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

Laure Cazabat, Bruno Ragazzon, Audrey Varin, Marie Potier-cartereau, Christophe Vandier, Delphine Vezzosi, Marthe Risk-rabin, Aziz Guellich, Julia Schittl, Patrick Lechêne, Wito Richter, Viacheslav O. Nikolaev, Jin Zhang, Jérôme Bertherat, Grégoire Vandecasteele

Research output: Contribution to journalArticle

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.

Original languageEnglish (US)
Article numberddt510
Pages (from-to)1163-1174
Number of pages12
JournalHuman Molecular Genetics
Volume23
Issue number5
DOIs
StatePublished - Mar 2014

ASJC Scopus subject areas

  • Genetics
  • Genetics(clinical)
  • Molecular Biology

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    Cazabat, L., Ragazzon, B., Varin, A., Potier-cartereau, M., Vandier, C., Vezzosi, D., Risk-rabin, M., Guellich, A., Schittl, J., Lechêne, P., Richter, W., Nikolaev, V. O., Zhang, J., Bertherat, J., & Vandecasteele, G. (2014). 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. Human Molecular Genetics, 23(5), 1163-1174. [ddt510]. https://doi.org/10.1093/hmg/ddt510