Vav proteins are guanine nucleotide exchange factors for Rho GTPases that regulate cell adhesion, motility, spreading and proliferation in response to growth factor signalling. In this work, we show that Vav2 expression delayed epidermal growth factor receptor (EGFR) internalization and degradation, and enhanced EGFR, ERK and Akt phosphorylations. This effect of Vav2 on EGFR degradation is dependent on its guanine nucleotide exchange function. Knockdown of Vav2 in HeLa cells enhanced EGFR degradation and reduced cell proliferation. 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 EGFR stability is modulated by its interaction with two endosome-associated proteins and require RhoA function. Thus, in this work, we report for the first time that Vav2 can regulate growth factors receptor signalling by slowing receptor internalization and degradation through its interaction with endosome-associated proteins.
Vav family proteins are guanine nucleotide exchange factors (GEF) for Rho family GTPases. They regulate cytoskeletal dynamics in response to stimuli such as growth factor receptor activation, leading to modulation of adhesion, motility and proliferation of both normal and cancer cells (Marcoux and Vuori, 2003; Patel et al., 2007). Vav1, the best characterized of the three human Vav family members, is expressed in hematopoietic cells and their derivatives whereas the other two proteins, Vav2 and Vav3, show wider expression patterns (Hornstein et al., 2004). Vav proteins are potent oncogenes that can induce cell transformation by activating Rho GTPase function and modulating cell signaling (Bustelo et al., 1994; Zeng et al., 2000; Servitja et al., 2003; Fernandez-Zapico et al., 2005). It has been reported that Vav-induced cell transformation require its GEF function. In addition to their function as regulators of Rho proteins, Vav family proteins regulate gene expression as components of transcription complexes (Doody et al., 2000; Houlard et al., 2002). Role of Vav proteins in lymphocyte function is well characterized in both cellular and animal models. Studies using mice lacking all three Vav family members showed a role for these proteins in T-cell and B-cell responses and in adaptive immune response (Swat and Fujikawa, 2005).
Vav family proteins are conserved multi-domain proteins containing: a Calpolin homology domain, a Dbl homology domain, a pleckstrin homology domain, a zinc-finger domain and two SH3 domains flanking a SH2 domain. Regulation of Rho Family GTPases, the primary function attributed to Vav proteins, is mediated by their Dbl homology domain (Romero and Fischer, 1996). Deletion of their N-terminal region leads to constitutive activation of these GTPases resulting in an enhanced oncogenicity (Abe et al., 2000; Bustelo, 2002). The similar domain organization and high sequence similarity shared between the Vav proteins results in overlapping functions, however, several studies have reported differences in their functions (Schuebel et al., 1996).
Growth factor stimulation leads to SH2 domain-mediated binding of Vav proteins to phosphotyrosine residues on activated receptors such as epidermal growth factor receptor (EGFR) and platelet-derived growth factor receptor (Pandey et al., 2000; Tamas et al., 2003). Vav proteins phosphorylation by these receptors and Src kinase family leads to inhibition of intramolecular interactions leading to their activation (Servitja et al., 2003; Tamas et al., 2003; Tu et al., 2003). Membrane translocation of Vav proteins and PH domain-mediated phospholipid binding are also crucial to 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 stimulation and are potent regulators of cell signaling (Abe et al., 2000; Liu and Burridge, 2000). Interestingly, during pancreatic carcinogenesis, Vav-mediated activation of mitogen-activated protein kinase signaling is required for Src-induced cell transformation (Servitja et al., 2003; Fernandez-Zapico et al., 2005). These observations suggest a pivotal role for Vav proteins in regulation of cell growth.
