Materials and methods 8

polyacrylamide gel electrophoresis (PAGE) and visualised by fluorography or autoradiography, respectively at -70 °C on XAR-5 film (Kodak).

Cos7 cells: For phosphoinositide analysis Cos7 cells were metabolically labelled 36 h (to 40 h) after transfection. Cells were washed extensively in P, free DMEM and labelled with p2p]orthophosphate (1mCi per 10 cm dish) in P j free DMEM containing

0.8% dialysed FCS and 20 mM Hepes, pH7.4 for 8 h. Phospholipids were extracted as described below.

Transient transfections

Cos7 cells: Cos7 cells were transfected by electroporation with 5 to 10 pg of plasmid DNA. Amounts of plasmid DNA were kept constant per cuvette by adding vector DNA. Cos7 cells (4 x 10®/0.8 ml) were pulsed at 450 V and 250 pF using a Gene Puiser (Bio- Rad). After 42 h to 48 h cells were collected.

293 cells: 293 cells were seeded at 3 x 10® cells per 10-cm dish in DMEM containing 10% FCS the day before transfection. Cells were washed and further cultured in serum-free DMEM when cells were transfected using lipofectamine reagent (GibcoBRL). Total amount of plasmid DNA was adjusted to 11 pg per dish with vector DNA. After 32 h cells were stimulated by addition of FCS as indicated and cells were collected. (In some experiments, 293 cells were transfected using a CaP0 4-based method and N-,N-

Bis(2-hydroxy-ethyl)-2-amino-ethanesulfonic acid (pH6.95) as transfection buffer (Chen and Okayama, 1988)).

NIH 3T3 cells: NIH 373 cells were transfected by the diethylaminoethyl (DEAE)- Dextran method (Hill et al., 1993). DNA concentrations in each experiment were constant (generally 8 pg) by adding vector DNA. Transfected cells were serum deprived for 36 h, stimulated by UV irradiation or addition of FCS as indicated and harvested after 8 h to 10 h. (In some experiments, NIH 3T3 cells were transfected using a CaP0 4-based method and Hepes buffered saline (pH7.05) as transfection buffer.)

Chapter 2 Materials and methods 8 1

Jurkat T cells: Jurkat T cells were transfected by electroporation with 20 to 40 pg of plasmid DNA. Amounts of plasmid DNA were kept constant per cuvette by adding vector DNA. Jurkat cells (1.0 to 1.2 x 10^ cells/0.5 ml) were pulsed at 310 V and 960 |iF. For gene reporter assays cells were stimulated 3h to 6h after transfection. Fourteen hours to 20 h after transfections Jurkat cells were harvested.

Kit225 cells: Kit225 were transfected by electroporation with 20 to 40 |ig of plasmid DNA. Amounts of plasmid DNA were kept constant per cuvette by adding vector DNA. Kit225 cells (1.5 x I O ? cells/0.625 ml) were pulsed at 320 V and 960 pF. For gene reporter assays cells were stimulated as indicated 2 h to 4 h after transfection. Cells were collected 14 h to 18 h after transfection.

Amounts of plasmid DNA used for transfections

The following amounts of plasmid were used unless indicated otherwise: Cos7 cells: 5 pg of rCD2-PI 3-kinase chimeras (pcDNA3)

293 cells: 2 pg of HA-p70S6k, 2 pg of H A -E rki, I p g o f vector plasmid (control), 1 pg of rCD2p85 (pRK5), 0.5 pg of rCD2p110 (pRK5), 0.5 pg of rCD2p110-R/P (pRK5) or 1 pg of Ha-v-ras.

NIH 3T3 cells: 0.5 pg rCD2p110 (pRK5), 0.5 pg rCD2p110-R/P (pRK5), 1 pg VI4Rho, 1 pg V12Rac, 1 pg Dbl or 2 pg 03 transferase. Four pg of CAT reporter constructs, 1 pg of lacZ plasmids and 0.25 pg of Nlex.elk-1 (pMLV) were used.

Kit225 cells: 7.5 pg of HA-p70S6k; 12.5 pg of HA-PKB; 10 pg of HA-Erk2; 20 pg of the following plasmids: pEF empty, rCD2p110 (pEF), rCD2p110-R/P (pEF), Ha-v-ras, V12Rac, V12Cdc42, gagPKB, or rCD2p85 (pEF); 7.5 pg of 2lexoptk.CAT and 15 pg of Nlex.elk-1 (pEF).

