Catalytic Subunit Of Protein Kinase A Research Articles

Activating astrocytic α2A adrenoceptors in hippocampus reduces glutamate toxicity to attenuate sepsis-associated encephalopathy in mice

BackgroundGlutamate metabolism disorder is an important mechanism of sepsis-associated encephalopathy (SAE). Astrocytes regulate glutamate metabolism. In septic mice, α2A adrenoceptor (α2A-AR) activation in the central nervous system provides neuroprotection. α2A-ARs are expressed abundantly in hippocampal astrocytes. This study was performed to determine whether hippocampal astrocytic α2A-AR activation confers neuroprotection against SAE and whether this protective effect is astrocyte specific and achieved by the modulation of glutamate metabolism. MethodsMale C57BL/6 mice with and without α2A-AR knockdown were subjected to cecal ligation and puncture (CLP). They were treated with intrahippocampal guanfacine (an α2A-AR agonist) or intraperitoneal dexmedetomidine in the presence or absence of dihydrokainic acid [DHK; a glutamate transporter 1 (GLT-1) antagonist] and/or UCPH-101 [a glutamate/aspartate transporter (GLAST) antagonist]. Hippocampal tissue was collected for the measurement of astrocyte reactivity, GLT-1 and GLAST expression, and glutamate receptor subunit 2B (GluN2B) phosphorylation. In vivo real-time extracellular glutamate concentrations in the hippocampus were measured by ultra-performance liquid chromatography tandem mass spectrometry combined with microdialysis, and in vivo real-time hippocampal glutamatergic neuron excitability was assessed by calcium imaging. The mice were subjected to the Barnes maze and fear conditioning tests to assess their learning and memory. Golgi staining was performed to assess changes in the hippocampal synaptic structure. In vitro, primary astrocytes with and without α2A-AR knockdown were stimulated with lipopolysaccharide (LPS) and treated with guanfacine or dexmedetomidine in the presence or absence of 8-bromo- cyclic adenosine monophosphate (8-Br-cAMP, a cAMP analog). LPS-treated primary and BV2 microglia were also treated with guanfacine or dexmedetomidine. Astrocyte reactivity, PKA catalytic subunit, GLT-1 an GLAST expression were determined in primary astrocytes. Interleukin-1β, interleukin-6 and tumor necrosis factor-alpha in the medium of microglia culture were measured. ResultsCLP induced synaptic injury, impaired neurocognitive function, increased astrocyte reactivity and reduced GLT-1 and GLAST expression in the hippocampus of mice. The extracellular glutamate concentration, phosphorylation of GluN2B at Tyr-1472 and glutamatergic neuron excitability in the hippocampus were increased in the hippocampus of septic mice. Intraperitoneal dexmedetomidine or intrahippocampal guanfacine administration attenuated these effects. Hippocampal astrocytes expressed abundant α2A-ARs; expression was also detected in neurons but not microglia. Specific knockdown of α2A-ARs in hippocampal astrocytes and simultaneous intrahippocampal DHK and UCPH-101 administration blocked the neuroprotective effects of dexmedetomidine and guanfacine. Intrahippocampal administration of DHK or UCPH-101 alone had no such effect. In vitro, guanfacine or dexmedetomidine inhibited astrocyte reactivity, reduced PKA catalytic subunit expression, and increased GLT-1 and GLAST expression in primary astrocytes but not in primary astrocytes that received α2A-AR knockdown or were treated with 8-Br-cAMP. Guanfacine or dexmedetomidine inhibited microglial reactivity in BV2 but not primary microglia. ConclusionsOur results suggest that neurocognitive protection against SAE after hippocampal α2A-AR activation is astrocyte specific. This protection may involve the inhibition of astrocyte reactivity and alleviation of glutamate neurotoxicity, thereby reducing synaptic injury. The cAMP/protein kinase A (PKA) signaling pathway is a potential cellular mechanism by which activating α2A-AR modulates astrocytic function.

