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Vol. 12, Issue 9, 2711-2720, September 2001
Department of Cell and Developmental Biology and Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, North Carolina 27599
Submitted April 20, 2001; Revised May 22, 2001; Accepted June 26, 2001  |
ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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The binding of extracellular matrix proteins to integrins triggers rearrangements in the actin cytoskeleton by regulating the Rho family of small GTPases. The signaling events that mediate changes in the activity of Rho proteins in response to the extracellular matrix remain largely unknown. We have demonstrated in previous studies that integrin signaling transiently suppresses RhoA activity through stimulation of p190RhoGAP. Here, we investigated the biological significance of adhesion-dependent RhoA inactivation by manipulating p190RhoGAP signaling in Rat1 fibroblasts. The inhibition of RhoA activity that is induced transiently by adhesion was antagonized by expression of dominant negative p190RhoGAP. This resulted in impaired cell spreading on a fibronectin substrate, reduced cell protrusion, and premature assembly of stress fibers. Conversely, overexpression of p190RhoGAP augmented cell spreading. Dominant negative p190RhoGAP elevated RhoA activity in cells on fibronectin and inhibited migration, whereas overexpression of the wild-type GAP decreased RhoA activity, promoted the formation of membrane protrusions, and enhanced motility. Cells expressing dominant negative p190RhoGAP, but not control cells or cells overexpressing the wild-type GAP, were unable to establish polarity in the direction of migration. Taken together, these data demonstrate that integrin-triggered RhoA inhibition by p190RhoGAP enhances spreading and migration by regulating cell protrusion and polarity.
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Rho family GTPases serve as molecular switches, transducing
signals from the extracellular environment to elicit cellular responses
such as changes in gene expression, morphology, and migration (Hall,
1998
; Sastry and Burridge, 2000). Of the 14 known Rho proteins, Cdc42,
Rac1, and RhoA are the most thoroughly characterized (Takai et
al., 2001). Each of these GTPases contributes to cell motility by
stimulating rearrangements in the actin cytoskeleton. Cdc42 and Rac1
stimulate the formation of filopodia and lamellipodia, respectively
(Ridley et al., 1992; Kozma et al., 1995; Nobes
and Hall, 1995). Both of these protrusive structures allow cells to extend into new areas. Accordingly, filopodia and lamellipodia are
commonly found along the circumference of spreading cells and at the
leading edge of migrating cells (Hall, 1998). In addition, Cdc42 is
necessary for the establishment of polarity (Nobes and Hall, 1999;
Erickson and Cerione, 2001).
Active RhoA stimulates the formation of focal adhesions and stress
fibers (Ridley and Hall, 1992). Induction of these structures is
thought to occur via the concerted activities of the RhoA effectors Dia
and Rho kinase/ROCK/ROK but may involve other targets (Kimura et
al., 1996; Watanabe et al., 1999). Rho kinase
stimulates myosin-based contractility by directly and indirectly
elevating phosphorylation of the regulatory myosin light chain
(Kaibuchi et al., 1999). The resulting activation of myosin
triggers myosin filament formation and bundling of filamentous actin
into stress fibers. The tension exerted by stress fibers is responsible
for the maturation of focal complexes into focal adhesions by
aggregating integrins and their associated proteins
(Chrzanowska-Wodnicka and Burridge, 1996). Precise regulation of RhoA
is crucial for efficient cell migration. Although some RhoA activity is
required for migration, possibly to maintain sufficient adhesion to the
substrate, high RhoA activity inhibits movement (Takaishi et
al., 1994; Ridley et al., 1995; Nobes and Hall, 1999).
Because Rho proteins are involved in dynamic cellular processes, such
as migration, their activity is tightly controlled by both positive and
negative regulators (Van Aelst and D'Souza-Schorey, 1997). These
GTPases are activated by guanine nucleotide exchange factors (GEFs),
which catalyze the release of GDP, allowing GTP to bind and thereby
activate the proteins. Rho proteins return to an inactive state upon
hydrolysis of GTP to GDP, a reaction that is potentiated by
GTPase-activating proteins (GAPs). Rho GTPases are also regulated by
guanine nucleotide disassociation inhibitors, which extract Rho
proteins from the plasma membrane (Olofsson, 1999). Deciphering how
extracellular signals alter the activity of GEFs, GAPs, and guanine
nucleotide disassociation inhibitors is critical to understanding the
regulation of Rho family GTPases. Originally it was postulated that
adhesion was necessary, but not sufficient, for activation of RhoA
(Hotchin and Hall, 1995). Subsequent studies demonstrated that adhesion to fibronectin or integrin engagement with arginine, glycine, and aspartic acid (RGD)-containing peptides is sufficient for stimulating RhoA-dependent stress fibers (Barry et al.,
1997). Ren et al. (1999) directly measured RhoA activity,
revealing that adhesion to fibronectin regulates RhoA activity in a
triphasic manner. RhoA is rapidly and transiently inhibited (10-30
min) when cells first bind to fibronectin. This initial inactivation is
followed by an activation phase that peaks between 60 and 90 min. In
the final phase, RhoA activity gradually decreases, by 2-3 h, to a
level slightly higher than that of the initial inhibition.
