Inhibition of Mammalian Target of Rapamycin Signaling by 2-(Morpholin-1-yl)pyrimido[2,1-α]isoquinolin-4-one
2007; Elsevier BV; Volume: 282; Issue: 33 Linguagem: Inglês
10.1074/jbc.m704741200
ISSN1083-351X
AutoresLisa M. Ballou, Elzbieta S. Selinger, Jun‐Yong Choi, Dale G. Drueckhammer, Richard Z. Lin,
Tópico(s)Synthesis of Organic Compounds
ResumoSignaling through the mammalian target of rapamycin (mTOR) is hyperactivated in many human tumors, including hamartomas associated with tuberous sclerosis complex (TSC). Several small molecules such as LY294002 inhibit mTOR kinase activity, but they also inhibit phosphatidylinositol 3-kinase (PI3K) at similar concentrations. Compound 401 is a synthetic inhibitor of DNA-dependent protein kinase (DNA-PK) that also targets mTOR but not PI3K in vitro (Griffin, R. J., Fontana, G., Golding, B. T., Guiard, S., Hardcastle, I. R., Leahy, J. J., Martin, N., Richardson, C., Rigoreau, L., Stockley, M., and Smith, G. C. (2005) J. Med. Chem. 48, 569-585). We used 401 to test the cellular effect of mTOR inhibition without the complicating side effects on PI3K. Treatment of cells with 401 blocked the phosphorylation of sites modified by mTOR-Raptor and mTOR-Rictor complexes (ribosomal protein S6 kinase 1 Thr389 and Akt Ser473, respectively). By contrast, there was no direct inhibition of Akt Thr308 phosphorylation, which is dependent on PI3K. Similar effects were also observed in cells that lack DNA-PK. The proliferation of TSC1-/- fibroblasts was inhibited in the presence of 401, but TSC1+/+ cells were resistant. In contrast to rapamycin, long-term treatment of TSC1-/- cells with 401 did not up-regulate phospho-Akt Ser473. Because increased Akt activity promotes survival, this may explain why the level of apoptosis was increased in the presence of 401 but not rapamycin. These results suggest that mTOR kinase inhibitors might be more effective than rapamycins in controlling the growth of TSC hamartomas and other tumors that depend on elevated mTOR activity. Signaling through the mammalian target of rapamycin (mTOR) is hyperactivated in many human tumors, including hamartomas associated with tuberous sclerosis complex (TSC). Several small molecules such as LY294002 inhibit mTOR kinase activity, but they also inhibit phosphatidylinositol 3-kinase (PI3K) at similar concentrations. Compound 401 is a synthetic inhibitor of DNA-dependent protein kinase (DNA-PK) that also targets mTOR but not PI3K in vitro (Griffin, R. J., Fontana, G., Golding, B. T., Guiard, S., Hardcastle, I. R., Leahy, J. J., Martin, N., Richardson, C., Rigoreau, L., Stockley, M., and Smith, G. C. (2005) J. Med. Chem. 48, 569-585). We used 401 to test the cellular effect of mTOR inhibition without the complicating side effects on PI3K. Treatment of cells with 401 blocked the phosphorylation of sites modified by mTOR-Raptor and mTOR-Rictor complexes (ribosomal protein S6 kinase 1 Thr389 and Akt Ser473, respectively). By contrast, there was no direct inhibition of Akt Thr308 phosphorylation, which is dependent on PI3K. Similar effects were also observed in cells that lack DNA-PK. The proliferation of TSC1-/- fibroblasts was inhibited in the presence of 401, but TSC1+/+ cells were resistant. In contrast to rapamycin, long-term treatment of TSC1-/- cells with 401 did not up-regulate phospho-Akt Ser473. Because increased Akt activity promotes survival, this may explain why the level of apoptosis was increased in the presence of 401 but not rapamycin. These results suggest that mTOR kinase inhibitors might be more effective than rapamycins in controlling the growth of TSC hamartomas and other tumors that depend on elevated mTOR activity. The mammalian target of rapamycin (mTOR) 2The abbreviations used are: mTOR, mammalian target of