Serodiagnosis of multiple cancers using an extracellular protein kinase A autoantibody-based lateral flow platform

Extracellular protein kinase A autoantibody (ECPKA-AutoAb) has been suggested as a universal cancer biomarker due to its higher amounts in serum of several types of cancer patients than that of normal individuals. Herein, we first developed a lateral flow immunoassay (LFIA) tool, using a sandwich format, toward ECPKA-AutoAb in human serum. For this format, 3G2 as a capture antibody was identified using hybridoma technique and a series of screenings where it showed superior capacity to recognize Enzo PKA catalytic subunit alpha (Cα), compared to other PKA antibodies and antigens. Using these components, we performed sandwich ELISA toward a mimic and real sample of ECPKA-AutoAb. As per the results, limit of detection (LOD) was found to be 135 ng/mL and ECPKA-AutoAb levels were higher in various cancer patients than in normal individuals like previous studies. Based on these results, we applied this sandwich format into LFIA tool and found that the LOD of the fabricated LFIA tool showed about 3.8 ng/mL using spiked PKA-Ab, which is significantly improved compared to the LOD of sandwich ELISA. Also, the developed LFIA tool demonstrated a remarkable ability to detect significant differences in ECPKA-AutoAb levels between normal and cancer patients within 15 min, showing a potential for point-of-care (PoC) detection. One interesting point is that our LFIA strip contains an additional conjugation pad II, named because of its position behind the conjugation pad, in which PKA Cα is dried, enabling a sandwich format.

Secreted aspartyl protease 3 regulated by the Ras/cAMP/PKA pathway promotes the virulence of Candida auris.

The surge of multidrug-resistant fungal pathogens, especially Candida auris, poses significant threats to global public health. Candida auris exhibits resistance to multiple antifungal drugs, leading to major outbreaks and a high mortality rate. With an urgent call for innovative therapeutic strategies, this study focused on the regulation and pathobiological significance of secreted aspartyl proteinases (SAPs) in C. auris, as these enzymes play pivotal roles in the virulence of some fungal species. We delved into the Ras/cAMP/PKA signaling pathway's influence on SAP activity in C. auris. Our findings underscored that the Ras/cAMP/PKA pathway significantly modulates SAP activity, with PKA catalytic subunits, Tpk1 and Tpk2, playing a key role. We identified a divergence in the SAPs of C. auris compared to Candida albicans, emphasizing the variation between Candida species. Among seven identified secreted aspartyl proteases in C. auris (Sapa1 to Sapa7), Sapa3 emerged as the primary SAP in the pathogen. Deletion of Sapa3 led to a significant decline in SAP activity. Furthermore, we have established the involvement of Sapa3 in the biofilm formation of C. auris. Notably, Sapa3 was primarily regulated by Tpk1 and Tpk2. Deletion of SAPA3 significantly reduced C. auris virulence, underscoring its pivotal role in C. auris pathogenicity. The outcomes of this study provide valuable insights into potential therapeutic targets, laying the groundwork for future interventions against C. auris infection.

The psychosis risk factor RBM12 encodes a novel repressor of GPCR/cAMP signal transduction

RBM12 is a high-penetrance risk factor for familial schizophrenia and psychosis, yet its precise cellular functions and the pathways to which it belongs are not known. We utilize two complementary models, HEK293cells and human iPSC-derived neurons, and delineate RBM12 as a novel repressor of the G protein-coupled receptor/cAMP/PKA (GPCR/cAMP/PKA) signaling axis. We establish that loss of RBM12 leads to hyperactive cAMP production and increased PKA activity as well as altered neuronal transcriptional responses to GPCR stimulation. Notably, the cAMP and transcriptional signaling steps are subject to discrete RBM12-dependent regulation. We further demonstrate that the two RBM12 truncating variants linked to familial psychosis impact this interplay, as the mutants fail to rescue GPCR/cAMP signaling hyperactivity in cells depleted of RBM12. Lastly, we present a mechanism underlying the impaired signaling phenotypes. In agreement with its activity as an RNA-binding protein, loss of RBM12 leads to altered gene expression, including that of multiple effectors of established significance within the receptor pathway. Specifically, the abundance of adenylyl cyclases, phosphodiesterase isoforms, and PKA regulatory and catalytic subunits is impacted by RBM12 depletion. We note that these expression changes are fully consistent with the entire gamut of hyperactive signaling outputs. In summary, the current study identifies a previously unappreciated role for RBM12 in the context of the GPCR-cAMP pathway that could be explored further as a tentative molecular mechanism underlying the functions of this factor in neuronal physiology and pathophysiology.

