Experimental Procedures
2.4 Methods to detect protein interactions
2.4.1 Protein-protein affinity chromatography
2.4.1.1 Preparation of rat brain and liver extracts
Ten rat brains or livers were homogenised in 40 ml of ice-cold 10 mM sodium phosphate (pH 6.9) buffer, 70 mM sodium glutamate, 2 mM EDTA, 2 mM MgSO^, 1 mM DTT, containing a cocktail of protease inhibitors [2 pM, 10 pg/ml leupeptin, 2 pg/ml aprotinin and 2 mM phenylmethylsulfonyl fluoride (PMSF)]. The homogenate was centrifuged (10,000 rpm, 10 min, 4°C) using a Sorval SS34 rotor and the supernatant recentrifuged (50,000 rpm, 45 min, 4°C) in a Beckman TL-100 centrifuge with a TLA 100.2 rotor. Liver lysates were additionally precleared with 0.5 ml of glutathione sepharose beads. The supernatants were collected and used for affinity binding studies.
2.4.1.2 Preparation of cell extracts of U937 and HELA cells
Cells were washed twice with PBS and resuspended in 10 X cell pellet volume of ice- cold lysis solution [50 mM Tris HCl [pH 7.4], 150 mM NaCl, 50 mM NaF, 5 mM EDTA, 1% Triton X-100, 500 pM sodium orthovanadate, 2 mM PMSF, and 100 kallikrein inhibitor units of Aprotinin]. The lysate was incubated on ice for 30 min, after which it was centrifuged for 20 min at 4°C at 14,000 rpm to pellet the cell debris. The supernatant was then utilised in affinity binding studies.
2.4.1.3 Affinity screening of extracts
Cell extracts were prepared as described in sections 2.4.1.1 and 2.4.1.2. 50 pi of 50% (w/v) glutathione sepharose 4B pre-equilibrated in the appropriate cell extract buffer was incubated (20 min at 4°C) with 5 pg of GST or GST-fusion proteins in final volume of 750 pi. After washing to remove unbound protein, the affinity matrix was incubated with cell lysates for 1 hour at 4°C. The matrix was washed 4 times with ice-cold cell extract buffer, by repeated centrifugation (500 rpm, 3 min), and bound proteins analysed by SDS-PAGE using both high (15%) and low (7%) percentage polyacrylamide gels. Proteins were visualised by silver staining.
2.4.1.4 PKC binding assay
5 n g o f G ST and G ST-PH dom ains (Btk, dynam in, I R S l, Sos and V av) w ere im m obilised to Glutathione sepharose 4B in coupling buffer [50 mM Tris-H C L (pH 8.0), 150 mM NaCl, 1 mM DTT] using the same procedure described in 2.4.1.3. The m atrix was incubated with 3 pg of either a, pi, pH, y or ; PKC isoforms (Dr Parker, ICRF), in a volum e adjusted to 750 pi with coupling buffer, for 1 hour at 4°C. The beads were collected by centrifugation and washed 4 times with ice-cold coupling buffer by repeated centrifugation (500 rpm, 3 min) and proteins resolved by 10% SD S-PA G E. Binding of PK C isoform s to GST-PH domains was analysed by Im m unoblotting (section 2.3.4)
2.4.2 Real-time analysis of protein-phosphoinositide interactions
2.4.2.1 Liposom e preparation
P hosphatidylcholine [PtdCho], Phosphatidylserine [PtdSer], Phosphatidylethanolam ine [PtdEth], sphingom yelin [SM] and cholesterol were obtained from Sigm a Chem ical Co. L td. P h o sp h a tid y lin o s ito l [P dtlns] and P h o sp h a tid y lin o s ito l 4 ,5 -b is p h o s p h a te [PtdIns(4,5)Pi] w ere o b tain e d from L ip id P ro d u c ts (R e d h ill, S u rre y , U K ). Phosphatidylinositol 4-m onophosphate [PtdIns(4)P] was obtained from C albiochem . Synthetic Phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P3] w as prepared by Dr G iggs (Desai et al., 1996).
