Activation of phosphoinositide 3-kinase is required for PDGF-stimulated membrane ruffling

Activation of phosphoinositide 3-kinase isrequired for PDGF-stimulated membrane ruffling Stefan Wennstrim*, Phillip Hawkinst, Frank Cooket, Kenta Hara*, Kazuyoshi Yonezawa*, Masato Kasuga*, Trevor Jacksont, Lena Claesson-Welsh* and Len Stephens t 'Ludwig Institute for Cancer Research, Uppsala Branch, Box 595, S-751 24 Uppsala, Sweden. tDepartment of Development and Signalling, AFRC Babraham Institute, Cambridge CB2 4AT, UK. The Second Department, Internal Medicine, Kobe University, School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650, Japan.

Background: There is substantial evidence that phosphoinositide 3-kinase (PI 3-kinase) is a critical component of signalling pathways used by the cellsurface receptors for a variety of mammalian growth factors and other hormones. The physiological product of this enzyme is a highly polar membrane lipid called phosphatidylinositol (3,4,5)-trisphosphate This lipid has been postulated to act as a secondmessenger in cells but its putative targets are still unknown. Results: A particular rearrangement of actin filaments, which results in membrane ruffling, is elicited by the activation of PDGF -receptors expressed in cultured porcine aortic endothelial cells. We have found that this consequence of PDGF 3-receptor activation is inhibited by three independent manipulations of

PI 3-kinase activity: firstly, by the deletion of tyrosine residues in the PDGF 3-receptor to which PI 3-kinase binds; secondly, by the overexpression of a mutant 85 kD PI 3-kinase regulatory subunit to which the catalytic kinase subunit cannot bind; and thirdly, by the addition of the fungal metabolite wortmannin, which is a potent inhibitor of the catalytic activity of PI 3-kinase. Conclusions: These results argue strongly that phosphatidylinositol (3,4,5)-trisphosphate synthesis is required for growth-factor-stimulated membrane ruffling in porcine aortic endothelial cells, and suggest that synthesis of this lipid may be part of a signalling pathway leading to direct or indirect activation of the small GTP-binding protein Rac.

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Background Ligand binding to a variety of cell-surface receptors leads to rapid activation of an enzyme known as phosphoinositide 3-kinase (PI3-kinase) [1-3]. In vivo, this enzyme is thought to phosphorylate the head group of phosphatidylinositol (4,5)-bisphosphate (Ptdlns(4,5)-P 2) in the 3 position to yield Ptdlns (3,4,5)-P 3 [4-7]. Thus, activation of appropriate receptors leads to the rapid appearance of Ptdlns (3,4,5)-P 3 in cells, and this lipid has been postulated to act as a second messenger conveying information from the receptor to various, as yet unidentified, intracellular targets [2,3]. Cell-surface receptors using a variety of signal-transduction mechanisms are known to stimulate synthesis of Ptdlns (3,4,5)-P3 in target cells. However, most of the work elucidating the mechanism of activation of P13kinase has concentrated on those receptors that use protein tyrosine kinase activity to transduce their signals. This is not only because PI'3-kinase was initially discovered as an enzyme that translocates into a tight complex with protein tyrosine kinases on stimulation, but also because many of the protein tyrosine kinases implicated in the activation of PI3-kinase (such as growth factor receptors and members of the Src-family of kinases) are known to be critical regulators of mitogenesis [1].