1549-P: scEMC10 Promotes Glucose Intolerance via the Inhibition of PKA-Mediated Insulin Secretion

It has been shown recently that overexpression of the secreted isoform of endoplasmic reticulum membrane complex subunit 10 (scEMC10) impairs, whereas Emc10 knockout (KO) or antibody neutralization of circulating scEMC10 improves glucose tolerance in mice fed with high fat diet. In humans, serum scEMC10 levels correlate positively with fasting plasma glucose and HbA1c. The mechanism underlying the regulation of glucose metabolism by scEMC10 remains largely unknown. In this study, alation of EMC10 significantly improved glucose tolerance in chow diet-fed mice, which was attributed to increased glucose-stimulated insulin secretion (GSIS), but not improved insulin sensitivity evaluated by euglycemic-hyperinsulinemic clamp. Using an adeno-associated virus, either reconstitution of scEMC10 in Emc10 KO mice or overexpression of scEMC10 in wildtype mice significantly decreased glucose tolerance accompanied by blunted GSIS. Recombinant scEMC10 inhibited GSIS in both INS-1 cells and isolated primary islets in vitro. Mechanistically, we presented evidence that extracellular scEMC10 can be transported into beta cells where it binds to the catalytic subunit of PKA and inhibits phosphorylations of PKA substrates including phosphorylated CREB, along with reduced PDX-1 expression. Furthermore, antibody neutralization of circulating scEMC10 alleviated hyperglycemia in both STZ-treated mice and ob/ob mice. This work identifies scEMC10 as a suppressor of insulin secretion via the inhibition of PKA signaling in islet beta cells and suggests scEMC10 neutralizing antibody as a promising pharmaceutical agent to treat diabetes. Disclosure J.Dai: None. S.Liu: None. K.Chen: None. S.Jin: None. Y.Wang: None. L.Chen: None. X.Wang: None. Funding National Natural Science Foundation of China (81873645); Science and Technology Commission of Shanghai Municipality (22140902700)

Conformational changes in protein kinase A along its activation cycle are rooted in the folding energetics of cyclic-nucleotide binding domains

Cyclic-nucleotide binding (CNB) domains are structurally and evolutionarily conserved signaling modules that regulate proteins with diverse folds and functions. Despite a wealth of structural information, the mechanisms by which CNB domains couple cyclic-nucleotide binding to conformational changes involved in signal transduction remain unknown. Here we combined single-molecule and computational approaches to investigate the conformation and folding energetics of the two CNB domains of the regulatory subunit of protein kinase A (PKA). We found that the CNB domains exhibit different conformational and folding signatures in the apo state, when bound to cAMP, or when bound to the PKA catalytic subunit, underscoring their ability to adapt to different binding partners. Moreover, we show while the two CNB domains have near-identical structures, their thermodynamic coupling signatures are divergent, leading to distinct cAMP responses and differential mutational effects. Specifically, we demonstrate mutation W260A exerts local and allosteric effects that impact multiple steps of the PKA activation cycle. Taken together, these results highlight the complex interplay between folding energetics, conformational dynamics, and thermodynamic signatures that underlies structurally conserved signaling modules in response to ligand binding and mutational effects.

Transgenic overexpression of heat shock protein (HSP83) enhances protein kinase A activity, disrupts GP63 surface protease expression and alters promastigote morphology in Leishmania amazonensis

Leishmania parasites undergo morphological changes during their infectious life cycle, including developmental transitions within the sandfly vector, culminating in metacyclic stages that are pre-adapted for infection. Upon entering vertebrate host phagocytes, Leishmania differentiate into intracellular amastigotes, the form that is ultimately transmitted back to the vector to complete the life cycle. Although environmental conditions that induce these cellular transitions are well-established, molecular mechanisms governing Leishmania morphologic differentiation in response to these cues remain largely uncharacterized. Previous studies indicate a key role for HSP83 in both promastigote metacyclogenesis and amastigote differentiation. To further elucidate HSP83 functions in the Leishmania lifecycle, we examined the biological impact of experimentally elevating HSP83 gene expression in Leishmania. Significantly, HSP83 overexpression was associated with altered metacyclic morphology, increased protein kinase A (PKA) activity and decreased expression of the Leishmania major surface protease, GP63. Corroborating these findings, overexpression of the L. amazonensis PKA catalytic subunit resulted in a largely similar phenotype. Our findings demonstrate for the first time in Leishmania, a functional link between HSP83 and PKA in the control of Leishmania gene expression, replication and morphogenesis.

Construction of Candida albicans Strains with ATP-Analog-Sensitive Protein Kinase A and Hog1.

