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Faciogenital dysplasia (FGD1) and Vav, two related required for normal embryonic development, are upstream regulators of Rho GTPases Michael F. Olson*†, N. German Pasteris‡, Jerome L. Gorski‡ and Alan Hall*

Background: Dbl, a guanine nucleotide exchange factor (GEF) for members of Addresses: *CRC Oncogene and Signal the Rho family of small GTPases, is the prototype of a family of 15 related Transduction Group, MRC Laboratory for Molecular Cell Biology and Department of proteins. The majority of proteins that contain a DH (Dbl ) domain were Biochemistry, University College London, Gower isolated as oncogenes in transfection assays, but two members of the DH family, Street, London WC1E 6BT, UK. ‡Department of FGD1 (the product of the faciogenital dysplasia or Aarskog–Scott syndrome Human Genetics and Department of Pediatrics, locus) and Vav, have been shown to be essential for normal embryonic University of Michigan, Ann Arbor, Michigan development. Mutations to the FGD1 result in a human developmental 48109-0688, USA. disorder affecting specific skeletal structures, including elements of the face, †Present address: Chester Beatty Laboratories, cervical vertebrae and distal extremities. Homozygous Vav–/– knockout mice Institute of Cancer Research, Fulham Road, embryos are not viable past the blastocyst stage, indicating an essential role of London SW3 6JB, UK. Vav in embryonic implantation. Correspondence: Alan Hall E-mail: [email protected] Results: Here, we show that the microinjection of FGD1 and Vav into Swiss 3T3 fibroblasts induces the polymerization of actin and the assembly of clustered Received: 6 August 1996 integrin complexes. FGD1 activates Cdc42, whereas Vav activates Rho, Rac and Revised: 6 September 1996 Accepted: 30 September 1996 Cdc42. In addition, FGD1 and Vav stimulate the mitogen activated protein kinase cascade that leads to activation of the c-Jun kinase SAPK/JNK1. Current Biology 1996, Vol 6 No 12:1628–1633

Conclusions: We conclude that FGD1 and Vav are regulators of the Rho © Current Biology Ltd ISSN 0960-9822 GTPase family. Along with their target proteins Cdc42, Rac and Rho, FGD1 and Vav control essential signals required during embryonic development.

Background known as the Jun N-terminal protein kinase or JNK) The Rho GTPase family of small molecular weight GTP- [4,7–10]. In addition, Rho, Rac and Cdc42 have been shown binding proteins control important aspects of cell shape, to be involved in mediating the activity of the serum adhesion, movement and growth. Rho, Rac and Cdc42 response factor (SRF) signalling pathway, which links a control three distinct signal transduction pathways in variety of stimuli to the transcriptional activation of Swiss 3T3 fibroblast cells: Rho regulates the assembly of that contain the serum response element (SRE) [11]. actin stress fibres and focal adhesion complexes; Rac con- trols the formation of lamellipodia and membrane ruffles Small molecular weight GTPases are switched on by by stimulating peripheral actin polymerization and the guanine nucleotide exchange factors (GEFs), which convey assembly of small integrin complexes; and Cdc42 induces information from upstream by promoting the exchange of actin-rich filopodial extensions, which are also associated GDP for GTP. In the case of Rho GTPases, it is believed with clustered integrin complexes [1–3]. that the Dbl homology (DH) protein family are the cellular Rho GEFs; indeed, a number of DH proteins have been In addition to their roles in regulating the actin shown to act as GEFs in vitro [12]. The DH family com- cytoskeleton, it has been demonstrated recently that Rho prises a number of proteins that share two conserved GTPases are involved in the regulation of cell growth. The domains — a DH domain of approximately 250 amino microinjection of activated forms of Rho, Rac and Cdc42 acids, and a pleckstrin homology (PH) domain of approxi- into quiescent fibroblasts stimulates progression through mately 100 amino acids. Two members of the DH protein the cell cycle, leading to new DNA synthesis [4], and all family, FGD1 and Vav, play essential roles in embryonic three GTPases are required for the serum-stimulated mito- development: FGD1 was determined by positional cloning genic response of fibroblasts [4–6]. One mechanism to be the genetic locus responsible for faciogenital dysplasia whereby the Rho GTPases could control growth is by their or Aarskog–Scott syndrome, a multisystemic developmental newly described function as regulators of RNA transcrip- disease involving the skeletal and urogenital systems [13]; tion. Cdc42 and Rac, but not Rho, regulate a mitogen-acti- and homozygous Vav–/– knockout mice embryos could not vated protein (MAP) kinase cascade that leads to the be detected at mid-gestation stages E7.5 to E12.5, indicat- activation of the stress-activated protein kinase (SAPK, also ing the importance of Vav for normal embryogenesis [14]. Research Paper Regulation of Rho GTPases by FGD1 and Vav Olson et al. 1629