We demonstrated in earlier studies that the initial
integrin-triggered inhibition of RhoA is mediated by
c-Src-dependent activation of p190RhoGAP (Arthur et al.,
2000). Here we have examined the biological significance of this RhoA
inactivation in cell spreading and migration. Our results suggest that
localized RhoA inactivation by p190RhoGAP in response to
integrin-mediated adhesion contributes to efficient cell
spreading and migration by enhancing both membrane protrusion and cell polarity.
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MATERIALS AND METHODS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Generation of Cell Lines
Rat1 fibroblasts were transfected with the use of LipofectAMINE
PLUS (Life Technologies, Rockville, MD) with 1.05 µg of pPUR (Clontech, Palo Alto, CA) only or cotransfected with 0.05 µg of pPUR
and 1.0 µg of pKH3-p190RhoGAP or
pKH3-p190RhoGAPR1283A (Tatsis et al.,
1998) and then selected in 10 µg/ml puromycin (Sigma, St. Louis, MO).
Clonal lines were established and screened for expression by
immunoblotting with monoclonal antibodies against hemagglutinin epitope (HA; Covance, Richmond, CA) and p190RhoGAP (BD
Transduction Laboratories, Lexington, KY). Ten pPUR clones (mock), five
HA-p190RhoGAPR1283A clones (p190-RA), and three
HA-p190RhoGAP clones (wt-p190) of similar expression levels were
pooled. Cells were maintained in DMEM supplemented with 10% fetal
bovine serum (Life Technologies), antibiotics, and 10 µg/ml puromycin.
For all experiments, cells were replated in the absence of
puromycin the day before experiments, trypsinized, washed twice in
DMEM, and then suspended before plating for 1 h in DMEM and 0.5%
fatty acid-free bovine serum albumin (Sigma). Plating surfaces were
coated with 20 µg/ml fibronectin (Life Technologies) overnight at
4°C, followed by blocking for 1 h with 0.5% fatty acid-free bovine serum albumin. RhoA inhibition was achieved by introduction of
C3 transferase as previously described (Renshaw et al.,
1996). Briefly, 12 µg (spreading assays) or 34 µg (RhoA assays) of
C3 transferase or glutathione S-transferase (GST; as a
negative control) were mixed with 25 µl of LipofectAMINE and 200 µl
of DMEM for 15 min and then added to a 10-cm dish of subconfluent cells
in 5 ml of DMEM for 90 min. RhoA was activated by transfecting cells with 0.5 µg of PAX142-LscD4-HA (Whitehead et al., 1996),
an expression vector encoding a constitutively active mutant of the
RhoA exchange factor, Lsc. As a negative control for transfection,
cells were transfected with pEGFP-C1 (Clontech).
RhoA Activity Assay
RhoA activity was measured with the use of a technique similar
to the method described by Ren et al. (1999). Briefly, cells were lysed in 300 µl of 50 mM Tris, pH 7.4, 10 mM
MgCl2, 500 mM NaCl, 1% Triton X-100, 0.1% SDS,
0.5% deoxycholate, 10 µg/ml each of aprotinin and leupeptin, 1 mM
phenylmethylsulfonyl fluoride, and 200 µM vanadate. Lysates (500-750
µg) were cleared at 16,000 × g for 5 min, and the
supernatant was rotated for 30 min with 30 µg of GST-RBD (GST fusion
protein containing the RhoA-binding domain [amino acids 7-89] of
Rhotekin) bound to glutathione-Sepharose beads (Amersham Pharmacia
Biotech, Uppsala, Sweden). Samples were washed three times in 50 mM
Tris, pH 7.4, 10 mM MgCl2, 150 mM NaCl, 1%
Triton X-100, and inhibitors and then immunoblotted with RhoA monoclonal antibodies (BD Transduction Laboratories). Whole cell
lysates were also immunoblotted for RhoA as loading controls.
