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Human Molecular Genetics Advance Access originally published online on May 17, 2007
Human Molecular Genetics 2007 16(13):1639-1647; doi:10.1093/hmg/ddm113
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© The Author 2007. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

EGFR regulates RhoA-GTP dependent cell motility in E-cadherin mutant cells

Ana Rita Mateus1,2, Raquel Seruca1,3, José Carlos Machado1,3, Gisela Keller2, Maria José Oliveira1, Gianpaolo Suriano1,3,* and Birgit Luber2

1 Instituto de Patologia e Imunologia Molecular da Universidade do Porto (IPATIMUP), 4200-465 Porto, Portugal, 2 Technische Universität München, Klinikum rechts der Isar, Institut für Allgemeine Pathologie und Pathologische Anatomie, D-81675 München, Germany and 3 Faculdade de Medicina da Universidade do Porto, 4200-465 Porto, Portugal

* To whom correspondence should be addressed at: Instituto de Patologia e Imunologia Molecular da Universidade do Porto, Rua Dr Roberto Frias s/n, 4200-465 Porto, Portugal. Tel: +351 225570700; Fax: +351 225570799; Email: gsuriano{at}ipatimup.pt

Received March 9, 2007; Revised April 16, 2007; Accepted April 25, 2007


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Gastric cancer associated E-cadherin germline missense mutations lead to significant functional consequences, in both the structural and signalling properties of the protein. In this study, we have characterized the effect of four E-cadherin germline missense mutations (T340A, A634V, P799R and V832M) in the interaction with the epidermal growth factor receptor (EGFR). We challenged the hypothesis that E-cadherin mutations perturb its ability to bind to EGFR, leading to constitutional activation of the EGFR, triggering activation of downstream effectors. We verified that missense mutations localized in the extracellular domain of the protein (T340A and A634V) exhibited reduced stability of the EGFR/E-cadherin heterodimers in contrast to germline mutations localized at the cytoplasmatic domain of the protein (P799R and V832M). We observed that cells expressing E-cadherin extracellular mutants displayed increased levels of phosphorylated EGFR upon ligand stimulation, when compared with cells expressing wild-type E-cadherin or intracellular mutants. We showed that upon treatment of E-cadherin extracellular mutant cells with the EGFR inhibitor, the increase of RhoA activation is abrogated and accompanied by decreased migratory behaviour, supporting the idea that Rho-like proteins are EGFR downstream effectors. Our results bring new insights into the understanding of the distinct in vitro behaviours observed for E-cadherin missense mutations localized in different domains of the protein. Furthermore, we demonstrate that E-cadherin-dependent EGFR activation contributes to enhanced cell motility, in a mechanism involving RhoA activation.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
E-cadherin is a transmembrane protein critical for establishing and maintaining polarized and differentiated epithelia during development and in adult tissues. Adherens junctions cluster, via homophilic interactions, through the extracellular domains of calcium-dependent E-cadherin molecules on the surface of homotypic neighbour cells (1). Beside its adhesive function, E-cadherin plays a key role in multiple processes, including cell migration, morphology and polarity (2), and has been implicated as a membrane receptor involved in signal regulation (3–5). In this regard, attention has been drawn on the existence of multicomponent complexes between E-cadherin and receptor protein tyrosine kinases (RTKs), namely the epidermal growth factor receptor (EGFR), at basolateral areas of polarized epithelial cells (6–8). Increasing evidence suggests that diverse classes of RTKs can deregulate E-cadherin-dependent adhesion during epithelial to mesenchymal transition (9,10). Further, EGF-dependent activation of EGFR has been also reported to be inhibited in an E-cadherin adhesion-dependent manner (11,12) implying a bidirectional cross-talk between E-cadherin and EGFR.

