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INTRODUCTION |
Rho family GTPases are key regulators of diverse cellular
functions including actin-based morphological changes, gene induction, cell motility, and intracellular membrane trafficking (1, 2). They act
as binary molecular switches that are turned on and turned off in
response to a variety of extracellular stimuli. Rho proteins in the
GTP-bound active state can interact with a number of effector targets
that transduce signals leading to biological response (3). When the
bound GTP is hydrolyzed to GDP, the Rho protein-mediated signaling is
turned off. Three classes of regulatory proteins are involved in
balancing Rho GTPases between the on- and off-states: the guanine
nucleotide exchange factors that promote the release of bound GDP and
facilitate GTP binding (4), the GTPase-activating proteins
(GAPs)1 that increase the
intrinsic GTPase activity of Rho GTPases to accelerate the return of
the proteins to the inactive state (5, 6), and the guanine nucleotide
dissociation inhibitors that sequester the GDP-bound form of Rho
GTPases and may also regulate their intracellular localization (7). One
emerging theme from recent studies of Rho GTPase regulation is that a
balanced act between the activation and the deactivation signals
is required for effective signal flow through Rho GTPases, and this may
involve concerted action of all classes of regulatory proteins (8). Understanding how such a balance is achieved in various
physiological situations represents a major challenge in elucidating
the regulatory mechanism of Rho GTPases.
Together with Rho family GTPases, RhoGAPs are found in eukaryotes
ranging from yeast to human, suggesting an evolutionarily conserved
role in eukaryotic cell regulation. So far over 30 RhoGAP family
members have been reported, and more are found in mammalian genomes (9,
10). RhoGAPs may therefore far outnumber their cellular substrates,
i.e. the Rho family GTPases that stand with 20 members. The
overabundance of RhoGAPs evidently suggests that each RhoGAP may play a
specialized role in regulating individual Rho GTPase activity and/or in
mediating their specific functions. Moreover, the biochemical GAP
activities of each RhoGAP must also be fine-tuned in cells by tight
regulation in a spacial and temporal manner such that Rho GTPases would
not be turned off all the time.
In neuronal systems, RhoGAPs and their substrate Rho GTPases have
been implicated in regulating multiple processes of the morphological
development of neurons, including axon growth, guidance, branch
stabilization, and dendritic elaboration (6, 11). For example, the
Rho-specific RhoGAP p190 has been shown to dissolve actin stress fibers
when introduced into fibroblasts (12) and was found to be essential for
exon stability in mushroom body neurons in Drosophila (13).
Deletion of p190 in mice led to deregulation of Rho GTPase function and
neuronal malfunction that is stemmed from Src kinase-regulated adhesion
signaling defect (14). The discovery of a RhoGAP, oligophrenin-1, that
is associated with X-linked mental retardation (15), further underlines
the significance of RhoGAP in the nervous system. Thus, it is of
particular interest to examine what role each individual RhoGAP might
play in complex biological systems and how the RhoGAP activity is
controlled in response to cellular stimulation.
In the present work, we describe the identification and initial
characterization of a novel member of the RhoGAP family, p200RhoGAP. p200RhoGAP is brain-specific and acts as a GAP to down-regulate RhoA
and Rac1 activities in vitro and in vivo. In
N1E-115 neuroblastoma cells it is localized at the cortical actin site
in the undifferentiated state and at the ends of the neurite extensions
in the differentiated state. Overexpression of the RhoGAP domain or
full-length p200RhoGAP in N1E-115 cells induced a differentiation
phenotype. Moreover we found that p200RhoGAP binds to the SH3 domain of
Src in vitro and is tyrosine-phosphorylated upon association
with active Src in cells. These results suggest that this novel RhoGAP
may have a role in neuronal differentiation, and its cellular function may be subject to the regulation by Src or Src-like kinases.
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EXPERIMENTAL PROCEDURES |
Antibodies--
Rabbit antisera against p200RhoGAP were raised
using an N-terminal polypeptide derived from p200RhoGAP (residues
11-25; KQRGILKERVFGCDL) that was conjugated to KLH as an
immunogen. Immunization and enzyme-linked immunosorbent assay analysis
for the verification of the specificity of the antibody were performed
by Sigma-Genosys. Antiserum was purified by protein A affinity
chromatography according to the manufacturer's protocol (Pierce). The
anti-GST monoclonal antibody was purchased from Sigma, and the anti-HA
antibody was purchased from Roche Molecular Biochemicals.
Fluorescein isothiocyanate-conjugated antibodies and
rhodamine-conjugate phalloidin were purchased from Molecular Probes.
Monoclonal anti-RhoA antibody was obtained from Santa Cruz
Biotechnology, Inc., anti-Rac1 was from Upstate Biotechnology, Inc.,
and anti-Cdc42 was from BD Biosciences. Anti-phospho-tyrosine and
anti-Src antibodies were obtained from Upstate Biotechnology, Inc.
