Background: The DNA-binding activity of p53 is essential to its function as a tumour suppressor. Point mutations that abolish this activity have been found to occur frequently in the p53 genes of human cancer cells. Wild-type p53 protein assembles into oligomers with latent DNA-binding activity that can be activated in vitro by phosphorylation of a carboxy-terminal regulatory region, catalyzed by protein kinase C or casein kinase II. We have investigated the mechanism underlying this post-translational regulation of p53. Specifically, we have asked the following questions. First, whether the carboxy-terminal regulatory site contributes to p53's ability to form tetramers. Second, whether the latent DNA-binding activity of p53 can be activated in vivo. And third, whether the activation of p53 is reversible.
Results: Biophysical molecular-sizing analysis shows that both latent and activated forms of p53 are tetramers. Using a novel method, we have further established that p53 remains tetrameric when bound to DNA. We have also found that p53 can indeed be activated in vivo: p53 prepared from cells can be separated into activated and latent forms. Finally, we generated a monoclonal antibody specific for the casein kinase II target site in the carboxy-terminal regulatory region of p53, and used it to demonstrate the allosteric inhibition of in vitro and in vivo activated forms of p53.
Conclusions: p53 protein assembles naturally as a tetramer that can be converted between latent and activated forms by a concerted, allosteric transition. The highly purified, reconstituted system that we have developed, in which the DNA-binding activity of p53 can be reversibly regulated, should facilitate the discovery of agents that can modulate the DNA-binding activity of p53--particularly those that can activate mutant p53 proteins and that may have potential in the design of anti-cancer drugs.