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

动粒相互作用蛋白的鉴定 (KNL-1/KNL-3/KNL-2)

• 批准号: 8171385
• 负责人: Arshad Desai
• 金额: $ 0.24万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-01 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8171385
• 关键词: Biological Assay; Caenorhabditis elegans; Cell division; Chromosome Segregation; Chromosomes; Collaborations; Computer Retrieval of Information on Scientific Projects Database; Congenital Abnormality; Consensus; Embryo; Funding; Grant; In Vitro; Institution; Kinetochores; Location; Malignant Neoplasms; Maps; Mass Spectrum Analysis; Mitosis; Mitotic; Mitotic Checkpoint; N-terminal; Phosphorylation; Phosphotransferases; Physiological; Proteins; Proteomics; Radioactive; Regulation; Research; Research Institute; Research Personnel; Resources; Role; Sampling; Series; Signal Transduction; Site; Source; Testing; Time; United States National Institutes of Health; human PLK1 protein; melting; prevent

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.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目和 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 Polo 样激酶 1 (Plk1) 是一种中央有丝分裂激酶，参与有丝分裂检查点的调节，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，这是出生缺陷的已知原因，也是癌症发生的一个促成因素。 Plk1 功能的多样性使其难以解析其在细胞分裂中的功能。解决这个问题的一个潜在方法是识别着丝粒特异性 PLK-1 底物。我们确定了一系列 N 末端“MELT”一种名为 KNL-1（动粒空蛋白-1）的蛋白质中的重复序列，适合 PLK-1 磷酸化的共有位点。 KNL-1 是一种位于染色体上的蛋白质，对于着丝粒组装和染色体分离是必需的。我们正在测试 KNL-1 是 PLK-1 底物的假设，并研究这种磷酸调节在染色体分离和检查点信号传导中的作用。根据 MELT 重复序列的位置预测，KNL-1 (KNL-11-505) 的 N 末端片段是极好的体外 PLK-1 底物（放射性激酶测定）。为了评估 KNL-1 PLK-1 磷酸化的功能，我们与斯克里普斯研究所生理蛋白质组学中心合作，通过结合体外激酶测定和质谱分析，在 KNL-1 上绘制了 PLK-1 靶位点。在我们提交分析的 3 个样品中，分别多次鉴定出 6 个磷酸位点，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中有丝分裂进展和染色体分离中的作用。