An ATP-competitive Mammalian Target of Rapamycin Inhibitor Reveals Rapamycin-resistant Functions of mTORC1
The mammalian target of rapamycin (mTOR) kinase is the catalytic subunit of two functionally distinct complexes, mTORC1 and mTORC2, that coordinately promote cell growth, proliferation, and survival. Rapamycin is a potent allosteric mTORC1 inhibitor with clinical applications as an immunosuppressant and anti-cancer agent. Here we find that Torin1, a highly potent and selective ATP-competitive mTOR inhibitor that directly inhibits both complexes, impairs cell growth and proliferation to a far greater degree than rapamycin. Surprisingly, these effects are independent of mTORC2 inhibition and are instead because of suppression of rapamycin-resistant functions of mTORC1 that are necessary for cap-dependent translation and suppression of autophagy. These effects are at least partly mediated by mTORC1-dependent and rapamycin-resistant phosphorylation of 4E-BP1. Our findings challenge the assumption that rapamycin completely inhibits mTORC1 and indicate that direct inhibitors of mTORC1 kinase activity may be more successful than rapamycin at inhibiting tumors that depend on mTORC1. The mammalian target of rapamycin (mTOR) kinase is the catalytic subunit of two functionally distinct complexes, mTORC1 and mTORC2, that coordinately promote cell growth, proliferation, and survival. Rapamycin is a potent allosteric mTORC1 inhibitor with clinical applications as an immunosuppressant and anti-cancer agent. Here we find that Torin1, a highly potent and selective ATP-competitive mTOR inhibitor that directly inhibits both complexes, impairs cell growth and proliferation to a far greater degree than rapamycin. Surprisingly, these effects are independent of mTORC2 inhibition and are instead because of suppression of rapamycin-resistant functions of mTORC1 that are necessary for cap-dependent translation and suppression of autophagy. These effects are at least partly mediated by mTORC1-dependent and rapamycin-resistant phosphorylation of 4E-BP1. Our findings challenge the assumption that rapamycin completely inhibits mTORC1 and indicate that direct inhibitors of mTORC1 kinase activity may be more successful than rapamycin at inhibiting tumors that depend on mTORC1. Correction: An ATP-competitive mammalian target of rapamycin inhibitor reveals rapamycin-resistant functions of mTORC1.Journal of Biological ChemistryVol. 295Issue 9PreviewVOLUME 284 (2009) PAGES 8023–8032 Full-Text PDF Open Access The mammalian target of rapamycin (mTOR) 3The abbreviations used are: mTOR, mammalian target of rapamycin; Raptor, regulatory associated protein of mTOR; Rictor, rapamycin-insensitive companion of mTOR; PI3K, phosphatidylinositol 3-kinase; LC3, light chain 3; 4E-BP1, eIF4E-binding protein 1; eIF4E, eukaryotic initiation factor 4E; GβL, G-β subunit-like; MEF, mouse embryonic fibroblast; mTORC1, mTOR complex 1; mTORC2, mTOR complex 2; HEK, human embryonic kidney; DMSO, dimethyl sulfoxide; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; ATM, ataxia telangiectasia, mutated; PI, phosphatidylinositol; BSA, bovine serum albumin; PBS, phosphate-buffered saline; shRNA, short hairpin RNA. pathway is considered a major regulator of cell growth. The mTOR serine/threonine kinase is the founding component of the pathway and the catalytic subunit of two functionally distinct protein complexes, mTORC1 and mTORC2. mTORC1 contains the large protein Raptor, as well as mLST8/GβL and PRAS40, whereas mTORC2 is defined by the protein Rictor and also includes Sin1, Protor, and mLST8/GβL (1Guertin D.A. Sabatini D.M. Cancer Cell. 2007; 12: 9-22Abstract Full Text Full Text PDF PubMed Scopus (2433) Google Scholar). Growth factors, such as insulin and IGF, activate both complexes, and they are important downstream effectors of the PI3K/PTEN signaling network (2Manning B.D. Cantley L.C. Cell. 2007; 129: 1261-1274Abstract Full Text Full Text PDF PubMed Scopus (4763) Google Scholar). Additionally, the availability of nutrients, like amino acids and glucose, regulates mTORC1. Many insights into mTOR signaling have come from investigations into