From the Instituto de Investigaciones Biomedicas, Consejo Superior de Investigaciones Científicas, Madrid, Spain 28029
Received for publication, December 18, 2000, and in revised form, March 26, 2001
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ABSTRACT |
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The function of the Ras guanine nucleotide
exchange factor Ras-GRF/cdc25Mn is subject to tight
regulatory processes. We have recently shown that the activation
of the Ras/MAPK pathway by Ras-GRF is controlled by the Rho family
GTPase Cdc42 through still unknown mechanisms. Here, we report that
retaining Cdc42 in its GDP-bound state by overexpressing Rho-GDI
inhibits Ras-GRF-mediated MAPK activation. Conversely, Ras-GRF basal
and LPA- or ionomycin-stimulated activities were unaffected by a
constitutively active GTP-bound Cdc42. Moreover, the Cdc42 downstream
effectors MLK3, ACK1, PAK1, and WASP had no detectable influence on
Ras-GRF-mediated MAPK activation. In contrast, promoting GDP release
from Cdc42 with the Rho family GEF Dbl or with ionomycin suppressed the
restraint exerted by Cdc42 on Ras-GRF activity. We conclude that
Cdc42-GDP inhibits Ras-GRF-induced MAPK activation, but neither
Cdc42-GTP nor the Cdc42 downstream effectors affect Ras-GRF
performance. Interestingly, the loss of the GDP-bound state by Cdc42
abolishes its inhibitory effects on Ras-GRF function. These results
suggest that the Cdc42 mechanism of action may not be solely restricted
to activation of downstream signaling cascades when GTP-loaded.
Furthermore, the GDP-bound form may be acting as an
inhibitory molecule down-modulating parallel signaling routes such as
the Ras/MAPK pathway.
Small GTP-binding proteins act as key molecular switches in signal
transduction routes through which stimuli received through cell surface
receptors are conveyed to the nucleus. The hallmark of small
GTP-binding protein function, the transition from an inactive GDP-bound
state to an activated GTP-bound state, is tightly regulated by three
types of regulatory proteins: GTPase-activating proteins (GAPs),1
potentiators of the intrinsic capacity of small G proteins to hydrolyze
GTP; guanine nucleotide dissociation
inhibitors (GDIs) that regulate the nucleotide interchange
process by preventing GDP release; and guanine nucleotide
exchange factors (GEFs) that initiate the
nucleotide replacement cycle by catalyzing the exchange of GDP for GTP
(1).
Ras-GRF/Cdc25mn is a GEF for the Ras family of small
GTP-binding proteins cloned by virtue of its homology with the CDC25
gene product that stimulates nucleotide exchange on Saccharomyces
cerevisiae Ras (2-5). The primary structure of Ras-GRF reveals
the presence of a number of regulatory motifs presumably involved in
diverse signaling control mechanisms and protein-protein interactions. These include a Dbl homology (DH) domain (6), generally present in GEFs
for the Rho family of small G proteins (7). The DH domain is flanked by
two Pleckstrin homology (PH) domains, the function of which remains
largely unclear, although it has been suggested that it may play a role
in membrane targeting (8).
With regard to regulation, it is known that Ras-GRF exchange activity
over Ras is augmented by G protein-coupled receptors but is largely
unaffected by receptors of the tyrosine kinase type (9-13). Calcium
can also regulate Ras-GRF activity by a mechanism mediated through a
calmodulin-binding motif (IQ domain) present in its N terminus (14,
15). As such, calcium ionophores, like ionomycin, can greatly enhance
the activation of the MAPK pathway by Ras-GRF (15, 16). Interestingly,
we have recently shown that the ability of Ras-GRF to activate the
Ras/MAPK pathway is regulated by the Rho family GTPase Cdc42 that
apparently controls the translocation of Ras-GRF to the membrane
(17).
In this study we have taken a step further in the investigation of the
mechanisms by which Cdc42 regulates the activation of the Ras/MAPK
pathway by Ras-GRF. We show that Cdc42, retained in a GDP-bound state
by the action of the guanine dissociation inhibitor Rho-GDI, acts as a
down-regulator of Ras-GRF. Conversely, ectopic expression of either a
constitutively activated, GTP-loaded Cdc42 or Cdc42 downstream effector
proteins does not affect Ras-GRF-mediated activation of MAPK.
Interestingly, inducing GDP release from Cdc42 by the overexpression of
Dbl, a bona fide Cdc42 exchange factor, relieves the
inhibitory effects exerted by Cdc42 on the activation of the MAPK
pathway by Ras-GRF.