Thus far, most is known about how receptors with intrinsic protein tyrosine kinase activity, such as the receptors for platelet derived growth factor (PDGF) and insulin, stimulate P1 3-kinase. A ligand binding to these receptors stimulates autophosphorylation of tyrosine residues, which then act as specific recruitment sites for the tight binding of signalling molecules that bear a Src homology region 2 (SH2) domain (Fig. 1) [1,8]. Recent work indicates that the SH2 domains of different signalling molecules show great specificity for the amino-acid sequence surrounding their phosphotyrosine targets: thus, tyrosine residues at positions 740/751, 579/581, 771 and 1009/1021 of the PDGF 13-receptor have been implicated as major determinants for the binding of PI 3-kinase, Src, Ras GTPase-activating protein (GAP) and phospholipase Cy (PLCy), respectively [9-12]. The PI3-kinase that associates with activated receptors in this manner is a heterodimer of tightly bound 85kD regulatory (p8 5 ) [13-151 and 110kD catalytic (pl10) [16] subunits. The 85kD subunit possesses two SH2 domains, and hence confers upon P113-kinase the ability to interact with activated receptor tyrosine kinases. Tyrosine-phosphorylated peptides, which can bind tightly to PI 3-kinase, stimulate this enzyme in vitro, implying that SH2-domain docking may be a physiologically relevant mechanism for activating 1' 3-kinase [17,18].

Correspondence to: Len Stephens.

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Current Biology 1994, Vol 4 No 5 Despite progress in understanding the mechanism by which receptor tyrosine kinases activate PI 3-kinase, little is known about the putative signalling functions of the response. There is good evidence that in some cell types removal of the tyrosine phosphate residues in the PDGF -receptor to which PI 3-kinase binds dramatically impairs the normal mitogenic response elicited by this receptor [12,19]. However, these studies have not yet identified the precise biochemical step(s) controlled by PI 3-kinase and, whilst potentially very exciting, it is clear that PI 3-kinase is also activated in non-mitogenic contexts as well (for example, on stimulation of neutrophils [41 and platelets [20] by formylpeptide and thrombin, respectively). We show here that activation of PI 3-kinase is essential for a PDGF-stimulated rearrangement of actin filaments that causes cell-surface membrane ruffling, and suggest that this may provide an insight into a common function for this pathway in many cells, namely the activation of the small GTP-binding protein Rac.

Results and discussion PDGF -receptors lacking their major PI 3-kinase binding site do not stimulate membrane ruffling Wild-type PDGF -receptors and mutated receptors with the tyrosine residues at positions 740 and 751

replaced by phenylalanine residues (Y740/751F receptors) were expressed in porcine aortic endothelial cells (PAE) that had been stably transfected with the appropriate cDNAs. As predicted from previous work, Y740/751F receptors were unable to support substantial PDGF-stimulated recruitment of PI 3-kinase to the receptor (Fig. 2b and c). Furthermore, PDGF was unable to stimulate accumulation of PtdIns (3,4,5)P3 in these cells (Fig. 3a). However, although the response of these mutated receptors was reduced compared with the responses elicited by wild-type PDGF -receptors, both their tyrosine kinase activity (Fig. 2a) and their capacity to stimulate synthesis of inositol phosphates (Fig. 3b) remained substantially PDGF-sensitive. Collectively, these results indicate that PI 3-kinase is indeed the enzyme responsible for PDGF-stimulated synthesis of PtdIns (3,4,5)-P 3 and that 'SH2 domain-docking' is essential for activation of the PI 3-kinase. PAE cells expressing Y740/751F receptors failed to support PDGF-stimulated membrane ruffling (Fig. 4) [21]. Membrane ruffling [22] appears as a result of a rearrangement of cortical actin filaments, and is thought to represent an integral part of directed cellular movement. Many different growth-factor receptor tyrosine kinases induce this response in a variety of adherent cell types [23-251.

Fig. 1. Specificity of tyrosine phosphate autophosphorylation sites in the PDGF receptor for SH2-domain bearing signalling molecules. The tyrosine autophosphorylation sites are indicated, the SH2 domain on the signalling molecules is shown in red, the receptor kinase catalytic region is shown in green and the non-catalytic regions in grey. (Adapted from [9].)

involvement of P 3-kinase in ruffling Wennstr~m et al. e a. InolemntofPH3-inseinruflngWensro

