A dominant-negative UBC12 mutant sequesters NEDD8 and inhibits NEDD8 conjugation in vivo.
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.
Wada
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Division of Molecular Medicine, Department of Internal Medicine, Research Center for Cardiovascular Diseases, Institute of Molecular Medicine for the Prevention of Human Diseases, the University of Texas-Houston Health Science Center, Houston, USA.
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Kamitani
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D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002352:Carrier Proteins / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000378:metabolism; D044842:Cullin Proteins; D005799:Genes, Dominant; D006801:Humans; D000075687:NEDD8 Protein; D017354:Point Mutation; D029701:Saccharomyces cerevisiae Proteins; D014452:Ubiquitins / Q000235:genetics / Q000378:metabolism*
C105153:CUL2 protein, human; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D044842:Cullin Proteins; D000075687:NEDD8 Protein; C082860:NEDD8 protein, human; D029701:Saccharomyces cerevisiae Proteins; C112595:UBC12 protein, S cerevisiae; D014452:Ubiquitins