title: 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.
abstract: 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 (RIα). Although PKA activity is enhanced in CNC, the mechanisms linking PKA dysregulation to endocrine tumorigenesis are poorly understood. In this study, we used Förster resonance energy transfer (FRET)-based sensors for cAMP and PKA activity to define the role of RIα in the spatiotemporal organization of the cAMP/PKA pathway. RIα knockdown in HEK293 cells increased basal as well as forskolin or prostaglandin E1 (PGE1)-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 PGE1 in the cytoplasm and at the outer mitochondrial membrane. In contrast, at the plasma membrane, the response to PGE1 was decreased along with an increase in basal FRET ratio. These results were confirmed by western blot analysis of basal and PGE1-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 RIα inactivating mutation. RIα 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 RIα inactivation leads to multiple, compartment-specific alterations of the cAMP/PKA pathway revealing new aspects of signaling dysregulation in tumorigenesis.
authors.affiliation: INSERM U1016, Paris F-75014, France.
authors.forename: Laure
authors.initials: L
authors.lastname: Cazabat
authors.forename: Bruno
authors.initials: B
authors.lastname: Ragazzon
authors.forename: Audrey
authors.initials: A
authors.lastname: Varin
authors.forename: Marie
authors.initials: M
authors.lastname: Potier-Cartereau
authors.forename: Christophe
authors.initials: C
authors.lastname: Vandier
authors.forename: Delphine
authors.initials: D
authors.lastname: Vezzosi
authors.forename: Marthe
authors.initials: M
authors.lastname: Risk-Rabin
authors.forename: Aziz
authors.initials: A
authors.lastname: Guellich
authors.forename: Julia
authors.initials: J
authors.lastname: Schittl
authors.forename: Patrick
authors.initials: P
authors.lastname: Lechêne
authors.forename: Wito
authors.initials: W
authors.lastname: Richter
authors.forename: Viacheslav O
authors.initials: VO
authors.lastname: Nikolaev
authors.forename: Jin
authors.initials: J
authors.lastname: Zhang
authors.forename: Jérôme
authors.initials: J
authors.lastname: Bertherat
authors.forename: Grégoire
authors.initials: G
authors.lastname: Vandecasteele
mesh_terms: D000527:Alprostadil / Q000494:pharmacology; D056733:Carney Complex / Q000235:genetics / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism*; D054756:Cyclic AMP-Dependent Protein Kinase RIalpha Subunit / Q000235:genetics* / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects / Q000235:genetics; D020868:Gene Silencing; D057809:HEK293 Cells; D006801:Humans; D042541:Intracellular Space / Q000378:metabolism; D021381:Protein Transport; D034622:RNA Interference; D015398:Signal Transduction
chemical_list: D054756:Cyclic AMP-Dependent Protein Kinase RIalpha Subunit; C517834:PRKAR1A protein, human; D005576:Colforsin; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D000527:Alprostadil