REGULATORY ROLES OF CASEIN KINASE 2 (CK2) IN KINETOCHORE FUNCTIONS

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 着丝点是一种多蛋白质复合体，在着丝粒上组装，在细胞分裂过程中将染色体附着在纺锤体微管上。它与染色体的附着受到严密监控，以确保忠实的染色体分离。最近对有丝分裂纺锤体功能的系统酵母双杂交筛选发现动粒蛋白Mif2与酪蛋白激酶2(CK2)亚基Cka2和Ckb2之间存在相互作用。CK2是真核生物中普遍存在且高度保守的丝氨酸/苏氨酸蛋白激酶。尽管CK2与细胞周期调控有关，但CK2如何调控这一事件在很大程度上仍不清楚。此外，Mif2和另一种动粒核心蛋白Ndc10(Cbf2)先前被确定为CK2的可能底物。NDC10是CBF3复合体的一个组成部分。Mif2是人CENP-C的酿酒酵母同源物。这两种蛋白都与着丝粒结合，并招募其他内部和外部动粒蛋白。以前的研究表明，Mif2和Ndc10都被酵母Aurora B激酶Ipl1磷酸化。然而，磷酸化的作用还没有得到充分的研究。 我这个项目的总体目标是确定CK2是如何调节动粒功能的。我假设CK2通过调节Mif2和Ndc10的Aurora B磷酸化来调节有丝分裂进程。为了验证这些假设，我将首先确定Cka2激酶活性的丧失是否会影响有丝分裂纺锤体和染色体分离。我们将研究Ka2-ts和Ipl1-ts之间的遗传互作。除了KAA2-TS突变体外，还将创建和测试KAA2-AS(类似物敏感)突变体。我还将在体外和体内测试Mif2和Ndc10是否被CK2磷酸化，并用质谱仪绘制它们的磷酸化位置。Mif2和Ndc10都有多个可能的Ipl1磷酸化位点。一种酶的磷酸化如何影响另一种的磷酸化，将在体外和体内进行研究。此外，Mif2和Ndc10的磷酸化位点将被突变，这些修饰的作用将被检测。最后，使用KKA2-作为突变体，将对动粒及其调节蛋白进行筛选，以确定额外的CK2底物。了解CK2是如何调节动粒的，将为深入了解CK2在细胞周期调控中的作用提供新的见解。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. The kinetochores is a multi-protein complex that assemblies on the centromere and attaches chromosomes to spindle microtubules during cell division. Its attachment to chromosomes is tightly monitored to ensure faithful chromosome segregation. A recent systematic yeast two-hybrid screen focusing on mitotic spindle function identified interactions between the kinetochore protein Mif2 and two Casein Kinase 2 (CK2) subunits, Cka2 and Ckb2. CK2 is a ubiquitous and highly conserved Ser/Thr kinase in eukaryotes. Although CK2 has been linked to cell cycle regulation, it has remained largely unclear how CK2 regulates this event. Additionally, Mif2 and another kinetochore protein Ndc10 (Cbf2) were previously identified as putative substrates of CK2. Ndc10 is a component of the CBF3 complex. Mif2 is the S. cerevisiae homolog of human CENP-C. Both proteins bind to centromeres and recruit other inner and outer kinetochore proteins. Previous studies have demonstrated that both Mif2 and Ndc10 are phosphorylated by Ipl1, the yeast Aurora B kinase. However, the role of phosphorylation has not been fully explored. The overall goal of my project is to determine how CK2 regulates kinetochore function. I hypothesize that CK2 regulates mitotic progression by regulating Aurora B phosphorylation of Mif2 and Ndc10. To test these hypotheses, I will first determine if loss of Cka2 kinase activity affects mitotic spindle and chromosome segregation. Genetic interaction between cka2-ts and ipl1-ts will be examined. In addition to cka2- ts mutants, cka2-as (analogue sensitive) mutants will be created and tested. I will also test if Mif2 andNdc10 are phosphorylated by CK2 in vitro and in vivo, and their phosphorylation sites will be mapped by mass spectrometry. Both Mif2 and Ndc10 have multiple putative Ipl1 phosphorylation sites. How phosphorylation by one kinase affects phosphorylation by the other will be investigated in vitro and in vivo. Moreover, phosphorylation sites of Mif2 and Ndc10 will be mutagenized and the role of these modifications will be examined. Finally, using cka2-as mutants, a screen focusing on kinetochores and their regulatory proteins will be conducted to identify additionalCK2 substrates. Understanding how CK2 regulates kinetochores will provide novel insights into the role of CK2 in cell cycle regulation.