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.

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 Polo-like kinase1(Plk1)是一种中央有丝分裂激酶，参与有丝分裂检查点的调控，是一种控制染色体分离和防止整个染色体的丢失或获得的机制，是已知的出生缺陷原因和癌症发生的促成因素。Plk1功能的多样性使其在细胞分裂中的功能很难去卷积。规避这个问题的一个潜在方法是确定动粒特异的PLK-1底物。我们鉴定了一系列N-末端？熔体？在一种名为Knl-1(Kinetochore Null Protein-1)的蛋白质中重复，该蛋白质符合PLK-1磷酸化的共识位点。Knl-1是一种位于染色体上的蛋白质，它是动粒组装和染色体分离所必需的。我们正在验证Knl-1是PLK-1底物的假设，并研究这种磷酸调节在染色体分离和检查点信号转导中的作用。根据熔体重复序列的位置预测，Kn1-1的N-末端片段(Kn1-11-505)在体外是一个很好的PLK-1底物。为了评估Knl-1的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，通过体外激酶分析和质谱分析相结合的方法，定位了Knl-1上的PLK-1靶点。在我们提交分析的3个样本中，每个样本都多次鉴定出6个亚磷酸盐，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。