Protein Kinase A Phosphorylation Research Articles

The Intellectual Disability and Schizophrenia Associated Transcription Factor TCF4 Is Regulated by Neuronal Activity and Protein Kinase A.

Transcription factor 4 (TCF4 also known as ITF2 or E2-2) is a basic helix-loop-helix (bHLH) protein associated with Pitt-Hopkins syndrome, intellectual disability, and schizophrenia (SCZ). Here, we show that TCF4-dependent transcription in cortical neurons cultured from embryonic rats of both sexes is induced by neuronal activity via soluble adenylyl cyclase and protein kinase A (PKA) signaling. PKA phosphorylates TCF4 directly and a PKA phosphorylation site in TCF4 is necessary for its transcriptional activity in cultured neurons and in the developing brain in vivo We also demonstrate that Gadd45g (growth arrest and DNA damage inducible gamma) is a direct target of neuronal-activity-induced, TCF4-dependent transcriptional regulation and that TCF4 missense variations identified in SCZ patients alter the transcriptional activity of TCF4 in neurons. This study identifies a new role for TCF4 as a neuronal-activity-regulated transcription factor, offering a novel perspective on the association of TCF4 with cognitive disorders.SIGNIFICANCE STATEMENT The importance of the basic helix-loop-helix transcription factor transcription factor 4 (TCF4) in the nervous system is underlined by its association with common and rare cognitive disorders. In the current study, we show that TCF4-controlled transcription in primary cortical neurons is induced by neuronal activity and protein kinase A. Our results support the hypotheses that dysregulation of neuronal-activity-dependent signaling plays a significant part in the etiology of neuropsychiatric and neurodevelopmental disorders.

Effects of Zinc Alpha2 Glycoprotein on Lipid Metabolism of Liver in High-Fat Diet-Induced Obese Mice.

Zinc alpha2 glycoprotein (ZAG) is a new type of adipokine involved in adipose tissue mobilization, however, little is known about its lipid metabolism effect in liver. Therefore, we investigated the effects of ZAG in the regulation of hepatic lipid accumulation. Mice were randomly divided into two groups; one was fed a normal diet and another was fed a high-fat diet for eight weeks to establish obesity model. After that, the normal diet group was divided into ND (injection of pcDNA3.1) and NDZ (injection of ZAG recombinant plasmid) and the high-fat diet group was divided into HF (injection of pcDNA3.1) and HFZ (injection of ZAG recombinant plasmid). The mice were weighed once per week and injected with plasmid once every three days for eight times. The results showed that body weight and hepatic TG content were decreased dramatically in HFZ group compared with HF group. The stearoyl-CoAdesaturase1 (SCD1) and Acyl-CoA Synthetase-1 (ACSS1) protein levels in HFZ group were significantly decreased. Furthermore, phosphorylated hormone sensitive lipase (P-HSL) was significantly higher in HFZ group. In HFZ group, hepatic fatty acid translocase (CD36) and fatty acids binding protein-1 (FABP1) protein levels were reduced. In addition, the expression of phosphorylated protein kinase A (PPKA) in HFZ group was higher than the HF group. Meanwhile, NDZ group showed significantly decreased body weight and increased P-HSL level though the hepatic TG content showed no significantly changes compared with the ND group. Therefore, we conclude that ZAG may be beneficial for preventing high-fat-diet-induced hepatic lipid metabolic disorders.

Effects of phosphorylation on μ-calpain activity at different incubation temperature

The study investigated the effects of alkaline phosphatase (AP) dephosphorylation and protein kinase A (PKA) phosphorylation on μ-calpain activity and its sensitivity to temperature. The purified μ-calpain was treated with AP or PKA for 30min at 30°C to modulate its phosphorylation level. Samples were then incubated at controlled freezing point (−1), 4, 25 and 37°C, respectively. The results showed that PKA and AP had no influence on pH values of incubation solution. At −1 and 4°C, the degradation rate of μ-calpain was maximum in AP group and minimum in control group. Low temperature of controlled freezing point prevented dephosphorylation and phosphorylation progression and delayed μ-calpain degradation. Increased incubation temperature of 4, 25 and 37°C increased μ-calpain degradation. Two about 50kDa degradation products from μ-calpain were identified, of which the intensity was also lower in control group than in the other two groups. These observations demonstrated that AP dephosphorylation and PKA phosphorylation of μ-calpain promoted μ-calpain autolysis and activation.

