From the Division of Cellular Biochemistry, The Netherlands Cancer
Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands and the
Max-Planck-Institut fur Molekulare Physiologie,
Otto-Hahn-Strasse 11, 44227 Dortmund, Germany
Received for publication, May 5, 2000, and in revised form, September 12, 2000
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ABSTRACT |
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Rho family GTPases control numerous
cellular processes including cytoskeletal reorganization and
transcriptional activation. Rho GTPases are activated by guanine
nucleotide exchange factors (GEFs) which stimulate the exchange of
bound GDP for GTP. We recently isolated a putative GEF, termed
p190RhoGEF that binds to RhoA and, when overexpressed in neuronal
cells, induces cell rounding and inhibits neurite outgrowth. Here we
show that the isolated tandem Dbl homology/pleckstrin homology
domain of p190RhoGEF activates RhoA in vitro, but not Rac1
or Cdc42, as determined by GDP release and protein binding assays. In
contrast, full-length p190RhoGEF fails to activate RhoA in
vitro. When overexpressed in intact cells, however, p190RhoGEF
does activate RhoA with subsequent F-actin reorganization and serum
response factor-mediated transcription. Immunofluorescence studies show
that endogenous p190RhoGEF localizes to distinct RhoA-containing
regions at the plasma membrane, to the cytosol and along microtubules.
In vitro and in vivo binding experiments show
that p190RhoGEF directly interacts with microtubules via its C-terminal
region adjacent to the catalytic Dbl homology/pleckstrin homology
domain. Our results indicate that p190RhoGEF is a specific activator of
RhoA that requires as yet unknown binding partners to unmask its
GDP/GTP exchange activity in vivo, and they suggest that
p190RhoGEF may provide a link between microtubule dynamics and RhoA signaling.
Rho family GTPases act as molecular switches by cycling between an
inactive GDP-bound and an active GTP-bound state. In the GTP-bound
state they bind to effector proteins resulting in multiple downstream
responses. Among the best studied effects of Rho GTPases is the
regulation of the actin cytoskeleton. In fibroblasts, RhoA, Rac, and
Cdc42 induce the formation of stress fibers, lamellipodia, and
filopodia, respectively (1-3). Furthermore, Rho GTPases have been
implicated in the control of diverse responses such as cell adhesion,
motility, transcription activation, cell cycle progression, cytokinesis, and cell fate determination (4).
Rho GTPases are activated by the Dbl family of guanine nucleotide
exchange factors (GEFs),1
that stimulate the exchange of GDP for GTP and are characterized by a
Dbl homology (DH) domain in tandem with a pleckstrin homology (PH)
domain. The DH domain is responsible for catalytic activity (5), while
the PH domain is essential either for proper localization (6, 7) or for
full catalytic activity (8).
The Dbl family consists of more than 30 members, showing
tissue-specific distribution patterns and distinct specificity for Rho,
Rac, or Cdc42. Dbl family members contain various conserved structural
motifs, like Ca2+-binding EF-hands,
Src-homology 2 (SH2),
Src-homology 3 (SH3), and
Psd95/Dlg/ZO-1 (PDZ) homology
domains (9). Most of these domains presumably mediate protein-protein
or protein-lipid interactions, thereby coupling GEFs to upstream
regulators or downstream effectors.
Some GEFs have been shown to function in specific biological processes:
for example, Ect2 regulates cytokinesis (10) while Vav plays a role in
adaptive immunity (11). Furthermore, genetic studies have revealed the
importance of GEFs in development: DRhoGEF mediates cell shape changes
during gastrulation of Drosophila embryos (12), and UNC-73A
is required for cell and growth cone migration in Caenorhabditis
elegans (13). However, the biological roles of most Dbl family
GEFs remain unclear.
In neuronal cells, RhoA mediates neurite retraction and cell rounding
in response to G protein-coupled receptor agonists such as
lysophosphatidic acid and thrombin (14). Receptor stimulation induces translocation of RhoA from the cytosol to the plasma membrane (15) and subsequent activation of RhoA. RhoA activation and neurite
retraction are mediated by the G12/13 subfamily of trimeric G proteins (16) whose (17) activated We recently isolated a novel RhoA-binding protein of 190 kDa that
contains a DH/PH domain and, hence, is a putative GEF for Rho family
GTPases (20). We called this protein p190RhoGEF, and showed that it is
ubiquitously expressed. When overexpressed in neuronal cells,
p190RhoGEF mimics activated RhoA in stimulating cytoskeletal
contraction and preventing neurite outgrowth. It remains unclear,
however, to what extent the DH/PH catalytic domain of p190RhoGEF is a
bona fide activator of RhoA and/or other Rho family members.
p190RhoGEF contains several potential regulatory motifs, including an
N-terminal leucine-rich region, a cysteine-rich zinc-finger domain and
a C-terminal region that may form an In the present study, we have characterized p190RhoGEF in further
biochemical and cell biological detail. We show that p190RhoGEF is a
specific activator of RhoA both in vitro and in
vivo. We find that full-length p190RhoGEF, unlike its isolated
DH/PH domain, is inactive in vitro, indicating that
additional factors are required to activate p190RhoGEF in
vivo. Furthermore, we show that p190RhoGEF binds to and
colocalizes with microtubules, suggesting that p190RhoGEF may provide a
link between microtubule dynamics and RhoA signaling.