Activated EGFR undergoes Cbl-mediated mono-ubiquitination and is internalized to early endosomes (Sorkin and Goh, 2008). The best characterized EGFR internalization mechanism involves its translocation to clathrin-coated pits, although other mechanisms have also been described (Sorkin and Goh, 2008). Once internalized, the endosome-associated EGFR continues the signaling leading to cell survival until it is addressed to the multivesicular bodies and subsequently degraded in the lysosome (Wang et al., 2002). This trafficking from early endosomes to multivesicular bodies and lysosomes is a complex process mediated by different endosomal proteins including Rab proteins and the endosomal sorting complex required for transport complexes that regulate trafficking by binding to the ubiquitinated EGFR (Ceresa, 2006; Williams and Urbe, 2007). This process is tightly controlled in cells as it can affect cell proliferation. For example, deregulation of endosomal sorting complex required for transport proteins have been implicated in cancers suggesting a tumor-suppressor role for them (Stuffers et al., 2009). Similarly, recycling of the activated receptor also leads to enhanced EGFR stability and signaling. Thus, deregulation of these processes that leads to abnormal EGFR internalization, trafficking or recycling can affect its activity causing enhanced cell division and conferring growth advantages often observed in cancer cells (Mosesson et al., 2008).
Vav2 and EGFR localize to the same regions at the cell membrane and share interactions with several proteins including Src kinases family, Ras, Cbl and Grb2. Several of these proteins co-localize with EGFR on endosomes and regulate both its signaling and trafficking (de Melker et 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 a role in EGFR endocytosis or trafficking. Recently, in a proteomic study on multi-SH3 domain containing proteins, we identified two endosome-associated proteins that interacts with Vav2: the Rab regulatory protein Gapvd1 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 better understand the role of Vav2 in EGFR endocytosis and degradation in association with the endosomal proteins (Gapvd1 and Tom1L1).
Vav2 expression affects EGFR degradation and signalling
Although Vav family proteins have been implicated in Eph receptor endocytosis and signalling, any direct endosomal role for these proteins has not been 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 proteins that regulate endocytosis and vesicle trafficking (Thalappilly et al., 2008). These proteins function in ligand-induced endocytosis and ordered trafficking of transmembrane receptors to multivesicular bodies and lysosomes in which they are depredated. As Vav2 also interacts with EGFR and is regulated by it, we first analyzed the effect of Vav2 expression on total EGFR level. HeLa cells were transfected with Vav2–GFP or GFP (as control) expression plasmids. The cells were lysed 48 h later and total EGFR level was determined by western blot. As shown in Figure 1a, Vav2 expressing cells had more than two times higher level of EGFR than control cells. This suggested that Vav2 expression may affect EGFR degradation in these cells.
To further analyze the effect of Vav2 on EGFR stability and signalling, HeLa cells were transfected with Vav2 and stimulated with epidermal growth factor (EGF) for different spans of time. To discriminate between protein degradation and synthesis, translation was blocked by treating the cells with Cycloheximide. As shown in Figure 1b, Vav2 expression delays EGFR degradation significanly. Interestingly, EGFR and both ERK and Akt (EGFR downstream effectors) were phosphorylated more in Vav2 transfected cells compared with control. This observation suggests an increase in EGFR activation induced by Vav2 expression. This effect of Vav2 on EGFR stability and activity correlates with a significantly enhanced proliferation of HeLa cell (P<0.05) (Figure 1c).
The best characterized role for Vav2 is its function as guanine nucleotide exchange factor for Rho GTPases. To analyze whether the Vav2 GEF function is required for the effect observed on EGFR stability, we used a mutant Vav2 lacking the Dbl homology domain (ΔGEF-Vav2). As shown in Figure 1d, when EGF-stimulated cells were transfected with the mutant Vav2, the EGFR level was similar to the control level obtained after the empty vector transfection (P<0.05). This observation suggests that the GEF activity of Vav2 is important for the EGFR stabilization. Previous studies have identified the Vav2-mediated activation of different members of the Rho GTPases family (Abe et al., 2000; Liu and Burridge, 2000). To identify the role of Rho proteins in the regulation of EGFR stability downstream of Vav2, EGFR level was analyzed in HeLa cells transfected with Vav2 along with control or RhoA-specific small interference RNA (siRNA). As shown in Figure 1e, RhoA knockdown led to enhanced degradation of EGFR in Vav2 expressing cells compared with control cells. This showed that RhoA function is necessary for Vav2-mediated EGFR stability in Vav2 expressing HeLa cells. We further analyzed whether expression of Vav2 affected ubiquitination of EGFR after EGF stimulation. Analysis of EGFR levels in anti-ubiquitin immunoprecipites of HeLa cells transfected with Vav2 expression plasmids or control plasmids did not identify appreciable difference in ubiquitinated EGFR levels (Supplementary Figure 1a).
