ORIGINAL ARTICLE

VAV2 regulates epidermal growth factor receptor endocytosis

and degradation

S Thalappilly1,2, P Soubeyran1,2, JL Iovanna1,2 and NJ Dusetti1,2

1INSERM U624, Stress Cellulaire, Marseille, France and 2Aix-Marseille Universite, Campus de Luminy, Marseille, France

Vav proteins are guanine nucleotide exchange factors forRho GTPases that regulate cell adhesion, motility,spreading and proliferation in response to growth factorsignalling. In this work, we show that Vav2 expressiondelayed epidermal growth factor receptor (EGFR) inter-nalization and degradation, and enhanced EGFR, ERKand Akt phosphorylations. This effect of Vav2 on EGFRdegradation is dependent on its guanine nucleotideexchange function. Knockdown of Vav2 in HeLa cellsenhanced EGFR degradation and reduced cell prolifera-tion. epidermal growth factor stimulation led to co-localization of Vav2 with EGFR and Rab5 in endosomes.We further show that the effect of Vav2 on EGFRstability is modulated by its interaction with twoendosome-associated proteins and require RhoA function.Thus, in this work, we report for the first time that Vav2can regulate growth factors receptor signalling by slowingreceptor internalization and degradation through itsinteraction with endosome-associated proteins.Oncogene (2010) 29, 2528–2539; doi:10.1038/onc.2010.1;published online 8 February 2010

Keywords: VAV2; EGFR; endosome; receptorinternalization

Introduction

Vav family proteins are guanine nucleotide exchangefactors (GEF) for Rho family GTPases. They regulatecytoskeletal dynamics in response to stimuli such asgrowth factor receptor activation, leading to modulationof adhesion, motility and proliferation of both normaland cancer cells (Marcoux and Vuori, 2003; Patel et al.,2007). Vav1, the best characterized of the three humanVav family members, is expressed in hematopoietic cellsand their derivatives whereas the other two proteins,Vav2 and Vav3, show wider expression patterns(Hornstein et al., 2004). Vav proteins are potentoncogenes that can induce cell transformation byactivating Rho GTPase function and modulating cell

signaling (Bustelo et al., 1994; Zeng et al., 2000; Servitjaet al., 2003; Fernandez-Zapico et al., 2005). It has beenreported that Vav-induced cell transformation requireits GEF function. In addition to their function asregulators of Rho proteins, Vav family proteins regulategene expression as components of transcription com-plexes (Doody et al., 2000; Houlard et al., 2002). Role ofVav proteins in lymphocyte function is well character-ized in both cellular and animal models. Studies usingmice lacking all three Vav family members showed a rolefor these proteins in T-cell and B-cell responses and inadaptive immune response (Swat and Fujikawa, 2005).

Vav family proteins are conserved multi-domainproteins containing: a Calpolin homology domain, aDbl homology domain, a pleckstrin homology domain,a zinc-finger domain and two SH3 domains flankinga SH2 domain. Regulation of Rho Family GTPases, theprimary function attributed to Vav proteins, is mediatedby their Dbl homology domain (Romero and Fischer,1996). Deletion of their N-terminal region leads toconstitutive activation of these GTPases resulting in anenhanced oncogenicity (Abe et al., 2000; Bustelo, 2002).The similar domain organization and high sequencesimilarity shared between the Vav proteins results in over-lapping functions, however, several studies have reporteddifferences in their functions (Schuebel et al., 1996).

Growth factor stimulation leads to SH2 domain-mediated binding of Vav proteins to phosphotyrosineresidues on activated receptors such as epidermalgrowth factor receptor (EGFR) and platelet-derivedgrowth factor receptor (Pandey et al., 2000; Tamaset al., 2003). Vav proteins phosphorylation by thesereceptors and Src kinase family leads to inhibition ofintramolecular interactions leading to their activation(Servitja et al., 2003; Tamas et al., 2003; Tu et al., 2003).Membrane translocation of Vav proteins and PHdomain-mediated phospholipid binding are also crucialto their activation (Tamas et al., 2001; Aoki et al., 2005).Activated Vav proteins mediate the activation of Rac,RhoA and cdc42 in response to growth factor stimula-tion and are potent regulators of cell signaling (Abeet al., 2000; Liu and Burridge, 2000). Interestingly,during pancreatic carcinogenesis, Vav-mediated activa-tion of mitogen-activated protein kinase signaling isrequired for Src-induced cell transformation (Servitjaet al., 2003; Fernandez-Zapico et al., 2005). Theseobservations suggest a pivotal role for Vav proteins inregulation of cell growth.

Received 12 June 2009; revised 19 November 2009; accepted 7 December2009; published online 8 February 2010

Correspondence: Dr NJ Dusetti, INSERM U624, Stress Cellulaire,Parc Scientifique de Luminy, Case 915, Bouches du Rhones, Marseille,F-13288, France.E-mail: nelson.dusetti@inserm.fr

