PKA Kinase Activity Research Articles

CAMP/PKA signaling regulates TDP-43 aggregation and mislocalization

Cytoplasmic mislocalization and aggregation of TDP-43 protein are hallmarks of amyotrophic lateral sclerosis (ALS) and are observed in the vast majority of both familial and sporadic cases. How these two interconnected processes are regulated on a molecular level, however, remains enigmatic. Genome-wide screens for modifiers of the ALS-associated genes TDP-43 and FUS have identified the phospholipase D (Pld) pathway as a key regulator of ALS-related phenotypes in the fruit fly Drosophila melanogaster [M. W. Kankel et al., Genetics 215, 747-766 (2020)]. Here, we report the results of our search for downstream targets of the enzymatic product of Pld, phosphatidic acid. We identify two conserved negative regulators of the cAMP/PKA signaling pathway, the phosphodiesterase dunce and the inhibitory subunit PKA-R2, as modifiers of pathogenic phenotypes resulting from overexpression of the Drosophila TDP-43 ortholog TBPH. We show that knockdown of either of these genes results in a mitigation of both TBPH aggregation and mislocalization in larval motor neuron cell bodies, as well as an amelioration of adult-onset motor defects and shortened lifespan induced by TBPH. We determine that PKA kinase activity is downstream of both TBPH and Pld and that overexpression of the PKA target CrebA can rescue TBPH mislocalization. These findings suggest a model whereby increasing cAMP/PKA signaling can ameliorate the molecular and functional effects of pathological TDP-43.

Live-cell fluorescence imaging and optogenetic control of PKA kinase activity in fission yeast Schizosaccharomyces pombe.

The cAMP-PKA signaling pathway plays a crucial role in sensing and responding to nutrient availability in the fission yeast Schizosaccharomyces pombe. This pathway monitors external glucose levels to control cell growth and sexual differentiation. However, the temporal dynamics of the cAMP-PKA pathway in response to external stimuli remains unclear mainly due to the lack of tools to quantitatively visualize the activity of the pathway. Here, we report the development of the kinase translocation reporter (KTR)-based biosensor spPKA-KTR1.0, which allows us to measure the dynamics of PKA activity in fission yeast cells. The spPKA-KTR1.0 is derived from the transcription factor Rst2, which translocates from the nucleus to the cytoplasm upon PKA activation. We found that spPKA-KTR1.0 translocates between the nucleus and cytoplasm in a cAMP-PKA pathway-dependent manner, indicating that the spPKA-KTR1.0 is a reliable indicator of the PKA activity in fission yeast cells. In addition, we implemented a system that simultaneously visualizes and manipulates the cAMP-PKA signaling dynamics by introducing bPAC, a photoactivatable adenylate cyclase, in combination with spPKA-KTR1.0. This system offers an opportunity for investigating the role of the signaling dynamics of the cAMP-PKA pathway in fission yeast cells with higher temporal resolution.

RNP components condense into repressive RNP granules in the aging brain

Cytoplasmic RNP condensates enriched in mRNAs and proteins are found in various cell types and associated with both buffering and regulatory functions. While a clear link has been established between accumulation of aberrant RNP aggregates and progression of aging-related neurodegenerative diseases, the impact of physiological aging on neuronal RNP condensates has never been explored. Through high-resolution imaging, we uncover that RNP components progressively cluster into large yet dynamic granules in the aging Drosophila brain. We further show that age-dependent clustering is caused by an increase in the stoichiometry of the conserved helicase Me31B/DDX6, and requires PKA kinase activity. Finally, our functional analysis reveals that mRNA species recruited to RNP condensates upon aging exhibit age-dependent translational repression, indicating that co-clustering of selected mRNAs and translation regulators into repressive condensates may contribute to the specific post-transcriptional changes in gene expression observed in the course of aging.

