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

Recently, it was shown that lipoprotein lipase (LPL) was produced in neonatal but not in adult rat liver. In an attempt to further define the mechanism involved in liver LPL expression, we identified a neonatal mouse hepatoma cell line, BWTG3, capable of producing LPL. The regulation of LPL expression by various extracellular stimuli was investigated in this cell line. Progesterone caused a rise in LPL production by BWTG3 cells. Other hormones tested, such as insulin, glucagon, adrenalin, testosterone, and thyroid hormone, had no effect on LPL production. The effects of progesterone on LPL production showed slow kinetics reaching a maximum 24 h after addition. Cotransfection of a progesterone receptor expression vector with a 5'-LPL-CAT reporter construct resulted in an induction of CAT activity, suggesting that the increase in LPL accumulation after progesterone was linked to transcriptional induction of the LPL gene. Stimuli causing an elevation of protein kinase A activity in the cells also increased LPL production. Three agents capable of elevating intracellular cAMP levels, i.e., forskolin, dBcAMP, and choleratoxin, caused an elevation of LPL production. The increase in LPL activity caused by forskolin and choleratoxin was paralleled by an elevation of LPL mRNA levels, while dBcAMP only induced a small elevation of LPL mRNA levels. The increase in LPL production was shown to be linked to the stimulation of the PKA signal transduction pathway and was apparently transmitted via the transcription factor CREB. No effect of the stimulation of protein kinase C or calcium/calmodulin-dependent kinase on LPL production was detected.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Lipoprotein lipase expression in undifferentiated hepatoma cells is regulated by progesterone and protein kinase A. 132 33

The transactivation of genes through the cAMP-regulated enhancer (CRE) is proposed to occur by the binding and phosphorylation of the transcription factor CREB (CRE-binding protein). Originally believed to be a single protein, more than 10 different CREB proteins have been cloned. The contributions of each of these factors to gene regulation have yet to be determined unambiguously. We have isolated a CREB cDNA that contains a mutation of a single amino acid in the DNA-binding domain. In gel shift assays, this mutant, designated KCREB, is unable to bind to the somatostatin (SS) CRE. In addition, KCREB acts as a dominant repressor of the wild-type factor, blocking the ability of wild-type CREB to bind to the CRE when present as a KCREB:CREB heterodimer. The KCREB mutant also acts as a dominant repressor in vivo, completely blocking the ability of wild-type CREB to mediate induction by protein kinase-A of a SS CRE reporter gene in F9 teratocarcinoma cells. We have used this mutant to analyze the participation of CREB in the induction of the SS promoter in CA-77 cells, a medullary thyroid carcinoma cell line that produces high levels of SS. Although KCREB can block a portion of the cAMP induction of the SS promoter in CA-77 cells, approximately 45% of the induction remains insensitive to the mutant. These data support the paradigm that CREB is involved in the cAMP induction of SS in vivo. Furthermore, the inability of KCREB to completely block cAMP-mediated SS expression in CA-77 cells suggests that additional factors may contribute to the cAMP regulation of CRE function.
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PMID:A dominant repressor of cyclic adenosine 3',5'-monophosphate (cAMP)-regulated enhancer-binding protein activity inhibits the cAMP-mediated induction of the somatostatin promoter in vivo. 135 57

Many eukaryotic genes are regulated by cAMP through a conserved cAMP response element (CRE). Here we show that, in the pancreatic islet cell line Tu6, a well-characterized CRE in the somatostatin gene does not provide cAMP responsiveness but functions as an essential element for its basal activity. DNA-binding and functional analyses indicate that the cAMP-responsive factor CREB regulates somatostatin expression in these cells without requirement for phosphorylation at the protein kinase A-regulated Ser-133 phosphorylation site. In addition to the CRE site, cell-specific expression of the somatostatin gene requires a second promoter element, which binds the recently characterized LIM family protein Isl-1. Thus, Isl-1 and CREB appear to synergize on the somatostatin promoter to stimulate high-level expression in Tu6 cells. The ability of CREB to function in a phosphorylation-independent manner suggests a mechanism by which this protein can regulate gene transcription.
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PMID:The LIM family transcription factor Isl-1 requires cAMP response element binding protein to promote somatostatin expression in pancreatic islet cells. 135 85

