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Functional analysis and consequences of Mdm2 E3 ligase inhibition in human tumor cells

Oncogene volume 31, pages 4789–4797 (2012)Cite this article

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Abstract

Mdm2 is the major negative regulator of p53 tumor-suppressor activity. This oncoprotein is overexpressed in many human tumors that retain the wild-type p53 allele. As such, targeted inhibition of Mdm2 is being considered as a therapeutic anticancer strategy. The N-terminal hydrophobic pocket of Mdm2 binds to p53 and thereby inhibits the transcription of p53 target genes. Additionally, the C-terminus of Mdm2 contains a RING domain with intrinsic ubiquitin E3 ligase activity. By recruiting E2 ubiquitin-conjugating enzyme(s), Mdm2 acts as a molecular scaffold to facilitate p53 ubiquitination and proteasome-dependent degradation. Mdmx (Mdm4), an Mdm2 homolog, also has a RING domain and hetero-oligomerizes with Mdm2 to stimulate its E3 ligase activity. Recent studies have shown that C-terminal residues adjacent to the RING domain of both Mdm2 and Mdmx contribute to Mdm2 E3 ligase activity. However, the molecular mechanisms mediating this process remain unclear, and the biological consequences of inhibiting Mdm2/Mdmx co-operation or blocking Mdm2 ligase function are relatively unexplored. This study presents biochemical and cell biological data that further elucidate the mechanisms by which Mdm2 and Mdmx co-operate to regulate p53 level and activity. We use chemical and genetic approaches to demonstrate that functional inhibition of Mdm2 ubiquitin ligase activity is insufficient for p53 activation. This unexpected result suggests that concomitant treatment with Mdm2/Mdmx antagonists may be needed to achieve therapeutic benefit.

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Acknowledgements

This work was funded by a National Institutes of Health R01 Grant CA61449 and a Discovery Research Grant from sanofi-aventis awarded to GMW. We thank all members of the Wahl lab for input on various aspects of this project, Dimitris Xirodimas (Dundee University) for advice on the ubiquitylation assays and Masha Poyurovsky (Columbia University) for productive discussions. We also thank Rachel Klevit and Chris Pierini (University of Washington) for their comments and insight into ubiquitylation during this project.

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  1. S M G Braun

    Present address: 4Current address: Institute of Cell Biology, Swiss Federal Institute of Technology (ETH), CH-8093 Zurich, Switzerland,

Authors and Affiliations

  1. Gene Expression Laboratory, Salk Institute for Biological Studies, La Jolla, CA, USA

    M Wade, Y C Li, A S Matani, S M G Braun, L W Rodewald & G M Wahl

  2. Center for Genomic Science of IIT@SEMM, Milan, Italy

    M Wade & F Milanesi

  3. Division of Biological Sciences, University of California, San Diego, CA, USA

    A S Matani

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Wade, M., Li, Y., Matani, A. et al. Functional analysis and consequences of Mdm2 E3 ligase inhibition in human tumor cells. Oncogene 31, 4789–4797 (2012). https://doi.org/10.1038/onc.2011.625

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