rapamycin; PI, phosphatidylinositol; PI3K, PI 3-kinase; PIKK, PI3K-related kinase; ATM, ataxia telangiectasia-mutated; ATR, ATM and Rad3-related; DNA-PK, DNA-dependent protein kinase; PDGF, platelet-derived growth factor; IC50, concentration that gives 50% inhibition; S6K1, ribosomal protein S6 kinase 1; 4E-BP1, eukaryotic initiation factor 4E-binding protein 1; GST, glutathione S-transferase; PDK1, 3-phosphoinositide-dependent protein kinase-1; MEF, mouse embryo fibroblast; TSC, tuberous sclerosis complex; IGF-I, insulin-like growth factor-I; PLD, phospholipase D; PA, phosphatidic acid; AMPK, AMP-activated kinase. 2The abbreviations used are: mTOR, mammalian target of rapamycin; PI, phosphatidylinositol; PI3K, PI 3-kinase; PIKK, PI3K-related kinase; ATM, ataxia telangiectasia-mutated; ATR, ATM and Rad3-related; DNA-PK, DNA-dependent protein kinase; PDGF, platelet-derived growth factor; IC50, concentration that gives 50% inhibition; S6K1, ribosomal protein S6 kinase 1; 4E-BP1, eukaryotic initiation factor 4E-binding protein 1; GST, glutathione S-transferase; PDK1, 3-phosphoinositide-dependent protein kinase-1; MEF, mouse embryo fibroblast; TSC, tuberous sclerosis complex; IGF-I, insulin-like growth factor-I; PLD, phospholipase D; PA, phosphatidic acid; AMPK, AMP-activated kinase. is a protein kinase that occupies a central position in a signaling network that regulates cell proliferation, size and survival. This signaling network integrates growth factor-activated signals with permissive signals in the presence of sufficient amino acids and energy to result in mTOR activation. Many genetic defects found in human cancers lead to hyperactivation of mTOR signaling (1Guertin D.A. Sabatini D.M. Trends Mol. Med. 2005; 11: 353-361Abstract Full Text Full Text PDF PubMed Scopus (440) Google Scholar). It is believed that certain tumor cells become dependent on this pathway because it provides a growth or survival advantage. Therefore, drugs that inhibit mTOR have great therapeutic potential for the treatment of cancer. Rapamycin, a macrocyclic antibiotic produced by the bacterium Streptomyces hygroscopicus, binds to its intracellular receptor FKBP12 to form a complex that inhibits mTOR function. Rapamycin and its analogs are under clinical development as anti-cancer agents, but these drugs have two disadvantages: they block only some of the functions of mTOR, and they can activate the protein kinase Akt that promotes cell survival. By contrast, a small molecule designed to compete with ATP in the catalytic site of mTOR would be expected to inhibit all of the kinase-dependent signaling functions of the enzyme without enhancing the survival pathway. The mTOR kinase domain is most closely related to the one found in phosphatidylinositol (PI) 3-kinases (PI3Ks) (2Crespo J.L. Hall M.N. Microbiol. Mol. Biol. Rev. 2002; 66: 579-591Crossref PubMed Scopus (280) Google Scholar). However, unlike PI3Ks, mTOR phosphorylates proteins, not lipids. The unusual mTOR kinase domain defines the PIKK (PI3K-related kinase) family of protein serine/threonine kinases, which includes ATM (ataxia telangiectasia-mutated), ATR (ATM and Rad3-related), and DNA-PK (DNA-dependent protein kinase) (3Abraham R.T. DNA Repair (Amst). 2004; 3: 883-887Crossref PubMed Scopus (377) Google Scholar). Until recently, rapamycin sensitivity was the major criterion used in mammalian systems to identify mTOR-controlled events. However, it is now known that mTOR binds to different regulatory subunits to produce multiprotein complexes with distinct signaling functions and rapamycin sensitivity (4Sarbassov D.D. Ali S.M. Sabatini D.M. Curr. Opin. Cell Biol. 2005; 17: 596-603Crossref PubMed Scopus (1289) Google Scholar). Complexes containing mTOR and Raptor (mTORC1) phosphorylate ribosomal protein S6 kinase 1 (S6K1) and eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) and are rapamycin-sensitive (5Hara K. Maruki Y. Long X. Yoshino K. Oshiro N. Hidayat S. Tokunaga C. Avruch J. Yonezawa K. Cell. 2002; 110: 177-189Abstract Full Text Full Text PDF PubMed Scopus (1416) Google Scholar). Complexes that contain mTOR and Rictor (mTORC2) phosphorylate Akt and are insensitive to the drug (6Sarbassov D.D. Guertin D.A. Ali S.M. Sabatini D.M. Science. 