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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 influences β-catenin phosphorylation and subcellular localizationThe indicated cell lines were analyzed using: a and d. Sequential western blotting with the indicated Abs for total cell extracts (a) or after subcellular fractionation between membrane, cytoplasmic and nuclear fractions (d). b. TCF/Lef-luciferase reporter activation was evaluated in 293T cells 48h after transfection with the indicated constructs and incubation (LiCl, grey bars) or not (NT, black bars) with 30 mM LiCl. Expression was calculated relative to levels detected in control cells. Results are means ±SD of three independent experiments performed in triplicates. c. Immunofluorescence microscopy after immunostaining with Alexa 647-conjugated anti-β-catenin (upper panel) and anti-Myc tag Ab followed by Alexa 488-conjugated secondary Ab (Vav, lower panel). Fluorescence intensity of β-catenin labelling was analysed all along the membrane using the Analyze-Plot profile function (graphs in middle panel). Arrows indicate the peaks of intensity observed in the upper panel.
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Figure 6: Vav1 influences β-catenin phosphorylation and subcellular localizationThe indicated cell lines were analyzed using: a and d. Sequential western blotting with the indicated Abs for total cell extracts (a) or after subcellular fractionation between membrane, cytoplasmic and nuclear fractions (d). b. TCF/Lef-luciferase reporter activation was evaluated in 293T cells 48h after transfection with the indicated constructs and incubation (LiCl, grey bars) or not (NT, black bars) with 30 mM LiCl. Expression was calculated relative to levels detected in control cells. Results are means ±SD of three independent experiments performed in triplicates. c. Immunofluorescence microscopy after immunostaining with Alexa 647-conjugated anti-β-catenin (upper panel) and anti-Myc tag Ab followed by Alexa 488-conjugated secondary Ab (Vav, lower panel). Fluorescence intensity of β-catenin labelling was analysed all along the membrane using the Analyze-Plot profile function (graphs in middle panel). Arrows indicate the peaks of intensity observed in the upper panel.

Mentions: Since subcellular localization and phosphorylation on particular residues of β-catenin are crucial events that modulate its different functions, we further investigated whether Vav1 proteins could have an impact on these modifications. Western blot analysis with anti pS675 β-catenin, a residue phosphorylated by the JNK kinase [29, 40] confirmed increased phosphorylation in Vav1 expressing cells, and more significantly with the two oncogenic forms. Similar increases of pY654- and non phosphorylated- β-catenin were also observed for all Vav1 expressing cells (Figure 6a). A non specific increase of all phosphorylated forms in these cells was ruled out by using anti pY142-, pS552 and pY489- β-catenin that remained with constant levels in all the transfectants (data not shown).


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 influences β-catenin phosphorylation and subcellular localizationThe indicated cell lines were analyzed using: a and d. Sequential western blotting with the indicated Abs for total cell extracts (a) or after subcellular fractionation between membrane, cytoplasmic and nuclear fractions (d). b. TCF/Lef-luciferase reporter activation was evaluated in 293T cells 48h after transfection with the indicated constructs and incubation (LiCl, grey bars) or not (NT, black bars) with 30 mM LiCl. Expression was calculated relative to levels detected in control cells. Results are means ±SD of three independent experiments performed in triplicates. c. Immunofluorescence microscopy after immunostaining with Alexa 647-conjugated anti-β-catenin (upper panel) and anti-Myc tag Ab followed by Alexa 488-conjugated secondary Ab (Vav, lower panel). Fluorescence intensity of β-catenin labelling was analysed all along the membrane using the Analyze-Plot profile function (graphs in middle panel). Arrows indicate the peaks of intensity observed in the upper panel.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 6: Vav1 influences β-catenin phosphorylation and subcellular localizationThe indicated cell lines were analyzed using: a and d. Sequential western blotting with the indicated Abs for total cell extracts (a) or after subcellular fractionation between membrane, cytoplasmic and nuclear fractions (d). b. TCF/Lef-luciferase reporter activation was evaluated in 293T cells 48h after transfection with the indicated constructs and incubation (LiCl, grey bars) or not (NT, black bars) with 30 mM LiCl. Expression was calculated relative to levels detected in control cells. Results are means ±SD of three independent experiments performed in triplicates. c. Immunofluorescence microscopy after immunostaining with Alexa 647-conjugated anti-β-catenin (upper panel) and anti-Myc tag Ab followed by Alexa 488-conjugated secondary Ab (Vav, lower panel). Fluorescence intensity of β-catenin labelling was analysed all along the membrane using the Analyze-Plot profile function (graphs in middle panel). Arrows indicate the peaks of intensity observed in the upper panel.
Mentions: Since subcellular localization and phosphorylation on particular residues of β-catenin are crucial events that modulate its different functions, we further investigated whether Vav1 proteins could have an impact on these modifications. Western blot analysis with anti pS675 β-catenin, a residue phosphorylated by the JNK kinase [29, 40] confirmed increased phosphorylation in Vav1 expressing cells, and more significantly with the two oncogenic forms. Similar increases of pY654- and non phosphorylated- β-catenin were also observed for all Vav1 expressing cells (Figure 6a). A non specific increase of all phosphorylated forms in these cells was ruled out by using anti pY142-, pS552 and pY489- β-catenin that remained with constant levels in all the transfectants (data not shown).

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