Fig. 2. Effect of wortmannin, overexpression of Ap85 and replacement of tyrosine residues 740/751 on PDGF 3-receptor autophosphorylation and PI 3-kinase translocation. PAE cells stably expressing wild-type PDGF 3-receptors, PAE cells stably expressing wild-type PDGF 3-receptors and transiently overexpressing Ap85, and PAE cells stably expressing Y740/751F PDGF 3-receptors were treated with 100 nM wortmannin or vehicle (DMSO) for 5 min, then with PDGF (100 ng ml-') or salts for a further 4 min. Cells were then lysed and PDGF 3-receptor immunoprecipitates prepared and assayed for in vitro kinase activity or presence of P 3-kinase immunoreactivity, as described in the Materials and methods. (a) In vitro kinase assays of PDGF 13-receptor immunoprecipitates. This experiment has been repeated three times and in each case the degree of PDGFstimulated autophosphorylation of the wild-type receptor was unaffected by the presence of wortmannin, and that of the Y740/751 F receptor reduced to approximately 40-60 % of that of the wild-type receptor. (b) Western blots of PDGF 13-receptor immunoprecipitates using an anti p85a-specific monoclonal antibody. (c) Western blots of PDGF 13-receptor immunoprecipitates using an anti-pl 10 monoclonal antibody; this blot was performed directly after the p85a-blot shown in panel (b) without 'stripping' and thus the p85a immunoreactive bands are still visible (the p85 bands are in the same relative proportions as that shown in (b) but the film has been exposed for longer). WT, wild type; Wort, wortmannin.

Wortmannin inhibits PDGF-stimulated membrane ruffling We sought further evidence for the involvement of PI 3kinase in PDGF-stimulated membrane ruffling by the use of the fungal metabolite wortmannin. Wortmannin has recently been shown to potently inhibit PI 3-kinase catalytic activity in vitro by binding directly to p110 ([26] and M. Thelen, personal communication). The addition of 100 nM wortmannin to PAE cells expressing wildtype PDGF receptors dramatically reduced PDGFstimulated synthesis of PtdIns (3,4,5)-P 3 (Fig. 3a). Wortmannin may be a specific inhibitor of the catalytic activity of PI 3-kinase, as the wortmannin treatment

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Fig. 3. Effects of wortmannin and replacement of tyrosine residues 740/751 on PDGF 3-receptor stimulated Ptdlns (3,4,5)P3-synthesis and inositol phosphate production. (a) PAE cells expressing wild-type or Y740/751 F receptors were labelled with 32p, pretreated with 100 nM wortmannin or vehicle for 5 min, stimulated with PDGF (100 ng mi- 1) or salts for a further 50 sec, and then their 32P-labelled lipids quantified as described in the Materials and methods. The values shown are for [32p] Ptdlns (3,4,5)-P3 contents per bottle ( range, n=2). The levels of [32P]Ptdlns (4,5)-P2 (dpm per bottle; ± range n=2) in the various incubations were: wild-type, control and PDGF-stimulated cells, 1 905 366 ± 96 850 and 1 813 167 + 10 349, respectively; wortmannin-treated wild-type, control and PDGF-stimulated cells, 1 956 322 + 88 408 and 1 843 962 + 63 900, respectively: Y740/751F, control and PDGF-stimulated cells, 1 302 453 ± 80 961 and 1 493 545 ±122 000, respectively. Similar results were obtained in an independent experiment. (b) PAE cells expressing wild-type or Y740/751F receptors were labelled with [3H]inositol, pretreated with 100 nM wortmannin or vehicle for 6 min, stimulated with PDGF (100 ng ml-1) or salts for a further 10 min and then their [3H]inositol phosphate content measured as described in the Materials and methods. The values shown are for the amount of radioactivity per well eluted from the columns under conditions which elute InsP to InsP 6 (mean ± SEM, n=3).

had little effect on PDGF-stimulated translocation of p85 (Fig. 2b) or p110 (Fig. 2c) to the PDGF receptor, PDGFstimulated receptor autophosphorylation (Fig. 2a), PDGF-stimulated tyrosine phosphorylation of cell proteins (data not shown), PDGF-stimulated synthesis