2005; 307: 1098-1101Crossref PubMed Scopus (5076) Google Scholar). Because mTOR has rapamycin-insensitive functions, it is thought that direct inhibitors of mTOR kinase activity will display broader anti-tumor activity than rapamycins. Most kinase inhibitors that have been developed for clinical applications work by competing with ATP in the catalytic site. LY294002 (Fig. 1) is a synthetic morpholino compound that was designed as a PI3K inhibitor (7Vlahos C.J. Matter W.F. Hui K.Y. Brown R.F. J. Biol. Chem. 1994; 269: 5241-5248Abstract Full Text PDF PubMed Google Scholar). Kinetic studies showed that this compound behaves as a competitive inhibitor with ATP (7Vlahos C.J. Matter W.F. Hui K.Y. Brown R.F. J. Biol. Chem. 1994; 269: 5241-5248Abstract Full Text PDF PubMed Google Scholar), and x-ray crystallographic stuctures have revealed how LY294002 fits into the active site of the p110γ isoform of PI3K (8Walker E.H. Pacold M.E. Perisic O. Stephens L. Hawkins P.T. Wymann M.P. Williams R.L. Mol. Cell. 2000; 6: 909-919Abstract Full Text Full Text PDF PubMed Scopus (1006) Google Scholar). Not surprisingly, considering the structural similarity between the PI3K and mTOR catalytic domains, the concentration of LY294002 that gives 50% inhibition (IC50) of mTOR (IC50 ∼ 5 μm) is almost the same as for PI3K (IC50 ∼ 3 μm) (9Brunn G.J. Williams J. Sabers C. Wiederrecht G. Lawrence Jr., J.C. Abraham R.T. EMBO J. 1996; 15: 5256-5267Crossref PubMed Scopus (615) Google Scholar). This compound also blocks the activity of DNA-PK (10Griffin R.J. Fontana G. Golding B.T. Guiard S. Hardcastle I.R. Leahy J.J. Martin N. Richardson C. Rigoreau L. Stockley M. Smith G.C. J. Med. Chem. 2005; 48: 569-585Crossref PubMed Scopus (143) Google Scholar). PI3Ks regulate a wide range of cellular functions including growth, metabolism and motility, and PIKKs regulate processes such as cell cycle progression and genome maintenance. Therefore, use of LY294002 as a non-selective mTOR inhibitor might have undesirable toxic side effects. Recently, Griffin et al. (10Griffin R.J. Fontana G. Golding B.T. Guiard S. Hardcastle I.R. Leahy J.J. Martin N. Richardson C. Rigoreau L. Stockley M. Smith G.C. J. Med. Chem. 2005; 48: 569-585Crossref PubMed Scopus (143) Google Scholar) used LY294002 as a template for the design of DNA-PK inhibitors. One derivative (compound 13 in Ref. 10Griffin R.J. Fontana G. Golding B.T. Guiard S. Hardcastle I.R. Leahy J.J. Martin N. Richardson C. Rigoreau L. Stockley M. Smith G.C. J. Med. Chem. 2005; 48: 569-585Crossref PubMed Scopus (143) Google Scholar; compound 401 in Fig. 1) was reported to be a poor inhibitor of PI3K, ATM, and ATR in vitro, but it was active against mTOR. 401 was not subjected to a biological evaluation. In this study, we tested the ability of compound 401 to inhibit mTOR signaling in vivo and evaluated its effect on proliferation and apoptosis in cells with hyperactivated mTOR signaling. Our results support the idea that inhibitors of mTOR kinase activity might be more effective than rapamycins in treating some proliferative disorders. Materials—Platelet-derived growth factor (PDGF) A/B, phenylephrine, and insulin-like growth factor I (IGF-I) were from Sigma. Rapamycin, AMA37, Akt inhibitor VIII and LY294002 were from Calbiochem. Phosphospecific antibodies were from Cell Signaling Technology (Danvers, MA). Antibodies to Akt1/2, S6K1, and Erk2 were from Santa Cruz Biotechnology (Santa Cruz, CA). Raptor antibody was from Bethyl Laboratories (Montgomery, TX) and AU1 antibody was from Covance (Berkeley, CA). 