the mechanism of action of rapamycin, a bacterially produced macrolide inhibitor of mTOR that has diverse clinical applications as an anti-fungal, immunosuppressant, and anti-cancer drug (3Sehgal S.N. Transplant. Proc. 2003; 35: S7-S14Crossref PubMed Scopus (600) Google Scholar). Rapamycin acts through an unusual allosteric mechanism that requires binding to its intracellular receptor, FKBP12, for inhibition of its target. Under acute treatment, rapamycin is thought to selectively inhibit mTORC1, which is often referred to as the rapamycin-sensitive complex. Conversely, mTORC2 is considered rapamycin-insensitive, although its assembly can be inhibited by prolonged rapamycin treatment in some cell types (4Sarbassov D.D. Ali S.M. Sengupta S. Sheen J.H. Hsu P.P. Bagley A.F. Markhard A.L. Sabatini D.M. Mol. Cell. 2006; 22: 159-168Abstract Full Text Full Text PDF PubMed Scopus (2207) Google Scholar). Because of its perceived potency and selectivity, rapamycin is commonly used in research experiments as a test of the involvement of mTORC1 in a particular process. Two downstream mTORC1 substrates that were identified, in part, by their sensitivity to rapamycin are the S6 kinases (S6K1 and S6K2) and the translational inhibitor 4E-BP1. Both proteins mediate important links between mTORC1 and the cell growth machinery, largely through their influence on cap-dependent translation (reviewed in Ref. 5Richter J.D. Sonenberg N. Nature. 2005; 433: 477-480Crossref PubMed Scopus (761) Google Scholar). All nuclear-encoded mRNAs possess a 5′,7-methyl guanosine cap, which is recognized and bound by the small protein eIF-4E. Under growth-promoting conditions, eIF-4E also associates with the large scaffolding protein eIF-4G, the eIF-4A helicase, and the eIF-4B regulatory protein, together forming the eIF-4F complex. This complex, in conjunction with the eIF3 preinitiation complex, delivers the mRNA to the 40 S ribosomal subunit and primes the translational apparatus. 4E-BP1 interferes with this process by binding to eIF-4E and preventing the formation of a functional eIF-4F complex. However, its ability to do this is blocked by phosphorylation at four sites, two of which are considered rapamycin-sensitive. S6K1 also plays a role in regulating translational initiation by phosphorylating the S6 protein of the 40 S ribosomal subunit and by stimulating eIF-4A helicase activity (6Holz M.K. Ballif B.A. Gygi S.P. Blenis J. Cell. 2005; 123: 569-580Abstract Full Text Full Text PDF PubMed Scopus (913) Google Scholar, 7Raught B. Peiretti F. Gingras A.C. Livingstone M. Shahbazian D. Mayeur G.L. Polakiewicz R.D. Sonenberg N. Hershey J.W. EMBO J. 2004; 23: 1761-1769Crossref PubMed Scopus (368) Google Scholar, 8Shahbazian D. Roux P.P. Mieulet V. Cohen M.S. Raught B. Taunton J. Hershey J.W. Blenis J. Pende M. Sonenberg N. EMBO J. 2006; 25: 2781-2791Crossref PubMed Scopus (407) Google Scholar). Despite the connections of mTORC1 to the translational machinery, the effects of rapamycin on mammalian cell growth and proliferation are, oddly, less severe than its effects in yeast. In Saccharomyces cerevisiae, rapamycin treatment induces a starvation-like state that includes a severe G1/S cell cycle arrest and suppression of translation initiation to levels below 20% of nontreated cells (9Barbet N.C. Schneider U. Helliwell S.B. Stansfield I. Tuite M.F. Hall M.N. Mol. Biol. Cell. 1996; 7: 25-42Crossref PubMed Scopus (603) Google Scholar). Moreover, in yeast rapamycin strongly promotes induction of autophagy (self-eating), a process by which cells consume cytoplasmic proteins, ribosomes, and organelles, such as mitochondria, to maintain a sufficient supply of amino acids and other nutrients (10Noda T. Ohsumi Y. J. Biol. Chem. 1998; 273: 3963-3966Abstract Full Text Full Text PDF PubMed Scopus (1052) Google Scholar). The effects of rapamycin in mammalian cells are similar to those in yeast, but typically much less dramatic and highly dependent on cell type. For instance, rapamycin only causes cell cycle arrest in a limited number of cell types and has modest effects on protein synthesis (11Neshat M.S. Mellinghoff I.K. Tran C. Stiles B. Thomas G. Petersen R. Frost P. Gibbons J.J. Wu H. Sawyers C.L. