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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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Identification of a domain targeting p190A to cell protrusions. (A) A region of p190A containing the two last FF motifs and the MD (2F-MD, 380–1,060 aa) was generated (see Table S3). Huh7 cells were transfected with HA-2F-MD, fixed, and stained for HA (green) and F-actin (red). Bottom panels show membrane outgrowth at higher magnification. Arrowheads show cell edge where p190A constructs and F-actin colocalize. (B) Schematic representation of N and C terminus truncations of 2F-MD. (C) Immunostaining of Huh7 cells transfected with HA-2F-MD3, fixed, and stained as in A. (D) Quantification of the number of cells presenting a localization of p190A constructs at actin-rich edges. Values are expressed as the mean ± SEM (n > 200 for each construct; three to four independent experiments). ****, P < 0.0001 when compared with p190AWT condition, by ANOVA followed by Tukey’s multiple-comparison test. (E) Huh7 cells were transfected with GFP or GFP-PLS, fixed, and stained for actin. Arrowheads point out colocalization of p190A constructs and F-actin. (F) Quantification of cells showing localization of GFP or GFP-PLS at actin-rich edges. Values are expressed as the mean ± SEM (n = 360; three independent experiments). P-value from the unpaired t test is indicated. ****, P < 0.0001. (G) Schematic representation of the p190AΔPLS protein compared with the full-length p190A protein (p190AWT). (H) Western blot analysis of Huh7 cells transiently expressing the recombinant proteins HA-p190AWT and HA-p190AΔPLS. (I) Huh7 cells were transfected with HA-p190AWT or HA-p190AΔPLS, fixed, and immunostained for HA (green) and F-actin (red). Arrowheads show actin-rich edges with construct localization; * indicates the cytoplasmic localization of p190AΔPLS. (J) Quantification of (I), indicating percentage of cells showing HA-p190AWT or HA-p190AΔPLS at actin-rich edges. Values are expressed as the mean ± SEM (n = 715; three independent experiments). P-value from the unpaired t test is indicated. ****, P < 0.0001.
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fig2: Identification of a domain targeting p190A to cell protrusions. (A) A region of p190A containing the two last FF motifs and the MD (2F-MD, 380–1,060 aa) was generated (see Table S3). Huh7 cells were transfected with HA-2F-MD, fixed, and stained for HA (green) and F-actin (red). Bottom panels show membrane outgrowth at higher magnification. Arrowheads show cell edge where p190A constructs and F-actin colocalize. (B) Schematic representation of N and C terminus truncations of 2F-MD. (C) Immunostaining of Huh7 cells transfected with HA-2F-MD3, fixed, and stained as in A. (D) Quantification of the number of cells presenting a localization of p190A constructs at actin-rich edges. Values are expressed as the mean ± SEM (n > 200 for each construct; three to four independent experiments). ****, P < 0.0001 when compared with p190AWT condition, by ANOVA followed by Tukey’s multiple-comparison test. (E) Huh7 cells were transfected with GFP or GFP-PLS, fixed, and stained for actin. Arrowheads point out colocalization of p190A constructs and F-actin. (F) Quantification of cells showing localization of GFP or GFP-PLS at actin-rich edges. Values are expressed as the mean ± SEM (n = 360; three independent experiments). P-value from the unpaired t test is indicated. ****, P < 0.0001. (G) Schematic representation of the p190AΔPLS protein compared with the full-length p190A protein (p190AWT). (H) Western blot analysis of Huh7 cells transiently expressing the recombinant proteins HA-p190AWT and HA-p190AΔPLS. (I) Huh7 cells were transfected with HA-p190AWT or HA-p190AΔPLS, fixed, and immunostained for HA (green) and F-actin (red). Arrowheads show actin-rich edges with construct localization; * indicates the cytoplasmic localization of p190AΔPLS. (J) Quantification of (I), indicating percentage of cells showing HA-p190AWT or HA-p190AΔPLS at actin-rich edges. Values are expressed as the mean ± SEM (n = 715; three independent experiments). P-value from the unpaired t test is indicated. ****, P < 0.0001.

