Figure 4: Nedd8 is attached to Smurf1 through C426-catalysed autoneddylation. | Nature Communications

Figure 4: Nedd8 is attached to Smurf1 through C426-catalysed autoneddylation.

From: The covalent modifier Nedd8 is critical for the activation of Smurf1 ubiquitin ligase in tumorigenesis

Figure 4

(a) Covalent neddylation of Smurf1 in vitro. Purified His-Smurf1-WT or C699A proteins were incubated with Nedd8 and Nedd8-E1/E2. Reactions were performed as described in the Methods section. Samples were analysed by western blotting with an anti-His antibody. (b) Deneddylation enzyme NEDP1 abolished covalent neddylation of Smurf1 in vitro. Neddylated Smurf1 was purified on Ni2+-NTA Superflow Cartridges (QIAGEN) and then incubated with GST-NEDP1 WT or C163A. Reactions were performed as described in the Methods. Samples were analysed by western blotting with an anti-His antibody. (c) In vivo neddylation and ubiquitylation assay were used to detect covalent modification of endogenous Smurf1. HCT116 cells (1 × 109) were solubilized in modified lysis buffer and immunoprecipitated with the Nedd8 and ubiquitin antibodies. (d,e) Smurf1 was neddylated in mammalian cells. Smurf1 neddylation was significantly attenuated by the NAE inhibitor MLN4924 or depletion of Uba3 (one subunit of NAE) or Ubc12. The indicated plasmids or siRNAs were transfected into HCT116 cells. Flag antibody-immunoprecipitated Smurf1 proteins were analysed by immunoblotting with an anti-Myc antibody to detect conjugated Nedd8. (f) Smurf1-WT and the indicated CA mutants were co-transfected with Nedd8 WT, K0 or ΔGG to detect covalently modified Smurf1 in HCT116 cells. (g) In vivo Smurf1 neddylation assay. HEK293T cells were transfected with Flag-Nedd8, Myc-tagged Smurf1 WT and mutants, including K0 (all 38 Lys residues mutated to Arg), 1–14 K (only retained lysines in the C2 domain), 15–17 K (in the WW-HECT linker), 18–38 K (in the HECT domain), 18–22 K (in the N-terminal region of HECT), 23–38 K (in the resting region of HECT). The cells were harvested and subjected to neddylation asssy 48 h after transfection. A schematic diagram of the lysine sites on Smurf1 is shown. * means none-specific band. (h,i) In vitro Nedd8-thioester bond assays. Smurf1 WT and K0 mutants were expressed in bacteria and purified.

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