Endocrine Abstracts

We previously identified mutations in PRKACA, coding for the catalytic a (Ca) subunit of protein kinase A (PKA), as the main genetic alteration in cortisol-producing adrenal adenomas (CPAs) responsible for Cushing’s syndrome. Here, we further investigated the mechanism of action of all PRKACA mutations identified so far by our team (L206R, L199_C200_insW, S213R_L212_K214insIILR, C200_GlyinsV, W197R, del244-248+E249Q and E32V). Five out of seven mutants showed reduced binding to at least one of the two tested regulatory subunits (RIa and RIIb). Similarly, not all mutants show increased basal PKA activity. This suggested that the reported mechanism of increased basal activity due to interference with holoenzyme formation was unlikely to be the sole mechanism of action of PRKACA mutations. Since most of these mutations lie close to the active site of PKA, we hypothesized that they might alter substrate specificity. Consistent with this hypothesis, a Western blot analysis of PKA phosphorylated substrates using an antibody recognizing the phosphorylated PKA consensus suggested that each PRKACA mutation induced specific changes in PKA phosphorylation pattern. These findings were further corroborated by an in silico prediction of substrate specificity. Thus, we used a quantitative mass spectrometry method (NanoLC-MS/MS) to precisely analyze and compare the phosphorylation patterns induced by the different mutants. We found that all three tested mutants (L206R, del244-248+E249Q, C200_G201insV) induced relevant changes in substrate specificity. Among all PKA substrates with increased phosphorylation with the mutants compared to wild-type Ca subunit, histone H1.4 was hyper-phosphorylated at Ser36 by all three mutants. Importantly, we found that CPAs harboring the L206R PRKACA mutation (n=3) had increased H1.4 phosphorylation at Ser36 compared to non-mutated adenomas (n=4). Since H1.4 Ser36 phosphorylation has been shown to be required for mitosis and chromatin condensation, H1.4 hyperphosphorylation might play a relevant part in the mechanisms linking PRKACA mutations to increased proliferation of adrenocortical cells. Altogether, these findings indicate that several mechanisms, including a change in substrate specificity, contribute to the development of CPAs caused by PRKACA mutations.