Candida albicans is an opportunistic human fungal pathogen and a member of the mucosal microbiota. To survive in the host and cause disease, C. albicans utilizes several virulence traits, including the ability to respond and adapt to diverse stressors, as well as the morphogenetic switch between yeast and filamentous morphologies. While complex cellular circuitry governs these virulence attributes, the following two kinase-mediated signaling pathways play particularly critical roles in controlling these processes: the Hog1 mitogen-activated protein kinase (MAPK) cascade and the protein kinase A (PKA) pathway. Here, we describe the construction of C. albicans strains harboring substitutions in the ATP-binding pockets of Hog1 and the catalytic subunits of PKA, Tpk1, and Tpk2 to render their activities sensitive to the addition of bulky ATP analogs. Specifically, inhibition by the ATP analog 1NM-PP1 resulted in phenotypes characteristic of the corresponding homozygous deletion mutants for each kinase gene. These strains represent a toolset for the rapid and specific inhibition of PKA and Hog1 kinase activity to further understand their roles in regulating C. albicans morphogenesis and stress responses. IMPORTANCE As an opportunistic pathogen in humans, the fungus Candida albicans relies on virulence traits to cause disease. They include the ability to transition from yeast to filamentous morphologies and the ability to grow in diverse environmental stress conditions, including nutrient limitation, as well as osmotic and heat shock. Previous work identified the following two kinases that play a critical role in regulating these responses: Hog1 and PKA. Here, we generated versions of each kinase that are sensitive to inhibition by a bulky ATP analog, 1NM-PP1. In the presence of the analog, kinase activity is inhibited rapidly and specifically, facilitating the analysis of both kinases in regulating C. albicans morphogenesis and stress responses. Together, these strains represent an important toolset to further our understanding of C. albicans biology and virulence.

Upregulated TGF-β1 contributes to hyperglycaemia in type 2 diabetes by potentiating glucagon signalling.

Glucagon-stimulated hepatic gluconeogenesis contributes to endogenous glucose production during fasting. Recent studies suggest that TGF-β is able to promote hepatic gluconeogenesis in mice. However, the physiological relevance of serum TGF-β levels to human glucose metabolism and the mechanism by which TGF-β enhances gluconeogenesis remain largely unknown. As enhanced gluconeogenesis is a signature feature of type 2 diabetes, elucidating the molecular mechanisms underlying TGF-β-promoted hepatic gluconeogenesis would allow us to better understand the process of normal glucose production and the pathophysiology of this process in type 2 diabetes. This study aimed to investigate the contribution of upregulated TGF-β1 in human type 2 diabetes and the molecular mechanism underlying the action of TGF-β1 in glucose metabolism. Serum levels of TGF-β1 were measured by ELISA in 74 control participants with normal glucose tolerance and 75 participants with type 2 diabetes. Human liver tissue was collected from participants without obesity and with or without type 2 diabetes for the measurement of TGF-β1 and glucagon signalling. To investigate the role of Smad3, a key signalling molecule downstream of the TGF-β1 receptor, in mediating the effect of TGF-β1 on glucagon signalling, we generated Smad3 knockout mice. Glucose levels in Smad3 knockout mice were measured during prolonged fasting and a glucagon tolerance test. Mouse primary hepatocytes were isolated from Smad3 knockout and wild-type (WT) mice to investigate the underlying molecular mechanisms. Smad3 phosphorylation was detected by western blotting, levels of cAMP were detected by ELISA and levels of protein kinase A (PKA)/cAMP response element-binding protein (CREB) phosphorylation were detected by western blotting. The dissociation of PKA subunits was measured by immunoprecipitation. We observed higher levels of serum TGF-β1 in participants without obesity and with type 2 diabetes than in healthy control participants, which was positively correlated with HbA1c and fasting blood glucose levels. In addition, hyperactivation of the CREB and Smad3 signalling pathways was observed in the liver of participants with type 2 diabetes. Treating WT mouse primary hepatocytes with TGF-β1 greatly potentiated glucagon-stimulated PKA/CREB phosphorylation and hepatic gluconeogenesis. Mechanistically, TGF-β1 treatment induced the binding of Smad3 to the regulatory subunit of PKA (PKA-R), which prevented the association of PKA-R with the catalytic subunit of PKA (PKA-C) and led to the potentiation of glucagon-stimulated PKA signalling and gluconeogenesis. The hepatic TGF-β1/Smad3 pathway sensitises the effect of glucagon/PKA signalling on gluconeogenesis and synergistically promotes hepatic glucose production. Reducing serum levels of TGF-β1 and/or preventing hyperactivation of TGF-β1 signalling could be a novel approach for alleviating hyperglycaemia in type 2 diabetes.

