Molecular Biology of the Cell
Vol. 11, 3703–3721, November 2000
Activation of the Small GTPase Rac Is Sufficient to
Disrupt Cadherin-dependent Cell-Cell Adhesion in
Normal Human Keratinocytes
Vania M.M. Braga,*†Martha Betson,* Xiaodong Li,‡and Nathalie Lamarche-Vane‡
*Medical Research Council Laboratory for Molecular Cell Biology and the Department of Biochemistry
and Molecular Biology, University College London, London WC1E 6BT, United Kingdom; and
‡Department of Anatomy and Cell Biology, McGill University, Montreal, Canada H3A 2B2
Submitted January 21, 2000; Revised August 1, 2000; Accepted August 10, 2000
Monitoring Editor: Joan S. Brugge
To achieve strong adhesion to their neighbors and sustain stress and tension, epithelial cells develop
many different specialized adhesive structures. Breakdown of these structures occurs during tumor
progression, with the development of a fibroblastic morphology characteristic of metastatic cells.
During Ras transformation, Rac-signaling pathways participate in the disruption of cadherin-depen-
dent adhesion. We show that sustained Rac activation per se is sufficient to disassemble cadherin-
mediated contacts in keratinocytes, in a concentration- and time-dependent manner. Cadherin recep-
tors are removed from junctions before integrin receptors, suggesting that pathways activated by Rac
can specifically interfere with cadherin function. We mapped an important region for disruption of
junctions to the putative second effector domain of the Rac protein. Interestingly, although this region
overlaps the domain necessary to induce lamellipodia, we demonstrate that the disassembly of
cadherin complexes is a new Rac activity, distinct from Rac-dependent lamellipodia formation.
Because Rac activity is also necessary for migration, Rac is a good candidate to coordinately regulate
cell-cell and cell-substratum adhesion during tumorigenesis.
Cell-cell adhesion is an essential feature of epithelia that
ensures their polarized status and therefore their differenti-
ation and physiological function. During tumorigenesis, the
breakdown of intercellular adhesion has two main conse-
quences: loss of epithelial characteristics and, as dedifferen-
tiation proceeds, increased migration and metastasis of the
dissociated cells. Cell-cell adhesion receptors of the cadherin
family have been implicated in these cellular processes.
First, it is well established that cadherin receptors play an
important role in the development and maintenance of the
differentiated epithelial phenotype during organogenesis
and adult life (reviewed by Gumbiner, 1996). Second, cad-
herins participate in the contact inhibition of growth shown
by nonimmortalized cells (St Croix et al., 1998) and alter-
ations in cadherin function are frequently found, and have a
causal role, during tumor progression (Perl et al., 1998).
Cadherins are transmembrane proteins that promote cal-
cium-dependent intercellular adhesion between cells con-
taining the same type of receptor (homophilic binding;
Gumbiner, 1996). At the intracellular side, cadherin mole-
cules associate with cytoplasmic proteins known collectively
as catenins. At the surface of individual cells, it is believed
that cadherin complexes are found as dimers in a lateral
association mediated by their extracellular domains (Shapiro
et al., 1995; Brieher et al., 1996; Nagar et al., 1996; Yap et al.,
1997). Dimers from two opposing cells interact in an antipa-
rallel manner (adhesive association) to form the structural
unit of cadherin-mediated cell-cell adhesion (Chitaev and
Troyanovsky, 1998). This adhesive interaction requires and
is stabilized by extracellular calcium ions and, at the cyto-
plasmic side, by the association of cadherin receptors with
the catenins and actin cytoskeleton (reviewed by Kemler,
1993; Yap et al., 1997; Chitaev and Troyanovsky, 1998). Al-
though nonadhesive cadherin complexes can weakly inter-
act with the cytoskeleton (Sako et al. 1998), the cytoskeletal
interaction is greatly enhanced during the formation of cell-
cell adhesion, by the clustering of the adhesive complexes at
contact sites (reviewed by Kemler, 1993; Brieher et al., 1996;
Yap et al., 1997; Chitaev and Troyanovsky, 1998).
Over the past few years, much effort has been put into
understanding how cadherin function adhesion is regulated
from the cytoplasm. Recently, we and others have demon-
strated that cadherin-mediated adhesion requires the activ-
ity of the cytosolic proteins of the Rho family of small
GTPases (Braga et al., 1997, 1999; Hordijk et al., 1997; Takai-
shi et al., 1997; Zhong et al., 1997). They belong to the Ras
superfamily of small GTPases, proteins whose function is
†Corresponding author. E-mail address: firstname.lastname@example.org.
© 2000 by The American Society for Cell Biology3703
regulated depending on the type of guanine nucleotide
bound (reviewed by Van Aelst and D’Souza-Schorey, 1997).
When GTP is associated, the small GTPases are in an acti-
vated form and competent for signaling. Upon GTP hydro-
lysis and liberation of phosphate, the small GTPases are
inactivated, in a cycle that is tightly modulated by regula-
tory proteins (Van Aelst and D’Souza-Schorey, 1997). The
output signal is dependent on the amount of time that GTP
remains associated as well as the localization of the GTP-
bound protein within the cell (brought about by GDP-GTP
exchange factors or GEF) (Bokoch et al., 1994; Michiels et al.,
The RHO subfamily members, Rho, Rac, and Cdc42, par-
ticipate in a variety of cellular processes primarily involving
actin cytoskeleton reorganization, such as cell-cell adhesion,
cell-extracellular matrix adhesion, cytokinesis, and cell mo-
tility (Van Aelst and D’Souza-Schorey, 1997). Rho is in-
volved in cell contractility and stress fiber formation,
whereas Rac drives actin polymerization and formation of
lamellipodia (Ridley and Hall; 1992; Ridley et al., 1992). In
epithelial cells, the activity of small GTPases is required both
for the formation of new cadherin-mediated contacts and for
the maintenance of stable junctions (Braga et al., 1997, 1999;
Hordijk et al., 1997; Takaishi et al., 1997; Zhong et al., 1997).
Rho and Rac effects on cadherin receptors are modulated by
the maturation of the junctions and the cellular context in
which the cadherin molecule is expressed (Braga et al., 1999).
In simple epithelial cells such as Madin-Darby canine
kidney (MDCK), exogenously expressed myc-tagged Rho
and Rac localize at sites of cell-cell adhesion (Adamson et al.,
1992; Takaishi et al., 1995, 1997; Jou and Nelson, 1998).
Moreover, proteins that can interact with the small GTPase
Rac can also localize at intercellular junctions, but the func-
tional significance of their localization is not known (re-
viewed by Van Aelst and D’Souza Schorey, 1997; Hordijk et
al., 1997; Kuroda et al., 1998). In MDCK cells, Rac activation
correlates with an increased staining of cadherin receptors
and actin at cell-cell borders, suggesting that Rac may
strengthen cadherin-dependent adhesion (Hordijk et al.,
1997; Takaishi et al., 1997).
