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:2.7.11.1 (protein kinase)
81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have initiated an analysis of protein kinase A (PKA) in Drosophila using transgenic techniques to modulate PKA activity in specific tissues during development. We have constructed GAL4/UAS-regulated transgenes in active and mutant forms that encode PKAc, the catalytic subunit of PKA, and PKI(1-31), a competitive inhibitor of PKAc. We present evidence that the wild-type transgenes are active and summarize the phenotypes produced by a number of GAL4 enhancer-detector strains. We compare the effects of transgenes encoding PKI(1-31) with those encoding PKAr*, a mutant regulatory subunit that constitutively inhibits PKAc because of its inability to bind cyclic AMP. Both inhibitors block larval growth, but only PKAr* alters pattern formation by activating the Hedgehog signaling pathway. Therefore, transgenic PKI(1-31) should provide a tool to investigate the role of PKAc in larval growth regulation without concomitant changes in pattern formation. The different effects of PKI(1-31) and PKAr* suggest two distinct roles, cytoplasmic and nuclear, for PKAc in Hedgehog signal transduction. Alternatively, PKAr* may target proteins other than PKAc, suggesting a role for free PKAr in signal transduction, a role inhibited by PKAc in reversal of the classical relationship of these subunits.
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PMID:Transgenic inhibitors identify two roles for protein kinase A in Drosophila development. 1022 60

Bovine cholesterol side-chain cleavage cytochrome P450 (P450scc; product of the CYP11A gene) gene expression is regulated by gonadotropins via cAMP in the ovary, and by ACTH via cAMP in adrenal cortical cells. Previously, we characterized response elements located at -57/-32 and at -111/-101 bp in the 5'-flanking region of the bovine CYP11A gene required for cAMP-stimulated transcription in both mouse Y-1 adrenal tumor cells and bovine ovarian cells in primary culture, which bind SF-1 (or Ad4-BP) and Sp1, respectively. The role of these transcription factors in CYP11A transcription was further confirmed by deletion and mutation analyses. In addition, results obtained employing a double mutation of the Sp1- and SF-1-binding sites and a mammalian two-hybrid system indicate that Sp1 and SF-1 function cooperatively in the transactivation of the bovine CYP11A promoter in both bovine luteal cells and Y-1 cells. Here we report that SF-1 and Sp1 are able to associate with one another in vitro and in vivo. The NH2-terminal region of SF-1, especially the DNA-binding domain, is the binding site for Sp1. In addition, as CBP is a common coactivator required for the transcriptional activity of numerous transcription factors including nuclear receptors, we investigated whether CBP functions as a cofactor for the regulation of bovine CYP11A promoter activity. We show here that CBP enhanced the PKA-induced CYP11A promoter activity, while a double mutation of both Sp1 and SF-1 sites within the CYP11A promoter region abolished CBP-induced activity. Furthermore, CBP stimulated Sp1-dependent transactivation, and a CBP/Sp1 complex in vivo was demonstrated by a co-immunoprecipitation assay. Also, CBP potentiated the transcriptional activity of GAL4-SF-1 in the presence of PKA. Thus, the cooperation between SF-1 and Sp1, required for the regulation of bovine CYP11A gene expression, is mediated by a direct protein-protein interaction and/or the common coactivator CBP.
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PMID:Molecular mechanism for cooperation between Sp1 and steroidogenic factor-1 (SF-1) to regulate bovine CYP11A gene expression. 1045 66