Cyclic AMP treatment of hepatoma cells leads to increased protein binding at the cyclic AMP response element (CRE) of the tyrosine aminotransferase (TAT) gene in vivo, as revealed by genomic footprinting, whereas no increase is observed at the CRE of the phosphoenolpyruvate carboxykinase (PEPCK) gene. Several criteria establish that the 43 kDa CREB protein is interacting with both of these sites. Two classes of CRE with different affinity for CREB are described. One class, including the TATCRE, is characterized by asymmetric and weak binding sites (CGTCA), whereas the second class containing symmetrical TGACGTCA sites shows a much higher binding affinity for CREB. Both classes show an increase in binding after phosphorylation of CREB by protein kinase A (PKA). An in vivo phosphorylation-dependent change in binding of CREB increases the occupancy of weak binding sites used for transactivation, such as the TATCRE, while high affinity sites may have constitutive binding of transcriptionally active and inactive CREB dimers, as demonstrated by in vivo footprinting at the PEPCK CRE. Thus, lower basal level and higher relative stimulation of transcription by cyclic AMP through low affinity CREs should result, allowing finely tuned control of gene activation.
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PMID:Phosphorylation of CREB affects its binding to high and low affinity sites: implications for cAMP induced gene transcription. 135 12

Transcription of the ornithine decarboxylase (ODC) gene is rapidly elevated by activation of protein kinase A (PKA). The additive influence of three cis-acting elements is responsible for this regulation in an adrenal carcinoma cell line. Two sites, CRE2 at -48 base pairs (bp) relative to the start of transcription and CRE3 at +95 bp, are identical to the core motif of the cAMP-responsive element (CRE) of the somatostatin gene and are conserved in the mouse, rat, and human ODC genes. Mutation of CRE2 resulted in a substantial decrease in basal promoter activity, as well as a 5-fold decrease in inducibility of the ODC promoter by PKA. CRE3 did not contribute to the basal activity of the ODC promoter, but mutation of this site resulted in a 2-fold decrease in inducibility by PKA. Deletion of a 45-bp sequence (GC-box) located 5' of CRE2, also resulted in a 2-fold decrease in inducibility of the ODC promoter. DNase I protection revealed the presence of protein binding at CRE2, the TATA box, and the GC-box of the ODC promoter. Mutation of CRE2 resulted in loss of protection of this sequence, as well as the 3' extension of the footprint over the TATA box, without affecting interactions at the GC box. Antibodies to the well characterized CRE-binding protein CREB recognized proteins binding to CRE2, suggesting that binding of CREB, or an antigenically related protein, is important for the activity of CRE2. Additionally, recombinant CREB bound to a DNA probe containing the CRE2 sequence.
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PMID:Multiple DNA elements responsible for transcriptional regulation of the ornithine decarboxylase gene by protein kinase A. 135 8

