Fig. 3: Smurf1 and Smurf2 target RNF220 for K48-linked polyubiquitination.
A, B In vivo ubiquitination assays showing the ubiquitination status of RNF220 when wild-type or E3 ubiquitin ligase inactive form of Smurf1 or Smurf2 was co-expressed in HEK293 cells. Overexpression of wild-type Smurf1 or Smurf2, but not their mutants promoted the ubiquitination of RNF220. The statistics of relative protein levels of polyubiquitinated RNF220 in each group was shown in (B), and the control group was set to 1. C–F In vivo ubiquitination assays showing the ability of Smurf1 (C, D) or Smurf2 (E, F) to ubiquitinate RNF220 when the indicated ubiquitin mutants were used. Smurf1 and Smurf2 promote K48-linked ubiquitination of RNF220. For ubiquitin mutants: K48 the K48 ubiquitin mutant with all lysines except the K48 mutated to arginines, K48R the K48R ubiquitin mutant with only the K48 lysine mutated to arginine, WT the wild-type ubiquitin construct. The statistics of relative protein levels of polyubiquitinated RNF220 in each group was shown in (D, F), and the control group was set to 1. G, H In vivo ubiquitination analysis showing the level of endogenous ubiquitinated RNF220 protein when Smurf1 or Smurf2 was knocked down in HEK293 cells by siRNA transfection. The statistics of relative protein levels of ubiquitinated RNF220 in each group was shown in (H), and the control group was set to 1. I, J Western blot analysis showing the protein level of wild-type or KR mutated form of RNF220 protein when co-expressed with Smurf1 or Smurf2 in HEK293 cells. The statistics of relative protein levels of RNF220 in each group was shown in (J), and the respective controls were set to 1. K–N In vivo ubiquitination analysis showing the ubiquitinated status of wild-type or KR mutated form of RNF220 protein when co-expressed with Smurf1 (K, L) or Smurf2 (M, N). O–Q In vitro ubiquitination analysis showing that Smurf1 or Smurf2 promotes polyubiquitination of wild-type, but not KR mutated, RNF220 protein. The statistics of relative protein levels of RNF220 in each group was shown in (P, Q), and the respective controls were set to 1. IB immunoblot, IP immunoprecipitation, WCL whole cell lysate, WT wild type, KR RNF220K321/323/326R mutant; CA Smurf1 E3 ubiquitin ligase-defective mutant, CG Smurf2 E3 ubiquitin ligase-defective mutant; NC negative control, ns not significant. p > 0.05 and **p < 0.01.