Abstract 13122: STK25 Regulates the Cardiac PKA Pathway in Human Cardiomyocytes

Hypothesis: STK25 regulates cell survival, metabolism and cardiac function through phosphorylating PKA R1α and modulating PKA signaling pathway in human cardiomyocytes. Background: Dysregulation of cardiac kinases are related with many cardiovascular diseases including hypertrophy, remodeling and ischemia injury. STK25 is a stress response kinase that regulates cell survival through modulating several signaling pathways and is involved in the metabolism regulation. However, the direct substrates and mechanism of STK25 remain undefined. Methods: CRISPR-Cas9 was used to generate a homozygous knockout (KO) of STK25 in induced pluripotent stem cell (iPSC) which were then differentiated into cardiomyocytes (iPSC-CMs) and used for engineered human cardiac tissue generation. Role of STK25 in gene expression, cell proliferation/apoptosis, metabolism and cardiac function were characterized. STK25 was quantified in human left ventricular tissue from heart failure (HF) patients. Results: In iPSC-CMs, phospho-proteomic analysis identified PKA signaling as the most significantly upregulated pathway by STK25 deletion. PKA regulatory subunit I α (R1α) was identified as a downstream substrate of STK25 and validated by IP. Loss of STK25 resulted in elevated PKA kinase activity and decreased phosphorylation of PKA targets implicated in cardiac contractility and Ca2+ handling, such as cardiac myosin-binding protein C (cMyBPC), cardiac troponin (cTnT), ryanodine receptor-2 (RyR2) and L-type calcium channel (CaV1.2). Further mechanistic studies revealed that STK25 mediates PKA activity through phosphorylation of the R1α regulatory subunit at S77/83 sites, leading to inhibition of PKA kinase activity. In functional assays, STK25 modulated cell cycle progression, proliferation, hypoxia induced apoptosis, beta adrenergic response, and mitochondrial respiration in iPSC-CMs. We also detected significant increase in STK25 expression and PKA R1α phosphorylation in HF patients versus normal control, demonstrating STK25 altered regulation in heart diseases. Conclusions: STK25 kinase is a critical regulator of the PKA pathway in human cardiomyocytes modulating cell growth, metabolism and cardiac function and is aberrantly expressed in heart failure.

Abstract 1213: PDE10A as a novel target to suppress Wnt/β-catenin signaling and other oncogenic pathways in ovarian cancer

Abstract Objective: A leading theory for ovarian carcinogenesis proposes that the inflammation associated with incessant ovulation is a driver of oncogenesis. Consistent with this theory, epidemiological studies have shown that nonsteroidal anti-inflammatory drugs (NSAIDs) decrease ovarian cancer risk. Previous studies suggest, however, that the antineoplastic activity of NSAIDs does not require the traditional cyclooxygenase (COX) enzyme inhibition, and rather may be exerted through phosphodiesterase (PDE) inhibition. PDEs represent a potentially unique chemopreventive target for ovarian cancer given that ovulation is regulated by cyclic nucleotide signaling. This study evaluates the effects of phosphodiesterase 10A (PDE10A) inhibition as a novel chemopreventive and therapeutic approach for ovarian cancer. Methods: We investigated the effects of PDE10A small molecule inhibitors, Pf-2545920 and MCI-030 (a novel non-COX inhibitory NSAID-derivative), and PDE10A knockout by CRISPR/Cas9 gene editing in various ovarian cancer cell lines using in vitro assays that measured cell proliferation, cell viability, cell cycle arrest, apoptosis, cell migration and invasion. Downstream signaling pathways affected by PDE10A inhibition and gene knockout (KO) were assessed by western-blotting, confocal microscopy, and RNA sequencing. Results: Analysis of The Cancer Genome Atlas (TCGA) ovarian cancer database and an institutional cohort of ovarian cancer patients revealed that high PDE10A mRNA expression was associated with significantly worse overall survival. PDE10A expression was also positively correlated with oncogenic and inflammatory signaling pathways in the TCGA dataset. PDE10A inhibition with Pf-2545920 or MCI-030 decreased ovarian cancer cell proliferation, while inducing cell cycle arrest and apoptosis. Using inhibitors to block PKA (H89) and PKG (KT5823) kinase activity, we demonstrated that the pro-apoptotic effects of PDE10A inhibition were mediated by activation of cGMP/PKG and cAMP/PKA signaling. SKOV3 and OV-90 PDE10A KO cells showed decreased colony formation, cell proliferation, and migration and invasion properties compared to their wild-type (WT) counterparts. Moreover, PDE10A inhibition decreased Wnt-induced β-catenin nuclear translocation as well as decreased EGF-mediated activation of RAS/MAPK and AKT pathways in ovarian cancer cells. RNA sequencing of SKOV3 PDE10A KO clones revealed that pathways associated with cancer, Wnt and TGF-β signaling were downregulated compared to WT control cells. Conclusions: Our data demonstrate that PDE10A has pro-tumorigenic effects in ovarian cancer cells and its high expression in ovarian cancer patients is associated with poor prognosis. Altogether, our results warrant future studies of PDE10A as a novel target for ovarian cancer chemoprevention and/or treatment. Citation Format: Rebecca M. Barber, Elaine Gavin, Alla Musiyenko, Wito Richter, Kevin J. Lee, Annelise Wilhite, Joel F. Andrews, Steve McClellan, Ileana Aragon, Antonio Ward, Xi Chen, Adam Keeton, Kristy Berry, Gary A. Piazza, Jennifer M. Scalici, Luciana Madeira da Silva. PDE10A as a novel target to suppress Wnt/β-catenin signaling and other oncogenic pathways in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1213.

