The Role of Cks Proteins in Mammalian Meiosis

描述（由申请人提供）：背景：Cks蛋白是与细胞周期蛋白依赖性激酶（Cdks）相关的小的（9 kD）、高度保守的蛋白.细胞周期蛋白B1、Cdk 1和Cks蛋白形成称为成熟促进因子（MPF）的异源三聚体，其调节有丝分裂和减数分裂细胞分裂周期中的M期。虽然Cks蛋白的精确功能还不清楚，但据信它们起“衔接子”的作用，将细胞周期蛋白-Cdk复合物连接到多种细胞分裂调节过程。我们在小鼠中功能性失活了直系同源物Cks 2，发现它在哺乳动物减数分裂中起着重要作用。CKS 2-/-雄性和雌性小鼠由于精母细胞和卵母细胞在减数分裂I（Ml）中期停滞而不育。目的/假设：在低等真核生物中的研究表明，Cks蛋白参与了几个重要的M期调节过程，包括：1）MPF激酶活性的激活; 2）多亚基泛素连接酶后期促进复合物/细胞周期体（APC/C）的激活; 3）细胞周期蛋白B1的蛋白水解;和4）APC/C激活蛋白Cdc 20的转录激活。我们的假设是Cks 2在哺乳动物Ml中执行类似的调节功能。具体目标：目标1。确定Cks 2在Ml中调节MPF和APC/C活性中的作用。目标2。明确Cks 2在Ml中调节26 S蛋白酶体功能的作用。目标3。通过产生CKS 2Cks 1/Cks 1敲入小鼠来研究哺乳动物M1中Cks 2的直系同源物特异性功能。研究设计：目的1）确定Cks 2在调节MPF活性中的作用，我们将：1）使用H1激酶测定法分析Cdk 1激酶活性; 2）使用Western印迹分析法分析Cdk 1磷酸化状态。Cks 2在APC/C活化中的作用将使用精母细胞提取物的体外泛素化测定来确定。目的2）Cks 2在介导26 S蛋白酶体功能中的作用将通过以下方法进行评估：1）使用精母细胞提取物和重组细胞周期蛋白B1底物的体外蛋白水解测定;和2）表达分析和卵母细胞显微注射实验以确定Cks 2对Cdc 20转录的影响。目的3）我们将通过产生CKS 2（Cks 1/Cks 1）敲入小鼠来评估Cks 2在哺乳动物减数分裂I中的直系同源物特异性功能，所述小鼠通过同源靶向在CKS 2启动子的调控下表达Cks 1。与公共卫生的相关性：从基础科学和临床的角度来看，阐明哺乳动物减数分裂I的分子控制是高度相关的。关于减数分裂在分子水平上是如何调节的，以及这个过程的失调如何导致人类不育，我们知之甚少。Ml的中期到后期的转变在临床上是特别相关的，因为其失调与女性中的卵母细胞非整倍性和男性和女性中的不育症相关。非整倍性（三体或单体）是人类妊娠中最常见的遗传异常（5-25%的病例），也是人类智力迟钝的主要原因。揭示Cks 2在哺乳动物减数分裂中的功能可以更好地理解Ml是如何调节的，并有助于确定人类不育和出生缺陷的根本原因。

DESCRIPTION (provided by applicant): Background: Cks proteins are small (9 kD), highly conserved proteins that associate with cyclin- dependent kinases (Cdks). Cyclin B1, Cdk1, and Cks protein form a hetero-trimer known as maturation- promoting factor (MPF) that regulates M phase in both mitotic and meiotic cell division cycles. Although the precise function(s) of Cks proteins is not understood, it is believed that they function as "adaptors," linking cyclin-Cdk complexes to a variety of cell division regulatory processes. We functionally inactivated the ortholog Cks2 in mice and found that it plays an essential role in mammalian meiosis. CKS2-/- male and female mice are sterile due to an arrest of spermatocytes and oocytes in metaphase of meiosis I (Ml). Objective/Hypothesis: Studies in lower eukaryotes have implicated Cks proteins in several important M phase regulatory processes including: 1) activation of MPF kinase activity; 2) activation of the multi-subunit ubiquitin ligase anaphase-promoting complex/cyclosome (APC/C); 3) proteolysis of cyclin B1; and 4) transcriptional activation of APC/C activator protein Cdc20. Our hypothesis is that Cks2 performs analogous regulatory functions in mammalian Ml. Specific Aims: Aim1. Determine the role of Cks2 in regulating MPF and APC/C activity in Ml. Aim2. Define the role of Cks2 in regulating 26S proteasome function in Ml. Aim3. Investigate the ortholog-specific function(s) of Cks2 in mammalian Ml through generation of CKS2Cks1/Cks1 knock-in mice. Study Design: Aim1) determine the role of Cks2 in regulating MPF activity, we will: 1) analyze Cdk1 kinase activity using H1 kinase assays; and 2) analyze Cdk1 phosphorylation status by Western blot analysis. The role of Cks2 in APC/C activation will be determined using in vitro ubiquitination assays on spermatocyte extracts. Aim2) the role of Cks2 in mediating 26S proteasome functions will be evaluated by: 1) in vitro proteolysis assays using spermatocyte extracts and a recombinant cyclin B1 substrate; and 2) expression analysis and oocyte microinjection experiments to determine the effect of Cks2 on Cdc20 transcription. Aim3) we will evaluate the ortholog-specific function(s) of Cks2 in mammalian meiosis I by generating CKS2(Cks1/Cks1) knock-in mice, which express Cks1 under regulation of the CKS2 promoter, by homologous targeting. Relevance to Public Health: Clarifying the molecular controls of meiosis I in mammals is highly relevant from both a basic science and clinical perspective. Very little is known regarding how meiosis is regulated at the molecular level and how dysregulation of this process leads to human infertility. The metaphase-to- anaphase transition of Ml is particularly relevant clinically because its dysregulation is associated with oocyte aneuploidy in women and infertility in both men and women. Aneuploidy (trisomy or monosomy) is the most common genetic abnormality in human pregnancies (5-25% of cases) and the predominant cause of mental retardation in humans. Uncovering the function(s) of Cks2 in mammalian meiosis could lead to a better understanding about how Ml is regulated, and help to define the underlying causes of infertility and birth defects in humans.