However, although Rac function is necessary for cad-
herin-dependent adhesion, there is evidence in the literature
that Rac can play a role during tumor progression. Rac
activation is required for the full transformed phenotype
induced by oncogenes such as Tiam-1, Ras, and Mas (Habets
et al., 1994; Khosravi-Far et al., 1995; Qiu et al., 1995; van
Leeuwen et al., 1995; Roux et al., 1997; Zohn et al., 1998). In
addition, it has been shown that Rac activation can promote
invasion of carcinoma and lymphoma cell lines (Habets et
al., 1994; Keely et al., 1997; Shaw et al., 1997). Although Rac
clearly participates in cell migration, the question remains of
how to reconcile its role in migration with the “strengthen-
ing” effect on cell-cell contacts.
In this article, we investigated in more detail the effects of
Rac activation on the stability of cadherin receptors in hu-
man keratinocytes. Rac activation does not lead to increased
levels of cadherin staining at the keratinocyte junctions,
contrary to what has been shown for MDCK cells. Moreover,
our results suggest that sustained Rac activation can specif-
ically remove cadherin receptors from newly formed and
stable cell-cell contacts in a concentration- and time-depen-
dent manner. Interestingly, although the Rac-dependent loss
of cadherin function was accompanied by changes in cell
shape and protusion formation, we demonstrate that this is
a new Rac activity, distinct from its reported cytoskeletal
role in lamellipodia formation.
MATERIALS AND METHODS
Normal human keratinocytes (strain Kb, passages 3 to 7) were
cultured on a mitomycin C-treated monolayer of 3T3 fibroblasts at
37°C and 5% CO2as reported previously (Rheinwald, 1989). Cells
were routinely cultured in standard medium (DMEM:F-12 medium,
1:3 mixture; Imperial Laboratories, Hampshire, United Kingdom)
containing 1.8 mM calcium ions and supplements as described, but
with 5% fetal calf serum. Cultures grown in the absence of calcium-
dependent cell-cell contacts used the same medium formulation as
described above, but with 0.1 mM calcium ions and serum depleted
of divalent ions by treatment with Chelex-100 resin (Bio-Rad, Rich-
mond, CA; Hodivala and Watt, 1994). HaCat cells (immortalized,
nontumorigenic human keratinocytes) were a kind gift from N.
Fusenig, Deutches Krebsforschungszentrum, Heidelberg, Germany
(Ryle et al., 1989). In experiments in which the calcium switch was
performed, HaCat cells were transferred to low-calcium medium
(1–2 days after plating) and cultured until confluence as described
above. Swiss 3T3 cells were routinely cultured as described previ-
ously (Ridley and Hall, 1992; Ridley et al., 1992); cells were allowed
to reach confluence and become quiescent for 6–10 d before seeding
E-cadherin staining was performed using either ECCD-2 antibody
(rat monoclonal) (Hirai et al., 1989) or HECD-1 (mouse monoclonal;
gift from M. Takeichi, Kyoto University, Japan; Shimoyama et al.,
1989). Integrin labeling was done using the anti-?1 integrin anti-
body P5D2 (mouse monoclonal) (Dittel et al., 1993). The other mono-
clonal antibody used was anti-myc (mouse monoclonal 9E10). Sec-
ondary antibodies were bought from Jackson ImmunoResearch
Laboratories, West Grove, PA (Stratech Scientific, Luton, United
Kingdom): indodicarbocyanine (Cy5)-conjugated donkey anti-
mouse IgG; fluorescein isothiocyanate-conjugated goat anti-mouse
IgG and FITC-conjugated donkey anti-rat IgG. FITC-phalloidin was
purchased from Sigma (Poole, United Kingdom).
Mutagenesis and Subcloning
Point mutations were introduced into constitutively active Rac
(L61Rac) by polymerase chain reaction (PCR) with 5? primers and 3?
primers containing the respective alanine-alanine substitutions in
the putative second effector domain as follows: A147 A148 (lys147
glu148converted to ala147ala148, respectively; or using single letter
code; KE to AA); A162 A163 (glu162arg163to ala162ala163, respec-
tively; QR to AA); A166 A167 (lys166thr167to ala166ala167, respec-
tively; KT to AA), and A170 A171 (asp170glu171to ala170ala171,
respectively; DE to AA). The PCR fragments were then subcloned as
NcoI-EcoRI inserts into pGEX-2T-L61Rac. All L61Rac mutants were
fully sequenced using the Stratagene kit.
L61Rac second effector domain mutants were PCR amplified and
subcloned into the EcoRI/BamHI sites of the yeast two hybrid vector
pYTH9. The constructs were sequenced to confirm that the Rac
sequence was fused in frame with the sequence encoding the GAL4
DNA-binding domain. To create pACTII-NIQGAP2, the sequence
corresponding to amino acids 711-1579 of IQGAP2 was amplified
using the primers GTG CTA CAT CAT CAT CGG AAG AG and
CCT TGA TTG GAG ACT TGA CC and subcloned into the NcoI-
BamHI site of the GAL4-activation domain vector pACTII. Rac
targets (ROK-?, MLK2, MLK3, and PAK) and RhoGAP subcloned
V.M.M. Braga et al.
Molecular Biology of the Cell3704
into pACTII vector were a kind gift from Alan Hall (Aspenstrom
and Olson, 1995; Nagata et al., 1998).
Recombinant proteins were purified as glutathione S-transferase-
fusion proteins from Escherichia coli by using glutathione beads,
thrombin cleaved (unless otherwise stated), dialysed, and concen-
trated essentially as described (Ridley et al., 1992). The protein
concentration of each batch was determined by bicinchoninic acid
assay (Pierce, Rockford, IL), by using bovine serum albumin as
standard, and the purity of the preparation was evaluated by sep-
aration in SDS-PAGE followed by Coomassie blue staining. Biolog-
ical activity was determined beforehand in fibroblasts and keratin-
ocytes as reported (Ridley and Hall, 1992; Ridley et al., 1992; Braga
et al., 1997).
Recombinant proteins used were as follows: C3 transferase (used
at 0.1 mg/ml), constitutively active forms of Rac (L61Rac, 4 mg/ml),
Rho (L63Rho, 3.76 mg/ml), or H-Ras (V12Ras, 3.77 mg/ml). RacRho
chimeras used were as follows: Rac73Rho (3.35 mg/ml), Rac126Rho
(0.53 mg/ml), Rac143Rho (0.43 mg/ml), and Rac175Rho (2.39 mg/
ml). L61Rac second effector domain mutant recombinant proteins
were also prepared (see above for details): A147 A148 (0.89 mg/ml),
A162 A163 (2.26 mg/ml), A166 A167 (2.91 mg/ml), and A170 A171
(3.57 mg/ml). In addition to GST, the following proteins were used
uncleaved: RhoGAP (1.84 mg/ml); ROK-? (GBD, GTPase binding
domain only, 4.44 mg/ml; gift from David Drechsel, Heidelberg,
Germany; Burbelo et al., 1995); PAK (GBD only, 4.42 mg/ml; Sander
et al., 1998); MLK2 (leucine-zipper and GBD domain; Nagata et al.,
1998); and POSH (GBD only, 2 mg/ml, kind gift from Anne Bishop
(MRC-LMCB, London, UK); Tapon et al., 1998).
