scholarly journals Cushing’s syndrome driver mutation disrupts protein kinase A allosteric network, altering both regulation and substrate specificity

2019 ◽  
Vol 5 (8) ◽  
pp. eaaw9298 ◽  
Author(s):  
Caitlin Walker ◽  
Yingjie Wang ◽  
Cristina Olivieri ◽  
Adak Karamafrooz ◽  
Jordan Casby ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Substrate Specificity ◽  
Protein Kinase A ◽  
Cushing’S Syndrome ◽  
Genetic Alterations ◽  
Cushing's Syndrome ◽  
Single Mutation ◽  
Α Subunit ◽  
Adrenocortical Adenomas ◽  
Kinase A

Genetic alterations in the PRKACA gene coding for the catalytic α subunit of the cAMP-dependent protein kinase A (PKA-C) are linked to cortisol-secreting adrenocortical adenomas, resulting in Cushing’s syndrome. Among those, a single mutation (L205R) has been found in up to 67% of patients. Because the x-ray structures of the wild-type and mutant kinases are essentially identical, the mechanism explaining aberrant function of this mutant remains under active debate. Using NMR spectroscopy, thermodynamics, kinetic assays, and molecular dynamics simulations, we found that this single mutation causes global changes in the enzyme, disrupting the intramolecular allosteric network and eliciting losses in nucleotide/pseudo-substrate binding cooperativity. Remarkably, by rewiring its internal allosteric network, PKA-CL205R is able to bind and phosphorylate non-canonical substrates, explaining its changes in substrate specificity. Both the lack of regulation and change in substrate specificity reveal the complex role of this mutated kinase in the formation of cortisol-secreting adrenocortical adenomas.

scholarly journals Alterations in Protein Kinase A Substrate Specificity as a Potential Cause of Cushing Syndrome

Endocrinology ◽  
2019 ◽  
Vol 160 (2) ◽  
pp. 447-459 ◽  
Author(s):  
Kerstin Bathon ◽  
Isabel Weigand ◽  
Jens T Vanselow ◽  
Cristina L Ronchi ◽  
Silviu Sbiera ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Substrate Specificity ◽  
Protein Kinase A ◽  
Cushing Syndrome ◽  
Gene Encoding ◽  
Α Subunit ◽  
Adrenocortical Adenomas ◽  
Molecular Events ◽  
Shed Light ◽  
Kinase A

Abstract Cushing syndrome is a severe endocrine disorder of cortisol excess associated with major metabolic and cardiovascular sequelae. We recently identified somatic mutations in PRKACA, the gene encoding the catalytic (C) α subunit of protein kinase A (PKA), as being responsible for cortisol-producing adrenocortical adenomas (CPAs), which are a major cause of Cushing syndrome. In spite of previous studies on the two initially identified mutations (L206R, 199_200insW), the mechanisms of action of the clinically highly relevant PRKACA mutations remain poorly understood. Here, by investigating a large panel of PRKACA mutations, including all those identified so far in Cushing syndrome, we unexpectedly found that not all mutations interfere with the binding of regulatory (R) subunits as previously hypothesized. Because several mutations lie in a region of PKA Cα involved in substrate recognition, we investigated their consequences on substrate specificity by quantitative phosphoproteomics. We found that all three mutations analyzed (L206R, 200_201insV, and d244−248+E249Q) cause major changes in the preference of PKA for its targets, leading to hyperphosphorylation of several PKA substrates, most notably including histone H1.4 at Ser36, which is required for and promotes mitosis. This is reflected by a ninefold hyperphosphorylation of H1.4 in CPAs carrying the L206R mutation. Thus, our findings suggest that in addition to hampering binding to R subunits, PRKACA mutations act by altering PKA substrate specificity. These findings shed light on the molecular events leading to Cushing syndrome and illustrate how mutations altering substrate specificity of a protein kinase may cause human disease.

scholarly journals Molecular Genetic and Genomic Alterations in Cushing’s Syndrome and Primary Aldosteronism

