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The small Cdc42Hs, Rac1 and RhoG delineate Raf- independent pathways that cooperate to transform NIH3T3 cells Pierre Roux, Cécile Gauthier-Rouvière, Sandrine Doucet-Brutin and Philippe Fort

Background: Ras-mediated transformation of mammalian cells has been Address: Institut de Génétique Moléculaire, shown to activate multiple signalling pathways, including those involving C.N.R.S. UMR5535, 1919 route de Mende, F-34293 Montpellier cedex 5, France. mitogen-activated kinases and the small GTPase Rho. Members of the Rho family affect cell morphology by controlling the formation of - Correspondence: Pierre Roux dependent structures: specifically, filopodia are induced by Cdc42Hs, E-mail: [email protected] lamellipodia and ruffles by Rac, and stress fibers by RhoA. In addition, Rho Received: 27 February 1997 GTPases are involved in progression through the G1 phase of the cell cycle, Revised: 4 June 1997 and Rac1 and RhoA have recently been directly implicated in the morphogenic Accepted: 23 June 1997 and mitogenic responses to transformation by oncogenic Ras. In order to 30 July 1997 examine the cross-talk between Ras and Rho , we investigated the Published: effects on focus-forming activity and cell growth of the Rho-family members Current Biology 1997, 7:629–637 Cdc42Hs, Rac1 and RhoG by expressing constitutively active or dominant- http://biomednet.com/elecref/0960982200700629 negative forms in NIH3T3 cells. © Current Biology Ltd ISSN 0960-9822 Results: Expression of Rac1 or RhoG modulated the saturation density to which the cells grew, probably by affecting the level of contact inhibition. Although all three GTPases were required for cell transformation mediated by Ras but not by constitutively active Raf, the selective activation of each GTPase was not sufficient to induce the formation of foci. The coordinated activation of Cdc42Hs, RhoG and Rac1, however, elicited a high focus-forming activity, independent of the mitogen-activated ERK and JNK protein kinase pathways.

Conclusions: Ras-mediated transformation induces extensive changes in cell morphology which require the activity of members of the Rho family of GTPases. Our data show that the pattern of coordinated Rho family activation that elicits a focus-forming activity in NIH3T3 cells is distinct from the regulatory cascade that has been proposed for the control of actin-dependent structures in Swiss 3T3 cells.

Background cells, Cdc42Hs regulates the formation of filopodia, Rac In mammalian cells, the expression of oncogenic Ras pro- the formation of membrane ruffles and lamellipodia, and teins has been shown to activate mitogenesis, as well as to Rho the formation of focal adhesion plaques and actin cause extensive changes in cell morphology. Activation of stress fibres [10–15]. More recently, Rho family members mitogenesis relies on the pathways involving Raf, Mek have also been shown to be essential for cell growth: and the extracellular signal regulated kinases (ERKs, also RhoA, Rac1 and Cdc42Hs promote cell cycle progression called mitogen-activated protein or MAP kinases), the through the G1 phase, up to the point of S phase entry individual components of which have also been described [16]. Furthermore, three other Rho family members have as capable of transforming cells [1]. Analysis of Ras been shown to be encoded by activated upon extra- mutants impaired in binding to Raf has revealed that Ras- cellular signal stimulation: Rac2, a hemopoietic lineage- induced transformation requires the activation of the Raf specific GTPase closely related to Rac1 [17]; RhoB, which pathway, and also of two Raf-independent pathways, at is closely related to RhoA and encoded by an immediate- least one of which is Rho-dependent [2,3]. early growth response [18]; and RhoG, which is related to Rac1 and Cdc42Hs, and whose mRNA accumu- Members of the Rho family of small GTPases have been lates during the late G1 phase in growth-stimulated implicated in the signal transduction pathways involved in fibroblasts [9]. In addition, Cdc42Hs and Rac1 activate the the control of cell morphology and motile activity. The Jun N-terminal kinase/stress-activated protein kinase Rho family includes Rho (A, B, C), RhoE, RhoL, Rac (1 (JNK/SAPK) [19,20], leading to subsequent gene activa- and 2), RhoG, Cdc42Hs and TC10 [4–9]. In fibroblastic tion driven by the serum response factor (SRF) [21]. 630 Current Biology, Vol 7 No 9

