IDENTIFICATION AND POST-TRANSLATIONAL MODIFICATION OF MITOTIC REGULATORY PROTEI

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。在细胞周期中，染色体乘客蛋白的分布发生了巨大的变化，在有丝分裂的不同阶段定位于着丝点、中间区、分裂沟和中间体。CPs在增殖和细胞周期检查点信号通路中起着关键作用，由于其作为有丝分裂调节因子的重要性，因此受到广泛研究。使用针对磷酸化肽产生的抗体（P190），我们观察到9个主要蛋白在有丝分裂期间被磷酸化，在那里它们表现为CPs。其中一种蛋白质已被确定为Plk3，它与已知的CP Plk1密切相关。Plk1被广泛研究，是有丝分裂的关键调控因子之一，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。我们对Plk3的功能知之甚少，但我们假设它与Plk1起着类似的作用，并在细胞周期中受到翻译后修饰的调节。本项目的目的是：(1)利用HMEC细胞的亲和下拉蛋白鉴定新的染色体乘客蛋白；(2)绘制Plk3和其他CPs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损伤和氧化应激的功能。

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.