Enhancement of Monascus yellow pigments production by activating the cAMP signalling pathway in Monascus purpureus HJ11

BackgroundMonascus azaphilone pigments (MonAzPs), which were produced by Monascus species, have been used as important food colorant and food supplements for more than one billion people during their daily life. Moreover, MonAzPs recently have received more attention because of their diverse physiological activities. However, the high microbial production of MonAzPs is still not always guaranteed. Herein, the aim of this study was to develop an efficient biotechnological process for MonAzPs production.ResultsIn this study, exogenous cyclic adenosine monophosphate (cAMP) treatment not only induced MonAzPs production, but also stimulated the expression of a cAMP phosphodiesterase gene, named as mrPDE, in M. purpureus HJ11. Subsequently, MrPDE was identified as a cAMP phosphodiesterase by in vitro enzymatic reaction with purified enzyme. Further, a gene knockout mutant of mrPDE was constructed to verify the activation of cAMP signalling pathway. Deletion of mrPDE in M. purpureus HJ11 improved cAMP concentration by 378% and enhanced PKA kinase activity 1.5-fold, indicating that activation of cAMP signalling pathway was achieved. The ΔmrPDE strain produced MonAzPs at 8563 U/g, with a 2.3-fold increase compared with the WT strain. Moreover, the NAPDH/NADP+ ratio of the ΔmrPDE strain was obviously higher than that of the wild type strain, which led to a higher proportion of yellow MonAzPs. With fed-batch fermentation of the ΔmrPDE strain, the production and yield of MonAzPs achieved 332.1 U/mL and 8739 U/g.ConclusionsA engineered M. purpureus strain for high MonAzPs production was successfully developed by activating the cAMP signalling pathway. This study not only describes a novel strategy for development of MonAzPs-producing strain, but also provides a roadmap for engineering efforts towards the production of secondary metabolism in other filamentous fungi.