Cell Culture and Transfection--
COS-7 and N1E-115 cells were
cultured in Dulbecco's modified Eagle's medium containing 10% fetal
calf serum and antibiotics and NIH3T3 cells were cultured in
Dulbecco's modified Eagle's medium containing 10% newborn calf
serum and antibiotics. COS-7 cells were transfected using the
DEAE-dextran method, whereas NIH3T3 cells were transfected using
LipofectAMINE PLUS (Life Technologies) as described by the
manufacturer. For luciferase assays, transfection was stopped after
3 h by switching to culture medium containing 0.5% newborn calf serum.
Plasmids--
Cloning of full-length p190RhoGEF (bp 1-5400) and
Antibodies--
A polyclonal anti-RhoGEF serum, termed antibody
40, was made by immunizing rabbits with a GST fusion protein containing
the DH/PH domain of p190RhoGEF (bp 2534-3734). This plasmid was
generated by subcloning a SalI-EcoRI fragment
from pMT2smHA-DH/PH cDNA into pRP261, a derivative of pGEX-3X. Rho
and Rac proteins were detected with 26C4 (Santa Cruz Biotechnology) and
23A8 (Upstate Biotechnology) monoclonal antibodies. GST was detected
with the 2F3 monoclonal antibody. Cytoskeletal structures were analyzed
using rhodamine-conjugated phalloidin (Molecular probes) and monoclonal
anti-tubulin antibodies (Sigma). A polyclonal anti-tubulin antibody
(Sigma) was used for immunoprecipitation.
GDP/GTP Exchange Assay--
Recombinant Rho was prepared from
Escherichia coli using a bacterial expression system as
described previously (25). Single 9-cm dishes of COS-7 cells were
transiently transfected with 5 µg of expression plasmid encoding
HA-tagged DH/PH domain. In case full-length p190RhoGEF was assayed, 20 dishes were transfected. After 48 h cells were lysed in 300 µl
of 100 mM NaCl, 20 mM Tris-HCl, pH 8.0, 1 mM EDTA, 1 mM dithiothreitol, 0.2% Triton
X-100 and protease inhibitors. After clearance (13,000 rpm, 10 min),
cell lysates were precleared with nonspecific mouse immunoglobulins precoupled to protein A-Sepharose CL-4B (Amersham Pharmacia Biotech) and subsequently incubated with 12CA5 monoclonal antibodies coupled to
protein A-Sepharose. GDP dissociation from RhoA was assayed exactly as
described (26) using 30 µl of slurry (50% beads/buffer) per assay.
Control immune complexes were prepared from mock-transfected COS-7 cells.
Fluorescence Measurements--
Recombinant proteins of DH/PH
domain, truncated RhoA (amino acid residues 1-181), Rac1 and Cdc42
were produced as GST fusion proteins in E. coli strain BL21
(DE3) and purified by glutathione-Sepharose affinity chromatography
(Amersham Pharmacia Biotech, Uppsala, Sweden) as described (27). The
fluorescent derivative of GDP, mGDP
(2',3'-O-(N-methylanthraniloyl)guanosine
5'-diphosphate), in complex with the respective GTPases was prepared
(28). The nucleotide exchange activity of the DH/PH domain was
determined on an LS50B Perkin-Elmer spectrofluorometer (Norwalk, CT)
using 0.1 µM mGDP-bound GTPase, 20 µM GDP,
and different concentration of DH/PH domain in 30 mM
Tris-HCl, 5 mM MgCl2, 10 mM
KPO4, 3 mM dithioerythritol, pH 7.5, at
25 °C as described for Cdc25 (28). Exponential fits to the data were
done using the program Grafit (Erithacus software).
GTPase Pull-down Assay--
Preparation of GST-C21 and GST-PBD
and analysis of cellular activation of Rho and Rac was performed as
described previously (21, 22). In brief, transfected COS-7 cells were
lysed in Nonidet P-40 fish buffer. Lysates were centrifuged to remove
debris, and incubated for 45 min at 4 °C with 20 µl of
GSH-Sepharose loaded with 20 µg of either GST-C21 or GST-PBD. The
beads were washed three times with the Nonidet P-40 buffer and the
bound proteins were separated by SDS-PAGE and Western blotting.
Complex Formation Assay--
To determine interactions between
p190RhoGEF and GST-GTPases, transfected COS-7 cells were lysed in a
buffer containing 0.1% Triton X-100, 150 mM NaCl, 50 mM Tris-HCl, pH 7.4, 10 mM EDTA, and protease
inhibitors. Lysates were incubated for 2 h at 4 °C with
E. coli expressed, GST-RhoA, GST-Rac, or GST-Cdc42 bound to
GSH-Sepharose. Samples were washed three times in the 0.1% Triton
X-100 buffer and the bound proteins were subjected to SDS-PAGE and
Western blotting using monoclonal antibody 12CA5. Experiments testing
the nucleotide dependence of the interactions were performed similarly,
except that GST-RhoA was preloaded in a buffer containing 20 mM Tris-HCl, 1 mM dithiothreitol, 10 mM EDTA, 5 mM MgCl2, and 50 µM of either GDP or GTP Immunofluorescence--
N1E-115 and NIH3T3 cells were grown on
glass coverslips. After overnight culturing in serum-free medium, the
cells were fixed in 3.7% formaldehyde and were processed for
immunofluorescence as described (15) using 12CA5, antibody 40, or
anti-RhoA antibodies as indicated. Rhodamine-conjugated phalloidin and
anti-tubulin antibody were used to stain F-actin and tubulin, respectively.
Luciferase Assay--
Luciferase activities were measured
24 h after the start of the transfection by using the
Dual-Luciferase Reporter Assay System (Promega) as described by the
manufacturer. In the Dual-Luciferase Reporter assay the activities of
firefly and Renilla luciferases are measured sequentially
from a single sample. Luminescence intensities were measured using the
TD 20/20 luminometer (Turner Designs model, Promega) counting the ratio
of luminescence between the firefly and the Renilla
luciferase reactions.