It is well known that the loss of E-cadherin is associated to increased cell migration and invasion. We have previously shown that hereditary diffuse gastric cancer associated E-cadherin germline missense mutations induce in vitro and in vivo cell invasion (13–15). In particular, we showed a genotype–phenotype correlation between the site of E-cadherin mutations and cellular effects. E-cadherin mutations clustering on the extracellular domain of the protein exhibit enhanced cell motility (15,16). There is also evidence supporting the idea that EGFR-mediated signalling plays a major role in cell migration and invasion besides its well-known growth stimulatory activity. Recently, using a set of deletion constructs along the E-cadherin gene (E-cad {Delta}ß-catenin, Ecad (AAA(764)) and E-cad {Delta}NT), it has been suggested that the interaction of E-cadherin with EGFR would require an intact extracellular E-cadherin domain (12). In this work, we challenged the hypothesis that hereditary diffuse gastric cancer associated E-cadherin germline missense mutations, in particular those clustering on the extracellular domain of the protein, also perturb its ability to bind to EGFR, leading to increased activation of the receptor, triggering downstream signalling pathways associated to increased cell motility.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
To test the hypothesis that E-cadherin deregulation leads to activation of the EGF receptor, triggering downstream signalling pathways associated to increased cell motility, we chose Chinese hamster ovary (CHO) cells stably expressing either wild-type E-cadherin or its hereditary diffuse gastric cancer-associated germline missense mutations T340A, A634V, P799R and V832M as in vitro model. The four mutations were previously shown to impair in vitro the E-cadherin ability to mediate cell-to-cell adhesion and suppress cell invasion, supporting their pathogenic relevance (16). T340A and A634V localize on the extracellular domain of the protein, whereas P799R and V832M belong to the cytoplasmic E-cadherin tail. The two mutations T340A and A634V show high motile phenotype in contrast to both mutations localized in the cytoplasmic domain of the protein (P799R and V832M). None of the mutations affected the E-cadherin localization at the cell membrane.

EGFR binds to the membrane-unbound fraction of ß-catenin independently of E-cadherin
It has been reported that EGFR and E-cadherin are found in complexes in which ß-catenin is also present (17). The first question we asked was whether EGFR could interact independently with both E-cadherin and ß-catenin and whether the selected E-cadherin germline missense mutations would affect the interaction between E-cadherin and EGFR. To this end, the affinity of EGFR to ß-catenin and/or the different E-cadherin variants was determined by performing immunoprecipitation analysis. As shown in Figure 1A, in all samples ß-catenin/EGFR immunocomplexes were obtained independently of the E-cadherin mutational status. This observation together with the fact that positive immunoprecipitation was obtained also for mock E-cadherin-negative cells, suggests that EGFR is able to interact with ß-catenin independently of E-cadherin. Moreover, since in mock cells ß-catenin protein is mainly found into the cytoplasm (14), our results suggest that EGFR binds to the membrane-unbound fraction of ß-catenin.


Figure 1
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  		Figure 1. Western-blot and immunoprecipitation showing that EGFR binds to ß-catenin independently of E-cadherin and suggesting a reduced stability of the EGFR/E-cadherin heterodimers in the presence of E-cadherin extracellular mutations. Cells were cultured on collagen I coated dishes and protein extracted 24 h after. Five hundred microgram of protein was immunoprecipitated with anti-EGFR, resolved by SDS–PAGE and transferred to nitrocellulose. (A) Staining with ß-catenin antibody; (B) staining for E-cadherin. lc—loading control ({alpha}-tubulin). The intensity of the bands was normalized in function of the loading control and the fold of increase calculated in comparison with mock cells. (C) Thirty microgram of total lysate protein were separated by SDS–PAGE and immunoblotted for E-cadherin, ß-catenin, EGFR and {alpha}-tubulin (as a loading control).

