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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
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