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)

Type II secreted phospholipase A(2) (sPLA(2)) releases precursors of important inflammatory lipid mediators from phospholipids. Some observations have indicated that the sPLA(2), which has been implicated in chronic inflammatory conditions such as arthritis, contributes to atherosclerosis in the arterial wall. sPLA(2) was not detected in control vascular smooth muscle cells (VSMC). Treatment of VSMC with agents that increase intracellular cAMP (eg, forskolin, dibutyryl [db]-cAMP) resulted in a time- and concentration-dependent increase in sPLA(2) gene expression. Semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR) showed a marked dose-dependent inhibition of forskolin-induced mRNA by protein kinase A inhibitor. Electrophoretic mobility shift analysis of nuclear proteins from forskolin-treated and db-cAMP-treated VSMC with C/EBP consensus oligonucleotides and C/EBP oligonucleotides from the rat promoter revealed greater binding than in control VSMC. Incubation of VSMC with H89, a specific protein kinase inhibitor, also blocked the binding of nuclear C/EBP to the C/EBP site of the rat promoter induced by db-cAMP and forskolin. Binding was unchanged with the use of CRE consensus oligonucleotides. Antibodies revealed the specific formation of C/EBP/DNA complexes, the majority of which were supershifted by C/EBP-ss and -delta antibodies. Functional activation of C/EBP was confirmed by a luciferase reporter gene assay. A construct comprising 4 tandem repeat copies of the C/EBP element from the rat sPLA(2) promoter linked to luciferase was transcriptionally activated in VSMC by cotransfection with expression vector for the protein kinase A catalytic subunit. It was also significantly activated in transfected VSMC treated by forskolin or db-cAMP. H89 inhibited this activations. We therefore conclude that the increases in sPLA(2) mRNA and enzyme activity produced by cAMP-elevating agents is controlled by a mechanism involving nuclear C/EBP-ss and -delta acting through a protein kinase A signaling pathway.
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PMID:Protein kinase A-dependent stimulation of rat type II secreted phospholipase A(2) gene transcription involves C/EBP-beta and -delta in vascular smooth muscle cells. 1111 53

The lysozyme gene is activated in myelomonocytic HD11 cells in response to LPS. In this study, we described the involvement of LPS-activated signal transduction pathways in activation of the lysozyme gene. Pre-treatment of HD 11 cells with H-89, H-7, TMB-8, or KN-93 resulted in inhibition of the LPS-enhanced lysozyme expression, suggesting that PKA, PKC, and Ca2+-dependent protein kinases participate in the LPS activation. CaMKII seems to be required for the processing of lysozyme transcripts. TPA and calcium ionophore A23187, when separately added to HD11 cells, stimulated the lysozyme expression effectively, and forskolin was ineffective. It is interesting that simultaneous treatment of cells with forskolin and calcium ionophore A23187 resulted in a potentiated increase in lysozyme mRNA expression, indicating a synergistic cooperation of PKA and Ca2+. This synergistic effect of PKA and Ca2+ was observed on the expression of a stably integrated CAT construct, controlled by the lysozyme promoter and the -6.1-kb enhancer containing binding sites for C/EBP and NF-kappaB/Rel. Therefore, we discussed the role of C/EBPbeta(NF-M), CREB, and NF-kappaB/Rel as possible targets for phosphorylation mediated by PKA, PKC, and Ca2+.
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PMID:Involvement of PKA, PKC, and Ca2+ in LPS-activated expression of the chicken lysozyme gene. 1131 Aug 53

CCAAT/enhancer binding protein beta (C/EBP beta) also named liver-enriched transcriptional activating protein (LAP) is a member of the C/EBP family of transcription factors and is involved in hepatocyte-specific gene expression and in the process of tissue differentiation. The activity of LAP/C/EBP beta can be regulated at the transcriptional and posttranslational level or by protein-protein interaction with other transcription factors. In this study we show that LAP/C/EBP beta can stimulate its own transcription. Deletion analysis of the rat LAP/C/EBP beta promoter in luciferase reporter gene experiments demonstrated that the region located between nucleotide -121 to -71, comprising two recently characterized cAMP responsive element (CRE)-like elements, is important for autoregulation. Gel shift experiments using oligonucleotides with overlapping point mutations identified the sequence GCAATGA (beta-site) adjacent to and partially overlapping the first CRE-like site as core motif for LAP/C/EBP beta binding. Analysis of a mutated beta-site in reporter gene experiments showed the functional relevance of this site for autoregulation. The composite C/EBP beta-CRE-element in the promoter enables synergistic activation of transcription by LAP/C/EBP beta and the protein kinase A (PKA)/cAMP responsive element binding protein (CREB) pathway in a cell-type specific manner. In hepatoma cells nuclear factor kappa B (NF-kappa B) increased autoregulation and therefore could mediate enhanced activation during inflammatory responses. In summary, our results demonstrated that the assembly of the three binding sites in the promoter and thus the interaction between LAP/C/EBP beta and members of the CREB or NF-kappa B family allows the control of LAP/C/EBP beta gene transcription as a response to different stimuli in a tissue specific manner.
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PMID:Autoregulation enables different pathways to control CCAAT/enhancer binding protein beta (C/EBP beta) transcription. 1139 64

