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)

sgk is a novel member of the serine/threonine protein kinase family that is transcriptionally regulated by serum and glucocorticoids in Rat2 fibroblasts and in mammary epithelial cells. 5'-Deletion analysis of the sgk promoter, using a series of sgk-CAT. (chloramphenicol acetyltransferase) chimeric reporter gene plasmids, defined a glucocorticoid-responsive region that contains a glucocorticoid response element (sgkGRE) between -1000 and -975 bp. The sgkGRE is specifically bound by glucocorticoid receptors and is sufficient to confer glucocorticoid responsiveness to a heterologous promoter in several cell lines. Strikingly, cotransfection of either the murine or human wild type p53, but not a mutant p53, repressed the dexamethasone-stimulated transactivation of reporter plasmids containing either the sgkGRE or a consensus GRE. Gel shift analysis revealed that in vitro synthesized p53 prevented binding of the glucocorticoid receptor both to the sgkGRE as well as to a consensus GRE. The p53-mediated repression of dexamethasone-induced sgkGRE activity required both the DNA binding and transactivation functions of the p53 protein. Activation of endogenous p53, by exposure to UV light, repressed the glucocorticoid receptor transactivation of a consensus GRE-CAT reporter plasmid in transfected cells. Conversely, activated glucocorticoid receptors suppressed the transactivation function of p53, while transrepression by p53 was largely unaffected. The presented data demonstrate that sgk is a primary glucocorticoid-responsive protein kinase gene that implicates a new pathway of cross-talk between steroid receptor signaling and cellular phosphorylation cascades. In addition, our study provides the first evidence of mutual interference of transactivation functions of p53 and the glucocorticoid receptor, possibly through their direct interaction.
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PMID:Repression of glucocorticoid receptor transactivation and DNA binding of a glucocorticoid response element within the serum/glucocorticoid-inducible protein kinase (sgk) gene promoter by the p53 tumor suppressor protein. 905 78

We have characterized the biosynthetic origin of somatostatin-14 (SS-14), SS-28, and pro-SS[1-10] from pro-SS (PSS) in 1027B2 rat islet tumor cells. Because these cells lack regulated secretion and show unresponsiveness of the SS gene to cAMP, we have additionally carried out morphological and functional studies to elucidate the molecular defect in cAMP signalling and to localize the sites of PSS maturation along the secretory pathway. Cell extracts and secretion media were analysed by high performance liquid chromatography and specific C- and N-terminal radioimmunoassays. Electron microscopic sampling of 1027B2 cell cultures showed that most cells had very few dense core secretory granules for heterogeneous sizes. The cells expressed the endoproteases furin, PC1, and PC2 and contained large quantities of fully processed SS-14 and SS-28 with very little unprocessed PSS (ratio SS-14:SS-28:PSS = 39:51:10%). They secreted high concentrations of SS-14, SS-28, and PSS[1-10] constitutively along with PC1 and PC2. Pulse-chase studies demonstrated that PSS is rapidly (within 15 min), and efficiently processed to SS-14, SS-28, and PSS[1-10] via separate biosynthetic pathways: PSS --> SS-14 + 8 kDa; PSS --> SS-28 + 7 kDa; PSS --> PSS[1-10]. Monensin reduced intracellular SS-like immunoreactivity without altering processing efficiency. Transfection with the catalytic subunit of protein kinase A (PKA-C) activated SS promoter-CAT activating indicating that the defect in cAMP-dependent signaling in 1027B2 cells lies at the level of PKA-C. PKA-C overexpression failed to alter the ratio of processed SS-14 and SS-28. These results demonstrate that SS-14, SS-28, and PSS[1-10] are independently synthesized from PSS and that efficient precursor processing can occur within the constitutive secretory pathway in the relative absence of dense core secretory vesicles.
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PMID:Somatostatin-14, somatostatin-28, and prosomatostatin[1-10] are independently and efficiently processed from prosomatostatin in the constitutive secretory pathway in islet somatostatin tumor cells (1027B2). 929 77

