Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.3.1.21 (CPT)
4,580 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Elevating cyclic AMP with a combination of forskolin and IBMX (Fskn/IBMX) was found as the cause of G1 growth arrest in Jurkat T-cells, concomitant with an induction of the cyclin-dependent kinase inhibitor, p27Kip1. The protein kinase inhibitor H-89, which can discriminate between EPAC and PKA pathways, blocked the inhibition in cell growth and induction of p27Kip1, indicating an involvement of PKA, but not EPAC. The EPAC-specific cyclic AMP analogue, 8-CPT-2Me-cAMP was able to activate Rap1, but failed to induce growth arrest or induction p27Kip1. These results demonstrate that PKA, and not EPAC, mediates cyclic AMP-dependent growth arrest in Jurkat T-cells. To further investigate a role for EPAC in these cells, we carried out cDNA microarray analysis of cells stimulated with 8-CPT-2Me-cAMP. We identified separate groups of genes whose expression was either induced or repressed in response to 8-CPT-2Me-cAMP. This provides the first demonstration that EPAC can regulate gene expression, although it may not be involved in cell cycle control. Finally, we identify c-Jun as a transcription factor whose activity is specifically down-regulated following EPAC activation, but not PKA. The control of gene expression by cyclic AMP in Jurkat T-cells therefore requires input from the EPAC signalling cascade.
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PMID:Elevation of cyclic AMP in Jurkat T-cells provokes distinct transcriptional responses through the protein kinase A (PKA) and exchange protein activated by cyclic AMP (EPAC) pathways. 1596 1

The CD94/NKG2A heterodimer is a natural killer receptor (NKR), which inhibits cell-mediated cytotoxicity upon interaction with MHC class I gene products. It is expressed by NK cells and by a small fraction of activated T cells, predominantly of CD8+ phenotype. Abnormal upregulation of the CD94/NKG2A inhibitory NKR on cytotoxic T cells (CTLs) could be responsible for a failure of immunosurveillance in cancer or HIV infection. In an attempt to identify the mechanisms leading to inhibitory NKR upregulation on T cells, we analyzed the expression of the CD94/NKG2A heterodimer on human CTLs activated with anti-CD3 mAb in the presence of PGE2 or with 8-CPT-cAMP, an analogue of cyclic AMP. As previously described, anti-CD3 mAb-mediated activation induced the expression of CD94/NKG2A on a small fraction of CD8+ T cells. Interestingly, when low concentrations of PGE2 or 8-CPT-cAMP were present during the culture, the proportion of CD8+ T cells expressing CD94/NKG2A was two- to five-fold higher. This upregulation was partially prevented by PKA inhibitors, such as KT5720 and Rp-8-Br-cAMP (type I selective). We also report that cAMP induces upregulation of NKG2A at the mRNA level. We further demonstrated that cross-linking of CD94 on CD8+ T cells expressing the CD94/NKG2A heterodimer inhibits their cytotoxic activity in a bispecific antibody redirected lysis assay. Our findings clearly demonstrate that the PGE2/cAMP/PKA type I axis is involved in the expression of CD94/NKG2A receptor on human CD8+ T lymphocytes.
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PMID:Prostaglandin E2 induces the expression of functional inhibitory CD94/NKG2A receptors in human CD8+ T lymphocytes by a cAMP-dependent protein kinase A type I pathway. 1597 47

PGE(2) and PGI(2) stimulate renin secretion and cAMP accumulation in juxtaglomerular granular (JG) cells. We addressed, at the single-cell level, the receptor subtypes and intracellular transduction mechanisms involved. Patch clamp was used to determine cell capacitance (C(m)), current, and membrane voltage in response to PGE(2), EP2 and EP4 receptor agonists, and an IP receptor agonist. PGE(2) (0.1 micromol/l) increased C(m) significantly, and the increase was abolished by intracellular application of the protein kinase A antagonist Rp-8-CPT-cAMPS. EP2-selective ligands butaprost (1 micromol/l), AE1-259-01 (1 nmol/l), EP4-selective agonist AE1-329 (1 nmol/l), and IP agonist iloprost (1 micromol/l) significantly increased C(m) mediated by PKA. The EP4 antagonist AE3-208 (10 nmol/l) blocked the effect of EP4 agonist but did not alter the response to PGE(2). Application of both EP4 antagonist and EP2-antagonist AH-6809 abolished the effects of PGE(2) on C(m) and current. EP2 and EP4 ligands stimulated cAMP formation in JG cells. PGE(2) rapidly stimulated renin secretion from superfused JG cells and diminished the membrane-adjacent granule pool as determined by confocal microscopy. The membrane potential hyperpolarized significantly after PGE(2), butaprost, AE1-329 and AE1-259 and outward current was augmented in a PKA-dependent fashion. PGE(2)-stimulated outward current, but not C(m) change, was abolished by the BK(Ca) channel inhibitor iberiotoxin (300 nmol/l). EP2 and EP4 mRNA was detected in sampled JG cells, and the preglomerular and glomerular vasculature was immunopositive for EP4. Thus IP, EP2, and EP4 receptors are associated with JG cells, and their activation leads to rapid PKA-mediated exocytotic fusion and release of renin granules.
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PMID:Prostaglandin E2 EP2 and EP4 receptor activation mediates cAMP-dependent hyperpolarization and exocytosis of renin in juxtaglomerular cells. 1598 51