Mentions: p190A is a large multidomain protein including an N-terminal GTP-binding segment (GBD) followed by four FF domains (protein–protein-interacting modules harboring two strictly conserved phenylalanine residues), a middle domain (MD), a p120RasGAP binding domain (p120BD), and the C-terminal RhoGAP domain (GAP). To functionally characterize p190A domains, we generated 12 HA-tagged truncated versions of p190A overlapping the different domains as described in Table S3. Each construct was then transfected into Huh7 cells, and actin remodeling and recombinant protein localization were analyzed by immunofluorescence microscopy (Fig. S1). Due to its RhoGAP activity, overexpression of HA-p190AWT in cells induces stress fiber loss (Ridley et al., 1993). The same pattern was observed for each construct containing the GAP domain (Fig. S1; p120BD-GAP, GAP, ΔGBD, and ΔGBD-2F). On the contrary, all of the constructs without this catalytic domain left the stress fibers unaffected (Fig. S1; Δp120BD-GAP, GBD-4F, 4F, 4F-MD, 2F-MD-p120BD, p120BD, 2F-MD, and 2F), confirming that the GAP domain alone is sufficient to promote actin cytoskeleton reorganization. This analysis also led to the identification of a construct composed of the two last FF domains and the MD of p190A (2F-MD) as the minimal p190A protein that colocalizes with F-actin at membrane protrusions (Fig. 2 A; Fig. S1; and Table S3). This enrichment at actin-rich edges was not observed for many other soluble HA-tagged truncated constructs, ruling out artifactual staining attributable to an increased membrane thickness in this region (Fig. S1). To determine the minimal PLS, N- and C-terminal truncations of the 2F-MD construct were performed (Fig. 2 B). A deletion of 90 aa from the C-terminal end of the 2F-MD construct did not alter its membrane targeting (2F-MD3; Fig. 2, C and D). However, further C-terminal truncation dramatically decreased its localization to actin-rich edges (Fig. 2, B and D; and Fig. S2). Furthermore, truncation from the N-terminal end (1F-MD2 and MD2) altered the specificity of the localization (Fig. S2). Indeed, the suppression of the FF domains led to the nonspecific localization of p190A mutants to all F-actin structures, including stress fibers and focal adhesions (Fig. S2). We can therefore hypothesize that the MD mediates the localization of p190A to polymerized actin, whereas the FF domains coupled to the MD specify actin-rich protrusion targeting. Hence, our results demonstrate that the 2F-MD3 construct, corresponding to aa 380–971, is the minimal domain sufficient to target p190A to membrane protrusions and is therefore p190A PLS.


Cancer-associated mutations in the protrusion-targeting region of p190RhoGAP impact tumor cell migration
Identification of a domain targeting p190A to cell protrusions. (A) A region of p190A containing the two last FF motifs and the MD (2F-MD, 380–1,060 aa) was generated (see Table S3). Huh7 cells were transfected with HA-2F-MD, fixed, and stained for HA (green) and F-actin (red). Bottom panels show membrane outgrowth at higher magnification. Arrowheads show cell edge where p190A constructs and F-actin colocalize. (B) Schematic representation of N and C terminus truncations of 2F-MD. (C) Immunostaining of Huh7 cells transfected with HA-2F-MD3, fixed, and stained as in A. (D) Quantification of the number of cells presenting a localization of p190A constructs at actin-rich edges. Values are expressed as the mean ± SEM (n > 200 for each construct; three to four independent experiments). ****, P < 0.0001 when compared with p190AWT condition, by ANOVA followed by Tukey’s multiple-comparison test. (E) Huh7 cells were transfected with GFP or GFP-PLS, fixed, and stained for actin. Arrowheads point out colocalization of p190A constructs and F-actin. (F) Quantification of cells showing localization of GFP or GFP-PLS at actin-rich edges. Values are expressed as the mean ± SEM (n = 360; three independent experiments). P-value from the unpaired t test is indicated. ****, P < 0.0001. (G) Schematic representation of the p190AΔPLS protein compared with the full-length p190A protein (p190AWT). (H) Western blot analysis of Huh7 cells transiently expressing the recombinant proteins HA-p190AWT and HA-p190AΔPLS. (I) Huh7 cells were transfected with HA-p190AWT or HA-p190AΔPLS, fixed, and immunostained for HA (green) and F-actin (red). Arrowheads show actin-rich edges with construct localization; * indicates the cytoplasmic localization of p190AΔPLS. (J) Quantification of (I), indicating percentage of cells showing HA-p190AWT or HA-p190AΔPLS at actin-rich edges. Values are expressed as the mean ± SEM (n = 715; three independent experiments). P-value from the unpaired t test is indicated. ****, P < 0.0001.
