PBS for 1 h (PBS) and further incubated in D-PBS containing 4.5 g/l of glucose (+glucose) or 4.5 g/l of glucose and 1× amino acid mixture (+glucose, +amino acids) for 30 min. The amount of total or phosphorylated S6K1 was analyzed by Western blot.

2.1.1. Cell culture and transfection

HEK293 and HeLa cells are maintained in Dulbecco’s Modified Eagle’s Medium (DMEM, Invitrogen) supplemented with 10% (v/v) fetal bovine serum and cultured at 37° in a 5% CO2 incubator. Transfection in these cells can be performed either by calcium phosphate method or lipofection method. Here we show the former method. The latter method requires reagents that are commercially available from several sources and transfection is performed according to the manufacturer’s protocol.

  1. Prepare following reagents for the calcium phosphate transfection.

    0.25 M CaCl2: Dissolve CaCl2 in water to the concentration of 0.25 M, and filter through a 0.45-µm membrane filter.

    2× BBS: Make a 2× BBS solution (50 mM BES (N, N-bis [2-hydro-xyethyl]-2-aminoethane sulfonic acid), 280 mM NaCl, 1.5 mM Na2HPO4), adjust the pH to 6.95 with NaOH, and filter through a 0.45-µm membrane filter.

    Cell: Plate 1 × 106 HEK293 cells in a 3.5-cm dish the day before transfection. Growth medium is replaced before adding transfection mixture.

  2. Add 20 µg of plasmid DNA in 500 µl of 0.25 M CaCl2 and vortex well.

  3. Add 500 µl of 2× BBS, vortex well, and incubate for 20 min at room temperature.

  4. Pour this transfection mixture directly to each dish, mix gently, and incubate cells at 37°.

  5. Replace transfection medium with fresh medium at 18 to 24 h after transfection.

  6. Incubate at 37° for a total of 48 to 72 h until target proteins are expressed.

Maximum Mtor Activation

2.1.2. Serum and amino acid starvation

  1. Incubate cells in DMEM containing 0.1% bovine serum albumin at 37° in a 5% CO2 incubator for 24 h.

  2. Remove medium and wash cells two times with Dulbecco’s Phosphate-Buffered Saline (D-PBS, Invitrogen) containing 100 mg/l each CaCl2 and MgCl2. Incubate them in D-PBS for 1 h.

2.1.3. Preparation and addition of amino acid mixture

To make amino acid mixture, add several amino acids in D-PBS at the following concentrations: L-Arg, 84 mg/l; L-Cys, 48 mg/l; L-Glu, 584 mg/l; L-His, 42 mg/l; L-Ile, 105 mg/l; L-Leu, 105 mg/l; L-Lys, 146 mg/l; L-Met, 30 mg/l; L-Phe, 66 mg/l; L-Thr, 95 mg/l; L-Trp, 16 mg/l; L-Tyr, 72 mg/l; L-Val, 94 mg/l. Stir until dissolved and then filter through 0.22-µm membrane filter. To activate mTOR, 4.5 g/l of glucose is mixed in this mixture.

For mTOR stimulation, cells are incubated in this amino acid mixture containing 4.5 g/l of glucose for 30 min at 37° in 5% CO2 after serum and amino acid starvation.

2.1.4. Detection of phospho-S6K1 and phospho-4E-BP1

Lyse the cells with lysis buffer (1% Triton X-100, 20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 50 mM β-glycerophosphate, 1× protease inhibitor cocktail from Roche. After protein quantification, add 1 volume of 2× SDS sample buffer (6% SDS, 10% glycerol, 124.7 mM Tris-HCl) (pH 6.7), 2% 2-mercaptoethanol, 0.02% bromophenol blue), and incubate them at 95° for 5 min. These samples are resolved by 10% polyacrylamide gel for S6K1 detection or 14% polyacrylamide gel for 4E-BP1 detection, and analyzed by Western blot. The anti-phospho-S6K at Ser 389, anti-phospho-4E-BP1 at Thr 37/46, and anti-phospho-4E-BP1 at Thr 70 antibodies are available from several sources. In addition, since hyperphosphorylated 4E-BP1 is separated from less- or non-phosphorylated 4E-BP1 in SDS-polyacrylamide gel electrophoresis, total 4E-BP1 can be resolved into three bands; α, β, and γ from the top. Transiently expressed S6K1 and 4E-BP1 are used because the expression and phosphorylation levels of endogenous S6K1 and 4E-BP1 are low in HEK293 cells.