Oncogene (2010) 29, 2528–2539& 2010 Macmillan Publishers Limited All rights reserved 0950-9232/10 $32.00

www.nature.com/onc

Activated EGFR undergoes Cbl-mediated mono-ubi-quitination and is internalized to early endosomes (Sorkinand Goh, 2008). The best characterized EGFR inter-nalization mechanism involves its translocation to cla-thrin-coated pits, although other mechanisms have alsobeen described (Sorkin and Goh, 2008). Once internalized,the endosome-associated EGFR continues the signalingleading to cell survival until it is addressed to themultivesicular bodies and subsequently degraded in thelysosome (Wang et al., 2002). This trafficking from earlyendosomes to multivesicular bodies and lysosomes is acomplex process mediated by different endosomal pro-teins including Rab proteins and the endosomal sortingcomplex required for transport complexes that regulatetrafficking by binding to the ubiquitinated EGFR (Ceresa,2006; Williams and Urbe, 2007). This process is tightlycontrolled in cells as it can affect cell proliferation. Forexample, deregulation of endosomal sorting complexrequired for transport proteins have been implicated incancers suggesting a tumor-suppressor role for them(Stuffers et al., 2009). Similarly, recycling of the activatedreceptor also leads to enhanced EGFR stability andsignaling. Thus, deregulation of these processes that leadsto abnormal EGFR internalization, trafficking or recy-cling can affect its activity causing enhanced cell divisionand conferring growth advantages often observed incancer cells (Mosesson et al., 2008).

Vav2 and EGFR localize to the same regions at the cellmembrane and share interactions with several proteinsincluding Src kinases family, Ras, Cbl and Grb2. Severalof these proteins co-localize with EGFR on endosomesand regulate both its signaling and trafficking (de Melkeret al., 2001; Jiang and Sorkin, 2002; Yamazaki et al., 2002;Donepudi and Resh, 2008; Sorkin and Goh, 2008).However, it is not known whether Vav proteins have arole in EGFR endocytosis or trafficking. Recently, in aproteomic study on multi-SH3 domain containing pro-teins, we identified two endosome-associated proteinsthat interacts with Vav2: the Rab regulatory proteinGapvd1 and the endosome-associated protein Tom1L1(Puertollano, 2005; Bache et al., 2006; Hunker et al., 2006;Thalappilly et al., 2008). The aim of this work is to betterunderstand the role of Vav2 in EGFR endocytosis anddegradation in association with the endosomal proteins(Gapvd1 and Tom1L1).

Results

Vav2 expression affects EGFR degradation and signallingAlthough Vav family proteins have been implicated inEph receptor endocytosis and signalling, any directendosomal role for these proteins has not beencharacterized (Cowan et al., 2005). We have previouslyreported, in a yeast two hybrid-based study, theinteraction of Vav2 with Gapvd1 and Tom1L1, twoproteins that regulate endocytosis and vesicle trafficking(Thalappilly et al., 2008). These proteins function inligand-induced endocytosis and ordered trafficking oftransmembrane receptors to multivesicular bodies andlysosomes in which they are depredated. As Vav2 also

interacts with EGFR and is regulated by it, we firstanalyzed the effect of Vav2 expression on total EGFRlevel. HeLa cells were transfected with Vav2–GFP orGFP (as control) expression plasmids. The cells werelysed 48 h later and total EGFR level was determined bywestern blot. As shown in Figure 1a, Vav2 expressingcells had more than two times higher level of EGFRthan control cells. This suggested that Vav2 expressionmay affect EGFR degradation in these cells.

To further analyze the effect of Vav2 on EGFRstability and signalling, HeLa cells were transfected withVav2 and stimulated with epidermal growth factor(EGF) for different spans of time. To discriminatebetween protein degradation and synthesis, translationwas blocked by treating the cells with Cycloheximide.As shown in Figure 1b, Vav2 expression delays EGFRdegradation significanly. Interestingly, EGFR and bothERK and Akt (EGFR downstream effectors) werephosphorylated more in Vav2 transfected cells com-pared with control. This observation suggests anincrease in EGFR activation induced by Vav2 expres-sion. This effect of Vav2 on EGFR stability and activitycorrelates with a significantly enhanced proliferation ofHeLa cell (Po0.05) (Figure 1c).

The best characterized role for Vav2 is its functionas guanine nucleotide exchange factor for RhoGTPases. To analyze whether the Vav2 GEF functionis required for the effect observed on EGFR stability,we used a mutant Vav2 lacking the Dbl homologydomain (DGEF-Vav2). As shown in Figure 1d, whenEGF-stimulated cells were transfected with the mutantVav2, the EGFR level was similar to the control levelobtained after the empty vector transfection (Po0.05).This observation suggests that the GEF activity of Vav2is important for the EGFR stabilization. Previousstudies have identified the Vav2-mediated activation ofdifferent members of the Rho GTPases family (Abeet al., 2000; Liu and Burridge, 2000). To identify the roleof Rho proteins in the regulation of EGFR stabilitydownstream of Vav2, EGFR level was analyzed in HeLacells transfected with Vav2 along with control or RhoA-specific small interference RNA (siRNA). As shown inFigure 1e, RhoA knockdown led to enhanced degrad-ation of EGFR in Vav2 expressing cells comparedwith control cells. This showed that RhoA function isnecessary for Vav2-mediated EGFR stability in Vav2expressing HeLa cells. We further analyzed whetherexpression of Vav2 affected ubiquitination of EGFRafter EGF stimulation. Analysis of EGFR levels in anti-ubiquitin immunoprecipites of HeLa cells transfectedwith Vav2 expression plasmids or control plasmids didnot identify appreciable difference in ubiquitinatedEGFR levels (Supplementary Figure 1a).

