Centrosomes and BRCA1

描述（由申请人提供）：BRCA1基因突变导致乳腺癌和卵巢癌的原因尚不清楚。该实验室最近的数据表明，BRCA1肿瘤抑制因子的泛素化活性在组织培养的乳腺上皮细胞中特异性地调节着中心体的复制和功能。使用瞬时测定法抑制BRCA1功能，我们未观察到源自非乳腺组织的细胞系中中心体扩增。在早期和最具侵袭性的乳腺癌病变中观察到中心体扩增，并且BRCA1的这种功能很可能在这些肿瘤的病因学中起关键作用。我们的数据揭示了一种新的生物学途径，它控制着中心体的数量和功能，这取决于BRCA1在乳腺细胞中的功能。本项目将通过三个目标剖析BRCA1的功能。1)。BRCA1泛素化的多肽靶点将从乳腺细胞系纯化的中心体中鉴定。通过brca1依赖性泛素化活性修饰的蛋白质将被检测对中心体复制的影响。2)。BRCA1依赖性泛素化对中心体微管成核功能的影响将在体外和体内进行分析，并且在aim 1中鉴定的蛋白质和泛素化底物也将测试BRCA1对该细胞器的功能调节。3)。我们将在非乳腺细胞中确定使BRCA1泛素化活性冗余的因子，并测试新发现的因子加BRCA1是否调节这些细胞类型中的中心体扩增。该项目将确定BRCA1调节乳腺细胞中心体复制和中心体功能的机制。早期乳腺癌细胞最早的变化之一是在乳腺细胞分裂时分离DNA的细胞机器出现了问题。这个实验室的研究发现，这个被称为中心体的机器的功能在乳腺细胞中是由BRCA1蛋白控制的。该项目将剖析中心体的工作原理，并确定BRCA1如何在正常细胞中控制其功能，我们将了解当BRCA1功能因突变而丧失时，这一过程是如何改变的，就像乳腺癌中发生的那样。

DESCRIPTION (provided by applicant): Why mutations in the BRCA1 gene result in breast and ovarian cancer is unknown. Recent data from this laboratory suggest that the ubiquitination activity of the BRCA1 tumor suppressor regulates the duplication and function of centrosomes specifically in mammary epithelial cells in tissue culture. Using a transient assay to inhibit BRCA1 function, we have not observed centrosome amplification in cell lines derived from non- breast tissue. Centrosome amplification has been observed in the earliest and most aggressive breast cancer lesions, and it is likely that this function of BRCA1 is critical in the etiology of these tumors. Our data has revealed a new biological pathway, which controls centrosome number and function and which depends on the function of BRCA1 in breast cells. This project will dissect the BRCA1 function via three aims. 1.) Polypeptide targets of BRCA1 ubiquitination will be identified from centrosomes purified from breast cell lines. Proteins modified by the BRCA1-dependent ubiquitination activity will be assayed for effects on centrosome duplication. 2.) The effects of BRCA1-dependent ubiquitination on centrosome microtubule nucleation function will be assayed in vitro and in vivo, and the proteins and ubiquitinated substrates identified in aim 1 will also be tested for modulating the function of BRCA1 on this organelle. 3.) We will identify the factors in non-breast cells which render the BRCA1 ubiquitination activity redundant, and test whether the newly identified factor plus BRCA1 regulate centrosome amplification in these cell types. This project will identify the mechanism by which BRCA1 regulates centrosome duplication and centrosome function in breast cells. One of the earliest changes in the cells in an incipient breast cancer is a problem with the cellular machine that segregates the DNA when the breast cell divides. Research in this laboratory has found that the function of this machine, called a centrosome, is controlled in breast cells by the BRCA1 protein. This project will dissect the workings of the centrosome and determine how BRCA1 controls its function in normal cells, and we will learn how this process is changed when BRCA1 function is lost by mutation as happens in breast cancer.