Predominant Nuclear Localization of Mammalian Target of Rapamycin in Normal and Malignant Cells in Culture
2002; Elsevier BV; Volume: 277; Issue: 31 Linguagem: Inglês
10.1074/jbc.m202625200
ISSN1083-351X
AutoresXiongwen Zhang, Lili Shu, Hajime Hosoi, K. Gopal Murti, Peter J. Houghton,
Tópico(s)Toxin Mechanisms and Immunotoxins
ResumoMammalian target of rapamycin (mTOR) controls initiation of translation through regulation of ribosomal p70S6 kinase (S6K1) and eukaryotic translation initiation factor-4E (eIF4E) binding protein (4E-BP). mTOR is considered to be located predominantly in cytosolic or membrane fractions and may shuttle between the cytoplasm and nucleus. In most previous studies a single cell line, E1A-immortalized human embryonic kidney cells (HEK293), has been used. Here we show that in human malignant cell lines, human fibroblasts, and murine myoblasts mTOR is predominantly nuclear. In contrast, mTOR is largely excluded from the nucleus in HEK293 cells. Hybrids between HEK293 and Rh30 rhabdomyosarcoma cells generated cells co-expressing markers unique to HEK293 (E1A) and Rh30 (MyoD). mTOR distribution was mainly nuclear with detectable levels in the cytoplasm. mTOR isolated from Rh30 nuclei phosphorylated recombinant GST-4E-BP1 (Thr-46) in vitro and thus has kinase activity. We next investigated the cellular distribution of mTOR substrates 4E-BP, S6K1, and eIF4E. 4E-BP was exclusively detected in cytoplasmic fractions in all cell lines. S6K1 was localized in the cytoplasm in colon carcinoma, HEK293 cells, and IMR90 fibroblasts. S6K1 was readily detected in all cellular fractions derived from rhabdomyosarcoma cells. eIF4E was detected in all fractions derived from rhabdomyosarcoma cells but was not detectable in nuclear fractions from colon carcinoma HEK293 or IMR90 cells. Mammalian target of rapamycin (mTOR) controls initiation of translation through regulation of ribosomal p70S6 kinase (S6K1) and eukaryotic translation initiation factor-4E (eIF4E) binding protein (4E-BP). mTOR is considered to be located predominantly in cytosolic or membrane fractions and may shuttle between the cytoplasm and nucleus. In most previous studies a single cell line, E1A-immortalized human embryonic kidney cells (HEK293), has been used. Here we show that in human malignant cell lines, human fibroblasts, and murine myoblasts mTOR is predominantly nuclear. In contrast, mTOR is largely excluded from the nucleus in HEK293 cells. Hybrids between HEK293 and Rh30 rhabdomyosarcoma cells generated cells co-expressing markers unique to HEK293 (E1A) and Rh30 (MyoD). mTOR distribution was mainly nuclear with detectable levels in the cytoplasm. mTOR isolated from Rh30 nuclei phosphorylated recombinant GST-4E-BP1 (Thr-46) in vitro and thus has kinase activity. We next investigated the cellular distribution of mTOR substrates 4E-BP, S6K1, and eIF4E. 4E-BP was exclusively detected in cytoplasmic fractions in all cell lines. S6K1 was localized in the cytoplasm in colon carcinoma, HEK293 cells, and IMR90 fibroblasts. S6K1 was readily detected in all cellular fractions derived from rhabdomyosarcoma cells. eIF4E was detected in all fractions derived from rhabdomyosarcoma cells but was not detectable in nuclear fractions from colon carcinoma HEK293 or IMR90 cells. mammalian target of rapamycin FKBP12-rapamycin-associated protein phosphatidylinositol 3′-kinase eukaryotic initiation factor 4E eIF4E binding protein 1 ribosomal p70 S6 kinase mouse monoclonal anti-mTOR antibody growth medium differentiation medium phosphate-buffered saline Dulbecco's modified Eagle's medium insulin-like growth factor I receptor β chain 4-morpholinepropanesulfonic acid glutathione S-transferase AU1 epitope-tagged kinase-dead rapamycin-resistant mTOR epidermal growth factor receptor The mammalian target of rapamycin (mTOR,1 also designated FRAP, RAFT1, and RAPT1) is a 289-kDa serine/threonine kinase (1Brown E.J. Albers M.W. Shin T.B. Ichikawa K. Keith C.T. Lane W.S. Schreiber S.L. 