Further evidence for a role of Rho family members in Ras- Figure 1 mediated transformation was provided by the observation that dominant-negative mutants of Rac1, RhoA and RhoB (a) inhibit Ras-induced transformation [22–25]. Activated ver- sions of these proteins have a low transforming potential, eliciting foci that are morphologically distinct from those C1 RhoG-V12RhoG-N1720x RhoG-N17200xC2 RhoG-N17Cdc42Hs-V12Cdc42Hs-N17Rac1-V12Rac1-N17C3 kDa induced by Ras and are formed of aggregates of non- 36 refractile, piled-up cells. Foci of similar morphology are 30 Cdc42Hs RhoG Rac1 also produced — at a higher frequency — by the onco- 16 genic versions of guanine exchange factors (GEFs), such Relative intensities: 1 4 11 as Vav or Dbl, that act on Rho family members [26]. In the present study, we investigated the effects of RhoG (b) 10000 protein, containing either an activating or an inhibitory mutation, on cell proliferation and on Ras-mediated and ) RhoG-V12 –3 1000 Raf-mediated transformation, as well as the relationships NIH3T3 10

RhoG-N17 20x RhoG-N17 of RhoG with Rac1 and Cdc42Hs. 200x RhoG-N17 100 Results Activation of RhoG and Rac1 but not Cdc42Hs increases 10 cell saturation density Cell number (x It was recently reported that cells expressing constitu- 1 tively active Rac1, which contains a valine substitution at 0123456 position 12 (Rac1-V12), grew at a higher saturation Time (days) (c) density than normal cells as a result of a partial loss of 15 contact inhibition [25]. In order to compare the effects of

RhoG, Rac and Cdc42Hs, we produced retroviruses ) directing the expression of activated (V12) and inhibitory –5 10 10 (N17) RhoG proteins tagged with the hemagglutinin epitope (HA), and Rac1 and Cdc42Hs proteins tagged with the Myc epitope, and used them to infect NIH3T3 5 cells. The specificity of the N17 dominant-negative forms of Rac1 and Cdc42Hs was previously demonstrated by Cell number (x their effects on the Rac-dependent membrane ruffling 0 induced by epidermal growth factor (EGF), the Cdc42Hs- dependent induction of filopodia by bradykinin, and the NIH3T3 RhoA-dependent stress fiber formation induced by RhoG-V12Rac1-V12 RhoG-N17Rac1-N17 lysophosphatidic acid (LPA). Using a similar approach, we Cdc42Hs-V12 Cdc42Hs-N17 showed that expression of RhoG-N17 did not impair the Changes in cell saturation density upon expression of RhoG, Rac1 or formation of ruffles, filopodia or stress fibers (data not Cdc42Hs. (a) NIH3T3 cells (105) were infected with 105 colony forming shown). An extensive study of the actin structure and units (cfu) pLXSN wild-type particles (C1, C2 and C3) or particles expressing HA-tagged RhoG-V12 or RhoG-N17, or Myc-tagged morphology of fibroblasts expressing activated and domi- Cdc42Hs-V12, Cdc42Hs-N17, Rac1-V12 or Rac1-N17. Total proteins nant-negative RhoG will be described elsewhere (C.G-R., from infected cells (40 µg) were analysed by gel electrophoresis unpublished observations). followed by western blotting with 12CA5 anti-HA and 9E10 anti-Myc monoclonal antibodies. The increase in RhoG-N17 protein expression in cells infected with 2 × 106 cfu (20x RhoG-N17) and 2 × 107 cfu (200x Expression of the retrovirally expressed proteins was mon- RhoG-N17) is shown. Signals, quantified with the Intelligent Quantifier itored by western blotting (Figure 1a). Expression of software (Millipore), are indicated below the lanes, and size markers are RhoG-V12 led to an overall increased proliferation of the indicated on the left. (b,c) Infected cells were seeded in 6-well dishes at infected cells (Figure 1b). Time-course analysis indicated 5 × 103 cells per well. Cell number was determined after trypsinization. that the infected cells displayed a slightly longer doubling (b) Growth kinetics of RhoG-expressing NIH3T3 cells. Results are the mean of four independent experiments; SEMs (all lower than 5%) are not time but grew at a higher saturation density (60% shown. (c) Cells expressing RhoG, Rac1 or Cdc42Hs were grown to increase) than NIH3T3 control cells infected with wild- saturation, corresponding to day 5 in (b). The means and SEMs type pLXSN particles (Table 1). Conversely, cells calculated for six independent experiments are shown. expressing RhoG-N17 had an increased doubling time during the first day, then grew at the same rate as control higher viral particle concentrations for the infection cells, and stopped growing at a 45% lower saturation elicited a 4-fold and 11-fold increase in RhoG-N17 expres- density (Figure 1b). The use of 20-fold and 200-fold sion (Figure 1a), which led to a further 10% and 50% Research Paper Cooperative transformation by RhoG, Rac1 and Cdc42Hs Roux et al. 631

