Reagents were obtained from the following sources: antibodies to phospho-T389 S6K1, phospho-S235/S236 S6, phospho-T37/T46 4E-BP1, phospho-S65 4E-BP1, phospho-S70 4E-BP1, phospho-T183 PRAS40, phospho-S758 ULK1, phospho-S150 Grb10, phospho-S476 Grb10, phospho-S106 Lipin1, phosphor-S472 Lipin1, phosphor-S1135 Rictor, S6K1, 4E-BP1, PRAS40, FLAG, S6, and Rictor from Cell Signaling Technology; an antibody to Grb10 and HRP-labeled anti-mouse and anti-rabbit secondary antibodies from SantaCruz Biotechnology; an antibody to p62 from Progen; antibodies to ULK1, FLAG and β-actin (clone AC-15), FLAG M2 affinity gel, ATP, FKBP12, amino acids, and insulin from Sigma-Aldrich; [γ-32P] ATP from Perkin-Elmer; FuGENE 6, PhosSTOP, and Complete Protease Cocktail from Roche; rapamycin from LC Laboratories; DMEM from SAFC Biosciences; Inactivated Fetal Calf Serum (IFS), Fetal Bovine Serum (FBS) and SimplyBlue Coomassie G from Invitrogen; amino acid-free RPMI from US Biological; Superose 6 10/300 GL from GE Healthcare; BCA assay reagent, protein G-sepharose, streptavidin agarose, and immobilized glutathione beads from Thermo Scientific; Whatman grade P81 ion exchange chromatography paper from Fisher Scientific; QIAamp DNA Mini Kit, QuikChange XLII mutagenesis kit and XL10-Gold Competent Cells from Stratagene; SYBR Green PCR Master Mix from AB Applied Biosystems; and SAM2 Biotin Capture Membrane from Promega. Torin1 was provided by Nathanael Gray (Harvard Medical School) (18
Cell lines and tissue culture
HEK-293E and MEFs were cultured in DMEM with 10% FBS and antibiotics. HEK-293T was cultured in DMEM with 10% IFS and antibiotics. HEK-293Es were generously provided by John Blenis (Harvard Medical School), p53−/− MEFs by David Kwiatkowski (Harvard Medical School), S6K1+/+S6K2+/+ and S6K1−/−S6K2−/− MEFs by Mario Pende (INSERM U845, Medical School, Paris Descartes University), ULK1+/+ and ULK1−/− shULK2 MEFs by Reuben Shaw (Salk Institute), and ULK1+/+ULK2+/+ and ULK1−/−ULK2−/− by Craig Thompson (Memorial Sloan-Kettering Cancer Center).
To generate stable cell lines, mRNA-encoding plasmids were co-transfected with Delta VPR (pLJM60/61 lentivirus) or Gag-pol envelope (pQCXIP/N retrovirus) and CMV VSV-G packaging plasmids into actively growing HEK-293T using FuGENE 6 transfection reagent as previously described (45
). Virus containing supernatants were collected at 48 hr post transfection, centrifuged to eliminate floating cells, and target cells (100,000–1,000,000) infected in the presence of 8 mg/ml polybrene. 24 hr post infection, the cells were given or split into fresh media 2 μg/mL puromycin or 1 mg/mL neomycin. mRNA-expressing cells were analyzed 2–7 days post-infection.
cDNA manipulations and mutagenesis
The mTOR truncation mutant (1295–2549) and LARP1 cDNAs were amplified by PCR, and the products were subcloned into the SalI and XhoI sites of the FLAG-tagged pQCXIP (puromycin resistant) retroviral vector for stable expression. The mLST8 cDNA was amplified by PCR, and the product was subcloned into the NotI and EcoRI sites of the pQCXIN (neomycin resistant) vector for stable expression. The S6K1 and ULK1 cDNAs were amplified by PCR, and the products were subcloned into the SalI and NotI sites of the pLJM60 (puromycin resistant) or pLJM61 (neomycin resistant) lentiviral vector for stable expression. The pLJM60 S6K1, pLJM60/61 ULK1 and pRK5 GST-tagged mouse Akt1 plasmids were mutagenized with the QuikChange XLII mutagenesis kit with oligonucleotides obtained from Integrated DNA Technologies. The S6K1, ULK1 and Akt1 mutants used in our experiments were T389S, S758T and S473T, respectively. For barcoding pLJM60 S6K1 constructs, GGATCC (BamHI) and GGTACC (KpnI) sequences were inserted in front of the start codons of wild-type and T389S S6K1, respectively, using the QuikChange XLII mutagenesis kit with oligonucleotides obtained from Integrated DNA Technologies.
