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）：在减数分裂期间，同源染色体互相寻找，然后由Synaptonemal复合物（SC）和姐妹染色质凝聚力束缚在一起，没有任何同源重组和减数分裂分裂。编码任何SC成分或粘着素的基因中具有突变的小鼠表现出减数分裂的缺陷，并且在大多数情况下是无菌的。 SC蛋白FKBP6对于雄性小鼠的减数分裂预言I至关重要，它与一种新型的减数分裂激酶NEK1（NIMA相关激酶1）相互作用。 NEK1是一种双重活性丝氨酸/苏氨酸和酪氨酸激酶，在生殖细胞中高度表达，尤其是狭窄的窗口，包括进入预言的狭窄窗口。NEK1突变小鼠的出现严重的发育缺陷，不仅在其生育方面都表现出严重的发育缺陷，而且还显示出生长缺陷，颅面异常和多种疾病。该提案的中心假设是，NEK1是前期I需要中期进展所必需的，因为它将关键SC事件与涉及姐妹染色质凝聚的人联系起来。为了检验这一假设，我将分析具有单个核苷酸插入和随后的过早停止的Nek1kat2J小鼠系中的减数分裂进程，从而导致蛋白质产物和无效表型的截断。 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小鼠生殖细胞。这些实验将提供有关NEK1在减数分裂进程中的作用的新颖而令人兴奋的数据，以及在预言I结束时去除粘蛋白的机制，该主题报道的数据很少。我在分子生物学和细胞遗传学方面都具有强大的背景（重点是哺乳动物配子发生）；我的目标是增强我的蛋白质组学技能，以执行该项目并朝着独立研究发展。四年来，我一直是康奈尔大学的博士后研究员，其中两个是由高清基金会博士后奖资助的。在康奈尔大学，我在Paula Cohen博士的实验室进行了研究，Paula Cohen博士的实验室在记录了主要的跨界途径小鼠减数分裂方面发挥了作用。我的直接职业目标包括在高影响力期刊上发布，在康奈尔（Cornell）之外建立科学网络，并向学术机构介绍研究；我的长期职业目标是在高素质的机构中获得终身制职位，在那里我可以专注于独立研究以及对未来研究人员的培训。我在这个方向上的进步将通过与我的委托人的定期会议，关于我的研究的室内研讨会以及我的出版记录来评估。公共卫生相关性：不孕症影响了美国生殖种群的15％，许多基本原因是未知的。减数分裂的预言I期间的错误可能导致人类的生育能力或出生缺陷。了解关键基因在减数分裂过程中所涉及的关键基因在确保正确的染色体隔离方面的作用将增加我们对控制这些事件的机制的理解和不育的复杂原因。