The transcriptional repressor for rat vasoactive intestinal polypeptide receptor 1 (VIPR-RP) is a recently isolated transcription factor. In this study, we have characterized the functional domains of VIPR-RP and the importance of phosphorylation on VIPR-RP function. Using various regions of VIPR-RP in gel mobility shift assays, we show that the amino acid sequences between positions 367 and 475 play an essential role for VIPR-RP DNA binding. Transient transfection of fusion constructs containing GAL4 DNA binding domain and different parts of VIPR-RP indicated that there are two separate transcriptional repression domains in VIPR-RP, located between amino acids 50 and 101 and between 469 and 527. We demonstrated that VIPR-RP is phosphorylated in vitro by casein kinase II on Ser-69/71 and Thr-110, and by cAMP-dependent kinase on Ser-245/361. Furthermore, by site-directed mutagenesis, we show that phosphorylation of the casein kinase II sites potentiates VIPR-RP transcriptional repression activity by enhancing its nuclear translocation, and that phosphorylation by cAMP-dependent kinase inhibits VIPR- RP transcriptional repression function without affecting its subcellular localization. These observations suggest that phosphorylation plays an important role in regulating VIPR-RP function.
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PMID:Phosphorylation modulates the function of the vasoactive intestinal polypeptide receptor transcriptional repressor protein. 1062 61

In liver and kidney, the terminal step in gluconeogenesis is catalyzed by glucose-6-phosphatase. To examine the effect of the cAMP signal transduction pathway on transcription of the gene encoding the catalytic subunit of glucose-6-phosphatase (G6Pase), G6Pase-chloramphenicol acetyltransferase (CAT) fusion genes were transiently transfected into either the liver-derived HepG2 or kidney-derived LLC-PK cell line. Co-transfection of an expression vector encoding the catalytic subunit of cAMP-dependent protein kinase (PKA) markedly stimulated G6Pase-CAT fusion gene expression, and mutational analysis of the G6Pase promoter revealed that multiple regions are required for this PKA response in both the HepG2 and LLC-PK cell lines. A sequence in the G6Pase promoter that resembles a cAMP response element is required for the full PKA response in both HepG2 and LLC-PK cells. However, in LLC-PK cells, but not in HepG2 cells, a hepatocyte nuclear factor-1 (HNF-1) binding site was critical for the full induction of G6Pase-CAT expression by PKA. Changing this HNF-1 motif to that for the yeast transcription factor GAL4 reduces the PKA response in LLC-PK cells to the same degree as deleting the HNF-1 site. However, co-transfection of this mutated construct with chimeric proteins comprising the GAL4-DNA binding domain ligated to the coding sequence for HNF-1alpha, HNF-1beta, HNF-3, or HNF-4 completely restored the PKA response. Thus, we hypothesize that, in LLC-PK cells, HNF-1 is acting as an accessory factor to enhance PKA signaling through the cAMP response element by altering G6Pase promoter conformation or accessibility rather than specifically affecting some component of the PKA signal transduction pathway.
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PMID:Differential role of hepatocyte nuclear factor-1 in the regulation of glucose-6-phosphatase catalytic subunit gene transcription by cAMP in liver- and kidney-derived cell lines. 1076 45

Recently, cyclic GMP-dependent protein kinase (cGK) was shown to translocate to the nucleus and regulate gene transcription. To determine whether cGK I proteins function as transcriptional activators, we produced the constructs of cGK Ialpha or Ibeta fused with the DNA binding domain of the yeast transcriptional activator GAL4. Here, we demonstrate that the amino-terminal region of cGK Ibeta (amino acids 1-107) exhibits transcriptional activation in yeast. However, full-length cGK Ialpha and Ibeta and the amino-terminal region of cGK Ialpha had no transcriptional activation function. Amino acid replacement in the leucine zipper motif of the amino-terminal region of cGK Ibeta substantially reduced transcriptional activation. These results suggest that the Ibeta-specific region in cGK I proteins may interact with other proteins by way of the leucine zipper motif and has a transcriptional activation function.
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PMID:Specific domain of cGMP-dependent protein kinase Ibeta but not Ialpha functions as a transcriptional activator in yeast. 1077 36

