Mdm2 Is a RING Finger-dependent Ubiquitin Protein Ligase for Itself and p53*
- From the ‡Laboratory of Immune Cell Biology, Division of Basic Sciences, NCI, National Institutes of Health, Bethesda, Maryland 20892-1152 and §ABL-Basic Research Program, NCI-Frederick Cancer Research and Development Center, Frederick, Maryland 21702-1201
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.
Footnotes
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↵* This work was supported in part by the NCI, DHHS, under contract with ABL.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵¶ To whom correspondence should be addressed: Laboratory of Immune Cell Biology, Division of Basic Sciences, NCI, Bldg. 10, Rm. 1B34, National Institutes of Health, 9000 Rockville Pike, Bethesda, MD 20892-1152. Tel.: 301-496-3557; Fax: 301-402-4844; E-mail: amw@nih.gov.
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↵2 J. P. Jensen and A. M. Weissman, unpublished observations.
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↵3 S. Fang, K. H. Vousden, R. L. Ludwig, and A. M. Weissman, unpublished data.
- Abbreviations:
- Ub
-
ubiquitin
- NEM
-
N-ethylmaleimide
- HECT
-
homologous to E6-AP C terminus
- GFP
-
green fluorescent protein
- TPEN
-
N,N,N′,N′-tetrakis(2-pyridylmethyl)-ethylenediamine
- PML
-
promyelocytic leukemia protein
- Ubc
-
ubiquitin-conjugating enzyme
- GST
-
glutathione S-transferase
- GS
-
glutathione-Sepharose beads
- PBS
-
phosphate-buffered saline
- DTT
-
dithiothreitol
- PAGE
-
polyacrylamide gel electrophoresis
- RT
-
room temperature
-
- Received October 7, 1999.
- Revision received December 23, 1999.
- The American Society for Biochemistry and Molecular Biology, Inc.