Proteolytic cleavage and PKA phosphorylation of α 1C subunit are not required for adrenergic regulation of Ca V 1.2 in the heart

Calcium influx through the voltage-dependent L-type calcium channel (CaV1.2) rapidly increases in the heart during "fight or flight" through activation of the β-adrenergic and protein kinase A (PKA) signaling pathway. The precise molecular mechanisms of β-adrenergic activation of cardiac CaV1.2, however, are incompletely known, but are presumed to require phosphorylation of residues in α1C and C-terminal proteolytic cleavage of the α1C subunit. We generated transgenic mice expressing an α1C with alanine substitutions of all conserved serine or threonine, which is predicted to be a potential PKA phosphorylation site by at least one prediction tool, while sparing the residues previously shown to be phosphorylated but shown individually not to be required for β-adrenergic regulation of CaV1.2 current (17-mutant). A second line included these 17 putative sites plus the five previously identified phosphoregulatory sites (22-mutant), thus allowing us to query whether regulation requires their contribution in combination. We determined that acute β-adrenergic regulation does not require any combination of potential PKA phosphorylation sites conserved in human, guinea pig, rabbit, rat, and mouse α1C subunits. We separately generated transgenic mice with inducible expression of proteolytic-resistant α1C Prevention of C-terminal cleavage did not alter β-adrenergic stimulation of CaV1.2 in the heart. These studies definitively rule out a role for all conserved consensus PKA phosphorylation sites in α1C in β-adrenergic stimulation of CaV1.2, and show that phosphoregulatory sites on α1C are not redundant and do not each fractionally contribute to the net stimulatory effect of β-adrenergic stimulation. Further, proteolytic cleavage of α1C is not required for β-adrenergic stimulation of CaV1.2.

CAMP-dependent regulation of IKs single-channel kinetics.

The delayed potassium rectifier current, IKs , is composed of KCNQ1 and KCNE1 subunits and plays an important role in cardiac action potential repolarization. During β-adrenergic stimulation, 3'-5'-cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA) phosphorylates KCNQ1, producing an increase in IKs current and a shortening of the action potential. Here, using cell-attached macropatches and single-channel recordings, we investigate the microscopic mechanisms underlying the cAMP-dependent increase in IKs current. A membrane-permeable cAMP analog, 8-(4-chlorophenylthio)-cAMP (8-CPT-cAMP), causes a marked leftward shift of the conductance-voltage relation in macropatches, with or without an increase in current size. Single channels exhibit fewer silent sweeps, reduced first latency to opening (control, 1.61 ± 0.13 s; cAMP, 1.06 ± 0.11 s), and increased higher-subconductance-level occupancy in the presence of cAMP. The E160R/R237E and S209F KCNQ1 mutants, which show fixed and enhanced voltage sensor activation, respectively, largely abolish the effect of cAMP. The phosphomimetic KCNQ1 mutations, S27D and S27D/S92D, are much less and not at all responsive, respectively, to the effects of PKA phosphorylation (first latency of S27D + KCNE1 channels: control, 1.81 ± 0.1 s; 8-CPT-cAMP, 1.44 ± 0.1 s, P < 0.05; latency of S27D/S92D + KCNE1: control, 1.62 ± 0.1 s; cAMP, 1.43 ± 0.1 s, nonsignificant). Using total internal reflection fluorescence microscopy, we find no overall increase in surface expression of the channel during exposure to 8-CPT-cAMP. Our data suggest that the cAMP-dependent increase in IKs current is caused by an increase in the likelihood of channel opening, combined with faster openings and greater occupancy of higher subconductance levels, and is mediated by enhanced voltage sensor activation.

Protein kinase A-mediated phosphorylation of the Broad-Complex transcription factor in silkworm suppresses its transcriptional activity

