Genetic analysis of p53 activation

描述(由申请人提供)：P53肿瘤抑制因子的激活是细胞对各种遗传毒性和非遗传毒性刺激反应的重要部分，而激活过程中的关键步骤是P53在受影响细胞的细胞核中积累。调控P53亚细胞定位的机制还知之甚少。然而，观察到一些肿瘤细胞中的一些P53被排除在细胞核之外，这表明这可能是P53失活的另一种机制，并表明了解P53亚细胞定位是如何控制的重要性。为了深入了解P53蛋白的运输，我们采用了一个模型系统，在该系统中，TSP53在细胞核中的积累抑制了细胞的增殖，并选择了对这些影响具有抗性的突变体。初步研究表明，突变的细胞系在P53核运输方面存在缺陷，并且对热休克致死明显更敏感。结合热休克蛋白在蛋白质转运到细胞核中的作用的报道，我们提出了热休克蛋白在应激刺激下细胞中p53转运到细胞核中的作用的假说。为了阐明P53亚细胞定位的调控，我们将1)通过确定该基序的活性是否受磷酸化和/或与hsc70的相互作用控制来表征P53核定位的功能。2)确定在P53核运输缺陷的突变细胞中P53的磷酸化或与hsc70的相互作用是否异常3)确定失活P53的突变是否促进P53与热休克蛋白之间的相互作用，或者热休克蛋白是否通过锚定在细胞质中而抑制P53的活性。

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.