2-(Morpholin-1-yl)pyrimido[2,1-α]isoquinolin-4-one (compound 401) was synthesized as described earlier (10Griffin R.J. Fontana G. Golding B.T. Guiard S. Hardcastle I.R. Leahy J.J. Martin N. Richardson C. Rigoreau L. Stockley M. Smith G.C. J. Med. Chem. 2005; 48: 569-585Crossref PubMed Scopus (143) Google Scholar). Its identity and purity were confirmed by 1H NMR. Akt T308D-myc-His and Akt S473D-myc-His were described earlier (11Ballou L.M. Lin H.Y. Fan G. Jiang Y.P. Lin R.Z. J. Biol. Chem. 2003; 278: 23472-23479Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). Purification of the p110α/p85α complex from insect cells was previously described (11Ballou L.M. Lin H.Y. Fan G. Jiang Y.P. Lin R.Z. J. Biol. Chem. 2003; 278: 23472-23479Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). The cDNA for the human p110β PI3K catalytic subunit was purchased from Invitrogen. It was found to contain a mutation (C at 2772 was changed to A) that rendered the enzyme catalytically inactive. The mutation was repaired by PCR and the coding region was subcloned into pBlueBacHis2B (Invitrogen). A baculovirus was produced using the Bac-N-Blue kit (Invitrogen). A baculovirus expressing the p85α regulatory subunit of PI3K was purchased from Orbigen (San Diego, CA). The p110β/p85α PI3K complex was purified from infected insect cells as described for p110α/p85α (11Ballou L.M. Lin H.Y. Fan G. Jiang Y.P. Lin R.Z. J. Biol. Chem. 2003; 278: 23472-23479Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). Cell Culture—FreeStyle 293-F cells (Invitrogen) were grown in FreeStyle 293 Expression Medium. Rat-1 fibroblasts stably transfected with the human α1A-adrenergic receptor (12Kenny B.A. Miller A.M. Williamson I.J. O'Connell J. Chalmers D.H. Naylor A.M. Br. J. Pharmacol. 1996; 118: 871-878Crossref PubMed Scopus (103) Google Scholar), COS7 cells and mouse embryo fibroblasts (MEFs) expressing or lacking the tuberous sclerosis complex 1 (TSC1) gene (a gift from Dr. D. Kwiatkowski, Harvard University) were maintained in Dulbecco’s modified Eagle’s medium (DMEM; Sigma) containing antibiotics and 10% fetal bovine serum. M059J human glioma cells (American Type Culture Collection, Manassas, VA) were kept in complete growth medium (a 1:1 mixture of DMEM and Ham’s F12 medium plus supplements) according to the vendor’s instructions. mTOR Assays—FreeStyle 293-F cells were transfected with cDNA for AU1-mTOR (a gift from Dr. R. T. Abraham, The Burnham Institute, La Jolla, CA) using 293fectin (Invitrogen). Two days later, the cells were lysed and mTOR immunoprecipitates were prepared as described (13Chiang G.G. Abraham R.T. Methods Mol. Biol. 2004; 281: 125-141PubMed Google Scholar) using AU1 antibody. Alternatively, the mTORC1 complex was immunoprecipitated from untransfected cells using Raptor antibody under conditions described earlier (14Sarbassov D.D. Ali S.M. Kim D.H. Guertin D.A. Latek R.R. Erdjument-Bromage H. Tempst P. Sabatini D.M. Curr. Biol. 2004; 14: 1296-1302Abstract Full Text Full Text PDF PubMed Scopus (2089) Google Scholar). Kinase activity in the immunoprecipitates was assayed in the presence of vehicle (dimethyl sulfoxide) or compound as described (13Chiang G.G. Abraham R.T. Methods Mol. Biol. 2004; 281: 125-141PubMed Google Scholar) using bacterially expressed glutathione S-transferase (GST)-4E-BP1 (15Jiang Y.P. Ballou L.M. Lin R.Z. J. Biol. Chem. 2001; 276: 10943-10951Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar) as a substrate. Kinase reactions were stopped by boiling in SDS sample buffer and the samples were subjected to SDS-PAGE. Phosphorylated 4E-BP1 was detected by autoradiography. Radioactivity in the bands was quantified by scintillation counting. PI3K Assays—Vehicle (dimethylformamide) or compound was added to assay tubes and the solvent was evaporated under vacuum. 