2021 ◽  
Vol 12 ◽  
Author(s):  
Crystal D. C. Kamilaris ◽  
Constantine A. Stratakis ◽  
Fady Hannah-Shmouni
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Cushing’S Syndrome ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Genetic Alterations ◽  
Cushing's Syndrome ◽  
Adrenocortical Tumors ◽  
Pathogenic Variants ◽  
Kinase A

The genetic alterations that cause the development of glucocorticoid and/or mineralocorticoid producing benign adrenocortical tumors and hyperplasias have largely been elucidated over the past two decades through advances in genomics. In benign aldosterone-producing adrenocortical tumors and hyperplasias, alteration of intracellular calcium signaling has been found to be significant in aldosterone hypersecretion, with causative defects including those in KCNJ5, ATP1A1, ATP2B3, CACNA1D, CACNA1H, and CLCN2. In benign cortisol-producing adrenocortical tumors and hyperplasias abnormal cyclic adenosine monophosphate-protein kinase A signaling has been found to play a central role in tumorigenesis, with pathogenic variants in GNAS, PRKAR1A, PRKACA, PRKACB, PDE11A, and PDE8B being implicated. The role of this signaling pathway in the development of Cushing’s syndrome and adrenocortical tumors was initially discovered through the study of the underlying genetic defects causing the rare multiple endocrine neoplasia syndromes McCune-Albright syndrome and Carney complex with subsequent identification of defects in genes affecting the cyclic adenosine monophosphate-protein kinase A pathway in sporadic tumors. Additionally, germline pathogenic variants in ARMC5, a putative tumor suppressor, were found to be a cause of cortisol-producing primary bilateral macronodular adrenal hyperplasia. This review describes the genetic causes of benign cortisol- and aldosterone-producing adrenocortical tumors.

scholarly journals Structural insights into mis-regulation of protein kinase A in human tumors

2015 ◽  
Vol 112 (5) ◽  
pp. 1374-1379 ◽  
Author(s):  
Jonah Cheung ◽  
Christopher Ginter ◽  
Michael Cassidy ◽  
Matthew C. Franklin ◽  
Michael J. Rudolph ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Cushing’S Syndrome ◽  
Biochemical Characterization ◽  
Cushing's Syndrome ◽  
Regulatory Subunit ◽  
Recurrent Mutations ◽  
Distinct Disease ◽  
Genomic Studies ◽  
Kinase A

The extensively studied cAMP-dependent protein kinase A (PKA) is involved in the regulation of critical cell processes, including metabolism, gene expression, and cell proliferation; consequentially, mis-regulation of PKA signaling is implicated in tumorigenesis. Recent genomic studies have identified recurrent mutations in the catalytic subunit of PKA in tumors associated with Cushing’s syndrome, a kidney disorder leading to excessive cortisol production, and also in tumors associated with fibrolamellar hepatocellular carcinoma (FL-HCC), a rare liver cancer. Expression of a L205R point mutant and a DnaJ–PKA fusion protein were found to be linked to Cushing's syndrome and FL-HCC, respectively. Here we reveal contrasting mechanisms for increased PKA signaling at the molecular level through structural determination and biochemical characterization of the aberrant enzymes. In the Cushing’s syndrome disorder, we find that the L205R mutation abolishes regulatory-subunit binding, leading to constitutive, cAMP-independent signaling. In FL-HCC, the DnaJ–PKA chimera remains under regulatory subunit control; however, its overexpression from the DnaJ promoter leads to enhanced cAMP-dependent signaling. Our findings provide a structural understanding of the two distinct disease mechanisms and they offer a basis for designing effective drugs for their treatment.

scholarly journals Genetic Landscape of Sporadic Unilateral Adrenocortical Adenomas Without PRKACA p.Leu206Arg Mutation

2016 ◽  
Vol 101 (9) ◽  
pp. 3526-3538 ◽  
Author(s):  
Cristina L. Ronchi ◽  
Guido Di Dalmazi ◽  
Simon Faillot ◽  
Silviu Sbiera ◽  
Guillaume Assié ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Signaling Pathway ◽  
Hormone Secretion ◽  
Genetic Alterations ◽  
Tissue Samples ◽  
Adrenocortical Adenomas ◽  
Genetic Landscape ◽  
Fresh Frozen ◽  
Kinase A

