title: MDM2 protein-mediated ubiquitination of numb protein: identification of a second physiological substrate of MDM2 that employs a dual-site docking mechanism.
abstract: The E3 ubiquitin ligase, MDM2, uses a dual-site mechanism to ubiquitinate and degrade the tumor suppressor protein p53, involving interactions with the N-terminal hydrophobic pocket and the acidic domain of MDM2. The results presented here demonstrate that MDM2 also uses this same dual-site mechanism to bind to the cell fate determinant NUMB with both the N-terminal hydrophobic pocket and the acidic domain of MDM2 also involved in forming the interaction with NUMB. Furthermore, the acidic domain interactions are crucial for MDM2-mediated ubiquitination of NUMB. Contrary to p53, where two separate domains form the interface with MDM2, only one region within the phosphotyrosine binding domain of NUMB (amino acids 113-148) mediates binding to both these regions of MDM2. By binding to both domains on MDM2, NUMB disrupts the MDM2-p53 complex and MDM2-catalyzed ubiquitination of p53. Therefore, we have identified the mechanism NUMB uses to regulate the steady-state levels of the p53 in cells. By targeting the acidic domain of MDM2 using acid domain-binding ligands we can overcome MDM2-mediated ubiquitination and degradation of NUMB impacting on the stabilization of p53 in cells. Furthermore, delivery of MDM2 acid domain-binding ligands to cancer cells promotes p53-dependent growth arrest and the induction of apoptosis. This highlights the dual-site mechanism of MDM2 on another physiological substrate and identifies the acid domain as well as N terminus as a potential target for small molecules that inhibit MDM2.
authors.affiliation: The Roslin Institute and Royal Dick School of Veterinary Science, University of Edinburgh, Easter Bush EH25 9RG, United Kingdom.
authors.forename: Matylda
authors.initials: M
authors.lastname: Sczaniecka
authors.forename: Karen
authors.initials: K
authors.lastname: Gladstone
authors.forename: Susanne
authors.initials: S
authors.lastname: Pettersson
authors.forename: Lorna
authors.initials: L
authors.lastname: McLaren
authors.forename: Anne-Sophie
authors.initials: AS
authors.lastname: Huart
authors.forename: Maura
authors.initials: M
authors.lastname: Wallace
mesh_terms: D017209:Apoptosis; D001665:Binding Sites; D045744:Cell Line, Tumor; D019070:Cell Lineage; D049109:Cell Proliferation; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D005434:Flow Cytometry / Q000379:methods; D006801:Humans; D008024:Ligands; D008565:Membrane Proteins / Q000737:chemistry*; D009419:Nerve Tissue Proteins / Q000737:chemistry*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D051736:Proto-Oncogene Proteins c-mdm2 / Q000737:chemistry*; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism; D025801:Ubiquitin / Q000737:chemistry*; D044767:Ubiquitin-Protein Ligases / Q000737:chemistry; D054875:Ubiquitination
chemical_list: D008024:Ligands; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; C113026:NUMB protein, human; C495901:TP53 protein, human; D016159:Tumor Suppressor Protein p53; D025801:Ubiquitin; C497206:MDM2 protein, human; D051736:Proto-Oncogene Proteins c-mdm2; D044767:Ubiquitin-Protein Ligases