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Journal of Cell Science 108, 225-233 (1995)Printed in Great Britain © The Company of Biologists Limited 1995

Activation of the small GTP-binding proteins rho and rac by growth factor

receptors

Catherine D. Nobes1, Phillip Hawkins2, Len Stephens2 and Alan Hall1,3,*1CRC Signal Transduction and Oncogene Group, MRC Laboratory for Molecular Cell Biology, University College London, LondonWC1E 6BT, UK 2The Inositide Laboratory, the Babraham Institute, Babraham, Cambridge CB2 4AT, UK3Department of Biochemistry, University College London, London WC1E 6BT, UK

*Author for correspondence

The small GTP-binding proteins, rho and rac, controlsignal transduction pathways that link growth factorreceptors to the activation of actin polymerization. In Swiss3T3 cells, rho proteins mediate the lysophosphatidic acidand bombesin-induced formation of focal adhesions andactin stress fibres, whilst rac proteins are required for theplatelet-derived growth factor-, insulin-, bombesin- andphorbol ester (phorbol 12-myristate 13-acetate)-stimulatedactin polymerization at the plasma membrane that resultsin membrane ruffling. To investigate the role of p85/p110phosphatidylinositol 3-kinase in the rho and rac signallingpathways, we have used a potent inhibitor of this activity,wortmannin. Wortmannin has no effect on focal adhesionor actin stress fibre formation induced by lysophosphatidicacid, bombesin or microinjected recombinant rho protein.

In contrast, it totally inhibits plasma membrane edge-ruffling induced by platelet-derived growth factor andinsulin though not by bombesin, phorbol ester or microin-jected recombinant rac protein. We conclude that phos-phatidylinositol 3,4,5 trisphosphate mediates activation ofrac by the platelet-derived growth factor and insulinreceptors. The effects of lysophosphatidic acid on the Swiss3T3 actin cytoskeleton can be blocked by the tyrosinekinase inhibitor, tyrphostin. Since tyrphostin does notinhibit the effects of microinjected rho protein, we concludethat lysophosphatidic acid activation of rho is mediated bya tyrosine kinase.

Key words: actin stress fibre, membrane ruffle, PI3-kinase,wortmannin, tyrosine kinase

SUMMARY

INTRODUCTION

The rho-related small GTP-binding proteins play a key role inregulating the assembly and organization of the actincytoskeleton in response to extracellular growth factors. Theaddition of serum (the active constituent of which is lysophos-phatidic acid, LPA) to quiescent, serum-starved Swiss 3T3cells induces the rapid formation of actin stress fibres and theassembly of focal adhesions and this can be completelyblocked if the cells are first microinjected with C3 transferase,a bacterial exoenzyme that ADP-ribosylates and inactivatesendogenous rho proteins (Paterson et al., 1990; Ridley andHall, 1992). Growth factors such as platelet-derived growthfactor (PDGF) and insulin, on the other hand, stimulate poly-merization of actin at the plasma membrane of many cell typesto produce lamellipodia and edge-ruffles (Nistér et al., 1988;Ridley et al., 1992). In Swiss 3T3 cells, this can be inhibitedby a dominant negative mutant of rac, N17rac1, establishing adistinct rac-regulated signal transduction pathway linkinggrowth factor receptors to the polymerization of actin at theplasma membrane (Ridley et al., 1992).

The signals required for initiating actin polymerization inresponse to extracellular factors are not known, but there has