Constructs--
Plasmids encoding WASP and MLK3 were provided by
J. S. Gutkind and PAK1 (L83, L86) by J. Field. A constitutively
activated mutant (L543F) of ACK1 and Rho-GDI Cell Culture--
COS-7 cells were regularly grown in
Dulbecco's modified Eagle's medium supplemented with 10% fetal calf
serum. Subconfluent cells were transfected by the DEAE-dextran
technique (18). Total amount of transfected plasmid DNA was equalized
with vector DNA when necessary.
Kinase Assays--
MAPK and JNK kinase activities were
determined as described previously (19) in anti-HA
immunoprecipitates using myelin basic protein (MBP) (Sigma) or
GST·ATF2 as substrates for MAPK or JNK, respectively. Reactions were
terminated by the addition of 5× Laemmli buffer and boiled and
electrophoresed in 12% SDS-polyacrylamide gels. Gels were visualized
by autoradiography and quantitated by phosphorimager.
Immunoblotting--
Immunoprecipitations were performed as
described previously (20). Total lysates and immunoprecipitates were
fractionated in SDS-polyacrylamide gels and transferred onto
nitrocellulose filters. Immunocomplexes were visualized by ECL
detection (Amersham Pharmacia Biotech) using horseradish
peroxidase-conjugated secondary antibody (Cappel). Mouse monoclonal
anti-AU5, anti-HA, and anti-Myc antibodies were from Babco. Rabbit
polyclonal antibodies anti-Ras-GRF, anti-Cdc42, anti-MLK3, anti-Dbl,
and anti-RHO-GDI were from Santa Cruz Laboratories. Subcellular
fractionation was performed in 20 mM HEPES, pH 7.4 buffer,
as described (17).
Ras GTP Loading--
Ras GTP loading was performed basically as
described by Taylor and Shalloway (21). Briefly, cells cotransfected
with AU5-H-Ras and the indicated plasmids were lysed in HEM buffer (25 mM HEPES, pH 7.3, 10 mM MgCl2, 150 mM NaCl, 0.5 mM EGTA, 20 mM
GTP-Cdc42 Pull-down Assay--
AU5-Cdc42-transfected cells
were lysed as above. GTP-bound AU5-Cdc42 was affinity sequestered
with bacterially synthesized GST·PAK CRIB (Cdc42/Rac Interaction
Binding) domain (amino acids 70-106) basically as described (22).
Immunoblots were performed as described above using anti-AU5 antibody.
MAPK Activation by Ras-GRF Is Inhibited by Rho-GDI--
We have
recently demonstrated that the activation of Ras/MAPK by Ras-GRF could
be efficiently prevented by the Cdc42 dominant interfering mutant,
Cdc42 N17 (17). It is currently believed that GTPases N17 mutants work
by competing with the endogenous proteins for binding to GEFs, thus
retaining them in an inactive, GDP-bound form. However, potential
pitfalls exist when interpreting results obtained with N17 mutants
(23). Because of this, we tested whether other means of
maintaining Cdc42 in its GDP-bound state would also restrain
Ras-GRF function. For this purpose, we utilized the guanine nucleotide
dissociation inhibitor Rho-GDI (24) that has been shown to prevent GDP
dissociation from Cdc42 and other Rho GTPases (25, 26).
The direct method for analyzing the activity of an exchange factor is
by assaying its capacity to catalyze nucleotide exchange on its target
GTPase. For this purpose Ras-GRF was cotransfected with AU5-tagged
H-Ras into COS-7 cells, in the presence of Cdc42 N17 or Rho-GDI, and
Ras-GTP was affinity-precipitated from the resulting cellular lysates
with the aid of GST-Raf RBD (see "Materials and Methods"). As shown
in Fig. 1A, Ras-GRF induced a
potent incorporation of GTP into Ras. However Ras-GTP levels dropped
over 60% when Ras-GRF was cotransfected with Cdc42 N17 or Rho-GDI,
indicating that these proteins were effectively blocking
Ras-GRF-induced nucleotide exchange on Ras. These results closely
matched those obtained when MAPK activation was used to monitor the
effects of Rho-GDI on Ras-GRF function. As shown in Fig. 1B,
Rho-GDI blocked Ras-GRF-induced MAPK activation almost as efficiently
as Cdc42 N17 when expressed at similar levels. On the other hand, the
activation of MAPK by RasV12 was unaltered by Rho-GDI (Fig.
1B), thereby demonstrating that Rho-GDI is affecting the
activation of the Ras/MAPK pathway upstream from Ras. These data,
together with our previous findings (17), clearly demonstrate that the
results obtained by direct measurement of Ras GDP/GTP exchange are
identical to those obtained by assaying the activation of MAPK. Thus,
we have chosen MAPK activation as a valid readout for further
experimentation.
