Loss or inactivation of p53 is an important step in cancer development. It is therefore important to determine how the levels and activity of p53 are normally regulated, and how this regulation is altered in cancer. p53 degradation occurs, at least in part, through the ubiquitin-proteolysis system. Our laboratory can readily detect ubiquitin: p53 conjugates in cells that express wild-type p53, and in cells transfected with p53 expression DNAs. This ability affords us an excellent system to study p53 ubiquitination in response to diverse stimuli. Preliminary studies indicate that p53 ubiquitination is regulated during release from quiescence and in response to DNA damage. We will determine conditions when high or low levels of p53 ubiquitination are observed. We will then determine whether high levels of p53 ubiquitination are correlated with a decreased protein half-life. Finally, the potential role of MDM2 in mediating p53 ubiquitination will be assessed. These studies will be done in normal cells and in cancer cells to determine whether the regulation of p53 ubiquitination is altered in cancer. An assay system has been established in which wild-type p53 is efficiently ubiquitinated in transfected cells when coexpressed with MDM2. Deletion analysis and mutagenesis of p53 will be done to define the determinants within p53 required for ubiquitination. Cancer-derived p53 mutants vary in their susceptibility to ubiquitination. We will determine the molecular basis for this variation. In addition, two MDM2 mutants will be examined to address the mechanism by which MDM2 promotes p53 ubiquitination. The recent cloning of p73 and p63 has revealed a family of p53-related proteins that may carry out redundant functions. We wish to determine whether the levels and stability of these newly identified p53 family members are regulated through the same pathways that regulate p53. Preliminary studies indicate that certain cancer-derived p53 mutants can promote the rapid degradation of p73. We will elaborate the molecular basis of this effect. In addition, we will determine whether the ability to promote p73 degradation is associated with a gain-of-function phenotype for mutant p53.
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