 
Missense mutations clustering in the extracellular region of E-cadherin reduce the stability of the EGFR–E-cadherin complex
Recently, using a set of deletion constructs along the E-cadherin gene (E-cad {Delta}ß-catenin, Ecad (AAA(764) and E-cad {Delta}NT), it has been suggested that the interaction of E-cadherin with EGFR would require an intact extracellular E-cadherin domain (12). When we characterized the interaction between E-cadherin and EGFR, we observed a reduced affinity of EGFR to the extracellular mutant forms of E-cadherin T340A and A634V, when compared to the wild-type E-cadherin protein (Fig. 1B). No differences were observed when comparing cells expressing the wild-type protein to the intracellular mutants P799R and V832M. No positive immunostaining was obtained for mock cells after immunoprecipitation, confirming the absence of the E-cadherin adhesion complex in the parental cell line. Western blot analysis demonstrated that all cell lines expressed comparable amount of E-cadherin (as expected negative in mock cells), EGFR and ß-catenin (Fig. 1C), thus supporting the hypothesis of a reduced stability of the EGFR–E-cadherin complex in T340A and A634V expressing cells. Altogether, these results support the role of the extracellular region of E-cadherin in the establishment of the complex with EGFR.

Missense mutations clustering in the extracellular region of E-cadherin show increased phosphorylation of EGFR
It was previously reported that EGF-dependent activation of EGFR is inhibited in an E-cadherin adhesion-dependent manner (11,12). To characterize the ability of EGFR to be activated by its ligand in the context of E-cadherin mutations, cells expressing either wild-type E-cadherin or the aforementioned mutations were stimulated with EGF 100 ng/ml (8,12,18) and the activated form of EGFR characterized by immunofluorescence. A kinetic of stimulation was obtained upon quantification of the phospho-EGFR signal intensity at each time point, peaking at 15 min upon stimulation in all cell lines (Fig. 2A). At each time point analysed T340A and A634V expressing cells, as well as mock E-cadherin negative cells, displayed a stronger signal of the activated form of EGFR when compared with the wild-type expressing cells or the intracellular mutants expressing cells (Fig. 2B and C). No differences were observed when comparing cells expressing the wild-type protein to the mutants P799R and V832M, suggesting a close relation between the ability of EGFR to be activated by its ligand and the extent of binding of the receptor to E-cadherin. Moreover, since all mutations in this study were previously shown to impair the E-cadherin ability to promote cell–cell adhesion, our results indicate that the inhibitory effect exerted by E-cadherin on the EGFR activation is adhesion-independent but regulated by the stability of the E-cadherin–EGFR hetero-complexes.


Figure 2
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  		Figure 2. EGFR phosphorylation in response to EGF in function of the E-cadherin status. (A) A kinetic of stimulation points was performed to determine the time point of maximum EGFR phosphorylation for all cell lines. Panels show T340A cells stimulated with EGF 100 ng/ml for different time points: a—no stimulation; b to h—1, 5, 10, 15, 30, 45 and 60 min of stimulation prior to fixation, respectively. All pictures were taken with a 40x objective. (B) Cells were stimulated with EGF 100 ng/ml 15 min prior to fixation. Panels a–f show Mock, wild-type, A340T, A634V, P799R and V832M, respectively. (C) Western-blot using the phospho-specific antibody Tyr1086 phospho-EGFR shows a stronger activation of the EGFR for Mock and T340A E-cadherin expressing cells, when compared with wild-type or V832M E-cadherin transfectants. {alpha}-tubulin was used as a loading control.