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Burnett P.E. Lai M.M. Field M.E. Bahr B.A. Kirsch J. Betz H. Snyder S.H. Science. 1999; 284: 1161-1164Crossref PubMed Scopus (160) Google Scholar) thus raising the possibility that formation of protein complexes may direct mTOR distribution within the cell. In mammalian cells two translational components, ribosomal p70S6 kinase (S6K1) and eukaryotic translation initiation factor-4E (eIF4E) binding protein 1 (4E-BP1), are the best characterized downstream effector molecules of mTOR. However, the full spectrum of cellular events controlled by mTOR extends beyond these pathways. Increasing evidence has implicated mTOR as a sensor that integrates extracellular and intracellular events, coordinating growth and proliferation. mTOR may directly or indirectly regulate translation initiation, actin organization, membrane traffic, protein degradation, protein kinase C signaling, ribosome biogenesis, tRNA synthesis, and transcription (reviewed in (6Schmelzle T. Hall M.N. 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Chem. 1997; 272: 26457-26463Abstract Full Text Full Text PDF PubMed Scopus (408) Google Scholar). eIF4E regulates initiation of translation of mRNA species that encode cell cycle regulators, such as cyclin D1 (20Rosenwald I.B. Kaspar R. Rousseau D. Gehrke L. Leboulch P. Chen J.J. Schmidt E.V. Sonenberg N. London I.M. J. Biol. Chem. 1995; 270: 21176-21180Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar), and ornithine decarboxylase (21Shantz L.M. Pegg A.E. Cancer Res. 1994; 54: 2313-2316PubMed Google Scholar). Thus, one might anticipate that mTOR localizes in the cytoplasm or to the plasma membrane. This would result in signals from putative upstream components, PI3K and Akt/PKB, both of which are considered to localize to the cytoplasm or plasma membrane (22Kulik G. Klippel A. Weber M.J. Mol. Cell. Biol. 1997; 17: 1595-1606Crossref PubMed Scopus (965) Google Scholar), to downstream components, S6K1 or 4E-BP, which are considered to localize to the cytoplasm (23Reinhard C. Fernandez A. Lamb N.J.C. Thomas G. EMBO J. 1994; 13: 1557-1565Crossref PubMed Scopus (179) Google Scholar). However, at least a proportion of eIF4E has been demonstrated to localize to the nucleus. It is suggested that nuclear eIF4E is involved in nuclear functions, including splicing and/or nucleocytoplasmic transport of a specific subset of mRNAs (24Lejbkowicz F. Goyer C. Darveau A. Neron S. Lemieux R. Sonenberg N. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 9612-9616Crossref PubMed Scopus (164) Google Scholar, 25Dostie J. Lejbkowicz F. Sonenberg N. J. Cell Biol. 2000; 148: 239-247Crossref PubMed Scopus (108) Google Scholar) that include those highly growth regulated proteins described above (26Rousseau D. Kasper R. Rosenwald I. Gehrke L. Sonenberg N. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1065-1070Crossref PubMed Scopus (358) Google Scholar). Indeed, mTOR has been reported to be a cytoplasmic protein localized to intracellular membranes. In fractionated rat brain, mTOR was localized to presynaptic and synaptic vesicles (10Sabatini D.M. Barrow R.K. Blackshaw S. Burnett P.E. Lai M.M. Field M.E. Bahr B.A. Kirsch J. Betz H. Snyder S.H. Science. 