Activation of G1 Progression, JNK Mitogen-activated Protein Kinase, and Actin Filament Assembly by the Exchange Factor FGD1*

Koh-ichi NagataDagger §, Mariette DriessensDagger , Nathalie LamarcheDagger , Jerome L. Gorskiparallel , and Alan Hall**Dagger Dagger

From the Dagger  Medical Research Council Laboratory for Molecular Cell Biology, Cancer Research Campaign Oncogene and Signal Transduction Group and the ** Department of Biochemistry, University College London, Gower Street, London WC1E 6BT, United Kingdom and the parallel  Departments of Human Genetics and Pediatrics, University of Michigan, Ann Arbor, Michigan 48109-0680

    ABSTRACT
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Abstract
Introduction
Procedures
Results & Discussion
References

Cdc42 has been shown to control bifurcating pathways leading to filopodia formation/G1 cell cycle progression and to JNK mitogen-activated protein kinase activation. To dissect these pathways further, the cellular effects induced by a Cdc42 guanine nucleotide exchange factor, FGD1, have been examined. All exchange factors acting on the Rho GTPase family have juxtaposed Dbl homology (DH) and pleckstrin homology (PH) domains. We report here that FGD1 triggers G1 cell cycle progression and filopodia formation in Swiss 3T3 fibroblasts as well as JNK mitogen-activated protein kinase activation in COS cell transfection assays. FGD1-induced filopodia formation is Cdc42-dependent, and both the DH and PH domains are essential. Although expression of the FGD1 DH domain alone does not activate Cdc42 and induce filopodia, it does trigger both the JNK cascade in COS cells and G1 progression in quiescent Swiss 3T3 cells. We conclude that FGD1 can trigger G1 progression independently of actin polymerization or integrin adhesion complex assembly. Furthermore, since FGD1 activates JNK and G1 progression in a Cdc42-independent manner, it must have additional, as yet unidentified, targets.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results & Discussion
References

It is well established that Rho, Rac, and Cdc42, three members of the Rho family of small GTPases, control both the organization of the actin cytoskeleton and signal transduction pathways leading to gene transcription. In fibroblasts, Rho controls the assembly of actin stress fibers and associated focal adhesion complexes, whereas Rac and Cdc42 control the formation of lamellipodia and filopodia, respectively (1-4). In addition to these effects, the three GTPases have been reported to trigger a number of additional cellular activities, including activation of the JNK and p38 mitogen-activated protein kinase cascades, NF-kappa B activation, G1 cell cycle progression, and transcription from defined promoter elements upstream of the c-fos and cyclin D genes (5-10). The biochemical relationship between these varied responses is not yet clear, but genetic analysis of dorsal closure in Drosophila strongly argues that the ability of Rac to coordinately control both the JNK mitogen-activated protein kinase cascade and actin reorganization is physiologically important (11).

Mutational analysis of Rac and Cdc42 has revealed that stimulation of JNK activity and of actin polymerization is triggered by the interaction of each GTPase with two distinct target proteins (12, 13). In these studies, stimulation of G1 cell cycle progression correlated well with actin filament assembly, whereas JNK activation correlated well with activation of a Ser/Thr target kinase, p65PAK (12, 13). More recent work, however, has raised some doubt as to whether p65PAK is required for JNK activation (10, 14, 15).

To dissect further the Cdc42-induced responses, we have now examined the cellular effects induced by an upstream regulator of Cdc42, the guanine nucleotide exchange factor (GEF) 1 FGD1 (16-18). FGD1 was originally isolated by positional cloning as the gene responsible for faciogenital dysplasia (Aarskog-Scott syndrome), a human X-linked developmental disorder characterized by disproportionately short stature and by facial, skeletal, and urogenital anomalies (16). The FGD1 gene product belongs to a family of some 15 GEFs active on members of the Rho GTPase family (19). Like all members of the Rho GEF family, FGD1 contains a Dbl homology (DH) domain and a closely associated pleckstrin homology (PH) domain (16, 19). The DH domain encodes GEF catalytic activity and is required to stimulate GDP release from the GTPases. The function of the PH domain is unclear, although it has been suggested that it might serve to localize GEFs to discrete intracellular locations (19).

In this study, we have compared the effects of expressing FGD1 constructs containing either both the DH and PH domains or just the DH domain alone. We found that FGD1-DH can induce G1 progression and activate the JNK cascade, but both the DH and PH domains are essential for Cdc42-dependent actin polymerization. This suggests that FGD1 can stimulate G1 progression independently of its effects on the actin cytoskeleton and that FGD1 can activate JNK and G1 progression through Cdc42-dependent and -independent mechanisms.

