Related Experiment Videos
Regulation of Mdm2-directed degradation by the C terminus of p53
M H Kubbutat1, R L Ludwig, M Ashcroft
1ABL-Basic Research Program, National Cancer Institute-Frederick Cancer Research and Development Center, Frederick, Maryland 21702-1201, USA.
Abstract:
The stability of the p53 tumor suppressor protein is regulated by interaction with Mdm2, the product of a p53-inducible gene. Mdm2-targeted degradation of p53 depends on the interaction between the two proteins and is mediated by the proteasome. We show here that in addition to the N-terminal Mdm2 binding domain, the C terminus of p53 participates in the ability of p53 to be degraded by Mdm2. In contrast, alterations in the central DNA binding domain of p53, which change the conformation of the p53 protein, do not abrogate the sensitivity of the protein to Mdm2-mediated degradation. The importance of the C-terminal oligomerization domain to Mdm2-targeted degradation of p53 is likely to reflect the importance of oligomerization of the full-length p53 protein for interaction with Mdm2, as previously shown in vitro. Interestingly, the extreme C-terminal region of p53, outside the oligomerization domain, was also shown to be necessary for efficient degradation, and deletion of this region stabilized the protein without abrogating its ability to bind to Mdm2. Mdm2-resistant p53 mutants were not further stabilized following DNA damage, supporting a role for Mdm2 as the principal regulator of p53 stability in cells. The extreme C terminus of the p53 protein has previously been shown to contain several regulatory elements, raising the possibility that either allosteric regulation of p53 by this domain or interaction between this region and a third protein plays a role in determining the sensitivity of p53 to Mdm2-directed degradation.
Insights
The C terminus of p53, not just the Mdm2 binding domain, is crucial for Mdm2-mediated degradation. This finding highlights the C terminus
Area of Science:
- Molecular Biology
- Cell Biology
- Cancer Research
Background:
- p53 tumor suppressor protein stability is tightly regulated by its interaction with Mdm2.
- Mdm2, a p53-inducible gene product, targets p53 for proteasomal degradation.
- The N-terminal Mdm2 binding domain of p53 is known to be critical for this interaction.
Purpose of the Study:
- To investigate the role of other p53 domains in Mdm2-mediated degradation.
- To determine if alterations in the DNA binding domain affect p53 sensitivity to Mdm2.
- To elucidate the contribution of the C-terminal region to p53 stability regulation.
Main Methods:
- Analysis of p53 mutants with alterations in the N-terminal, central DNA binding, and C-terminal domains.
- Assessment of p53 degradation by Mdm2 in the presence of these mutations.
- Evaluation of p53-Mdm2 binding affinity in vitro and in cells.
- Investigation of p53 stability following DNA damage in Mdm2-resistant mutants.
Main Results:
- The C terminus of p53, in addition to the N-terminal binding domain, is essential for Mdm2-targeted degradation.
- Conformational changes in the central DNA binding domain do not abolish p53's sensitivity to Mdm2.
- The extreme C-terminal region, outside the oligomerization domain, is necessary for efficient degradation; its deletion stabilizes p53 without affecting Mdm2 binding.
- Mdm2-resistant p53 mutants show no further stabilization after DNA damage, reinforcing Mdm2's role in regulating p53 stability.
Conclusions:
- p53 stability is regulated by both its N-terminal and C-terminal domains interacting with Mdm2.
- The C-terminal oligomerization domain and the extreme C-terminal region are critical for Mdm2-mediated p53 degradation.
- The C terminus may be involved in allosteric regulation or interaction with other proteins to control p53 sensitivity to Mdm2.