Vav2 expression alters internalization and endosomallocalization of EGFRLigand-induced receptor endocytosis leads to a punctu-ate cytoplamic localization for EGFR (endocyticvesicles) in contrast to the diffused membrane stainingobserved in unstimulated cells. To study the effect of

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Vav2 on EGFR internalization and trafficking, welocalized EGFR in the presence or not of transfectedVav2 by immunofluorescence in HeLa cells. On serumstarvation, cells expressing Vav2 (Vav2–GFP) as well asthe control cells (GFP) showed weak staining for EGFR

(Figure 2a at 0min). After 15min of EGF stimulation,Vav2 expressing cells showed less EGFR-containingendocytic vesicles than control cells (not expressingVav2 or expressing GFP). This suggested that Vav2expression slowed down the internalization of stimu-

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lated EGFR. Finally, after 45min of EGF stimulation,we observed that Vav2 expressing cells had muchhigher levels of EGFR localized on cytoplasmic vesiclesthan control cells. This last observation suggested thatVav2 not only delayed internalization but also EGFRtargeting to lysosomal degradation. Similar results wereobtained in the human pancreatic cell line Panc1 aswell. As shown in Figure 2b, Panc-1 cells expressingVav2, showed reduced cytosolic staining for EGFRafter 15min of EGF stimulation compared with controlcells. After 45min of EGF stimulation, EGFR stainingwas observed as vesicles at the cellular periphery in Vav2expressing cells compared with more perinuclear stain-ing in untransfected cells. These observations suggesteda generic, cell-type-independent delay in EGFR inter-nalization and degradation in Vav2 expressing cells.Interestingly, we did not observe any difference intransferrin receptor internalization between Vav2 ex-pressing or control cells treated with transferringsuggesting that Vav2 specifically regulates growth factorreceptor internalization (Supplementary Figure 1B).To confirm endosomal co-localization of EGFR andVav2, HeLa cells were cotransfected with Vav2 andRab5-GFP (early endosome marker) expression plas-mids and stimulated with EGF. As shown in Figure 2c,Vav2 partially co-localize with Rab5-GFP and EGFR inEGF-stimulated HeLa cells. The co-localization wasconfirmed by analysis using Co-localization Finder, anImageJ software plugin (http://rsb.info.nih.gov/ij/).

To validate the effect of Vav2 on EGFR internaliza-tion, we used biotinilation of cell surface proteins atdifferent times after EGF stimulation. As shown in theFigure 2d, Vav2 expressing cells had significantly higherlevel of biotinylated EGFR after 5 and 15min of EGFtreatment (Po0.05). This result indicates an increase incell surface retention of EGFR in these cells, confirmingour previous observation that Vav2 expression delayedEGFR internalization.

The Vav2 effect on EGFR stability is regulated by itsinteraction with endosomal proteinsWe have recently shown that Vav2 interacts with Gapvd1and Tom1L1, two endosome-associated proteins (Tha-lappilly et al., 2008). As Vav2 interaction with theseproteins was identified initially by yeast two hybrid, wefirst confirmed these interactions in mammalian cells byco-immunoprecipitation. As shown in Figure 3a, bothGapvd1 and Tom1L1 specifically co-immunoprecipitatedwith Vav2. These interactions suggest a role for them inmediating the Vav2 effect on EGFR stability. Therefore,we used siRNA-mediated knockdown of Gapvd1 andTom1L1 to study their role in Vav2 function (Figure 3b).Interestingly, after EGF stimulation, Gapvd1 andTom1L1 knockdown had opposite effects on Vav2-induced EGFR stabilization. Gapvd1 knockdown sig-nificantly enhanced the Vav2-induced stabilization ofEGFR while Tom1L1 knockdown enhanced EGFRdegradation, compared with cells transfected with Vav2and control siRNA (Figure 3c). Control experiments,carried out in cells transfected only with siRNA forGapvd1 or Tom1L1, do not show significant differencesin EGFR stability (data not shown). This suggests thatVav2 interaction with endosomal proteins (Gapvd1 andTom1L1) modulates its effect on EGFR stability.

Vav2 knockdown affects EGFR degradationWe have shown that forced expression of Vav2 delayedendocytosis and degradation of activated EGFR.To further confirm the role of Vav2 on EGFR stability,we knocked down Vav2 expression in HeLa cells usinga specific siRNA. Transfection of the siRNA into HeLacells efficiently blocked Vav2 protein expression asshown in Figure 4a. Vav2 knockdown in EGF-stimulatedHeLa cells significantly enhanced the degradation ofEGFR compared with control cells (Figure 4b). Thisdata validate the EGFR stabilization effect of Vav2.Importantly, we have shown that this finding was