Granulin-epithelin precursor (GEP/progranulin) is an autocrine growth factor for ovarian cancer. We examined the production and function of GEP and report that: (1) GEP production is regulated by endothelin (ET-1), lysophosphatidic acid (LPA), and cAMP; (2) cAMP signals GEP production through exchange protein activated by cAMP (EPAC); (3) ET-1 and cAMP/EPAC induce GEP through ERK1/2; and (4) neutralization of GEP results in apoptosis. Exposure of HEY-A8 and OVCAR3 ovarian cancer cells to LPA and ET-1 yielded GEP production and secretion in a dose- and time-dependent fashion; neither stimulated significant concentrations of cAMP directly. Stimulation of cAMP production with pertussis and cholera toxin, or forskolin induced GEP in a PKA-independent fashion. EPAC, an intracellular cAMP receptor, is activated specifically by the cAMP analog, 8-CPT-2'-O-Me-cAMP (8-CPT); 8-CPT treatment stimulated GEP production and secretion. The MEK inhibitor, U0126, abrogated GEP production in response to ET-1 and 8-CPT, confirming involvement of MAPK. A partial inhibition of basal and stimulated GEP production was observed when cells were treated with a internal calcium chelator, BAPTA. Neutralizing anti-GEP antibody reversed basal as well as LPA, ET-1 and 8-CPT-induced ovarian cancer cell growth and induced apoptosis as demonstrated by caspase-3 and PARP cleavage, DNA fragmentation, and nuclear condensation. These results indicate that GEP is a growth and survival factor for ovarian cancer, induced by LPA and ET-1 and cAMP/EPAC through ERK1/2.
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PMID:Lysophosphatidic acid and endothelin-induced proliferation of ovarian cancer cell lines is mitigated by neutralization of granulin-epithelin precursor (GEP), a prosurvival factor for ovarian cancer. 1604 62

Disturbances in Galpha(s-L) levels and function have been implicated in the pathophysiology of bipolar disorder, but the role of these changes in the development of the illness is not clear. In view of the critical role of Galpha(s)-mediated cAMP signaling in regulating cell survival, we investigated the potential role of Galpha(s-L) in modulating susceptibility to cellular stressors in human SH-SY5Y neuroblastoma cells. Overexpression of Galpha(s-L) to a level twice that of the vector-transfected cells did not directly affect cell viability but significantly increased the sensitivity to induction of cell death by serum deprivation and other apoptotic stimuli, including staurosporine, H(2)O(2), and tunicamycin. This enhanced sensitivity was associated with increased caspase-3 activation and appearance of fragmented nuclei (Hoechst 33342 staining). The broad-spectrum caspase inhibitor z-VAD-fmk completely suppressed cell death evoked by these apoptotic insults in both vector-transfected and Galpha(s-L)-overexpressing cells. The increased vulnerability conferred by increased Galpha(s-L) expression was neither mimicked by cAMP analogs 8-Br-cAMP, 8-CPT-cAMP, and 8-CPT-2Me-cAMP nor attenuated by PKA inhibitors Rp-cAMPS and KT5720. These data indicate that Galpha(s-L) may modulate apoptotic processes in a caspase-dependent manner through a signaling cascade that is independent of the cAMP/PKA or cAMP/Epac pathway. These results suggest that enhanced Galpha(s-L) expression, as was observed in post-mortem brain of bipolar patients, may impair cellular resilience in response to intracellular stress signals resulting from mitochondrial and/or endoplasmic reticulum dysfunction implicated in this disorder.
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PMID:Galpha(s) sensitizes human SH-SY5Y cells to apoptosis independently of the protein kinase A pathway. 1667 98