Microtubule Binding Assay--
Transfected COS-7 cells were
lysed in tubulin buffer (Cytoskeleton) containing 0.1% Triton X-100.
Lysates were centrifuged at 100,000 × g for 1 h
in a Beckman airfuge. Supernatant fractions were incubated for 30 min
at room temperature with purified, Taxol-stabilized microtubules, which
were generated using the Microtubule/Tubulin Biochem Kit from
Cytoskeleton. Alternatively, purified GST fusion proteins were prepared
and incubated with microtubules. Microtubules were pelleted by
high-speed centrifugation (100,000 × g for 1 h)
and supernatant and pellet fractions were subjected to SDS-PAGE and
immunoblotting using anti-HA or anti-tubulin antibodies.
Metabolic Labeling--
p190RhoGEF was expressed in COS-7 cells
as described above. Two days after transfection cells were
serum-starved for 1 h, labeled with
[35S]methionine/cysteine for 4 h, and then lysed in
a buffer containing 0.2% Triton X-100, 100 mM NaCl, 20 mM Tris, pH 7.4, 1 mM dithiothreitol, 1 mM EGTA, and 5 mM MgCl2. Lysates
were clarified and precipitated with 12CA5 or tubulin antibodies
precoupled to protein A-Sepharose. Reprecipitation was performed by
boiling the immunoprecipitates for 5 min in lysis buffer containing
0.1% SDS, followed by adding 9 volumes of lysis buffer and antibodies
precoupled to protein A-Sepharose. Precipitates were washed
extensively, subjected to SDS-PAGE, and analyzed by autoradiography.
p190RhoGEF: Homology to Rho-Specific GEFs--
Fig.
1 shows the structural features of
p190RhoGEF in comparison to other Dbl family GEFs. The DH/PH domain of
p190RhoGEF is most closely related to the DH/PH domains in the
Rho-specific GEFs Brx and proto-Lbc (52% identity), Lfc (50%), GEF-H1
(48%), and p115RhoGEF (25%) (22-26). Aside from its DH/PH domain,
p190RhoGEF contains an N-terminal leucine-rich region and a
cysteine-rich domain that could form a zinc finger. C-terminal of the
DH/PH domain, p190RhoGEF contains a region with high propensity to form coiled coils, as is also observed in GEF-H1. We note that p190RhoGEF lacks an apparent LH domain that can interact directly with G protein
p190RhoGEF Activates RhoA, but neither Rac nor Cdc42--
We
examined whether p190RhoGEF can stimulate GDP/GTP exchange on Rho
family GTPases in vitro. Specific exchange activity was determined using an assay in which the dissociation of fluorescently labeled GDP from RhoA, Rac1, or Cdc42 is monitored in real time (28).
As shown in Fig. 2A, the
isolated DH/PH domain promotes nucleotide exchange on RhoA, but not on
Rac-1 or Cdc42. The p190RhoGEF catalytic domain stimulates the rate of
nucleotide exchange on RhoA about 40-fold (from 0.0048 min
To examine whether p190RhoGEF acts as a Rho-specific GEF in
vivo, we measured endogenous RhoA activation by affinity
precipitation. In this assay, the Rho-binding domain of rhotekin
(GST-C21) is used to specifically precipitate GTP-bound RhoA from cell
extracts (30). Similarly, the Cdc42/Rac interactive binding domain of the Rac/Cdc42 effector protein PAK (GST-PBD) was used to pull-down activated Rac (31). Fig. 2B shows that expression of the
isolated DH/PH domain of p190RhoGEF in COS-7 cells activates RhoA but
not Rac1. Conversely, the Rac-specific GEF Tiam-1 (32) activates Rac-1
but not RhoA in COS-7 cells (Fig. 2B). Both RhoA and Rac1 appear as doublets, presumably representing differently isoprenylated forms of these GTPases (33). Taken together, these results demonstrate that the catalytic domain of p190RhoGEF acts specifically on RhoA both
in vitro and in vivo.
Interaction of p190RhoGEF with Rho GTPases--
GEF-mediated
stimulation of GDP/GTP exchange involves a direct interaction between
GEF and the small GTPase. Although these complexes form transiently,
they can be isolated from cells (5). Since p190RhoGEF shows in
vitro and in vivo exchange activity toward RhoA, we
examined whether the two proteins can form a complex. To this end, we
precipitated full-length p190RhoGEF (HA-tagged) from COS-7 cell lysates
using immobilized GST fusion proteins of RhoA, Rac1, or Cdc42. As shown
in Fig. 3A, we find that
p190RhoGEF binds to RhoA but not to Rac1 or Cdc42.
GEFs stimulate GDP/GTP exchange by inducing a conformational change in
the GTPase, resulting in the release of bound GDP. GEFs stabilize a
transition state, in which interaction occurs with nucleotide-free
GTPase. Since cellular GTP levels are much higher than GDP levels, this
leads to net GDP/GTP exchange on the GTPase (34). We examined the
binding of p190RhoGEF to RhoA in both nucleotide-bound and
nucleotide-free states. Nucleotide was depleted from GST-RhoA by
chelating magnesium from the assay buffer. Alternatively, GST-RhoA was
loaded with either GDP In Vitro Activity of Full-length Versus Truncated
p190RhoGEF--
Aside from the tandem DH/PH domain, p190RhoGEF
contains several structural elements that may play a role in regulating
its activity and/or intracellular localization. We compared the
in vitro exchange activity of the full-length protein to
that of the isolated catalytic DH/PH domain. Fig.
