title: PKA catalytic subunit mutations in adrenocortical Cushing's adenoma impair association with the regulatory subunit.
abstract: We recently identified a high prevalence of mutations affecting the catalytic (Cα) subunit of protein kinase A (PKA) in cortisol-secreting adrenocortical adenomas. The two identified mutations (Leu206Arg and Leu199_Cys200insTrp) are associated with increased PKA catalytic activity, but the underlying mechanisms are highly controversial. Here we utilize a combination of biochemical and optical assays, including fluorescence resonance energy transfer in living cells, to analyze the consequences of the two mutations with respect to the formation of the PKA holoenzyme and its regulation by cAMP. Our results indicate that neither mutant can form a stable PKA complex, due to the location of the mutations at the interface between the catalytic and the regulatory subunits. We conclude that the two mutations cause high basal catalytic activity and lack of regulation by cAMP through interference of complex formation between the regulatory and the catalytic subunits of PKA.
authors.affiliation: 1] Institute of Pharmacology and Toxicology, University of Würzburg, Versbacher Strasse 9, 97078 Würzburg, Germany [2] Rudolf Virchow Center, Josef-Schneider-Strasse 2, 97080 Würzburg, Germany.
authors.forename: Davide
authors.initials: D
authors.lastname: Calebiro
authors.affiliation: Institute of Pharmacology and Toxicology, University of Würzburg, Versbacher Strasse 9, 97078 Würzburg, Germany.
authors.forename: Annette
authors.initials: A
authors.lastname: Hannawacker
authors.affiliation: 1] Institute of Pharmacology and Toxicology, University of Würzburg, Versbacher Strasse 9, 97078 Würzburg, Germany [2] Rudolf Virchow Center, Josef-Schneider-Strasse 2, 97080 Würzburg, Germany.
authors.forename: Sandra
authors.initials: S
authors.lastname: Lyga
authors.affiliation: 1] Institute of Pharmacology and Toxicology, University of Würzburg, Versbacher Strasse 9, 97078 Würzburg, Germany [2] Rudolf Virchow Center, Josef-Schneider-Strasse 2, 97080 Würzburg, Germany.
authors.forename: Kerstin
authors.initials: K
authors.lastname: Bathon
authors.affiliation: Institute of Pharmacology and Toxicology, University of Würzburg, Versbacher Strasse 9, 97078 Würzburg, Germany.
authors.forename: Ulrike
authors.initials: U
authors.lastname: Zabel
authors.affiliation: Comprehensive Cancer Center Mainfranken, University of Würzburg, Josef-Schneider-Strasse 6, 97080 Würzburg, Germany.
authors.forename: Cristina
authors.initials: C
authors.lastname: Ronchi
authors.affiliation: Medizinische Klinik und Poliklinik IV, Ludwig-Maximilians-Universität München, Ziemssenstraße 1, 80336 München, Germany.
authors.forename: Felix
authors.initials: F
authors.lastname: Beuschlein
authors.affiliation: Medizinische Klinik und Poliklinik IV, Ludwig-Maximilians-Universität München, Ziemssenstraße 1, 80336 München, Germany.
authors.forename: Martin
authors.initials: M
authors.lastname: Reincke
authors.affiliation: 1] Institute of Pharmacology and Toxicology, University of Würzburg, Versbacher Strasse 9, 97078 Würzburg, Germany [2] Comprehensive Heart Failure Center, Universitätsklinikum Würzburg, Straubmühlweg 2a, 97078 Würzburg, Germany.
authors.forename: Kristina
authors.initials: K
authors.lastname: Lorenz
authors.affiliation: Department of Medicine I, Endocrine and Diabetes Unit, University Hospital, University of Würzburg, Oberdürrbacher Strasse 6, 97080 Würzburg, Germany.
authors.forename: Bruno
authors.initials: B
authors.lastname: Allolio
authors.affiliation: Rudolf Virchow Center, Josef-Schneider-Strasse 2, 97080 Würzburg, Germany.
authors.forename: Caroline
authors.initials: C
authors.lastname: Kisker
authors.affiliation: 1] Comprehensive Cancer Center Mainfranken, University of Würzburg, Josef-Schneider-Strasse 6, 97080 Würzburg, Germany [2] Department of Medicine I, Endocrine and Diabetes Unit, University Hospital, University of Würzburg, Oberdürrbacher Strasse 6, 97080 Würzburg, Germany.
authors.forename: Martin
authors.initials: M
authors.lastname: Fassnacht
authors.affiliation: 1] Institute of Pharmacology and Toxicology, University of Würzburg, Versbacher Strasse 9, 97078 Würzburg, Germany [2] Rudolf Virchow Center, Josef-Schneider-Strasse 2, 97080 Würzburg, Germany [3] Comprehensive Heart Failure Center, Universitätsklinikum Würzburg, Straubmühlweg 2a, 97078 Würzburg, Germany.
authors.forename: Martin J
authors.initials: MJ
authors.lastname: Lohse
mesh_terms: D000306:Adrenal Cortex Neoplasms / Q000201:enzymology* / Q000235:genetics / Q000473:pathology; D018246:Adrenocortical Adenoma / Q000201:enzymology* / Q000235:genetics / Q000473:pathology; D020134:Catalytic Domain; D045744:Cell Line, Tumor; D003480:Cushing Syndrome / Q000201:enzymology* / Q000235:genetics / Q000473:pathology; D000242:Cyclic AMP / Q000378:metabolism; D054751:Cyclic AMP-Dependent Protein Kinase Catalytic Subunits / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D054757:Cyclic AMP-Dependent Protein Kinase RIIbeta Subunit / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D054756:Cyclic AMP-Dependent Protein Kinase RIalpha Subunit / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006801:Humans; D009154:Mutation; D011485:Protein Binding; D055550:Protein Stability
chemical_list: D054757:Cyclic AMP-Dependent Protein Kinase RIIbeta Subunit; D054756:Cyclic AMP-Dependent Protein Kinase RIalpha Subunit; C517834:PRKAR1A protein, human; C517837:PRKAR2B protein, human; D000242:Cyclic AMP; D054751:Cyclic AMP-Dependent Protein Kinase Catalytic Subunits; D017868:Cyclic AMP-Dependent Protein Kinases; C517757:PRKACA protein, human