Microinjection was performed essentially as described (Braga et al.,
1997). Confluent patches of keratinocytes grown in the absence of
contacts were microinjected with the different recombinant proteins
mixed with Dextran Texas-Red (Molecular Probes, Eugene, OR) to
visualize the injected patches. Within 5 to 15 min after injection, cells
were transferred to standard medium to induce calcium-dependent
cell-cell contacts for additional 1 to 5 h. Alternatively, medium-sized
colonies of keratinocytes cultured in standard medium (mature
junctions) were injected with distinct recombinant proteins and
incubated for different amounts of time in the same medium. Swiss
3T3 cells were seeded onto coverslips subconfluent and prepared
for microinjection as reported (Puls et al., 1999). After microinjec-
tion, cells were incubated for 15 to 30 min in the same medium.
Recombinant proteins were injected either neat or at the stated
dilutions to better assess their effects on junction disassembly in
keratinocytes or lamellipodia formation in Swiss 3T3 cells. Quanti-
fication of the effects of Rac mutants on cadherin-mediated adhesion
was performed using the following criteria. Patches containing
three or more cell-cell borders with perturbed cadherin staining
between at least two different injected cells were scored and ex-
pressed as a percentage of the total number of microinjected
patches. Between 30 and 50 patches (containing 4 to 10 cells each)
were analyzed for any given mutant. Quantification of lamellipo-
dium formation in Swiss 3T3 cells is expressed as the percentage of
injected cells with lamellipodia/ruffles. Between 60 and 180 injected
cells (Swiss 3T3) were scored for each recombinant protein tested.
Statistical analysis was performed using Student’s t test, assuming
Lamarche et al., 1996) was microinjected into the nucleus of HaCat
cells grown in low-calcium medium. After 2 h of expression, cells
were transferred to standard calcium medium to induce junction
formation for 4 h. Activated H-Ras (V12 Ras-pRK5myc) and domi-
nant-negative Rac (N17Rac-pRK5myc) were injected in HaCat cells
grown in standard medium (mature junctions) and incubated for
5 h. DNA was injected at 0.1 mg/ml.
Cells were fixed in 3% paraformaldehyde for 10 min at room tem-
perature, permeabilized, and stained as described previously (Braga
et al., 1997). In some experiments, cells were extracted with CSK
buffer containing 0.5% Triton X-100 for 10 min at room temperature
before fixation (Braga et al., 1995). Single labeling for E-cadherin was
performed by using the mouse monoclonal HECD-1 and FITC-
conjugated anti-mouse IgG. Double labeling for cadherins and in-
tegrins was performed by sequential incubation with rat anti-E-
cadherin monoclonal (ECCD-2), FITC-conjugated anti-rat IgG,
followed by mouse anti-1 integrin antibody (P5D2), and Cy5-con-
jugated anti-mouse IgG. Stainining for myc-tagged proteins was
performed using the mouse monoclonal 9E10 and Cy5-conjugated
anti-mouse IgG. Filamentous actin in Swiss 3T3 cells was labeled
with FITC-phalloidin. Confocal images were obtained (1-?m slices)
at the plane in which the majority of cadherin staining in the
injected patch was found and processed as reported (Braga et al.,
1999). For the Dextran-Texas Red image, the optical section is taken
at a different plane (usually a few microns below) to show that the
cells are still touching each other and not retracted at the end of the
Fusion proteins were immobilized onto PVDF membranes (Milli-
pore, Bedford, MA) by using a slot blot apparatus (Hoeffer, San
Francisco, CA). Equal amounts of L61Rac and L61Rac containing
additional mutations in the second effector region (A162 A163, and
A170 A171) were loaded with radioactive GTP ([?-32P]GTP, 6000
Ci/mmol; NEN-DuPont, Boston, MA) and allowed to interact with
the immobilized proteins as described (Lamarche et al., 1996).
Yeast Two-Hybrid Interactions
Integrated yeast strains were created containing L61Rac and the
L61Rac second effector domain mutants fused to the GAL-4 DBD
(Aspenstrom and Olson, 1995). Yeast strains were transformed with
cDNAs encoding various Rac binding partners in a GAL4-activation
domain vector: pACTII-RhoGAP, pACTII-PAK, pACT?-ROK-?
(GTPase binding domain only), pACTII-IQGAP2, pACTII-MLK2,
and pACTII-MLK3 (Nagata et al., 1998). Interactions were assayed
by testing growth of colonies in the presence of 3-amino-1,2,4-
triazole and by filter-lift ?-galactosidase assay (Aspenstrom and
Olson, 1995). IQGAP2 interactions were tested on 10 mM 3AT
plates, as the association with activated Rac was barely detectable at
the standard concentration used for the other targets (25 mM).
Mammalian Cell Transfections and c-Jun NH2-
Terminal Kinase 1(JNK1) Activation Assay
Cos-7 cells were transfected by DEAE-dextran method as described
(Lamarche et al., 1996). Plasmid amounts per 10-cm Petri dish were
as follows: 5 ?g of pCMV-FLAG-JNK1 with 1 ?g each of pRK5myc,
pRK5myc-RacL61, or the various RacL61 mutants. Twenty-four
hours later, transfected cells were serum-starved for 16 h before
lysis in 25 mM HEPES (pH 7.6), 1% (vol/vol) Triton X-100, 1%
(wt/vol) sodium deoxycholate, 0.1% (wt/vol) SDS, 0.3 M NaCl, 50
mM NaF, 0.1 mM vanadate, 5 mM EDTA, 5 mM EGTA, 40 mM
sodium pyrophosphate, and protease inhibitors. To quantitate the
amount of JNK1 present in each experiment, one-tenth of each
lysate was loaded onto 15% SDS-PAGE and transferred to nitrocel-
lulose membrane. Flag-tagged JNK1 was visualized with an anti-
FLAG monoclonal antibody (Sigma) and 0.1 Ci/ml protein A-125I
and quantitated by phosphorimage analysis. An equal amount of
JNK1 protein was loaded onto 7.5% SDS-PAGE and transferred to
nitrocellulose membrane. Activated JNK1 was determined with an
antiphospho-JNK1 (Thr 183/Thr185) monoclonal antibody (New
England Biolabs, Beverly, MA) and protein A-125I and revealed by
autoradiography. The relative levels of activated JNK1 were deter-
Rac Activation and Cadherin Adhesion
Vol. 11, November 20003705
mined by phosphorimage analysis. To determine the amount of
JNK1 in each lane, the membrane was stripped in 100 mM 2-mer-
captoethanol, 2% SDS, 62.5 mM Tris-HCl, pH 6.7, at 50°C for 30 min
and incubated with an anti-FLAG antibody and revealed by chemi-
A Role for Rac during Tumorigenesis
As suggested by experiments in MDCK cells, Rac activation
may strengthen cadherin-dependent contacts because it in-
duces an increased localization of the receptors and actin at
junctions (Hordijk et al., 1997; Takaishi et al., 1997). To test
this possibility in normal keratinocytes, L61Rac was micro-
injected into cells grown in standard medium (mature con-
tacts) and incubated for 2 h (0.5 mg/ml, Figure 1, a and b).