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 10314-10319Crossref PubMed Scopus (913) Google Scholar, 12Pedersen S. Celis J.E. Nielsen J. Christiansen J. Nielsen F.C. Eur. J. Biochem. 1997; 247: 449-456Crossref PubMed Scopus (40) Google Scholar, 13Shor B. Zhang W.G. Toral-Barza L. Lucas J. Abraham R.T. Gibbons J.J. Yu K. Cancer Res. 2008; 68: 2934-2943Crossref PubMed Scopus (114) Google Scholar). Moreover, rapamycin is a relatively poor inducer of autophagy, and it is often used in combination with LY294002, an inhibitor of PI3K and mTOR (14Takeuchi H. Kondo Y. Fujiwara K. Kanzawa T. Aoki H. Mills G.B. Kondo S. Cancer Res. 2005; 65: 3336-3346Crossref PubMed Scopus (473) Google Scholar). These inconsistent effects may explain why, despite high expectations, rapamycin has had only limited success as a clinical anti-cancer therapeutic. We have hypothesized that the effectiveness of rapamycin against a particular cancer might be determined by its ability to inhibit mTORC2 in addition to mTORC1 (15Sabatini D.M. Nat. Rev. Cancer. 2006; 6: 729-734Crossref PubMed Scopus (1139) Google Scholar). To test this hypothesis, we developed the ATP-competitive inhibitor Torin1 that suppresses both complexes. In contrast to rapamycin, Torin1 treatment recapitulates in mammalian cells many of the phenotypes caused by TOR inhibition in yeast. Surprisingly, however, we find that these effects are independent of mTORC2 and are instead caused by inhibition of rapamycin-resistant functions of mTORC1. Materials-Reagents were obtained from the following sources: antibodies to phospho-Thr-389 S6K, phospho-Ser-473 Akt, phospho-Thr-308 Akt, pan-Akt, phospho-Thr-36/47 4E-BP1, phospho-Ser-65 4E-BP1, phospho-Thr-70 4E-BP1, 4E-BP1, α-tubulin, Raptor, eIF-4E, phospho-S51 eIF2α, cyclin D1, cyclin D3 and p27/Kip1 from Cell Signaling Technology (note: we have not confirmed that the phospho-Thr-70 4E-BP1 antibody does not detect unphosphorylated 4E-BP1); antibodies to mTOR, S6K, and horseradish peroxidase-labeled anti-mouse, anti-goat, and anti-rabbit secondary antibodies from Santa Cruz Biotechnology; anti-Rictor antibodies from Bethyl Laboratories; FuGENE 6 and Complete Protease Mixture from Roche Applied Science; FLAG M2 antibody, FLAG M2-agarose, and from from from from rapamycin from Laboratories; from and from phosphatidylinositol and from protein and from from serum from as well as were by and in with and have D.A. D.M. J. M. Sabatini D.M. Cell. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar). Torin1 and in the and is Cell with were in and and of inhibitors The of cell were by at for in a were obtained from the of The at the J. D.A. G. B. B. N. Sabatini D.M. Cell. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar). These are with the at the mouse shRNA, and mouse Rictor shRNA, were with the and into FuGENE 6 as S.M. Sabatini D.M. J. Biol. Chem. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar, D.D. D.A. Ali S.M. Sabatini D.M. 2005; PubMed Scopus Google Scholar). were at and to and target cells were in the of cells were with and on the were in and were with for with and in of and of protein cells were and were by at for To protein, were on with two for in two for in for in and at The of into protein a mTORC1 and mTORC2 in mTORC1, we cell that For mTORC2, we cells that Both were by cells in were at for and the by at for were with for and with and with a of mTORC1 with FLAG in can be and at S6K1 and were as D.A. D.M. J. M. Sabatini D.M. Cell. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar, Y. Sabatini D.M. Mol. Cell. 2007; 25: Full Text Full Text PDF PubMed Scopus Google Scholar). were for at in a of of the kinase and of S6K1 as were by the addition of of and for were by and PI3K and for were determined were with of for and of by a In for were determined as B. D.D. R. D. A. B. T. Cell. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar). of phosphatidylinositol and were in in to a and and at For kinase with and of in kinase and at for were with with a of and on phosphatidylinositol by as a protein from T. M. M. P. F. Thomas G. Proc. Natl. Acad. Sci. U. S. A. 2005; PubMed Scopus Google and the and kinase with from the of and in kinase and for at were with and were for in and for in and by in kinase were to B. D.D. R. D. A. B. T. Cell. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar). Cell were in and to cells were by in a with and cell determined a with Cell with the Cell were with cells well and cells were with the and on For were for at of to and were on an for on a Cell were in and were to the treatment for and in and at in were in and in PBS, BSA, and at for were in BSA, in PBS, and a Cell cycle determined the Torin1 a and mTOR small ATP-competitive inhibitors of mTOR, we a for inhibitors of mTOR kinase activity in a of this we a that through a to Torin1, a of the of kinase S. in In in kinase mTORC1 mTORC2, Torin1 inhibits both with between and and acts through an ATP-competitive mechanism We also the potency of Torin1 in were with of Torin1 the inhibitors and and the activity of complex determined by the phosphorylation of at and at mTORC1 and mTORC2 in the for Torin1 in cells is also between and rapamycin, Torin1 had on the of mTORC1 mTORC2. We determined the of Torin1 for mTOR other Because mTOR to the kinase a of protein kinases that is defined by a high degree of to PI3K the catalytic many inhibitors of PI3K, such as LY294002, and are also mTOR inhibitors B. D.D. R. D. A. B. T. Cell. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar, J. C. G. Abraham R.T. EMBO J. 1996; PubMed Scopus Google Scholar, S.M. F. J. P. C. C. S. P. A. K. D. D. M. L. P. C. Mol. Cancer 2008; 7: PubMed Scopus Google Scholar). To PI3K inhibition in we of the that the phosphorylation of at on two that directly PI3K phosphatidylinositol of to the and of the kinase that directly this In phosphorylation of is also by phosphorylation at D.D. D.A. Ali S.M. Sabatini D.M. 2005; PubMed Scopus Google Scholar, A. M. EMBO J. 2001; PubMed Scopus Google Scholar, Mol. Cell. Biol. 22: PubMed Scopus Google Scholar). To this we in an mTORC2 is and is Because is in a state in these cell phosphorylation at only PI3K this we determined the of Torin1 for PI3K to be to in of the for We also against other PI3K the in which confirmed a high degree of for mTOR that inhibit PI3K and mTOR also have the to inhibit other the kinases and For and ATM, we the of Torin1 in We also inhibition of the PI3K have that acts of mTORC1, and we to be that with this kinase not mTORC1 activity in cells T. M. M. P. F. Thomas G. Proc. Natl. Acad. Sci. U. S. A. 2005; PubMed Scopus Google Scholar). Torin1 at least selective for mTOR of these we Torin1 at a of against a of diverse kinases the which the binding of the target to and we of effects in These that Torin1 is a highly selective inhibitor of mTOR against an of and Torin1 Cell through a of to test the role of mTOR signaling in mTORC1 inhibition cell proliferation and cell and we that inhibition have similar but more severe effects T. M. M. P. F. Thomas G. Proc. Natl. Acad. Sci. U. S. A. 2005; PubMed Scopus Google Scholar). with to rapamycin to at a and In Torin1 completely inhibited proliferation and and caused a G1/S cell cycle arrest Moreover, Torin1 cell to a greater degree than rapamycin on the assumption that rapamycin completely mTORC1 kinase we hypothesized that the of Torin1 because of mTORC2 To test this hypothesis, we experiments that mTORC2 activity because Rictor has D.A. D.M. J. M. Sabatini D.M. Cell. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar). We that Torin1 have the as rapamycin on the proliferation and growth of these cells because mTORC2 is in rapamycin but not proliferation However, we were to find that Torin1 to proliferation and cell that the effects of this with to rapamycin were not to mTORC2 mTOR has functions that are for cell growth and proliferation and that are and independent of mTORC2. Torin1 mTORC1-dependent than the assumption to the for is that rapamycin inhibits some but not of the functions of mTORC1. To this we the effects of Torin1 on other growth and proliferation that are commonly associated with mTORC1 such process is often referred to as autophagy. considered a to conditions, autophagy the formation of large that cytoplasmic both proteins and (reviewed in Ref. N. B. Nature. 