Jurkat cells: 7.5 pg of HA-p54p Sapk; 15 pg of the following plasmids: pEF empty, rCD2p110 (pEF), rCD2p110-R/P (pEF), rCD2p85 (pEF), rCD2 (pEF), Ha-v-ras, V12Cdc42 or gagPKB; 10 pg of the following plasmids: HA-PKB, p85A, V12Rac, or Dbl;

Chapter 2 Materials and methods

82

3 |xg of 2lexoptk.CAT; 6 |ig of Nlex.elk-1 (pMLV); 6 ^ig of NIex.jun (pMLV); 2 p,g of

API .CAT; 15 pg of NF-AT.CAT; and 5 pg of SRE.CAT.

Figure 2.1 G radient profile used to elute deacylated

phosphoinositides during anion- exchange high-pressure liquid chrom atography. 5 0 - oo o 3 0 - 10- \r> cJ 50 time (min) 75 100 125 25

Extraction of phosphoiipids from Cos7 ceiis and HPLC anaiysis of inositol- phosphates

Phospholipid extractions were based on the method described previously (Carter and Downes, 1992). Briefly, cells were washed with PBSA, collected in 1 ml 2.4

M HCI and transferred to a polypropylene test tube. CHCIg/MeOH (1:2 v/v) (1.5 ml) containing 10 pg phosphoinositides (Sigma) to act as carrier lipids were added followed by 1 ml CHCI3. After vortexing and centrifugation the lower phase was removed to a

fresh tube, the upper phase was re-extracted twice with 1.5 ml of CHCI3 and the

CHCI3 phases were combined. Dry lipid films were deacylated with 0.5 ml methylamine

at 50 °C for 1 h in the presence of 50 pi n-butanol. The resulting glycerophosphoinositol esters were re-dissolved in 0.5 ml H2O and once extracted with an equal volume of n-

butanol. The aqueous phase was mixed with ^H-labelled lns(1,3,4 ,5)P4 as an internal

standard (Stephens et al., 1991) and resolved by anion-exchange high-pressure liquid chromatography (HPLC) using a S5 SAX column (Phase Separations). The column was eluted at 1ml min’ i with a linear gradient of Milli Q water versus 2.5 M NaH2P0 4

Chapter 2 Materials and methods 8 3

(pHS.B with NaOH (%B)); times were t=0, %B=0; t=10, %B=0; t=60, %B=12; t=70, %B=19; t=110, %B=50, t=130, %B=50; t=140, %B=0 (Fig. 2.1); fractions were collected every 0.5 min. The data were collected as values of 32p and radioactivity measured using a calibrated dual-label program on a Beckman LS6000 series scintillation counter.

Immunofluorescent staining and fiow cytometry

Cell populations were washed twice in RPM11640 or DMEM medium containing 2% PCS (wash medium, WM) and resuspended at a concentration of 1 x I O ? cells per ml in WM at 4 °C. All antibody staining procedures were carried out on ice. Cells (5 x 105) were incubated with either 6 pg/ml rCD2 mAb 0x34 or for control staining mIgG (Sigma) in 50 pi WM. After 30 to 60 min incubation, the cells were washed twice in WM, then 50 pi of a dilution (1:40) of FITC-conjugated rabbit anti-mouse IgG (DAKO) in WM was added. After 30 to 60 min, the cells were washed three times in WM. The cells were analysed by flow cytometry on a FACScan (Becton Dickinson & Co.) using CellQuest™ software.

Microinjection experiments

Serum-starved sub-confluent Swiss 3T3 cells were prepared for microinjection as described above. Plasmid DMAs were microinjected at 200 pg/ml in PBS into the nucleus. For vinculin-staining cells were co-injected with Texas Red-lysinated dextran (2 mg/ml) as an injection marker. After 3 h to 4 h cells were fixed with 4% paraformaldehyde (w/v). To prevent activation of endogenous Rac and Rho proteins, rCD2p110 microinjected cells were re-injected with recombinant N17Rac proteins (0.7 mg/ml) and C3 transferase (100 pg/ml), respectively together with rat IgG (0.5 mg/ml) as an injection marker and after 30 min cells were fixed. Recombinant N17Rac proteins and C3 transferase were prepared as described (Ridley et al., 1992). Vinculin or actin was visualised by immunofluorescence as detailed below.