Vav2 expression alters internalization and endosomal localization of EGFR
Ligand-induced receptor endocytosis leads to a punctuate cytoplamic localization for EGFR (endocytic vesicles) in contrast to the diffused membrane staining observed in unstimulated cells. To study the effect of Vav2 on EGFR internalization and trafficking, we localized EGFR in the presence or not of transfected Vav2 by immunofluorescence in HeLa cells. On serum starvation, cells expressing Vav2 (Vav2–GFP) as well as the control cells (GFP) showed weak staining for EGFR (Figure 2a at 0 min). After 15 min of EGF stimulation, Vav2 expressing cells showed less EGFR-containing endocytic vesicles than control cells (not expressing Vav2 or expressing GFP). This suggested that Vav2 expression slowed down the internalization of stimulated EGFR. Finally, after 45 min of EGF stimulation, we observed that Vav2 expressing cells had much higher levels of EGFR localized on cytoplasmic vesicles than control cells. This last observation suggested that Vav2 not only delayed internalization but also EGFR targeting to lysosomal degradation. Similar results were obtained in the human pancreatic cell line Panc1 as well. As shown in Figure 2b, Panc-1 cells expressing Vav2, showed reduced cytosolic staining for EGFR after 15 min of EGF stimulation compared with control cells. After 45 min of EGF stimulation, EGFR staining was observed as vesicles at the cellular periphery in Vav2 expressing cells compared with more perinuclear staining in untransfected cells. These observations suggested a generic, cell-type-independent delay in EGFR internalization and degradation in Vav2 expressing cells. Interestingly, we did not observe any difference in transferrin receptor internalization between Vav2 expressing or control cells treated with transferring suggesting that Vav2 specifically regulates growth factor receptor internalization (Supplementary Figure 1B). To confirm endosomal co-localization of EGFR and Vav2, HeLa cells were cotransfected with Vav2 and Rab5-GFP (early endosome marker) expression plasmids and stimulated with EGF. As shown in Figure 2c, Vav2 partially co-localize with Rab5-GFP and EGFR in EGF-stimulated HeLa cells. The co-localization was confirmed by analysis using Co-localization Finder, an ImageJ software plugin (http://rsb.info.nih.gov/ij/).
To validate the effect of Vav2 on EGFR internalization, we used biotinilation of cell surface proteins at different times after EGF stimulation. As shown in the Figure 2d, Vav2 expressing cells had significantly higher level of biotinylated EGFR after 5 and 15 min of EGF treatment (P<0.05). This result indicates an increase in cell surface retention of EGFR in these cells, confirming our previous observation that Vav2 expression delayed EGFR internalization.
The Vav2 effect on EGFR stability is regulated by its interaction with endosomal proteins
We have recently shown that Vav2 interacts with Gapvd1 and Tom1L1, two endosome-associated proteins (Thalappilly et al., 2008). As Vav2 interaction with these proteins was identified initially by yeast two hybrid, we first confirmed these interactions in mammalian cells by co-immunoprecipitation. As shown in Figure 3a, both Gapvd1 and Tom1L1 specifically co-immunoprecipitated with Vav2. These interactions suggest a role for them in mediating the Vav2 effect on EGFR stability. Therefore, we used siRNA-mediated knockdown of Gapvd1 and Tom1L1 to study their role in Vav2 function (Figure 3b). Interestingly, after EGF stimulation, Gapvd1 and Tom1L1 knockdown had opposite effects on Vav2-induced EGFR stabilization. Gapvd1 knockdown significantly enhanced the Vav2-induced stabilization of EGFR while Tom1L1 knockdown enhanced EGFR degradation, compared with cells transfected with Vav2 and control siRNA (Figure 3c). Control experiments, carried out in cells transfected only with siRNA for Gapvd1 or Tom1L1, do not show significant differences in EGFR stability (data not shown). This suggests that Vav2 interaction with endosomal proteins (Gapvd1 and Tom1L1) modulates its effect on EGFR stability.
