GENETIC MUTATIONS OF PROTEIN KINASE INHIBITOR

蛋白激酶抑制剂的基因突变

• 批准号: 2668592
• 负责人: REJEAN L IDZERDA
• 金额: $ 8.77万
• 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/2668592
• 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：一系列激素，在与它们的同源物结合后， 细胞表面上的受体，引起细胞内cAMP的增加， 其主要靶点是cAMP依赖性蛋白激酶（PKA）。 这一系统构成了一个主要的信号通路， 生理是有规律的。 沃尔什抑制剂，或PKI，是一种有效的， PKA的高度特异性抑制剂是近30年前发现的 在骨骼肌中。 第二种抑制剂，称为睾丸PKI， 随后被确认。 现在已知PKI的这两种异构体 由独特的基因编码，并以独特的，但 组织的重叠和组装。 重要的是，两者都表达在 睾丸，在那里它们受到不同的调节。 尽管有广泛的生化特征，包括最近的 发现PKI展示了核出口活动，但鲜为人知 PKI的生理作用 该项目的目标是 审查抑制性活动和核出口活动的重要性 PKI在男性生殖功能中的作用 这个项目使用胚胎干细胞 细胞介导的基因打靶技术开发小鼠品系 在每个PKI基因中携带失活突变，或者在每个PKI基因中携带细微的 使PKI的核输出信号失活的突变。 的 将评估这些突变的表型和生化结果。 初步研究将确定哪种细胞类型（生殖细胞 vs.非生殖细胞）表达两种PKI同种型， 为后续突变小鼠品系的分析提供基础。 此外，PKI的核出口信号将被识别， 培养细胞中的瞬时转染研究，为 小鼠的基因打靶 这些研究将确定缺陷， 睾丸功能丧失或功能改变导致的睾丸功能 在PKI中，以及为未来的研究提供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 关于PKI生理功能的丰富新颖信息， 并将大大有助于我们对 cAMP第二信使系统在男性生殖和生育中的作用 全球关注。