Reagents were obtained from the following sources: protein A-Sepharose CL4B and protein G-Sepharose Fast Flow were from GE Healthcare; 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS) was from Pierce; Immobilon-P polyvinylidene difluoride (PVDF) membrane (0.45 μm) was from Millipore; autoradiography film (HyBlot CL) was from Denville Scientific; reagents for enhanced chemiluminescence (ECL) were from Millipore (Immobilon Western chemiluminescent horseradish peroxidase [HRP] substrate); all chemicals were from either Fisher Chemicals or Sigma.
AU1, Myc (9E10), and HA (HA.11) antibodies were from Covance. Flag-M2 antibody was from Sigma, and Rheb antibody was from AbCam. Donkey anti-rabbit antibody-HRP and sheep anti-mouse antibody-HRP were from GE Healthcare. The following antibodies were from Cell Signaling Technology: P-S6K1 (Thr389; rabbit monoclonal 108D2), S6, mTOR P-S2481, P-Akt (S473), total Akt, P-4EBP1 (T37/46), P-4EBP1 (S65), P-4EBP1 (T70), and total 4EBP1. Tubulin monoclonal antibody was a gift from K. Verhey (University of Michigan Medical School, Ann Arbor).
Custom generation of antibodies to raptor, mTOR, P-S1261-mTOR, rictor, S6K1, and P-S6.
To generate immunogen, peptides, and P-peptides (70% pure; synthesized by Boston Biomolecules, Woburn, MA [now called Advanced Peptides, Inc., Boston, MA]) were coupled via an N-terminal cysteine to maleimide-activated mariculture keyhole limpet hemocyanin (mcKLH; Pierce). Antipeptide antibodies were affinity purified by positive selection on antigen peptide that was coupled to Affigel matrix (Bio-Rad). Phospho-peptide antibodies were affinity purified by positive selection on antigen phospho-peptide followed by negative selection on cognate antigen non-phospho-peptide and irrelevant phospho-peptide (either a P-Ser Jak2 or a P-Thr-Jak2 peptide). The following peptides were used to generate antibodies (“p” indicates a phosphorylated residue): (i) raptor (amino acids 885 to 901; human; CSSSLTNDVAKQPVSRDL); (ii) mTOR (amino acids 221 to 237; rat; CTQREPKEMQKPQWYRHT); (iii) P-S1261-mTOR [amino acids 1256 to 1266; rat; CKKLHV(pS)TINLQ]; (iv) rictor (amino acids 6 to 20; human; CRGRSLKNLRVRGRND); (v) S6K1 (C-terminal 17 amino acids 485 to 502 of the 70-kDa rat isoform; CKQAFPMISKRPEHLRMNL); (vi) P-S6 [amino acids 232 to 249; CRRL(pS)(pS)LRA(pS)TSK(pS)EE(pS)QK].
The pcDNA3/AU1-mTOR wild-type (WT), rapamycin-resistant (RR; S2035I), kinase-dead (KD; S2338A), and RR/KD (S2035I; D2338A) plasmids were generated by R. Abraham (Wyeth, Pearl River, NY); the pRK5/Myc-raptor plasmid was provided by D. Sabatini (MIT, Boston, MA); pRK5/Myc-mTOR and pRK5/Myc-mTOR-KD were obtained from D. Sabatini via Addgene (nos. 1861 and 8482, respectively); the pRK7/HA-S6K1, pKH3/HA-mLST8/GβL, and pRK7/Flag-Rheb plasmids were from J. Blenis (Harvard Medical School, Boston, MA), and pACTAG2/3HA-4EBP1 was from N. Sonenberg (McGill, Montreal, Canada).
Mass spectrometric analysis to identify mTOR P-sites.
HEK293 cells (20 15-cm plates per condition; ~15 × 106 cells/15-cm plate) were untransfected or transiently transfected with AU1-tagged mTOR (25 μg) and cultured in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS) or in growth factor and amino acid-stimulated medium (DMEM plus insulin; 30 min) after factor deprivation (via serum withdrawal for 24 h followed by incubation in Dulbecco's phosphate-buffered saline [D-PBS]-glucose for 60 min). AU1-mTOR was immunoprecipitated overnight, resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and stained with Coomassie blue R-250. AU1-mTOR protein was excised from the gel, digested with trypsin, and analyzed by tandem mass spectrometry.
In-gel digestion, mass spectrometry, and data analysis.