Table 1 Figure 2

Saturation densities of NIH3T3 cells expressing active or dominant-negative GTPases. 450 Saturation density (cell number on day 5) 400 Cells Mean (× 10–3) SEM (× 10–3) 350 Control 779.5 7.5 300 RhoG-V12 1121.9 6.0 Rac1-V12 1319.6 17.6 250 Cdc42Hs-V12 806.2 3.9 200 RhoG-N17 (1×) 301.4 1.3 × RhoG-N17 (20 ) 273.2 1.8 150

RhoG-N17 (200×) 230.0 1.6 Relative number of foci (%) Rac1-N17 (1×) 393.1 1.4 100 Cdc42Hs-N17 (1×) 406.1 3.8 50 Infected cells were seeded in 6-well dishes at 5 × 103 cells per well and grown for 5 days to saturation. The means and SEMs were 0 calculated from six independent experiments. g) g) g) ng) ng) ng) ng) ng) ng) ng) ng) ng) ng) µ µ µ

Control reduction of the saturation density, respectively (Figure 1b). Identical results were observed using NIH3T3 cells Ras-V12 (2 Rac1-N17 (1

stably transfected with plasmid DNA directing the expres- RhoG-N17 (1 Rac1-V12 (50 RhoG-V12 (50 Rac1-V12 (500 Rac1-V12 (500 RhoG-V12 (500 sion of the same mutant RhoG proteins, indicating that the RhoG-V12 (500 Cdc42Hs-N17 (1 Cdc42Hs-V12 (50 observed effects were independent of the expression pro- Cdc42Hs-V12 (500 Cdc42Hs-V12 (500 cedure. Expression of Rac1-V12 led to a 90% increase in + Ras-V12 (2 ng) the cell density at the plateau, whereas expression of Rac1-N17 led to a 40–45% drop in cell density (Figure 1c), Effects of activated and dominant-negative forms of RhoG, Rac1 and in agreement with previously published data [25]. In con- Cdc42Hs on Ras-V12 focus-forming activity. NIH 3T3 cells (105) were trast, Cdc42Hs-V12 expression led to a very limited effect either transfected with 500 ng vector expressing RhoG-V12, (less than 10%), whereas a 40–45% drop in cell density was Cdc42Hs-V12 or Rac1-V12, or cotransfected with 1µg DNA containing observed upon Cdc42Hs-N17 expression, which is within 2 ng pECEG12V-ras (encoding Ras-V12) and 50 ng or 500 ng of construct expressing RhoG-V12, Cdc42Hs-V12 or Rac1-V12, or 1µg the range of the effects observed using RhoG and Rac1. plasmid DNA expressing the dominant-negative forms. Empty pCDNA3 was used as a negative control. After the cells reached confluence, each Focus formation induced by Ras-V12 requires and is culture was split into four dishes and the concentration of fetal calf potentiated by RhoG, Rac1 and Cdc42Hs activity serum was reduced from 10% to 4%. The number of foci was scored 14 days later, and is expressed as a proportion of the foci induced by We next examined the role of RhoG, Rac1 and Cdc42Hs Ras-V12 (100%). The mean of two independent assays done in in focus formation by NIH3T3 cells, using transfection triplicate is shown; error bars indicate the SEM. and retroviral infection procedures. Rac activity has previ- ously been shown to be insufficient to promote focus for- mation but necessary for Ras-induced transformation RhoG-V12 and Cdc42Hs-V12. On the other hand, co- [23,25]. We therefore investigated whether RhoG and expression of the N17 dominant-negative mutant forms Cdc42Hs had similar properties. Transfected Ras-V12 led to a 50% (RhoG), 60% (Cdc42Hs) or 80% (Rac1) alone produced approximately 5 × 103 transformed foci per decrease in the number of foci, indicating that the activity µg of transfected DNA (corresponding to 100% in Figure of each GTPase is required for focus formation mediated 2), which is within the range of previously published data by Ras-V12. The values obtained for both Rac1 mutants [23,25,27]. RhoG-V12, Rac1-V12 and Cdc42Hs-V12 had are within the range of published results [23,25]. very little intrinsic focus-forming activity, if any (less than four transformed foci per µg transfected DNA). Cotrans- Cdc42Hs, RhoG and Rac1 cooperate in focus formation fection of 2 ng of vector encoding Ras-V12 with vectors The preceding results suggested that RhoG, Rac1 and encoding the activated versions of RhoG, Rac1 or Cdc42Hs might mediate several Ras-V12-dependent Cdc42Hs (50 ng or 500 ng) led to a two-fold, four-fold and pathways that lead to focus formation. This notion is three-fold increase in the number of foci, respectively, supported by the oncogenic potential of many GEFs that indicating a cooperative effect of each Rho GTPase with activate several members of the Rho family [15,28–32]. In Ras-V12. The cooperation was particularly efficient with order to determine whether RhoG, Rac1 and Cdc42Hs Rac1-V12, which required a DNA input 10-fold lower than might activate distinct cooperative pathways, we first 632 Current Biology, Vol 7 No 9