1999; 284: 1161-1164Crossref PubMed Scopus (160) Google Scholar). However, immunostaining of rat hippocampal neurons shows distribution of mTOR, 4E-BP1, and eIF4E throughout the cell body and nucleus (27Tang S.J. Reis G. Kang H. Gingras A.-C. Sonenberg N. Schuman E.M. Proc. Natl. Acad. Sci. U. S. A. 2002; 93: 467-472Crossref Scopus (609) Google Scholar). Cellular fractionation of E1A-immortalized human embryonic kidney cells (HEK293) and 3T3-L1 adipocytes (28Withers D.J. Ouwens D.M. Nave B.T. van der Zon G.C. Alarcon C.M. Cardenas M.E. Heitman J. Maassen J.A. Shepherd P.R. Biochem. Biophys. Res. Commun. 1997; 241: 704-709Crossref PubMed Scopus (64) Google Scholar) showed that mTOR localized to membranes, although immunoblots of nuclear fractions were not presented. Overexpression of epitope-tagged (green fluorescent protein) mTOR in HEK293 and HeLa cells (10Sabatini D.M. Barrow R.K. Blackshaw S. Burnett P.E. Lai M.M. Field M.E. Bahr B.A. Kirsch J. Betz H. Snyder S.H. Science. 1999; 284: 1161-1164Crossref PubMed Scopus (160) Google Scholar, 30Kim J.E. Chen J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 14340-14345Crossref PubMed Scopus (187) Google Scholar) results in predominantly cytoplasmic staining. Furthermore, mTOR becomes nuclear in HEK293 cells treated with leptomycin B, a specific inhibitor of nuclear export receptor Crm1, suggesting that mTOR is a cytoplasmic-nuclear shuttling protein (30Kim J.E. Chen J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 14340-14345Crossref PubMed Scopus (187) Google Scholar). However, data demonstrating cellular localization of endogenous mTOR is limited to HEK293 cells or derived from experiments in which epitope-tagged mTOR has been overexpressed. Here we have used immunofluorescence/confocal immunostaining in conjunction with cell fractionation and Western blot analysis to examine distribution of mTOR and its putative substrates in four human cell types, colon carcinoma, rhabdomyosarcoma, fibroblasts, and HEK293 cells. In addition, the distribution of mTOR was examined in murine C2C12 myoblasts during differentiation. The human rhabdomyosarcoma cell lines Rh1, Rh30, and Rh41 have been described previously (31Dilling M.B. Dias P. Shapiro D.N. German G.S. Johnson R.K. Houghton P.J. Cancer Res. 1994; 54: 903-907PubMed Google Scholar). Rhabdomyosarcoma cells were obtained from the American Type Culture Collection (Manassas, VA). Briefly, cells were grown in antibiotic-free RPMI 1640 medium (BioWhittaker, Walkersville, MD) supplemented with 10% fetal bovine serum (HyClone Laboratories, Logan, UT) and 2 mml-glutamine (BioWhittaker) at 37 °C in an atmosphere of 5% CO2. Human colon carcinoma cell lines (HCT8, HCT29, and HCT116) and normal human fibroblasts (IMR90) were cultured under the same conditions as the rhabdomyosarcoma cell lines. Human embryonic kidney cells (HEK293) were grown in antibiotic-free DMEM (BioWhittaker) containing 10% fetal bovine serum and 2 mml-glutamine at 37 °C in an atmosphere of 10% CO2. Mouse C2C12 myoblasts were purchased from the American Type Culture Collection and were routinely grown in antibiotic-free DMEM with 15% fetal calf serum and 4 mml-glutamine (growth medium, GM) at 37 °C and 5% CO2. Cells were induced to differentiate by growth in differentiation medium (DM, DMEM with 2% horse serum supplemented with 4 mml-glutamine) at 37 °C and 5% CO2. Mouse monoclonal antibody 26E3 was raised against a synthetic peptide (KPQWYRHTFEE) representing amino acid residues from 230 to 240 in the N terminus of mTOR, using procedures reported previously (32Hosoi H. Dilling M.B. Liu L.N. Danks M.K. Shikata T. Sekulic A. Abraham R.T. Lawrence J.C., Jr. Houghton P.J. Mol. Pharmacol. 