    EXPERIMENTAL PROCEDURES
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Procedures
Results & Discussion
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Materials-- The following colleagues kindly provided the indicated constructs: Drs. M. Karin (University of California) and J. Ham (Eisai London Research Laboratories), pCMV-FLAG-JNK1 and pGEX-c-Jun, respectively; Dr. P. Aspenstrom (Uppsala University), pGEX-WASP-BD, an active Cdc42-binding domain (amino acids 201-321) of WASP (Wiscott-Aldrich syndrome protein (20); Dr. S. Moss (University College London), anti-Myc antibody (9E10); and Dr. J. Chant (Harvard University), pRK5-Myc-PAK and a constitutively active mutant, pRK5-Myc-PAK-L107F (21). Anti-FLAG tag antibody (M2) was purchased from Eastman Kodak Co. FGD1-DH (amino acids 375-560) and FGD1-PH (amino acids 561-710) were synthesized using the polymerase chain reaction and subcloned into pRK5 vector containing an Myc tag. FGD1-DH/PH (amino acids 375-710), an activated version of Cdc42 (L61Cdc42), and WASP-BD were also introduced into pRK5-Myc (17). All constructs were verified by DNA sequencing.

Cell Transfections-- NIH 3T3 focus assays were carried out using the NIH 3T3 subclone D4 (gift of C. J. Marshall) as described (22). COS-1 cells were transfected by the DEAE-dextran method (23). Plasmid amounts used were as follows: (i) 4 µg of pCMV-FLAG-JNK1 with 4 µg each of pRK5-Myc, pRK5-Myc-FGD1-DH/PH, pRK5-Myc-FGD1-DH, and pRK5-Myc-FGD1-PH or 1 µg of pRK5-Myc-L61Cdc42; and (ii) 5 µg of pRK5-Myc-PAK with 5 µg each of pRK5-Myc, pRK5-Myc-FGD1-DH/PH, and pRK5-Myc-FGD1-DH or 1.5 µg of pRK5-FLAG-L61Cdc42. pRK5-Myc-WASP-BD (3 µg) was cotransfected where indicated.

JNK Kinase Assays-- JNK1 kinase activity in transfected COS-1 cell extracts was measured after immunoprecipitation with anti-FLAG M2 antibody, using 0.4 µg of GST-c-Jun as a substrate (23). The relative phosphorylation levels of c-Jun were determined by a Phospho-Imager (Molecular Dynamics). The amount of immunoprecipitated JNK1 was evaluated on Western blots using M2 antibody and revealed by chemiluminescence using an ECL kit (Amersham Pharmacia Biotech).

p65PAK Kinase Assay-- Transfected COS-1 cells were lysed, and p65PAK kinase activity was measured using myelin basic protein as a substrate as described (12). The relative levels of myelin basic protein phosphorylation were determined by Phospho-Imager analysis. Levels of p65PAK immunoprecipitated were checked by Western blotting with anti-p65PAK antibody (rabbit).

Swiss 3T3 Microinjections-- Serum-starved quiescent Swiss 3T3 cells were prepared for microinjection as described (3). After microinjecting FGD1 expression constructs with or without 0.2 mg/ml GST-WASP-BD into the nucleus, cells were incubated for the indicated times. For detection of Myc-tagged FGD1 constructs and filamentous actin, cells were fixed in 4% paraformaldehyde for 15 min, permeabilized with 0.2% Triton X-100 for 5 min, and incubated with mouse monoclonal antibody 9E10 for 1 h, followed by incubation with fluorescein isothiocyanate-labeled anti-mouse IgG and rhodamine-phalloidin. Cells were viewed on a Zeiss Axioplan fluorescence microscope.

DNA Synthesis-- Confluent quiescent Swiss 3T3 cells were microinjected with DNA constructs and rat IgG as an injection marker and incubated with 10 µg/ml bromodeoxyuridine (BrdUrd) for 40 h. Cells were fixed and stained for rat IgG to localize injected cells, and BrdUrd incorporation was monitored using anti-BrdUrd antibody (7).