Cubed Coomassie-stained gel bands of immunoprecipitated AU1-tagged rat mTOR were rinsed with water, destained with 50% acetonitrile (MeCN), 50% ammonium bicarbonate, dehydrated with 100% MeCN, and subjected to in-gel digestion with 6 ng/μl sequencing-grade modified trypsin (Promega) in 50 mM ammonium bicarbonate for 16 h at 37°C. Peptides were extracted once with 50% MeCN, 2.5% formic acid (FA), and once with 100% MeCN. Dried peptides were resuspended in 2.5% MeCN, 2.5% FA, and loaded using a Micro AS autosampler (Thermo Electron) and a Surveyor MS Pump Plus (Thermo Electron) onto a nano-electrospray microcapillary column packed with 14 cm of reverse-phase MagicC18 material (5 μm; 200Å; Michrom Bioresources, Inc.). Elution was performed with a 5 to 35% MeCN (0.15% FA) gradient over 45 min, after a 15-min isocratic loading at 2.5% MeCN, 0.15% FA. Solvent A was 2.5% MeCN, 0.15% FA, and solvent B was 99.85% MeCN, 0.15% FA. Mass spectra were acquired in an LTQ linear ion trap mass spectrometer (Thermo Electron). Throughout the entire run, 10 data-dependent tandem mass spectrometry (MS/MS) scans were acquired on the most abundant ions in dynamic exclusion mode following a precursor survey (MS1) scan. Mass spectral data were searched against a rat mTOR protein database using Turbo SEQUEST (Thermo Electron, version 27, revision 12) requiring no enzyme specificity and a 2-Da precursor mass tolerance. Cysteine residues were required to have a static increase of 71.0 Da for acrylamide (C3H5ON) adduction. Differential modification of 16.0 Da on methionine and 80.0 Da on serine, threonine, and tyrosine was permitted.
cDNA mutagenesis and sequencing.
Site-directed mutagenesis was performed using QuikChange II XL (Stratagene), and cDNA inserts in mutated plasmids were fully sequenced. We generated the following mutations in WT and RR pcDNA3/AU1-mTOR backbones: S1261A, S1261D, and S2481A. We generated the following mutations in pRK5/Myc-mTOR: RR (S2035I), S1261A, RR/S1261A, RR/S1261D, and S2481A. The following oligonucleotides were used to create point mutations in the rat mTOR cDNA (accession number L37085) (capital letters indicate mismatches, and the three underlined nucleotides represent the codon mutated): S1261A, primer 1, 5′-gaagaaactgcatgtcGCcaccatcaacctcc-3′, and primer 2, 5′-ggaggttgatggtgGCgacatgcagtttcttc-3′; S1261D, primer 1, 5′-gaagaaactgcatgtcGAcaccatcaacctcc-3′, and primer 2, 5′-ggaggttgatggtgTCgacatgcagtttcttc-3′; S2481A, primer 1, 5′-tgccagaatccatccatgcCttcattggagatggtttgg-3′, and primer 2, 5′-ccaaaccatctccaatgaagGcatggatggattctggca-3′; S2035I, primer 1, 5′-ggcctagaagaggccATtcgcttgtactttggg-3′, and primer 2, 5′cccaaagtacaagcgaATggcctcttctaggcc-3′.
Generation of shRNA-encoding lentiviruses and lentiviral infection.
shRNA-encoding lentiviruses were generated via the protocol posted on the Addgene website (http://www.addgene.org/pgvec1
). Briefly, 7 × 105
HEK293T cells on 60-mm plates were cotransfected with pLKO.1 shRNA plasmid (1 μg), pCMV-dR8.2 dvpr (Addgene number 8455) packaging plasmid (750 ng), and pCMV-VSVG (Addgene number 8454) envelope plasmid (250 ng) using FuGENE transfection reagent. The next morning, the cells were refed with 5 ml fresh medium (DMEM-10% FBS). After 24 h, the virus-containing medium was harvested, and another 5 ml of fresh medium was added. After 24 h, the viral-containing medium was harvested a second time, pooled with the first collection, and filtered (0.45 μm). Viral infection was performed by adding 500 μl of virus-containing medium to a ~70% confluent plate of TSC1−/−
mouse embryonic fibroblasts (MEFs) that were fed with fresh medium containing 8 μg/ml Polybrene. After 24 h, cells were split 1:10 (due to the high proliferation rate) and selected for ~6 days in fresh medium containing 2.5 μg/ml puromycin. The following plasmids were used: pLKO.1-scramble shRNA (Addgene number 1864), pLKO.1 Rheb-1 (Open Biosystems number TRCN0000075603), and pLKO.1 Rheb-3 (Open Biosystems number TRCN0000075605) (the Rheb shRNA plasmids were kindly shared by John Blenis, Harvard Medical School, Boston, MA). The hairpin sequence for the mouse-specific Rheb-1 shRNA was the following: 5′-CCGGCCCGTCATCCTTGAAGATAAACTCGAGTTTATCTTCAAGGATGACGGGTTTTTG-3′. The hairpin sequence for the mouse-specific Rheb-3 shRNA was the following: 5′-CCGGCAGACATACTCCATAGATATTCTCGAGAATATCTATGGAGTATGTCTGTTTTTG-3′.
