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. 2021 Jan 13;295(49):16897–16904. doi: 10.1074/jbc.AC120.014698

Figure 2.

Figure 2

Hotspot mutations in Gα13 Arg-200 cause constitutive G-protein activation and lead to enhanced YAP/TAZ-dependent and MRTF-A/B-dependent transcription.A, top, lollipop plot of Gα13 residues mutated in bladder cancer. Bottom, alignment of Gα switch I region showing in red the fully conserved arginine that corresponds to Gα13 Arg-200. B, diagram of a G12/13 signaling cascade culminating in the activation of transcriptional regulators and specific luciferase-based reporters used to measure their activity. Manipulations implemented in other panels of this figure to inhibit specific steps of the pathway are indicated in red. C, Gα13 Arg-200 mutants activate YAP/TAZ-dependent transcription. HEK293T cells were transfected with plasmids for the expression of the indicated Gα13 constructs and TEAD reporter (8xGTIIC) activity determined as described under “Experimental procedures.” Results are mean ± S. E. (error bars), n = 4. *, p < 0.05; **, p < 0.01, analysis of variance with Tukey post hoc test. D and G, YAP/TAZ depletion abolishes TEAD reporter (D) but not SRE.L reporter (G) activation caused by Gα13 Arg-200 mutants. HEK293T cells were transfected with plasmids for the expression of the indicated Gα13 constructs and with the indicated siRNAs, and TEAD reporter or SRE.L reporter activity was determined as described under “Experimental procedures.” Results are mean ± S. E., n = 3. *, p < 0.05; **, p < 0.01; ***, p < 0.001, n.s., not significant, Student's t test. E and H, Rho GTPase blockade abolishes TEAD reporter (E) and SRE.L reporter (H) activation caused by Gα13 Arg-200 mutants. HEK293T cells were transfected with plasmids for the expression of the indicated Gα13 constructs with or without a plasmid for the expression of C3 toxin. TEAD reporter or SRE.L reporter activity was determined as described under “Experimental procedures.” Results are mean ± S. E., n = 3–4. *, p < 0.05; **, p < 0.01; ***, p < 0.001, Student's t test. F and I, blocking Gα13-mediated activation of RhoGEFs with a dominant-negative construct (p115RH) inhibits TEAD reporter (F) and SRE.L reporter (I) activation caused by Gα13 Arg-200 mutants. HEK293T cells were transfected with plasmids for the expression of the indicated Gα13 constructs and a plasmid for the expression of mCherry-p115RH or mCherry as negative control. TEAD reporter or SRE.L reporter activity was determined as described under “Experimental procedures.” Results are mean ± S. E., n = 3–4. *, p < 0.05; **, p < 0.01; ***, p < 0.001, Student's t test. For all panels showing reporter activation results, an immunoblot of lysates of cells used in one of the experiments is shown below the graph.