4A shows that, while the DH/PH
domain promotes GDP/GTP exchange on RhoA, the full-length protein is
completely inactive. About equal amounts of proteins were precipitated
from the cell lysates (Fig. 4A, lower panel). Even under
conditions where the full-length protein was used in excess of the
DH/PH domain, exchange activity was only detected with the isolated
DH/PH domain (Fig. 4B). Although the Rho-specific GEFs Ect-2
and GEF-H1 are active as full-length proteins (10, 36), the activities
of most GEFs have only been tested with the isolated DH/PH domains. Of
note, in fibroblast transformation assays, Lbc, Dbl, Ost, and
p115RhoGEF are only active when truncated (5, 29, 37-39). The striking
difference in in vitro activity between full-length and
truncated p190RhoGEF suggests that the full-length protein contains an
intrinsic autoinhibitory domain and requires cellular factors to be
activated.
p190RhoGEF Induces Actin Stress Fibers and Activates SRF-mediated
Transcription--
When activated, RhoA, Rac, and Cdc42 induce
specific rearrangements of the actin cytoskeleton. In fibroblasts, RhoA
induces stress fiber formation, whereas lamellipodia and filopodia are formed in response to activated Rac and Cdc42, respectively. We examined the cytoskeletal changes induced by full-length p190RhoGEF in
NIH-3T3 fibroblasts. As shown in Fig.
5A, p190RhoGEF mimics active
RhoA in inducing the formation of actin stress fibers, with no sign of
the formation of membrane ruffles or filopodia. These findings in 3T3
cells are consistent with the observation that p190RhoGEF induces
cytoskeletal contraction and inhibits neurite outgrowth in N1E-115
neuroblastoma cells (20).
In addition to their effects on the actin cytoskeleton, Rho GTPases can
modulate gene transcription. In particular, activated forms of RhoA,
Rac1, and Cdc42 can activate the serum response factor (SRF) (40).
We examined whether p190RhoGEF could activate SRF-mediated
transcription by using a reporter plasmid in which the luciferase gene
is under the control of a c-Fos serum-responsive promotor
element (pSRE.L). This modified SRE element depends on SRF, but not on
Elk-1 activity, making it a specific reporter for signaling by Rho
family GTPases (24). NIH-3T3 cells were transiently co-transfected with
pSRE.L and full-length p190RhoGEF. As shown in Fig. 5B,
expression of p190RhoGEF results in a 5-fold activation of SRF, similar
to what is observed after serum stimulation (Fig. 5B).
We also used a point mutant of p190RhoGEF in which residue
Tyr1003 is replaced by an alanine (Y1003A mutant).
Tyr1003 is a conserved residue in the catalytic DH domain
of all known GEFs; its mutation in Lbc renders this GEF biologically
inactive (37). Similarly, mutant p190RhoGEF(Y1003A) is biologically
inactive as it fails to promote cytoskeletal contraction when expressed in N1E-115 cells.2 As shown
in Fig. 5B, co-transfection of the inactive Y1003A mutant with pSRE.L does not lead to enhanced SRF-mediated transcription, indicating that p190RhoGEF exchange activity (i.e.
activation of RhoA) is required for the transcriptional
response. Taken together, our findings demonstrate that
full-length p190RhoGEF is active in vivo, and acts as a
Rho-specific GEF that promotes stress fiber formation, cytoskeletal
contraction, and activation of SRF-mediated gene transcription.
Intracellular Localization of p190RhoGEF--
To obtain possible
clues to the biological role(s) of p190RhoGEF, we analyzed its
intracellular localization in neuronal N1E-115 cells by
immunofluorescence using polyclonal antibodies (number 40) against the
catalytic DH/PH domain. N1E-115 cells were used because they contain
relatively high levels of p190RhoGEF mRNA (20). Antibody
specificity was demonstrated by the following control experiments: (i)
preimmune sera gave only a faint nuclear and cytosolic background
staining; (ii) signals were blocked by preincubation of the antibodies
with the GST-DH/PH antigen; and (iii) transfected HA-tagged p190RhoGEF
was detected by both antibody 40 and anti-HA in exactly overlapping
staining patterns (results not shown).
Immunofluorescence analysis shows that endogenous p190RhoGEF in N1E-115
cells is found at various intracellular locations: (i) in the
cytoplasm; (ii) at distinct regions of the plasma membrane (Fig.
6, A, arrowheads, B,
left panel); and (iii) along filamentous structures (Fig.
6C). Plasma membrane and cytosol staining was observed in
the majority of the cells, while about 20% of the cells showed
filamentous staining. We analyzed the p190RhoGEF localization pattern
in further detail by examining its colocalization with RhoA, F-actin,
and tubulin.
Since RhoA is also found in the cytosol and at the plasma membrane
(15), we performed double labeling immunofluorescence experiments using
anti-RhoA and anti-p190RhoGEF antibodies. Although RhoA staining did
not fully overlap with p190RhoGEF staining, it is seen that both
proteins localize to the same regions at the plasma membrane (Fig.
6B). It seems likely that these are the sites where RhoA is
activated by p190RhoGEF. Through double labeling experiments, we find
that these regions are distinct from CD44- or caveolin-containing
plasma membrane domains (results not shown). We previously reported
that lysophosphatidic acid activates RhoA and induces its translocation
from the cytosol to the plasma membrane in N1E-115 cells (15, 16). In
preliminary experiments, we did not observe clear translocation of
cytosolic p190RhoGEF to the plasma membrane in response to
lysophosphatidic acid (results not shown).
