IDENTIFICATION AND POST-TRANSLATIONAL MODIFICATION OF MITOTIC REGULATORY PROTEI

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。染色体乘客蛋白在细胞周期中的分布发生了巨大的变化，在有丝分裂的不同阶段定位于着丝点、中带、卵裂沟和中体。CP在细胞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而成为研究的热点。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被专门磷酸化，在有丝分裂中它们表现为CP。其中一种蛋白被鉴定为Plk3，它与已知的CP Plk1密切相关。PLK1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期转录上调和有丝分裂完成时蛋白酶体降解的控制。对Plk3的功能知之甚少，但我们假设它扮演着与Plk1类似的角色，并在细胞周期中受到翻译后修饰的调节。该项目的目标是：(1)利用亲和力下拉从HMEC细胞中鉴定新的染色体乘客蛋白；(2)定位Plk3和其他CP上的磷酸化位点；以及(3)检测含有特定磷酸化CP的蛋白质复合体的组成随细胞周期、DNA损伤和氧化应激的变化。

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.