Mutation of Vav1 adaptor region reveals a new oncogenic activation.
Bottom Line: We show that the oncogenicity of activated Vav1 proto-oncogene is associated with a non-degradative phosphorylation of β-catenin at residues important for its functions and its redistribution along the cell membrane in fibroblasts.In these cells, Vav1 is also involved in the modulation of β-catenin phosphorylation.Altogether, our data highlight that only a single mutation in the proto-oncogene Vav1 enhances tumorigenicity.
Affiliation: INSERM, UMR 978, Bobigny, France.
Vav family members function as remarkable scaffold proteins that exhibit both GDP/GTP exchange activity for Rho/Rac GTPases and numerous protein-protein interactions via three adaptor Src-homology domains. The exchange activity is under the unique regulation by phosphorylation of tyrosine residues hidden by intra-molecular interactions. Deletion of the autoinhibitory N-terminal region results in an oncogenic protein, onco-Vav, leading to a potent activation of Rac GTPases whereas the proto-oncogene barely leads to transformation. Substitution of conserved residues of the SH2-SH3 adaptor region in onco-Vav reverses oncogenicity. While a unique substitution D797N did not affect transformation induced by onco-Vav, we demonstrate that this single substitution leads to transformation in the Vav1 proto-oncogene highlighting the pivotal role of the adaptor region. Moreover, we identified the cell junction protein β-catenin as a new Vav1 interacting partner. We show that the oncogenicity of activated Vav1 proto-oncogene is associated with a non-degradative phosphorylation of β-catenin at residues important for its functions and its redistribution along the cell membrane in fibroblasts. In addition, a similar interaction is evidenced in epithelial lung cancer cells expressing ectopically Vav1. In these cells, Vav1 is also involved in the modulation of β-catenin phosphorylation. Altogether, our data highlight that only a single mutation in the proto-oncogene Vav1 enhances tumorigenicity.
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Mentions: The ectopic expression of Vav1 is observed in numerous non-hematopoietic cancer cells [10, 12, 13, 41]. As shown in Figure 7a, H358 and H441, two human lung carcinoma epithelial cell lines exhibit substantial amounts of Vav1 proteins compared to a lymphoid cell line (Raji). Our results obtained in fibroblasts prompted us to figure out whether Vav1 interacts with β-catenin in these cells. We confirmed the interaction between endogenous Vav1 and β-catenin in both cell lines. Analyses of Vav1 immunoprecipitates allowed also the specific detection of α-catenin and E-cadherin, two β-catenin well known partners at the cadherin-based adherens junctions (Figure 7b, . Remarkably, the two epithelial cell lines displayed pS33/37/T41β-catenin (Figure 7c). In order to confirm the impact of Vav1 expression on the stability and phosphorylation of β-catenin in these cells, we used RNA interference to reduce endogenous Vav1 expression or alternatively overexpression of wt-Vav1 or D797N. Transfection of Vav1 siRNA into H358 and H441 cells resulted in decreased levels of pS33/37/T41β-catenin as compared to control siRNA while no reduction of the overall β-catenin levels was observed (Figure 7c). This effect of Vav1 was further confirmed with stable overexpression of wt-Vav1 or D797N in H358 cells where higher levels of phosphorylation were detected in such conditions (Figure 8a). Moreover, we confirmed a differential impact of D797N compared to wt-Vav1 on pS33/37/T41-, pS675- and pY654-β-catenin intensities yet the mutant form was expressed at lower levels than wt-Vav1.