The Journal of Cell Biology, Volume 149, Number 4, May 15, 2000 775–781
The Rockefeller University Press, 0021-9525/2000/05/775/7 $5.00
Oncogenic Ras Downregulates Rac Activity, which Leads to Increased Rho
Activity and Epithelial–Mesenchymal Transition
Gerben C.M. Zondag, Eva E. Evers, J ean P. ten Klooster, Lennert J anssen, Rob A. van der Kammen,
and J ohn G. Collard
The Netherlands Cancer Institute, Division of Cell Biology, 1066 CX A msterdam, The Netherlands
etal rearrangements in response to receptor stimulation
and are involved in the establishment and maintenance
of epithelial cell morphology. We recently showed that
Rac is able to downregulate Rho activity and that the
reciprocal balance between Rac and Rho activity is a
major determinant of cellular morphology and motility
in NIH3T3 fibroblasts. Using biochemical pull-down as-
says, we analyzed the effect of transient and sustained
oncogenic Ras signaling on the activation state of Rac
and Rho in epithelial MDCK cells. In contrast to the ac-
tivation of Rac by growth factor-induced Ras signaling,
we found that sustained signaling by oncogenic RasV 12
permanently downregulates Rac activity, which leads to
upregulation of Rho activity and epithelial–mesenchy-
mal transition. Oncogenic Ras decreases Rac activity
Proteins of the Rho family regulate cytoskel- through sustained Raf/MA P kinase signaling, which
causes transcriptional downregulation of Tiam1, an ac-
tivator of Rac in epithelial cells. Reconstitution of Rac
activity by expression of Tiam1 or RacV 12 leads to
downregulation of Rho activity and restores an epithe-
lial phenotype in mesenchymal RasV 12- or RafCA A X -
transformed cells. The present data reveal a novel
mechanism by which oncogenic Ras is able to interfere
with the balance between Rac and Rho activity to
achieve morphological transformation of epithelial
Madin-Darby canine kidney cells • RaF/MA P kinase •
Ras signaling • Rho-like GTPases •
Transitions between epithelial and mesenchymal pheno-
types of cells are required for morphogenetic processes
and tissue remodeling during embryogenesis. These phe-
notypic conversions are regulated by cadherin-mediated
cell–cell adhesion, growth factors, and extracellular matrix
components (for recent reviews, see A dams and Nelson,
1998; V leminckx and Kemler, 1999). In addition, sustained
signaling by oncogenic Ras may result in morphological
transformation to a mesenchymal phenotype, which is as-
sociated with changes in gene expression, loss of E-cad-
herin-mediated cell–cell adhesions, and increased inva-
siveness of tumor cells (Behrens et al., 1989; V leminckx et
al., 1991). Rho-family proteins, in particular Rac and Rho,
mediate distinct cytoskeletal rearrangements in response
to receptor stimulation (Hall, 1998), and have been impli-
cated in the establishment and maintenance of cadherin-
based cell–cell adhesions (Braga et al., 1997; Hordijk et al.,
1997; Takaishi et al., 1997; Kuroda et al., 1998), as well as
in the migration of epithelial cells (Keely et al., 1997; Shaw
et al., 1997; Sander et al., 1998). We recently found that
Rac is able to downregulate Rho activity and that the bal-
ance between Rac and Rho activity determines the cellular
morphology and motile behavior of NIH3T3 fibroblasts
(Sander et al., 1999). The mechanism by which oncogenic
Ras induces morphological transformation of epithelial
cells is still poorly understood. Here, we used biochemical
pull-down assays to analyze the activation state of Rac
and Rho in response to Ras signaling in epithelial MDCK
cells. Our data indicate that, in contrast to growth factor-
induced Ras signaling, oncogenic Ras downregulates Rac
and upregulates Rho activity, leading to epithelial–mesen-
chymal transition. Oncogenic Ras-mediated downregula-
tion of Rac activity is caused by sustained activation of the