Calcium ionophores and certain agents acting through G protein-coupled
receptors such as lysophosphatidic acid (LPA) have been shown to boost
Ras-GRF-mediated MAPK activation (9, 15, 16). Thus, we also tested the
effects of Rho-GDI on the activity of Ras-GRF under the influence of
external stimuli. In agreement with our previous observations, Rho-GDI
potently inhibited Ras-GRF-mediated MAPK activation when stimulated by
ionomycin or LPA but had no effects on MAPK activation elicited by EGF
(Fig. 1C), which is known to be Ras-GRF-independent (10,
11).
An alternative mechanism through which a G protein can be kept in its
GDP form is by potentiating its GTPase activity, which can be
accomplished by the overexpression of adequate GAP proteins. Therefore,
we determined if p50Rho-GAP (27), known to have a potent GAP activity
against Cdc42 (28), could also prevent the activation of MAPK by
Ras-GRF. As expected, p50Rho-GAP could block the stimulation of
MAPK by Ras-GRF just as efficiently as Rho-GDI (Fig. 1D).
These effects could not be attributed to differences in the protein
levels of the HA·MAPK cotransfected in these assays, since the
protein was equally well expressed in all cases (Fig. 1C,
lower panel).
In summary, our results showing that Cdc42 N17, Rho-GDI, and p50Rho-GAP
can clearly abrogate the activation of MAPK by Ras-GRF, strongly point
to the maintenance of Cdc42 in its GDP-bound state as the underlying
cause of the Cdc42 inhibitory effects on Ras-GRF activity. Also, these
results suggest that the effects of Cdc42 N17 on Ras-GRF function
are not due to other indirect mechanisms. It should be noted
that both Rho-GDI and p50Rho-GAP are also active on the Rho family
GTPases Rho and Rac (25, 26, 28). However, we have previously described
that unlike Cdc42, the dominant inhibitory mutants of Rho and Rac have
no detectable effects on the activation of MAPK induced by Ras-GRF
(17). In light of this data, it is reasonable to attribute the effects
of Rho-GDI and p50Rho-GAP on Ras-GRF function, mainly to their
influence on Cdc42.
Analysis of Rho-GDI/Cdc42 Interactions--
It has been shown that
in vitro Rho-GDI is able to interact equally well with the
inactive GDP-bound state of Rho proteins and the active GTP-bound form
(29). This circumstance should be taken into account when interpreting
the effects of Rho-GDI on the functional relationship between Ras-GRF
and Cdc42. To clarify this point, we investigated the interaction of
Rho-GDI and Cdc42 in our cellular system. COS-7 cells were transfected
with wild-type AU5-tagged Cdc42 that is GDP-bound under
serum-starvation conditions or with AU5-Cdc42 Q61L (QL) found in a GTP
state under the same experimental conditions. Upon anti-AU5
immunoprecipitation, it was found that endogenous Rho-GDI associated
strongly with Cdc42-wt but was undetected in Cdc42-QL
immunoprecipitates (Fig. 2A).
This may reflect the observation that association of Rho-GDI with the GTP-bound form of Rho GTPases takes place mainly in vitro
(29). Other reports indicate that Rho-GDI interacts with the GTP-bound form of its cognate proteins much less efficiently than with the GDP-bound form (30), quite in line with our results. Moreover, in
absolute agreement with our observations, a recent in vivo study undertaken in COS-1 cells clearly demonstrates that Rho-GDI does
not bind to Cdc42-QL (31). Although it cannot be excluded that under
certain physiological circumstances Rho-GDI could bind to Cdc42-GTP,
under our experimental conditions and in our cellular environment,
Rho-GDI is preferentially associated to Cdc42-GDP.
In addition to its capability of inhibiting GDP release from Rho family
G-proteins, Rho-GDI can also bring about the extraction of Rho proteins
from cellular membranes, thus rendering the GTPases cytoplasmic and
therefore inactive (24, 25, 32). It was of interest to determine
whether Rho-GDI effects on Ras-GRF could be somehow associated with
this feature. For this purpose, AU5-Cdc42 was coexpressed with
Myc-tagged Rho-GDI, and its cellular distribution was examined.