Chronic angiotensin II signaling drives sustained PIP2 deficits and altered sarcolemmal Cav1.2 expression and function in cardiomyocytes.

Contractile dysfunction, hypertrophy, and cell death during heart failure are linked to altered Ca2+ handling and elevated levels of angiotensin II (AngII). This hormone signals through Gq-coupled AT1 receptors, initiating hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2). Previous work revealed acute AngII signaling and PIP2 depletion destabilizes CaV1.2 clusters in cardiomyocytes, triggering their internalization, and subsequent reduction of whole-cell Ca2+ current (ICa) and contractility. However, the effects of chronically elevated AngII on the phospholipid landscape and associated effects on ICa remain unknown. We subjected mice to sustained delivery of AngII to test the impact on phosphoinositides, ICa, and excitation-contraction coupling. We hypothesized that chronic AngII signaling leads to sustained PIP2 and CaV1.2 deficits, contributing to the diminished functional output associated with disease development. Phospholipid mass spectrometry revealed extensive alteration of phosphoinositide species, including a ∼44% reduction in PIP2 and ∼37% reduction in cardioprotective PIP3 in AngII-infused hearts compared to controls. To study the effects of this phospholipid imbalance on CaV1.2, we examined ICa, finding similar current density in both groups. However, super-resolution imaging revealed a ∼54% reduction in t-tubular CaV1.2 expression and a ∼23% reduction in cluster area. Conserved ICa density despite reduced t-tubular CaV1.2 suggests that either the channels have relocated to the sarcolemmal crest, or their Po has increased. We examined crest CaV1.2 and observed no change with AngII, thus the Po of the remaining channels must increase to maintain ICa. Western blots revealed enhanced phosphorylation of CaV1.2, phosphorylation of RyR2, and an increased expression of the catalytic subunit of PKA, suggesting a compensatory sympathetic activation underlying conserved ICa. These findings support a model wherein chronic elevation of AngII disrupts phosphoinositide and CaV1.2 levels, elucidating the role of chronic AngII in disease progression.

MiR-200a-3p Regulates PRKACB and Participates in Aluminium-Induced Tau Phosphorylation in PC12 Cells

Aluminium (Al) is an environmental neurotoxin that humans are widely exposed to, but the molecular mechanism of its toxic effects is not fully understood. Many studies have shown that exposure to Al can cause abnormal phosphorylation of the tau protein that is believed as one of pathological features of Alzheimer’s disease. Increasing evidence indicates that microRNAs (miRNAs) may be involved in the pathological processes of neurodegenerative diseases and are potential regulatory factors for related target genes. Phosphorylation at Ser-133 of cAMP response element-binding protein (CREB) is one of the major pathways of CREB activation, and phosphorylation at this site is controlled by protein kinase A (PKA). The catalytic subunit of PKA, cAMP-dependent protein kinase catalytic subunit beta (PRKACB), phosphorylates CREB. The target gene prediction software TargetScan showed that PRKACB was one of the target mRNAs of miR-200a-3p. The purpose of this study was to investigate whether miR-200a-3p regulates the PKA/CREB pathway by targeting PRKACB and leads to abnormal phosphorylation of the tau protein in nerve cells. The results showed that Al exposure increased the expression level of miR-200a-3p, and miR-200a-3p increased the expression of targeted downregulated PRKACB, and then decreased the PKA/CREB signalling pathway activity, leading to abnormal hyperphosphorylation of tau.Graphical

Secreted EMC10 is upregulated in human obesity and its neutralizing antibody prevents diet-induced obesity in mice

Secreted isoform of endoplasmic reticulum membrane complex subunit 10 (scEMC10) is a poorly characterized secreted protein of largely unknown physiological function. Here we demonstrate that scEMC10 is upregulated in people with obesity and is positively associated with insulin resistance. Consistent with a causal role for scEMC10 in obesity, Emc10-/- mice are resistant to diet-induced obesity due to an increase in energy expenditure, while scEMC10 overexpression decreases energy expenditure, thus promoting obesity in mouse. Furthermore, neutralization of circulating scEMC10 using a monoclonal antibody reduces body weight and enhances insulin sensitivity in obese mice. Mechanistically, we provide evidence that scEMC10 can be transported into cells where it binds to the catalytic subunit of PKA and inhibits its stimulatory action on CREB while ablation of EMC10 promotes thermogenesis in adipocytes via activation of the PKA signalling pathway and its downstream targets. Taken together, our data identify scEMC10 as a circulating inhibitor of thermogenesis and a potential therapeutic target for obesity and its cardiometabolic complications.

