The p53 tumor suppressor protein, is a key cellular regulatory component, that participate in controlling cell growth and death. As an essential determinant in p53's ability to mediate its multiple functions lies in its stability, we are studying the mechanisms that regulate p53 stability. We propose to test the hypothesis that the interplay between mdm2 and TAFs is an important regulator of p53 stability Being part of the transcription initiation complex, TAF displacement of mdm2 is expected to confer transcriptional activity of p53. It has been shown that the p53 interacting protein, mdm2, destabilizes p53 and our preliminary results indicate that mdm2 targets p53 ubiquitination. P53 stability is also affected by another interacting molecule, JNK (Jun-N-terminal kinase), that targets the ubiquitination and degradation of p53 in an mdm2-independent manner. The hypothesis that p53 stability is determined by a delicate balance between mdm2 and TAFs is supported by the finding that over-expression of the p53 co-activator, TAFII31 stabilizes p53 in mammalian cells. TO test our hypothesis we propose to: 1) evaluate the interplay between TAFII31 and mdm2 towards p53 stability using our novel in ubiquitination and in vitro degradation systems; 2) use an in vivo ubiquitination system to monitor the contribution of mdm2/TAFII31 to p53 stability in cells derived from the ubiquitination system to monitor the contribution of mdm2/TAFII31 to p53 stability is cells derived from p53+/+, p53-/-, p53/mdm2-/- knockout mice under non-stressed and DNA damaging conditions; and 3) identify the domains of mdm2 and TAFs that mediate regulation of p53 stability and subsequently identify mdm2 and TAFs-derived peptides that could alter p53 stability in vitro and subsequently in vivo. In our fourth aim, we plan to explore the possible involvement of deregulated TAFII31 in tumorigenesis. Tumor cells with varying levels of TAF expression would provide a relevant physiologic resource to study the effect of mdm2-TAFs imbalance on p53 stability and function. Because p53 stability is control to its regulatory activities, understanding the mechanism underlying its stability will be necessary to decipher its action as a tumor suppressor.
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