Interleukin-1 (IL-1) inhibits the proliferation of A375 human melanoma cells. We have demonstrated previously that p38 mitrogen-activated protein kinase (MAPK) mediated the antiproliferative effect of IL-1 partially through the downregulation of activity and protein level of ornithine decarboxylase (ODC). In this study, we investigated the role of CCAAT/enhancer-binding protein (C/EBP) homologous protein (CHOP), one of the p38 MAPK target transcriptional factors. The mRNA level of CHOP was not affected by IL-1 treatment in A375-6 cells. Unexpectedly, CHOP was constitutively phosphorylated, and IL-1 or p38 MAPK inhibitor, SB203580, did not affect the phosphorylation level. However, A375-6 cells exhibited enhanced sensitivity to IL-1 by transfecting CHOP expression plasmid and reduced sensitivity to IL-1 by antisense CHOP mRNA expression plasmid. Furthermore, CHOP appeared to regulate positively IL-6 production at the transcriptional level. The experiments using CHOP muteins revealed that dimerization ability - but not p38 MAPK-dependent phosphorylation or DNA binding activity - is important for the IL-6 inducing activity of CHOP. These results indicate that CHOP contributes to the IL-1 growth-inhibitory signal through augmenting IL-6 production.
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PMID:CHOP, a basic leucine zipper transcriptional factor, contributes to the antiproliferative effect of IL-1 on A375 human melanoma cells through augmenting transcription of IL-6. 1142 63

Structure/function analysis of CCAAT/enhancer binding proteins (C/EBP) alpha and beta have shown that they possess both constitutive and cAMP inducible activities. Three regions conserved between C/EBPalpha and beta were identified which lie within the cAMP inducible domains of each protein. Deletion analysis of these conserved regions within C/EBPalpha show that conserved region 2 plays a particularly critical role in mediating the PKA inducible activity of the protein, however, the constitutive activity of conserved region 2 depends on promoter context. This data supports previous findings that constitutive and cAMP responsiveness are mediated by domains of the protein that do not directly overlap, suggesting that they occur through distinct mechanisms.
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PMID:Characterization of domains in C/EBPalpha that mediate its constitutive and cAMP-inducible activities. 1147 38

There is positive feedback pathway in the ovine large luteal cell, such that prostaglandin (PG) F(2 alpha) stimulation induces intraluteal PGF(2 alpha) production as the result of induction of one of the rate-limiting enzymes in PG production, cyclooxygenase-2 (Cox-2). The objective of the present study was to evaluate the intracellular effector systems and important DNA transcriptional element(s) involved in regulating the Cox-2 gene in ovine large luteal cells. In transient transfection assays, Cox-2 promoter was rapidly induced (4 h) by phorbol didecanoate (a protein kinase [PK] C activator), ionomycin, and cloprostenol (PGF(2 alpha) analogue), with a peak induction at 12 h. Cloprostenol-mediated promoter activation was not blocked by inhibition of various second messenger systems, including PKA, calcium calmodulin kinase II, or mitogen-activated protein kinases. However, myristoylated PKC pseudosubstrate peptide inhibited cloprostenol stimulation of Cox-2 promoter, indicating the critical role of PKC in this stimulation. The Cox-2 promoter could be reduced to 282 base pairs (bp) of the 5' flanking sequence with retention of full inducibility by cloprostenol. Mutation of three critical cis-responsive elements within this 282-bp region (C/EBP, cAMP responsive element [CRE], and E-box) indicated that E-box was critical in both basal and cloprostenol-induced promoter activity. However, there was also significant but less dramatic inhibition of cloprostenol stimulation by mutation of C/EBP and CRE in the Cox-2 promoter, and mutation of all three elements eliminated cloprostenol induction of this promoter. Electrophoretic mobility shift assays of nuclear extracts from large luteal cells revealed that upstream stimulatory factor (USF)-1 and USF-2 bound to the E-box in Cox-2. Thus, PKC directly regulates transcription of the Cox-2 gene in large luteal cells by acting through DNA elements close to the putative transcriptional start point, particularly an E-box region at -50 bp.
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PMID:Transcriptional regulation of cyclooxygenase-2 gene in ovine large luteal cells. 1167 76