Figure 3 which decrease contact inhibition, had the lowest focus- forming activity, suggesting that these GTPases control some pathways in common. To address this latter issue, we coexpressed each pair of activated GTPases with the 100 dominant-negative version of the third one (grey boxes, Figure 3). Expression of Rac-N17 led to a significant inhi- 80 bition (up to 80%) of the focus-forming activity of Cdc42Hs-V12 plus RhoG-V12, whereas RhoG-N17 only partially reduced the number of foci elicited by Rac-V12 60 plus Cdc42Hs-V12. Although these data might fit several regulatory schemes, they rather suggest that the endoge- nous Rac1 protein might control an essential step down- 40 stream of RhoG-V12 and Cdc42Hs-V12. In contrast,

Relative number of foci (%) expression of Cdc42Hs-N17 did not reduce the focus- 20 forming activity of Rac1-V12 plus RhoG-V12, indicating that the action of these two GTPases is independent of endogenous Cdc42Hs. 0 + ––+ + + + + ––RhoG-V12 (50 ng) –+ –+ + + ––+ + Rac1-V12 (50 ng) Focus formation mediated by Cdc42Hs, RhoG and Rac1 Control ––+ + ––+ + + + Cdc42Hs-V12 (50 ng) uses pathways distinct from the Raf/Mek/ERK pathway –––––––––+ RhoG-N17 (1µg) Activation of the ERK pathway by the translocation and Ras-V12 (25 ng) µ –––––––+ ––Rac1-N17 (1 g) phosphorylation of cellular Raf-1 has long been known to –––––+ ––––Cdc42Hs-N17 (1µg) be necessary for cell transformation mediated by Ras-V12 Cdc42Hs, RhoG and Rac1 cooperate in focus formation. NIH3T3 [33,34]. We thus investigated the relationships between cells (105) were transfected with 1 µg DNA containing either empty the ERK pathway and the pathways controlled by pECE vector as a negative control, 25 ng pECEG12V-Ras (encoding Cdc42Hs, RhoG and Rac1. We first examined the effect Ras-V12) as a positive control, or various combinations of constructs of the activated and inhibitory forms of RhoG, Rac1 and expressing the V12-activated Rho GTPases (50 ng) and the N17- Cdc42Hs on focus formation in NIH3T3 cells expressing dominant-negative versions (1 µg), as indicated. Focus formation assays were performed as described in Figure 2 and the number of v-Raf, the viral oncogenic form of cellular Raf-1. The foci per µg is expressed as a proportion of those induced by 25 ng of average number of foci produced by v-Raf alone was about pECEG12V-Ras. Error bars indicate the SEM of two independent 102 per µg transfected DNA (indicated as 100% in Figure experiments done in triplicate. 4a), which corresponds to 2–5% of the number obtained with Ras-V12 (see Figure 3). Coexpression of v-Raf and the inhibitory forms of RhoG, Rac1 and Cdc42Hs did not performed focus formation assays on NIH3T3 cells con- reduce the number of foci. Conversely, coexpression of comitantly expressing the three activated RhoG, Rac1 and v-Raf with Rac1-V12 and RhoG-V12 elicited a three-fold Cdc42Hs mutants (Figure 3). For these assays, 50 ng to eight-fold increase in the foci number. The increase expression plasmid was used, a DNA input at which very observed for Rac1 is within the range of previously pub- low or no focus-forming activity was observed for each con- lished data [25]. RhoG requires a higher DNA input for struct (see Figure 2). Coexpression of all three Rho cooperating with Ras-V12 and v-Raf, a feature that might GTPases led to an average of ~103 foci per µg of input reflect a difference in protein level or activity. Interest- DNA — that is, about 20% of Ras-V12 activity and more ingly, coexpression of Cdc42Hs-N17 or Cdc42Hs-V12 had than two orders of magnitude higher than the focus-forming no effect on Raf transformation, a finding which further activity elicited by each GTPase alone. This prompted us strengthens the notion that Cdc42Hs controls pathways to examine the effects of pairwise combinations. distinct from RhoG and Rac1.