In this study, we sought to determine whether FGD1 and blocks all Lbc-induced effects [15]. The effects of injecting Vav could activate Rho GTPases in vivo. By microinjecting pRK5-Dbl or pRK5-Vav were indistinguishable from each plasmids encoding epitope-tagged versions of DH proteins other (Fig. 2): cells had prominent actin stress fibres and into Swiss 3T3 fibroblasts and examining the effects on the contained pronounced peripheral lamellipodia. Time-lapse polymerization of actin and the assembly of clustered inte- video recordings showed a dramatic induction of membrane grin complexes, we found that FGD1 activates Cdc42, ruffling by Dbl and Vav (data not shown). We conclude that whereas Vav activates Rho, Rac and Cdc42. FGD1 and Vav Vav, like Dbl, promotes guanine nucleotide exchange on also stimulate the MAP kinase cascade that leads to activa- Rho, and that both are potent activators of Rac — although tion of the c-Jun kinase SAPK/JNK1, but do not stimulate the mechanism could be direct, or indirect via Cdc42 [3]. the Ras-regulated MAP kinase cascade that leads to ERK2 activation. These results are consistent with the actions of The injection of cells with pRK5-FGD1 produced a FGD1 and Vav being specific for members of the Rho different and distinct effect from that induced by GTPase family. expression of Lbc, Vav or Dbl, as no stress fibres or overt lamellipodia were observed (Fig. 2). Instead, the injected Results and discussion cells showed large numbers of striking filopodial To determine whether FGD1 or Vav can activate members extensions; we conclude that FGD1 promotes nucleotide of the Rho GTPase family, sequences encoding the adja- exchange on Cdc42, but not Rac or Rho. Closer inspection cent DH and PH domains of the two proteins and an of pRK5-FGD1-injected cells revealed restricted lamel- amino-terminal Myc epitope tag were introduced into a lipodial extensions associated with filopodia, consistent eukaryotic expression vector (pRK5; see Fig. 1). As con- with a localized activation of Rac by Cdc42, as described trols, the corresponding regions of two additional GEFs, previously [3]. Time-lapse video recordings of pRK5- Dbl and Lbc, were introduced into epitope-tagged expres- FGD1-injected cells revealed a rather complicated pattern sion vectors (Fig. 1). (It has been shown, both in vitro and in of behaviour — filopodial extensions grew out of the cell vivo, that Lbc activates only Rho and that, in vitro, Dbl acti- periphery and then either retracted or gradually became vates Rho and Cdc42 and possibly Rac [15–18].) The result- subsumed under a growing lamellipodia, which then ing vectors were microinjected into the nuclei of quiescent detached and ‘ruffled’ back into the cell body. Swiss 3T3 cells. The FGD1 gene has been found to give rise to an additional As reported previously [15], uninjected quiescent cells alternatively spliced transcript in embryonic tissues, which contain very little organized polymerized actin (Fig. 2), but encodes a slightly shorter form of the protein lacking 36 injection of pSR-Lbc induces the formation of a dense amino acids near the amino-terminal end of the DH domain network of actin stress fibres (Fig. 2). Coinjection of pSR- (N.G. Pasteris and J.L. Gorski, unpublished observations). Lbc with the Rho inhibitor C3 transferase completely To determine whether this alternatively spliced form of

