1 SmithKline Beecham Laboratoires Pharmaceutiques,
Unité de Biologie Cardiovasculaire, 4 rue du
Chesnay Beauregard, BP 96205, 35760
Saint-Grégoire, France
2 Centre de Recherches en Biochimie
Macromoléculaire-Centre National de la
Recherche Scientifique, UPR 1086, 1919 Route de Mende, 34293 Montpellier Cedex
05, France
Authors for correspondence (e-mail:
t.calmels{at}bioprojet.com
;
debant{at}crbm.cnrs-mop.fr)
Accepted 26 October 2001
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Summary |
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Key words: GEF, RhoA, Cardiac sarcomere
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Introduction |
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The invariable association of a DH with a PH domain within the Rho GEF
family suggests that the DH-PH tandem represents the functional unit
responsible for activation of the Rho GTPases. In addition to this tandem
module, Rho GEFs often present other structural or functional domains,
predicted to play regulatory roles in the localization or the control of the
GEF activity (Stam and Collard,
1999) and in the involvment of Rho GEFs in specific signal
transduction networks.
Several 3D structures of GEF-DH domains have been resolved
(Liu et al., 1998) and
indicate that the DH domain is entirely composed of
helices. PH
domains show amino acid sequences with very low similarity although they share
a common 3D scaffold (Lemmon and Ferguson,
2000
). Outside the DH/PH tandem, the Rho-GEFs do not display
structural similarity (Whitehead et al.,
1997
).
Most of the Rho GEFs isolated so far display activity on Rho GTPases, at
least in vitro. Some Rho GEFs appear to exhibit in vivo selectivity for a
specific GTPase (e.g. Lbc for Rho, Tiaml for Rac, and FGD1 for Cdc42), whereas
others seem to act on several GTPases (e.g. Vav, Db1 and Trio)
(Stam and Collard, 1999).
Interestingly, the Rho GEF Trio possesses two GEF domains: the N-terminal
(Trio 1) shows a RhoG/Rac 1 specificity inducing ruffles formation whereas the
second domain (Trio2) is an exchange factor for RhoA and induces the formation
of stress fibers (Debant et al.,
1996
).
In the cardiovascular field, Rho GTPases play a key role in several
signaling pathways activated by G-protein-coupled receptors such as
lysophosphatidic acid (LPA) (Blomquist et
al., 2000), endothelin-1
(Gohla et al., 2000
;
Shome et al., 2000
),
angiotensin II (Aoki et al.,
1998
) and phenylephrin (Sah et
al., 1996
). It has been demonstrated that Rho is required for
1-adrenergic receptor-mediated hypertrophy in cardiomyocytes with an
increase of gene expression for ANF, MLC-2, ß-MHC, skeletal
-action (Sah et al.,
1996
). In addition, the Rho family of small G proteins plays a
critical role in mechanical stress-induced hypertrophic responses of cardiac
myocytes (Aikawa et al.,
1999
).
In the present study, we report the identification of a new member of the Rho GEF subfamily, called p63RhoGEF, from a proprietary detabase of human sequences. P63RhoGEF encodes a 63 kDa protein (580 amino acids) containing the conserved structural feature of a DH domain in tandem with a PH domain. P63RhoGEF is most closely related to the Rho GEF Trio2 with an identity score of 70%. Northern blot and in situ hybridization analysis have shown that p63RhoGEF is mainly expressed in heart and brain. P63RhoGEF functions as a GEF for RhoA in vitro and its expression induces an increase in stress fiber formation in REF-52 cells and H9C2 embryonic cardiac cells. Moreover, we show that the PH domain is necessary for p63RhoGEF-mediated RhoA activation in intact cells. Using a specific anti-p63RhoGEF antibody, we have detected the p63RhoGEF protein by immunocytochemistry experiments performed in human heart and brain tissue sections. Interestingly, p63RhoGEF protein was detected by confocal microscopy in the sarcomere of the cardiac fibers, more precisely located in the I-band mainly constituted of cardiac sarcomeric action.
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Materials and Methods |
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Bioinformatic or computational searches of both public and private EST and genomic databases (collaboration with Human Genome Sciences, Rockville, MD) were used to identify various sequences that encode structural characteristics of putative GEFs.