A ddress correspondence to John G. Collard, The Netherlands Cancer In-
stitute, Division of Cell Biology, Plesmanlaan 121, 1066 CX A msterdam,
The Netherlands. Tel.:
31 20 5121932. Fax:
31 20 5121944. E-mail:
The Journal of Cell Biology, Volume 149, 2000
Raf/MA P kinase pathway, which results in transcriptional
downregulation of the Rac-specific exchange factor, Tiam1
(Habets et al., 1994; Michiels et al., 1995). Reconstitution
of Rac activity in mesenchymal MDCK cells transformed
by activated mutants of Ras or Raf, downregulates Rho
activity and restores the epithelial phenotype. Our data
suggest that oncogenic Ras is able to induce a mesenchy-
mal phenotype in epithelial MDCK cells by influencing
the balance between the activation state of Rac and Rho
Materials and Methods
Cells and Retroviral Transductions
MDCK cells, MDCKf3 cells expressing RasV 12, and derivatives thereof
were grown in DME supplemented with 10% FCS and antibiotics. The
retroviral vector encoding C1199Tiam1 and transduced MDCK cells, and
RasV 12-transformed MDCKf3 cells have been described (Hordijk et al.,
1997). Constitutively active, myc-tagged R afCA A X and p110CA A X
cDNA s were cloned into a modified retroviral LZRS vector (Kinsella and
Nolan, 1996; Michiels et al., 2000) and stable cell lines were generated by
retroviral transduction and selection for neomycin resistance as described
(Sander et al., 1999). MDCK-RafCA A X cells expressing Tiam1 were gen-
erated by superinfection with retrovirus encoding C1199Tiam1 and a zeo-
cin-resistance marker and selected in medium containing G418 and zeocin
(Cayla). For Rac and Rho activity assays, cells were stimulated with 10 ng/
ml recombinant hepatocyte growth factor (HGF)
Where indicated, cells were pretreated for 4 h with 20
phatidylinositol 3 (PI3) kinase inhibitor, LY 294002 (Calbiochem).
for the indicated times.
M of the phos-
Rac and Rho Activity Assays
Rac and Rho activity assays were performed as previously described
(Sander et al., 1998, 1999; Reid et al., 1999). In brief, 10
in 10-cm dishes, washed in cold phosphate buffered saline, and lysed on
ice in lysis buffer (50 mM Tris-HCl, pH 7.4, 1% NP-40, 100 mM NaCl,
10% glycerol, 5 mM MgCl
, and protease inhibitors). Cleared lysates were
incubated for 30 min at 4
C with glutathione S transferase (GST)-PA K or
GST-rhotekin (Sander et al., 1998, 1999; Reid et al., 1999; Ren et al., 1999)
precoupled to glutathione-Sepharose beads (A mersham Pharmacia Bio-
tech) to precipitate GTP-bound Rac and Rho, respectively. Precipitated
complexes were washed three times in lysis buffer and boiled in sample
buffer. Total lysates and precipitates were analyzed on Western blot using
mA bs against Rac1 (Transduction Laboratories) and RhoA (Santa Cruz
cells were grown
Protein and RNA Analysis
A ctivated, phosphorylated forms of MA PK and PKB/A kt were detected
using antibodies from New England Biolabs, Inc. A nti-MA PK antibody
was provided by P. Hordijk (CLB, A msterdam, The Netherlands), and
anti-A kt1 antibody was obtained from Santa Cruz Biotechnology, Inc.
Tiam1 protein levels were analyzed by immunoprecipitation using anti-
Tiam1 (C16) polyclonal antibody from Santa Cruz Biotechnology, Inc.,
followed by Western blotting with anti-Tiam1 antibody
al., 1994). RafCA A X was detected with the myc-tag–specific antibody,
9E10. RNA analysis was performed using standard procedures (Habets et
DH (Habets et
For immunofluorescence, wild-type MDCK and transduced cell popula-
tions were stained with primary antibody recognizing
duction Laboratories) and rhodamine-conjugated phalloidin (Molecular
Probes, Inc.) to stain for F-actin. Images were collected by confocal mi-
HGF-induced Ras Activation Increases Rac and Rho
Activity through Independent Signaling Pathways
Growth factors like HGF and PDGF have been shown to
activate Rac through Ras-dependent PI3 kinase signaling.
