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Cancer-associated mutations in the protrusion-targeting region of p190RhoGAP impact tumor cell migration

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ABSTRACT

p190RhoGAP (p190A) is a negative regulator of RhoA and localizes to membrane protrusions, where its GAP activity is required for directional migration. Here, Binamé et al. identify the protrusion-localization sequence in p190A and show that cancer-associated mutations in this region affect p190A localization and function as well as tumor cell migration.

No MeSH data available.


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Regulation of p190A GAP activity. (A) Huh7 cells expressing p190AWT or p190AΔPLS were plated for 3 h on fibronectin and stained for HA (green), F-actin (red, phalloidin), and nuclei (blue, Hoechst). # indicates a cell without stress fibers. (B) Huh7 cells transfected with p190AWT or p190AΔPLS were used for a RhoA GTPase pulldown assay using GST-Rhotekin. RhoA is detected using an anti-RhoA antibody. Quantification of RhoA activity is represented as a histogram. (C) Huh7 cells were transfected with p190AWT or p190AΔPLS. Affinity purification of Huh7 cell lysate was performed with GST-RhoAQ63L and analyzed by immunoblot using anti-HA antibodies. Quantification of the affinity between p190A and active RhoA is represented as a histogram. (D) Huh7 cells were transfected or not (NT) with mcherry-cortactin. Affinity purification on Huh7 cell lysate was performed with GST-RhoAQ63L and analyzed with anti-p190A antibodies. Quantification of the affinity between endogenous p190A and active RhoA is represented as a histogram. On graphs, values were calculated by measuring the band intensity of pulldown/input and are represented as the mean ± SEM of three independent experiments. P-values from the ANOVA (B) or the unpaired t test (C and D) are indicated. *, P < 0.05; **, P < 0.01.
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fig5: Regulation of p190A GAP activity. (A) Huh7 cells expressing p190AWT or p190AΔPLS were plated for 3 h on fibronectin and stained for HA (green), F-actin (red, phalloidin), and nuclei (blue, Hoechst). # indicates a cell without stress fibers. (B) Huh7 cells transfected with p190AWT or p190AΔPLS were used for a RhoA GTPase pulldown assay using GST-Rhotekin. RhoA is detected using an anti-RhoA antibody. Quantification of RhoA activity is represented as a histogram. (C) Huh7 cells were transfected with p190AWT or p190AΔPLS. Affinity purification of Huh7 cell lysate was performed with GST-RhoAQ63L and analyzed by immunoblot using anti-HA antibodies. Quantification of the affinity between p190A and active RhoA is represented as a histogram. (D) Huh7 cells were transfected or not (NT) with mcherry-cortactin. Affinity purification on Huh7 cell lysate was performed with GST-RhoAQ63L and analyzed with anti-p190A antibodies. Quantification of the affinity between endogenous p190A and active RhoA is represented as a histogram. On graphs, values were calculated by measuring the band intensity of pulldown/input and are represented as the mean ± SEM of three independent experiments. P-values from the ANOVA (B) or the unpaired t test (C and D) are indicated. *, P < 0.05; **, P < 0.01.

Mentions: To functionally characterize the PLS domain, we analyzed the impact of its deletion on the RhoGAP activity of p190A. Because RhoA activity controls stress fibers and focal adhesion maturation and disassembly (Binamé et al., 2010), we analyzed the effect of p190A constructs expression on actin cytoskeleton (Fig. 5 A). 3 h after plating on a highly adhesive substrate of fibronectin, untransfected cells displayed many stress fibers, cells expressing HA-p190AWT less fibers, and cells expressing HA-p190AΔPLS no fibers at all (Figs. 5 A and 6 D for quantification), suggesting that the expression of this latter construct resulted in a higher RhoGAP activity in cells. We thus analyzed RhoA activation using a GST-Rhotekin pulldown assay in p190AWT- or p190AΔPLS-transfected cells (Fig. 5 B). The expression of both constructs resulted in the inhibition of RhoA, with a more important effect observed for the p190AΔPLS mutant, arguing for an enhanced GAP activity in cells. Hence, deletion of the PLS increases the protein capacity to inhibit cellular RhoA and remodel actin cytoskeleton. The strong effect of ΔPLS may result from increased intrinsic GAP catalytic activity toward RhoA. To analyze the level of activation of p190AWT and -ΔPLS, we performed an affinity precipitation assay with the activated form of RhoA (RhoAQ63L) that binds with high affinity to activated GAPs and effectors in the cell (García-Mata et al., 2006). Recombinant GST-RhoAQ63L was incubated with protein extracts of Huh7 cells expressing either HA-p190AWT or HA-p190AΔPLS. Interestingly, p190AΔPLS exhibited a higher affinity for active RhoA than p190AWT (Fig. 5 C), indicating that the GAP domain is more inclined to interact with active RhoA in the ΔPLS version of the protein. As cortactin is a partner of the PLS responsible for p190A localization, we then tested whether cortactin interaction affects the binding capacity of p190A to active RhoA. Surprisingly, we found that overexpression of cortactin decreases the ability of p190A to bind active RhoA (Fig. 5 D). Overall, our results show that besides its role in targeting p190A to cell protrusions, the PLS domain is important for the regulation of p190A interaction with active RhoA. Moreover, these data suggest that the PLS is committed in an autoinhibited conformation of p190A that is relieved upon PLS deletion and promoted by cortactin interaction.