The filamentous structures that are stained with anti-p190RhoGEF
antibody in a subpopulation of the cells (Fig. 6, A, C, and D) do not colocalize with actin filaments, as visualized by
rhodamine-conjugated phalloidin (Fig. 6C). When the cells
were double-stained with anti-p190RhoGEF and anti-tubulin antibodies,
p190RhoGEF is detected at the radial and cortical microtubule systems
(Fig. 6D).
p190RhoGEF Interacts with Microtubules via Its C Terminus--
We
next examined, by biochemical means, whether p190RhoGEF associates with
microtubules. To this end, we used an in vitro assay system
using purified, Taxol-stabilized microtubules and cytosolic COS-7 cell
extracts containing either full-length p190RhoGEF or deletion mutants
lacking either the N- or C-terminal regions. Since microtubules can be
pelleted by high-speed centrifugation, binding of p190RhoGEF to
microtubules can be tested in co-sedimentation assays. Fig.
7A (left panel)
shows that, in the absence of microtubules, full-length p190RhoGEF and
the
To determine whether the interaction of p190RhoGEF with microtubules is
direct or indirect, GST fusion proteins were made that encompass either
the complete C-terminal part (deleted in In Vivo Interaction of p190RhoGEF and Tubulin--
To determine
whether p190RhoGEF interacts with microtubules in intact cells, we
immunoprecipitated tubulin from metabolically labeled COS-7 cells,
overexpressing either full-length p190RhoGEF or the deletion mutant
which lacks the C-terminal domain (RhoGEF Microtubule Dynamics and p190RhoGEF Activity--
Microtubule
dynamics and F-actin reorganization are interconnected via Rho family
GTPases (41). For example, microtubule growth activates Rac1 (42),
whereas microtubule disruption leads to RhoA activation (43). Thus, the
observed interaction of p190RhoGEF with microtubules may serve to link
microtubule dynamics to RhoA activation. To test this, we employed the
RhoA activation pull-down assay. Full-length p190RhoGEF was expressed
in COS-7 cells and the cells were treated with nocodazole to disrupt
microtubules or were left untreated. As shown in Fig.
9, both nocodazole treatment and
expression of p190RhoGEF lead to activation of endogenous RhoA.
However, we find that microtubule disruption does not affect the
ability of overexpressed p190RhoGEF to activate RhoA. This is not due
to maximal RhoA activation in these cells since overexpression of the
isolated DH-PH domain leads to a more prominent activation of RhoA.
Similarly, the addition of Taxol-stabilized microtubules to either
full-length p190RhoGEF or the isolated DH/PH domain in in
vitro exchange reactions did not affect RhoGEF exchange activity
(not shown). Further work is needed to establish how microtubule
binding may influence p190RhoGEF action or vice versa.
We have shown that p190RhoGEF is a RhoA-specific GEF and thus
belongs to the same subclass of Dbl family GEFs as Lbc, Lfc, GEFH-1,
and p115RhoGEF. This conclusion is based upon various lines of
evidence. First, the catalytic core domain, RhoGEF-DH/PH, promotes GDP
release from RhoA, but not from Rac or Cdc42, in vitro.
Second, binding assays show that p190RhoGEF interacts with RhoA, but
not with Rac or Cdc42. Third, we find that RhoGEF-DH/PH activates
endogenous RhoA but not Rac1 in intact cells, as measured by pull-down
assays. Finally, p190RhoGEF mimics activated RhoA in stimulating stress
fiber formation and SRF-mediated transcription in 3T3 cells.
Dbl family GEFs show tissue-specific distribution patterns and distinct
substrate specificities (30). In addition, conserved structural motifs
in distinct GEF family members have presumably evolved to couple
specific upstream signals to activation of small GTPases. Since
full-length p190RhoGEF is inactive in vitro, but not
in vivo, p190RhoGEF activity must be regulated by cellular factors. We and others have shown that RhoA activation by G
protein-coupled receptors is mediated via the
G12/G13 family of heterotrimeric G proteins
(16, 44, 45). Indeed, G Our findings further indicate that a pool of p190RhoGEF may be
regulated through direct interaction with microtubules. We detect
interaction with microtubules in intact cells and find that it is
mediated via a C-terminal region in p190RhoGEF. Three other GEFs,
notably GEFH-1, Lfc, and Ect2, have also been reported to localize to
microtubules (10, 36, 47). Changes in microtubule stability are known
to impinge on Rho signaling (41); microtubule growth activates Rac
(42), whereas microtubule destabilization promotes RhoA activation
(43). It has been suggested that an unidentified RhoA activator resides
on microtubules from which it may be released upon microtubule
depolymerization (41). One obvious candidate is p190RhoGEF, although
our preliminary studies do not show enhanced p190RhoGEF-mediated
activation of RhoA in microtubule-disrupted cells. However, these
experiments should be interpreted with care. Subtle changes in
microtubule stability may locally regulate microtubule-bound p190RhoGEF
and such effects could easily be missed in nocodazole-treated cells
that overexpress p190RhoGEF. Moreover, nocodazole affects a number of
signaling pathways that may directly or indirectly influence p190RhoGEF or RhoA activity (48). An alternative possibility is that microtubule binding activates p190RhoGEF, although our preliminary experiments to
test this possibility have remained inconclusive. Interestingly, it has
recently been reported that activation of RhoA causes rapid stabilization of a subset of microtubules via an as-yet-unknown mechanism (49). One possibility that remains to be examined is that
p190RhoGEF may function as a "scaffold" to bring microtubules into
proximity with activated RhoA and thereby regulates RhoA-mediated stabilization of microtubules.