SMART method (Schultz et al.,
2000) was used for identification and cartoon representation of
the tandem DH-PH domain. Low compositional complexity was determined by the
SEG program available with SMART. Multiple alignments have been obtained with
Clustal W 1.7 method (Thompson et al.,
1994
) and other more sophisticated methods such as hydrophobic
cluster analysis (Callebaut et al.,
1997
). The evolutionary trace method
(Lichtarge et al., 1996
) was
used to generate a sequence identity dendrogram and is available from Binding
Site Analysis module implemented in InsightII 2000 program (Molecular
Simulations Inc., San Diego, CA).
Plasmid constructions
The cDNA sequence (2118 bp SrfI-XhoI fragment) containing
p63RhoGEF ORF (580 amino acids) was cloned into pBluescript KS(+) plasmid
(Stratagene) EcoRV-XhoI digested to give BSKS-p63RhoGEF
plasmid.
To perform mammalian cell transfection, the 1878 bp EheI-EcoRV fragment encoding p63RhoGEF was subcloned into pCDNA3-HisB expression vector (Invitrogen) linearized by EcoRV to generate pCDNA3-p63RhoGEF plasmid. This plasmid contains Xpress and His tags in frame with p63RhoGEF at the N-terminal. To perform guanine nucleotide exchange assay, the 663 bp BamHI-EcoRI fragment encoding p63RhoGEF-DH domain (amino acids 149-374) was subcloned into pGEX-6P3 GST-fusion vector (Pharmacia) BamHI-EcoRI digested to give GST-p63RhoGEF DH plasmid.
The mutant p63RhoGEF L301E was constructed using the Quick change site-directed mutagenesis kit from Stratagene according to the manufacturer's instructions. This mutation was performed both on mammalian expression vector pCDNA3-p63RhoGEF and on GST-p63RhoGEF pGEX plasmid and the constructs were verified by sequencing.
RNA hybridization
Human MTN blot (Clontech) was pre-hybridized for 30 minutes at 65°C in
5 ml of ExpressHyb buffer (Clontech), then hybridized for 1 hour at 65°C
in the same buffer containing denatured [-32P] dCTP-labeled
probe (1-2x106 cpm/ml). The filter was then washed twice for
15 minutes at 65°C in 50 ml of 2x SSC, 0.1% SDS, and washed once for
15 minutes at 52°C in 50 ml of 0.2x SSC, 0.1% SDS. It was
sequentially hybridized with p63RhoGEF and ß-actin cDNA probes.
C3 exoenzyme production and purification
C3 exoenzyme was expressed in the pET prokaryotic expression vector
(generous gift of P. Bocquet, INSERM U452, Nice, France) and purification of
the recombinant protein was performed by anion exchange chromatography as
previously described (Dillon and Feig,
1995).
Cell culture, transfections and immunofluorescence microscopy
REF-52 cells and H9C2 were maintained as described elsewhere
(Gauthier-Rouviere et al.,
1998; Kimes and Brandt,
1976
). Both cell lines were transfected with the lipofectamine
plus reagent according to Life's technology instructions. After fixation in
3.7% formaldehyde and permeabilization in 0.1% Triton in PBS for 3 minutes,
cells were stained with the appropriate primary antibodies followed by
FITC-coupled anti-mouse immunoglobulins and with rhodamine-conjugated
phalloidin for F-actin staining. Cells were observed under a DMR Leica
microscope using a 40x planapochromat lens. All transfections were
repeated at least three times, and an average of 100 cells were examined each
time.
Guanine nucleotide exchange assays
Recombinant GST-fusion proteins for p63RhoGEF DH domain (amino acids
149-374), Dbl and the Rho GTPases were produced using standard procedures. GDP
release and GTP binding assays were performed as described elsewhere
(Debant et al., 1996;
Vignal et al., 2000
).
Experiments were repeated at least three times, and each point was done in
duplicate.
