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: 6931528
• 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 2008-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6931528
• 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的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其在其余无p53突变的肿瘤中的功能。可以想象的是，由于上游调控基因的缺陷，肿瘤中明显的野生型p53对于一组确定的途径可能仍然是无功能的。此外，尽管频率较低，但在少数缺乏基因内p53改变的肿瘤中发现了p53上游调节剂Chk 1和Chk 2的突变，这意味着上游调节剂是p53途径失活的合理替代靶点。另一方面，几个调制器的p53功能已假定基于体外试验，但它们在体内的相关性在很大程度上是未知的。由于它仍然是一个主要的警告在p53的研究，我们已经开发了一种有效的检测酵母中，以确定在体内上游调节p53介导的反式激活。我们的屏幕上使用的人cDNA表达文库已经能够识别克隆，增强p53反式激活在一个公正的方式。在确认的克隆中，有两个是激酶。一个是ATM基因产物，它已被确立为p53的上游调节剂，以响应IR的DNA损伤。这一发现有力地验证了使用我们的酵母系统来分离p53的体内上游调节剂的基本原理。出乎意料的是，在筛选中分离的第二个推定激酶是hBUB 1。BUB 1最初是作为酵母中的纺锤体组装检查点基因分离的，并且在人类肿瘤的一个子集中具有突变。虽然p53长期以来一直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。我们鉴定hBUB 1作为p53的调节剂可以阐明p53维持基因组稳定性的能力的分子基础。我们证明，hBUB 1磷酸化p53的丝氨酸残基（S），包括丝氨酸15作为一个主要的网站，并表现出特异性p53在增强其能力的序列特异性反式激活。已知的效应基因如p21和GADD 45的基因表达增加，其调节细胞周期检查点也与hBUB 1-p53途径在维持基因组稳定性中的作用一致。缺失激酶结构域的显性负突变体hBUB 1或反义hBUB 1介导纺锤体组装破坏过程中有丝分裂检查点的逃逸。在这些条件下，hBUB 1和p53在细胞核中的共定位为它们的生化相互作用提供了额外的证据。基于这些观察，我们假设hBUB 1激酶是有丝分裂检查点期间p53活性的调节剂，并且肿瘤中p53或hBUB 1的失活是非整倍体的主要机制。尽管hBUB 1基因内突变水平较低，但最近的研究显示hBUB 1在肿瘤中下调，这强烈表明表观遗传机制导致的基因表达沉默可能在人类癌症中hBUB 1的功能失活中起关键作用。本研究拟从生物化学水平探讨hBUB对p53的调控作用，阐明hBUB对p53的诱导信号，鉴定特异性效应基因及其末端效应，并确定hBUB 1在肿瘤中的失活模式。这些分析可能导致对hBUB 1-p53通路在维持基因组稳定性方面的分子理解，从而有助于设计所有癌症的新诊断和治疗策略。