Fig. 5

The interaction between MAST3 and YAP promotes the phosphorylation of YAP (S127). (A–B): Endogenous and exogenous immune coprecipitation experiments were conducted to verify the interaction between MAST3 and YAP. (C): Immunofluorescence experiments confirmed the co-localization of MAST3 and YAP in the cytoplasm of MCF-7 cell lines. Scale bar: 25 μm. (D): Schematic diagram of the structure of MAST3 and YAP splicing bodies. (E–F): MAST3 interacts with YAP’s PDZ-BD domain through its PDZ domain. Co-transfection of GFP-YAP and Myc MAST3-WT and Myc MAST3 in MCF-7-Δ PDZ; immunoprecipitation showed that MAST3 could not interact with YAP in the absence of the PDZ domain (E); Similarly, YAP lacking the PDZ-BD domain cannot bind to MAST3 (F). (G–H): Western blot detection of changes in YAP phosphorylation levels after the bidirectional regulation of MAST3 expression in MDA-MB-468 and MCF-7. After MAST3 overexpression, the phosphorylation level of YAP-ser127 was significantly upregulated, whereas after knockdown, the results showed the opposite effect (phosphorylation levels at other sites did not show significant changes). GAPDH served as an internal reference. (I): The MDA-MB-468 cell line was co-transfected with MAST3 as well as YAP wild-type and mutant YAP-S127A plasmids, and immunofluorescence experiments were performed to detect the changes in YAP nuclear translocation. Scale bar: 25 μm. (J): MAST3 wild-type, MAST3-delta kinase, and MAST3-delta PDZ mutant plasmids were transfected into MDA-MB-468 cells, and western blot was performed to verify that MAST3 upregulates the phosphorylation levels of the YAP-ser127 site, whereas the mutants abrogated this effect. (K): MAST3 plasmids were transfected into MDA-MB-468. After 36 h, cycloheximide (CHX, 532.5 nM) was added and proteins at 0 h and 12 h were collected for immunoprecipitation analysis. GAPDH served as an internal reference protein. IgH: heavy chain