Reagents were obtained from the following sources: rabbit polyclonal antibodies to DEPTOR (09–463), raptor (09–217), Akt (05–591), and phospho-S877 raptor (09–107) from Upstate/Millipore; mouse monoclonal antibody to DEPTOR from Novus Biologicals; mouse monoclonal antibodies to rictor and raptor from Assay Designs; antibodies to mTOR, β-catenin, actin, S6K1, c-MAF, as well as HRP-labeled anti-mouse, anti-goat, and anti-rabbit secondary antibodies from Santa Cruz Biotechnology; antibodies to phospho-T389 S6K1, phospho-T37/T46 4E-BP1, phospho-S473 Akt/PKB, phospho-T308-Akt, phospho-T346-NDRG1, phospho-S307-IRS-1, IRS-1, PTEN, TSC2, 4E-BP1, cleaved caspase-3, PARP, PDGFR- β, and the c-MYC epitope from Cell Signaling Technology; antibodies to rictor, HA, c-MYC from Bethyl Laboratories; Flag M2 affinity gel, Flag M2 antibody and SGK1 antibodies, ATP, and SYBR Green JumpStart Taq ReadyMix from Sigma Aldrich; mouse monoclonal antibody to mTOR and recombinant IL-6 from BD Pharmingen; protein G Sepharose and anti-sheep secondary antibody from Pierce; DMEM from SAFC Biosciences; rapamycin from LC Labs; MG-132 from Biomol; PreScission protease from Amersham Biosciences; pTREQ Tet-On vector from Clontech; Adenoviral CRE and GFP from University of Iowa Gene Transfer Vector Core; FuGENE 6 and Complete Protease Cocktail from Roche; 4E-BP1 from A.G. Scientific; SuperScript II Reverse Transcriptase, Platinum Pfx Polymerase, SimplyBlue Coomassie G, Silverquest Staining kit, and inactivated fetal calf serum (IFS) from Invitrogen. An antibody to NDRG1 was kindly provided by Dario Alessi (University of Dundee, UK). We have found that the phospho-S877 raptor antibody also recognizes immunoprecipitated phosphorylated DEPTOR and can be used to read out the DEPTOR phosphorylation state.
Cell Lines and Cell Culture
HEK-293E cells were kindly provided by John Blenis (Harvard Medical School). HeLa, HEK-293E, HEK-293T, HT-29, U87, PC3, MD-MBA-435, and MEFs were cultured in DMEM with 10% Inactivated Fetal Bovine Serum (IFS). The Human Multiple Myeloma cell lines: FR4, XG-7, U266, KMS-12BM, KMS-12PE, PE2, 8226, OCI-MY5, KMS-28BM were provided by the Kuehl lab. The EJM, MM-1S, JJN-3, and Δ47 Human Multiple Myeloma cell lines were kindly provided by Ken Anderson (Dana Farber Cancer Institute). Human Multiple Myeloma cell lines were cultured in RPMI-1640 with 10% Fetal Bovine Serum (FBS) supplemented with 2 mM glutamine. XG-7 cells were additionally supplemented with 2 ng/ml IL-6. TSC2−/−, p53−/− and TSC2+/+, p53−/− MEFs were kindly provided by David Kwitakowski (Harvard Medical School). PTEN LoxP/LoxP MEFs were generated from PTEN LoxP/LoxP mice kindly provided by Hong Wu (UCLA). To produce PTEN −/− and PTEN +/+ MEFs, 1 µl of Adenoviral CRE or Adenoviral GFP at a titer of 1×1010 PFU/mL was added to 500,000 PTEN LoxP/LoxP MEFs. Cell lysates were generated 5 days post-infection. The HeLa cell line with doxycycline-inducible DEPTOR expression was generated by retroviral transduction of HeLa that were previously modified to express rtTA with an inducible DEPTOR cDNA.
cDNA Manipulations, Mutagenesis, and Sequence Alignments
The cDNA for DEPTOR (NCBI gene symbol: DEPDC6) in the pCMV6-XL4 vector was obtained from Origene. The DEPTOR cDNA was amplified by PCR and the product subcloned into the Sal 1 and Not 1 sites of pRK5, the Xho I and Not I sites of pMSCV, or the BsiWI and BstB I sites of pTREQ. All constructs were verified by DNA sequencing. The DEPTOR cDNA in pRK5 was mutagenized using the QuikChange XLII mutagenesis kit (Stratagene) with oliogonucleotides obtained from Integrated DNA Technologies. NCBI Blosum62 Blast searches were used to identify blocks of similar sequence between DEPTOR orthologues. Amino acid sequence alignment of the phosphorylated region of DEPTOR was performed using ClustalX v1.81.
