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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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Rac1 and RhoA GTPases are effectors for D797N Vav1 mutant-induced transformationa. Cytoskeleton analysis. The indicated stable NIH3T3 cells lines were observed using light microscopy (Leica DMI6000, Camera MicroMAX 1300Y/HS, magnification 20x, first left panel) or were immunostained with Alexa 647-conjugated-phalloidin (right and middle panels) and anti-Myc Tag Ab followed by Alexa 488-conjugated anti-mouse Ig Ab (Vav1, second left panel). Pseudocolor images indicate the intensity gradients of cytoskeleton labelling (right panel). Bar = 12μm. Confocal images were acquired with a BD Pathway 855 Bioimaging System with a 40x magnification and analyzed by ImageJ software. A line was drawn across at least 15 cells from each different clone and linescan profiles of fluorescence intensity were obtained using the Analyze-Plot profile function. The numbers of cells presenting the indicated profiles are indicated for each cell line under the plot. b Rac1 activation. Rac1 activity was assessed in stable NIH3T3 cell lines using GST-Pak1 pull-down experiments (n=3). A representative experiment is shown (right panel). Cellular extracts were also analysed by immunoblottting for Rac1 and Vav1 expression (left panel) c. Rac1/RhoA are required for foci formation. NIH3T3 cells were transfected with Vav1 constructs altogether with empty, RacN17 or RhoN19 pRK5-myc based vectors as indicated. The number of foci was scored for each transfection. Columns represent the mean ± SD of 3 independent experiments performed in duplicate (left panel). Expression of Vav1, RacN17 and RhoN19 proteins was monitored by immunoblotting (right panel).
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Figure 3: Rac1 and RhoA GTPases are effectors for D797N Vav1 mutant-induced transformationa. Cytoskeleton analysis. The indicated stable NIH3T3 cells lines were observed using light microscopy (Leica DMI6000, Camera MicroMAX 1300Y/HS, magnification 20x, first left panel) or were immunostained with Alexa 647-conjugated-phalloidin (right and middle panels) and anti-Myc Tag Ab followed by Alexa 488-conjugated anti-mouse Ig Ab (Vav1, second left panel). Pseudocolor images indicate the intensity gradients of cytoskeleton labelling (right panel). Bar = 12μm. Confocal images were acquired with a BD Pathway 855 Bioimaging System with a 40x magnification and analyzed by ImageJ software. A line was drawn across at least 15 cells from each different clone and linescan profiles of fluorescence intensity were obtained using the Analyze-Plot profile function. The numbers of cells presenting the indicated profiles are indicated for each cell line under the plot. b Rac1 activation. Rac1 activity was assessed in stable NIH3T3 cell lines using GST-Pak1 pull-down experiments (n=3). A representative experiment is shown (right panel). Cellular extracts were also analysed by immunoblottting for Rac1 and Vav1 expression (left panel) c. Rac1/RhoA are required for foci formation. NIH3T3 cells were transfected with Vav1 constructs altogether with empty, RacN17 or RhoN19 pRK5-myc based vectors as indicated. The number of foci was scored for each transfection. Columns represent the mean ± SD of 3 independent experiments performed in duplicate (left panel). Expression of Vav1, RacN17 and RhoN19 proteins was monitored by immunoblotting (right panel).

Mentions: The transforming activity of all N-terminal deleted Vav oncoproteins has been ascribed to uncontrolled GEF activity on GTPases of the Rho family [6, 9, 20, 32]. Therefore, we investigated whether D797N-induced transformation might similarly depend on Rho GTPases activation. Cell morphology and cytoskeleton organization of onco-Vav- or D797N-transformed cells suggested activation of the GTPases with typical formation of ruffles (lamellipodia and filipodia) (Figure 3a; [33]). These cells also exhibited a large reduction of stress fibers that were abundantly present in control or wt-Vav1-expressing fibroblasts (Figure 3a, Pseudocolor and quantification of stress fibers and/or ruffles). Using pull-down experiments with a glutathione S-transferase (GST) fusion protein containing the Rac1 binding domain of p21protein-activated protein kinase 1 (PAK), we observed a significant activation of Rac1 GTPase in onco-Vav and D797N-expressing cells and a faint activation in wt-Vav1-expressing cells. These results also confirmed the differential transforming potential of onco-Vav and D797N (Figure 3b). Additionally, overexpression of the dominant-negative form of Rac1, RacN17, strongly repressed foci formation for both constructs. Comparable repression was obtained using the dominant-negative form of RhoA, RhoN19, albeit to a lesser extent that might be attributed to a weaker expression of the GTPase (Figure 3c).