PRKAR1A deficiency delays postnatal heart growth

AimsProtein kinase A (PKA) activity is pivotal for proper functioning of the human heart, and its dysregulation has been implicated in a variety of cardiac pathologies. PKA regulatory subunit 1α (R1α, encoded by the PRKAR1A gene) is highly expressed in the heart, and controls PKA kinase activity by sequestering the catalytic subunits. Patients with PRKAR1A mutations are often diagnosed with Carney complex (CNC) in young adults and may die prematurely from cardiac complications such as heart failure. However, no cardiac defect has been reported in adult animal models of PRKAR1A deficiency.Methods and ResultsTo investigate the impact of PRKAR1A deficiency on the heart, we generated cardiac‐specific PRKAR1A heterozygous knockout (cPRKAR1A+/−) mice by breeding the floxed PRKAR1A mice with the Mlc2v‐Cre mice. We also studied a cohort of young CNC patients with PRKAR1A mutations or deletions. Ablation of the PRKAR1A gene in mice increased cardiac PKA activity, reduced heart weight to body weight ratio (Control: 5.3±0.19, cPRKAR1A+/−: 4.7±0.11 mg/g, P<0.05) and cardiomyocyte size (Control: 298.5±9.8 μm2, cPRKAR1A+/−: 191.3±4.5 μm2, P<0.005) without altering contractile function at 3 months of age. Importantly, left ventricular mass was reduced in young patients diagnosed with CNC (Female control: 80.6 ± 12.8g, CNC: 70.8 ± 8.1, P<0.05). Cardiomyocyte hypertrophy in response to activation of the α1‐adrenergic receptor, which is necessary for heart growth after birth, was completely abolished by silencing of PRKAR1A, or stimulation with the PKA activator forskolin. Mechanistically, depletion of PRKAR1A provoked PKA‐dependent phosphorylation of the mitochondrial fission protein Drp1 at S637, leading to suppression of mitochondrial fission and inhibition of cardiomyocyte hypertrophy.ConclusionsCombined with previous findings that mitochondrial fission is required for hypertrophic growth of cardiomyocytes, our results suggest that PRKAR1A deficiency impedes postnatal myocardial development and physiological hypertrophy through modulation of mitochondrial dynamics. These findings provide a potential novel mechanism for the cardiac manifestations associated with CNC.Support or Funding InformationThis study was supported by intramural program of National Institute of Child Health and Human Development (NICHD), National Institutes of Health, National Heart, Lung, and Blood Institute (NIH R00HL119605, R56HL145034); WSU College of Pharmacy and Pharmaceutical Sciences.

Informatics-guided drug repurposing for Autosomal Dominant Polycystic Kidney Disease (ADPKD).

Informatics-guided drug repurposing for Autosomal Dominant Polycystic Kidney Disease (ADPKD).

Electroacupuncture ameliorates learning and memory deficits via hippocampal 5-HT1A receptors and the PKA signaling pathway in rats with ischemic stroke.

Hippocampal 5-HT1A receptors and the PKA signaling pathway have been implicated in learning and memory. This study aimed to investigate whether PKA signaling mediated by 5-HT1A receptors was involved in the electroacupuncture (EA)-mediated learning and memory in a rat model of middle cerebral artery occlusion-induced cognitive deficit (MICD). Compared to no treatment or non-acupoint EA treatment, EA at DU20 and DU24 acupoints improved the neurological deficit of scores, shortened escape latency and increased the frequency of crossing the platform in the Morris water maze test. T2-weighted imaging demonstrated that the MICD rat brain lesions were mainly located in the cortex and hippocampus, and injured volumes were reduced after EA. Furthermore, we found that these behavioral changes were concomitant with the deficit of the 5HT1A and PKA signaling pathways in the hippocampus, as the activation of the 5-HT1A receptor, the reduction of PKA kinase activity, and AMPA and NMDA receptor phosphorylation occurred in the injured hippocampus at Day 14 after MICD. Additionally, EA dramatically elevated the activation of PKA. Moreover, EA significantly increased intracellular calcium concentrations regulated by the activation of NMDA receptors. Therefore, PKA kinase and NMDA receptors mediated by 5-HT1A receptors in the hippocampus might contribute to improving learning and memory during the recovery process following ischemic stroke with an EA intervention.

G-Protein-Coupled Receptor 5 (LGR5) Overexpression Activates β-Catenin Signaling in Breast Cancer Cells via Protein Kinase A.

