PKA & PKC Targeting Mechanisms

总体描述(由申请人提供)： 我们计划项目赠款的总体主题是阐明利用cAMP和脂质第二信使的信号事件的空间和时间特征。协调这些细胞过程需要将信号分子协调整合到高度调控和动态的多蛋白质复合体中。每个项目的一个共同目标是了解PKA和PKC亚型是如何针对这些复合体的，以及第二信使信号是如何被处理并传递到PKA、PKC及其选定底物的锚定池中的。在过去的授权期内，我们更加认识到这些进程的复杂性。具体地说，我们已经确定了(1)由PKA、cAMP选择性磷酸二酯酶(PDE4D3)和EPAC-1鸟核苷酸交换因子(Scott)组成的锚定cAMP信号模块(MAKAP)，(2)利用PDZ结构域将PKA和AKAP招募到信号复合体(Taylor)中的模块化支架蛋白，(3)通过激酶B、C和D(牛顿)进行信号传递的时空动态，(4)PKA催化亚单位(Taylor)N端的靶向作用，以及(5)Pin1在调节PKC周转中的作用(Jennings，牛顿)。我们还使用核磁共振和结晶学(Jennings，Taylor，Scott)解决了RL(和RLL(二聚化/对接结构域)自由和与AKAP肽结合的结构)。我们还设计和设计了新的重组FRET报告，用于监测活细胞(钱氏、泰勒、牛顿)中PKA、PKC、PKD和Akt/PKB活性以及DAG的活性。最后，我们利用多肽阵列设计了针对AKAP的PKA靶向的异构体选择性干扰多肽。在此基础上，我们在下一个授权期的目标集中在三个方面。(1)我们将实时和空间地监测PKA、PKC和PKD信号，重点关注它们在线粒体中的功能。(2)利用核磁共振和结晶学的方法，阐明通过AKAPs和PDZ结构域进行靶向的分子基础。(3)我们将设计新的荧光探针和靶向的异构体选择性干扰肽来跟踪线粒体上和线粒体内的PKA和PKC信号事件。所有四个项目都被整合到核磁共振核心(核心A)中。为了加强生物学研究，我们还将必要的专门知识合并为三个核心。蛋白质组学/Beta细胞核心(核心B：King)提供分析蛋白质资源以及获取β细胞生物学的途径，成像核心(核心C：Ellisman)提供成像和电子显微镜专业知识，线粒体核心(核心D：Murphy)提供线粒体分离和功能分析方面的专业知识。这些核心将相互结合，并与项目相结合。因此，拟议的工作通过深入线粒体中第二信使信号的未知领域，完善我们对蛋白质支架的结构理解，并揭示新的支架机制，将我们对激酶靶向的研究带到了一个新的水平。

DESCRIPTION, OVERALL (provided by applicant): The overall theme of our Program Project Grant is to elucidate the spatial and temporal features signaling events that utilize cAMP and lipid second messengers. Orchestrating these cellular processes requires the coordinated integration of signaling molecules into highly regulated and dynamic multi-protein complexes. A common objective in each project is to understand how PKA and PKC isoforms are targeted to these complexes and how second messenger signals are processed and disseminated to anchored pools of PKA, PKC, and their selected substrates. During the past granting period our recognition of the complexity of these processes has increased. Specifically, we have identified (1) an anchored cAMP signaling module (mAKAP) comprised of PKA, a cAMP-selective phosphodiesterase (PDE4D3) and the Epac-1 guanine nucleotide exchange factor (Scott), (2) modular scaffolding proteins that utilize PDZ domains to recruit both PKA and AKAPs, into signaling complexes (Taylor), (3) the spatial and temporal dynamics of signaling by kinases B, C, and D (Newton), (4) a targeting role for the N terminus of the PKA catalytic subunit (Taylor),and (5) the role of Pin1 in regulating the turnover of PKC (Jennings, Newton). We also have solved structures of the Rl( and Rll( Dimerization/Docking domains free and bound to AKAP peptides using both NMR and crystallography (Jennings, Taylor, Scott). We also designed and engineered novel recombinant FRET reporters for monitoring PKA, PKC, PKD, and Akt/PKB activity, as well as DAG, in live cells (Tsien, Taylor, Newton). Finally, we engineered isoform-selective disrupter peptides for PKA targeting to AKAPs using peptide arrays. Building on this foundation, our goals over the next granting period are focused in three areas. (1) We will monitor PKA, PKC, and PKD signaling in real time and space, focusing specifically on their function in mitochondria. (2) Using NMR and crystallography we will elucidate the molecular basis for targeting through AKAPs and PDZ domains. (3) We will design novel fluorescent probes as well as targeted isoform-selective disrupting peptides to follow PKA and PKC signaling events on and inside mitochondria. All four projects are integrated into the NMR Core (Core A). To augment the biological studies we also consolidated essential expertise into three cores. The Proteomics/Beta Cell Core (Core B: King) provides analytical protein resources as well as access to beta cell biology, the Imaging Core (Core C: Ellisman) provides imaging and electron microscopy expertise, and the Mitochondria Core (Core D: Murphy) provides expertise in mitochondria isolation and functional analysis. These cores will be integrated with each other as well as with the projects. Thus, proposed work takes our studies on kinase targeting to the next level by delving into the unexplored territory of second messenger signaling in mitochondria, refining our structural understanding of protein scaffolds, and unveiling novel scaffolding mechanisms.