Autophagy regulates neuronal excitability by controlling cAMP/protein kinase A signaling at the synapse.

Autophagy provides nutrients during starvation and eliminates detrimental cellular components. However, accumulating evidence indicates that autophagy is not merely a housekeeping process. Here, by combining mouse models of neuron-specific ATG5 deficiency in either excitatory or inhibitory neurons with quantitative proteomics, high-content microscopy, and live-imaging approaches, we show that autophagy protein ATG5 functions in neurons to regulate cAMP-dependent protein kinase A (PKA)-mediated phosphorylation of a synapse-confined proteome. This function of ATG5 is independent of bulk turnover of synaptic proteins and requires the targeting of PKA inhibitory R1 subunits to autophagosomes. Neuronal loss of ATG5 causes synaptic accumulation of PKA-R1, which sequesters the PKA catalytic subunit and diminishes cAMP/PKA-dependent phosphorylation of postsynaptic cytoskeletal proteins that mediate AMPAR trafficking. Furthermore, ATG5 deletion in glutamatergic neurons augments AMPAR-dependent excitatory neurotransmission and causes the appearance of spontaneous recurrent seizures in mice. Our findings identify a novel role of autophagy in regulating PKA signaling at glutamatergic synapses and suggest the PKA as a target for restoration of synaptic function in neurodegenerative conditions with autophagy dysfunction.

MHY4571, a novel diarylcyclohexanone derivative, exerts anti-cancer activity by regulating the PKA-cAMP-response element-binding protein pathway in squamous cell lung cancer

BackgroundThe protein kinase A (PKA)/cAMP response element-binding protein (CREB) has been suggested to be related to the inhibition of the proliferation of non-small cell lung cancer (NSCLC) cells. This study aimed to investigate the efficacy of a novel diarylcyclohexanone derivative, MHY4571, in regulating the PKA-CREB pathway and to study its anti-tumor role in squamous NSCLC.MethodsWe designed MHY4571 as a novel PKA inhibitor with acceptable in silico ADME properties and tested it in vitro in lung cancer cell lines and in vivo in xenograft and orthotopic mouse models of squamous cell lung carcinoma.ResultsMHY4571 inhibited PKA activity (> 70% inhibition) and suppressed the expression of p-PKA and p-CREB dose-dependently. MHY4571 treatment reduced lung cancer cell viability and promoted caspase 3-dependent apoptotic cell death. Orally administered MHY4571 significantly suppressed lung tumor growth in xenograft and orthotopic mouse models. PKA catalytic subunit alpha-silencing by siRNA (siPKA) strongly attenuated CREB phosphorylation; siCREB did not alter PKA protein levels or its phosphorylation, suggesting that PKA is an upstream regulator of CREB activity. MHY4571 acted synergistically with cisplatin (on co-treatment) to induce apoptotic cell death in lung cancer cells.ConclusionsOur results imply that MHY4571 may be a potential drug candidate for squamous cell lung cancer treatment.

Phosphorylation of Jhd2 by the Ras-cAMP-PKA(Tpk2) pathway regulates histone modifications and autophagy

Cells need to coordinate gene expression with their metabolic states to maintain cell homeostasis and growth. How cells transduce nutrient availability to appropriate gene expression remains poorly understood. Here we show that glycolysis regulates histone modifications and gene expression by activating protein kinase A (PKA) via the Ras-cyclic AMP pathway. The catalytic subunit of PKA, Tpk2 antagonizes Jhd2-catalyzed H3K4 demethylation by phosphorylating Jhd2 at Ser321 and Ser340 in response to glucose availability. Tpk2-catalyzed Jhd2 phosphorylation impairs its nuclear localization, reduces its binding to chromatin, and promotes its polyubiquitination and degradation by the proteasome. Tpk2-catalyzed Jhd2 phosphorylation also maintains H3K14 acetylation by preventing the binding of histone deacetylase Rpd3 to chromatin. By phosphorylating Jhd2, Tpk2 regulates gene expression, maintains normal chronological life span and promotes autophagy. These results provide a direct connection between metabolism and histone modifications and shed lights on how cells rewire their biological responses to nutrient signals.

The cAMP-PKA pathway regulates prey sensing and trap morphogenesis in the nematode-trapping fungus Arthrobotrys oligospora.