Figure 1

Schematic diagram of the DH proteins Dbl, Vav and FGD1, and the eukaryotic expression DH PH 1 925 vectors constructed. For Lbc, the structure of Dbl only onco-Lbc is available. Structural domains pRK5-myc-Dbl 495 826 of the proteins are abbreviated as follows: DH, Dbl homology; PH, pleckstrin homology; Cys, cysteine-rich, zinc finger-like; SH3, Src DH PH homology 3; SH2, Src homology 2; Pro, pSRneo-FLAG-Lbc 1 424 proline-rich. Numbers refer to amino-acid residues. In the text, the vectors are abbreviated: for example, pRK5-myc-Dbl is DH PH Cys SH3 SH2 SH3 denoted as pRK5-Dbl, and pSRneo-FLAG- Vav 1 845 Lbc as pSR-Lbc. pRK5-myc-Vav 145 598

Pro DH PH Cys FGD1 1 961 pRK5-myc-FGD1 391 710 pRK5-myc-FGD1∆ 391 710 397 434 1630 Current Biology 1996, Vol 6 No 12

Figure 2

Lbc, Dbl, Vav and FGD1 promote polymerization of actin in quiescent Swiss 3T3 fibroblasts. Serum-starved Swiss 3T3 cells were microinjected with plasmid DNA encoding epitope-tagged DH proteins, as indicated. Actin cytoskeletal structures in cells expressing the DH protein constructs (data not shown) were visualized with TRITC- conjugated phalloidin. Scale bar in (a) represents 20 ␮m and refers to all panels in all figures.

FGD1 could also affect the actin cytoskeleton, the corre- Cdc42-binding domain of WASP (the protein product of sponding sequences were introduced into the pRK5 expres- the Wiskott–Aldrich syndrome locus). This domain inter- sion vector (pRK5-FGD1⌬, Fig. 1) and injected into cells. acts specifically with the GTP-bound form of Cdc42 Although the product was clearly expressed, no induction of [19,20] and, when injected into cells, inhibits the Cdc42- polymerized actin could be observed, consistent with the mediated signal transduction pathway [21]. Microinjection DH domain being responsible for activation of Cdc42. of WASP markedly attenuated FGD1-induced effects, but had no inhibitory effect on Lbc (Fig. 4). We conclude that In addition to their effects on actin, Rho, Rac and Cdc42 FGD1 specifically promotes guanine nucleotide exchange induce the assembly of discrete integrin complexes that on Cdc42. can be visualized using an anti-vinculin antibody [1,3]. Uninjected cells had very few vinculin-containing com- As both Vav and Dbl produced such dramatic lamellipodial plexes (data not shown), but the injection of pRK5-Lbc spreading compared with FGD1 (Fig. 2), it seemed likely induced numerous focal adhesions, consistent with the that these two proteins were able to activate Rac directly. In activation of Rho (Fig. 3). Dbl- and Vav-expressing cells agreement with this prediction, the co-injection of WASP showed a combination of focal adhesions and Rac- with DNA encoding Dbl or Vav had no significant dependent peripheral focal complexes, whereas FGD1 inhibitory effect on either stress fibre or lamellipodia induced vinculin complexes localized predominantly along formation (Fig. 4); we conclude that Dbl and Vav can acti- filopodial extensions [3]. vate both Rho and Rac directly. In vitro assays have demon- strated that Dbl catalyzes nucleotide exchange on Rho and To confirm that FGD1 acts only upon Cdc42, and to deter- Cdc42, although there have been conflicting reports as to mine whether Vav activates Rac and/or Cdc42, we used the whether it has activity on Rac [17,18]. Although no obvious

Figure 3

Lbc, Dbl, Vav and FGD1 induce the assembly of clustered integrin complexes in quiescent Swiss 3T3 cells. Serum-starved Swiss 3T3 fibroblasts were microinjected with plasmid DNA encoding epitope-tagged DH proteins, as indicated, along with Texas-Red conjugated dextran as an injection marker. Vinculin distribution was visualized with a monoclonal anti-vinculin antibody (VIN-11-5). Research Paper Regulation of Rho GTPases by FGD1 and Vav Olson et al. 1631

Figure 4

Inhibition of Cdc42 and Rac with a fragment of WASP and N17Rac, respectively. Serum- starved Swiss 3T3 fibroblasts were microinjected with plasmid DNA encoding epitope-tagged DH proteins, along with the bacterially expressed Cdc42-binding domain of WASP (residues 201–321) or bacterially expressed N17Rac as indicated. Actin cytoskeletal structures in cells expressing the DH protein constructs (data not shown) were visualized with TRITC-conjugated phalloidin.