Chapter 2 Materials and methods 8 4

Immunofluorescence microscopy

Cell staining of Swiss 3T3 and Cos7 cells and immunofluorescence microscopy was carried out as described (Dieckmann et al., 1995). Briefly, cells were permeabilised for 5 min at room temperature with 0.2% Triton X-100 in PBSA. Permeabilised cells were labelled for protein expression (Cos7 cells) or double-labelled for actin and protein expression by first incubating with 0x34 mAb (2.4 pg/ml) to detect rCD2-PI 3-kinase chimeras. Then cells were incubated with FITC-conjugated goat anti-mouse Ab, and for detection of actin, with rhodamine-phalloidin (0.1 pg/ml). Permeabilised cells were labelled for vinculin by incubating with anti-vinculin mAb (Sigma, VIN 11-5) for 60 min in PBS, then with FITC-conjugated goat anti-mouse Ab for 30 minutes. Finally, an additional antibody layer (FITC-conjugated donkey anti-goat) was used for enhancement of the vinculin signal. Injected cells were visualised via the Texas Red-lysinated dextran injection marker. For re-injection experiments with recombinant proteins, co-injected cells was detected via the injection marker rat IgG which was visualised using a cascade blue- conjugated goat anti-rat IgG.

The coverslips were mounted by inverting them onto 5 pi moviol mountant containing p-phenylenediamine (1 mg/ml) as an antibleach agent and cells were examined on a Zeiss Axioskop microscope using Zeiss 63 x 1.4 (Cos7 cells), 40 x 1.3, and 100 X 1.3 oil immersion objectives. Fluorescence images were recorded on Kodak T-

max 400 ASA film or Kodak Ectachrome 200 ASA film (Cos7 cells).

Inductions

The following concentration of agonists were used unless stated otherwise: 10 pg/ml UCHT-1 for activation of the TCR/CD3 complex (< 1 h) 5 pg/ml UCHT-1 for activation of the TCR/CD3 complex (> 1 h) 1.6 pg/ml OKT3F(ab')2 for activation of the TCR/CD3 complex

50 ng/ml PdBu for activation of PKC

500 ng/ml ionomycin for activation of Ca^+-regulated pathways 20 ng/ml rlL-2 for activation of the IL-2R

Chapter 2 Materials and methods 8 5

10% FCS 40 J/m2 UV-C

for p70S6k assays in adherent cells for activation of Sapks in adherent cells

General induction times 2 min UCHT-1

5 min UCHT-1 15 min PdBu -+- lono 15 min rlL-2 15 min PdBu 5 min PdBu 5 min rlL-2 60 min FCS 5 min FCS

for adaptor association assays in Jurkat cells for PKB assays in Jurkat cells

for Jnk assays in Jurkat cells for p70S6k assays in Kit225 cells for p70S6k assays in Kit225 cells for Erk assays in Kit225 cells for PKB assays in Kit225 cells for p70S6k assays in 293 cells for Erk assays in 293 cells

Ceii iysis

When not stated otherwise cells were lysed in the following buffers:

T cells: T cells (1 x 10^ to 2 x 10^ cells per ml) were lysed in 0.5 ml lysis buffer 1: 1% NP40, 150 mM NaCI, 50 mM HEPES pH7.4,10 mM NaF, 10 mM lodoacetamide, 100 |xM sodium orthovanadate (Na3V0 4), 1 mM phenylmethylsulfonyl fluoride (PMSF), and the

small peptide protease inhibitors leupeptin, pepstatin A, and chymostatin, all at 1 pg/ml. Rat-1 cells: Rat-1 cells plated onto 9 cm cell culture dishes (Nunc) were lysed in 1 ml lysis buffer 1.

Cos7 cells: Cos7 cells plated onto 10 cm cell culture dishes (Falcon) were lysed in 0.5

ml Cos7 lysis buffer. 1 %(w/v) Triton X-100, 20 mM Tris pH7.5, 137 mM NaCI,

15%(v/v) glycerol, 2 mM EDTA, 2 mM PMSF, 20 pg/ml aprotinin, 2 mM benzamidine, 1 mM Na3V0 4 , and the small peptide inhibitors leupeptin, pepstatin A, and chymostatin all

at 2 pg/ml.

293 cells: 293 cells plated onto 10 cm cell culture dishes (Falcon) were lysed in 0.4 ml

lysis buffer 2: 120 mM NaCI, 50 mM Tris pHS.O, 20 mM NaF, 1 mM benzamidine, 1 mM

Chapter 2 Materials and methods 8 6

0.1 mM PMSF, and 0.1 mM Na3V0 4 . Extracts were prepared as described (Ferrari et

a!., 1993).