BackgroundTargeting cancer stem cells (CSCs) in breast cancer (BrCa) may improve treatment outcome and patient prognosis. Leucine-rich repeat-containing G-protein-coupled receptor 5 (LGR5) is a well-recognized adult stem cell and CRC marker, and previous reports have suggested the cancer-promoting role of LGR5 in breast cancer, but the mechanism remains unclear.Material/MethodsPotential LGR5-associating genes were explored using STRING database, and LGR5 overexpression and knockdown was constructed in MCF-7 and MDA-MB-453 human BrCa cells, respectively. PKA catalytic subunit activation and PKA kinase activity in human BrCa cells was examined by Western blot and PKA kinase activity assay, respectively. Protein expression level or activation of β-catenin and GSK-3β in human BrCa cells was investigated by Western blot. Cell proliferation, colony formation, Transwell migration, cisplatin sensitivity, and in vivo tumor formation of human BrCa cells were examined.ResultsLGR5 overexpression increased PKA activation and its kinase activity in human BrCa cells, which was decreased by LGR5 knockdown. LGR5 expression level or PKA kinase activity were correlated with β-catenin Ser 552 phosphorylation but inversely correlated with GSK-3β Ser9 phosphorylation in human BrCa cells in vitro. LGR5/PKA increased cell proliferation, colony formation, Transwell migration, and cisplatin resistance in vitro, as well as tumor formation in vivo, of human BrCa cells.ConclusionsLGR5 activates the Wnt/β-catenin signaling pathway in human BrCa cells in vitro via PKA.

Propofol reduced myocardial contraction of vertebrates partly by mediating the cyclic AMP-dependent protein kinase phosphorylation pathway.

Propofol inhibits myocardial contraction in a dose dependent manner. The present study is designed to examine the effect of propofol on PKA mediated myocardial contraction in the absence of adrenoreceptor agonist. The contraction of isolated rat heart was measured in the presence or absence of PKA inhibitor H89 or propofol, using a pressure transducer. The levels of cAMP and PKA kinase activity were detected by ELISA. The mRNA and total protein or phosphorylation level of PKA and downstream proteins were tested in the presence or absence of PKA inhibitor H89 or propofol, using RT-PCR, QPCR and western blotting. The phosphorylation level of PKA was examined thoroughly using immunofluorescence and PKA activity non-radioactive detection kit. Propofol induced a dose-dependent negative contractile response on the rat heart. The inhibitory effect of high concentration propofol (50μM) with 45% decease of control could be partly reversed by the PKA inhibitor H89 (10μM) and the depressant effect of propofol decreased from 45% to 10%. PKA kinase activity was inhibited by propofol in a dose-dependent manner. Propofol also induced a decrease in phosphorylation of PKA, which was also inhibited by H89, but did not alter the production of cAMP and the mRNA levels of PKA. The downstream proteins of PKA, PLN and RyR2 were phosphorylated to a lesser extent with propofol or H89 than control. These results demonstrated that propofol induced a negative myocardial contractile response partly by mediating the PKA phosphorylation pathway.

EGF-like peptide-enhanced cell movement in Dictyostelium is mediated by protein kinases and the activity of several cytoskeletal proteins

DdEGFL1, a synthetic epidermal growth factor-like (EGFL) peptide based on the first EGFL repeat of the extracellular matrix, cysteine-rich, calmodulin-binding protein CyrA, has previously been shown to sustain the threonine phosphorylation of a 210kDa protein during the starvation of Dictyostelium cells. Immunoprecipitation coupled with a LC/MS/MS analysis identified the 210kDa protein as vinculin B (VinB). VinB shares sequence similarity with mammalian vinculin, a protein that links the actin cytoskeleton to the plasma membrane. Both threonine phosphorylated VinB (P-VinB) and VinB-GFP localized to the cytoplasm and cytoskeleton of Dictyostelium amoebae. VinB-GFP was also shown to be threonine phosphorylated and co-immunoprecipitated with established vinculin-binding cytoskeletal proteins (e.g. myosin II heavy chain, actin, alpha-actinin, talin). P-VinB and VinB-GFP were detected in DdEGFL1 pull-down assays, which also identified a 135kDa phosphothreonine protein and two phosphotyrosine proteins (35 and 32kDa) as potential components of the DdEGFL1 signaling pathway. DdEGFL1-enhanced cell movement required the cytoskeletal proteins talin B and paxillin B and tyrosine kinase activity mediated by PKA signaling, however VinB threonine phosphorylation was shown to be independent of PI3K/PLA2 signaling and PI3K and PKA kinase activity. Finally, VinB-GFP over-expression suppressed DdEGFL1-enhanced random cell movement, but not folic acid-mediated chemotaxis. Together, this study provides the first evidence for VinB function plus new insight into the signaling pathway(s) mediating EGFL repeat/peptide-enhanced cell movement in Dictyostelium. This information is integrated into an emerging model that summarizes existing knowledge.