DNA Constructs--
A human cDNA clone containing a RhoGAP
homology domain, KIAA0712, was obtained from Dr. Takahiro Nagase at the
Kazusa DNA Research Institute (Chiba, Japan). The cDNA clone was
sequenced to verify the reported sequences, and a missing guanine at
the position 414 was detected and corrected. Genomic DNA searches were
performed by using the BLAST program in NCBI genomic data base for the
human p200RhoGAP and in the Celera genomic data base for the murine
p200RhoGAP. The exon sequences were identified manually by the
GT-AG rule (16). Recombinant RhoGAP domain of p200RhoGAP
(residues 1-251) was generated by polymerase chain reaction of the
coding cDNA and subsequent subcloning of the PCR fragment into the
BamHI and EcoRI sites of pGEX-2T vector (Amersham Biosciences). GAP(R58K) that bears a mutation of arginine to
lysine at the predicted catalytic arginine (residue 58) was generated by introducing a point mutation using PCR-based site-directed mutagenesis (17) and was cloned into pGEX-2T vector similarly. GST
fusion constructs of Cdc42, Rac1, and RhoA were described before (17,
18). GST fusion constructs of SH3 domains of Src, PLC
, p85
, Crk,
Abl, spectrin, and Grb2 (C) were kind gifts from Dr. Steve Taylor
(University of California). Production and purification of the GST
fusion proteins were as described (17, 18). The purity of the
recombinant proteins was estimated at
90% by Coomassie Blue
staining of the polyacrylamide gel electrophoresis of the purified proteins.
The cDNAs encoding RhoGAP domain (residues 1-251), the GAP domain
containing the arginine mutation (GAP(R58K)), full-length p200RhoGAP,
the full-length p200RhoGAP containing the arginine mutation
(p200(R58K)), and the C-terminal region of p200RhoGAP (p200-C; residues
224-1738) were cloned into the mammalian expression vector pCEFL-GST
to be expressed as GST fusions and into the pKH3 vector to be expressed
as (HA)3 fusions. The cDNAs were also cloned into a retroviral
vector pMX-GFP that bicistronically expresses green fluorescent protein
(GFP) to be expressed by retroviral infection (19). The pKH3 constructs
of the HA-tagged Rho GTPases were described before (19), and the
pCDNA3 constructs expressing kinase-deficient Src
(Src(K295R/Y527F)), wild type c-Src, or constitutively active Src
(Src(Y527F)) were obtained from Dr. Steve Taylor (University of California).
Northern and Western Blotting--
For Northern blot analysis,
representative organs were harvested from two adult mice. Each organ
was briefly washed with phosphate-buffered saline (PBS), pH 7.4, and
was resuspended in RNAzol B solution (Tel-Test). The tissues were
homogenated by using a Teflon homogenizer in gentle strokes, and the
RNA extraction was performed by adding chloroform to 1/10 of the
volume of the homogenate. After the extraction, the aqueous phase was
taken for alcohol precipitation of RNA. Equal amount of RNA (~30
µg) was loaded on each lane for gel electrophoresis and subsequent
transfer to a nitrocellulose membrane. The membrane was probed with
32P radiolabeled cDNA probes prepared by the random
priming method (Amersham Biosciences). A 668-bp PCR fragment
corresponding to the residue's C terminus of the RhoGAP domain of
p200RhoGAP (residues 758-1002) was used as a template for the random
priming labeling. The hybridization conditions were as recommended by
Amersham Biosciences. A human multiple tissue RNA blot was obtained
from Clontech, Inc. for the Northern blot analysis
following similar protocols.
For Western blot analysis, cell lysates or co-precipitates were
separated by SDS-PAGE and transferred onto polyvinylidene difluoride
membranes (Bio-Rad). The membranes were blocked with 5% skim milk in
TBS-T (20 mM Tris-HCl, pH 8.0, 150 mM NaCl,
0.05% Tween 20) for 1 h and probed with primary antibodies
followed by horseradish peroxidase-coupled secondary antibody for
enhanced chemiluminescence analysis (Amersham Biosciences).
In Vitro GTPase Activity Assay--
The intrinsic and
GAP-stimulated GTPase activities of Cdc42, Rac1, and RhoA were measured
as described by the nitrocellulose filter-binding method (17, 18).
Briefly, recombinant G-proteins were preloaded with
[
-32P]GTP (10 µCi, 6000 Ci/mmol; PerkinElmer Life
Sciences) in a 100-µl buffer containing 50 mM
HEPES, pH 7.6, 0.2 mg/ml bovine serum albumin, and 0.5 mM
EDTA for 10 min at ambient temperature before the addition of
MgCl2 to a final concentration of 5 mM. An
aliquot of the [
-32P]GTP-loaded RhoA was mixed with
GAP assay buffer containing 50 mM HEPES, pH 7.6, 100 mM NaCl, 0.2 mg/ml bovine serum albumin, and 5 mM MgCl2 in the presence or absence of GAP. At
different time points the reaction was terminated by filtering the
reaction mixture through nitrocellulose filters followed by washing
with 10 ml of ice-cold buffer with 50 mM HEPES, pH 7.6, and
10 mM MgCl2. The radioactivities retained on
the filters were then subjected to quantification by scintillation counting.
