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.
We recently identified a high prevalence of mutations affecting the catalytic (Ca) 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.
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.
We describe for the first time PRKACA mutations as rare findings associated with unilateral primary aldosteronism. As cortisol cosecretion occurs in a subgroup of APAs, other molecular mechanisms are likely to exist.
The cAMP signaling pathway is one of the major players in the regulation of growth and hormonal secretion in adrenocortical cells. Although its role in the pathogenesis of adrenocortical hyperplasia associated with Cushing's syndrome has been clarified, a clear involvement of the cAMP signaling pathway and of one of its major downstream effectors, the protein kinase A (PKA), in sporadic adrenocortical adenomas remained elusive until recently. During the last year, a report by our group and three additional independent groups showed that somatic mutations of PRKACA, the gene coding for the catalytic subunit a of PKA, are a common genetic alteration in patients with Cushing's syndrome due to adrenal adenomas, occurring in 35-65% of the patients. In vitro studies revealed that those mutations are able to disrupt the association between catalytic and regulatory subunits of PKA, leading to a cAMP-independent activity of the enzyme. Despite somatic PRKACA mutations being a common finding in patients with clinically manifest Cushing's syndrome, the pathogenesis of adrenocortical adenomas associated with subclinical hypercortisolism seems to rely on a different molecular background. In this review, the role of cAMP/PKA signaling in the regulation of adrenocortical cell function and its alterations in cortisolproducing adrenocortical adenomas will be summarized, with particular focus on recent developments.
Somatic mutations in protein kinase A catalytic α subunit (PRKACA) were found to be causative for 30–40% of cortisol-producing adenomas (CPA) of the adrenal gland, rendering PKA signalling constitutively active. In its resting state, PKA is a stable and inactive heterotetramer, consisting of two catalytic and two regulatory subunits with the latter inhibiting PKA activity. The human genome encodes three different PKA catalytic subunits and four different regulatory subunits that are preferentially expressed in different organs. In normal adrenal glands all regulatory subunits are expressed, while CPA exhibit reduced protein levels of the regulatory subunit IIβ. In this study, we linked for the first time the loss of RIIβ protein levels to the PRKACA mutation status and found the down-regulation of RIIβ to arise post-transcriptionally. We further found the PKA subunit expression pattern of different tumours is also present in the zones of the normal adrenal cortex and demonstrate that the different PKA subunits have a differential expression pattern in each zone of the normal adrenal gland, indicating potential specific roles of these subunits in the regulation of different hormones secretion.
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