ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

Endocrine Abstracts (2008) 16 P656

Consequences of PRKAR1A (Carney complex gene) inactivation on cellular and subcellular PKA activity monitored by FRET-based reporters

Laure Cazabat1, Bruno Ragazzon1, Audrey Varin2, Karine Perlemoine1, Jin Zhang3, Jerome Bertherat1 & Gregoire Vandecasteele2

1INSERM, U567, Institut Cochin, Département Endocrinologie Métabolisme and Cancer, CNRS, UMR8104, Université Paris 5, Faculté de Médecine René Descartes, Paris F-75014, France; 2INSERM U769, Université Paris-Sud 11, Faculté de Pharmacie, Châtenay-Malabry F-92296, France; 3Department of Pharmacology and Molecular Sciences, The Johns Hopkins University School of Medicine, Baltimore, MD 21205, United States.

cAMP/PKA pathway activation is frequently involved in endocrine tumors with overactivity. The Carney complex (CNC) is an autosomal dominant multiple endocrine neoplasia syndrome which associates cardiac myxomas, spotty skin pigmentation and endocrine overactivity. Mutations in the PRKAR1A gene located at 17q22-24 and encoding for the R1A regulatory subunit of protein kinase A have been found in about 60% of CNC. These mutations are heterozygous germline mutations leading to abnormal mRNA generally degraded by NMD (nonsense mediated decay) and thus no abnormal protein is expressed. Loss of heterozygosity (LOH) at 17q22-24 can be observed in tumors, suggesting that PRKAR1A is a tumor suppressor gene.

The aim of this study is to investigate the consequences of PRKAR1A mutations on PKA activity at the cellular and subcellular levels. We use the silencing RNA method to inactivate R1A and obtain at least a 80% decrease in PRKAR1A protein content in HEK293 cells. PKA activity is monitored by fluorescent resonance energy transfer (FRET) using A-kinase reporters (AKARs) in the whole cell (AKAR3) and in different subcellular compartments (cytosol: ELS-AKAR3, nucleus: NLS-AKAR3, mitochondria: dAKAP-AKAR3, plasma membrane: PM-AKAR3). The FRET ratio is measured in basal conditions and after cAMP pathway activation with forskolin or prostaglandin E1 (PGE1). R1A inactivation is associated with a two-fold up to four-fold increase of basal and stimulated PKA activity in whole cells compared with control. Targeted fluorescent probes show different subcellular patterns of PKA activity alterations after R1A inactivation.These results demonstrate at the cellular level that PRKAR1A mutations augment PKA activity and suggest that this effect differ between subcellular compartments.

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