Subcellular Localization and Functional Characterization of cAMP‐dependent Protein Kinase A Isoforms: Painting Specificity by Mosaic Brain Mapping

cAMP dependent Protein kinase (PKA) plays a critical role in numerous neuronal functions. Specificity in PKA signaling is achieved in part by the four functionally non‐redundant regulatory (R) subunits. The inactive holoenzyme has a dimeric R‐subunit bound to two Catalytic (C) subunits. The full‐length holoenzyme crystal structure allowed me to understand how isoform‐specific assembly can create distinct holoenzyme structures that each defines its allosteric regulation. High‐resolution large‐scale mosaic images provide global views of brain sections and allow identification of subcellular features. Analysis of multiple regions demonstrates that the R‐isoforms are concentrated within discrete regions and express unique patterns of subcellular localization. Using correlated light and electron microscopy I confirmed mitochondrial and nuclear localization of RIb isoform, modifying the existing dogma of cAMP‐PKA in the nucleus. To show the functional significance of nuclear localization, I demonstrated that RIb down‐regulation, but not RIIb, decreased CREB phosphorylation in primary neuronal cultures, consistent with deficits observed in RIb mutant mice. Furthermore, I show that a point mutation in the RIb gene, found in patients diagnosed with a neurodegenerative disease, abolishes dimerization while retaining robust interaction with its catalytic subunit. As a consequence, the interaction with A Kinase Anchoring Proteins (AKAPs) is also abolished. Quantitative mass spectroscopy analysis of patient samples identified a set of proteins that were up or down regulated in this disease. This point mutation further emphasizes the importance of precisely controlled PKA isoform subcellular localization as defined by my mosaic maps.