VAV2 Regulates Epidermal Growth Factor Receptor Endocytosis and Degradation

Oncogene (2010) 29, 2528–2539 & 2010 Macmillan Publishers Limited All rights reserved 0950-9232/10 $32.00 www.nature.com/onc 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 for signaling (Bustelo et al., 1994; Zeng et al., 2000; Servitja Rho GTPases that regulate cell adhesion, motility, et al., 2003; Fernandez-Zapico et al., 2005). It has been spreading and proliferation in response to growth factor reported that Vav-induced cell transformation require signalling. In this work, we show that Vav2 expression its GEF function. In addition to their function as delayed epidermal growth factor receptor (EGFR) inter- regulators of Rho proteins, Vav family proteins regulate nalization and degradation, and enhanced EGFR, ERK gene expression as components of transcription com- and Akt phosphorylations. This effect of Vav2 on EGFR plexes (Doody et al., 2000; Houlard et al., 2002). Role of degradation is dependent on its guanine nucleotide Vav proteins in lymphocyte function is well character- exchange function. Knockdown of Vav2 in HeLa cells ized in both cellular and animal models. Studies using enhanced EGFR degradation and reduced cell prolifera- mice lacking all three Vav family members showed a role tion. epidermal growth factor stimulation led to co- for these proteins in T-cell and B-cell responses and in localization of Vav2 with EGFR and Rab5 in endosomes. adaptive immune response (Swat and Fujikawa, 2005). We further show that the effect of Vav2 on EGFR Vav family proteins are conserved multi-domain stability is modulated by its interaction with two proteins containing: a Calpolin homology domain, a endosome-associated proteins and require RhoA function. Dbl homology domain, a pleckstrin homology domain, Thus, in this work, we report for the first time that Vav2 a zinc-finger domain and two SH3 domains flanking can regulate growth factors receptor signalling by slowing a SH2 domain. Regulation of Rho Family GTPases, the receptor internalization and degradation through its primary function attributed to Vav proteins, is mediated interaction with endosome-associated proteins. by their Dbl homology domain (Romero and Fischer, Oncogene (2010) 29, 2528–2539; doi:10.1038/onc.2010.1; 1996). Deletion of their N-terminal region leads to published online 8 February 2010 constitutive activation of these GTPases resulting in an enhanced oncogenicity (Abe et al., 2000; Bustelo, 2002). Keywords: VAV2; EGFR; endosome; receptor The similar domain organization and high sequence internalization similarity shared between the Vav proteins results in over- lapping functions, however, several studies have reported differences in their functions (Schuebel et al., 1996). Growth factor stimulation leads to SH2 domain- Introduction mediated binding of Vav proteins to phosphotyrosine residues on activated receptors such as epidermal Vav family proteins are guanine nucleotide exchange growth factor receptor (EGFR) and platelet-derived factors (GEF) for Rho family GTPases. They regulate growth factor receptor (Pandey et al., 2000; Tamas cytoskeletal dynamics in response to stimuli such as et al., 2003). Vav proteins phosphorylation by these growth factor receptor activation, leading to modulation receptors and Src kinase family leads to inhibition of of adhesion, motility and proliferation of both normal intramolecular interactions leading to their activation and cancer cells (Marcoux and Vuori, 2003; Patel et al., (Servitja et al., 2003; Tamas et al., 2003; Tu et al., 2003). 2007). Vav1, the best characterized of the three human Membrane translocation of Vav proteins and PH Vav family members, is expressed in hematopoietic cells domain-mediated phospholipid binding are also crucial and their derivatives whereas the other two proteins, to their activation (Tamas et al., 2001; Aoki et al., 2005). Vav2 and Vav3, show wider expression patterns Activated Vav proteins mediate the activation of Rac, (Hornstein et al., 2004). Vav proteins are potent RhoA and cdc42 in response to growth factor stimula- oncogenes that can induce cell transformation by tion and are potent regulators of cell signaling (Abe activating Rho GTPase function and modulating cell et al., 2000; Liu and Burridge, 2000). Interestingly, during pancreatic carcinogenesis, Vav-mediated activa- Correspondence: Dr NJ Dusetti, INSERM U624, Stress Cellulaire, tion of mitogen-activated protein kinase signaling is Parc Scientifique de Luminy, Case 915, Bouches du Rhones, Marseille, required for Src-induced cell transformation (Servitja F-13288, France. et al., 2003; Fernandez-Zapico et al., 2005). These E-mail: [email protected] Received 12 June 2009; revised 19 November 2009; accepted 7 December observations suggest a pivotal role for Vav proteins in 2009; published online 8 February 2010 regulation of cell growth. Vav2 regulates EGFR stability S Thalappilly et al 2529 Activated EGFR undergoes Cbl-mediated mono-ubi- interacts with EGFR and is regulated by it, we first quitination and is internalized to early endosomes (Sorkin analyzed the effect of Vav2 expression on total EGFR and Goh, 2008). The best characterized EGFR inter- level. HeLa cells were transfected with Vav2–GFP or nalization mechanism involves its translocation to cla- GFP (as control) expression plasmids. The cells were thrin-coated pits, although other mechanisms have also lysed 48 h later and total EGFR level was determined by been described (Sorkin and Goh, 2008). Once internalized, western blot. As shown in Figure 1a, Vav2 expressing the endosome-associated EGFR continues the signaling cells had more than two times higher level of EGFR leading to cell survival until it is addressed to the than control cells. This suggested that Vav2 expression multivesicular bodies and subsequently degraded in the may affect EGFR degradation in these cells. lysosome (Wang et al.,2002).Thistraffickingfromearly To further analyze the effect of Vav2 on EGFR endosomes to multivesicular bodies and lysosomes is a stability and signalling, HeLa cells were transfected with complex process mediated by different endosomal pro- Vav2 and stimulated with epidermal growth factor teins including Rab proteins and the endosomal sorting (EGF) for different spans of time. To discriminate complex required for transport complexes that regulate between protein degradation and synthesis, translation trafficking by binding to the ubiquitinated EGFR (Ceresa, was blocked by treating the cells with Cycloheximide. 2006; Williams and Urbe, 2007). This process is tightly As shown in Figure 1b, Vav2 expression delays EGFR controlled in cells as it can affect cell proliferation. For degradation significanly. Interestingly, EGFR and both example, deregulation of endosomal sorting complex ERK and Akt (EGFR downstream effectors) were required for transport proteins have been implicated in phosphorylated more in Vav2 transfected cells com- cancers suggesting a tumor-suppressor role for them pared with control. This observation suggests an (Stuffers et al., 2009). Similarly, recycling of the activated increase in EGFR activation induced by Vav2 expres- receptor also leads to enhanced EGFR stability and sion. This effect of Vav2 on EGFR stability and activity signaling. Thus, deregulation of these processes that leads correlates with a significantly enhanced proliferation of to abnormal EGFR internalization, trafficking or recy- HeLa cell (Po0.05) (Figure 1c). cling can affect its activity causing enhanced cell division The best characterized role for Vav2 is its function and conferring growth advantages often observed in as guanine nucleotide exchange factor for Rho cancer cells (Mosesson et al., 2008). GTPases. To analyze whether the Vav2 GEF function Vav2 and EGFR localize to the same regions at the cell is required for the effect observed on EGFR stability, membrane and share interactions with several proteins we used a mutant Vav2 lacking the Dbl homology including Src kinases family, Ras, Cbl and Grb2. Several domain (DGEF-Vav2). As shown in Figure 1d, when of these proteins co-localize with EGFR on endosomes EGF-stimulated cells were transfected with the mutant and regulate both its signaling and trafficking (de Melker Vav2, the EGFR level was similar to the control level et al., 2001; Jiang and Sorkin, 2002; Yamazaki et al.,2002; obtained after the empty vector transfection (Po0.05). Donepudi and Resh, 2008; Sorkin and Goh, 2008). This observation suggests that the GEF activity of Vav2 However, it is not known whether Vav proteins have a is important for the EGFR stabilization. Previous role in EGFR endocytosis or trafficking. Recently, in a studies have identified the Vav2-mediated activation of proteomic study on multi-SH3 domain containing pro- different members of the Rho GTPases family (Abe teins, we identified two endosome-associated proteins et al., 2000; Liu and Burridge, 2000). To identify the role that interacts with Vav2: the Rab regulatory protein of Rho proteins in the regulation of EGFR stability Gapvd1 and the endosome-associated protein Tom1L1 downstream of Vav2, EGFR level was analyzed in HeLa (Puertollano, 2005; Bache et al., 2006; Hunker et al.,2006; cells transfected with Vav2 along with control or RhoA- Thalappilly et al., 2008). The aim of this work is to better specific small interference RNA (siRNA). As shown in understand the role of Vav2 in EGFR endocytosis and Figure 1e, RhoA knockdown led to enhanced degrad- degradation in association with the endosomal proteins ation of EGFR in Vav2 expressing cells compared (Gapvd1 and Tom1L1). with control cells. This showed that RhoA function is necessary for Vav2-mediated EGFR stability in Vav2 expressing HeLa cells. We further analyzed whether Results expression of Vav2 affected ubiquitination of EGFR after EGF stimulation. Analysis of EGFR levels in anti- Vav2 expression affects EGFR degradation and signalling ubiquitin immunoprecipites of HeLa cells transfected Although Vav family proteins have been implicated in with Vav2 expression plasmids or control plasmids did Eph receptor endocytosis and signalling, any direct not identify appreciable difference in ubiquitinated endosomal role for these proteins has not been EGFR levels (Supplementary Figure 1a). characterized (Cowan et al., 2005). We have previously reported, in a yeast two hybrid-based study, the interaction of Vav2 with Gapvd1 and Tom1L1, two Vav2 expression alters internalization and endosomal proteins that regulate endocytosis and vesicle trafficking localization of EGFR (Thalappilly et al., 2008). These proteins function in Ligand-induced receptor endocytosis leads to a punctu- ligand-induced endocytosis and ordered trafficking of ate cytoplamic localization for EGFR (endocytic transmembrane receptors to multivesicular bodies and vesicles) in contrast to the diffused membrane staining lysosomes in which they are depredated. As Vav2 also observed in unstimulated cells. To study the effect of

