This proposal is designed to study the effects of disrupting PKA localization by the neural specific A-kinase anchoring protein, AKAP150. Elevations of cAMP within a cell occur as a result of the opposing actions of adenylyl cyclase and phosphodiestrerases. As a result, cAMP concentration fluctuations occur within rather limited microdomains of a cell. These facts, coupled with the very broad PKA specificity, make it reasonable that restricted subcellular localization is important in PKA signaling fidelity. One of our approaches to study the importance of localized docking of PKA is to create a line of knock-in mice, which carry a mutation in AKAP150. This mutation introduces a premature stop codon in the open reading frame and results in a 36 amino acid truncation of the C-terminal end of the protein, effectively eliminating any PKA docking site. The remainder of this scaffolding AKAP150 protein will remain intact. In a second approach, we will generate a line of mice carrying a complete knock-out of the AKAP150 gene. We anticipate that this will give rise to mice with a more marked phenotype as the complex knock-out will disrupt the signalling fidelity of several kinase and phosphatase pathways.
