Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.11.1 (protein kinase)
 81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The cAMP responsiveness of the phosphoenolpyruvate carboxykinase (PEPCK) gene promoter is mediated by a cAMP response unit, which includes three CCAAT/enhancer-binding protein (C/EBPs) sites, and a cAMP response element (CRE). Because both the CRE-binding protein and several C/EBP isoforms can to bind to the CRE with similar affinity, a variety of transcription factor bindings arrays in the cAMP response unit are possible that may affect the protein kinase A (PKA) responsivity of the promoter. To explore this issue, we have designed PEPCK promoter variants that have the native cis-elements within the cAMP response unit replaced with one or more LexA- and/or GAL4-binding sites. We also engineered the corresponding C/EBP and CRE-binding protein chimeras, which have their basic region leucine zipper domains replaced with LexA or GAL4 DNA-binding domains. Using this approach, we have reconstituted the PKA responsiveness of permissive PEPCK promoters in hepatoma cells and have characterized the PKA responsivity of the promoter under defined transcription factor occupancy patterns. Furthermore, analysis of deletion mutants of C/EBPalpha indicated that the domains that mediate its constitutive and PKA-inducible activities vary depending on which cis-element it occupies on the PEPCK promoter. These results suggest that promoter context may influence which domains within a transcription factor are employed to mediate transactivation.
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PMID:Different transcription factor binding arrays modulate the cAMP responsivity of the phosphoenolpyruvate carboxykinase gene promoter. 1223 88

Secretin evokes catecholamine secretion from PC12 pheochromocytoma cells. We tested whether secretin activates transcription of the major vesicular core protein chromogranin A (CgA). Secretin stimulated both endogenous CgA gene transcription (approximately 4-6-fold) as well as transfected CgA promoter activity (approximately 8-10-fold; EC50, approximately 7 nm) in PC12 cells. Studies on CgA promoter 5'-deletion mutant/luciferase reporter constructs, point mutations of the CgA cAMP response element (CRE), and their transfer to a heterologous promoter implicated CRE in cis as both necessary and sufficient for secretin-stimulated CgA gene transcription. Secretin-induced CgA gene transcription was inhibited/abolished by cytosolic Ca2+ chelation, chemical blockade of phospholipase C, protein kinase A (PKA), or mitogen-activated protein (MAP) kinase extracellular signal regulated kinase (ERK) 1/2 and the expression of dominant negative mutants of ERK1/2, CRE binding protein (CREB) kinase RSK2, or CREB. Secretin also augmented (approximately 4-fold) phosphorylation of ERK1/2. Trans-activation (approximately 21-fold) of GAL4-CREB fusion protein by secretin indicates involvement of CREB in secretin signaling to gene transcription. Electrophoretic mobility shift assays also identified CREB as the mediator of secretin-induced CgA gene transcription, and pCREB supershifts indicated Ser-133 as the active CREB moiety in vitro. This conclusion was reinforced in vivo by results of chromatin pCREB immunoprecipitation assays. We conclude that secretin signals to CgA gene transcription through the CRE domain in cis and through cAMP, Ca2+, PKA, MAP kinase, and the transcription factor CREB in trans. Thus, multiple signal transduction pathways seem to subserve the function of stimulus-transcription coupling after this peptidergic stimulus to chromaffin cells.
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PMID:Secretin activation of chromogranin A gene transcription. Identification of the signaling pathways in cis and in trans. 1264 81

17 beta-Estradiol (E2) induces E2F-1 gene expression in ZR-75 and MCF-7 human breast cancer cells. Analysis of the E2F-1 gene promoter in MCF-7 cells previously showed that hormone-induced transactivation required interactions between estrogen receptor alpha (ER alpha)/Sp1 bound to upstream GC-rich sites and NFYA bound to downstream CCAAT sites within the -169 to -54 region of the promoter. This same region of the E2F-1 promoter was also E2 responsive in ER alpha-positive ZR-75 cells; however, further analysis of the promoter showed that cooperative ER alpha/Sp1/NFY interactions were not necessary for hormone-induced transactivation in ZR-75 cells. The upstream GC-rich motifs (-169 to -111) are activated independently by ER alpha/Sp1 in ZR-75 but not MCF-7 cells, and a construct (pE2F-1j(m1)) containing the -122 to -54 downstream CCAAT site that bound NFYA was also E2 responsive. E2 also induced reporter gene activity in ZR-75 cells transfected with an expression plasmid for a chimeric protein containing the DNA-binding domain of the yeast GAL4 protein fused to NFYA (pM-NFYA) and a construct containing five tandem GAL4 response elements. Subsequent studies showed that hormonal activation of pE2F-1j(m1) and pM-NFYA are dependent on nongenomic pathways in which E2 activates cAMP/protein kinase A. Hormone-dependent regulation of E2F-1 gene expression in ZR-75 and MCF-7 involves the same cis elements and interacting transcription factors but different mechanisms, demonstrating the importance of cell context on transactivation pathways, even among ER-positive breast cancer cell lines.
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PMID:Cell context-dependent differences in the induction of E2F-1 gene expression by 17 beta-estradiol in MCF-7 and ZR-75 cells. 1269 71

