title: Phosphomimetic mutation of the N-terminal lid of MDM2 enhances the polyubiquitination of p53 through stimulation of E2-ubiquitin thioester hydrolysis.
abstract: Mouse double minute 2 (MDM2) has a phosphorylation site within a lid motif at Ser17 whose phosphomimetic mutation to Asp17 stimulates MDM2-mediated polyubiquitination of p53. MDM2 lid deletion, but not Asp17 mutation, induced a blue shift in the λ(max) of intrinsic fluorescence derived from residues in the central domain including Trp235, Trp303, Trp323, and Trp329. This indicates that the Asp17 mutation does not alter the conformation of MDM2 surrounding the tryptophan residues. In addition, Phe235 mutation enhanced MDM2 binding to p53 but did not stimulate its ubiquitination function, thus uncoupling increases in p53 binding from its E3 ubiquitin ligase function. However, the Asp17 mutation in MDM2 stimulated its discharge of the UBCH5a-ubiquitin thioester adduct (UBCH5a is a ubiquitin-conjugating enzyme E2D 1 UBC4/5 homolog yeast). This stimulation of ubiquitin discharge from E2 was independent of the p53 substrate. There are now four known effects of the Asp17 mutation on MDM2: (i) it alters the conformation of the isolated N-terminus as defined by NMR; (ii) it induces increased thermostability of the isolated N-terminal domain; (iii) it stimulates the allosteric interaction of MDM2 with the DNA-binding domain of p53; and (iv) it stimulates a novel protein-protein interaction with the E2-ubiquitin complex in the absence of substrate p53 that, in turn, increases hydrolysis of the E2-ubiquitin thioester bond. These data also suggest a new strategy to disrupt MDM2 function by targeting the E2-ubiquitin discharge reaction.
authors.affiliation: Institute of Genetics and Molecular Medicine, Cell Signalling Unit, Edinburgh Cancer Research Centre, University of Edinburgh, Edinburgh EH8 9YL, United Kingdom.
authors.forename: Jennifer A
authors.initials: JA
authors.lastname: Fraser
authors.affiliation: Institute of Genetics and Molecular Medicine, Cell Signalling Unit, Edinburgh Cancer Research Centre, University of Edinburgh, Edinburgh EH8 9YL, United Kingdom.
authors.forename: Erin G
authors.initials: EG
authors.lastname: Worrall
authors.affiliation: Institute of Genetics and Molecular Medicine, Cell Signalling Unit, Edinburgh Cancer Research Centre, University of Edinburgh, Edinburgh EH8 9YL, United Kingdom.
authors.forename: Yao
authors.initials: Y
authors.lastname: Lin
authors.affiliation: Institute of Genetics and Molecular Medicine, Cell Signalling Unit, Edinburgh Cancer Research Centre, University of Edinburgh, Edinburgh EH8 9YL, United Kingdom.
authors.forename: Vivien
authors.initials: V
authors.lastname: Landre
authors.affiliation: Institute of Genetics and Molecular Medicine, Cell Signalling Unit, Edinburgh Cancer Research Centre, University of Edinburgh, Edinburgh EH8 9YL, United Kingdom.
authors.forename: Susanne
authors.initials: S
authors.lastname: Pettersson
authors.affiliation: Edinburgh Centre for Chemical Biology, University of Edinburgh, Edinburgh EH8 9YL, United Kingdom.
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Blackburn
authors.affiliation: Edinburgh Centre for Chemical Biology, University of Edinburgh, Edinburgh EH8 9YL, United Kingdom.
authors.forename: Malcolm
authors.initials: M
authors.lastname: Walkinshaw
authors.affiliation: RECAMO, Masaryk Memorial Cancer Institute, 656 53 Brno, Czech Republic.
authors.forename: Petr
authors.initials: P
authors.lastname: Muller
authors.affiliation: RECAMO, Masaryk Memorial Cancer Institute, 656 53 Brno, Czech Republic.
authors.forename: Borek
authors.initials: B
authors.lastname: Vojtesek
authors.affiliation: Institute of Genetics and Molecular Medicine, Cell Signalling Unit, Edinburgh Cancer Research Centre, University of Edinburgh, Edinburgh EH8 9YL, United Kingdom.
authors.forename: Kathryn
authors.initials: K
authors.lastname: Ball
authors.affiliation: Institute of Genetics and Molecular Medicine, Cell Signalling Unit, Edinburgh Cancer Research Centre, University of Edinburgh, Edinburgh EH8 9YL, United Kingdom. Electronic address: Ted.Hupp@ed.ac.uk.
authors.forename: Ted R
authors.initials: TR
authors.lastname: Hupp
mesh_terms: D000494:Allosteric Regulation; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D045744:Cell Line, Tumor; D006801:Humans; D006868:Hydrolysis; D051379:Mice; D008969:Molecular Sequence Data; D017354:Point Mutation; D025821:Polyubiquitin / Q000378:metabolism*; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D051736:Proto-Oncogene Proteins c-mdm2 / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D016159:Tumor Suppressor Protein p53 / Q000737:chemistry / Q000378:metabolism*; D025801:Ubiquitin / Q000378:metabolism; D044763:Ubiquitin-Conjugating Enzymes / Q000378:metabolism*; D054875:Ubiquitination
chemical_list: D016159:Tumor Suppressor Protein p53; D025801:Ubiquitin; D025821:Polyubiquitin; C097308:UBE2D1 protein, human; D044763:Ubiquitin-Conjugating Enzymes; D051736:Proto-Oncogene Proteins c-mdm2
keywords: MDM2; RING domain; allostery; linear motifs; ubiquitination