The PKA-CREB1 axis regulates coronavirus proliferation by viral helicase nsp13 association.

The COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has posed a worldwide threat in the past 3 years. Although it has been widely and intensively investigated, the mechanism underlying the coronavirus-host interaction requires further elucidation, which may contribute to the development of new antiviral strategies. Here, we demonstrated that the host cAMP-responsive element-binding protein (CREB1) interacts with the non-structural protein 13 (nsp13) of SARS-CoV-2, a conserved helicase for coronavirus replication, both in cells and in lung tissues subjected to SARS-CoV-2 infection. The ATPase and helicase activity of viral nsp13 were shown to be potentiated by CREB1 association, as well as by Protein kinase A (PKA)-mediated CREB1 activation. SARS-CoV-2 replication is significantly suppressed by PKA Cα, cAMP-activated protein kinase catalytic subunit alpha (PRKACA), and CREB1 knockdown or inhibition. Consistently, the CREB1 inhibitor 666-15 has shown significant antiviral effects against both the WIV04 strain and the Omicron strain of the SARS-CoV-2. Our findings indicate that the PKA-CREB1 signaling axis may serve as a novel therapeutic target against coronavirus infection. IMPORTANCE: In this study, we provide solid evidence that host transcription factor cAMP-responsive element-binding protein (CREB1) interacts directly with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) helicase non-structural protein 13 (nsp13) and potentiate its ATPase and helicase activity. And by live SARS-CoV-2 virus infection, the inhibition of CREB1 dramatically impairs SARS-CoV-2 replication in vivo. Notably, the IC50 of CREB1 inhibitor 666-15 is comparable to that of remdesivir. These results may extend to all highly pathogenic coronaviruses due to the conserved nsp13 sequences in the virus.

GalNAc-conjugated siRNA targeting the DNAJB1-PRKACA fusion junction in fibrolamellar hepatocellular carcinoma.

Fibrolamellar hepatocellular carcinoma (FLC) is a rare liver cancer caused by a dominant recurrent fusion of the heat shock protein (DNAJB1) and the catalytic subunit of protein kinase A (PRKACA). Current therapies such as chemotherapy and radiation have limited efficacy, and new treatment options are needed urgently. We have previously shown that FLC tumors are dependent on the fusion kinase DNAJB1::PRKACA, making the oncokinase an ideal drug target. mRNA degrading modalities such as antisense oligonucleotides or small interfering RNAs (siRNAs) provide an opportunity to specifically target the fusion junction. Here, we identify a potent and specific siRNA that inhibits DNAJB1::PRKACA expression. We found expression of the asialoglycoprotein receptor in FLC to be maintained at sufficient levels to effectively deliver siRNA conjugated to the GalNAc ligand. We observe productive uptake and siRNA activity in FLC patient-derived xenografts (PDX) models in vitro and in vivo. Knockdown of DNAJB1::PRKACA results in durable growth inhibition of FLC PDX in vivo with no detectable toxicities. Our results suggest that this approach could be a treatment option for FLC patients.

ASIC1 promotes migration and invasion of hepatocellular carcinoma via the PRKACA/AP-1 signaling pathway.

Hepatocellular carcinoma (HCC) exhibits a high mortality rate due to its high invasion and metastatic nature, and the acidic microenvironment plays a pivotal role. Acid-sensing ion channel 1 (ASIC1) is upregulated in HCC tissues and facilitates tumor progression in a pH-dependent manner, while the specific mechanisms therein remain currently unclear. Herein, we aimed to investigate the underlying mechanisms by which ASIC1 contributes to the development of HCC. Using bioinformatics analysis, we found a significant association between ASIC1 expression and malignant transformation of HCC, such as poor prognosis, metastasis and recurrence. Specifically, ASIC1 enhanced the migration and invasion capabilities of Li-7 cells in the in vivo experiment using an HCC lung metastasis mouse model, as well as in the in vitro experiments such as wound healing assay and Transwell assay. Furthermore, our comprehensive gene chip and molecular biology experiments revealed that ASIC1 promoted HCC migration and invasion by activating the PRKACA/AP-1 signaling pathway. Our findings indicate that targeting ASIC1 could have therapeutic potential for inhibiting HCC progression.

The 3-phosphoinositide-dependent protein kinase 1 is an essential upstream activator of protein kinase A in malaria parasites.

Cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA) signalling is essential for the proliferation of Plasmodium falciparum malaria blood stage parasites. The mechanisms regulating the activity of the catalytic subunit PfPKAc, however, are only partially understood, and PfPKAc function has not been investigated in gametocytes, the sexual blood stage forms that are essential for malaria transmission. By studying a conditional PfPKAc knockdown (cKD) mutant, we confirm the essential role for PfPKAc in erythrocyte invasion by merozoites and show that PfPKAc is involved in regulating gametocyte deformability. We furthermore demonstrate that overexpression of PfPKAc is lethal and kills parasites at the early phase of schizogony. Strikingly, whole genome sequencing (WGS) of parasite mutants selected to tolerate increased PfPKAc expression levels identified missense mutations exclusively in the gene encoding the parasite orthologue of 3-phosphoinositide-dependent protein kinase-1 (PfPDK1). Using targeted mutagenesis, we demonstrate that PfPDK1 is required to activate PfPKAc and that T189 in the PfPKAc activation loop is the crucial target residue in this process. In summary, our results corroborate the importance of tight regulation of PfPKA signalling for parasite survival and imply that PfPDK1 acts as a crucial upstream regulator in this pathway and potential new drug target.

Smoothened transduces Hedgehog signals via activity-dependent sequestration of PKA catalytic subunits.

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.

Gα/GSA-1 works upstream of PKA/KIN-1 to regulate calcium signaling and contractility in the Caenorhabditis elegans spermatheca.

Correct regulation of cell contractility is critical for the function of many biological systems. The reproductive system of the hermaphroditic nematode C. elegans contains a contractile tube of myoepithelial cells known as the spermatheca, which stores sperm and is the site of oocyte fertilization. Regulated contraction of the spermatheca pushes the embryo into the uterus. Cell contractility in the spermatheca is dependent on actin and myosin and is regulated, in part, by Ca2+ signaling through the phospholipase PLC-1, which mediates Ca2+ release from the endoplasmic reticulum. Here, we describe a novel role for GSA-1/Gαs, and protein kinase A, composed of the catalytic subunit KIN-1/PKA-C and the regulatory subunit KIN-2/PKA-R, in the regulation of Ca2+ release and contractility in the C. elegans spermatheca. Without GSA-1/Gαs or KIN-1/PKA-C, Ca2+ is not released, and oocytes become trapped in the spermatheca. Conversely, when PKA is activated through either a gain of function allele in GSA-1 (GSA-1(GF)) or by depletion of KIN-2/PKA-R, the transit times and total numbers, although not frequencies, of Ca2+ pulses are increased, and Ca2+ propagates across the spermatheca even in the absence of oocyte entry. In the spermathecal-uterine valve, loss of GSA-1/Gαs or KIN-1/PKA-C results in sustained, high levels of Ca2+ and a loss of coordination between the spermathecal bag and sp-ut valve. Additionally, we show that depleting phosphodiesterase PDE-6 levels alters contractility and Ca2+ dynamics in the spermatheca, and that the GPB-1 and GPB-2 Gß subunits play a central role in regulating spermathecal contractility and Ca2+ signaling. This work identifies a signaling network in which Ca2+ and cAMP pathways work together to coordinate spermathecal contractions for successful ovulations.

Novel protein kinase cAMP-Activated Catalytic Subunit Alpha (PRKACA) inhibitor shows anti-tumor activity in a fibrolamellar hepatocellular carcinoma model.

Fibrolamellar hepatocellular carcinoma (FL-HCC) is known as a highly aggressive liver cancer that typically affects young adults without virus infection. Since this type of cancer does not respond to chemotherapy, surgery is the only known effective therapeutic option. Most FL-HCC patients express the fusion gene DNAJB1-PRKACA, which has been recognized as the signature of FL-HCC. It has also been reported that PRKACA kinase activity is essential for its oncogenic activity, suggesting that PRKACA kinase inhibition could be considered as an useful therapeutic target. In this study, we established an evaluation system for PRKACA kinase inhibitors and synthesized DS89002333, a novel PRKACA inhibitor. DS89002333 showed potent PRKACA inhibitory activity and inhibited fusion protein-dependent cell growth both in vitro and in vivo. Furthermore, this compound showed anti-tumor activity in an FL-HCC patient-derived xenograft model expressing the DNAJB1-PRKACA fusion gene. Our data suggest that DS89002333 could be considered as a potential therapeutic agent for FL-HCC.

PKA Cß: a forgotten catalytic subunit of cAMP-dependent protein kinase opens new windows for PKA signaling and disease pathologies.