Liposom es contained 30% (w/w) PtdCho, 15% (w/w) SM, 20% (w/w) cholesterol, 15% (w/w) PtdEth, 10% (w/w) PtdSer and 10% (w/w) of the test phosphoinositide. Large unilam ellar liposom es were generated according to published procedures (Philippot et al., 1983). In sum m ary lipids (dissolved in chloroform /m ethanol [1:2]) were dried under a nitrogen stream with vortexing and resuspended in 750 pi of liposom e b u ffe r'[10 mM HEPES (pH 7.4); 80 mM KCl; 15 mM NaCl; 0.7 mM NaHzPOa; 1 mM EGTA; 0.466 m M C a C l2; 2.1 mM M g C l]], c o n ta in in g 50 m M n-octyl-p-D -glucopyranoside (C albiochem ). The tubes were rotated for 20 m ins at 25°C to solubilise the lipids and then dialysed (12-14,000 Da Spectra/Por, Spectrum) for 16 hrs against 3 changes of 500 ml Lt>. SM -2 Bio-Beads (Bio Rad) were included during the second dialysis exchange to enhance the rem oval of n-octyl-(3-D-glucopyranoside. Liposom es were stored at 4°C for up to 2 weeks.
2.4.2.2 BIAcore analysis
The basic operating procedures of the BIAcore biosensor (Pharmacia) have been published (Jonsson et ah, 1991). The interaction matrix was activated by injecting 40 pi of a mixture of equal volumes of 11.5 mg/ml N-Hydroxysuccinimide (NHS) and 75 mg/ml N-Ethyl-N'-(3-dimethylaminopropyl)-carbodimide (EDC) to enable it to covalently bind free amino groups of proteins. Goat anti-GST antibody (Pharmacia) in acetate buffer (pH 4.8) was applied according to the manufacturer's instructions. Any remaining unreacted sites were blocked by injecting 40 pi of a 1 M ethanolamine solution. GST-PH domain fusion proteins exchanged into liposome buffer were coupled to the antibody-coated sensorchip surface by injecting saturating amounts of protein (approximately 5-10 pg). 1-5 pi of liposomes were diluted into 100 pi Lb and injected over the surface. All reactions were carried out at 25°C and under a constant flow-rate of 5 pl/min. To facilitate the repeated analysis of liposome and covalently immobilised GST-fusion protein interactions the liposomes were removed with one pulse 4 pi of 25 mM n-octyl-p-D-glucopyranoside. The anti-GST antibodies were regenerated by injecting 10 pi of 0.2 M glycine, pH 2.2 and could be used for several cycles of interaction.
2.5
Dynamin GTPase assays
2.5.1.1 Phospholipid preparation
In addition to the phosphoinositide sources described in section 2.4.2.1, Ptdlns, Ptdlns(4)P and Ptdlns(4,5)P% were obtained from Sigma chemical Co. Ltd. 90% (w/v) PtdCho and 10% (w/v) of the test phosphoinositide (both dissolved in chloroform/methanol [1:2]) were mixed and dried under a nitrogen stream on ice. The phosphoinositide mixture was then resuspended to a final concentration of 1 mg/ml in 50 mM Tris-HCl (pH 8.0), 5 mM MgCh, 0.1 mM DTT and sonicated (three, 45 s cycles) using an MSE Soniprep 150 sonicator. Vesicles were stored at 4°C for up to 48 hours.
2.5.1.2 GTPase assay
Dynamin GTPase assays were performed using 0.5 |ig of dynamin in 50 mM Tris-HCl (pH 8.0), 5 mM MgClz, 0.1 mM DTT, 130 pM GTP, 13 nM [a-32p]GTP (1 |iCi, 3,000 Ci/mmol; Amersham Corp.) in a total volume of 25 |il for 1 hour at 37°C. Grb2, Grb2 N- and C-terminal SH3 domains, phospholipids or inositol phosphates were added to the reaction mix to assess their effect on the GTPase activity of dynamin. Reactions were
terminated by heating at 65°C for 10 min. The products were resolved by thin layer chromatography on polyethyleneimine-cellulose plates (Merck) in 1.6 M LiCl for 1 hour. The extent of GTP hydrolysis was assessed with a phosphorimager (Molecular Dynamics).