Vav2 knockdown affects EGFR degradation
We have shown that forced expression of Vav2 delayed endocytosis and degradation of activated EGFR. To further confirm the role of Vav2 on EGFR stability, we knocked down Vav2 expression in HeLa cells using a specific siRNA. Transfection of the siRNA into HeLa cells efficiently blocked Vav2 protein expression as shown in Figure 4a. Vav2 knockdown in EGF-stimulated HeLa cells significantly enhanced the degradation of EGFR compared with control cells (Figure 4b). This data validate the EGFR stabilization effect of Vav2. Importantly, we have shown that this finding was physiologicaly relevant as Vav2 knockdown in HeLa cells showed reduced proliferation (but no cell death, when analyzed by Trypan blue staining) under normal growth conditions (Figure 4c). Taken together these data suggest that Vav2 regulates EGFR function through modulation of its internalization and subsequent degradation.
Vav proteins are well characterized as regulators of actin cytoskeleton, cell motility, adhesion and signaling. Their translocation to the cell membrane in response to growth factor receptors activation (EGFR and platelet-derived growth factor receptor) leading to activation of Rho GTPases have been studied (Pandey et al., 2000; Tamas et al., 2001). In this study, we show that Vav2 expression in HeLa cells affect internalization and degradation of EGFR. We report that this function of Vav2 is modulated by two endosome-associated proteins, Gapvd1 and Tom1L1, with which it interacts. Overexpression of Vav2 in both HeLa and Panc1 cells caused delayed internalization and increased stability of EGFR leading to an enhanced signaling cascade for this receptor. Moreover, siRNA-mediated knockdown of Vav2 affected EGFR degradation and cell growth.
EGFR endocytosis and trafficking are complex processes that are regulated by several proteins associated with activated EGFR. Hence, the fact that Vav2 interacts with two proteins (Gapvd1 and Tom1L1) described as part of different endosomal complexes involved in EGFR trafficking attracted our attention. Gapvd1, is an activator of the early endosome regulator protein Rab. It regulates vesicular trafficking processes such as endocytosis of activated transmembrane proteins and exocytosis of GLUT4 vesicles (Hunker et al., 2006; Lodhi et al., 2007). Recent studies have shown that it can regulate EGFR endocytosis and degradation independently of Rab5 function (Su et al., 2007). Tom1L1 has also been shown to localize on endosomes and interact with proteins of the endosomal sorting complex required for transport machinery (Puertollano, 2005). Interaction of Vav2 with these two proteins (Figure 3) suggested that they have a role in the Vav2-mediated modulation of EGFR endocytosis, trafficking and stability.
We found that overexpression of Vav2 in HeLa cells delayed EGF-induced EGFR internalization and degradation. These cells showed enhanced phosphorylation of EGFR as well, compared with control cells. Phosphorylated EGFR induces survival and proliferative signaling by activating different downstream signaling pathways. Akt and mitogen-activated protein kinase pathways are important among them and have been implicated in cancer cell growth and survival (Goel et al., 2007). We have found that HeLa cells expressing Vav2 showed enhanced phosphorylation of ERK and Akt proteins (Figure 1b). In accordance with these results, expression of Vav2-enhanced HeLa cells proliferation (Figure 1c). Regulation of EGFR degradation can thus be a novel mechanism for Vav2-induced cell survival and proliferation. The role of Vav2 in the EGFR trafficking can contribute to the complex modulation of this process because the activity of Vav2 itself can be regulated by EGFR (Abe et al., 2000).
