Using binding assays, we find that RNF11 can directly compete with SMAD7 for SMURF2 and that binding is mutually exclusive and dependent on a proline-rich domain. that the choice of binding partners for SMURF2 can sustain or repress TGF signaling, and RNF11 may promote TGF-induced cell migration. encodes a protein of 154 amino acids with several potential interaction domains, including a PPis subject to regulation by RNF11 when the expression of the latter vastly exceeds endogenous levels. We note that high levels of RNF11 have been reported in reported in late-stage breast and pancreatic cancers (7, 22). Open in a separate window Figure 3. RNF11 suppresses B2M SMURF2 but not SMURF1 polyubiquitylation in the cell. and and Fig. 7. ubiquitylation assay. The reaction was resolved by SDS-PAGE and immunoblotted. serves as a negative control and contains only the recombinant E3 and no E1 or E2. ubiquitylation assay. The reaction was resolved by SDS-PAGE and immunoblotted. For each protein complex, there is a lane without E1 or E2 to detect unmodified protein. UBCH5C was used as the E2 in this experiment. ubiquitylation assay using wild-type or indicated ubiquitin mutants. The reaction was resolved by SDS-PAGE and immunoblotted. serves as a negative control and contains only the recombinant E3 and no E1 or E2. UBCH5C was used as the E2 in this experiment. RNF11 localization, but not its catalytic activity, is required for suppression of SMURF2 ubiquitylation To understand how RNF11 regulates SMURF2 activity, we asked whether myristoylation, palmitoylation, and ubiquitylation of RNF11, which regulate its sorting, also contribute to the stabilization of this complex. Second, we asked whether the regulation of SMURF2 activity was restricted to the endosomal compartment and whether this was impacted by its association with RNF11. Assembly of the SMURF2RNF11 complex is stabilized by robust protein-protein interactions involving the PPPY motif in RNF11 and two adjacent WW domains in SMURF2 (22). Therefore, because N-terminally tagged RNF11 delocalizes this protein (8,C11), we expressed untagged versions of wild type and selected RNF11 mutants, together with SMURF2 (Fig. 4and that this regulation requires stable complex assembly. We also tested our panel of RNF11 mutants that exhibit defects in acylation-dependent localization, catalytic activity, and sorting from the Ethoxzolamide and after co-expression and immunopurification of a complex of untagged RNF11 and FLAG-tagged wild-type or catalytically inactive (C716A) SMURF2 from insect cells (Fig. 6and and of the RNF11 blot). Importantly, however, these data suggest that in the wild-type complex, most of the activity emanates from SMURF2 rather than RNF11 (Fig. 6data establish that the SMURF2RNF11 Ethoxzolamide complex can indeed function as an E3 ligase and strongly suggest that it could also be active in the cell in some settings. SMURF2 complexes with RNF11 and SMAD7 assemble biochemically related E3 ligases in vitro To further biochemically characterize the RNF11/SMURF2 complex by incubating insect cell lysates expressing wild-type FLAG-SMURF2 or the C716A mutant of FLAG-SMURF2 together with those prepared from insect cells expressing recombinant SMAD7-HA or bacterially produced wild-type or I101A mutant GST-RNF11. After complex assembly, recombinant SMURF2 complexes were isolated by immunopurification and tested for E3 ligase activity and with and and with and (4). Next, we compared E2 usage for both SMURF2 complexes (Fig. 7ubiquitylation assay. For each protein or protein complex, there is a lane without E1 or E2 to detect unmodified protein. UBCH5C was used as the E2 in this experiment. ubiquitylation assay. Various E2s were used to determine preference. and serve as negative controls and contain only the recombinant E3 but no E1 or E2. Predicted molecular masses are as follows: RNF11 (untagged), 17.4 kDa; FLAG-SMURF2, 87 kDa; HA-SMAD7, 49.7 kDa; GST-RNF11, 44.4 kDa. *, mobility of unmodified RNF11. SMURF2 forms mutually exclusive complexes with RNF11 or SMAD7 To directly test this notion, a fixed amount of preassembled recombinant SMAD7-SMURF2 complexes was incubated with increasing amounts of recombinant GST-RNF11 or RNF11 Y40A, and complexes were purified through the GST moiety and detected by immunoblotting. As shown in Fig. 8alone is sufficient to suppress TGF signaling as compared with controls (Fig. 9expression alone could not Ethoxzolamide augment cellular responsiveness to TGF (Fig. 9antagonized SMURF2-mediated repression and, strikingly, augmented TGF responsiveness well above levels seen in the SMURF2 control. Importantly, an equivalent level of expression using the same N-terminally tagged form of RNF11 used in the previous study (22) was significantly less effective in antagonizing SMURF2 function in this assay. Open in a separate window Figure 9. RNF11 functionally antagonizes SMURF2 by sequestration.