Mass Spectrometric Analysis
mTOR or FLAG immunoprecipitates prepared from 30 million HEK-293E cells were resolved by SDS-PAGE, Coomassie G-stained, and gel bands were excised and processed as described in (Sancak et al., 2007
). In 4 independent experiments, a total of 12 peptides corresponding to DEPTOR were identified while no DEPTOR peptides were identified in control purifications.
DEPTOR phosphorylation sites were identified by mass spectrometry of trypsin digested FLAG-DEPTOR purified from HEK-293E or HEK-293T cells stably or transiently over-expressing FLAG-DEPTOR. All putative phosphorylated residues on DEPTOR (highlighted in BOLD
) were detected on the following peptides (amino acid position according to NCBI DEPDC6 Protein Sequence NP_073620):
- 234 KSPSSQETHDSPFCLR 249
- 257 KSTSFMSVSPSK 268
- 278 RSSMSSCGSSGYFSSSPTLSSSPPVLCNPK 311
Label-free quantification of DEPTOR phosphorylation sites were performed using BioWorks Rev3.3 software following methodology utilized in (Stokes et al., 2007
Mammalian Lentiviral shRNAs
Lentiviral shRNAs to human raptor, rictor, and mTOR were previously described (Sarbassov et al., 2005b
). All other shRNAs were obtained from the collection of The RNAi Consortium (TRC) at the Broad Institute (Moffat et al., 2006). These shRNAs are named with the numbers found at the TRC public website:
- Human DEPTOR_1 shRNA: TRC candidate; NM_022783.1-877s1c1
- Human DEPTOR_2 shRNA: TRC candidate; NM_022783.1-1101s1c1
- Human TSC2_1 shRNA: TRCN0000040178; NM_000548.2-4551s1c1
- Human PTEN_1 shRNA: TRCN0000002746; NM_000314.×-1320s1c1
- Mouse DEPTOR_1 shRNA: TRCN0000110157; NM_145470.1-1164s1c1
- Mouse DEPTOR_2 shRNA: TRCN0000110159; NM_145470.1-1165s1c1
- Mouse TSC2_1 shRNA: TRCN0000042727; NM_011647.1-1843s1c1
- Human SGK1_1 shRNA: TRCN0000040175; NM_005627.2-964s1c1
- Human SGK1_2 shRNA: TRCN0000040176; NM_005627.2-252s1c1
- Human c-MAF_1 shRNA: TRCN0000000255; NM_005360.×-1839s1c1
- Human c-MAF_2 shRNA: TRCN0000000257; NM_005360.×-1067s1c1
shRNA-encoding plasmids were co-transfected with the Delta VPR envelope and CMV VSV-G packaging plasmids into actively growing HEK-293T using FuGENE 6 transfection reagent as previously described (Sarbassov et al., 2005b
). Virus containing supernatants were collected at 48 hours after transfection, filtered to eliminate cells, and target cells (e.g., 300,000 HeLa cells or 500,000 8226 cells) infected in the presence of 8 µg/ml polybrene. For 8226 cells, infected cells were spun at 300g for 1.5 hours before incubating at 37°C for 24 hours. For all cell types, 24 hours after infection, the cells were split into fresh media (e.g., DMEM/10%IFS for HeLa/MEFs; RPMI/10%FBS for 8226/OCI-MY5), selected with 1 µg/ml puromycin. Five days post-infection, shRNA-expressing cells were analyzed or split again and analyzed 2–3 days later. For adherent cell lines, shRNA-expressing cells were analyzed at 50–75% confluence.