1998; 54: 815-824Crossref PubMed Scopus (165) Google Scholar). Rabbit polyclonal antibodies against FRAP (raised against the FKBP-rapamycin binding domain of mTOR), S6K1, c-Jun, insulin-like growth factor I receptor β chain (IGF-IRβ), E1A, protein A/G plus agarose, normal mouse IgG, normal rabbit IgG, and all secondary antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Anti-4E-BP1 was from Zymed Laboratories, Inc. (South San Francisco, CA); phospho-specific antibodies to the Thr-46 residue of 4E-BP1 were from Cell Signaling Technology (Beverly, MA); anti-eIF4E was from Transduction Laboratories (Lexington, KY); anti-β-tubulin was from Sigma Chemical Co. (St. Louis, MO); mouse monoclonal anti-MyoD was from BD PharMingen (San Diego, CA); monoclonal antibody against the epidermal growth factor receptor, ERBB1, was from Novacastra Laboratories (Newcastle, UK); and anti-AU1 was from BAbCO (Richmond, CA). Subconfluent cells grown in T-162 flasks (Corning Inc., Corning, NY) were trypsinized and washed twice with cold PBS. The cells were resuspended in 500 μl of ice-cold hypotonic buffer (10 mm HEPES, pH 7.9, 0.5 mmdithiothreitol, 0.5 mm phenylmethylsulfonyl fluoride, and one protease inhibitor mixture tablet (Roche Molecular Biochemicals, Mannheim, Germany)) and maintained on ice for 15 min. After addition of 20 μl of 10% Nonidet P-40, the samples were vortexed for 10 s and centrifuged to pellet the nuclei at 800 × g for 1 min. The supernatants were saved separately on ice. The pelleted nuclei were washed once with 500 μl of hypotonic buffer and 140 μl of Nonidet P-40, washed once with 500 μl of hypotonic buffer alone, and extracted in 200 μl of ice-cold hypertonic buffer (20 mmHEPES, pH 7.9, 25% glycerol, 0.42 m NaCl, 0.2 mm EDTA, 0.5 mm dithiothreitol, 0.5 mm phenylmethylsulfonyl fluoride, and one protease inhibitor mixture tablet (Roche Molecular Biochemicals)) on ice for 30 min. The cytoplasmic fraction was obtained as supernatant after centrifugation of the post-nuclear supernatant at 60,000 ×g for 30 min. The membrane fraction, obtained as the pellet, was dissolved in 200 μl of hypotonic buffer. Whole cell extracts were prepared directly in cell lysis buffer (Cell Signaling, Beverly, MA). Samples were maintained at −80 °C until analysis. After adding 4× SDS sample buffer, the samples containing equal protein concentration were heated for 5 min at 95 °C and resolved on a 7.5% Tris-HCl denaturing ready-gel (Bio-Rad, Hercules, CA) for detection of mTOR and ERBB1 or on a 12% Bio-Rad Tris-HCl denaturing ready-gel for detection of other proteins. Electrophoresis was performed at a constant 100 V at 4 °C for 1.5–2 h. The separated proteins were transferred to polyvinylidene difluoride membranes (Immobilon, Millipore, Bedford, MA) by electrophoresis at 4 °C for 1–2 h. Nonspecific binding was blocked by incubation with 5% nonfat milk at room temperature for 1 h, and the membranes were incubated overnight with primary antibody at 4 °C. The membranes were washed three times with PBS-T, incubated with secondary antibody conjugated to horseradish peroxidase at room temperature for 1 h, and again washed three times in PBS-T. Immunoreactive bands were visualized using Renaissance chemiluminescence reagent (PerkinElmer Life Sciences, Boston, MA) and Kodak Biomax MR film (Eastman Kodak Co., Rochester, NY). mTOR activity was assayed with a modification of the method of Dennis et al. (13Dennis P.B. Jaeschke A. Saitoh M. Fowler B. Kozma S.C. Thomas G. Science. 