Anti-Raptor antibody was purchased from Bethyl Laboratories. Antibodies pY20 and HA.11 were from BD Transduction Laboratories and Covance, respectively. All other antibodies were from Cell Signaling. Insulin, IBMX, dexamethasone, protein A, protein G, horseradish peroxidase-conjugated secondary antibodies, protease and phosphatase inhibitor cocktails, and metformin (1,1-dimethyl biguanide hydrochloride) were from Sigma. Rapamycin and fatty acid-free bovine serum albumin (catalog no. 126575) were obtained from Calbiochem. Tumor necrosis factor alpha (TNF-α) was from Research Diagnostics, lipids were from Avandi Polar Lipids, and Raptor small interfering RNA (siRNA) was from MWG-Biotech.
Rat wild-type pCMVhis-IRS-1 was a gift from M. White. For Myc-tagged IRS-1, the first 211 nucleotides of IRS-1 were PCR amplified with 5′-ATATGAATTCGCCGCCACCATGGAACAAAAACTTATTTCTGAAGAAGATCTGGCGAGCCCTCCGGATACCGATGGC-3′ (sense, includes the Myc epitope as well as a Kozak sequence for efficient translational initiation) and 5′-TTGAAACAGCTCTCGAGGGGG-3′ (antisense). The amplified fragment was pasted in the EcoRI and XhoI sites of pCMV-MCS vector (Stratagene). The full-length IRS-1 tagged with the Myc epitope generated by subcloning the XhoI/SalI fragment of wild-type (WT) IRS-1 in the XhoI site of the intermediate plasmid from the previous step to obtain the full-length pCMV-Myc-IRS-1 WT.
Site-specific mutagenesis of pCMVhis-IRS-1 was carried out with the help of the overlap extension method. Amino acid numbering refers to the human isoform. The following sense primers have been used: (i) for IRS-1 S636/639D, 5′-TATATGCCAATGGACCCCAAGGATGTATCTGCC-3′; (ii) for IRS-1 S636/639A, 5′-TATATGCCAATGGCTCCCAAGGCTGTATCTGCC-3′; (iii) for IRS-1 S616D, 5′-TACATGCCAATGGATCCCGGAGTG-3′; (iv) for IRS-1 S616/636D, 5′-CCAATGGATCCCGGAGTGGCTCCAGTGCCCAGCAAC CGCAAAGGAAATG GGGACTATATGCCAATGGAC-3′. Mutated nucleotides are underlined; silent mutagenized nucleotides are in boldface type. Outer primers spanned two NcoI restriction enzyme sites in the 5′ and 3′ ends of the aforementioned mutagenized sites.
Human LKB1/STK11 was amplified with the primers 5′-GAATTCGAGGTGGTGGACCCGCAGCAGCTG-3′ (sense) and 5′-TCTAGAAAATCACTGCTGCTTGCAGGCCGACAG-3′ (antisense) from the clone MGC-16254 (ATCC) and subcloned in frame in the EcoRI and XbaI sites of pCMV5-Myc vector to generate pCMV5-Myc-LKB1.
Wild-type pRK5-HA-Raptor was a gift from D. Sabatini. For the carboxy-terminal deletions, the following primers have been used: (i) for ΔWD40 (Δ1011 to 1335), 5′-TTGCCTTCTGGCCGGCCCGGC-3′ (sense) and 5′-GCGGCCGCACTACGTAATGCCCTTCTGAATGACTTG-3′ (antisense); (ii) for ΔWD40/Hinge (Δ678 to 1335), 5′-TGGCAGCCAAGGGCCCGGTACAAG-3′ (sense) and 5′-GCGGCCGCACTAATTGCTTTCATACTGAACCACAAG-3′ (antisense). PCR-amplified fragments were subcloned in the FseI and NotI and ApaI and NotI sites of digested pRK5-HA-Raptor, respectively.
Wild-type rat pCGN-HA-Rheb was a gift from J. Der and J. Clark. For the C181S point mutation, the following primers have been used: 5′-ATGGCTTCTAGCTATCCTTATGAC-3′ (sense) and 5′-ATGGATCCTCACATCACCGAGCTCGAAGACTTCCC-3′ (antisense).
Kinase-deficient rat pRK7-HA-S6K1 αII was a gift from J. Blenis. Wild-type rat pMT2-HA-S6K1 αI WT and the rapamycin-resistant mutant pMT2-HA-S6K1-Δ2-46/ΔCT104 were a gift from J. Avruch.