Cell Culture and Transfection--
Swiss 3T3 cells and COS-7
cells were cultured and maintained as described previously (19).
N1E-115 neuroblastoma cells were obtained from ATCC. The cells were
cultured in dishes coated with 20 µg/ml laminin and in Dulbecco's
modified Eagle's medium supplemented with 10% fetal bovine serum in
10% CO2 at 37 °C. For transient expression, COS-7 cells
were seeded in 6-cm dishes at a density of 5 × 105
cells/dish. The next day, plasmid constructs were transfected into the
cells by using LipofectAMINE Plus (Invitrogen) according to the
manufacturer's protocol. For transient expression in N1E-115 cells,
the cells were seeded in 6-cm dishes at a density of 6 × 105, and the transfections were carried out by using
Cytofectin (Bio-Rad) following the manufacturer's protocol. 48 h
after the transfections, cells were either fixed for immunofluorescence
analysis or washed with ice-cold PBS once, and the whole cell extracts
were prepared in a cell lysis buffer containing 50 mM Tris,
pH 8.0, 50 mM NaCl, 0.1 mM EDTA, 5 mM MgCl2, 0.5 mM
Na3VO4, 0.5% Nonidet P-40, 50 mM
NaF, 10% glycerol, 10 mM
-mercaptoethanol, 1 mM dithiothreitol, 0.4 mM phenylmethylsulfonyl
fluoride, and a protease inhibitor mixture (Sigma) for 30 min at
4 °C.
The recombinant retroviruses expressing various p200RhoGAP constructs
were generated by using the retroviral packaging Phoenix cell system
(20) with a retroviral construct that bicistronically expresses green
fluorescent protein as described (19). Swiss 3T3 and N1E-115 cells were
infected with the respective retroviruses according to an established
protocol (20). To observe ligand-induced actin structural changes,
Swiss 3T3 cells were serum-starved overnight and challenged for 10 min
with 20 ng/ml lysophosphotidyl acid (Sigma), 10 ng/ml PDGF (Upstate
Biotechnology, Inc.) or 100 ng/ml Bradykinin (Sigma) before fixation.
Immunocytochemistry--
For immunofluorescence staining, Swiss
3T3 cells were fixed with 3.8% formaldehyde in PBS for 10 min and
permeabilized with 0.1% Triton X-100 for 5 min. The cells were then
incubated with 1% bovine serum albumin in PBS for 30 min followed by
incubation with the TRITC-conjugated phalloidin (Molecular Probes) to
visualize the actin filaments. In N1E-115 cells, endogenous p200RhoGAP
was detected by staining the cells with anti-p200RhoGAP polyclonal antibody followed by incubation with fluorescein
isothiocyanate-conjugated anti-rabbit antibody. The actin filaments
were stained with TRITC-conjugated phalloidin (Molecular Probes). The
fluorescent images were obtained by using a Leica fluorescence microscope.
Rho GTPase Effector Pull-down Assay--
The p21-binding domain
(PBD) pull-down assays to determine the activation status of the Rho
family GTPases were performed as described (19). Briefly, COS-7 cells
were cotransfected with HA-tagged Rho GTPases expressing plasmid and a
vector expressing various GST-tagged p200RhoGAP cDNAs. 48 h
post-transfection, the cell lysates prepared in a lysis buffer
containing 50 mM Tris-HCl, pH 7.4, 150 mM NaCl,
10 mM MgCl2, 1% Triton X-100, protease
inhibitors, 1 mM phenylmethylsulfonyl fluoride were
incubated with GST fusion of the PBDs of PAK1, WASP, or Rhotekin that
were bound on glutathione beads. The expression of each protein was
confirmed on Western blots using either anti-HA or anti-GST antibodies.
The amount of RhoA-GTP, Rac1-GTP, or Cdc42-GTP that was pulled down by
the bead-associated PBD of PAK1, WASP, or Rhotekin was detected by immunoblotting using anti-HA antibody. Quantification of Western blots
was carried out using a Fujifilm LAS-1000 digital imaging system.
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RESULTS |
Identification of p200RhoGAP--
We have been interested in
identifying novel RhoGAPs that display distinctive tissue distribution
patterns. Data base searches using the conserved RhoGAP domain as a
probe led us to a few previously uncharacterized protein sequences that
show different extent of homology to the known RhoGAPs. Among these
proteins, we identified a cDNA clone predicted to encode 1738 amino
acids containing a RhoGAP domain at the N terminus
(GenBankTM accession numbers AB018255 and KIAA0712;
see Fig. 1A). Comparison of
this cDNA clone with the corresponding human genomic sequences in
the NCBI data base (accession number AP000751) revealed that a guanine
base at nucleotide position 414 was missing in the KIAA clone,
resulting in a loss of 17 amino acids at the N terminus and a reading
frameshift. This gene is located at chromosome 11q. We named this novel
putative RhoGAP p200RhoGAP based on its predicted molecular mass. Using
the human cDNA sequences, we further identified the mouse homolog
of p200RhoGAP by searching the Celera mouse genome data base
(GA_x5J8B7W6RL5; see Fig. 1A). Both the human and the mouse
cDNA sequences are composed of 12 exons, and mouse p200RhoGAP shows
86% sequence identity to human p200RhoGAP.