2.2. Overexpression of wildtype Rheb or constitutive active mTOR mutants confer mTOR activation in the absence of nutrients

Rheb activates mTOR and its downstream proteins in the presence of amino acids. Therefore, as described above, no activation is observed after nutrient starvation. However, overexpression of the wildtype or active mutant Rheb, Rheb-N153T, can induce the activation of mTOR that is identified by the high phosphorylation level of mTOR substrates, S6K1 or 4E-BP1, even in the absence of amino acids (Fig. 21.2).

Rheb overexpression confers phosphorylation of mTOR substrates in the absence of nutrients. HEK293 cells were transfected with pcDNA3 as a control (vector) , Rheb wildtype (wt), or N153Tactive mutant. To detect the phosphorylation of S6K1or 4E-BP1, FLAG-S6K1 or FLAG-4E-BP1was cotransfected. After serum and amino acid starvation, the cell lysates were analyzed by Western blot. (Adapted from . Point mutations in TOR confer Rheb-independent growth in fission yeast and nutrient-independent mammalian TOR signaling inmammalian cells. Proc. Natl. Acad. Sci. USA104, 3514–3519.)

It is important to note that a similar level of mTOR activation is observed with the wildtype and with the mutant Rheb. A possible reason why one can observe mTOR activation after the overexpression of the wildtype Rheb is that the overexpressed wildtype Rheb contains a high GTP level. Presumably, Rheb GAP activity is limiting in some types of cells, including HEK293. In fact, it is reported that the level of GTP bound to Rheb increases as more Rheb DNA is transfected (). Therefore, the overexpressed wildtype Rheb contains a high GTP level to begin with, and the differences between the wildtype and the mutant Rheb are not observed in this setting. However, if Tsc1/2 is overexpressed, clear differences are observed (). In this case, mTOR activation is observed only with the mutant Rheb.

The situation is different with mTOR. Activation of mTOR signaling in the absence of nutrients can only be observed when constitutive active mTOR mutants are overexpressed (Fig. 21.3). In contrast, little activation is observed with the wildtype mTOR. This result is consistent with the fact that mTOR has strict requirement for amino acids to be active.

Overexpression of mTOR mutants but not mTOR wildtype confers phosphorylation of mTOR substrates in the absence of nutrients. HEK293 cells were transfected with pcDNA3 as a control (vector), mTOR wildtype, E2419K active mutant, or L1460P active mutant. To detect the phosphorylation of S6K1 or 4E-BP1, FLAG-S6K1 or FLAG-4E-BP1was cotransfected. After serum and amino acid starvation, the cell lysates were analyzed by Western blot. (Adapted from . Point mutations in TOR confer Rheb-independent growth in fission yeast and nutrient-independent mammalian TOR signaling in mammalian cells. Proc. Natl. Acad. Sci. USA104, 3 51 4–3 519.)

Protocols for the experiments are similar to those described above except that the Rheb or mTOR construct is cotransfected with S6K1 or 4E-BP1 construct.

Activation Of Mtor Pathway

2.2.1. Rheb mutants

A variety of novel mutations in Rheb that confer constitutive activity have been identified (, ; ). We commonly use Rheb-N153T as an active mutant. This mutant shows a low GTP-binding activity and higher GTP-bound level than those of the wildtype protein in mammalian cells. Rheb-K120R is also reported to show constitutive activity, but it is unstable in mammalian cells and its expression level is low. Lamb and colleagues found the new active mutants, Rheb-S16N and -S16H, that show high GTP-bound levels and mTOR activation even in the cells overexpressing Tsc1/2 (). These mutants are expected to facilitate analysis of Rheb structure and elucidation of the activation mechanism of mTOR by Rheb. While the Rheb-Q64L mutant, analogous to the H-Ras Q61L, is reported to display a high basal GTP level (; ), it is unclear whether Rheb-Q64L is an active mutant, as Rheb-Q64L is sensitive to Tsc2GAP, and its GTP-bound level is decreased by the overexpression of Tsc1/2 ().