been much speculation that changes in plasma membranepolyphosphoinositides could directly influence actin polymer-ization (Stossel, 1989, 1993). Phosphatidylinositol 4,5 bispho-sphate (PtdInsP2), for example, can release monomeric actinfrom the actin binding protein profilin and this could be a signalfor polymerization (Lassing and Lindberg, 1985, 1988). Inmany cell types there is often a much better correlationbetween actin polymerisation and the production of theputative second messenger phosphatidylinositol 3,4,5 trispho-sphate (PtdInsP3) by activation of the enzyme phosphatidyli-nositol 3-kinase (PI3-kinase) (Eberle et al., 1990). PI3-kinaseis a dimer consisting of a regulatory 85 kDa subunit that asso-ciates with activated tyrosine kinase receptors through its srchomologous (SH2) domains and a 110 kDa subunit thatencodes the catalytic activity (Carpenter and Cantley, 1990;Panayotou and Waterfield, 1992). LPA, PDGF and insulinhave all been shown to affect the cellular concentrations ofPtdInsP3 (Cantley et al., 1991; Kumagai et al., 1993; Van Hornet al., 1994) and it has recently been shown that activation ofPI3-kinase is required for PDGF- and insulin-stimulatedmembrane ruffling (Wennström et al., 1994a,b; Kotani et al.,1994). However, it has not been possible to determine wherePI3-kinase is required on the signal transduction pathway.

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LPA is known to stimulate a number of signalling pathwaysincluding activation of ras, protein kinase C, Ca2+ mobilisationand a reduction in cAMP levels (van Corven et al., 1989, 1993;Moolenaar, 1994). However, none of these pathways appearsto be involved in the rapid effects of LPA on the actincytoskeleton (Ridley and Hall, 1994). An essential genistein-sensitive tyrosine kinase appears to act downstream of rho inthe formation of focal adhesions and actin stress fibres consis-tent with the observation that several components of the focaladhesion multiprotein complex are phosphorylated on tyrosineresidues (Turner and Burridge, 1991; Bockholt and Burridge,1993; Ridley and Hall, 1994; Schaller and Parsons, 1993).However the mechanism by which LPA leads to the activationof rho is currently unknown.

To define the role of PI3-kinase in the rho and rac signallingpathways we have analysed the effects of the inhibitor, wort-mannin, on cytoskeletal changes induced by the addition ofgrowth factors or by microinjection of recombinant rho and racprotein. Wortmannin is a fungal product that covalently bindsto and inhibits the p110 kinase subunit of PI3-kinase in the 1-10 nM range (Yano et al., 1993). It appears to be highlyspecific, though at concentrations above 1 µM it has beenreported to inhibit myosin light chain kinase (Nakanishi et al.,1992). Wortmannin blocks PI3-kinase activity stimulated bytyrosine kinase receptors, G protein-coupled receptors and thehigh affinity immunoglobulin E (FceR1) receptor (Arcaro andWymann, 1993; Kumagai et al., 1993; Yano et al., 1993). Inaddition we have used a combination of phosphotyrosine phos-phatase and tyrosine kinase inhibitors to determine if growthfactor induced activation of rho or rac is dependent upontyrosine kinases. We conclude that activation of rac by PDGFand insulin receptors is mediated by PI3-kinase whereas acti-vation of rho by LPA is mediated by a tyrosine kinase.

MATERIALS AND METHODS

MaterialsWortmannin, phorbol 12-myristate 13-acetate (PMA), LPA,bombesin, insulin, sodium orthovanadate and rhodamine-phalloidinwere obtained from Sigma Chemical Co. PDGFBB was from UBI.Tyrphostin A25 was from Gibco-BRL/Life Technologies. Vanadatewas activated as described by Mahadevan and Bell (1991).

Cell culture and microinjectionSwiss 3T3 cells were maintained in Dulbecco’s modified Eagle’smedium (DMEM) containing 10% foetal calf serum. Quiescent,serum-starved Swiss 3T3 cells were prepared for microinjection andtreatment with growth factors as follows: cells were seeded at adensity of 5×104 cells into 15 mm diameter wells containing 13 mmglass coverslips. Six to ten days after seeding, cells were serum-starved overnight (16 hours) in DMEM containing 2 g/l NaHCO3.Recombinant V14rhoA (0.1-0.15 mg/ml), V12rac1 (0.6 mg/ml) andC3 transferase (0.1 mg/ml) proteins were microinjected into thecytoplasm. Injected cells were localized after fixation by co-injectingcells with rat immunoglobulin (IgG) at 0.5 mg/ml and staining withFITC-conjugated rabbit anti-rat IgG. Pretreatment with wortmanninwas for 5 minutes prior to microinjection of recombinant V14rhoA orV12rac1 proteins or addition of growth factors. Cells were incubatedwith tyrphostin for 30 to 60 minutes prior to treatment with growthfactors for 15 or 30 minutes as indicated. Microinjection was for aperiod of 10 minutes after which the cells were returned to theincubator for a further 15 to 30 minutes before fixation.

