Abstract
Protein kinase A (PKA) are tetramers of two catalytic and two regulatory subunits, docked at precise intracellular sites to provide localized phosphorylating activity, triggered by cAMP binding to regulatory subunits and subsequent dissociation of catalytic subunits. It is unclear whether in the brain PKA dissociated subunits may also be found. PKA catalytic subunit was examined in various mouse brain areas using immunofluorescence, equilibrium binding and western blot, to reveal its location in comparison to regulatory subunits type RI and RII. In the cerebral cortex, catalytic subunits colocalized with clusters of RI, yet not all RI clusters were bound to catalytic subunits. In stria terminalis, catalytic subunits were in proximity to RI but separated from them. Catalytic subunits clusters were also present in the corpus striatum, where RII clusters were detected, whereas RI clusters were absent. Upon cAMP addition, the distribution of regulatory subunits did not change, while catalytic subunits were completely released from regulatory subunits. Unpredictably, catalytic subunits were not solubilized; instead, they re-targeted to other binding sites within the tissue, suggesting local macromolecular reorganization. Hence, the interactions between catalytic and regulatory subunits of protein kinase A consistently vary in different brain areas, supporting the idea of multiple interaction patterns.
Highlights
In vivo, intracellular molecules are precisely segregated in specific compartments, to accomplish specific functions at targeted locations
We focused on mouse brain areas that previously showed the most prominent differences in the distribution pattern of Protein kinase A (PKA) regulatory subunits, including the parietal cortex, hippocampus (Cornu Ammonis 1, CA1, subfield), amygdala, corpus striatum, stria terminalis and hypothalamus
Throughout the adult mouse cerebral cortex, immunolabeling showed that the PKA catalytic subunit was organized in discrete clusters
Summary
Intracellular molecules are precisely segregated in specific compartments, to accomplish specific functions at targeted locations. PKA are regulated dynamic multimolecular complexes, consisting of two regulatory subunits that reversibly bind two catalytic subunits, inhibiting their phosphorylating activity. Upon binding two cAMP molecules to each regulatory subunit, catalytic subunits dissociate from regulatory subunits, become enzymatically active and can phosphorylate target proteins [3]. Different non-redundant isoforms of regulatory, i.e., inhibitory, subunits have been described: RIalpha (RIA), RIbeta (RIB), RIIalpha (RIIA) and RIIbeta (RIIB). They display distinctive biochemical characteristics, including different binding affinities for cAMP and for catalytic subunits, or diverse ability to bind various intracellular anchoring proteins [4]. PKA regulatory subunits vary in different human diseases or animal models of disease, including depression, different brain tumors and Parkinson’s disease [12,13,14,15,16,17]
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