Determination of protein concentration

Aliquots of the supernatants were taken for a Bradford protein assay (Bradford, 1976), using the Coomassie Blue G-250 reagent solution from Pierce and bovine serum albumin as a standard.

Expression of recombinant proteins in E. coli

Desired recombinant proteins were expressed in E. coli as glutathione-S- tranferase (GST) fusion proteins to allow purification from bacterial lysates. An overnight culture of E. coli (XLI-Blue strain) carrying the pGEX expression plasmid which contains the desired cDNA were diluted in 400 ml L-broth/50 jig/ml ampicillin (see below). The culture was grown until an OÜ55o=0.5 to 0.6 was reached and expression of fusion protein was induced by adding isopropyl p-D-thiogalactopyranoside (Sigma) to a final concentration of 0.4 mM. The culture was left growing for an additional 2 h to 5 h at 37 oQ. A shorter incubation times yields less protein but the protein obtained may be more soluble and stable. E. co//were harvested by centrifugation at 4,000 rpm for 15 min at 4 °C, the pellet was resuspended in 10 ml of ice-cold GST iysis buffer {1% Triton X-100, 2 mM EDTA, 1 mM PMSF, in PBSA) and stored at -20 °C.

Purification of recombinant proteins from E. coli

The bacteria were lysed by sonication on ice (4 to 6 x 1 min) and cell debris were removed by centrifugation at 10,000 rpm for 10 min at 4 °C. The supernatant was incubated with 1 ml of 50%(w/v) suspension of glutathione agarose beads (Sigma) which had been washed thoroughly in PBSA containing 2 mM EDTA. After 1 h to 2 h at 4 °C, the suspension was centrifuged at room temperature in a bench top centrifuge and the beads were washed 2 times in GST lysis buffer and 3 times in PBSA containing 2

Chapter 2 Materials and methods 8 7

mM EDTA to remove unbound proteins. As an additional purification step the GST fusion proteins were eluted from the beads by competition with 25 mM reduced glutathione in 50 mM Tris pH8. Proteins were dialysed against PBSA containing 2 mM EDTA at 4 °C and the protein concentration was determined. GST fusion proteins were either re-coupled to glutathione agarose beads or left uncoupled and stored in 50% glycerol containing 50 mM Hepes pH7.4 and 50 mM NaCI at -20 °C.

Protein isolation from cell lysates

Postnuclear cell lysates were pre-cleared with protein A cell suspension (Sigma) prior to purification of cellular proteins with affinity resins. Protein A cell suspension (Staph A) (Sigma) (Kessler, 1975) was equilibrated, by washing four times in the appropriate lysis buffer. 1/10 volume of the washed Staph A suspension (10%) was added to the cell lysate (final conc. 1%) and lysates were precleared by rotating for 20 min at 4 °C and subsequent spinning at 15,000 x g for 5 min at 4 °C. The supernatant was transferred into a separate Eppendorf microcentrifuge tube and the pre-cleared cell lysates were subjected to protein isolation procedures. Precipitation procedures were carried out for two to four hours by gently rotating the tubes on a wheel at 4 °C as described below. Precipitated protein complexes were processed as detailed in following sections.

Affinity purification of cellular proteins: Proteins were affinity purified from precleared postnuclear lysates with either glutathione-agarose beads (Sigma) preloaded with approximately 5fig of GST fusion proteins or 25 pM of the various synthetic peptides precoupled to 'Affigel 10' activated ester agarose beads (Bio-Rad). 1 x 10? cell equivalents (0.5 ml volume of cell lysate) were used per sample.

Immunoprécipitations: To immunoprecipitate rCD2-PI 3-kinase chimeras 0.5 ml of precleared postnuclear lysates from transfected cells were incubated with 5 pg (to 10 pg) 0x34 anti-rCD2 mAbs. After 30 min 30 pi protein G-Sepharose beads (34% suspension of beads) (Sigma) were added and incubations were continued for 90 min.