 
E-cadherin dependent cell migration, in extracellular mutants, is mediated by activation of EGFR
We have previously shown that germline missense mutations of the E-cadherin gene affect cell motility by modulating the reorganization of the actin cytoskeleton (16). Considering a known involvement of EGFR in cell migration (19), as well as the effect of the E-cadherin extracellular missense mutations on the activation status of EGFR, we decided to investigate the contribution of EGFR to cell motility in our cell system. To this end, we assessed the ability of cells to move between the edges of an artificial wound upon modulation of the EGFR activity with its pharmacological inhibitor Tyrphostin AG 1478 (Fig. 3). As previously reported (16), T340A and A634V expressing cells display a 25% increased cell motility over the wild-type E-cadherin expressing cells, with cells randomly colonizing the wound 8 h after incision. In the presence of the EGFR inhibitor, migration of the T340A and A634V E-cadherin expressing cells is dramatically altered, with cells showing a centripetal migration resembling the wild-type E-cadherin expressing cells (Fig. 4). Measurement of the distance between wound edges at different time points upon inhibition revealed a comparable speed of wound closure in all analysed cell lines. These results suggest that the increased levels of active EGFR are responsible for the higher and random fashioned cell motility observed for the T340A and A634V mutant cells.


Figure 3
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  		Figure 3. Cell motility assay. The migratory behaviour of the cells transfected with wild-type E-cadherin and the different variants were assessed in the presence of the EGFR specific inhibitor Tyrphostin AG 1478. An artificial wound was created in monolayers of confluent cells and medium containing 9.45 µM of Tyrphostin AG 1478 or the equivalent volume of DMSO for the negative control was added to the cells after wounding. Migration was assessed by measuring the distance between wound edges in at least six different randomly chosen regions at time intervals (0, 3, 8 and 24 h). Panels show phase contrast pictures taken 3, 8 and 24 h after wounding for the wild-type, T340A and A634V cells.

 


Figure 4
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  		Figure 4. Effect of EGFR inhibition on RhoA activity. The level of active RhoA (GTP status) was assessed in the presence of the EGFR inhibitor Tyrphostin AG 1478 in wild-type E-cadherin, T340A and A634V expressing cells. The intensity of the bands was normalized in function of loading and the fold of increase calculated in comparison with wild-type untreated cells. (+9.45 µM Tyrphostin AG 1478; –DMSO negative control).

 
RhoA activation displayed by E-cadherin extracellular mutants, is abrogated by EGFR inhibition
We had previously reported the involvement of the Rho family of small-GTPases, RhoA in particular, in the enhanced cell motility displayed by cells expressing E-cadherin extracellular mutations. Because of our finding on the effect of EGFR activation on E-cadherin-dependent cell motility, we asked whether increased RhoA activation was dependent on EGFR activation. To this end, we determined the level of active RhoA by pull-down assay in cells expressing either the wild-type protein or the E-cadherin mutant T340A and A634V treated or not with the EGFR inhibitor Tyrphostin AG 1478. As expected, in the absence of the inhibitor, T340A and A634V expressing cells displayed increased active RhoA when compared with the wild-type expressing cells. Upon treatment of the cells with the EGFR inhibitor, no difference in the active level of GTPases could be observed between the different cell lines, confirming that the effect of EGFR on cell motility is mediated through RhoA activation (Fig. 4).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
E-cadherin is involved in a wide range of physiological and pathological processes (20). It is now well established that E-cadherin plays a pivotal role in tumour development, being its deregulation the major determinant of tumour progression and invasion in epithelial cancers (21). Besides its role in tumour progression, the study of early invasive cancers in carriers of E-cadherin germline mutations demonstrates that its deregulation is also an initiating event in tumorigenesis (22,23). E-cadherin has a structural role in cell–cell adhesion, but also acts as a cell membrane receptor (24,25). Many signalling molecules have been reported to interact with E-cadherin. RTKs have been found to localize at the basolateral areas of epithelial cells (6,7), and EGFR, in particular, has been reported to be involved in a bidirectional cross-talk with E-cadherin (12). Nevertheless, little is known about the role of this interaction in cell signalling and associated cellular effects. In this study, we have used hereditary diffuse gastric cancer-associated E-cadherin germline missense mutations (13,14,26) to confirm that the interaction of E-cadherin with EGFR requires an intact extracellular region of E-cadherin, as previously suggested by Qian et al. (12). Further, we aimed at identifying downstream effectors and associated cellular effects.

