Fig. 7

RBCK1 functions as one ubiquitin ligase to promote YAP K48-linked poly-ubiquitination A RBCK1 deletion decreases the total polyubiquitination of YAP. siControl or siRBCK1 were transfected into MDA-MB-231 cells. 24 h later, 1 mg HA-Ub plasmid and 2 µg YAP plasmid were transfected into cells. 24 h later, the cells were immunoprecipitated with an anti-HA antibody. B RBCK1 boosted total polyubiquitination of YAP. 2 µg of YAP plasmid, 0.5 µg of HA-Ub plasmid and 0.5 µg of Flag-tag or Flag -RBCK1 plasmids were transfected into HEK293T cells. After 24 h, the cells were immunoprecipitated with an anti-HA antibody. C RBCK1 deletion decreases the K48-linked polyubiquitination of YAP. siControl or siRBCK1 were transfected into MDA-MB-231 cells. 24 h later, 1 mg HA-K48 Ubi plasmid and 2 µg of YAP plasmid were transfected into cells. 24 h later, the cell were immunoprecipitated with an anti-HA antibody. D RBCK1 boosted the K48-linked polyubiquitination of YAP. 2 µg of YAP plasmid, 0.5 µg of HA-K48 Ubi plasmid and 0.5 µg of Flag-tag or Flag -RBCK1 plasmids were transfected into HEK293T cells. 24 h later, the cells were immunoprecipitated with an anti-HA antibody. E RBCK1 deletion boosted the K63-linked polyubiquitination of YAP. siControl or siRBCK1 were transfected into MDA-MB-231 cells. 24 h later, 1 mg HA-K63 Ubi plasmid and 2 µg of YAP plasmid were transfected into cells. 24 h later, the cell were immunoprecipitated with an anti-HA antibody. F RBCK1 decreases K63-linked polyubiquitination of YAP. 2 µg of YAP plasmid, 0.5 µg of HA-K63 Ubi plasmid and 0.5 µg of Flag-tag or Flag -RBCK1 plasmids were transfected into HEK293T cells. After 24 h, the cell extracts were immunoprecipitated with an anti-HA antibody. G RBCK1 inhibits YAP degradation by RBR domain. 2 µg Flag-RBCK1 full length or mutant plasmids (ΔUBL, ΔNZF, ΔRBR domains) were transfected into HEK293T cells. After 24 h, the cells were harvested directly and used for the western blot analysis. β-Actin was used as the internal reference. H The UBL domain or RBR domain of RBCK1 is important to improve total ubiquitination YAP. 2 µg, Myc-YAP, 1 µg HA-UB plasmid and 1 µg Flag-RBCK1 full length or mutants (ΔUBL, ΔNZF, ΔRBR domains) were transfected into HEK293 cells. The polyubiquitinated YAP was detected through the western blot analysis. I The RBR domain of RBCK1 is important to improve K48-linked ubiquitination of YAP. 2 µg Myc-YAP, 1 µg HA-K48 Ubi plasmid and 1 µg Flag-RBCK1 full length or mutants (ΔUBL, ΔNZF, ΔRBR domains) were transfected into HEK293 cells. The K48-specific polyubiquitinated YAP was detected through the western blot analysis. J The ability of RBCK1 to degrade YAP protein was impaired by the mutations of RBCK1 in MDA-MB-231 cells. K The mutations of RBCK1 that impaired ubiquitination of RBCK1 activity and the ability to increase the total polyubiquitination of YAP. HEK293 cells were transfected with 2 µg of YAP plasmid, 0.5 µg of HA-Ub plasmid and 0.5 µg of Flag-tag or Flag -RBCK1 plasmids or Flag-RBCK1 C460A. After 24 h, the cells were immunoprecipitated with an anti-HA antibody. The total polyubiquitinated YAP was detected via western blot analysis. L–M K76, K204, and K321 mutations (K76R, K204, and K321R) largely eliminated the ubiquitination effect of RBCK1 on YAP. The indicated vectors were transfected into HEK293 for ubiquitination analysis. Polyubiquitinated YAP was detected by Western blot analysis