Carnitine palmitoyltransferase (CPT) regulates the flux of long-chain fatty acids into the mitochondria for subsequent beta-oxidation. A 485 bp segment of the promoter for the gene encoding the 68 kDa CPT was isolated from a rat lambda DASH genomic library using the polymerase chain reaction. The promoter contained a consensus binding sequence for CREB (cyclic AMP response element binding protein) at -153 to -166, and for C/EBP alpha (CCAAT/enhancer binding protein) at -115 to -128. DNAase I footprinting using proteins isolated from rat liver nuclei indicated the presence of several regions of nuclear protein binding, most notably at -95 to -130, at -273 to -295, and at a wide region encompassing -395 to -465. DNAase I footprinting studies with purified CREB and C/EBP alpha confirmed that protein binding to DNA occurred at the sites predicted by the consensus sequences. The segment containing 481 bp of 5' flanking sequence plus 181 bp of untranslated mRNA was ligated to the structural gene for chloramphenicol acetyltransferase (CAT). When this plasmid was transfected into Hep G2 cells, CAT activity was stimulated 7-fold by addition of 1 mM-8-bromo-cyclic AMP (8-Br-cAMP) or co-transfection of the expression vector coding for the catalytic subunit of protein kinase A (PKA). The ability of several known second messengers and transcription factors to stimulate transcription of 68 kDa CPT promoter-CAT reporter was tested in co-transfection experiments. 68 kDa CPT promoter-CAT reporter transcription activity was stimulated 7-fold by addition of 8-Br-cAMP, and this induction was depressed 50% by the addition of phorbol esters. When the 68 kDa CPT promoter-CAT reporter was co-transfected with an expression vector for CREB or C/EBP alpha, transcription was increased 3- and 10-fold respectively. 8-Br-cAMP caused an additional 8-fold induction in the presence of each factor to yield 25- and 80-fold induction respectively. Co-transfection of the expression vector for c-jun also increased the CAT activity driven by the 68 kDa CPT promoter, while co-transfection with the expression vector for c-fos had no effect. When expression vectors for both c-jun and c-fos were co-transfected with the 68 kDa CPT promoter, c-fos depressed the induction seen with c-jun alone.
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PMID:Isolation and characterization of the promoter for the gene coding for the 68 kDa carnitine palmitoyltransferase from the rat. 825 Aug 54

CG is encoded by separate alpha- and beta-subunit genes. Expression of both genes is stimulated by cAMP, but the kinetics of activation are different, with cAMP stimulation of the alpha gene preceding that of the beta gene. The cAMP response element (CRE) in the alpha gene contains a palindromic DNA sequence, TGACGTCA, that binds the transcription factor CREB, a nuclear phosphoprotein that is activated by protein kinase-A. Previously, detailed characterization of a CRE in the CG beta gene had been difficult due to low levels of expression in transfected cells. In this study the 5'-flanking sequence of the CG beta gene was fused to a sensitive luciferase (LUC) reporter gene, allowing delineation of a CG beta CRE in transient expression assays performed in JEG-3 choriocarcinoma cells. The full-length CG beta promoter, -3700 to 362 basepairs (bp), was stimulated 8- to 14-fold by treatment with 1 mM 8-bromo-cAMP. Analyses of a series of deletion mutants in the CG beta promoter demonstrated that -311 CG beta LUC retained nearly complete cAMP stimulation, but deletion to -187 bp eliminated cAMP responsiveness. Overlapping DNA fragments between -311 and -30 bp were fused to a heterologous promoter (-99 alpha LUC) to further define the locations of basal elements and CREs. Basal expression required a combination of at least two distinct elements between -311 and -30 bp, whereas cAMP responsiveness was conferred by sequences between -311 and -202 bp. Shorter DNA sequences within this region were insufficient for cAMP stimulation, suggesting that more than one element may be required. DNase-I footprinting and gel mobility shift studies demonstrated at least three distinct protein-binding sites within the CG beta CRE sequence. Recombinant CREB (expressed in E. coli) did not bind to these sites, and they share no sequence homology with the alpha gene CRE, indicating that a cAMP-responsive transcription factor other than CREB interacts with the CG beta promoter.
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PMID:Novel cyclic adenosine 3',5'-monophosphate response element in the human chorionic gonadotropin beta-subunit gene. 164 92