Although interleukin-17 (IL-17) is the pre-eminent T-cell-derived pro-inflammatory cytokine, its cellular mechanism of action remains poorly understood. We explored novel signaling pathways mediating IL-17 induction of the cyclooxygenase-2 (COX-2) gene in human chondrocytes, synovial fibroblasts, and macrophages. In preliminary work, recombinant human (rh) IL-17 stimulated a rapid (5-15 min), substantial (>8-fold), and sustained (>24 h) increase in COX-2 mRNA, protein, and prostaglandin E2 release. Screening experiments with cell-permeable kinase inhibitors (e.g. SB202190 and p38 inhibitor), Western analysis using specific anti-phospho-antibodies to a variety of mitogen-activated protein kinase cascade intermediates, co-transfection studies using chimeric cytomegalovirus-driven constructs of GAL4 DNA-binding domains fused to the transactivation domains of transcription factors together with Gal-4 binding element-luciferase reporters, ectopic overexpression of activated protein kinase expression plasmids (e.g. MKK3/6), or transfection experiments with wild-type and mutant COX-2 promoter constructs revealed that rhIL-17 induction of the COX-2 gene was mediated exclusively by the stress-activated protein kinase 2/p38 cascade. A rhIL-17-dependent transcriptional pulse (1.76 +/- 0.11-fold induction) was initiated by ATF-2/CREB-1 transactivation through the ATF/CRE enhancer site in the proximal promoter. However, steady-state levels of rhIL-17-induced COX-2 mRNA declined rapidly (<2 h) to control levels under wash-out conditions. Adding rhIL-17 to transcriptionally arrested cells stabilized COX-2 mRNA for up to 6 h, a process compromised by SB202190. Deletion analysis using transfected chimeric luciferase-COX-2 mRNA 3'-untranslated region reporter constructs revealed that rhIL-17 increased reporter gene mRNA stability and protein synthesis via distal regions (-545 to -1414 bases) of the 3'-untranslated region. This response was mediated entirely by the stress-activated protein kinase 2/p38 cascade. As such, IL-17 can exert direct transcriptional and post-transcriptional control over target proinflammatory cytokines and oncogenes.
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PMID:T-cell-derived interleukin-17 regulates the level and stability of cyclooxygenase-2 (COX-2) mRNA through restricted activation of the p38 mitogen-activated protein kinase cascade: role of distal sequences in the 3'-untranslated region of COX-2 mRNA. 1274 33

Levels of the type IIbeta regulatory subunit (RIIbeta) of protein kinase A are abnormally high in the nuclei of T cells of some subjects with the autoimmune disorder systemic lupus erythematosus (SLE). However, the role of nuclear RIIbeta in the regulation of T cell function is unknown. Based on previous studies demonstrating that nuclear protein kinase A-RII subunits can modify cAMP response element (CRE)-dependent transcription, we tested the hypothesis that nuclear RIIbeta can alter CRE-directed gene expression in T cells through interaction with the nuclear transcription factor CRE-binding protein CREB. To test this hypothesis, we used the RIIbeta-deficient S49 and the Jurkat T cell lines. In both cell lines, transient transfection of RIIbeta resulted in nuclear localization of a portion of the ectopically expressed RIIbeta. In vitro and in vivo analyses revealed a novel, specific interaction between RIIbeta and CREB that mapped to the N-terminal 135 aa of RIIbeta. In functional studies, RIIbeta inhibited the transcriptional activity of a GAL4-CREB fusion protein by 67% in Jurkat T cells following activation with anti-CD3 and anti-CD28 mAbs. Importantly, deletion of the CREB-binding region of RIIbeta completely abrogated inhibition. Additionally, RIIbeta suppressed CRE-directed reporter gene expression and substantially reduced induction of promoter activity and endogenous protein levels of the CREB-dependent gene, c-fos, in activated T cells. We conclude that nuclear RIIbeta can act as a repressor of CREB transcriptional activity in T cells, providing a potential functional significance for aberrant levels of nuclear RIIbeta in systemic lupus erythematosus T cells.
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PMID:Protein kinase A regulatory subunit type II beta directly interacts with and suppresses CREB transcriptional activity in activated T cells. 1450 Jun 61