H 2 O 2 Activates the Nuclear Localization of Msn2 and Maf1 through Thioredoxins in Saccharomyces cerevisiae

The cellular response to hydrogen peroxide (H(2)O(2)) is characterized by a repression of growth-related processes and an enhanced expression of genes important for cell defense. In budding yeast, this response requires the activation of a set of transcriptional effectors. Some of them, such as the transcriptional activator Yap1, are specific to oxidative stress, and others, such as the transcriptional activators Msn2/4 and the negative regulator Maf1, are activated by a wide spectrum of stress conditions. How these general effectors are activated in response to oxidative stress remains an open question. In this study, we demonstrate that the two cytoplasmic thioredoxins, Trx1 and Trx2, are essential to trigger the nuclear accumulation of Msn2/4 and Maf1, specifically under H(2)O(2) treatment. Contrary to the case with many stress conditions previously described for yeast, the H(2)O(2)-induced nuclear accumulation of Msn2 and Maf1 does not correlate with the downregulation of PKA kinase activity. Nevertheless, we show that PP2A phosphatase activity is essential for driving Maf1 dephosphorylation and its subsequent nuclear accumulation in response to H(2)O(2) treatment. Interestingly, under this condition, the lack of PP2A activity has no impact on the subcellular localization of Msn2, demonstrating that the H(2)O(2) signaling pathways share a common route through the thioredoxin system and then diverge to activate Msn2 and Maf1, the final integrators of these pathways.

Big mitogen-activated protein kinase (BMK1)/ERK5 protects endothelial cells from apoptosis.

Blood flow that is steady and laminar is known to be atheroprotective. One likely mechanism is enhanced endothelial cell (EC) survival. Because the mitogen-activated protein kinases (MAPKs) are known regulators of cell survival, we investigated the role of Big MAPK-1 (BMK1 or ERK5), which is potently stimulated by fluid shear stress. To activate BMK1, we overexpressed constitutively active (CA)-MEK5 in bovine lung microvascular ECs (BLMECs). Cell apoptosis was induced by growth factor deprivation (0% serum for 24 hours). Analysis of cell viability with MTT assay showed that activation of BMK1 by CA-MEK5 significantly improved cell viability from 48% to 87% and decreased apoptotic cells from 49% to 10%. Growth factor deprivation induced caspase-3 activity 5.2-fold, which was inhibited (approximately 60%) by CA-MEK5 overexpression. In contrast, inhibiting BMK1 activity by overexpressing dominant-negative BMK1 (DN-BMK1) stimulated apoptosis in BLMECs. Steady laminar fluid shear stress inhibited BLMEC apoptosis, and this protective effect was also reduced significantly by overexpressing DN-BMK1. Analysis of antiapoptotic mechanisms showed that both shear stress and CA-MEK5 stimulated phosphorylation of Bad on Ser112 and Ser136, whereas DN-BMK1 inhibited phosphorylation. Phosphorylation of Bad induced by BMK1 activation was independent of Akt, PKA, or p90RSK kinase activity. These results suggest that BMK1 activation by steady laminar flow is atheroprotective by inhibiting EC apoptosis via phosphorylation of Bad.

Adeno-associated virus type 2 Rep78 inhibition of PKA and PRKX: fine mapping and analysis of mechanism.