Subcellular fractionation of the COS-7 lysates demonstrated that in the
absence of exogenous Rho-GDI, Cdc42 was found in the cell particulate
fraction as expected (Fig. 2B). However, despite high levels
of expression, as verified by anti-Myc immunoblotting, Rho-GDI did not
alter the cellular distribution of Cdc42, because no Cdc42 could be
detected in the soluble fraction (Fig. 2B). This
demonstrates that in our cellular system and under our experimental conditions, Rho-GDI did not provoke the release of Cdc42 from the cell
membranes. The fact that Rho-GDI effectively solubilizes Cdc42 in other
cell types such as human placental cells and epidermal carcinoma cells
(25) suggests that other unknown cell-specific factors may determine
whether Rho-GDI membrane-extracting activity is functional, depending
on the cell type and/or physiological circumstances. A recent report
suggests that the solubilization of Rho GTPases is strictly dependent
on the stoichiometric relationship between Rho-GDI and its cognate
GTPases (31). It is thus conceivable that the concentrations of Rho-GDI
utilized in our assays, although sufficient to inhibit nucleotide
exchange, are not high enough to solubilize Cdc42. Indeed, we have
observed that steadily increasing the concentration of Rho-GDI brings
about a gradual solubilization of Cdc42 (data not shown).
The possibility exists that Rho-GDI could inhibit Ras-GRF function
through a direct interaction by sequestering it unproductively in the
cytoplasm. To test this hypothesis, we cotransfected AU5-Rho-GDI with
Ras-GRF and assayed its ability to coimmunoprecipitate. As shown in
Fig. 2C, no Ras-GRF was detected in association with anti-AU5 immunoprecipitates, either under basal or ionomycin-stimulated conditions, indicating that no direct interaction exists between Rho-GDI and Ras-GRF.
On the other hand, it was found that Rho-GDI markedly inhibited Cdc42
GDP/GTP exchange induced by the GEF Dbl or by stimulation with
ionomycin (data not shown) as determined by Cdc42-GTP pull-down assays
using the GST·PAK CRIB domain (see "Materials and Methods"). To
further substantiate the notion that Rho-GDI down-regulatory effects
over Ras-GRF were mainly due to its function as an inhibitor of Cdc42
nucleotide exchange, we made use of Rho-GDI
Overall, our results demonstrate that under our experimental
conditions: (i) Rho-GDI does not associate with Cdc42 when in a
GTP-bound state; (ii) Rho-GDI is ineffective in rendering Cdc42 cytoplasmic; and (iii) Rho-GDI does not interact directly with Ras-GRF.
Furthermore, our data prove that the effects of Rho-GDI on Ras-GRF
function are primarily due to its role as a blocker of Cdc42 nucleotide
exchange, thus to maintain Cdc42 in a GDP-bound form.
Rho-GDI Blocks Ras-GRF Recruitment to the Membrane--
Our
previous findings demonstrate that the inhibition of Ras-GRF-mediated
MAPK activation by Cdc42 N17 correlates with the reduction of Ras-GRF
protein levels in the cell particulate fraction (17), suggesting that
Cdc42, when GDP-bound, would be interfering with Ras-GRF translocation
to the cell membranes. If this was the case, Rho-GDI, which as shown
before retains Cdc42 in its GDP-bound form, would be expected to
prevent a Ras-GRF presence in the membrane fraction. To test this
point, we analyzed Ras-GRF cellular distribution upon cotransfection
with Rho-GDI. As shown in Fig. 3, in the
absence of ectopic Rho-GDI, Ras-GRF was evenly distributed between the
particulate and soluble fractions. However, cotransfection of Rho-GDI
markedly diminished Ras-GRF levels in the membrane fraction. This
effect clearly correlated with the inhibition of Ras-GRF-mediated MAPK
activation by Rho-GDI (Fig. 3). On the other hand, the activation of
MAPK induced by a membrane-bound Ras-GRF-CAAX (17) was
unaffected by Rho-GDI. Likewise, the cellular distribution of this
membrane-targeted Ras-GRF was unaltered by Rho-GDI (Fig. 3). These
results are in perfect agreement with our previous observations using
Cdc42 N17 (17) and suggest that Cdc42 in its GDP state, regardless of
the mechanism by which this state is achieved, abrogates Ras-GRF
activation by preventing its localization to the cell particulate
fraction.
Ras-GRF Function Is Unaffected by Cdc42-GTP or by Cdc42 Downstream
Effectors--
A vast collection of data gathered hitherto indicates
that most biological processes that are inhibited or diminished by the action of a "switched off" GDP-bound GTPase are activated or
potentiated by the "switched on" GTP-bound form of the GTPase.
