Mammalian target of rapamycin complex 1-mediated phosphorylation of eukaryotic initiation factor 4E-binding protein 1 requires multiple protein–protein interactions for substrate recognition

Cellular Signalling 21 (2009) 1073–1084

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Cellular Signalling j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / c e l l s i g

Mammalian target of rapamycin complex 1-mediated phosphorylation of eukaryotic initiation factor 4E-binding protein 1 requires multiple protein–protein interactions for substrate recognition Elaine A. Dunlop, Kayleigh M. Dodd, Lyndsey A. Seymour, Andrew R. Tee ⁎ Institute of Medical Genetics, Cardiff University, Heath Park, Cardiff, Wales, CF14 4XN, UK

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Article history: Received 8 January 2009 Received in revised form 20 February 2009 Accepted 22 February 2009 Available online 9 March 2009 Keywords: mTOR Raptor 4E-BP1 Rag RAIP FKBP38

a b s t r a c t The mammalian target of rapamycin (mTOR) pathway is implicated in a number of human diseases, but the pathway details are not fully understood. Here we elucidate the interactions between various proteins involved in mTOR complex 1 (mTORC1). An in vitro mTORC1 kinase assay approach was used to probe the role of the mTORC1 component Raptor and revealed that certain Raptor mutations disrupt binding to eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) and prevent its subsequent phosphorylation by mTOR. Interestingly, we show that a point mutation in the highly conserved Raptor RNC domain still allows binding to mTOR but prevents Raptor association and mTOR-dependent phosphorylation of 4E-BP1, indicating that this Raptor domain facilitates substrate recognition by mTORC1. This Raptor RNC domain mutant also dominantly inhibits mTORC1 signalling to 4E-BP1, S6K1 and HIF1α in vivo. We further characterise the functions of the mTORC1 signalling (TOS) and RAIP motifs of 4E-BP1, which are involved in substrate recognition by Raptor and phosphorylation by mTORC1. We show that an mTOR mutant, L1460P, responds to insulin even in nutrient-deprived conditions and is resistant to inhibition by inactive RagB–RagC heterodimers that mimic nutrient withdrawal suggesting that this region of mTOR is involved in sensing the permissive amino acid input. We found that FKBP38 inhibits mTOR(L1460P), while the mTOR(E2419K) kinase domain mutant was resistant to FKBP38 inhibition. Finally, we show that activation of mTORC1 by both Rheb and RhebL1 is impaired by FKBP38. Our work demonstrates the value of an in vitro mTORC1 kinase assay to characterise cell signalling components of mTORC1 involved in recognition and phosphotransfer to mTORC1 substrates. © 2009 Elsevier Inc. All rights reserved.

1. Introduction The mammalian target of rapamycin (mTOR) kinase pathway coordinates the regulation of cell size through control of mRNA translation in response to growth factors and nutrients. mTOR exists in two complexes, termed mTORC1 when complexed with Raptor and mLST8, and mTORC2 when associated with Rictor, mLST8 and mSin1 [1]. The known downstream phosphorylated targets of mTORC1 are ribosomal protein S6 kinase 1 (S6K1) and eukaryotic initiation factor

Abbreviations: DMEM, Dulbecco's Modified Eagle's Medium; 4E-BP1, eukaryotic initiation factor 4E-binding protein 1; ECL, Enhanced Chemiluminescent; FAT, FRAP, ATM and TRRAP; FKBP38, FK506-binding protein 38; FRB, FKBP12/rapamycin binding (FRB); Rheb, Ras homolog enriched in brain; RhebL1, Rheb like-1 protein; GAP, GTPaseactivating protein; HEAT, Huntington, Elongation Factor 3, PR65/A, TOR; PI3K, phosphoinositide 3-kinase; HEK, human embryonic kidney; HRP, Horse Radish Peroxidase; MEM, Modified Eagle's Medium; mTOR, mammalian target of rapamycin; NF, Neurofibromatosis; PBS, phosphate buffered saline; PVDF, Polyvinylidene Difluoride; RNC, Raptor N-terminal conserved; S6K1, S6 kinase 1; TBS, tris buffered saline; TOS, TOR signalling; TSC, tuberous sclerosis complex. ⁎ Corresponding author. Tel.: +44 29 2074 4055; fax: +44 29 2074 6551. E-mail address: [email protected] (A.R. Tee). 0898-6568/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.cellsig.2009.02.024

4E-binding protein 1 (4E-BP1). Both substrates have a common mTORC1 signalling (TOS) motif which is essential for mTORC1-directed phosphorylation. This is thought to be because TOS motifs are crucial for substrate recognition by mTORC1 [2]. Additionally, 4E-BP1 contains a RAIP motif near its N-terminus, so called because of the amino acids of which it is composed [3]. This motif appears to play an accessory role in mTORC1-mediated phosphorylation of 4E-BP1 [4] and regulates the extent of 4E-BP1 phosphorylation in response to insulin [3]. Upstream of mTORC1 lies the small GTPase, Ras homolog enriched in brain (Rheb), which potently activates mTORC1 when GTP-bound [5]. Tuberous Sclerosis Complex (TSC) -1 and -2 act as a heterodimeric GTPase-activating protein (GAP) towards Rheb [6]. Inactivating mutations of TSC1/2 leads to heightened mTORC1 signalling that appears central to the pathogenesis of the inherited hamartoma syndrome TSC. Other hamartoma syndromes that involve mTORC1 upregulation include Peutz–Jeghers syndrome and Neurofibromatosis (NF) type 1 [7,8]. Despite mTOR's central role in regulating cell growth responses, many of the target molecules of mTOR that relay these growth signals are currently unknown or require further characterisation. The inability to purify an active mTOR kinase that resembles its physiological state using routine kinase assay procedures has been a

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limiting factor in elucidating novel downstream substrates of mTOR. Previous in vitro mTOR kinase assays required MnCl2, instead of physiological levels of MgCl2, to artificially enhance the weak phosphotransfer activity observed when mTOR was purified [9,10]. An adapted mTOR purification method using non-ionic detergents led to the discovery of Raptor [10] and the isolation of the mTORC1 complex that retains its kinase activity under physiological MgCl2 conditions [11]. By performing mTORC1 kinase assays based on the research carried out by Sancak et al. [11], which uses MgCl2, we are able to further characterise mTORC1. This approach enabled us to purify a highly active mTORC1 complex which was further enhanced by incubation with active GTP-bound Rheb. Using the assay we investigated mutations in mTOR and Raptor that affect downstream phosphorylation events. Kinase assays also allowed us to further characterise the roles of the TOS and RAIP motifs of 4E-BP1 and the mTORC1 inhibitor, FKBP38. 2. Materials and methods

2.4. Production of recombinant 4E-BP1 BL21 (DE3) pLys bacteria (Invitrogen) were transformed with the GST-tagged 4E-BP1/pGEX plasmid. Growing bacteria (O.D. 600 at 0.6–0.8), were induced with 0.5 mM IPTG for 3 h at 30 °C, before being pelleted by centrifugation at 4000 rpm for 30 min at 4 °C. The bacteria were lysed by one freeze/thaw cycle in the presence of phosphate buffered saline (PBS: 137 mM NaCl, 10 mM phosphate, 2.7 mM KCl, pH 7.4) supplemented with 10 mM EDTA, 0.1% (v/v) Triton and protease inhibitors (in all cases: 10 µM leupeptin, 2 µM antipain, 1 mM benzamidine, 1 µg/ml pepstatin, 100 µM PMSF, 1 mM sodium orthovanadate and 1 mM DTT). Bacterial DNA was sheared by pulse sonication. After centrifugation at 13,000 rpm for 10 min at 4 °C, GST4E-BP1 was purified from the bacterial supernatant using glutathioneSepharose beads (Amersham, GE Healthcare). The GST-4E-BP1 protein was dephosphorylated using 50 U shrimp alkaline phosphatase, washed in PBS, 10 mM EDTA, 0.1% (v/v) Triton, then eluted in 10 mM reduced glutathione in PBS pH 7.6. The eluate was desalted using a HiTrap Desalting Column (Amersham, GE Healthcare).

