Protein expression and analysis

2.2.4.1 E xpression and isolation o f recom binant proteins from Sf9 cells

Recombinant p 8 5 a /p llO a , p 8 5 a/p llO p and p 8 5 a /p ll0 ô heterodimers were expressed in baculovirally infected Sf9 insect cells as described above. The cells were lysed in lysis buffer (50mM Tris-HCl pH 7.4, 5mM EDTA, lOOmM NaCl, 50mM NaF, 40mM p-glycerophosphate, lOmM sodium pyrophosphate and 1% Triton X-100 supplemented with 2pg/ml aprotinin, l|iM pepstatin, lOpM leupeptin, ImM phenylmethylsulfonyl fluoride and 0.5mM sodium ortho vanadate), clarified by centrifugation at 12,000xg for lOmin at 4°C and purified using an affinity matrix

prepared by coupling the phosphopeptide YpVPMLG (Yp=phosphotyrosine), corresponding to the Tyr751 o f the human PDGF Receptor-p, to Actigel ALD (Sterogene, CA), according to the manufacturer’s instructions. In some experiments, the heterodimers were released from beads by incubating with soluble Tyr751 phosphopeptide at ImM for 15min at room temperature. Assays were performed using either Actigel-bound p85oc/pl 10 or free p85a/pl 10 complexes.

2.2.4.2 E xpression and purification o f GST fusion proteins

pG EX4T-l plasmids containing sequences for full-length p 8 5 a and the N-terminal SH2 domain (aminoacids 321-439) o f p 8 5 a were constructed as described above. These were used to transform competent E.coli XL 1-Blue cells. After selection on ampicillin-containing agar plates, a single colony was used to inoculate a 5ml starter culture in LB medium containing lOOjig/ ml ampicillin. This was grown overnight with shaking at 37°C and the next day used to inoculate a 500ml culture in LB medium suplemented with 100|ig/ ml ampicillin. The culture was grown at 37°C with shaking until it reached an OD^Qo^^=0.6. Expression o f the plasmids was induced by addition o f IPTG at a final concentration o f ImM, and following an incubation for further 3 hours, bacterial cells were pelleted at 10,000rpm, 4°C, for 10 minutes. Pelleted cells were resuspended in 10 volumes (ml/g cell pellet) ice-cold lysis buffer (50mM Tris-HCl pH 7.5, 1% Triton X-100 supplemented with ImM PMSF). The suspension was sonicated on ice by applying five rounds o f 10s pulses, with 10s intervals between pulses. The lysate was centrifiiged at 10,000rpm for 15 minutes to remove cell debris. Protein was isolated from the lysate by rotating with Glutathione-Sepharose 4B (Pharmacia) in a 15 ml Falcon tube at 4°C. Sepharose beads with bound GST fusion proteins were washed three times in lysis buffer, followed by three washes with 50mM Tris-HCl pH 8.0 and then bound GST fusion proteins were eluted by incubating with lOmM reduced glutathione. In the case o f the GST-N-terminal SH2 domain fusion protein, the eluate was concentrated using Vivaspin-20 filters (Sartorius) and then extensively dialyzed

against PBS, aliquoted, snap-frozen in liquid nitrogen and stored at -80°C. In the case o f the full-length G ST-p85a the eluate was concentrated as above and then thrombin (Pharmacia) was added at lOU/mg o f protein and incubated overnight at 4°C in order to cleave the GST tag. Subsequently, the mix was incubated with agarose conjugated p- aminobenzamidine (Sigma) for 30min to remove thrombin, centrifuged at 4,000rpm for 5min, and the supernatant was extensively dialysed against 50mM Tris-HCl pH 7.5, 150mM NaCl. After dialysis, the preparation was incubated again with Glutathione- Sepharose 4B to remove free GST, centrifuged at 4,000rpm for 5min, and the supernatant aliquoted, snap-frozen in liquid nitrogen and stored at -80°C.

2.2.4.3 P rotein A ssay

Determination o f protein concentration was performed using the BCA method (Pierce) according to the manufacturer instructions. BSA was used as a standard.

2 .2.4.4 Im m u n op récip itation

Cells monolayers were rinsed once with ice-cold PBS and lysed in a buffer containing 50mM Tris-HCl pH 7.4, 5mM EDTA, lOOmM NaCl, 50mM NaF, 40mM P- glycerophosphate, lOmM sodium pyrophosphate and 1% Triton X-100 supplemented with 2|ig/ml aprotinin, IpM pepstatin, lOpM leupeptin, ImM phenylmethylsulfonyl fluoride and 0.5mM sodium orthovanadate. 0.5ml o f lysis buffer was used per 10cm dish. Lysates were clarified from insoluble material by centrifugation at 12,000xg for

lOmin at 4°C.