Oncogene Vav2 regulates EGFR stability S Thalappilly et al 2530 Vav2 on EGFR internalization and trafﬁcking, we (Figure 2a at 0 min). After 15 min of EGF stimulation, localized EGFR in the presence or not of transfected Vav2 expressing cells showed less EGFR-containing Vav2 by immunoﬂuorescence in HeLa cells. On serum endocytic vesicles than control cells (not expressing starvation, cells expressing Vav2 (Vav2–GFP) as well as Vav2 or expressing GFP). This suggested that Vav2 the control cells (GFP) showed weak staining for EGFR expression slowed down the internalization of stimu-

2.5 2.0 1.5 1.0

EGFR/Tub 0.5 Tub 0.0 P min CHX +++ + + + + +

* *

Tub *

GFP Vav2-GFP

180 Vav2-GFP *

) 150 4 GFP * 120

90 * 60 * Cell Number (x 10 30

0 123456Days

Ctrl EGF (Min) 100 CHX + + + + + + + + + 80 60 Tub 40 EGFR/Tub Ctrl 20 Va v 2 ΔGEF-Vav2 0 EGF (min) 0 30 45 0 30 45 01545 Vav2 + + + + + + Time (min) EGFR RhoA Tubulin Ctrl si RhoA si

Oncogene Vav2 regulates EGFR stability S Thalappilly et al 2531 lated EGFR. Finally, after 45 min of EGF stimulation, The Vav2 effect on EGFR stability is regulated by its we observed that Vav2 expressing cells had much interaction with endosomal proteins higher levels of EGFR localized on cytoplasmic vesicles We have recently shown that Vav2 interacts with Gapvd1 than control cells. This last observation suggested that and Tom1L1, two endosome-associated proteins (Tha- Vav2 not only delayed internalization but also EGFR lappilly et al., 2008). As Vav2 interaction with these targeting to lysosomal degradation. Similar results were proteins was identified initially by yeast two hybrid, we obtained in the human pancreatic cell line Panc1 as first confirmed these interactions in mammalian cells by well. As shown in Figure 2b, Panc-1 cells expressing co-immunoprecipitation. As shown in Figure 3a, both Vav2, showed reduced cytosolic staining for EGFR Gapvd1 and Tom1L1 specifically co-immunoprecipitated after 15 min of EGF stimulation compared with control with Vav2. These interactions suggest a role for them in cells. After 45 min of EGF stimulation, EGFR staining mediating the Vav2 effect on EGFR stability. Therefore, was observed as vesicles at the cellular periphery in Vav2 we used siRNA-mediated knockdown of Gapvd1 and expressing cells compared with more perinuclear stain- Tom1L1 to study their role in Vav2 function (Figure 3b). ing in untransfected cells. These observations suggested Interestingly, after EGF stimulation, Gapvd1 and a generic, cell-type-independent delay in EGFR inter- Tom1L1 knockdown had opposite effects on Vav2- nalization and degradation in Vav2 expressing cells. induced EGFR stabilization. Gapvd1 knockdown sig- Interestingly, we did not observe any difference in nificantly enhanced the Vav2-induced stabilization of transferrin receptor internalization between Vav2 ex- EGFR while Tom1L1 knockdown enhanced EGFR pressing or control cells treated with transferring degradation, compared with cells transfected with Vav2 suggesting that Vav2 specifically regulates growth factor and control siRNA (Figure 3c). Control experiments, receptor internalization (Supplementary Figure 1B). carried out in cells transfected only with siRNA for To confirm endosomal co-localization of EGFR and Gapvd1 or Tom1L1, do not show significant differences Vav2, HeLa cells were cotransfected with Vav2 and in EGFR stability (data not shown). This suggests that Rab5-GFP (early endosome marker) expression plas- Vav2 interaction with endosomal proteins (Gapvd1 and mids and stimulated with EGF. As shown in Figure 2c, Tom1L1) modulates its effect on EGFR stability. Vav2 partially co-localize with Rab5-GFP and EGFR in EGF-stimulated HeLa cells. The co-localization was Vav2 knockdown affects EGFR degradation confirmed by analysis using Co-localization Finder, an We have shown that forced expression of Vav2 delayed ImageJ software plugin (http://rsb.info.nih.gov/ij/). endocytosis and degradation of activated EGFR. To validate the effect of Vav2 on EGFR internaliza- To further confirm the role of Vav2 on EGFR stability, tion, we used biotinilation of cell surface proteins at we knocked down Vav2 expression in HeLa cells using different times after EGF stimulation. As shown in the a specific siRNA. Transfection of the siRNA into HeLa Figure 2d, Vav2 expressing cells had significantly higher cells efficiently blocked Vav2 protein expression as level of biotinylated EGFR after 5 and 15 min of EGF shown in Figure 4a. Vav2 knockdown in EGF-stimulated treatment (Po0.05). This result indicates an increase in HeLa cells significantly enhanced the degradation of cell surface retention of EGFR in these cells, confirming EGFR compared with control cells (Figure 4b). This our previous observation that Vav2 expression delayed data validate the EGFR stabilization effect of Vav2. EGFR internalization. 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 was determined by Western blot. Three independent experiments were carried out and western blot bands corresponding to EGFR were quantified and normalized against tubulin level (graph on the right). Vav2 transfected cells had significantly higher level of EGFR compared with control cells (P ¼ 0.004) at Po0.05 significance level. (b) HeLa cells were transfected as in (a) and 24 h later were serum starved 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 and ERK level was determined by western blot. Three independent experiments were carried out and specific western blot bands corresponding to EGFR were quantified and normalized against tubulin level (graph on the right). The EGFR level in Vav2 transfected cells was significantly higher compared with control cells (15 min, P ¼ 0.0007; 30 min , P ¼ 0.0009; 45 min, P ¼ 0.0107) at Po0.05 significance level. The points in graph that represent significant difference are marked with *. Total Vav2–GFP western blot was 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 in 35 mm 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, P ¼ 0.0483; day 4, P ¼ 0.0164; day 5, P ¼ 0.0044; day 6, P ¼ 0.0141) at Po0.05 significance level (marked by *). GFP epifluorescence was used to monitor transfection efficiency and only experiments with equivalent transfection efficiencies were used for quantifications. (d) HeLa cells were transfected with Vav2, DGEF–Vav2 or empty (control) expression plasmids and processed as in Figure 1b. Western blot was carried out to detect EGFR, tubulin and total Vav2 levels. The graph on the right side shows EGFR intensities of bands normalized against tubulin from three independent experiments. After 45 min of EGF treatment, the cells transfected with Vav2 showed significant difference in EGF levels compared with control cells (P ¼ 0.001), whereas the DGEF–Vav2 transfected cells did not show significant difference (P ¼ 0.058) at Po0.05 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 siRNA or control siRNA. Forty-eight hours after transfection, the cells were serum-starved overnight and stimulated with EGF (10 ng/ml) for indicated times. Lysates prepared from these cells were analyzed by western blotting for EGFR, RhoA and tubulin levels.

Oncogene Vav2 regulates EGFR stability S Thalappilly et al 2532 physiologicaly relevant as Vav2 knockdown in HeLa Discussion cells showed reduced proliferation (but no cell death, when analyzed by Trypan blue staining) under normal Vav proteins are well characterized as regulators of actin growth conditions (Figure 4c). Taken together these cytoskeleton, cell motility, adhesion and signaling. Their data suggest that Vav2 regulates EGFR function translocation to the cell membrane in response to through modulation of its internalization and subse- growth factor receptors activation (EGFR and plate- quent degradation. let-derived growth factor receptor) leading to activation

