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(申请人提供)：背景：CKS蛋白是一种小分子(9kD)高度保守的蛋白，与细胞周期蛋白依赖性蛋白激酶(CDK)有关。细胞周期蛋白B1、CDK1和CKS蛋白形成一种被称为成熟促进因子(MPF)的异源三聚体，在有丝分裂和减数分裂周期中调节M期。尽管目前尚不清楚CKS蛋白的确切功能(S)，但人们认为它们作为“接头”发挥作用，将细胞周期蛋白-CDK复合体与各种细胞分裂调控过程联系起来。我们在小鼠中对同源基因Cks2进行了功能失活，发现它在哺乳动物减数分裂中起着至关重要的作用。CKS2-/-雄性和雌性小鼠是不育的，这是由于精母细胞和卵母细胞停止在减数分裂I中期(M1)。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期调控过程，包括：1)MPF激酶活性的激活；2)多亚单位泛素连接酶后期促进复合体/环体(APC/C)的激活；3)细胞周期蛋白B1的蛋白分解；以及4)APC/C激活蛋白CDC20的转录激活。我们的假设是Cks2在哺乳动物的ML中执行类似的调节功能。具体目标：目标1.确定Cks2在调节m1的MPF和APC/C活性中的作用。AIM2.明确Cks2在调节ML中26S蛋白酶体功能中的作用。Aim3.通过CKS2Cks1/CKS1敲入小鼠的研究，探讨CKS2在哺乳动物ML中的同源特异性功能(S)。研究设计：1)确定Cks2在调节MPF活性中的作用，我们将：1)用H1激酶分析CDK1的活性；2)用Western印迹分析CDK1的磷酸化状态。Cks2在APC/C激活中的作用将通过精母细胞提取液的体外泛素化试验来确定。目的：1)利用精母细胞提取液和重组细胞周期蛋白B1底物进行体外蛋白分解实验；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。目的：我们将通过同源打靶的方法产生CKS2(CKS1/CKS1)敲入小鼠，并在CKS2启动子的调控下表达CKS1，以此来评价CKS2在哺乳动物减数分裂I中的同源特异性功能(S)。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。关于减数分裂是如何在分子水平上调节的，以及这一过程的失调如何导致人类不孕，人们知之甚少。ML的中期到后期的转变特别具有临床意义，因为它的失调与女性的卵母细胞非整倍体和男性和女性的不孕不育有关。非整倍体(三体或单体)是人类妊娠中最常见的遗传异常(占病例的5%-25%)，也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(S)可能会更好地理解ML是如何调控的，并有助于确定人类不孕不育和出生缺陷的潜在原因。