METHODS

Plasmids.

Pact-Flag-Sin1 was obtained from Dr. Shunsuke Ishii 34 and Flag-Sin1-T86A, T398A, T86A/T398A, T86E, T398E, T86E/T398E were generated using the QuikChange XL Site-Directed Mutagenesis Kit (Stratagene) according to the manufacturer’s instructions. The HA-Sin1 plasmid was a kind gift from Dr. Kunliang Guan. MSCV-Sin1-Myc was obtained from Dr. Dos D. Sarbassov (UT M. D. Anderson Cancer Center). GST-Sin1, GST-Sin1-T86A, GST-Sin1-T398A and GST-Sin1-T86A/T398A were constructed by subcloning the corresponding Sin1 cDNAs from Pact-Flag-Sin1 into the pGEX-4T-1 vector. Myc-Rictor, HA-Rictor and Myc-mTOR have been described previously 32. Plasmids that express HA-Myr-Akt1/Akt2 and HA-SGK160 were described previously 63 and they are well-characterized constitutive active mutant forms for the AGC kinases in which the N-terminus of Akt1 and Akt2 are fused with the myristoylation signal peptide derived from Src, and the SGK1 N-terminal inhibitory domain was removed. These constructs have been widely used in identifying AGC kinase substrates 63,64. The HA-S6K1-CA, HA-S6K1-R3A, HA-S6K1-WT and HA-S6K1-KD constructs were described previously 63. Notably, the HA-S6K1-CA construct bears three mutations (F5A-T389E-R3A) and it is a constitutive active version of S6K1 in part due to its insensitivity to rapamycin inhibition. The short hairpin RNA (shRNA) construct against GFP was described previously 53. shSin1-resistant Pact-Flag-Sin1 and MSCV-Sin1-Myc constructs were generated using the QuikChange XL Site-Directed Mutagenesis Kit (Stratagene) according to the manufacturer’s instructions, with specific primer sequences listed below to generate the silent mutations: 5’-CGCAAATTGACATAGCCACCGTGCAAGATATGCAAGATATGCTTAGCAGC

C-3’ (sense) and 5’-GGCTGCTAAGCATATCTTGCACGGTGGCTATGTCAATTTGCG-3’ (anti-sense).

shRNAs.

shRNA vectors to deplete endogenous Rictor and endogenous Raptor were purchased from Addgene (plasmid number: #1584 for shRictor and #1858 for shRaptor, respectively). pLKO lenti-viral expression vectors to deplete endogenous S6K1 (denoted A with the targeted sequence as TATTGGAAGTGGTGCC

GATGC, B with the targeted sequence as TAAGCACCTTCATGGCAAATA and C with the targeted sequence as TAAGGTTGAGAAAGATGTCGC) were kind gifts from Dr. William Hahn. The shIRS-1 construct to deplete endogenous mouse origin of IRS-1 (targeted sequence as ATATTCACATATTCTCC

TGGG) was obtained from Dr. Stephen J. Elledge and the shGrb10 construct to deplete mouse Grb10 was described previously 30. To generate the lentiviral shRNA construct against human Sin1, the following sequences were cloned into the pLKO-puro and pLKO-hygro-lentiviral vectors (Sin1 sense: 5’GCCACAGTACAGGATATGCTT-3’; Sin1 anti-sense: 5’-AAGCATATCCTGTACTGTGGC-3’).

Antibodies.