Under these conditions, the concentration of cadherin recep-
tors at cell-cell contacts (Figure 1, a and b) and their deter-
gent solubility (Figure 1, c and d) were not increased in the
injected keratinocytes compared with neighboring cells.
In addition, our results indicate that Rac activation was
not sufficient to protect cadherin receptors from different
destabilizing effects. One such stimulus is the expression of
oncogenic Ras (V12H-Ras), which in keratinocytes interferes
with stable cell-cell adhesion (Figure 1, e and f; our unpub-
lished results; Espada et al., 1999). Controls showed that
when injected alone, H-Ras disrupted cadherin adhesion
within 2 h (Figure 1, e and f). This effect was not changed or
delayed by coinjection of activated Rac (Figure 1, g and h).
Similar results were observed if junctions were perturbed by
inhibition of endogenous Rho (our unpublished results;
Braga et al., 1997; Jou and Nelson, 1998). Our data support
the conclusion that Rac activation does not increase the
localization of cadherin receptors at junctions in normal
human keratinocytes as shown in MDCK cell lines.
If Rac is not providing a protective effect for the keratin-
ocyte junctions, what are the consequences of Rac activa-
tion? We asked whether Rac-signaling pathways could con-
tribute to the destabilization of cell-cell adhesion seen
during oncogenic transformation. Expression of activated
H-Ras (V12Ras) in HaCat cells, a keratinocyte cell line, dis-
rupted cadherin adhesion (Figure 2, d–f) similarly to what
was observed in normal human keratinocytes (Figure 1, e
and f). A dominant-negative form of Rac was expressed in
HaCats to levels not high enough to perturb junctions
(N17Rac, Figure 2, a–c). When N17Rac was coexpressed
with activated Ras (V12Ras), a reduction in the signaling
from Rac was sufficient to restore cadherin localization at
cell-cell contacts (Figure 2, g–i). In contrast, inhibition of
other pathways such as phosphoinositide 3-OH kinase (PI3
kinase) and mitogen-activated protein kinase (MAPK) dur-
ing Ras activation in normal keratinocytes could only par-
tially rescue the localization of cadherins at junctions (our
Rac Activation Specifically Perturbs Cadherin-
dependent Cell-Cell Contacts
The above-mentioned results suggested that Rac can be
activated during transformation and its activity may con-
tribute to destabilization of junctions. We next addressed
two questions: 1) whether Rac activation per se was suf-
ficient to disrupt cadherin-dependent adhesion in human
keratinocytes, and 2) whether Rac could specifically inter-
fere with cadherin-mediated adhesion. Keratinocytes
grown in standard medium (mature junctions) were mi-
croinjected with different concentrations of the same batch
of activated Rac (L61Rac, 0.25–2.0 mg/ml, Figure 3) and
incubated for 2 h. Cells were then fixed and double la-
beled for E-cadherin (Figure 3, b, e, h, and k) and ?1-in-
tegrins (Figure 3, c, f, i, and l). With increasing concen-
trations of activatedRac,
selectively removed from intercellular junctions (arrow-
heads, Figure 3), whereas localization of integrins re-
mained unchanged over 2 h (arrows, Figure 3). Our data
suggest that Rac activation can specifically destabilize
cadherin receptors from mature cell-cell junctions in nor-
mal keratinocytes, because it had no significant effect on
the localization of integrin receptors.
Activation of Rac Is Sufficient to Perturb Cadherin-
dependent Adhesion in Human Keratinocytes
We have previously shown that newly formed junctions
are more sensitive to the effects of the small GTPases
(Braga et al., 1999). The above-mentioned results with Rac
activation were also confirmed during induction of inter-
cellular junctions. Keratinocytes grown in low-calcium
medium were microinjected with different dilutions of the
same batch of activated Rac (L61Rac, 0.25–2 mg/ml, Fig-
ure 4). Cell-cell adhesion was induced for 2 h and cells
were labeled for E-cadherin (Figure 4, b, d, f, and h). Our
results showed that at lower concentration, L61Rac activ-
ity was compatible with stable cell-cell contacts (0.25 mg/
ml, Figure 4, a and b; Braga et al., 1997). However, increas-
ing amounts of activated Rac clearly perturbed cadherin
localization at junctions and cell morphology (0.5–2.0 mg/
ml, Figure 4, c–h), with a concomitant formation of pro-
tusions and lamellae (Figure 4, e–h). Thus, lower concen-
trations of active Rac disrupted newly formed junctions
(0.5 mg/ml) compared with the amount necessary to per-
turb mature junctions (1 mg/ml, Figure 3).
A time course was also performed by microinjecting
L61Rac at a given concentration (0.5 mg/ml) into keratino-
cytes without cell-cell contacts, and transferring the cells to
standard medium to induce intercellular adhesion for 1 to
5 h (Figure 5). Although a shorter incubation did not affect
the cadherin staining or cell morphology (1 h, Figure 5, a and
b), prolonged incubation after Rac activation interfered with
both (2 and 5 h, Figure 5, c–f). These results are consistent
with the data shown in Figure 4, and suggest that they did
not result from toxicity of the more concentrated L61Rac
protein solutions injected into keratinocytes.
To confirm the disruptive effect of activated Rac on junc-
tions and exclude the contribution of any bacterial protein
contaminants, we performed microinjections of L61Rac
DNA into the nucleus. We were unable to obtain good
expression levels when normal keratinocytes were used,
despite testing a variety of different expression vectors. In-
stead, we used HaCat cells, a human keratinocyte cell line.
After induction of cell-cell contacts for 4 h (total expression
time 6 h), we observed a qualitative disruption of cadherin
adhesion (Figure 5, g and h) as seen in primary keratinocytes
(see also Figures 4, c–f, and 5, e and f). This result can be
obtained with microinjection of either recombinant proteins
or DNA encoding activated Rac in HaCat cells. Taken to-
V.M.M. Braga et al.
Molecular Biology of the Cell3706
strengthen cadherin-dependent adhe-
sion in keratinocytes. Cells containing
mature intercellular junctions were mi-
croinjected with constitutively active
Rac (L61Rac, 0.5 mg/ml). Activated Rac
was injected either alone (a–d), or in
(V12Ras ? L61Rac, g and h). Controls
show keratinocytes injected with H-Ras
alone (V12Ras, e and f). After 2 h of
incubation, cells were fixed and stained
for E-cadherin (b, d, f, and h). In c and
d, keratinocytes were preextracted with
Triton X-100 before fixation (see MATE-
jected patches of cells are seen in a, c, e,
and g. Arrowheads (f and h) show ab-
sence of cadherin staining at junctions.