The insect-specific transcription factor Broad-Complex (BR-C) is transcriptionally activated by the steroid 20-hydroxyecdysone (20E) and regulates the expression of many target genes involved in insect growth and development. However, although the transcriptional regulation of BR-C proteins has been well studied, how BR-C is regulated at post-transcription and -translation levels is poorly understood. To this end, using liquid chromatography-tandem mass spectrometry analysis, we identified residue Ser-186 as a phosphorylation site of BR-C in silkworm. Site-directed mutagenesis and treatment with specific kinase activators and inhibitors indicated that the Ser-186 residue in silkworm BR-C is phosphorylated by protein kinase A (PKA). Immunostaining assays disclosed that PKA-mediated phosphorylation of silkworm BR-C has no effect on its nuclear import. However, luciferase reporter analysis, electrophoretic mobility shift assays, and chromatin immunoprecipitation revealed that the PKA phosphorylation event suppresses the transcriptional activation of silkworm BR-C target genes and that this inhibition was caused by repression of BR-C binding to its DNA targets. Of note, both in vitro and ex vivo experiments disclosed that a continuous 20E signal inhibits the PKA-mediated BR-C phosphorylation and also the cAMP/PKA pathway, indicating that 20E's inhibitory effect on PKA-mediated phosphorylation of silkworm BR-C contributes to maintaining BR-C transcriptional activity. In conclusion, our findings indicate that PKA-mediated phosphorylation inhibits silkworm BR-C activity by interfering with its binding to DNA and that 20E signaling relieves PKA-mediated phosphorylation of BR-C, thereby maintaining its transcriptional activity.

Protein kinase A activity leads to the extension of the acrosomal process in starfish sperm

Acrosomal vesicles (AVs) of sperm undergo exocytosis during the acrosome reaction, which is immediately followed by the actin polymerization-dependent extension of an acrosomal process (AP) in echinoderm sperm. In the starfish Asterias amurensis, a large proteoglycan, acrosome reaction-inducing substance (ARIS), together with asteroidal sperm-activating peptide (asterosap) and/or cofactor for ARIS, induces the acrosome reaction. Asterosap induces a transient elevation of intracellular cGMP and Ca2+ levels, and, together with ARIS, causes a sustained increase in intracellular cAMP and Ca2+ . Yet, the contribution of signaling molecules downstream of cAMP and Ca2+ in inducing AV exocytosis and AP extension remain unknown. A modified acrosome reaction assay was used here to differentiate between AV exocytosis and AP extension in starfish sperm, leading to the discovery that Protein kinase A (PKA) inhibitors block AP extension but not AV exocytosis. Additionally, PKA-mediated phosphorylation of target proteins occurs, and these substrates localize at the base of the AP, demonstrating that PKA activation regulates an AP extension step during the acrosome reaction. The major PKA substrate was further identified, from A. amurensis and Asterias forbesi sperm, as a novel protein containing six PKA phosphorylation motifs. This protein, referred to as PKAS1, likely plays a key role in AP actin polymerization during the acrosome reaction.

Insulin deficiency results in reversible protein kinase A activation and tau phosphorylation

Alzheimer's disease (AD) is a highly prevalent multifactorial disease for which Diabetes Mellitus (DM) is a risk factor. Abnormal phosphorylation and aggregation of tau is a key hallmark of AD. In animal models, DM induces or exacerbates the phosphorylation of tau, suggesting that DM may influence the risk at AD by directly facilitating tau pathology. Previously we reported that tau phosphorylation induced in response to metabolic stress is reversible. Since identification and understanding of early players in tau pathology is pivotal for therapeutic intervention, we here investigated the mechanism underlying tau phosphorylation in the diabetic brain and its potential for reversibility. To model DM we used streptozotocin-treatment to induce insulin deficiency in rats. Insulin depletion leads to increased tau phosphorylation in the brain and we investigated the activation status of known tau kinases and phosphatases in this model. We identified protein kinase A (PKA) as a tau kinase activated by DM in the brain. The potential relevance of this signaling pathway to AD pathogenesis is indicated by the increased level of active PKA in temporal cortex of early stage AD patients. Our data indicate that activation of PKA and tau phosphorylation are associated with insulin deficiency per se, rather than the downstream energy deprivation. In vitro studies confirm that insulin deficiency results in PKA activation and tau phosphorylation. Strikingly, both active PKA and induced tau phosphorylation are reversed upon insulin treatment in the steptozotocin animal model. Our data identify insulin deficiency as a direct trigger that induces the activity of the tau kinase PKA and results in tau phosphorylation. The reversibility upon insulin treatment underscores the potential of insulin as an early disease-modifying intervention in AD and other tauopathies.