filopodia were induced by Dbl, these are difficult to see in cotransfected with DNA encoding Myc-epitope-tagged Swiss 3T3 cells when Rac is strongly activated. To deter- ERK2 along with activated Ras (D12Ras), Dbl, Vav or mine whether Dbl and Vav activate Cdc42 in vivo, a domi- FGD1; we also used a membrane-targeted form of the nant-negative version of Rac (N17Rac) was used. When known Ras exchange factor, Sos. As expected, D12Ras and Dbl or Vav were expressed in cells in the presence of Sos stimulated the kinase activity of immunoprecipitated N17Rac, filopodial extensions were revealed in addition to ERK2, but despite comparable amounts of ERK2 being stress fibres (Fig. 4); in contrast, no filopodial extensions immunoprecipitated (data not shown), Dbl, Vav and FGD1 were observed when N17Rac was microinjected alone (data showed no increase in ERK2 activity (Fig. 5b). These not shown). We conclude, therefore, that Dbl and Vav results indicate that Dbl, Vav and FGD1 specifically acti- stimulate Rho, Rac and Cdc42 independently. vate members of the Rho GTPase family. Consistent with the results presented here, Crespo et al. [31] have recently It has been demonstrated that Cdc42 and Rac, but not reported that expression of Vav in COS-7 cells leads to the Rho, regulate a MAP kinase cascade that leads to the acti- activation of SAPK/JNK1 but not ERK2. However, this vation of SAPK/JNK [4,7–10]. In addition, it has been group reported that SAPK/JNK1 activation was decreased shown that Dbl is capable of stimulating this protein by coexpression of N17Rac but not by co-expression of kinase cascade, consistent with the activation of Rac and/or N17Cdc42, suggesting that Vav stimulates Rac and not Cdc42 [7]. We wished to determine whether FGD1 and Cdc42. Our data show that the effects of Vav and Dbl on Vav could also activate the SAPK/JNK signal transduction cascade. A transient transfection protocol was used in Figure 5 which COS-1 monkey kidney cells were co-transfected with DNA encoding Dbl, Vav or FGD1, together with (a) – MEKK L61Rac DbI Vav FGD1 DNA encoding SAPK/JNK1 tagged with a FLAG epitope (Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys). After 48 hours, GST-c-JUN SAPK/JNK1 was immunoprecipitated and its activity towards the substrate c-Jun determined in an in vitro (b) – D12Ras DbI Vav FGD1 Sos kinase assay. As shown previously, a constitutively acti- vated Cdc42 (L61Cdc42), Dbl and the carboxy-terminal domain of the upstream kinase MEKK each stimulated MBP kinase activity (Fig. 5a). FGD1 and Vav also stimulated SAPK/JNK1 activity. Effect of Dbl, Vav and FGD1 on SAPK/JNK1 and ERK2 MAP kinase Vav has been reported to be an activator of Ras [22–26], pathways. COS-1 cells were co-transfected with epitope-tagged SAPK/JNK1 (a) or ERK2 (b) along with expression vectors for DH although this has not been confirmed by others [27–30]. It proteins and controls as indicated. SAPK/JNK and ERK2 kinases were has also been reported that Vav- and Dbl-transformed cell immunoprecipitated from cell lysates and assayed in vitro for their lines have elevated ERK2 MAP kinase activity [27]. Using ability to phosphorylate GST-c-Jun or myelin basic protein (MBP), COS cell transfections, we tested the ability of Dbl, Vav respectively. Experiments were carried out in triplicate and representative assays are shown. Comparable amounts of SAPK/JNK1 and FGD1 to activate the Ras signal transduction pathway in (a) or ERK2 in (b) were immunoprecipitated (data not shown) leading to ERK2 MAP kinase activation. COS-1 cells were 1632 Current Biology 1996, Vol 6 No 12