cAMP markedly increases alpha 1B adrenergic receptor (alpha 1B-AR) expression in FRTL-5 and PC C13 rat thyroid cells, DDT1MF-2 smooth muscle cells, primary rat hepatocytes, and K9 rat liver cells. Here, we used DDT1MF-2 cells to evaluate further the mechanisms by which cAMP stimulates alpha 1B-AR expression. Receptor binding assays, Northern blotting, and nuclear run-on analyses demonstrated that forskolin (1 microM) in the presence of isobutylmethylxanthine (0.25 mM) increased alpha 1B-AR numbers, mRNA level, and gene transcription rate by 2.3 +/- 0.2-, 2.5 +/- 0.3-, and 3.5 +/- 0.2-fold over control, respectively. Dibutyryl cAMP (1 mM) plus isobutylmethylxanthine (0.25 mM) also enhanced alpha 1B-AR density by 2.7 +/- 0.1-fold over control. Further experiments demonstrated that the induction of alpha 1B-AR by forskolin requires new protein synthesis and is protein kinase A dependent. In DDT1MF-2 cells transfected with alpha 1B-AR gene P2 promoter/CAT constructs, both forskolin and dibutyryl cAMP significantly increased P2 promoter activity. The P2 promoter region of the rat alpha 1B-AR gene (-813 to -432) contains a cAMP response element (CRE) (-444 to -437) and an AP2 binding site (-647 to -638). Mutations in either one of these elements alone led to a decrease in both basal and cAMP-induced P2 promoter activity. Mutations in both elements caused a further inhibition of basal transcription and a complete block of cAMP-induced P2 promoter activity. Direct binding of purified activator protein 2 (AP2) to the AP2 element in the P2 promoter was reported previously. Gel mobility shift and super-shift assays using liver nuclear extracts from either rat liver or DDT1MF-2 cells demonstrated that the CRE in the alpha 1B-AR gene bound CRE binding protein. These data indicate that both the CRE and the AP2 element in the P2 promoter contribute to basal as well as cAMP-induced transcription of the alpha 1B-AR gene in DDT1MF-2 cells.
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PMID:Both the cyclic AMP response element and the activator protein 2 binding site mediate basal and cyclic AMP-induced transcription from the dominant promoter of the rat alpha 1B-adrenergic receptor gene in DDT1MF-2 cells. 941 11

Capacitation is a priming event that renders mammalian spermatozoa responsive to signals originating from the cumulus-oocyte complex. The attainment of a capacitated state is dependent upon an increase in tyrosine phosphorylation and results in the acquisition of responsiveness to physiological agonists such as progesterone and ZP3. In this study we have shown that this capacitation-dependent increase in tyrosine phosphorylation is controlled by a unique redox-regulated, cAMP-mediated, signal transduction cascade. Either stimulation of reactive oxygen species generation or elevation of intracellular cAMP induced increases in phosphotyrosine expression by human spermatozoa and enhanced their responsiveness to progesterone. Ultimate convergence of the redox- and cAMP-regulated pathways was indicated by the ability of the protein kinase A inhibitor, H89, to block both modes of signal transduction. Furthermore, the fact that the redox-regulated pathway could be silenced by catalase, while this enzyme had no effect on the cAMP-mediated response, indicated that oxidant generation must lie upstream from cAMP in the reaction sequence. In keeping with this conclusion, a functional association was demonstrated between the redox status of human spermatozoa and their cAMP content. The continuous production of reactive oxygen species was also shown to be necessary for the protein kinase A-tyrosine phosphorylation axis to remain functional. If the generation of oxidising conditions during capacitation was prevented with 2-mercaptoethanol, 2-deoxyglucose or the flavoprotein inhibitor, diphenylene iodonium, then cAMP could no longer trigger tyrosine phosphorylation. These data support a model for human sperm capacitation as a redox-regulated process, involving a unique sequence of interactive events including reactive oxygen species production, elevation of intracellular cAMP, stimulation of protein kinase A and the induction of tyrosine phosphorylation. This is the first report of such a signal transduction cascade and may have implications for the functional significance of reactive oxygen metabolites in other cell types.
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PMID:A novel signal transduction cascade in capacitating human spermatozoa characterised by a redox-regulated, cAMP-mediated induction of tyrosine phosphorylation. 945 38

