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Mdm2 Is a RING Finger-dependent Ubiquitin Protein Ligase for Itself and p53*

Open AccessPublished:March 24, 2000DOI:https://doi.org/10.1074/jbc.275.12.8945
      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.
      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
      Increases in cellular levels of p53 in response to stress induces cell growth arrest and apoptosis (
      • Giaccia A.J.
      • Kastan M.B.
      ,
      • Gottlieb T.M.
      • Oren M.
      ,
      • Bates S.
      • Vousden K.H.
      ). Accumulating evidence suggests that this crucial protein is regulated primarily through post-translational mechanisms (,
      • Ashcroft M.
      • Vousden K.H.
      ). Central to the regulation of p53 is Mdm2, which is itself a transcriptional target of p53 (,
      • Kubbutat M.H.
      • Vousden K.H.
      ). Two functional domains of Mdm2 are involved in regulating p53 levels. Its N terminus binds to p53 and targets p53 for degradation (
      • Kubbutat M.H.
      • Jones S.N.
      • Vousden K.H.
      ,
      • Haupt Y.
      • Maya R.
      • Kazaz A.
      • Oren M.
      ). Binding of p53 through this region also conceals the trans-activation domain of p53 and, therefore, inhibits its transcriptional activity (
      • Kubbutat M.H.
      • Vousden K.H.
      ). Diverse signals regulate p53 levels by regulating the interaction between Mdm2 and p53 (). Stress-induced phosphorylation of serines in the trans-activation domain of p53 attenuates the binding and stabilizes p53 (,
      • Shieh S.Y.
      • Taya Y.
      • Prives C.
      ,
      • Unger T.
      • Juven-Gershon T.
      • Moallem E.
      • Berger M.
      • Vogt Sionov R.
      • Lozano G.
      • Oren M.
      • Haupt Y.
      ). Several cellular kinases, including Raf, DNA-PK, ATM, CAK, and JNK, can catalyze this phosphorylation (
      • Jamal S.
      • Ziff E.B.
      ,
      • Meek D.W.
      • Campbell L.E.
      • Jardine L.J.
      • Knippschild U.
      • McKendrick L.
      • Milne D.M.
      ,
      • Shieh S.Y.
      • Ikeda M.
      • Taya Y.
      • Prives C.
      ,
      • Canman C.E.
      • Lim D.S.
      • Cimprich K.A.
      • Taya Y.
      • Tamai K.
      • Sakaguchi K.
      • Appella E.
      • Kastan M.B.
      • Siliciano J.D.
      ). Recent studies also indicate that nucleocytoplasmic shuttling of Mdm2 plays a significant role in p53 degradation (
      • Roth J.
      • Dobbelstein M.
      • Freedman D.A.
      • Shenk T.
      • Levine A.J.
      ,
      • Tao W.
      • Levine A.J.
      ,
      • Tao W.
      • Levine A.J.
      ,
      • Lain S.
      • Midgley C.
      • Sparks A.
      • Lane E.B.
      • Lane D.P.
      ). Mutation of the nuclear export signal of Mdm2 abrogates its ability to target p53 for degradation, raising the possibility that the shuttling of p53 by Mdm2 from nucleus to cytoplasm is required for p53 to be subject to proteasome-mediated degradation (
      • Roth J.
      • Dobbelstein M.
      • Freedman D.A.
      • Shenk T.
      • Levine A.J.
      ,
      • Tao W.
      • Levine A.J.
      ). This is supported by a recent finding that ARF sequesters Mdm2, but not p53, into nucleoli and blocks export of Mdm2 from the nucleus to cytoplasm (
      • Tao W.
      • Levine A.J.
      ,
      • Weber J.D.
      • Taylor L.J.
      • Roussel M.F.
      • Sherr C.J.
      • Bar-Sagi D.
      ,
      • Zhang Y.
      • Xiong Y.
      ). Several proteins including E2F1, Myc, Ras, and adenoviral protein E1A stabilize p53 through the ARF-mediated pathway (
      • Sherr C.J.
      ,
      • Zindy F.
      • Eischen C.M.
      • Randle D.H.
      • Kamijo T.
      • Cleveland J.L.
      • Sherr C.J.
      • Roussel M.F.
      ,
      • Bates S.
      • Phillips A.C.
      • Clark P.A.
      • Stott F.
      • Peters G.
      • Ludwig R.L.
      • Vousden K.H.
      ,
      • Palmero I.
      • Pantoja C.
      • Serrano M.
      ,
      • de Stanchina E.
