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Detection and Visualization of DNA Damage-induced Protein Complexes in Suspension Cell Cultures Using the Proximity Ligation Assay
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ATM-dependent activation of p53 involves dephosphorylation and association with 14-3-3 proteins

M J Waterman1, E S Stavridi, J L Waterman

  • 1Department of Molecular Genetics, The Wistar Institute, Philadelphia, Pennsylvania 19104-4268, USA.

Nature Genetics
|June 10, 1998
PubMed

Insights

DNA damage activates the p53 tumor suppressor protein by dephosphorylating Ser376, which enhances its DNA binding affinity. This mechanism is crucial for eliminating damaged cells and is impaired in ataxia telangiectasia.

Area of Science:

  • Molecular Biology
  • Cellular Biology
  • Cancer Research

Background:

  • The p53 protein is a crucial tumor suppressor involved in cell cycle arrest and apoptosis following genotoxic stress.
  • Its activation by DNA damage is vital for eliminating cells with damaged DNA and for cancer treatment responses.
  • The precise molecular mechanisms underlying DNA damage-induced p53 activation remain unclear.

Purpose of the Study:

  • To investigate the molecular mechanisms by which DNA damage activates the p53 transcription factor in vivo.
  • To determine if the carboxy-terminus of p53, known to regulate DNA binding in vitro, is targeted by DNA-damage signaling pathways.
  • To elucidate the role of specific p53 phosphorylation sites in response to genotoxic stress.

Main Methods:

  • Analysis of p53 protein phosphorylation status in irradiated and non-irradiated cells.
  • Investigation of the interaction between p53 and 14-3-3 proteins following DNA damage.
  • Comparison of p53 activation pathways in normal cells versus ataxia telangiectasia (AT) cells.

Main Results:

  • Ionizing radiation (IR) induced dephosphorylation of serine 376 (Ser376) on the p53 protein.
  • Dephosphorylation of Ser376 created a binding site for 14-3-3 proteins, leading to p53-14-3-3 complex formation.
  • This complex formation increased the affinity of p53 for sequence-specific DNA, enhancing its transcriptional activity.
  • In AT cells, IR failed to induce Ser376 dephosphorylation or p53-14-3-3 interaction, indicating a defect in the DNA damage response pathway.

Conclusions:

  • DNA damage signaling pathways target the carboxy-terminus of p53, specifically regulating Ser376 phosphorylation.
  • Dephosphorylation of Ser376 is a key event for p53 activation, promoting its DNA binding and transcriptional function.
  • The observed defects in AT cells highlight the critical role of this pathway in maintaining genomic integrity and suggest therapeutic implications for cancer treatment.

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