One of the mechanisms involved in EGFR stabilization by Vav2 is by delaying its internalization. We observed, by immunofluorescence, that the appearance of endosomal EGFR was delayed in Vav2 expressing cells compared with control cells (Figure 2a). In accordance with this, we found by cell surface protein biotinilation, enhanced retention of EGFR on cell surface in Vav2 expressing cells compared with control cells (Figure 2c). Interestingly, this delayed internalization was observed at very short times (5 min) when recycling of receptors have not still begun. This result suggests that the difference observed in the levels of biotinilated EGFR is due to an internalization delay and not to recycling. Furthermore, we found that Vav2 expression-induced delayed degradation of EGFR (Figures 1a and b). Endosomal EGFR staining was visible in Vav2 expressing cells even after 45 min of EGF stimulation whereas control cells had reduced, perinuclear staining probably because of lysosomal degradation (Figure 2a). These data suggest that Vav2 delay the internalization as well as degradation of EGFR in Vav2 expressing cells. Several studies have shown that signaling initiated from endosomal EGFR can sustain cell survival (Wang et al., 2002). Thus effect of Vav2 on EGFR might be important for its ability to induce cell survival and transformation. We have observed delayed EGFR internalization and degradation also in Panc-1 pancreatic cancer cells that overexpress Vav2. This result suggests that this Vav2 function is not cell-type dependent. It is interesting to note that in this context, deregulated Vav acts synergistically with EGFR during pancreatic carcinogenesis (Fernandez-Zapico et al., 2005). Expression of Vav2 in HeLa cells growing under normal conditions (serum-containing medium) led to enhanced levels of EGFR compared with control cells (Figure 1a). We suggest that this increase is due to reduced internalization and degradation of EGFR. However, at time 0 of EGF stimulation and under serum starvation, this difference was not apparent (Figures 1b, d and e at time 0); we hypothesized that under serum starvation the complete absence of EGF stops EGFR internalization leading to disruption of steady-state EGFR turnover. In this case, the effect of VAV2 on EGFR stability is abolished explaining this apparent discrepancy.
Vav family proteins share a high degree of similarity allowing us to hypothesize that they could also share functionalities. To verify this hypothesis, HeLa cells were transfected with vectors allowing overexpresion of VAV1 and VAV3 proteins and were cultured in serum-containing medium. Similarly, to Vav2 the other members of this family enhanced EGFR stability but in a lesser degree (data not shown). This fact can be explained by the high degree of similarity shared among the Vav proteins and because our overexpresion conditions used. This observation raised an interesting aspect given that Vav1 expression has been associated with pancreatic carcinogenesis (Fernandez-Zapico et al., 2005).
Activated EGFR and Vav family proteins localize to the same molecular complex assembled at the cell membrane and they share interactions with several proteins such as Grb2, Src and Cbl. These proteins were found to be associated with EGFR complex at endosomes as well and regulate both its signaling and degradation. For example, Cbl interaction at cell membrane was required for efficient internalization of EGFR and its function in the endosomes was necessary for efficient degradation of EGFR (Soubeyran et al., 2002; Umebayashi et al., 2008). Furthermore, interaction of Vav with dynamin 2, a protein that regulates EGFR internalization has also been described (Gomez et al., 2005; Orth et al., 2006).