      • McCurrach M.E.
      • Zindy F.
      • Shieh S.Y.
      • Ferbeyre G.
      • Samuelson A.V.
      • Prives C.
      • Roussel M.F.
      • Sherr C.J.
      • Lowe S.W.
      ). Other Mdm2-associated proteins also affect Mdm2-mediated p53 degradation; the interaction of p300 with Mdm2 promotes p53 degradation (
      • Grossman S.R.
      • Perez M.
      • Kung A.L.
      • Joseph M.
      • Mansur C.
      • Xiao Z.X.
      • Kumar S.
      • Howley P.M.
      • Livingston D.M.
      ), whereas the interaction of Mdm2 with pRB and c-Abl stabilizes p53 in cells (
      • Hsieh J.K.
      • Chan F.S.
      • O'Connor D.J.
      • Mittnacht S.
      • Zhong S.
      • Lu X.
      ,
      • Sionov R.V.
      • Moallem E.
      • Berger M.
      • Kazaz A.
      • Gerlitz O.
      • Ben-Neriah Y.
      • Oren M.
      • Haupt Y.
      ).
      It is becoming clear that Mdm2-mediated degradation of p53 is proteasome-dependent (
      • Kubbutat M.H.
      • Jones S.N.
      • Vousden K.H.
      ,
      • Haupt Y.
      • Maya R.
      • Kazaz A.
      • Oren M.
      ). Recent studies have suggested that Mdm2 has the capacity to function as a ubiquitin (Ub)1 protein ligase (E3) both for itself and for p53 in vitro (
      • Honda R.
      • Tanaka H.
      • Yasuda H.
      ,
      • Honda R.
      • Yasuda H.
      ). E3s provide specificity to Ub conjugation and are the final components in the multienzyme process that eventually leads to the covalent modification of proteins with Ub (
      • Hershko A.
      • Ciechanover A.
      ). The first step in Ub conjugation involves the ATP-dependent activation of Ub by a Ub-activating enzyme (E1), where a transient thiol ester linkage is formed with the C terminus of Ub. The activated Ub is then transferred to an active site cysteine of a Ub-conjugating enzyme (Ubc or E2). The final step requires an E3 that catalyzes the formation of an isopeptide linkage between the C terminus of Ub and ε-amino group of a lysine on a substrate (
      • Hershko A.
      • Ciechanover A.
      ). In some cases, such as the HECT (homologous to E6-AP Cterminus) family E3s (
      • Huibregtse J.M.
      • Scheffner M.
      • Beaudenon S.
      • Howley P.M.
      ), a thiol ester intermediate between Ub and the E3 is formed (
      • Hershko A.
      • Ciechanover A.
      ). In other instances it is believed that the E3 binds the E2-Ub complex and mediates the direct transfer of Ub from E2 to substrates (
      • Hershko A.
      • Ciechanover A.
      ).
      In this study, we demonstrate that Mdm2 is an E3 and that it functions both in ubiquitination of p53 and itself. The E3 activity of Mdm2 is dependent on its RING finger domain. Unlike the binding of the RING finger domain to RNA (
      • Lai Z.
      • Freedman D.A.
      • Levine A.J.
      • McLendon G.L.
      ), coordination of zinc by the RING finger is required for E3 activity of Mdm2. Furthermore, by substitution of the RING finger from a heterologous protein, we demonstrate that another RING finger can substitute for that of Mdm2 for mediating its own ubiquitination in vitro and proteasomal targeting in cells.

      DISCUSSION

      In this study we demonstrate that the intrinsic E3 activity of Mdm2 is dependent on its zinc-coordinated RING finger domain. The capacity to mediate Mdm2's own ubiquitination requires no eukaryotic protein other than E1 and E2. This finding is consistent with recent observations that multiple otherwise unrelated RING finger proteins, including AO7, BRCA1, NF-X1, Siah-1, TRC8, kf-1, and Praja1, have the intrinsic capacity to mediate ubiquitination (
      • Lorick K.L.
      • Jensen J.P.
      • Fang S.
      • Ong A.M.
      • Weissman A.M.
      ) and the finding of RING fingers within other E3s. Among known E3s, the N-end rule E3s (Ubr1, E3α) have RING finger domains, as does the Apc11 subunit of the anaphase-promoting complex (
      • Kwon Y.T.
      • Reiss Y.
      • Fried V.A.
      • Hershko A.
      • Yoon J.K.
      • Gonda D.K.
      • Sangan P.
      • Copeland N.G.