ImmunofluorescenceFor filamentous actin localization cells were fixed in 4%paraformaldehyde for 15 minutes, permeabilized with 0.2% Triton X-100 for 5 minutes and incubated with rhodamine-phalloidin (0.1µg/ml) for 45 minutes. Cells were viewed on a Zeiss microscope, andphotographed with Kodak T-MAX 400ASA film.

Expression and purification of recombinant proteinsRecombinant V14rhoA, V12rac1 and C3 transferase proteins wereexpressed as glutathione-S-transferase (GST) fusion proteins inEscherichia coli and purified on glutathione Sepharose beads (Smithand Johnson, 1988). The proteins were released from the beads bythrombin cleavage and purified as described by Ridley et al. (1992).For V14rhoA and V12rac1 proteins active protein concentrationswere determined by a filter binding assay using [3H]GDP or [3H]GTPas previously described (Hall and Self, 1986). Protein preparationsshowed only one band on Coomassie-stained SDS-polyacrylamidegels.

[32P]Pi labelling of intact cells and measurement of 32P-labelled lipids in cell extractsSwiss 3T3 cells were grown in 35 mm dishes to confluence over 7days and serum-starved for 16 hours in DMEM containing 2 g/lNaHCO3. The cells were washed and labelled in Hanks-type saltsolution for 70 minutes at 0.25 mCi/ml as described by Hawkins etal. (1992). The cells were then washed and left in 1 ml salts ± 100nM wortmannin (added from DMSO stock immediately prior to use)for approximately 10 minutes at room temperature. The cells werethen warmed for 3 minutes at 37°C and stimulated with 1 ml salts (atapproximately 37°C) containing agonists. After 3 minutes stimulationthe medium was poured off, the plates ‘blotted’ and 0.5 ml ice-cold1 M HCl added. The plates were scraped on ice and cell-debris/HCltransferred to a small glass bottle. The plates were washed with 1.367ml of HCl/MeOH (0.3 ml 1 M HCl, 5 mM tetrabutylammoniumsulphate + 1.067 ml MeOH) and residual debris/solution combinedwith the first extract. The combined scrapings were extracted using aFolch distribution of organic and aqueous phases; the lipids were thendeacylated with methylamine and the water-soluble head groupsresolved by HPLC on a Partisphere SAX column as described byJackson et al. (1992). All samples were counted for 20 minutes (i.e.180 dpm = 3600 counts above background).

RESULTS

Wortmannin inhibits PDGF and insulin but not rac-stimulated edge-ruffling Quiescent serum-starved Swiss 3T3 cells display only a finering of polymerised actin at their periphery when stained withrhodamine-labelled phalloidin (Fig. 1A). The addition ofPDGF (3 ng/ml) or insulin (1 µg/ml) caused an increase inpolymerized actin localized in plasma membrane edge-ruffles(Fig. 1B and D) and an increase in [32P]PtdIns(3,4,5)P3 levels(see Table 1). Membrane ruffling in response to these growthfactors was completely blocked by a 5 minute pretreatmentwith 100 nM wortmannin (Fig. 1C and E) as was the rise in[32P]PtdIns(3,4,5)P3 (Table 1). These results are consistentwith recent published data showing that activation of PI3-kinase is required for PDGF- and insulin-stimulated membraneruffling (Wennström et al., 1994a,b; Kotani et al., 1994).