Thus, it would be conceivable that the activation of MAPK by Ras-GRF,
which is inhibited by Cdc42-GDP, should be potentiated by Cdc42-GTP. To verify this point, we tested whether the Cdc42 constitutively active
mutant, Cdc42-QL, would be capable of boosting the MAPK response
induced by Ras-GRF. For this purpose Ras-GRF was cotransfected with
increasing amounts of Cdc42-QL into COS-7 cells. It was found that
Cdc42-QL strongly activated JNK (Fig.
4A, middle panel), known to be a downstream target of Cdc42 (18). However, it did not
affect MAPK activation elicited by different concentrations of Ras-GRF
(Fig. 4A, top and lower panels), thus
suggesting that basal Ras-GRF-induced MAPK activation is independent of
Cdc42-GTP levels. In a similar fashion, overexpression of Cdc42-QL did
not alter Ras-GRF-mediated MAPK activation in cells treated with
ionomycin or LPA (Fig. 4B), indicating that the externally
stimulated activation of MAPK mediated by Ras-GRF is also largely
insensitive to the amount of Cdc42-GTP present in the cell.
To further substantiate this point, we also tested whether several
proteins known to be activated by Cdc42-GTP and well proven as
bona fide Cdc42 effectors (7) would be capable of
potentiating Ras-GRF function. Hyperactive forms of ACK1 (L543F) (34),
MLK3 (35), PAK1 (L83, L86) (36), and WASP (37) were found to be well
expressed and/or to potently activate JNK upon transfection into COS-7
cells (Fig. 4C, middle and lower
panels). However, they were unable to augment even slightly the
activation of MAPK induced by Ras-GRF in an experimental setting in
which ionomycin strongly potentiated Ras-GRF-mediated MAPK activation
(Fig. 4C, top panel).
Overall, these results clearly demonstrate that the activation of MAPK
by Ras-GRF, although inhibited by Cdc42-GDP, is unaffected either by
GTP-loaded Cdc42 or by Cdc42 effector proteins. These data strongly
support our initial observations that Cdc42-QL does not affect the
presence of Ras-GRF in the cell particulate fraction (17), although
they conflict with a recent report in which ACK1 was found to synergize
with Ras-GRF in the activation of MAPK (34). However, the cellular
and/or experimental conditions utilized in the last study was such that
Ras-GRF activity was affected by stimuli signaled by receptors of the
tyrosine kinase type such as EGF, in contrast to previous results
(9-11).
Inhibition of Ras-GRF by Cdc42-GDP Can Be Rescued by Cdc42
GEFs--
We have previously shown that the intensifying effect of
ionomycin over Ras-GRF-mediated MAPK activation can be blocked by Cdc42
N17. We have also demonstrated that ionomycin induces nucleotide exchange on Cdc42 (17). This implies that relieving the blockade that
Cdc42-GDP exerts on Ras-GRF could be a requisite for fully enhancing
Ras-GRF potential to activate the Ras/MAPK pathway. To test this
hypothesis, we tested whether exchange factors for Cdc42 would be
capable of rescuing the restraint on Ras-GRF activity caused by
GDP-bound Cdc42. We have previously reported that high concentrations
of Cdc42-wt, mainly GDP-loaded under the serum-starved conditions used
in our assays, can be as efficient as Cdc42 N17 for inhibiting Ras-GRF
(17). Indeed, MAPK activation by Ras-GRF was remarkably diminished upon
cotransfection with 1 µg of Cdc42-wt (Fig.
5A). Interestingly, MAPK
activation levels were completely restored by coexpression of the GEF
Dbl (38) (Fig. 5A), in agreement with our previous findings
in which Dbl was shown to be capable of potentiating Ras-GRF-induced
MAPK activation (17). This effect could not be attributed to the
stimulation of MAPK through alternative routes, such as the
PAK>MEK>MAPK connection (39), as Dbl did not affect MAPK when acting
alone (Ref. 17 and data not shown). Likewise, ionomycin was also
capable of completely rescuing Ras-GRF activity. On the other hand, the
GEF Ost and Cdc42-QL were incapable of overcoming the inhibitory
effects of Cdc42-wt (Fig. 5A). The ineffectiveness of Ost,
despite high expression levels (not shown), is noteworthy, as Ost has
been reported to act as a Cdc42 GEF in vitro (40). A likely
explanation could be that Ost is not a GEF for Cdc42 in
vivo, at least in COS-7 cells. In a similar fashion, other Rho
family exchange factors such as Vav, which is not active over Cdc42
(41), also fail to affect Ras-GRF activity (17).
