Inactivation of the Carney complex gene 1 (PRKAR1A) alters spatiotemporal regulation of cAMP and cAMP-dependent protein kinase: a study using genetically encoded FRET-based reporters

Carney complex (CNC) is a hereditary disease associating cardiac myxoma, spotty skin pigmentation and endocrine overactivity. CNC is caused by inactivating mutations in the PRKAR1A gene encoding PKA type I alpha regulatory subunit (RI alpha). Although PKA activity is enhanced in CNC, the mechanisms linking PKA dysregulation to endocrine tumorigenesis are poorly understood. In this study, we used Forster resonance energy transfer (FRET)-based sensors for cAMP and PKA activity to define the role of RI alpha in the spatiotemporal organization of the cAMP/PKA pathway. RI alpha knockdown in HEK293 cells increased basal as well as forskolin or prostaglandin E-1 (PGE(1))-stimulated total cellular PKA activity as reported by western blots of endogenous PKA targets and the FRET-based global PKA activity reporter, AKAR3. Using variants of AKAR3 targeted to subcellular compartments, we identified similar increases in the response to PGE(1) in the cytoplasm and at the outer mitochondrial membrane. In contrast, at the plasma membrane, the response to PGE(1) was decreased along with an increase in basal FRET ratio. These results were confirmed by western blot analysis of basal and PGE(1)-induced phosphorylation of membrane-associated vasodilator-stimulated phosphoprotein. Similar differences were observed between the cytoplasm and the plasma membrane in human adrenal cells carrying a RI alpha inactivating mutation. RI alpha inactivation also increased cAMP in the cytoplasm, at the outer mitochondrial membrane and at the plasma membrane, as reported by targeted versions of the cAMP indicator Epac1-camps. These results show that RI alpha inactivation leads to multiple, compartment-specific alterations of the cAMP/PKA pathway revealing new aspects of signaling dysregulation in tumorigenesis.