Murine 3T3-L1 cells were grown in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% calf serum until confluence. Two days later, cells were transferred to the differentiation medium (DMEM containing 10% fetal bovine serum [FBS], 0.5 mM 3-isobutyl-1-methylxanthine, 1 μM dexamethasone, and 1.7 μM insulin). After 48 h, the differentiation medium was replaced with DMEM supplemented with 10% FBS. Cells were used after 6 to 8 days of differentiation. The myoblast line L6 was grown under subconfluent conditions in DMEM containing 20% FBS. Cells at ~80 to 90 confluence were maintained in DMEM supplemented with 2% calf serum for 5 to 7 days for differentiation into myotubes. HepG2, NIH 3T3, and 293HEK cells were grown in DMEM supplemented with 10% FBS. C2C12 fibroblasts were grown similar to L6 myoblasts. For stable expression of hemagglutinin (HA)-tagged Rheb, C2C12 fibroblasts were transfected with pCGN-HA-Rheb, and several clones were selected for 7 to 10 days in the medium containing 300 μg/ml hygromycin B (Invitrogen).
Cells were washed twice in ice-cold phosphate-buffered saline (PBS) and homogenized in buffer A (20 mM HEPES, pH 7.4, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride [PMSF], 2 mM Na3VO4, 30 mM NaF, supplemented with a mixture of protease and phosphatase inhibitors) by 16 strokes in a ball-bearing homogenizer (Isobiotec, Heidelberg, Germany) with a 12-μm clearance. The homogenate was centrifuged at 14,000 × g for 20 min at 4°C, and the supernatant was recentrifuged at 200,000 × g for 1 h at 4°C in a Beckman type Ti42.2 rotor. The cytosol and high-speed pellet were analyzed by Western blotting.
Cell lysis and immunoprecipitations.
Cells were washed twice in ice-cold PBS and solubilized in the lysis buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 1% Triton X-100, 10 mM Na3VO4, 50 mM NaF, 1 mM β-glycerophosphate, 1 mM sodium pyrophosphate, 1 mM EDTA, 1 mM EGTA, 1 mM PMSF, supplemented with a mixture of protease and phosphatase inhibitors). The lysate was cleared by centrifugation for 10 min at 14,000 × g at 4°C. For immunoprecipitation, 0.5 to 1 mg of the lysate was incubated overnight with 1 to 2 μg of the specific and nonspecific antibody coupled to protein A- or G-Sepharose beads under gentle agitation. After extensive washing with lysis buffer, beads were resuspended in sodium dodecyl suflate sample buffer supplemented with 5% β-mercaptoethanol, boiled for 5 min, and subjected to Western blot analysis.
In vitro measurement of IRS-1-associated PI3-kinase activity. IRS-1 was immunoprecipitated from cell lysates, and immune complexes were washed 3 times with lysis buffer and then with wash buffer A (100 mM Tris, pH 7.4, 500 mM LiCl, 2 mM Na3VO4). Then, immunoprecipitates were washed once with buffer B (10 mM Tris, pH 7.4, 100 mM NaCl, 1 mM EDTA, 2 mM Na3VO4), and twice with the kinase buffer (10 mM MgCl2 and 50 mM Tris, pH 7.4). After that, 45 μl of kinase buffer, 5 μl of sonicated lipid substrate (phosphatidylinositol and phosphatidylserine (1:1) in a concentration of 1 mg/ml in 25 mM HEPES, pH 7.4, and 1 mM EDTA), and 20 μCi of [γ-32P]ATP were added to washed beads for 20 min at room temperature. Reactions were stopped by the addition of 100 μl of 1 N HCl. Lipids were extracted and spotted on potassium oxalate-treated silica gel thin-layer chromatography (TLC) plates (250-μm layer, catalog no. 4420221; Whatman) and separated in chloroform, methanol, acetone, acetic acid, and H2O (60:20:23:18:12, vol/vol/vol/vol/vol). The plate was dried and exposed either to the X-ray film with an intensifying screen at −80°C or in Instant Imager (Packard) for quantification of radioactivity. Radioactive spots were identified by comigration with lipid standards detected by iodine vapors.
In vitro kinase assay.