Immunofluorescence
Cells on coverslips were fixed for 15 min in 3.7% formaldehyde and then permeabilized for 5 min in 0.5% Triton X-100. Texas Red-phalloidin and Hoechst 33342 from Molecular Probes (Eugene, OR) were used to label F-actin and nuclei, respectively. Monoclonal antibodies against the HA (Covance) or GM130 (BD Transduction Laboratories) were used, followed by incubation with fluorescein isothiocyanate-donkey anti-mouse antibody (Jackson ImmunoResearch Laboratories, West Grove, PA). Images were obtained on an Axiophot microscope (Zeiss, Thornwood, NY) with the use of a MicroMAX 5-MHz cooled charge-coupled device camera (Princeton Instrument, Trenton, NJ) and Metamorph Image software (Universal Imaging, West Chester, PA).
Spreading Assay
Suspended cells were plated on fibronectin-coated coverslips for 10, 20, 30, 45, 60, and 180 min. Coverslips were fixed and stained with Coomassie blue (2% Brilliant Blue [Sigma], 45% methanol, and 10% acetic acid) for 10 min and then washed with water and mounted. The relative areas of individual cells from Metamorph images were quantified with the use of National Institutes of Health Image software. Data from these and all other experiments were considered significantly different if the p values, as determined by two-tailed t-tests, were <0.05.
Migration Assay
The top and bottom surfaces of 8-µm pore, 6.5-mm polycarbonate
Transwell filters (Corning Costar, Corning, NY) were coated with
fibronectin. Cells (2.0 × 104) were seeded
on the upper surface of the filters and migration was allowed to
proceed for 3 h. Cells were fixed, stained with Coomassie blue,
and the number of cells per 25× field on the lower surface of the
filters was counted. For some experiments Rho kinase was inhibited with
3 µM Y-27632 (Uehata et al., 1997b; Welfide, Osaka, Japan).
Monolayer Wound Healing and Polarity Assay
Cells (4.0 × 105) in serum-free
medium were seeded on fibronectin-coated coverslips in 24-well plates
and allowed to adhere for 1 h. The monolayer was then wounded with
a rubber policeman. Cells were washed once, fresh serum-free media was
added, and the cells were allowed to invade the wound for 2 h
(polarity assay) or 3 h (morphology assay) before fixation.
Nuclei, F-actin, and the Golgi were labeled as described above with
Hoechst 33342, phalloidin, and GM130 antibodies, respectively. Cells
bordering the wound were considered polarized if the Golgi was
orientated toward the wound-side of the nucleus (Kupfer et
al., 1982).
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Adhesion-dependent Regulation of RhoA activity by p190RhoGAP
To investigate the biological function of p190RhoGAP in
integrin-mediated signaling, we generated pooled populations of
Rat1 fibroblasts stably expressing dominant negative, GAP-deficient HA-p190RhoGAPR1283A (p190-RA cells), empty vector
(mock cells), or overexpressing wild-type HA-p190RhoGAP (wt-p190
cells). Expression of GAP-deficient p190RhoGAP has previously been
shown to have a dominant negative effect on the endogenous protein
(Arthur et al., 2000). By generating stable lines, we
selected for cells expressing levels of p190RhoGAP below what is
required to induce the previously described severe phenotype
characterized by cell retraction and beaded extensions (Tatsis et
al., 1998). Expression levels of the exogenous p190RhoGAP variants
were approximately four to five times higher than endogenous p190RhoGAP
and had no effect on RhoA expression (Figure
1A).
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To examine the effect of exogenous wild-type or dominant negative
p190RhoGAP, we measured RhoA activity by precipitating active GTP-bound
RhoA with GST-RBD (Ren et al., 1999). In suspension, mock
and p190-RA cells had similar levels of RhoA activity, whereas RhoA
activity levels were reduced in wt-p190 cells (Figure 1B). RhoA
activity was also assessed in mock cells treated with the RhoA
inhibitor C3 transferase or transfected with the exchange factor Lsc as
negative and positive controls, respectively, for the assay. C3
transferase decreased RhoA activity to undetectable levels, whereas
RhoA activity was elevated in cells expressing Lsc (Figure 1B).
Adhesion of mock cells to fibronectin stimulated a rapid and transient
decrease in RhoA activity (Figure 1C). However, RhoA inactivation in
response to fibronectin was antagonized in p190-RA cells. Cells
expressing dominant negative p190RhoGAP continued to show a decrease in
RhoA activity in response to fibronectin adhesion, but this
inactivation was greatly attenuated. These data are consistent with our
previous studies (Arthur et al., 2000) and suggest that RhoA
inhibition by fibronectin-stimulated signaling is mediated by
p190RhoGAP. Furthermore, these data suggest that the activity of
endogenous p190RhoGAP requires adhesion, given that dominant negative
mutants of this GAP modify RhoA activity in adherent cells but not in
suspended cells.
