ECE2017 Guided Posters Adrenal 1 (10 abstracts)
PRKACA mutations in adrenal Cushing can alter substrate specificity
Kerstin Bathon 1, , Isabel Weigand 3 , Jens T Vanselow 4 , Cristina L Ronchi 3, , Guido Di Dalmazi 6, , Felix Beuschlein 6 , Silviu Sbiera 3 , Andreas Schlosser 4 , Martin Fassnacht 3, & Davide Calebiro 1,
1Institute of Pharmacology and Toxicology, University of Würzburg, Würzburg, Germany; 2Bioimaging Center, University of Würzburg, Würzburg, Germany; 3Division of Endocrinology and Diabetes, Department of Internal Medicine I, University Hospital, Würzburg, Germany; 4Rudolf Virchow Center, University of Würzburg, Würzburg, Germany; 5Comprehensive Cancer Center Mainfranken, University of Würzburg, Würzburg, Germany; 6Medizinische Klinik and Poliklinik IV, Ludwig-Maximilians University, München, Germany; 7Division of Endocrinology, Department of Medical and Surgical Sciences, University of Bologna, Bologna, Italy.
Previously, we identified seven mutations in the main catalytic subunit of protein kinase A (PKA Cα) to be responsible for cortisol-secreting adrenocortical adenomas (CPAs): L206R, L199_C200_insW, S213R_L212_K214insIILR, C200_GlyinsV, W197R, del244-248+E249Q, E32V. Here we performed a functional characterization of these mutants. Specifically, we evaluated the association between PKA regulatory and catalytic subunits using co-immunoprecipitation and PKA activity using a kemptide assay and a western blot analysis. Our results show that four mutations (L206R, L199_C200_insW, W197R, del244-248+E249Q) cause increased basal PKA activity. Interestingly, the measured activity varied considerably among mutants depending on the assay used, suggesting changes in substrate specificity. Binding to RIα and RIIβ subunits was lost for three and two mutants respectively. One mutant (E32V) showed no differences with the wild type. The S213R_L212_K214insIILR mutant was associated with a loss of PKA activity, but showed a strong accumulation in the nucleus. The C200_G201insV mutant was associated with a loss of activity in the kemptide assay but showed normal behavior in the western blot and nuclear translocation. For all mutants we performed an in silico analysis to predict the effect of the mutation on substrate specificity. The analysis suggested changes in substrate specificity for five mutants. Based on this, we performed phosphoproteomic experiments, which directly compare the phosphorylation of a large number of PKA substrates. The results directly demonstrated altered substrate specificity for three mutants (L206R, del244-248+E249Q, C200_G201insV). Taken together, these findings suggest that the PKA Cα mutations found in CPAs alter substrate specificity and that interference with the formation of a stable PKA holoenzyme is probably just one of the mechanisms through which these mutations affect PKA signaling, ultimately leading to increased cortisol production and cell proliferation. The results of the phosphoproteomics analysis might lead to the identification of new PKA targets involved in the pathogenesis of CPAs.
Volume 49
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Endocrine Abstracts
ISSN 1470-3947 (print) | ISSN 1479-6848 (online)
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