The role of hBUB1-p53 pathway in genomic stability.

hBUB1-p53 通路在基因组稳定性中的作用。

• 批准号: 6786057
• 负责人: Sam Thiagalingam
• 金额: $ 28.75万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-08-01 至 2008-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6786057
• 关键词: DNA binding protein; RNase protection assay; aneuploidy; apoptosis; carcinogenesis; clinical research; electrospray ionization mass spectrometry; enzyme activity; flow cytometry; fluorescent in situ hybridization; human tissue; immunoprecipitation; mass spectrometry; matrix assisted laser desorption ionization; northern blottings; p53 gene /protein; phosphorylation; polymerase chain reaction; posttranslational modifications; protein kinase; protein protein interaction; protein structure function; terminal nick end labeling; transcription factor; western blottings

DESCRIPTION (provided by applicant): The mutational inactivation of p53 is one of the most prevalent genetic changes in human cancer and hence is a critical target for diagnostic and therapeutic protocols. Although p53 is mutated in more than half of all human cancers, we believe that any effective therapeutic strategy based on p53 requires an understanding of its functionality in the remaining tumors without p53 mutations. It is conceivable that the apparent wild type p53 in tumors may still be non-functional for a defined set of pathways due to defects in upstream regulatory genes. Additionally, despite their lower frequency, mutations in Chk1 and Chk2, upstream modulators of p53, were found in a minority of tumors that lack intra-geneic p53 alterations which implicates upstream modulators as legitimate alternate targets for inactivation of p53 pathways. On the other hand, several modulators of p53 function have been postulated based on in vitro assays, but their in vivo relevance is largely unknown. Since it remains a major caveat in p53 research, we have developed an efficient assay in yeast to identify in vivo upstream modulators of p53-mediated transactivation. Our screen using a human cDNA expression library has enabled the identification of clones that augment p53 transactivation in an unbiased manner. Among the confirmed clones, two are kinases. One is the ATM gene product, which has been established as an upstream modulator of p53 in response to DNA damage by IR. This finding strongly validates the rationale for using our yeast system to isolate the in vivo upstream modulators of p53. Unexpectedly, the second putative kinase isolated in the screen is hBUB1. BUB1 was originally isolated as a spindle assembly checkpoint gene in yeast and harbors mutations in a subset of human tumors. Although p53 has long been implicated in the maintenance of genomic stability, the molecular mechanism of its involvement remains unresolved. Our identification of hBUB1 as a modulator of p53 could elucidate the molecular basis for the ability of p53 to maintain genomic stability. We demonstrate that hBUB1 phosphorylates p53 at serine residue(s), including serine 15 as a primary site and exhibits specificity to p53 in augmenting its ability for sequence specific transactivation. Increased gene expression of known effecter genes such as p21 and GADD45, which regulate cell cycle checkpoints are also consistent with the roles for the hBUB1-p53 pathways in maintaining genomic stability. Dominant negative mutant hBUB1 with a deleted kinase domain or antisense hBUB1 mediates escape from the mitotic checkpoint during spindle assembly disruption. Colocalization of hBUB1 and p53 in the nucleus under these conditions provides added credence to their biochemical interaction. Based on these observations, we hypothesize that the hBUB1 kinase is a regulator of p53 activity during the mitotic checkpoint, and inactivation of either p53 or hBUB1 in tumors is a major mechanism for aneuploidy. Despite the lower level of intra-genic mutations in hBUB1, recent studies showing down-regulation of hBUB1 in tumors strongly suggest that silencing of gene expression by epigenetic mechanisms may play a critical role in the functional inactivation of hBUB1 in human cancer. We propose to investigate the regulatory effect of hBUB on p53 at the biochemical level, delineate the induction signals, identify the specific effecter genes and their end effects, and determine the modes of inactivation of hBUB1 in tumors. These analyses could lead to the molecular understanding of the hBUB1-p53 pathway in maintaining genomic stability and thus help to design novel diagnostic and therapeutic strategies in all cancers.

描述（由申请人提供）：p53突变失活是人类癌症中最普遍的遗传变化之一，因此是诊断和治疗方案的关键靶点。尽管p53在超过一半的人类癌症中发生突变，但我们认为，任何基于p53的有效治疗策略都需要了解它在其余没有p53突变的肿瘤中的功能。可以想象，由于上游调控基因的缺陷，肿瘤中明显的野生型p53可能在一组确定的途径中仍然不起作用。此外，尽管频率较低，p53的上游调节剂Chk1和Chk2的突变在少数缺乏p53基因内改变的肿瘤中被发现，这意味着上游调节剂是p53通路失活的合法替代靶点。另一方面，基于体外实验已经假设了几种p53功能调节剂，但它们在体内的相关性在很大程度上是未知的。由于它仍然是p53研究中的一个主要警告，我们已经在酵母中开发了一种有效的测定方法，以确定p53介导的交易激活的体内上游调节剂。我们使用人类cDNA表达文库进行筛选，能够以无偏倚的方式鉴定增强p53转激活的克隆。在确认的克隆中，有两个是激酶。一种是ATM基因产物，它已被确定为p53响应IR DNA损伤的上游调节剂。这一发现有力地验证了使用我们的酵母系统分离p53的体内上游调节剂的基本原理。出乎意料的是，在筛选中分离出的第二个假定的激酶是hBUB1。BUB1最初是作为酵母中的纺锤体组装检查点基因分离出来的，在人类肿瘤的一个亚群中存在突变。尽管p53长期以来一直被认为与基因组稳定性的维持有关，但其参与的分子机制仍未得到解决。我们发现hBUB1是p53的调节因子，可以阐明p53维持基因组稳定性的分子基础。我们证明hBUB1在丝氨酸残基上磷酸化p53，包括作为主要位点的丝氨酸15，并在增强其序列特异性转激活能力方面表现出对p53的特异性。调节细胞周期检查点的已知效应基因如p21和GADD45的基因表达增加也与hBUB1-p53通路在维持基因组稳定性中的作用一致。显性负突变体hBUB1具有缺失的激酶结构域或反义hBUB1介导纺锤体组装中断期间有丝分裂检查点的逃逸。在这些条件下，hBUB1和p53在细胞核中的共定位为它们的生化相互作用提供了更多的证据。基于这些观察结果，我们假设hBUB1激酶在有丝分裂检查点是p53活性的调节剂，p53或hBUB1在肿瘤中的失活是非整倍体的主要机制。尽管hBUB1基因内突变水平较低，但最近的研究显示hBUB1在肿瘤中下调，这强烈表明通过表观遗传机制沉默基因表达可能在人类癌症中hBUB1功能失活中发挥关键作用。我们拟从生化水平研究hBUB对p53的调控作用，描绘诱导信号，鉴定特异性效应基因及其末端效应，确定hBUB1在肿瘤中的失活模式。这些分析可能导致hBUB1-p53途径在维持基因组稳定性方面的分子理解，从而有助于设计针对所有癌症的新的诊断和治疗策略。