Chapter 2 Materials and methods 8 8

The p85 subunit of PI 3-kinase was immunoprecipitated with 10 pg p85a (U5) mAb followed by protein G-Sepharose beads (see above). Lysates containing HA- or Myc- epitope tagged proteins (0.3 ml volume) were incubated with 2 pg 12CA5 mAbs or 2 pg 9E10 mAbs, respectively. After 90 min 30 pi of 34%(w/v) protein G-Sepharose beads were added and the immunocomplexes were precipitated after further 30 to 45 min. Endogenous p70S6k or endogenous PKB were isolated with 1 pg of M5 Abs or 2 pg of Rac-PK-CT Abs, respectively (150 pi volume and 90 min incubation). Immune complexes were precipitated with protein A-Sepharose beads (Pharmacia) (after an additional 45 min incubation).

In vitro protein kinase assay

The immunoprecipitates were washed three times in the appropriate lysis buffer and two times in protein kinase assay buffer (25 mM HEPES, pH7.4, 100 mM NaCI, 5 mM MnCl2, 10 mM MgCl2, 100 pM Na3V0 4 ). With the final wash the

immunoprecipitates were transferred into a separate tube and the rest of the solution was removed with a Hamilton syringe. The protein kinase assay was initiated by adding 100 nM [y-32p]ATP (10 pCi) and 30 pM ATP in 50 pi protein kinase assay buffer. After 20 min incubation at 25 °C in vitro phosphorylation was stopped by diluting the reaction mixture with 1 ml appropriate lysis buffer containing 20 mM EDTA. The immunoprecipitates were then washed two times in this buffer and the rest of the solution was removed with a Hamilton syringe. The immunoprecipitated proteins were eluted from the beads by boiling for 8 min in 60 to 90 pi reducing sample buffer and separated by SDS-PAGE as detailed below. The gels were fixed in 30% (v/v) methanol/10%(v/v) acetic acid for 30 min, dried and 32p_iabelled proteins were visualised by autoradiography at -70 °C.

Chapter 2 Materials and methods 8 9

PI kinase assays

The immunoprecipitates were washed once in the appropriate lysis buffer, twice in washing buffer 1 (100 mM Tris pH7.5, 0.5 M LiCI, 100 pM Na3V0 4 ) and twice in

washing buffer 2 {50 mM Hepes pH7.4, lOOmM NaCI, 1 mM EDTA, 100 pM Na3V0 4 ).

With the final wash the immunoprecipitates were transferred into a separate tube (Sarstedt, with a rubber sealed lid) and the rest of the washing buffer 2 was removed with a Hamilton syringe. The immunoprecipitates were assayed for inositol lipid kinase activity using unilam ellar liposom es composed of a 1:1 m ixture of L- a- phosphatidylinositol (PI) and L-a-phosphatidyl-L-serine (PS), which were prepared by sonication (30 s, 30 s break, 1 min, 30 s break, 30 s; 50% level) of the lipid mix (PI and PS, both at a concentration of 1 mg per ml) in 25 mM HEPES buffer, pH7.4 containing 1 mM EDTA on ice. The washed immunoprecipitates were resuspended in 10 pi lipid mixture and the reaction was initiated by the addition of 40 pi PI kinase reaction buffer

(25 nM [y-32p]ATP (5 pCi), 125 pM ATP, 12.5 mM MgClg, 125 mM NaCI, 25 mM Hepes pH7.4, 250 pM adenosine). The final concentrations in the assay were: 20 nM [y- 32p]ATP (5 pCi), 100 pM ATP, 10 mM MgCl2, 100 mM NaCI, 25 mM Hepes pH7.4, 200

pM EDTA, and 200 pM adenosine (to inhibit PI 4-kinase activity, (Whitman et al., 1988)). After 15 min at 25 °C the reaction was terminated by the addition of 500 pi chloroform : methanol (1:2) in 1% conc. HCI plus 125 pi chloroform and 125 pi HCI (10 mM). The mixture was vigorously mixed and then centrifuged to separate the phases. 200 pi of the organic (lower) layer was removed (with a chloroform sealed tip) and washed once with 400 pi methanol : 100 mM HCI plus 2 mM EDTA (1:1). The organic phase was extracted, dried in vacuo, and resuspended in 25 pi chloroform. The phospholipids were separated by thin layer chromatography (TLC) on 1 % potassium oxalate pre-treated Silica gel 60 plates (Whatman) in propanol-1 : 2 M acetic acid (65:35 (v/v)) developing solvents and visualised by exposure to iodine vapour and autoradiography at -70 °C. Phospholipids were identified by comparison with non-labelled standards. Incorporation of 32p into PI was quantified using a Phosphorlmager (Molecular Dynamics). The