3',5'-cyclic adenosine monophosphate (cAMP) dependent protein kinase or protein kinase A (PKA) has served as a prototype for the large family of protein kinases that are crucially important for signal transduction in eukaryotic cells. The PKA catalytic subunits are encoded by the two major genes PRKACA and PRKACB, respectively. The PRKACA gene encodes two known splice variants, the ubiquitously expressed Cα1 and the sperm-specifically expressed Cα2. In contrast, the PRKACB gene encodes several splice variants expressed in a highly cell and tissue-specific manner. The Cß proteins are called Cß1, Cß2, Cß3, Cß4 and so-called abc variants of Cß3 and Cß4. Whereas Cß1 is ubiquitously expressed, Cß2 is enriched in immune cells and the Cß3, Cß4 and their abc variants are solely expressed in neuronal cells. All Cα and Cß splice variants share a kinase-conserved catalytic core and a C-terminal tail encoded by exons 2 through 10 in the PRKACA and PRKACB genes, respectively. All Cα and Cß splice variants with the exception of Cα1 and Cß1 are hyper-variable at the N-terminus. Here, we will discuss how the PRKACA and PRKACB genes have developed as paralogs that encode distinct and functionally non-redundant proteins. The fact that Cα and Cß splice variant mutations are associated with numerous diseases further opens new windows for PKA-induced disease pathologies.

Vascular and Liver Homeostasis in Juvenile Mice Require Endothelial Cyclic AMP-Dependent Protein Kinase A.

During vascular development, endothelial cAMP-dependent protein kinase A (PKA) regulates angiogenesis by controlling the number of tip cells, and PKA inhibition leads to excessive angiogenesis. Whether this role of endothelial PKA is restricted to embryonic and neonatal development or is also required for vascular homeostasis later on is unknown. Here, we show that perinatal (postnatal days P1-P3) of later (P28-P32) inhibition of endothelial PKA using dominant-negative PKA expressed under the control of endothelial-specific Cdh5-CreERT2 recombinase (dnPKAiEC mice) leads to severe subcutaneous edema, hypoalbuminemia, hypoglycemia and premature death. These changes were accompanied by the local hypersprouting of blood vessels in fat pads and the secondary enlargement of subcutaneous lymphatic vessels. Most noticeably, endothelial PKA inhibition caused a dramatic disorganization of the liver vasculature. Hepatic changes correlated with decreased gluconeogenesis, while liver albumin production seems to be unaffected and hypoalbuminemia is rather a result of increased leakage into the interstitium. Interestingly, the expression of dnPKA only in lymphatics using Prox1-CreERT2 produced no phenotype. Likewise, the mosaic expression in only endothelial subpopulations using Vegfr3-CreERT2 was insufficient to induce edema or hypoglycemia. Increased expression of the tip cell marker ESM1 indicated that the inhibition of PKA induced an angiogenic response in the liver, although tissue derived pro- and anti-angiogenic factors were unchanged. These data indicate that endothelial PKA is a gatekeeper of endothelial cell activation not only in development but also in adult homeostasis, preventing the aberrant reactivation of the angiogenic program.

Integrated Phosphoproteomics for Identifying Substrates of Human Protein Kinase A (PRKACA) and Its Oncogenic Mutant DNAJB1-PRKACA.

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-PKAca (J-PKAca). 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-PKAca, 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-PKAca's oncogenic activity in a complex cellular system and may provide candidate targets for therapeutic follow-up.

Crosstalk between PKA and PKG controls pH-dependent host cell egress of Toxoplasma gondii.

Toxoplasma gondii encodes three protein kinase A catalytic (PKAc1-3) and one regulatory (PKAr) subunits to integrate cAMP-dependent signals. Here, we show that inactive PKAc1 is maintained at the parasite pellicle by interacting with acylated PKAr. Either a conditional knockdown of PKAr or the overexpression of PKAc1 blocks parasite division. Conversely, down-regulation of PKAc1 or stabilisation of a dominant-negative PKAr isoform that does not bind cAMP triggers premature parasite egress from infected cells followed by serial invasion attempts leading to host cell lysis. This untimely egress depends on host cell acidification. A phosphoproteome analysis suggested the interplay between cAMP and cGMP signalling as PKAc1 inactivation changes the phosphorylation profile of a putative cGMP-phosphodiesterase. Concordantly, inhibition of the cGMP-dependent protein kinase G (PKG) blocks egress induced by PKAc1 inactivation or environmental acidification, while a cGMP-phosphodiesterase inhibitor circumvents egress repression by PKAc1 or pH neutralisation. This indicates that pH and PKAc1 act as balancing regulators of cGMP metabolism to control egress. These results reveal a crosstalk between PKA and PKG pathways to govern egress in T. gondii.

