The long term objective of this laboratory is to understand the molecular and biochemical details of p53 regulation by its negative and positive regulators in response to physiological and pathological stimuli. Specifically, this proposal focuses on the MDM2-p53 autoregulatory feedback loop and its relationship with other positive p53 regulators. The core concept of the loop is that MDM2 is transcriptionally induced by p53 and in turn inhibits p53 function. In doing so, MDM2 is believed to utilize two main mechanisms: 1). Reducing p53 stability through a ubiquitin-mediated proteosome system; 2). Repressing p53 activity by probably """"""""interfering"""""""" with the interaction between p53 and its coactivators such as p300/CBP or PCAF. Although this model is currently accepted, its biochemical and molecular details are poorly understood. Some key questions still remain unanswered. For example, the """"""""interfering model"""""""" is challenged by our recent finding that MDM2 does not disrupt the p300-p53 interaction and instead forms a ternary complex with these proteins. Thus, how MDM2 represses p53 activity needs to be reinvestigated. Furthermore, p300/CBP acetylates the same C terminal lysine residues of p53 as those MDM2 ubiquitinates. This raises the possibility that these two types of modifications may be mutually exclusive. Indeed, our preliminary studies show that MDM2 inhibits p53 acetylation by p300/CBP and by PCAF, leading to p53 inactivation. However, it is unclear how MDM2 antagonizes these p300/CBP- or PCAF-mediated modifications and thus keep p53 in control. Therefore, a systematic and thorough investigation on these issues is not only necessary but also important for our better understanding of the MDM2-p53 feedback regulation. Compelling evidence from our initial studies provides a solid platform for our future research on these critical issues. Thus, we propose to address the two following specific aims using special approaches and quantitative methods established in this laboratory: 1. To determine how MDM2 negates p53 acetylation and activation by p300. We will determine whether forming a ternary complex with p300 and p53 is necessary for MDM2 to inhibit p53 acetylation. If so, we will assess whether acetylation of p53 by p300/CBP prevents its ubiquitination by MDM2 in vitro and in vivo, or vice versa. Finally, we will determine if the inhibition of p300-catalyzed p53 acetylation by MDM2 contributes to its repression activity on p53-dependent transcription in vivo and in cell-free transcription systems. 2. To determine the interplay between PCAF and MDM2 in p53 regulation. We will first determine if MDM2 inhibits p53 acetylation by PCAF in vivo. We will also determine whether the protein-protein interaction is required for the inhibition of PCAF-mediated p53 acetylation and activation by MDM2 and whether p14arf can reverse this inhibition. Finally, we will determine whether p300/CBP or PCAF needs to associate with p53 on its responsive DNA promoters, or only modify p53, to activate its activity, and whether MDM2 associates with promoter-bound p53 to repress its activity. Comparison between the results from aims 1 and 2 would offer important insight into the difference between MDM2-PACF and MDM2- p300 interplays in regulating p53 activity and/or stability. Also, the results from these studies will provide basic and practical information important for our understanding of cancer development as well as for anti-tumor drug designs.
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