2.2.2. mTOR mutants

We recently found a number of active mutants of yeast Tor2p (). Introducing these mutations to mTOR enabled identification of two mammalian TOR mutants, E2419K and L1460P. They exhibit mTORC1 activity in HEK293 cells starved for serum and amino acids. On the other hand, these mutants do not confer constitutive activity of mTORC2 toward Akt in HEK293 cells, since comparable amounts of phospho-Akt at Ser 473 are observed with the wildtype and mutant proteins. Interestingly, most of the active mutations identified in yeast Tor2p occur at residues conserved between yeast and human proteins. Further analysis may provide insight into the activation mechanism of mTOR. In addition, the finding that mTOR-activating mutants can be identified gives rise to the possibility that mutations in mTOR are involved in the uncontrolled growth of cancer cells.

2.3. Analysis of mTOR complexes and their in vitro kinase assay

The components of mTOR complexes can be examined by immunoprecipitating mTOR and performing Western blot analysis. Figure 21.4A shows detection of mTOR binding proteins; Raptor, a mTORC1 component, and Rictor, a mTORC2 component. Since mTOR complexes are unstable and disrupted in the presence of some detergents such as 1% NP-40 or Triton X-100, mTOR binding proteins cannot be observed in the mTOR immunoprecipitates containing these detergents (see Fig. 21.4A). Therefore, we commonly use lysis buffer containing CHAPS detergent to lyse cells and detect mTOR complexes. Endogenous mTOR can also be immunoprecipitated with anti-mTOR antibody.

Immunoprecipitation and in vitro kinase activity of mTOR complexes. (A) HEK293 cells were transfected with pcDNA3 as a control (vector), mTOR wildtype (wt), E2419K active mutant, or L1460P active mutant. After serum and amino acid starvation, mTOR complexes were immunoprecipitated using anti-AU1 antibody from each cell lysates containing 0.4%CHAPSor1%NP-40 as indicated and the immuno-precipitates were analyzed by Western blot. (B) mTOR immunoprecipitates were divided in two, and used for in vitro kinase assay with 4E-BP1as amTORC1substrate, and Akt as a mTORC2 substrate. Phosphorylation of substrates was analyzed by Western blot. (Adapted from . Point mutations in TOR confer Rheb-independent growth in fission yeast and nutrient-independent mammalian TOR signaling in mammalian cells. Proc. Natl. Acad. Sci. USA104, 3514–3519.)

mTOR complexes exist as a dimer or multimer in mammalian cells, and dimeric mTOR is reported to be the major form that responds to insulin (; ). The mTOR dimer is observed by the expression of two distinct epitope-tagged mTOR proteins. One epitope is used to immunoprecipitate mTOR, and then the other epitope is detected by the Western blot analysis of immunoprecipitates.

To directly assess the activity of mTOR complexes, in vitro kinase assay is performed. The in vitro kinase activity of wildtype mTOR, E2419K, and L1460P mutants are shown in Fig. 21.4B. This in vitro kinase assay is examined using mTOR immunoprecipitates and recombinant proteins, 4E-BP1, and Akt, which are used as substrates for mTORC1 and mTORC2, respectively. Active mutants of mTOR show higher activity with 4E-BP1 as the substrate compared with the wildtype mTOR in HEK293 cells under nutrient-starved condition. On the other hand, mTORC2 activities of mTOR mutant proteins are similar to that of wildtype mTOR protein. Alternatively, we can measure in vitro kinase activity after immunoprecipitation of mTORC1 or mTORC2, separately. Immunoprecipitation with anti-Raptor and anti-Rictor antibodies can isolate mTORC1 and mTORC2, respectively.

2.3.1. Immunoprecipitation of mTOR complex

  1. Lyse 1 × 107 HEK293 cells with lysis buffer for immunoprecipitation (0.4% CHAPS, 50 mM Tris-HCl [pH 7.4], 150 mM NaCl, 10 mM MgCl2, 50 mM β-glycerophosphate, 1× protease inhibitor). These lysates are incubated for 30 min at 4° and then centrifuged at 15,000 rpm for 15 min at 4°.

  2. Put the supernatants into new tubes and add 5 µg of anti-mTOR antibody (Santa Cruz, N-19), or other antibodies that recognize epitope-tagged mTOR as well as 30 µl of protein-G sepharose 4 fast-flow beads (GE Healthcare).