Dominant negative RasN17 was demonstrated to inhibit
HGF- or PDGF-induced membrane ruffling, whereas ac-
tive RasV 12 induces membrane ruffling in a PI3 kinase-
dependent manner (Ridley et al., 1995; Rodriguez-V iciana
et al., 1997; Scita et al., 1999). Consistent with this signal-
ing model, HGF/cMet receptor stimulation of MDCK cells
resulted in a rapid and transient increase in Rac activity
that returned to basal levels within ten minutes and was in-
hibited by pretreatment of cells with the PI3 kinase inhibi-
tor, LY 294002 (Fig. 1 a). These findings do not exclude
that a possible direct activation of PI3 kinase by HGF/
cMet receptor signaling may also contribute to Rac activa-
tion. The activation of Rac coincided with the induction of
membrane ruffles, but preceded HGF-induced cell scatter-
ing by a few hours (data not shown). HGF also stimulated
activation of Rho, which was much more prolonged than
the activation of Rac (Fig. 1 a). Increased Rho activity was
still observed four hours after stimulation, and may be re-
quired for the HGF-induced scatter response of MDCK
cells. Rho activation was not dependent on PI3 kinase ac-
tivity (Fig. 1 a), indicating that HGF/cMet receptor stimu-
lation activates Rac and Rho through independent signal-
Oncogenic Ras Signaling Downregulates Rac Activity
and Upregulates Rho Activity
In contrast to the activation of Rac by HGF, we found that
sustained Ras signaling in subclones and populations of
RasV 12-transformed MDCK cells resulted in a permanent
downregulation of Rac activity (Fig. 1 b). Others have
shown that microinjection of RasV 12 in different cell
hepatocyte growth factor; PI3, phosphatidylinositol 3.
Abbreviations used in this paper: GST, glutathione S transferase; HGF,
Figure 1. Differential regula-
tion of Rac and Rho activity
by transient and sustained
Ras signaling. a, Rac1 and
RhoA activity was analyzed
in MDCK cells stimulated for
the indicated times with 10
ng/ml HGF. Right, cells were
pretreated with the PI3 ki-
nase inhibitor LY 294002 as
indicated. b and c, Rac and
Rho activities in wild-type
MDCK cells or in MDCK
cells stably expressing acti-
vated mutants of Ras (b) or
Raf kinase (c).
Zondag et al.
Oncogenic Ras Downregulates Rac Activity
types is followed by membrane ruffling and lamellipodia
formation, indicative of Rac activation (Bar-Sagi and Fera-
misco, 1986; Ridley et al., 1992; Rodriguez-V iciana et al.,
1997; Scita et al., 1999). However, these experiments re-
flect short-term effects of Ras signaling, consistent with
the observed stimulation of Rac activity by HGF (Fig. 1 a).
Downregulation of Rac activity in RasV 12-transformed
cells is accompanied by increased Rho activity (Fig. 1 b)
and epithelial–mesenchymal transition (Fig. 2 a). Upregu-
lation of Rho activity alone by expression of activated
RhoV 14 in MDCK cells is not sufficient to induce transi-
tion to a mesenchymal phenotype (data not shown), indi-
cating that the downregulation of Rac represents a crucial
step in Ras-induced morphological cell transformation.
Epithelial cells expressing RasV 12 have lost E-cadherin–
mediated cell–cell adhesions and exhibit a fibroblast-like
and highly migratory phenotype (Fig. 2 a). Considering
the roles for Rac in cell spreading (van Leeuwen et al.,
1997; D’Souza-Schorey et al., 1998) and Rho in cell con-
traction (Zhong et al., 1997), the mesenchymal phenotype
of RasV 12-transformed MDCK cells is consistent with the
low Rac and high Rho activities, as observed in these cells.
Immunostaining with vinculin-specific antibodies and rho-
damine-labeled phalloidin revealed a characteristic Rho-
induced pattern of pronounced focal contacts at the end of
thick stress fibers in the fibroblastoid RasV 12-transformed
cells (not shown). In contrast, in epithelial, Tiam1-express-
ing RasV 12-transformed MDCK cells, which exhibit high
Rac and low Rho activity, we found adhesion complexes at
the cell periphery (presumably Rac-mediated), as well as
small focal contacts throughout the basal side of the cell at
the end of short thin stress fibers. Previously, we have ob-
served a similar difference in distribution of adhesion
complexes and stress fibers in fibroblastoid versus epithe-
lial-like NIH3T3 cells, which show opposing levels of Rac
and Rho activities (Sander et al., 1999).
