Activating PRKACB somatic mutation in cortisol-producing adenomas
Stéphanie Espiard, … , Constantine A. Stratakis, Jérôme Bertherat
Stéphanie Espiard, … , Constantine A. Stratakis, Jérôme Bertherat
Published April 19, 2018
Citation Information: JCI Insight. 2018;3(8):e98296. https://doi.org/10.1172/jci.insight.98296.
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Research Article Endocrinology Genetics

Activating PRKACB somatic mutation in cortisol-producing adenomas

Abstract

Mutations in the gene encoding the protein kinase A (PKA) catalytic subunit α have been found to be responsible for cortisol-producing adenomas (CPAs). In this study, we identified by whole-exome sequencing the somatic mutation p.S54L in the PRKACB gene, encoding the catalytic subunit β (Cβ) of PKA, in a CPA from a patient with severe Cushing syndrome. Bioluminescence resonance energy transfer and surface plasmon resonance assays revealed that the mutation hampers formation of type I holoenzymes and that these holoenzymes were highly sensitive to cAMP. PKA activity, measured both in cell lysates and with recombinant proteins, based on phosphorylation of a synthetic substrate, was higher under basal conditions for the mutant enzyme compared with the WT, while maximal activity was lower. These data suggest that at baseline the PRKACB p.S54L mutant drove the adenoma cells to higher cAMP signaling activity, probably contributing to their autonomous growth. Although the role of PRKACB in tumorigenesis has been suggested, we demonstrated for the first time to our knowledge that a PRKACB mutation can lead to an adrenal tumor. Moreover, this observation describes another mechanism of PKA pathway activation in CPAs and highlights the particular role of residue Ser54 for the function of PKA.

Authors

Stéphanie Espiard, Matthias J. Knape, Kerstin Bathon, Guillaume Assié, Marthe Rizk-Rabin, Simon Faillot, Windy Luscap-Rondof, Daniel Abid, Laurence Guignat, Davide Calebiro, Friedrich W. Herberg, Constantine A. Stratakis, Jérôme Bertherat

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Figure 5

The mutation S54L causes increased phosphotransferase activity under basal conditions.

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The mutation S54L causes increased phosphotransferase activity under bas...
PKA activity was determined by quantifying the phosphorylation of a synthetic fluorescently labeled peptide (kemptide) in HEK293 cell lysates (A) or with unlabeled kemptide for purified recombinant catalytic subunits (B). In detail, (A) PKA activity was tested in lysates of HEK293 cells cotransfected with a combination of RIα and either WT or S54L Cβ1. Under basal conditions (no cAMP stimulation), the mutant holoenzyme showed higher PKA activity than the WT. In the presence of cAMP, maximal activity was lower for the mutant enzyme. Transfection of the empty pcDNA vector was used as control (Ctrl). Data from 4 independent experiments are represented as dot plots and were analyzed by ANOVA with Sidak’s post hoc test for multiple comparisons. (B) PKA activity of purified recombinant RIα and catalytic subunits (Cβ-WT, Cβ-S54L, and Cα-WT) revealed an almost 3-fold-reduced phosphotransferase activity (15.6 ± 1.3 U/mg) for Cβ-S54L compared with Cβ-WT (45.1 ± 4.9 U/mg). The specific activity of the major isoform Cα1 was determined as a control (20.3 ± 2.0 U/mg). Data from 3 independent protein preparations are represented as dot plots and were analyzed by unpaired t test with Sidak’s post hoc test for multiple comparisons. *P < 0.05; ***P < 0.001.