Using the same amounts of each DNA (50 ng), coexpres- Although the endogenous activity of RhoG, Rac1 and sion of RhoG-V12 and Rac1-V12, RhoG-V12 and Cdc42Hs GTPases is not crucial for transformation medi- Cdc42Hs-V12, and Rac1-V12 and Cdc42Hs-V12 led to an ated by v-Raf, each of them is necessary for transformation average of 35, 80 and 480 foci per µg input DNA, respec- mediated by Ras-V12, suggesting that signal transduction tively. Cdc42Hs-V12 with Rac1-V12 therefore represents from Ras to Raf might be affected by Rho-dependent the most efficient combination (giving up to 10% of Ras- pathways. To address this issue, we examined the effects V12 focus-forming activity), suggesting that the two of RhoG, Rac1 and Cdc42Hs on the activity of the MAP GTPases control cooperative pathways, in agreement with kinase ERK2, activation of which is triggered by Raf. their differential effects on contact inhibition. Conversely, Coexpression of HA-tagged forms of ERK2 and the the combination of RhoG-V12 and Rac1-V12, both of mutated forms of either RhoG, Rac1 or Cdc42Hs was Research Paper Cooperative transformation by RhoG, Rac1 and Cdc42Hs Roux et al. 633

Figure 4

(a) (b) 800

700 ControlpCDNA3RhoG-N17Rac1-N17Cdc42Hs-N17 Ras-V12:– + + + + 600 MBP 500 Relative intensities: 1 5 13 12 8 400 (c) 300 HA–ERK2

Relative number of foci (%) 200 (d) 100 + – – + + – + RhoG-V12 – + – + – + + Rac1-V12 0

Control Ras-V12 – – + – + + + Cdc42Hs-V12 g) g) g) ng) ng) ng) ng) ng) ng) ng) µ µ µ

MBP Control

v-Raf (100 Relative intensities: 1 6 1 1.5 1.81.5 1.5 1.8 1.9 Rac1-N17 (1

RhoG-N17 (1 (e) Rac1-V12 (50 RhoG-V12 (50 Rac1-V12 (500 RhoG-V12 (500 HA–ERK2 Cdc42Hs-N17 (1 Cdc42Hs-V12 (50 Cdc42Hs-V12 (500

+ v-Raf (100 ng)

Cdc42Hs, RhoG and Rac1 cooperate independently of the Raf/Mek/ RhoG-N17, Rac1-N17 or Cdc42Hs-N17. (b) The ERK2 activities in the ERK2 pathway. (a) NIH3T3 cells (105) were transfected with 100 ng cell lysates were measured by immunocomplex kinase assays using pECEG-v-raf (encoding v-Raf), either alone or in combination with myelin basic protein (MBP) as a substrate, and the extent of the vectors expressing the V12-activated Rho GTPases (50 ng or 500 ng) stimulation was quantified with a phosphorimager and is expressed or the N17-dominant-negative versions (1µg). As a control, cells relative to the control cells. (c) HA–ERK2 expression in transfected cells transfected with empty pECE vector were used. Focus formation was was controlled by western blot analysis using an anti-HA antibody. (d,e) assayed as in Figure 2; error bars indicate the SEMs of two independent NIH3T3 cells were cotransfected with 1 ␮g total plasmid DNA, a experiments done in triplicate. (b,c) NIH3T3 cells were either transfected combination of pECEHAp44MAPK and either the empty vector (control) only with a vector directing the expression of HA–ERK2 (control) or or constructs expressing the indicated GTPases. (d) ERK2 activity and cotransfected with the HA–ERK2 vector, pECEG12V-ras (encoding (e) ERK2 expression were measured as in (b,c). The data shown in (b–e) Ras-V12) and either empty vector (pCDNA3) or constructs expressing are representative of two independent experiments. achieved by transient cotransfection of NIH3T3 cells, and RhoG-V12 and Rac1-V12 with v-Raf is not mediated by an kinase activity was assayed after immunoprecipitation increase in ERK2 activity. Besides, coexpression of any using an anti-HA antibody. As observed in Figure 4b, combination of the V12-GTPases failed to activate ERK2, expression of the N17-mutated GTPases did not reduced indicating that the high focus-forming activity elicited by Ras-V12-mediated ERK2 activation, demonstrating that the coordinate activation of Cdc42Hs, RhoG and Rac1 is the inhibitory effect of the N17 GTPases is not correlated independent of ERK2 activation. Similarly, no change in with an impairment of Raf activation. Instead, we ERK2 activity was detected in foci generated from the observed a two-fold to three-fold induction of the activity combination of RhoG-V12, Rac1-V12 and Cdc42Hs-V12 of the exogenous ERK2 upon expression of Rac1-N17 and (data not shown). RhoG-N17, suggesting that inhibition of either of the GTPases might increase the Ras/Raf signalling or Constitutively active RhoG, Rac1 and Cdc42Hs do not decrease its downregulation. cooperate to activate the JNK pathway Activation of Rac1 and Cdc42Hs has been previously rep- We next investigated the effect of the activated GTPases orted to activate the JNK pathway but to have no effect on on ERK2 activation (Figure 4c). Expression of any of the the ERK2 pathway [19,20,35]. In order to determine three GTPases had no significant effect on ERK2 activa- whether the focus formation controlled by the interaction tion in resting cells. Thus, the cooperative effect of of Cdc42Hs and RhoG or Rac1 could be associated with a 634 Current Biology, Vol 7 No 9