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Fig. 1.
A, the predicted amino acid
sequences of human and mouse p200RhoGAP. Human p200RhoGAP was
identified from a human cDNA library (KIAA0712). The mouse
p200RhoGAP sequences were identified from the Celera mouse genome data
base. The mouse homolog shares 86% identity with human p200RhoGAP. The
RhoGAP domain is in bold, and the proline-rich regions are
in bold italic. B, alignment of RhoGAP domains of
p200RhoGAP (human and mouse), mouse CdGAP, human
n-chimaerin, rat p190 RhoGAP, and human Cdc42GAP
(p50RhoGAP). Identical residues are indicated by stars, and
conserved residues are indicated by asterisks. C,
sequence alignment of the proline-rich regions in p200RhoGAP (human and
mouse), Cdc42GAP, BCR, 3BP-1, p85 , and CdGAP. X,
non-conserved residues; P, proline-preferred; ,
hydrophobic residue; P, conserved proline.
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A sequence comparison analysis indicated the existence of an N-terminal
RhoGAP domain, as well as five C-terminal proline-rich domains in human
p200RhoGAP (four proline-rich domains in the mouse homolog). In the
N-terminal RhoGAP domain, the critical residues that are required for
the GAP activity, including a highly conserved arginine (Arg-58), are
well represented, suggesting that the GAP domain might be involved in
the down-regulation of Rho GTPases (Fig. 1B). The RhoGAP
domain of p200RhoGAP is most similar to that of CdGAP with 70%
sequence identity (21) and less to that of other RhoGAPs (36% identity
to n-chimaerin, 27% to Cdc42GAP, and 28% to p190).
However, outside the RhoGAP region the sequences of p200RhoGAP diverge
from other RhoGAPs. Moreover, the proline-rich sequences present in all
five proline-rich regions of p200RhoGAP appear to confer to the class
II SH3 domain binding consensus (21) that are found in other signaling
RhoGAPs including p190, p85
, Cdc42GAP, Bcr, and 3BP-1 (Fig.
1C).
To examine the tissue expression pattern of p200RhoGAP, we prepared a
multiple tissue Northern blot using various mouse organs harvested from
a 6-week-old male C57/BL6 mouse. To ensure the specificity of the
signal, a radiolabeled probe was designed from the sequences outside of
the conserved N-terminal RhoGAP domain. The Northern blot analysis
showed that p200RhoGAP is specifically expressed in the brain but not
in other tissues examined (Fig. 2).
Similar Northern blot pattern was seen in a human multi-tissue mRNA
blot (data not shown). Because a single hybridization signal was
observed in a number of repeated hybridization experiments, it seems
that p200RhoGAP does not have closely related family members that
cross-react with the probe.

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Fig. 2.
Tissue distribution of p200RhoGAP mRNA
revealed by Northern blot analysis. Total cellular RNA was
extracted from adult mouse tissues using RNAzol B. The cDNA
fragment (2228-2336 bp) of p200RhoGAP was used as a probe.
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In Vitro and in Vivo GAP Activities of p200RhoGAP--
To
determine the function of the N-terminal RhoGAP domain of p200RhoGAP,
we performed an in vitro GTPase activity test using the
RhoGAP domain, as well as a mutant RhoGAP domain carrying a point
mutation (R58K) at the predicted catalytic arginine site. When
incubated with RhoA, Rac1, or Cdc42, the RhoGAP domain could significantly stimulate the GTPase activity of all three GTPases whereas the R58K mutant failed to do so (Fig.
3). The RhoGAP domain showed higher GAP
efficiency toward RhoA compared with Rac1 or Cdc42. In addition, the
presence of increasing concentrations of the RhoGAP domain further
enhanced the stimulatory effect (Fig. 3). These results indicate that
the RhoGAP domain of p200RhoGAP can function as a GAP for Rho GTPases
in vitro.

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Fig. 3.
The RhoGAP domain of p200RhoGAP stimulates
Cdc42, Rac1, and RhoA GTPase activities in vitro.
The GTPase activities of Cdc42, Rac1, and RhoA were measured in the
presence or absence of the recombinant RhoGAP domain (amino acid
residues 1-251) or the RhoGAP domain mutant (R58K). The intrinsic
GTPase activities are represented by open circles, GAP(R58K)
(60 nM)-stimulated activities are represented by
closed circles, GAP (20 nM)-stimulated
activities are represented by closed triangles, and GAP (60 nM)-stimulated activities are represented by open
triangles.
