The Role of Cks Proteins in Mammalian Meiosis

Cks 蛋白在哺乳动物减数分裂中的作用

• 批准号: 7405302
• 负责人: CHARLES H. SPRUCK
• 金额: $ 38.11万
• 依托单位: SIDNEY KIMMEL CANCER CENTER
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-04-15 至 2011-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7405302
• 关键词: 26S proteasome; Anaphase; Aneuploidy; Basic Science; Binding Proteins; Biological Assay; Biological Models; CDC2 Protein Kinase; Cell Cycle; Cell division; Chromatin; Clinical; Complex; Congenital Abnormality; Cyclin-Dependent Kinases; Cyclins; Development; Eukaryota; Eukaryotic Cell; Female; Generations; Genetic Transcription; Germ Cells; H1 kinase; Human; In Vitro; Infertility; Knock-in Mouse; Lead; Link; Mammals; Maturation-Promoting Factor; Mediating; Meiosis; Mental Retardation; Metaphase; Microinjections; Mitosis; Mitotic; Molecular; Molecular Abnormality; Monosomy; Mus; Oocytes; Orthologous Gene; Phosphorylation; Phosphotransferases; Play; Pregnancy; Process; Proteins; Proteolysis; Public Health; Recombinants; Regulation; Research Design; Role; Spermatocytes; Staging; Sterility; Transcriptional Activation; Transcriptional Regulation; Trisomy; Ubiquitin; Ubiquitination; Western Blotting; Woman; anaphase-promoting complex; clinically relevant; cyclin B1; male; men; promoter; protein function; research study; ubiquitin ligase

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.

描述（由申请人提供）：背景：CKS蛋白很小（9 kD），高度保守的蛋白质，与细胞周期蛋白依赖性激酶（CDKS）相关。 Cyclin B1，CDK1和CKS蛋白形成一种称为成熟 - 促进因子（MPF）的异质三聚体，可调节有丝分裂和减数分裂细胞分裂周期中M相。尽管尚不理解CKS蛋白的精确功能，但人们认为它们是“适配器”，将Cyclin-CDK复合物与各种细胞分裂调节过程联系起来。我们在功能上使小鼠的直系同源性CKS2失活，发现它在哺乳动物减数分裂中起着至关重要的作用。 CKS2 - / - 雄性和雌性小鼠是由于减数分裂I（ML）中期的精子细胞和卵母细胞的停滞而无菌的。客观/假设：较低真核生物的研究已在几个重要的M相调节过程中牵涉CKS蛋白，包括：1）激活MPF激酶活性； 2）激活多亚基泛素连接酶的复合体/循环体（APC/C）； 3）细胞周期蛋白B1的蛋白水解； 4）APC/C活化剂蛋白CDC20的转录激活。我们的假设是CKS2在哺乳动物ML中执行类似的调节功能。具体目的：AIM1。确定CKS2在调节ML中的MPF和APC活性中的作用。 AIM2。定义CKS2在调节ML中26S蛋白酶体功能中的作用。 AIM3。通过生成CKS2CKS1/CKS1敲击小鼠，研究哺乳动物ML中CKS2的直系同源性特异性功能。研究设计：AIM1）确定CKS2在调节MPF活性中的作用，我们将：1）使用H1激酶测定法分析CDK1激酶活性； 2）通过蛋白质印迹分析分析CDK1磷酸化状态。 CKS2在APC/C激活中的作用将使用精子提取物上的体外泛素化测定确定。 AIM2）CKS2在介导26S蛋白酶体功能中的作用将通过：1）使用精子提取物和重组细胞周期蛋白B1底物进行体外蛋白水解测定； 2）表达分析和卵母细胞显微注射实验，以确定CKS2对CDC20转录的影响。 AIM3）我们将通过产生CKS2（CKS1/CKS1）敲入小鼠来评估哺乳动物减数症I中CKS2的直系特异性功能，该敲门小鼠通过同源靶向在CKS2启动子调节下表达CKS1。与公共卫生的相关性：从基础科学和临床的角度来看，阐明哺乳动物中减数分裂I的分子控制是高度相关的。关于在分子水平上如何调节减数分裂以及该过程的失调如何导致人类不育症的方式，知之甚少。 ML的中期到后期过渡在临床上特别相关，因为其功能失调与女性的卵母细胞非整倍性相关，男性和女性的不孕症。非整倍性（三体或单切开术）是人类妊娠中最常见的遗传异常（占病例的5-25％），是人类智力低下的主要原因。揭示CKS2在哺乳动物减数分裂中的功能可能会更好地了解ML的调节，并有助于确定人类不孕和先天缺陷的潜在原因。