TARGETED DISRUPTION OF PKA LOCALIZATION BY AKAP150

AKAP150 有针对性地破坏 PKA 定位

• 批准号: 6207304
• 负责人: MARGARET L ALLEN
• 金额: $ 3.43万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-09-16 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6207304
• 关键词: biological signal transduction; enzyme activity; gene expression; gene mutation; gene targeting; genetically modified animals; isozymes; laboratory mouse; molecular genetics; mutant; neuroanatomy; neurons; neuropharmacology; neurophysiology; phenotype; phosphomonoesterases; polymerase chain reaction; protein binding; protein kinase A; tissue /cell culture

This proposal is designed to study the effects of disrupting PKA localization by the neural specific A-kinase anchoring protein, AKAP150. Elevations of cAMP within a cell occur as a result of the opposing actions of adenylyl cyclase and phosphodiestrerases. As a result, cAMP concentration fluctuations occur within rather limited microdomains of a cell. These facts, coupled with the very broad PKA specificity, make it reasonable that restricted subcellular localization is important in PKA signaling fidelity. One of our approaches to study the importance of localized docking of PKA is to create a line of knock-in mice, which carry a mutation in AKAP150. This mutation introduces a premature stop codon in the open reading frame and results in a 36 amino acid truncation of the C-terminal end of the protein, effectively eliminating any PKA docking site. The remainder of this scaffolding AKAP150 protein will remain intact. In a second approach, we will generate a line of mice carrying a complete knock-out of the AKAP150 gene. We anticipate that this will give rise to mice with a more marked phenotype as the complex knock-out will disrupt the signalling fidelity of several kinase and phosphatase pathways.

该提案旨在研究神经特异性 A 激酶锚定蛋白 AKAP150 破坏 PKA 定位的影响。细胞内 cAMP 升高是腺苷酸环化酶和磷酸二酯酶相反作用的结果。因此，cAMP 浓度波动发生在细胞相当有限的微区内。这些事实，加上非常广泛的 PKA 特异性，使得限制性亚细胞定位对于 PKA 信号保真度的重要作用是合理的。我们研究 PKA 局部对接重要性的方法之一是创建一系列携带 AKAP150 突变的敲入小鼠。该突变在开放阅读框中引入了提前终止密码子，导致蛋白质 C 末端截断 36 个氨基酸，有效消除了任何 PKA 对接位点。该支架 AKAP150 蛋白的其余部分将保持完整。在第二种方法中，我们将产生一系列携带 AKAP150 基因完全敲除的小鼠。我们预计这将产生具有更明显表型的小鼠，因为复杂的敲除将破坏几种激酶和磷酸酶途径的信号保真度。