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：
7962701
负责人：
Joanna Kim Holloway
金额：
$ 14.57万
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 2012-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7962701
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 Cytogenetics 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 Mus Mutant Strains Mice Mutation 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 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 public health relevance recombinational repair reproductive research study response segregation sexual dimorphism skills

项目摘要

DESCRIPTION (provided by applicant): 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 mice harboring 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. PUBLIC HEALTH RELEVANCE: Infertility affects about 15% of the reproductive population of the United States, with many of the underlying causes being unknown. Errors during prophase I of meiosis can lead to fertility or birth defects in humans. Understanding the role of key genes involved in ensuring proper chromosomal segregation during meiosis will increase our understanding of the mechanisms controlling these events and the complex causes of infertility.

描述（由申请人提供）：在减数分裂过程中，同源染色体相互寻找，然后通过联会复合体（SC）和姐妹染色单体内聚力将其束缚在一起，否则同源重组和减数分裂就无法发生。编码任何 SC 成分或粘连蛋白的基因发生突变的小鼠表现出减数分裂配对缺陷，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 的完成至关重要，它与一种新型减数分裂激酶 NEK1（NIMA 相关激酶 1）相互作用。 NEK1 是一种双活性丝氨酸/苏氨酸和酪氨酸激酶，在生殖细胞中高度表达，特别是在进入和进展到前期 I 的狭窄窗口中。Nek1 突变小鼠表现出严重的发育缺陷，不仅在生育能力方面，但也显示出生长缺陷、颅面部异常和多囊肾病。该提议的中心假设是 NEK1 是前期 I 到中期进展所必需的，因为它将关键 SC 事件与涉及姐妹染色单体凝聚力的事件联系起来。为了检验这一假设，我将分析一系列 Nek1kat2J 小鼠的减数分裂进展，这些小鼠含有单核苷酸插入和随后的过早停止，导致蛋白质产物截断和无效表型。提出了两个具体目标：（1）直接在小鼠精母细胞中深入研究 FKBP6、NEK1 和粘连蛋白之间的关系，并将其直接与卵母细胞中的作用进行比较，以确定减数分裂表型中的性别二态性(2)直接评估小鼠生殖细胞中野生型和突变型NEK1的丝氨酸/苏氨酸和酪氨酸激酶活性。这些实验将提供有关 NEK1 在减数分裂进程中的作用以及前期 I 结束时粘连蛋白去除机制的新颖且令人兴奋的数据，而关于这一主题的报道数据很少。我在分子生物学和细胞遗传学（重点是哺乳动物配子发生）方面拥有深厚的背景来完成这个项目；我的目标是加强我的蛋白质组学技能，以便执行这个项目并走向独立研究。四年来，我一直在康奈尔大学担任博士后研究员，其中两年由 HD 基金会博士后奖资助。在康奈尔大学，我在 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方面发挥了重要作用。我的近期职业目标包括在高影响力期刊上发表文章、在康奈尔大学以外建立科学网络以及在学术机构展示研究成果；我的长期职业目标是在高水平机构获得终身职位，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展将通过与我的共同导师的定期会议、关于我的研究的跨部门研讨会以及最终的出版记录来评估。公共卫生相关性：不孕症影响着美国约 15% 的生育人口，许多根本原因尚不清楚。减数分裂前期 I 期间的错误可能导致人类生育能力或出生缺陷。了解减数分裂过程中确保染色体正确分离的关键基因的作用将增加我们对控制这些事件的机制和不孕不育的复杂原因的理解。