HA-tagged Myr-Akt1 and T308A/S473A-Akt1 in the pCMV6 vector were the kind gift of Dr. A. Bellacosa (IRE, Roma, Italy); FLAG-tagged PDK1 was the kind gift of Dr H. Ha (Chungbuk National University, KOREA)
Cell lines and cell cultures
HCC1937 cells were grown in RPMI 1640 medium (Invitrogen-GIBCO, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS, Gibco), 2 mM glutamine, 100 U/ml penicillin and 100 ug/ml streptomycin.
SKBr3 cells were cultured in Dulbecco's modified Eagle's medium (Gibco) supplemented with 10% FBS (Gibco), 2 mM glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin. Akt inhibitors (pAkt1/2 and pAkt2) (Barnett et al, 2005) were kindly provided by Dr. DeFeo-Jones (Merck Research Laboratories, West Point, PA, USA). The pAkt1/2 and the selective pAkt2 inhibitors were used at a final concentration of 7 μM for 24 h, conditions at which pAkt was essentially undetectable in HCC1937 cells (see Additional File 6). Cells were treated when 60% confluent.
For immunoprecipitation of endogenous or ectopic HA-tagged Akt, lysates were precleared with protein G Agarose beads (Oncogene/Calbiochem Laboratories, Cambridge, MA) at 4°C for 45 min and incubated with specific antibodies using conditions suggested by the vendor. To achieve pAkt depletion, saturating anti-pAkt immunoprecipitation was performed for 5 h with an excess of anti-pAkt antibody-conjugated beads. After beads removal, lysates were incubated with the indicated antibody precoated with protein G Agarose beads for 2 h at 4°C. Beads were washed once with lysis buffer and twice with 10 mM Tris-HCL pH 7.4. For IPs, monodimensional electrophoresis and western blot analysis, beads were eluted with 2× Laemmli sample buffer supplemented with 100 μM DTT at 95°C for 7 min.; for 2D-MS analysis, elution was carried out using 2D rehydration buffer (8 M Urea, 2% CHAPS, 50 mM DTT, 30 min/37°C) and appropriate ampholytes (Bio-Rad).
Protein extraction and western blot
To obtain whole cell lysates, cells were suspended in detergent lysis buffer, disrupted for 20 min at 4°C on a rotary shaker and centrifuged at 13.000 × g for 30 min. The pellet was discarded and protein concentration in the supernatant measured using a Bredford Protein Assay kit (Bio-Rad, Richmond, CA, USA). For western blot analysis, equivalent amounts of proteins and IPs were resolved by SDS-PAGE, transferred to nitrocellulose membranes and blotted. The anti-Ser473-pAkt antibody (#4051, 1:1000 dilution) was from Cell Signaling Technology (Denvers, MA, USA), the anti-EF1α (#sc-12991, 1:800 dilution) antibody was from Santa Cruz Biotechnology (Santa Cruz, CA, USA), as were the anti-Akt1 and Akt-2 antibodies (#sc-5298 and #sc-5270, 1:1000 dilution). The anti EF1α2 antibody was kindly provided by Dr. Abbott (University of Edinburgh, Molecular Medicine Centre, Western General Hospital, Edinburgh). Anti-HA antibody (#MMS-101P, 1:000) was from Covance (Berkeley, CA, USA).
Secondary antibodies were peroxidase-labeled and peroxidase was detected with Enhanced Chemioluminescence Kit (ECL, Amersham Pharmacia Biotech, UK) or Immobilon Western (Millipore Corporation, Bedford, MA, USA).
Readystrip IPG strip (pH 4–7, 7 cm Bio-Rad) were reydrated in 2D rehydration buffer containing the protein sample. After IEF (Protean IEF cell, Bio-Rad) strips were subjected to reduction and alchylation reactions (15 min in rehydration buffer containing 10 mg/ml of DTT followed by 15 min in rehydration buffer containing 25 mg/ml of iodoacetamide). For second dimension's separation, 7 cm strips were dipped in the SDS running buffer and placed side by side on the top of the same 10% laboratory-made polyacrylamide gel (size 16 cm × 16 cm) to equalize the conditions of electrophoresis and stain. Gels were stained by the Silver Stain method.
In-gel protein digestion and mass spectrometry
After gel staining, bands or spots of interest were excided from gels with end-removed pipette tip and transferred into a microcentrifuge tube (0.5 mL). Briefly, protein pieces were destained by incubation with 200 μl of 1:1 solution 30 mM potassium hexacyano-ferrate (III) and 100 mM sodium thiosulphate, washed twice with 100 μl of water for 15 min and shrunk with 100% acetonitrile until the gels turned white. Proteins were then reduced adding 50 μl of a DTT solution (10 mM DTT in 50 mM ammonium bicarbonate) and sequentially alkylated using a IAA solution (55 mM IAA in 50 mM ammonium bicarbonate). A volume of 30 μl of trypsin (Promega, Madison, WI) solution (12.5 ng/μl in 25 mM ammonium bicarbonate) was then added, and the gel pieces were incubated at 4°C for 30 min. After digestion, trypsin solution was removed and the samples were incubated at 37° o/n in the same solution without trypsin. Resulting supernatant representing peptide solution were recoverd and concentrated in a vacuum drier (Savant Speed-Vac concentrator).