293HEK cells transiently transfected with Myc-mTOR and Myc-Raptor were harvested in lysis buffer which contained 0.3% 3-[(3-cholamidylpropyl)-dimethylammonio]-1-propanesulfonate (CHAPS) instead of Triton X-100. Ectopically expressed proteins were immunoprecipitated with anti-Myc antibody as described in the previous section. Beads were washed twice with mTOR kinase buffer (10 mM HEPES, pH 7.4, 50 mM NaCl, and 10 mM MnCl2), resuspended in 30 μl of kinase buffer containing 200 μM ATP with or without 20 μCi of [γ-32P]ATP, and recombinant IRS-1 (600 ng; catalog no. 13-124; Upstate) was immediately added to the kinase mixture. The phosphorylation reaction was carried out at 30°C for 30 min and terminated by the addition of an equal volume of 4× sample buffer. Phosphorylated proteins were separated by polyacrylamide gel electrophoresis and analyzed by Western blotting with phosphospecific antibody and/or autoradiography.
siRNA against human Raptor was transfected into 293HEK cells with the help of Lipofectamine 2000 as described previously by Kim and colleagues (27
). Transfected cells were further grown for 48 to 60 h before the experiment.
Measurements of GDP-bound Rheb.
293 HEK cells transiently transfected with pCGN-HA-Rheb were washed twice in ice-cold PBS and lysed in Rheb lysis buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 1% Triton X-100, 1 mM PMSF, 20 mM MgCl2 supplemented with the mixture of protease inhibitors). The lysate was cleared by centrifugation for 10 min at 14,000 × g at 4°C, and the NaCl concentration was adjusted to 500 mM to inhibit GTPase-activating protein activity in the lysate. HA-Rheb was immunoprecipitated with anti-HA antibody coupled to protein G-Sepharose beads under gentle agitation at 4°C. The beads were washed sequentially with high-salt buffer (50 mM Tris, pH 8, 500 mM NaCl, 10 mM MgCl2, and 0.5% Triton X-100) and with low-salt buffer (50 mM Tris, pH 8, 100 mM NaCl, 10 mM MgCl2, and 0.1% Triton X-100). The beads were resuspended in 30 μl elution buffer (5 mM Tris phosphate, pH 7.8, 2 mM EDTA, and 4 mM dithiothreitol), heated for 3 min at 100°C, cooled on ice, and pelleted for 3 min at 14,000 × g. The supernatant containing guanine nucleotides dissociated from bead-bound Rheb was collected and either analyzed immediately or stored at −80°C. The beads were resuspended in sodium dodecyl sulfate sample buffer, supplemented with 5% β-mercaptoethanol, boiled for 5 min, and subjected to Western blot analysis. For the measurement of GDP-bound Rheb, eluted GDP was converted to [γ-32P]GTP using nucleoside diphosphate (NDP) kinase (catalog no. N-2635; Sigma) and 1 μCi/μl [γ-32P]ATP for 90 min at 37°C in NDP reaction buffer (50 mM Tris-HCl, pH 7.4, 10 mM MgCl2 in the presence of 4 mU/μl of NDP kinase) in the following reaction: GDP + [γ-32P]ATP → [γ-32P]GTP + ADP. The labeled nucleotides were separated by chromatography on plastic-backed cellulose TLC plates (catalog no. 10089; Selecto Scientific) in saturated ammonium sulfate (53.1 g/100 ml H2O), H2O, 3 M sodium acetate, 10 N sodium hydroxide, isopropanol (80/10/6/2/2, vol/vol/vol/vol/vol). The Rf of GTP was 0.71, and that of ATP was 0.5. The proper migration was assessed by comigration of [α-32P]GTP and [γ-32P]ATP. The reaction was linear in the range of 1 to 50 pM of the GDP standard. Data were presented as the amount of GDP-bound Rheb normalized by the amount of immunoprecipitated HA-Rheb as determined by Western blotting.
Results are expressed as means ± standard errors of the means (SEM). Significance was analyzed with the help of Student's t test.
In all experiments, cells were incubated in either high-glucose (25 mM) serum-free DMEM (catalog no. 11995-065; Invitrogen) or in glucose-free Krebs-Ringer phosphate (KRP) buffer (12 mM HEPES, 120 mM NaCl, 6 mM KCl, 1.2 mM MgSO4, 1 mM CaCl2, 0.6 mM Na2HPO4, 0.4 mM NaH2PO4, and 0.1% fatty acid-free bovine serum albumin, pH 7.4) for 6 h unless indicated otherwise. Rapamycin, insulin, 2-deoxyglucose, leucine, glucose, and TNF-α were added to cells for 30 min unless indicated otherwise. In transfection experiments, the corresponding empty vector was used to balance the amounts of transfected DNA. Shifts in electrophoretic mobility of IRS-1 were analyzed in 6% gels.