Many promoters respond transcriptionally to elevated levels of cAMP through the cAMP-responsive enhancer (CRE). Several proteins have been characterized which bind to the CRE and presumably modulate CRE-dependent transcription. Of these CRE-binding proteins, only CREB has been shown to be activated by cAMP-dependent protein kinase A (PKA), and as such, CREB represents the only basis for our understanding of cAMP-regulated transcriptional activity. In this report, we describe the complete cDNA sequence of another CRE-binding protein, ATF-1. This protein contains a consensus phosphorylation site for PKA and shares extensive homology with CREB in the region surrounding and carboxyl-terminal to the PKA site. ATF-1 does not contain sequences homologous to the glutamine-rich amino-terminal domain found in CREB, however. ATF-1, like CREB, is expressed in a wide variety of cell types, and ATF-1 is capable of dimerizing with CREB. Both ATF-1 homodimers and ATF-1/CREB heterodimers bind to the CRE but not to the related phorbol ester response element. ATF-1 is as active as CREB in its ability to mediate the transcriptional effects of PKA, and, because ATF-1 has a smaller effect on basal expression, it is actually more responsive than CREB to cAMP. These findings indicate that CREB is not unique in its ability to mediate cAMP-dependent transcriptional regulation.
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PMID:The cAMP-regulated enhancer-binding protein ATF-1 activates transcription in response to cAMP-dependent protein kinase A. 165 49

Cyclic AMP mediates the hormonal stimulation of a number of eukaryotic genes by directing the protein kinase A (PK-A)-dependent phosphorylation of transcription factor CREB. We have previously determined that although phosphorylation at Ser-133 is critical for induction, this site does not appear to participate directly in transactivation. To test the hypothesis that CREB ultimately activates transcription through domains that are distinct from the PK-A site, we constructed a series of CREB mutants and evaluated them by transient assays in F9 teratocarcinoma cells. Remarkably, a glutamine-rich region near the N terminus appeared to be important for PK-A-mediated induction of CREB since removal of this domain caused a marked reduction in CREB activity. A second region consisting of a short acidic motif (DLSSD) C terminal to the PK-A site also appeared to synergize with the phosphorylation motif to permit transcriptional activation. Biochemical experiments with purified recombinant CREB protein further demonstrate that the transactivation domain is more sensitive to trypsin digestion than are the DNA-binding and dimerization domains, suggesting that the activator region may be structured to permit interactions with other proteins in the RNA polymerase II complex.
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PMID:Characterization of motifs which are critical for activity of the cyclic AMP-responsive transcription factor CREB. 167 8

We applied Southwestern and Western blotting and gel retardation techniques to investigate the changes that occur in the cyclic adenosine monophosphate (cAMP)-responsive element (CRE) binding (CREB) proteins in rapidly growing, chemically induced 5123tc and 5123D Morris hepatomas. Using the CRE sequences from the c-fos, E2A, and somatostatin gene promoters, we identified in the nuclear proteins from normal unstimulated or proliferating rat liver cells six different protein factors of Mr 34,000, 36,000, 40,000, 47,000, 56,000, and 72,000 capable of binding to the element. The Mr 47,000 protein had the highest specificity for the core CRE, suggesting its importance in cAMP-mediated gene expression. We could not find the Mr 47,000 CREB protein in the 5123tc and 5123D hepatomas. Our efforts to detect this protein in the tumors by (a) using the CRE sequence from different gene promoters, (b) altering the protocol for extracting nuclear proteins, or (c) attempting to restore its DNA-binding property by phosphorylation [with endogenous protein kinase(s), a catalytic subunit of cAMP-dependent protein kinase, and protein kinase C/dephosphorylation (with alkaline phosphatase)] were unsuccessful. The loss of tje Mr 47,000 CREB protein from solid tumors of the Morris hepatoma is likely to be related to the neoplastic properties of the tumor cell rather than to cell growth because the level of this protein remained unchanged during a 6-day period of liver regeneration. The nuclear extract from the Morris hepatoma that did not have the Mr 47,000 CRE-binding factor contained proteins immunologically related to the CREB, c-Jun, and c-Fos proteins. We conclude that the Mr 47,000 factor represents a distinct member of the CRE-binding protein family and that its absence from the hepatomas may lead to aberrant expression of cAMP-inducible genes.
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PMID:Changes in cyclic adenosine monophosphate-responsive element binding proteins in rat hepatomas. 182 83


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