      • Jenkins N.A.
      • Varshavsky A.
      ,
      • Zachariae W.
      • Shevchenko A.
      • Andrews P.D.
      • Ciosk R.
      • Galova M.
      • Stark M.J.
      • Mann M.
      • Nasmyth K.
      ). A protein containing a RING finger-like domain variously referred to as Rbx1, ROC1, or Hrt1 functions as a component of SCF (Skp1,Cdc53/cullin, F box) and VHL (vonHippel-Lindau tumor suppressor protein) E3 complexes (
      • Tyers M.
      • Willems A.R.
      ,
      • Kamura T.
      • Koepp D.M.
      • Conrad M.N.
      • Skowyra D.
      • Moreland R.J.
      • Iliopoulos O.
      • Lane W.S.
      • Kaelin W.G.J.
      • Elledge S.J.
      • Conaway R.C.
      • Harper J.W.
      • Conaway J.W.
      ,
      • Ohta T.
      • Michel J.J.
      • Schottelius A.J.
      • Xiong Y.
      ,
      • Tan P.
      • Fuchs S.Y.
      • Chen A.
      • Wu K.
      • Gomez C.
      • Ronai Z.
      • Pan Z.Q.
      ,
      • Seol J.H.
      • Feldman R.M.
      • Zachariae W.
      • Shevchenko A.
      • Correll C.C.
      • Lyapina S.
      • Chi Y.
      • Galova M.
      • Claypool J.
      • Sandmeyer S.
      • Nasmyth K.
      • Deshaies R.J.
      ,
      • Lisztwan J.
      • Imbert G.
      • Wirbelauer C.
      • Gstaiger M.
      • Krek W.
      ). More recently, the RING finger domain of Cbl has been implicated in ubiquitination of the epidermal growth factor receptor and the platelet-derived growth factor receptor (
      • Waterman H.
      • Levkowitz G.
      • Alroy I.
      • Yarden Y.
      ,
      • Joazeiro C.A.
      • Wing S.S.
      • Huang H.
      • Leverson J.D.
      • Hunter T.
      • Liu Y.C.
      ).
      The importance of the RING finger domain in ubiquitination is also supported by the fact that several RING finger proteins have been found to associate with Ubcs and/or to target specific proteins for proteasome-dependent degradation, although their E3 activities toward these proteins have not been shown. This is the case for the Drosophila RING finger protein SINA, which associates with UbcD1 and targets Tramtrack for degradation (
      • Li S.
      • Li Y.
      • Carthew R.W.
      • Lai Z.C.
      ,
      • Tang A.H.
      • Neufeld T.P.
      • Kwan E.
      • Rubin G.M.
      ). SINA and its mammalian homologues, Siah-1 and Siah-2, interact with Ubc9 through their N termini, which includes the RING finger domain. Siah-2 was found to target nuclear receptor corepressor for degradation (
      • Zhang J.
      • Guenther M.G.
      • Carthew R.W.
      • Lazar M.A.
      ). Furthermore, the RING finger is required for targeting Siah-1 itself and the deleted in colorectal cancer gene product for degradation in cells (
      • Hu G.
      • Zhang S.
      • Vidal M.
      • Baer J.L.
      • Xu T.
      • Fearon E.R.
      ,
      • Hu G.
      • Fearon E.R.
      ), which is consistent with the RING finger and E2-dependent auto-ubiquitination of Siah-1 (
      • Lorick K.L.
      • Jensen J.P.
      • Fang S.
      • Ong A.M.
      • Weissman A.M.
      ). Additionally, the Vmw110 protein of herpes simplex virus type 1 has the RING finger-dependent capacity to target several cellular proteins, such as DNA-PK, SUMO-conjugated PML, and CENP-C, for degradation after infection (
      • Everett R.D.
      • Freemont P.
      • Saitoh H.
      • Dasso M.
      • Orr A.
      • Kathoria M.
      • Parkinson J.
      ,
      • Everett R.D.
      • Earnshaw W.C.
      • Findlay J.
      • Lomonte P.
      ,
      • Parkinson J.
      • Lees-Miller S.P.
      • Everett R.D.
      ). In yeast, Hrd1p (Der3) is found to target misfolded ER proteins for degradation in RING finger-dependent manner (
      • Hampton R.Y.
      • Gardner R.G.
      • Rine J.
      ,
      • Bordallo J.
      • Wolf D.H.
      ). RAD6, an E2, associates with a RING finger protein RAD18 (
      • Bailly V.
      • Lauder S.
