Fig. 6: USP36 modulates YAP stability in a ubiquitination-dependent manner.

A The ubiquitination assay was performed with shUSP36 in ECA109 cells. After 48 h, the cells were treated with MG132 (20 µM) for 6 h. The ubiquitination assay demonstrated that depletion of USP36 promoted endogenous YAP total poly-ubiquitination in ECA109 cells. B–D HEK293 cells were co-transfected with YAP and USP36 constructs as indicated. After 48 h, the cells were treated with MG132 (20 µM) for 6 h. Global ubiquitination (B) K48- and K63-linked ubiquitination (C, D) of YAP were measured by ubiquitination assay. The ubiquitination assay demonstrated that USP36 inhibited YAP total poly-ubiquitination and K48-linked ubiquitination in HEK293 cells, while had little effect on YAP K63-linked ubiquitination. E Global ubiquitination of YAP in HEK293 cells transfected with WT or USP36 mutants was measured by ubiquitination assay. After 48 h, the cells were treated with MG132 (20 µM) for 6 h. The USP domain was essential for USP36 to regulate ubiquitination of YAP. F Global ubiquitination of YAP in HEK293 cells transfected with WT or USP36 mutant (C131A) was measured by ubiquitination assay. After 48 h, the cells were treated with MG132 (20 µM) for 6 h. The C131A mutation of USP36 impaired its ubiquitination activity and reduced the ability of USP36 to inhibit total YAP poly-ubiquitination in HEK293 cells. G USP36 associated with YAP and inhibited YAP K48-linked ubiquitination and degradation in ESCC cells, which promoted the activation of the Hippo/YAP axis and ESCC cancer progression.