Although we do not know which upstream signals regulate p190RhoGEF
activity, we find that in neuronal cells p190RhoGEF and RhoA localize
to the same regions at the plasma membrane. Presumably, these are the
sites of RhoA activation by p190RhoGEF. One possibility is that these
regions are equivalent to "puncta," as described in epithelial
cells (50), which are plasma membrane regions where microtubules terminate.
The localization of p190RhoGEF to microtubules could also allow it to
regulate microtubule-bound effectors distinct from RhoA. Such an
additional signaling mode has been reported for Lfc, which can activate
Jun N-terminal kinase (JNK) in a Rac-dependent manner without promoting GDP/GTP exchange on Rac (47). A possible link between
p190RhoGEF and JNK comes from a recent study showing that p190RhoGEF
can directly bind to a JNK-interacting protein, termed JIP-1 (51).
JIP-1 serves as a scaffold protein and selectively mediates signaling
by the "mixed-lineage" kinase to JNK activation (52). Like
p190RhoGEF and Lfc, mixed-lineage kinase and JNK both localize to
microtubules (47, 53). However, we have not been able to detect changes
in JNK activity following overexpression of p190RhoGEF in COS-7 cells.
Therefore, it remains unclear whether the localization of p190RhoGEF at
microtubules and its binding to JIP are functionally coupled. In any
case, the possibility that p190RhoGEF may control a wider range of
cellular processes than those predicted by its GDP/GTP exchange
activity should be taken into account.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
subunits can bind to and
activate at least two distinct RhoGEFs, notably p115RhoGEF (18) and
PDZ-RhoGEF (19).
-helical coiled-coil (Fig.
1).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Np190RhoGEF (bp 2156-5400) has been described (20).
pcDNA3-HA-p190RhoGEF was generated in two steps. First, a polymerase
chain reaction was performed on pBSKML4 using primers
gctctagaaggtaccccatggagttgagctgcagtg and T3, followed by digestion with
XbaI-ApaI and subcloning into pcDNA3-HA,
resulting in pcDNA3-HA-
RhoGEF. Subsequently, an
ApaI-ApaI fragment of full-length p190RhoGEF (bp
625-5400) was cloned into pcDNA3-HA-
RhoGEF resulting in the
final construct pcDNA3-HA-p190RhoGEF (bp 105-5400). Removal of
a BstEII-NotI fragment from
pcDNA3-HA-p190RhoGEF generated
Cp190RhoGEF (bp 105-4135). The
p190RhoGEF-DH/PH (bp 2534-3734) deletion mutant was generated by using
primers cccggtcgacttctctgtggatcgacct and ccccgcggccgcggcca. Polymerase
chain reaction was performed on the full-length p190RhoGEF cDNA,
followed by digestion with SalI and NotI and
subcloning into pMT2sm-HA. The Quick mutagenesis kit (Stratagene) was
used to make a single-point mutation, Y1003A, in
pcDNA3-HA-p190RhoGEF using the primers
cccagcgcatcacaaaggccccagtcttggtgg and
ccaccaagactggggcctttgtgatgcgctggg. The GST-C terminus (bp 4189-5400)
construct was prepared by subcloning a
BamHI-EcoRI fragment from
pcDNA3-HA-p190RhoGEF into pRP259, a derivative of pGEX-1N. The
construction of pGEX-C21 (GST-C21), pGST-PAK-CD (GST-PBD), pMT2smHA-C1199Tiam1 and SRE.L-luciferase plasmids are described elsewhere (21-24).
S for 1 h at 4 °C.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
subunits, as in p115RhoGEF (18).
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Fig. 1.
Schematic representation of the p190RhoGEF
protein and closely related Dbl family members. Amino acid
sequences of the different Dbl family exchange factors were aligned
with Clustal X and percentages of sequence identity were calculated
using Genedoc. Zn, zinc finger motif;
L-rich, leucine-rich region; COIL,
coiled-coil region; EF, EF-hand motif;
-H,
-helical region; P,
proline-rich region; LH, Lsc-homology domain.
1 to 0.193 min
1). We find a similar
stimulation using the DH/PH domain of p115RhoGEF (data not shown),
which is 4-fold higher than what has been reported for
p115RhoGEF-mediated GDP/GTP exchange (29).
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Fig. 2.
p190RhoGEF specificity. A,
the RhoGEF-DH/PH domain specifically stimulates GDP-release from RhoA.
Addition of 0.4 µM DH/PH to 0.1 µM
RhoA·mGDP and 20 µM GDP (upper panel)
resulted in a 40-fold stimulation of the nucleotide dissociation rate
(0.193 min 1) as compared with the intrinsic rate (0.0048 min
1). However, under the same conditions 0.6 µM DH/PH hardly affected the intrinsic rates of both
Rac1·mGDP (middle panel, 0.033 min
1 and
0.061 min
1) and Cdc42·mGDP (lower panel,
0.011 min
1 and 0.012 min
1) which revealed
7- and 2-fold higher intrinsic nucleotide dissociation rates,
respectively, as compared with that of RhoA. B, analysis of
in vivo GTP loading of RhoA and Rac by RhoGEF-DH/PH and
Tiam1. COS-7 cells were transiently transfected with empty vector,
HA-DH/PH, or HA-C1199Tiam1. Cells were serum-starved overnight and
lysed 48 h after transfection. Activated RhoA and Rac were
affinity precipitated using immobilized GST-C21 and GST-PBD,
respectively. Proteins were subjected to SDS-PAGE and immunoblotting
using anti-RhoA or anti-Rac antibodies. The amount of RhoA and Rac in
the total lysates is shown in the middle panels and the
amount of expressed GEF proteins (using anti-HA antibodies) in the
lower panels. p190RhoGEF acts as a Rho-specific exchange
factor in vivo.
