MOLECULAR BASIS OF P53 INDUCTION TO DNA DAMAGE

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Mdm2 plays a very crucial role in the regulation of p53 stability and p53-induction in response to DNA damage. The binding of Mdm2 to p53 is required for targeting p53 for degradation. p73, however, binds to Mdm2 but is resistant to degradation by Mdm2 and to the induction by DNA damage, indicating that binding to Mdm2 is not sufficient for degradation and there must be a sequence unique to p53 that determines its response to Mdm2-mediated degradation and to DNA damage. By utilizing the structural homology between p53 and p73, we generated p53-p73 chimeras, where each of p53 domains was replaced by the corresponding region of p73, to determine the sequence element of p53 essential for regulation of its stability. We found that replacing the amino acid 92-112 of p53 abolishes its ability to respond to Mdm2-mediated degradation and to the induction by damage. Significantly, the finding that swapping this fragment converts p73 from refractory into sensitive to Mdm2-mediated degradation and induction by DNA damage supports that the amino acid 92-112 of p53 is an essential element in the regulation of its stability and response to DNA damage. Preliminary studies also provide compelling evidences to indicate a presence of additional protein(s) that associates with the element and is essential for Mdm2-mediated degradation. This proposal aims at identifying the protein(s) and investigating its role in Mdm2-mediated degradation and in DNA damage-induced p53 accumulation. Potential involvement of the p53 determinant in the regulation of p53 nuclear import/export will also be examined. Results obtained from the proposed work will likely provide insights into mechanism of how p53 stability is regulated and how p53 is induced in response to DNA damage. We also propose to screen for inhibitors of Mdm2-mediated p53 degradation using our in vitro degradation assay system. The identified protein(s) and the inhibitors can be potential candidate for the development of therapeutic agent to reactivate p53 activity in cancer cells. The Specific Aims include: 1) to identify the p53 determinant-interacting protein essential for Mdm2-mediated p53 degradation and to examine role of the p53 determinant in regulation of p53 nuclear import/export: 2) To investigate role of the protein in Mdm2-targeted p53 degradation and p53-induction in response to DNA damage: 3) to search for inhibitors of p53 degradation by screening small compounds using our in vitro degradation assay system.