In situ hybridization
Probe synthesis
RNA probe synthesis was carried out by means of a RNA transcription Kit
(Stratagene, La Jolla, CA). Plasmid (pBluescript, Stratagene) containing
p63RhoGEF-DH domain (amino acids 149-374) was linearized to give rise to the
antisense and sense probes. 1 µg of the linearized DNA template was
incubated for 2 hours at 37°C in a solution containing transcription
buffer 1x, dithiothreitol (30 mM), rATP (0.4 mM), rGTP (0.4 mM), rCTP
(0.4 mM), [-35S]UTP (5 µCi/µl), RNAse inhibitor (1.6
U/µl) and RNA polymerase (0.4 U/µl) T7 (sense) and T3 (antisense). The
DNA template was then digested with RQ-1 DNAse (10 U) for 15 minutes at
37°C. After incubation, 10 µg yeast tRNA were added to the sample.
Probe isolation was achieved on a sephadex G50 column. After precipitation,
the probe was dissolved in hybridization mix (50% formamide, 0.3 M NaCl, 20 mM
Tris-HCl, pH 8.5, 5 mM EDTA, 10% dextran sulfate, 1x Denhardt's
solution, 0.5 µg/µl yeast tRNA and 10 mM DTT) at a final concentration
of 2.104 cpm/µl and stored at -70°C until hybridization.
Slide treatment
Wax sections (brain and heart) were obtained from Novagen (Hybrid Ready
Tissues). Radioactive in situ hybridization was performed on paraffin sections
as previously described (Mazurais et al.,
1999). After treatment with xylene (3x 5 minutes) to remove
paraffin and rehydratation through an ethanol series, sections were rinsed in
0.85% NaCl and PBS (0.1 M, pH 7.4), postfixed in 4% paraformaldehyde and then
treated with proteinase K (20 µg/ml) diluted in TE buffer (50 mM Tris-HCl,
5 mM EDTA, pH 8). After a rinse in PBS, the sections were refixed in 4%
paraformaldehyde to stop proteinase K activity and then rinsed in PBS before
acetylation with acetic anhydride (0.25% in triethanolamine 0.1 M, pH 8).
Finally, sections were rinsed in distilled water before dehydration through
ethanol series. After air drying, tissue sections were hybridized under
coverslips with radioactive probe diluted in hybridization buffer
(2.104 cpm/µl) overnight at 55°C in a humid chamber. After
hybridization, coverslips were removed in 5x standard saline citrate
(SSC 1x: trisodium citrate 15 mM, NaCl 150 mM, pH 7), 10 mM DTT at
55°C for 15 minutes and the slides were washed (30 minutes) with 2x
SSC, 50% formamide, 10 mM DTT at 65°C. After a rinse (10 minutes) in NTE
buffer (10 mM Tris-HCl, 0.5 mM NaCl, 5 mM EDTA, pH 8), sections were treated
with RNAse A (20 µg/ml in NTE) for 30 minutes at 37°C. Slides were than
washed in NTE for 15 minutes at room temperature and incubated for 30 minutes
in 2x SSC, 50% formamide, 10 mM DTT at 65°C. Before autoradiography,
the tissues were rinsed in 2x SSC and 0.1x SSC at room temperature
and dehydrated in an ethanol series containing 0.3 M ammonium acetate.
Autoradiography was performed by dipping the slides in Ilford K5 nuclear track
emulsion and exposing the slides in the dark for 28 days at 4°C. After
development, the sections were counterstained with toluidine blue and mounted
in Pertex (Microm, France).
Peptide synthesis and polyclonal antibody production
The deduced amino acid sequence of p63RhoGEF protein was analyzed for
highly antigenic regions using the Jameson-Wolf antigenic index and checked
for the absence of sequence homology with other proteins. Peptide 699,
comprising amino acids 428 to 442 (CRFALTSRGPEGGIQ) of p63RhoGEF, was
synthesized (Peptide Synthesizer Model 431A, Applied Biosystems), purified and
conjugated to keyhole limpet hemocyanin using
m-maleimidobenzoyl-N-hydroxysuccinimide as the coupling agent. Two
14-week-old New Zealand rabbits were injected with peptide-carrier conjugate
(150 µg/injection) in complete Freund's adjuvant on day 0 and every two
weeks with peptide-carrier conjugate (50 µg/injection) in incomplete
Freund's adjuvant. Animals were bled 7 days after boosts (J39, J69 and J95)
and their sera were tested at various dilutions on the unconjugated peptide
coated ELISA plates. The immunoglobulins fraction from the anti-p63RhoGEF
immune serums (Ab699) were obtained by affinity chromatography on protein
A-sepharose and used for immunocytochemistry. The antibody Ab699 was validated
by western immunoblot using a GST-p63RhoGEF fusion protein; Immunocomplex was
abolished in presence of the 699 peptide (from 0.5 µg/ml to 50 µg/ml) in
a concentration-dependent manner (data not shown).