Immunoprécipitations were performed from the Triton-soluble fraction (Im g of total protein) using the indicated antibodies diluted 1: 100 (1 jig o f Flag-M2 per lysate). Lysates were incubated with the antibody for Ih at 4°C under rotation followed by the addition o f protein A- or protein G-agarose beads (20pl o f 1:2 slurry) depending on the antibody used. Immune complexes were washed three times with “high salt” buffer

(lOOmM Tris-HCl pH 8.0, 0.5M LiCl), followed by three washes with kinase assay buffer and further processed for various assays.

2.2.4.5 T ris-glycin e-S D S -p olyacrylam id e gel electro p h o resis

SDS-polyacrylamide gel electrophoresis was performed according to the method o f Laemli (Laemmli, 1970). Vertical slab gels were prepared using either a Hoefer gel apparatus or BioRad mini gel apparatus. Samples were denatured by addition o f electrophoresis sample buffer (5x concentrated: 50% glycerol, 10% SDS, SOOmM DTT, SOOmM Tris-HCl pH 6.8) and heating at 95°C for 5min. Electrophoresis was performed under constant voltage o f 60V overnight for the Hoeffer or 150V for 90min for the BioRad apparatus. Gels were then either processed for Western blotting, stained with Coomassie blue or fixed, dried and exposed to autoradiography.

2.2.4.6 T ris-tricin e-S D S -p olyacrylam id e gel electrop h oresis

This system is suitable for resolving polypeptides in the range from 5 to 20 kDa. This is achieved by swapping tricine for glycine in the electrophoresis buffer, and lowering the gel pH. Here, it was used for the electrophoretic analysis o f low molecular weight proteins or peptides. Gels were prepared according to the method o f Schagger and von Jagow (Schagger and von Jagow, 1987). BioRad mini gel apparatus was used. 15% resolving gels were made up containing 12% glycerol, 15% acrylamide-0.5% bisacrylamide, 0.3% SDS, 0.1% ammonium persulphate, 0.2% TEMED in 0.125 M Tris-HCl pH 8.45. Stacking gels were made to a final concentration o f 4% acrylamide in O.IM Tris-HCl pH 8.45, 0.1% ammonium persulphate, 0.05% TEMED. The anode buffer consisted o f 200mM Tris-HCl pH 8.9 and the cathode buffer o f lOOmM Tris, lOOmM Tricine, 0.1% SDS. Electrophoresis was performed under constant voltage o f 80V. Following electrophoresis the gels were processed for western blotting, stained with Coomassie or fixed, dried and exposed to autoradiography.

2.2.4.7 C oom assie staining o f acrylam ide gels

Following electrophoresis, the gel is submerged in a solution o f 40% (v/v) methanol, 10% (v/v) acetic acid, 0.3% (w/v) Coomassie brilliant blue R 250 and incubated under constant agitation for Ih. Protein bands were then visualised by washing the gel in a destaining solution (20% (v/v) methanol, 7% (v/v) glacial acetic acid). Several changes o f the destaining solution are required to completely destain the background.

2.2.4.8 A utoradiographic exposure o f acrylam ide gels

Following electrophoresis, the gel is submerged in fixing solution o f 20% (v/v) ethanol, 10% (v/v) glacial acetic acid and incubated under constant agitation for Ih in order to fix proteins into the gel. The gels is then dried in vacuo and exposed to a phosphorimager screen. Images o f radiolabelled protein bands were acquired using a Fuji FLA-2000 phosphorimager and analysed with Fuji Image Gauge software.

2.2.4.9 W estern blotting and im m unodetection o f transferred proteins

Proteins separated by electrophoresis, as described in section 2.4.4.5/6, were transferred onto Immobilon-P polyvinylidene fluoride (PVDF) membrane (Millipore) using a Bio-Rad Trans-Blot Cell. The membrane was pre-wetted in methanol briefly and then rinsed in water for 2min. Both gel and membrane were equilibrated in transfer buffer (25mM Tris, 192mM glycine, 20% (v/v) methanol) for 5min. The transfer stack was assembled immersed in transfer buffer. To assemble the transfer stack a foam pad was placed on one side o f the transfer cassette and one sheet o f 3MM filter paper was placed on top. The gel was then placed on top o f the filter paper and the membrane on top o f the gel. One sheet o f 3MM filter paper was then placed on top o f the membrane and a foam pad was put on top o f the filter paper to complete the stack. The transfer cassette was closed and placed in the transfer tank containing transfer buffer such that the gel is facing the cathode. The transfer was run at 350mA for 75min.