Regulation of the Actin Cytoskeleton by p190RhoGAP during Cell Spreading
Because RhoA regulates the organization of filamentous actin, we
examined the actin cytoskeleton as cells initially adhered to
fibronectin. We found that mock and wt-p190 cells spreading on
fibronectin were nearly devoid of stress fibers at early time points
but had extensive F-actin-rich ruffles and lamellipodia around the
periphery of the cell (Figure 2A). In
contrast, p190-RA fibroblasts had few ruffles or other protrusions but
exhibited robust stress fibers. This phenotype was exaggerated in Rat1
cells transiently transfected with a vector encoding dominant negative HA-p190RhoGAPR1283A (Figure 2B) and found to be
sensitive to the RhoA-specific inhibitor C3 transferase (data not
shown). The precocious development of stress fibers in cells
expressing dominant negative p190RhoGAP at early time points is in
accord with the elevated RhoA activity observed in p190-RA cells
(Figure 1C). Stress fibers developed in mock cells after 60 min on
fibronectin (data not shown), consistent with the elevation in RhoA
activity in these cells at this time point (Figure 1C). These data
suggest that RhoA inhibition by p190RhoGAP is necessary for the
prevention of premature stress fiber formation as cells spread on
fibronectin.
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Regulation of Cell Spreading by p190RhoGAP
Given that RhoA-mediated contractility inhibits membrane
protrusion (Rottner et al., 1999; Cox et al.,
2001) and that RhoA inactivation by fibronectin is antagonized in cells
expressing dominant negative p190RhoGAP (Figure 1C), p190RhoGAP may
regulate cell spreading. To test this hypothesis, we measured the
relative area of individual cells during adhesion (Figure
3A). At early spreading time points (20 min), the area of p190-RA cells was significantly smaller than that of
mock cells. Furthermore, the area of wt-p190 cells was significantly
greater than the area of mock cells. After 3 h on fibronectin, we
observed no significant difference in the areas of mock, p190-RA, and
wt-p190 cells (data not shown). To determine whether the decreased
spreading by p190-RA cells was a result of high RhoA activity (Figure
1C), the RhoA-specific inhibitor C3 transferase was introduced into
cells. We found that RhoA inhibition by a low dose of C3 transferase
partially rescued the decrease in spreading by p190-RA cells (Figure
3B). These data are consistent with the described role of RhoA
inhibition in enhancing membrane protrusion (Rottner et al.,
1999) and suggest that RhoA inactivation by p190RhoGAP contributes to
efficient cell spreading on fibronectin.
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Regulation of Cell Migration by p190RhoGAP
The signaling and cytoskeletal dynamics that occur in spreading
cells are generally thought to mimic events at the leading edge of
migrating cells. Previous studies have demonstrated that p190RhoGAP
localizes to membrane protrusions found at both the periphery of
spreading cells and the leading edge of migrating cells (Nakahara
et al., 1997; Brouns et al., 2000). We found
similar subcellular localization patterns by endogenous and stably
expressed HA-tagged wild-type or GAP-deficient p190RhoGAP in Rat1 cells (data not shown). These findings suggest that p190RhoGAP may be active
at the leading edge of motile cells where they are actively engaging
the extracellular matrix. Consistent with this hypothesis, we found
that the RhoA activity in subconfluent p190-RA cells plated on
fibronectin for 3 h was elevated relative to mock cells (Figure
4). These data suggest that p190RhoGAP
signaling participates in the final decrease in RhoA activity observed
when cells are plated on fibronectin for 2-3 h or longer (Ren et
al., 1999). As expected, RhoA activity was low in wt-p190 cells.
The activity of Cdc42 and Rac1 were also examined with the use of a
previously described technique (Bagrodia et al., 1998);
however, no differences were detected among the mock, wt-p190, and
p190-RA cell lines (data not shown).
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To further explore the role of p190RhoGAP in motility, we examined the
morphology of cells as they migrated into a monolayer wound (Figure
5). We found that the p190-RA cells were
less able to extend membrane protrusions into the wound area, relative
to either mock or wt-p190 cells. In contrast, wt-p190 cells often extended elaborate growth cone-like protrusive structures (Figure 5,
arrowhead). Furthermore, both mock and wt-p190 cells, but not p190-RA
cells, elongated into the wound. These observations suggest that RhoA
inhibition by p190RhoGAP participates in cell elongation and the
formation or maintenance of membrane protrusions in migrating cells.