For that, we have used cell lines stably expressing two germline mutations localized in the extracellular domain of the protein (T340A and A634V) and two germline mutations localized at the cytoplasmic domain of the protein (P799R and V832M), in comparison to the wild-type E-cadherin. The extracellular mutations T340A and A634V locate at the end of the second extracellular repeat (EC2), immediately preceding a calcium-binding region, and in the fifth extracellular repeat (EC5) of E-cadherin, affecting a conserved sequence encoding one of the calcium binding motifs, respectively (13,27). T340A mutation was shown by computer modelling to disrupt a sequence motif likely to be involved in the stabilization of the active protein conformation (13). We verified that both T340A and A634V missense mutations cause a reduced stability of the EGFR/E-cadherin heterodimers; on the contrary no effect was observed for the mutants P799R and V832M. These results are in agreement with what previously described by Qian et al. (12), and could suggest that the formation of stable E-cadherin/EGFR heterodimers require an intact extracellular E-cadherin domain, so that mutations able to perturb the correct E-cadherin extracellular conformation would results in a reduced E-cadherin/EGFR affinity. Of notice, most somatic E-cadherin mutations identified in sporadic diffuse gastric cancer are found to cluster between exons 7 and 10 encoding part of the extracellular E-cadherin domain (28). As reported by Bremm et al. (data not shown; submitted for publication), somatic deletion of E-cadherin exon 8 affects a putative calcium-binding domain, having consequences on the conformation, surface localization and endocytosis of the protein. This somatic deletion of E-cadherin exon 8 also affects the heterodimerization of EGFR with E-cadherin, in accordance with our results.

Moreover, since all E-cadherin mutations used in this study (T340A, A634V, P799R and V832M) were previously shown to impair cell–cell adhesion, our results also demonstrate that the EGFR/E-cadherin interaction is adhesion independent and solely associated to the E-cadherin status.

For a long time, it has been proposed that the interaction between EGFR and E-cadherin would be mediated through ß-catenin (7). Our results also suggest that EGFR might interact independently with both E-cadherin and ß-catenin, where the EGFR/E-cadherin interaction would take place at the cell surface and EGFR/ß-catenin heterodimers could involve the membrane-unbound fraction of ß-catenin. Further studies are warranted to clarify the role of E-cadherin-bound ß-catenin in the formation and stability of the E-cadherin/EGFR heterodimers.

Prior to its activation, EGFR is found on the plasma as inactive monomers. Ligand binding to a monomeric unit of EGFR activates the cytoplasmic catalytic function, promoting receptor dimerization followed by autophosphorylation on tyrosine residues (29). Since EGFR binds to E-cadherin we asked whether this interaction would have any impact on the activation status of EGFR. Our results show that the E-cadherin extracellular mutant T340A and A634V display increased levels of phosphorylated EGFR upon ligand stimulation, when compared with the wild-type E-cadherin or P799R and V832M mutants. Since all cell lines tested express comparable amount of total EGFR, and considering that E-cadherin/EGFR heterodimers are less stable for extracellular E-cadherin mutants, this would suggest that E-cadherin exerts an inhibitory function on EGFR activity. In this model, EGFR/E-cadherin heterodimers would be insensitive to EGF activation, while loss of this interaction would result in more membranous EGFR available for EGF-dependent activation. Consistently, mock cells E-cadherin negative displayed the highest level of activated EGFR upon ligand stimulation. Ligand binding to a monomeric unit of EGFR induces the formation of receptor homodimers and activation of its intrinsic kinase domain, resulting in the autophosphorylation on specific tyrosine residues within the cytoplasmic tail. These phosphorylated residues serve as docking sites for various adaptor proteins and signalling molecules involved in the regulation of intracellular signalling pathways that promote cell proliferation and migration, among others (30).

