NEDD8, a novel ubiquitin-like protein, has been shown to conjugate to proteins in a manner analogous to ubiquitination and sentrinization. Recently, human UBC12 was identified as a putative NEDD8 conjugation enzyme (E2). While investigating the in vivo function of UBC12, we found that the point mutant, UBC12(C111S), showed a dominant-negative effect on NEDD8 conjugation. This mutant, with a single Cys-to-Ser substitution at the conserved Cys residue in the E2 family, could specifically inhibit NEDD8 conjugation. We observed the dominant-negative effect on NEDD8 conjugation to substrates, including the C-terminal fragment of cullin-2 (Cul-2-DeltaN), full-length cullin-1, and also other uncharacterized target proteins. Interestingly, UBC12(C111S) formed a heterodimeric conjugate with NEDD8. This conjugate was stable under stringent conditions, including 6 m guanidine HCl, 8 m urea, 2% SDS, or 5% beta-mercaptoethanol. Our results are consistent with the hypothesis that UBC12(C111S) sequesters the NEDD8 monomer by forming a UBC12(C111S)-NEDD8 conjugate and, in turn, inhibits the subsequent transfer of NEDD8 to its targets. To examine the biological role of NEDD8 conjugation, this dominant-negative form of UBC12 was applied to a cell growth assay. Overexpression of UBC12(C111S) led to inhibition of growth in U2OS and HEK293 cells. Thus, this dominant-negative form of UBC12 could be useful in defining the role of NEDD8 modification in other biological systems.
|Evidence ID||Analyze ID||Interactor||Interactor Systematic Name||Interactor||Interactor Systematic Name||Type||Assay||Annotation||Action||Modification||Phenotype||Source||Reference||Note|
|Evidence ID||Analyze ID||Gene||Gene Systematic Name||Gene Ontology Term||Gene Ontology Term ID||Qualifier||Aspect||Method||Evidence||Source||Assigned On||Annotation Extension||Reference|
|Evidence ID||Analyze ID||Gene||Gene Systematic Name||Phenotype||Experiment Type||Experiment Type Category||Mutant Information||Strain Background||Chemical||Details||Reference|
|Evidence ID||Analyze ID||Regulator||Regulator Systematic Name||Target||Target Systematic Name||Experiment||Assay||Construct||Conditions||Strain Background||Reference|