Therapeutic Targeting of Skeletal Muscle Nix in Early‐Onset Insulin Resistance

Fetal exposure to diabetes during pregnancy increases the risk for early‐onset insulin resistance in the offspring; however, the key molecular regulators responsible for fetal metabolic programming have not been characterized in muscle tissue. Previously, we demonstrated that the expression of a mitochondrial death gene Nix, was elevated in the skeletal muscle of rats exposed to gestational diabetes. Through detailed phospho‐peptide mapping of Nix, we identified a novel protein kinase‐A (PKA) phosphorylation residue within the transmembrane domain, suggesting that Nix function could be regulated post‐translationally. Therefore, we hypothesized that pharmacological activation of PKA by the adrenergic agonist clenbuterol could prevent Nix‐induced mitochondrial dysfunction in muscle cells. To investigate the cellular role of this phospho‐acceptor site, we engineered both neutral (S212A) and phospho‐mimetic (S212D) Nix mutants, and generated a custom phospho‐specific antibody targeted to serine‐212 (S212) of Nix. We confirmed that PKA activating agents led to Nix phosphorylation in intact cells, but not in the presence of S212A. In cultured C2C12 cells, either clenbuterol or PKA restored mitochondrial membrane potential following palmitate exposure and Nix expression. However, clenbuterol did not restore the mitochondria membrane potential in presence of the S212A mutant. Consistent with this finding, C2C12 cells transfected with Nix wild‐type and the S212A mutation decreased mitochondrial membrane potential, but the S212D mutant had no effect. Using organelle‐targeted calcium biosensors, we demonstrated that Nix wild‐type and the S212A mutation led to sarcoplasmic reticulum (SR) calcium release and mitochondrial calcium accumulation, which contribute to mitochondrial depolarization, whereas the phospho‐mimetic mutant of Nix had no effect on these biosensors. While investigating the effects of Nix phosphorylation on cellular function, we observed that Nix and S212 mutants equally contributed to induce autophagosome formation, determined by LC3‐GFP. Suggesting that Nix phosphorylation at S212 does not impact macro‐autophagy activation, which has been shown to be an important mechanism to maintain insulin sensitivity in muscle. Additionally, we showed that phosphorylated Nix is exclusively localized in the cytosol, and not in mitochondria. Furthermore, we demonstrate that Nix translocation to cytosol is dependent on a physical interaction with the molecular chaperone 14‐3‐3b, which is enhanced by PKA phosphorylation of Nix. Finally, we observed that Nix wild‐type and S212A mutation decreased insulin stimulated glucose uptake; however, the S212D mutant had no effect on insulin sensitivity. Our data supports the hypothesis that Nix regulates mitochondrial function and insulin sensitivity in differentiated myotubes and implicates PKA activating agents as possible therapeutic approach to restore mitochondrial dysfunction and insulin sensitivity in skeletal muscle of offspring exposed to gestational diabetes.

Synergy of cAMP and calcium signaling pathways in CFTR regulation

Cystic fibrosis results from mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel, leading to defective apical chloride transport. Patients also experience overactivation of inflammatory processes, including increased calcium signaling. Many investigations have described indirect effects of calcium signaling on CFTR or other calcium-activated chloride channels; here, we investigate the direct response of CFTR to calmodulin-mediated calcium signaling. We characterize an interaction between the regulatory region of CFTR and calmodulin, the major calcium signaling molecule, and report protein kinase A (PKA)-independent CFTR activation by calmodulin. We describe the competition between calmodulin binding and PKA phosphorylation and the differential effects of this competition for wild-type CFTR and the major F508del mutant, hinting at potential therapeutic strategies. Evidence of CFTR binding to isolated calmodulin domains/lobes suggests a mechanism for the role of CFTR as a molecular hub. Together, these data provide insights into how loss of active CFTR at the membrane can have additional consequences besides impaired chloride transport.

Enhanced Photoelectrochemical Method for Sensitive Detection of Protein Kinase A Activity Using TiO2/g-C3N4, PAMAM Dendrimer, and Alkaline Phosphatase.

A novel photoelectrochemical (PEC) assay is developed for sensitive detection of protein kinase A (PKA) activity based on PKA-catalyzed phosphorylation reaction in solution and signal amplification strategy triggered by PAMAM dendrimer and alkaline phosphatase (ALP). In this strategy, it is noteworthy at this point that PKA phosphorylation was achieved in solution instead of on the surface of the electrode, which has advantages of the good contact in reactants and simple experimental procedure. For immobilizing the phosphorylated peptide (P-peptide) on electrode surface, graphite-like carbon nitride (g-C3N4) and titanium dioxide (TiO2) complex is synthesized and characterized, which plays a significant role for TiO2 conjugating phosphate groups and g-C3N4 providing PEC signal. Subsequently, PAMAM dendrimer and ALP can be captured on P-peptide and TiO2/g-C3N4 modified ITO electrode via interaction between the -COOH groups on the surface of PAMAM dendrimer and the -NH2 groups of peptide and ALP, which can lead to the increase of ALP amount on the modified electrode surface assisted with the PAMAM dendrimer. As a result, the amount of ALP catalyzes of L-ascorbic acid 2-phosphate trisodium salt (AAP) to produce electron donor of ascorbic acid (AA), resulting in an increased photocurrent. The proposed detection assay displays high selectivity and low detection limit of 0.048 U/mL (S/N = 3) for PKA activity. This biosensor can also be applied for the evaluation of PKA inhibition and PKA activity assay in cell samples. Therefore, the fabricated PEC biosensor is potentionally well in PKA activity detection and inhibitor screening.