2001; 294: 1102-1105Crossref PubMed Scopus (800) Google Scholar). To pre-clear cell lysates 20 μl of protein A/G plus agarose beads (Santa Cruz Biotechnology) and 2 μg of normal mouse IgG were added to the cell fractions, and the samples were rotated at 4 °C for 1 h. The complexes were pelleted at 2000 × g for 5 min. 2 μg of mouse monoclonal 26E3 antibody, 2 μg of anti-AU1 mouse monoclonal antibody, or 2 μg of normal mouse IgG (as negative control) was added to the supernatant, and the samples were rotated at 4 °C overnight. 30 μl of protein A/G plus agarose beads was added, and the samples were rotated for 3 h at 4 °C. After centrifuging, the beads were washed once with 500 μl of ice-cold 1m NaCl in assay buffer (30 mm MOPS, pH 7.5, 5 mm NaF, 20 mm β-glycerophosphate, 1 mm dithiothreitol, 0.1% Triton X-100, and 10% glycerol) and twice with 500 μl of cold assay buffer alone. The pellets were resuspended in 30 μl of assay buffer containing 10 mmMnCl2, 2 mm ATP, and 1 μg of GST-4E-BP1 recombinant protein. After incubation for 30 min at 30 °C the assay was terminated by the addition of 10 μl of 4× SDS sample buffer, and the samples were heated for 5 min at 95 °C. Proteins were separated on a 10% Bio-Rad Tris-HCl denaturing ready-gel and transferred to polyvinylidene difluoride membranes. Membranes were probed with mouse monoclonal antibody 26E3, rabbit polyclonal anti-phospho-4E-BP1 (Thr-46), or rabbit polyclonal anti-4E-BP1, followed by incubation with goat anti-rabbit IgG-conjugated horseradish peroxidase. Membranes were incubated with chemiluminescence substrate and exposed to Kodak Biomax film. Similar procedures were used to immunoprecipitate AU1 epitope-tagged kinase-dead rapamycin-resistant mTOR (SIDA), which was stably expressed in Rh30 cells, but only using anti-AU1 antibody. A modified method of Wright (33Wright W.E. Exp. Cell Res. 1978; 112: 395-407Crossref PubMed Scopus (57) Google Scholar) was used with slight modification for preparing hybrids between Rh30 and HEK293 cells as previously reported (34Sosinski J. Thakar J.H. Germain G.S. Dias P. Harwood F.C. Kuttesch J.F. Houghton P.J. Mol. Pharmacol. 1994; 45: 962-970PubMed Google Scholar). Briefly, 1 × 107 Rh30 and HEK293 cells were plated in separate T-162 flasks and grown for 24 h. Cells were washed twice with bicarbonate-free Hanks' solution (Cellgro, Herndon, VA), trypsinized, and pelleted. Rh30 cells were resuspended in 30 ml of freshly prepared cold Hanks' solution containing 0.001% diethylpyrocarbonate (Sigma, St. Louis, MO). HEK293 cells were resuspended in 30 ml of freshly prepared cold Hanks' solution containing 0.5 mmiodoacetamide (Sigma). To determine that individual treatments with diethylpyrocarbonate and iodoacetamide were highly toxic, a sample of cells (0.5 ml) from each treatment were plated in 100-mm culture dishes and grown as described below. Suspensions of Rh30 and HEK293 cells were added together and mixed by gently inverting the tube and then centrifuged at 200 × g for 5 min and fused with polyethylene glycol 1000 (final concentration of 50% v/v, Sigma) that was diluted in serum-free DMEM containing 15% Me2SO. Cells growing on four-chamber well slides (Nunc Inc., Naperville, IL) were fixed in freshly prepared 1% paraformaldehyde for 30 min at room temperature, rinsed, and permeabilized with 0.4% Triton X-100 in PBS for 30 min. Fixed cells were then incubated with 10% swine serum in PBS to block nonspecific binding of antibodies. After thorough rinsing, cells were incubated with the mouse monoclonal 26E3 antibody (in 1% swine