Figure 5

+ – – + + – + RhoG-V12

ControlpCDNA3RhoG-N17Rac1-N17Cdc42Hs-N17 – + – + – + + Rac1-V12

(a)Ras-V12:– + + + + (c) Control Ras-V12 – – + – + + + Cdc42Hs-V12 GST–c-Jun GST–c-Jun

Relative intensities: 1 3 1 1 1 Relative intensities: 1 5 8 6 17 9 17 20 18 (b) (d) HA–JNK HA–JNK

Cdc42Hs, RhoG and Rac1 cooperate independently of the JNK/SAPK of the stimulation was quantified using phosphorimager screens and pathway. (a,b) NIH3T3 cells were cotransfected with 1 µg total expressed as a multiple of the signal from control cells. (b) HA–JNK plasmid DNA containing pSRα.3HA.JNK1 (encoding HA–JNK), expression in transfected cells was analysed by western blot using an pECEG12V-ras (encoding Ras-V12) and either empty pCDNA3 or anti-HA antibody. (c,d) NIH3T3 cells were cotransfected with constructs expressing the indicated N17-mutated GTPases. As a pSRα.3HA.JNK1 and either empty pCDNA3 (control), pECEG12V-ras, control, cells were transfected with DNA containing pSRα.3HA.JNK1 or different combinations of constructs expressing the V12-mutated only. (a) JNK activities in the cell lysates were measured by GTPases, as indicated. (c) JNK activity and (d) the level of JNK immunocomplex kinase assays using, as a substrate, a fusion protein expression were analysed as for (a,b). Data shown in (a–d) are of glutathione-S-transferase and cellular Jun (GST–c-Jun). The extent representative of two experiments.

quantitative change in JNK activity, we performed the progression from G0 through to the G1 phase [16] and experiments presented in Figure 5. We first checked modulate transcriptional activity mediated by SRF [21]. whether Ras-V12-mediated JNK activation was depen- In addition, Rac1 and Cdc42Hs activate the JNK dent on RhoG, Rac1 and Cdc42Hs activity. NIH3T3 cells pathway, inducing changes in the pattern of gene tran- were transiently cotransfected to express HA-tagged ver- scription [19,20,35]. Second, the members of the Dbl sions of JNK and each of the inhibitory versions of RhoG, family of oncogenes all contain a common domain that Rac1 or Cdc42Hs. As shown in Figure 5a, expression of stimulates GDP–GTP exchange for Rho GTPases and is either one of the mutated GTPases prevented the Ras- necessary for the oncogenic properties of the Dbl family mediated JNK activation, as has been described previ- [15,29]. And third, Ras-induced, but not Raf-induced, ously for Rac1 and Cdc42Hs [19,20]. We next analysed transformation is blocked by the expression of dominant- the effect of expressing the activated GTPases, either negative mutants of Rac1, RhoA and RhoB, whereas alone or in combination (Figure 5b). Cdc42Hs-V12 and expression of constitutively active Rac1 or RhoA acts syn- Rac1-V12 led to a 17-fold and a 6-fold increase in JNK ergistically with activated Raf to transform cells [22–25]. activity, respectively, in agreement with previous reports In this study, we investigated the contributions of [19,20]. Expression of RhoG-V12 led to an 8-fold Cdc42Hs and RhoG to the control of cell proliferation and increase, which is within the range of that elicited by transformation, as well as their regulatory cross-talks with Rac1-V12. When different combinations were used, Rac1, Ras and Raf. however, the effects on JNK activity were not additive: the resulting JNK activity was identical to that induced Our data showed that activation of RhoG led to a modula- by the most potent GTPase used in the combination. tion of cell saturation density, as has previously been This indicates that no major change in JNK activation is shown for Rac1 [25]. This might account for the low co- associated with the focus-forming activity elicited by the operativity in focus formation displayed by these two association of Cdc42Hs, Rac1 and RhoG. Similar levels of GTPases when expressed together, and might suggest JNK activity were observed in cells derived from foci gen- that they are involved in a common pathway controlling erated from the coexpression of the three V12-GTPases cell contact inhibition. RhoG might act usptream of Rac1, (data not shown). as expression of RhoG-N17 has little effect on the focus- forming activity of Rac1/Cdc42Hs, whereas Rac1-N17 Discussion inhibits the focus-forming activity of RhoG/Cdc42Hs. Several reports have implicated Rho family members in This suggestion agrees well with our unpublished results the processes of cell proliferation and transformation. showing that RhoG requires endogenous Rac1 activity to First, Rac1, Cdc42Hs and RhoA are required for cell cycle elicit ruffling in Swiss 3T3 cells (C.G-R., unpublished Research Paper Cooperative transformation by RhoG, Rac1 and Cdc42Hs Roux et al. 635