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Fig. 3.
Characterization of p190RhoGEF binding to
RhoA. A, binding of full-length p190RhoGEF to GST-RhoA,
GST-Rac, and GST-Cdc42. COS-7 cells were transiently transfected with
HA-tagged full-length p190RhoGEF. Cells were lysed 48 h after
transfection and GST-RhoA, GST-Rac, and GST-Cdc42 were added to the
lysates. After washing, the pull-downs were analyzed for the presence
of p190RhoGEF by SDS-PAGE and Western blotting using anti-HA antibody.
Blots were reprobed with anti-GST antibody to assess that equal amounts
of the GST-GTPases were used. Full-length p190RhoGEF binds to RhoA, but
not to Rac or Cdc42. B, binding of full-length p190RhoGEF to
nucleotide-depleted (ND), GDP- or GTP S- (GTP) loaded
GST-RhoA. Binding of full-length p190RhoGEF to GST-RhoA was tested as
in A. GST-RhoA was depleted from nucleotide by preincubation
in a buffer containing 10 mM EDTA (ND).
Alternatively, GST-RhoA was loaded in a buffer containing 10 mM EDTA, 5 mM MgCl2, and 50 µM of either GDP or GTP
S (GTP). It is seen
that full-length p190RhoGEF has a slight preference for interaction
with the nucleotide-depleted form of RhoA.
S or GTP
S. Fig. 3B shows that
p190RhoGEF associates with all three forms of RhoA, although it appears
that there is a slight preference for binding to nucleotide-depleted
RhoA. This is in agreement with previous findings on the binding of
other GEFs to small GTPases (5, 35).
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Fig. 4.
In vitro exchange activity of
full-length and truncated p190RhoGEF. COS-7 cells were transiently
transfected with empty vector, HA-tagged full-length p190RhoGEF or
DH/PH domain. Lysates were prepared and HA-tagged proteins were
immunoprecipitated using anti-HA antibodies. The precipitates were
tested for GEF activity by measuring the dissociation of
[3H]GDP from E. coli expressed GST-RhoA, in a
nitrocellulose filter-binding assay (see "Experimental
Procedures"). To control for the amount of immunoprecipitated
p190RhoGEF, precipitates were separated by SDS-PAGE and analyzed by
Western blotting using anti-HA antibody (lower panels). The
bands observed at about 68 and 80 kDa are background signals of the
anti-HA antibody and are also observed in the control
immunoprecipitates. A, time course of p190RhoGEF and DH/PH
domain-mediated release of [3H]GDP from GST-RhoA.
B, p190RhoGEF and DH/PH domain-mediated
[3H]GDP dissociation from GST-RhoA measured at
t = 30 min. The experiment was repeated three times
with similar results. In contrast to the isolated DH/PH domain,
full-length p190RhoGEF is completely inactive in
vitro.
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Fig. 5.
p190RhoGEF effects on the actin cytoskeleton
and on gene transcription. A, NIH3T3 cells, grown on
glass coverslips, were transfected with HA-tagged full-length
p190RhoGEF or constitutive active RhoA (RhoL63). Cells were cultured in
1% serum overnight and analyzed by immunofluorescence using anti-HA
antibodies and rhodamine-conjugated phalloidin to stain filamentous
actin. It is seen that overexpressed p190RhoGEF, like activated RhoA,
induces the formation of actin stress fibers. B, NIH3T3
cells were co-transfected with 1 µg of SRE.L-luciferase
reporter plasmid, 0.1 µg of Renilla luciferase, and 2 µg
of empty vector or HA-tagged full-length p190RhoGEF or
p190RhoGEF(Y1003A). One day after transfection the cells were
stimulated with 10% NCS for 6 h, where indicated, lysed
and luciferase activities were measured. Values were normalized to the
Renilla luciferase activity. Experiments were carried out in
duplicate and were repeated twice. p190RhoGEF activates SRF-mediated
gene transcription via RhoA.
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Fig. 6.
Intracellular localization of
p190RhoGEF. A, N1E-115 cells were grown on glass
coverslips and analyzed by indirect immunofluorescence for endogenous
p190RhoGEF using antibody 40. Both panels show that endogenous
p190RhoGEF is localized in the cytoplasm, at membrane patches
(arrowheads) and along filamentous structures. B,
double labeling of N1E-115 cells with antibody 40 (left
panel) and anti-RhoA (middle panel) shows both proteins
being localized at the same plasma membrane subdomains. C,
costaining of N1E-115 cells with antibody 40 (left panel)
and rhodamine-conjugated phalloidin (right panel) shows
that the p190RhoGEF positive filaments do not colocalize with actin
filaments. D, double labeling with antibody 40 (left
panels) and anti-tubulin antibody (right panels) shows
that a pool of p190RhoGEF is localized to the microtubular network. In
B, C, and D, the left panels show
p190RhoGEF staining, middle panels RhoA, F-actin, or tubulin
staining, as indicated; the merged staining pattern is shown in the
right panels.
N and
C truncation mutants remain in the high-speed
supernatant. In the presence of microtubules, however, we find that
both full-length p190RhoGEF and the
N mutant, but not the
C
mutant, cosediment with microtubules during high-speed centrifugation
(Fig. 7A). These results suggest that p190RhoGEF binds to
microtubules, either directly or indirectly, and that the C-terminal
region is essential for this interaction.
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Fig. 7.