2008; PubMed Scopus Google Scholar). These with to that their the cell with a of amino acids and other nutrients these are not from the In yeast, rapamycin is a potent of autophagy (10Noda T. Ohsumi Y. J. Biol. Chem. 1998; 273: 3963-3966Abstract Full Text Full Text PDF PubMed Scopus (1052) Google Scholar). The is less in mammalian rapamycin at an inconsistent of autophagy and requires combination with other such as LY294002, for We that autophagy might also be in by rapamycin-resistant functions of mTORC1. commonly used of autophagy is the protein light chain which from the to it is autophagy is Y. N. T. A. T. T. Ohsumi Y. T. EMBO J. PubMed Scopus Google Scholar). a we that Torin1 causes a of from the to in both and whereas rapamycin caused only a we that Torin1 treatment, like amino causes of and of the in both and cells and An in in a similar as Torin1 treatment these that mTORC1 inhibition is sufficient to autophagy. the signaling that mTORC1 to autophagy are ATP-competitive like Torin1, for mTORC1 that have because of their to rapamycin. The mTORC1 pathway also has many connections to the of cap-dependent However, rapamycin often has only modest effects on of protein To test Torin1 might inhibit protein synthesis more we cells in the of Torin1 rapamycin. Surprisingly, whereas rapamycin had Torin1 caused a in protein synthesis in both and with autophagy, these indicate that mTORC1 is a far more important regulator of protein synthesis than experiments with rapamycin have of mTORC1 for mTORC1 and 4E-BP1, are important of mRNA we considered is in the of mTORC1-dependent but rapamycin-resistant activity has to be completely inhibited by rapamycin treatment, and we considered it to be the target of rapamycin-resistant activity of mTORC1. 4E-BP1, however, is to a more complex regulatory process. The ability of 4E-BP1 to and inhibit eIF-4E is by the phosphorylation of four and of and is thought to be a that the phosphorylation of the other from eIF-4E and the formation of a functional eIF-4F complex A.C. Gygi S.P. Raught B. Polakiewicz R.D. Abraham R.T. M.F. R. Sonenberg N. PubMed Scopus Google Scholar). mTORC1 has in the of 4E-BP1, but are of the of this as well as the For instance, mTORC1 the and in but these are considered rapamycin-insensitive in cells Cohen Sabatini D.M. Proc. Natl. Acad. Sci. U. S. A. 1998; PubMed Scopus Google Scholar, L. C. Blenis J. Mol. Cell. Biol. 2004; PubMed Scopus Google Scholar, A. M. S. Mol. Cell. Biol. 2005; 25: PubMed Scopus Google Scholar). Conversely, mTORC1 has in on the phosphorylation of that are considered and Moreover, a in 4E-BP1, as the TOR signaling and to mediate binding to mTORC1, and the are for phosphorylation of J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar, Sabatini D.M. Blenis J. Biol. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar, Mol. Cell. Biol. 22: PubMed Scopus Google Scholar). although rapamycin causes a in protein translation in some cell types L. Gingras A.C. Hall M.N. Sonenberg N. EMBO J. 1996; PubMed Scopus Google it has in B. Zhang W.G. Toral-Barza L. Lucas J. Abraham R.T. Gibbons J.J. Yu K. Cancer Res. 2008; 68: 2934-2943Crossref PubMed Scopus (114) Google Scholar). is that rapamycin completely inhibit mTORC1-dependent phosphorylation of 4E-BP1. To test this hypothesis, we with of Torin1 rapamycin and the phosphorylation of and by Rapamycin completely phosphorylation of S6K1 and caused a in the phosphorylation of of 4E-BP1, but it had on the phosphorylation of at as high as greater than its for inhibition of mTORC1 In Torin1 phosphorylation of and at as as and it completely at Torin1 had effects in with the that these effects are because of inhibition of mTORC1 Surprisingly, by Torin1 rapamycin, that it may be the target of a such as J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). it is that the 4E-BP1 antibody is not The inhibitors and caused similar effects as Torin1 on 4E-BP1 phosphorylation Additionally, Torin1 had much greater effects than rapamycin on 4E-BP1 phosphorylation in