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Mutation of Vav1 adaptor region reveals a new oncogenic activation.

Razanadrakoto L, Cormier F, Laurienté V, Dondi E, Gardano L, Katzav S, Guittat L, Varin-Blank N - Oncotarget (2015)

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.

View Article: PubMed Central - PubMed

Affiliation: INSERM, UMR 978, Bobigny, France.

ABSTRACT
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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Vav1 interacts with β-catenin in lung epithelial cancer cell linesa. Vav1 protein expression in pulmonary and lymphoid cell lines. Total cell extracts were analysed by sequential immunoblotting with the indicated Abs. b. Total cell extracts from the indicated cells were immunoprecipitated (IP) with anti-Vav1 Ab or control IgG. Immune complexes or total extracts (Input) were analysed by sequential immunoblotting with the indicated Abs. c. Η358 and Η441 cells were transfected with the indicated siRNA and proteins levels assessed 72 hours after transfection. Ratio between phospho-β-catenin and total β-catenin was calculated relative to transfection of control siRNA.
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Figure 7: Vav1 interacts with β-catenin in lung epithelial cancer cell linesa. Vav1 protein expression in pulmonary and lymphoid cell lines. Total cell extracts were analysed by sequential immunoblotting with the indicated Abs. b. Total cell extracts from the indicated cells were immunoprecipitated (IP) with anti-Vav1 Ab or control IgG. Immune complexes or total extracts (Input) were analysed by sequential immunoblotting with the indicated Abs. c. Η358 and Η441 cells were transfected with the indicated siRNA and proteins levels assessed 72 hours after transfection. Ratio between phospho-β-catenin and total β-catenin was calculated relative to transfection of control siRNA.

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, [40]. 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.


Mutation of Vav1 adaptor region reveals a new oncogenic activation.

Razanadrakoto L, Cormier F, Laurienté V, Dondi E, Gardano L, Katzav S, Guittat L, Varin-Blank N - Oncotarget (2015)

Vav1 interacts with β-catenin in lung epithelial cancer cell linesa. Vav1 protein expression in pulmonary and lymphoid cell lines. Total cell extracts were analysed by sequential immunoblotting with the indicated Abs. b. Total cell extracts from the indicated cells were immunoprecipitated (IP) with anti-Vav1 Ab or control IgG. Immune complexes or total extracts (Input) were analysed by sequential immunoblotting with the indicated Abs. c. Η358 and Η441 cells were transfected with the indicated siRNA and proteins levels assessed 72 hours after transfection. Ratio between phospho-β-catenin and total β-catenin was calculated relative to transfection of control siRNA.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4385868&req=5

Figure 7: Vav1 interacts with β-catenin in lung epithelial cancer cell linesa. Vav1 protein expression in pulmonary and lymphoid cell lines. Total cell extracts were analysed by sequential immunoblotting with the indicated Abs. b. Total cell extracts from the indicated cells were immunoprecipitated (IP) with anti-Vav1 Ab or control IgG. Immune complexes or total extracts (Input) were analysed by sequential immunoblotting with the indicated Abs. c. Η358 and Η441 cells were transfected with the indicated siRNA and proteins levels assessed 72 hours after transfection. Ratio between phospho-β-catenin and total β-catenin was calculated relative to transfection of control siRNA.
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, [40]. 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.

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.

View Article: PubMed Central - PubMed

Affiliation: INSERM, UMR 978, Bobigny, France.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus