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Comparative Strategies for Ubiquitination Detection in Mammalian Cell Lysates Using SMAD2/SMURF2 as a Model
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Comparative Strategies for Ubiquitination Detection in Mammalian Cell Lysates Using SMAD2/SMURF2 as a Model

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Mdm2 induces mono-ubiquitination of FOXO4

Arjan B Brenkman1, Peter L J de Keizer, Niels J F van den Broek

  • 1Department of Physiological Chemistry and Centre for Biomedical Genetics, University Medical Centre Utrecht, Utrecht, The Netherlands.

Plos One
|July 31, 2008
PubMed
Abstract

Insights

Mdm2, a known E3 ligase for p53, also targets Forkhead box O (FOXO) transcription factors for ubiquitination. This discovery reveals a new regulatory mechanism for FOXOs and highlights similarities in how FOXO and p53 are controlled.

Area of Science:

  • Molecular Biology
  • Cellular Biology
  • Biochemistry

Background:

  • Forkhead box O (FOXO) transcription factors regulate vital cellular processes like apoptosis and cell cycle progression.
  • FOXO proteins function as tumor suppressors and influence aging in model organisms.
  • Similarities exist between FOXO and p53 regulatory proteins, including shared de-ubiquitinating enzymes like USP7.

Purpose of the Study:

  • To investigate the role of Mdm2, an E3 ligase primarily known for p53 regulation, in the ubiquitination of FOXO transcription factors.
  • To elucidate the mechanism by which Mdm2 interacts with and modifies FOXO proteins.
  • To explore the functional consequences of Mdm2-mediated FOXO ubiquitination on FOXO transcriptional activity.

Main Methods:

  • In vitro ubiquitination assays using purified Mdm2 and FOXO proteins.
  • In vivo co-expression studies in cells to assess FOXO ubiquitination.
  • siRNA-mediated knockdown of Mdm2 to evaluate its role in regulating FOXO ubiquitination.
  • Co-immunoprecipitation assays to confirm direct interaction between Mdm2 and FOXO.
  • Analysis of FOXO transcriptional activity following Mdm2-mediated ubiquitination.

Main Results:

  • Mdm2 directly ubiquitinates FOXO proteins in an ATP-dependent manner, similar to its action on p53.
  • Co-expression of Mdm2 and FOXO in vivo leads to FOXO mono-ubiquitination.
  • Depletion of Mdm2 reduces hydrogen peroxide-induced FOXO mono-ubitination.
  • Mdm2 and FOXO proteins co-immunoprecipitate, indicating a direct interaction.
  • Mdm2-mediated ubiquitination impacts FOXO transcriptional activity.

Conclusions:

  • Mdm2 is identified as a novel E3 ligase for FOXO transcription factors.
  • These findings establish an analogous regulatory pathway for FOXO proteins, mirroring that of p53.
  • The study expands the understanding of post-translational modification and regulation of FOXO factors.

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