Functional Analysis of the Dual Specificity Kinase NEK1 in Mammalian Meiosis

项目摘要 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，没有它同源重组和 减数分裂不可能发生。具有任何SC成分或粘附素编码基因突变的小鼠 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，它是 雄性小鼠完成减数分裂前期I，与新的减数分裂激酶NEK1(NIMA相关)相互作用 激酶1)。NEK1是一种具有丝氨酸/苏氨酸和酪氨酸双活性的蛋白，在生殖细胞中高度表达， 尤其是包括进入和通过第一阶段的狭窄窗口 突变小鼠表现出严重的发育缺陷，不仅在生育方面，而且还表现出生长缺陷， 头面部异常和多囊肾病。这一提议的中心假设是NEK1 是前期I到中期进展所必需的，因为它将关键SC事件与涉及的事件联系起来 姐妹染色单体凝聚力。为了验证这一假设，我将分析一系列Nek1kat2J的减数分裂进程 小鼠携带单核苷酸插入和随后的过早停止，导致截断 蛋白质产物和零表型。提出了两个具体目标：(1)深入研究 FKBP6、NEK1与小鼠精母细胞粘附素蛋白的直接关系及其比较 直接在卵母细胞中的作用，以确定减数分裂表型中的任何性别二型性和(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。这些实验将提供新的和令人兴奋的数据，说明NEK1在 减数分裂进程，以及在前期I结束时粘连蛋白去除的机制，这是一个关于 这方面的报道数据非常少。 我对这个项目有很强的分子生物学和细胞遗传学背景(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 这一项目并朝着独立研究的方向发展。四年来，我一直是哈佛大学的博士后研究员 康奈尔大学，其中两年由HD基金会博士后奖项资助。在康奈尔，我有 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。我近期的职业目标包括出版有影响力的作品。 期刊，在康奈尔大学以外建立一个科学网络，并在学术机构展示研究；我的 长期的职业目标是在一所高水平的机构获得一个终身教职的职位，在那里我可以专注于 自主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 通过定期与共同导师的会议、关于我的研究的跨部门研讨会和 最终，根据我的出版记录。

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.