HeLa Cells HeLa Cells

Panc1 Cells

Co-localization

15 min

HeLa Cells

Ctrl

EGFR Tub

1.0 0.8 * * 0.6 0.4

EGFR/Tub 0.2 Ctrl Vav2 0 0 515 Time (min) HeLa Cells

Oncogene Vav2 regulates EGFR stability S Thalappilly et al 2533 of Rho GTPases have been studied (Pandey et al., 2000; et al., 2007). We have found that HeLa cells expressing Tamas et al., 2001). In this study, we show that Vav2 Vav2 showed enhanced phosphorylation of ERK and expression in HeLa cells affect internalization and Akt proteins (Figure 1b). In accordance with these degradation of EGFR. We report that this function of results, expression of Vav2-enhanced HeLa cells pro- Vav2 is modulated by two endosome-associated proliferation (Figure 1c). Regulation of EGFR degradation teins, Gapvd1 and Tom1L1, with which it interacts. can thus be a novel mechanism for Vav2-induced cell Overexpression of Vav2 in both HeLa and Panc1 cells survival and proliferation. The role of Vav2 in the caused delayed internalization and increased stability of EGFR trafficking can contribute to the complex EGFR leading to an enhanced signaling cascade for this modulation of this process because the activity of receptor. Moreover, siRNA-mediated knockdown of Vav2 itself can be regulated by EGFR (Abe et al., 2000). Vav2 affected EGFR degradation and cell growth. One of the mechanisms involved in EGFR stabiliza- EGFR endocytosis and trafficking are complex tion by Vav2 is by delaying its internalization. We processes that are regulated by several proteins asso- observed, by immunofluorescence, that the appearance ciated with activated EGFR. Hence, the fact that Vav2 of endosomal EGFR was delayed in Vav2 expressing interacts with two proteins (Gapvd1 and Tom1L1) cells compared with control cells (Figure 2a). In described as part of different endosomal complexes accordance with this, we found by cell surface protein involved in EGFR trafficking attracted our attention. biotinilation, enhanced retention of EGFR on cell Gapvd1, is an activator of the early endosome regulator surface in Vav2 expressing cells compared with control protein Rab. It regulates vesicular trafficking processes cells (Figure 2c). Interestingly, this delayed internaliza- such as endocytosis of activated transmembrane proteins tion was observed at very short times (5 min) when and exocytosis of GLUT4 vesicles (Hunker et al.,2006; recycling of receptors have not still begun. This result Lodhi et al., 2007). Recent studies have shown that it can suggests that the difference observed in the levels of regulate EGFR endocytosis and degradation indepen- biotinilated EGFR is due to an internalization delay and dently of Rab5 function (Su et al.,2007).Tom1L1has not to recycling. Furthermore, we found that Vav2 also been shown to localize on endosomes and interact expression-induced delayed degradation of EGFR with proteins of the endosomal sorting complex required (Figures 1a and b). Endosomal EGFR staining was for transport machinery (Puertollano, 2005). Interaction visible in Vav2 expressing cells even after 45 min of EGF of Vav2 with these two proteins (Figure 3) suggested that stimulation whereas control cells had reduced, peri- they have a role in the Vav2-mediated modulation of nuclear staining probably because of lysosomal degra- EGFR endocytosis, trafficking and stability. dation (Figure 2a). These data suggest that Vav2 delay We found that overexpression of Vav2 in HeLa cells the internalization as well as degradation of EGFR in delayed EGF-induced EGFR internalization and de- Vav2 expressing cells. Several studies have shown that gradation. These cells showed enhanced phosphoryla- signaling initiated from endosomal EGFR can sustain tion of EGFR as well, compared with control cells. cell survival (Wang et al., 2002). Thus effect of Vav2 on Phosphorylated EGFR induces survival and prolifera- EGFR might be important for its ability to induce cell tive signaling by activating different downstream survival and transformation. We have observed delayed signaling pathways. Akt and mitogen-activated protein EGFR internalization and degradation also in Panc-1 kinase pathways are important among them and have pancreatic cancer cells that overexpress Vav2. This result been implicated in cancer cell growth and survival (Goel suggests 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-supplemented Dulbecco’s modified Eagle’s medium (DMEM) (100 ng/ml) was added to the cells and incubated for the indicated times. The cells were then fixed and stained using anti-EGFR antibody. The coverslips were mounted and visualized by confocal microscopy. Green color indicates cells expressing Vav2–GFP or GFP (control) and red color indicates endogenous EGFR staining. Three independent experiments were carried out showing similar results. (b) Panc-1 cells were transfected with Vav2–GFP expression plasmids and grown under normal conditions for 24 h. After 24 h, the cells were serum-starved overnight and then treated with EGF (10 ng/ml) for indicated times. The cells were then fixed and immunostained for EGFR. The coveslips were mounted on slides and analyzed using confocal microscopy. Green cells indicate Vav2–GFP expression and EGFR staining is shown in red. (c) HeLa cells were plated on coverslips and transfected with Vav2 and Rab5–GFP expression plasmids. Twenty-four hours post-transfection the cells were serum starved for a period of 12 h. The cells were then incubated in EGF-supplemented DMEM (100 ng/ml) and cycloheximide (CHX, 10 mg/ml) for 30 min at 37 1C, fixed and stained with anti-Vav2 and anti-EGFR antibodies (Vav2 in red, and EGFR in blue). The cells were observed using confocal microscopy. Green color shows epifluorescence of Rab5–GFP. White colored spots on the merge image indicate the position in which all the three proteins co-localize. The co-localization of the three proteins was further confirmed using the ImageJ plugin Co-localization Finder. (d) HeLa cells were transfected with Vav2 or pCDNA3 (Ctrl) plasmids. The cells were grown under normal conditions for 24 h and then were serum starved for 12 h. They were then treated with EGF-containing DMEM (50 ng/ml) for the indicated times. Total cell surface proteins were biotinylated as described in experimental procedures. The cells were subsequently lysed and EGFR was immunoprecipitated. The immunoprecipitated proteins were resolved on sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and the level of biotinylated EGFR was detected by western blot using streptavidin-conjugated horseradish peroxidase. The western blot shown is representative of three experiments. The graph bellow represents the quantification of western blot bands corresponding to EGFR from three different experiments normalized against tubulin (Tub). EGFR levels in Vav2 transfected cells, as analyzed by t-tests, were significantly higher compared with control cells (5 min, P ¼ 0.009 and 15 min, P ¼ 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.

Oncogene Vav2 regulates EGFR stability S Thalappilly et al 2534 Co-IP 100 100 - 80 80

60 60 Anti - Anti - 40 40 Gapvd1/Tub Tom1L1/Tub Expression (%) Anti - Expression (%) 20 20 Anti - 0 0

Tub Tub - + - + Ctrl + - + - Ctrl

EGF (min) CHX +++ +++ +++

Tub

100 Ctrl 90 * Tom1L1 si Gapvd1 si 80 * 70 60 EGFR/Tub 40 50 30 03045

Time (min) 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 the indicated proteins in the co-immunoprecipitates (TCL: total cell lysate, Ctrl: control). (b) Western blots showing siRNA knockdown for Gapvd1 and Tom1L1. The western blot presented is representative of three independent experiments. The graph shows the densitometric quantification (three different experiments) for the remaining expression of Gapvd1 and Tom1L1 in percentage to the total. (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 and lysed. Western blots were carried out to determine the EGFR level. The graphs show quantified intensities (three independent experiments) of the western blot bands normalized against tubulin. Analysis using one-way analysis of variance (ANOVA) showed that all the values are significantly different at both 30 (P ¼ 0.0002) and 45 (P ¼ 0.0002) minutes at significance level Po0.05 (marked in the figure as *). Vav2 level measured by western blot were used to control transfection efficiency. Only experiments with equivalent transfection efficiencies were used for quantifications.

It is interesting to note that in this context, deregulated In this case, the effect of VAV2 on EGFR stability is Vav acts synergistically with EGFR during pancreatic abolished explaining this apparent discrepancy. carcinogenesis (Fernandez-Zapico et al., 2005). Expression Vav family proteins share a high degree of similarity of Vav2 in HeLa cells growing under normal conditions allowing us to hypothesize that they could also share (serum-containing medium) led to enhanced levels of functionalities. To verify this hypothesis, HeLa cells were EGFR compared with control cells (Figure 1a). We transfected with vectors allowing overexpresion of VAV1 suggest that this increase is due to reduced internaliza- and VAV3 proteins and were cultured in serum-contain- tion and degradation of EGFR. However, at time 0 ing medium. Similarly, to Vav2 the other members of this of EGF stimulation and under serum starvation, this family enhanced EGFR stability but in a lesser degree difference was not apparent (Figures 1b, d and e at time (data not shown). This fact can be explained by the high 0); we hypothesized that under serum starvation the degree of similarity shared among the Vav proteins and complete absence of EGF stops EGFR internalization because our overexpresion conditions used. This observa- leading to disruption of steady-state EGFR turnover. tion raised an interesting aspect given that Vav1 expres-

Oncogene Vav2 regulates EGFR stability S Thalappilly et al 2535 Ctrl

100 CHX ++++++ + + 80 Tub 60 Vav2 40

Vav2/Tub Vav2/Tub (%) expression 20 100 0 Ctrl si 80 Vav2 si * Tub 60 * _ + 40 Ctrl +_ * EGFR/Tub 20

0 0 15 30 45 Time (min)