In particular, EGFR activation has been reported to trigger cytoskeleton reorganization during cell movement (31). We have previously shown that mutations on the extracellular domain of E-cadherin are responsible for increased cell motility in a mechanism involving RhoA activation (16). In the present work, we demonstrated that these E-cadherin mutant expressing cells show a reversion of the motile phenotype upon EGFR pharmacological inhibition. This is correlated with decreased RhoA activation, supporting the idea that Rho-like proteins are downstream effectors of EGFR activation. We propose a model in which upon disruption of EGFR/E-cadherin heterodimers, EGF activation of the receptor takes place which in turn causes RhoA activation ultimately leading to increased cell motility (Figs 4 and 5).


Figure 5
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  		Figure 5. Schematic proposed model. Extracellular mutations of E-cadherin disturb the stability of E-cadherin/EGFR heterodimers, allowing receptor activation by the ligand and consequent activation of RhoA signalling pathway, accompanied by enhanced cell motility.

 
In conclusions, (1) we demonstrated that EGFR/E-cadherin interaction requires an intact E-cadherin extracellular domain. (2) Upon interaction with EGFR, E-cadherin exerts an inhibitory function modulating the kinase activity of the receptor in an adhesion-independent manner. (3) The extracellular E-cadherin mutants, by reducing its affinity for EGFR, increase the fraction of unbound EGFR, which can thus be activated resulting in enhanced cell motility. (4) This effect is transmitted through the activation of RhoA.

On one hand, these results bring new insights into the understanding of the role of E-cadherin as cell membrane receptor. On the other hand, we believe that the distinct in vitro behaviours we observed for the different missense mutations are likely to be relevant during the onset of cancers associated to these mutations, which might have important implication in the prediction of the clinical outcome of carriers of E-cadherin germline missense mutations.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Cell culture
CHO-K1 cells transfected with the pcDNA3 vector alone (mock cells) or stably expressing the E-cadherin cDNA variants (wild-type, two derived HDGC germline missense mutations localized at the extracellular domain of the protein -T340A, A634V, and two localized at the cytoplasmic domain of the protein- P799R and V832M) were established as previously described (13,14). Cells were grown at 37°C under 5% CO2 in humidified air, in {alpha}-MEM (+) medium (Gibco-BRL) supplemented with 5% foetal bovine serum (HyClone), 2 mM L-glutamine, 1% penicillin/streptomycin and 500 µg/ml geneticin (Gibco-BRL). At least two independent clones for each cell line were used in each experiment, to exclude clonal dependence of the results; mock cells were used as negative control.

Immunoprecipitation and immunoblotting
Immunoprecipitation studies were performed by using the nProtein A SepharoseTM Fast Flow (Amersham Bioscience) and the ‘Catch and Release® v2.0’ kit (Upstate). About 2 x 106 cells were seeded per 10 cm tissue culture dish coated with Collagen type I 50 µg/ml in 0.02 N acetic acid and lysed 24 h later with 300 µl of L-CAM buffer (140 mM NaCl, 4.7 mM KCl, 0.7 mM MgSO4, 1.2 mM CaCl2, 10 mM Hepes pH 7.4, containing 1% (v/v) Triton-X-100 (17). The lysis buffer contained 2 mM phenylmethylsulfonylfluoride, 2 mM orthovanadate, 19 mg/ml aprotinin, 20 mg/ml leupeptin, 10 mM sodium phosphate and 100 mM sodium fluoride. Total protein was quantified by following the Bradford dye-binding procedure (32). An aliquot of 500 µg of protein was immunoprecipitated, according to the manufacturer's instructions; 2 µg of monoclonal antibody against EGFR (Santa Cruz) were used. Immunoprecipitated proteins were separated on a 7.5% SDS–polyacrylamide gel electrophoresis, followed by transfer onto a nitrocellulose membrane (Hybond C-extra, Amersham Bioscience). Immunoblotting was performed with antibodies against E-cadherin (BD Biosciences, 1:5000) and ß-catenin (BD Biosciences, 1:2000).