Figure 6 Materials and methods DNA constructs Myc-epitope tagged Dbl (residues 495–826), Vav (residues 145–598), Dbl FGD1 (residues 391–710) and FGD1⌬ (residues 391–710, Lbc FGD1 Vav ⌬398–433) were introduced into the cytomegalovirus (CMV) promoter based vector, pRK5 [35], by PCR using the following sets of oligonu- cleotide pairs: Dbl, GGGGGATCCTTAAAGAACCACGTA and GGGGATCCTCATAATTGATCCTGTTG; Vav, GGGGGATCC- CTTTCAGACCAGATT, and GGGAATTCTCACACTTCCATCTTAGG; Cdc42 Rac Rho FGD1 and FGD1⌬, CTGGGATCCTTTCACATTGCCAATGAGCTC, and GCAGGAATTCTAGTCTTCATCTTCCCTGTT. pRK5 was modi- fied by ligating the following annealed oligos into the EcoRI and PstI sites to create a Myc-epitope tag followed by a multiple cloning site: sense oligo, AATTGGCCACCATGGAGCAGAAGCTGATCTCCGAG- Filopodia Lamellipodia Stress Fibres GAGGACCTGGGATCCCGGGTCTAGAATTCCTGCA; antisense SAPK/JNK SAPK/JNK oligo GGAATTCTAGACCCGGGATCCCAGGTCCTCCTCGGA- GATCAGCTTCTGCTCCATGGTGGCC. All constructs were verified by DNA sequencing. The pSRneo-FLAG-Lbc plasmid was a gift from D. Schematic representation of the Rho GTPases activated by Lbc, Dbl, Toksoz [15]. DNA for microinjection was prepared by a standard CsCl Vav and FGD1. Dbl and Vav appear to regulate Rho, Rac and Cdc42. gradient purification method. All DNA manipulations followed standard The specificity of FGD1 and Lbc is more restricted: Lbc only activates protocols [36]. Rho, and the actions of FGD1 are limited to Cdc42. Mammalian cell culture Swiss 3T3 fibroblasts were maintained in DMEM (BRL/Life Technolo- the actin cytoskeleton are indistinguishable and, as Dbl has gies) supplemented with 10 % fetal calf serum, penicillin and strepto- been shown to be an exchange factor for Cdc42 in vitro, we mycin (Sigma), as described [3]. Cells were allowed to reach conclude that Vav activates Cdc42 as well as Rac and Rho. confluence and, 7–10 days after seeding, cells were serum-starved for 16 h by removing medium and replacing with serum-free medium con- taining 0.2 % NaHCO3. Quiescent serum-starved Swiss 3T3 fibrob- Conclusions lasts were briefly trypsinized, resuspended in serum-free medium The results presented here show that, like Dbl and Lbc, containing 0.5 mg ml–1 soybean trypsin inhibitor (Sigma), plated at low the DH family proteins FGD1 and Vav specifically density in serum-free medium onto 13 mm glass coverslips that had promote guanine nucleotide exchange on members of the been coated with 50 ␮gml–1 fibronectin (Sigma) and allowed to attach for 30–60 min prior to microinjection. Rho GTPase family. Because of the indirect nature of the assays used in this study, it remains to be seen whether the COS-1 cells were cultured in DMEM containing 10 % fetal calf serum activation of the Rho GTPases is mediated solely by FGD1 as described [37]. For transient transfections, cells were trypsinized and plated at 5 × 105 cells per 100 mm dish in DMEM supplemented and Vav, or whether additional accessory proteins are with 10 % NuSerum (Collaborative Biomedical Products). required. We have been unable to express recombinant Vav or FGD1 protein in Escherichia coli for direct examination of Microinjection and immunofluorescence guanine nucleotide exchange activity in vitro. Plasmid DNA (0.1 mg ml–1) was microinjected into the nuclei of Swiss 3T3 fibroblasts and, 2–3 h later, cells were fixed in 4 % paraformalde- hyde for 15 min, and permeabilized with 0.5 % Triton X-100 for 15 min. DH proteins appear to regulate Rho GTPases with either Expressed protein was visualized by incubating for 1 h with the Myc- a restricted specificity (FGD1 and Lbc) or with a broad epitope antibody (9E10) or the FLAG-epitope antibody (M2; Kodak) specificity (Dbl and Vav) (Fig. 6). Mutations to FGD1, followed by incubation for 45 min with FITC-conjugated goat anti- either by interruption of the gene by chromosomal translo- mouse antibody (Sigma). Actin structures were visualized by incubating with 0.1 ␮gml–1 TRITC-conjugated phalloidin (Sigma) for 45 min. cation or by insertion of a single resulting in pre- mature translational termination within the DH domain Texas-Red conjugated dextran (2 mg ml–1; Molecular Probes) was [13], have been detected in human patients suffering from included with DNA as an injection marker in experiments examining vin- culin distribution. Cells were fixed and permeabilized as described faciogenital dysplasia or Aarskog–Scott syndrome. The above, then treated with 1 mg ml–1 sodium borohydride (Sigma) for predicted loss of FGD1 is associated with a specific and 10 min. Vinculin was visualized by incubation for 1 h with a monoclonal characteristic pattern of skeletal anomalies, which affects anti-vinculin antibody (anti-vinculin clone VIN-11-5, Sigma) followed embryogenesis and alters the size of small bones and sequentially by FITC-conjugated goat anti-mouse antibody (1 h) and then FITC donkey anti-goat antibody (1 h). cartilage elements in the face, distal extremities, and vertebrae [32,33]. Our results suggest that a signal trans- Plasmid DNA encoding Dbl, Lbc, Vav or FGD1 (0.1 mg ml–1) were duction pathway controlled by FGD1/Cdc42 may play a coinjected with bacterially expressed GST–WASP (6 mg ml–1) or –1 –/– N17Rac (0.4 mg ml ) and stained for protein expression and actin crucial role in skeletal development. Vav mouse structures as described above. embryos die at around E7.5 prior to implantation. As tro- phoblast cells must migrate from embryonic to maternal Cells were photographed using a Zeiss Axiophot microscope with tissue and invade the uterine wall it is possible that the TMY-400 film (Kodak). activation of Rac and Cdc42 by Vav may be required for Protein expression cell migration or invasion, two processes that have been N17Rac and the Cdc42-binding domain of WASP (residues associated with Rac activation in other circumstances [34]. 201–321) were produced as recombinant GST-fusion proteins in Research Paper Regulation of Rho GTPases by FGD1 and Vav Olson et al. 1633