All antibodies were used at a 1:1000 dilution in 5% non-fat milk. Anti-Sin1 antibody for western blots was purchased from Millipore (07-2276). Anti-Sin1 antibody for endogenous Sin1 IP was purchased from Bethyl (S300-910). Anti-mTOR antibody (2972), anti-pSer2481-mTOR antibody (2974), anti-Raptor antibody (2280), anti-GβL antibody (3274), anti-phospho-Ser473-Akt antibody (4051), anti-phospho-Thr308-Akt antibody (2965), anti-phospho-Thr450-Akt antibody (9267), anti-Akt1 antibody (2938), anti-Akt total antibody (4691), anti-phospho-Thr389-S6K antibody (9205), anti-S6K antibody (9202), anti-phospho-Akt substrate antibody (9614), anti-phospho-S6 antibody (4858), anti-pTSC2-antibody (3617), anti-TSC2 antibody (3990), anti-phospho-T24/32-FOXO1/3a antibody (9464), anti-phospho-S318/321-FOXO3a antibody (9465), anti-FOXO1 antibody (2880), anti-FOXO3a antibody (2497), anti-Skp2 antibody (4313), anti-pS9-GSK3β antibody (9323), anti-GSK3β antibody (9315) were purchased from Cell Signaling. The anti-IRS-1 antibody was obtained from Dr. Stephen J. Elledge, anti-mouse-Grb10 antibody (sc 13955), polyclonal anti-HA antibody (sc-805) were purchased from Santa Cruz. Anti-Tubulin antibody (T-5168), anti-Vinculin antibody (V-4505), polyclonal anti-Flag antibody (F-2425), monoclonal anti-Flag antibody (F-3165, clone M2), anti-Flag agarose beads (A-2220), anti-HA agarose beads (A-2095), peroxidase-conjugated anti-mouse secondary antibody (A-4416) and peroxidase-conjugated anti-rabbit secondary antibody (A-4914) were purchased from Sigma. Monoclonal anti-HA antibody (MMS-101P) was purchased from Covance. The anti-pT86-Sin1 and anti-pT398-Sin1 antibodies as well as the anti-pS72-Skp2 antibody were produced by Cell Signaling Technology.

Immunoblots and immunoprecipitation.

Cells were lysed in EBC buffer (50 mM Tris pH 7.5, 120 mM NaCl, 0.5% NP-40) or CHAPS buffer (for immunoprecipitation) (25 mM HEPES pH7.4, 150 mM NaCl, 1 mM EDTA, 0.3% CHAPS) supplemented with protease inhibitors (Complete Mini, Roche) and phosphatase inhibitors (phosphatase inhibitor cocktail set I and II, Calbiochem). The total protein concentrations of whole cell lysates were measured by the Beckman Coulter DU-800 spectrophotometer using the Bio-Rad protein assay reagent. Same amounts of whole cell lysates were resolved by SDS-PAGE and immunoblotted with indicated antibodies. For immunoprecipitation, 1000 g lysates were incubated with the indicated antibody (1-2 g) for 3-4 h at 4 C followed by 1 h incubation with Protein A sepharose beads (GE Healthcare). Immunoprecipitants were washed five times with NETN buffer (20 mM Tris, pH 8.0, 100 mM NaCl, 1 mM EDTA and 0.5% NP-40) or CHAPS buffer (25 mM HEPES pH7.4, 150 mM NaCl, 1 mM EDTA, 0.3% CHAPS) before being resolved by SDS-PAGE and immunoblotted with indicated antibodies. Please be noted that for the immunoblot analysis results presented in Figure 6b, Supplementary Figures S1e-f, S3f, S4g-k and S7c, due to the large sample sizes to be compared side by side that are not feasibly done in one SDS-PAGE gel of 15 lanes, the protein samples were, alternatively, loaded on separate SDS-PAGE gels, but transferred and blotted on the same PVDF membranes. In this experimental condition, cares have been given to ensure that all the samples are subjective to same immunoblotting and exposure conditions, which can be directly compared to each other, as we described previously 32,63,65.

Cell culture and cell viability assays.

Cell culture and transfection have been described previously 66. The WT and Sin1-/-MEFs were described before 13. TSC2-WT and TSC2-/- MEFs were from Dr. Brendan D. Manning. Lentiviral shRNA virus packaging and subsequent infection of various cell lines were performed according to the protocol described previously67.Kinase inhibitors Akt 1/2 Inhibitor VIII (Calbiochem, 124018), PP242 (Sigma, P0037), rapamycin (Calbiochem, 553210) and S6K1-I (TOCRIS, 4032) were used as indicated. For cell viability assays, cells were plated at 10,000 per well in 96-well plates, and incubated with complete DMEM medium containing different concentrations of etoposide (Sigma, E1383) for 48 hours. Assays were performed with CellTiter-Glo Luminescent Cell Viability Assay Kit according to the manufacturer’s instructions (Promega).

In vitro kinase assays.