Figure 1 Vav2 expression affects EGFR degradation and signalling. (a) HeLa cells were transfected with Vav2–GFP or GFP(as control) expression plasmids. Forty-eight hours later the cells were lysed and EGFR, Vav2–GFP and tubulin (Tub) level wasdetermined by Western blot. Three independent experiments were carried out and western blot bands corresponding to EGFR werequantified and normalized against tubulin level (graph on the right). Vav2 transfected cells had significantly higher level of EGFRcompared with control cells (P¼ 0.004) at Po0.05 significance level. (b) HeLa cells were transfected as in (a) and 24 h later were serumstarved for 12 h. They were then stimulated with EGF (100 ng/ml) and protein synthesis blocked with cycloheximide (CHX, 10 mg/ml)for the indicated times. Cells were lysed and EGFR, Vav2–GFP, GFP, tubulin (Tub), P-EGFR, P-ERK, P-Akt and total Akt andERK level was determined by western blot. Three independent experiments were carried out and specific western blot bandscorresponding to EGFR were quantified and normalized against tubulin level (graph on the right). The EGFR level in Vav2transfected cells was significantly higher compared with control cells (15min, P¼ 0.0007; 30min , P¼ 0.0009; 45min, P¼ 0.0107) atPo0.05 significance level. The points in graph that represent significant difference are marked with *. Total Vav2–GFP western blotwas used to control transfection efficiency. Only experiments with comparable transfection efficiencies were used for quantifications.(c) HeLa cells were transfected with Vav2–GFP or GFP expression plasmid. Twenty-four hours later 10 000 of these cells were plated in35mm of diameter dishes and counted daily during 6 days. The data in the graph represent average of three independent experiments.The Vav2 expressing cells showed higher proliferation compared with control cells (day 3, P¼ 0.0483; day 4, P¼ 0.0164; day 5,P¼ 0.0044; day 6, P¼ 0.0141) at Po0.05 significance level (marked by *). GFP epifluorescence was used to monitor transfectionefficiency and only experiments with equivalent transfection efficiencies were used for quantifications. (d) HeLa cells were transfectedwith Vav2, DGEF–Vav2 or empty (control) expression plasmids and processed as in Figure 1b. Western blot was carried out to detectEGFR, tubulin and total Vav2 levels. The graph on the right side shows EGFR intensities of bands normalized against tubulin from threeindependent experiments. After 45min of EGF treatment, the cells transfected with Vav2 showed significant difference in EGF levelscompared with control cells (P¼ 0.001), whereas the DGEF–Vav2 transfected cells did not show significant difference (P¼ 0.058) atPo0.05 significance level. Transfection efficiency for Vav2 and DGEF–Vav2 were determined by western blot with anti-Vav2 antibodies.Only experiments with similar transfection efficiencies were considered. (e) HeLa cells were transfected with Vav2 and either RhoA siRNAor control siRNA. Forty-eight hours after transfection, the cells were serum-starved overnight and stimulated with EGF (10ng/ml) forindicated times. Lysates prepared from these cells were analyzed by western blotting for EGFR, RhoA and tubulin levels.

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physiologicaly relevant as Vav2 knockdown in HeLacells showed reduced proliferation (but no cell death,when analyzed by Trypan blue staining) under normalgrowth conditions (Figure 4c). Taken together thesedata suggest that Vav2 regulates EGFR functionthrough modulation of its internalization and subse-quent degradation.

Discussion

Vav proteins are well characterized as regulators of actincytoskeleton, cell motility, adhesion and signaling. Theirtranslocation to the cell membrane in response togrowth factor receptors activation (EGFR and plate-let-derived growth factor receptor) leading to activation

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of Rho GTPases have been studied (Pandey et al., 2000;Tamas et al., 2001). In this study, we show that Vav2expression in HeLa cells affect internalization anddegradation of EGFR. We report that this function ofVav2 is modulated by two endosome-associated pro-teins, Gapvd1 and Tom1L1, with which it interacts.Overexpression of Vav2 in both HeLa and Panc1 cellscaused delayed internalization and increased stability ofEGFR leading to an enhanced signaling cascade for thisreceptor. Moreover, siRNA-mediated knockdown ofVav2 affected EGFR degradation and cell growth.

EGFR endocytosis and trafficking are complexprocesses that are regulated by several proteins asso-ciated with activated EGFR. Hence, the fact that Vav2interacts with two proteins (Gapvd1 and Tom1L1)described as part of different endosomal complexesinvolved in EGFR trafficking attracted our attention.Gapvd1, is an activator of the early endosome regulatorprotein Rab. It regulates vesicular trafficking processessuch as endocytosis of activated transmembrane proteinsand exocytosis of GLUT4 vesicles (Hunker et al., 2006;Lodhi et al., 2007). Recent studies have shown that it canregulate EGFR endocytosis and degradation indepen-dently of Rab5 function (Su et al., 2007). Tom1L1 hasalso been shown to localize on endosomes and interactwith proteins of the endosomal sorting complex requiredfor transport machinery (Puertollano, 2005). Interactionof Vav2 with these two proteins (Figure 3) suggested thatthey have a role in the Vav2-mediated modulation ofEGFR endocytosis, trafficking and stability.

We found that overexpression of Vav2 in HeLa cellsdelayed EGF-induced EGFR internalization and de-gradation. These cells showed enhanced phosphoryla-tion of EGFR as well, compared with control cells.Phosphorylated EGFR induces survival and prolifera-tive signaling by activating different downstreamsignaling pathways. Akt and mitogen-activated proteinkinase pathways are important among them and havebeen implicated in cancer cell growth and survival (Goel

et al., 2007). We have found that HeLa cells expressingVav2 showed enhanced phosphorylation of ERK andAkt proteins (Figure 1b). In accordance with theseresults, expression of Vav2-enhanced HeLa cells pro-liferation (Figure 1c). Regulation of EGFR degradationcan thus be a novel mechanism for Vav2-induced cellsurvival and proliferation. The role of Vav2 in theEGFR trafficking can contribute to the complexmodulation of this process because the activity ofVav2 itself can be regulated by EGFR (Abe et al., 2000).