Cell treatments and lysis and immunoprecipitations
For rapamycin and Torin1 treatments, 70–80% confluent cells were treated with DMSO or inhibitors as indicated in figure legends. Amino acids and serum were titrated as indicated in figure legends. Cells rinsed once with ice-cold PBS (Phosphate Buffered Saline) and lysed in ice-cold lysis buffer (50 mM HEPES pH 7.4, 40 mM NaCl, 2 mM EDTA, 1 mM orthovanadate, 50 mM NaF, 10 mM pyrophosphate, 10 mM glycerophosphate, and 1% Triton X-100 or 0.3% CHAPS (for immunoprecipitations) with one PhosSTOP tablet and one tablet of EDTA-free protease inhibitors per 25 mL. The soluble fractions of cell lysates were isolated by centrifugation at 13,000 rpm for 10 min. For FLAG immunoprecipitations, 50% slurry of FLAG M2 affinity agarose was added to the lysates and the mixtures incubated with rotation for 2–6 hr at 4°
C. Immunoprecipitates were washed three times with lysis buffer containing 150 mM NaCl. Immunoprecipitated proteins were denatured by the addition of sample buffer, boiled for 5 min, resolved by SDS-PAGE, and analyzed by immunoblotting as previously described (28
Purifications of mTORC1 and truncated mTOR
mTORC1 purification from HEK-293T cells stably expressing FLAG-raptor was performed as described previously (29
). Purification of the truncated mTOR mutant from HEK-293T cells stably expressing FLAG-mTOR (1295–2549) and mLST8 was also performed as described previously without a gel filtration step (29
). Purified recombinant proteins were aliquoted and stored at −80°C.
In vitro kinase assays
Individual peptide substrates (GYXXXX[S/T]XXXXGRRRRR) were synthesized by the MIT Koch Institute Biopolymers and Proteomics Core Facility and purified by reversed phase HPLC. In vitro kinase activity of mTORC1 or truncated mTOR toward peptides was determined by incubating 0.1–0.2 mM peptide with ~100 ng mTORC1 or ~20 ng truncated mTOR in reaction buffer (25 mM HEPES pH 7.4, 50 mM KCl, 5 mM MgCl2 and 5 mM MnCl2) containing 50 μM cold ATP and 2–5 μCi [γ-32P] ATP for 20–30 min at room temperature. Aliquots (3.3 μL) of each reaction were spotted onto P81 ion exchange chromatography paper in triplicates and quenched in 0.42% H3PO4. Paper was washed 8–10 times in same solution and dried. Resulting radioactivity was determined by phosphoimager. For kinase assays with rapamycin, 100 nM rapamycin was preincubated with 50 ng FKBP12 for 30 min and added to reaction mixtures. FKBP12 was added in excess to ensure that most of rapamycin would be in an FKBP12-rapamcyin complex.
For S6K1 kinase assays, recombinant S6K1 proteins were purified from HEK-293T stably expressing WT or T389S S6K1 using the same method as for truncated mTOR. S6 peptide substrate (AKRRRLSSLRA) was incubated in 20 μL of reaction mixture consisting of kinase assay buffer (25 mM HEPES, pH 7.4, 50 mM KCl, 5 mM MgCl2 and 5 mM MnCl2), recombinant S6K1, 50 μM ATP and 2–5 μCi [γ-32P]ATP for 30 min at room temperature. Aliquots (3.3 μL) of each reaction were spotted onto P81 ion exchange chromatography paper in triplicates and quenched in 0.42% H3PO4. Paper was washed 8–10 times in same solution and dried. Resulting radioactivity was determined by phosphoimager.