Activation of the Ae4 (Slc4a9) cation-driven Cl-/HCO3- exchanger by the cAMP-dependent protein kinase in salivary gland acinar cells.

Ae4 transporters are critical for Cl- uptake across the basolateral membrane of acinar cells in the submandibular gland (SMG). Although required for fluid secretion, little is known about the physiological regulation of Ae4. To investigate whether Ae4 is regulated by the cAMP-dependent signaling pathway, we measured Cl-/HCO3- exchanger activity in SMG acinar cells from Ae2-/- mice, which only express Ae4, and found that the Ae4-mediated activity was increased in response to ß-adrenergic receptor stimulation. Moreover, pretreatment with H89, an inhibitor of the cAMP-activated kinase (PKA), prevented the stimulation of Ae4 exchangers. We then expressed Ae4 in CHO-K1 cells and found that the Ae4-mediated activity was increased when Ae4 is coexpressed with the catalytic subunit of PKA (PKAc), which is constitutively active. Ae4 sequence analysis showed two potential PKA phosphorylation serine residues located at the intracellular NH2-terminal domain according to a homology model of Ae4. NH2-terminal domain Ser residues were mutated to alanine (S173A and S273A, respectively), where the Cl-/HCO3- exchanger activity displayed by the mutant S173A was not activated by PKA. Conversely, S273A mutant kept the PKA dependency. Together, we conclude that Ae4 is stimulated by PKA in SMG acinar cells by a mechanism that probably depends on the phosphorylation of S173.NEW & NOTEWORTHY We found that Ae4 exchanger activity in secretory salivary gland acinar cells is increased upon ß-adrenergic receptor stimulation. The activation of Ae4 was prevented by H89, a nonselective PKA inhibitor. Protein sequence analysis revealed two residues (S173 and S273) that are potential targets of cAMP-dependent protein kinase (PKA). Experiments in CHO-K1 cells expressing S173A and S273A mutants showed that S173A, but not S273A, is not activated by PKA.

Aberration of the modulatory functions of intronic microRNA hsa-miR-933 on its host gene ATF2 results in type II diabetes mellitus and neurodegenerative disease development.

BACKGROUND: MicroRNAs are ~ 22-nucleotide-long biological modifiers that act as the post-transcriptional modulator of gene expression. Some of them are identified to be embedded within the introns of protein-coding genes, these miRNAs are called the intronic miRNAs. Previous findings state that these intronic miRNAs are co-expressed with their host genes. This co-expression is necessary to maintain the robustness of the biological system. Till to date, only a few experiments are performed discretely to elucidate the functional relationship between few co-expressed intronic miRNAs and their associated host genes. RESULTS: In this study, we have interpreted the underlying modulatory mechanisms of intronic miRNA hsa-miR-933 on its target host gene ATF2 and found that aberration can lead to several disease conditions. A protein-protein interaction network-based approach was adopted, and functional enrichment analysis was performed to elucidate the significantly over-represented biological functions and pathways of the common targets. Our approach delineated that hsa-miR-933 might control the hyperglycemic condition and hyperinsulinism by regulating ATF2 target genes MAP4K4, PRKCE, PEA15, BDNF, PRKACB, and GNAS which can otherwise lead to the development of type II diabetes mellitus. Moreover, we showed that hsa-miR-933 can regulate a target of ATF2, brain-derived neurotrophic factor (BDNF), to modulate the optimal expression of ATF2 in neuron cells to render neuroprotection for the inhibition of neurodegenerative diseases. CONCLUSIONS: Our in silico model provides interesting resources for experimentations in a model organism or cell line for further validation. These findings may extend the common perception of gene expression analysis with new regulatory functionality.

Involvement of Protein Kinase A in Oxytocin Neuronal Activity in Rat Dams with Pup Deprivation.