Vav proteins associate with several receptor tyrosine kinases in addition to EGFR including platelet-derived growth factor, Eph, vascular endothelial growth factor and HGF receptors (Kodama et al., 2000; Pandey et al., 2000; Seye et al., 2004; Cowan et al., 2005; Gavard and Gutkind, 2006). Vav2 also associated with B-cell-specific CD19 and CD44v3 proteins (Doody et al., 2000; Bourguignon et al., 2001). As most of these proteins are internalized on activation, we suggest that Vav2 acts as one of their downstream effectors that may also regulate their endocytosis and sorting. Sustaining this hypothesis, Ephrin-induced Eph receptor endocytosis during axon guidance has been shown to be dependent on Vav family proteins (Cowan et al., 2005). However, we found that Vav2 expression did not alter the kinetics of transferrin receptor internalization (Supplementary Figure 1B). This might be due to the localization of Vav proteins proximal to EGFR and its interaction with proteins involved in EGFR internalization and degradation. Interestingly, in our conditions, we found that transfected Vav2 protein undergoes phosphorylation in serum-starved HeLa cells stimulated with EGF (Supplementary Figure 2). This observation suggests that Vav2 could be phosphorylated and activated by EGFR in our model as was already described by Pandley and Tamas (Pandey et al., 2000; Tamas et al., 2003).
Deletion of the Dbl homology domain of Vav2 abrogated almost totally its effect on EGFR stability. This links, at least in part, the effect of Vav2 on EGFR stability to Rho GTPases. A role for Vav2 in the activation of these proteins downstream of EGFR activation has already been described (Abe et al., 2000; Liu and Burridge, 2000). Furthermore, recent findings have implicated several Rho GTPases in EGFR endocytosis and trafficking. It has been described that Rac1 and RhoA proteins are required for internalization of EGFR, RhoD localizes to early endosomes and RhoB to late endosome- multivesicular bodies vesicles (Ellis and Mellor, 2000). Interestingly, expression of constitutively active forms of these proteins interferes with endocytosis at the respective stages of their function. Another Rho family protein, cdc42 has also been described as an important regulator of EGFR degradation (Wu et al., 2003). When we analyzed the role of RhoA protein on EGFR stability in cells overexpressing Vav2, we observed that delayed EGFR degradation depends on RhoA function (Figure 1e). This observation explained, at least in part, the requirement of GEF domain for Vav2-mediated EGFR stabilization. In accord with this, a role for ROCK, a RhoA effector in EGFR endocytosis has already been described (Ung et al., 2008). Furthermore, a recent report has linked Vav2 to EGFR-induced activation of RhoA in mesenchymal cells (Peng et al., 2010).
It has been suggested that in addition to their role as regulators of Rho GTPases, Vav proteins might also function as adapter proteins (Bustelo, 2001). The large size and multi-domain structure of these proteins supports this hypothesis. Interaction of Vav2 with endosomal proteins might be an example of such a function. Gapvd1 (Gapex-5), one of the endosomal proteins that interacted with Vav2, has been shown to regulate stability of growth factor receptors. Its expression enhanced the internalization and degradation of insulin receptor while specific siRNA inhibited EGFR degradation independently of its effect on Rab5 (Hunker et al., 2006; Su et al., 2007). Tom1L1 associates with endosomes and has been shown to regulate the activity of Src kinase as well (Puertollano, 2005; Franco et al., 2006). Interaction of Vav2 with Tom1L1 and Gapvd1 suggested a role for these proteins in Vav2 function. Knockdown of Gapvd1 and Tom1L1 had opposing effects on EGFR stability in Vav2 overexpressing cells (Figure 3c). Gapvd1 knockdown enhanced the stability of EGFR in Vav2 overexpressing cells while Tom1L1 siRNA abrogated the effect of Vav2 expression. This showed that these endosomal proteins regulate the effect of Vav2 on EGFR stability. Recent studies have shown that EGFR trafficking and degradation requires monoubiquitination of its lysine residues. Perturbation of these ubiquitination processes led to inhibition of EGFR internalization and degradation (Clague and Urbe, 2006). Hence, we analyzed the levels of EGFR ubiquitination in cells transfected with Vav2 or control plasmids. EGFR levels in anti-ubiquitin immunoprecipitates from control or Vav2 expressing cells did not show significant difference (Supplementary Figure 1A). This observation suggests that the effect of Vav2 on EGFR is independent of its ubiquitination status.
