Fig. 3: RNF214 promotes nonproteolytic polyubiquitylation of TEADs.

a, b RNF214 promotes TEADs ubiquitylation. HEK293T cells were transfected with HA-Ub, Flag-TEAD2 or Flag-TEAD3 and Myc-RNF214 or the Myc-RNF214 RD mutant plasmids. Flag-tagged TEAD proteins were pulled out using anti-Flag beads by denaturing immunoprecipitation (d-IP) and the ubiquitylated TEAD proteins were detected using anti-HA antibody. c RNF214 ubiquitylates TEAD2. A biotinylated Avi-tagged TEAD2 (Bio-TEAD2) was expressed in HLF cells. Flag-RNF214 was then transfected into the Bio-TEAD2 HLF cells and biotin (2 μg/mL) was added to culture medium overnight before cell harvest. Biotinylated-TEAD2 proteins were isolated through Streptavidin beads under a denaturing buffer condition and ubiquitylated TEAD2 proteins were then detected using an anti-ubiquitin antibody. d Depletion of RNF214 attenuates TEAD2 ubiquitylation. Bio-TEAD2 HLF cells were transfected with RNF214 siRNA or control siRNA. After transfection, the ubiquitylation of TEAD2 was detected using the same procedure as shown in (c). e Knockout of RNF214 decreases ubiquitylation of TEADs. Halo-ThUBDs proteins were expressed and purified, and then incubated with HLF cell lysates. Ubiquitylated TEADs were detected using a pan-TEAD antibody. Two independent clones were selected from sgRNF214-3 HLF pools. f RNF214 has little effect to the TEADs protein stability. Hep3b cells were transfected with siRNF214 for 72 h, and then treated with cycloheximide (CHX 20 μg/mL) for the indicated time. c-Myc was employed as a positive control for CHX chase experiments. g–i RNF214 promotes the K27 polyubiquitylation of TEAD2. HEK293T cells were transfected with Flag-TEAD2, and wild type, lysine less (K0), or K-only ubiquitin mutants (g) or KR mutants of HA-Ub (h). 24 h post-transfection, Flag-TEAD2 proteins were pulled out using anti-Flag beads under a denaturing buffer condition and the ubiquitylated TEAD2 proteins were reviewed using anti-HA antibody. i Flag-TEAD2, HA-Ub WT or the K27-only mutant with or without Myc-RNF214 were co-expressed in HEK293T cells. Experiments in these figures (a–i) were repeated twice. Source data are provided as a Source Data file.