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.
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