2.1. Plasmid details 2.5. Preparation of mTORC1 Myc-tagged mTOR/pRK5 (Addgene plasmid 1861) and HA-tagged raptor/pRK5 (Addgene plasmid 8513) plasmids were kindly obtained from Dr. D. M. Sabatini [12]. The mutant HA-Raptor vectors contain the following changes: mutant 1 194YDC196-AAA, mutant 2 261DLF263-AAA, mutant 3 313 NWIF 316 -AAAA, mutant 4 391 SQ 392 -PA, mutant 7 738SLQN741-PAAA and mutant 9 1191RVYDRR1196-DAAADD, as described in [10]. GST-4E-BP1/pGEX vectors were generated as previously described [13], with I15A and P16A mutations introduced by sitedirected mutagenesis. The L1460P and E2419K mTOR mutants were based on the active mutants identified by Urano et al. [14]. RagB and RagC constructs were from Addgene and subcloned into a V5-tagged Gateway vector (Invitrogen). Site-directed mutagenesis was used to create the mutants: RagBGTP Q99L, RagBGDP T54L, RagCGTP Q120L and RagCGDP S75L [15]. All site-directed mutagenesis was performed using Phusion DNA polymerase (New England Biolabs (UK) Ltd.) and TOP 10 cells (Invitrogen, Paisley, UK). The FKBP38 vector was a kind gift from Dr G. Camenisch (for construction see [16]) and was further subcloned into a GST-tagged Gateway vector (Invitrogen) according to the manufacturer's protocol. TSC2/pcDNA3.1 was a kind gift from Mark Nellist (Erasmus Medical Centre, The Netherlands).

Myc-mTOR and HA-Raptor transfected cells were treated with 10 µg/ml insulin for 30 min (as indicated in the figures) or left untreated. Cells were washed once in ice cold PBS, then lysed in mTORC1 Lysis Buffer (40 mM HEPES pH 7.4, 2 mM EDTA, 10 mM β-glycerophosphate, 0.3% (w/v) CHAPS and protease inhibitors). Insoluble material was removed by centrifugation and anti-Myc antibody (Sigma) was incubated with the lysates for 2 h at 4 °C. Protein-G Sepharose beads were added and lysates incubated for a further 1 h at 4 °C to immunoprecipitate MycmTOR complexes. Beads were washed once in low salt buffer (40 mM HEPES pH 7.4, 2 mM EDTA, 10 mM β-glycerophosphate, 150 mM NaCl, 0.3% (w/v) CHAPS and protease inhibitors), then either twice in high salt buffer (40 mM HEPES pH 7.4, 2 mM EDTA, 10 mM β-glycerophosphate, 400 mM NaCl, 0.3% (w/v) CHAPS and protease inhibitors) to remove the inhibitory PRAS40 protein [11], or low salt buffer as indicated in the figures. Following two further washes in HEPES/KCl buffer (25 mM HEPES pH 7.4, 20 mM KCl) the beads were split into Eppendorf tubes for the in vitro assay (3 Eppendorf tubes from each original transfected 10 cm plate). 2.6. Preparation of Rheb

2.2. Antibodies and other biochemicals Clone 9E10 anti-Myc antibodies (Sigma, Gillingham, Dorset, UK) were used for immunoprecipitation, while clone 9B11 anti-Myc antibodies (Cell Signalling, Hitchen, Hertfordshire, UK) were used for western blotting. Anti-HA antibody was from Roche (Welwyn Garden City, Hertfordshire, UK), anti-GST from Sigma, anti-Rheb (C19) from Santa Cruz (Heidelberg, Germany) while anti-TSC2, mLST8, phospho-4E-BP1 (Thr 37/46, Ser 65 and Thr 70) and total 4E-BP1 antibodies were obtained from Cell Signalling. Rapamycin was purchased from Calbiochem (Beeston, Nottingham, UK).

GST-Rheb transfected HEK293 cells were washed once in ice cold PBS, then lysed in Rheb lysis buffer (40 mM HEPES pH 7.4, 10 mM β-glycerophosphate, 10 mM pyrophosphate, 5 mM MgCl2, 0.3% (w/v) CHAPS and protease inhibitors (but no DTT)). GST-Rheb was captured using a GST spin trap module (GE Healthcare, UK), washed three times in Rheb lysis buffer, then Rheb storage buffer (20 mM HEPES pH 8, 200 mM NaCl, 5 mM MgCl2) and finally eluted in 10 mM reduced glutathione made up in Rheb storage buffer. Rheb was then loaded with either GDP or non-hydrolysable GTPγS as previously described [11].

2.3. Cell culture and transfection

2.7. Performing the mTOR kinase assay

HEK293 cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% foetal calf serum, 100 U/ml penicillin and 100 µg/ml streptomycin (Gibco, Paisley, UK). CaCl2-mediated transfection was carried out as previously described [2]. Cells were either co-transfected with Myc-tagged mTOR and HA-tagged Raptor constructs or transfected with a GST-tagged Rheb construct (kindly provided by Dr. J. Blenis (Harvard Medical School, Boston)). At 36 h posttransfection, after overnight serum-starvation, these cells were harvested. Cells requiring insulin stimulation were treated with 10 µg/ml insulin (Sigma) for 30 min prior to lysis.

75 ng of Rheb, with or without inhibitors (as indicated in the figures) was made up in 3× mTOR Kinase Assay Buffer (75 mM HEPES pH 7.4, 60 mM KCl, 30 mM MgCl2), then added to the mTOR/Raptor complexed beads. A preincubation at 30 °C for 5 min was performed before the addition of start buffer (25 mM HEPES pH 7.4, 10 mM MgCl2, 140 mM KCl, 0.5 mM ATP and 150 ng dephosphorylated recombinant 4E-BP1). Reactions were performed at 30 °C for 30 min (or longer if indicated in the figures) with constant shaking and stopped by the addition of 4× sample buffer (0.5 M Tris pH 6.8, 2.86 M β-mercaptoethanol, 30% (v/v) glycerol, 0.4 mM bromophenol blue).

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2.8. Western blotting Following boiling at 95 °C for 5 min, mTOR kinase assay samples were resolved by SDS-PAGE. Proteins were transferred to Polyvinylidene Difluoride (PVDF) membranes (Millipore), blocked in 5% (w/v) dry milk powder/Tris buffered saline 0.1% (v/v) Tween (TBS-T), then probed using the required primary antibody and Horse Radish Peroxidase (HRP)-conjugated secondary antibody (Sigma). Proteins were visualised using the Enhanced Chemiluminescent (ECL) solution and Hyperfilm (both GE Healthcare). All western blots shown are representative of at least three independent experiments. 2.9. Raptor overlay assays Raptor-containing lysates were prepared by the transfection of HEK293 cells with HA-tagged Raptor, followed by lysis in Raptor Lysis buffer (50 mM β-glycerol phosphate, 1 mM EDTA, 1 mM EGTA, 1% (v/v) Triton X-100 plus protease inhibitors, pH 7.4) after 48 h. Insoluble material was removed by centrifugation. 50 ng of recombinant 4E-BP1 (or a 4E-BP1 mutant) purified from bacteria was dotted onto PVDF membrane and the membrane then blocked in 5% (w/v) dry milk powder/TBS-T. The membrane was incubated overnight at 4 °C in 5% (w/v) dry milk powder made up in Raptor lysis buffer supplemented with the lysate containing over-expressed HA-Raptor at 10% (v/v). The next day, the membrane was washed then probed using anti-HA antibody, followed by processing and visualisation using the final steps of the standard western blotting protocol. The same principle was applied for analysis of Raptor and Raptor mutant 7 binding to 4E-BP1 mutants (Fig. 4B), however the purified 4E-BP1 protein samples were resolved on a gel rather than dotted onto PVDF membranes. 2.10. Amino acid deprivation and analysis of the mTOR mutants HEK293 cells were transfected with Myc-mTOR mutants and Myc4E-BP1 using Lipofectamine 2000 transfection reagent (Invitrogen) according to the manufacturer's protocol. Cells were changed to serum-