Age-related changes in the activities of acid and neutral cholesteryl ester hydrolases (ACEH and NCEH) and their activation by protein kinase A (PKA) and also by protein kinase C (PKC) were examined in the aortae of 4-, 8-, 12- and 20-week-old rats in relation to their aortic lipid and lipid peroxides and lipid contents. The physiological basal activity as well as total activities of the ACEH and NCEH activated by the two kinases, which were high in the aortae of the 4- and 8-week-old rats, decreased gradually with increasing age to about 40% (ACEH) and 50% (NCEH) by 20 weeks of age. The vitamin E intake and ad libitum-diet intake of the rats each modified the age-related decline of CEH activities. The aortic PKA and PKC activities were reflected by the CEH activities to some degree. The in vitro exposure of the aortic CEH to active oxygen (AO) generators revealed the PKC-mediated activation of CEH, which was inhibited by superoxide dismutase and catalase. These results suggested that the activities of ACEH and NCEH and their regulatory enzymes may be modulated by the dual effect of endogenous AO; an activation of CEH at low doses and an inactivation at high doses, or upon a long-term exposure in aging to a low level of endogenous AO.
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PMID:Age-related changes in the activation of aortic cholesteryl ester hydrolases by protein kinases in rats. 951 48

Complement receptor 2 (CR2) has been implicated as a regulator of B cell function. In this study, we sought to identify mechanisms that control the expression of the CR2 gene in human B cells. Dibutyryl cAMP increased the DNA-binding activity of a nuclear protein that recognized specifically a CR2 promoter-defined oligonucleotide in human B cell lines. The nuclear protein was subsequently purified from B cell nuclear extracts using a biotinylated CR2 promoter-defined oligonucleotide. Partial amino acid sequence analysis of internal peptides revealed that the 42-kDa protein belongs to a family of heterogeneous nuclear ribonucleoproteins (hnRNP). Using a set of mutated double-stranded oligonucleotides, we demonstrated that the purified protein displayed sequence specificity for the CR2 promoter-defined oligonucleotide. Like some hnRNP, this protein was found to bind to single-stranded DNA. The DNA-binding activity of the purified protein increased after in vitro phosphorylation with protein kinase A. Using a CAT reporter gene driven by a single recognition site in B cell lines, dibutyryl cAMP caused a 3-fold induction of reporter gene expression. The highest induction (6.7-fold) was achieved with a combination of dibutyryl cAMP and PMA. The involvement of the nuclear protein in regulating the expression of the CR2 gene is supported by our finding that dibutyryl cAMP increased the levels of the CR2 mRNA and CR2 surface membrane protein in human B cell lines. These data strongly suggest that a cAMP-inducible hnRNP, which can recognize a novel DNA-motif, controls the expression of the CR2 gene.
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PMID:Transcriptional regulation of the complement receptor 2 gene: role of a heterogeneous nuclear ribonucleoprotein. 954 89