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To quantitate migration, assays were performed with the use of
Transwell filters. The top and bottom surfaces of the filters were
coated with fibronectin, cells were added to the upper chamber, and
migration was allowed to proceed for 3 h (Figure
6A). We found that migration was enhanced
in wt-p190 cells. Conversely, significantly fewer p190-RA cells were
able to migrate through the filters compared with mock cells. To
determine whether the decrease in migration by p190-RA cells was due to
excessive RhoA signaling, the RhoA effector Rho kinase was inhibited
with Y-27632 (Uehata et al., 1997a). Although Y-27632 did
not significantly alter the motility of mock cells, treatment with this
inhibitor partially rescued the migration defect exhibited by p190-RA
cells (Figure 6B). These data, together with our finding that dominant
negative p190RhoGAP elevated basal RhoA activity in adherent cells
(Figure 4), support our hypothesis that localized RhoA inhibition at
the leading edge by p190RhoGAP enhances cell migration.
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Requirement of p190RhoGAP for the Establishment of Polarity
Although our spreading and wound healing data implicate p190RhoGAP
in the regulation of membrane protrusion, we investigated additional
factors that might contribute to the migration defect exhibited by
p190-RA cells. We initially observed that the nuclei in p190-RA cells
were often positioned centrally within the cell, unlike mock and
wt-p190 cells (not shown). Accordingly, we sought to determine whether
p190RhoGAP is necessary for the establishment of cell polarity, i.e.,
the ability to orient in the direction of migration. To address this
issue, the nuclei and Golgi were labeled in wounded monolayers (Figure
7). Cells along the edge of monolayer
wounds were considered polarized if the Golgi was localized to the
wound-side of the nucleus, as previously described (Kupfer et
al., 1982; Nobes and Hall, 1999). Approximately two-thirds of the
Golgi in the mock and wt-p190 cells were facing the wound. However, we
found that significantly fewer p190-RA cells were polarized relative to
either the mock or wt-p190 cells. In fact, polarization of p190-RA
cells on the edge of wounds was not significantly different from the
random distribution observed in a confluent monolayer. These findings
suggest that localized RhoA inhibition by p190RhoGAP is necessary for
cells to establish polarity in the direction of migration, in addition
to enhancing membrane protrusion.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
In this study, we examined the biological consequence of
adhesion-mediated RhoA inactivation by p190RhoGAP. The impetus for this
work came from several earlier studies that implicated p190RhoGAP signaling in response to adhesion. p190RhoGAP is tyrosine
phosphorylated by the binding of antibodies to 
1 integrins
(Nakahara et al., 1997), translocates to a
detergent-insoluble fraction upon adhesion to fibronectin (Sharma,
1998), and colocalizes with F-actin in lamellipodial protrusions
(Nakahara et al., 1997; Brouns et al., 2000).
Furthermore, we have previously described a signaling mechanism in
which integrin ligation triggers RhoA inhibition via
c-Src-dependent activation of p190RhoGAP (Arthur et al.,
2000).
Here we show that adhesion-dependent RhoA inhibition by p190RhoGAP is necessary for efficient cell spreading and migration. Cells expressing dominant negative p190RhoGAP failed to decrease RhoA activity from high basal levels, resulting in premature stress fiber formation and impaired spreading. In contrast, overexpression of wild-type p190RhoGAP promoted cell spreading. The observation that p190RhoGAP localizes to membrane protrusions at the leading edge of migrating cells suggests that this GAP is active during migration. Consistent with this hypothesis, RhoA activity was elevated in subconfluent adherent cells expressing dominant negative p190RhoGAP. In migrating cells, expression of dominant negative p190RhoGAP inhibited both cell elongation and membrane protrusion, whereas these behaviors were enhanced in cells overexpressing wild-type p190RhoGAP. Accordingly, expression of dominant negative p190RhoGAP inhibited migration, whereas overexpression of p190RhoGAP enhanced motility. Interestingly, dominant negative p190RhoGAP also blocked the ability of cells to establish polarity in the direction of migration. We propose that RhoA inhibition by p190RhoGAP in response to adhesion to fibronectin contributes to both spreading and migration by enhancing cell protrusion, elongation, and polarity.