    RESULTS AND DISCUSSION
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Introduction
Procedures
Results & Discussion
References

Since many members of the Dbl family of Rho GEFs have been shown to behave as oncogenes, we first tested whether FGD1 or Cdc42 has transforming capability using an NIH 3T3 focus assay (22). As shown in Fig. 1A, transfection of FGD1 or L61Cdc42 into NIH 3T3 cells failed to induce focus formation under conditions where Lbc, a GEF specific for Rho, and L61Ras yielded characteristic transformed foci (22). L61Rac induced foci, but only when cotransfected with Raf-CAAX, an activator of the ERK mitogen-activated protein kinase pathway (Fig. 1A). Neither FGD1 nor L61Cdc42 induced foci when cotransfected with Raf-CAAX (Fig. 1A). We conclude that FGD1 does not behave as an oncogene in this assay. However, our results do not exclude the possibility that FGD1 behaves as an oncogene in NIH 3T3 cells with regard to other parameters of transformation, such as growth in agar, tumor growth in nude mice, or serum-dependent growth. Indeed, it has recently been reported that Cdc42 can induce an anchorage-independent phenotype in Rat1 cells (24), and it will be interesting to see whether FGD1 causes the same effects; this work is currently in progress.


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Fig. 1.   FGD1 does not induce transformed foci, but does induce G1 progression. A, NIH 3T3 cells were seeded at 1.3 × 105 cells/100-mm dish in Dulbecco's modified Eagle's medium and 10% newborn calf serum, and the next day, they were transfected with 0.5 mg of plasmid. Transfectants were fed Dulbecco's modified Eagle's medium and 10% newborn calf serum every third day and stained with crystal violet at days 12-14 post-transfection. The foci induced by Lbc were small and densely piled up, whereas the foci induced by L61Ras or L61Rac plus Raf-CAAX were spread out. Each experiment was performed three times in duplicate. Error bars correspond to S.D. B, quiescent Swiss 3T3 cells were microinjected with 0.1 mg/ml pRK5-Myc control vector or pRK5-Myc-FGD1 constructs along with rat IgG (0.5 mg/ml) to localize injected cells. BrdUrd incorporation was monitored 40 h later by immunofluorescence. Values are the percentage of injected cells positive for BrdUrd staining and correspond to the averages of three independent experiments in which ~50 injected cells were scored per assay. Error bars correspond to S.D.

Cdc42 has also been shown to stimulate G1 cell cycle progression when introduced into quiescent Swiss 3T3 cells (7). To examine whether FGD1 can induce G1 progression and entry into S phase, an expression vector encoding the DH and PH domains of FGD1 (FGD1-DH/PH) was injected into the nuclei of quiescent Swiss 3T3 cells, and DNA synthesis was monitored using BrdUrd incorporation. As shown in Fig. 1B, FGD1-DH/PH induced G1 progression and entry into S phase in ~60% of injected cells. This is as efficient as constitutively active Cdc42 injections (7). To examine whether both the DH and PH domains are required, cells were also injected with the FGD1 DH domain alone (FGD1-DH). As shown in Fig. 1B, FGD1-DH also triggered a signal leading to G1 progression in ~60% of injected cells. FGD1-PH had no effect on BrdUrd incorporation. To determine whether FGD1-induced G1 progression is mediated by Cdc42, FGD1 was co-injected with an expression plasmid encoding the Cdc42-binding domain of WASP (WASP-BD). However, within 5 h after injection, WASP-BD-expressing cells began to retract, and by 16 h, almost all cells had detached (data not shown). We were therefore unable to assess the long-term effects of FGD1 on serum-starved cells depleted of Cdc42 activity.

FGD1-DH/PH induces filopodia formation when expressed in quiescent Swiss 3T3 cells as shown in Fig. 2 (panels a and b) (16). To confirm that this is Cdc42-dependent, we co-injected FGD1 with WASP-BD. As shown in Fig. 2 (panels c and d), WASP-BD completely blocked FGD1-induced actin filament assembly, confirming that FGD1 induces filopodia through activation of Cdc42. Next, to determine whether both the DH and PH domains of FGD1 are required for this effect, we microinjected quiescent Swiss 3T3 cells with an expression vector encoding the DH domain alone (pRK5-Myc-FGD1-DH) and examined actin reorganization. As shown in Fig. 3A (panels a and b), under conditions where FGD1-DH was expressed at the same level as FGD1-DH/PH (compare Fig. 3A (panel a) with Fig. 2 (panel a)), the DH domain alone was unable to induce any actin filament assembly. FGD1-DH/PH and Cdc42 both induce integrin adhesion complexes associated with filopodia (3, 17). FGD1-DH did not induce any integrin complex assembly (data not shown). We conclude that activation of Cdc42 is dependent on both the DH and PH domains of FGD1.


