Mdm2 is a RING finger-dependent ubiquitin protein ligase for itself and p53.
Mdm2 has been shown to regulate p53 stability by targeting the p53 protein for proteasomal degradation. We now report that Mdm2 is a ubiquitin protein ligase (E3) for p53 and that its activity is dependent on its RING finger. Furthermore, we show that Mdm2 mediates its own ubiquitination in a RING finger-dependent manner, which requires no eukaryotic proteins other than ubiquitin-activating enzyme (E1) and an ubiquitin-conjugating enzyme (E2). It is apparent, therefore, that Mdm2 manifests an intrinsic capacity to mediate ubiquitination. Mutation of putative zinc coordination residues abrogated this activity, as did chelation of divalent cations. After cation chelation, the full activity could be restored by addition of zinc. We further demonstrate that the degradation of p53 and Mdm2 in cells requires additional potential zinc-coordinating residues beyond those required for the intrinsic activity of Mdm2 in vitro. Replacement of the Mdm2 RING with that of another protein (Praja1) reconstituted ubiquitination and proteasomal degradation of Mdm2. However, this RING was ineffective in ubiquitination and proteasomal targeting of p53, suggesting that there may be specificity at the level of the RING in the recognition of heterologous substrates.
Fang
S
S
Laboratory of Immune Cell Biology, Division of Basic Sciences, NCI, National Institutes of Health, Bethesda, Maryland 20892-1152, USA.
Jensen
J P
JP
Ludwig
R L
RL
Vousden
K H
KH
Weissman
A M
AM
D000478:Alkylation; D000595:Amino Acid Sequence; D002614:Chelating Agents / Q000494:pharmacology; D016384:Consensus Sequence; D003546:Cysteine Endopeptidases / Q000378:metabolism; D008025:Ligases / Q000187:drug effects / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000378:metabolism; D009154:Mutation; D009687:Nuclear Proteins; D046988:Proteasome Endopeptidase Complex; D011499:Protein Processing, Post-Translational; D011518:Proto-Oncogene Proteins / Q000187:drug effects / Q000378:metabolism*; D051736:Proto-Oncogene Proteins c-mdm2; D011994:Recombinant Proteins / Q000378:metabolism; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism*; D044764:Ubiquitin-Activating Enzymes; D044767:Ubiquitin-Protein Ligases; D014452:Ubiquitins / Q000378:metabolism*; D015032:Zinc; D016335:Zinc Fingers / Q000235:genetics
D002614:Chelating Agents; D009097:Multienzyme Complexes; D009687:Nuclear Proteins; D011518:Proto-Oncogene Proteins; D011994:Recombinant Proteins; D016159:Tumor Suppressor Protein p53; D014452:Ubiquitins; D051736:Proto-Oncogene Proteins c-mdm2; D044767:Ubiquitin-Protein Ligases; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; D008025:Ligases; D044764:Ubiquitin-Activating Enzymes; D015032:Zinc