150 Vav2 si *

) 125 Ctrl si 4 100 *

Cell Number (x 10 25

0 1 2 3 4 5 6 Days Figure 4 Vav2 knockdown affects EGFR degradation and cell growth. (a) HeLa cells were transfected with siRNA targeting Vav2 or scramble siRNA (Ctrl). In all, 24–48 h later, cells were lysed and Vav2 and tubulin levels determined by western blot. The graph represents the western blot band quantification for Vav2 relative to tubulin (tub) from three independent experiments. (b) HeLa cells were transfected with control siRNA or Vav2–siRNA and grown for 48 h. They were serum starved for 12 h and then Dulbecco’s modified Eagle’s medium (DMEM) containing 100 ng/ml EGF and 10 mg/ml cycloheximide (CHX) was added. Cells were incubated for the indicated times, lysed and EGFR level determined by western blot. Tubulin blot is shown as a control. The graph on the right side shows the normalized values (EGFR/tubulin) from three independent experiments. EGF level in Vav2 siRNA transfected cells was significantly lower compared with control cells (15 min, P ¼ 0.001; 30 min, P ¼ 0.013 and 45 min, P ¼ 0.009) at significance level Po0.05 (marked in the graph as *). (c) HeLa cells were transfected with Vav2 siRNA or scrambled siRNA (Ctrl Si). Twenty-four hours later 10 000 cells were plated in 35-mm dishes and grown for the indicated times, they were then trypsinized and counted. The experiment represents mean of independent triplicates. Vav2 siRNA transfected cells showed reduced proliferation compared with control cells. Statistical analysis revealed difference between the two populations at day 5 (P ¼ 0.024) and day 6 (P ¼ 0.011) at significance level of P ¼ 0.05. sion has been associated with pancreatic carcinogenesis Vav proteins associate with several receptor tyrosine (Fernandez-Zapico et al., 2005). kinases in addition to EGFR including platelet-derived Activated EGFR and Vav family proteins localize to growth factor, Eph, vascular endothelial growth factor the same molecular complex assembled at the cell and HGF receptors (Kodama et al., 2000; Pandey et al., membrane and they share interactions with several 2000; Seye et al., 2004; Cowan et al., 2005; Gavard and proteins such as Grb2, Src and Cbl. These proteins were Gutkind, 2006). Vav2 also associated with B-cell-specific found to be associated with EGFR complex at endo- CD19 and CD44v3 proteins (Doody et al., 2000; somes as well and regulate both its signaling and Bourguignon et al., 2001). As most of these proteins degradation. For example, Cbl interaction at cell are internalized on activation, we suggest that Vav2 acts membrane was required for efficient internalization of as one of their downstream effectors that may also EGFR and its function in the endosomes was necessary regulate their endocytosis and sorting. Sustaining this for efficient degradation of EGFR (Soubeyran et al., hypothesis, Ephrin-induced Eph receptor endocytosis 2002; Umebayashi et al., 2008). Furthermore, interac- during axon guidance has been shown to be dependent tion of Vav with dynamin 2, a protein that regulates on Vav family proteins (Cowan et al., 2005). However, EGFR internalization has also been described (Gomez we found that Vav2 expression did not alter the kinetics et al., 2005; Orth et al., 2006). of transferrin receptor internalization (Supplementary

Oncogene Vav2 regulates EGFR stability S Thalappilly et al 2536 Figure 1B). This might be due to the localization of regulate the effect of Vav2 on EGFR stability. Recent Vav proteins proximal to EGFR and its interaction studies have shown that EGFR trafficking and degrada- with proteins involved in EGFR internalization and tion requires monoubiquitination of its lysine residues. degradation. Interestingly, in our conditions, we found that Perturbation of these ubiquitination processes led to transfected Vav2 protein undergoes phosphorylation in inhibition of EGFR internalization and degradation serum-starved HeLa cells stimulated with EGF (Supple- (Clague and Urbe, 2006). Hence, we analyzed the levels mentary Figure 2). This observation suggests that Vav2 of EGFR ubiquitination in cells transfected with Vav2 or could be phosphorylated and activated by EGFR in our control plasmids. EGFR levels in anti-ubiquitin immu- model as was already described by Pandley and Tamas noprecipitates from control or Vav2 expressing cells did (Pandey et al., 2000; Tamas et al., 2003). not show significant difference (Supplementary Figure Deletion of the Dbl homology domain of Vav2 1A). This observation suggests that the effect of Vav2 on abrogated almost totally its effect on EGFR stability. EGFR is independent of its ubiquitination status. This links, at least in part, the effect of Vav2 on EGFR Recent studies have identified the presence of endoso- stability to Rho GTPases. A role for Vav2 in the mal proteins including endosomal sorting complex activation of these proteins downstream of EGFR required for transport proteins and Tom1L1 at the activation has already been described (Abe et al., 2000; midbody during cytokinesis in which they function as Liu and Burridge, 2000). Furthermore, recent findings regulators of mitotic cell division (Morita et al.,2007; have implicated several Rho GTPases in EGFR Yanagida-Ishizaki et al.,2008).Interestingly,weobserved endocytosis and trafficking. It has been described that that endogenous Vav2 protein localized to the central Rac1 and RhoA proteins are required for internalization spindles of mitotic cells at telophase stage as well as at the of EGFR, RhoD localizes to early endosomes and midbody during cytokinesis in which it co-localized with RhoB to late endosome- multivesicular bodies vesicles the Tom1L1 protein (Thalappilly and Dusetti, unpub- (Ellis and Mellor, 2000). Interestingly, expression of lished results). Rho GTPases have also been shown to constitutively active forms of these proteins interferes regulate both the mitotic and cytokinesis phases of cell with endocytosis at the respective stages of their division (Wadsworth, 2005; Narumiya and Yasuda, function. Another Rho family protein, cdc42 has also 2006). This suggests that Vav2 might have wider roles in been described as an important regulator of EGFR cellular processes than currently understood. degradation (Wu et al., 2003). When we analyzed the Finally, the role of Vav2 in EGFR degradation was role of RhoA protein on EGFR stability in cells confirmed by knockdown of its expression in HeLa cells overexpressing Vav2, we observed that delayed EGFR (Figure 4). The silencing of Vav2 reduced proliferation of degradation depends on RhoA function (Figure 1e). these cells as well. The observed effect on EGFR stability This observation explained, at least in part, the for Vav2 knockdown corresponds to the effect observed for requirement of GEF domain for Vav2-mediated EGFR the deletion of the Vav2–GEF domain. These observations stabilization. In accord with this, a role for ROCK, a suggest a specific role for Vav2 in this process. We report RhoA effector in EGFR endocytosis has already been here that Vav2 regulates EGFR signaling by delaying its described (Ung et al., 2008). Furthermore, a recent internalization and degradation through interaction with report has linked Vav2 to EGFR-induced activation of endosome-associated proteins. As both Vav2 and these RhoA in mesenchymal cells (Peng et al., 2010). endosomal proteins can associate with other growth factor It has been suggested that in addition to their role as receptors, Vav2 may be implicated in the internalization regulators of Rho GTPases, Vav proteins might also and degradation of a wide spectrum of receptors, function as adapter proteins (Bustelo, 2001). The large enhancing its oncogenic potential. size and multi-domain structure of these proteins supports this hypothesis. Interaction of Vav2 with endosomal proteins might be an example of such a Materials and methods function. Gapvd1 (Gapex-5), one of the endosomal proteins that interacted with Vav2, has been shown to Materials regulate stability of growth factor receptors. Its expres- Rabbit anti-EGFR, rabbit anti-Vav2, mouse anti-Myc, mouse sion enhanced the internalization and degradation of anti-EGFR, anti-ubiquitin and mouse anti-HA antibodies insulin receptor while specific siRNA inhibited EGFR were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Mouse anti-beta tubulin and anti-FLAG M2 antibodies degradation independently of its effect on Rab5 were from Sigma (St Louis, MO, USA). Anti-phospho-Akt (Hunker et al., 2006; Su et al., 2007). Tom1L1 associates and anti-phospho ERK antibodies were obtained from Cell with endosomes and has been shown to regulate the Signaling Technology (Beverly, MA, USA). Human recombi- activity of Src kinase as well (Puertollano, 2005; Franco nant EGF and Texas-Red-conjugated transferrin was from et al., 2006). Interaction of Vav2 with Tom1L1 and Invitrogen (Groningen, The Netherlands) and Biotin-X-NHS Gapvd1 suggested a role for these proteins in Vav2 was purchased from Calbiochem (Nottingham, UK). function. Knockdown of Gapvd1 and Tom1L1 had opposing effects on EGFR stability in Vav2 over- Cell culture and transfections expressing cells (Figure 3c). Gapvd1 knockdown en- HeLa, HEK293T and Panc-1 cells were grown in Dulbecco’s hanced the stability of EGFR in Vav2 overexpressing modified Eagle’s medium containing 10% fetal calf serum at cells while Tom1L1 siRNA abrogated the effect of Vav2 37 1Cin5%CO2 environment. HeLa cells were transfected using expression. This showed that these endosomal proteins FuGENE-HD (Roche, Indianapolis, IN, USA) and HEK293T