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A number of effector proteins are known to be able to form a tight
complex exclusively with the active form of RhoGTPases through their
PBD (3). We next examined the in vivo substrate specificity
of p200RhoGAP toward Rho GTPases using the immobilized GST fusion PBDs
of Rhotekin, WASP, and PAK1 as bait for selective binding of RhoA-GTP,
Cdc42-GTP, and Rac1-GTP, respectively. In COS-7 cells, wild type RhoA,
constitutively active mutant of RhoA (V14RhoA), or dominant negative
mutant of RhoA (N19RhoA) was cotransfected with a plasmid expressing
the RhoGAP domain of p200RhoGAP (GAP), the catalytic arginine mutant of
the RhoGAP domain (GAP(R58K)), the full-length p200RhoGAP (p200), or
the full-length containing the arginine residue mutation (p200(R58K)).
The lysates of the transfectants were incubated with immobilized
p21-binding domain of Rhotekin, and the co-precipitates were
immunoblotted with anti-RhoA antibody. As shown in Fig.
4A, constitutively active RhoA
(V14RhoA) readily associated with GST-Rhotekin in the presence or
absence of the RhoGAP domain, whereas dominant negative RhoA (N19RhoA) did not interact with this probe. The amount of active RhoA was significantly reduced (a 76% reduction) by the presence of the RhoGAP
domain in the cells in comparison to the conditions in which the RhoGAP
was absent. Furthermore, the catalytic arginine residue mutant of the
RhoGAP domain, GAP(R58K), was only partially (24% reduction) effective
in down-regulating RhoA activity (Fig. 4A). On the other
hand, RhoA-GTP was slightly reduced by the full-length p200RhoGAP (a
30% reduction) and by the full-length molecule bearing the arginine
mutation, p200(R58K) (a 22% reduction). These results suggest that the
RhoGAP domain is an active GAP toward RhoA, and the full-length
p200RhoGAP can weakly deactivate RhoA in vivo. Similar sets
of experiments were also performed to examine the in vivo
GTPase-activating efficacy of the RhoGAP domain and full-length p200RhoGAP toward Rac1 and Cdc42. As shown in Fig. 4B,
neither the RhoGAP domain nor the full-length p200RhoGAP can inactivate Cdc42 effectively. In contrast, expression of the RhoGAP domain significantly reduced the Rac1-GTP species (a 67% reduction) whereas the full-length had only a minor effect (29% reduction) (Fig. 4C). Again, mutation of the catalytic arginine residue in
the RhoGAP domain, Arg-58, to lysine reduced the negative regulatory effect of the GAP (a 23% reduction of Rac1-GTP). Taken together, these
results indicate that p200RhoGAP preferentially regulates RhoA and Rac1
activity through the catalytic GAP domain in cells.

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Fig. 4.
p200RhoGAP specifically activates the GTPase
activities of RhoA and Rac1 in vivo. COS-7 cells
were co-transfected with HA-tagged wild type, constitutively active or
inactive mutant of Cdc42, Rac1 or RhoA, and the pCEFL vector, the
vector containing the RhoGAP domain, the RhoGAP domain mutant
(GAP(R58K)), the full-length, or the full-length mutant (p200(R58K)) of
p200RhoGAP. GTP-bound Cdc42, Rac1, and RhoA were detected by the
effector domain pull-down assay using the GBDs of
His6-tagged WASP, PAK1, and Rhotekin immobilized on beads,
respectively. The representative Western blots of the pull-down assays
are shown on the left in each panel, and the
statistical results from three independent experiments are plotted on
the right.
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The Effects of the GAP Activity of p200RhoGAP on the Actin
Structure of Fibroblasts--
To investigate the cellular role of
p200RhoGAP, the RhoGAP domain was expressed in Swiss 3T3 cells, and the
effect on actin cytoskeleton was examined by staining actin filaments
with TRITC-conjugated phalloidin. An expression vector that
bicistronically expresses GFP as a marker was used to track the RhoGAP
expressing cells in the culture dish. As shown in Fig.
5, the cells expressing RhoGAP domain
became insensitive to lysophosphatidic acid stimulation under
conditions that readily induced heavy stress fibers in the GFP
expressing cells. When the RhoGAP domain expressing cells were
stimulated with PDGF, minimal membrane ruffles were observed under
conditions at which the GFP expressing cells displayed significant membrane ruffles on the cell surface (Fig. 5). Further, the RhoGAP domain of p200 did not affect Bradykinin-induced actin microspike formation that is mediated by Cdc42 activation (Fig. 5). These effects
are similar to those observed in the case of CdGAP (22), another RhoGAP
family member, and are consistent with a negative role of the RhoGAP
domain in the regulation of Rac and Rho activities in these cells,
because one of direct consequences of the inactivation of RhoA and Rac1
in fibroblasts is loss of stress fibers or cell rounding (1, 12). The
effects of the RhoGAP domain, i.e. disruption of actin
stress fibers and membrane ruffles stimulated by lysophosphatidic acid
(LPA) or PDGF, suggests that it favor RhoA and Rac1 as a
substrate in this cellular context.