E.coli as described [3,19] and N17Rac released from GST by but not for hematopoietic development in vitro. EMBO J 1995, thrombin treatment. 14:1–11. 15. Zheng Y, Olson MF, Hall A, Cerione RA, Toksoz D: Direct involvement of the small GTP-binding protein Rho in lbc Transient transfection and kinase assays oncogene function. J Biol Chem 1995, 270:9031–9034. Transient transfection of COS-1 cells was carried out as described 16. Hart MJ, Eva A, Evans T, Aaronson SA, Cerione RA: Catalysis of [36]. Plasmid concentrations per 10 cm petri dish were as follows: for guanine nucleotide exchange on the CDC42Hs protein by the dbl SAPK/JNK1 assays: 10 ␮g pCMV-FLAG-JNK1 and 5 ␮g each of pMT- oncogene product. Nature 1991, 354:311–314. myc-MEKK carboxy-terminal domain, pEXV-myc-L61cdc42, pRK5-myc- 17. Hart MJ, Eva A, Zangrilli D, Aaronson SA, Evans T, Cerione RA, et al.: Cellular transformation and guanine nucleotide exchange activity Dbl, pRK5-myc-Vav and pRK5-myc-FGD1; for ERK assays: 10 ␮g are catalyzed by a common domain on the dbl oncogene product. pEXVmyc-ERK2 and 5 ␮g each of pEXV-myc-D12ras, pRK5-myc-Dbl, J Biol Chem 1994, 269:62–65. pRK5myc-Vav, pRK5-myc-FGD1 and pRK5-Sos. After incubation for 18. Yaku H, Sasaki T, Takai Y: The Dbl oncogene product as a 16 h in serum-free DMEM, cell lysates were harvested and assayed for GDP/GTP exchange protein for the Rho family: its properties in SAPK/JNK1 activity and for ERK2 activity as described [4]. Quantita- comparison with those of Smg GDS. Biochem Biophys Res tive western-blot analysis of the epitope-tagged proteins was carried Commun 1994, 198:811–817. 19. Aspenstrom P, Lindberg U, Hall A: Two GTPases, Cdc42 and Rac, out using the monoclonal antibodies anti-myc 9E10 and anti-FLAG M2 bind directly to a protein implicated in the immunodeficiency 125 and I-labelled protein A (Amersham). The levels of SAPK/JNK1 or disorder Wiskott–Aldrich syndrome. Curr Biol 1996, 6:70–75. ERK2 immunoprecipitated were determined on western blots with M2 20. Kolluri R, Fuchs Tolias K, Carpenter C, Rosen FS, Kirchhausen T: anti-FLAG antibody or 9E10 anti-myc antibody and alkaline-phos- Direct interaction of the Wiskott–Aldrich syndrome protein with phatase conjugated anti-mouse antibody (Pierce). Experiments were the GTPase Cdc42. Proc Natl Acad Sci USA 1996, 93:5615–5618. carried out in triplicate and representative assays are shown. 21. Symons M, Derry JMJ, Karlak B, Jiang S, Lemahieu V, McCormick F, et al.: Wiskott–Aldrich syndrome protein, a novel effector for the GTPase CDC42Hs is implicated in actin polymerization. Cell 1996, Acknowledgements 84:723–734. We thank D. Toksoz for pSRneo-FLAG-Lbc, P. Chardin for pRK5-Sos, P. 22. Gulbins E, Coggeshall KM, Baier G, Katzav S, Burn P, Altman A: Aspenstrom for pGEX-WASP and T. Bridges for recombinant proteins. M.F.O. Tyrosine kinase stimulated guanine nucleotide exchange activity and A.H. were supported by a programme