Oncogene Vav2 regulates EGFR stability S Thalappilly et al 2537 and Panc-1 cells were transfected using Lipofectamine 2000 were then resolved on sodium dodecyl sulfate–polyacrylamide (Invitrogen) according to manufacturer’s suggestion. Fetal calf gel electrophoresis and transferred to nirocellulose filters. serum, Dulbecco’s modified Eagle’s medium and OptiMEM The filters were blocked for 1 h at 47 1C in 5% nonfat milk in medium for cell culture were purchased from Invitrogen. For TBS (50 mM Tris, 150 mM NaCl) containing 0.1% Tween-20 EGF treatment, the cells were serum starved for 12 h and (Sigma). They were then incubated for 2 h with primary then treated with 100 ng/ml EGF and 10 mg/ml cycloheximide antibodies in blocking solution. After extensive washes in TBS in Dulbecco’s modified Eagle’s medium for required time. 0.1% Tween-20, the filters were incubated for 1 h Full-length Tom1L1 complementary DNA was cloned in with horseradish peroxidase-conjugated secondary antibody pcDNA3-FLAG vector after PCR amplification using (Serotech, Du¨sseldorf, Germany) diluted 1–5000 in TBS 5% primers 50-AGAATTCGCGTTTGGCAAGAGTCACC-30 nonfat milk solution. After final washes with TBS-Tween, and 50-ATGTGCGGCCGCGTGGTAGTGAGCTGATCA western blots were developed using the ECL kit from TC-30. GAPex-5 constructs were kindly provided by Dr Alan Amersham Biosciences (Saclay, France). The bands obtained Saltiel (University of Michigan, Ann Arbor, MI, USA) and from three independent experiments were quantified using Vav2–GFP plasmids were gift from Dr La´szlo´ Buday ImageJ software and normalized with respect to the control (Semmelweis University, Budapest, Hungary). XtremeGENE bands. These values were plotted using SigmaPlot (Systat, transfection reagent from Roche was used for siRNA San Jose, CA, USA). Statistical analysis of the values were transfection as per manufacture’s suggestions. siRNA carried out using t-tests (Po0.05) for two samples or one-way Vav2 (50-AGUCCGGUCCAUAGUCAACDTDT-30), RhoA analysis of variance (Po0.05) for more than two samples. (50-GAACUAUGUGGCAGAUAUCUUDTDT-30), Tom1L1 (50-CATGTGTGTGCAGAACTGTGGTCDTDT-30) and Gapvd1 (50-AAGAAUCGAUUACCUAUAGCADTDT-30) EGFR internalization assay were obtained from (Eurogentec, Seraing, Belgium). Vav2 and pCDNA3 (empty vector as control) were transfected A scrambled siRNA was used as control. in HeLA cells. The cells were serum starved for 12 h and Myc-tagged full-length Vav2 expression plasmid has been treated with EGF-containing Dulbecco’s modified Eagle’s described earlier (Thalappilly et al., 2008). The Delta-GEF medium as indicated and then washed three times with cold mutant of Vav2 was created using PCR-based mutagenesis. PBS and incubated with 0.5 mg/ml biotin-X-NHS dissolved Briefly, full-length Vav2 expression plasmid was used as the in a borate buffer (10 mM boric acid, 150 mM NaCl, pH 8.0) 1 template for PCR with complementary primer pairs that for 1 h at 4 C. The reaction was then quenched using ice-cold whose sequence corresponds to the flanking regions of part to 15 mM glycine-containing PBS and cells were washed three be deleted (50-GGAGGTGCAGCAGCCCATGAAACCA- times with cold PBS. They were then lysed and EGFR GACAAAGCCAATGCCAACCAC-30). The original tem- immunoprecipitated using anti-EGFR antibody. Protein plate plasmids were then removed by Dpn1 digestion before biotinylation was detected by western blotting using horse- transformation into Escherichia coli for amplification. The radish peroxidase-conjugated streptavidin (Calbiochem). deletion of the resultant plasmid was confirmed by sequencing.

