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.

An aberrant fusion of the DNAJB1 and PRKACA genes generates a chimeric protein kinase (PKA-CDNAJB1) in which the J-domain of the heat shock protein 40 is fused to the catalytic α subunit of cAMP-dependent protein kinase A (PKA-C). Deceivingly, this chimeric construct appears to be fully functional, as it phosphorylates canonical substrates, forms holoenzymes, responds to cAMP activation, and recognizes the endogenous inhibitor PKI. Nonetheless, PKA-CDNAJB1 has been recognized as the primary driver of fibrolamellar hepatocellular carcinoma and is implicated in other neoplasms for which the molecular mechanisms remain elusive. Here we determined the chimera’s allosteric response to nucleotide and pseudo-substrate binding. We found that the fusion of the dynamic J-domain to PKA-C disrupts the internal allosteric network, causing dramatic attenuation of the nucleotide/PKI binding cooperativity. Our findings suggest that the reduced allosteric cooperativity exhibited by PKA-CDNAJB1 alters specific recognitions and interactions between substrates and regulatory partners contributing to dysregulation.

In fibrolamellar hepatocellular carcinoma a single genetic deletion results in the fusion of the first exon of the heat shock protein 40, DNAJB1, which encodes the J domain, with exons 2–10 of the catalytic subunit of protein kinase A, PRKACA. This produces an enzymatically active chimeric protein J-PKAcα. We used molecular dynamics simulations and NMR to analyze the conformational landscape of native and chimeric kinase, and found an ensemble of conformations. These ranged from having the J-domain tucked under the large lobe of the kinase, similar to what was reported in the crystal structure, to others where the J-domain was dislodged from the core of the kinase and swinging free in solution. These simulated dislodged states were experimentally captured by NMR. Modeling of the different conformations revealed no obvious steric interactions of the J-domain with the rest of the RIIβ holoenzyme.

The DNAJB1−PRKACA fusion is the signature genetic event of fibrolamellar hepatocellular carcinoma (FL-HCC), a rare but lethal liver cancer that primarily affects adolescents and young adults. A deletion fuses the first exon of the HSP40 gene (DNAJB1), with exons 2−10 of protein kinase A (PRKACA), producing the chimeric kinase DNAJB1-PKA ca (J-PKA ca ). The HSP40 portion's scaffolding/chaperone function has been implicated in redirecting substrate recognition to upregulate oncogenic pathways, but the direct substrates of this fusion are not fully known. We integrated cell-based and in vitro phosphoproteomics to identify substrates targeted directly by PKA and J-PKA ca , comparing phosphoproteome profiles from cells with in vitro rephosphorylation of peptides and proteins from lysates using recombinant enzymes. We identified a subset of phosphorylation sites in both cell-based and in vitro experiments, as well as altered pathways and proteins consistent with observations from related studies. We also treated cells with PKA inhibitors that function by two different mechanisms (rpcAMPs and PKI) and examined phosphoproteome profiles, finding some substrates that persisted in the presence of inhibitors and revealing differences between WT and chimera. Overall, these results provide potential insights into J-PKA ca 's oncogenic activity in a complex cellular system and may provide candidate targets for therapeutic follow-up.