Recent studies have identified the presence of endosomal proteins including endosomal sorting complex required for transport proteins and Tom1L1 at the midbody during cytokinesis in which they function as regulators of mitotic cell division (Morita et al., 2007; Yanagida-Ishizaki et al., 2008). Interestingly, we observed that endogenous Vav2 protein localized to the central spindles of mitotic cells at telophase stage as well as at the midbody during cytokinesis in which it co-localized with the Tom1L1 protein (Thalappilly and Dusetti, unpublished results). Rho GTPases have also been shown to regulate both the mitotic and cytokinesis phases of cell division (Wadsworth, 2005; Narumiya and Yasuda, 2006). This suggests that Vav2 might have wider roles in cellular processes than currently understood.
Finally, the role of Vav2 in EGFR degradation was confirmed by knockdown of its expression in HeLa cells (Figure 4). The silencing of Vav2 reduced proliferation of these cells as well. The observed effect on EGFR stability for Vav2 knockdown corresponds to the effect observed for the deletion of the Vav2–GEF domain. These observations suggest a specific role for Vav2 in this process. We report here that Vav2 regulates EGFR signaling by delaying its internalization and degradation through interaction with endosome-associated proteins. As both Vav2 and these endosomal proteins can associate with other growth factor receptors, Vav2 may be implicated in the internalization and degradation of a wide spectrum of receptors, enhancing its oncogenic potential.
Materials and methods
Rabbit anti-EGFR, rabbit anti-Vav2, mouse anti-Myc, mouse anti-EGFR, anti-ubiquitin and mouse anti-HA antibodies were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Mouse anti-beta tubulin and anti-FLAG M2 antibodies were from Sigma (St Louis, MO, USA). Anti-phospho-Akt and anti-phospho ERK antibodies were obtained from Cell Signaling Technology (Beverly, MA, USA). Human recombinant EGF and Texas-Red-conjugated transferrin was from Invitrogen (Groningen, The Netherlands) and Biotin-X-NHS was purchased from Calbiochem (Nottingham, UK).
Cell culture and transfections
HeLa, HEK293T and Panc-1 cells were grown in Dulbecco's modified Eagle's medium containing 10% fetal calf serum at 37 °C in 5% CO2 environment. HeLa cells were transfected using FuGENE-HD (Roche, Indianapolis, IN, USA) and HEK293T and Panc-1 cells were transfected using Lipofectamine 2000 (Invitrogen) according to manufacturer's suggestion. Fetal calf serum, Dulbecco's modified Eagle's medium and OptiMEM medium for cell culture were purchased from Invitrogen. For EGF treatment, the cells were serum starved for 12 h and then treated with 100 ng/ml EGF and 10 μg/ml cycloheximide in Dulbecco's modified Eagle's medium for required time. Full-length Tom1L1 complementary DNA was cloned in pcDNA3-FLAG vector after PCR amplification using primers 5′-IndexTermAGAATTCGCGTTTGGCAAGAGTCACC-3′ and 5′-IndexTermATGTGCGGCCGCGTGGTAGTGAGCTGATCATC-3′. GAPex-5 constructs were kindly provided by Dr Alan Saltiel (University of Michigan, Ann Arbor, MI, USA) and Vav2–GFP plasmids were gift from Dr László Buday (Semmelweis University, Budapest, Hungary). XtremeGENE transfection reagent from Roche was used for siRNA transfection as per manufacture's suggestions. siRNA Vav2 (5′-IndexTermAGUCCGGUCCAUAGUCAACDTDT-3′), RhoA (5′-IndexTermGAACUAUGUGGCAGAUAUCUUDTDT-3′), Tom1L1 (5′-IndexTermCATGTGTGTGCAGAACTGTGGTCDTDT-3′) and Gapvd1 (5′-IndexTermAAGAAUCGAUUACCUAUAGCADTDT-3′) 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-GEF mutant of Vav2 was created using PCR-based mutagenesis. Briefly, full-length Vav2 expression plasmid was used as the template for PCR with complementary primer pairs that whose sequence corresponds to the flanking regions of part to be deleted (5′-IndexTermGGAGGTGCAGCAGCCCATGAAACCAGACAAAGCCAATGCCAACCAC-3′). The original template plasmids were then removed by Dpn1 digestion before transformation into Escherichia coli for amplification. The deletion of the resultant plasmid was confirmed by sequencing.