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free medium 4 h post-transfection. For amino acid deprivation, cells were treated for 4 h in Dulbecco's Phosphate Buffered Saline (D-PBS) supplemented with 100 mg/l CaCl2, 0.1 mg/l Fe(NO3), 97.68 mg/l Mg SO4, 200 mg/l KCl, 3.7 g/l NaHCO3, 125 mg/l NaH2PO4, 3.5 g/l D-glucose, 0.67 nM sodium pyruvate (Gibco, Paisley, UK), and 4% Modified Eagle's Medium (MEM) vitamins (pH 8.06) that was equilibrated overnight at 37 °C, 5% (v/v) CO2. For amino acid-stimulation, cells were treated in the above modified D-PBS media that were further supplemented with 30 mg/l glycine, 42 mg/l L-serine, and 0.2 mM L-glutamine (Gibco). Cells were lysed after being stimulated with 100 nM insulin for 30 min, where indicated, in NP-40 Lysis buffer (1 mM EDTA, 5 mM EGTA 10 mM MgCl2, 50 mM β-glycerol phosphate, 0.5% NP-40, 0.1% Brij-35, to pH 7.4.) supplemented with protease inhibitors. 2.11. S6K1 assay HEK293 cells were transfected with Myc-mTOR mutants and HAS6K1 using polyfect transfection reagent (Qiagen, West Sussex, UK) according to the manufacturer's protocol. 4 h post-transfection cells were changed to serum-free medium and incubated overnight prior to insulin stimulation (100 nM insulin for 30 min). Cells were lysed using 10 mM K2PO4 (pH 7.4), 1 mM EDTA pH 7.05, 5 mM EGTA, 10 mM MgCl2, 50 mM β-glycerol phosphate, 1 mM Na3VO4, 100 mM, and 2 mM DTT, plus protease inhibitors. Lysates were incubated for 2 h at 4 °C with protein G-Sepharose beads, and anti-HA antibody. HA immunoprecipitates were washed once each with Buffer A (10 mM Tris,1% nonidet P-40, 0.5% sodium deoxycholate, 100 mM NaCl, 1 mM EDTA and protease inhibitors, pH 7.2), Buffer B (10 mM Tris, 0.1% nonidet P-40, 0.5% sodium deoxycholate, 1 M NaCl, 1 mM EDTA, plus protease inhibitors pH 7.2), and ST Buffer (50 mM Tris–HCL, 5 mM Tris-base, 150 mM NaCl, plus protease inhibitors, pH 7.2). S6K1 complexed beads were incubated for 10 min at 30 °C in 20 mM HEPES, 10 mM MgCl2, 50 µM ATP unlabelled, 5 µCi of [γ-32P]ATP (PerkinElmer Life Sciences), and 3 ng/µl PKI, pH 7.2, in the presence of recombinant GST-S6 peptide (32 final amino acids of ribosomal S6). Reactions were subjected to SDS-PAGE, and the relative levels of [32P]-labelled GST-S6 were determined by autoradiography.

Fig. 1. Single I15A and P16A mutations of the RAIP motif in 4E-BP1 partially affect Raptor binding but do not alter 4E-BP1 phosphorylation by mTOR in vitro. Recombinant GST-4E-BP1 wild-type and F114A, I15A, P16A were either (A), dotted on PVDF membrane at 50 ng or (B), resolved on SDS-PAGE and transferred to PVDF membrane at 3 µg. Raptor overlay assays were performed as detailed in ‘Materials and methods’. Raptor binding was assessed using α-HA antibody. α-GST antibody was used to show levels of GST-4E-BP1 protein. (C), HEK293 lysates transfected with Myc-mTOR and HA-Raptor were subjected to the mTOR kinase assay as described in ‘Materials and methods’ in the presence of Rheb-GTPγS. Levels of phospho-4E-BP1 were analysed using both Thr37/46 and Thr70 phospho-specific antibodies. Levels of Myc-mTOR, HA-Raptor and total GST-4E-BP1 are shown as loading controls. (D) Control blot of the mTORC1 complex shows that the three key components, mTOR, Raptor and mLST8, are present in the mTORC1 kinase assay.

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2.12. HIF1α activity assay These assays were carried out as before [17] using TSC2−/− MEF cells (a kind gift from Dr. D. J. Kwiatkowski) that were transfected using Polyfect Transfection Reagent with a HIF-inducible luciferase reporter. Luminescence was measured after the addition of luciferase reagent (50 mM tricine, 15 mM MgS04, 15 mM KH2PO4, 4 mM EGTA, 2 mM ATP, 1 mM luciferin, pH 7.8). 50 µl of buffer was added to 20 µl of lysate for luminometer analysis. 3. Results 3.1. The influence of the RAIP motif on 4E-BP1 phosphorylation The RAIP motif contained at the N-terminus of 4E-BP1 has been suggested to be important for optimal phosphorylation and regulation of 4E-BP1 by insulin [3]. We wished to verify whether mutations within this motif would affect Raptor association and mTOR-directed phosphorylation of 4E-BP1 in our kinase assay. I15A and P16A mutants were prepared from bacterial cells and used as a substrate for Raptor interaction and

mTOR kinase activity. They were compared to wild-type 4E-BP1 and 4EBP1(F114A) which has the phenylalanine within the TOS motif mutated to an alanine. The TOS motif within 4E-BP1 is required for mTOR-dependent phosphorylation of 4E-BP1 in vivo and in vitro [13]. Far western analysis revealed that Raptor could not bind to 4E-BP1(F114A) but could still associate with the RAIP mutant forms of 4E-BP1 that were dotted onto PDVF membrane (Fig. 1A). To be more precise when comparing these 4EBP1 mutants, we carried out the Raptor overlay assay on 3 µg of these purified GST-4E-BP1 proteins that had first been subjected to SDS-PAGE and then transferred to PVDF membrane (Fig. 1B). Interestingly the I15A and P16A forms of 4E-BP1 showed slightly weaker interaction with Raptor when compared to that of wild-type (37% and 7% reduction in binding, respectively) (Fig. 1B). Kinase assay data showed that both 4E-BP1 RAIP mutants could be phosphorylated at Thr 37/46 by mTORC1 in vitro to a level similar to wild-type, while the F114A mutant could not be phosphorylated (Fig. 1C). As there are discrepancies in the literature as to the importance of the RAIP motif for phosphorylation of the other 4EBP1 sites [4,18–20], we also analysed the phosphorylation of Thr 70 by mTOR in our assay. Analysis showed that phosphorylation at Thr 70 was weaker than that at Thr 37/46, but it showed the same pattern, namely

Fig. 2. mTOR-mediated phosphorylation of 4E-BP1 is modulated by interaction of the RNC domain of Raptor with the TOS motif of 4E-BP1. (A) A schematic of Raptor indicating where the mutations are located is shown and refers to Raptor mutants 1, 2, 3, 4, 7 and 9. These Raptor mutants were used in the Raptor overlay assay and were performed using 50 ng of recombinant GST-4E-BP1 wild-type and the F114A mutant as detailed in ‘Materials and methods’. Raptor binding and total Raptor levels were assessed using α-HA antibody. To show protein loading of GST-4E-BP1, α-GST antibody was employed. (B) HEK293 cells were transfected with Myc-mTOR and one of the HA-Raptor constructs (wild-type, or mutants 1, 2, 3, 4, 7, and 9), then subjected to the mTOR kinase assay as detailed in ‘Materials and methods’, with the exception that the immunoprecipitation step was performed using α-HA antibody rather than α-Myc. Phosphorylation of 4E-BP1 was detected using the phospho-4E-BP1 Thr 37/46 antibody. Levels of Myc-mTOR, HA-Raptor and total GST-4E-BP1 are shown as loading controls. (C) A Raptor overlay assay was performed using Raptor mutant 7 lysate on wild-type 4E-BP1 and the mutants indicated. Total loading is shown using an α-GST antibody.

