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Interaction of p53 with MDM2 is independent of E6 and does not mediate wild type transformation suppressor function
N J Marston1, T Crook, K H Vousden
1Ludwig Institute for Cancer Research, St Mary's Hospital Medical School, London, UK.
Abstract:
Using a new series of p53 mutants targeting the conserved regions we have analysed the relationship of various activities of the protein. Mdm-2 and human papillomavirus (HPV) E6, two proteins which interact with and abrogate p53 function, were shown to bind independently. Deletion of the conserved regions of the protein in which most of the naturally occurring mutations are found (boxes II-V) abrogated transcriptional activity and the ability to interact with E6, supporting the importance of this DNA binding domain to these activities. Nevertheless, these mutants retained the ability to interact with mdm2. One mutant, deleted of all the C-terminal sequences, showed loss of mdm2 binding, E6 binding and transcriptional activity. More subtle mutations within the C-terminus of the protein, including alterations of the cdc2 and CKII phosphorylation sites, had no effect on the transcriptional trans-activation, mdm-2 or E6 binding functions, indicating that phosphorylation of these sites is not essential for these activities. Deletion of conserved box I sequences abolished the interaction with mdm-2 without loss of transcriptional activation or transformation suppressor activity, suggesting that mdm-2 is not a downstream effector of p53 function.
Insights
This study analyzes p53 protein functions using mutants. Key findings show that while some mutations disrupt DNA binding and E6 interaction, others do not affect mdm-2 binding, suggesting mdm-2 is not a direct downstream effector of p53.
Area of Science:
- Molecular Biology
- Cancer Research
- Protein Biochemistry
Background:
- The p53 protein is a critical tumor suppressor.
- Its function is regulated by interactions with proteins like Mdm-2 and HPV E6.
- Understanding these interactions is key to cancer biology.
Purpose of the Study:
- To investigate the relationship between different p53 protein activities.
- To analyze the role of conserved regions and C-terminal sequences in p53 function.
- To elucidate the interaction mechanisms of p53 with Mdm-2 and HPV E6.
Main Methods:
- Generation and analysis of a series of p53 mutants targeting conserved regions.
- Assessing transcriptional activity, Mdm-2 binding, and HPV E6 binding capabilities of mutants.
- Investigating the impact of specific mutations, including phosphorylation site alterations.
Main Results:
- Deletion of conserved regions (boxes II-V) abolished transcriptional activity and E6 binding but retained Mdm-2 binding.
- A C-terminal deletion mutant lost all tested functions (Mdm-2 binding, E6 binding, transcriptional activity).
- Mutations in C-terminal phosphorylation sites did not affect transcriptional activity, Mdm-2, or E6 binding.
Conclusions:
- The DNA binding domain (conserved regions II-V) is crucial for p53's transcriptional activity and E6 interaction.
- Mdm-2 interaction is independent of the DNA binding domain but dependent on Box I sequences.
- Phosphorylation of specific C-terminal sites is not essential for p53's transcriptional, Mdm-2, or E6 binding functions.
- Mdm-2 may not be a direct downstream effector of p53 function.