Immunohistochemistry
Wax human sections were obtained from Novagen (Hybrid-Ready Tissues). After
treatment with xylene and rehydratation through an ethanol series and PBS, the
specimens were heated in target retrieval solution, pH 9.9 (Dako), 40 minutes
at 95°C and then left to cool for 20 minutes on the bench. Competition was
performed by overnight incubation of the primary antibody (1:750 and 1:20
dilutions for p63RhoGEF and ß-myosin heavy chain (ß-MHC) detection,
respectively) with or without related 699 peptide (three times in excess
compared with antibody concentration) used to generate anti-p63RhoGEF rabbit
polyclonal antibody Ab699. After two washes of 2 minutes in TBS solution
(Dako), sections were incubated for two hours at room temperature with primary
antibody. Secondary antibody and the tyramide signal amplification peroxydase
immunohistochemistry detection kit (Dako) were used according to the
manufacter's instructions with diaminobenzidine as a substrate. Stained
immunocytochemical sections were analyzed on a Nikon (Eclipse E800, Sony
camera DXC-950P) microscope.
Immunofluorescence studies were performed on confocal microscope (Olympus Fluoview IX70) using primary antibodies: either Ab699 (1:750 dilution), monoclonal antibody anti-ß-MHC at 1:20 dilution (Chemicon International) and monoclonal antibody anti-vinculin at 1:250 dilution (Sigma-Aldrich). For detection of p63RhoGEF, biotin-SP-conjugated AffiniPure F(ab')2 fragment Donkey anti-Rabbit IgG (H+L) (Jackson ImmunoResearch Laboratories) at 1:200 dilution followed by Cy2-conjugated streptavidin (1:360 dilution) were used. For detection of ß-MHC, Rhodamine (TRITC)-conjugated AffiniPure F (ab')2 fragment Donkey anti-Mouse IgG (H+L) (Jackson ImmunoResearch Laboratories) was used at 1:50 dilution.
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Results |
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Protein sequence analysis performed on p63RhoGEF led to the identification
of the characteristic GEF tandem domain composed of the DH and PH domains
(Fig. 1A). P63RhoGEF was then
compared with well characterized GEFs (Fig.
1B). Moreover, the DH domain was classified with SMART
(Schultz et al., 2000) as Rho
GEF domain and thus, was putatively considered as an upstream regulator of the
Rho GTPases family. The evolutionary trace method, which allows partitioning
of groups of sequences (Lichtarge et al.,
1996
) highlighted the high identity score of 70% between p63RhoGEF
and Trio2 (Fig. 1C). This
result emphasized the putative linkage between p63RhoGEF and Rho GTPases, as
Trio2 has been reported to be a RhoA-specific GEF
(Debant et al., 1996
).
|
P63RhoGEF is expressed in human brain and heart
We next examined the pattern of expression of p63RhoGEF mRNA. Hybridization
of a 1.9 Kb p63RhoGEF probe to a northern blot membrane from multiple human
tissues revealed the presence of a single mRNA product of about 2.6 Kb mainly
in heart and brain tissues (Fig.