Immunofluorescence Abbreviations The cells grown on coverslips were fixed with 4% paraformal- dehyde in phosphate-buffered saline (PBS) for 15 min. Cells EGF, epidermal growth factor; EGFR, epidermal growth were the permeabilized with 0.1% Triton X-100 in PBS for factor receptor; PDGFR, platelet-derived growth factor 5 min and blocked with 2% bovine serum albumin in PBS for receptor; MVB, multivesicular bodies; ESCRT, endosomal 1 h. They were incubated with primary antibodies for 1 h, sorting complex required for transport; GFP, green fluorescent followed by Chromio 640- (Active Motif, Rixensart, Belgium), protein; CXM, cycloheximide. Alexa-Fluor 594-, or 488-goat anti-mouse or rabbit secondary antibodies (Invitrogen) for 1 h at room temperature. After washing the coverslips were mounted on glass slides using Conflict of interest ProLong Gold mounting medium (Invitrogen). The authors declare no conflict of interest. Immunoprecipitation and western blot HEK293T cells were transfected using Lipofectamine 2000 as suggested by the manufacturer. Twenty-four hours after trans- Acknowledgements fection, the cells were lysed in ice-cold 1% Triton X-100 buffer (pH 7.5) containing protease inhibitors. The lysates were cleared We gratefully acknowledge M Seux for help throughout by centrifugation at 13 000 r.p.m for 15 min at 4 1C. Immuno- the work and P Spotto for technical help. Dr Alan Saltiel precipitations using the cleared cell lysates were performed at (University of Michigan, Ann Arbor, MI, USA) kindly 4 1C for 2 h with appropriate antibody. Immune-complexes were provided Gapvd1 constructs and Dr La´szlo´Buday (Semmelweis precipitated with protein A or G-Sepharose (Zymed Labora- University, Budapest, Hungary) Vav2–GFP plasmids. This work tories, San Francisco, CA, USA) for an additional 1 h and was supported in part by INSERM and grants from the Ligue washed three times with lysis buffer. They were then resuspended Contre le Cancer. ST was supported by a postdoctoral fellowship in Laemmli sample buffer and boiled for 5 min. The proteins from ARC (Association pour la Recherche sur le Cancer).

References

Abe K, Rossman KL, Liu B, Ritola KD, Chiang D, Campbell SL et al. Aoki K, Nakamura T, Fujikawa K, Matsuda M. (2005). Local (2000). Vav2 is an activator of Cdc42, Rac1, and RhoA. J Biol phosphatidylinositol 3,4,5-trisphosphate accumulation recruits Chem 275: 10141–10149. Vav2 and Vav3 to activate Rac1/Cdc42 and initiate neurite

Oncogene Vav2 regulates EGFR stability S Thalappilly et al 2538 outgrowth in nerve growth factor-stimulated PC12 cells. Mol Biol Liu BP, Burridge K. (2000). Vav2 activates Rac1, Cdc42, and RhoA Cell 16: 2207–2217. downstream from growth factor receptors but not beta1 integrins. Bache KG, Stuffers S, Malerod L, Slagsvold T, Raiborg C, Lechardeur Mol Cell Biol 20: 7160–7169. D et al. (2006). The ESCRT-III subunit hVps24 is required for Lodhi IJ, Chiang SH, Chang L, Vollenweider D, Watson RT, Inoue M degradation but not silencing of the epidermal growth factor et al. (2007). Gapex-5, a Rab31 guanine nucleotide exchange factor receptor. Mol Biol Cell 17: 2513–2523. that regulates Glut4 trafficking in adipocytes. Cell Metab 5: 59–72. Bourguignon LY, Zhu H, Zhou B, Diedrich F, Singleton PA, Marcoux N, Vuori K. (2003). EGF receptor mediates adhesion- Hung MC. (2001). Hyaluronan promotes CD44v3-Vav2 interaction dependent activation of the Rac GTPase: a role for phosphatidy- with Grb2-p185(HER2) and induces Rac1 and Ras signaling linositol 3-kinase and Vav2. Oncogene 22: 6100–6106. during ovarian tumor cell migration and growth. J Biol Chem Morita E, Sandrin V, Chung HY, Morham SG, Gygi SP, Rodesch CK 276: 48679–48692. et al. (2007). Human ESCRT and ALIX proteins interact with Bustelo XR. (2001). Vav proteins, adaptors and cell signaling. proteins of the midbody and function in cytokinesis. EMBO J 26: Oncogene 20: 6372–6381. 4215–4227. Bustelo XR. (2002). Regulation of Vav proteins by intramolecular Mosesson Y, Mills GB, Yarden Y. (2008). Derailed endocytosis: an events. Front Biosci 7: d24–d30. emerging feature of cancer. Nat Rev Cancer 8: 835–850. Bustelo XR, Suen KL, Leftheris K, Meyers CA, Barbacid M. (1994). Narumiya S, Yasuda S. (2006). Rho GTPases in animal cell mitosis. Vav cooperates with Ras to transform rodent fibroblasts but is not a Curr Opin Cell Biol 18: 199–205. Ras GDP/GTP exchange factor. Oncogene 9: 2405–2413. Orth JD, Krueger EW, Weller SG, McNiven MA. (2006). A novel Ceresa BP. (2006). Regulation of EGFR endocytic trafficking by rab endocytic mechanism of epidermal growth factor receptor seques- proteins. Histol Histopathol 21: 987–993. tration and internalization. Cancer Res 66: 3603–3610. Clague MJ, Urbe S. (2006). Endocytosis: the DUB version. Trends Cell Pandey A, Podtelejnikov AV, Blagoev B, Bustelo XR, Mann M, Biol 16: 551–559. Lodish HF. (2000). Analysis of receptor signaling pathways by mass Cowan CW, Shao YR, Sahin M, Shamah SM, Lin MZ, Greer PL et al. spectrometry: identification of vav-2 as a substrate of the epidermal (2005). Vav family GEFs link activated Ephs to endocytosis and and platelet-derived