The p53 tumor suppressor protein plays an important role in the cellular response to stress such as DNA damage. The biochemical activity of p53 that is required for this relies on its ability to bind to specific DNA sequences and to function as a transcription factor. Recently, we identified Thr-55 as a novel phosphorylation site on human p53 and showed that TAFII250, the largest subunit of transcription factor TFIID complex, is responsible for the phosphorylation. TAFII250 is identical to CCG1, a cell cycle regulatory protein important for progression through the G1 phase and apoptosis. We observed that the phosphorylation of Thr-55 by TAFII250 results in p53 degradation and leads to a decrease in G1 arrest. Importantly, the Thr-55 phosphorylation was much reduced following DNA damage, which suggests that this phosphorylation contributes to the stabilization of p53 in response to DNA damage. Thus, phosphorylation by TAFII250 and the resultant destabilization of p53 provide a novel mechanism for regulation of the cell cycle in response to DNA damage. We propose to characterize the molecular mechanisms by which TAFII250 regulates the stability of p53 in response to DNA damage. First, we will identify the domains of TAFII250 required for p53 interaction and phosphorylation and we will examine whether TAFII250 double bromodomains (DBrD) are required for the interaction. Second, we will further characterize the role of Thr-55 phosphorylation in TAFII250-mediated cell G1 progression and apoptosis. Third, as p53 is a DNA-bound transcription activator and TAFII250 is a component of general transcription machinery, we will examine whether phosphorylation of p53 by TAFII250 occurs during the act of transcription. Fourth, we will define the underlying molecular mechanisms of Thr-55 phosphorylation in the regulation of p53 by Mdm2. Finally, we will explore how DNA damage affects Thr-55 phosphorylation and explore the effect of other phosphorylation and acetylation on Thr-55 phosphorylation.