Oxytocin (OT) neuronal activity is the key factor for breastfeeding and it can be disrupted by mother-baby separation. To explore cellular mechanisms underlying OT neuronal activity, we studied the role of protein kinase A (PKA) in OT neuronal activity in the supraoptic nucleus (SON) using a rodent model of pup deprivation (PD) Intermittent (IPD) or continuous (CPD) PD significantly reduced suckling duration and number of milk ejections in lactating rats, particularly those with CPD. In Western blots of the SON, PD increased expressions of OT receptor (OTR) and its immediate downstream effectors, Gαq and Gß subunits, particularly IPD, but reduced the expression of catalytic subunit of PKA (cPKA). In brain slices, inhibition of PKA blocked prostaglandin E2-evoked increase in firing activity including burst firing in OT neurons. In IPD dams, filamentous actin formed ring-like structures in the cytoplasmic region of OT neurons, which was reduced in CPD. Moreover, molecular association between actin and cPKA also reduced in PD dams. Incubation of brain slices with OT reduced the expression of cPKA, which was blocked by pretreatment with atosiban, an antagonist of OTR. These results indicate that PD disrupts OT neuronal activity through dissociating the Gq proteins and PKA in OTR-associated signaling cascade, which couples with reduced interactions between filamentous actin and PKA in OT neurons in the SON. This study highlights that PKA can be a novel target treating abnormal OT neuronal activity and its associated diseases.

Water-mediated conformational preselection mechanism in substrate binding cooperativity to protein kinase A.

Substrate binding cooperativity in protein kinase A (PKA) seems to involve allosteric coupling between the two binding sites. It received significant attention, but its molecular basis still remains not entirely clear. Based on long molecular dynamics of PKA and its complexes, we characterized an allosteric pathway that links ATP binding to the redistribution of states adopted by a protein substrate positioning segment in favor of those that warrant correct binding. We demonstrate that the cooperativity mechanism critically depends on the presence of water in two distinct, buried hydration sites. One holds just a single water molecule, which acts as a switchable hydrogen bond bridge along the allosteric pathway. The second, filled with partially disordered solvent, is essential for providing a smooth free energy landscape underlying conformational transitions of the peptide binding region. Our findings remain in agreement with experimental data, also concerning the cooperativity abolishing effect of the Y204A mutation, and indicate a plausible molecular mechanism contributing to experimentally observed binding cooperativity of the two substrates.

Protein kinase A catalytic-α and catalytic-ß proteins have nonredundant regulatory functions.

Vasopressin regulates osmotic water transport in the renal collecting duct by protein kinase A (PKA)-mediated control of the water channel aquaporin-2 (AQP2). Collecting duct principal cells express two seemingly redundant PKA catalytic subunits, PKA catalytic α (PKA-Cα) and PKA catalytic ß (PKA-Cß). To identify the roles of these two protein kinases, we carried out deep phosphoproteomic analysis in cultured mpkCCD cells in which either PKA-Cα or PKA-Cß was deleted using CRISPR-Cas9-based genome editing. Controls were cells carried through the genome editing procedure but without deletion of PKA. TMT mass tagging was used for protein mass spectrometric quantification. Of the 4,635 phosphopeptides that were quantified, 67 phosphopeptides were significantly altered in abundance with PKA-Cα deletion, whereas 21 phosphopeptides were significantly altered in abundance with PKA-Cß deletion. However, only four sites were changed in both. The target proteins identified in PKA-Cα-null cells were largely associated with cell membranes and membrane vesicles, whereas target proteins in PKA-Cß-null cells were largely associated with the actin cytoskeleton and cell junctions. In contrast, in vitro incubation of mpkCCD proteins with recombinant PKA-Cα and PKA-Cß resulted in virtually identical phosphorylation changes. In addition, analysis of total protein abundances in in vivo samples showed that PKA-Cα deletion resulted in a near disappearance of AQP2 protein, whereas PKA-Cß deletion did not decrease AQP2 abundance. We conclude that PKA-Cα and PKA-Cß serve substantially different regulatory functions in renal collecting duct cells and that differences in phosphorylation targets may be due to differences in protein interactions, e.g., mediated by A-kinase anchor proteins, C-kinase anchoring proteins, or PDZ binding.

Meprin-ß activity modulates the ß-catalytic subunit of protein kinase A in ischemia-reperfusion-induced acute kidney injury.

