GENETIC MUTATIONS OF PROTEIN KINASE INHIBITOR

蛋白激酶抑制剂的基因突变

• 批准号: 2378544
• 负责人: REJEAN L IDZERDA
• 金额: $ 10.19万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-03-01 至 2001-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2378544
• 关键词: cell nucleus; embryonic stem cell; enzyme inhibitors; fertility; gene expression; gene mutation; gene targeting; genetic strain; genetically modified animals; isozymes; laboratory mouse; male; phenotype; protein kinase A; protein structure function; reporter genes; testis; tissue /cell culture; transfection

DESCRIPTION: A vast array of hormones, upon binding to their cognate receptors on the cell surface, cause an increase in intracellular cAMP, the principal target of which is cAMP-dependent protein kinase (PKA). This system constitutes a major signaling pathway by which animal physiology is regulated. The Walsh inhibitor, or PKI, is a potent and highly specific inhibitor of PKA that was discovered nearly 30 years ago in skeletal muscle. A second inhibitor, referred to as testis PKI, was subsequently identified. It is now known that these two isoforms of PKI are encoded by unique genes, and are expressed in a distinct, but overlapping, assembly of tissues. Importantly, both are expressed in testis, where they are differentially regulated. Despite extensive biochemical characterization, including the recent discovery that PKI exhibits a nuclear export activity, little is known about the physiological role of PKI. The goal of this project is to examine the importance of the inhibitory and nuclear export activities of PKI in male reproductive function. This project uses embryonic stem cell mediated gene targeting technology to develop mouse strains carrying either an inactivating mutation in each PKI gene or a subtle mutation that inactivates the nuclear export signal of PKI. The phenotypic and biochemical outcome of these mutations will be assessed. Preliminary studies will identify which of the cell types (germ cells vs. non-germ cells) of the testis express the two PKI isoforms, and will provide the basis for subsequent analysis of the mutant mouse lines. In addition, the nuclear export signal of PKI will be identified by transient transfection studies in cultured cells in preparation for the gene targeting in mice. These studies will identify defects in testicular function resulting from a loss of, or a functional alteration in PKI, as well as provide PKI-deficient mice for future studies on the role of PKI in non- reproductive tissues. It is anticipated that these molecular genetic approaches will provide a wealth of novel information about the physiological function of PKI, and will contribute significantly to our overall understanding of the cAMP second messenger system in male reproduction and fertility, an area of global concern.

描述：大量激素与同源激素结合后 细胞表面的受体，导致细胞内cAMP增加， 其主要靶点是 cAMP 依赖性蛋白激酶 (PKA)。 该系统构成了动物的主要信号传导途径 生理机能受到调节。 Walsh 抑制剂（PKI）是一种有效且 近 30 年前发现的高度特异性的 PKA 抑制剂 在骨骼肌中。 第二种抑制剂，称为睾丸 PKI，是 随后确定。 现在已知 PKI 的这两种亚型 由独特的基因编码，并以独特的方式表达，但 重叠，组织的组装。 重要的是，两者都表达为 睾丸，它们在那里受到不同的调节。 尽管有广泛的生化特征，包括最近的 发现 PKI 表现出核​​出口活动，但鲜为人知 关于PKI的生理作用。 该项目的目标是 审查抑制和核出口活动的重要性 PKI 对男性生殖功能的影响。 该项目使用胚胎干 细胞介导的基因打靶技术开发小鼠品系 每个 PKI 基因都携带失活突变或微妙的突变 突变使 PKI 的核输出信号失活。 这 将评估这些突变的表型和生化结果。 初步研究将确定哪些细胞类型（生殖细胞） 睾丸的（相对于非生殖细胞）表达两种 PKI 同工型，并且会 为后续突变小鼠系的分析提供基础。 此外，PKI的核输出信号将通过 在培养细胞中进行瞬时转染研究，为 小鼠中的基因靶向。 这些研究将识别缺陷 睾丸功能丧失或功能改变所致 并为未来的研究提供 PKI 缺陷小鼠 PKI 在非生殖组织中的作用。 预计这些分子遗传学方法将提供 有关 PKI 生理功能的大量新颖信息， 并将极大地有助于我们对 cAMP 第二信使系统在男性生殖和生育能力中的作用 全球关注的。