One of the mechanisms involved in EGFR stabiliza-tion by Vav2 is by delaying its internalization. Weobserved, by immunofluorescence, that the appearanceof endosomal EGFR was delayed in Vav2 expressingcells compared with control cells (Figure 2a). Inaccordance with this, we found by cell surface proteinbiotinilation, enhanced retention of EGFR on cellsurface in Vav2 expressing cells compared with controlcells (Figure 2c). Interestingly, this delayed internaliza-tion was observed at very short times (5min) whenrecycling of receptors have not still begun. This resultsuggests that the difference observed in the levels ofbiotinilated EGFR is due to an internalization delay andnot to recycling. Furthermore, we found that Vav2expression-induced delayed degradation of EGFR(Figures 1a and b). Endosomal EGFR staining wasvisible in Vav2 expressing cells even after 45min of EGFstimulation whereas control cells had reduced, peri-nuclear staining probably because of lysosomal degra-dation (Figure 2a). These data suggest that Vav2 delaythe internalization as well as degradation of EGFR inVav2 expressing cells. Several studies have shown thatsignaling initiated from endosomal EGFR can sustaincell survival (Wang et al., 2002). Thus effect of Vav2 onEGFR might be important for its ability to induce cellsurvival and transformation. We have observed delayedEGFR internalization and degradation also in Panc-1pancreatic cancer cells that overexpress Vav2. This resultsuggests that this Vav2 function is not cell-type dependent.

Figure 2 Vav2 expression altered EGFR endocytosis. (a) HeLa cells were plated on coverslips and transfected with Vav2 or GFP(control) expression plasmids. Cells were then grown in normal medium for 24 h and serum starved for 12 h. EGF-supplementedDulbecco’s modified Eagle’s medium (DMEM) (100 ng/ml) was added to the cells and incubated for the indicated times. The cells werethen fixed and stained using anti-EGFR antibody. The coverslips were mounted and visualized by confocal microscopy. Green colorindicates cells expressing Vav2–GFP or GFP (control) and red color indicates endogenous EGFR staining. Three independentexperiments were carried out showing similar results. (b) Panc-1 cells were transfected with Vav2–GFP expression plasmids and grownunder normal conditions for 24 h. After 24 h, the cells were serum-starved overnight and then treated with EGF (10 ng/ml) for indicatedtimes. The cells were then fixed and immunostained for EGFR. The coveslips were mounted on slides and analyzed using confocalmicroscopy. Green cells indicate Vav2–GFP expression and EGFR staining is shown in red. (c) HeLa cells were plated on coverslipsand transfected with Vav2 and Rab5–GFP expression plasmids. Twenty-four hours post-transfection the cells were serum starved for aperiod of 12 h. The cells were then incubated in EGF-supplemented DMEM (100ng/ml) and cycloheximide (CHX, 10mg/ml) for30min at 37 1C, fixed and stained with anti-Vav2 and anti-EGFR antibodies (Vav2 in red, and EGFR in blue). The cells were observedusing confocal microscopy. Green color shows epifluorescence of Rab5–GFP. White colored spots on the merge image indicate theposition in which all the three proteins co-localize. The co-localization of the three proteins was further confirmed using the ImageJplugin Co-localization Finder. (d) HeLa cells were transfected with Vav2 or pCDNA3 (Ctrl) plasmids. The cells were grown undernormal conditions for 24 h and then were serum starved for 12 h. They were then treated with EGF-containing DMEM (50ng/ml) forthe indicated times. Total cell surface proteins were biotinylated as described in experimental procedures. The cells were subsequentlylysed and EGFR was immunoprecipitated. The immunoprecipitated proteins were resolved on sodium dodecyl sulfate–polyacrylamidegel electrophoresis (SDS–PAGE) and the level of biotinylated EGFR was detected by western blot using streptavidin-conjugatedhorseradish peroxidase. The western blot shown is representative of three experiments. The graph bellow represents the quantificationof western blot bands corresponding to EGFR from three different experiments normalized against tubulin (Tub). EGFR levels inVav2 transfected cells, as analyzed by t-tests, were significantly higher compared with control cells (5min, P¼ 0.009 and 15min,P¼ 0.001) at Po0.05 significance levels (indicated in the figure by *). Vav2 level was determined by western blot in the total cell lysates(TCL) and serve to determine transfection efficiency, only experiments with similar transfection efficiencies were considered.

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It is interesting to note that in this context, deregulatedVav acts synergistically with EGFR during pancreaticcarcinogenesis (Fernandez-Zapico et al., 2005). Expressionof Vav2 in HeLa cells growing under normal conditions(serum-containing medium) led to enhanced levels ofEGFR compared with control cells (Figure 1a). Wesuggest that this increase is due to reduced internaliza-tion and degradation of EGFR. However, at time 0of EGF stimulation and under serum starvation, thisdifference was not apparent (Figures 1b, d and e at time0); we hypothesized that under serum starvation thecomplete absence of EGF stops EGFR internalizationleading to disruption of steady-state EGFR turnover.

In this case, the effect of VAV2 on EGFR stability isabolished explaining this apparent discrepancy.

Vav family proteins share a high degree of similarityallowing us to hypothesize that they could also sharefunctionalities. To verify this hypothesis, HeLa cells weretransfected with vectors allowing overexpresion of VAV1and VAV3 proteins and were cultured in serum-contain-ing medium. Similarly, to Vav2 the other members of thisfamily enhanced EGFR stability but in a lesser degree(data not shown). This fact can be explained by the highdegree of similarity shared among the Vav proteins andbecause our overexpresion conditions used. This observa-tion raised an interesting aspect given that Vav1 expres-