35 μl of PI3K assay buffer (20 mm HEPES, pH 7.4, 100 mm NaCl, and 0.5 mm EGTA) was added to each tube, and they were placed on a shaker for 10 min at 25 °C to dissolve the compounds. Then 5 μl of enzyme was added, and the mixtures were incubated for 10 min at 25 °C. Kinase assays were started by adding 5 μl of 10 mg/ml L-α-PI (Sigma) sonicated in assay buffer and 5 μl of reaction mix containing 400 μm ATP, 0.25 μl of [γ-32P]ATP (3000 Ci/mmol; PerkinElmer Life Sciences) per assay, and 100 mm MgCl2 in assay buffer. Tubes were heated at 25 °C for 20 min with shaking. The reactions were stopped with 120 μl of CHCl3:CH3OH:HCl (10:20:0.2), and the tubes were put on a shaker for 10 min. After centrifuging for 5 min, 15 μl of the lower phase was spotted onto a silica gel thin layer plate and the reaction product was separated by chromatography for 1.5 h in CHCl3:CH3OH:NH4OH:H2O (86:76:10:14). Radioactive spots containing PI 3-phosphate were visualized by autoradiography, cut out of the plate and quantified by scintillation counting. For PI3K autophosphorylation, 20 μl of autophosphorylation buffer (20 mm HEPES, pH 7.4, and 100 mm NaCl) was added to tubes containing dried vehicle or compound and they were placed on a shaker for 10 min at 25 °C. Then 5 μl of p110α/p85α PI3K was added, and the mixtures were incubated for 10 min at 25 °C. Reactions were started by adding 5 μl of autophosphorylation buffer containing 60 μm ATP, 0.25 μl of [γ-32P]ATP per assay, and 60 mm MnCl2. Tubes were heated at 25 °C for 20 min. The reactions were stopped by boiling in SDS sample buffer, and the samples were subjected to SDS-PAGE. Phosphorylated p85α was detected by autoradiography. Western Blots—After treatments, cells were rinsed on ice with cold phosphate-buffered saline and scraped into lysis buffer containing 50 mm HEPES, pH 7.5, 1% Triton X-100, 50 mm NaCl, 5 mm EDTA, 50 mm NaF, 10 mm sodium pyrophosphate, 1 mm sodium orthovanadate, and a mixture of protease inhibitors. After centrifugation at 20,000 × g, equal quantities of supernatant protein were subjected to SDS gel electrophoresis and Western blotting. Signals were detected using horseradish peroxidase-linked second antibodies (GE Healthcare) and chemiluminescence reagents (PerkinElmer Life Sciences). Protein was determined by a Bradford assay (Bio-Rad). Apoptosis—After treatments, cells were lysed and nucleosomes in the cytoplasmic fraction were assayed using the Cell Death Detection ELISAPLUS kit (Roche Diagnostics GmbH, Mannheim). The nucleosome level was calculated as (A405 nm - A490 nm) according to the kit instructions. Effect of 401 on Kinase Activities in Vitro—Several small molecules have been reported to inhibit mTOR kinase activity at low micromolar concentrations or better, but they show unfavorable selectivity between mTOR and PI3K (9Brunn G.J. Williams J. Sabers C. Wiederrecht G. Lawrence Jr., J.C. Abraham R.T. EMBO J. 1996; 15: 5256-5267Crossref PubMed Scopus (615) Google Scholar, 10Griffin R.J. Fontana G. Golding B.T. Guiard S. Hardcastle I.R. Leahy J.J. Martin N. Richardson C. Rigoreau L. Stockley M. Smith G.C. J. Med. Chem. 2005; 48: 569-585Crossref PubMed Scopus (143) Google Scholar, 16Hardcastle I.R. Cockcroft X. Curtin N.J. El-Murr M.D. Leahy J.J. Stockley M. Golding B.T. Rigoreau L. Richardson C. Smith G.C. Griffin R.J. J. Med. Chem. 2005; 48: 7829-7846Crossref PubMed Scopus (144) Google Scholar, 17Knight Z.A. Gonzalez B. Feldman M.E. Zunder E.R. Goldenberg D.D. Williams