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Role of Mdm2 acid domain interactions in recognition and ubiquitination of the transcription factor IRF-2

Susanne Pettersson1, Michael Kelleher, Emmanuelle Pion

  • 1CRUK Interferon and Cell Signalling Group, Cell Signalling Unit, IGMM, University of Edinburgh, Edinburgh, UK.

The Biochemical Journal
|November 27, 2008
PubMed

Insights

Murine double minute 2 (Mdm2) ligase targets p53 for ubiquitination via two binding sites. Researchers identified Interferon regulatory factor-2 (IRF-2) as a novel Mdm2 substrate, confirming dual-site interaction is crucial for Mdm2 E3-ligase activity.

Area of Science:

  • * Molecular Biology
  • * Biochemistry
  • * Cancer Research

Background:

  • * Murine double minute 2 (Mdm2) is an E3 ubiquitin ligase that targets the p53 tumor suppressor for ubiquitination.
  • * Mdm2-mediated p53 ubiquitination requires interaction at two distinct binding sites on p53, involving its transactivation and DNA-binding domains.
  • * The acid domain of Mdm2 plays a critical role in recognizing a ubiquitination signal within p53's core structure.

Purpose of the Study:

  • * To identify novel Mdm2 substrates and elucidate the fundamental requirements for Mdm2-mediated ubiquitination.
  • * To investigate the role of Mdm2's acid domain in substrate recognition and E3-ligase activity.
  • * To explore the implications of dual-site substrate interaction for Mdm2 E3-ligase function.

Main Methods:

  • * Bioinformatics search for proteins with homology to Mdm2-binding sites in p53.
  • * Biochemical assays to assess Mdm2-IRF-2 interactions and ubiquitination.
  • * Site-directed mutagenesis of Mdm2-binding sites on IRF-2.
  • * Cellular complex formation assays between Mdm2 and IRF-2.

Main Results:

  • * Interferon regulatory factor-2 (IRF-2) was identified as a novel Mdm2-binding protein and substrate.
  • * IRF-2 requires interaction with both the hydrophobic pocket and the acid domain of Mdm2 for ubiquitination.
  • * Mutation of either Mdm2-binding site on IRF-2 attenuated substrate ubiquitination, confirming a dual-site mechanism.
  • * Ligands binding to the hydrophobic pocket alone were insufficient to inhibit Mdm2 activity, while acid domain ligands acted as inhibitors.

Conclusions:

  • * A dual-site substrate interaction mechanism is essential for Mdm2 E3-ligase activity.
  • * The acid domain of Mdm2 is a key determinant of its E3-ligase inhibitory activity.
  • * IRF-2 represents a novel substrate for studying E3 ubiquitin ligase-substrate interactions in vitro and in cells.

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