S6K and mTORC2 invitro kinase assays were adapted from described previously 68. Briefly, for S6K kinase assay, 3 µg of indicated GST-Sin1 fusion proteins or GST-Rictor-C-tail (aa1390-1708) were incubated with 50 ng commercially obtained recombinant active S6K proteins (R&D system, Catalog number: 896-KS), in the presence of 5 µCi [g-32P]-ATP and 200 µM cold ATP in the kinase reaction buffer for 30 min. For mTORC2 kinase assay, 3 µg of indicated GST-Akt1-Tail (408-480) fusion proteins were incubated with 1/5 of Flag-Sin1 immunoprecipitation products from 2mg whole cell lysates expressing the Flag-Sin1 construct, in the presence of 5 µCi [g-32P]-ATP and 200 µM cold ATP in the kinase reaction buffer for 30 min. The reaction was stopped by the addition of SDS containing lysis buffer and resolved by SDS-PAGE. Phosphorylation of GST-Sin1 or GST-Akt1-Tail was detected by autoradiography.

Mass spectrometry analysis to detect Sin1 T86 phosphorylation invivo.

The procedure was performed as described previously 32 with minor modifications. Briefly, 293T cells were transiently transfected with the Flag-Sin1 plasmid, and 24hours post-transfection, cells were treated with insulin for 30 min before harvesting. Whole-cell lysates were collected to perform Flag immunoprecipitation. The Flag immunoprecipitates were then resolved on SDS-PAGE and visualized by colloidal coomassie blue. The band containing Flag-Sin1 was excised and washed with 50% acetone. In-gel digestion of the protein was performed with chymotrypsin and analyzed by reversed-phase microcapillary/tandemmass spectrometry (LC/MS/MS) using a LTQ Orbitrap XL (Thermo Fisher Scientific) hybrid ion trap-orbitrap mass spectrometer as described previously35. MS/MS centroid spectra collected via CID in Top 6 data-dependent acquisition 69 mode were searched against the concatenated target and decoy (reversed) Swiss-Prot protein database (version 2012_01) using the Sequest search engine via Proteomics Browser Software (PBS) (W.S. Lane, Harvard University) with differential modifications for Ser/Thr/Tyr phosphorylation (+79.97) and differential modification of Met oxidation (+15.99, Msx). Phosphorylated and unphosphorylated peptide sequences were identified if they initially passed the following Sequest scoring thresholds against the target database: 1+ ions, Xcorr ³ 2.0 Sf ³ 0.4, P ³ 5; 2+ ions, Xcorr ³ 2.0, Sf ³ 0.4, P ³ 5; 3+ ions, Xcorr ³ 2.60, Sf ³ 0.4, P ³ 5 against the target protein database with mass accuracy less than 15 ppm. Manual inspection and determination of the exact sites of phosphorylation was confirmed using FuzzyIons and GraphMod software (PBS, W.S. Lane, Harvard University). False discovery rates (FDR) of peptide hits (phosphorylated and non-phosphorylated) were estimated below 1.0% based on reversed database hits.

Mass spectrometry analysis to detect Sin1 T398 phosphorylation invitro.

The mass spectrometry procedure was performed as described in the above section. Phosphorylated GST-Sin1 samples were prepared as described in the kinase assay section. Specifically, 1 g of phosphorylated GST-Sin1 proteins were subjected to trypsin digestion before being analyzed by mass spectrometry.

Gel filtration chromatography analysis.

Sin1 constructs were transiently transfected into HeLa cells (three 10 cm dishes for each sample per gel filtration experiment). 48 hours post transfection, cells were washed with phosphate-buffered saline, lysed in 0.5ml of CHAPS lysis buffer (25mM HEPES [pH 7.4], 150mM NaCl, 1mM EDTA, and 0.3% CHAPS) containing protease inhibitors (Complete Mini, Roche) and phosphatase inhibitors (phosphatase inhibitor cocktail set I and II, Calbiochem), and filtered through a 0.45 μm syringe filter. Total protein concentration was then adjusted to 8mg/mL with CHAPS buffer and 500μL of the lysate was loaded onto a Superdex 200 10/300 GL column (GE Lifesciences Cat. No. 17-5175-01). Chromatography was performed on the AKTA-FPLC (GE Lifesciences Cat. No. 18-1900-26) with CHAPS buffer as described previously 32. One column volume of elutes were fractionated with 500 mL in each fraction, at the elution speed of 0.5ml/min. 30μL aliquots of each fraction were loaded onto SDS-PAGE and detected with indicated antibodies.

