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

该副本是利用资源的众多研究子项目之一 由NIH/NCRR资助的中心赠款提供。对该子弹的主要支持 而且，副投影的主要研究员可能是其他来源提供的 包括其他NIH来源。 列出的总费用可能 代表subproject使用的中心基础架构的估计量， NCRR赠款不直接向子弹或副本人员提供的直接资金。 蛋白激酶A（PKA）是一种中央细胞内激酶，可调节许多参与细胞代谢的蛋白质的活性。 PKA活性通过与激酶锚定蛋白（AKAP）的相互作用来控制。 AKAP通过与PKA调节亚基结合，将PKA定位在细胞中。 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同工型是高度保守的，因此很难研究同工型之间选择性的分子决定因素。 我们将噬菌体显示与高通量测序结合使用，以识别AKAP选择性的序列决定因素。 我们在T7噬菌体表面显示了数百万个诱变的AKAP蛋白的库，然后对PKA调节亚基的α或β同工型进行选择。 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比。 大多数变体在针对α和β同工型的选择中的表现相似。 但是，某些变体对α或β同工型表现出很强的选择性。 我们正在使用该测定的结果来开发高度α-和β特异性AKAP，该AKAP应仅与同源调节同工型与PKA结合。 如果以高浓度引入细胞，它们应破坏其同源型的正常调节相互作用，从而使我们能够研究同工型的生物学意义