UNDERSTANDING THE MOLECULAR BASIS OF SELECTIVITY IN AKAP

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 蛋白激酶A（PKA）是一种调节参与细胞代谢的许多蛋白质的活性的中心细胞内激酶。 PKA活性通过与A激酶介导蛋白（AKAP）的相互作用来控制。 AKAP通过与PKA调节亚基结合，将PKA定位在细胞内来发挥作用。 AKAP可以与PKA的调节亚基的或同种型相互作用，或者它们可以与两者相互作用。 和异构体是高度保守的，使得很难研究异构体之间的选择性的分子决定因素。 我们使用噬菌体展示结合高通量测序来鉴定AKAP选择性的序列决定因素。 我们在T7噬菌体表面展示了数百万个诱变AKAP蛋白的文库，然后对该文库进行PKA调节亚基或亚型的选择。 通过比较选择前后每个变体的丰度，我们得出了几十万个变体的富集比。 大多数变体在针对和同种型的选择中表现相似。 然而，一些变体显示出对或同种型的强选择性。 我们正在使用该测定的结果，努力开发高度特异性的AKAP，其应仅与具有同源调节同种型的PKA结合。 如果以高浓度引入细胞，它们应该破坏其同源异构体的正常调节相互作用，使我们能够研究异构体的生物学意义

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