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 C-terminal SH3 adaptor region of Vav1 contains several conserved residues present in Src, Grb2 or Abl SH3 domains but also unique residues specific for Vav family members (Figure 1a). We analysed a selection of Vav1 proto-oncogene CSH3 mutants: D797N, A789N and G830V, for their capacity to induce foci of transformed cells when expressed in NIH3T3 cells and compared them to onco-Vav (Δ1-66). Interestingly, mutation of D797 residue activates significant transformation potential, inducing foci similar albeit to a lesser extent than Δ1-66 onco-Vav. Despite equivalent transfection efficiencies, as verified by the comparable amounts of G418-resistant clones obtained with the various constructs, none of the other mutants as well as wt-Vav1 induced transformed foci (Figure 1b, c and d). Immunoblot analysis confirmed that the transforming potential of the D797N mutant could not be ascribed to higher levels of protein expression. Two days after transfection or in established stable cell lines, D797N mutant and wt-Vav1 showed similar levels of expression and higher levels than onco-Vav that displayed the greatest transforming capacity (Figure 1e and f). Kinetic analysis of the mutated proteins in the presence of cycloheximide validated the steady state levels observed in stable cell lines showing a weaker stability for A789N or G830V when compared to D797N, onco-Vav or wt-Vav1 (Figure 1f and supplemental Figure S1). Moreover, we noticed higher levels of phosphorylated D797N and onco-Vav1 proteins compared to wt-Vav1 reflecting an increased activation of both mutants in these cell lines (Figure 1g).