Direct binding of p190RhoGEF to
microtubules. A, cosedimentation of p190RhoGEF
(mutants) and microtubules. COS-7 cells were transiently transfected
with HA-tagged full-length, C-, or
N-RhoGEF. Cells were lysed
48 h after transfection and cytosolic extracts were prepared by
high-speed clearance of the lysates. Purified, Taxol stabilized
microtubules were added where indicated and p190RhoGEF (mutants) were
allowed to bind for 30 min at room temperature. Microtubules were then
pelleted by high-speed centrifugation. Supernatant (S) and
pellet (P) fractions were subjected to SDS-PAGE and
immunoblotting using anti-HA antibodies to detect p190RhoGEF and
anti-tubulin. It is seen that full-length and
N-, but not
C-,
RhoGEF cosediment with microtubules. B, GST fusion proteins
encompassing either the complete C-terminal region or the isolated
DH/PH domain of p190RhoGEF (see schematic representation) were
incubated with purified microtubules and used in a cosedimentation
assay as in A. The C terminus of p190RhoGEF cosediments with
microtubules.
C; residues 1343-1693) or
the catalytic DH/PH domain (residues 811-1210). Using the purified
fusion proteins in a microtubule cosedimentation assay, we find that
the C terminus of p190RhoGEF cosediments with microtubules, whereas the
DH/PH domain does not (Fig. 7B). Taken together, these
experiments indicate that a pool of p190RhoGEF directly interacts with
microtubules in vivo, and that microtubule interaction
is mediated by the C-terminal domain of p190RhoGEF.
C). Fig.
8 shows that the tubulin antibody
coprecipitates p190RhoGEF, but not the
C deletion mutant. To verify
that the 190-kDa protein is indeed HA-tagged RhoGEF, the
immunoprecipitates were denatured and re-precipitated using anti-HA
antibodies. Fig. 8 shows that anti-HA antibodies indeed re-precipitate
full-length p190RhoGEF from tubulin immunoprecipitates. Thus, in
agreement with the immunofluorescence studies and the in
vitro assays, p190RhoGEF interacts with tubulin in intact cells
via its C-terminal domain.
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Fig. 8.
Coimmunoprecipitation of p190RhoGEF and
tubulin. COS-7 cells were transiently transfected with empty
vector, HA-tagged full-length p190RhoGEF, or deletion mutant
RhoGEF C. Cells were metabolically labeled with
[35S]methionine/cysteine and lysed 48 h after
transfection. Tubulin and HA-RhoGEF were immunoprecipitated
(IP) using anti-tubulin (T) and anti-HA
(HA) antibodies, respectively, and either subjected to
SDS-PAGE or denatured and then re-precipitated using anti-HA
antibodies, as described under "Experimental Procedures." It is
seen that full-length p190RhoGEF, but not RhoGEF
C,
coimmunoprecipitates with tubulin.
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Fig. 9.
Effect of microtubule disruption on
p190RhoGEF-mediated RhoA activation. COS-7 cells were transfected
with empty vector, HA-tagged full-length p190RhoGEF, or DH/PH domain as
indicated. Cells were cultured overnight in serum-free medium and were
treated with nocodazole where indicated (1 h, 10 µM).
Immunofluorescence analysis using anti-tubulin antibodies showed that
this results in a complete disappearance of the microtubular network
(not shown). The activity state of RhoA was determined as in Fig.
2B. Both p190RhoGEF as well as nocodazole induce activation
of RhoA but p190RhoGEF-mediated activation of RhoA is not potentiated
in cells lacking microtubules.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
12 and G
13 can
directly bind to both p115RhoGEF and PDZ-RhoGEF via the N-terminal LH
domain present in both GEFs. The interaction of p115RhoGEF with the
G
13 subunit results in an activation of p115RhoGEF
exchange activity (18, 46). The p190RhoGEF sequence, however, does not
contain an LH domain. Moreover, we have not been able to detect an
interaction between p190RhoGEF and either G
12 or
G
13 in transfected COS-7 cells.3 It thus seems
unlikely that p190RhoGEF is activated by G
12/13 subunits.
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ACKNOWLEDGEMENTS |
---|
We thank J. Collard for providing GST-C21, GST-PBD, and C1199Tiam1, D. Wu for providing the SRE.L-luciferase reporter plasmid, and B. Giepmans for subcloning pRP-GST-CT. M. R. A. acknowledges the continuous support of F. Wittinghofer.
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FOOTNOTES |
---|
* This work was supported by the Dutch Cancer Society and the Netherlands Organization for Scientific Research.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.
§ To whom correspondence should be addressed: Div. of Cellular Biochemistry, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands. Tel.: 31-20-512-1971; Fax: 31-20-512-1989; E-mail: wmoolen@nki.nl.
Published, JBC Papers in Press, October 31, 2000, DOI 10.1074/jbc.M003839200
2 F. P. G. van Horck, W. H. Moolenaar, and O. Kranenburg, manuscript in preparation.
3 F. P. G. van Horck, W. H. Moolenaar, and O. Kranenburg, unpublished observations.
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ABBREVIATIONS |
---|
The abbreviations used are:
GEF, guanine
nucleotide exchange factor;
SRF, serum-response factor;
GTPS, guanosine 5'-3-O-(thio)triphosphate;
DH, Dbl homology;
PH, pleckstrin homology;
SH2, Src homology domain 2;
SH3, Src homology
domain 3;
bp, base pair(s);
PAGE, polyacrylamide gel electrophoresis;
GST, glutathione S-transferase;
GDP
S, guanyl-5'-yl
thiophosphate;
HA, hemagglutinin;
JNK, Jun N-terminal kinase.
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