Cholecystokinin (CCK) is one of the most abundant peptide transmitters in the mammalian brain. Despite the physiological significance of CCK expression in long-term memory and psychiatric disorders, little is known about the factors that regulate the expression of CCK peptides. Here, we report that KCl and forskolin synergistically increase CCK gene transcription via protein kinase A (PKA) and extracellular signal-regulated kinase (ERK) signalling pathways, activating cAMP response element-binding protein (CREB) associated with the CRE(- 80) element of the CCK promoter. Whereas, CREB Ser133 phosphorylation was essential for transcriptional activation, the synergistic stimulation was not correlated to the level of Ser133 phosphorylation, indicating that recruitment and/or activation of additional downstream factors were required for maximal stimulation. Transcriptional activation was reduced by co-expression of adenovirus 12S E1A, that inhibits binding of CREB-binding protein (CBP) to CREB. Moreover GAL4-CREB-DIEDML, which mediates the phosphorylation-independent binding of CBP, and the C-terminal domain of CBP was synergistically activated by forskolin and KCl. Taken together the results imply that neuronal CCK gene transcription is regulated by the cumulative action of calcium and cAMP via stimulation of the PKA and ERK signalling pathways and that synergy is accomplished by the coordinate activation of CREB and CBP.
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PMID:KCl and forskolin synergistically up-regulate cholecystokinin gene expression via coordinate activation of CREB and the co-activator CBP. 1503 Mar 85

cGMP-dependent protein kinase (cGK) forms encoded by the dg2 (for) gene are implicated in behavior and epithelial transport in Drosophila melanogaster. Here, we provide the first biochemical characterization and cellular localization of cGKs encoded by the major transcripts of dg2: dg2P1 and dg2P2. cGMP stimulates kinase activity of DG2P1 (EC(50): 0.13 +/- 0.039 microm) and DG2P2 (EC(50): 0.32 +/- 0.14 microm) in Malpighian tubule and S2 cell extracts. DG2P1 and DG2P2 are magnesium-requiring enzymes and were inhibited by 10 and 100 microm of a cGK inhibitor, 8-(4-chlorophenylthio)guanosine-3',5'-cyclic monophosphorothioate, Rp isomer; whereas DG1, the cGK encoded by the D. melanogaster dg1 gene, was unaffected. DG2P1 and DG2P2 were localized in the plasma membrane in S2 cells, whereas DG1 was localized in the cytosol. The D. melanogaster fluid-transporting Malpighian tubule was used as an organotypic model to analyze cGK localization and function in vivo. Targeted expression of DG2P2, DG2P1, and DG1 in tubule cells via the UAS/GAL4 system in transgenic flies revealed differential localization of all three cGKs in vivo: DG2P2 expression at the apical membrane; DG2P1 expression at both the apical and basolateral membranes; and DG1 expression at the basolateral membrane and in the cytosol. Transgenic tubules for all three cGKs displayed enhanced cGK activity compared with wild-type tubules. The physiological impact of targeted expression of individual cGKs in tubule principal cells was assessed by measuring basal and stimulated rates of fluid transport. DG1 expression greatly enhanced fluid transport by the tubule in response to exogenous cGMP, whereas DG2P2 expression significantly increased fluid transport in response to the nitridergic neuropeptide, capa-1. Thus, dg2-encoded proteins are bona fide cGKs, which have differential roles in epithelial fluid transport, as assessed by in vivo studies. Furthermore, a novel epithelial role is suggested for DG1, which is considerably more responsive to cGMP than to capa-1 stimulation.
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PMID:Analysis of Drosophila cGMP-dependent protein kinases and assessment of their in vivo roles by targeted expression in a renal transporting epithelium. 1521 25