Cell culture, drug treatment, and transfection.
HEK293 cells and immortalized TSC1+/+
mouse embryonic fibroblasts (originally from D. Kwiatkowski, Brigham and Women's Hospital, Boston, MA) were cultured in DMEM that contained high glucose (4.5 g/liter), glutamine (584 mg/liter), and sodium pyruvate (110 mg/liter; Gibco/Invitrogen) supplemented with 10% FBS (HyClone). 3T3-L1 fibroblasts were differentiated into adipocytes by a standard protocol (41
). All cells were incubated at 37°C in a humidified atmosphere containing 5% CO2
. All cells were serum deprived via incubation in DMEM supplemented with 20 mM HEPES (pH 7.2) for ~20 h. Insulin (100 nM; Invitrogen) was added to serum-deprived cells for 30 min. For drug treatment, serum-deprived cells were pretreated with rapamycin (20 ng/ml; Calbiochem) or wortmannin (100 nM; Upstate/Millipore) for 30 min prior to the addition of insulin; cells proliferating in DMEM-FBS (steady state) were pretreated with drugs for 1 to 2 h. To effect amino acid deprivation, HEK293 cells were incubated in Dulbecco's PBS containing d
-glucose (1 g/liter) and sodium pyruvate (36 mg/liter; D-PBS/Glc) for 60 min. HEK293 cells on 60-mm plates were transfected using TransIT-LT1 (Mirus) and a total of 4 to 5 μg of DNA per plate; the specific amounts of experimental plasmid transfected are stated in the figure legends. Cells were lysed ~24 to 48 h posttransfection.
Cell lysis, immunoprecipitation, and immunoblotting.
Cells were washed twice with ice-cold PBS and scraped into ice-cold lysis buffer A (10 mM KPO4
, pH 7.2, 1 mM EDTA, 5 mM EGTA, 10 mM MgCl2
, 50 mM β-glycerophosphate, 1 mM sodium orthovanadate [Na3
], 5 μg/ml pepstatin A, 10 μg/ml leupeptin, 40 μg/ml phenylmethylsulfonyl fluoride [PMSF]) containing CHAPS (0.3%) (to preserve the mTOR-Raptor interaction postlysis) (24
) or NP-40 (0.5%) and Brij35 (0.1%). Lysates were spun at 13,200 rpm for 5 min at 4°C, and the postnuclear supernatants were collected. The Bradford assay was used to normalize protein levels for immunoprecipitation and immunoblot analyses. For immunoprecipitation, whole-cell lysates (WCL) were incubated with antibodies for ~2 h (overnight for AU1 immunoprecipitations) at 4°C, incubated with protein A- or G-Sepharose beads for 1.5 h, washed three times in lysis buffer, and resuspended in 1× sample buffer (50 mM Tris-HCl, pH 6.8, 10% glycerol, 2% SDS, 2% β-mercaptoethanol, 0.02% bromophenol blue). Samples were resolved on SDS-PAGE gels and transferred to PVDF membranes by using Towbin transfer buffer (25 mM Tris, 192 mM glycine, 10% methanol, 0.02% SDS). Immunoblotting was performed by blocking PVDF membranes in Tris-buffered saline with Tween 20 (TBST; 40 mM Tris-HCl, pH 7.5, 0.9% NaCl, 0.1% Tween 20) containing 3% nonfat milk and incubating the membranes in TBST with 2% bovine serum albumin containing primary antibodies or secondary HRP-conjugated antibodies. Blots were developed by ECL.
Treatment of mTOR with λ-phosphatase.
