Identification and Characterization of Novel Mutations in the Human Gene Encoding the Catalytic Subunit Calpha of Protein Kinase A (PKA)

Søberg, Kristoffer; Larsen, Anja C V; Diskar, Mandy; Backe, Paul Hoff; Bjørås, Magnar; Jahnsen, Tore; Laerdahl, Jon K.; Rognes, Torbjørn; Herberg, Friedrich W.; Skålhegg, Bjørn Steen

doi:10.1371/journal.pone.0034838

Cited by 10 publications

(6 citation statements)

References 58 publications

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“…We introduced two patient mutations into the PKAc-F[2] R luc PCA constructs. It has been described that the PKAc mutation G187V diminished, whereas the hotspot mutation L206R of PKAc in adrenal Cushing's syndrome promoted PKAc activity (Soberg et al, 2012 ; Cao et al, 2014 ; Di Dalmazi et al, 2014 ; Sato et al, 2014 ). Following co-expression of PCA pairs in HEK293 cells we observed a significant reduction of complex formation of PKAc mutants with all three PKA-inhibitory proteins.…”

Section: Resultsmentioning

confidence: 99%

Impact of kinase activating and inactivating patient mutations on binary PKA interactions

et al. 2015

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The second messenger molecule cAMP links extracellular signals to intracellular responses. The main cellular cAMP effector is the compartmentalized protein kinase A (PKA). Upon receptor initiated cAMP-mobilization, PKA regulatory subunits (R) bind cAMP thereby triggering dissociation and activation of bound PKA catalytic subunits (PKAc). Mutations in PKAc or RIa subunits manipulate PKA dynamics and activities which contribute to specific disease patterns. Mutations activating cAMP/PKA signaling contribute to carcinogenesis or hormone excess, while inactivating mutations cause hormone deficiency or resistance. Here we extended the application spectrum of a Protein-fragment Complementation Assay based on the Renilla Luciferase to determine binary protein:protein interactions (PPIs) of the PKA network. We compared time- and dose-dependent influences of cAMP-elevation on mutually exclusive PPIs of PKAc with the phosphotransferase inhibiting RIIb and RIa subunits and the protein kinase inhibitor peptide (PKI). We analyzed PKA dynamics following integration of patient mutations into PKAc and RIa. We observed that oncogenic modifications of PKAc(L206R) and RIa(Δ184-236) as well as rare disease mutations in RIa(R368X) affect complex formation of PKA and its responsiveness to cAMP elevation. With the cell-based PKA PPI reporter platform we precisely quantified the mechanistic details how inhibitory PKA interactions and defined patient mutations contribute to PKA functions.

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Section: Resultsmentioning

confidence: 99%

Impact of kinase activating and inactivating patient mutations on binary PKA interactions

et al. 2015

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“…It should be noted that PRKACA mutations that were predicted to lose interaction with the regulatory subunit while maintaining enzymatic activity had been studied in vitro in the early 1990s 22–24 . Additional PRKACA sequences that had similar effects in vitro were described recently, and the first PRKACA mutations in humans with predicted functional effects were reported in 2012 with no association with an obvious phenotype 25 . The first study to investigate the presence of PRKACA mutations in endocrine lesions failed to show any defects in a large collection of thyroid and pituitary adenomas 26 .…”

Section: Discussionmentioning

confidence: 77%

Germline PRKACA amplification causes variable phenotypes that may depend on the extent of the genomic defect: molecular mechanisms and clinical presentations