      • Prakash S.
      • Prakash L.
      ). However, the significance of this association remains to be determined.
      Whereas several RING finger proteins have been directly shown to coordinate zinc (
      • Lai Z.
      • Freedman D.A.
      • Levine A.J.
      • McLendon G.L.
      ,
      • Roehm P.C.
      • Berg J.M.
      ), for the most part assignment of a RING finger domain to proteins has been based on the presence of consensus sequences, with neither direct evidence of metal binding nor functional correlations (
      • Borden K.L.
      • Freemont P.S.
      ,
      • Saurin A.J.
      • Borden K.L.
      • Boddy M.N.
      • Freemont P.S.
      ). The determination that RING finger proteins mediate E2-dependent ubiquitination (
      • Lorick K.L.
      • Jensen J.P.
      • Fang S.
      • Ong A.M.
      • Weissman A.M.
      ) now allows an assessment of structure-function relationships for the RING finger domain. Based on primary amino acid sequence, Mdm2 does not fall within the limits of RING finger consensus sequences, as there is no appropriately spaced cysteine-histidine pair in the region of the third and fourth coordination sites. This led Freemont and colleagues (
      • Boddy M.N.
      • Freemont P.S.
      • Borden K.L.
      ) to postulate, based on sequence alignment, that Thr-455 and His-457 represent the third and fourth coordination sites. However, metal binding studies in which Cys-449 and His-452 were simultaneously mutated have implicated at least one of these residues as a zinc ligand at the third and/or fourth coordination sites (
      • Lai Z.
      • Freedman D.A.
      • Levine A.J.
      • McLendon G.L.
      ). Our in vitro analysis of Mdm2 auto-ubiquitination implicates His-452 and His-457 as being required for activity. However mutation of Thr-455 alters both the pattern of auto-ubiquitination and the efficiency of this process, markedly decreases in vitro ubiquitination of p53, and results in the stabilization of both Mdm2 and p53 in cells. Mutation of Cys-449 also stabilizes both Mdm2 and p53 in vivo. Thus, mutation of any of the four amino acids postulated to be the third and fourth zinc ligands adversely affects the function of Mdm2. Whether only two of these four residues actually participate in metal coordination, with the others affecting the structure or stability of this atypical RING, or whether there is an additional degree of complexity to metal coordination in this region requires further analysis. Regardless, these results demonstrate that cysteine- and histidine-rich RING-like regions that mediate ubiquitination can vary significantly from canonical RING consensus sequences.
      Although in vitro ubiquitination of p53 by Mdm2 requires addition of only E1, E2, and Ub, we cannot exclude the participation of additional proteins in the binding to, or ubiquitination of, p53. Indeed, it has been suggested that p300 may function as a platform, bringing together the necessary catalytic and regulatory factors needed for p53 ubiquitination (
      • Grossman S.R.
      • Perez M.
      • Kung A.L.
      • Joseph M.
      • Mansur C.
      • Xiao Z.X.
      • Kumar S.
      • Howley P.M.
      • Livingston D.M.
      ). However, it is clear from the present study that RING finger-dependent ubiquitination of Mdm2 requires only E1 and E2.
      Whereas otherwise unrelated RING finger proteins have the capacity to mediate ubiquitination, it is also the case that the RING fingers of these proteins vary substantially in sequence (
      • Lorick K.L.
      • Jensen J.P.
      • Fang S.
      • Ong A.M.
      • Weissman A.M.
      ). This raises the possibility that, in addition to serving as regions that interact with and activate E2s, some degree of substrate specificity also resides within the RING finger. Consistent with this, Mdm2 binds to the p53-related molecule p73 but does not target it for degradation (
      • Zeng X.
      • Chen L.
      • Jost C.A.
      • Maya R.
      • Keller D.
      • Wang X.
      • Kaelin W.G.J.
      • Oren M.
      • Chen J.
      • Lu H.
      ,
      • Dobbelstein M.
      • Wienzek S.
      • Konig C.
      • Roth J.
      ,
      • Balint E.
      • Bates S.
      • Vousden K.H.