observations). The case for Rac1 being downstream of is required, in addition to Rho pathways, to produce a fully RhoG is further strengthened by the observation that transformed phenotype. dominant-negative Rac1 is the most potent inhibitor of Ras-V12-mediated transformation. The failure of RhoG Conclusions and Rac1 to elicit focus formation is consistent with the Our present analysis shows that coordinated activation of results of Tiam1 characterizations. Tiam1 is an exchange the Cdc42Hs, Rac1 and RhoG GTPases elicited a high factor for Rac1, and oncogenic versions of Tiam1 elicit focus-forming activity in NIH3T3 cells, whereas expres- tumours with a high invasive and metastatic potential sion of each of them alone failed to induce foci. This [15]. Tiam1 was shown to induce relatively dense areas of effect of coordinated activation cannot be explained by epithelium-like cells that grew as monolayers [30], indicat- the regulatory linear cascade proposed for actin-depen- ing that although Rac1 activation leads to a reduction in dent cytoskeletal reorganisation in Swiss 3T3 cells, in contact inhibition, additional signals are required for the which Cdc42Hs activation was shown to activate Rac1 formation of multilayers by NIH3T3 cells. Such signals which in turn activates RhoA [10–13,16]. We propose that might be mediated through Cdc42Hs, which delineates a additional pathways, independent of the ERK2 and JNK distinct pathway, as it has no effect on saturation density kinases, are involved in the process of focus formation. and promotes focus-forming activity when coexpressed One pathway modulates cell contact inhibition and is gov- with Rac1, reaching 10% of the activity of Ras-V12. A erned by Rac1 and, to a lesser extent, RhoG. A second third pathway might be controlled by RhoA, which was pathway, acting independently, is controlled by Cdc42Hs. previously reported to be involved in Ras-mediated trans- Detailed analysis of the morphological changes induced in formation: although it has a very low transforming poten- the ‘Rho-like’ foci, as well as knowledge of the cross-talks tial on its own, RhoA strongly cooperates with weakly between RhoA and the RhoG/Rac1/Cdc42Hs GTPases, transforming Raf mutants, and expression of dominant- are now required, in order to determine which additional negative RhoA-N19 reverts Ras-mediated transformation pathways are involved and what impact they have on the [23,25]. Cooperativity between Rho GTPases in focus for- process of focus formation. mation is in keeping with the densely packed morphology of foci induced by exchange factors such as Dbl, Vav, Ect2 Materials and methods or Ost, which might act simultaneously on distinct DNA constructs and site directed mutagenesis GTPases [26,28,32,36]. The wild-type RhoG open reading frame (ORF) was amplified using the polymerase chain reaction (PCR) and the amplimers 5′-dCG- GAATTCATGCAGAGCATCAAGTG-3′ and 5′-dGCTCTAGAGGTCA- Interestingly, the focus-forming activity elicited by the CAAGAGGATGCA-3′; PCR products were cloned into the EcoR1 association of RhoG, Rac1 and Cdc42Hs uses pathways and Xba1 sites of pT7T318U (Pharmacia). Mutated ORFs (Gly12→Val independent of the ERK2 and JNK MAP kinases. JNK and Thr17→Asn) were generated by site-directed mutagenesis of uridylated phagemid single-stranded DNA, as described by Wang et al. activity is enhanced in cells expressing Cdc42Hs-V12 or [40] using the oligonucleotides 5′-dCTTGCCCACAGCGACGTCAC- Rac1-V12, as previously reported [16,19,20], and we have CCACCACp-3′ (Gly12→Val), 5′-dGCAGGCAATTCTTGCCCACAG- shown that this is also the case for RhoG-V12. However, CGCCGTCACCp-3′ (Thr17→Asn) and 5’-dCGAACACGGCCGGG- no increase in JNK activity was detected in cells express- ATGTACTCp-3′ (Thr35→Ala). The presence of mutations was ing any focus-forming combination of GTPases when com- checked by DNA sequencing. The mutated ORFs were subcloned into the EcoRI and XbaI sites of pCDFT [41], a derivative of pCDNA3 allow- pared to cells expressing only Cdc42Hs, the most potent ing expression of HA-tagged proteins, to give pCDNA-rhoG-V12, and JNK activator. This is in agreement with the finding that pCDNA-rhoG-N17. JNK activation is not required for Rac-induced prolifera- tion, for G1 cell cycle progression induced by Rac or To express HA-tagged RhoG proteins by retroviral infection, the oligonucleotide 5′-dAATTGCCACCATGTATGATGTTCCTGATTATG- Cdc42, or for actin polymerization [37,38]. Ras-dependent CTAGCCTCGAATTCAAC-3′ encoding the HA epitope was inserted transformation was shown to be impaired upon inhibition into pLXSN, a MoMuLV-derived vector expressing resistance to the of the activity of ERK1 or ERK2 [23] or Mek [1,39]. Nev- drug G418, giving pLXSN-HA. RhoG mutated ORFs were then sub- ertheless, a doubly mutated Ras (Glu12→Val, cloned into the EcoRI and SalI sites of pLXSN-HA, to give pLXSN- → RhoG-V12 and pLXSN-RhoG-N17. The ORFs encoding Myc-epitope Thr35 Ser) was reported to be defective for its transform- tagged mutated versions of Rac1 and Cdc42Hs were excised from ing properties, although it could nevertheless activate the pMT90-derived constructs (kindly provided by Philippe Chavrier, CIML, ERK cascade and stimulate SRE-dependent gene tran- Marseille) and subcloned into a pLXSN-derived vector, containing a scription [2]. Our own data show that inhibition of NotI site instead of its HpaI site, to give pLXSN-Cdc42HS-V12, Cdc42Hs, Rac1 or RhoG decreases Ras-mediated transfor- pLXSN-Cdc42HS-N17, pLXSN-Rac1-V12 and pLXSN-Rac1-N17. mation without any inhibition of ERK2 activation. Con- Cell culture and retroviral particle production versely, the coordinated activation of Rac1, RhoG and NIH3T3 and virus producer GP+E-86 were cultured in 4.5 g/l Dulbec- Cdc42Hs gives a high focus-forming activity without any co’s Modified Eagle’s Medium (DMEM) containing 10% fetal calf serum change in ERK2 activation. The resulting foci have a mor- (FCS; Bio Media) at 37°C in a humidified atmosphere (95% air; 5% CO2). Transfections into GP+E-86 cells were performed using the Lipo- phology distinct from Ras-V12-transformed cells, however, fectamin method, as recommended by the supplier (Gibco-BRL). suggesting that activation of the Raf/Mek/ERK2 pathway Neomycin-resistant clones were selected with 600µg/ml G418 and then 636 Current Biology, Vol 7 No 9