Arrows (b and d) point to similar levels
of cadherin staining in microinjected
cells. Bar, 50 ?m.
Rac activation does not
Rac Activation and Cadherin Adhesion
Vol. 11, November 20003707
gether, our data indicate that sustained Rac activation dur-
ing junction formation resulted in changes in cell shape and
E-cadherin localization in a time- and concentration-depen-
Although the L61Rac effects on cadherin adhesion in ma-
ture junctions were similar to those observed when new
cell-cell contacts were established, epithelial cell shape was
not significantly perturbed when keratinocytes have stable
junctions (Figures 3, j–l, and 4, g and h) and higher levels of
active Rac were required to disrupt the intercellular contacts
(Figures 3, d and f, and 4, c and d). The same differences
were observed in HaCat cells: although formation of junc-
tions was readily affected by expression of active Rac, it was
more difficult to disrupt mature contacts (our unpublished
with an expression vector containing dominant-negative Rac (N17Rac, a–c), oncogenic Ras (V12Ras, d–f), or both together (g–i). After 5 h,
cells were fixed and double labeled for the myc tag (b, e, and h) and E-cadherin (c, f, and i). Injected patches were identified by coinjection
of Dextran-Texas Red (a, d, and g). Arrows (c and i) point to the presence of cadherin staining at junctions; arrowhead (f) shows the absence
of cadherin staining between two expressing cells. Bar, 50 ?m.
Rac signaling pathways contribute to the destabilization of cadherin receptors after Ras activation. HaCat cells were microinjected
V.M.M. Braga et al.
Molecular Biology of the Cell3708
(mature cell-cell contacts) were microinjected with different concentrations of constitutively active Rac (L61Rac) as follows: 0.25 mg/ml (a–c),
0.5 mg/ml (d–f), 1.0 mg/ml (g–i), and 2.0 mg/ml (j–l). After a 2-h incubation in the same medium, cell were fixed and double labeled for
E-cadherin (b, e, h, and k) and integrins (c, f, i, and l). Injected cells are seen in a, d, g, and j. Arrows (i and l) show integrin staining at
junctions; arrowheads (h and k) show absence of cadherin receptors at junctions. Bar, 50 ?m.
Increased Rac activation perturbs the stability of cadherin receptors in mature junctions. Keratinocytes grown in standard medium
Rac Activation and Cadherin Adhesion
Vol. 11, November 20003709
of constitutively active Rac (L61Rac)
perturb newly formed cell-cell adhe-
sion and epithelial cell shape. Differ-
ent concentrations of activated Rac
(L61Rac) were microinjected into ker-
atinocytes in the absence of intercel-
lular contacts as follows: 0.25 mg/ml
(a and b), 0.5 mg/ml (c and d), 1.0
mg/ml (e and f), and 2.0 mg/ml (g
and h). Calcium-dependent cell-cell
adhesion was induced for 2 h; cells
were then fixed and labeled for E-
cadherin, followed by an FITC-conju-
gated anti-mouse IgG (b, d, f, and h).
Injected cells are seen in a, c, e, and g.
Bar, 50 ?m.
V.M.M. Braga et al.
Molecular Biology of the Cell 3710
with the stability of newly formed cadherin-de-
pendent contacts. Constitutively active Rac
(L61Rac, 0.5 mg/ml) was microinjected into cells
grown without contacts, and cadherin-depen-
dent cell-cell adhesion was induced for 1 h (a
and b), 2 h (c and d), or 5 h (e and f). Alterna-
tively, L61Rac-pRK5myc expression vector was
injected into the nucleus of HaCat cells (g and h),
and after a 2-h expression, cell-cell contacts were
induced for further 4 h. Cells were fixed and
labeled for E-cadherin (b, d, f, and h) as stated in
Figure 2; injected patches of cells are visualized
in a, c, e, and g. In g, cells are labeled for the
myc-tag epitope. Bar, 75 ?m for g and h; 50 ?m
for all the other images.
Sustained Rac activation interferes
Rac Activation and Cadherin Adhesion
Vol. 11, November 20003711
observations). The reasons for the distinct effects of Rac on
mature junctions versus newly formed junctions are not
Rac Domain Important for Disruption of Cadherin-
We next determined which domain in the Rac protein is
required for its inhibitory activity on cadherin function.
Different chimeric molecules containing an activated Rac
N-terminal domain and Rho C-terminal domain were mi-
croinjected into keratinocytes or Swiss 3T3 fibroblasts (sum-
marized in Figure 6 a; Diekmann et al., 1995; Kwong et al.,
1995; Nisimoto et al., 1997). To demonstrate the purity of the
microinjected proteins used in this study, the different re-
combinant proteins are shown in Figure 6b. After injection
into keratinocytes without cell-cell contacts, intercellular
junctions were induced for 4 h, and cells stained for E-
cadherin (Figure 7, b, d, and f). As opposed to full-length
activated Rac (0.5 mg/ml, Figure 5, e–h), activated Rho did not
cause significant disruption of intercellular junctions at the
concentration tested (0.5 mg/ml, our unpublished results).
Rac73Rho (3.35 mg/ml, our unpublished results), Rac126Rho
(0.53 mg/ml, Figure 7, a and b) and Rac143Rho (0.43 mg/ml;
Figure 7, c and d) had no significant effect on cell shape or the
localization of cadherin receptors. In contrast, Rac175Rho (2.39
mg/ml, Figure 7, e and f) interfered with cadherin stability and
induced formation of lamellae in a similar manner to constitu-
tively active, full-length Rac (0.5 mg/ml, Figure 5, e–h).
important for the disruption of cad-
herin-dependent adhesion. (a) Com-
parison of the Rac domains relevant
for disruption of cadherin-mediated
contacts in keratinocytes and lamella
formation in Swiss 3T3 cells (Diek-
mann et al., 1995; data not shown).
Rac protein (?), Rho protein (f), and
the small GTPase functional domains
are represented: N-terminal effector
domain 1, insert region, and the C-
terminal effector domain 2. Chimeras
containing portions of the Rac se-
quence (?) and Rho sequence (f) are
also shown: Rac73Rho (3.35 mg/ml);
Rac126Rho (0.53 mg/ml); Rac143Rho
(0.43 mg/ml); Rac175Rho (2.39 mg/
ml) (see text for details). Activated
Rac and Rho were tested at 0.5 mg/
ml. (b) Recombinant proteins used in
this study were separated in SDS-
PAGE and visualized by Coomassie
markers are shown on the left (top to
bottom): 36.5, 26, and 20 kDa.
Mapping the Rac domain
V.M.M. Braga et al.
Molecular Biology of the Cell3712
cules were injected into keratinocytes grown in
the absence of cell-cell contacts, and calcium-
dependent adhesion was induced for 4 h (a–f).