Oncogenic Ras Induces Morphological Transformation
of MDCK Cells through Sustained Raf/MAP
To investigate the signaling pathways activated by sus-
tained Ras signaling that lead to phenotypic changes and
downregulation of Rac activity, we introduced activated
forms of the Ras effectors Raf kinase (RafCA A X ) and
PI3 kinase (p110CA A X ) into epithelial MDCK cells by
retroviral transduction. A ctivation of both effector path-
ways in transduced cell populations was examined using
antibodies directed against phosphorylated MA P kinase
and PKB/A kt, two downstream components of Raf and
PI3 kinase signaling, respectively (Marshall, 1996). A s ex-
pected, both the MA P kinase and the PKB/A KT pathways
were activated in RasV 12-transformed cells, whereas Raf-
CA A X or p110CA A X expression resulted in specific acti-
vation of their downstream targets (Fig. 2 b). In contrast to
sustained PI3 kinase signaling, constitutive activation of
the Raf/MA P kinase pathway by RafCA A X was sufficient
to induce transition to a mesenchymal phenotype (Fig. 2
a). Similar to oncogenic RasV 12 (Fig. 1 b), sustained Raf
signaling (Fig. 1 c), but not PI3 kinase signaling (not
shown) resulted in decreased Rac and increased Rho ac-
tivity. We thus conclude that oncogenic Ras regulates Rac
and Rho activities through sustained activation of the Raf/
MA P kinase pathway.
Sustained Raf/MAP Kinase Signaling Results in
Transcriptional Downregulation of the Rac-specific
Exchange Factor, Tiam1
MA P kinase controls cellular behavior by regulating the
transcription of a large number of genes. Therefore, we
examined whether downregulation of Rac activity was
caused by altered expression of Rac-specific exchange fac-
tors, like Tiam1, that is endogenously expressed in MDCK
cells (Michiels et al., 1995; Hordijk et al., 1997). In both
RasV 12- and RafCA A X -transformed cells, the levels of
Tiam1 protein were strongly reduced or completely absent
(Fig. 3, a and c). Consistent with the effects of MA P kinase
on gene transcription, downregulation of Tiam1 occurred
at the transcriptional level, since Tiam1 mRNA transcripts
were barely detectable in RasV 12-transformed cells (Fig. 3
b). We did not find decreased expression of other putative
Rac exchange factors, such as Sos1, PIX , and V av-2, in
RasV 12- or RafCA A X -transformed cells, whereas the
Tiam1 homologue Stef (Hoshino et al., 1999) was not ex-
pressed in MDCK cells (data not shown). However, this
does not exclude that other as yet unidentified exchange
factors for Rac may be downregulated in RasV 12- or Raf-
CA A X -transformed cells.
Figure 2. Ras-induced transformation of epithelial MDCK cells
is mediated by the Raf/MA P kinase pathway. a, Phase-contrast
images of wild-type MDCK cells and MDCK cell populations ex-
pressing activated mutants of Ras, Raf kinase, or PI3 kinase. b,
A ctivation of the Raf/MA P kinase and PI3 kinase pathways in
the different lines was determined by Western blotting of total
cell lysates using antibodies against phospho-MA PK and phos-
The Journal of Cell Biology, Volume 149, 2000
To substantiate the role of Raf/MA P kinase signaling
in the transcriptional downregulation of Tiam1 and de-
creased Rac activity, we isolated six individual clones from
the RafCA A X -transformed cell population. The mesen-
chymal cell clones 1 and 2, which express relatively high
levels of RafCA A X , displayed high levels of MA P kinase
phosphorylation and loss of Tiam1 expression (Fig. 3 c). In
contrast, the epithelial cell clones 4, 5, and 6, expressed
very low levels of RafCA A X , exhibited control levels of
MA P kinase activation, and expressed normal levels of
Tiam1 (Fig. 3 c). Clone 3 showed an intermediate pheno-
type and moderate levels of MA P kinase activation that
were associated with intermediate levels of Tiam1. The de-
gree of MA P kinase activation in the cell clones thus cor-
related with decreased Tiam1 protein levels and transition
to a mesenchymal phenotype. Similar results were ob-
tained using subclones derived from RasV 12-transformed
cells (not shown).