Previous work has shown that PDGF- and insulin-stimulatedruffling is mediated by rac since prior microinjection of a dom-inant negative rac mutant, N17rac1, into Swiss 3T3 cells blocksgrowth factor-induced ruffling (Ridley et al., 1992). Further-

227Receptor-mediated activation of rho and rac

Fig. 1. Wortmannin inhibits membrane ruffling stimulated byPDGF and insulin but not by microinjected rac. Actin filamentsare shown after treatment of serum-starved Swiss 3T3 cells. Noaddition (A), 3 ng/ml PDGF (B,C), 1 µg/ml insulin (D,E),microinjected with V12rac1 protein (600 µg/ml) (F,G). Cells inC,E and G were given a 5 minute preincubation with wortmannin(100 nM). Cells were stained and fixed for actin filaments withrhodamine-phalloidin. Bar, 50 µm.

228 C. D. Nobes and others

Table 1. Effect of wortmannin on the levels of 32P-labelled inositol lipids and [32P] PtdOH in Swiss 3T3 cells stimulatedwith PDGF, LPA and bombesin

PtdOH PtdIns3P PtdIns4P PtdIns(3,4)P2 PtdIns(4,5)P2 PtdIns(3,4,5)P3

Control 977±30 490±23 16,839±659 42±13 29,717±603 17±2PDGF (30 ngml−1) 2658±106 441±36 17,327±952 117±6 26,615±2037 178±5LPA (200 ngml−1) 2979±50 399±18 23,196±141 38±1 28,723±967 17±1Bombesin (100 nM) 5035±63 315±6 10,944±65 26±10 21,363±1058 10±0Control + Wortmannin (100 nM) 1058±98 119±2 14,246±278 33±6 31,553±2300 0±1PDGF (30 ngml−1) + Wortmannin (100 nM) 2780±411 133±9 18,969±1015 48±5 28,437±1800 37±8LPA (200 ngml−1) + Wortmannin (100 nM) 2756±245 112±11 23,814±1423 40±1 29,571±274 8±0

Cells were prelabelled with [32P]Pi, washed and incubated±wortmannin (100 nM) for 10 minutes at room temperature. The cells were then stimulated withPDGF (30 ng/ml), LPA (200 ng/ml) or bombesin (100 nM) for 3 minutes at 37°C. Incubations were terminated and lipids extracted and analysed as described inMaterials and Methods. The values presented are dpm ± range for two independent dishes of cells.

more, microinjection of recombinant, activated rac (V12rac1)into quiescent cells induces membrane ruffling in the absenceof growth factors (Fig. 1F, and Ridley et al., 1992). In order todistinguish whether the wortmannin-sensitive step in mem-brane ruffling occurs upstream or downstream of rac, we havemicroinjected V12rac1 into cells that have been treated withwortmannin. As shown in Fig. 1F and G, membrane rufflingstimulated by microinjection of recombinant V12rac1 protein isnot blocked by pretreatment with wortmannin.

Bombesin- and PMA-stimulated membrane rufflingis not blocked by wortmanninIn order to investigate further the relationship of activation ofPI3-kinase by growth factors to cortical actin polymerizationand membrane ruffling we have looked at the effect of wort-mannin on bombesin, phorbol 12-myristate 13-acetate (PMA)and activated ras-induced membrane ruffling. Bombesin,acting through a G-protein coupled receptor, is able tostimulate the accumulation of actin filaments in membraneruffles via rac-activation (Ridley et al., 1992), but this neu-ropeptide does not activate PI3-kinase in Swiss 3T3 cells(Jackson et al., 1992, and Table 1). Since bombesin can alsostimulate rho-dependent actin stress fibre formation, we havemicroinjected C3 transferase into cells prior to stimulation withbombesin in order to inhibit the rho-mediated pathway (Ridleyet al., 1992). We find that bombesin (10 nM)-stimulatedmembrane ruffling is not inhibited by wortmannin (100 nM)(Fig. 2A and B) and we conclude that rac can be activated byPI3-kinase independent pathways. This result also indicatesthat wortmannin does not have a non-specific effect onreceptor-induced membrane actin polymerization in Swiss 3T3cells.