Rac126Rho (a and b); Rac143Rho (c, d, g, and h);
and Rac175Rho (e and f). Injected cells (a, c, e,
and g) and E-cadherin staining (b, d, and f) are
visualized. In g and h, Rac143Rho was injected
into Swiss 3T3 fibroblasts and cells were
stained for filamentous actin (h). Bar, 50 ?m.
Different chimeric RacRho mole-
Rac Activation and Cadherin Adhesion
Vol. 11, November 2000 3713
Rac175Rho diluted to 1 mg/ml showed the same disruptive
effect (our unpublished results). These results suggest that the
Rac sequence between amino acid residues 143 and 175 was
necessary to perturb cell-cell contacts.
The chimeric molecules were also evaluated for their abil-
ity to induce ruffles by injection into serum starved Swiss
3T3 cells seeded onto fibronectin coverslips for 2 days (Puls
et al., 1999). Under these conditions, Rac175Rho induced
ruffles and lamellipodia as previously reported (Diekmann
et al., 1995). However Rac143Rho was also able to promote
lamellipodia formation (Figure 7, g and h; our unpublished
results). A previous study did not demonstrate ruffling ac-
tivity for Rac143Rho (Diekmann et al., 1995). It is not clear
why, but we attribute this difference to the culturing condi-
tions, which may affect the nature of the response. In the
former study, microinjection was performed on freshly
plated, serum-starved fibroblasts (2 h, Diekmann et al.,
1995). In our study, we used cells seeded 48 h before in
serum-free medium supplemented with 1/50 dilution of
conditioned medium (Puls et al., 1999). It is also possible that
the protein batch used in our study is more active than the
batch used before. The data are summarized in Figure 6a
and, taken together, indicate that the Rac domains respon-
sible for lamella formation and cadherin disruption did not
Two Distinct Pathways?
We confirmed the above-mentioned results by an alternative
approach, site-directed mutagenesis. This approach allowed
us 1) to dissociate between the Rac-induced lamellipodia
activity and the Rac-dependent perturbation of cadherin
adhesion, and 2) to map the important domain more pre-
cisely between amino acids 143 and 175. Mutations were
introduced into L61Rac at different positions: A147 A148
(single letter code KE to AA); A162 A163 (QR to AA); A166
A167 (KT to AA); and A170 A171 (DE to AA) (see MATE-
RIALS AND METHODS for details). These mutants were
tested for their ability to disrupt cadherin-dependent con-
tacts in keratinocytes or to induce lamella formation in Swiss
3T3 cells and representative pictures are shown in Figure 8.
During formation of intercellular adhesion in keratinocytes,
all the mutants showed qualitatively the same phenotype
(but see below): no changes in cell morphology or significant
decrease in cadherin staining at cell-cell borders in keratin-
ocytes (Figure 8, a–d, and our unpublished results). Inter-
estingly, induction of lamellipodia was not impaired in any
of the mutants (as assessed by actin staining in fibroblasts,
Figure 8, e–h; our unpublished results).
The quantification of these effects is shown in Figure 9.
Control L61Rac at 0.5 mg/ml disrupted newly formed junc-
tions in ?85% of the injected patches after 4–5 h of incubation
(Figure 9a). On the other hand, when L61Rac second effector
domain mutants were injected at concentrations between 0.9 to
2.9 mg/ml, cadherin-dependent contacts were perturbed in
only 10% of injected patches (Student’s t test, p ? 0.005). The
mutant A170 A171 (3.5 mg/ml) was the exception, showing
perturbed junctions in 35% of the patches (Student’s t test, p ?
0.01; Figure 9a). On the other hand, all mutants were able to
induce lamellipodia to a similar extent as L61Rac (at 2 mg/ml),
and no significant difference was detected (Figure 9b; Student’s
t test). Further experiments using dilutions of two of the mu-
tants, A162 A163 and A170 A171, revealed that their ruffling
activity could be titrated down in a similar pattern to L61Rac
(Figure 9c). These results are summarized in Figure 9d and
taken together suggest that disruption of cadherin adhesion
and lamella formation are two independent activities triggered
To assess whether the Rac mutants could interact with
known Rac targets, in vitro binding assays were performed
using recombinant proteins. Fusion proteins containing the
GTPase binding domain of known Rac targets were immo-
bilized onto membranes and probed with radioactively la-
beled Rac or the second effector domain mutants (Figure 10
a; Burbelo et al., 1995; reviewed by Van Aelst and D’Souza
Schorey, 1997). In addition, interactions were tested by yeast
two-hybrid technique and evaluated by growth on 3AT
plates (Figure 10b and Table 1) or ?-galactosidase filter assay
(our unpublished results). Both techniques produced similar
results: no binding was detected to the negative controls
(GST, Figure 10a; empty vector, Figure 10b). All GTPases
interacted similarly with RhoGAP, PAK, ROK-?, and IQ-
GAP2 (Figure 10; our unpublished observation). Two Rac
mutants (A147 A148 and A162 A163) were able to interact
with POSH and MLK2. The only target that showed limited
binding to all 4-s effector mutants is MLK3 (Table 1). In
addition, with the exception of A170 A171, all other Rac
mutants were able to activate the JNK kinase pathway (Ta-
ble 1; our unpublished results). Because of the similar prop-
erties of L61Rac and second effector mutants, we concluded
that these mutations did not affect the overall shape and
activity of the mutants. Instead, the double alanine muta-
tions interfered with the interaction with a particular subset
of target(s) (among them MLK3).
In this article, we identified Rac as a key regulator of cad-
herin-mediated adhesion in human keratinocytes. Our ma-
jor findings are as follows: Rac-signaling pathways contrib-
ute to the destabilization of cadherin receptors at junctions
during Ras transformation in keratinocytes. During epithe-
lial tumorigenesis, sustained levels of Rac activation can be
achieved in vivo (Mira et al., 2000) and we demonstrate that
Rac activation is sufficient to specifically disrupt cadherin-
dependent adhesion. In addition, we demonstrate that per-
turbation of cell-cell contacts is a new Rac activity, distinct
from lamellipodia formation. We mapped the putative sec-
ond effector domain of Rac as an important domain for
disruption of cadherin receptor localization, and produced
mutants that can be useful tools to identify putative Rac
targets. These results are discussed below.
Transfection of activated Rac in MDCK cells induces en-
hanced levels of cadherin and actin at junctions (Hordijk et
al., 1997; Takaishi et al., 1997). In addition, transfection of
Tiam-1, a Rac activator, can cause a reversion of the fibro-
blastoid morphology of Ras-transformed in MDCK (Hordijk
et al., 1997; Sander et al., 1998). Contrary to the above-
mentioned reports, Rac activation in normal human keratin-
ocytes does not result in an increased localization or stability
of cadherin receptors at cell-cell contacts. Our results are
consistent with published data that Rac activation promotes
cell-cell adhesion breakdown and migration of different car-
cinoma cells (Keely et al., 1997). In other epithelial cell lines,
Rac activation also plays a role in scattering after distinct
V.M.M. Braga et al.