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that the TOS mutant was weakly phosphorylated but the RAIP mutants were phosphorylated to the same extent as the wild-type 4E-BP1. To confirm that endogenous mLST8 was a component within our in vitro mTORC1 kinase assays we carried out a western blot on purified mTORC1 for mLST8 (Fig. 1D). We show that both endogenous mLST8 and exogenously expressed HA-Raptor co-purifies with immunoprecipitated Myc-mTOR. 3.2. Raptor mutant 4 fails to interact with and phosphorylate 4E-BP1 Previous work by the Sabatini lab characterised a series of Raptor mutants with point mutations that affected their ability to interact with mTOR [10]. We wanted to determine whether these Raptor mutants could still interact with 4E-BP1, so using a far-western

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approach, the interaction of Raptor mutants 1, 2, 3, 4, 7 and 9 (see Fig. 2A) with 4E-BP1 was analysed. We observed that only wild-type Raptor and mutant 7 retained their ability to interact with 4E-BP1 (Fig. 2A). The 4E-BP1(F114A) TOS mutant was unable to interact with Raptor (Fig. 2A). To examine these mutants in more detail, HA-Raptor immunoprecipitations were performed to investigate both the association of mTOR with Raptor as well as Raptor's ability to enhance the activity of mTOR against 4E-BP1 in vitro (Fig. 2B). As previously reported [10], we only saw co-immunoprecipitation of mTOR with wild-type and mutants 4 and 7 of Raptor (Fig. 2B, upper panel). We observed strong levels of phosphorylation of 4E-BP1 when kinase assays using wild-type Raptor or mutant 7 were carried out (Fig. 2B, lower panel, lanes 2 and 7 respectively). Although Raptor mutant 4 still associated with mTOR, we observed only a trace amount of 4E-BP1

Fig. 3. Raptor mutant 4 disrupts normal mTOR signalling to 4E-BP1 in vivo. (A) HEK293 cells over-expressing Myc-mTOR wild-type or mutant (either L1460P or E2419K) along with HA-Raptor (wild-type or mutant 4) were serum-starved, then treated with insulin for 30 min as indicated. To demonstrate insulin stimulation, phosphorylation levels and total PKB are shown. Lysates were analysed by western blotting for total 4E-BP1 and phosphorylation of 4E-BP1 at Thr37/46 and Thr70. Myc-mTOR was immunoprecipitated using NP40 lysis buffer and Myc-mTOR and co-purified HA-Raptor are shown. (B) An S6K assay was performed on serum-starved or insulin-stimulated HEK293 cells transfected with wild-type mTOR along with HA-Raptor (wild-type or mutant 4) and HA-S6K1. Levels of S6K1 phosphorylation on Thr389 and [32P]-radiolabelled GST-S6 were determined by western blotting and autoradiography, respectively. (C) TSC2−/− MEFS over-expressing wild-type Raptor or mutant 4 and a HIF reporter construct were assayed for luciferase activity. The HIF1α transcriptional activity from the pcDNA3.1 empty vector DMOG was standardized to 100%. n = 3. Western blotting was carried out to detect the expression levels of exogenously transfected TSC2 and HA-Raptor.

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phosphorylation. Therefore, only expression of Raptor protein capable of binding 4E-BP1 could facilitate a robust level of mTOR-mediated phosphorylation of 4E-BP1, i.e., both wild-type and mutant 7 (see Fig. 2A and B). The mutation of Raptor mutant 4 (391SQ392-PA) lies within the Raptor N-terminal conserved (RNC) domain (see schematic in Fig. 2A) suggesting that this region of the RNC domain could be involved in mTORC1 substrate recognition rather than mTOR association. Raptor mutant 7, which we previously showed to interact with wild-type 4E-BP1 (Fig. 2A), was found to have further reduced binding to the I15A and P16A 4E-BP1 mutants (Fig. 2C) than wild-type Raptor suggesting that mTORC1 substrate recognition is weaker with Raptor mutant 7. 3.3. Raptor mutant 4 dominantly inhibits mTOR substrate phosphorylation Although Raptor mutant 4 was able to interact with mTOR, we did not detect 4E-BP1 phosphorylation in vitro reflecting its inability to interact with 4E-BP1. These results suggested that Raptor mutant 4 over-expression should prevent mTORC1 substrate recognition. To test this possibility, we examine if Raptor mutant 4 over-expression prevented substrate recognition by mTORC1 in vivo. In the presence of wild-type mTOR and Raptor co-expression, treatment with insulin resulted in phosphorylation of 4E-BP1 at Thr37/46 and Thr70 (Fig. 3A, lanes 1 and 2). Raptor mutant 4 dominantly impaired phosphorylation of 4E-BP1 when cells were stimulated with insulin (Fig. 3A, lanes 3 and 4). The experiment was repeated in the presence of activating mutants of mTOR (L1460P and E2419K) to see if a higher activity of mTOR could overcome the inhibitory effect of Raptor mutant 4. These two point mutations in TOR were previously shown to confer Rheb-independent growth in fission yeast and nutrient-independent activity in mammalian cells [14]. The L1460P and E2419K points mutations are located within the FRAP, ATM and TRRAP (FAT) and kinase domains of mTOR, respectively. While the basal level of 4E-BP1 phosphorylation was increased when the more active form of mTOR was present (Fig. 3A, lanes 1, 5 and 9), Raptor mutant 4 still ablated phosphorylation of 4E-

BP1, both basally and after insulin stimulation (Fig. 3A, lanes 7, 8, 11 and 12). This analysis was extended to other known mTORC1 substrates by examining the effect of Raptor mutant 4 on S6K1 phosphorylation and activity and by analysing the transcriptional activity of HIF1α. Similarly to the observations made with 4E-BP1, Raptor mutant 4 dominantly inhibited insulin induced activation of S6K1 on Thr389 and the activity of S6K1 when cells were insulin-treated (Fig. 3B). Comparing insulin-stimulated conditions with cells over-expressing wild-type Raptor (Fig. 3B, lane 4) versus Raptor mutant 4 (Fig. 3B, lane 6), we observed robust inhibition of Thr389 phosphorylation of S6K1 and S6K1 activity when assayed against the substrate ribosomal protein S6 (rpS6). To analyse HIF1α we utilised TSC2−/− MEFs as they exhibit heightened mTOR activity. As expected, when empty vector pcDNA3.1 (as a negative control) was co-transfected with the HIF1α luciferase reporter construct, luminescence analysis demonstrated high levels of HIF1α transcriptional activity (Fig. 3C, lane 1). This heightened activity of HIF1α was inhibited significantly with rapamycin (Fig. 3C, compare lane 1 versus lane 2, 72% mean reduction) confirming dependency upon mTORC1. Reintroducing functional TSC2 into these cells almost completely repressed HIF1α activity (Fig. 3C, lane 3), which showed an average reduction of 96.3% when compared with empty vector control. Expression of wild-type Raptor showed no significant difference in activity when compared with empty vector (Fig. 3C, lanes 1 and 4), however expression of Raptor mutant 4 markedly impaired the transcriptional activity of HIF1α to a level comparable to that of cells treated with rapamycin (Fig. 3C, compare lanes 4 and 5). 3.4. Active mutants of mTOR are responsive to Rheb and sensitive to FKBP38 To further characterise the mTOR active mutants, L1460P and E2419K, we co-immunoprecipitated the active Myc-mTOR mutant protein with HA-Raptor from serum-starved cells and analysed their response to Rheb in the mTORC1 kinase assay. It was found that both

