UBIQUITINATION AND STABILITY OF P53 AND P73

P53的丢失或失活是肿瘤发生发展的重要步骤。因此，重要的是要确定P53的水平和活性是如何正常调节的，以及这种调节在癌症中是如何改变的。P53的降解至少部分是通过泛素-蛋白分解系统发生的。我们的实验室可以很容易地在表达野生型P53的细胞中检测到泛素：P53结合物，以及在转染有P53表达DNA的细胞中检测到泛素：P53结合物。这种能力为我们提供了一个很好的系统来研究P53泛素化对不同刺激的反应。初步研究表明，P53泛素化在从静止状态释放和对DNA损伤的反应中受到调节。我们将确定观察到高水平或低水平的p53泛素化的条件。然后我们将确定高水平的P53泛素化是否与蛋白质半衰期的减少相关。最后，将评估MDM2在介导P53泛素化中的潜在作用。这些研究将在正常细胞和癌细胞中进行，以确定癌症中P53泛素化的调节是否发生了变化。已经建立了一种检测系统，在该系统中，野生型p53与MDM2共表达时，在转基因细胞中有效地泛素表达。将进行p53的缺失分析和突变，以确定泛素化所需的p53内的决定因素。癌症来源的p53突变体对泛素化的敏感性不同。我们将确定这种变异的分子基础。此外，将研究两个MDM2突变体，以解决MDM2促进p53泛素化的机制。最近p73和p63的克隆揭示了一个与P53相关的蛋白家族，它们可能具有多余的功能。我们希望确定这些新发现的P53家族成员的水平和稳定性是否通过与调节P53相同的途径来调节。初步研究表明，某些肿瘤来源的p53突变体可以促进p73的快速降解。我们将详细阐述这种效应的分子基础。此外，我们将确定促进p73降解的能力是否与突变型p53的功能获得表型相关。

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.