To carry out experiments presented in Fig. 5c-f, cells transiently transfected with Flag-Sin1-WT or Flag-Sin1-R81T were treated with 100 nM insulin for 45 minutes before harvesting in CHAPS buffer (25mM HEPES [pH 7.4], 150mM NaCl, 1mM EDTA, and 0.3% CHAPS) containing protease inhibitors (Complete Mini, Roche) and phosphatase inhibitors (phosphatase inhibitor cocktail set I and II, Calbiochem) and filter cleared. 25 L of each gel filtration fraction was incubated with 2 g of GST-Akt-tail proteins and the in vitro kinase assays were performed as described previously 32.

Annexin-V/7-AAD double staining.

For detection of apoptosis, cells treated with various drugs were co-stained with Annexin-V-PE and 7-AAD (Annexin V-PE Apoptosis Detection Kit I, BD Bioscience) according to the manufacturer's instructions. Stained cells were sorted with a Dako-Cytomation MoFlo sorter (Dako).

Soft agar assays.

The solid medium consists of two layers. The bottom layer contains 0.8% noble agar and the top layer contains 0.4% agar. Briefly, 3x105 cells were plated in the top layer. 500 L complete DMEM medium was added every other day to keep the top layer moistured and 3 weeks later the cells were stained with iodonitrotetrazolium chloride for colony visualization and counting. Three independent experiments were performed to generate the error bar.

Mouse xenograft assays.

Tumorigenesis assay was performed as described previously 65. Briefly, 3x106 cells were mixed with matrigel (1:1) and injected into the flank of 10 male nude mice (NCRNU-M-M from Taconic, 4-5 weeks of age at purchase) for OVCAR5 cells stably expressing either Sin1-WT or Sin1-R81T mutation. Tumor size was measured weekly with a caliper, and the tumor volume was determined with the formula: L*W2*0.52, where L is the longest diameter and W is the shortest diameter. After 28 days, mice were sacrificed and in vivo solid tumors were dissected and tumor weights were measured and recorded.

IHC of tissue microarray (immunohistochemistry and immunostaining analyses).

Formalin-fixed and paraffin-embedded tissue microarrays of human ovarian tissues and ovarian cancer tissues were purchased from Imgenex (IMH-347; San Diego, CA). Immunohistochemical stainings for Akt-pS473 and Sin1-pT86 were performed as described previously 70. The Akt-pS473 antibody for IHC was purchased from Cell Signaling Technology (#4060) and the Sin1-pT86 antibody was generated by collaboration with the Cell Signaling Technology. Statistical analysis was performed by the Fisher’s exact analysis.

Sample size, randomization and blinding for animal studies.

For mouse xenograft assays in Fig. 8e-g and Supplementary Fig. S8h, 10 mice were included in each group to determine the statistical significance. No method of randomization was used to determine how samples/animals were allocated to experimental groups and processed. The investigators were not blinded to allocation during experiments and outcome assessment.

Repeatability of experiments.

Figures 1a, 1f, 1g, 2f, 2h, 3a, 3c, 4a, 4b, 4f, 5d, 5f, 7b, 7e, 7g, S1a, S1l, S1m, S2c, S2p, S3a-d, S4c-d, S4g, S4i, S6d-e, S7a-b, S8c-d are representative images from two independent experiments. Figures 1b, 1e, 2a, 3b, 5a and 6a are representative images from three independent experiments. Error bars in figures 6d, 6e, 6g, 7a, 7b, 7dS6b, S6c, S8e, S8e-g and S8i-k are obtained from three independent experiments. Figure S8m is a representative image from 4 independent experiments. For the xenograft experiments presented in figure 8e-g and S8h, each cell line was injected into 10 mice to obtain the statistical significance. The western blot images from Fig. 8h were derived from a single experiment due to the limited amount of mice; however, 4 mice in each group were included as internal controls.

Supplementary References:

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