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.
03 - 07 May 2008
European Society of Endocrinology