It could be argued that Tiam1 downregulation is due to
the phenotypic changes induced by RasV 12 or RafCA A X
expression in cells (see Fig. 2), rather than by sustained
R af/MA P kinase signaling. To exclude this possibility,
we analyzed Tiam1 protein levels in RasV 12-transformed
cells that reverted towards an epithelial phenotype upon
introduction of constitutively active RacV 12 (Hordijk et
al., 1997). Despite their epithelioid morphology, these
cells still showed a RasV 12-mediated increase in MA P ki-
nase activity and failed to restore normal Tiam1 levels
(Fig. 3 a). This strongly suggests that constitutive activa-
tion of the Raf/MA P kinase pathway by oncogenic Ras
causes transcriptional downregulation of Tiam1 expres-
sion, leading to decreased R ac activity and transition to
a mesenchymal phenotype. Treatment of fibroblastoid
RasV 12-transformed MDCK cells with the MEK inhibitor
PD98059 (18 h, 20
M) showed an epithelial-like appear-
ance due to inhibition of polarization and migration of the
cells. However, PD-treated cells hardly formed E-cad-
herin–based adhesions, and no changes in Rac and Rho
activity or Tiam1 were found in response to PD treatment
(not shown). These data do not exclude that, in addition to
the Raf–MA P kinase pathway, other events play a role in
epithelial–mesenchymal transition, such as changes in
phosphorylation of the myosin II light chain or heavy
chain (Klemke et al., 1997; van Leeuwen et al., 1999),
which affect cell spreading and cell contraction, or changes
in phosphorylation of proteins involved in the formation
of E-cadherin adhesions (Kinch et al., 1995).
Reconstitution of Rac Activity Is Sufficient to Revert
Ras or Raf-transformed Cells to an
We next examined whether downregulation of Rac activ-
ity by sustained Raf/MA P kinase signaling could explain
the mesenchymal transition. Reconstitution of Rac activity
to approximately the level of wild-type MDCK cells by ex-
ogenous expression of Tiam1 reverted the fibroblastoid
RafCA A X -expressing cells towards an epithelioid pheno-
type (Fig. 4 a). In these cells, adherens junctions were re-
stored, as illustrated by the relocalization of the marker
proteins E-cadherin (not shown) and
In addition, the tight junction marker ZO-1 was re-
distributed to the sites of cell–cell contact (not shown).
Restoration of Rac activity by Tiam1 in RasV 12- and Raf-
CA A X -transformed MDCK cells resulted in strong down-
regulation of Rho activity (Fig. 4 a), a phenomenon that we
also observed in Tiam1- and RacV 12-expressing NIH3T3
fibroblasts (Sander et al., 1999). From these data we con-
clude that Rac negatively regulates Rho activity, and that
the lack of this negative regulation in Ras- and Raf-trans-
formed cells is the major cause of increased Rho activity.
Therefore, our data suggest that oncogenic Ras is able to
induce a mesenchymal phenotype in epithelial MDCK
cells by influencing the balance between Rac and Rho ac-
-catenin (Fig. 4, b–d).
A s illustrated in Fig. 5, our data indicate complex cross-
talk between Ras, Rac, and Rho in epithelial MDCK cells.
Figure 3. Downregulation of Tiam1 expression by sustained sig-
naling of the Raf/MA P kinase pathway. a, Tiam1 was immuno-
precipitated from wild-type MDCK cells and from cells express-
ing RasV 12 alone or RasV 12 together with RacV 12. MA P kinase
activation was analyzed by phospho-MA PK blotting as described
in Materials and Methods. b, Total RNA isolated from wild-type
MDCK and MDCK cells expressing RasV 12 was isolated and
probed for Tiam1 and GA PDH mRNA . c, Six independent cell
clones were selected from the RafCA A X -transduced population
on a phenotypic basis and analyzed for Tiam1 expression and
MA PK activation as described above. Expression of RafCA A X
was determined by Western blotting using a Myc-tag–specific an-
tibody. M, mesenchymal cells; E, epithelial cells; and M/E, inter-
mediate phenotype of the cells.
