Genetic analysis of p53 activation

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DESCRIPTION (provided by applicant): Activation of the p53 tumor suppressor is an important part of the response that cells mount to a variety of genotoxic and nongenotoxic stimuli and a key step in that activation process is the accumulation of p53 in the nuclei of affected cells. The mechanism that regulates p53 subcellular localization is only poorly understood. However, the observation that some p53 is excluded from the nucleus in the cells of some tumors suggests that this may be another mechanism by which p53 can be inactivated and points to the importance of understanding how p53 subcellular localization is controlled. In order to gain insight into the p53 protein trafficking we adopted a model system in which the accumulation of a tsp53 in the nucleus inhibited cell proliferation and selected for mutants that were resistant to these affects. Preliminary studies indicate that the mutant cell lines are defective for p53 nuclear trafficking and are significantly more sensitive to killing by heat shock. These observations combined with reports indicating that heat shock proteins play a role in the trafficking of proteins into the nucleus lead us to propose the hypothesis that heat shock proteins function in transportation of p53 into the nucleus in cells exposed to stressful stimuli. To elucidate regulation of p53 subcellular localization we will 1) characterize functioning of the p53 nuclear localization signally by determining whether activity of this motif is controlled by phosphorylation and/or by interaction with hsc70 2) determine whether p53 phosphorylation or interaction with hsc70 is aberrant in our mutant cells where p53 nuclear trafficking is defective 3) determine whether mutations that inactivate p53 promote interaction between p53 and heat shock proteins or whether heat shock proteins suppress p53 activity by anchoring the protein in the cytoplasm.