Synergistic effects of metformin with liraglutide against endothelial dysfunction through GLP-1 receptor and PKA signalling pathway

Metformin or glucagon-like peptide-1 (GLP-1) analogue liraglutide has cardiovascular benefits. However, it is not clear whether their combined treatment have additive or synergistic effects on the vasculature. In this study, human umbilical vein endothelial cells (HUVECs), exposed to palmitic acid (PA) to induce endothelial dysfunction, were incubated with metformin, liraglutide or their combination. High fat diet (HFD)-fed ApoE−/− mice were randomized into control, metformin, liraglutide, and combination treatment groups. Results showed that in PA-treated HUVECs and HFD-fed ApoE−/− mice, combination of metformin and liraglutide at lower dose significantly improved endothelial dysfunction compared with the single treatment. Metformin upregulated GLP-1 receptor (GLP-1R) level and protein kinase A (PKA) phosphorylation. However, PKA inhibition but not GLP-1R blockade eliminated the protective effects of metformin on endothelial function. Furthermore, AMPK inhibitor compound C abolished the metformin-mediated upregulation of GLP-1R level and PKA phosphorylation. In conclusion, combination of metformin and liraglutide has synergistic protective effects on endothelial function. Moreover, metformin stimulates GLP-1R and PKA signalling via AMPK-dependent pathway, which may account for its synergistic protective effects with liraglutide. Our findings provide new insights on the interaction between metformin and GLP-1, and provide important information for designing new GLP-1-based therapy strategies in treating type 2 diabetes.

Calmodulin Regulation of Ryanodine Receptors (RyR2) Depends on RyR2 Phosphorylation at Residues S2808 and S2814

Introduction: Calmodulin (CaM) is a calcium-binding protein that regulates target proteins directly or via their regulatory kinases. CaM binds RyR2 and forms part of the RyR2 macromolecular complex that includes Protein Kinase A (PKA), CaM-dependent kinase II (CaMKII) and the phosphatases, PP1 and PP2A. In the complex, CaM influences SR Ca2+ release either by directly binding RyR2 (inhibition) or by phosphorylation of the RyR2 at S2814 through CaMKII (activation). CaM binding to RyR2 is critical for normal cardiac function and loss of CaM inhibiting efficacy leads to pathological activation of RyR2 that produce spontaneous Ca2+ releases from the SR. These in themselves can trigger miss-timed APs, cardiac arrhythmias and sudden death. Several recent studies have reported that CaM binding affinity to RyR2 is reduced by oxidation of both CaM and RyR2. Here we test the hypothesis that the action of CaM on the activity of RyR2 also depends on the phosphorylation state of the channel. Methods and Results: CaM effect on Ca2+ sparks (and hence RyR2 activity) was tested on permeabilized cardiomyocytes from transgenic mice where CaMKII or PKA phosphorylation sites in RyR2 were ablated (S2814A and S2808A, respectively) or where CaMKII phosphorylation site in RyR2 was fully active (S2814D). RyR2 phosphorylation was confirmed by Western Blot. CaM only inhibited Ca2+ sparks frequency in cardiomyocytes with high phosphorylation levels of RyR2 in CaMKII site (S2814D), in PKA site (S2814A+cAMP or S2808A+cAMP) or in both residues (RyR2 S2814D+cAMP). In contrast, CaM did not have an inhibitory effect in cardiomyocytes with low phosphorylation levels of RyR2 (S2814A and S2808A). Conclusion: Our results show that CaM inhibitory efficacy is dependent on RyR2 phosphorylation in CaMKII and PKA sites S2814 and S2808 in mouse ventricular myocytes.