A-kinase can inhibit RhoA activation through phosphorylating ser188 of RhoA. AKAP is a novel protein that can target PKA to different subcellular compartment. Evidence has been presented that PKA anchorage by AKAP is important for the kinase to exert its function. This study analyzed the role of PKA anchorage in PKA-induced antagonism against RhoA activity and function. The cells transfected with pcDNA HT31wt/mut were treated with LPA and/or CPT-cAMP. The amount of GTP-RhoA and phosphorylation of RhoA was detected by Western blotting with specific antibodies. The formation of stress fiber was visualized under fluorescent microscope. The gene expression activity was analyzed by luciferase reporter gene assay. The motility and the anchorage-independent growth assays were carried out with stably transfected cells expressing the AKAP inhibitory peptide HT31. The results showed that HT31 not only blocked the PKA-induced phosphorylation of RhoA but also prevented the PKA-induced inhibition on RhoA activation. The disruption of PKA anchorage abolished its inhibition on the LPA-induced expression of reporter gene SRE-luciferase. The ability of PKA to antagonize the LPA-induced stress fiber formation was partly impaired upon the disruption of the PKA anchorage. The control of PKA on migration and the proliferation excited by LPA disappeared in stably transfected cells highly expressing HT31. The results revealed that PKA anchorage was necessary for the kinase to exert its inhibitory effect on RhoA activation and RhoA-dependent biological activities.
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PMID:AKAPs competing peptide HT31 disrupts the inhibitory effect of PKA on RhoA activity. 1696 90

Urocortin, a peptide hormone related to the corticotropin releasing factor, is suggested to be involved in blood pressure regulation by dilating the peripheral blood vessels. In rat tail arteries, urocortin-induced vasodilation is due to a decrease in myofilament Ca2+ sensitivity the mechanism of which is still unclear. In this study, the hypothesis was tested that the decrease in Ca2+ sensitivity in mouse tail arteries results from the activation of myosin light chain phosphatase. The relaxation of KCl-precontracted (42 mM) intact mouse tail arteries by urocortin (1 nM and 10 nM) was significantly inhibited by 1 microM antisauvagine30, a CRF-2 receptor antagonist (p < 0.05, n = 3). The addition of 1 microM KT 5720, an inhibitor of PKA, to intact rat tail arteries did not affect the KCl-induced force but significantly attenuated the urocortin-induced relaxation (n = 5). In alpha-toxin permeabilized mouse tail arteries, urocortin relaxed submaximally activated preparations at constant pCa 6.1 by 37.6 +/- 8.2% (n = 5) as compared to control vessels (n = 5, p < 0.001). The relaxation in permeabilized vessels was inhibited by pre-treatment with 30 microM Rp-8-CPT-cAMPS, an inactive analogue of cAMP. In permeabilized mouse tail arteries, treatment with 100 nM urocortin was associated with dephosphorylation of MLC20(Ser19) and MYPT1(Thr696/Thr850). The effect of urocortin on MYPTI dephosphorylation was completely abolished by 30 M Rp-8-CPT-cAMPS and mimicked by the cAMP analogue Sp-5,6-DCI-cBiMPS. Based on these findings, we propose that the urocortin-induced relaxation is due to a decrease in calcium sensitivity mediated by a cAMP-dependent increase in the activity of MLCP.
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PMID:[Urocortin decreases phosphorylation of MYPT1 and increases the myosin phosphatase activity via elevation of the intracellular level of cAMP]. 1713 11