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Figure 3 Vav2 interacts with endosomal proteins that regulate its effect. (a) HEK293T cells were co-transfected with Vav2 and HA-Gapvd1 or FLAG-Tom1L1 expression plasmids. The cells were grown under normal conditions for 24 h and then lysed. Co-immunoprecipitations (Co-IP) were carried out using anti-Vav2 antibody or PBS (control). Western blot was used to detect theindicated proteins in the co-immunoprecipitates (TCL: total cell lysate, Ctrl: control). (b) Western blots showing siRNA knockdownfor Gapvd1 and Tom1L1. The western blot presented is representative of three independent experiments. The graph shows thedensitometric quantification (three different experiments) for the remaining expression of Gapvd1 and Tom1L1 in percentage to thetotal. (c) HeLa cells were co-transfected with Vav2 expression plasmid, scramble (Ctrl.) or specific siRNAs for the indicated proteins.Cells were grown for 72 h under normal conditions and then serum starved for 12 h. Subsequently, they were treated with EGF-containing Dulbecco’s modified Eagle’s medium (DMEM) (100 ng/ml) and cycloheximide (CHX, 10 mg/ml) for the indicated times andlysed. Western blots were carried out to determine the EGFR level. The graphs show quantified intensities (three independentexperiments) of the western blot bands normalized against tubulin. Analysis using one-way analysis of variance (ANOVA) showed thatall the values are significantly different at both 30 (P¼ 0.0002) and 45 (P¼ 0.0002) minutes at significance level Po0.05 (marked in thefigure as *). Vav2 level measured by western blot were used to control transfection efficiency. Only experiments with equivalenttransfection efficiencies were used for quantifications.

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sion has been associated with pancreatic carcinogenesis(Fernandez-Zapico et al., 2005).

Activated EGFR and Vav family proteins localize tothe same molecular complex assembled at the cellmembrane and they share interactions with severalproteins such as Grb2, Src and Cbl. These proteins werefound to be associated with EGFR complex at endo-somes as well and regulate both its signaling anddegradation. For example, Cbl interaction at cellmembrane was required for efficient internalization ofEGFR and its function in the endosomes was necessaryfor efficient degradation of EGFR (Soubeyran et al.,2002; Umebayashi et al., 2008). Furthermore, interac-tion of Vav with dynamin 2, a protein that regulatesEGFR internalization has also been described (Gomezet al., 2005; Orth et al., 2006).

Vav proteins associate with several receptor tyrosinekinases in addition to EGFR including platelet-derivedgrowth factor, Eph, vascular endothelial growth factorand HGF receptors (Kodama et al., 2000; Pandey et al.,2000; Seye et al., 2004; Cowan et al., 2005; Gavard andGutkind, 2006). Vav2 also associated with B-cell-specificCD19 and CD44v3 proteins (Doody et al., 2000;Bourguignon et al., 2001). As most of these proteinsare internalized on activation, we suggest that Vav2 actsas one of their downstream effectors that may alsoregulate their endocytosis and sorting. Sustaining thishypothesis, Ephrin-induced Eph receptor endocytosisduring axon guidance has been shown to be dependenton Vav family proteins (Cowan et al., 2005). However,we found that Vav2 expression did not alter the kineticsof transferrin receptor internalization (Supplementary

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Figure 4 Vav2 knockdown affects EGFR degradation and cell growth. (a) HeLa cells were transfected with siRNA targeting Vav2 orscramble siRNA (Ctrl). In all, 24–48 h later, cells were lysed and Vav2 and tubulin levels determined by western blot. The graphrepresents the western blot band quantification for Vav2 relative to tubulin (tub) from three independent experiments. (b) HeLa cellswere transfected with control siRNA or Vav2–siRNA and grown for 48 h. They were serum starved for 12 h and then Dulbecco’smodified Eagle’s medium (DMEM) containing 100 ng/ml EGF and 10 mg/ml cycloheximide (CHX) was added. Cells were incubatedfor the indicated times, lysed and EGFR level determined by western blot. Tubulin blot is shown as a control. The graph on the rightside shows the normalized values (EGFR/tubulin) from three independent experiments. EGF level in Vav2 siRNA transfected cells wassignificantly lower compared with control cells (15min, P¼ 0.001; 30min, P¼ 0.013 and 45min, P¼ 0.009) at significance levelPo0.05 (marked in the graph as *). (c) HeLa cells were transfected with Vav2 siRNA or scrambled siRNA (Ctrl Si). Twenty-fourhours later 10 000 cells were plated in 35-mm dishes and grown for the indicated times, they were then trypsinized and counted. Theexperiment represents mean of independent triplicates. Vav2 siRNA transfected cells showed reduced proliferation compared withcontrol cells. Statistical analysis revealed difference between the two populations at day 5 (P¼ 0.024) and day 6 (P¼ 0.011) atsignificance level of P¼ 0.05.

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Figure 1B). This might be due to the localization ofVav proteins proximal to EGFR and its interactionwith proteins involved in EGFR internalization anddegradation. Interestingly, in our conditions, we found thattransfected Vav2 protein undergoes phosphorylation inserum-starved HeLa cells stimulated with EGF (Supple-mentary Figure 2). This observation suggests that Vav2could be phosphorylated and activated by EGFR in ourmodel as was already described by Pandley and Tamas(Pandey et al., 2000; Tamas et al., 2003).

Deletion of the Dbl homology domain of Vav2abrogated almost totally its effect on EGFR stability.This links, at least in part, the effect of Vav2 on EGFRstability to Rho GTPases. A role for Vav2 in theactivation of these proteins downstream of EGFRactivation has already been described (Abe et al., 2000;Liu and Burridge, 2000). Furthermore, recent findingshave implicated several Rho GTPases in EGFRendocytosis and trafficking. It has been described thatRac1 and RhoA proteins are required for internalizationof EGFR, RhoD localizes to early endosomes andRhoB to late endosome- multivesicular bodies vesicles(Ellis and Mellor, 2000). Interestingly, expression ofconstitutively active forms of these proteins interfereswith endocytosis at the respective stages of theirfunction. Another Rho family protein, cdc42 has alsobeen described as an important regulator of EGFRdegradation (Wu et al., 2003). When we analyzed therole of RhoA protein on EGFR stability in cellsoverexpressing Vav2, we observed that delayed EGFRdegradation depends on RhoA function (Figure 1e).This observation explained, at least in part, therequirement of GEF domain for Vav2-mediated EGFRstabilization. In accord with this, a role for ROCK, aRhoA effector in EGFR endocytosis has already beendescribed (Ung et al., 2008). Furthermore, a recentreport has linked Vav2 to EGFR-induced activation ofRhoA in mesenchymal cells (Peng et al., 2010).