Molecular Biology of the Cell 3714
stimuli such as growth factor stimulation or integrin engage-
ment (Takaishi et al., 1994; Ridley et al., 1995; Shaw et al.,
1997; Potempa and Ridley, 1998; Gimond et al., 1999).
This controversy found in the literature might reflect the
distinct cellular context, methodology used or different lev-
els of Rac activation achieved. It is also conceivable that
with respect to disruption of cadherin-de-
pendent adhesion and lamella formation.
Different positions within the putative sec-
ond effector domain were mutated to ala-
nine to generate four mutants in a consti-
tutively active Rac background. Similar
results were obtained for all mutants, and
representative pictures are shown for the
mutants A162 A163 (a, b, e, and f) and A170
A171 (c, d, g, and h). To evaluate disrup-
tion of cadherin function in keratinocytes
(a–d), cell-cell contacts were induced for
4–5 h after microinjection, and cells were
then fixed and stained for E-cadherin (b
and d). The same mutants were also ana-
lyzed for their ability to induce formation
of lamellae/ruffles in Swiss 3T3 cells, after
staining with FITC-phalloidin (e–h). Bar,
Second effector domain analysis
Rac Activation and Cadherin Adhesion
Vol. 11, November 20003715
activation of Rac-dependent pathways by a constitutively
active form (L61Rac) or via Tiam-1 might differ, because the
latter also provides a localization signal (Sander et al., 1998;
this work). More experimental work is necessary to under-
stand and reconcile the distinct phenotypes produced by
Rac activation in different cell types.
Nevertheless, it is clear that in keratinocytes, sustained
Rac activation does not alter the detergent solubility of the
receptors nor the amount of actin recruited to junctions (our
unpublished results). If Rac activation is not promoting the
localization of cadherin receptors to junctions, what are the
consequences of Rac activity? In keratinocytes, when junc-
second effector domain mutants. (a)
Quantification of the effects of the Rac
mutants on cadherin-dependent adhe-
sion. Patches of keratinocytes microin-
jected with the different mutants were
scored for the presence of perturbed
cadherin staining at intercellular junc-
tions and expressed as a percentage of
the total number of patches (see MA-
TERIALS AND METHODS). (b) Quan-
tification of the lamella-inducing activ-
ity of Rac mutants. Swiss 3T3 cells
were injected with the different pro-
teins and scored as a percentage of
cells showing ruffles/lamellae. (c) Ti-
tration of lamellipodia formation in-
duced by L61Rac, A162 A163, and
A170 A171. The same amount of re-
combinant protein (2, 1, or 0.5 mg/ml)
was injected into Swiss 3T3 cells and
the percentage of injected cells with
ruffles/lamellae were scored. (d) Sum-
mary of Rac mutants’ ability to perturb
cadherin adhesion in keratinocytes or
induce lamellipodia in fibroblasts. A
active Rac containing the relevant do-
mains (effector domains 1 and 2, insert
domain), and the different mutants
generated in the second effector do-
main. Unless otherwise stated, in the
were used at the following concentra-
tions: A147 A148, 0.89 mg/ml; A162
A163, 2.26 mg/ml; A166 A167, 2.91
mg/ml; and A170 A171, 3.57 mg/ml.
L61Rac was tested at 0.5 mg/ml in ker-
atinocytes and at 2 mg/ml in Swiss 3T3
cells. ?p ? 0.005;@p ? 0.01 (Student’s t
test). Results are the mean of at least
three independent experiments; Figure
9c shows the mean of at least two ex-
periments for each concentration of
distinct mutants. Error bars represent
Characterization of the Rac
V.M.M. Braga et al.
Molecular Biology of the Cell3716
Figure 9. Continued
mutants with known Rac targets. (a) In
vitro interaction: L61Rac, A162 A163,
and A170 A171 were labeled with radio-
active GTP and allowed to interact with
RhoGAP, GST-PAK, GST-ROK-?, GST-
POSH, and GST-MLK2.
amounts of recombinant protein were
spotted onto the membranes (10, 5, or
1 ?g). GST was used as a negative con-
trol; positive (RhoGAP and PAK) and
negative controls were used at 10 ?g. (b)
Yeast two-hybrid interaction by using
L61Rac or the second effector domain
mutants as baits and the following tar-
gets as prey: PAK, MLK2, MLK3, and
IQGAP2. Empty vector was used as a
negative control. ?, IQGAP interaction
was tested at lower concentration of 3AT
(10 mM) because the association with
activated Rac was barely detectable with
the standard concentration used for the
other targets (25 mM). A summary of the
yeast two-hybrid results with all the tar-
gets tested is shown in Table 1.
Binding properties of Rac
Rac Activation and Cadherin Adhesion
Vol. 11, November 20003717
tion stability is challenged by either Rho inhibition (our
unpublished results) or H-Ras activation, coinjection of ac-
tived Rac cannot protect cadherin receptors from these de-
stabilizing stimuli (Braga et al., 1997). Indeed, Rac activation
is necessary for the disassembly of cadherin contacts in-
duced by the oncogene H-Ras. These results are also in
agreement with published data, in which Rac activation
contributes to the H-Ras-dependent perturbation of cell-cell
contacts in breast cancer cell lines (Quillan, 1999). In kera-
tinocytes, we demonstrate that inhibition of Rac signaling
pathways prevents the Ras-dependent perturbation of cell-
cell adhesion, whereas blocking PI3 kinase and mitogen-
activated protein kinase pathways have only a partial effect
(our unpublished results).
Rac Can Specifically Destabilize Cadherin-
A previous report has shown activation of Rac 3 in breast
cancer cells, suggesting that Rac activation during tumori-
genesis might be a widespread process (Mira et al., 2000).
Together with our results, the above-mentioned results dem-
onstrate that activation of Rac can occur after transformation
and that this pathway may contribute to the Ras-dependent
disassembly of junctions. Moreover, our data show that Rac
activation is sufficient to disassemble cadherin-dependent
contacts in a time- and concentration-dependent manner in
human keratinocytes. Activated Rac specifically interferes
with the localization of cadherin but not integrin receptors in
the time frame examined. Disruption of cadherin adhesion is
observed in both newly formed and mature junctions, but it
is more clearly seen during the induction of cell-cell adhe-
sion (see RESULTS). However, formation of new cell-cell
contacts is not prevented by activated Rac, but rather the
stability of cadherin receptors at intercellular contacts is
compromised (after 1 h).
Our results that Rac activation may promote junction
disassembly are intriguing. It is possible that, as for other
biological stimuli, the cellular response to Rac activation
may follow a bell-shaped curve: too little Rac is inhibitory to
junctions as is too much Rac activity. We think that the Rac
destabilization of junctions is unlikely to be a nonspecific
effect of bacterial contaminants or overexpression for the
following reasons: 1) the same effect is obtained by expres-
sion of activated Rac DNA in HaCat cells; 2) microinjection
of distinct proteins at much higher concentration cannot
disrupt cell-cell contacts; and 3) injected keratinocytes do
not show any signs of apoptosis (i.e. annexin V staining, our
unpublished results). In addition, there is specificity in the
response because cadherin molecules are removed from
junctions before other cell-cell adhesion receptors.