Fig. 4. Characterising activating mutants of mTOR and their sensitivity to FKBP38 and Rheb-GTPγS. (A) HEK293 cells over-expressing Myc-mTOR wild-type or mutant (either L1460P or E2419K) along with HA-Raptor were serum-starved prior to lysis. The mTOR kinase assay was performed as detailed in ‘Materials and methods’, with or without the addition of Rheb-GTPγS. mTOR activity was analysed by detection of 4E-BP1 phosphorylation at Thr37/46. (B) The assay was performed as described in (A), but the cells were insulin stimulated and the assays were carried out in the presence of Rheb-GTPγS, with or without a preincubation with the inhibitors FKBP12/Rapamycin or FKBP38, as indicated. Phosphorylation of 4E-BP1 at Thr37/46 was determined.

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mTOR mutants had an increased activity towards 4E-BP1 in the presence of Rheb-GTPγS than with no Rheb present (Fig. 4A). Of interest, we found enhanced phosphorylation of 4E-BP1 with the E2419K mutant compared to wild-type, even in the absence of Rheb (Fig. 4A, lanes 1 versus 5). In contrast, the L1460P mutant did not reveal a higher level of activity when compared to wild-type mTOR in this mTORC1 in vitro assay. It should be noted that Urano et al. [14] had previously shown that the L1460P mutant was more active than wild-type mTOR in nutrientdeprived conditions, while the mTOR examined in our assay was purified from serum-starved cell lysates which still contained nutrients. Interestingly, in preparation of the mTORC1 assays, immunoprecipitation of mTOR(L1460P) consistently co-immunoprecipitated a lower level of Raptor than that found complexed with wild-type mTOR or the E2419K mutant (Fig. 4A and also see Fig. 4B below). FK506-binding protein 38 (FKBP38) was recently described as an endogenous mTOR inhibitor [9]. It was postulated that FKBP38 inhibits mTORC1 activity, by a mechanism similar to that of FKBP12/rapamycin. FKBP38 was proposed to inhibit mTORC1 through binding to the FKBP12–rapamycin binding (FRB) domain (that lies proximal to the kinase domain of mTOR) and unlike FKBP12/rapamycin does not require rapamycin for this interaction. We wanted to determine whether these active mutants of mTOR were still sensitive to the inhibitory action of

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FKBP38 in our in vitro mTORC1 kinase assay. To do this, we purified GSTtagged FKBP38 from HEK293 cells and compared its ability to inhibit mTORC1 activity to that of FKBP12/rapamycin. It was found that FKBP38 potently inhibited mTOR-directed phosphorylation of 4E-BP1 to a level similar to that of FKBP12/rapamycin when both wild-type and L1460P mutant mTOR were analysed (Fig. 4B). However, even in the presence of either the FKBP12/rapamycin or FKBP38 inhibitors, the E2419K mutant retained a higher level of activity than the uninhibited wild-type mTOR (compare lanes 1 versus 8 and 9 in Fig. 4B). These findings suggest that the E2419K mutant possesses resistance to the inhibitory action of FKBP38. 3.5. Activation through mTOR(L1460P) is resistant to nutrient withdrawal and inactive RagB–RagC complexes To clarify the differences between our analysis of L1460P and E2419K mTOR mutants and the data presented by Urano et al. [14], we examined whether the L1460P or E2419K mTOR mutants had altered activity (compared to wild-type) in vivo under conditions of nutrient-deprivation and insulin stimulation (Fig. 5A). To analyse mTOR activity, we cotransfected Myc-tagged mTOR with Myc-tagged 4E-BP1 and examined its phosphorylation. 4E-BP1 typically resolves as three isoforms on SDS-

Fig. 5. Analysis of the nutrient sensitivity of mTOR activating mutants. (A) HEK293 cells were transfected with both Myc-4E-BP1 and Myc-mTOR (wild-type, L1460P or E2419K). The cells were treated for 4 h in either serum-free DMEM or supplemented PBS in the presence or absence of amino acids (labelled ‘AA’) (described in ‘Materials and methods’), then treated with insulin before lysis, where indicated. Total Myc-mTOR was then analysed using α-Myc antibodies. The phosphorylation of Myc-4E-BP1 was determined with α-Myc antibodies and phospho-specific antibodies for 4E-BP1 at Thr-37 and/or 46, Ser-65, and Thr-70, as indicated. The α-, β-, and γ-species of 4E-BP1 are labelled. Densitometry analysis was carried out on the α-, β-, and γ-species of 4E-BP1 using ImageJ v1.41 where the protein abundance between these three species is set to 100% (labelled as ‘% 4E-BP1 Isoforms’). (B) HEK 293 cells were transfected with Myc-mTOR (wild-type or L1460P), HA-Raptor, HA-S6K1 and either a control plasmid, an active Rag complex (RagBGTP/RagCGDP) or an inactive Rag complex (RagBGDP/RagCGTP). S6K1 assays were carried on lysates prepared from serum-starved or insulin-stimulated cells. Levels of S6K1 phosphorylation on Thr389 and [32P]-incorporation into GST-S6 were determined by western blotting and autoradiography, respectively.

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PAGE, where the lowest migrating band (referred to as the α-isoform) is hypophosphorylated while the upper migrating band (referred to as the γ-isoform) is hyperphosphorylated. Both the L1460P and E2419K mutants showed higher levels of activity towards 4E-BP1 when compared to the wild-type under serum-starved conditions, as observed by a marked increase in the proportion of 4E-BP1 that resolved as the βisoform in the cells that expressed the L1460P and E2419K mutants (25% for wild-type compared to 50% for both mutants, Fig. 5A, lanes 1, 6 and 11). Interestingly, maximal activation of both wild-type and E2419K mTOR upon insulin stimulation was fully dependent on the presence of amino acids, while the L1460P mutant did not require the permissive amino acid input to mTOR to facilitate 4E-BP1 phosphorylation upon treatment with insulin. For instance, in the mTOR(L1460P) expressing cells which were amino acid-deprived, insulin was sufficient to induce a marked mobility shift to the hyperphosphorylated γ- and β-isoforms, accounting for 15% and 47% of 4E-BP1 isoforms respectively. This was confirmed by the marked 4E-BP1 phosphorylation seen using phosphospecific antibodies for Thr 37/46, Ser65 and Thr 70 (Fig. 5A, compare lanes 4 and 9). Under conditions of amino acid resupply and insulin stimulation, both the L1460P and E2419K mutants demonstrate a higher level activity when compared to that of wild-type, as observed by increased phosphorylation of 4E-BP1 at all sites.

