p63 belongs to the emerging family of proteins that are homologous to the tumor suppressor protein p53. The importance of p53 can be seen from the fact that more than half of all human primary tumors contain mutations that inactivate p53. In cell culture experiments p63 binds to p53 sites and induces apoptosis. However, despite these similarities knock-out mouse studies have demonstrated that both proteins have very different biological functions. While p53 is a key player in cell cycle control, p63 is involved in the development of epithelial tissue. These results are surprising based on the fact that the DNA-binding domain is 65% identical with all amino acids known to be important for p53 DNA binding being conserved. The key to understanding the different functions of both proteins lies in their C-termini. The C-terminus of p53 forms an open, protease-sensitive domain of 26 amino acids, while p63 exists in three C-terminal isoforms differing in the length of their C-termini between 6 kD and 27 kD. These three different forms show remarkably different characteristics in their transactivation potential. The largest version of p63 (p63 with the α C-terminus) shows only very low activity and seems to mainly act as a negative regulator of transactivating p63 isoforms and possibly p53. In contrast, the other two forms (p63α and p63α) are transcriptionally active. Because the differences in the biological activities between p53 and p63 appear to be linked to the specific regulatory mechanisms of both proteins, we want to investigate how the α C-terminus regulates the transcriptional activity of p63 using a combination of cell biology, biochemistry and structure determination. So far we have identified the domain in p63 that is responsible for transcriptional suppression (transcriptional inhibitory domain or TID). Moreover, we have shown that this domain binds to the transactivation domain. In this grant application we propose to investigate the exact mechanism of transcriptional regulation. In the three specific aims described below we outline a sequence of experiments that will provide increasingly detailed structural information about the largest form of p63, TAp63α which contains both the transactivation domain as well as the inhibitory domain. Starting with biochemical experiments we will map the exact domain boundaries as well as structurally important amino acids. In a second step we will use cryo-electron microscopy in combination with chemical cross-linking to obtain a low resolution model of the entire complex. In the final step we want to improve the quality of our current crystals of the entire TAp63α tetramer to obtain a high resolution structure.
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