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Fig. 4. Effects of wortmannin and deleting PDGF -receptor binding sites for PI 3-kinase on PDGF-stimulated membrane ruffling. Coverslips of PAE cells expressing either wild-type PDGF 3-receptors or Y740/751 F receptors were pretreated with either wortmannin (100 nM) or vehicle (DMSO) for 5 min, challenged with PDGF (100 ng ml- 1) or salts for a further 3-4 min, then fixed with paraformaldehyde and processed to visualize actin filaments using TRITC-labelled phalloidin as described in the Materials and methods. The cells were viewed with a Zeiss Axiophot at x 400 magnification and the cells displaying membrane ruffling were counted (see arrowed examples). Typical fields of PAE cells are: (a) cells overexpressing wild-type PDGF p-receptors but unstimulated; (b) cells overexpressing wild-type PDGF 13-receptors and stimulated with PDGF; (c) cells overexpressing wild-type PDGF 13-receptors and stimulated with PDGF in the presence of 100 nM wortmannin. (d) Shows the numbers of cells displaying edge ruffling under various conditions (cells displaying one or more clear ruffles were taken as positive; N > 100 for each condition).

Involvement of P 3-kinase in rufling Wennstr~m et al.

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Fig. 5. Effects of expression of a p85 unable to bind PI 3-kinase activity on PDGF-stimulated membrane ruffling. PAE cells expressing the wild-type PDGF 13-receptor were transiently transfected with a vector containing the cDNA for either Ap85 or an irrelevant but immuno-detectable protein (the E. coli lac repressor protein); the transfection frequencies for the two vectors were not significantly different (5-8 %). 12 hours post-transfection, the cells were plated onto coverslips and after an additional 24 hours the cells were challenged with vehicle, (a) and (b), or PDGF, (c) and (d), and fixed as described in the Materials and methods. Cells were double-stained for total p85 protein (FITC fluorescence in panels (a) and (c)) and phalloidin-binding proteins (TRITC fluorescence in (b) and (d)). This enabled cells over-expressing Ap85 to be examined for effects on PDGF-stimulated membrane ruffling. Thus, (a) and (b)show the FITC and TRITC fluorescence of the same field of unstimulated cells and (c) and (d) a field of PDGF-stimulated cells. Inspection of coverslips from 2 independent experiments showed that only 5 % of cells that were expressing Ap85 and stimulated with PDGF, exhibited edge ruffling (n=427), compared with 87 % of their non-overexpressing neighbours. In contrast, 84 % (n=338) of cells expressing an irrelevant protein activated edge ruffling in the presence of PDGF.

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Current Biology 1994, Vol 4 No 5 of inositol phosphates (Fig. 3b) or the endogenous levels of PtdIns (4,5)P 2 (Fig. 3). Wortmannin potently inhibited PDGF-stimulated membrane ruffling in these cells: a half-maximal effect was seen at approximately 25 nM and complete inhibition at 60 nM (Fig. 4 and data not shown). Wortmannin also inhibited PDGF-stimulated membrane ruffling in human foreskin fibroblasts AG1518 (50 % inhibition at approximately 10 nM and complete inhibition at 40 nM; S.W. and L.C-W., unpublished observations). These effects of wortmannin occur over a similar dose range (2-40 nM) to those described for the inhibition of PI 3-kinase in cells ([26,27] and data not shown). Transient expression of a mutant PI 3-kinase regulatory subunit blocks PDGF-stimulated membrane ruffling We also attempted to block PDGF-stimulated activation of PI 3-kinase by overexpression of an altered bovine p85 (Ap85), which lacks a region of approximately 30 amino acids that is required for tight association with p1 10, but is still able to bind to appropriate phosphotyrosine targets [28]. A Chinese hamster ovary (CHO) cell line stably overexpressing Ap85 shows markedly reduced insulin-stimulated PtdIns (3,4,5)-P 3 accumulation (K.Y., M.K., L.S., P.T.H. and T.J., unpublished observations). PAE cells expressing wild-type PDGF [3-receptors and overexpressing Ap85 showed PDGFsensitive translocation (Fig. 2b) and phosphorylation (Fig. 2a) of Ap85, but failed to form membrane ruffles in response to PDGF (Fig. 5).

