The RAN GTPase

描述（由申请人提供）：该资助的持续目标是在分子水平上了解Ran GT酶和Ran相互作用蛋白在核转运和有丝分裂中的功能。除了它们的基本生物学相关性之外，这些蛋白质在病毒感染、衰老和癌症中发挥关键作用。特别是，我们最近对Ran交换因子RCC 1的研究表明，它对维持基因组稳定性至关重要。在上一个资助期间，我们确定了Exportin-5和Importin-11的功能，并深入了解了核运输的全球监管。我们还发现了一种新的、完全出乎意料的RCC 1翻译后修饰类型，它调节与染色质的结合。鉴于这项新修改的关键重要性，我们计划在下一个资助期内通过以下4个目标专注于这一领域：1。确定调节RCC 1功能的机制。我们已经发现RCC 1在其N-末端α-氨基上被甲基化。我们还发现，核提取物含有以前未被识别的α-N甲基转移酶活性，并且RCC 1的甲基化增强了染色质缔合。关键目标是确定这种不寻常的修饰是否对RCC 1有额外的影响;鉴定α-N-甲基转移酶（a-N-MT）;并确定其活性是否受到调节。2.通过RCC 1进行构象转换。RCC 1与染色质的结合受Ran调节。为了了解潜在的机制和α-N-甲基化的作用，我们将使用FRET生物传感器测试RCC 1的N-末端是否在Ran结合后经历构象变化。3.确定RCC 1在有丝分裂中的Ran独立功能。突变RCC 1，D182 A，是有缺陷的Ranbinding，核苷酸交换活性，和染色质结合，但仍然诱导有丝分裂缺陷时，在细胞中表达。这些数据表明，区域合作中心1具有独立于其全球环境基金活动的职能。我们将测试是否N-末端尾巴是必要的或足以诱导有丝分裂缺陷，确定关键的残基内的尾巴，并寻求结合的合作伙伴，介导这些影响的RCC 1。4.确定未甲基化的RCC 1是否诱导基因组不稳定性。将从原代鼠胚胎成纤维细胞产生稳定的细胞系，以表达野生型RCC 1或甲基化缺陷突变体。将对这些细胞系进行核型分析以鉴定非整倍性，并将测试体外遗传不稳定性和小鼠中的肿瘤形成。RCC 1突变体将为检验肿瘤发生的非整倍体假说提供一种严格的新方法。

DESCRIPTION (provided by applicant): The continuing goal of this grant is to understand at a molecular level the functions of the Ran GTPase and of Ran-interacting proteins in nuclear transport and mitosis. In addition to their fundamental biological relevance, these proteins play key roles in viral infection, aging, and cancer. In particular, our recent work on the Ran exchange factor, RCC1, suggests that it is essential for maintaining genomic stability. In the previous funding period we determined the functions of Exportin-5 and Importin-11, and gained insight into the global regulation of nuclear transport. We also discovered a new, entirely unanticipated type of post-translational modification of RCC1 that regulates association with chromatin. Given the pivotal importance of this novel modification, we plan to focus on this area during the next funding period, through the following 4 aims: 1. Identify the mechanisms that regulate RCC1 function. We have discovered that RCC1 is methylated on its N-terminal a-amino group. We also found that nuclear extracts contain a previously unrecognized a-Nmethyltransferase activity, and that the methylation of RCC1 enhances chromatin association. Key goals are to detemine whether this unusual modification has additional effects on RCC1; to identify the a-Nmethyltransferase (a-N-MT); and to determine whether its activity is regulated. 2. Conformational switching by RCC1. RCC1 binding to chromatin is modulated by Ran. To understand the underlying mechanisms and the role of a-N-methylation, we will test whether the N-terminus of RCC1 undergoes a conformational change upon Ran binding, using FRET biosensors. 3. Identify Ran-independent functions of RCC1 in mitosis. A mutant RCC1, D182A, is defective in Ranbinding, nucleotide exchange activity, and chromatin binding, but nonetheless still induces mitotic defects when expressed in cells. These data argue that RCC1 possesses a function that is independent of its GEF activity. We will test whether the N-terminal tail is necessary or sufficient to induce mitotic defects, identify critical residues within the tail, and seek binding partners that mediate these effects of RCC1. 4. Determine whether unmethylated RCC1 induces genomic instability. Stable cell lines will be generated from primary murine embryonic fibroblasts, to express either wild-type RCC1 or a methylation-defective mutant. These cell lines will be karyotyped to identify aneuploidies, and will be tested for genetic instability in vitro, and for tumor formation in mice. The RCC1 mutants will provide a rigorous new way to test the aneuploidy hypothesis of tumorigenesis.