Our overall goals are to understand the biochemical and molecular basis for the targeting of PKA and PKC and the cellular consequences of targeting (1) these kinases, and other signaling molecules, in close proximity to their substrates. To achieve this goal, we have assembled a diverse, highly interdisciplinary team of 5 investigators and 3 scientific cores. During our first 3 years our collaborative efforts have built a strong foundation in structural, biochemical, and cellular studies of kinase anchoring. By solving structures of the docking domains of RI and RII, we established a molecular understanding of PKA targeting to AKAPs. Parallel studies have shown the importance of the C-terminal tail of PKC for its assembly, maturation, and subcellular location. We also defined higher levels of complexity where anchoring proteins coordinate the location of PKA or PKC with signal termination such as phosphodiesterases and phosphatases. These signaling networks provide focal points for the bi-directoinal regulation of second messenger signaling events. The development of novel fluorescent probes provides a means to explore the temporal and spatial dynamics of compartmentalized signaling units in living cells. During the next granting period we shall build on this foundation. Project 1 (Taylor/Tsien) focuses on 2 dual-specific AKAPs, DAKAP1 and 2. In addition to structure analysis, including a newly discovered phosphatase binding site, they will use H/D exchange coupled with mass spectrometry to map global architecture, domain boundaries, and protein interaction sites. Novel fluorescent tools will be used to measure PKA activity and localization. Project 2 (Scott) focuses on mAKAP that binds to PDE4 and ERK5 and provides an integrated complex. In collaboration with Tsien, fluorescent tools will be used to evaluate the physiological consequences of targeting PKA in close proximity to a PKA-regulated PDE.
A second aim i s to use transgenic mice to explore the importance of PKA targeting on the secretion of insulin in pancreatic beta cells. Project 3 (Newton) will characterize biochemical, structural, and cellular mechanisms of targeting of PKC and Akt/PKB to pericentrin, a protein Newton and Scott independently discovered as a PKC and PKA binding scaffold. In addition, the molecular and cellular mechanism for regulating PKC signalling by docking of the stress protein HSP70 to the C-terminal tail of PKC will be explored. In Project 4 (Jennings) structures of AKAP:PKA complexes, PKC:pericentrin complexes, and the C-terminal tail of PKC will be solved by heteronuclear NMR. In addition to the scientific projects we have 3 scientific cores that integrate all of the projects. The NMR core (Jennings) integrated with Scott, Taylor, and Newton; the Protein Expression and Xray Crystallography Core (Xuong) collaborates with Taylor, Newton, and Jennings; the Imaging and Microscopy Core (Ellisman) is working with Tsien to develop fluorescent tools that will have wide applicability for Projects 1, 2, and 3.
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