Cell migration is a complex process involving extension of membrane
protrusions at the leading edge, the formation of adhesions, detachment, and retraction at the rear of the cell (Lauffenburger and
Horwitz, 1996). Although it is well established that Cdc42 and Rac1
activity are crucial for protrusion (Van Aelst and D'Souza-Schorey, 1997), other studies have shown that RhoA inhibition is also involved. Introduction of the RhoA inhibitor C3 transferase into cells stimulates membrane ruffling (Rottner et al., 1999). Furthermore,
expression of wild-type RhoA (Cox et al., 2001) or
constitutively active RhoA (E.A. Cox and A. Huttenlocher, personal
communication) inhibits the extension of membrane protrusions. These
studies support our hypothesis that localized RhoA inactivation by
p190RhoGAP is a permissive step that allows membrane extension. Without
this inhibition, RhoA activity would functionally antagonize the
formation of protrusions through excessive contractility. Consequently,
RhoA inhibition by p190RhoGAP activity contributes to the protrusive
activity required for both spreading and migration.
Recent studies have shown that the nonreceptor tyrosine kinase FAK (Ren
et al., 2000), in addition to c-Src and p190RhoGAP (Arthur
et al., 2000), is necessary for the initial
fibronectin-stimulated RhoA inactivation. Interestingly, the phenotype
we observed in spreading Rat1 cells expressing dominant negative
p190RhoGAP resembles that of spreading cells that lack FAK (Sieg
et al., 1999). It is presently unclear whether FAK
participates in RhoA inhibition by the
integrin 
c-Src p190RhoGAP-signaling mechanism or if it lies in a parallel pathway. Consistent with the idea that FAK participates in c-Src-mediated p190RhoGAP activation, in FAK null cells adhesion activates c-Src and yet fails to inhibit RhoA (Sieg et al., 1998; Ren et al., 2000). In addition, a
scaffolding role has been assigned to the FAK-related kinase
Pyk2/CAK/CADTK/RAFTK because it binds both c-Src and p190RhoGAP and
mediates p190RhoGAP tyrosine phosphorylation by c-Src in response to
heregulin (Zrihan-Licht et al., 2000). Thus, it seems likely
that FAK mediates tyrosine phosphorylation of p190RhoGAP by c-Src in
response to adhesion, just as Pyk2 does downstream of heregulin
stimulation. Intriguingly, Masiero et al. (1999) recently
showed that FAK coimmunoprecipitates with p190RhoGAP in adherent cells.
An unexpected finding in this work was that inhibition of p190RhoGAP
resulted in a failure of cells to develop the polarity that is
associated with migration into a wounded monolayer. Polarity of
migration is associated with selective protrusion of lamellipodia and
filopodia at the leading edge, with persistence of a leading edge, and
suppression of lateral protrusions (Nabi, 1999). It is also associated
with orientation of the Golgi and centrosome to a region in front of
the nucleus in the direction of migration (Kupfer et al.,
1982; Euteneuer and Schliwa, 1992). Little is also known about the
signaling pathways that are initiated by wounding a monolayer that give
rise to polarized cytoskeletal organization and directed migration,
although Cdc42 activity is necessary (Nobes and Hall, 1999; Erickson
and Cerione, 2001). We first suspected that the effect of the dominant
negative form of p190RhoGAP on polarity might be mediated directly or
indirectly by diminished Cdc42 activity. However, we did not detect any
change in activity of this Rho family protein. One possibility is that the elevated RhoA activity inhibits signaling downstream from Cdc42.
Alternatively, it could be that RhoA-mediated contractility exerts its
inhibitory effect on polarity by opposing the formation or maintenance
of protrusive structures (Rottner et al., 1999; Cox et
al., 2001). In this latter scheme of functional antagonism, an
implication is that the protrusive structures contribute themselves to
the development of polarity and are upstream of the polarized orientation of the Golgi. In future work it will be interesting to
explore the hierarchy of these structures with respect to their role in
governing polarity of cell migration.
Further evidence that p190RhoGAP is important for mediating cell
polarity has arisen from studies of the p190RhoGAP-binding partner,
p120RasGAP. The observation that recruitment of p120RasGAP to
p190RhoGAP correlates with stress fiber disassembly in a Src-dependent manner has led to the conclusion that p120RasGAP is important for the
activation of p190RhoGAP (Ellis et al., 1990; Settleman et al., 1992; Chang et al., 1995; van der Geer
et al., 1997; Roof et al., 1998; Fincham et
al., 1999). If p120RasGAP contributes to the activation of
p190RhoGAP, then it would be predicted that neutralization of
p120RasGAP or p190RhoGAP would produce similar phenotypes. Strikingly,
p120RasGAP null cells have defects in cell elongation and polarity,
which result in impaired migration (Kulkarni et al., 2000),
similar to our observations of Rat1 cells expressing dominant negative
p190RhoGAP. The importance of the p190RhoGAP-p120RasGAP association is
further underscored by the demonstration that disruption of this
complex with inhibitory peptides impairs migration and polarization
(Kulkarni et al., 2000).
