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Mdm2 is a RING finger-dependent ubiquitin protein ligase for itself and p53
S Fang1, J P Jensen, R L Ludwig
1Laboratory of Immune Cell Biology, Division of Basic Sciences, NCI, National Institutes of Health, Bethesda, Maryland 20892-1152, USA.
Abstract:
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.
Insights
Mdm2 acts as an E3 ubiquitin ligase for p53, targeting it for degradation. Mdm2 also ubiquitinates itself intrinsically, requiring zinc for activity, and shows substrate specificity via its RING finger.
Area of Science:
- Molecular Biology
- Biochemistry
- Cell Biology
Background:
- Mdm2 protein regulates p53 stability by targeting it for proteasomal degradation.
- The precise mechanism of Mdm2's E3 ubiquitin ligase activity and substrate specificity is under investigation.
Purpose of the Study:
- To elucidate the role of Mdm2's RING finger in its E3 ubiquitin ligase activity towards p53.
- To investigate the intrinsic ubiquitination capacity of Mdm2 and its dependence on zinc.
- To determine the basis for Mdm2's substrate specificity.
Main Methods:
- In vitro ubiquitination assays using purified Mdm2 and p53.
- Site-directed mutagenesis of Mdm2's RING finger and zinc-coordinating residues.
- Chelation and restoration of divalent cations (zinc).
- Reconstitution experiments using a heterologous RING finger (Praja1).
Main Results:
- Mdm2 functions as a RING finger-dependent E3 ubiquitin ligase for p53.
- Mdm2 possesses intrinsic E3 ubiquitin ligase activity, mediating its own ubiquitination.
- This intrinsic activity is zinc-dependent and requires specific zinc-coordinating residues.
- Degradation of p53 and Mdm2 in cells requires additional zinc-coordinating residues not essential for in vitro activity.
- The Mdm2 RING finger confers specificity for p53 ubiquitination, as a Praja1 RING could not substitute.
Conclusions:
- Mdm2 is an intrinsic E3 ubiquitin ligase for p53, with its activity modulated by zinc.
- The Mdm2 RING finger plays a critical role in both Mdm2 auto-ubiquitination and p53 ubiquitination, suggesting substrate specificity at the RING level.