serum-PBS) for 1 h at 37 °C. Slides were rinsed with PBS and incubated with fluorescein isothiocyanate-coupled anti-mouse antibody (in 1% swine serum-PBS). After another thorough rinse, the samples were incubated with rabbit polyclonal anti-FRAP antibody followed by rhodamine-coupled anti-rabbit antibody. The cells were rinsed with PBS, incubated with 0.1 mg/ml RNase for 30 min at 37 °C, and mounted inp-phenylenediamine medium containing 1 mmTO-PRO-3 (far-red DNA dye excitable with a helium-neon laser; Molecular Probes, Eugene, OR) to stain DNA. Appropriate controls were maintained by substituting the primary antibodies with normal mouse and rabbit IgGs to check for nonspecific binding. The cells were examined in a Leica TCS NT SP confocal laser scanning microscope equipped with argon (488 nm), krypton (568 nm), and helium-neon (633 nm) lasers; the three lasers permitted the imaging of fluorescein isothiocyanate (green), rhodamine (red), and TO-PRO-3 (far red), respectively. Single optical sections (0.5 μm) were obtained through the center of the cell, and the images were sequentially scanned (to reduce cross-talk between channels) on the three channels. The TO-PRO-3 image (DNA fluorescence) was pseudo-colored blue to discriminate it from the rhodamine image. The TOPRO-3 staining in the blue channel is shown to indicate the outlines of the nuclei. The three channel images and an overlay image of red and green channels were recorded using the Leica imaging software. The images were re-scaled and gamma-corrected with Adobe Photoshop. The mouse monoclonal antibody against the N-terminal sequence of mTOR was characterized as previously reported for another antibody produced against this synthetic peptide (32Hosoi H. Dilling M.B. Liu L.N. Danks M.K. Shikata T. Sekulic A. Abraham R.T. Lawrence J.C., Jr. Houghton P.J. Mol. Pharmacol. 1998; 54: 815-824Crossref PubMed Scopus (165) Google Scholar). As shown in Fig.1, 26E3 detects a single protein (∼220 kDa) by Western blot analysis that is competed by the cognate peptide (Peptide 1, Fig. 1 a), but not by another peptide sequence from mTOR (Peptide 3, residues 920–929, Fig.1 b). 26E3 immunoprecipitates a single protein of ∼220 kDa that is competed by the cognate peptide (peptide 1) added either during immunoprecipitation or during subsequent immunoblotting (Fig.1 c). Furthermore, the protein detected by 26E3 is retained on an FKBP affinity column only in the presence of rapamycin (Fig.1 d). These data are consistent with 26E3 specifically binding to mTOR. To determine the cellular localization of mTOR in neoplastic and normal cells we used immunofluorescence staining in conjunction with confocal microscopy. Because mTOR localization in the cytosol of HEK293 cells has been reported previously, we used this cell line as a control. As shown in Fig. 2, staining of HEK293 by 26E3 monoclonal or rabbit anti-FRAP polyclonal antibodies showed similar predominant cytoplasmic distribution of mTOR thus confirming previous reports (30Kim J.E. Chen J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 14340-14345Crossref PubMed Scopus (187) Google Scholar). Cellular distributions of mTOR in rhabdomyosarcomas (Rh30 and Rh41), IMR90 human fibroblasts, and HCT8 colon carcinoma cells are shown in Fig.3. Appropriate controls (normal mouse or rabbit IgG) are presented in Fig. 4. In contrast to the results obtained in HEK293 cells, mTOR is localized predominantly in the nucleus of each of the other cell lines. Similar results were obtained with both antibodies in murine C2C12 myoblasts cultured in growth medium or differentiation medium in the presence or absence of rapamycin (Fig. 5). In all conditions nuclear mTOR was readily detected and distribution was not altered during myogenic differentiation or by rapamycin treatment.Figure 3Immunofluorescent images of rhabdomyosarcoma (Rh30 and Rh41), normal human fibroblasts (IMR90), and HCT8 colon carcinoma cells. Staining with 26E3 (green, top row), anti-FRAP (red, row 2), TO-PRO-3 (TOPRO, blue, row 3), and merged images from antibody staining (yellow, row 4). Bar, 25 μm.