Together, our results and those of previous studies lead us to propose
the following signaling mechanism by which adhesion to fibronectin
controls cell behavior via regulation of RhoA (Figure 8). The binding of the fibronectin RGD
motif to integrins initially triggers RhoA inactivation by
c-Src-dependent activation of p190RhoGAP (Arthur et al.,
2000), a mechanism that likely includes FAK (Ren et al.,
2000). RhoA inhibition by p190RhoGAP alleviates contractile forces that
would otherwise collapse protrusions required for spreading. Next, RhoA
is activated (Ren et al., 1999), resulting in the formation
of stress fibers and strong adhesion through clustering of
integrins to form focal adhesions (Chrzanowska-Wodnicka and
Burridge, 1996). The delayed activation of RhoA by adhesion to
fibronectin may be triggered by the activation of a GEF in response to
binding of the RGD site to integrins (Barry et al., 1997) or by the heparin-binding domain of fibronectin triggering syndecan-4 signaling (Woods and Couchman, 1992; Bloom et
al., 1999; Saoncella et al., 1999), or both.
Subsequently, RhoA activity decreases to a lower basal level (Ren
et al., 1999). The mechanism by which RhoA activity declines
with time has not been determined, but our observation that dominant
negative p190RhoGAP elevates RhoA activity at longer time points
suggests that p190RhoGAP is involved in this decrease. Localized RhoA
inhibition by p190RhoGAP at the leading edge allows cells to polarize
and extend the membrane protrusions that are essential for cell
migration. In ongoing studies, we are investigating the signaling
components responsible for activation of RhoA by fibronectin binding to
syndecans and integrins.
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ACKNOWLEDGMENTS |
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The authors are grateful to Dr. Ian Macara for providing the wild-type and GAP-deficient p190RhoGAP expression vectors. We also wish to thank Christy Cloonan for excellent technical assistance, Dr. Channing Der for the Lsc construct, Geneva Arthur for preparation of the manuscript, and the members of the Burridge laboratory and Dr. Leslie Petch for thoughtful discussions. This work was supported by National Institutes of Health grant GM29860.
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FOOTNOTES |
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* Corresponding author. E-mail address: warthur{at}med.unc.edu.
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ABBREVIATIONS |
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Abbreviations used: GEF, guanine nucleotide exchange factor; GAP, GTPase-activating protein; GST, glutathione S-transferase; HA, hemagglutinin epitope; p190-RA, Rat1 fibroblasts stably expressing dominant negative HA-p190RhoGAP; RBD, the RhoA-binding domain (amino acids 7-89) of Rhotekin; RGD, single letter amino acid code for arginine, glycine, and aspartic acid; wt-p190, Rat1 fibroblasts stably expressing wild-type HA-p190RhoGAP.
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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I. D. Coghill, S. Brown, D. L. Cottle, M. J. McGrath, P. A. Robinson, H. H. Nandurkar, J. M. Dyson, and C. A. Mitchell FHL3 Is an Actin-binding Protein That Regulates {alpha}-Actinin-mediated Actin Bundling: FHL3 LOCALIZES TO ACTIN STRESS FIBERS AND ENHANCES CELL SPREADING AND STRESS FIBER DISASSEMBLY J. Biol. Chem., June 20, 2003; 278(26): 24139 - 24152. [Abstract] [Full Text] [PDF]
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K. A. DeMali and K. Burridge Coupling membrane protrusion and cell adhesion J. Cell Sci., June 15, 2003; 116(12): 2389 - 2397. [Abstract] [Full Text] [PDF]
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 A. Masszi, C. Di Ciano, G. Sirokmany, W. T. Arthur, O. D. Rotstein, J. Wang, C. A. G. McCulloch, L. Rosivall, I. Mucsi, and A. Kapus Central role for Rho in TGF-beta 1-induced alpha -smooth muscle actin expression during epithelial-mesenchymal transition Am J Physiol Renal Physiol, May 1, 2003; 284(5): F911 - F924. [Abstract] [Full Text] [PDF]
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T. Wakatsuki, R. B. Wysolmerski, and E. L. Elson Mechanics of cell spreading: role of myosin II J. Cell Sci., April 15, 2003; 116(8): 1617 - 1625. [Abstract] [Full Text] [PDF]