After resuspensin in 5% formic acid, extracted peptides were sent to C.I.G.S. (Centro Interdipartimentale Grandi Strumenti, Università di Modena, Italy) and identificated by ESI/Q-Tof Mass Spectrometry (Waters-Micromass, Manchester, UK). Peptide identity was determined by searching the Swiss-Prot database.
Glutathione S-Transferase (GST) Fusion Proteins
To synthesize GST fusion proteins, cDNA segments encoding parts of EF1α protein (amino acids 1 – 137) were amplified by RT-PCR from HCC1937 cells and subcloned into the EcoRI-XhoI-digested pGEX5.1 plasmid. Proteins were expressed in BL21 cells by IPTG induction (1 mM, 3 h), and purified on glutathione-agarose beads (Sigma). GST-proteins were eluted in 50 mM Tris-HCl pH 9.0, 30 mM GSH.
Synthetic peptide KAERERGITID (EFtide) corresponding to amino acids 64–74 of EF1α and RPRAATF (AKTide) a specific substrate of Akt were synthesized by solid phase peptide synthesis method using an automatized peptide synthesizer (model 431-A, Applied Biosystems, Foster City, CA). Crude peptides were purified by preparative reverse phase HPLC and purity was evaluated by analytical reverse phase HPLC (about 95%). Molecular masses of the peptides were confirmed by mass spectroscopy with direct infusion on a Micromass ZMD-4000 Mass Spectrometer (Waters- Micromass).
In vitro kinase assay
Phosphorylation reactions were performed by incubating the phosphorylatable protein or peptide substrate in 30 μl of a medium containing 20 mM HEPES (pH 7,5), 10 mM MgCl2, 10 mM MnCl2, 1 mM DTT, 50 μM [γ-33P]ATP (specific activity, 2000 cpm/pmol) and 100 ng of full-length recombinant active Akt1 (specific activity 124 nmol/min/mg) or Akt2 (specific activity 43 nmol/min/mg), expressed in Sf9 cells (from SignalChem, Richmond, BC, Canada) (active Akt1 # A16-10G-05, active Akt2 # A17-10H-05) for the indicated time at 30°C.
The phosphate incorporated into substrates was evaluated either by subjecting samples to SDS/PAGE, staining and autoradiography or using phosphocellulose filters . Values obtained represent the mean of at least three independent experiments.
siRNA duplex oligoribonucleotides corresponding to the EF1α gene were designed by Block-iT RNAi Designer (Invitrogen). Best results of EF1α downregulation were obtained by using the following EF1α gene-specific sequences beginning at nt 607 (named "EFsiRNA"): sense 5' Flo-GCGCCUACAUCAAGAAGAUdTdT 3', antisense 5' Flo-AUCUUCUUGAUGUAGGCGCTT 3'.
Transfection of siRNA oligos was carried out with Lipofectamine™ 2000 (Invitrogen). Cells were incubated for 24, 48, 72 and 96 h post-transfection and EF1α expression was detected by western blotting. Fluorescent oligos were used to determine uptake efficiency and set optimal lipofection conditions.
Colony formation and soft agar assay
24 h after transfection, HCC1937 cells were trypsinized, counted, suspended in 15% conditioned medium and seeded for colony formation assays in 60-mm dishes at 3,000 cells per dish. To test the effect of Akt inhibitors, cells were seeded in medium supplemented with pAkt inhibitors (7 μM in DMSO) in the dark; equal volume of DMSO was used in control experiments. After incubation for 14 days, colonies (>30 cells) were stained with crystal violet and counted. Three different experiments were performed in triplicate.
For soft-agar assays, 5,000 cells were pleated in 60-mm-diameter tissue culture plates containing 0.35% top low-melt agarose-0.5% bottom low-melt agarose. After 2 weeks of incubation at 37°C in 5% CO2 and 95% humidified air, colonies that contained 30 or more cells were counted. Experiments were performed in triplicate.
Cell migration was analyzed with the BioCoat Matrigel Invasion Chambers (BD, Becton-Dickinson, San Jose, CA, USA). Control, siRNA-transfected and Akt inhibitor-treated HCC1937 cells were seeded at 40,000 cells in each upper chamber in RPMI 1640 supplemented with 5% FBS. Lower chambers were filled with 750 μl of 5% conditioned medium of confluent HCC1937 cells containing 10% of FBS as a chemoattractant. Chambers were incubated for 22 h at 37°C in a humidified 5% CO2 atmosphere, then cells in the upper chamber were removed. Cells adherent to the lower surface of the membrane were fixed with methanol and stained with crystal violet. Cells migrated to the lower surface of the filter were considered to have invaded through the overlying matrix and were counted.
Adherent cells were trypsinized, centrifuged at 1000 g for 5 min, fixed in ice-cold 70% ethanol and incubated at -20°C for 2 h. Cells were centrifuged, resuspended in PBS and incubated with RNAse A (200 μg/ml) for 30 min at room temperature; then, cells were incubated with propidium iodide (50 μg/ml) for 30 min at room temperature in the dark. Quantification of sub-2N DNA was determined by flow cytometry using Coulter Epics XL (Beckman Coulter, Fullerton, CA).