The involvement of serine/threonine protein phosphatases in signaling pathways that control the expression of the cyclooxygenase-2 (COX-2) gene in human chondrocytes was examined. Okadaic acid (OKA), an inhibitor of protein phosphatases 1 (PP-1) and 2A (PP-2A), induced a delayed, time-dependent increase in the rate of COX-2 gene transcription (runoff assay) resulting in increased steady-state mRNA levels and enzyme synthesis. The latter response was dose dependent over a narrow range of 1-30 nmol/L with declining expression and synthesis of COX-2 at higher concentrations due to cell toxicity. The delayed increase in COX-2 mRNA expression was accompanied by the induction of the proto-oncogenes c-jun, junB, junD, and c-fos (but not FosB or Fra-1). Increased phosphorylation of CREB-1/ATF-1 transcription factors was observed beginning at 4 h and reached a zenith at 8 h. Gel-shift analysis confirmed the up-regulation of AP-1 and CRE nuclear binding proteins, though there was little or no OKA-induced nuclear protein binding to SP-1, AP-2, NF-kappaB or NF-IL-6 regulatory elements. OKA-induced nuclear protein binding to 32P-CRE oligonucleotides was abrogated by a pharmacological inhibitor of protein kinase A (PKA), KT-5720; the latter compound also inhibited OKA-induced COX-2 enzyme synthesis. Calphostin C (CalC), an inhibitor of PKC isoenzymes, had little effect in this regard. Inhibition of 12P-CRE binding was also observed in the presence of an antibody to CREB-binding protein (265-kDa CBP), an integrator and coactivator of cAMP-responsive genes. The binding to 32P-CRE was unaffected in the presence of excess radioinert AP-1 and COX-2 NF-IL-6 oligonucleotides, although a COX-2 CRE-oligo competed very efficiently. 32P-AP-1 consensus sequence binding was unaffected by incubation of chondrocytes with KT-5720 or CalC, but was dramatically diminished by excess radioinert AP-1 and CRE-COX-2 oligos. Supershift analysis in the presence of antibodies to c-Jun, c-Fos, JunD, and JunB suggested that AP-1 complexes were composed of c-Fos, JunB, and possibly c-Jun. OKA has no effect on total cellular PKC activity but caused a delayed time-dependent increase in total PKA activity and synthesis. OKA suppressed the activity of the MAP kinases, ERK1/2 in a time-dependent fashion, suggesting that the Raf-1/MEKK1/MEK1/ERK1,2 cascade was compromised by OKA treatment. By contrast, OKA caused a dramatic increase in SAPK/JNK expression and activity, indicative of an activation of MEKK1/JNKK/SAPK/JNK pathway. OKA stimulated a dose-dependent activation of CAT activity using transfected promoter-CAT constructs harboring the regulatory elements AP-1 (c-jun promoter) and CRE (CRE-tkCAT). We conclude that in primary phenotypically stable human chondrocytes, COX-2 gene expression may be controlled by critical phosphatases that interact with phosphorylation dependent (e.g., MAP kinases:AP-1, PKA:CREB/ATF) signaling pathways. AP-1 and CREB/ATF families of transcription factors may be important substrates for PP-1/PP-2A in human chondrocytes.
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PMID:Transcriptional induction of cyclooxygenase-2 gene by okadaic acid inhibition of phosphatase activity in human chondrocytes: co-stimulation of AP-1 and CRE nuclear binding proteins. 962 Jan 67

The present study was undertaken to investigate the role of protein kinase A (PKA) in the control of human T-cell leukemia virus type-I (HTLV-I) long terminal repeat (LTR) expression, since this issue is still controversial. For this purpose we employed two human T-cell lines; the Jurkat cells in which long exposure to diBu-cAMP severely down-regulated the catalytic subunit of PKA (PKA-C), and H-9 cells in which such exposure markedly increased PKA-C level. Transient transfection assays revealed that addition of diBu-cAMP 1 h before or after transfection profoundly increased HTLV-I LTR directed CAT expression and synergistically enhanced its stimulation by the viral transactivator tax gene product in both cell lines. However longer exposure to diBu-cAMP before transfection reduced LTR-CAT expression to below its basal level and completely abolished its stimulation by tax in Jurkat cells, and this diBu-cAMP inhibitory effect could be abrogated by co-transfection of a PKA-C expressing vector. By contrast, in H-9 cells, this long exposure to diBu-cAMP continued enhancing LTR-CAT expression and its tax-mediated transactivation, and this stimulatory effect of diBu-cAMP could be diminished by the PKA-specific inhibitor N-12-(p-bromocinnamylamine)ethyll-5-isoquinolinsulfonamid e (H-89). Notably, in the absence of diBu-cAMP treatment H-89 reduced LTR-CAT expression to below its basal level and prevented its stimulation by tax in both cell lines. Together these findings indicate not only that cAMP-activated PKA stimulates HTLV-I LTR expression and its transactivation by tax, but even in the absence of PKA activating signals the basal HTLV-I LTR expression as well as its stimulation by tax are both dependent on a basal PKA activity.
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PMID:Evidence that protein kinase A activity is required for the basal and tax-stimulated transcriptional activity of human T-cell leukemia virus type-I long terminal repeat. 965 31

