Functional Analysis of the Dual Specificity Kinase NEK1 in Mammalian Meiosis

双特异性激酶 NEK1 在哺乳动物减数分裂中的功能分析

基本信息

批准号：
8686013
负责人：
Joanna Kim Holloway
金额：
$ 34.32万
依托单位：
CORNELL UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-08-16 至 2014-09-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8686013
关键词：
Affect Amino Acid Sequence Antibodies Award Biochemical Biological Assay Caliber Cell Culture Techniques Cell Cycle Progression Cell physiology Cells Cephalic Chromosome Arm Chromosome Pairing Chromosome Segregation Chromosomes Cloning Complement Complementary DNA Complex Congenital Abnormality Data Data Reporting Defect Development Diploidy Diplotene Stage Ensure Event Excision Exhibits Exons Face Female Fertility Foundations Funding Future G2/M Transition Gametogenesis Generations Genes Genetic Recombination Germ Cells Goals Growth Haploidy Homologous Gene Human In Vitro Infertility Institution Journals Knock-in Mouse Knowledge Laboratories Lead Link Longevity Mammalian Cell Meiosis Meiotic Prophase I Mentors Metaphase Molecular Biology Molecular Cytogenetics Mus Mutant Strains Mice Mutation NIMA Nonsense Codon Nucleotides Oocytes Pachytene Stage Pathway interactions Peptide Sequence Determination Peptides Phase Phenotype Phosphorylation Phosphotransferases Play Point Mutation Polycystic Kidney Diseases Population Positioning Attribute Preparation Process Property Protein Truncation Protein Tyrosine Kinase Protein-Serine-Threonine Kinases Proteins Proteomics Publications Publishing Regulation Reporting Research Research Personnel Role Science Serine Signal Transduction Sister Chromatid Specificity Spermatocytes Staging Sterility Synaptonemal Complex Techniques Testing Testis Threonine Tissues Training Transcript Tyrosine Tyrosine Kinase Domain United States Universities Work career cohesin cohesion daughter cell design homologous recombination in vivo male meetings mutant novel null mutation precursor cell premature protein complex recombinational repair reproductive research study response segregation sexual dimorphism skills

项目摘要

Project Summary During meiosis, homologous chromosomes seek each other out and are then tethered together by the synaptonemal complex (SC) and sister chromatid cohesion, without which homologous recombination and meiotic division cannot occur. Mice with mutations in genes encoding any of the SC components or cohesins show meiotic pairing defects and, in most cases, are sterile. The SC protein, FKBP6, which is essential for completion of meiotic prophase I in male mice, interacts with a novel meiotic kinase, NEK1 (NIMA-related kinase 1). NEK1 is a dual activity serine/threonine and tyrosine kinase, and is highly expressed in germ cells, particularly the narrow window encompassing the entry into, and progression through, Prophase I. Nek1 mutant mice show severe developmental defects, not only in their fertility, but also show growth defects, cranial-facial abnormalities and polycystic kidney disease. The central hypothesis of this proposal is that NEK1 is required for prophase I to metaphase progression, as it links key SC events with those involving sister chromatid cohesion. To test this hypothesis, I will analyze meiotic progression in a line of Nek1kat2J miceharboring a single nucleotide insertion and a subsequent premature stop, resulting in truncation of the protein product and a null phenotype. Two specific aims are proposed: (1) to perform an in-depth study of the relationship between FKBP6, NEK1 and cohesin proteins directly in mouse spermatocytes, and compare this directly with the action in oocytes, to determine any sexual dimorphism in the meiotic phenotype and (2) to assess the serine/threonine and tyrosine kinase activities of both the wild type and mutant forms of NEK1 directly in mouse germ cells. These experiments will provide novel and exciting data on the role of NEK1 in meiotic progression, as well as on the mechanisms of cohesin removal at the end of prophase I, a subject on which there is very little reported data. I approach this project with a strong background in both molecular biology and cytogenetics (with an emphasis on mammalian gametogenesis); my goal is to strengthen my proteomics skills in order to execute this project and move toward independent research. For four years I have been a postdoctoral researcher at Cornell University, two of these years funded by an HD foundation postdoctoral award. At Cornell I have conducted research in the laboratory of Dr. Paula Cohen, whose lab has been instrumental in documenting the major crossover pathways mouse meiosis. My immediate career goals include publishing in high-impact journals, establishing a science network outside of Cornell and to present research at academic institutions; my long-term career goal is to obtain a tenure-track position at a high caliber institution where I can focus on independent research as well as the training of future researchers. My progress in this direction will be assessed by regular meetings with my co-mentors, inter-departmental seminars on my research and, ultimately, by my publication record.

项目概要在减数分裂过程中，同源染色体互相寻找，然后通过联会复合体（SC）和姐妹染色单体的内聚力，没有它们同源重组和减数分裂不能发生。编码任何 SC 成分或粘连蛋白的基因发生突变的小鼠显示减数分裂配对缺陷，并且在大多数情况下是不育的。 SC 蛋白 FKBP6，对于雄性小鼠减数分裂前期 I 的完成，与一种新型减数分裂激酶 NEK1（NIMA 相关激酶1)。 NEK1 是一种具有丝氨酸/苏氨酸和酪氨酸双重活性的激酶，在生殖细胞中高度表达，特别是包含进入和进展到前期 I 的狭窄窗口。 Nek1 突变小鼠表现出严重的发育缺陷，不仅在生育能力方面，而且还表现出生长缺陷，颅面部异常和多囊肾病。该提案的中心假设是 NEK1 是前期 I 到中期进展所必需的，因为它将关键 SC 事件与涉及的事件联系起来姐妹染色单体凝聚力。为了检验这个假设，我将分析 Nek1kat2J 系的减数分裂进展小鼠携带单个核苷酸插入和随后的过早停止，导致截断蛋白质产物和无效表型。提出两个具体目标：（1）深入研究 FKBP6、NEK1 和粘连蛋白直接在小鼠精母细胞中的关系，并比较了这一点直接与卵母细胞中的作用，以确定减数分裂表型中的任何性别二态性和（2）评估 NEK1 野生型和突变型的丝氨酸/苏氨酸和酪氨酸激酶活性直接在小鼠生殖细胞中进行。这些实验将为 NEK1 在减数分裂进展，以及前期 I 结束时粘连蛋白去除的机制，这是一个主题报道的数据很少。我在分子生物学和细胞遗传学方面拥有深厚的背景（具有强调哺乳动物配子发生）；我的目标是加强我的蛋白质组学技能，以便执行该项目并走向独立研究。四年来，我一直在以下机构担任博士后研究员：康奈尔大学，其中两年由 HD 基金会博士后奖资助。在康奈尔大学我有在 Paula Cohen 博士的实验室进行了研究，他的实验室在记录小鼠减数分裂的主要交叉途径。我近期的职业目标包括发表高影响力的文章期刊，在康奈尔大学之外建立科学网络，并在学术机构展示研究成果；我的长期职业目标是在高素质机构获得终身职位，在那里我可以专注于独立研究以及未来研究人员的培训。我在这个方向上的进步将是通过与我的共同导师定期会面、关于我的研究的跨部门研讨会进行评估，最终，根据我的发表记录。