2). P63RhoGEF mRNA was also detected to a small extent in human
small intestine but not in colon, thymus, spleen or peripheral blood
leukocytes (data not shown).
|
P63RhoGEF displays in vitro exchange activity towards RhoA
To identify the Rho GTPase targets of p63RhoGEF, we performed in vitro
guanine nucleotide release assays using bacterially expressed GST-p63RhoGEF DH
domain (amino acids 149-374), GST-Dbl as a positive control and
GST-Rho-GTPases. The guanine nucleotide exchange activity of p63RhoGEF was
measured on various recombinant [3H]GDP-loaded GTPases in the
presence of GTP. As described elsewhere, Dbl preferentially stimulated guanine
nucleotide release on RhoA, Cdc42, and RhoG, and to a lesser extent on Rac
(Hart et al., 1994). As shown
in Fig. 3A, p63RhoGEF
specifically displayed exchange activity towards RhoA. p63RhoGEF did not
promote nucleotide exchange on the other tested GTPases, but rather stabilized
GDP binding on Rac and Cdc42. This observation suggests that p63Rho-GEF may
bind ineffectively to these two GTPases in vitro, therefore preventing GDP
release.
|
p63RhoGEF stimulated complete GDP dissociation from RhoA within 20 minutes
(Fig. 3C) and consistently
stimulated [35S]GTPS binding on the GDP-loaded GTPase
(Fig. 3D). We also produced a
p63RhoGEF protein containing a point mutation on leucine 301, residue located
in one
-helix of the DH domain facing the GTPase partner
(Liu et al., 1998
) and highly
conserved among the known Rho GEFs. This mutation is predicted to strongly
affect exchange activity leading to an inactive protein
(Alberts and Treisman, 1998
).
Indeed, the mutation L301E in p63RhoGEF completely abolished its exchange
activity on RhoA (Fig. 3B).
P63RhoGEF induces stress fiber formation via RhoA activation in
fibroblasts
In fibroblasts, Cdc42 and Rac induce filopodia and lamellipodia formation,
respectively, while RhoA promotes stress fiber formation
(Hall, 1998). We then measured
the effect of p63RhoGEF expression on the actin cytoskeleton reorganization of
REF-52 cells. REF-52 cells present basal stress fiber formation as illustrated
by phalloidin staining of non-transfected cells. The expression of p63RhoGEF
strongly induced cell retraction and an enhancement of stress fiber formation
in 70% of the transfected cells, as did an activated form of RhoA (RhoAV14)
(Fig. 4A,B). This effect was
shown to be dependent on the p63RhoGEF GEF activity, since the L301E p63RhoGEF
mutant-expressing cells do not exhibit enhanced stress fiber formation.
Indeed, only 20% of the transfected cells by the p63RhoGEF mutant presented
the p63RhoGEF wild-type phenotype. Similarly, treatment of the cells with the
Rho-specific inhibitor exoenzyme C3 dramatically inhibits the induction of
stress fiber formation by the wild-type p63RhoGEF. In addition, deletion of
the PH domain abrogates the capacity of p63RhoGEF to induce an increase in
stress fiber formation, suggesting that this domain is absolutely required for
p63RhoGEF function in vivo. All together, these data indicate that RhoA is
likely to be the target of p63RhoGEF in vivo.
|
P63RhoGEF induces stress fiber formation in cardiac myoblasts
In order to further investigate the putative role of p63RhoGEF in cardiac
tissue, we investigated the effect of p63RhoGEF expression on the actin
cytoskeleton reorganization of the rat cardiomyocytes-derived cell line H9C2.
As it was observed in REF-52 fibroblasts, the expression of p63RhoGEF strongly
induced an increase of stress fiber formation in the transfected cardiac
myoblasts, as did an activated from of RhoA (RhoAV14)
(Fig. 5). These data suggest
that p63RhoGEF may be able to promote RhoA activation in intact cardiac
myoblasts.
|
Detection of p63RhoGEF transcript in human heart and brain by in situ
hybridization
Sections of human cerebellar cortex and heart were subjected to an in situ
hybridization using a p63RhoGEF antisense probe. The expression pattern was
remarkably consistent from one experiment to the other. In all cases, with
respect to the northern blot analysis, the p63RhoGEF mRNA was shown to be
expressed in brain and in heart. No specific signal could be detected in the
liver, lung and kidney (data not shown).