PMA is one of the most potent activators of membraneruffling we have found in Swiss 3T3 cells, though unlike otherstimulators of ruffling, no actin stress fibres are formed evenat later (30 minute) times (Ridley et al., 1992). Oncogenic ras(V12H-ras) is also able to stimulate rac-dependent rufflingthough the mechanism is unclear (Ridley et al., 1992). Wort-mannin had no effect on PMA-induced membrane ruffles (Fig.2C and 2D) or membrane ruffling that was stimulated bymicroinjection of recombinant V12H-ras (not shown).

Wortmannin does not block LPA or rho-inducedactin stress fibre formationThe addition of LPA to quiescent Swiss 3T3 cells stimulatesthe formation of actin stress fibres and new focal contacts by

activating a rho-dependent signal transduction pathway andmicroinjection of recombinant, activated rho protein(V14rhoA) induces actin stress fibres in the absence of addedgrowth factors (Ridley and Hall, 1992). In addition to tyrosinekinase receptors, PI3-kinase activity can also be stimulated byheterotrimeric G protein-coupled receptors. For example, thechemoattractant fMLP acting through a G-protein coupledreceptor in neutrophils activates a PI3-kinase. Wortmanninblocks this response and prevents fMLP stimulation of the res-piratory burst and of actin polymerization (Arcaro andWymann, 1993). LPA is also thought to act through a G proteincoupled receptor, though this has not yet been cloned (Van derBend et al., 1992). A recent report suggests that LPA not onlyactivates the p85/p110 PI3-kinase in Swiss 3T3 cells, but thatthis is dependent upon a functional rho protein (Kumagai et al.,1993).

In contrast, we find that LPA-induced actin polymerisationis not accompanied by a rise in [32P]PtdIns(3,4,5)P3 (see Table1) indicating that, in this system at least, LPA does not activatePI3-kinase. We do not know the reason for this discrepancybut it could reflect differences in Swiss 3T3 subclones. Insupport of the conclusion that PI3-kinase activity is notrequired for rho signalling, we find that 100 nM wortmanninhas no effect on the ability of LPA (Fig. 3A and B) or microin-jected rho proteins (Fig. 3C and D) to stimulate actin stressfibre formation. Even a ten-fold higher concentration of wort-mannin (1 µM) had no effect on LPA-induced stress fibres orfocal adhesion formation (not shown). Interestingly, LPA stim-ulates a significant increase in [32P]PtdIns4P which is similarto the effects of EGF on these cells (Jackson et al., 1992).

Evidence that a tyrosine kinase is required for theactivation of rhoIn order to further investigate the mechanism by which LPAactivates rho leading to stress fibre formation we have used thephosphotyrosine phosphatase inhibitor sodium orthovanadate.Vanadate can increase intracellular phosphotyrosine levels incells thereby acting as a signalling agonist of pathways that areregulated by tyrosine kinases. Indeed vanadate is able to causefibroblast transformation (Smith, 1983; Klarlund, 1985) and isknown to have insulin-like actions on some cells by stimula-tion of insulin receptor tyrosine kinase activity (Tamura et al.,1984). We find that vanadate (100 µM) strongly stimulates theformation of actin stress fibres but not membrane ruffles inserum-starved Swiss 3T3 cells (Fig. 4A). The response to thisconcentration of vanadate is maximal at 20-30 minutes con-