Clarifying a Novel PKA Phosphorylation Site on the L-type Calcium Channel

Persistent sympathetic activation leads to the development of hypertrophy and heart failure. Activation of the L-type calcium channel is essential for the increase in contractility and heart rate however the mechanism for the increase in channel function has remained controversial for over 40 years. The downstream effector of β-adrenergic signalling pathway is the cyclic AMP-dependent protein kinase A (PKA). We recently identified a novel candidate PKA phosphorylation site on Cav1.2 protein (the main pore forming and ion conducting alpha subunit). We reconstituted the purified channel isoforms in liposomes. Using single channel patch-clamp analysis, mutation of serine 1458 to alanine attenuated the effect of PKA on channel function (Po = 0.052 ± 0.01 vs 0.043 ± 0.007, n = 5; p = NS). In vitro thermal stability assay performed on the S1458A mutant demonstrated similar melting peak profiles. Following 1 hr in vitro PKA treatment the melting temperature of the WT isoform was altered, but not the mutant isoform, demonstrating that protein-protein interactions between PKA and the Cav1.2 had occurred. Mass spectrometry analysis was performed to demonstrate that serine 1458 could be phosphorylated by PKA. We performed in vitro PKA phosphorylation of the non-phosphorylated synthetic peptide YLTRDWSILGPHHL, corresponding to aa 1452-1465 of the short NT isoform then performed MS/MS analysis. Peptide ions for YLTRDWSILGPHHL were observed with a mass of 1706.86 in control and 1786.84 in PKA treated samples, and we could track the mass difference to the serine residue in the MS/MS spectra. In conclusion, we identified a novel, PKA specific phosphorylation site on the proximal C terminus of Cav1.2 protein.

The impact of tau hyperphosphorylation at Ser262 on memory and learning after global brain ischaemia in a rat model of reversible cardiac arrest

An increase in phosphorylated tau (p-tau) is associated with Alzheimer's disease (AD), and brain hypoxia. Investigation of the association of residue-specific tau hyperphosphorylation and changes in cognition, leads to greater understanding of its potential role in the pathology of memory impairment. The aims of this study are to investigate the involvement of the main metabolic kinases, Liver Kinase B1 (LKB1) and Adenosine Monophosphate Kinase Protein Kinase (AMPK), in tau phosphorylation-derived memory impairment, and to study the potential contribution of the other tau kinases and phosphatases including Glycogen Synthase Kinase (GSK-3β), Protein kinase A (PKA) and Protein Phosphatase 2A (PP2A). Spatial memory and learning were tested in a rat global brain ischemic model of reversible cardiac arrest (CA). The phosphorylation levels of LKB1, AMPK, GSK-3β, PP2A, PKA and tau-specific phosphorylation were assessed in rats, subjected to ischaemia/reperfusion and in clinically diagnosed AD and normal human brains. LKB1 and AMPK phosphorylation increased 4 weeks after CA as did AMPK related p-tau (Ser262). The animals showed unchanged levels of GSK-3β specific p-tau (Ser202/Thr205), phospho-PP2A (Tyr307), total GSK-3β, PP2A, phospho-cAMP response element-binding protein (CREB) which is an indicator of PKA activity, and no memory deficits. AD brains had hyperphosphorylated tau in all the residues of Ser262, Ser202 and Thr205, with increased phosphorylation of both AMPK (Thr172) and GSK-3β (Ser9), and reduced PP2A levels. Our data suggests a crucial role for a combined activation of tau kinases and phosphatases in adversely affecting memory and that hyperphosphorylation of tau in more than one specific site may be required to create memory deficits.

A Role for cAMP and Protein Kinase A in Experimental Necrotizing Enterocolitis

Necrotizing enterocolitis (NEC) is a devastating intestinal disease that has been associated with Cronobacter sakazakii and typically affects premature infants. Although NEC has been actively investigated, little is known about the mechanisms underlying the pathophysiology of epithelial injury and intestinal barrier damage. Cyclic adenosine monophosphate (cAMP) and protein kinase A (PKA) are important mediators and regulators of apoptosis. To test the hypothesis that C. sakazakii increases cAMP and PKA activation in experimental NEC resulting in increased epithelial apoptosis, we investigated the effects of C. sakazakii on cAMP and PKA invitro and invivo. Specifically, rat intestinal epithelial cells and a human intestinal epithelial cell line were infected with C. sakazakii, and cAMP levels and phosphorylation of PKA were measured. An increase in cAMP was demonstrated after infection, as well as an increase in phosphorylated PKA. Similarly, increased intestinal cAMP and PKA phosphorylation were demonstrated in a rat pup model of NEC. These increases were correlated with increased intestinal epithelial apoptosis. The additional of a PKA inhibitor (KT5720) significantly ameliorated these effects and decreased the severity of experimental NEC. Findings were compared with results from human tissue samples. Collectively, these observations indicate that cAMP and PKA phosphorylation are associated with increased apoptosis in NEC and that inhibition of PKA activation protects against apoptosis and experimental NEC.

