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Multiple C-terminal lysine residues target p53 for ubiquitin-proteasome-mediated degradation
M S Rodriguez1, J M Desterro, S Lain
1School of Biology, University of St. Andrews, St. Andrews Fife KY16 9ST, Scotland, United Kingdom.
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.
Insights
The p53 C-terminal lysine residues are key sites for ubiquitination, controlling its degradation. Mutating these sites enhances p53 transcriptional activity and resistance to degradation by Mdm2.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- p53 protein levels are tightly regulated by ubiquitin-mediated proteolysis.
- Mdm2 acts as a ubiquitin ligase, targeting p53 for degradation.
- The C-terminal domain of p53 influences its stability and degradation.
Purpose of the Study:
- To investigate the role of p53 C-terminal lysine residues in ubiquitination and degradation.
- To analyze how mutations in these residues affect p53 transcriptional activity and stability.
Main Methods:
- Generated p53 mutants with lysine-to-arginine substitutions in the C-terminal domain (residues 370-386).
- Assessed subcellular distribution, transcriptional activity, and susceptibility to Mdm2-mediated degradation of WT and mutant p53.
- Examined p53 ubiquitination in HeLa cells expressing HPV E6 protein.
Main Results:
- p53 mutants exhibited higher transcriptional activity than wild-type (WT) p53.
- A specific mutant (6KR p53) showed potent transcriptional activity, resistance to Mdm2-induced degradation, and was refractory to Mdm2-mediated ubiquitination.
- The C-terminal lysine residues are implicated in both Mdm2- and E6-AP-mediated ubiquitination.
Conclusions:
- p53 C-terminal lysine residues are primary sites for ubiquitin ligation, targeting p53 for proteasomal degradation.
- Modulating these residues can enhance p53 stability and transcriptional function.
- These findings offer insights into p53 regulation and potential therapeutic strategies.