Zondag et al.
Oncogenic Ras Downregulates Rac Activity
Short-term activation of Ras by HGF/cMet receptor sig-
naling induces a transient, PI3 kinase-dependent Rac acti-
vation, presumably by PI3 kinase-mediated activation or
membrane translocation of exchange factors for Rac, as
shown for Sos, V av1, and Tiam1 (Han et al., 1998; Nim-
nual et al., 1998; Sander et al., 1998). Of note, Eps8 and
E3b1, two downstream targets of receptor tyrosine ki-
nases, also have been implicated in growth factor-induced
activation of Rac by Ras through regulating the activity of
Sos (Scita et al., 1999). HGF induces a more prolonged ac-
tivation of Rho that occurs independently of PI3 kinase,
indicating that distinct signaling pathways are involved in
HGF-induced activation of Rac and Rho. Stimulation of
NIH3T3 cells with PDGF also results in a transient activa-
tion of Rac. However, in contrast to HGF stimulation of
MDCK cells, Rho is transiently inactivated by PDGF re-
ceptor signaling (Sander et al., 1999). This indicates that
regulation of Rac and Rho activity is dependent on the
growth factor receptor involved, and possibly also on the
cell type studied.
In RasV 12-transformed cells, persistent Ras signaling
results in a R af/MA P kinase-mediated downregulation
of the Rac-specific exchange factor Tiam1, leading to de-
creased Rac activity (Fig. 5). A lthough the expression of
other putative Rac exchange factors like Sos1, PIX , V av2,
and Stef was not affected, we cannot exclude that the
expression of as yet unidentified Rac exchange factors is
influenced by oncogenic Ras signaling. Decreased Rac ac-
tivity in RasV 12- or RafCA A X -transformed cells is ac-
companied by increased Rho activity, whereas restoration
of Rac activity results in decreased Rho activity in these
cells. This indicates that Rac negatively influences Rho ac-
tivity under conditions where Rho is not activated by sepa-
rate pathways, such as HGF-receptor stimulation. A ntago-
nistic roles for Rac and Rho have been demonstrated in
the control of neuronal morphology (Kozma et al., 1997;
van Leeuwen et al., 1997) by regulating myosin light chain
or heavy chain phosphorylation (Sanders et al., 1999; van
Leeuwen et al., 1999). The present data indicate that Rac
can also antagonize Rho in a more direct manner by inhib-
iting Rho at the GTPase level. Previously, we found that
Tiam1-expressing RasV12-transformed MDCK cells showed
a fibroblast-like migratory phenotype when seeded on col-
lagen, but an epithelial phenotype when seeded on fi-
bronectin or laminin (Sander et al., 1998). These pheno-
types were associated with similar levels of Rac activity on
Figure 4. Reconstitution of Rac
activity in RafCA A X -trans-
formed MDCK cells by Tiam1
expression restores their epithe-
lial phenotype and decreases
Rho activity. a, Rac and Rho ac-
tivities in MDCK cells express-
ing RasV 12 or RafCA A X alone
or together with Tiam1. b–d,
Immunofluorescent staining of
?-catenin (green) and F-actin
(red) in wild-type MDCK cells
(b), MDCK cells expressing Raf-
CA A X (c), and RafCA A X -
transformed MDCK cells ex-
pressing Tiam1 (d).