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Fig. 5.
Effect of the RhoGAP domain of p200RhoGAP on
actin structural changes induced by LPA, PDGF, or Bradykinin in Swiss
3T3 fibroblasts. GFP or the RhoGAP domain, together with GFP, were
expressed by retroviral infection. The cells were isolated by
fluorescence-activated cell sorter based on the GFP marker. After a
16-h serum withdrawal, LPA (20 ng/ml), PDGF (10 ng/ml), or
Bradykinin (100 ng/ml) was added to the cells for 10 min prior to cell
fixation. Filamentous actin was visualized by TRITC-conjugated
phalloidin staining.
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Intracellular Localization and Neurite Induction in Neuroblastoma
Cells--
Because the p200RhoGAP mRNA was found exclusively in
the brain, we wished to determine the role of p200RhoGAP in a neuronal cell line. For this purpose, we raised a polyclonal antibody against p200RhoGAP by using a peptide fragment derived from a unique N-terminal region of the protein (residues 11-25; KQRGILKERVFGCDL) as an immunogen. As shown in Fig.
6A, Western blot analysis
confirmed the expression of p200RhoGAP in a neuroblastoma cell line,
N1E-115. The endogenous p200RhoGAP migrated on a 4-15% gradient
SDS-PAGE as a band greater than 204 kDa, higher than the predicted
200-kDa size, suggesting potential post-translational modifications of this molecule. To examine the intracellular localization of p200RhoGAP, N1E-115 cells were immunostained with anti-p200RhoGAP antibody followed
by incubation with fluorescein isothiocyanate-conjugated anti-rabbit
antibody. In naïve cells, p200RhoGAP was seen predominantly co-stained with cortical actin (Fig. 6B). When the N1E-115
cells were allowed to differentiate by a 24-h serum withdrawal,
p200RhoGAP was detected in the ends of neurite extensions, which were
also heavy in actin content (Fig. 6B). Moreover,
overexpression of the RhoGAP domain or the full-length p200RhoGAP, but
not their respective catalytic arginine residue mutants, could induce
differentiation of N1E-115 cells (Fig.
7). Therefore, it is possible that
p200RhoGAP acts as a Rho GTPase regulator that is involved in the
differentiation of neuronal cells during the formation of neurite
extensions.

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Fig. 6.
A, detection of endogenous p200RhoGAP in
neuroblastoma N1E-115 cell lysates. Rabbit antiserum against p200RhoGAP
was raised using the N-terminal polypeptide of p200RhoGAP
(KQRGILKERVFGCDL). The purified polyclonal antibody was used to detect
endogenous expression of p200RhoGAP in different cell lines.
B, p200RhoGAP colocalizes with actin at the neurite
extension in differentiated N1E-115 cells. Differentiation of N1E-115
cells was induced by serum withdrawn from medium for 24 h.
Cellular distribution of endogenous p200RhoGAP was detected by
immunostaining with the anti-p200RhoGAP antibody. Filamentous actin was
visualized by TRITC-conjugated phalloidin staining.
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Fig. 7.
The GAP activity of p200RhoGAP induces the
differentiation of N1E-115 cells. Wild type RhoGAP domain, the
RhoGAP domain mutant (GAP(R58K)), wild type full-length
p200RhoGAPm and full-length mutant (p200(R58K)) were cloned into a
retroviral vector that bicistronically expresses GFP. Changes of
morphology of GFP-positive N1E-115 cells were observed under a
phase/fluorescent microscope.
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Interaction of p200RhoGAP with SH3 Domains and Active Src
Kinase--
Because the C-terminal region of p200RhoGAP contains
multiple proline-rich motifs that confer to the class II SH3 domain
binding consensus (Fig. 1C) (21), we examined the
possibility that p200RhoGAP may interact with various SH3-containing
proteins. To this end, HA-tagged p200RhoGAP was transiently expressed
in COS-7 cells, and the cell lysates were incubated with GST-fusions of
SH3 domains derived from Abl, Crk, Grb2(C), p85
, Src, spectrin, or
PLC
. As shown in Fig. 8A,
p200RhoGAP could preferentially interact with the SH3 domains of Crk,
p85
, PLC
, and Src, but not with that of Abl, Grb2(C), or
spectrin. The relative stronger in vitro interaction between
the SH3 domain of Src and p200RhoGAP prompted us to further test
whether Src and p200RhoGAP might associate with each other in
vivo. Fig. 8B shows that the constitutively active Src
kinase (Src(Y527F)), but not the kinase-deficient Src (Src(K295R/Y527F)) or wild type c-Src, co-precipitated with
GST-p200RhoGAP when they were co-expressed in COS-7 cells. This
interaction was mediated through the C terminus of p200RhoGAP, because
the RhoGAP domain deletion mutant, p200-C, was able to interact with
the active Src similarly like the full-length molecule (Fig.
