Figure 2.
WW domains are required for YAP1 transactivation and nuclear translocation. (A) The co-IP experiment shows the interaction of MYC-tagged TEAD1 with HA-tagged YAP1 or the YAP1-W4A mutant in HEK293T cells. TCL, total cell lysates. (B) The W4A mutant loses the transcriptional activity in a TEAD-dependent luciferase reporter (representative of three independent experiments, N = 3, all data shown as the average values ± SD, two-tailed Student’s t test). The representative western blotting shows expressions of YAP1 and the mutant in reporter assays. The inserted diagram illustrates the construct of the TEAD-dependent luciferase reporter. TBE, the TEAD binding element. (C) Substitution of the disordered sequence between the WW domains by five tandem flexible linkers (Gly-Gly-Gly-Gly-Ser) does not affect YAP1 transcriptional activity in HEK293T cells (representative of three independent experiments, N = 3, all data shown as the average values ± SD, two-tailed Student’s t test). The representative western blotting shows expressions of YAP1 and the mutant. (D) tdWWs facilitate YAP1 transcriptional activity in a conformational position-independent manner (representative of three independent experiments, N = 3, all data shown as the average values ± SD, two-tailed Student’s t test). The representative western blotting shows expressions of YAP1 and the mutants. (E) The mRNA levels of CYR61 and CTGF genes in YAP1-knockout (KO) HeLa cells are rescued by the wild-type YAP1 and the tdWWs−ΔWWs chimera, but not by the ΔWWs mutant (representative of three independent experiments, N = 3, all data shown as the average values ± SD, two-tailed Student’s t test). The representative western blotting shows the expression of YAP1 and the mutants in YAP1-KO HeLa cells. (F) Representative images show the subcellular distributions of YAP1 and YAP1 mutants in YAP1-KO HeLa cells, and the scatter chart shows their nucleocytoplasmic ratios (representative of three independent experiments, n represents the total number of the measured cells, two-tailed ANOVA test). The nucleocytoplasmic (N/C) ratios of the subcellular distributions of YAP1 as well as the mutants are measured as in the inserted diagram. Briefly, three areas in the nucleus or the cytoplasm of each cell are randomly selected and then the mean fluorescent intensities in these areas are obtained by ImageJ software. The mean values of fluorescent intensities in three areas are representative of nuclear or cytoplasmic YAP1 levels, respectively. The relative subcellular distribution of YAP1 proteins in each cell is indicated by the ratio of the nuclear and the cytoplasmic YAP1 levels. To plot the scatter chart, the ratios of each mutant are collected from at least three independent images. (G) Representative images show the subcellular localization of 3xGFP and tdWWs-fused 3xGFP in HEK293T cells, and the scatter chart shows their nucleocytoplasmic ratios (representative of three independent experiments, n represents the total number of the measured cells, two-tailed ANOVA test). The inserted schematic diagram illustrates the coding sequence (cds) of the artificial 3xGFP. Source data are available for this figure: SourceData F2.

WW domains are required for YAP1 transactivation and nuclear translocation. (A) The co-IP experiment shows the interaction of MYC-tagged TEAD1 with HA-tagged YAP1 or the YAP1-W4A mutant in HEK293T cells. TCL, total cell lysates. (B) The W4A mutant loses the transcriptional activity in a TEAD-dependent luciferase reporter (representative of three independent experiments, N = 3, all data shown as the average values ± SD, two-tailed Student’s t test). The representative western blotting shows expressions of YAP1 and the mutant in reporter assays. The inserted diagram illustrates the construct of the TEAD-dependent luciferase reporter. TBE, the TEAD binding element. (C) Substitution of the disordered sequence between the WW domains by five tandem flexible linkers (Gly-Gly-Gly-Gly-Ser) does not affect YAP1 transcriptional activity in HEK293T cells (representative of three independent experiments, N = 3, all data shown as the average values ± SD, two-tailed Student’s t test). The representative western blotting shows expressions of YAP1 and the mutant. (D) tdWWs facilitate YAP1 transcriptional activity in a conformational position-independent manner (representative of three independent experiments, N = 3, all data shown as the average values ± SD, two-tailed Student’s t test). The representative western blotting shows expressions of YAP1 and the mutants. (E) The mRNA levels of CYR61 and CTGF genes in YAP1-knockout (KO) HeLa cells are rescued by the wild-type YAP1 and the tdWWs−ΔWWs chimera, but not by the ΔWWs mutant (representative of three independent experiments, N = 3, all data shown as the average values ± SD, two-tailed Student’s t test). The representative western blotting shows the expression of YAP1 and the mutants in YAP1-KO HeLa cells. (F) Representative images show the subcellular distributions of YAP1 and YAP1 mutants in YAP1-KO HeLa cells, and the scatter chart shows their nucleocytoplasmic ratios (representative of three independent experiments, n represents the total number of the measured cells, two-tailed ANOVA test). The nucleocytoplasmic (N/C) ratios of the subcellular distributions of YAP1 as well as the mutants are measured as in the inserted diagram. Briefly, three areas in the nucleus or the cytoplasm of each cell are randomly selected and then the mean fluorescent intensities in these areas are obtained by ImageJ software. The mean values of fluorescent intensities in three areas are representative of nuclear or cytoplasmic YAP1 levels, respectively. The relative subcellular distribution of YAP1 proteins in each cell is indicated by the ratio of the nuclear and the cytoplasmic YAP1 levels. To plot the scatter chart, the ratios of each mutant are collected from at least three independent images. (G) Representative images show the subcellular localization of 3xGFP and tdWWs-fused 3xGFP in HEK293T cells, and the scatter chart shows their nucleocytoplasmic ratios (representative of three independent experiments, n represents the total number of the measured cells, two-tailed ANOVA test). The inserted schematic diagram illustrates the coding sequence (cds) of the artificial 3xGFP. Source data are available for this figure: SourceData F2.

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