title: A second p53 binding site in the central domain of Mdm2 is essential for p53 ubiquitination.
abstract: Mdm2 negatively regulates p53 by inhibiting its transcriptional activity and promoting its degradation by functioning as an E3 ubiquitin ligase. The primary p53 binding site on mdm2 is located in its N-terminal domain. Through binding to p53 at its N-terminal transactivation domain, mdm2 directly blocks the transcriptional activation function of p53. We discovered that truncated mdm2 protein constructs without the N-terminal p53 binding domain are at least as active as full-length mdm2 in catalyzing p53 ubiquitination. Furthermore, the deletion of the central acidic domain significantly reduces the E3 ligase activity of mdm2 toward p53. We have also performed GST pull-down experiments to probe the direct binding of various mdm2 domain constructs toward full length p53 and found that mdm2 constructs without the N-terminal p53 binding domain retain the ability to bind to p53. Our kinetic and binding data localize the second p53 binding site between amino acids 211 and 361, including the acidic domain and the zinc finger region. Our work, consistent with other reports, suggests that the p53 tetramer interacts with at least two sites on mdm2. Although the interaction between the N-termini of mdm2 and p53 blocks the transactivation activity of p53, the interaction between the central domain of mdm2 and the core domain of p53 is critical for the ubiquitination and degradation of p53. This second mdm2-p53 interaction site represents an alternative target for small molecule modulators of the mdm2-p53 pathway.
authors.affiliation: Enzymology & Mechanistic Pharmacology, GlaxoSmithKline, 1250 South Collegeville Road, Collegeville, Pennsylvania 19426, USA.
authors.forename: Jianhong
authors.initials: J
authors.lastname: Ma
authors.forename: John D
authors.initials: JD
authors.lastname: Martin
authors.forename: Hong
authors.initials: H
authors.lastname: Zhang
authors.forename: Kurt R
authors.initials: KR
authors.lastname: Auger
authors.forename: Thau F
authors.initials: TF
authors.lastname: Ho
authors.forename: Robert B
authors.initials: RB
authors.lastname: Kirkpatrick
authors.forename: Michael H
authors.initials: MH
authors.lastname: Grooms
authors.forename: Kyung O
authors.initials: KO
authors.lastname: Johanson
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Tummino
authors.forename: Robert A
authors.initials: RA
authors.lastname: Copeland
authors.forename: Zhihong
authors.initials: Z
authors.lastname: Lai
mesh_terms: D000818:Animals; D001665:Binding Sites / Q000235:genetics; D002460:Cell Line; D003546:Cysteine Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007093:Imidazoles / Q000737:chemistry; D007700:Kinetics; D010879:Piperazines / Q000737:chemistry; D025941:Protein Interaction Mapping; D051736:Proto-Oncogene Proteins c-mdm2 / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017384:Sequence Deletion; D018411:Spodoptera; D016159:Tumor Suppressor Protein p53 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D025801:Ubiquitin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism
chemical_list: D007093:Imidazoles; D010879:Piperazines; D016159:Tumor Suppressor Protein p53; D025801:Ubiquitin; C482205:nutlin 3; D051736:Proto-Oncogene Proteins c-mdm2; D044767:Ubiquitin-Protein Ligases; D003546:Cysteine Endopeptidases; C118083:Ulp1 protease