Lodish

Yuan

Levy

et al. 2015

European Journal of Endocrinology

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Objective We reported recently 5 patients with bilateral adrenocortical hyperplasia (BAH) and Cushing syndrome (CS) caused by constitutive activation of the catalytic subunit of protein kinase A (PRKACA). By doing new, in depth analysis of their cytogenetic abnormality, we attempt a better genotype-phenotype correlation of their PRKACA amplification. Design Case series. Methods Molecular cytogenetic, genomic, clinical and histopathologic analyses were performed in 5 patients with CS. Results Reinvestigation of the defects of previously described patients by state-of-the-art molecular cytogenetics showed complex genomic rearrangements in the chromosome 19p13.2p13.12 locus resulting in copy number gains encompassing the entire PRKACA; three patients (one sporadic case and two related cases) were observed with gains consistent with duplications, while two sporadic patients were observed with gains consistent with triplications. Although all five patients presented with ACTH-independent CS, the three sporadic patients had micronodular BAH and underwent bilateral adrenalectomy in early childhood whereas the two related patients, a mother and a son, presented with macronodular BAH as adults. In at least one patient, PRKACA triplication was associated with a more severe phenotype. Conclusions Constitutional chromosomal PRKACA amplification is a recently identified genetic defect associated with CS, a trait that may be inherited in an autosomal dominant manner or occur de novo. Genomic rearrangements can be complex and can result in different copy number states of dosage sensitive genes; e.g. duplication and triplication. PRKACA amplification can lead to variable phenotypes clinically and pathologically, and both micro- and macro-nodular BAH, the latter of which we speculate may depend on the extent of amplification.

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“…We and others have shown the crucial importance of the Gly186 residue in Cα (Cα1 numbering) for catalytic activity. We performed a thorough search for naturally occurring mutations in the human PRKACA gene using both publicly available databases as well as through sequencing of exons 2–10 in 498 individuals ( 183 ). The search revealed several missense mutations, including Arg45Gln, Ser109Pro, Gly186Val, and Ser263Cys.…”

Section: Catalytic Subunit Structural Features As Determinant For Pkamentioning

confidence: 99%

The Molecular Basis for Specificity at the Level of the Protein Kinase a Catalytic Subunit

Søberg

Skålhegg

2018

Front. Endocrinol.

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Assembly of multi enzyme complexes at subcellular localizations by anchoring- and scaffolding proteins represents a pivotal mechanism for achieving spatiotemporal regulation of cellular signaling after hormone receptor targeting [for review, see (1)]. In the 3′ 5′-cyclic adenosine monophosphate (cAMP) dependent protein kinase (PKA) signaling pathway it is generally accepted that specificity is secured at several levels. This includes at the first level stimulation of receptors coupled to heterotrimeric G proteins which through stimulation of adenylyl cyclase (AC) forms the second messenger cAMP. Cyclic AMP has several receptors including PKA. PKA is a tetrameric holoenzyme consisting of a regulatory (R) subunit dimer and two catalytic (C) subunits. The R subunit is the receptor for cAMP and compartmentalizes cAMP signals through binding to cell and tissue-specifically expressed A kinase anchoring proteins (AKAPs). The current dogma tells that in the presence of cAMP, PKA dissociates into an R subunit dimer and two C subunits which are free to phosphorylate relevant substrates in the cytosol and nucleus. The release of the C subunit has raised the question how specificity of the cAMP and PKA signaling pathway is maintained when the C subunit no longer is attached to the R subunit-AKAP complex. An increasing body of evidence points toward a regulatory role of the cAMP and PKA signaling pathway by targeting the C subunits to various C subunit binding proteins in the cytosol and nucleus. Moreover, recent identification of isoform specific amino acid sequences, motifs and three dimensional structures have together provided new insight into how PKA at the level of the C subunit may act in a highly isoform-specific fashion. Here we discuss recent understanding of specificity of the cAMP and PKA signaling pathway based on C subunit subcellular targeting as well as evolution of the C subunit structure that may contribute to the dynamic regulation of C subunit activity.

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Identification and Characterization of Novel Mutations in the Human Gene Encoding the Catalytic Subunit Calpha of Protein Kinase A (PKA)

Cited by 10 publications

References 58 publications

Impact of kinase activating and inactivating patient mutations on binary PKA interactions

Impact of kinase activating and inactivating patient mutations on binary PKA interactions

Germline PRKACA amplification causes variable phenotypes that may depend on the extent of the genomic defect: molecular mechanisms and clinical presentations

The Molecular Basis for Specificity at the Level of the Protein Kinase a Catalytic Subunit

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