229Receptor-mediated activation of rho and rac

Fig. 2. Bombesin- and PMA-stimulated membrane ruffling are not blocked by wortmannin. Serum-starved Swiss 3T3 cells stimulated withbombesin (10 nM) were first microinjected with C3 transferase (100 µg/ml) to block the rho-activated stress fibre pathway that is stimulated bybombesin. C3 transferase injection reveals more clearly membrane ruffling induced by bombesin in Swiss 3T3 cells. Actin filaments are shownin cells microinjected with C3 transferase and stimulated with bombesin (10 nM) without (A) or with (B) wortmannin (100 nM), or stimulatedwith PMA (100 nM) without (C) or with (D) wortmannin. Cells were fixed and stained for actin filaments with rhodamine-phalloidin. Bar, 20 µm.

sistent with a lag required for entry of vanadate into the cell.As for LPA, vanadate-stimulated stress fibres are inhibited incells microinjected with C3 transferase, which inactivates rhoproteins (Fig. 4B), indicating that activation of a tyrosinekinase can lead to activation of rho and the induction of stressfibre formation.

We have shown previously that the tyrosine kinase inhibitorgenistein inhibits the formation of actin stress fibres not onlyby LPA but also by microinjected rho (Ridley and Hall, 1994).It was not possible therefore to analyse the role of tyrosinekinases upstream of rho. Neither the erbstatin analogue, methyl2,5-dihydroxycinnamate, nor herbimycin A (a potent inhibitorof pp60src activation) significantly inhibited stress fibreformation in response to growth factor stimulation in Swiss3T3 cells (Ridley and Hall, 1994). We now report here, thatthe tyrosine kinase inhibitor, tyrphostin A25 (150 µM) blocksnew stress fibre formation stimulated by LPA (Fig. 4C and D)but not by microinjected rho protein (Fig. 4E and F). Further-more, tyrphostin A25 also inhibits the ability of vanadate toactivate rho (Fig. 4G and H).

DISCUSSION

In this paper we have examined components of growth factor-stimulated signalling pathways that activate the small GTP-binding proteins rho and rac. Firstly we have investigated therole of PI3-kinase in rho and rac mediated signal transductionpathways by using a recently characterised inhibitor of PI3-kinase, wortmannin. Our results are consistent with recentlypublished data showing that activation of PI3-kinase isessential for membrane edge-ruffling stimulated by PDGF andinsulin (Wennström et al., 1994a,b; Kotani et al., 1994). Wehave previously shown that rac is required for membraneruffling stimulated by PDGF and insulin and we have nowshown using the microinjection technique, that wortmannindoes not inhibit rac-induced ruffling. We conclude thatPtdInsP3 generated by the p85/p110 PI3-kinase is required foractivation of rac. One possiblity is that the lipid can activate aguanine nucleotide exchange factor, or inhibit a GTPase acti-vating protein (GAP). At least five mammalian GAPs activeon rac have been identified so far, rhoGAP, bcr, n-chimerin,

230 C. D. Nobes and others

Fig. 3. LPA and rho-induced actin stress fibre formation are not blocked by wortmannin. Actin filamemts are shown in serum-starved Swiss3T3 cells stimulated with LPA (200 ng/ml) (A,B), microinjected with V14rhoA protein (150 µg/ml) (C,D). Cells in B and D were pretreated for5 minutes with wortmannin (100 nM). Cells were stained for actin filaments with rhodamine-phalloidin. Bar, 20 µm.

Fig. 4. Activation of rho by a tyrosine kinase. Actin filaments areshown after treatment of serum-starved Swiss 3T3 cells. 100 µMsodium orthovanadate (A,B,G,H), 100 ng/ml LPA (C,D),microinjected with V14rhoA (100 µg/ml) (E,F). Cells weremicroinjected with C3 transferase (100 µg/ml) 15 minutes prior toaddition of vanadate (B) or given a 40 minutes preincubation withtyrphostin (150 µM) (D,F,H). In (B) two cells in the centre of thefield were microinjected with C3 transferase, in (E) all cells shownwere microinjected with V14rhoA apart form the cell in the top leftcorner and in (F) three cells in the centre and bottom right of the fieldwere microinjected with V14rhoA. Bar, 50 µm.

