IDENTIFICATION OF KINETOCHORE INTERACTING PROTEINS (KNL-1/KNL-3/KNL-2)

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。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 Polo样激酶1（Plk 1）是一种参与有丝分裂检查点调控的中心有丝分裂激酶，这是一种控制染色体分离和防止整个染色体丢失或获得的机制，是出生缺陷的已知原因，也是癌症发生的一个促成因素。Plk 1功能的多样性使得其在细胞分裂中的功能难以去卷积。一个潜在的方法来规避这个问题是确定kinetochore特异性PLK-1基板。我们发现了一系列的N末端？融化？在称为KNL-1（动粒蛋白-1）的蛋白质中重复，其适合PLK-1磷酸化的共有位点。KNL-1是位于染色体上的蛋白质，其对于着丝粒的组装和染色体分离是必需的。我们正在验证KNL-1是PLK-1底物的假设，并研究这种磷酸化调节在染色体分离和检查点信号传导中的作用。正如MELT重复序列的位置所预测的，KNL-1的N-末端片段（KNL-11-505）是体外（放射性激酶测定）极好的PLK-1底物。为了评估PLK-1磷酸化KNL-1的功能，与Scripps研究所生理蛋白质组学中心合作，我们通过使用体外激酶测定和质谱分析的组合来绘制KNL-1上PLK-1的靶位点。在我们提交分析的3个样品中，6个磷酸化位点分别被多次鉴定，表明它们是KNL-1上PLK-1磷酸化的主要位点。现在我们正在研究这些磷酸化在控制线虫胚胎有丝分裂进程和染色体分离中的作用。

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. Polo-like kinase 1 (Plk1) is a central mitotic kinase implicated in the regulation of the mitotic checkpoint, a mechanism controlling chromosomes segregation and preventing loss or gain of whole chromosomes, which is a known cause of birth defects and a contributing factor in the genesis of cancer. The multiplicity of Plk1 functions for has made it difficult to deconvolve its function in cell division. A potential approach to circumventing this problem is to identify kinetochore-specific PLK-1 substrates. We identified a series of N-terminal ?MELT? repeats in a protein called KNL-1 (Kinetochore Null protein-1) that fit the consensus site for PLK-1 phosphorylation. KNL-1 is a protein located on the chromosomes necessary for the assembly of the kinetochore and chromosomes segregation. We are testing the hypothesis that KNL-1 is a PLK-1 substrate and investigate the role of this phosphoregulation in chromosome segregation and checkpoint signaling. As predicted by the location of the MELT repeats, the N-terminal fragment of KNL-1 (KNL-11-505) is an excellent PLK-1 substrate in vitro (radioactive kinase assays). To assess the function of PLK-1 phosphorylation of KNL-1, in collaboration with the Scripps Research Institute Center for Physiological Proteomics, we mapped PLK-1 target sites on KNL-1 by using a combination of in vitro kinase assays and analysis by mass spectrometry. 6 phosphosites were each identified multiple times in the 3 samples we submitted to analysis, indicating that they are major sites on KNL-1 for PLK-1 phosphorylation. Now we are investigating the role of these phosphorylations in the control of mitosis progression and chromosomes segregation in C.elegans embryos.