Two recent papers identified the Rag family of GTPases as mediators of amino acid signalling to mTORC1 [15,21]. To determine whether the L1460P mutation altered the sensitivity of mTOR to the Rag input, we generated V5-tagged mutants of RagB and RagC in order to create active and inactive Rag complexes. The active complex (RagBGTP/RagCGDP) causes robust phosphorylation of S6K1, while the inactive complex (RagBGDP/RagCGTP) acts as a dominant inhibitor mimicking nutrient withdrawal [15]. We confirm these findings in the presence of wild-type mTOR (Fig. 5B). However, in contrast to wildtype mTOR, mTOR(L1460P) induced phosphorylation and activation of S6K1 was not dominantly inhibited in the presence of the inactive Rag heterodimer (Fig. 5B, compare lanes 6 and 12). This data shows that the L1460P mutant is resistant to the inhibitory RagBGDP/RagCGTP heterodimers, suggesting that RagB/RagC might signal through the FAT domain, where this L1460P mutant is contained. 3.6. RhebL1 activates mTOR in vitro and is inhibited by FKBP38 After establishing and characterising the mTORC1 assay, we set out to re-address the role of RhebL1 and FKBP38 in regulating mTORC1 activation and signalling. Rheb like-1 protein (RhebL1) has been shown to promote mTOR signalling in a nutrient and rapamycin sensitive

Fig. 6. RhebL1 activates mTOR in vitro, and similar to Rheb is inhibited by FKBP38. (A) An mTOR kinase assay was performed from unstimulated Myc-mTOR and HA-Raptor transfected HEK293 cells as described in ‘Materials and methods’ with the addition of increasing amounts of Rheb (10–75 ng) or RhebL1 (30–250 ng). mTOR activity was determined by analysis of 4E-BP1 phosphorylation on Thr37/46. (B) 75 ng of both Rheb and RhebL1 was incubated in the mTOR kinase assay (based on (A)) along with increasing amounts of FKBP38 (0–25 ng). Phosphorylation of 4E-BP1 at Thr37/46 was used to determine the activity of mTOR. (C) HEK293 cells were transfected with HA-S6K1 and the tagged forms of PRAS40 or FKBP38 indicated. mTORC1 activity was determined by phosphorylation of S6K1 at T389 and phosphorylation of S6 by autoradiography. Long and short exposures of the western blot using α-V5 antibodies are shown to reveal higher levels of V5-FKBP38 protein expression when compared to V5-PRAS40.

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manner, although RhebL1 is a weaker activator of mTOR when compared to Rheb [22]. In our assay we reveal that RhebL1 behaved similarly to Rheb and robustly enhanced mTORC1-mediated phosphorylation of 4E-BP1 (Fig. 6A). It was interesting that we only required a trace amount of either Rheb or RhebL1 to incur mTORC1 activation. It was previously shown that FKBP38 binds to and inhibits Rheb and thus mTORC1 activity [9]. We, therefore, wanted to examine whether FKBP38 could inhibit Rheb as well as RhebL1 in our mTORC1 in vitro assays (Fig. 6B). The action of the inhibitory protein, FKBP38, was tested against Rheb and RhebL1 in vitro (Fig. 6B), with doses of Rheb and RhebL1 based on that shown in Fig. 6A to obtain similar levels of mTORC1-mediated 4E-BP1 phosphorylation. Increasing amounts of FKBP38 (approximately 0 ng, 6.25 ng, 12.5 ng and 25 ng) were incubated with the mTORC1 complexes and levels of phospho-4E-BP1 analysed. It was found that FKBP38 inhibited both Rheb and RhebL1-stimulated mTOR activity in a dose-dependent manner. 3.7. FKBP38 inhibits mTORC1 both in vitro and in vivo The scientific literature currently reports contrasting observations about the inhibitory action of FKBP38 on mTORC1. It was initially identified as an endogenous inhibitor of the complex both in vitro and in vivo [9], but two recent reports have contradicted this finding [23,24], with neither group able to detect any inhibition of mTORC1 signalling in vivo by FKBP38 over-expression. Having shown FKBP38 as an inhibitor of mTORC1 activity in our in vitro assay (Figs. 4B and 6B), we wanted to confirm whether we could also observe inhibition of mTORC1 by FKBP38 in vivo. GST-tagged proteins are known to sometimes cause erroneous results due to the large GST-tag, so in addition to the GST-FKBP38 which we found to be inhibitory in vitro, we also generated a vector to express FKBP38 with a smaller V5 tag to be analysed alongside. To compare the level of mTORC1 repression by these FKBP38 constructs we also analysed two tagged forms of PRAS40, a verified mTORC1 inhibitor [11,25]. The phosphorylation status of S6K1 at Thr389 and the activity of S6K1 against rpS6 following expression of Flag-PRAS40, V5-PRAS40, GST-FKBP38 or V5-FKBP38 were determined (Fig. 6C). Expression of these exogenous proteins was capable of inhibiting signal transduction through mTORC1 in vivo, as observed by the reduction of S6K1 activity with Flag-PRAS40 inducing the strongest inhibition (Fig. 6C, compare lane 4 with the other even-numbered lanes). The GSTFKBP38 and V5-FKBP38 had matched potency to suppress S6K1 activation by insulin stimulation, indicating that their ability to inhibit mTORC1 was specific for FKBP38 and not caused by the tag. This supports the previous study [9] showing FKBP38 as an mTORC1 inhibitor. However, from our over-expression data, it is evident that FKBP38 is a weaker mTORC1 inhibitor when compared to PRAS40. For instance, we observe inhibition of mTORC1 by FKBP38 but the expression of V5-tagged FKBP38 protein is far greater than that of expressed V5-tagged PRAS40 protein (Fig. 6C, compare long and short exposure of V5-tagged PRAS40 and FKBP38). Our data is in accordance with the more recent studies on FKBP38 where lower protein expression of FKBP38 may account for not observing any detectable inhibition of mTORC1 [23,24]. 4. Discussion In this study, we aimed to further characterise the interactions of regulatory protein components involved in mTORC1 signalling as well as to further develop the mTORC1 protein kinase assay so that these techniques could be used to better reflect the regulation of mTORC1 in vitro and in vivo. We utilised various techniques when purifying an active mTORC1 for our in vitro kinase assays. Historically, when studying the in vitro kinase activity of mTOR, it was necessary to employ unphysiological MnCl2 conditions to artificially enhance the weak phosphotransfer reaction that was typically observed when

