Activating hotspot L205R mutation in PRKACA and adrenal Cushing’s syndrome

Abstract

Adrenal Cushing's syndrome is caused by excess production of glucocorticoid from adrenocortical tumors and hyperplasias, which leads to metabolic disorders. We performed whole-exome sequencing of 49 blood-tumor pairs and RNA sequencing of 44 tumors from cortisol-producing adrenocortical adenomas (ACAs), adrenocorticotropic hormone-independent macronodular adrenocortical hyperplasias (AIMAHs), and adrenocortical oncocytomas (ADOs). We identified a hotspot in the PRKACA gene with a L205R mutation in 69.2% (27 out of 39) of ACAs and validated in 65.5% of a total of 87 ACAs. Our data revealed that the activating L205R mutation, which locates in the P+1 loop of the protein kinase A (PKA) catalytic subunit, promoted PKA substrate phosphorylation and target gene expression. Moreover, we discovered the recurrently mutated gene DOT1L in AIMAHs and CLASP2 in ADOs. Collectively, these data highlight potentially functional mutated genes in adrenal Cushing's syndrome.

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

“…Measurements of the intrinsic activities of purified C subunits ( Fig. S1) also show elevated PKAc L205R activity, as previously reported (10,11). Although it is not obvious how the mutation increases the catalytic rate, minor perturbations are observed in the packing of residues near the site of mutation and this may alter the dynamics of the protein in solution.…”

“…1, Table 1, and Table S1): the full-length wild-type C subunit (PKAc), the predominant point mutant (PKAc L205R), the predominant chimeric fusion protein (DnaJ-PKAc), and PKAc with exon 1 residues deleted (PKAc Δexon1). All structures were determined as complexes with ATP and the protein kinase inhibitor (PKI) peptide (5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24). In each, ATP is bound with two metal ions within the cleft formed between the smaller N-terminal and larger C-terminal lobes of the C subunit, associated as previously described (14).…”

“…Previous studies (8)(9)(10)(11) have suggested that the mutation may abolish the binding of PKAc L205R to the R subunit. The structure of PKAc L205R provides an atomic basis for this effect, where steric clash between R205 and the P+1 residue of the regulatory subunit inhibitory sequence is expected (Fig.…”

“…In Cushing's syndrome, a disorder arising from overproduction of cortisol from tumors and hyperplasia of the adrenal cortex, mutations in PRKACA have been discovered in tumor specimens from patients with this disease (8)(9)(10)(11), including the predominant L205R mutation in the PKA C subunit. In fibrolamellar hepatocellular carcinoma (FL-HCC), a rare liver tumor affecting young adults, ∼400-kb deletions have been discovered in chromosome 19.…”

DNAJB1-PRKACA is specific for fibrolamellar carcinoma

“…Honeyman et al 3 proposed the chimera formed by DNAJB1 and PRKACA leads to upregulation of PRKACA activity by a promoter switch mechanism. Interestingly, point mutations [24][25][26] in PRKACA and copy number gain 24 of PRKACA have been shown as recurrent genetic abnormalities underlying functional adrenal adenomas and adrenal hyperplasia. The discovery of a translocation involving PRKACA is a third mechanism of activation of the PRKACA gene in tumors.…”