It has been suggested that in addition to their role asregulators of Rho GTPases, Vav proteins might alsofunction as adapter proteins (Bustelo, 2001). The largesize and multi-domain structure of these proteinssupports this hypothesis. Interaction of Vav2 withendosomal proteins might be an example of such afunction. Gapvd1 (Gapex-5), one of the endosomalproteins that interacted with Vav2, has been shown toregulate stability of growth factor receptors. Its expres-sion enhanced the internalization and degradation ofinsulin receptor while specific siRNA inhibited EGFRdegradation independently of its effect on Rab5(Hunker et al., 2006; Su et al., 2007). Tom1L1 associateswith endosomes and has been shown to regulate theactivity of Src kinase as well (Puertollano, 2005; Francoet al., 2006). Interaction of Vav2 with Tom1L1 andGapvd1 suggested a role for these proteins in Vav2function. Knockdown of Gapvd1 and Tom1L1 hadopposing effects on EGFR stability in Vav2 over-expressing cells (Figure 3c). Gapvd1 knockdown en-hanced the stability of EGFR in Vav2 overexpressingcells while Tom1L1 siRNA abrogated the effect of Vav2expression. This showed that these endosomal proteins

regulate the effect of Vav2 on EGFR stability. Recentstudies have shown that EGFR trafficking and degrada-tion requires monoubiquitination of its lysine residues.Perturbation of these ubiquitination processes led toinhibition of EGFR internalization and degradation(Clague and Urbe, 2006). Hence, we analyzed the levelsof EGFR ubiquitination in cells transfected with Vav2 orcontrol plasmids. EGFR levels in anti-ubiquitin immu-noprecipitates from control or Vav2 expressing cells didnot show significant difference (Supplementary Figure1A). This observation suggests that the effect of Vav2 onEGFR is independent of its ubiquitination status.

Recent studies have identified the presence of endoso-mal proteins including endosomal sorting complexrequired for transport proteins and Tom1L1 at themidbody during cytokinesis in which they function asregulators of mitotic cell division (Morita et al., 2007;Yanagida-Ishizaki et al., 2008). Interestingly, we observedthat endogenous Vav2 protein localized to the centralspindles of mitotic cells at telophase stage as well as at themidbody during cytokinesis in which it co-localized withthe Tom1L1 protein (Thalappilly and Dusetti, unpub-lished results). Rho GTPases have also been shown toregulate both the mitotic and cytokinesis phases of celldivision (Wadsworth, 2005; Narumiya and Yasuda,2006). This suggests that Vav2 might have wider roles incellular processes than currently understood.

Finally, the role of Vav2 in EGFR degradation wasconfirmed by knockdown of its expression in HeLa cells(Figure 4). The silencing of Vav2 reduced proliferation ofthese cells as well. The observed effect on EGFR stabilityfor Vav2 knockdown corresponds to the effect observed forthe deletion of the Vav2–GEF domain. These observationssuggest a specific role for Vav2 in this process. We reporthere that Vav2 regulates EGFR signaling by delaying itsinternalization and degradation through interaction withendosome-associated proteins. As both Vav2 and theseendosomal proteins can associate with other growth factorreceptors, Vav2 may be implicated in the internalizationand degradation of a wide spectrum of receptors,enhancing its oncogenic potential.

Materials and methods

MaterialsRabbit anti-EGFR, rabbit anti-Vav2, mouse anti-Myc, mouseanti-EGFR, anti-ubiquitin and mouse anti-HA antibodieswere from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA,USA). Mouse anti-beta tubulin and anti-FLAGM2 antibodieswere from Sigma (St Louis, MO, USA). Anti-phospho-Aktand anti-phospho ERK antibodies were obtained from CellSignaling Technology (Beverly, MA, USA). Human recombi-nant EGF and Texas-Red-conjugated transferrin was fromInvitrogen (Groningen, The Netherlands) and Biotin-X-NHSwas purchased from Calbiochem (Nottingham, UK).

Cell culture and transfectionsHeLa, HEK293T and Panc-1 cells were grown in Dulbecco’smodified Eagle’s medium containing 10% fetal calf serum at37 1C in 5% CO2 environment. HeLa cells were transfected usingFuGENE-HD (Roche, Indianapolis, IN, USA) and HEK293T

