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The Hedgehog (Hh) pathway is essential for organ development, homeostasis, and regeneration. Dysfunction of this cascade drives several cancers. To control expression of pathway target genes, the G protein–coupled receptor (GPCR) Smoothened (SMO) activates glioma-associated (GLI) transcription factors via an unknown mechanism. Here, we show that, rather than conforming to traditional GPCR signaling paradigms, SMO activates GLI by binding and sequestering protein kinase A (PKA) catalytic subunits at the membrane. This sequestration, triggered by GPCR kinase (GRK)-mediated phosphorylation of SMO intracellular domains, prevents PKA from phosphorylating soluble substrates, releasing GLI from PKA-mediated inhibition. Our work provides a mechanism directly linking Hh signal transduction at the membrane to GLI transcription in the nucleus. This process is more fundamentally similar between species than prevailing hypotheses suggest. The mechanism described here may apply broadly to other GPCR- and PKA-containing cascades in diverse areas of biology.

We recently identified a high prevalence of mutations affecting the catalytic (Cα) 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’s adenoma is associated with somatic mutations in the gene encoding the Cα subunit of protein kinase A. Calebiro et al. reveal that these mutations increase protein kinase A activity by preventing proper assembly of the protein kinase A holoenzyme.

Cushing's syndrome is caused by excess cortisol production from the adrenocortical gland. In corticotropin-independent Cushing's syndrome, the excess cortisol production is primarily attributed to an adrenocortical adenoma, in which the underlying molecular pathogenesis has been poorly understood. We report a hotspot mutation (L206R) in PRKACA, which encodes the catalytic subunit of cyclic adenosine monophosphate (cAMP)–dependent protein kinase (PKA), in more than 50% of cases with adrenocortical adenomas associated with corticotropin-independent Cushing's syndrome. The L206R PRKACA mutant abolished its binding to the regulatory subunit of PKA (PRKAR1A) that inhibits catalytic activity of PRKACA, leading to constitutive, cAMP-independent PKA activation. These results highlight the major role of cAMP-independent activation of cAMP/PKA signaling by somatic mutations in corticotropin-independent Cushing's syndrome, providing insights into the diagnosis and therapeutics of this syndrome.

Allosteric cooperativity between ATP and substrates is a prominent characteristic of the cAMP-dependent catalytic subunit of protein kinase A (PKA-C). This long-range synergistic action is involved in substrate recognition and fidelity, and it may also regulate PKA’s association with regulatory subunits and other binding partners. To date, a complete understanding of this intramolecular mechanism is still lacking. Here, we integrated NMR(Nuclear Magnetic Resonance)-restrained molecular dynamics simulations and a Markov State Model to characterize the free energy landscape and conformational transitions of PKA-C. We found that the apoenzyme populates a broad free energy basin featuring a conformational ensemble of the active state of PKA-C (ground state) and other basins with lower populations (excited states). The first excited state corresponds to a previously characterized inactive state of PKA-C with the αC helix swinging outward. The second excited state displays a disrupted hydrophobic packing around the regulatory (R) spine, with a flipped configuration of the F100 and F102 residues at the αC-β4 loop. We validated the second excited state by analyzing the F100A mutant of PKA-C, assessing its structural response to ATP and substrate binding. While PKA-C F100A preserves its catalytic efficiency with Kemptide, this mutation rearranges the αC-β4 loop conformation, interrupting the coupling of the two lobes and abolishing the allosteric binding cooperativity. The highly conserved αC-β4 loop emerges as a pivotal element to control the synergistic binding of nucleotide and substrate, explaining how mutations or insertions near or within this motif affect the function and drug sensitivity in homologous kinases.

PRKACB (Protein Kinase CAMP-Activated Catalytic Subunit Beta) is predominantly expressed in the brain, and regulation of this gene links to neuroprotective effects against tau and Aβ-induced toxicity. Here we studied a (GCC)-repeat spanning the core promoter and 5′ UTR of this gene in 300 human subjects, consisting of late-onset neurocognitive disorder (NCD) (N = 150) and controls (N = 150). We also implemented several models to study the impact of this repeat on the three-dimensional (3D) structure of DNA. While the PRKACB (GCC)-repeat was strictly monomorphic at 7-repeats, we detected two 7/8 genotypes only in the NCD group. In all examined models, the (GCC)7 and its periodicals had the least range of divergence variation on the 3D structure of DNA in comparison to the 8-repeat periodicals and several hypothetical repeat lengths. A similar inert effect on the 3D structure was not detected in other classes of short tandem repeats (STRs) such as GA and CA repeats. In conclusion, we report monomorphism of a long (GCC)-repeat in the PRKACB gene in human, its inert effect on DNA structure, and enriched divergence in late-onset NCD. This is the first indication of natural selection for a monomorphic (GCC)-repeat, which probably evolved to function as an “epigenetic knob”, without changing the regional DNA structure.