Pull-down assay with biotinylated peptides
Biotinylated peptides were dissolved in kinase assay buffer and soluble fractions of cell lysates were collected by centrifugation at 13,000 rpm for 10 minutes. Preincubated mixtures of peptides and 50% slurry of streptavidin agarose were added to FLAG-tagged mTOR (1295–2549) and incubated in the presence of 500 nM AMP-PNP for 4–12 hr at 4°C. Pull-down mixtures were washed three times with lysis buffer containing 150 mM NaCl. Recombinant mTOR protein was denatured by addition of sample buffer, boiled for 5 min, resolved by SDS-PAGE, and analyzed by immunoblotting as previously described (28
). For pull-down assays with rapamycin, 100 nM rapamycin was preincubated with 50 ng FKBP12 for 30 min and added to pull-down mixtures.
Steady-state kinetic measurements
To determine the kinetic parameters for peptide phosphorylation, assays were conducted in the presence of 40 nM mTORC1, various concentrations of peptide substrates (0, 10, 100, 250, 500 and 1000 μ) and an ATP mixture containing 500 μM cold ATP (at least 10-fold above Km), and 2–5 μCi [γ-32P] ATP in a 30 μL reaction mixture. The reaction was initiated via the addition of the ATP mixture. After incubation at room temperature, aliquots (3 μL) of each reaction were spotted onto P81 ion exchange chromatography paper and quenched in 0.42% H3PO4. The paper was washed 8–10 times in same solution and dried. Resulting radioactivity was determined by phosphoimager. For kinetic measurements with rapamycin, 100 nM rapamycin was preincubated with 50 ng FKBP12 for 30 min and added to reaction mixtures. The steady-state kinetic parameters were obtained by fitting the reaction rates to the Michaelis-Menten equation using GraphPad Prism version 5.0 (GraphPad Inc.)
Mass Spectrometric Analyses
LARP1 phosphorylation sites were identified by mass spectrometric analysis of trypsin-digested FLAG-LARP1 purified from HEK293T cells stably overexpressing FLAG-LARP1. The amino acid positions of all LARP1 phosphorylation sites were numbered according to NCBI. Label-free quantification of LARP1 phosphorylation sites was performed with BioWorks Rev3.3 software according to the methodology previously described (31
Positional scanning peptide library screening and PWM generation
PSPL screening was performed and analyzed with the truncated mTOR mutant as previously described (47
Phosphopeptide recognition by phosphospecific antibodies
1 μL of biotinylated phosphopeptides at the indicated concentrations were spotted on a SAM2 Biotin Capture Membrane (Promega) and washed 3 times in PBST (PBS with Tween-20). Subsequently, the washed membrane was analyzed by immunoblotting as previously described (28
). Phosphopeptide sequences used are as follows:
- T389 S6K1: GGYFLGF[pT]YVAPGRRRRR
- T389S S6K1: GGYFLGF[pS]YVAPGRRRRR
- S758 ULK1: GGYFTVG[pS]PPSGGRRRRR
- S758T ULK1: GGYFTVG[pT]PPSGGRRRRR
Competitive proliferation assay
S6K1−/−S6K2−/− MEFs stably expressing barcoded wild-type and T389S S6K1 were mixed in equal number (100,000) and placed in 10-cm culture dishes. The mixture of cells was cultured in either 100% amino acid RPMI with 10% FBS and antibiotics or 20% amino acid RPMI with 10% dialyzed FBS and antibiotics. After 32 population doublings, cells were harvested and genomic DNA was isolated using QIAamp DNA Mini Kit. The concentration and purity of DNA were determined by absorbance at 260/280 nm. Primers for real-time PCR were obtained from Integrated DNA Technologies. Reactions were run on an Applied Biosystems Prism machine using Sybr Green Master Mix (Applied Biosystems) and relative abundance of wild-type and T389S S6K1 was calculated. Primer sequences used to produce barcode-specific amplicons are as follows:
- WT S6K1 forward: GTGGTGGTGCGTCGACGGGAT
- WT S6K1 reverse: CACAATGTTCCATGCCAAGT
- T389S S6K1 forward: GTGGTGGTGCGTCGACGGGTA
- T389S S6K1 reverse: CACAATGTTCCATGCCAAGT