8B). Moreover, p200RhoGAP association with active Src
correlated with its tyrosine phosphorylation pattern (Fig.
8B), suggesting that it may serve as an Src kinase substrate
upon complex formation. These results raise the possibility that in
response to extracellular stimuli such as nerve growth factor,
activated Src or Src-like kinases could recruit p200RhoGAP through the
SH3 domain-proline-rich motif interaction that leads to p200RhoGAP
phosphorylation, contributing to its regulation.

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Fig. 8.
Interaction of p200RhoGAP with SH3 domains
and active Src kinase. A, HA-tagged p200RhoGAP was
transiently expressed in COS-7 cells. The cell lysates were incubated
with GST-tagged SH3 domains immobilized on beads from Abl, Crk, Grb(C),
p85, PLC , spectrin, and Src. The co-precipitated p200RhoGAP was
detected by anti-HA Western blotting. B, the GST fusion of
p200RhoGAP or the GAP domain-deleted p200RhoGAP (p200-C) was
co-expressed with kinase-deficient Src (Src(K295R/Y527F)), wild type
c-Src, or constitutively active Src (Src(Y527F)) in COS-7 cells. The
expression of GST fusion constructs was probed by anti-GST Western
blotting, and the associated Src was detected by anti-Src blotting of
the glutathione-agarose co-precipitates. The phosphorylation state of
p200RhoGAP was probed by anti-phosphotyrosine blotting.
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DISCUSSION |
The RhoGAP family is defined by the presence of a conserved RhoGAP
domain in the primary sequences that consist of ~150 amino acids and
share at least 20% sequence identity with other RhoGAPs (5, 6).
Previous studies have established that RhoGAP domain is sufficient for
the binding to GTP-bound Rho proteins and for accelerating their GTPase
activity. To identify novel RhoGAP family members, we have performed a
data base search in both cDNA and genomic DNA data banks
using the conserved features of RhoGAP domain as a probe. Among several
potentially interesting clones, we analyzed the p200RhoGAP
sequences in detail and further identified its murine homolog. Further
characterization of p200RhoGAP led us to conclude that 1) p200RhoGAP is
specifically expressed in the brain; 2) p200RhoGAP is capable of
stimulating the GTPase activities of RhoA and Rac1 in vivo
and in vitro, and a conserved catalytic arginine residue in
its RhoGAP domain is necessary for the GAP activity; 3) endogenous
p200RhoGAP in N1E-115 cells colocalizes with cortical actin in the
undifferentiated state and at the ends of the neurite extension
in the differentiated state; 4) overexpression of the RhoGAP domain or
full-length of p200RhoGAP induces a differentiation phenotype in
N1E-115 cells; and 5) p200RhoGAP binds to class II SH3 domains and is
tyrosine-phosphorylated upon association with active Src. Taken
together, our studies suggest that this novel RhoGAP is involved in the
regulation of neurite outgrowth by exerting its RhoGAP activity and
that its cellular activity may be regulated through interaction with
Src or Src-like tyrosine kinases.
One of the established physiological roles of Rho GTPases is the
regulation of actin cytoskeleton during neuronal migration, axonal
growth and guidance, and formation of synapses (11). Consequently,
regulators and effectors of Rho GTPases are found to play key roles in
neuronal morphogenesis. Recent studies of p190RhoGAP-deficient mice
showed that this RhoGAP plays an important role in axon outgrowth,
guidance and fasciculation, and neuronal morphogenesis (14). In cells
of the neural tube floor plate of p190RhoGAP mutant mice, excessive
accumulation of polymerized actin were found, suggesting a role in the
regulation of Rho-mediated actin assembly within the neuroepithelium
(23). p190RhoGAP was co-enriched with F-actin at the distal end of
axon, and overexpression of p190RhoGAP induced neurite formation in a
neuronal cell line (14). Furthermore, deletion of neuronal adhesion
molecules in mice causes similar defects as seen in p190RhoGAP null
mice, and p190RhoGAP appears to be one of the major Src kinase
substrates in neuron (14). These studies implicate p190RhoGAP in the
neuronal development and neuritogenesis by mediating
Src-dependent adhesion and Rho GTPase regulation. Such a
role is further strengthened by an RNA interference study of p190RhoGAP
in Drosophila, where blockage of p190RhoGAP leads to the
retraction of axonal branches by affecting the RhoA-Drok-MRLC signaling
chain (13). The exclusive presence of p200RhoGAP in the brain suggests
a neuronal-specific role. Although p200RhoGAP is capable of inducing
neurite formation in N1E-115 cells similar to p190, apparently it is
non-redundant to p190 in vivo. One way to address the issue
of the in vivo function of p200RhoGAP would be to decipher
its physiological role in neurons through gene targeting approach.