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Fig. 2.   FGD1-induced filopodia formation is dependent on Cdc42. Serum-starved subconfluent Swiss 3T3 cells were fixed 1.5 h after injection of nuclei with 0.1 mg/ml pRK5-Myc-FGD1-DH/PH (panels a and b) or 0.1 mg/ml pRK5-Myc-FGD1-DH/PH plus 0.2 mg/ml GST-WASP-BD protein (panels c and d). Uninjected cells are shown in panels e and f. Expressed FGD1-DH/PH (panels a, c, and e) was visualized by indirect immunofluorescence with anti-Myc epitope monoclonal antibody 9E10. Actin filaments (panels b, d, and f) were visualized with fluorescently tagged phalloidin. The experiment was repeated three times, and 50-100 cells were microinjected in each experiment. GST-WASP-BD blocked filopodia formation in all injected cells. Scale bars represent 20 µm. Panels c and d and panels e and f are in the same scale.


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Fig. 3.   The PH domain of FGD1 is essential for filopodia formation. A, serum-starved subconfluent Swiss 3T3 cells were fixed 4 h after injection of nuclei with 0.1 mg/ml pRK5-Myc-FGD1-DH (panels a and b) or 0.1 mg/ml FGD1-DH-CAAX (panels c and d). Expressed FGD1-DH (panel a) and FGD1-DH-CAAX (panel c) were visualized by indirect immunofluorescence with 9E10 antibody. Actin filaments (panels b and d) were visualized with fluorescently tagged phalloidin. Between 50 and 100 cells were microinjected in each of three experiments. No cells expressing FGD1-DH or FGD1-DH-CAAX were observed showing induced filopodia formation. The scale bar represents 20 µm. B, cells in a monolayer of epithelial Caco-2 cells were microinjected with pRK5-Myc-FGD1-DH (panel a) or pRK5-Myc-FGD1-DH-CAAX (panel b), and Myc-tagged FGD1 was visualized after 2 h with 9E10 antibody.

Recent experiments with Lbc, a Rho-specific GEF, have revealed that its PH domain is not required for G1 progression or actin stress fiber assembly, at least when overexpressed in cells (25), whereas for Tiam-1, a Rac-specific GEF, an intact PH domain is required for both actin polymerization and JNK activation (26, 27). The role of the PH domain is therefore not clear, but it has been suggested that it might play a role in localizing exchange factors to the correct intracellular location, presumably the plasma membrane in the case of Cdc42 and Rac. In agreement with this, some PH domains have been shown to interact with phospholipids, which could act as a signal for translocation to the plasma membrane (28). To test whether the role of the PH domain of FGD1 is to induce plasma membrane localization, we added the "CAAX box" plasma membrane-localizing motif to the C terminus of FGD1-DH and analyzed the effects of this construct on the actin cytoskeleton. As shown in Fig. 3A (panels c and d), FGD1-DH-CAAX did not induce any actin filament assembly. To confirm that FGD1-DH-CAAX localizes to the plasma membrane, the construct was expressed in the epithelial cell line Caco-2 since it is very difficult to see plasma membrane localization in the flat fibroblast cells. As shown in Fig. 3B, expressed FGD1-DH (panel a) was found predominantly in the cytosol of Caco-2 cells, whereas FGD1-DH-CAAX (panel b) clearly localized to the plasma membrane. We conclude that the role of the FGD1 PH domain is unlikely to be simply that of a plasma membrane targeting signal. Perhaps the PH domain is required for the recruitment of other components of the Cdc42 signaling machinery. Analysis of Cdc42 pathways in Saccharomyces cerevisiae, for example, revealed that Cdc24, the GEF for Cdc42, also interacts with Bem1, a scaffold-like protein (29). Cdc24 therefore appears to be part of a large signaling complex and may have other roles in addition to its Cdc42 GEF activity.

FGD1 has previously been shown to act as a GEF for Cdc42 and to activate the JNK1 mitogen-activated protein kinase pathway (17, 18). Furthermore, Cdc42 interacts with and activates a Ser/Thr kinase, p65PAK, and this has been suggested to mediate activation of JNK (12, 13, 21, 30-32). To determine whether FGD1 can activate p65PAK, wild-type p65PAK and FGD1 were cotransfected into COS-1 cells. Fig. 4 shows that under conditions where L61Cdc42 activated p65PAK ~12-fold, the FGD1-DH/DH and FGD1-DH constructs did not activate p65PAK. Under these conditions, a constitutively activated p65PAK (PAK-LF) had ~62-fold increased activity compared with the wild type (Fig. 4). We conclude that FGD1-mediated activation of the JNK pathway previously reported is unlikely to be via p65PAK.