growth factor receptors. Proc Natl Acad Sci axon guidance. Neuron 46: 205–217. USA 97: 179–184. de Melker AA, van der Horst G, Calafat J, Jansen H, Borst J. (2001). Patel V, Rosenfeldt HM, Lyons R, Servitja JM, Bustelo XR, Siroff M c-Cbl ubiquitinates the EGF receptor at the plasma membrane and et al. (2007). Persistent activation of Rac1 in squamous carcinomas remains receptor associated throughout the endocytic route. J Cell of the head and neck: evidence for an EGFR/Vav2 signaling axis Sci 114: 2167–2178. involved in cell invasion. Carcinogenesis 28: 1145–1152. Donepudi M, Resh MD. (2008). c-Src trafficking and co-localization Peng F, Zhang B, Ingram AJ, Gao B, Zhang Y, Krepinsky JC. (2010). with the EGF receptor promotes EGF ligand-independent EGF Mechanical stretch-induced RhoA activation is mediated by the receptor activation and signaling. Cell Signal 20: 1359–1367. RhoGEF Vav2 in mesangial cells. Cell Signal 22: 34–40. Doody GM, Billadeau DD, Clayton E, Hutchings A, Berland R, Puertollano R. (2005). Interactions of TOM1L1 with the multi- McAdam S et al. (2000). Vav-2 controls NFAT-dependent vesicular body sorting machinery. J Biol Chem 280: 9258–9264. transcription in B- but not T-lymphocytes. EMBO J 19: 6173–6184. Romero F, Fischer S. (1996). Structure and function of vav. Cell Signal Ellis S, Mellor H. (2000). Regulation of endocytic traffic by rho family 8: 545–553. GTPases. Trends Cell Biol 10: 85–88. Schuebel KE, Bustelo XR, Nielsen DA, Song BJ, Barbacid M, Fernandez-Zapico ME, Gonzalez-Paz NC, Weiss E, Savoy DN, Goldman D et al. (1996). Isolation and characterization of murine Molina JR, Fonseca R et al. (2005). Ectopic expression of VAV1 vav2, a member of the vav family of proto-oncogenes. Oncogene 13: reveals an unexpected role in pancreatic cancer tumorigenesis. 363–371. Cancer Cell 7: 39–49. Servitja JM, Marinissen MJ, Sodhi A, Bustelo XR, Gutkind JS. (2003). Franco M, Furstoss O, Simon V, Benistant C, Hong WJ, Roche S. Rac1 function is required for Src-induced transformation. Evidence (2006). The adaptor protein Tom1L1 is a negative regulator of Src of a role for Tiam1 and Vav2 in Rac activation by Src. J Biol Chem mitogenic signaling induced by growth factors. Mol Cell Biol 26: 278: 34339–34346. 1932–1947. Seye CI, Yu N, Gonzalez FA, Erb L, Weisman GA. (2004). Gavard J, Gutkind JS. (2006). VEGF controls endothelial-cell The P2Y2 nucleotide receptor mediates vascular cell adhesion permeability by promoting the beta-arrestin-dependent endocytosis molecule-1 expression through interaction with VEGF receptor-2 of VE-cadherin. Nat Cell Biol 8: 1223–1234. (KDR/Flk-1). J Biol Chem 279: 35679–35686. Goel S, Hidalgo M, Perez-Soler R. (2007). EGFR inhibitor-mediated Sorkin A, Goh LK. (2008). Endocytosis and intracellular trafficking of apoptosis in solid tumors. J Exp Ther Oncol 6: 305–320. ErbBs. Exp Cell Res 314: 3093–3106. Gomez TS, Hamann MJ, McCarney S, Savoy DN, Lubking CM, Soubeyran P, Kowanetz K, Szymkiewicz I, Langdon WY, Dikic I. Heldebrant MP et al. (2005). Dynamin 2 regulates T cell activation (2002). Cbl-CIN85-endophilin complex mediates ligand-induced by controlling actin polymerization at the immunological synapse. downregulation of EGF receptors. Nature 416: 183–187. Nat Immunol 6: 261–270. Stuffers S, Brech A, Stenmark H. (2009). ESCRT proteins in Hornstein I, Alcover A, Katzav S. (2004). Vav proteins, masters of the physiology and disease. Exp Cell Res 315: 1619–1626. world of cytoskeleton organization. Cell Signal 16: 1–11. Su X, Kong C, Stahl PD. (2007). GAPex-5 mediates ubiquitination, Houlard M, Arudchandran R, Regnier-Ricard F, Germani A, trafficking, and degradation of epidermal growth factor receptor. Gisselbrecht S, Blank U et al. (2002). Vav1 is a component of J Biol Chem 282: 21278–21284. transcriptionally active complexes. J Exp Med 195: 1115–1127. Swat W, Fujikawa K. (2005). The Vav family: at the crossroads of Hunker CM, Galvis A, Kruk I, Giambini H, Veisaga ML, signaling pathways. Immunol Res 32: 259–265. Barbieri MA. (2006). Rab5-activating protein 6, a novel endosomal Tamas P, Solti Z, Bauer P, Illes A, Sipeki S, Bauer A et al. (2003). protein with a role in endocytosis. Biochem Biophys Res Commun Mechanism of epidermal growth factor regulation of Vav2, a guanine 340: 967–975. nucleotide exchange factor for Rac. JBiolChem278: 5163–5171. Jiang X, Sorkin A. (2002). Coordinated traffic of Grb2 and Ras during Tamas P, Solti Z, Buday L. (2001). Membrane-targeting is critical for epidermal growth factor receptor endocytosis visualized in living the phosphorylation of Vav2 by activated EGF receptor. Cell Signal cells. Mol Biol Cell 13: 1522–1535. 13: 475–481. Kodama A, Matozaki T, Fukuhara A, Kikyo M, Ichihashi M, Takai Thalappilly S, Suliman M, Gayet O, Soubeyran P, Hermant A, Lecine Y. (2000). Involvement of an SHP-2-Rho small G protein pathway P et al. (2008). Identification of multi-SH3 domain-containing in hepatocyte growth factor/scatter factor-induced cell scattering. protein interactome in pancreatic cancer: a yeast two-hybrid Mol Biol Cell 11: 2565–2575. approach. Proteomics 8: 3071–3081.