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 4Immunofluorescent images after staining the same cell lines with isotype-matched control mouse or rabbit IgG or TO-PRO-3. Bar, 25 μm.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 5Immunostaining of endogenous mTOR in C2C12 murine myoblasts with 26E3 anti-FRAP. Cells were grown 3 days in growth medium (GM) or differentiation medium (DM) with (+) or without (−) rapamycin (100 ng/ml). The 26E3 signal (green, left column), FRAP signal (red, column 2), DNA fluorescence (TOPRO, column 3), and merged imagesfrom antibody staining (yellow, column 4) were analyzed by confocal microscopy. Negative control cells were co-stained with normal mouse IgG and rabbit IgG. Staining with isotype control antibodies or TO-PRO-3 is shown in the top row. Rows 2–5 show immunostaining with the reagent listed at thetop of each column of images. The right columnshows merged images. Bar, 25 μm.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To independently determine the cellular distribution of mTOR, cells were fractionated into nuclear, cytoplasmic, and membrane fractions. Staining for MyoD (for rhabdomyosarcomas) or c-Jun transcription factors was used to mark nuclear fractions, β-tubulin as a cytosolic marker, and IGF-IRβ or ERBB1 as a membrane marker, where appropriate. mTOR was detected by both 26E3 and anti-FRAP antibodies. As shown in Fig.6 a, mTOR was detected in nuclear fractions of rhabdomyosarcoma cells by both antibodies. In addition, mTOR was detected in the membrane fraction by 26E3 and to a lesser extent by anti-FRAP in all of these cells. mTOR was not detected in cytoplasmic fractions. The relative purity of each fraction is shown by the localization of the marker proteins. For rhabdomyosarcomas MyoD was predominantly or exclusively detected in the nuclear fraction (Rh1 cells do not express MyoD (35Morton C.L. Potter P.M.J. Pharmacol. Exp. Ther. 1998; 286: 1066-1073PubMed Google Scholar)). Similarly, the transcription factor c-Jun was predominantly nuclear, β-tubulin was exclusively cytoplasmic, and IGF-IRβ was predominantly associated with the membrane fraction. Thus, results of cellular fractionation show disposition of mTOR that is consistent with results obtained from the immunofluorescence/confocal microscopy studies. Cellular fractionation of colon adenocarcinoma cells also demonstrated predominantly nuclear detection of mTOR using either antibody (Fig.6 b). Using the 26E3 monoclonal antibody, mTOR was detected as a single band in nuclear fractions prepared from HCT8 and HCT116 cells but not in cytoplasmic or membrane fractions. A small fraction of mTOR was detected in membranes from HCT29 cells. The polyclonal anti-FRAP reagent detected several bands in nuclear fractions but did not detect mTOR in membrane fractions from any colon cell line. The relative purity of each fraction is demonstrated by detection of ERBB1, β-tubulin, and c-Jun exclusively in the membrane, cytoplasmic, and nuclear fractions, respectively. The cellular distribution of mT
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