UNDERSTANDING THE MOLECULAR BASIS OF SELECTIVITY IN AKAP

了解 AKAP 选择性的分子基础

• 批准号: 8365785
• 负责人: STANLEY FIELDS
• 金额: $ 2.18万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-01 至 2012-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8365785
• 关键词: A kinase anchoring protein; Bacteriophage T7; Binding; Biological; Biological Assay; Biology; Cells; Cyclic AMP-Dependent Protein Kinases; Funding; Fungal Genome; Grant; Libraries; Metabolism; Molecular; National Center for Research Resources; Phage Display; Phosphotransferases; Principal Investigator; Protein Isoforms; Proteins; Research; Research Infrastructure; Resources; Source; Surface; United States National Institutes of Health; Variant; base; cost; protein function

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Protein Kinase A (PKA) is a central intracellular kinase that regulates the activity of many proteins involved in cellular metabolism. PKA activity is controlled via interactions with A Kinase Anchoring Proteins (AKAPs). AKAPs function by binding to the PKA regulatory subunit, localizing PKA within the cell. AKAPs can interact with either the α or the β isoform of the regulatory subunit of PKA, or they can interact with both. The α and β isoforms are highly conserved, making it difficult to study the molecular determinants of selectivity between isoforms. We are using phage display in combination with high-throughput sequencing to identify the sequence determinants of AKAP selectivity. We displayed a library of millions of mutagenized AKAP proteins on the surface of T7 phage and then subjected this library to selection against either the α or β isoform of the regulatory subunit of PKA. By comparing the abundance of each variant before and after selection, we derived enrichment ratios for several hundred thousand variants. Most variants performed similarly in selections against both the α and β isoforms. However, some variants displayed strong selectivity for either the α or β isoform. We are using the results of this assay in an effort to develop highly α- and β-specific AKAPs, which should bind only to PKAs with the cognate regulatory isoform. If introduced into cells at high concentrations, they should disrupt the normal regulatory interaction for their cognate isoform, enabling us to study the biological significance of the isoforms

该子项目是利用资源的众多研究子项目之一 由 NIH/NCRR 资助的中心拨款提供。子项目的主要支持 并且子项目的主要研究者可能是由其他来源提供的， 包括其他 NIH 来源。 子项目可能列出的总成本 代表子项目使用的中心基础设施的估计数量， NCRR 赠款不直接向子项目或子项目工作人员提供资金。 蛋白激酶 A (PKA) 是一种中心细胞内激酶，可调节参与细胞代谢的许多蛋白质的活性。 PKA 活性通过与 A 激酶锚定蛋白 (AKAP) 的相互作用来控制。 AKAP 通过与 PKA 调节亚基结合，将 PKA 定位在细胞内发挥作用。 AKAP 可以与 PKA 调节亚基的 α 或 β 同工型相互作用，也可以与两者相互作用。 α 和 β 同工型高度保守，因此很难研究同工型之间选择性的分子决定因素。 我们将噬菌体展示与高通量测序相结合来识别 AKAP 选择性的序列决定因素。 我们在 T7 噬菌体表面展示了包含数百万诱变 AKAP 蛋白的文库，然后针对 PKA 调节亚基的 α 或 β 同工型对该文库进行选择。 通过比较选择前后每个变体的丰度，我们得出了数十万个变体的富集率。 大多数变体在针对 α 和 β 同工型的选择中表现相似。 然而，一些变体对 α 或 β 同工型表现出很强的选择性。 我们正在利用该测定的结果来开发高度 α 和 β 特异性的 AKAP，该 AKAP 应该仅与具有同源调节亚型的 PKA 结合。 如果以高浓度引入细胞，它们应该会破坏其同源亚型的正常调节相互作用，使我们能够研究亚型的生物学意义