Rac activation perturbs cadherin contacts with a concom-
itant change in cell shape, including formation of lamellae/
protusions and a clear conversion to a more fibroblastic
morphology. Although lamellae are also present upon Rac
activation in keratinocytes containing mature junctions, the
change in cell shape is not observed over a 2-h incubation.
Interestingly, levels of Rac activation that disrupt newly
formed cadherin contacts very efficiently (0.5 mg/ml, 85% of
injected patches) can only induce ruffling in 30% of injected
Both lamella formation and cadherin adhesion require
actin polymerization dependent on Rac activity (Machesky
and Hall, 1997; Braga et al., 1999). Because our data suggest
that lamella formation may antagonize cadherin-mediated
adhesion, two possibilities can be envisaged. First, induction
of lamellipodia may cause the destabilization of cadherin
receptors at junctions, or second, lamella formation and
perturbation of cadherin adhesion may be two independent
activities triggered by Rac. Our results support the latter
possibility because we are able to dissect these two Rac
Important Domain in the Rac Molecule for
Interfering with Cadherin-dependent Contacts
Previous work has identified three functional domains in the
Rho subfamily of small GTPases: the N-terminal effector
domain, an insert region, and a putative C-terminal effector
domain (Diekmann et al., 1995; Joseph and Pick, 1995;
Kwong et al., 1995; Nisimoto et al., 1997). We restricted an
important region for destabilizing intercellular junctions to
the putative second effector domain of Rac, between amino
acids 143 and 175. This domain overlaps with, but is not
identical to, the domain necessary to induce lamellae (Diek-
mann et al., 1995; our unpublished results). In addition, by
mutating specific amino acids within residues 143 and 175 of
activated Rac, we obtained mutants that are impaired in
their ability to perturb cadherin adhesion in keratinocytes,
but are still able to promote ruffling and lamellipodia in
Swiss 3T3 cells.
Characterization and quantification of these effects indi-
cate that the second effector domain mutants (A147 A148,
A162 A163, A166 A167, and A170 A171) showed around
fourfold reduction in the destabilization of cell-cell adhesion
compared with L61Rac, in spite of being microinjected at
much higher concentration (2- to 8-fold more concentrated).
We think it unlikely that the double alanine mutations in-
terfere with the overall stability or activity of the Rac mole-
cule for the following reasons. First, like constitutively active
Rac, all second effector domain mutants were able to induce
Table 1. Characterization of the Rac second effector mutants
L61Rac and the double alanine Rac mutants were evaluated for
activation of the JNK pathway and their binding with distinct
targets in the yeast two-hybrid assay. The Rac targets tested are
RhoGAP, PAK, ROK-?, MLK2, MLK3 and IQGAP2: (?), negative;
(?), positive; (?/?), weakly positive as revealed by growth in 3AT
plates (see MATERIALS AND METHODS for details). Empty vector
was used as a negative control.
V.M.M. Braga et al.
Molecular Biology of the Cell3718
lamellae. Normalization of their concentrations and micro-
injection of dilutions produced a proportional decline in
their ability to induce lamellae, as for L61Rac. Second, their
GTP binding ability was not significantly perturbed (our
unpublished results). Finally, the mutants can associate with
RhoGAP and other Rac targets such as PAK, ROK-?, and
IQGAP2 to a similar extent as activated Rac (reviewed by
Van Aelst and D’Souza-Schorey, 1997).
To our knowledge, this is the first report showing exten-
sive mutagenesis analysis of the putative second effector
domain. This domain forms an exposed loop in the Rac
three-dimensional structure, suggesting good access for tar-
get interaction (Hirshberg et al., 1997). Interestingly, the
binding of the small GTPases Rac and Cdc42 to distinct
targets also requires and is stabilized by residues within the
second effector domain (Abdul-Manan et al., 1999, Mott et al.,
1999; Tolias et al., 2000).
In an attempt to investigate the mechanism by which Rac
activation may disturb cadherin function, preliminary re-
sults show that new protein synthesis is not required. The
process does not involve down-regulation of Rho (Izawa et
al., 1998, Rottner et al., 1999; Sander et al., 1999; van Leeuwen
et al., 1999; Zondag et al., 2000) nor does Rac activation
interfere with the recycling compartment in keratinocytes
(our unpublished results; Lamaze et al., 1996). Together with
our analyses of putative Rac targets, these results suggest
that Rac is able to activate specific pathways that perturb the
stability of cadherin receptors at the keratinocyte junction.
If this hypothesis is true, the Rac second effector domain
mutants can be useful tools to identify which pathway is
important for junction disassembly. Two possibilities can be
envisaged. First, the mutants may display a reduced binding
to specific targets. Alternatively, the binding interactions
may be the same, but the ability to activate the target is
compromised. We began to test these two possibilities with
known Rac targets. We found that at least activation of the
JNK pathway is not affected by the double alanine mutations
in the second effector domain. By screening Rac targets for
their ability to differentially interact with activated Rac and
the second effector mutants, we identified MLK3 (mixed
lineage kinase 3) as a putative effector candidate (Burbelo et
al., 1995; Nagata et al., 1998; Hartkamp et al., 1999) because it
shows reduced binding to all mutants tested. We are cur-
rently performing experiments to address the question of
whether MLK3 activation per se is sufficient to disturb cad-
In summary, we demonstrate that Rac is a key regulator of
cadherin-dependent cell-cell contacts as sustained Rac acti-
vation is sufficient to destabilize normal keratinocyte junc-
tions. An important question that remains to be addressed
experimentally is the threshold level of Rac activation that is
necessary to perturb cell adhesion during tumor progres-
sion. However, it is conceivable other pathways triggered by
oncogenes may cooperate with Rac to promote cytoskeletal
changes and junction breakdown. Because Rac plays an
important role in cell migration, our study sheds light on the
biological problem of how cells are able to integrate cell-cell
and cell-substratum adhesion during tumorigenesis. More-
over, our data suggest that downstream signaling pathways
activated by Rac could be potential therapeutical targets for
preventing cell-cell disassembly.
We thank Alan Hall for continuous support and encouragement.
We also thank M. Takeichi, D. Kwiatkowiski, and Anne Bishop for
generous gifts of antibodies and recombinant proteins; J. Collard, H.
Daub, and David Dreschel for plasmids; N. Fusenig for sending cell
lines; and Axel Puls and Lars Kjoller for Swiss 3T3 cells and advice
on how to grow them. M.B. is in the Medical Research Council
Graduate Program at the Medical Research Council Laboratory for
Molecular Cell Biology. V.M.M.B is supported by Cancer Research
Campaign. N.L.-V. is a Junior Scholar from Fonds de la Recherche
en Sante ´ du Que ´bec.
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