Hormones and neurotransmitters utilize cyclic AMP (cAMP) as a second messenger in signal transduction pathways to regulate cell growth and division, differentiation, gene expression, and metabolism. Adeno-associated virus type 2 (AAV-2) nonstructural protein Rep78 inhibits members of the cAMP signal transduction pathway, the protein kinases PKA and PRKX. We mapped the kinase binding and inhibition domain of Rep78 for PRKX to amino acids (aa) 526 to 561 and that for PKA to aa 526 to 621. These polypeptides were as potent as full-length Rep78 in kinase inhibition, which suggests that the kinase-inhibitory domain is entirely contained in these Rep peptides. Steady-state kinetic analysis of Rep78-mediated inhibition of PKA and PRKX showed that Rep78 appears to increase the K(m) value of the peptide kinase substrate, while the maximal velocity of the reaction was unaffected. This indicates that Rep78 acts as a competitive inhibitor with respect to the peptide kinase substrate. We detected homology between a cellular pseudosubstrate inhibitor of PKA, the protein kinase inhibitor PKI, and the PRKX and PKA inhibition domains of Rep78. Due to this homology and the competitive inhibition mechanism of Rep78, we propose that Rep78 inhibits PKA and PRKX kinase activity by pseudosubstrate inhibition.

Tyrosine kinase-mediated serine phosphorylation of adenylyl cyclase.

Receptor tyrosine kinase (RTK) activation is associated with modulation of heptahelical receptor-stimulated adenylyl cyclase responses. The mechanisms underlying the RTK-mediated enhancement of adenylyl cyclase function remain unclear. In the present studies, we show that the tyrosine kinase-dependent enhancement of adenylyl cyclase isoform VI function parallels an enhancement in serine phosphorylation of the enzyme. This effect was mediated by both RTK activation, with IGF-1, and by tyrosine phosphatase inhibition, with sodium orthovanadate. This enhancement of adenylyl cyclase function was not attenuated by inhibitors of ERK, PKC, PKA, or PI3 kinase activity but was blunted by inhibition of endogenous p74(raf-1)() activity. To characterize the molecular site of this effect we identified multiple candidate serine residues in and adjacent to the adenylyl cyclase VI C1b catalytic region and performed serine-to-alanine site-directed mutagenesis using adenylyl cyclase VI as a template. Mutation of serine residues 603 and 608 or serine residues 744, 746, 750, and 754 attenuated both the tyrosine kinase-mediated enhancement of enzyme phosphorylation as well as the sensitization of function. Together, these data define a novel tyrosine kinase-mediated mechanism leading to serine phosphorylation of adenylyl cyclase isoform VI and the sensitization of adenylyl cyclase responsiveness.

Memory Consolidation for Contextual and Auditory Fear Conditioning Is Dependent on Protein Synthesis, PKA, and MAP Kinase

Fear conditioning has received extensive experimental attention. However, little is known about the molecular mechanisms that underlie fear memory consolidation. Previous studies have shown that long-term potentiation (LTP) exists in pathways known to be relevant to fear conditioning and that fear conditioning modifies neural processing in these pathways in a manner similar to LTP induction. The present experiments examined whether inhibition of protein synthesis, PKA, and MAP kinase activity, treatments that block LTP, also interfere with the consolidation of fear conditioning. Rats were injected intraventricularly with Anisomycin (100 or 300 microg), Rp-cAMPS (90 or 180 microg), or PD098059 (1 or 3 microg) prior to conditioning and assessed for retention of contextual and auditory fear memory both within an hour and 24 hr later. Results indicated that injection of these compounds selectively interfered with long-term memory for contextual and auditory fear, while leaving short-term memory intact. Additional control groups indicated that this effect was likely due to impaired memory consolidation rather than to nonspecific effects of the drugs on fear expression. Results suggest that fear conditioning and LTP may share common molecular mechanisms.

Regulation of stress-induced cytokine production by pyridinylimidazoles; inhibition of CSBP kinase

Members of three classes of pyridinylimidazoles bind with varying affinities to CSBP (p38) kinase which is a member of a stress-induced signal transduction pathway. Based upon SAR and protein homology modeling, the pharmacophore and three potential modes of binding to the enzyme are presented. For a subset of pyridinylimidazoles, binding is shown to correlate with inhibition of CSBP kinase activity, whereas no significant inhibition of PKA, PKCα and ERK kinase activity is observed.