Sensing environmental factors and responding swiftly to them is essential for all living organisms. For instance, predators must act rapidly once prey is sensed. Nematode-trapping fungi (NTF) are predators that use “traps” differentiated from vegetative hyphae to capture, kill, and consume nematodes. These traps undergo drastic and rapid morphological changes upon nematode induction. Multiple signaling hubs have been shown to regulate this remarkable process. Here, we demonstrate that the conserved cAMP-PKA signaling pathway exerts a crucial role in trap morphogenesis of the nematode-trapping fungi Arthrobotrys oligospora. A gene deletion mutant of the PKA catalytic subunit TPK2 proved insensitive toward nematode presence. Moreover, we show that the G protein alpha subunit GPA2 acts upstream of adenylate cyclase, with GPA2 deletion resulting in substantially reduced trap formation, whereas exogenous provision of cAMP rescued the prey-sensing and trap morphogenesis defects of a gpa2 mutant. Thus, we show that cAMP production triggered by G protein signaling and downstream PKA activity are vital for prey-sensing and trap development in A. oligospora, demonstrating that this highly conserved signaling pathway is critical for nematode-trapping fungi and nematode predator–prey interactions.

Protein kinase A participates in hyphal and appressorial development by targeting Efg1-mediated transcription of a Rab GTPase in Setosphaeria turcica.

The cyclic adenosine monophosphate (cAMP) signalling pathway plays an important role in the regulation of the development and pathogenicity of filamentous fungi. cAMP‐dependent protein kinase A (PKA) is the conserved element downstream of cAMP, and its diverse mechanisms in multiple filamentous fungi are not well known yet. In the present study, gene knockout mutants of two catalytic subunits of PKA (PKA‐C) in Setosphaeria turcica were created to illustrate the regulatory mechanisms of PKA‐Cs on the development and pathogenicity of S. turcica. As a result, StPkaC2 was proved to be the main contributor of PKA activity in S. turcica. In addition, it was found that both StPkaC1 and StPkaC2 were necessary for conidiation and invasive growth, while only StPkaC2 played a negative role in the regulation of filamentous growth. We reveal that only StPkaC2 could interact with the transcription factor StEfg1, and it inhibited the transcription of StRAB1, a Rab GTPase homologue coding gene in S. turcica, whereas StPkaC1 could specifically interact with a transcriptional regulator StFlo8, which could rescue the transcriptional inhibition of StEfg1 on StRAB1. We also demonstrated that StRAB1 could positively influence the biosynthesis of chitin in hyphae, thus changing the filamentous growth. Our findings clarify that StPkaC2 participates in chitin biosynthesis to modulate mycelium development by targeting the Efg1‐mediated transcription of StRAB1, while StFlo8, interacting with StPkaC1, acts as a negative regulator during this process.

Yeast Protein Kinase A Isoforms: A Means of Encoding Specificity in the Response to Diverse Stress Conditions?

Eukaryotic cells have developed a complex circuitry of signalling molecules which monitor changes in their intra- and extracellular environments. One of the most widely studied signalling pathways is the highly conserved cyclic AMP (cAMP)/protein kinase A (PKA) pathway, which is a major glucose sensing circuit in the yeast Saccharomyces cerevisiae. PKA activity regulates diverse targets in yeast, positively activating the processes that are associated with rapid cell growth (e.g., fermentative metabolism, ribosome biogenesis and cell division) and negatively regulating the processes that are associated with slow growth, such as respiratory growth, carbohydrate storage and entry into stationary phase. As in higher eukaryotes, yeast has evolved complexity at the level of the PKA catalytic subunit, and Saccharomyces cerevisiae expresses three isoforms, denoted Tpk1-3. Despite evidence for isoform differences in multiple biological processes, the molecular basis of PKA signalling specificity remains poorly defined, and many studies continue to assume redundancy with regards to PKA-mediated regulation. PKA has canonically been shown to play a key role in fine-tuning the cellular response to diverse stressors; however, recent studies have now begun to interrogate the requirement for individual PKA catalytic isoforms in coordinating distinct steps in stress response pathways. In this review, we discuss the known non-redundant functions of the Tpk catalytic subunits and the evolving picture of how these isoforms establish specificity in the response to different stress conditions.