Finally, we explored whether GEFs for Cdc42 could also alleviate the
inhibition of Ras-GRF activity brought about by Rho-GDI. For this
purpose, Ras-GRF was cotransfected with equal concentrations of
Cdc42-wt and Rho-GDI. Both constructs were able to markedly diminish
the activation of MAPK induced by Ras-GRF (Fig. 5B). The
coexpression of increasing concentrations of Dbl, gradually counteracted the repression on Ras-GRF caused by Cdc42-wt. This effect
was also observed in Rho-GDI-transfected cells, although in this case,
the amount of Dbl necessary to initiate the rescue of Ras-GRF activity
was 2-fold higher (Fig. 5B). This can be explained as
Rho-GDI induces a potent inhibitory effect on GDP release catalyzed by
Dbl, which can only be overcome by high concentrations of
this GEF (25). On the other hand, the wild-type version of Dbl,
proto Dbl, known to have a weaker nucleotide exchange activity on Cdc42 (38) was less effective for rescuing Ras-GRF-mediated MAPK activation (Fig. 5B).
In conclusion, our data demonstrate that Cdc42 in its GDP-bound form,
acts as a potent down-regulator of Ras-GRF-induced activation of the
Ras/MAPK pathway. However, Cdc42 in a GTP-bound state does not have the
expected potentiating effect on Ras-GRF function. This is clearly
envisioned by the unresponsiveness of Ras-GRF to Cdc42-activated
downstream effector proteins and to hyperactive Cdc42-QL, and by the
inability of this mutant to rescue the restraint exerted on Ras-GRF
activity by the overexpression of Cdc42-wt. Despite this, we show that
the stimulation of GDP release from Cdc42, with the aid of agents that
promote GDP/GTP exchange such as an external stimulus like ionomycin or
the ectopic expression of a specific GEF for Cdc42, strongly
facilitates the activation of MAPK by Ras-GRF.
The most plausible model that emerges from these data is one in which
the loss of Cdc42 GDP-bound structural characteristics rather than the
acquisition of a GTP-bound structural conformation, would be the key
step for relieving the inhibitory effect that Cdc42 imposes on Ras-GRF
function. Based on this hypothesis, the conformational features of
Cdc42 when GDP-loaded would enable it to exert an inhibitory effect
over Ras-GRF activity by still unknown mechanisms. This inhibition
would be unaffected by the presence of ectopic Cdc42-GTP or
Cdc42-activated effectors. However, catalyzing GDP release from Cdc42
would bring about the structural change, concomitant with the
acquisition of a GTP-bound conformation, which would relieve the
restraint exerted over Ras-GRF functions.
If this were the case, the mode of action of Cdc42 would not be
restricted solely to the stimulation of downstream effector proteins,
such as those bearing CRIB domains (7), when in an activated, GTP-bound
state. In addition, when GDP-loaded, Cdc42 may be acting as a
down-regulator of the Ras/MAPK pathway by inhibiting the activity of
Ras-specific GEFs. This double mechanism of action, although hitherto
unprecedented among small GTP-binding proteins, is no stranger in the
world of G proteins. Indeed, heterotrimeric G proteins function in that
way. As such, G
At present, whether this model can be applicable to Cdc42 needs to be
fully validated. As yet, we ignore the precise mechanism by which Cdc42
inhibits Ras-GRF. Our previous results (17) do not indicate a direct
physical interaction between Ras-GRF and Cdc42, at least in
vitro. That suggests the participation of additional components,
in a fashion also reminiscent of heterotrimeric G-proteins. Elucidating
the exact mechanism of action and the identity of these components is
necessary in future investigations.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
60 were generated by
polymerase chain reaction-directed mutagenesis and subcloned in
pCDNA3. The sequences of the oligonucleotides utilized are
available upon request. All constructs used the CMV promoter (18).
-glycerophosphate, 0.5% Nonidet-P40, 4% glycerol, 2 mM
sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride,
25 µg/ml leupeptin, and 25 µg/ml aprotinin. AU5-H-Ras was affinity
sequestered with bacterially synthesized GST·Raf Ras Binding Domain
(RBD) (amino acids 1-149). Immunoblots were performed as
described above using anti-AU5 antibody and quantitated by
densitometry, using the program NIH Image 1.60. Ras GTP levels were
related to the total Ras protein levels as determined by anti-AU5
immunoblotting in the corresponding cellular lysates.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
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Fig. 1.