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and Panc-1 cells were transfected using Lipofectamine 2000(Invitrogen) according to manufacturer’s suggestion. Fetal calfserum, Dulbecco’s modified Eagle’s medium and OptiMEMmedium for cell culture were purchased from Invitrogen. ForEGF treatment, the cells were serum starved for 12 h andthen treated with 100 ng/ml EGF and 10 mg/ml cycloheximidein Dulbecco’s modified Eagle’s medium for required time.Full-length Tom1L1 complementary DNA was cloned inpcDNA3-FLAG vector after PCR amplification usingprimers 50-AGAATTCGCGTTTGGCAAGAGTCACC-30

and 50-ATGTGCGGCCGCGTGGTAGTGAGCTGATCATC-30. GAPex-5 constructs were kindly provided by Dr AlanSaltiel (University of Michigan, Ann Arbor, MI, USA) andVav2–GFP plasmids were gift from Dr Laszlo Buday(Semmelweis University, Budapest, Hungary). XtremeGENEtransfection reagent from Roche was used for siRNAtransfection as per manufacture’s suggestions. siRNAVav2 (50-AGUCCGGUCCAUAGUCAACDTDT-30), RhoA(50-GAACUAUGUGGCAGAUAUCUUDTDT-30), Tom1L1(50-CATGTGTGTGCAGAACTGTGGTCDTDT-30) andGapvd1 (50-AAGAAUCGAUUACCUAUAGCADTDT-30)were obtained from (Eurogentec, Seraing, Belgium).A scrambled siRNA was used as control.Myc-tagged full-length Vav2 expression plasmid has been

described earlier (Thalappilly et al., 2008). The Delta-GEFmutant of Vav2 was created using PCR-based mutagenesis.Briefly, full-length Vav2 expression plasmid was used as thetemplate for PCR with complementary primer pairs thatwhose sequence corresponds to the flanking regions of part tobe deleted (50-GGAGGTGCAGCAGCCCATGAAACCA-GACAAAGCCAATGCCAACCAC-30). The original tem-plate plasmids were then removed by Dpn1 digestion beforetransformation into Escherichia coli for amplification. Thedeletion of the resultant plasmid was confirmed by sequencing.

ImmunofluorescenceThe cells grown on coverslips were fixed with 4% paraformal-dehyde in phosphate-buffered saline (PBS) for 15min. Cellswere the permeabilized with 0.1% Triton X-100 in PBS for5min and blocked with 2% bovine serum albumin in PBS for1 h. They were incubated with primary antibodies for 1 h,followed by Chromio 640- (Active Motif, Rixensart, Belgium),Alexa-Fluor 594-, or 488-goat anti-mouse or rabbit secondaryantibodies (Invitrogen) for 1 h at room temperature. Afterwashing the coverslips were mounted on glass slides usingProLong Gold mounting medium (Invitrogen).

Immunoprecipitation and western blotHEK293T cells were transfected using Lipofectamine 2000 assuggested by the manufacturer. Twenty-four hours after trans-fection, the cells were lysed in ice-cold 1% Triton X-100 buffer(pH 7.5) containing protease inhibitors. The lysates were clearedby centrifugation at 13 000 r.p.m for 15min at 4 1C. Immuno-precipitations using the cleared cell lysates were performed at4 1C for 2h with appropriate antibody. Immune-complexes wereprecipitated with protein A or G-Sepharose (Zymed Labora-tories, San Francisco, CA, USA) for an additional 1 h andwashed three times with lysis buffer. They were then resuspendedin Laemmli sample buffer and boiled for 5min. The proteins

were then resolved on sodium dodecyl sulfate–polyacrylamidegel electrophoresis and transferred to nirocellulose filters.The filters were blocked for 1 h at 47 1C in 5% nonfat milk inTBS (50mM Tris, 150mM NaCl) containing 0.1% Tween-20(Sigma). They were then incubated for 2 h with primaryantibodies in blocking solution. After extensive washes in TBS0.1% Tween-20, the filters were incubated for 1 hwith horseradish peroxidase-conjugated secondary antibody(Serotech, Dusseldorf, Germany) diluted 1–5000 in TBS 5%nonfat milk solution. After final washes with TBS-Tween,western blots were developed using the ECL kit fromAmersham Biosciences (Saclay, France). The bands obtainedfrom three independent experiments were quantified usingImageJ software and normalized with respect to the controlbands. These values were plotted using SigmaPlot (Systat,San Jose, CA, USA). Statistical analysis of the values werecarried out using t-tests (Po0.05) for two samples or one-wayanalysis of variance (Po0.05) for more than two samples.

EGFR internalization assayVav2 and pCDNA3 (empty vector as control) were transfectedin HeLA cells. The cells were serum starved for 12 h andtreated with EGF-containing Dulbecco’s modified Eagle’smedium as indicated and then washed three times with coldPBS and incubated with 0.5mg/ml biotin-X-NHS dissolvedin a borate buffer (10mM boric acid, 150mM NaCl, pH 8.0)for 1 h at 4 1C. The reaction was then quenched using ice-cold15mM glycine-containing PBS and cells were washed threetimes with cold PBS. They were then lysed and EGFRimmunoprecipitated using anti-EGFR antibody. Proteinbiotinylation was detected by western blotting using horse-radish peroxidase-conjugated streptavidin (Calbiochem).

Abbreviations

EGF, epidermal growth factor; EGFR, epidermal growthfactor receptor; PDGFR, platelet-derived growth factorreceptor; MVB, multivesicular bodies; ESCRT, endosomalsorting complex required for transport; GFP, green fluorescentprotein; CXM, cycloheximide.

Conflict of interest

The authors declare no conflict of interest.

Acknowledgements

We gratefully acknowledge M Seux for help throughoutthe work and P Spotto for technical help. Dr Alan Saltiel(University of Michigan, Ann Arbor, MI, USA) kindlyprovided Gapvd1 constructs and Dr Laszlo Buday (SemmelweisUniversity, Budapest, Hungary) Vav2–GFP plasmids. This workwas supported in part by INSERM and grants from the LigueContre le Cancer. ST was supported by a postdoctoral fellowshipfrom ARC (Association pour la Recherche sur le Cancer).

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