Regulation of Sororin by Cdk1-mediated Phosphorylation.

描述（由申请人提供）： 在有丝分裂过程中，复制的染色体被分离到细胞的两极，必须准确地发生，以确保子细胞包含完整的基因组拷贝。这个过程中的缺陷会导致染色体数量的变化，这是癌症形成的驱动力。Sororin通过与cohesin复合物相互作用在维持姐妹染色单体凝聚中起关键作用。对Sororin的监管知之甚少。我们已经创建了Sororin突变体，其中9个推定的Cdk 1磷酸化位点已转化为丙氨酸（“Sororin 9A”）。Sororin 9A保持与染色体组成性相关，并在有丝分裂期间增加姐妹染色单体之间的凝聚力。我们建议分析Cdk 1介导的磷酸化调节Sororin在姐妹染色单体凝聚中的功能的机制。这一建议有两个具体目标。目标1. Cdk 1和PP 2A对Sororin磷酸化的调控Sororin中9个假定Cdk 1位点中每一个的磷酸化相对效率尚不清楚。磷酸化胰蛋白酶图谱将用于确定9个Cdk 1位点中的哪一个是体内主要磷酸化位点。有了这些信息，我们将着手对Sororin功能中特定磷酸化位点的作用进行更直接的分析。Sororin的磷酸化被预测抑制其稳定姐妹染色单体凝聚的能力。我们计划通过分析Sororin突变体的细胞效应来直接测试这一想法，其中单个丝氨酸/苏氨酸已转化为谷氨酸以模拟组成性磷酸化状态。最后，PP 2A似乎在有丝分裂退出期间使Sororin去磷酸化。将确定内着丝粒处的Sororin通过动粒处的PP 2A的调节。目标2. Sororin磷酸化在姐妹染色单体凝聚中的作用。Sororin 9A增加姐妹染色单体的凝聚力，这种效应被假设是由于染色体臂上的凝聚素复合物的持久性。免疫荧光将用于测量Sororin 9A过表达后与染色体相关的粘着蛋白亚基的量。爪蟾提取物的研究表明，Sororin磷酸化的影响是由于Sororin与粘附素辅助因子Pds 5的关联的变化。我们建议，以确定突变的Pds 5对接网站Sororin，一个区域，包含的基本基序“FGF”的效果。在Sororin 9A中将该基序突变为阿加应该阻断Sororin 9A引起的内聚增加。Sororin在姐妹染色单体凝聚中的作用的当前模型表明它与Wapl竞争结合Pds 5。Wapl与Pds 5的结合在前期期间沿着染色体臂释放粘附素环沿着，而Sororin可以拮抗这种作用。我们预测过表达Wapl将逆转Sororin 9A对凝聚力的影响。确定Sororin磷酸化对姐妹染色单体凝聚力的作用将填补我们对这一重要过程理解的重要空白。 公共卫生相关性： 在美国，每年有50多万人死于癌症，这种疾病的特征是细胞分裂失控和染色体分离不准确。Sororin蛋白在细胞分裂中起着至关重要的作用，了解它是如何调节的将为人类癌细胞如何分裂以及如何杀死它们提供重要的见解。成纤维细胞和平滑肌细胞的增殖增加也与心血管系统疾病有关，了解Sororin是如何调节的也可以提供对这种疾病谱的了解。

DESCRIPTION (provided by applicant): Mitosis, during which duplicated chromosomes are segregated to opposite poles of the cell must occur accurately to ensure that daughter cells contain an intact copy of the genome. Defects in this process can lead to changes in chromosome number, a driving force behind cancer formation. Sororin plays a key role in maintaining sister chromatid cohesion by interacting with the cohesin complex. The regulation of Sororin is poorly understood. We have created a Sororin mutant in which nine putative Cdk1 phosphorylation sites have been converted to alanine ("Sororin9A"). Sororin9A remains constitutively associated with chromosomes and increases cohesion between sister chromatids during mitosis. We propose to analyze the mechanism by which Cdk1-mediated phosphorylation regulates the function of Sororin in sister chromatid cohesion. This proposal entails two specific aims. Aim 1. Regulation of Sororin phosphorylation by Cdk1 and PP2A. The relative efficiency of phosphorylation at each of nine putative Cdk1 sites in Sororin is not known. Phospho-tryptic mapping will be used to determine which of the nine Cdk1 sites are major phosphorylation sites in vivo. With this information, we will embark on a more directed analysis of the role of specific phosphorylation sites in Sororin function. Phosphorylation of Sororin is predicted to inhibit its ability to stabilize sister chromatid cohesion. We plan to test this idea directly by analyzing the cellular effects of Sororin mutants in which individual serines/threonines have been converted to glutamic acid to mimic the constitutively phosphorylated state. Finally, PP2A appears to dephosphorylate Sororin during mitotic exit. The regulation of Sororin at the inner centromere by PP2A at the kinetochore will be determined. Aim 2. Role of Sororin phosphorylation in sister chromatid cohesion. Sororin9A increases sister chromatid cohesion, an effect that is hypothesized to be due to the persistence of the cohesin complex on chromosome arms. Immunofluorescence will be used to measure the amount of cohesin subunits associated with chromosomes after overexpression of Sororin9A. Studies in Xenopus extracts suggest that the effect of Sororin phosphorylation is due to changes in the association of Sororin with the cohesin accessory factor Pds5. We propose to determine the effect of mutating the Pds5-docking site on Sororin, a region that contains the essential motif "FGF". Mutating this motif to AGA in Sororin9A should block the increase in cohesion caused by Sororin9A. The current model for the role of Sororin in sister chromatid cohesion suggests that it competes with Wapl for binding to Pds5. Binding of Wapl to Pds5 releases the cohesin ring along chromosome arms during prophase, while Sororin may antagonize this effect. We predict that overexpressing Wapl will reverse the effects of Sororin9A on cohesion. Determining the role of Sororin phosphorylation on sister chromatid cohesion will fill an important gap in our understanding of this essential process. PUBLIC HEALTH RELEVANCE: Project Narrative More than half a million people in the US die every year due to cancer, a disease characterized by uncontrolled cell division, and inaccurate segregation of chromosomes. The protein Sororin plays an essential role in cell division and understanding how it is regulated will provide important insight into how human cancer cells divide and possibly how to kill them. Increased proliferation of fibroblasts and smooth muscle cells has also been implicated in diseases of the cardiovascular system, and knowing how Sororin is regulated may also provide insight into this spectrum of diseases.