Twenty microgram of the same lysates were immunoblotted with antibodies against E-cadherin (1:5000), ß-catenin (1:2000) and EGFR (1:2000), all from BD Biosciences, Tyr1086 phospho-EGFR (Zymed Laboratories 1:2000) and {alpha}-tubulin (Sigma 1:20 000) as a loading control. For signal detection, the ECL western blotting detection kit (Amersham Bioscience) was used.

Immunofluorescence staining
To assess the activation profile of the EGFR, cells were grown on top of glass coverslips at a density of 3.5 x 105 cells/ml and starved overnight 24 h after seeding. Prior to fixation, cells were stimulated with EGF 100 ng/ml for different time points in intervals of 1 and 5 min until a maximum of 1 h. Fixation was done in 4% Formaldehyde in PBS, for 30 min at 4°C followed by blocking of the aldehyde groups with NH4Cl 50 mM in PBS for 10 min at room temperature. Permeabilization was done with Triton X-100 0.25% in PBS for 5 min at room temperature before blocking with BSA 1% for 30 min at room temperature. The Tyr1086 phospho-EGFR (Zymed Laboratories) was used at 1:150 dilution in PBS with BSA 1% and incubated over-night at 4°C. An anti-rabbit FITC-coupled antibody (Dako) was applied as secondary antibody at 1:40 dilution together with DAPI (1:1000) to counterstain the nucleus. Antibody was diluted in PBS containing 1% BSA and incubated for 1 h at room temperature in the dark. Coverslips were mounted on slides using an antifading reagent (Vectashield, Vector) and examined on a LeicaDM IRE2 Fluorescence Microscope using Leica, 20x, 40x and 100x oil immersion objectives. Images were taken with a DC-300F Leica camera and processed with LeicaFW4000 software and Adobe PhotoShop.

Cell motility assay
The migratory/motility behaviour of the transfected cells was analysed in an in vitro wound healing assay, as previously described (33). Cells were grown to confluence in the 6-well plates, an artificial wound was created with a blue Gilson pipette tip and each culture was carefully washed twice with PBS to remove detached cells. Migration was assessed by measuring the distance between wound edges in at least six different randomly chosen regions at time intervals (0, 3, 8 and 24 h), in the presence of the EGFR inhibitor Tyrphostin AG 1478 (9.45 µM) (SIGMA). To exclude the effect of the DMSO in which the inhibitor is solved, the same assay was repeated in the presence of the same volume of DMSO as it had been used of Tyrphostin. Phase contrast photographs were taken with a digital camera using the 10x objective.

RhoA activity assay
RhoA activity assay was performed as previously reported (34). To take into account the effect of wound healing on the activation level of RhoA, confluent monolayers of cells in 10 cm plates were wounded with a blue Gilson tip, 4 h prior to lysis. Cells were treated with the EGFR inhibitor Tyrphostin AG 1478 (9.45 µM) after wounding and with the equivalent volume of DMSO in the negative control plates. Equal amount of proteins were then engaged in the pull-down assays. GTPbound RhoA was precipitated by the C21 domain of Rhotekin, fused to GST and pre-coupled to glutathione-sepharose beads (Upstate Biotechnology). Precipitated complexes were analysed on western blot using monoclonal antibody against RhoA (Pierce, 1:1000). Immunoblots were quantified with the Scion Image Analysis, taking as 1 the ratio between RhoA-GTP and Rhotekin bands for wild-type E-cadherin cells. Using the same antibody, the total amount of RhoA in whole-cell lysates (2.5% of input) was analysed in parallel.


   ACKNOWLEDGEMENTS
 
This study was funded by grants from the Fundação para a Ciência e a Tecnologia, Portugal (SFRH/BD/16747/2004 and POCI/SAU - OBS/57670/2004), ADI-Agência de Inovação, SA 70/353 (INV-ONC-DPN) and Marie Curie Fellowship association.

Conflict of Interest statement. None declared.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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