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Fig. 4.   FGD1 does not activate p65PAK. COS-1 cells were transfected with a constitutively activated p65PAK construct (pRK5-Myc-PAK-L107F) or wild-type p65PAK (pRK5-Myc-PAK) together with pRK5 vectors containing FLAG-L61Cdc42, Myc-FGD1-DH/PH, or Myc-FGD1-DH. The kinase activity of immunoprecipitated p65PAK (upper panel) was determined using myelin basic protein (MBP) as a substrate. The amount of p65PAK used in each kinase assay (middle panel) was checked by Western blotting using 9E10 antibody. The expression level of each protein in the transfection assay (lower panel) was checked by Western blotting with a mixture of 9E10 (anti-Myc) and M2 (anti-FLAG) antibodies. Experiments were carried out four times with similar results, and representative assays are shown.

To analyze the role of Cdc42 in FGD1-induced JNK activation more carefully, we examined the effect of cotransfecting FGD1 with WASP-BD. As a control and to show the effectiveness of this approach, Fig. 5A shows that WASP-BD completely inhibited the ability of L61Cdc42 to induce JNK activation. However, as shown in Fig. 5B, WASP-BD only partially blocked JNK activation by FGD1-DH/PH. Furthermore, Fig. 5B shows that the FGD1 DH domain alone was capable of activating the JNK pathway and that this was resistant to coexpression of WASP-BD. FGD1-PH itself did not activate JNK (data not shown). We conclude that in addition to a Cdc42-dependent activation of JNK, FGD1 can stimulate a Cdc42-independent pathway leading to JNK activation and that this pathway depends only on the DH domain and not the PH domain of FGD1 (Fig. 6). The nature of this additional pathway is unclear, although one obvious possibility is that FGD1 can act as a GEF for other Rho family GTPases. We have already shown that neither Rho nor Rac is a target for FGD1 (Fig. 2 (panel d) and Ref. 17). We have also found that an activated RhoD (33) does not activate JNK in COS cell transfections and that a dominant-negative RhoD does not interfere with FGD1-induced JNK activation (data not shown). We are currently examining other known members of the mammalian Rho GTPase family.


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Fig. 5.   FGD1-DH/PH and FGD1-DH activate JNK. A, Cdc42-induced JNK activation was blocked by the Cdc42-binding domain of WASP (amino acids 201-321; WASP-BD). FLAG-tagged JNK was expressed in COS-1 cells either alone or with pRK5-Myc-L61Cdc42. pRK5-Myc-WASP-BD (amino acids 201-321; 3 µg) or empty plasmid was cotransfected where indicated . JNK activity was assessed by immunocomplex kinase assays using c-Jun as a substrate and revealed by autoradiography (upper panel). The levels of FLAG-tagged JNK used in the assays were determined with M2 antibody (middle panel). Expression levels of L61Cdc42 and WASP-BD were determined by Western blotting using 9E10 antibody (lower panel). B, shown is the effect of WASP-BD on FGD1-induced JNK activation. COS-1 cells were transfected with pCMV-FLAG-JNK plus pRK5-Myc-FGD1-DH/PH or pRK5-Myc-FGD1-DH. pRK5-Myc-WASP-BD (amino acids 201-321; 3 µg) or empty plasmid was cotransfected where indicated. The amount of JNK used in each kinase assay was determined as described for A (middle panel). The expression level of each protein was revealed by Western blotting using a mixture of 9E10 and M2 antibodies (lower panel). Experiments were carried out five times with similar results, and representative assays are shown.


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Fig. 6.   FGD1 mediates Cdc42-dependent and -independent signal transduction pathways.

In conclusion, FGD1 acts as an exchange factor for Cdc42, and when overexpressed in cells, it can induce filopodia formation, G1 progression, and JNK activation. Activation of Cdc42 requires both the DH and PH domains of FGD1, but expression of the FGD1 DH domain alone activates JNK and stimulates G1 progression. It therefore appears that FGD1-induced changes to the actin cytoskeleton are Cdc42-dependent, but that FGD1-induced effects on gene transcription are mediated by both Cdc42-dependent and -independent signaling pathways.

    FOOTNOTES

* This work was supported in part by Cancer Research Campaign Program Grant SP2249 (United Kingdom).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ Recipient of fellowships from the Japan Society for Promotion of Science and the Uehara Memorial Foundation.

Supported by the Medical Research Council (United Kingdom).

Dagger Dagger To whom correspondence should be addressed: MRC Lab. for Molecular Cell Biology, University College London, Gower Street, London WC1E 6BT, UK. Tel.: 44-171-380-7909; Fax: 44-171-380-7805; E-mail: Alan.Hall{at}ucl.ac.uk.

1 The abbreviations used are: GEF, guanine nucleotide exchange factor; DH, Dbl homology; PH, pleckstrin homology; GST, glutathione S-transferase; BrdUrd, bromodeoxyuridine.

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Abstract
Introduction
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Results & Discussion
References

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