In the brain, the expression was detected in cell bodies of astrocytes and oligodendrocytes localized in the cerebellar cortex (Fig. 6Aa,b,e). Absence of labelling on adjacent sections hybridized with the sense probe consistently demonstrated the specificity of the p63RhoGEF probe (Fig. 6Ac,d,f).
|
In the heart, the results obtained by light microscopy showed that the p63RhoGEF mRNA had a widespread distribution within the left ventricle but that the labeling was restricted to the cardiomyocytes (Fig. 6Ba,b,e) as no specific signal could be detected in vessels and fibroblasts (data not shown). The specificity of the signal was confirmed by the absence of signal on the control slides (Fig. 6Bc,d,f).
P63RhoGEF protein is detected in human brain tissue sections
Analysis of human cerebellar cortex using Ab699 polyclonal antiserum
revealed that p63RhoGEF protein is present at relatively high levels in the
different cell populations (Fig.
7A). The staining was strong and uniformly distributed through the
sections. Homogenous punctuated staining was observed in the external
hypocellular layer containing astrocytes and proximal dendrites, in the
intermediate Purkinje cell layer (Fig.
7Ab, arrow) and in the deep hypercellular granular cell layer. By
contrast, no appreciable staining in the different cell populations was
detected in the section using rabbit control nonspecific IgG.
|
P63RhoGEF protein is detected in human heart tissue sections
Analysis of human heart left ventricular section using Ab699 polyclonal
antiserum revealed that p63RhoGEF protein was present at high levels in the
vast majority of cardiomyocytes, when observing the fibers in transversal and
longitudinal orientations (Fig.
7Ba,b), whereas a control rabbit IgG showed no appreciable
staining (Fig. 7Bc). Most of
the signal was abolished by pre-incubation with the immunogenic 699 peptide
that was used to generate the anti-p63RhoGEF rabbit polyclonal antibody
(Fig. 7Bd).
Moreover, at the transversal incidence, a homogenous dotted staining was detected (Fig. 7Ba). Interestingly, the p63RhoGEF staining depicted in longitudinal fibers was represented by a brown repetitive striated staining, consistent with a sarcomeric organization (Fig. 7Bb,e). ß-MHC monoclonal antibody used as a control of the endogenous sarcomeric striated pattern exhibited a similar arrangement (Fig. 7Bf). Different layers of blood vessels showed faint or no detectable staining with Ab699 (Fig. 7Ba, asterisk).
The striated pattern of immunolabelling exhibited by p63RhoGEF led us to
analyze its sarcomeric distribution by means of double immunostaining.
Confocal microscopic analysis was performed using rabbit p63RhoGEF polyclonal
antibody and either mouse anti-ß-MHC or mouse anti-vinculin in human left
ventricle (Fig. 7C). Myosin,
the principal component of the thick filaments, and vinculin localized in the
extrasarcomeric cytoskeleton are good markers of the cardiac sarcomeric
structure (Chen and Chien,
1999).
Fluorescence immunohistochemistry demonstrated that p63RhoGEF exhibit striated labelling organized in doublets (Fig. 7Ca). Moreover, with respect to myosin distribution, ß-MHC immunolabelling was detected in doublets in transverse A-bands on both sides of the M-line (Fig. 7Cb, labeled as a and m, respectively). To determine potential colocalization between myosin and p63RhoGEF, dual immunostaining of p63RhoGEF and ß-MHC in the double channel scan was performed. This experiment shows the alternative localization of the two proteins in the sarcomere indicating potential localization of p63RhoGEF in the I-band (Fig. 7Cc).
To confirm this hypothesis, double immunolabelling using anti-vinculin antibody was performed. With respect to extrasarcomeric cytoskeleton organization, vinculin was found to be localized in Z-disks and at the level of the intercalated disk (Fig. 7Ce). Double immunostaining analysis revealed that vinculin signal is found between p63RhoGEF immunolabelling (Fig. 7Cf). These results could indicate that p63RhoGEF is localized in the I-band of human cardiac sarcomeres.