Cancer-associated mutations in the protrusion-targeting region of p190RhoGAP impact tumor cell migration
Regulation of p190A GAP activity. (A) Huh7 cells expressing p190AWT or p190AΔPLS were plated for 3 h on fibronectin and stained for HA (green), F-actin (red, phalloidin), and nuclei (blue, Hoechst). # indicates a cell without stress fibers. (B) Huh7 cells transfected with p190AWT or p190AΔPLS were used for a RhoA GTPase pulldown assay using GST-Rhotekin. RhoA is detected using an anti-RhoA antibody. Quantification of RhoA activity is represented as a histogram. (C) Huh7 cells were transfected with p190AWT or p190AΔPLS. Affinity purification of Huh7 cell lysate was performed with GST-RhoAQ63L and analyzed by immunoblot using anti-HA antibodies. Quantification of the affinity between p190A and active RhoA is represented as a histogram. (D) Huh7 cells were transfected or not (NT) with mcherry-cortactin. Affinity purification on Huh7 cell lysate was performed with GST-RhoAQ63L and analyzed with anti-p190A antibodies. Quantification of the affinity between endogenous p190A and active RhoA is represented as a histogram. On graphs, values were calculated by measuring the band intensity of pulldown/input and are represented as the mean ± SEM of three independent experiments. P-values from the ANOVA (B) or the unpaired t test (C and D) are indicated. *, P < 0.05; **, P < 0.01.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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fig5: Regulation of p190A GAP activity. (A) Huh7 cells expressing p190AWT or p190AΔPLS were plated for 3 h on fibronectin and stained for HA (green), F-actin (red, phalloidin), and nuclei (blue, Hoechst). # indicates a cell without stress fibers. (B) Huh7 cells transfected with p190AWT or p190AΔPLS were used for a RhoA GTPase pulldown assay using GST-Rhotekin. RhoA is detected using an anti-RhoA antibody. Quantification of RhoA activity is represented as a histogram. (C) Huh7 cells were transfected with p190AWT or p190AΔPLS. Affinity purification of Huh7 cell lysate was performed with GST-RhoAQ63L and analyzed by immunoblot using anti-HA antibodies. Quantification of the affinity between p190A and active RhoA is represented as a histogram. (D) Huh7 cells were transfected or not (NT) with mcherry-cortactin. Affinity purification on Huh7 cell lysate was performed with GST-RhoAQ63L and analyzed with anti-p190A antibodies. Quantification of the affinity between endogenous p190A and active RhoA is represented as a histogram. On graphs, values were calculated by measuring the band intensity of pulldown/input and are represented as the mean ± SEM of three independent experiments. P-values from the ANOVA (B) or the unpaired t test (C and D) are indicated. *, P < 0.05; **, P < 0.01.
Mentions: To functionally characterize the PLS domain, we analyzed the impact of its deletion on the RhoGAP activity of p190A. Because RhoA activity controls stress fibers and focal adhesion maturation and disassembly (Binamé et al., 2010), we analyzed the effect of p190A constructs expression on actin cytoskeleton (Fig. 5 A). 3 h after plating on a highly adhesive substrate of fibronectin, untransfected cells displayed many stress fibers, cells expressing HA-p190AWT less fibers, and cells expressing HA-p190AΔPLS no fibers at all (Figs. 5 A and 6 D for quantification), suggesting that the expression of this latter construct resulted in a higher RhoGAP activity in cells. We thus analyzed RhoA activation using a GST-Rhotekin pulldown assay in p190AWT- or p190AΔPLS-transfected cells (Fig. 5 B). The expression of both constructs resulted in the inhibition of RhoA, with a more important effect observed for the p190AΔPLS mutant, arguing for an enhanced GAP activity in cells. Hence, deletion of the PLS increases the protein capacity to inhibit cellular RhoA and remodel actin cytoskeleton. The strong effect of ΔPLS may result from increased intrinsic GAP catalytic activity toward RhoA. To analyze the level of activation of p190AWT and -ΔPLS, we performed an affinity precipitation assay with the activated form of RhoA (RhoAQ63L) that binds with high affinity to activated GAPs and effectors in the cell (García-Mata et al., 2006). Recombinant GST-RhoAQ63L was incubated with protein extracts of Huh7 cells expressing either HA-p190AWT or HA-p190AΔPLS. Interestingly, p190AΔPLS exhibited a higher affinity for active RhoA than p190AWT (Fig. 5 C), indicating that the GAP domain is more inclined to interact with active RhoA in the ΔPLS version of the protein. As cortactin is a partner of the PLS responsible for p190A localization, we then tested whether cortactin interaction affects the binding capacity of p190A to active RhoA. Surprisingly, we found that overexpression of cortactin decreases the ability of p190A to bind active RhoA (Fig. 5 D). Overall, our results show that besides its role in targeting p190A to cell protrusions, the PLS domain is important for the regulation of p190A interaction with active RhoA. Moreover, these data suggest that the PLS is committed in an autoinhibited conformation of p190A that is relieved upon PLS deletion and promoted by cortactin interaction.

View Article: PubMed Central - HTML - PubMed

ABSTRACT

p190RhoGAP (p190A) is a negative regulator of RhoA and localizes to membrane protrusions, where its GAP activity is required for directional migration. Here, Binam&eacute; et al. identify the protrusion-localization sequence in p190A and show that cancer-associated mutations in this region affect p190A localization and function as well as tumor cell migration.

No MeSH data available.


Related in: MedlinePlus