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Updated: Aug 8, 2026
Detection of Protein Ubiquitination
Published on: August 19, 2009
Multiple lysine mutations in the C-terminal domain of p53 interfere with MDM2-dependent protein degradation and
S Nakamura1, J A Roth, T Mukhopadhyay
1Section of Thoracic Molecular Oncology, Departments of Thoracic and Cardiovascular Surgery, The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030, USA.
Abstract:
To investigate the effect of mutations in the p53 C-terminal domain on MDM2-mediated degradation, we introduced single and multiple point mutations into a human p53 cDNA at four putative acetylation sites (amino acid residues 372, 373, 381, and 382). Substitution of all four lysine residues by alanines (the A4 mutant) and single lysine-to-alanine substitutions were functional in sequence-specific DNA binding and transactivation; however, the A4 mutant protein was resistant to MDM2-mediated degradation, whereas the single lysine substitutions were not. Although the A4 mutant protein and the single lysine substitutions both bound MDM2 reasonably well, the single lysine substitutions underwent normal MDM2-dependent ubiquitination, whereas the A4 protein was inefficiently ubiquitinated. In addition, the A4 mutant protein was found in the cytoplasm as well as in the nucleus of a subpopulation of cells, unlike wild-type p53, which is mostly nuclear. The partially cytoplasmic distribution of A4 mutant protein was not due to a defect in nuclear import because inhibition of nuclear export by leptomycin B resulted in nuclear accumulation of the protein. Taken together, the data suggest that mutations in the putative acetylation sites of the p53 C-terminal domain interfere with ubiquitination, thereby regulating p53 degradation.
Insights
Mutations in the p53 C-terminal domain
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- The p53 protein is a tumor suppressor crucial for cellular response to stress.
- MDM2 is a key regulator of p53 stability, promoting its degradation.
- Post-translational modifications, like acetylation, can influence p53 stability and function.
Purpose of the Study:
- To investigate how mutations at putative p53 acetylation sites affect MDM2-mediated degradation.
- To determine the role of specific lysine residues in the p53 C-terminal domain in regulating p53 stability.
Main Methods:
- Introduction of single and multiple point mutations into human p53 cDNA at residues 372, 373, 381, and 382.
- Functional analysis of p53 mutants for DNA binding and transactivation.
- Assessment of MDM2-mediated degradation, ubiquitination, and subcellular localization of p53 mutants.
Main Results:
- The A4 mutant (all four lysines substituted with alanines) was resistant to MDM2-mediated degradation.
- Single lysine substitutions did not confer resistance to degradation.
- The A4 mutant showed inefficient ubiquitination and partially cytoplasmic localization, unlike wild-type p53.
- Nuclear import was not defective; nuclear export inhibition led to nuclear accumulation.
Conclusions:
- Mutations in p53 C-terminal acetylation sites disrupt ubiquitination, impacting p53 degradation.
- Acetylation sites are critical for regulating p53 stability and preventing aberrant cytoplasmic localization.
- Targeting these sites may offer novel strategies for modulating p53 stability in cancer therapy.
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