a of human cell that rapamycin of mTORC1 is a of not mammalian We the of 4E-BP1 by Torin1 to with eIF-4E. to eIF-4E from cell we that Torin1 causes more binding of 4E-BP1 to eIF-4E than does rapamycin and Torin1 not the phosphorylation of Because the effects of Torin1 were in and we that they not be dependent on mTORC2. However, it that mTOR an were To that mTORC1 inhibition is sufficient to explain the effects of Torin1 on 4E-BP1 we used to Raptor, an mTORC1 in of in these cells and phosphorylation and 4E-BP1 to a degree that the effects of Torin1 and those of rapamycin, the that mTORC1, at least a mTOR complex, regulates 4E-BP1 phosphorylation through a rapamycin-insensitive in cap-dependent translation are also to cell cycle This is thought to through translation of cap-dependent mRNAs that that promote cell cycle such as cyclin and cyclin and translation of mRNAs that that such as R.T. A. Hall J. Biol. Chem. Full Text PDF PubMed Google Scholar, H. J. R. Cancer Cell 2003; PubMed Scopus Google Scholar, A. Sonenberg N. Mol. Cell. Biol. PubMed Scopus Google Scholar). Moreover, has that the of cyclin that is caused by amino and rapamycin is mediated by 4E-BP1 J. B.D. 2008; PubMed Scopus Google Scholar). We that the cell cycle arrest caused by Torin1 might be by in the of these with both and for with Torin1, but not rapamycin, had levels of cyclin and and a induction of p27/Kip1 The ability of cells to from this arrest the of Torin1 highly dependent on cell not Rapamycin has an the of TOR research and as a mTORC1 inhibitor in both research and clinical in yeast, it is a of the of In mammalian mTOR substrates were and rapamycin as a Rapamycin a complex with the intracellular protein FKBP12, which to the of mTOR and inhibits phosphorylation of substrates through a is for rapamycin in a complex with and the of mTOR, it this phosphorylation of direct mTOR kinase substrates J. J. J. 1996; 273: PubMed Scopus Google Scholar). to explain findings is that rapamycin to only a of mTORC1 whereas Torin1, because of its ATP-competitive of phosphorylation of Additionally, as Torin1 is much than it its target in more than of the that mTORC1 is to rapamycin. B. Zhang W.G. Toral-Barza L. Lucas J. Abraham R.T. Gibbons J.J. Yu K. Cancer Res. 2008; 68: 2934-2943Crossref PubMed Scopus (114) Google that high of rapamycin inhibit mTOR directly through an both mTORC1 and mTORC2. the commonly used and to Torin1, rapamycin suppresses cap-dependent translation and inhibits proliferation in a of cell these that these effects are because of mTORC2 findings indicate that they are more because of inhibition of rapamycin-resistant mTORC1-dependent from J. B.D. 2008; PubMed Scopus Google that amino caused a more of cyclin than rapamycin treatment and that this mediated through 4E-BP1. on the assumption that rapamycin completely mTORC1, these that amino to 4E-BP1 through mTORC1. We that it is more that amino to a more inhibition of mTORC1 functions than does rapamycin. Roux P.P. Blenis J. Proc. Natl. Acad. Sci. U. S. A. 2008; PubMed Scopus Google that phosphorylation on 4E-BP1 that are to rapamycin in some cell of rapamycin Moreover, the of 4E-BP1 phosphorylation on the mTORC1 component Raptor, the to that prolonged rapamycin treatment on mTORC1 the to 4E-BP1 in a rapamycin-resistant We find that mTORC1 has rapamycin-resistant functions in cell Because prolonged rapamycin treatment is to the PI3K which is of mTORC1, for the of Roux P.P. Blenis J. Proc. Natl. Acad. Sci. U. S. A. 2008; PubMed Scopus Google is that rapamycin to the of the rapamycin-resistant of mTORC1, the inhibition caused by rapamycin. Because many important of TOR signaling are between yeast and that mTORC1 but rapamycin-resistant functions is the indicate that the for signaling in protein synthesis and cell are more similar between yeast and mammalian than had we have on the rapamycin-insensitive of 4E-BP1, we it that other similar mTORC1 substrates the of autophagy. The of Torin1 and a more of mTOR the for rapamycin as a it is that ATP-competitive inhibitors of mTOR have clinical as We D. for and of for and for kinase We also of the Sabatini and for and for