grant (SP2249) from the Cancer of vav in T cell activation. Science 1993, 260:822–825. Research Campaign (UK). This work was also supported, in part, by March of 23. Gulbins E, Coggeshall KM, Baier G, Telford D, Langlet C, Baier BG, Dimes-Birth Defects Foundation Basic Science Grants 1-91-176 and 1-95- et al.: Direct stimulation of Vav guanine nucleotide exchange 0715, and National Institutes of Health (NIH) grant NS-30771 to J.L.G. activity for Ras by phorbol esters and diglycerides. Mol Cell Biol 1994, 14:4749–4758. 24. Gulbins E, Langlet C, Baier G, Bonnefoy BN, Herbert E, Altman A, et References al.: Tyrosine phosphorylation and activation of Vav GTP/GDP 1. Ridley AJ, Hall A: The small GTP-binding protein rho regulates the exchange activity in antigen receptor-triggered B cells. J Immunol assembly of focal adhesions and actin stress fibers in response 1994, 152:2123–2129. to growth factors. Cell 1992, 70:389–399. 25. Gulbins E, Coggeshall KM, Langlet C, Baier G, Bonnefoy BN, Burn P, 2. Ridley AJ, Paterson HF, Johnston CL, Diekmann D, Hall A: The small et al.: Activation of Ras in vitro and in intact fibroblasts by the Vav GTP-binding protein rac regulates growth factor-induced guanine nucleotide exchange protein. Mol Cell Biol 1994, membrane ruffling. Cell 1992, 70:401–410. 14:906–913. 3. Nobes CD, Hall A: Rho, Rac and Cdc42 GTPases regulate the 26. Gulbins E, Schlottmann K, Brenner B, Lang F, Coggeshall KM: assembly of multimolecular focal complexes associated with Molecular analysis of Ras activation by tyrosine phosphorylated actin stress fibers, lamellipodia and filopodia. Cell 1995, 81:1–20. Vav. Biochem Biophys Res Commun 1995, 217:876–885. 4. Olson MF, Ashworth A, Hall A: An essential role for Rho, Rac, and 27. Khosravi-Far R, Chrzanowska WM, Solski PA, Eva A, Burridge K, Der Cdc42 GTPases in cell cycle progression through G1. Science CJ: Dbl and Vav mediate transformation via mitogen-activated 1995, 269:1270–1272. protein kinase pathways that are distinct from those activated by 5. Nishiki T, Narumiya S, Morii N, Yamamoto M, Fujiwara M, Kamata Y, et oncogenic Ras. Mol Cell Biol 1994, 14:6848–6857. al.: ADP-ribosylation of the rho/rac proteins induces growth 28. Bustelo XR, Suen KL, Leftheris K, Meyers CA, Barbacid M: Vav inhibition, neurite outgrowth and acetylcholine esterase in cooperates with Ras to transform rodent fibroblasts but is not a cultured PC-12 cells. Biochem Biophys Res Commun 1990, Ras GDP/GTP exchange factor. Oncogene 1994, 9:2405–2413. 167:265–272. 