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14-3-3gamma binds to MDMX that is phosphorylated by UV-activated Chk1, resulting in p53 activation

Yetao Jin1, Mu-Shui Dai, Steven Z Lu

  • 1Department of Biochemistry and Molecular Biology, Oregon Health and Science University, Portland, OR 97239, USA.

The EMBO Journal
|March 3, 2006
PubMed

Insights

14-3-3gamma and Chk1 overcome MDMX inhibition of tumor suppressor p53. This study identifies these proteins as novel regulators of MDMX activity, particularly in response to UV irradiation, impacting p53 levels and cell cycle arrest.

Area of Science:

  • Molecular Biology
  • Cell Biology
  • Oncology

Background:

  • MDMX (also known as MDM4) inhibits the tumor suppressor p53, often in conjunction with MDM2.
  • Understanding regulators of MDMX is crucial for developing cancer therapies targeting the p53 pathway.

Purpose of the Study:

  • To identify novel proteins that regulate MDMX activity.
  • To investigate the role of 14-3-3gamma and Chk1 in modulating the MDMX-p53 interaction.
  • To elucidate the mechanism by which MDMX inhibition of p53 can be overcome.

Main Methods:

  • Immuno-affinity purification coupled with mass spectrometry to identify MDMX-associated proteins.
  • In vitro and in-cell binding assays to confirm protein interactions.
  • Site-directed mutagenesis and kinase assays to study phosphorylation events.
  • UV irradiation, siRNA-mediated gene silencing, and Western blotting to assess protein levels and cellular responses.
  • Overexpression studies of wild-type and mutant proteins.

Main Results:

  • 14-3-3gamma was identified as an MDMX-binding protein, with interaction enhanced by MDMX phosphorylation.
  • Chk1 phosphorylates MDMX at serine 367 in response to UV irradiation, promoting 14-3-3gamma binding and MDMX cytoplasmic retention.
  • Overexpression of 14-3-3gamma suppressed MDMX-mediated p53 ubiquitination, leading to p53 stabilization and activation.
  • Ablation of 14-3-3gamma reduced UV-induced p53 levels and G1 cell cycle arrest.

Conclusions:

  • 14-3-3gamma and Chk1 are novel regulators of MDMX.
  • These proteins counteract MDMX-mediated inhibition of p53, particularly under UV stress.
  • The findings reveal a new mechanism for controlling p53 activity via MDMX modulation.

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