PKA-Mediated Phosphorylation of APOBEC3B Suppresses Its DNA Mutagenic Potential in Myeloma Cells

Multiple myeloma (MM) is an incurable plasma cell malignancy and the reason for this is not yet completely understood. It might be partially attributable to heterogeneity and clonal evolution through DNA mutation accumulation. Recent next generation sequencing (NGS) studies have revealed that apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like (APOBEC) 3B (A3B) is one of the cause of clonal evolution in MM. APOBEC protein family consists of 11 proteins and most of them, including A3B, have cytidine deaminase (CDA) activity on single-strand DNA, which catalyzes deamination of cytosine to uridine, resulting in C-to-T or C-to-G mutations. We have previously shown in human cell line models that A3B induces genomic mutations (Shinohara et al., Sci Rep, 2012). Therefore, controlling A3B CDA activity is an attractive therapeutic strategy to overcome the unfavorable prognosis of patients with these malignancies.Enzymatic activity of other APBEC family proteins, activation-induced cytidine deaminase (AID) and APOBEC3G (A3G) is regulated by protein kinase A (PKA)-mediated phosphorylation. A3B also has two consensus PKA phosphorylation motifs conserved upstream of each CDA domain. Thus, we hypothesized that PKA phosphorylates A3B which may alter A3B CDA activity.Using NetPhosK1.0 and ScanSite programs, two A3B residues (serine at the 46th and threonine at the 214th position) are predicted to be PKA phosphorylation sites. Firstly, we examined the binding between A3B and PKA. We transfected HEK293T cells with expression vectors for C-terminal HA-tagged A3B and N-terminal FLAG-tagged PKA catalytic subunit a (PKACA) and used CoIP assays with anti-HA or anti-FLAG antibodies to demonstrate physical binding between A3B and PKACA. Secondly, to determine whether the putative residues are phosphorylated, we overexpressed PKACA and A3B alanine mutants in HEK293T cells and performed immunoblotting analysis with anti-phospho PKA motif antibodies and found that threonine-214 is phosphorylated by PKA. To confirm that PKA phosphorylates A3B directly, we employed in vitro phosphorylation assays using purified C-terminal A3B and PKACA and found that PKACA phosphorylated A3B, but not the A3B T214A mutant, thus we concluded that PKACA directly phosphorylates threonine-214 in A3B.Next, to investigate the effect of this phosphorylation on the CDA activity of A3B, we constructed phosphomimetic mutants of these residues, T214D and T214E. We carried out in vitro CDA assays using cell lysates from HEK293T cells overexpressing A3B or mutants (T214A, T214D, T214E), and found that T214D and T214E almost completely lost their CDA activity. We also performed in vitro CDA assays using purified C-terminal A3B and mutant proteins. The purified A3B T214D mutant also lost CDA activity, suggesting that PKA-mediated phosphorylation of A3B might also lead to loss of its CDA activity.To confirm this result in cell lines, we used differential DNA denaturation PCR (3D-PCR) of DNA from HEK293T cells transfected with vectors for uracil DNA glycosylase inhibitor, EGFP and A3B wild type or mutants. 3D-PCR products of the EGFP sequence were amplified at a lower denaturation temperature in cells expressing the wild type A3B compared to those in cells expressing phosphomimetic A3B or catalytically inactivated A3B. These results indicate that phosphomimetic A3B no longer has exogenous DNA editing activity. We further plan to evaluate the PKA phosphorylation of A3B and the consequences on its mutagenic potential in myeloma cell lines.In conclusion, PKA induces phosphorylation at threonine-214 in A3B, and this phosphorylation suppresses its CDA activity. PKA-mediated phosphorylation of A3B may be an attractive therapeutic target for inhibiting A3B-induced clonal evolution in lymphoid malignancies, especially in MM. DisclosuresTakaori-Kondo:Kyowa Kirin: Research Funding; Alexion Pharmaceuticals: Research Funding; Eisai: Research Funding; Shionogi: Research Funding; Chugai Pharmaceutical: Research Funding; Mochida Pharmaceutical: Research Funding; Takeda Pharmaceutical: Research Funding; Astellas Pharma: Research Funding; Pfizer: Research Funding; Janssen Pharmaceuticals: Speakers Bureau; Merck Sharp and Dohme: Speakers Bureau; Bristol-Myers Squibb: Speakers Bureau; Toyama Chemical: Research Funding; Cognano: Research Funding.