The results of the current studies define the major elements whereby glucose metabolism in islet beta-cells leads to transcriptional activation of an early response gene in insulinoma cell lines and in rat islets. Glucose stimulation (2-20 mm) resulted in a 4-fold increase in Egr-1 mRNA at 30 min, as did the depolarizing agents KCl and tolbutamide. This response was inhibited by diazoxide and EGTA, indicating that beta-cell depolarization and Ca(2+) influx, respectively, are essential. Pharmacological inhibition of the Egr-1 induction by H89 (48%) and calmidazolium (35%), but not by mitogen-activated protein kinase/extracellular signal-regulated kinase kinase 1 and 2 or phosphatidylinositol 3-kinase inhibitors, implied that protein kinase A and Ca(2+)/calmodulin pathways are involved. Deletion mapping of the Egr-1 promoter revealed that the proximal -198 base pairs containing two serum response elements (SREs) and one cAMP-response element retained the depolarization response. Depolarization resulted in phosphorylation of cAMP-response element-binding protein, yet partial inhibition by a dominant negative cAMP-response element-binding protein, along with a robust response of a cAMP-response element-mutated Egr-1 promoter suggested the presence of a second Ca(2+)-responsive element. Depolarization activation of 5XSRE-LUC and serum response factor (SRF)-GAL4 constructs, along with activation of SRF-GAL4 by co-transfection with constitutively active calmodulin kinase IV and protein kinase A, and binding of Ser(103)-phosphorylated SRF in nuclear extracts, indicated that the SRE.SRF complexes contribute to the Ca(2+)-mediated transcriptional regulation of Egr-1. The results of the current experiments demonstrate for the first time SRE-dependent transcription and the role of SRF, a transcription factor known to be a major component of growth responses, in glucose-mediated transcriptional regulation in insulinoma cells.
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PMID:Activation of serum response factor in the depolarization induction of Egr-1 transcription in pancreatic islet beta-cells. 1082 28

The mechanisms by which excitable cells adapt and respond to changes in O2 levels remain largely unknown. We have investigated the effect of hypoxia on the cyclic AMP response element binding protein (CREB) transcription factor. PC12 cells were exposed to moderate levels of hypoxia (5% O2) for various times between 20 min and 6 hr. We found that hypoxia rapidly and persistently induced ser133 phosphorylation of CREB. This effect was more robust than that produced by exposing PC12 cells to either forskolin, KCl, or NGF. This effect was not due to activation of any of the previously known CREB kinases, including PKA, CaMK, PKC, p70s6k, or MAPKAP kinase-2. Thus, hypoxia may induce activation of a novel CREB kinase. To test whether phosphorylation of CREB was associated with an activation of CRE-dependent gene expression, cells were transfected with wild type and mutated regions of the 5'-flanking region of the tyrosine hydroxylase (TH) gene fused to a CAT reporter gene. Mutation of the CRE element in a TH reporter gene reduced, but did not abolish, the effects of hypoxia on TH gene expression. However, hypoxia did not induce transactivation of a GAL4-luciferase reporter by a GAL4-CREB fusion protein. Thus, the mechanism by which hypoxia regulates CREB is distinct, and more complex, than that induced by forskolin, depolarization, or nerve growth factor.
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PMID:Regulation of CREB by moderate hypoxia in PC12 cells. 1084 56

The transcription factor serum amyloid A (SAA)-activating factor (SAF), a family of zinc finger proteins, plays a significant role in the induced expression of the SAA gene. Activity of SAF is regulated by a phosphorylation event involving serine/threonine protein kinase (Ray, A., Schatten, H., and Ray, B. K. (1999) J. Biol. Chem. 274, 4300-4308; Ray, A., and Ray, B. K. (1998) Mol. Cell. Biol. 18, 7327-7335). However, the identity of the protein kinase has so far remained unknown. Induction of SAA by phorbol 12-myristate 13-acetate, a known agonist of protein kinase C (PKC), suggested a potential role of the PKC signaling pathway in the activation process. The DNA binding activity of endogenous SAF was increased by agonists of PKC. In vitro phosphorylation of SAF-1 by PKC-beta markedly increased its DNA binding ability. Consistent with these findings, treatment of cells with activators of PKC or overexpression of PKC-betaII in transfected cells increased expression of an SAF-regulated promoter. Further analysis with a GAL4 reporter system indicated that PKC-mediated phosphorylation mostly increases the DNA binding activity of SAF-1. Together these data indicated that the PKC signaling pathway plays a major role in controlling expression of SAF-regulated genes by increasing the interaction between promoter DNA and phosphorylated SAF.
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PMID:Activation of transcription factor SAF involves its phosphorylation by protein kinase C. 1099 78

