Figure 5
Fbxw7 binds to YAP and promotes the ubiquitin-mediated proteolysis of YAP. A) YAP and Fbxw7 coimmunoprecipitate with each other. Flag-Fbxw7 and HA-YAP plasmids were transfected into HEK293 cells. YAP or Fbxw7 was immunoprecipitated with anti-HA or anti-Flag antibody. Western blotting was performed to detect the specific proteins indicated on the left side of each panel. B) HA-YAP was precipitated from HA-YAP overexpressing HepG2 cells by immunoprecipitation using an anti-HA antibody; YAP ubiquitination was detected by western blotting. Fbxw7 overexpression markedly promoted YAP ubiquitination. C) The Fbxw7 turnover rate was shorter in Fbxw7 overexpressing HepG2 cells. The protein half-life of YAP was analyzed following treatment with cycloheximide. The YAP band intensity was normalized to GAPDH and then normalized to t = 100 controls. The half-life of YAP in Flag-Fbxw7 = 5.2 h (R2 = 0.96) and in EV = 16.5 h (R2 = 0.92). Additionally, treatment with MG132 (a proteasome inhibitor) inhibited Fbxw7 induced YAP degradation in HepG2 cells. The data are representative of multiple independent experiments.