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fig2: Identification of a domain targeting p190A to cell protrusions. (A) A region of p190A containing the two last FF motifs and the MD (2F-MD, 380–1,060 aa) was generated (see Table S3). Huh7 cells were transfected with HA-2F-MD, fixed, and stained for HA (green) and F-actin (red). Bottom panels show membrane outgrowth at higher magnification. Arrowheads show cell edge where p190A constructs and F-actin colocalize. (B) Schematic representation of N and C terminus truncations of 2F-MD. (C) Immunostaining of Huh7 cells transfected with HA-2F-MD3, fixed, and stained as in A. (D) Quantification of the number of cells presenting a localization of p190A constructs at actin-rich edges. Values are expressed as the mean ± SEM (n > 200 for each construct; three to four independent experiments). ****, P < 0.0001 when compared with p190AWT condition, by ANOVA followed by Tukey’s multiple-comparison test. (E) Huh7 cells were transfected with GFP or GFP-PLS, fixed, and stained for actin. Arrowheads point out colocalization of p190A constructs and F-actin. (F) Quantification of cells showing localization of GFP or GFP-PLS at actin-rich edges. Values are expressed as the mean ± SEM (n = 360; three independent experiments). P-value from the unpaired t test is indicated. ****, P < 0.0001. (G) Schematic representation of the p190AΔPLS protein compared with the full-length p190A protein (p190AWT). (H) Western blot analysis of Huh7 cells transiently expressing the recombinant proteins HA-p190AWT and HA-p190AΔPLS. (I) Huh7 cells were transfected with HA-p190AWT or HA-p190AΔPLS, fixed, and immunostained for HA (green) and F-actin (red). Arrowheads show actin-rich edges with construct localization; * indicates the cytoplasmic localization of p190AΔPLS. (J) Quantification of (I), indicating percentage of cells showing HA-p190AWT or HA-p190AΔPLS at actin-rich edges. Values are expressed as the mean ± SEM (n = 715; three independent experiments). P-value from the unpaired t test is indicated. ****, P < 0.0001.
Mentions: p190A is a large multidomain protein including an N-terminal GTP-binding segment (GBD) followed by four FF domains (protein–protein-interacting modules harboring two strictly conserved phenylalanine residues), a middle domain (MD), a p120RasGAP binding domain (p120BD), and the C-terminal RhoGAP domain (GAP). To functionally characterize p190A domains, we generated 12 HA-tagged truncated versions of p190A overlapping the different domains as described in Table S3. Each construct was then transfected into Huh7 cells, and actin remodeling and recombinant protein localization were analyzed by immunofluorescence microscopy (Fig. S1). Due to its RhoGAP activity, overexpression of HA-p190AWT in cells induces stress fiber loss (Ridley et al., 1993). The same pattern was observed for each construct containing the GAP domain (Fig. S1; p120BD-GAP, GAP, ΔGBD, and ΔGBD-2F). On the contrary, all of the constructs without this catalytic domain left the stress fibers unaffected (Fig. S1; Δp120BD-GAP, GBD-4F, 4F, 4F-MD, 2F-MD-p120BD, p120BD, 2F-MD, and 2F), confirming that the GAP domain alone is sufficient to promote actin cytoskeleton reorganization. This analysis also led to the identification of a construct composed of the two last FF domains and the MD of p190A (2F-MD) as the minimal p190A protein that colocalizes with F-actin at membrane protrusions (Fig. 2 A; Fig. S1; and Table S3). This enrichment at actin-rich edges was not observed for many other soluble HA-tagged truncated constructs, ruling out artifactual staining attributable to an increased membrane thickness in this region (Fig. S1). To determine the minimal PLS, N- and C-terminal truncations of the 2F-MD construct were performed (Fig. 2 B). A deletion of 90 aa from the C-terminal end of the 2F-MD construct did not alter its membrane targeting (2F-MD3; Fig. 2, C and D). However, further C-terminal truncation dramatically decreased its localization to actin-rich edges (Fig. 2, B and D; and Fig. S2). Furthermore, truncation from the N-terminal end (1F-MD2 and MD2) altered the specificity of the localization (Fig. S2). Indeed, the suppression of the FF domains led to the nonspecific localization of p190A mutants to all F-actin structures, including stress fibers and focal adhesions (Fig. S2). We can therefore hypothesize that the MD mediates the localization of p190A to polymerized actin, whereas the FF domains coupled to the MD specify actin-rich protrusion targeting. Hence, our results demonstrate that the 2F-MD3 construct, corresponding to aa 380–971, is the minimal domain sufficient to target p190A to membrane protrusions and is therefore p190A PLS.

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