ECE2017 Guided Posters Adrenal 1 (10 abstracts)
Protein Kinase A signaling saves regulatory subunit IIβ from PRKACA mutation-mediated degradation
Isabel Weigand 1 , Kerstin Bathon 2 , Cristina Ronchi 1 , Marthe Rizk-Rabin 3 , Guido Di Dalmazi 4 , Vanessa Wild 5 , Beatrice Rubin 6 , Jens T Vanselow 7 , Andreas Schlosser 7 , Davide Calebiro 2 , Felix Beuschlein 4 , Jérôme Bertherat 3 , Martin Fassnacht 1 & Silviu Sbiera 1
1Division of Endocrinology and Diabetes, University Hospital Wuerzburg, Wuerzburg, Germany; 2Institute of Pharmacology and Toxicology, Bioimaging Centre, University of Wuerzburg, Wuerzburg, Germany; 3Institut Cochin, Department of Endocrinology, Hôpital Cochin, Paris, France; 4Medizinische Klinik and Poliklinik IV, Ludwig-Maximilians University, Munich, Germany; 5Institute of Pathology, University of Wuerzburg, Wuerzburg, Germany; 6Department of Medicine, University of Padua, Padua, Italy; 7Rudolf Virchow Center for Experimental Biomedicine, University of Wuerzburg, Wuerzburg, Germany.
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 (34 Cα-WT and 23 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. By performing RIIβ co-IP experiments in the presence of Cα-WT or Cα-L206R, followed by nanoLC-MS/MS analysis, 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. LC-MS/MS revealed putative RIIβ interaction partners affecting its stability in the presence of Cα L206R only after stimulation of PKA signaling.
Article tools
My recent searches
My recently viewed abstracts
Authors
Endocrine Abstracts
ISSN 1470-3947 (print) | ISSN 1479-6848 (online)
© Bioscientifica 2024 |
Privacy policy |
Cookie settings
BiosciAbstracts
Bioscientifica Abstracts is the gateway to a series of products that provide a permanent, citable record of abstracts for biomedical and life science conferences.
Find out more