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Discussion |
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We demonstrated that p63RhoGEF is a RhoA-specific GEF by in vitro guanine
nucleotide exchange assays. Moreover, p63RhoGEF activated the formation of
stress fibers in fibroblasts and cardiomyocyte-derived H9C2 cells, indicating
that p63RhoGEF can stimulate RhoA activity in intact cells. The inhibition of
p63RhoGEF-induced stress fiber formation in fibroblasts treated by the
Rho-specific inhibitor C3-exoenzyme confirmed the RhoA specificity for
p63RhoGEF. This is consistent with the fact that p63RhoGEF is highly
homologous to Trio2, which has been shown previously to be a RhoA-specific GEF
(Debant et al., 1996).
However, we cannot formally exclude the possibility that p63RhoGEF catalysed
guanine nucleotide exchange activity on other C3-exoenzyme substrates such as
the GTPases RhoB and RhoC.
Heart and brain-specific expression of p63RhoGEF was shown by northern blot
analysis and confirmed by results from in situ hybridization and
immunohistochemistry. Interestingly, the distribution pattern of the p63RhoGEF
protein in heart was found to be organized in characteristic striated doublets
in the sarcomere of cardiac cells. Confocal analysis of p63RhoGEF pattern with
both ß-MHC and vinculin patterns (specific markers of the cardiac
sarcomeric structure characterizing the A-band and the Z-disk, respectively)
was performed. From dual channel scan, we could conclude that p63RhoGEF is
located in the sarcomeric I-band mainly constituted of cardiac
-sarcomeric actin. This result, together with the formation of F-actin
stress fibres in H9C2 myoblasts and REF-53 cells, suggests that p63RhoGEF may
be connected directly or indirectly to actin thin filaments. Indeed, Rho GEFs
have been shown to be linked to actin or to actin-binding proteins. For
example, Trio PH1 binds to the actin binding protein filamin
(Bellanger et al., 2000
), and
the Rho GEF frabin possesses an actin-binding domain
(Umikawa et al., 1999
). The
human Trio-like protein Duet, a close analog of human p63RhoGEF (63%
identity), was localized to actin-associated cytoskeletal elements
(Kawai et al., 1999
). It would
be of interest to investigate putative binding of p63RhoGEF via its PH domain
to such structural proteins, given the fact that the p63RhoGEF PH domain is
absolutely required for p63RhoGEF-mediated RhoA activation in fibroblasts and
H9C2 myoblasts (data not shown). Other cytoskeletal proteins found in the
Z-disk at the proximity of I-band, such as talin, vinculin, titin and
-actinin, may also contribute to the p63RhoGEF function. Interestingly,
a myosin-M protein carrying a putative Rho GEF domain has been identified in
Dictyostelium (Oishi et al.,
2000
). Such findings suggest that an association of a Rho GEF with
a contractile protein might be required to execute appropriate function, and
may be a way of regulating p63RhoGEF activity.
Indeed, it is of crucial importance to determine how the activity of these
Rho-GEFs is regulated. Several Rho-GEFs have been directly involved in
coupling heterotrimeric G protein to Rho. Cell surface receptors that transmit
signal through heterotrimeric G protein activate Rho pathways
(Sah et al., 2000) mainly by
stimulating the activity of members of the G
-12 family, which in turn,
activate a GEF acting on Rho. These GEFs contain a
G
12/G
13-binding region such as a LH and/or PDZ domains
(Mao et al., 1998
). These
specific domains were not found in p63RhoGEF, suggesting that there is no
direct evidence of its activation by G
12/G
13. This may indicate
an alternative pathway for the regulation of p63RhoGEF activity, such as a
phosphorylation event, as in the case of vav
(Lopez-Lago et al., 2000
) or
such as a cytoskeletal targeting mediated by its PH domain. This latter
hypothesis is reinforced by the observation that the PH domain of p63RhoGEF is
absolutely required for its function.
Considering that overexpression of p63RhoGEF in H9C2 cardiac myoblasts resulted in the cytoskeletal reorganization of the F-actin filaments, it is tempting to postulate that p63RhoGEF is involved in the contractile process of cardiac cells. However, the precise functional role of p63RhoGEF at the sarcomeric level in cardiac cells and in other tissues such as brain remains to be determined.
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Acknowledgments |
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Footnotes |
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References |
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