Fig 2. Deletion or depletion of PPM1A enhances Hippo signaling and inactivates YAP/TAZ.

(A, B) PPM1A KO HEK293 cells were generated by CRISPR-based genome editing and verified by immunofluorescence (A) and immunoblotting (B). Genetic ablation of PPM1A resulted in a substantial increase of cytoplasmic YAP/TAZ (A), an up-regulation of phospho-YAP (S127) (B, second panel), a marked accumulation of highly phosphorylated YAP in a Phos-Tag electrophoresis (B, first panel), and a decrease of TAZ proteins (B, third panel). Glucose deficiency resulted in a phosphorylation status of YAP/TAZ similar to a degree of those cells with PPM1A deletion, as evidenced by the assays of mobility shift and phospho-YAP (S127) (B). (C) A compromised responsiveness to the TEAD-driven promoter was detected in cells without PPM1A. (D) Reconstitution of PPM1A in PPM1A KO cells via a viral-based delivery entirely recovered the expression of YAP/TAZ target genes. (E) Reintroduction of ectopic PPM1A in PPM1A KO cells alleviated the 2-DG-induced phosphorylation of endogenous YAP. The expression of endogenous and ectopic PPM1A was indicated by an anti-PPM1A immunoblotting. (F) Immunofluorescence imaging showed a profound increase of cytoplasmic YAP/TAZ in MEFs isolated from PPM1A KO mice, which were generated by a conventional HR strategy. (G) qRT-PCR assays for the TTFs of PPM1A KO mice showed a significant decrease of CTGF and CYR61 mRNAs. (H–K) Depletion of PPM1A in HEK293 cells (H) and HaCaT cells (I) by siRNA interferences resulted in an increased level of phospho-YAP (S127), either in resting state or states with nutrient deficiency. PPM1A depletion also down-regulated the transactivation of the YAP/TAZ-TEAD complex (J) and mRNAs of their target genes (K). (L) The levels of CTGF and CYR61 mRNAs in PPM1A-depleted cells were comparable to those cells under nutrient deficiency. Unprocessed images of blots are shown in S1 Raw Images. Statistics source data are provided in S1 Data. HR, homozygous recombination; KO, knockout; MEF, mouse embryonic fibroblast; PPM1A, protein phosphatase magnesium-dependent 1A; qRT-PCR, real-time quantitative PCR; siRNA, small interfering RNA; TAZ, transcriptional coactivator with PDZ-binding motif; TEAD, transcriptional enhanced associate domain; TTF, tail-tip fibroblast; YAP, Yes-associated protein.