  3. Rotate the samples at 4° for 2 h.

  4. Centrifuge at 3000 rpm for 2 min at 4°, and then wash the beads three times with 1 ml of washing buffer (0.4% CHAPS, 50 mM Tris-HCl [pH 7.4], 150 mM NaCl, 10 mM MgCl2, 50 mM β-glycerophosphate). After the wash, add 30 µl of 2 × SDS sample buffer (6% SDS, 10% glycerol, 124.7 mM Tris-HCl (pH 6.7), 2% 2-mercaptoethanol), and incubate them at 95° for 5 min. Free program latest proxifier configuration file. These samples are resolved by 8% polyacrylamide gel to detect mTOR, Raptor, and Rictor, and 13% gel to detect GβL and PRAS40. The antibodies for Western blot analysis can be obtained from the following vendors: anti-mTOR and anti-Raptor antibody, Cell Signaling; anti-GβL antibody, BETHYL Lab; and anti-PRAS40 antibody, Biosource.

2.3.2. Dimer formation

To examine the formation of dimeric mTOR, two distinct epitope-tagged mTORs are coexpressed in HEK 293 cells. After one epitope-tagged mTOR is immunoprecipitated using antibody against its epitope, the presence of the other epitope-tagged mTOR is analyzed by Western blot. Coimmunoprecipitation of the two epitope-tagged mTORs indicate the dimer formation. The protocols for transfection and immunoprecipitation are same as above.

2.3.3. In vitro mTORC1 and mTORC2 kinase assay

  1. Immunoprecipitate mTOR complex using anti-Raptor antibody (BETHYL Lab) for mTORC1 or anti-Rictor antibody (BETHYL Lab) for mTORC2.

  2. After the wash, immunoprecipitates are washed one time with 1 × kinase buffer (20 mM Tris-HCl [pH 7.4], 10 mM MgCl2, 0.2 mM ATP)

  3. Add 8 µl of 5× kinase buffer and 1 µg of recombinant 4E-BP1 for mTORC1 kinase assay or 1 µg of recombinant unactive Akt (Millipore) for mTORC2 kinase assay, and bring the total volume to 40 µl with sterile water.

  4. Incubate immunoprecipitates at 37° for 30 min. To stop the reaction, immediately add 40 µl of 2 × SDS sample buffer and incubate at 95° for 5 min.

  5. These samples are resolved by 10% polyacrylamide gel for Akt detection or 14% polyacrylamide gel for 4E-BP1 detection, and analyzed by Western blot. The anti-phospho-Akt at Ser 473, anti-Akt, anti-phospho-4E-BP1 at Thr 37/46, and anti-4E-BP1 antibodies are available from several sources. Phosphorylation of 4E-BP1 at Thr 70 is not detected in this assay.

2.4. Rapamycin treatment

Rheb Activation Of Mtor

Rapamycin suppresses mTOR kinase activity by the formation of a ternary complex with FKBP12 and mTOR (; ). It has been suggested that rapamycin inhibits mTORC1 activity, but does not affect mTORC2 activity. However, recent studies show that a prolonged treatment of rapamycin can suppress mTORC2 as well as mTORC1 in some cells such as PC3 and Jurkat cells (). The mechanism by which rapamycin inhibits mTOR activity is still unknown, but the rapamycin binding to mTOR may perturb the mTOR complexes and lead to the disruption of mTOR interaction with its substrates.

Before treating with rapamycin, cells are cultured in DMEM containing 0.1% bovine serum albumin at 37° in a 5% CO2 incubator for 24 h. The cells are then treated with 20 nM rapamicin in DMEM containing 0.1% bovine serum albumin. Although mTORC1 kinase activity is suppressed by 1 h of treatment, 24 h of treatment are required for the inhibition of mTORC2 kinase activity. Decreased phosphorylation levels of S6K1 and 4E-BP1 are observed in these cells.

Activation Of Mtor Survival Pathway

3. Conclusion

Mtorc1 Activation By Rheb

The Rheb-mTOR signaling pathway has been extensively studied from yeast to human. This pathway plays a pivotal role in the regulation of cell growth, proliferation, cell size, etc. We presented here the protocols commonly used to examine the Rheb-mTOR signaling pathway. In addition, we showed protocols to evaluate Rheb and mTOR mutants that we recently identified as constitutively active mutants (, ). The findings of these activating mutants are significant as it raises the possibility that one mutation in these genes may disrupt homeostasis such as contact inhibition in mammalian cells and cause tumor progression. In fact, activation of the mTOR pathway is implicated in a number of human diseases associated with benign tumors, including tuberous sclerosis. Inhibitors of mTOR, including rapamycin and its derivatives, are under clinical evaluation as anticancer drugs. Further studies on the Rheb-mTOR signaling pathway may provide important insights into the activation mechanism of the Rheb-mTOR signaling pathway.

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Rheb Activation Of Mtorc1

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Mtor Activation Training

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