Effects of Rho-GDI on the activation of the
Ras/MAPK pathway by Ras-GRF. A, Ras GTP loading was
determined as described under "Materials and Methods," in COS-7
cells cotransfected with Ras-GRF and AU5-H-Ras (1 µg), in the
presence of Cdc42 N17 or Rho-GDI (0.5 µg each) as shown. Data shows
average ± S.E. of six independent experiments. B,
effects of Rho-GDI on MAPK activation by Ras-GRF. Top,
HA-MAPK (ERK2) (1 µg) was cotransfected with constructs encoding
Ras-GRF (1 µg) or Ras V12 (0.2 µg) in addition to AU5-tagged
Rho-GDI and AU5-Cdc42 N17 (0.5 µg each) where indicated. Kinase
assays were performed in anti-HA immunoprecipitates using MBP as
substrate (see "Materials and Methods"). The figures show average
values of three independent experiments, expressed relative to the MAPK
activation levels detected in control cells, as quantitated by
phosphorimager. Bottom, expression levels of AU5 Rho-GDI and
Cdc42 N17 as determined by anti-AU5 immunoblotting. C,
effects of Rho-GDI on Ras-GRF-induced activation of MAPK triggered by
external stimuli. Suboptimal concentrations (0.1 µg) of Ras-GRF (+)
were cotransfected with constructs expressing Rho-GDI and Cdc42 N17
(0.5 µg each), as indicated. After 48 h, the cells were starved
for 12 h and treated with 1 µM ionomycin, 10 µM LPA, or EGF (100 ng/ml) for 5 min. D,
effects of p50Rho-GAP on MAPK activation by Ras-GRF. Ras-GRF (1 µg)
or Ras V12 (0.2 µg) were cotransfected with Rho-GDI or p50Rho-GAP
(0.5 µg each) as indicated. Bottom, expression levels of
the cotransfected HA-MAPK (1 µg) as determined by anti-HA
immunoblotting.
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Fig. 2.
Interactions of Rho-GDI and Cdc42.
A, association of Rho-GDI with Cdc42. AU5-Cdc42-wt and
AU5-Cdc42-QL (1 µg each) were transfected into COS-7 cells. After
12 h of serum-starvation, cells were lysed in HEM buffer.
Immunoprecipitation with anti-AU5 antibodies or preimmune
(PI) mouse serum was performed as described under
"Materials and Methods." Associated Rho-GDI was detected in the
immunoprecipitates by anti-Rho-GDI immunoblotting. Cdc42 expression
levels were determined by anti-AU5 Western blotting. TL,
total lysate. B, effects of Rho-GDI on Cdc42 subcellular
distribution. AU5-tagged Cdc42 was transfected into COS-7 cells with or
without Myc-tagged Rho-GDI as indicated. Cellular fractionation was
performed as described under "Materials and Methods." Cdc42 and
Rho-GDI cellular distributions were ascertained by anti-AU5 and
anti-Myc immunoblotting respectively. S, S100 soluble
fraction; P, P100 particulate fraction. C, lack
of interaction between Rho-GDI and Ras-GRF. Ras-GRF and AU5- Rho-GDI (1 µg each) were cotransfected into COS-7 cells as indicated (+).
Serum-starved and ionomycin-treated cells (1 µM/5 min)
were lysed in HEM buffer. Immunoprecipitation with anti-AU5 antibody or
preimmune (PI) mouse serum was performed as described.
Rho-GDI and Ras-GRF protein levels in total lysates (TL) and
immunoprecipitates (IP) were determined by anti-AU5 and
anti-Ras-GRF Western blotting. D, effects of Rho-GDI 60 on
the activation of MAPK by Ras-GRF. Ras-GRF (1 µg) was cotransfected
with AU5-tagged Rho-GDI or Rho-GDI
60 (0.5 µg each) as indicated.
Middle, expression levels of the cotransfected HA-MAPK (1 µg) as determined by anti-HA immunoblotting. Bottom,
expression levels of AU5-tagged Rho-GDI and Rho-GDI
60 as determined
by anti-AU5 immunoblotting.
60. This mutant lacks the
60 N-terminal amino acids, a region that contributes little to binding
but is necessary to inhibit nucleotide dissociation from Cdc42 (33). As
shown in Fig. 2D, the activation of MAPK brought about by
Ras-GRF was clearly inhibited by AU5-Rho-GDI, but was largely
unaffected by AU5-Rho-GDI
60 when expressed at similar levels. These
results clearly demonstrate that Rho-GDI inhibitory effects over
Ras-GRF reside with its ability to repress the dissociation of GDP from Cdc42.
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Fig. 3.
Effects of Rho-GDI on Ras-GRF cellular
distribution. Top, MAPK activation by Ras-GRF-wt and
membrane-bound Ras-GRF-CAAX in the absence ( ) or presence
(+) of Rho-GDI. Bottom, cellular distribution of Ras-GRF-wt
and Ras-GRF-CAAX in the absence (
) or presence (+) of
Rho-GDI, as determined by anti-Ras-GRF Western blotting. S,
S100 soluble fraction; P, P100 particulate fraction.
