From the 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.
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- 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.
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).
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.
Medical Research Council Laboratory for
Molecular Cell Biology,
Departments of Human Genetics and Pediatrics, University of
Michigan, Ann Arbor, Michigan 48109-0680
ABSTRACT
Top
Abstract
Introduction
Procedures
Results & Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results & Discussion
References
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).
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results & Discussion
References
RESULTS AND DISCUSSION
Top
Abstract
Introduction
Procedures
Results & Discussion
References
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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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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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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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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.
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FOOTNOTES |
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* 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).
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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