Evidence for a distinct inhibitory factor in the regulation of p53 functional activity

Under normal conditions, tumor suppressor protein p53 exists in the cell in its latent form and is unable to function as a transcription factor. The a llosteric model of p53 regulation postulates that the extreme portion of p5 3 carboxyl terminus (aa 364-393) binds to the core domain of the protein, t hereby abrogating specific DNA binding in that region. In this study we pro pose an alternative mechanism of p53 functional regulation, which involves a separate molecule acting in trans to inhibit p53 transcriptional activity . Through the use of chimeric proteins of p53, p63 gamma and p73 beta, we s how that the extreme COOH-terminal domain of p53 exerts a powerful and spec ific inhibitory effect on the p73- and p63-driven expression of a reporter gene. Moreover, fusion of p53 extreme COOH terminus to a completely unrelat ed transcriptional activator Gal4-VP16 also results in significant inhibiti on of transactivation. activity. Since p73, p63, or Gal4-VP16 cannot associ ate with any part of the p53 molecule, we conclude that p53(aa 364-393) rep resses transcriptional activity of chimeric proteins and p53 itself through the binding of external negative modulator(s) in that region and not by th e allosteric mechanism of regulation. In accordance with the "distinct inhi bitor" hypothesis, the activity of wild type p53 is substantially increased by overexpression of chimeric proteins bearing p53(aa 364-393), which migh t be due to the competitive removal of trancriptional inhibitor(s). Our fin dings provide the basis for the identification of such negative modulators of p53 transcriptional activity.