Functional Analysis of the Dual Specificity Kinase NEK1 in Mammalian Meiosis

描述(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)和姐妹染色单体凝聚力连接在一起，没有这些联系，同源重组和减数分裂就不可能发生。编码任何SC成分或粘附素基因突变的小鼠表现出减数分裂配对缺陷，在大多数情况下是不育的。SC蛋白FKBP6是雄性小鼠完成减数分裂前期I所必需的，它与一种新的减数分裂激酶NEK1(NIMA相关激酶1)相互作用。NEK1是一种丝氨酸/苏氨酸和酪氨酸激酶，在生殖细胞中高度表达，特别是在进入和通过早期I期的狭窄窗口中。Nek1突变小鼠不仅在生育方面表现出严重的发育缺陷，而且还表现出生长缺陷、头面部异常和多囊肾病。这一建议的中心假设是，NEK1是前期I到中期进展所必需的，因为它将关键的SC事件与涉及姐妹染色单体凝聚力的事件联系在一起。为了验证这一假设，我将分析一组Nek1kat2J小鼠的减数分裂进程，这些小鼠携带单核苷酸插入，随后过早停止，导致蛋白质产物截断和零表型。提出了两个具体的目标：(1)深入研究FKBP6、NEK1和粘附素蛋白在小鼠精母细胞中的直接关系，并将其与在卵母细胞中的作用进行直接比较，以确定减数分裂表型中是否存在性别二型性；(2)直接在小鼠生殖细胞中评估NEK1野生型和突变型的丝氨酸/苏氨酸和酪氨酸激酶活性。这些实验将提供新的和令人兴奋的数据，关于NEK1在减数分裂过程中的作用，以及关于前期I结束时粘附素去除的机制，这是一个报道很少的课题。我在分子生物学和细胞遗传学(重点是哺乳动物配子发生)方面都有很强的背景，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，我一直是康奈尔大学的博士后研究员，其中两年是由HD基金会博士后奖资助的。在康奈尔大学，我在保拉·科恩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。我近期的职业目标包括在影响力很大的期刊上发表文章，在康奈尔大学以外建立一个科学网络，并在学术机构发表研究；我的长期职业目标是在一家高素质的机构获得一个终身教职的职位，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。我在这方面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我的出版记录来评估。 公共卫生相关性：不孕不育影响了美国约15%的生殖人口，许多潜在原因尚不清楚。减数分裂前期I的错误可能会导致人类生育或出生缺陷。了解在减数分裂过程中确保适当染色体分离的关键基因的作用将增加我们对这些事件的控制机制和不孕不育的复杂原因的理解。

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.