Meprin metalloproteases have been implicated in the progression of kidney injury. Previous work from our group has shown that meprins proteolytically process the catalytic subunit of protein kinase A (PKA-C), resulting in decreased PKA-C kinase activity. The goal of the present study was to determine the PKA-C isoforms impacted by meprin-ß and whether meprin-ß expression affects downstream mediators of the PKA signaling pathway in ischemia-reperfusion (IR)-induced kidney injury. IR was induced in 12-wk-old male wild-type (WT) and meprin-ß knockout (ßKO) mice. Madin-Darby canine kidney cells transfected with meprin-ß cDNA were also subjected to 2 h of hypoxia. Western blot analysis was used to evaluate levels of total PKA-C, PKA-Cα, PKA-Cß, phosphorylated (p-)PKA-C, and p-ERK1/2. Meprin-ß expression enhanced kidney injury as indicated by levels of neutrophil gelatinase-associated lipocalin and cystatin C. IR-associated decreases were observed in levels of p-PKA-C in kidney tissue from WT mice but not ßKO mice, suggesting that meprin-ß expression/activity is responsible for the in vivo reduction in kinase activity. Significant increases in levels of PKA-Cß were observed in kidney lysates for WT mice but not ßKO mice at 6 h post-IR. Proximal tubule PKA-Cß increases in WT but not ßKO kidneys were demonstrated by fluorescent microscopy. Furthermore, IR-induced injury was associated with significant increases in p-ERK levels for both genotypes. The present data demonstrate that meprin-ß enhances IR-induced kidney injury in part by modulating mediators of the PKA-Cß signaling pathway.

Interaction of AIP with protein kinase A (cAMP-dependent protein kinase).

Germline mutations in the aryl hydrocarbon receptor-interacting protein (AIP) gene cause mostly somatotropinomas and/or prolactinomas in a subset of familial isolated pituitary adenomas (FIPA). AIP has been shown to interact with phosphodiesterases (PDEs) and G proteins, suggesting a link to the cyclic AMP (cAMP)-dependent protein kinase (PKA) pathway. Upregulation of PKA is seen in sporadic somatotropinomas that carry GNAS mutations, and those in Carney complex that are due to PRKAR1A mutations. To elucidate the mechanism of AIP-dependent pituitary tumorigenesis, we studied potential functional and physical interactions of AIP with PKA's main subunits PRKAR1A (R1α) and PRKACA (Cα). We found that AIP physically interacts with both R1α and Cα; this interaction is enhanced when all three components are present, but maintained during Cα-R1α dissociation by PKA activation, indicating that AIP binds Cα/R1α both in complex and separately. The interaction between AIP and R1α/Cα is reduced when the frequent AIP pathogenic mutation p.R304* is present. AIP protein levels are regulated both by translation and the ubiquitin/proteasome pathway and Cα stabilizes both AIP and R1α protein levels. AIP reduction by siRNA leads to an increase of PKA activity, which is disproportionately enhanced during PDE4-inhibition. We show that AIP interacts with the PKA pathway on multiple levels, including a physical interaction with both the main regulatory (R1α) and catalytic (Cα) PKA subunits and a functional interaction with PDE4-dependent PKA activation. These findings provide novel insights on the mechanisms of AIP-dependent pituitary tumorigenesis.

Deletion of yeast TPK1 reduces the efficiency of non-homologous end joining DNA repair.

Non-homologous end joining (NHEJ) is a highly conserved mechanism of DNA double-stranded break (DSB) repair. Here we utilize a computational protein-protein interaction method to identify human PRKACB as a potential candidate interacting with NHEJ proteins. We show that the deletion of its yeast homolog, TPK1 that codes for the protein kinase A catalytic subunit reduces the efficiency of NHEJ repair of breaks with overhangs and blunt ends in plasmid-based repair assays. Additionally, tpk1Δ mutants showed defects in the repair of chromosomal breaks induced by HO-site specific endonuclease. Our double deletion mutant analyses suggest that TPK1 and YKU80, a key player in NHEJ could function in parallel pathways. Altogether, here we report a novel involvement for TPK1 in NHEJ.

Inhibitors and fluorescent probes for protein kinase PKAcß and its S54L mutant, identified in a patient with cortisol producing adenoma.

Recently, a mutation was discovered in the gene PRKACB encoding the catalytic subunit ß of PKA (PKAcß) from a patient with severe Cushing's syndrome. This mutation, S54L, leads to a structural change in the glycine-rich loop of the protein. In the present study, an inhibitor with six-fold selectivity toward S54L-PKAcß mutant over the wild-type enzyme was constructed. Moreover, we developed a fluorescent assay allowing to determine side by side the affinity of commercially available PKA inhibitors, newly synthesized compounds, and fluorescent probes toward PKAcß and S54L-PKAcß.