Cell Lysis and Immunoprecipitations
All cells were rinsed with ice-cold PBS before lysis. All cells, with the exception of those used to isolate mTOR-containing complexes, were lysed with Triton-X 100 containing lysis buffer (40 mM HEPES [pH 7.4], 2 mM EDTA, 10 mM sodium pyrophosphate, 10 mM sodium glycerophosphate, 150 mM NaCl, 50 mM NaF, 1% Triton-X 100, and one tablet of EDTA-free protease inhibitors [Roche] per 25 ml). The soluble fractions of cell lysates were isolated by centrifugation at 13,000 arpm for 10 min in a microcentrifuge. For immunoprecipitations, primary antibodies were added to the lysates and incubated with rotation for 1.5 hr at 4°C. A 50% slurry of protein G Sepharose (60 µl) was then added, and the incubation continued for an additional 1 hr. Immunoprecipitated proteins were denatured by the addition of 20 µl of sample buffer and boiling for 5 min, resolved by 4%–12% SDS-PAGE, and analyzed by immunoblotting as described (Kim et al., 2002). To observe gel mobility shifting in DEPTOR, 8% Tris Glycine gels (Invitrogen) were used. For all other applications, 4–12% Bis-Tris gels (Invitrogen) were used. For immunoprecipitations of mTOR containing complexes, cells were lysed in ice-cold CHAPS-containing lysis buffer lacking added NaCl (40 mM HEPES [pH 7.4], 2 mM EDTA, 10 mM pyrophosphate, 10 mM glycerophosphate, 0.3% CHAPS, and one tablet of EDTA-free protease inhibitors [Roche] per 25 ml). Immunoprecipitates were washed once each in the CHAPS lysis buffer and twice with CHAPS lysis buffer containing 150 mM NaCl such that the immunopurified material would be rinsed in a solution with a phyisiologically-relevant salt concentration. When specified, the latter two washes contained 0 mM or 500 mM NaCl.
In Vitro Kinase Assay For mTORC1 and mTORC2 Activities
For kinase assays, immunoprecipitates were washed once in CHAPS lysis buffer followed by two additional washes in CHAPS lysis buffer containing 150 mM NaCl. Immunoprecipitates were then washed twice in 25 mM HEPES (pH 7.4), 20 mM KCl. Kinase assays were performed as described (Sancak et al., 2007
cDNA Transfection-based Experiments
To examine the effects on mTOR signaling of DEPTOR overexpression, 500,000 HeLa; 300,000 p53−/− or rictor −/−, p53−/−; or 200,000 TSC2−/−, p53−/− regularly passaged (every 2–3 days) cells were plated in 6 cm culture dishes in DMEM/10%IFS. 12 hours later, cells were transfected with the pRK5-based cDNA expression plasmids indicated in the figures in the following amounts: 2 µg for all FLAG tagged cDNAs and 200 ng of HA-GST S6k1 or Akt1. All cells were lysed at 50–75% confluence 24 hours after transfection.
Gene Expression and Mutation Analysis in Human Cancers and Cancer Cell Lines
For quantification of DEPTOR, Integrin β7, and GAPDH mRNA expression in HeLa, PC3, or 8226 cell lines, total RNA was isolated from cells grown in the indicated conditions and reverse-transcription was performed. The resulting cDNA was diluted in DNase-free water (1:25) before quantification by real-time PCR. mRNA transcript levels were measured using Applied Biosystems 7900HT Sequence Detection System v2.3 software. Data are expressed as the ratio between the expression of DEPTOR or Integrin β7 and the housekeeping gene GAPDH. The following primers were used for quantitative real-time PCR:
- DEPTOR (H. sapiens):
- Forward: TTTGTGGTGCGAGGAAGTAA
- Reverse: CATTGCTTTGTGTCATTCTGG
- GAPDH (H. sapiens):
- Forward: CTCTCTGCTCCTCCTGTTCGAC
- Reverse: TGAGCGATGTGGCTCGGCT
- Integrin β7 (H. sapiens):
- Forward: TGGAGCGCTGCCAGTCACCATT
- Reverse: CGTCTGAAGTGAACACCAGCAGC
For meta-analysis of DEPTOR mRNA expression in human cancers, “DEPDC6” was searched in NCBI GEO and Oncomine gene expression data repositories. Only those studies where data from primary human tumors could be compared with matched unaffected tissue were considered further. Fold change in DEPTOR mRNA was measured by taking the quotient of the mean level of DEPTOR mRNA in unaffected tissue versus that of the tumor sample. Statistical significance was measured by one-tailed, unequal variance T test. Only those studies with p<0.05 were included in the final analysis.
RNA isolation from primary Multiple Myelomas has been described (Zhan et al., 2002
). Normalized DEPTOR mRNA expression was clustered according to the translocation/Cyclin D groups classified in (Bergsagel et al., 2005
). The Multiple Myeloma gene expression data used in this study was generated on an Affymetrix U133_Plus_2 platform and can be found in its entirety in the NCBI GEO database with the following identifiers: GSE2658 for 559 newly diagnosed, untreated tumors and GSE5900 for 22 normal plasma cells, 12 smoldering myeloma, and 44 MGUS. Human Myeloma cell line mRNA data have been deposited in an MMRC genomics portal website that is sponsored by the MMRF (www.broad.mit.edu/mmgp
Live Cell Imaging
8226 cells grown in 12 well dishes were imaged at 20× magnification using a Canon Powershot 5 Megapixel digital camera.