title: An iTRAQ proteomics screen reveals the effects of the MDM2 binding ligand Nutlin-3 on cellular proteostasis.
abstract: Mouse double minute 2 (MDM2) participates in protein synthesis, folding, and ubiquitin-mediated degradation and is therefore a proteostasic hub protein. The MDM2 interactome contains over 100 proteins, yet stratification of dominant MDM2-interacting proteins has not been achieved. 8-plex iTRAQ (nanoLC-MS/MS) of MCF7 cells treated with the MDM2-binding ligand Nutlin-3 identified the most abundant cellular protein changes over early time points; 1,323 unique proteins were identified including 35 with altered steady-state levels within 2 h of Nutlin-3 treatment, identifying a core group of MDM2 related proteins. Six of these proteins were previously identified MDM2 interactors, and the effects of Nutlin-3 on the MDM2-nucleophosmin interaction (NPM) was further validated. This revealed that Nutlin-3 mediates the in vivo conversion of NPM from an oligomer to a monomer as an MDM2-dependent phenomenon, with Nutlin-3 stimulating MDM2 binding to a peptide motif derived from the oligomerization interface of NPM. These data form the first proteomic screen of Nutlin-3 in cells whereby we (i) identify the most abundant MDM2-interacting proteins whose steady-state levels change early after Nutlin-3 treatment; (ii) identify the first protein apart from p53, nucleophosmin (NPM), whose interaction with MDM2 can be stimulated allosterically by Nutlin-3; and (iii) raise the possibility that Nutlin-3 might act as a general agonist of other MDM2 protein-protein interactions.
authors.affiliation: Cell Signalling Unit, p53 Signal Transduction Laboratories, Edinburgh Cancer Research Centre, University of Edinburgh, Crewe Road South, Edinburgh EH4 2XR, United Kingdom.
authors.forename: Judith
authors.initials: J
authors.lastname: Nicholson
authors.forename: Kalainanghi
authors.initials: K
authors.lastname: Neelagandan
authors.forename: Anne-Sophie
authors.initials: AS
authors.lastname: Huart
authors.forename: Kathryn
authors.initials: K
authors.lastname: Ball
authors.forename: Mark P
authors.initials: MP
authors.lastname: Molloy
authors.forename: Ted
authors.initials: T
authors.lastname: Hupp
mesh_terms: D001704:Biopolymers / Q000378:metabolism; D015153:Blotting, Western; D045744:Cell Line, Tumor; D002853:Chromatography, Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D007093:Imidazoles / Q000378:metabolism*; D008024:Ligands; D010879:Piperazines / Q000378:metabolism*; D011485:Protein Binding; D040901:Proteomics; D051736:Proto-Oncogene Proteins c-mdm2 / Q000378:metabolism*; D053719:Tandem Mass Spectrometry
chemical_list: D001704:Biopolymers; D007093:Imidazoles; D008024:Ligands; D010879:Piperazines; C482205:nutlin 3; C497206:MDM2 protein, human; D051736:Proto-Oncogene Proteins c-mdm2