Protein Kinase A (PKA) is the major receptor for the cyclic adenosine monophosphate (cAMP) secondary messenger in eukaryotes. cAMP binds to two tandem cAMP-binding domains (CBD-A and -B) within the regulatory subunit of PKA (R), unleashing the activity of the catalytic subunit (C). While CBD-A in RIα is required for PKA inhibition and activation, CBD-B functions as a \"gatekeeper\" domain that modulates the control exerted by CBD-A. Preliminary evidence suggests that CBD-B dynamics are critical for its gatekeeper function. To test this hypothesis, here we investigate by Nuclear Magnetic Resonance (NMR) the two-domain construct RIα (91-379) in its apo, cAMP2, and C-bound forms. Our comparative NMR analyses lead to a double conformational selection model in which each apo CBD dynamically samples both active and inactive states independently of the adjacent CBD within a nearly degenerate free energy landscape. Such degeneracy is critical to explain the sensitivity of CBD-B to weak interactions with C and its high affinity for cAMP. Binding of cAMP eliminates this degeneracy, as it selectively stabilizes the active conformation within each CBD and inter-CBD contacts, which require both cAMP and W260. The latter is contributed by CBD-B and mediates capping of the cAMP bound to CBD-A. The inter-CBD interface is dispensable for intra-CBD conformational selection, but is indispensable for full activation of PKA as it occludes C-subunit recognition sites within CBD-A. In addition, the two structurally homologous cAMP-bound CBDs exhibit marked differences in their residual dynamics profiles, supporting the notion that conservation of structure does not necessarily imply conservation of dynamics.

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.

Tatsuhiro Shibata and colleagues molecularly characterize 260 biliary tract cancer samples by a combination of exome and transcriptome sequencing. They find genomic alterations that could potentially be therapeutic targets. The incidence of biliary tract cancer (BTC), including intrahepatic (ICC) and extrahepatic (ECC) cholangiocarcinoma and gallbladder cancer, has increased globally; however, no effective targeted molecular therapies have been approved at the present time. Here we molecularly characterized 260 BTCs and uncovered spectra of genomic alterations that included new potential therapeutic targets. Gradient spectra of mutational signatures with a higher burden of the APOBEC-associated mutation signature were observed in gallbladder cancer and ECC. Thirty-two significantly altered genes, including ELF3 , were identified, and nearly 40% of cases harbored targetable genetic alterations. Gene fusions involving FGFR2 and PRKACA or PRKACB preferentially occurred in ICC and ECC, respectively, and the subtype-associated prevalence of actionable growth factor–mediated signals was noteworthy. The subgroup with the poorest prognosis had significant enrichment of hypermutated tumors and a characteristic elevation in the expression of immune checkpoint molecules. Accordingly, immune-modulating therapies might also be potentially promising options for these patients.

Fibrolamellar hepatocellular carcinoma (FL-HCC) is a rare liver tumor affecting adolescents and young adults with no history of primary liver disease or cirrhosis. We identified a chimeric transcript that is expressed in FL-HCC but not in adjacent normal liver and that arises as the result of a ∼400-kilobase deletion on chromosome 19. The chimeric RNA is predicted to code for a protein containing the amino-terminal domain of DNAJB1, a homolog of the molecular chaperone DNAJ, fused in frame with PRKACA, the catalytic domain of protein kinase A. Immunoprecipitation and Western blot analyses confirmed that the chimeric protein is expressed in tumor tissue, and a cell culture assay indicated that it retains kinase activity. Evidence supporting the presence of the DNAJB1-PRKACA chimeric transcript in 100% of the FL-HCCs examined (15/15) suggests that this genetic alteration contributes to tumor pathogenesis.

Allosteric effects of mutations, ligand binding, or post-translational modifications on protein function occur through changes to the protein's shape, or conformation. In a cell, there are many copies of the same protein, all experiencing these perturbations in a dynamic fashion and fluctuating through different conformations and activity states. According to the \"conformational selection and population shift\" theory, ligand binding selects a particular conformation. This perturbs the ensemble and induces a population shift. In a new PLOS Biology paper, Melacini and colleagues describe a novel model of protein regulation, the \"Double-Conformational Selection Model\", which demonstrates how two tandem ligand-binding domains interact to regulate protein function. Here we explain how tandem domains with tuned interactions-but not single domains-can provide a blueprint for sensitive activation sensors within a narrow window of ligand concentration, thereby promoting signaling control.