      ). Therefore, physical association alone is apparently not sufficient to target Mdm2-associated proteins for degradation. One explanation for this is that the tertiary structure of the Mdm2 RING specifically facilitates the juxtaposition of a RING-associated complex of E2 and Ub with target lysines in p53. The finding that Mdm2PR mediates its own ubiquitination in vitroand proteasomedependent degradation in cells, but is ineffective in ubiquitinating p53 in vitro or in targeting p53 for degradation in cells, is in agreement with the concept of substrate specificity at the level of the RING. An alternative explanation for the failure of Mdm2PR to ubiquitinate p53 is that unanticipated steric constraints generated by fusing the Praja1 RING to Mdm2 through the Praja1-derived linker precludes access to target lysines within p53. However, this latter possibility seems less likely as a second chimera, in which the Mdm2 RING was replaced by the Praja1 RING without the Praja1-derived linker, also targets itself, but not p53, for degradation in cells.
      S. Fang, K. H. Vousden, R. L. Ludwig, and A. M. Weissman, unpublished data.
      In addition to playing roles in ubiquitination, RING fingers have other cellular functions. The RING finger of PML has been implicated either directly or indirectly in its association with Ubc9 and in modification of PML with the Ub-like molecule Sumo-1 (
      • Duprez E.
      • Saurin A.J.
      • Desterro J.M.
      • Lallemand-Breitenbach V.
      • Howe K.
      • Boddy M.N.
      • Solomon E.
      • de The H.
      • Hay R.T.
      • Freemont P.S.
      ). Zinc-coordinated RING fingers have been shown to mediate dimerization of proteins such as BARD1 and BRCA1, Mdm2 and MdmX, and constitutive photomorphogenic protein 1 and CIP8 (
      • Meza J.E.
      • Brzovic P.S.
      • King M.C.
      • Klevit R.E.
      ,
      • Tanimura S.
      • Ohtsuka S.
      • Mitsui K.
      • Shirouzu K.
      • Yoshimura A.
      • Ohtsubo M.
      ,
      • Torii K.U.
      • Stoop-Myer C.D.
      • Okamoto H.
      • Coleman J.E.
      • Matsui M.
      • Deng X.W.
      ). Recently a RING finger-like domain, the FYVE domain, has been determined to bind specifically phosphatidylinositol 3-phosphate (
      • Kutateladze T.G.
      • Kenyon D.
      • Ogburn K.D.
      • Watson W.T.
      • Beer T.D.
      • Emr S.D.
      • Burd C.G.
      • Overduin M.
      ). The FYVE domain contains a highly conserved motif that provides specificity for binding to phosphatidylinositol 3-phosphate (
      • Kutateladze T.G.
      • Kenyon D.
      • Ogburn K.D.
      • Watson W.T.
      • Beer T.D.
      • Emr S.D.
      • Burd C.G.
      • Overduin M.
      ). This ability to bind phosphatidylinositol 3-phosphate is also dependent on zinc coordination, and the removal of zinc results in the unfolding of this domain (
      • Kutateladze T.G.
      • Kenyon D.
      • Ogburn K.D.
      • Watson W.T.
      • Beer T.D.
      • Emr S.D.
      • Burd C.G.
      • Overduin M.
      ). In the case of Mdm2 the RING finger not only mediates ubiquitination (this study) but also binds to RNA (
      • Lai Z.
      • Freedman D.A.
      • Levine A.J.
      • McLendon G.L.
      ). Notably, this RNA binding has been found to occur whether or not zinc is present (
      • Lai Z.
      • Freedman D.A.
      • Levine A.J.
      • McLendon G.L.
      ). An interesting question that now arises is whether the physical association of Mdm2 with RNA affects the ability of this RING finger protein to mediate ubiquitination.
      The mdm2 proto-oncogene is overexpressed and amplified in a variety of human tumors (
      • Freedman D.A.
      • Wu L.
      • Levine A.J.
      ). The tumorigenic activity associated with a high level of Mdm2 may be due to its ability to target p53 for degradation, leading to inhibition of p53-induced cell growth arrest and apoptosis (
      • Kubbutat M.H.
      • Vousden K.H.
      ). Mdm2 has also been found to induce spontaneous tumorigenesis independent of p53 (
      • Jones S.N.
      • Hancock A.R.
      • Vogel H.
      • Donehower L.A.
      • Bradley A.
      ). Identification of the requirement for a zinc-coordinated RING finger in the E3 activity of Mdm2 toward p53 may provide a potential new target for assessing the tumorigenic activity of Mdm2 and for re-activation of p53 in tumor therapy.

      Acknowledgments

      We thank Drs. P. Howley, J. Huibregtse, K. Iwai, L. Mishra, and R. Vierstra for reagents; Dr. J. D. Ashwell for critical review of the manuscript; Drs. J. D. Ashwell, K. L. Lorick, A. Magnifico, S. Twari, and Y. L. Yang for helpful discussions.

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