17beta-estradiol (E2) induces ornithine decarboxylase (ODC) activity in several E2-responsive tissues/cells, and this study investigated the mechanism of hormone-induced transactivation in MCF-7 human breast cancer cells. E2-induced reporter gene (luciferase) activity in MCF-7 cells transfected with a construct (pODC1) containing the -164 to +29 region of the human ODC gene promoter linked to bacterial luciferase. This promoter sequence contains GC-rich Sp1 binding sites, CAAT, LSF, cAMP response element (CRE), and TATA motifs. Deletion and mutational analysis of the ODC promoter showed that both CAAT and LSF sites were required for hormone-induced transactivation. Gel mobility shift and DNA footprinting assays indicated that NFYA and LSF bound the CAAT and LSF motifs, respectively, and GAL4-NFYA/GAL4-LSF chimeras were also activated by E2, 8-bromo-cAMP, and protein kinase A (PKA) expression plasmid. However, E2-induced transactivation of GAL4-NFYA and GAL4-LSF was blocked by the PKA inhibitor SQ22356 indicating that the mechanism of ODC induction by E2 involves upregulation of cAMP/PKA through nongenomic pathways of estrogen action.
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PMID:Estrogen-dependent regulation of ornithine decarboxylase in breast cancer cells through activation of nongenomic cAMP-dependent pathways. 1522 48

Transcriptional induction of cyclooxygenase-2 (COX-2) occurs early after T cell receptor triggering and has functional implications in inflammation. Here, we show that phosphodiesterase (PDE)-4 inhibitors block COX-2 induction and prostaglandin synthesis in activated T cells. COX-2 inhibition by PDE4 inhibitors occurs mainly at the transcriptional level. Two response elements for the nuclear factor of activated T cells (NFAT) in the COX-2 promoter were required for inhibition by these drugs. PDE4 inhibitors did not affect NFAT nuclear translocation upon T cell activation; rather they prevented NFAT binding to DNA and induction of the transactivation function of GAL4-NFAT. These effects seem to be cAMP/PKA independent as they were not mimicked by the permeable analog dBcAMP or by forskolin, neither can be reverted by the PKA inhibitors H89 or KT-5720. These results may explain some of the anti-inflammatory properties of PDE4 inhibitors through the blockade of NFAT-mediated transactivation of pro-inflammatory genes such as COX-2.
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PMID:Effect of phosphodiesterase 4 inhibitors on NFAT-dependent cyclooxygenase-2 expression in human T lymphocytes. 1538 Dec 52

Cardiomyocyte hypertrophy is transcriptionally controlled and inhibited by glycogen synthase kinase 3beta (GSK3beta). Myocardin is a muscle-specific transcription factor with yet unknown relation to hypertrophy. Therefore, we investigated whether myocardin is sufficient to induce cardiomyocyte hypertrophy and whether myocardin is regulated by GSK3beta through site-specific phosphorylation. Adenoviral myocardin overexpression induced cardiomyocyte hypertrophy in neonatal rat cardiomyocytes, with increased cell size, total protein amount, and transcription of atrial natriuretic factor (ANF). In vitro and in vivo (HEK 293 cells) kinase assays with synthetic peptides and full-length myocardin demonstrated that myocardin was a "primed" GSK3beta substrate, with serines 455 to 467 and 624 to 636 being the major GSK3beta phosphorylation sites. Myocardin-induced ANF transcription and increase in total protein amount were enhanced by GSK3beta blockade (10 mmol/L LiCl), indicating that GSK3beta inhibits myocardin. A GSK3beta phosphorylation-resistant myocardin mutant (8xA) activated ANF transcription twice as potently as wildtype myocardin under basal conditions with GSK3beta being active. Conversely, a GSK3beta phospho-mimetic myocardin mutant (8xD) was transcriptionally repressed after GSK3beta blockade, indicating that GSK3beta phosphorylation at the sites identified inhibits myocardin transcriptional activity. GAL4-myocardin fusion constructs demonstrated that GSK3beta phosphorylation reduced the intrinsic myocardin transcriptional activity. A cell-permeable (Antennapedia protein transduction tag) peptide containing the mapped myocardin GSK3beta motifs 624 to 636 induced hypertrophy of cultured cardiomyocytes, suggesting that the peptide acted as substrate-based GSK3beta inhibitor in cardiomyocytes. Therefore, we conclude that the GSK3beta-myocardin interaction constitutes a novel molecular control of cardiomyocyte hypertrophy. Phosphorylation by GSK3beta comprises a novel post-transcriptional regulatory mechanism of myocardin.
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PMID:Glycogen synthase kinase 3beta inhibits myocardin-dependent transcription and hypertrophy induction through site-specific phosphorylation. 1614 10


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