IDENTIFICATION AND POST-TRANSLATIONAL MODIFICATION OF MITOTIC REGULATORY PROTEI

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。列出的机构是 研究中心，而研究中心不一定是研究者所在的机构。 染色体过客蛋白在细胞周期中的分布发生了巨大的变化，在有丝分裂的各个阶段，定位于着丝粒、中间区、卵裂沟和中间体。CP在增殖和细胞周期检查点信号传导途径中发挥关键作用，并且由于其作为有丝分裂调节剂的重要性而成为深入研究的主题。使用针对磷酸化肽产生的抗体（P190），我们观察到9种主要蛋白质在有丝分裂期间专门磷酸化，其中它们表现为CP。这些蛋白质之一已被确定为Plk 3，其与已知的CP Plk 1密切相关。Plk 1已被广泛研究，是有丝分裂的关键调节因子之一，其活性受G2期间转录上调和有丝分裂完成时蛋白酶体降解的控制。关于Plk 3的功能知之甚少，但我们假设它与Plk 1起着类似的作用，并在细胞周期中受到翻译后修饰的调控。该项目的目的是：（1）使用HMEC细胞的亲和下拉来鉴定新的染色体乘客蛋白;（2）绘制Plk 3和其他CP上的磷酸化位点;（3）检查含有特异性磷酸化CPS的蛋白质复合物的组成作为细胞周期，DNA损伤和氧化应激的函数。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Chromosomal passenger (CP) proteins undergo dramatic changes in their distribution during the cell cycle, localizing to kinetochores, midzone, cleavage furrow and midbody at various stages of mitosis. CPs play critical roles in the proliferation and cell cycle checkpoint signaling pathways and are the subject of intense research due to their importance as mitotic regulators. Using an antibody (P190) generated against a phosphorylated peptide, we have observed 9 major proteins that are phosphorylated exclusively during mitosis where they behave as CPs. One of these proteins has been positively identified as Plk3 which is closely related to a known CP, Plk1. Plk1 has been extensively studied and is one of the key regulators of mitosis whose activity is controlled by transcriptional upregulation during G2 and proteasomal degradation at the completion of mitosis. Less is known about Plk3 function, but we hypothesize that it plays a similar role as Plk1 and is regulated by post-translational modifications during the cell cycle. The aims of this project are: (1) Identify novel chromosomal passenger proteins using affinity pulldowns from HMEC cells; (2) Map phosphorylation sites on Plk3 and other CPs; and (3) Examine the composition of protein complexes containing specifically phosphorylated CPS as a function of the cell cycle, DNA damage, and oxidative stress.