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traditional MgCl2 kinase assay buffers were used. More recently, work carried out by Sancak et al. [11] showed that an active mTOR/Raptor complex could be co-purified from cells and was able to phosphorylate mTORC1 substrates in the presence of MgCl2. Furthermore, this high level of specific kinase activity of mTORC1 could be markedly improved upon following preincubation with Rheb-GTP [26]. Our adapted mTORC1 kinase assay under physiological MgCl2 conditions comprised of exogenous mTOR/Raptor bound to endogenous mLST8 (Fig. 1D) that retained high activity towards 4E-BP1 (Fig. 1C). Phosphorylation of 4E-BP1 required the presence of the TOS motif, which is concurrent with previous work [13,18]. Our Raptor interaction studies with 4E-BP1 (Fig. 1A and B) is also in agreement with previously published data where the TOS motif present in mTORC1 substrates is necessary for the binding of Raptor [2,13,17,18]. The TOS motif is essential for the scaffold protein Raptor to recruit the mTORC1 substrate in the proper conformation to facilitate optimal mTOR-mediated phosphorylation. Our data disagrees with that published by others which indicated 4E-BP1 substrates containing mutations in the TOS motif could still be phosphorylated in vitro, at least at the Thr37/46 site [4,18,19]. Our data, taken together with that published by Kim et al. [10], has allowed us to conclude that the direct interaction of Raptor with 4E-BP1 through the TOS motif is vital for maximal phosphotransfer by mTORC1. Indeed, we saw no phosphorylation of the 4E-BP1(F114A) TOS mutant when the Raptor mutants in mTORC1 kinase assays were analysed in parallel (data not shown). Additionally, we did not detect any binding of wildtype HA-Raptor to 4E-BP1(F114A) in our Raptor overlay assay (Fig. 2A). This reiterates the importance of the TOS motif in the binding of Raptor to 4E-BP1. The RAIP motif is reported as another region of 4E-BP1 involved in directing mTORC1 phosphorylation [3]. By using our Raptor binding assay we found that the 4E-BP1(I15A) mutant showed reduced Raptor binding when compared to wild-type 4E-BP1 (Fig. 1B). This effect was accentuated further when the wild-type Raptor was replaced with Raptor mutant 7 which still retains its ability to interact with both mTOR and 4E-BP1 (Fig. 2C). However, mutation of I15A or P16A still allowed mTORC1 to phosphorylate both the Thr37/46 and Thr70 sites, confirming previous observations [4]. Other work shows that when all four residues are mutated (i.e. AAAA) phosphorylation by mTOR no longer occurs [18,19] which suggests that while individual residues of the RAIP motif can be altered with minor impact on function, mutations of the whole motif are not well-tolerated. Therefore, while an intact RAIP motif is not essential for Raptor binding, it may play a more subtle role in the complex interaction between Raptor and 4E-BP1. Given that Raptor mutant 7 lost some of its ability to bind to the two 4E-BP1 RAIP mutants also suggests that the region between the HEAT and WD40 repeats of Raptor may be involved in 4E-BP1 interactions. Although it is speculated that Raptor is the substrate recognition component of mTORC1 [18], it has never been conclusively shown that mTOR alone does not play a role in substrate recognition. By utilising our validated Raptor interaction and mTORC1 kinase assays we show that Raptor mutant 4, containing a RNC domain mutation (391SQ392-PA), still associated with mTOR but had lost its ability to bind to and phosphorylate 4E-BP1 (Fig. 2A and B). This data reveals that Raptor binding with the mTORC1 substrates is responsible for facilitating substrate recognition and phospho-transfer by mTOR. For instance, the presence of mTOR alone without functional Raptor in the kinase assay is not sufficient to phosphorylate 4E-BP1 (Fig. 2B). RNC domains are predicted to form α-helices and are unrelated to any other sequences in the public databases [10]. Their conservation between Raptor homologs suggests that these RNC domains play an important role in the function of Raptor, and our data indicates for the first time a role for the RNC domain in substrate binding. This data, together with that published by Kim et al. [10] strongly supports the idea that mutations in Raptor are poorly tolerated and its association with mTOR and downstream targets

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relies on multiple protein–protein interactions, which includes an important role for these RNC domains. Based on the above in vitro findings, we predicted that over-expression of Raptor mutant 4 might prevent mTORC1 substrate recognition in cells. Indeed, when we analysed Raptor mutant 4 in vivo, we confirmed our in vitro findings and identified Raptor mutant 4 as a dominant inhibitor of endogenous mTORC1 signalling. For instance, Raptor mutant 4 potently inhibited 4EBP1 phosphorylation (Fig. 3A), S6K1 activation (Fig. 3B) and HIF activity (Fig. 3C) in mammalian cells, three known mTOR-dependent cell signalling events that require Raptor interaction (see review [27]). Presumably, mTOR/Raptor mutant 4 complexes are favoured in cells when Raptor mutant 4 is over-expressed. Given that these mTOR/Raptor mutant 4 complexes are unable to bind to downstream mTORC1 substrates containing TOS motifs, Raptor mutant 4 over-expression would lead to the dominant inhibition of mTORC1-mediated downstream cell signalling events. The mutants L1310P and E2221K in yeast TOR have been shown to confer Rhb1p independent growth, while their mammalian equivalents, L1460P and E2419K, show constitutive activation when cells are starved of nutrients [14]. We wanted to further characterise these activating mTOR mutants to better understand how mTORC1 was regulated in cells. By using these activating mutants in our mTORC1 kinase assays, we showed that E2419K exhibited vastly enhanced kinase activity towards 4E-BP1, while L1460P showed a level of activity that was lower than that of wild-type mTOR (Fig. 4A). In vivo analysis of 4E-BP1 phosphorylation showed both mTOR mutants exhibited higher basal 4E-BP1 phosphorylation than wild-type under conditions of nutrient availability (Fig. 3A) and nutrient-deprivation (Fig. 5A). At first we were surprised that the activity of the L1460P mutant was so low in the in vitro mTORC1 kinase assay yet in cells the L1460P mutant resulted in a higher basal level of 4E-BP1 phosphorylation when compared to wild-type mTOR. The reduced level of activity of the L1460P mutant in the mTORC1 kinase assay (Fig. 4A) might be a result of losing Raptor protein during preparation of the mTORC1 assays. Under wash conditions used to generate mTORC1 for the kinase assay, we found that mTOR(L1460P) co-purified with less Raptor when compared to that of wild-type mTOR or the E2419K mutant (Fig. 4A and B). It is known that nutrient-rich conditions result in a weaker interaction of Raptor with mTOR [10] and suggests that mTOR(L1460P)-Raptor exists in an active but loose-conformation that mimics a nutrient-supplied status. This notion is reinforced by our in vivo analysis of the L1460P mTOR mutant where we see that mTOR (L1460P) responds to insulin stimulation, even under nutrientdeprived conditions (Fig. 5A). This increased activity of mTOR (L1460P) under nutrient-deprived conditions is also concurrent with the original observations by Urano et al. [14]. It is considered that mTOR acts as a nutrient sensor within the cell. This is because mTOR requires a permissive nutrient input that is a prerequisite before it is activated in response to hormones and growth factors, such as insulin via the PI3K–Akt signalling pathway. The L1460P mutant appears to be responsive to insulin treatment even in an amino acid-deprived state, which suggests that the L1460P mutation within the FAT domain affects the ability of mTOR to respond to cellular nutrient levels. The precise mechanism of how mTORC1 senses amino acid supply is still not fully understood. However, a milestone to understanding this elusive mechanism was recently made with the discovery of the RagGTPases [15,21]. These Rag proteins function as a heterodimer that only bind to Raptor and promote mTORC1 signalling when the Rag heterodimer is reverted to an active state when amino acids are in supply [15]. Furthermore, Sabatini's group revealed that these active Rag heterodimers were responsible for mTORC1 activation in cells by amino acid dependant intracellular localisation of mTOR to endomembrane compartments where Rheb was also localised [15]. These studies show that these Rag heterodimers function upstream of mTORC1 and downstream of an amino acid signal. In our study, we utilised the active RagBGTP/RagCGDP heterodimers that were pre-