O. Loewith R. Stokoe D. Balla A. Toth B. Balla T. Weiss W.A. Williams R.L. Shokat K.M. Cell. 2006; 125: 733-747Abstract Full Text Full Text PDF PubMed Scopus (945) Google Scholar). By contrast, compound 401 shows activity against mTOR (IC50 = 5.3 μm) but not p110α/p85α PI3K (IC50 > 100 μm, Ref. 10Griffin R.J. Fontana G. Golding B.T. Guiard S. Hardcastle I.R. Leahy J.J. Martin N. Richardson C. Rigoreau L. Stockley M. Smith G.C. J. Med. Chem. 2005; 48: 569-585Crossref PubMed Scopus (143) Google Scholar). We speculated that 401 might be a useful tool to probe mTOR signaling without the complicating side effect of PI3K inhibition. First, we performed in vitro kinase assays to confirm that 401 selectively inhibits mTOR over PI3K. In agreement with the results of Griffin et al. (10Griffin R.J. Fontana G. Golding B.T. Guiard S. Hardcastle I.R. Leahy J.J. Martin N. Richardson C. Rigoreau L. Stockley M. Smith G.C. J. Med. Chem. 2005; 48: 569-585Crossref PubMed Scopus (143) Google Scholar), we found that 401 inhibited immunoprecipitated epitope-tagged mTOR or endogenous mTOR in Raptor immunoprecipitates (Fig. 2A). In both cases, inhibition of 67% or 78% was obtained at 5 μm or 10 μm 401, respectively. By contrast, dose response curves showed that the p110α/p85α or p110β/p85α PI3K complexes were poorly inhibited by 401 at these concentrations (Fig. 2B). LY294002 was much more potent than 401 at inhibiting both PI3Ks (Fig. 2B). We also tested if 401 inhibits the protein kinase activity of p110α by examining autophosphorylation of p85α in the PI3K complex. Compound 401 at 25 μm had no effect on this reaction, whereas inhibition was seen with 5 μm LY294002 (Fig. 2C). Together, these results confirm that compound 401 has increased selectivity for mTOR over PI3K. The sensitivity of 40 different protein kinases to 5 μm 401 was also determined. Most of these enzymes were inhibited less than 20% by 401 at this concentration (Table 1).TABLE 1Inhibition of protein kinases by 401Protein kinase% InhibitionProtein kinase% InhibitionABL1−5LCK−3AKT1 (PKBα)5MAP2K1 (MEK1)−8BTK9MAP4K4 (HGK)3CDK1/cyclin B3MAPK14 (p38α)17CHEK1 (CHK1)−9MAPK3 (ERK1)−3CSNK1G2 (CK1γ2)2MAPKAPK240CSNK2A1 (CK2α1)3MET (cMet)19DYRK35NTRK1 (TRKA)−17EGFR (ErbB1)−3PDGFRB (PDGFRβ)−4EPHA2−13PHKG2−6ERBB2 (HER2)3PIM144FGFR10PRKACA (PKA)−11FLT336PRKCB1 (PKCβ1)8GSK3β (GSK3β)0RET−4IGF1R−8ROCK113INSR−3RPS6KA3 (RSK2)7IRAK4−5SRC−14JAK32STK6 (Aurora A)44KDR (VEGFR2)0SYK14KIT13TEK (Tie2)12 Open table in a new tab Effect of 401 on mTOR Signaling—S6K1 and Akt are two of the best-studied effectors of mTOR signaling. Both kinases possess highly conserved phosphorylation sites in the T loop of the kinase catalytic domain and in a C-terminal hydrophobic motif. Thr389 in the hydrophobic motif of S6K1 is phosphorylated by mTORC1 (5Hara K. Maruki Y. Long X. Yoshino K. Oshiro N. Hidayat S. Tokunaga C. Avruch J. Yonezawa K. Cell. 2002; 110: 177-189Abstract Full Text Full Text PDF PubMed Scopus (1416) Google Scholar, 18Burnett P.E. Barrow R.K. Cohen N.A. Snyder S.H. Sabatini D.M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 1432-1437Crossref PubMed Scopus (917) Google Scholar). Phosphorylation of Thr389 allows 3-phosphoinositide-dependent protein kinase-1 (PDK1) to phosphorylate Thr229 in the T loop to activate the enzyme (19Pullen N. Dennis P.B. Andjelkovic M. Dufner A. Kozma S.C. Hemmings B.A. Thomas G. Science. 1998; 279: 707-710Crossref PubMed Scopus (718) Google Scholar). Similarly, mTORC2 phosphorylates Akt at Ser473 in the hydrophobic motif and facilitates PDK1 phosphorylation of Thr308 in the T loop (6Sarbassov D.D. Guertin D.A. Ali S.M. Sabatini D.M. Science. 