Conclusions The three experimental approaches described above, which imply that PI 3-kinase activation is required for PDGF-stimulated membrane ruffling, can each be criticized for the specificity with which PI 3-kinase has been perturbed. Replacement of tyrosine residues 740 and 751 in the PDGF 3-receptor may affect the activation of other, as yet unidentified, signalling proteins. The expression of very large amounts of a mutant 85 kD PI 3-kinase subunit in cells may lead to competition with other SH2-domain-bearing molecules for phosphotyrosines that are not normal sites of PI 3-kinase interaction. The specificity of wortmannin is still unclear and it has documented effects at high concentrations on other relevant enzyme activities, for example myosin light chain kinase [291. However, the fact that all three approaches correlated PI 3-kinase activation with membrane ruffling and yet had relatively little effect on the activation of PLC-y (a signalling pathway not only operating in parallel to PI 3-kinase, but also requiring the same substrate, PtdIns (4,5)-1' 2) strongly suggests that PI 3kinase has a major role in the ruffling response. Furthermore, because wortmannin does not prevent translocation of p85 or p110 to the PDGF-receptor, and Ap85, which can translocate to the receptor, includes

Fig. 6. A flow diagram illustrating the possible consequences of PI 3-kinase activation. Agonist binding to appropriate heterotrimeric G-protein or tyrosine kinase coupled receptors leads to the activation of PI 3-kinasets) and production of Ptdlns (3,4,5)-P 3 [3,37]. There is evidence that synthesis of Ptdlns (3,4,5)-P is important for PDGF-stimulated membrane ruffling (see text), PDGF-stimulated mitogenesis [12,19], FMLP-stimulated superoxide production [27,30], IgE-stimulated histamine secretion [261 and insulin-stimulated glucose transport (K.Y., K.H., M.K., T.J., P.H. and L.S., unpublished observations). These processes have been shown to depend upon the function of small GTP-binding proteins and, in some cases, the identity of the protein involved is thought to be Rac (see text). Therefore, either directly, or via intermediates, Rac is a potential target for regulation by Ptdlns (3,4,5)-P3 (see ?). Rac may be activated either by regulation of guanine nucleotide exchange or by posttranslational modification and translocation [38,391. It can be envisaged that Rac-GTP may be a critical component in the stimulated assembly of a matrix of cortical actin filaments and that this process is central to a number of cell responses such as directed cell movement, pinocytosis, exocytosis, insertion of new membrane components into the cell membrane and, more generally, cell growth and differentiation. (Those steps in the pathway requiring further clarification are shown in boxes with a dashed outline.) the SH3, BCR (breakpoint cluster region) and SH3binding domains of the wild-type protein, it appears that this role in membrane ruffling requires the synthesis of Ptdlns (3,4,5)-P 3.

involvement of P 3-kinase in ruffling Wennstr~m et al.

Involvement of P1 3-kinase in ruffling Wennstrom et al. Wortmannin is a potent inhibitor of both PI 3-kinase and the formylpeptide-stimulated oxidative burst in neutrophils [27,30]. Neither the mechanism of activation of the oxidase nor the step(s) where wortmannin acts to prevent oxidase activation are known, but recent evidence suggests that the small GTP-binding protein, Racl, may play an important role in the assembly of oxidase components at the correct membrane location [31]. Racl has also been implicated in the mechanism by which PDGF stimulates membrane ruffling in fibroblasts [32]. It therefore seems highly plausible that the synthesis of Ptdlns (3,4,5)-P 3 may trigger the activation of Rac in these cells, perhaps via the regulation of specific guanine nucleotide exchange inhibitors or stimulators. Thus, in the context of the mariy responses that appear to be regulated by highly homologous small GTP-binding proteins, this hypothesis may provide a common basis for understanding the apparently diverse role of PI 3-kinase in cell signalling (Fig. 6).