Adipocyte HIF2α functions as a thermostat via PKA Cα regulation in beige adipocytes

Thermogenic adipocytes generate heat to maintain body temperature against hypothermia in response to cold. Although tight regulation of thermogenesis is required to prevent energy sources depletion, the molecular details that tune thermogenesis are not thoroughly understood. Here, we demonstrate that adipocyte hypoxia-inducible factor α (HIFα) plays a key role in calibrating thermogenic function upon cold and re-warming. In beige adipocytes, HIFα attenuates protein kinase A (PKA) activity, leading to suppression of thermogenic activity. Mechanistically, HIF2α suppresses PKA activity by inducing miR-3085-3p expression to downregulate PKA catalytic subunit α (PKA Cα). Ablation of adipocyte HIF2α stimulates retention of beige adipocytes, accompanied by increased PKA Cα during re-warming after cold stimuli. Moreover, administration of miR-3085-3p promotes beige-to-white transition via downregulation of PKA Cα and mitochondrial abundance in adipocyte HIF2α deficient mice. Collectively, these findings suggest that HIF2α-dependent PKA regulation plays an important role as a thermostat through dynamic remodeling of beige adipocytes.

Differential Roles of the α and β Catalytic Subunits of PKA in the Regulation of Formoterol‐modulated Gene Expression Changes in Human Airway Epithelial Cells

IntroductionProtein kinase A (PKA) is the most studied cAMP effector and phosphorylates different downstream proteins to control various cellular functions including gene expression. PKA is a heterotetramer composed of two regulatory (R) subunits and two catalytic (C) subunits of which there are three main variants: Cα, Cβ and Cγ. Whereas PKA‐Cα and Cβ are expressed ubiquitously, Cγ appears to be restricted to the testis. The specificity of PKA to phosphorylate different substrates has been attributed to the C subunits; however, this suggestion remains controversial given that several common features are shared between Cα and Cβ including the approximately 90.5% sequence homology in the catalytic core region.HypothesisPKA Cα and Cβ subunits differentially regulate gene changes induced by the β2‐ adrenoceptor agonist, formoterol, in the BEAS‐2B human airway epithelial cell line.Summary of ResultsRNA‐sequencing determined that PRKACA transcripts encoding Cα were abundantly expressed in BEAS‐2B cells at a level that was 9.3‐fold higher than PRKACB, which encodes Cβ. Cα and Cβ were also identified at the protein level by western blotting. siRNA mediated knockdown of C subunits was employed to assess their role in regulating cAMP response element (CRE)‐dependent transcription following β2‐adrenoceptor stimulation. In BEAS‐2B cells depleted of Cα or Cβ, formoterol‐induced CRE binding protein (CREB) phosphorylation was inhibited by 66.1% and 31.4%, respectively and by 89.6% when both catalytic subunits were knocked‐down simultaneously. Similarly, the increase in formoterol‐induced CRE reporter luciferase activity was reduced by 38.5% and 21.5% in Cα‐ and Cβ‐depleted cells, respectively and by 66.5% following knockdown of both PKA catalytic subunits. These data were confirmed by using CRISPR/Cas9‐ mediated deletion of Cα in BEAS‐2B cells wherein formoterol induced CREB phosphorylation was reduced by 81%. However, transfection of Cα‐deficient cells with Cβ siRNAs abolished formoterolinduced CREB phosphorylation indicating that both C subunits regulated CRE‐dependent transcription. Formoterol also promoted the PKA‐dependent phosphorylation of c‐Raf in BEAS‐2B cells at Ser43 and Ser259, which inhibited the transcription of the Elk‐1‐regulated gene, EGR1. However, unlike CREB phosphorylation, formoterol‐induced c‐Raf phosphorylation was unaffected by Cα depletion but, nevertheless, abolished in cell concurrently depleted of Cβ. Such a discrepancy was investigated further by examining the regulation of 14 formoterol‐sensitive genes using qPCR. Of those, the expression of five was attenuated by >50% in Cα knockout cells whereas the remainder was considerably more resistant with NR4A3 (70% inhibition, CREB‐dependent) and EGR1 (13% inhibition, ElK‐1‐dependent) lying at the extremes of this continuum. The expression of all 14 genes was abrogated in Cα‐deficient BEAS‐2B cells depleted of Cβ.ConclusionsThese data suggest that PKA Cα and Cβ subunits serve distinct, non‐redundant roles in BEAS‐2B cells that cooperatively regulate β2‐adrenoceptor‐mediated gene expression changes in a transcript‐dependent manner that may depend on the substrate (e.g., c‐Raf vs. CREB) targeted by PKA