Protein Kinase A (PKA) isoform specificity: Multiscale imaging of PKA from individual molecules to the brain

Phosphorylation mediated by cAMP dependent protein kinase (PKA) is critically involved in cell growth, differentiation, apoptosis and neurotransmitters. 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 structures allow us to understand how isoform-specific assembly can create distinct holoenzyme structures that each defines its allosteric regulation. High-resolution large-scale mosaic images (HRMI) 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 and consistent patterns of subcellular localization. Using the miniSOG technique for correlating fluorescent microscopy with electron microscopy we find RIb in the mitochondria within the cristae and the inner membrane, and in the nucleus, modifying the existing dogma of cAMP-PKA in the nucleus. To understand PKA isoform-specific function, we use human patient sampes of individulas that carry RIb mutation and were diangosed with a neurodegenerative disease. We show that the mutation selectively affects AKAP-mediated compartmentalization of RI holoenzymes to specific cellular compartments, as evidenced in patients by accumulation of RIb into neuronal inclusions. We performed quantitavie multiplex phospho-proteomics to characterize phospho-protein network perturbations in the hippocampus of these patiants. This study emphasizes the importance of precisely controlled PKA isoform subcellular localization and shows how we utilize the mosaic maps to drive new hypotheses in a PKA disease associated mutation where cAMP signaling is aberrant.