Materials and methods Construction of stable PAE cell lines expressing wild-type and Y740/751F PDGF p-receptors cDNA encoding the PDGF 13-receptor [33] was subcloned into the pAlter vectorTM (Promega Corp.) and site-directed mutagenesis was performed to substitute phenylalanine residues for tyrosine residues 740 and 751 using the Altered sites in vitro Mutagenesis System (Promega Corp.). Wild-type and mutated cDNAs were inserted into the expression vector pcDNA1 neo (Invitrogen) and PAE cells transfected with the constructs by electroporation. Stable cell lines expressing wild-type (48 x 104 receptors per cell) and Y740/751F receptors (30 x 104 receptors per cell) were selected with neomycin (G-418 sulphate) and continuously cultured as described in [34]. Measurement of PDGF 1-receptor autophosphorylation and PI 3-kinase translocation Clones of PAE cells stably expressing either wild-type or Y740/751F PDGF -receptors were grown to confluence in 75 cm2 flasks in Earle's minimal medium (MEM) with 10 % FBS. The cells were serum-starved by incubation for an additional 16 hr in Earle's MEM with 1 % FBS and then treated with either wortmannin (Sigma; 15 Itl of a 100 ixM stock in DMSO was added to give final concentrations of 100 nM wortmannin and 0.1 % DMSO) or an equivalent volume of DMSO for 5 min at 37 °C, followed by either salts (-) or PDGF (+) (BB, 100 ng ml-') for a further 4 min. Cells were then washed, lysed and antibody-PDGF receptor immunoprecipitates prepared and collected on Protein A Sepharose beads as described previously [25], except that samples derived from cells initially treated with wortmannin had 100 nM wortmannin included in all the relevant buffers throughout the experiment. The beads were then divided, one half were subjected to an ill vitro kinase assay with [y- 32 P]ATI' and separated by polyacrylamide gel electrophoresis after treatment with sodium dodecyl sulphate (SDS PAGE) as described previously [25]. Proteins on the other half of the beads were separated by SDS PAGE, transferred to immobilon (Millipore) and blotted (in phosphate buffered saline, PBS, containing 0.1 % (w/v) Tween 20, 4 % (w/v) dried milk) with a mouse anti-p85a-specific monoclonal antibody (U13, a gift from M. Waterfield and I. Gout, Ludwig Institute, London). The presence of p85 antibody