Following mTOR immunoprecipitation, beads were washed three times in CHAPS lysis buffer followed by two additional washes in ST buffer (50 mM Tris-HCl, pH 7.2; 150 mM NaCl). Beads were then resuspended in 1× phosphatase buffer (50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 2 mM dithiothreitol, 0.1 mM EGTA, 0.01% Brij 35) that contained 2 mM MnCl2. Samples were incubated at 30°C for 30 min in the absence or presence of λ-phosphatase (Invitrogen), and reactions were terminated by adding EDTA, pH 8.0, to a 50 mM final concentration and sample buffer to a 1× final concentration. Six percent SDS-PAGE gels were run at 8 mA for ~16 h.
S6K1 in vitro kinase assays.
Lysates from HA-S6K1 transfected cells were immunoprecipitated with anti-hemagglutinin (HA) antibodies for 2 h and incubated with protein G-Sepharose beads for 1.5 h. Immune complexes were each washed in 1 ml of ice-cold buffer A (10 mM Tris-HCl, pH 7.2, 100 mM NaCl, 1 mM EDTA, 1.0% NP-40, 0.5% sodium deoxycholate, 1 mM Na3VO4, 40 μg/ml PMSF), buffer B (10 mM Tris-HCl, pH 7.2, 1 M NaCl, 1 mM EDTA, 0.1% NP-40, 1 mM Na3VO4, 40 μg/ml PMSF), ST (see above), and 1× kinase buffer (20 mM HEPES, pH 7.2, 100 mM MgCl2, 1 mg/ml bovine serum albumin). Immune complexes were then resuspended in 20 μl 1.5× kinase buffer. Kinase reactions were initiated with the addition of 10 μl of “start” solution containing cold ATP (50 μM final), [32P]ATP (10 μCi/reaction mixture), cyclic AMP-dependent protein kinase inhibitor (0.03 μg/reaction mixture; Sigma), and recombinant glutathione S-transferase (GST)-S6 purified from bacteria (2 μg/reaction mixture) and incubated at 30°C for 10 min. Kinase reactions were terminated by the addition of 30 μl of 2× sample buffer. The amount of 32P incorporated into GST-S6 was measured using a PhosphorImager (Typhoon 9400; Amersham Biosciences) and quantitated using the software ImageQuant TL.
Flow cytometry to determine relative cell size.
HEK293 cells on 60-mm plates (80% confluent) were cotransfected with 1 μg green fluorescent protein (GFP)-spectrin and 10 μg Myc-mTOR plasmids. The next day, one-quarter of a 60-mm plate was transferred to a 10-cm plate and allowed to proliferate in DMEM-FBS in the absence or presence of rapamycin (20 ng/ml) for 72 h. For immunoblot analysis, one-half of the 60-mm culture was plated to a 10-cm plate and cultured for ~24 h. For cell size analysis, plates of subconfluent cells were washed once with PBS and incubated in 3 ml PBS-0.1% EDTA for ~5 min at room temperature. Cells were then gently triturated off the plastic and transferred to conical tubes on ice, centrifuged for 5 min at 1,000 rpm, and washed with 3 ml of PBS-1% calf serum. Cells were again centrifuged at 1,000 rpm for 5 min, resuspended in 0.5 ml PBS, fixed in 80% ethanol by adding 5 ml 88% ethanol, and stored overnight at 4°C. Immediately before fluorescence-activated cell sorting analysis, fixed cells were centrifuged at 1,600 rpm for 5 min, washed in 3 ml PBS-1% calf serum, and centrifuged at 1,000 rpm for 5 min. DNA was stained by resuspending fixed cells in 1 to 1.5 ml PI-RNase A solution and incubating them at 37°C for 30 min. The PI-RNase A solution contained the following: 10 μg/ml propidium iodide in 0.76 mM sodium citrate (pH 7.0), 250 μg/ml RNase A in 10 mM Tris-HCl (pH 7.5), 15 mM NaCl diluted in PBS-1% calf serum. For cell size analysis by flow cytometry, the mean forward scatter height (FSC-H) of 5,000 GFP-positive, G1-phase cells was determined by gating on GFP and PI fluorescence using a BD Biosciences FACSCalibur with CellQuest software.
For some figures, irrelevant lanes were removed from a scanned autoradiograph and flanking lanes juxtaposed using Adobe Photoshop. The presence of a thin, vertical black line indicates such a modification.
To quantitate ECL Western blot band densities, the software NIH Image J (version 1.41) was used after scanning/digitizing the autorads; a Student's unpaired t test (two-sample, assuming equal variances; two tails) was used (a P value of <0.05 was considered significant). For the cell size experiments, analysis of variance (ANOVA) followed by Tukey's post hoc tests was used to determine the significance of the population means (a P value of <0.01 was considered significant). The software Prism 5 from GraphPad was used for the ANOVA.