Fig. 5

WWP2 and Itch might adopt the same multi-lock regulation mechanism as WWP1. a Schematic of WWP2 domains showing an enzymatic activity summary derived from the autoubiquitination assay in b. b Autoubiquitination assay of Trx-tagged full-length (FL) WWP2 and various fragments. The weight markers belong to all separate gels. c–d GST pull-down assay of WWP2 GST-WW with Trx-HECT. Mutations that were predicted to impair interactions at the “Re” site in WW2-HECT (W358A^WW2 and M575E^HECT) or the “Le” site in WW4-HECT (Y491A^HECT and H465A^WW4) weakened the WW–HECT interaction of WWP2, and the double mutation of both sites (Y491A^HECT, M575E^HECT, and W358A^WW2, H465A^WW4) completely disrupted the interaction. e The in vitro autoubiquitination assay of WWP2 12L34HECT Y491A^HECT and M575E^HECT mutants. f Schematic of Itch domains showing a summary of enzymatic activity derived from the autoubiquitination assay in g. g Autoubiquitination assay of various Trx-tagged Itch fragments. The weight markers belong to all separate gels. h–j GST pull-down assay of Itch GST-WW with Trx-HECT. Mutations that were predicted to impair interactions at the “Re” site in WW2-HECT (W347A^WW2 and M569E^HECT) or the “Le” site in WW4-HECT (Y485A^HECT and H460A^WW4) weakened the WW–HECT interaction with Itch, and double mutation of both sites (Y485A^HECT, M569E^HECT, and W347^WW2, H460A^WW4) completely disrupted the interaction. j In vitro autoubiquitination assay of Itch 12L34HECT Y485A^HECT and M569E^HECT mutants. For all ubiquitination assays, the statistics showing enzymatic activity are shown below. Data are presented as the mean ± SD of triplicate experiments; ns, not significant, *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 based on one-way analysis of variance (ANOVA) with Tukey’s multiple comparison test. Source data are provided as a Source Data file