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)

Rac1 and RhoA GTPases are effectors for D797N Vav1 mutant-induced transformationa. Cytoskeleton analysis. The indicated stable NIH3T3 cells lines were observed using light microscopy (Leica DMI6000, Camera MicroMAX 1300Y/HS, magnification 20x, first left panel) or were immunostained with Alexa 647-conjugated-phalloidin (right and middle panels) and anti-Myc Tag Ab followed by Alexa 488-conjugated anti-mouse Ig Ab (Vav1, second left panel). Pseudocolor images indicate the intensity gradients of cytoskeleton labelling (right panel). Bar = 12μm. Confocal images were acquired with a BD Pathway 855 Bioimaging System with a 40x magnification and analyzed by ImageJ software. A line was drawn across at least 15 cells from each different clone and linescan profiles of fluorescence intensity were obtained using the Analyze-Plot profile function. The numbers of cells presenting the indicated profiles are indicated for each cell line under the plot. b Rac1 activation. Rac1 activity was assessed in stable NIH3T3 cell lines using GST-Pak1 pull-down experiments (n=3). A representative experiment is shown (right panel). Cellular extracts were also analysed by immunoblottting for Rac1 and Vav1 expression (left panel) c. Rac1/RhoA are required for foci formation. NIH3T3 cells were transfected with Vav1 constructs altogether with empty, RacN17 or RhoN19 pRK5-myc based vectors as indicated. The number of foci was scored for each transfection. Columns represent the mean ± SD of 3 independent experiments performed in duplicate (left panel). Expression of Vav1, RacN17 and RhoN19 proteins was monitored by immunoblotting (right panel).
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Figure 3: Rac1 and RhoA GTPases are effectors for D797N Vav1 mutant-induced transformationa. Cytoskeleton analysis. The indicated stable NIH3T3 cells lines were observed using light microscopy (Leica DMI6000, Camera MicroMAX 1300Y/HS, magnification 20x, first left panel) or were immunostained with Alexa 647-conjugated-phalloidin (right and middle panels) and anti-Myc Tag Ab followed by Alexa 488-conjugated anti-mouse Ig Ab (Vav1, second left panel). Pseudocolor images indicate the intensity gradients of cytoskeleton labelling (right panel). Bar = 12μm. Confocal images were acquired with a BD Pathway 855 Bioimaging System with a 40x magnification and analyzed by ImageJ software. A line was drawn across at least 15 cells from each different clone and linescan profiles of fluorescence intensity were obtained using the Analyze-Plot profile function. The numbers of cells presenting the indicated profiles are indicated for each cell line under the plot. b Rac1 activation. Rac1 activity was assessed in stable NIH3T3 cell lines using GST-Pak1 pull-down experiments (n=3). A representative experiment is shown (right panel). Cellular extracts were also analysed by immunoblottting for Rac1 and Vav1 expression (left panel) c. Rac1/RhoA are required for foci formation. NIH3T3 cells were transfected with Vav1 constructs altogether with empty, RacN17 or RhoN19 pRK5-myc based vectors as indicated. The number of foci was scored for each transfection. Columns represent the mean ± SD of 3 independent experiments performed in duplicate (left panel). Expression of Vav1, RacN17 and RhoN19 proteins was monitored by immunoblotting (right panel).
Mentions: The transforming activity of all N-terminal deleted Vav oncoproteins has been ascribed to uncontrolled GEF activity on GTPases of the Rho family [6, 9, 20, 32]. Therefore, we investigated whether D797N-induced transformation might similarly depend on Rho GTPases activation. Cell morphology and cytoskeleton organization of onco-Vav- or D797N-transformed cells suggested activation of the GTPases with typical formation of ruffles (lamellipodia and filipodia) (Figure 3a; [33]). These cells also exhibited a large reduction of stress fibers that were abundantly present in control or wt-Vav1-expressing fibroblasts (Figure 3a, Pseudocolor and quantification of stress fibers and/or ruffles). Using pull-down experiments with a glutathione S-transferase (GST) fusion protein containing the Rac1 binding domain of p21protein-activated protein kinase 1 (PAK), we observed a significant activation of Rac1 GTPase in onco-Vav and D797N-expressing cells and a faint activation in wt-Vav1-expressing cells. These results also confirmed the differential transforming potential of onco-Vav and D797N (Figure 3b). Additionally, overexpression of the dominant-negative form of Rac1, RacN17, strongly repressed foci formation for both constructs. Comparable repression was obtained using the dominant-negative form of RhoA, RhoN19, albeit to a lesser extent that might be attributed to a weaker expression of the GTPase (Figure 3c).

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