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

该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。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其他NIH来源。 为子项目列出的总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 着丝粒是一种多蛋白质复合物，在细胞分裂过程中组装在着丝粒上并将染色体附着在纺锤体微管上。它对染色体的附着受到严格监控，以确保染色体分离可靠。最近的一个系统的酵母双杂交筛选有丝分裂纺锤体的功能，确定了动粒蛋白Mif 2和两个酪蛋白激酶2（CK 2）亚基，Cka 2和Ckb 2之间的相互作用。CK 2是真核生物中普遍存在的高度保守的丝氨酸/苏氨酸激酶。虽然CK 2与细胞周期调控有关，但CK 2如何调控这一事件仍不清楚。此外，Mif 2和另一种动粒蛋白Ndc 10（Cbf 2）先前被鉴定为CK 2的推定底物。Ndc 10是CBF 3复合物的组分。Mif 2是S.人CENP-C的酿酒酵母同源物。这两种蛋白质都与着丝粒结合，并招募其他内部和外部动粒蛋白。先前的研究已经证明Mif 2和Ndc 10都被酵母Aurora B激酶Ipl 1磷酸化。然而，磷酸化的作用尚未得到充分的研究。 我的项目的总体目标是确定CK 2如何调节动粒功能。我推测CK 2通过调节Mif 2和Ndc 10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Cka 2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。将检查cka 2-ts和ipl 1-ts之间的遗传相互作用。除cka 2- ts突变体外，还将创建和检测cka 2-as（类似物敏感性）突变体。我还将在体外和体内检测Mif 2和Ndc 10是否被CK 2磷酸化，并通过质谱法绘制它们的磷酸化位点。Mif 2和Ndc 10都具有多个推定的Ipl 1磷酸化位点。将在体外和体内研究一种激酶的磷酸化如何影响另一种激酶的磷酸化。此外，Mif 2和Ndc 10的磷酸化位点将被诱变，并将检查这些修饰的作用。最后，使用cka 2-作为突变体，筛选集中在着丝粒和它们的调节蛋白将进行以确定额外的CK 2底物。了解CK 2如何调节动粒将提供新的见解CK 2在细胞周期调控中的作用。

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.