The Journal of Cell Biology, Volume 149, 2000
all substrates, and appeared to be due to a substrate-
induced relocalization of the Tiam1/Rac signaling com-
plex to lamellipodia. The possible formation of different
Rac-dependent signaling complexes escapes detection of
the GTPase pull-down assay, which allows to monitor
overall changes in GTPase activity in cells, but not alter-
ations in localization and/or composition of GTPase com-
The observed Rac–Rho antagonism as found in epithe-
lial cells is consistent with our earlier findings in NIH3T3
fibroblasts, where Rac was also shown to downregulate
Rho activity (Sander et al., 1999). High Rac and low Rho
activity is associated with an epithelial-like phenotype in
NIH3T3 fibroblasts, characterized by the formation of
cadherin-based cell–cell adhesions and inhibition of cell
migration. In contrast, elevated levels of Rac and Rho ac-
tivities are associated with a fibroblastoid and migratory
phenotype of these fibroblasts. The present data show that
in epithelial MDCK cells, the balance between Rac and
Rho activity determines the cellular phenotype. Onco-
genic Ras is able to shift this balance by decreasing Rac
and increasing Rho activity, leading to mesenchymal
MDCK cells that have lost the capacity to establish E-cad-
herin–based cell–cell adhesions. It should be noted that the
observed downregulation of Rac activity by RasV 12 does
not exclude a requirement for Rac in invasion and migra-
tion of cells. Rather, it appears that the balance between
Rac and Rho activity is a major determinant of epithelial–
mesenchymal transition. Blocking Ras or Rac pathways
using dominant negative mutants inhibits motility (Ridley
et al., 1995; Keely et al., 1997; Shaw et al., 1997), and Rac
activity has been shown to be required for the formation
and maintenance of E-cadherin–based cell–cell adhesions
(Braga et al., 1997; Takaishi et al., 1997; Zhong et al., 1997;
Sander et al., 1998). Both processes appear to be regulated
by Rac-mediated signaling pathways.
How Rac is able to downregulate Rho activity remains a
challenge for future research. The signaling pathway in-
volved lies downstream of Rac and upstream of Rho, since
expression of RacV 12 downregulates Rho activity at the
GTPase level. Studies with Rac effector mutants in
NIH3T3 fibroblasts suggest that Rac-mediated signaling
pathways leading to reorganization of the cytoskeleton or
to stimulation of Jun kinase are not involved in downregu-
lation of Rho activity (Sander et al., 1999).
A ctivated mutants of Rac and Rho have been shown to
cooperate with Ras in the transformation of fibroblasts
(Qiu et al., 1995a,b; Khosravi-Far et al., 1995). Here, we
show that in epithelial MDCK cells, RasV 12-induced mor-
phological transformation involves downregulation of Rac
and upregulation of Rho activity. In addition to a role in
cytoskeletal reorganization, the observed increase in Rho
activity may also contribute to uncontrolled growth by
suppressing Ras-mediated induction of the cyclin-depen-
dent kinase inhibitor p21
cle progression (Olson et al., 1998).
, thereby allowing cell cy-
We thank T. Reid and S. van Delft for generation and testing of GST-
PA K and GST-rhotekin fusion proteins; M. Mareel and W. Birchmeier
for providing Ras-transformed MDCK cells for initial studies; G. Nolan
for the retroviral vector and packaging cells; M. van Dijk and J. Down-
ward for providing RafCA A X and p110CA A X constructs; and F. van
Leeuwen and other members of the department for stimulating discus-
sions and critical reading of the manuscript.
This work was supported by grants from the Dutch Cancer Society to
John G. Collard. Eva E. Evers was supported by the Deutsche For-
Submitted: 21 December 1999
Revised: 20 March 2000
A ccepted: 30 March 2000
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Figure 5. Schematic model depicting the differential effects of
transient and sustained Ras signaling towards Rac and Rho.
Short-term activation of Ras by HGF/cMet receptor signaling
leads to a transient PI3 kinase-dependent activation of Rac and a
more prolonged, PI3 kinase-independent activation of Rho,
which is accompanied by membrane ruffling and cell scattering.
In contrast, sustained signaling by oncogenic Ras results in de-
creased Rac activity through Raf/Map kinase mediated transcrip-
tional downregulation of the Rac exchange factor Tiam1. Onco-
genic Ras-mediated downregulation of Rac activity is
accompanied by upregulation of Rho activity and transition to a
mesenchymal phenotype. Reconstitution of Rac activity down-
regulates Rho activity, and restores an epithelial phenotype. See
Discussion for further details.
Zondag et al. Download full-text
Oncogenic Ras Downregulates Rac Activity
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C. Hama. 1999. Identification of the stef gene that encodes a novel guanine
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