Abstract Context: Adrenocortical adenomas (ACAs) are among the most frequent human neoplasias. Genetic alterations affecting the cAMP/protein kinase A signaling pathway are common in cortisol-producing ACAs, whereas activating mutations in the gene encoding β-catenin (CTNNB1) have been reported in a subset of both benign and malignant adrenocortical tumors. However, the molecular pathogenesis of most ACAs is still largely unclear. Objective: The aim of the study was to define the genetic landscape of sporadic unilateral ACAs. Design and Setting: Next-generation whole-exome sequencing was performed on fresh-frozen tumor samples and corresponding normal tissue samples. Patients: Ninety-nine patients with ACAs (74 cortisol-producing and 25 endocrine inactive) negative for p.Leu206Arg PRKACA mutation. Main Outcome Measures: Identification of known and/or new genetic alterations potentially involved in adrenocortical tumorigenesis and autonomous hormone secretion, genotype-phenotype correlation. Results: A total of 706 somatic protein-altering mutations were detected in 88 of 99 tumors (median, six per tumor). We identified several mutations in genes of the cAMP/protein kinase A pathway, including three novel mutations in PRKACA, associated with female sex and Cushing's syndrome. We also found genetic alterations in different genes involved in the Wnt/β-catenin pathway, associated with larger tumors and endocrine inactivity, and notably, in many genes of the Ca2+-signaling pathway. Finally, by comparison of our genetic data with those available in the literature, we describe a comprehensive genetic landscape of unilateral ACAs. Conclusions: This study provides the largest sequencing effort on ACAs to date. We thereby identified somatic alterations affecting known and novel pathways potentially involved in adrenal tumorigenesis.

Is disrupted nucleotide-substrate cooperativity a common trait for Cushing's syndrome driving mutations of protein kinase A?

2021 ◽  
pp. 167123
Author(s):  
Caitlin Walker ◽  
Yingjie Wang ◽  
Cristina Olivieri ◽  
Manu V.S ◽  
Jiali Gao ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Cushing’S Syndrome ◽  
Cushing's Syndrome ◽  
Kinase A

scholarly journals PKA Cα subunit mutation triggers caspase-dependent RIIβ subunit degradation via Ser114 phosphorylation

2021 ◽  
Vol 7 (8) ◽  
pp. eabd4176
Author(s):  
Isabel Weigand ◽  
Cristina L. Ronchi ◽  
Jens T. Vanselow ◽  
Kerstin Bathon ◽  
Kerstin Lenz ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Cushing’S Syndrome ◽  
Molecular Mechanisms ◽  
Cushing's Syndrome ◽  
Regulatory Subunit ◽  
Cortisol Secretion ◽  
Adrenocortical Cells ◽  
Protein Levels ◽  
Adrenocortical Adenomas ◽  
Kinase A

Mutations in the PRKACA gene are the most frequent cause of cortisol-producing adrenocortical adenomas leading to Cushing’s syndrome. PRKACA encodes for the catalytic subunit α of protein kinase A (PKA). We already showed that PRKACA mutations lead to impairment of regulatory (R) subunit binding. Furthermore, PRKACA mutations are associated with reduced RIIβ protein levels; however, the mechanisms leading to reduced RIIβ levels are presently unknown. Here, we investigate the effects of the most frequent PRKACA mutation, L206R, on regulatory subunit stability. We find that Ser114 phosphorylation of RIIβ is required for its degradation, mediated by caspase 16. Last, we show that the resulting reduction in RIIβ protein levels leads to increased cortisol secretion in adrenocortical cells. These findings reveal the molecular mechanisms and pathophysiological relevance of the R subunit degradation caused by PRKACA mutations, adding another dimension to the deregulation of PKA signaling caused by PRKACA mutations in adrenal Cushing’s syndrome.

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