Expression of progesterone receptor (PR) mRNA in granulosa cells of ovarian preovulatory follicles is induced by LH (1, 2) and is essential for ovulation (3). Although 17beta-estradiol (E) can induce PR mRNA and activate PR promoter-reporter constructs in other cell types, the effects of E in granulosa cells appear to be indirect. We show herein that E alone does not induce the expression of PR mRNA in preovulatory granulosa cells. Rather, induction of PR mRNA depends on the differentiation of granulosa cells in response to E and a physiological amount of FSH followed by exposure to agonists (elevated levels of LH, FSH, and forskolin) that markedly increase cAMP. Induction of PR mRNA by forskolin is blocked by the A-kinase inhibitor H89 and cycloheximide but not by the E antagonist, ICI 164,384. These results indicate that phosphorylation and synthesis of some regulatory factor(s) other than or in addition to the estrogen receptor (ER) are essential for transactivation of the PR gene. When distal and proximal PR promoter-reporter constructs that are responsive to E in other cell types were transiently transfected into differentiated granulosa cells, forskolin, but not E, induced activity. Likewise, when a vector containing the consensus vitellogenin B1 gene estrogen response element (ERE) was transfected into differentiated granulosa cells, forskolin, but not E, induced activity. Using electrophoretic mobility shift assays, the consensus ERE was shown to bind ERbeta, the predominant subtype present in rat granulosa cells, and ERalpha, the predominant subtype present in luteal cells, whereas the putative ERE-like region (ERE3) of the proximal PR promoter did not bind either ER subtype. Although the identity of the specific factors binding to the ERE3 site remain to be determined, mutation of this region abolished forskolin-induced activity of ERE3-PR-CAT constructs. The GC-rich region of the distal PR promoter bound Sp1 and Sp3 but not C/EBPalpha/beta, indicating that factors binding to ERE3 interact synergistically with Sp1/Sp3 to confer increased responsiveness of the distal promoter to forskolin. Taken together, these results indicate that activation of the A-kinase pathway leads to the phosphorylation of some transcription factor(s) other than or in addition to ER that is (are) critical for the transactivation of the PR gene and that this mechanism is selectively activated in differentiated granulosa cells possessing a preovulatory phenotype.
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PMID:Hormone induction of progesterone receptor (PR) messenger ribonucleic acid and activation of PR promoter regions in ovarian granulosa cells: evidence for a role of cyclic adenosine 3',5'-monophosphate but not estradiol. 971 46

Azide, in the absence of other stimuli, enhanced neutrophil migration in a chemotactic way. The effect of azide on migration was significant at concentrations > or = 1 microM and maximal at 10 microM azide. Although azide itself could not induce exocytosis, at concentrations > or = 10 microM azide enhanced exocytosis induced by a combination of the chemotactic peptide f-methionyl-leucyl-phenylalanine (fMLP) and cytochalasin B (CB). Azide can be oxidized by catalase and myeloperoxidase in the presence of H2O2, resulting in the generation of nitric oxide (NO). Formation of NO from azide was detected by ESR spectroscopy with carboxy-PTIO as a NO-selective probe, and by measurement of nitrite formation. Azide-induced migration, and the enhancement by azide of fMLP/CB-induced exocytosis, were blocked by pre-incubating cells with aminotriazole, an inhibitor of catalase and myeloperoxidase, suggesting that the effect of azide was mediated by NO. Azide-induced migration, but not the enhancement by azide of fMLP/CB-induced exocytosis, was inhibited to a large extent by inhibitors of soluble guanylate cyclase and by inhibitors of cGMP-dependent protein kinase. These observations suggest that azide-induced migration is mediated via cGMP and cGMP-dependent protein kinase, while the enhancement of fMLP/CB-induced exocytosis is not. Azide caused a sustained elevation of the intracellular Ca2+-concentration of neutrophils stimulated with fMLP/CB, which was not affected by inhibitors of the cGMP-signalling cascade. Since neutrophil exocytosis has been shown to be closely correlated with increases in intracellular Ca2+, a further increase by azide of the intracellular Ca2+-level of cells stimulated with fMLP/CB provides a likely mechanism for the enhancement of fMLP/CB-induced exocytosis by azide.
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PMID:Sodium azide enhances neutrophil migration and exocytosis: involvement of nitric oxide, cyclic GMP and calcium. 971 94


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