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N. K. Noren, W. T. Arthur, and K. Burridge Cadherin Engagement Inhibits RhoA via p190RhoGAP J. Biol. Chem., April 11, 2003; 278(16): 13615 - 13618. [Abstract] [Full Text] [PDF]
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R. A. Worthylake and K. Burridge RhoA and ROCK Promote Migration by Limiting Membrane Protrusions J. Biol. Chem., April 4, 2003; 278(15): 13578 - 13584. [Abstract] [Full Text] [PDF]
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J. C. Hodge, J. Bub, S. Kaul, A. Kajdacsy-Balla, and P. F. Lindholm Requirement of RhoA Activity for Increased Nuclear Factor {kappa}B Activity and PC-3 Human Prostate Cancer Cell Invasion Cancer Res., March 15, 2003; 63(6): 1359 - 1364. [Abstract] [Full Text] [PDF]
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R. M. Wolf, N. Draghi, X. Liang, C. Dai, L. Uhrbom, C. Eklof, B. Westermark, E. C. Holland, and M. D. Resh p190RhoGAP can act to inhibit PDGF-induced gliomas in mice: a putative tumor suppressor encoded on human Chromosome 19q13.3 Genes & Dev., February 15, 2003; 17(4): 476 - 487. [Abstract] [Full Text] [PDF]
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B. Butler, M. P. Williams, and S. D. Blystone Ligand-dependent Activation of Integrin alpha vbeta 3 J. Biol. Chem., February 7, 2003; 278(7): 5264 - 5270. [Abstract] [Full Text] [PDF]
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A. S. Maddox and K. Burridge RhoA is required for cortical retraction and rigidity during mitotic cell rounding J. Cell Biol., January 21, 2003; 160(2): 255 - 265. [Abstract] [Full Text] [PDF]
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L. Zeng, X. Si, W.-P. Yu, H. T. Le, K. P. Ng, R. M.H. Teng, K. Ryan, D. Z.-M. Wang, S. Ponniah, and C. J. Pallen PTP{alpha} regulates integrin-stimulated FAK autophosphorylation and cytoskeletal rearrangement in cell spreading and migration J. Cell Biol., January 2, 2003; 160(1): 137 - 146. [Abstract] [Full Text] [PDF]
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H.-B. Guo, I. Lee, M. Kamar, S. K. Akiyama, and M. Pierce Aberrant N-Glycosylation of {beta}1 Integrin Causes Reduced {alpha}5{beta}1 Integrin Clustering and Stimulates Cell Migration Cancer Res., December 1, 2002; 62(23): 6837 - 6845. [Abstract] [Full Text] [PDF]
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A. Tsubouchi, J. Sakakura, R. Yagi, Y. Mazaki, E. Schaefer, H. Yano, and H. Sabe Localized suppression of RhoA activity by Tyr31/118-phosphorylated paxillin in cell adhesion and migration J. Cell Biol., November 25, 2002; 159(4): 673 - 683. [Abstract] [Full Text] [PDF]
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A. L. Berrier, R. Martinez, G. M. Bokoch, and S. E. LaFlamme The integrin {beta} tail is required and sufficient to regulate adhesion signaling to Rac1 J. Cell Sci., November 15, 2002; 115(22): 4285 - 4291. [Abstract] [Full Text] [PDF]
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J. M. Dunty and M. D. Schaller The N Termini of Focal Adhesion Kinase Family Members Regulate Substrate Phosphorylation, Localization, and Cell Morphology J. Biol. Chem., November 15, 2002; 277(47): 45644 - 45654. [Abstract] [Full Text] [PDF]
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W. T. Arthur, S. M. Ellerbroek, C. J. Der, K. Burridge, and K. Wennerberg XPLN, a Guanine Nucleotide Exchange Factor for RhoA and RhoB, But Not RhoC J. Biol. Chem., November 1, 2002; 277(45): 42964 - 42972. [Abstract] [Full Text] [PDF]
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 Y. Chang, B. Ceacareanu, M. Dixit, N. Sreejayan, and A. Hassid Nitric Oxide-Induced Motility in Aortic Smooth Muscle Cells: Role of Protein Tyrosine Phosphatase SHP-2 and GTP-Binding Protein Rho Circ. Res., September 6, 2002; 91(5): 390 - 397. [Abstract] [Full Text] [PDF]
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T. Tsuji, T. Ishizaki, M. Okamoto, C. Higashida, K. Kimura, T. Furuyashiki, Y. Arakawa, R. B. Birge, T. Nakamoto, H. Hirai, et al. ROCK and mDia1 antagonize in Rho-dependent Rac activation in Swiss 3T3 fibroblasts J. Cell Biol., May 28, 2002; 157(5): 819 - 830. [Abstract] [Full Text] [PDF]
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