viously showed to activate mTORC1 and the inactive RagBGDP/RagCGTP heterodimers that were unable to interact with Raptor and inhibit mTORC1 activity by mimicking a nutrient-starved status [15]. Our data supports Sancak et al.'s findings [15], showing that the inactive RagBGDP/RagCGTP heterodimers potently repress mTORC1 signalling in vivo (Fig. 5B). Upon further characterisation of the active L1460P mutant of mTOR, which this study (Fig. 5A) and another study [14] observed as being resistant to inhibition by nutrient withdrawal, we observed that mTOR(L1460P) was also resistant to the dominant inhibitory effects of the inactive RagBGDP/RagCGTP complexes (Fig. 5B). This finding indicates that the L1460P mutation renders mTOR less sensitive to nutrient-deprivation and involves Rag heterodimer signalling. This finding suggests for the first time that a protein region within mTOR, i.e., the FAT domain where the L1460P mutation is located, could be involved in responding to the permissive nutrient input. The role of the FAT domain has not yet been elucidated, although it has previously been postulated to mediate protein–protein interactions [28]. It remains to be determined how the nutrient input to the FAT domain of mTOR is coordinated, but our data highlights that activation of mTORC1 by Rag heterodimers is likely to involve signal transduction through the FAT domain. Unlike the L1460P mutant, mTOR(E2419K) still required the presence of amino acids to be responsive to insulin stimulation (Fig. 5A). Another difference was apparent when we compared these active mTOR mutants in our mTORC1 assays; we saw that the E2419K mutant was resistant to the inhibitory actions by both FKBP12/ rapamcyin and FKBP38 while the L1460P mutant was robustly repressed (Fig. 4B). Rapamycin is a well-characterised exogenous inhibitor of mTOR which functions as a rapamycin/FKBP12 complex in cells. FKBP38 is another member of this family and recently was discovered as an endogenous inhibitor of the pathway [9]. Our data supports this inhibitory function of FKBP38, which is currently under scrutiny in the literature. During our investigations, we revealed that FKBP38 potently repressed mTORC1 activity to a level equivalent to that observed with rapamycin/FKBP12, at least in vitro (Fig. 4B). Although rapamycin/FKBP12 or FKBP38 inhibits mTORC1 signalling via interaction with the FRB domain of mTOR, the exact mechanism of how this causes inhibition is currently unknown. Given that the FRB domain lies proximal to the kinase domain, one could postulate that either rapamycin/FKBP12 or FKBP38 binding to the FRB region could cause a conformational change that would repress the kinase domain within mTORC1. This notion could explain the higher levels of kinase activity that we observe when using the E2419K mutant during our FKBP38 study (Fig. 4B). As the E2419K point mutation lies within the kinase domain and results in a change of charge, this could enhance the phosphotransfer observed in our kinase assay and also render this E2419K mutant less susceptible to inhibition by FKBP38. It is well documented that mTOR is regulated by the GTPaseactivating protein (GAP) activity of the TSC1/2 heterodimer towards Rheb, that reverts Rheb to an inactive state [6]. GTP-bound Rheb is a potent activator of mTORC1, as is demonstrated in our mTORC1 assay (Fig. 6A) and by previous in vitro studies [11,26]. RhebL1, a similar protein from the Ras family of GTPases, is known to activate mTOR in vivo, although less potently than Rheb [22]. We wanted to examine whether RhebL1 was as potent at enhancing mTORC1 in vitro as Rheb. By substituting RhebGTP with RhebL1GTP in the in vitro mTORC1 kinase assay, we demonstrated that low levels of either RhebGTP or RhebL1GTP was sufficient to induce high levels of 4E-BP1 phosphorylation (Fig. 6A), with both appearing to be equally as potent as the other. Higher doses of RhebGTP or RhebL1GTP did not further enhance 4E-BP1 phosphorylation, indicating that a trace amount of these small G-proteins was ample to potently activate mTORC1 in vitro. While it is clear that activation of mTORC1 requires GTP-loaded Rheb or RhebL1 [22], it is not completely understood how these small G-proteins activate mTORC1. Rheb has been shown to interact with mTOR independent of its nucleotide binding status [26,29] and has also been shown to interact with Raptor in vitro

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Fig. 7. Schematic of mTORC1 signalling. A number of negative regulators are involved in repressing the activity of the mTORC1 complex. The binding of PRAS40 to Raptor acts to negatively regulate the complex, as does the inhibition of the mTOR kinase domain by FKBP38. Both FKBP38 and FKBP12/rapamycin bind to the FRB region of mTOR. The FAT domain of mTOR appears to be at least partially responsible for sensing nutrient levels, with the Rag family of proteins being involved in amino acid sensing. Upon amino acid resupply and insulin stimulation mTORC1 becomes active through a series of signalling events: (i) dissociation of FKBP38 from mTOR, which requires interaction of either RhebGTP or RhebL1GTP to FKBP38. Although we do not show this in the model, we cannot exclude the possibility that Rheb might not be modulating mTORC1 activity via direct interaction with either mTOR or Raptor or both. (ii) Dissociation of PRAS40 from Raptor that is dependent on PKB-dependent phosphorylation of PRAS40 and (iii) recruitment of 4E-BP1 to mTOR which involves residues within the second RNC domain of Raptor and the RAIP and TOS motif of 4E-BP1 to mediate the interaction.

[30]. Furthermore, Both Rheb and mTOR were observed to co-localise to endomembrane compartments [15]. It is possible that both Rheb and RhebL1 enhance mTORC1 activity via direct binding of this kinase complex, which occurs predominantly in endomembrane compartments. Another possibility is via indirect activation of mTORC1 that involves the displacement of the negative regulator, FKBP38, from mTOR by Rheb [9]. The study by Bai et al. [9] showed that Rheb interacted with FKBP38 and prevented FKBP38–mTORC1 association. We support these findings by demonstrating that FKBP38 could efficiently inhibit both Rheb and RhebL1-enhanced mTORC1 activation in vitro (Fig. 6B). Although the inhibition we observed by FKBP38 was robust (Fig. 4B) and was sufficient to over-ride Rheb and RhebL1-induced activation of mTORC1 in vitro (Fig. 6B), we observed a weaker level of inhibition in vivo (Fig. 6C). From our investigation in cells, it was apparent that FKBP38 was a weaker inhibitor than that of PRAS40, which is known to bind to Raptor and inhibit mTORC1 signalling [11,25]. During preparation of this manuscript, several studies on FKBP38 revealed no evidence that FKBP38 inhibited the phosphorylation of mTORC1 substrates when FKBP38 was over-expressed cells [23,24]. The discrepancy between the studies on FKBP38 is most likely to be due to the levels of FKBP38 protein over-expression, where it appears necessary to over-express FKBP38 to a higher degree than that of PRAS40 to observe mTORC1 inhibition. From our data generated in combination with previously published work by other groups [9–11,15], we propose that mTORC1 converts from the inactive to the active form via the dissociation of FKBP38 from mTOR through binding to either Rheb-GTP or RhebL1-GTP, the dissociation of PRAS40 from Raptor by PKB phosphorylation and the recruitment of 4E-BP1 to the complex, mediated at least in part through the RNC domain of Raptor (Fig. 7). The permissive nutrient input is sensed by an unknown mechanism but is dependent on the RagGTPase proteins [15,21] where signal transduction from RagGTPase heterodimers through mTORC1 involves residues within the FAT domain of mTOR.

5. Conclusions Our in vitro mTORC1 kinase and Raptor interaction assays have allowed us to explore various elements of the mTORC1 signal transduction pathway. Our study enabled us to identify that the RNC region of Raptor is involved in substrate recognition by mTORC1 and sequentially these investigations led to the finding that Raptor mutant 4 could dominantly inhibit endogenous mTORC1 signal transduction in vivo. Our techniques also allowed us to further characterise the RAIP and TOS motifs of 4E-BP1. Further characterisation of the constitutively activated mTOR mutants revealed the importance of the FAT domain, which when mutated, leads to a kinase activity that is more resistant to nutrient-deprivation. These investigations suggest that this FAT domain within mTOR is involved in the permissive nutrient input and is dependent on cell signal transduction from RagGTPases. This work has also strengthened the original hypothesis that FKBP38 appears to function an mTORC1 inhibitor. Importantly, the procedures used in this manuscript will help facilitate the identification and characterisation of further proteins involved in the regulation of the mTORC1 pathway and will enhance our understanding of mTORC1-mediated signalling. Acknowledgements This research was supported by the Association for International Cancer Research Career Development Fellowship [No. 06-914/915] (to A. Tee), and funding from the Tuberous Sclerosis Association (to A. Tee and L. Seymour). We also thank Wales Gene Park. References [1] E. Jacinto, R. Loewith, A. Schmidt, S. Lin, M.A. Ruegg, A. Hall, M.N. Hall, Nat. Cell Biol. 6 (2004) 1122.

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