Bivalent binding of p14ARF to MDM2 RING and acidic domains inhibits E3 ligase function

Kowalczyk, D., Nakasone, M. A., Smith, B. O. and Huang, D. T. (2022) Bivalent binding of p14ARF to MDM2 RING and acidic domains inhibits E3 ligase function. Life Science Alliance, 5(12), e202201472. (doi: 10.26508/lsa.202201472) (PMID:35944929) (PMCID:PMC9366199)

Abstract

ARF tumor suppressor protein is a key regulator of the MDM2-p53 signaling axis. ARF interferes with MDM2-mediated ubiquitination and degradation of p53 by sequestering MDM2 in the nucleolus and preventing MDM2-p53 interaction and nuclear export of p53. Moreover, ARF also directly inhibits MDM2 ubiquitin ligase (E3) activity, but the mechanism remains elusive. Here, we apply nuclear magnetic resonance and biochemical analyses to uncover the mechanism of ARF-mediated inhibition of MDM2 E3 activity. We show that MDM2 acidic and zinc finger domains (AD-ZnF) form a weak intramolecular interaction with the RING domain, where the binding site overlaps with the E2∼ubiquitin binding surface and thereby partially reduces MDM2 E3 activity. Binding of human N-terminal 32 residues of p14ARF to the acidic domain of MDM2 strengthens the AD-ZnF-RING domain interaction. Furthermore, the N-terminal RxFxV motifs of p14ARF participate directly in the MDM2 RING domain interaction. This bivalent binding mode of p14ARF to MDM2 acidic and RING domains restricts E2∼ubiquitin recruitment and massively hinders MDM2 E3 activity. These findings elucidate the mechanism by which ARF inhibits MDM2 E3 activity.

• Item Type: Articles
• Additional Information: This work was supported by Beatson Institute core funding from Cancer Research UK (A17196 and A31287), Cancer Research UK funding to DT Huang (A23278/ A29256), and the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (grant agreement no 647849).
• Status: Published
• Refereed: Yes
• Glasgow Author(s) Enlighten ID: Kowalczyk, Dominika and Nakasone, Dr Mark and Huang, Professor Danny and Smith, Dr Brian
• Authors: Kowalczyk, D., Nakasone, M. A., Smith, B. O., and Huang, D. T.
• College/School: College of Medical Veterinary and Life Sciences > School of Cancer Sciences; College of Medical Veterinary and Life Sciences > School of Molecular Biosciences
• Journal Name: Life Science Alliance
• Publisher: Life Science Alliance
• ISSN: 2575-1077
• ISSN (Online): 2575-1077
• Published Online: 09 August 2022
• Copyright Holders: Copyright © 2022 Kowalczyk et al.
• First Published: First published in Life Science Alliance 5(12): e202201472
• Publisher Policy: Reproduced under a Creative Commons License