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Purification of Ubiquitinated p53 Proteins from Mammalian Cells
10:55

Purification of Ubiquitinated p53 Proteins from Mammalian Cells

Published on: March 21, 2022

Modulation of p53 C-terminal acetylation by mdm2, p14ARF, and cytoplasmic SirT2

Ingeborg M M van Leeuwen1, Maureen Higgins, Johanna Campbell

  • 1Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Nobels väg 16, Stockholm 171 77, Sweden.

Molecular Cancer Therapeutics
|February 19, 2013
PubMed

Insights

p14ARF enhances p53 acetylation at lysine 382, particularly when mdm2 is present. This suggests p14ARF sequesters p53-mdm2 complexes on chromatin, preventing deacetylation by enzymes like SirT2.

Area of Science:

  • Molecular Biology
  • Cancer Research
  • Epigenetics

Background:

  • Acetylation of the p53 tumor suppressor at C-terminal lysine residues enhances its stability and transcriptional activity.
  • The p53 protein's function, level, and acetylation are negatively regulated by mdm2, which is itself inhibited by the p14ARF tumor suppressor.

Purpose of the Study:

  • To investigate the effect of p14ARF on the acetylation status of p53, specifically at lysine 382.
  • To elucidate the mechanism by which p14ARF influences p53 acetylation and stability in the context of mdm2 interaction.

Main Methods:

  • Analysis of p53 acetylation levels at lysine 382 in nuclear chromatin-rich fractions.
  • Experimental manipulation involving ectopic expression of mdm2 and p14ARF.
  • Investigation of p53 deacetylation in cytoplasmic fractions and identification of catalyzing sirtuins.

Main Results:

  • p14ARF increases the level of p53 acetylated at lysine 382 (p53AcK382) within the nuclear chromatin-rich fraction.
  • The accumulation of p53AcK382 is unexpectedly enhanced in the presence of ectopic mdm2.
  • p53AcK382 can be deacetylated in the cytoplasm, a reaction catalyzed by sirtuin SirT2.

Conclusions:

  • p14ARF may increase the binding of p53-mdm2 complexes to chromatin, limiting deacetylase access to p53.
  • These findings suggest a mechanism where p14ARF promotes p53 acetylation and stability by chromatin sequestration.
  • Understanding the role of SirT2 in p53 deacetylation highlights the need to inhibit both SirT1 and SirT2 for effective p53 activation by small-molecule inhibitors.

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