2005; 307: 1098-1101Crossref PubMed Scopus (5076) Google Scholar). Examination of the phosphorylation state of S6K1 Thr389 and Akt Ser473 is a convenient way to assess intracellular mTORC1 and mTORC2 activity, respectively. We expected that treatment of cells with an mTOR kinase inhibitor such as 401 would block phosphorylation of both sites, unlike rapamycin, which only affects S6K1. To test the effect of 401 on growth factor-activated mTOR signaling, serum-starved Rat-1 fibroblasts were pretreated with inhibitors and then stimulated with PDGF. The phosphorylation of S6K1 and Akt was analyzed on Western blots probed with antibodies that recognize S6K1 phospho-Thr389 or Akt phospho-Ser473. As expected for an mTOR kinase inhibitor, 401 decreased the phosphorylation of both sites (Fig. 3A). Inhibition was nearly complete at a concentration of 401 (10 μm) that has little effect on PI3K activity (see Fig. 2B). Consistent with its ability to target mTOR, LY294002 also reduced the phosphorylation of both sites (Fig. 3A). By contrast, rapamycin affected only the mTORC1 site (S6K1 Thr389, Fig. 3A). Compound 401 did not inhibit the phosphorylation of Erk induced by PDGF (Fig. 3A), indicating that it does not block all PDGF receptor-induced responses. The blots were reprobed with general antibodies to S6K1, Akt, or Erk2 to show that similar amounts of each protein were present in each lane (Fig. 3A). These results are consistent with 401 acting as an mTOR kinase inhibitor in vivo. Treatment of these Rat-1 cells with phenylephrine to stimulate the α1A adrenergic receptor also induced S6K1 Thr389 phosphorylation that was inhibited in the presence of 401 or LY294002 (Fig. 3B). Because phenylephrine activation of S6K1 occurs without an increase in PI3K activity (20Ballou L.M. Cross M.E. Huang S. McReynolds E.M. Zhang B.X. Lin R.Z. J. Biol. Chem. 2000; 275: 4803-4809Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar), it is likely that the inhibitory effect of 401 is due to inhibition of mTOR and not PI3K. The PI3K inhibitor LY294002 blocked the phosphorylation of Akt Thr308 (Fig. 3A), which was expected because PI 3-phosphates promote the colocalization of Akt and PDK1 at the plasma membrane (21Anderson K.E. Coadwell J. Stephens L.R. Hawkins P.T. Curr. Biol. 1998; 8: 684-691Abstract Full Text Full Text PDF PubMed Scopus (301) Google Scholar). Surprisingly, Akt Thr308 phosphorylation was also inhibited in the presence of 401 (Fig. 3A). Two possible explanations for this observation are that 401 inhibits PDK1 or that Thr308 phosphorylation depends on prior phosphorylation of Ser473, as has been reported by others (22Scheid M.P. Marignani P.A. Woodgett J.R. Mol. Cell. Biol. 2002; 22: 6247-6260Crossref PubMed Scopus (268) Google Scholar). To examine these possibilities, COS7 cells were transfected with Akt mutants that contain the acidic amino acid Asp at either Thr308 or Ser473 to mimic phosphorylation. Cells were then treated with insulin to induce phosphorylation of the intact site. Phosphorylation of Ser473 was inhibited in the presence of 401, but phosphorylation of Thr308 was not affected (Fig. 4). These results indicate that 401 does not inhibit PDK1, and suggest that decreased Akt Thr308 phosphorylation is an indirect effect due to inhibition of mTOR-catalyzed Ser473 phosphorylation. Because 401 is a potent inhibitor of DNA-PK (IC50 = 0.28 μm, Ref. 10Griffin R.J. Fontana G. Golding B.T. Guiard S. Hardcastle I.R. Leahy J.J. Martin N. Richardson C. Rigoreau L. Stockley M. Smith G.C. J. Med. Chem. 2005; 48: 569-585Crossref PubMed Scopus (143) Google Scholar), the possibility exists that its effects on Akt and S6K1 phosphorylation might be due either directly or indirectly to inhibition of DNA-PK. This possibility was tested in M059J glioma cells, which lack DNA-PK (23Lees-Miller S.P. Godbout R. Chan D.W. Weinfeld M. Day R.S. Barron 3rd, G.M. Allalunis-Turner J. Science. 