pooled. Ecotropic retroviral particles were prepared by harvesting 10 ml 3. Khosravi-Far R, White MA, Westwick JK, Solski PA, Chrzanowska- normal medium, applied 18 h previously, from a confluent monolayer of a Wodnicka M, Van Aelst L, et al.: Oncogenic Ras activation of 100 mm dish of GP+E-86 virus-producing cells and then filtering the Raf/mitogen-activated protein kinase- independent pathways is medium through a 0.45 µm filter (Millipore). To estimate retroviral titers, sufficient to cause tumorigenic transformation. Mol Cell Biol 1996, 16:3923-3933. NIH3T3 cells were infected by incubating a 100 mm plate containing 4. Symons M: Rho family GTPases: the and beyond. × 5 5 10 cells with 2 ml viral supernatant, or a dilution thereof, in the pres- Trends Biochem Sci 1996, 21:178-181. µ ence of 5 g/ml Polybrene (Aldrich) for 5 h. Medium (8 ml) was added 5. Marshall CJ: Ras effectors. 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J Cell Biol 1996, 133:617-630. the mutated versions of RhoG, Rac and Cdc42Hs, using lipofection (8µl 9. Vincent S, Jeanteur P, Fort P: Growth-regulated expression of Lipofectamin in 800 ␮l OptiMEM (Gibco-BRL) for 5 h) on 6 cm dishes. RhoG, a new member of the Ras homolog gene family. Mol Cell Alternatively, cells were first transfected then, 24 h later, infected Biol 1992, 12:3138-3148. overnight with retroviral particles (2× 105 cfu) expressing the different 10. Nobes CD, Hawkins P, Stephens L, Hall A: Activation of the small mutant forms of RhoG, Rac1 and Cdc42Hs in the presence of 5␮g/ml GTP-binding proteins Rho and Rac by growth factor receptors. J Polybrene. After cells reached confluency, each 6 cm dish was split into Cell Sci 1995, 108:225-233. 11. Nobes CD, Hall A: Rho, Rac, and Cdc42 GTPases regulate the four 6 cm diameter dishes and the FCS concentration was reduced from assembly of multimolecular focal complexes associated with 10% to 4%. Medium was changed every 2.5 days. 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