29. Holsinger LJ, Spencer DM, Austin DJ, Schreiber SL, Crabtree GR: 6. Yamamoto M, Marui N, Sakai T, Morii N, Kozaki S, Ikai K, et al.: ADP- Signal transduction in T lymphocytes using a conditional allele of ribosylation of the rhoA gene product by botulinum C3 Sos. Proc Natl Acad Sci USA 1995, 92:9810–9814. exoenzyme causes Swiss 3T3 cells to accumulate in the G1 30. Clevenger CV, Ngo W, Sokol DL, Luger SM, Gewirtz AM: Vav is phase of the cell cycle. J Biol Chem 1993, 268:21509–21512. necessary for prolactin-stimulated proliferation and is 7. Minden A, Lin A, Claret FX, Abo A, Karin M: Selective activation of translocated into the nucleus of a T-cell line. J Biol Chem 1995, the JNK signaling cascade and c-Jun transcriptional activity by the 270:13246–13253. small GTPases Rac and Cdc42Hs. Cell 1995, 81:1147–1157. 31. Crespo P, Bustelo XR, Aaronson DS, Coso OA, Lopez-Barahona M, 8. Coso OA, Chiariello M, Yu JC, Teramoto H, Crespo P, Xu N, et al.: Barbacid M, et al:: Rac-1 dependent stimulation of the JNK/SAPK The small GTP-binding proteins Rac1 and Cdc42 regulate the signaling pathway by Vav. Oncogene 1996, 13:455–460. activity of the JNK/SAPK signaling pathway. Cell 1995, 32. Grier RE, Farrington FH, Kendig R, Mamunes P: Autosomal 81:1137–1146. dominant inheritance of the Aarskog syndrome. Am J Med Genet 9. Zhang S, Han J, Sells MA, Chernoff J, Knaus UG, Ulevitch RJ, et al.: 1983, 15:39–46. Rho family GTPases regulate p38 mitogen-activated protein 33. Fryns JP: Aarskog syndrome: the changing phenotype with age. kinase through the downstream mediator Pak1. J Biol Chem Am J Med Genet 1992, 43:420–427. 1995, 270:23934–23936. 34. van Leeuwen FN, van der Kammen RA, Habets GGM, Collard JG: 10. Bagrodia S, Derijard B, Davis RJ, Cerione RA: Cdc42 and PAK- Oncogenic activity of TIAM1 and Rac1 in NIH3T3 cells. Oncogene mediated signaling leads to Jun kinase and p38 mitogen- 1995, 11:2215–2221. activated protein kinase activation. J Biol Chem 1995, 35. Guo CH, Janovick JA, Kuphal D, Conn PM: Transient transfection of 270:27995–27998. GGH3-1’ cells [GH3 cells stably transfected with the 11. Hill CS, Wynne J, Treisman R: The Rho family GTPases RhoA, Rac1, gonadotropin-releasing hormone (GnRH) receptor and CDC42Hs regulate transcriptional activation by SRF. Cell complementary deoxyribonucleic acid] with the carboxyl-terminal 1995, 81:1159–1170. of betaadrenergic receptor kinase 1 blocks prolactin release: 12. Cerione RA, Zheng Y: The Dbl family of oncogenes. Curr Op Cell evidence for a role of the G protein beta gamma-subunit complex Biol 1996, 8:216–222. in GnRH signal transduction. Endocrinology 1995, 136:3031–3036. 13. Pasteris NG, Cadle A, Logie LJ, Porteous MEM, Glover TW, Wilroy 36. Sambrook J, Fritsch EF, Maniatis T: Molecular Cloning: A Laboratory RS, et al.: Isolation and characterisation of the faciogenital Manual, 2nd edn. New York: Cold Spring Harbor Laboratory Press; dysplasia (Aarskog–Scott Syndrome) gene: a putative Rho/Rac 1989. guanine nucleotide exchange factor. Cell 1994, 79:669–678. 37. Howe LR, Leevers SJ, Gomez N, Nakielny S, Cohen P, Marshall CJ: 14. Zmuidzinas A, Fischer K-D, Lira SA, Forrester L, Bryant S, Bernstein A, Activation of the MAP kinase pathway by the protein kinase raf. et al.: The vav proto-oncogene is required early in embryogenesis Cell 1992, 71:335–342.