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was visualized using horseradish peroxidase-conjugated antimouse antibody and ECL reagent (Amersham). Some blots were then washed in PBS and reprobed with an anti-pll0 mouse monoclonal antibody ([11], a gift from M. Waterfield and I. Gout) and visualized as described above. Measurement of PDGF-stimulated Ptdlns (3,4,5)P3 synthesis in intact PAE cells Clones of PAE cells expressing either wild-type or Y740/751F PDGF -receptors were grown to confluence in small glass bottles (Wheaton 'shorty vials', precoated with gelatin) and then serum-starved (in Earle's MEM with 1 % FBS; 1 ml per bottle). After 16 hours, the cells were washed twice with phosphate-free HEPES-buffered Hanks solution (containing 0.2 % fatty acid free BSA, 0.1 mM pyruvate and 10 mM glucose; solution A), then incubated with 32p (0.85 mCi ml-1 in 0.4 ml of solution A) for 70 min at 37 C, washed twice and rewarmed to 37 C in 0.4 ml of solution A as described previously [7]. After 3 min at 37 °C, cells were treated with either wortmannin (added directly from a stock in DMSO to final concentrations of 100 nM wortmannin and 0.25 % DMSO) or an equivalent volume of DMSO for 5 min and then incubated with either salts or PDGF (100 ng ml-') for a further 50 sec. Incubations were terminated, and lipids extracted, deacylated and quantified by HPLC as described in [6,7]. Measurement of PDGF-stimulated inositol phosphate production in intact PAE cells Clones of PAE cells expressing either wild-type or Y740/751F PDGF receptors were grown to confluence in 6 x well plates and then labelled with [3 H]inositol during serumstarvation (inositol-free Ham's F12, 1% dialysed FBS, 25 Ci- ml-' [3 H]inositol; 2 ml per well). Cells were then washed and warmed to 37 C in solution A (2 ml per well) supplemented with 10 mM LiCl and either 100 nM wortmannin (added directly from a 1 000-fold concentrated stock in DMSO, immediately prior to use) or 0.1% DMSO. After 6 min, PDGF (100 ng ml-1) or its vehicle were added. After a further 10 min, incubations were quenched by aspirating the medium and adding 1 ml ice-cold 4 % perchloric acid (containing phytate hydrolysate, 12 ixg phosphorus). The acid extracts were neutralized and inositol phosphates quantified by chromatography on small Dowex-1 columns, as described previously [35]. Measurement of membrane ruffling in PAE cells expressing wild-type and Y740/75 I1F PDGF 1-receptors Coverslips of PAE cells stably expressing either wild-type PDGF 13-receptors or Y740/751F receptors were cultured in Earle's MEM with 10 % FBS. 16-24 hrs after plating, they were serum-starved by incubation for 12-16 hrs in F12 nutrient mix with 1% FBS, challenged with PDGF (100 ng ml-1) for 3-4 mins then fixed with 4 % paraformaldehyde (20 min) and acetone (-20 C, 2 mins). The samples were processed to visualize actin filaments using TRITC-labelled phalloidin (Sigma) as described previously [25]. Some coverslips were treated with either wortmannin (100 nM) or vehicle alone (DMSO for 5 mins before the addition of the agonist). Measurement of membrane ruffling in PAE cells transiently expressing Ap85 PAE cells expressing wild-type P'DGF -receptors were grown to confluence in 75 cm2 flasks in Earle's MEM with 10 % FBS and then transfected using either calcium phosphate or liposome- (Gibco BRL) based protocols, with TM 20 .g of plasmid DNA (prepared using Promega Magic

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Current Biology 1994, Vol 4 No 5 Resin columns). The constructs were based on the vector pcDNA3 (Invitrogen) in which the original 0.8 kb promotor was replaced with the 2.1 kb hCMVIE1 promotor [36] (a gift from A. Venkitaraman, LMB, Cambridge). The Ap85 cDNA or cDNA for the lac repressor protein (Stratagene) were ligated into the multiple cloning site of this expression vector. 12-16 hr post-transfection, cells were replated on glass coverslips and grown for a further 16-24 hr in F12 nutrient mix with 1% FBS before challenging them with PDGF (see above). p85 and phalloidin-binding proteins were simultaneously detected by incubating the fixed cells with an anti-p85 monoclonal antibody (U13) in hybridoma supernatant supplemented with 5 % goat serum and approximately 25 ng ml-1 phalloidin. After 1 hr, the cells were washed with 4 % (w/v) BSA in PBS then incubated for a further 1 hr with FITC-coupled anti-mouse antibody (from goat, 20 Rg ml-1, Sigma) in the continued presence of phalloidin. The cells were finally washed in PBS, H2 0, mounted in Citifluor (Citifluor Ltd) and viewed with a Zeiss Axiophot at x 400 magnification. The expression of the lac repressor protein was detected by substituting the anti-p85 reagent with a 1/1 000 diluted anti-lac repressor rabbit polyclonal serum (a gift from D. Wyborski, Stratagene) and the secondary detection reagent with a FITC-coupled anti-rabbit serum (from goat, Pierce). Acknowledgements: P.T.H. is a Lister Institute Fellow, TJ. is a Royal Society Mr &Mrs J. Jaffe Fellow and F. Cooke is an MRC student.

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STEPHENS L, SMRCKA A, COOKE FT, JACKSON TR, STERNWEIS PC,

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