WW domains are dispensable to YAP1 phosphorylation catalyzed by LATS1/2. (A) The schematic diagram illustrates the functional modules of YAP1, the linear feature of the WW domain, and the three-dimensional structures of the WW domains of YAP1 (Protein Data Bank [PDB]: 6JK1). TAD, transcriptional activity domain. (B) The co-IP experiment shows the interaction of FLAG-tagged LATS2 with HA-tagged YAP1 or W4A mutant in HEK293T cells. W4A, alanine substitutions of four conserved tryptophan residues (W177, W199, W236, and W258) in the WW domains of YAP1; TCL, total cell lysates. (C) The co-IP experiment shows the interaction of HA-tagged LATS1 with MYC-tagged YAP1 or YAP1-ΔWWs mutant, which lacks all WW domains, in HEK293T cells. (D) The co-IP experiment shows that the ectopic restoration of the WW domains at the amino terminus of the YAP1-ΔWWs mutant rescues the interaction of LATS2 with YAP1-ΔWWs mutant in HEK293T cells. (E) The representative western blotting shows MST2–LATS2 cascade-mediated phosphorylation of Ser-127 of the wild-type YAP1, the ΔWWs mutant, and the tdWW−ΔWWs chimera in HEK293T cells, and the column chart shows normalized quantitative Ser-127 phosphorylation (quantitative data are obtained from three independent experiments, N = 3, all data shown as the average values ± SD, two-tailed Student’s t test). (F) The co-IP experiment shows that deletion of the WW domains does not affect phosphorylation-mediated interaction of YAP1 and 14-3-3 in HEK293T cells. (G) The CRISPR/Cas9-mediated depletion of YAP1 gene expression in HEK293T cells. The representative western blotting shows YAP1 protein content in the wild-type and the knockout (KO) cells. (H) The representative western blotting shows endogenous Ser-127 phosphorylation of YAP1 in HEK293T/YAP1-KO cells, which are stably restored to the expression of the wild-type YAP1, the W4A mutant, the ΔWWs mutant, or the S127A substitution mutant, respectively. Source data are available for this figure: SourceData F1.