The growing neurites possess growth cones at the tip where both
positive and negative molecular guidance cues are detected and by which
the path to the target cells is found during neural development.
Recently, Wong et al. (24) have obtained compelling evidence
showing the involvement of a RhoGAP in the intracellular signaling
pathway connecting the extracellular guidance cue to the actin
cytoskeleton in neuronal cells. The Slit proteins, by binding to the
Robo receptor, control the migration of neurons by repelling axons and
migrating neurons. One of the Robo cytoplasmic domain interacting
proteins is Slit-Robo GAP (srGAP). Binding of Slit to Robo leads to the
activation of srGAP, which in turn inactivates Cdc42. The differential
cellular localization of srGAP induced by recruitment to the Robo
receptor could generate a gradient of Cdc42 activity and uneven actin
polymerization, providing a mechanism of Slit-initiated repulsive
effects in neuronal migration (24). It will be of particular interest
to see whether p200RhoGAP is involved in a similar manner in modulating
Rho and/or Rac activity in response to neurotransmitters and/or growth
or differentiation factors such as nerve growth factor and to further
examine whether it may have a role in neuronal guidance.
RhoGAP may also play essential roles in the neuronal synaptic
transmission. Nadrin, a neuron-specific RhoGAP, is involved in the
regulation of Ca2+-dependent exocytosis (25).
Nadrin is colocalized in the neurite termini with cortical actin
filaments. It has been proposed that Nadrin could regulate Rho GTPases
to disassemble cortical actin filaments for the regulated exocytosis
through its RhoGAP activity. Given the similar intracellular
distribution pattern of p200RhoGAP to Nadrin, it will be of interests
to determine whether, like Nadrin, p200RhoGAP could be involved in
regulating neurotransmitter production.
Although we have observed potent GAP activity of the RhoGAP domain of
p200RhoGAP toward RhoA, Rac1, and Cdc42 in purified protein assay
systems (Fig. 3), we could detect the GAP activity on RhoA and Rac1,
but not Cdc42, in COS-7 and Swiss 3T3 cells (see Figs. 4 and 5).
Further, the actin stress fiber disassembly and neuronal
differentiation phenotype induced by p200RhoGAP overexpression are
consistent with a Rho-specific GAP activity by p200RhoGAP. Although the
differences between the RhoGAP domain and full-length molecule may be
explained by the potential regulatory role of the C terminus of
p200RhoGAP (discussed below), the cause for the difference between the
specificity of RhoGAP domain action in vitro and in cells is
not clear at this time. Such in vitro and in vivo
discrepancies in GAP specificity were seen previously in the cases of
the RhoGAP domain of Cdc42GAP and p190RhoGAP, which displayed a
predominantly Rho-specific GAP activity in cells but acted well as a
GAP on Cdc42 in vitro (12).
It appears that many RhoGAPs, including p200RhoGAP, contain SH3 domain
or SH3 domain binding motifs that may contribute to the regulation of
the RhoGAP activity. Previously Cdc42GAP and p85
were shown to
interact with multiple signaling SH3 domains through the proline-rich
motifs (26, 27). CdGAP, possibly through its multiple proline-rich
sequences, binds to the endocytic protein Intersectin (28). Graf and
PSGAP may interact with FAK or proline-rich tyrosine kinase 2 with their respective SH3 domains (29-31). Given the ability of
p200RhoGAP to interact with multiple SH3 domains, one working
hypothesis we are pursuing is that the proline-rich motifs in the
C-terminal region of p200RhoGAP may be involved in connecting to the
Src, Crk, PLC
, or phosphatidylinositol 3-kinase network.
Accumulating evidence indicates that RhoGAP activities can be modulated
by protein kinases (6). One prominent example is the p190RhoGAP
regulation by Src family tyrosine kinases (32, 33). Activation of Src
in cells leads to phosphorylation of tyrosines 1087 or 1105 of
p190RhoGAP that are located close to the RhoGAP domain (34, 35). Upon
phosphorylation, p190RhoGAP recruits p120RasGAP through an
SH2-phosphotyrosine interaction (36). This effectively activates the
Rho-specific GAP activity of p190, causing disruption of actin stress
fibers, reduction of focal contacts, and impairing the ability of the
cell to adhere to fibronectin, the cellular effects consistent with
decreased active Rho GTPase species in cells. In this context, our
observation that p200RhoGAP binds to active Src in cells and becomes
tyrosine-phosphorylated upon association with active Src may provide an
analogy to the case of p190 regulation by Src, although the initial
recognition of p200RhoGAP by Src is likely through a proline-rich
motif-SH3 domain interaction instead of the phosphotyrosine-SH2 domain
interaction. It remains to be seen whether Src SH3 domain binding
and/or phosphorylation by the activated Src may lead to an enhanced GAP
biochemical activity of p200RhoGAP. However, it is possible that this
interaction and modification simply alter the intracellular
localization pattern of p200RhoGAP and serve to fine-tune the local Rho
GTPase activity in neuronal cells.