The cells grown on coverslips were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) for 15 min. Cells were the permeabilized with 0.1% Triton X-100 in PBS for 5 min and blocked with 2% bovine serum albumin in PBS for 1 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 secondary antibodies (Invitrogen) for 1 h at room temperature. After washing the coverslips were mounted on glass slides using ProLong Gold mounting medium (Invitrogen).
Immunoprecipitation and western blot
HEK293T cells were transfected using Lipofectamine 2000 as suggested by the manufacturer. Twenty-four hours after transfection, the cells were lysed in ice-cold 1% Triton X-100 buffer (pH 7.5) containing protease inhibitors. The lysates were cleared by centrifugation at 13 000 r.p.m for 15 min at 4 °C. Immunoprecipitations using the cleared cell lysates were performed at 4 °C for 2 h with appropriate antibody. Immune-complexes were precipitated with protein A or G-Sepharose (Zymed Laboratories, San Francisco, CA, USA) for an additional 1 h and washed three times with lysis buffer. They were then resuspended in Laemmli sample buffer and boiled for 5 min. The proteins were then resolved on sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to nirocellulose filters. The filters were blocked for 1 h at 47 °C in 5% nonfat milk in TBS (50 mM Tris, 150 mM NaCl) containing 0.1% Tween-20 (Sigma). They were then incubated for 2 h with primary antibodies in blocking solution. After extensive washes in TBS 0.1% Tween-20, the filters were incubated for 1 h with horseradish peroxidase-conjugated secondary antibody (Serotech, Düsseldorf, Germany) diluted 1–5000 in TBS 5% nonfat milk solution. After final washes with TBS-Tween, western blots were developed using the ECL kit from Amersham Biosciences (Saclay, France). The bands obtained from three independent experiments were quantified using ImageJ software and normalized with respect to the control bands. These values were plotted using SigmaPlot (Systat, San Jose, CA, USA). Statistical analysis of the values were carried out using t-tests (P<0.05) for two samples or one-way analysis of variance (P<0.05) for more than two samples.
EGFR internalization assay
Vav2 and pCDNA3 (empty vector as control) were transfected in HeLA cells. The cells were serum starved for 12 h and treated with EGF-containing Dulbecco's modified Eagle's medium as indicated and then washed three times with cold PBS and incubated with 0.5 mg/ml biotin-X-NHS dissolved in a borate buffer (10 mM boric acid, 150 mM NaCl, pH 8.0) for 1 h at 4 °C. The reaction was then quenched using ice-cold 15 mM glycine-containing PBS and cells were washed three times with cold PBS. They were then lysed and EGFR immunoprecipitated using anti-EGFR antibody. Protein biotinylation was detected by western blotting using horseradish peroxidase-conjugated streptavidin (Calbiochem).
epidermal growth factor
epidermal growth factor receptor
platelet-derived growth factor receptor
endosomal sorting complex required for transport
green fluorescent protein
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We gratefully acknowledge M Seux for help throughout the work and P Spotto for technical help. Dr Alan Saltiel (University of Michigan, Ann Arbor, MI, USA) kindly provided Gapvd1 constructs and Dr László Buday (Semmelweis University, Budapest, Hungary) Vav2–GFP plasmids. This work was supported in part by INSERM and grants from the Ligue Contre le Cancer. ST was supported by a postdoctoral fellowship from ARC (Association pour la Recherche sur le Cancer).
The authors declare no conflict of interest.
Supplementary Information accompanies the paper on the Oncogene website
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Thalappilly, S., Soubeyran, P., Iovanna, J. et al. VAV2 regulates epidermal growth factor receptor endocytosis and degradation. Oncogene 29, 2528–2539 (2010). https://doi.org/10.1038/onc.2010.1
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