View larger version (44K):
[in a new window]
Fig. 4.
Ras-GRF function is unaffected by
constitutively active Cdc42. A, basal MAPK activation by
Ras-GRF is unaffected by activated Cdc42. Top, suboptimal
concentrations of Ras-GRF (0.2 and 0.5 µg), were cotransfected with
vector ( ) or with increasing concentrations of Cdc42-QL (0.5 and 1 µg), and MAPK assays were performed as described under "Materials
and Methods." The figures show average values of three independent
experiments, expressed relative to the MAPK activation levels detected
in control cells. Middle, levels of JNK activation under the
same circumstances. Bottom, Ras-GRF expression levels as
ascertained by anti-Ras-GRF immunoblotting. B,
Ras-GRF-mediated MAPK activation triggered by external stimuli is
unaffected by constitutively active Cdc42. Top, cells
transfected with suboptimal concentrations (0.2 µg) of Ras-GRF were
cotransfected with increasing concentrations of Cdc42-QL (0.5 and 1 µg). After 12 h of serum-starvation, cells were stimulated with
1 µM ionomycin or 10 µM LPA for 5 min, and
MAPK assays were performed. Bottom, expression levels of
Ras-GRF and Cdc42-QL. C, Cdc42 effector proteins do not
affect the activation of MAPK by Ras-GRF. Top, MAPK
activation in cells transfected with vector (
) or suboptimal
concentrations (0.2 µg) of Ras-GRF (+) were cotransfected with
constructs expressing activated forms of the indicated Cdc42 effector
proteins (1 µg each) or treated with 1 µM ionomycin for
5 min. Middle, JNK activation levels under the same
circumstances. Bottom, Expression levels of MLK3 and
AU5-tagged WASP.
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[in a new window]
Fig. 5.
Inhibition of Ras-GRF by Cdc42 can be rescued
by Rho-family GEFs. A, effects of Rho-family GEFs on
Ras-GRF-induced MAPK activation when down-regulated by Cdc42-wt. MAPK
activity levels in COS-7 cells transfected with Ras-GRF and Cdc42 wt (1 µg each) as shown (+), in addition to Dbl, Ost, Cdc42-QL, or empty
vector ( ) (1 µg each) and treated with 1 µM ionomycin
for 5 min where indicated. Data show average ± S.E. of three
independent experiments, expressed relative to the MAPK levels detected
in cells transfected with Ras-GRF only. B, effects of Dbl on
Ras-GRF-mediated MAPK activation when inhibited by Cdc42-wt or by
Rho-GDI. Ras-GRF (1 µg) (+) was cotransfected with Cdc42 wt or
Rho-GDI (1 µg each), and increasing concentrations (0.5, 1, and 2 µg) of Dbl or 2 µg of proto Dbl were also transfected where
indicated. Bottom, expression levels of Dbl and
proto-Dbl.
subunits when GTP-loaded directly stimulate
downstream effectors like PLC
or adenylate cyclase (42). Also, when
in a GDP-bound state, G
subunits act by sequestering G
dimers,
thereby preventing this component from interacting with its own
effectors (43). It is interesting to note that one of the main pathways
triggered by G
dimers, thus inhibited by GDP-bound G
subunits
and unaffected by the ectopic expression of constitutively active G
subunits, is the Ras/MAPK pathway (20). It is also noteworthy that in
the case of heterotrimeric G proteins the connection between the
G
complex and Ras exchange factors is not direct and implies the
presence of many intermediaries (44).
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ACKNOWLEDGEMENTS |
---|
We thank J. S. Gutkind and J. Field for providing us with reagents.
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FOOTNOTES |
---|
* This work was supported by Grant PM 98-0131 from the Spanish Ministry of Education and a grant from Fundación Marcelino Botín.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.
A predoctoral fellow of the Spanish Ministry of Education.
§ To whom correspondence should be addressed. Tel.: 34-91-5854886; Fax: 34-91-5854587; E-mail: pcrespo@iib.uam.es.
Published, JBC Papers in Press, April 2, 2001, DOI 10.1074/jbc.M011383200
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ABBREVIATIONS |
---|
The abbreviations used are: GAP, GTPase-activating protein; GEF, guanine nucleotide exchange factor; GDI, guanine nucleotide dissociation inhibitor; MAPK, mitogen-activated protein kinase; HA, hemagglutinin; GST, glutathione S-transferase; JNK, Jun N-terminal kinase; MBP, myelin basic protein; LPA, lysophosphatidic acid; EGF, epidermal growth factor; wt, wild type.
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