Multiple C-terminal Lysine residues target p53 for ubiquitin-proteasome-mediated degradation

Manuel S Rodriguez, Joana M P Desterro, Sonia Lain, David P. Lane, Ronald Thomas Hay

Research output: Contribution to journalArticlepeer-review

286 Citations (Scopus)

Abstract

In normal cells, p53 is maintained at a low level by ubiquitin-mediated proteolysis, but after genotoxic insult this process is inhibited and p53 levels rise dramatically. Ubiquitination of p53 requires the ubiquitin-activating enzyme Ubc5 as a ubiquitin conjugation enzyme and Mdm2, which acts as a ubiquitin protein ligase. In addition to the N-terminal region, which is required for interaction with Mdm2, the C-terminal domain of p53 modulates the susceptibility of p53 to Mdm2-mediated degradation. To analyze the role of the C-terminal domain in p53 ubiquitination, we have generated p53 molecules containing single and multiple lysine-to-arginine changes between residues 370 and 386. Although wild-type (WT) and mutant molecules show similar subcellular distributions, the mutants display a higher transcriptional activity than WT p53. Simultaneous mutation of lysine residues 370, 372, 373, 381, 382, and 386 to arginine residues (6KR p53 mutant) generates a p53 molecule with potent transcriptional activity that is resistant to Mdm2-induced degradation and is refractory to Mdm2-mediated ubiquitination. In contrast to WT p53, transcriptional activity directed by the 6KR p53 mutant fails to be negatively regulated by Mdm2. Those differences are also manifest in HeLa cells which express the human papillomavirus E6 protein, suggesting that p53 C-terminal lysine residues are also implicated in E6-AP-mediated ubiquitination. These data suggest that p53 C-terminal lysine residues are the main sites of ubiquitin ligation, which target p53 for proteasome-mediated degradation.

Original languageEnglish
Pages (from-to)8458-8467
Number of pages10
JournalMolecular and Cellular Biology
Volume20
Issue number22
DOIs
Publication statusPublished - Nov 2000

Keywords

  • Tumor-suppressor p53
  • Human-papillomavirus e6
  • Nuclear Export signal
  • DNA-binding domain
  • Casein Kinase-II
  • In-vivo
  • Sumo-1 modification
  • Oncoprotein mdm2
  • Activates p53
  • Protein

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