abr and p190 but as yet their functions are unclear (Lancasteret al., 1994; Diekmann et al., 1991; Heisterkamp et al., 1993;Settleman et al., 1992). No exchange factors for rac have yetbeen described though members of the the family of dbl relatedproteins are likely candidates (for review see Nobes and Hall,1994). Interestingly, p85, the regulatory subunit of PI3-kinasecontains a domain with significant homology to the rhoGAPfamily of proteins (Otsu et al., 1991). p85 does not have GAPactivity in vitro towards rac but it is possible that the twoproteins could directly interact through this domain. Anothercandidate regulatory protein for rho-related GTPases isrhoGDI. This protein is thought to regulate the cycling of theGTPases on and off the plasma membrane and there is somesuggestion that this can be affected by lipids (Chung et al.,1993). In addition, however, it is clear from the work describedhere, that rac can also be activated through a PI3-kinase inde-pendent route in Swiss 3T3 cells, since rac activation bybombesin, PMA or oncogenic ras is unaffected by wortman-nin.

The rho-mediated signal transduction pathway activated byLPA or by bombesin in Swiss 3T3 cells does not require PI3-kinase activity since we show that neither of these growth

factors stimulates a rise in PtdInsP3. Furthermore, stress fibreformation is not blocked by wortmannin. We have previouslyshown that a genistein-sensitive tyrosine kinase is involved inthis signal transduction pathway but since genistein blockedthe activity of microinjected rho, we were unable to addressthe possibility that a tyrosine kinase also acted upstream aswell as downstream of rho (Ridley and Hall, 1994). Here weprovide evidence that a tyrosine kinase is indeed required foractivation of rho by LPA. We have found that tyrphostin blocksstress fibre formation stimulated by LPA but not by microin-

231Receptor-mediated activation of rho and rac

232 C. D. Nobes and others

jected rho. Tyrphostins are competitive inhibitors of tyrosinekinases that have been engineered to mimic the structure of aphosphorylated tyrosine residue in a peptide chain (Levitski,1990). They do not affect the activity of protein kinase A,protein kinase C, serine kinases or threonine kinases (Levitski,1990). Presumably tyrphostin in this system has a differentsubstrate specificity to that of genistein thereby allowing us todefine at least two candidate tyrosine kinases in theLPA/rho/stress fibre pathway; one upstream and one down-stream of rho. It is noteworthy that tyrphostins have beenshown to interfere with adhesion associated tyrosine phospho-rylation of pp125FAK in endothelial cells in addition toreducing focal adhesion and stress fibre formation (Romer etal., 1994).

We have also found that orthovanadate, a phosphotyrosinephosphatase inhibitor, stimulates actin stress fibre formation inquiescent Swiss 3T3 cells. Furthermore this is blocked bymicroinjection of cells with C3 transferase, that ADP-ribosy-lates and inactivates rho, and by tyrphostin. It is likelytherefore that vanadate leads to the activation of the sametyrosine kinase upstream of rho, that is activated by LPA. Sincephosphatase inhibition leads to kinase activation even inquiescent cells, it seems likely that a tyrosine kinase/phospho-tyrosine phosphatase cycle is being maintained in a low basalactive state. This nicely explains the observation that inhibi-tion of rho in quiescent cells, by microinjection of C3 trans-ferase, is not without effect; cells round up and begin to detachfrom the substrate. We propose therefore, that activation of therho GTPase switch is not an all or nothing event, but thatincreasing concentrations of rho can lead to a gradation ofcellular effects.

This work was generously supported by the Cancer ResearchCampaign (UK) and the Medical Research Council (UK) (C.D.N. andA.H). P.H. is an AFRC postdoctoral fellow. We would like to thankShuh Narimiya for suggesting to us the use of wortmannin to look atruffling.

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(Received 5 July 1994 - Accepted 19 September 1994)

Note added in proofActivation of stress fibre formation by vanadate has also beenreported recently by Barry and Critchley (1994) J. Cell Sci.107, 2033-2045.