The essential role of CCAAT/enhancer binding proteins (C/EBPs) beta and delta for adipocyte differentiation has been clearly established. In preadipocytes, their expression is up-regulated by the activation of leukemia inhibitory factor receptor (LIF-R) and prostacyclin receptor (IP-R) via the extracellular signal-regulated kinase (ERK) pathway and cAMP production, respectively. However, the molecular mechanisms by which LIF and prostacyclin-induced signals are propagated to the nucleus and the transcription factors mediating ERK and cAMP-induced C/EBP gene expression were unknown. Here we report that both pathways share cAMP responsive element binding protein/activation transcription factor 1 (CREB/ATF-1) as common downstream effectors. LIF-R and IP-R activation induced binding of CREB and/or ATF-1 to C/EBP promoters and CREB-dependent transcription. Expression of dominant negative forms of CREB dramatically reduced the LIF- and prostacyclin-stimulated C/EBP beta and C/EBP delta expression. Upon stimulation of the IP-R, the ERK pathway was activated in a PKA-dependent manner. ERK activation by the PKA pathway was not required for CREB/ATF-1 phosphorylation but rather was necessary for CREB-dependent up-regulation of C/EBPs expression. Our findings suggest that ERK activation is required for CREB transcriptional activity, possibly by recruitment of a coactivator.
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PMID:Activation of extracellular signal-regulated kinases and CREB/ATF-1 mediate the expression of CCAAT/enhancer binding proteins beta and -delta in preadipocytes. 1168 32

This study was designed to elucidate the molecular mechanism(s) mediating cyclooxygenase-2 (Cox-2) regulation during differentiation of the granulosa cell. The 5' flanking sequence of the Cox-2 gene was linked to a vector with a luciferase reporter gene, and this vector was transfected into freshly isolated bovine granulosa cells or granulosa cells after culture with or without forskolin to induce luteinization in vitro. The Cox-2 promoter was inducible by 8-bromo cAMP but not by phorbol esters in fresh granulosa cells, and maximal expression by cAMP was delayed until 48 h after treatment. In contrast, after luteinization of granulosa cells by 8-day treatment with forskolin, the Cox-2 promoter was immediately inducible by phorbol esters but not by cAMP. In granulosa cells cultured for 8 days without forskolin, the Cox-2 promoter continued to be inducible only by cAMP and not by phorbol esters. Unexpectedly, no delay was observed in the induction of Cox-2 by cAMP in granulosa cells that were cultured without forskolin, compared with an approximately 1 day delay in Cox-2 induction by cAMP in fresh granulosa cells. Myristoylated protein kinase (PK) A and PKC inhibitory peptides were utilized to further confirm the PKA- or PKC-dependence of Cox-2 induction. Time-course experiments showed that only 2 days of forskolin treatment could induce PKC-responsiveness of the Cox-2 promoter, although maximal responsiveness was not observed until 10 days of luteinization. Promoter activity was also analyzed in a series of deletion mutants as well as site-directed mutants of C/EBP, CRE, and E-box. A 282-base pair sequence in the Cox-2 5' flanking region maintained full inducibility by PKA in granulosa cells and by PKC in luteinized granulosa cells. The E-box element was found to be the critical regulatory element for Cox-2 induction by either PKA in granulosa cells or by PKC in luteinized granulosa cells. Electrophoretic mobility shift assays were performed on nuclear extracts from fresh or luteinized granulosa cells. Upstream stimulatory factor (USF)-1 and USF-2 bound to the E-box of the Cox-2 gene, and binding was similar for nuclear extracts from fresh, cultured, or luteinized granulosa cells. Thus, although luteinization changes transcriptional regulation of Cox-2 from PKA- to PKC-dependence, the crucial role of the E-box element in this transcriptional activation is conserved.
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PMID:Transcriptional regulation of the cyclooxygenase-2 gene changes from protein kinase (PK) A- to PKC-dependence after luteinization of granulosa cells. 1196 17

The abundant secretion of type IIA secreted phospholipase A(2) (sPLA(2)) is a major feature of the inflammatory process of atherosclerosis. sPLA(2) is crucial for the development of inflammation, as it catalyses the production of lipid mediators and induces the proliferation of smooth muscle cells. We have analysed the activation of sPLA(2) transcription by cAMP and interleukin-1beta (IL-1beta), and shown that the 500 bp region upstream of the transcription start site of the rat sPLA(2) gene is implicated in activation by synergistically acting cAMP and IL-1beta. We transiently transfected and stimulated rat smooth muscle cells in primary culture and measured the promoter activities of serial and site-directed deletion mutants of sPLA(2)-luciferase constructs. A distal region, between -488 and -157 bp, bearing a CAAT/enhancer binding protein (C/EBP)-responsive element (-242 to -223) was sufficient for cAMP/protein kinase A-mediated sPLA(2) promoter activation. We find evidence for the first time that activation of the sPLA(2) promoter by IL-1beta requires activation of an Ets-responsive element in the -184 to -180 region of the distal promoter via the Ras pathway and a nuclear factor-kappaB site at positions -141 to -131 of the proximal promoter. We also used electrophoretic mobility shift assays to identify five binding sites for the Sp1 factor; a specific inhibitor of Sp1, mithramycin A, showed that this factor is crucial for the basal activity of the sPLA(2) promoter.
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PMID:Transcriptional regulation of the rat type IIA phospholipase A2 gene by cAMP and interleukin-1beta in vascular smooth muscle cells: interplay of the CCAAT/enhancer binding protein (C/EBP), nuclear factor-kappaB and Ets transcription factors. 1218 23

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


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