1995; 267: 1183-1185Crossref PubMed Scopus (501) Google Scholar). Treatment of M059J cells with 401 decreased the phosphorylation of Akt Ser473 and S6K1 Thr389 at the same doses that were effective in Rat-1 cells (Fig. 5). Thus, the inhibitory effect of 401 on phosphorylation of these mTOR sites is not due to DNA-PK inhibition. Effect of 401 on Growth and Survival of TSC1-/- MEFs—Tuberous sclerosis complex (TSC) is a disease characterized by the development of benign tumor-like growths called hamartomas in multiple tissues. This disorder is caused by mutations in the tsc1 or tsc2 tumor suppressor genes. The TSC1 and TSC2 proteins form a complex that suppresses mTOR activity. Loss of either protein leads to constitutive elevation of mTOR signaling (reviewed in Ref. 24Crino P.B. Nathanson K.L. Henske E.P. N. Engl. J. Med. 2006; 355: 1345-1356Crossref PubMed Scopus (1281) Google Scholar). Recent studies indicate that Akt activity in TSC1-/- or TSC2-/- MEFs is abnormally low as compared with control MEFs due to feedback inhibition from the hyperactivated mTORC1/S6K1 pathway (25Shah O.J. Wang Z. Hunter T. Curr. Biol. 2004; 14: 1650-1656Abstract Full Text Full Text PDF PubMed Scopus (644) Google Scholar, 26Yang Q. Inoki K. Kim E. Guan K.L. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 6811-6816Crossref PubMed Scopus (137) Google Scholar). Long term treatment of these cells with rapamycin turns off the negative feedback program and up-regulates Akt, which can provide a survival signal (25Shah O.J. Wang Z. Hunter T. Curr. Biol. 2004; 14: 1650-1656Abstract Full Text Full Text PDF PubMed Scopus (644) Google Scholar). To determine if rapamycin and 401 have different effects on Akt, TSC1-/- MEFs were incubated with drugs for increasing times for up to 24 h. Cells cultured in the presence of rapamycin exhibited an increase in Akt Ser473 phosphorylation, whereas Akt phosphorylation remained low in cells cultured with 401 (Fig. 6A). Both drugs completely blocked S6K1 Thr389 phosphorylation (Fig. 6A). Several breast cancer cell lines (BT-474, MCF-7, and ZR-754) that up-regulated phospho-Akt Ser473 after 24 h in the presence of rapamycin also maintained low Akt phosphorylation levels in the presence of 401 (Ref. 27O'Reilly K.E. Rojo F. She Q.B. Solit D. Mills G.B. Smith D. Lane H. Hofmann F. Hicklin D.J. Ludwig D.L. Baselga J. Rosen N. Cancer Res. 2006; 66: 1500-1508Crossref PubMed Scopus (2121) Google Scholar and data not shown). Next, we determined if rapamycin and 401 differentially affect the proliferation of TSC1-/- cells. Rapamycin treatment for 1 day did not cause a significant decrease in cell number as compared with the control cells, but after 2 days a significant decrease (57%) was evident (Fig. 6B). By contrast, proliferation was strongly inhibited in cells exposed to 401 for 1 day (54%) or 2 days (84%) (Fig. 6B). Growth inhibition by 401 was dose-dependent, with a half-maximal effect at roughly 2 μm (Fig. 6C). The growth inhibitory effect of 401 is not due to general toxicity, as TSC1+/+ MEFs cultured for 2 days in the presence of the drug did not show a significant decrease in cell number (Fig. 6B). The effect of 401 on cell growth is also probably not due to DNA-PK inhibition, because TSC1-/- MEFS were resistant to AMA37 (Fig. 6D), a compound that inhibits DNA-PK (IC50 = 0.27 μm) but not mTOR (IC50 > 100 μm) (28Knight Z.A. Chiang G.G. Alaimo P.J. Kenski D.M. Ho C.B. Coan K. Abraham R.T. Shokat K.M. Bioorg. Med. Chem. 2004; 12: 4749-4759Crossref PubMed Scopus (133) Google Scholar). To determine if the 401-induced decrease in cell number in the proliferation experiments above might be due in part to apoptosis, we measured the level of nucleosom
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