Protein Kinase a Signaling Saves Regulatory Subunit IIβ from Prkaca Mutation-Mediated Degradation

Presentation Number: LB MON 51
Date of Presentation: April 3rd, 2017

Isabel Weigand1, Kerstin Bathon2, Cristina Lucia Ronchi3, Marthe Rizk-Rabin4, Guido Di Dalmazi5, Vanessa Wild6, Beatrice Rubin7, Davide Calebiro2, Felix Beuschlein8, Jerome Yves Bertherat9, Martin Fassnacht3 and Silviu Sbiera*3
1Endocrine and Diabetes Unit, University Hospital, University of Wuerzburg, Würzburg, Germany, 2University of Würzburg, Würzburg, Germany, 3University Hospital Wuerzburg, Wuerzburg, Germany, 4Institut Cochin, 5Medizinische Klinik und Poliklinik IV, Munich, Germany, 6University of Wuerzburg, 7Endocrinology Unit, University Hospital of Padua, Padova, Italy, 8Klinikum der Universität München, Ludwig-Maximilian University, Munich, Germany, 9INSERM U 1016, Cochin Institute, Paris Descartes University, Paris, France


Protein Kinase A (PKA) consists of two catalytic and two regulatory subunits with several isoforms (Cα, β, γ, RIα, IIα, Iβ, IIβ). In 30-40% of cortisol-producing adrenocortical adenomas (CPA) heterozygous activating somatic mutations in the catalytic subunit α (Cα) of PKA have been found. Previous reports found strikingly reduced levels of RIIβ in CPA compared to other adrenocortical tumors.

Here, we investigated the correlation between Cα mutational status, RIIβ expression levels and the underlying regulation mechanisms in CPA and the adrenal cell line NCI-H295R.

RIIβ expression was strongly reduced in Cα-mutated CPAs, especially in tumors harboring the frequent L206R mutation (22 Cα-WT and 18 Cα-mutated CPA) by both immunohistochemistry (mean expression: 1.5±0.7 vs 0.4±0.5, p<0.05) and WB. Similar results were observed for RIα (1.8±0.9 vs 2.6±0.6, p<0.05) but not for the other regulatory subunits. Notably, mRNA expression of all subunits was unchanged in Cα-WT compared to Cα-mutated CPA. In NCI-H295R cells co-transfected with RIIβ and Cα-WT or different Cα mutants, only the L206R mutation led to a full degradation of RIIβ and this degradation could not be abolished by proteasome and lysosome inhibition but, surprisingly, by stimulating PKA signaling. Same co-transfections with RIα did not lead to its degradation. Performing LC-MS/MS we could identify possible novel RIIβ interaction partners mediating RIIβ stability in NCI-H295R cells.

In conclusion, our data demonstrate that mutations in PKA Cα lead to post-transcriptional downregulation of the main regulatory subunit in CPA. In NCI-H295R cells, transfection with the L206R mutant led to full degradation of RIIβ, which could not be rescued by different degradation mechanisms, suggesting another mechanism that can be abrogated by stimulating PKA signaling. By LC-MS/MS we could identify putative RIIβ interaction partners affecting its stability in the presence of Cα L206R.


Nothing to Disclose: IW, KB, CLR, MR, GD, VW, BR, DC, FB, JYB, MF, SS