Tyrosine hydroxylase (TH) is the rate-limiting enzyme in catecholamine synthesis. Its activity is controlled by PACAP, acutely by phosphorylation at Ser40 and chronically by protein synthesis. Using bovine adrenal chromaffin cells we found that PACAP, acting via the continuous activation of PACAP 1 receptors, sustained the phosphorylation of TH at Ser40 and led to TH activation for up to 24 h in the absence of TH protein synthesis. The sustained phosphorylation of TH at Ser40 was not mediated by hierarchical phosphorylation of TH at either Ser19 or Ser31. PACAP caused sustained activation of PKA, but did not sustain activation of other protein kinases including ERK, p38 kinase, PKC, MAPKAPK2 and MSK1. The PKA inhibitor H89 substantially inhibited the acute and the sustained phosphorylation of TH mediated by PACAP. PACAP also inhibited the activity of PP2A and PP2C at 24 h. PACAP therefore sustained TH phosphorylation at Ser40 for 24 h by sustaining the activation of PKA and causing inactivation of Ser40 phosphatases. The PKA activator 8-CPT-6Phe-cAMP also caused sustained phosphorylation of TH at Ser40 that was inhibited by the PKA inhibitor H89. Using cyclic AMP agonist pairs we found that sustained phosphorylation of TH was due to both the RI and the RII isotypes of PKA. The sustained activation of TH that occurred as a result of TH phosphorylation at Ser40 could maintain the synthesis of catecholamines without the need for further stimulus of the adrenal cells or increased TH protein synthesis.
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PMID:PACAP stimulates the sustained phosphorylation of tyrosine hydroxylase at serine 40. 1726 61

Smac/DIABLO is a mitochondrial protein that participates in apoptotic cell death by means of sequestering several members of the inhibitor of apoptosis protein family. This action allows caspase activation, cleavage of key cellular substrates and death. Release from mitochondria is considered the main regulatory step of Smac/DIABLO activity. Nevertheless, the fact that at least one isoform, Smac-beta, does not reside in this organelle implies that transcriptional regulation could also be important. cAMP is a well known second messenger with important apoptotic effects. To analyze if cAMP could be involved in Smac/DIABLO gene regulation, we analyzed 2903 base pairs upstream of the coding sequence and characterized the minimal promoter, which contains a consensus CRE site. We found that cAMP/PKA/CREB pathway is indeed an important regulator of Smac/DIABLO transcription, since exposure to the cAMP analog 8-CPT-cAMP, the adenylyl cyclase activator forskolin, the inhibitor of phosphodiesterase isobutylmethylxanthine or by hindering PKA activation with H89, regulated the promoter activity, as shown by gene reporter and RT-PCR assays. Additionally, the results of site-directed mutagenesis revealed that the consensus CRE site was biologically functional and required for cAMP-induced promoter activity in human HeLa cells. Supporting these results, a negative dominant version of the protein kinase A responsive factor, KCREB, reduced basal Smac/DIABLO expression and rendered the promoter unresponsive to cAMP. Reducing Smac expression using an antisense approach blocked the apoptosis effects of cAMP in cervical cancer cells. These results show that cAMP is an important modulator of the apoptotic threshold in cancer cell by means of regulating Smac/DIABLO expression.
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PMID:Apoptosis induced by cAMP requires Smac/DIABLO transcriptional upregulation. 1732 Mar 50

Sensitization of purinergic P2X receptors is one of the mechanisms responsible for exaggerated pain responses to inflammatory injuries. Prostaglandin E2 (PGE2), produced by inflamed tissues, is known to contribute to abnormal pain states. In a previous study, we showed that PGE2 increases fast inactivating ATP currents that are mediated by homomeric P2X3 receptors in dorsal root ganglion (DRG) neurons isolated from normal rats. Protein kinase A (PKA) is the signalling pathway used by PGE2. Little is known about the action of PGE2 on ATP currents after inflammation, although the information is crucial for understanding the mechanisms underlying inflammation-induced sensitization of P2X receptors. We therefore studied the effects of PGE2 on P2X3 receptor-mediated ATP currents in DRG neurons dissociated from complete Freund's adjuvant (CFA)-induced inflamed rats. We found that PGE2 produces a large increase in ATP currents. PKCepsilon, in addition to PKA, becomes involved in the modulatory action of PGE2. Thus, PGE2 signalling switches from a solely PKA-dependent pathway under normal conditions to both PKA- and PKC-dependent pathways after inflammation. Studying the mechanisms underlying the switch, we demonstrated that cAMP-responsive guanine nucleotide exchange factor 1 (Epac1) is up-regulated after inflammation. The Epac agonist CPT-OMe mimics the potentiating effect of PGE2 and occludes the PKC-mediated PGE2 action on ATP currents. These results suggest that Epac plays a critical role in P2X3 sensitization by activation of de novo PKC-dependent signalling of PGE2 after inflammation and would be a useful therapeutic target for pain therapies.
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PMID:A critical role of the cAMP sensor Epac in switching protein kinase signalling in prostaglandin E2-induced potentiation of P2X3 receptor currents in inflamed rats. 1770 20


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