A cannabinoid link between mitochondria and memory.

Cellular activity in the brain depends on the high energetic support provided by mitochondria, the cell organelles which use energy sources to generate ATP. Acute cannabinoid intoxication induces amnesia in humans and animals, and the activation of type-1 cannabinoid receptors present at brain mitochondria membranes (mtCB1) can directly alter mitochondrial energetic activity. Although the pathological impact of chronic mitochondrial dysfunctions in the brain is well established, the involvement of acute modulation of mitochondrial activity in high brain functions, including learning and memory, is unknown. Here, we show that acute cannabinoid-induced memory impairment in mice requires activation of hippocampal mtCB1 receptors. Genetic exclusion of CB1 receptors from hippocampal mitochondria prevents cannabinoid-induced reduction of mitochondrial mobility, synaptic transmission and memory formation. mtCB1 receptors signal through intra-mitochondrial Gαi protein activation and consequent inhibition of soluble-adenylyl cyclase (sAC). The resulting inhibition of protein kinase A (PKA)-dependent phosphorylation of specific subunits of the mitochondrial electron transport system eventually leads to decreased cellular respiration. Hippocampal inhibition of sAC activity or manipulation of intra-mitochondrial PKA signalling or phosphorylation of the Complex I subunit NDUFS2 inhibit bioenergetic and amnesic effects of cannabinoids. Thus, the G protein-coupled mtCB1 receptors regulate memory processes via modulation of mitochondrial energy metabolism. By directly linking mitochondrial activity to memory formation, these data reveal that bioenergetic processes are primary acute regulators of cognitive functions.

Current insights into the role of PKA phosphorylation in CFTR channel activity and the pharmacological rescue of cystic fibrosis disease-causing mutants.

Cystic fibrosis transmembrane conductance regulator (CFTR) channel gating is predominantly regulated by protein kinase A (PKA)-dependent phosphorylation. In addition to regulating CFTR channel activity, PKA phosphorylation is also involved in enhancing CFTR trafficking and mediating conformational changes at the interdomain interfaces of the protein. The major cystic fibrosis (CF)-causing mutation is the deletion of phenylalanine at position 508 (F508del); it causes many defects that affect CFTR trafficking, stability, and gating at the cell surface. Due to the multiple roles of PKA phosphorylation, there is growing interest in targeting PKA-dependent signaling for rescuing the trafficking and functional defects of F508del-CFTR. This review will discuss the effects of PKA phosphorylation on wild-type CFTR, the consequences of CF mutations on PKA phosphorylation, and the development of therapies that target PKA-mediated signaling.

Genetically Encoded Biosensors Reveal PKA Hyperphosphorylation on the Myofilaments in Rabbit Heart Failure.

In heart failure, myofilament proteins display abnormal phosphorylation, which contributes to contractile dysfunction. The mechanisms underlying the dysregulation of protein phosphorylation on myofilaments is not clear. This study aims to understand the mechanisms underlying altered phosphorylation of myofilament proteins in heart failure. We generate a novel genetically encoded protein kinase A (PKA) biosensor anchored onto the myofilaments in rabbit cardiac myocytes to examine PKA activity at the myofilaments in responses to adrenergic stimulation. We show that PKA activity is shifted from the sarcolemma to the myofilaments in hypertrophic failing rabbit myocytes. In particular, the increased PKA activity on the myofilaments is because of an enhanced β2 adrenergic receptor signal selectively directed to the myofilaments together with a reduced phosphodiesterase activity associated with the myofibrils. Mechanistically, the enhanced PKA activity on the myofilaments is associated with downregulation of caveolin-3 in the hypertrophic failing rabbit myocytes. Reintroduction of caveolin-3 in the failing myocytes is able to normalize the distribution of β2 adrenergic receptor signal by preventing PKA signal access to the myofilaments and to restore contractile response to adrenergic stimulation. In hypertrophic rabbit myocytes, selectively enhanced β2 adrenergic receptor signaling toward the myofilaments contributes to elevated PKA activity and PKA phosphorylation of myofilament proteins. Reintroduction of caveolin-3 is able to confine β2 adrenergic receptor signaling and restore myocyte contractility in response to β adrenergic stimulation.