Following transfer to PVDF membranes, the membrane was first incubated in blocking buffer (5% (w/v) dried skimmed milk in 0.1% (v/v) Tween 20 in phosphate buffered saline (PBS-T)) for 1 hour at room temperature to saturate the binding sites on the membrane. For immunoblotting using phosphospecific antibodies, bovine serum albumin (BSA) and Tris-buffered saline were used instead o f skimmed milk and PB S, respectively, throughout the procedure.

The primary antibody was then added at the appropriate dilution in 10ml o f 1% (w/v) dried milk powder in PBS-T and incubated with the membrane on a rocking platform at room temperature for Ih or at 4°C overnight. The membrane was then washed three times with PBS-T for 5min each. The secondary antibody, conjugated to horseradish peroxidase (HRP) was diluted at 1:2000 in 10ml o f 1% milk in PBS-T and incubated with the membrane for 1 hour at room temperature. The membrane was then washed three times in PBS-T for 5min each. Antibody-antigen complexes were detected by Enhanced Chemiluminescence (KPL) according to the manufacturer’s instructions. Images were captured by a Fuji LA S-1000 Luminescent Image Analyser and analysed with Fuji Image Gauge software.

2.2.4.10 P rotein kin ase assays

PI 3-kinase autophosphorylation assays were performed in a final volume o f 30|il in a buffer containing 50mM Tris-HCl pH 7.4, 50mM NaCl, lOmM MnCl2 and 50jiM ATP (plus 5|xCi [y-^^P]ATP/assay). Reactions were initiated by adding the ATP/Mn^^ mix, run for 20min at 25°C and terminated by addition o f 5x electrophoresis sample buffer and boiling. When recombinant proteins were used as exogenous substrates (as in the case o f 4EBP1 and H-Ras), l|ig o f each was added per assay. When peptides were used as substrates, they were added at a final concentration o f ImM. Protein kinase CK2 kinase assays were performed as for PI 3-kinase using 20U o f recombinant enzyme per assay. In some experiments, OTP was used instead o f ATP as a phosphoryl donor for protein kinase CK2. For DNA-PK assays, 2U o f purified DNA-

PK and 500ng o f linearized double-stranded plasmid DNA were used per assay. The reaction products were analysed by SDS-PAGE and either autoradiography or immunoblotting with phosphospecific antibodies.

2.2.4.11 PI 3-kinase lipid kinase assays

PI 3-kinase lipid kinase assays were performed in a final volume o f 50pl in a buffer containing 50mM HEPES pH 7.4, lOOmM NaCl, ImM DTT, 5mM MgClj, lOOpM ATP (plus 0.1 pCi o f [y-^^P]ATP/assay) using 200|ig/ml phosphatidylinositol or phosphatidylinositol-4,5-bisphosphate as a substrate. Reactions were initiated by adding a mix o f the ATP and Mg^^, run for 20min at 25°C and terminated by the addition o f lOCjxl O.IM HCl. 200|xl chloroform: methanol (1: 1) were added, the mixture was vortexed and the phases separated by centrifugation at 10,000xg for 2min. The aqueous phase was discarded and the lower organic phase washed with 80|il o f methanol: IM HCl (1: 1). After centrifugation the aqueous phase was discarded again and the lower organic phase evaporated to dryness. The dried lipids were resuspended in 30|il o f chloroform: methanol (4: 1) and spotted onto thin layer Silica Gel-60 plates (Merck), pre-treated with 1% oxalic acid, ImM EDTA in water: methanol (6:4). TLC plates were developed in chloroform: methanol: 4M ammonia (9: 7: 4) for analysis o f phosphatidylinositol-3-phosphate or n-propanol: 2M acetic acid (13: 7) for analysis o f phosphatidylinositol-3,4,5-trisphosphate. After completion o f the separation, the plates were dried and exposed to autoradiography. Images o f radiolabelled lipid products were acquired using a Fuji FLA-2000 phosphorimager and analysed with Fuji Image Gauge software.

3. Role of PI 3-kinase protein kinase