Cyclic AMP-response element modulator alpha (CREMalpha) is a transcription factor that is highly related to cAMP-response element-binding protein (CREB) but represses cAMP-induced gene expression from simple artificial promoters containing a cAMP-response element (CRE). CREMalpha lacks two glutamine-rich Q regions that, in CREB, are thought to be necessary for transcriptional activation. Nevertheless, protein kinase A stimulation induces CREMalpha to activate the complex native promoter in the phosphoenolpyruvate carboxykinase (PEPCK) gene. To study this phenomenon in the absence of protein kinase A stimulation, we introduced a mutation into CREMalpha to allow constitutive binding to the coactivator CREB-binding protein. This mutant, CREMalpha(DIEDML), constitutively activated the PEPCK promoter. By engineering the leucine zipper regions of CREMalpha(DIEDML) and CREB(DIEDML) to direct their patterns of dimerization, we found that only CREMalpha(DIEDML) homodimers fully activated the PEPCK promoter. By using a series of deletion and block mutants of the PEPCK promoter, we found that activation by CREMalpha(DIEDML) depended on the CRE and two CCAAT/enhancer-binding protein (C/EBP) sites. A dominant negative inhibitor of C/EBP, A-C/EBP, suppressed activation by CREMalpha(DIEDML). Furthermore, a GAL4-C/EBPalpha fusion protein and CREMalpha(DIEDML) cooperatively activated a promoter containing three GAL4 sites and the PEPCK CRE. Thus, we propose that the C/EBP sites in the PEPCK promoter allow CREMalpha to activate transcription despite its lack of Q regions.
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PMID:Cooperative mechanism of transcriptional activation by a cyclic AMP-response element modulator alpha mutant containing a motif for constitutive binding to CREB-binding protein. 1109 86

The conversion of L-tyrosine to 3,4-dihydroxy-L-phenylalanine by tyrosine hydroxylase (TH) is the first and rate-limiting step in biosynthesis of catecholamine neurotransmitters. TH gene expression is regulated in a cell type-specific and cAMP-dependent manner. Evidence from this laboratory and others indicates that the cAMP response element (CRE), residing at -45 to -38 bp upstream of the transcription initiation site, is essential for both basal and cAMP-inducible transcription of the TH gene. To understand the control mechanisms of TH gene transcription in greater detail, we sought to identify and characterize the transcription factors involved in recognition and activation of the CRE of the TH gene. Remarkably, electrophoretic mobility shift assay and antibody supershift experiments indicated that all three major CRE-binding protein factors, i.e. CREB, ATF1, and CREM, may participate in forming specific DNA/protein complexes with the CRE of the TH gene. To address the transcriptional activation function of individual factors, we replaced the TH CRE with a GAL4-binding site and cotransfected this modified TH promoter-reporter gene with an effector plasmid that encodes GAL4-fused transcription factor. Our results indicate that CREB but not ATF1 can support basal promoter activity while both can robustly induce the promoter activity in response to co-expression of the catalytic subunit of cAMP-dependent protein kinase (PKA). We further show that the coactivator CBP up-regulates PKA-mediated activation of the TH promoter and, if tethered to the TH promoter by a GAL4-fusion, can robustly transactivate the TH promoter even in the absence of PKA. Collectively, our results suggest that multiple CRE-binding factors interact with the CRE and regulate, in conjunction with the coactivator CBP, the transcriptional activity of the TH gene.
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PMID:Regulation of tyrosine hydroxylase gene transcription by the cAMP-signaling pathway: involvement of multiple transcription factors. 1110 36


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