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: 7598982
• 负责人: CHARLES H. SPRUCK
• 金额: $ 14.54万
• 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 2009-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7598982
• 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; 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 蛋白是小 (9 kD)、高度保守的蛋白，与细胞周期蛋白依赖性激酶 (Cdks) 相关。细胞周期蛋白 B1、Cdk1 和 Cks 蛋白形成称为成熟促进因子 (MPF) 的异源三聚体，可调节有丝分裂和减数分裂细胞分裂周期中的 M 期。尽管 Cks 蛋白的精确功能尚不清楚，但据信它们起到“适配器”的作用，将细胞周期蛋白-Cdk 复合物与多种细胞分裂调节过程连接起来。我们在小鼠体内功能性地灭活了直系同源物 Cks2，并发现它在哺乳动物减数分裂中发挥着重要作用。 CKS2-/-雄性和雌性小鼠由于精母细胞和卵母细胞在减数分裂I(M1)中期的停滞而不育。目的/假设：对低等真核生物的研究表明 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/环体(APC/C)的激活； 3) 细胞周期蛋白B1的蛋白水解； 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 M1 中发挥类似的调节功能。具体目标： 目标1。确定 Cks2 在调节 M1 中 MPF 和 APC/C 活性中的作用。目标2。定义 M1 中 Cks2 在调节 26S 蛋白酶体功能中的作用。目标3。通过生成 CKS2Cks1/Cks1 敲入小鼠来研究哺乳动物 M1 中 Cks2 的直系同源特异性功能。研究设计：目的1) 确定Cks2 在调节MPF 活性中的作用，我们将： 1) 使用H1 激酶测定分析Cdk1 激酶活性； 2) 通过Western blot分析Cdk1磷酸化状态。 Cks2 在 APC/C 激活中的作用将通过对精母细胞提取物进行体外泛素化测定来确定。 Aim2) Cks2 在介导 26S 蛋白酶体功能中的作用将通过以下方式进行评估： 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) 我们将通过同源靶向产生 CKS2(Cks1/Cks1) 敲入小鼠来评估 Cks2 在哺乳动物减数分裂 I 中的直系同源特异性功能，这些小鼠在 CKS2 启动子的调节下表达 Cks1。与公共健康的相关性：从基础科学和临床角度来看，阐明哺乳动物减数分裂 I 的分子控制都具有高度相关性。关于减数分裂如何在分子水平上进行调节以及该过程的失调如何导致人类不育，人们知之甚少。 M1的中期到后期的转变在临床上特别相关，因为它的失调与女性卵母细胞非整倍性以及男性和女性的不育有关。非整倍体（三体性或单体性）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可以让我们更好地了解 Ml 是如何调节的，并有助于确定人类不孕和出生缺陷的根本原因。