Oncogene Vav2 regulates EGFR stability S Thalappilly et al 2539 Tu S, Wu WJ, Wang J, Cerione RA. (2003). Epidermal growth factor- Williams RL, Urbe S. (2007). The emerging shape of the ESCRT dependent regulation of Cdc42 is mediated by the Src tyrosine machinery. Nat Rev Mol Cell Biol 8: 355–368. kinase. J Biol Chem 278: 49293–49300. Wu WJ, Tu S, Cerione RA. (2003). Activated Cdc42 sequesters Umebayashi K, Stenmark H, Yoshimori T. (2008). Ubc4/5 and c-Cbl c-Cbl and prevents EGF receptor degradation. Cell 114: continue to ubiquitinate EGF receptor after internalization to 715–725. facilitate polyubiquitination and degradation. Mol Biol Cell 19: Yamazaki T, Zaal K, Hailey D, Presley J, Lippincott-Schwartz J, 3454–3462. Samelson LE. (2002). Role of Grb2 in EGF-stimulated EGFR Ung CY, Li H, Ma XH, Jia J, Li BW, Low BC et al. (2008). Simulation internalization. J Cell Sci 115: 1791–1802. of the regulation of EGFR endocytosis and EGFR-ERK signaling Yanagida-Ishizaki Y, Takei T, Ishizaki R, Imakagura H, Takahashi S, by endophilin-mediated RhoA-EGFR crosstalk. FEBS Lett 582: Shin HW et al. (2008). Recruitment of Tom1L1/Srcasm to 2283–2290. endosomes and the midbody by Tsg101. Cell Struct Funct 33: Wadsworth P. (2005). Cytokinesis: Rho marks the spot. Curr Biol 15: 91–100. R871–R874. Zeng L, Sachdev P, Yan L, Chan JL, Trenkle T, McClelland M et al. Wang Y, Pennock S, Chen X, Wang Z. (2002). Endosomal signaling of (2000). Vav3 mediates receptor protein tyrosine kinase signaling, epidermal growth factor receptor stimulates signal transduction regulates GTPase activity, modulates cell morphology, and induces pathways leading to cell survival. Mol Cell Biol 22: 7279–7290. cell transformation. Mol Cell Biol 20: 9212–9224.

Supplementary Information accompanies the paper on the Oncogene website (http://www.nature.com/onc)

Oncogene