EC:2.7.11.13 - FACTA Search

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Query: EC:2.7.11.13 (protein kinase C)
49,245 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In LLC-PK1 cells urokinase-type plasminogen activator (uPA) mRNA has a short half-life. It is stabilized by inhibition of protein synthesis and by downregulation of protein kinase C (PKC). In the present study on uPA mRNA metabolism, we focused our attention on the 3' untranslated region (3'UTR) of the uPA mRNA, as this region is long and highly conserved among several mammalian species, including mice and humans. To investigate the possible role of the 3'UTR of uPA mRNA in mRNA metabolism, we inserted this region into the 3'UTR of the rabbit beta-globin gene that is linked to the cytomegalovirus promoter and stably transfected it into LLC-PK1 cells. While the parental globin mRNA was stable, the chimeric mRNA was degraded as rapidly as endogenous uPA mRNA, suggesting that the 3'UTR of uPA mRNA contains most of the information required for its rapid turnover. Further analysis showed that there are at least three independent determinants of instability in the 3'UTR; one is an AU-rich sequence located immediately 3' of the poly(A) addition signal, and one is a sequence containing a stem structure. One determinant seems to require ongoing RNA synthesis for its activity. All chimeric unstable globin mRNAs became stable in the presence of cycloheximide, a protein synthesis inhibitor, suggesting that the stabilization of mRNA by protein synthesis inhibition is not through a specific sequence in the mRNA. In PKC-downregulated cells, globin mRNAs with the complete 3'UTR or the AU-rich sequence were stabilized, suggesting that PKC downregulation stabilizes uPA mRNA through the AU-rich sequence. Here we discuss the significance of multiple, independently acting instability determinants in the regulation of uPA mRNA metabolism.
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PMID:Multiple instability-regulating sites in the 3' untranslated region of the urokinase-type plasminogen activator mRNA. 800 88

Ribonucleotide reductase catalyses the reaction that eventually provides the four deoxyribonucleotides required for the synthesis and repair of DNA. U.v.-cross-linking and band-shift experiments have identified in COS 7 monkey cells an approx. 57 kDa ribonucleotide reductase R1 mRNA-binding protein called R1BP, which binds specifically to a 49-nt region of the R1 mRNA 3'-untranslated region (3'UTR). The R1BP-RNA binding activity was down-regulated by the tumour promoters phorbol 12-myristate 13-acetate (PMA; 'TPA') and okadaic acid, and up-regulated by the protein kinase C inhibitor staurosporine, in a dose-dependent fashion. Furthermore, staurosporine treatment decreased the stability of R1 and CAT (chloramphenicol acetyltransferase)/R1 hybrid mRNAs, whereas PMA and okadaic acid increased the stability of these messages, in a dose-dependent manner. In contrast, treatment of cells with forskolin, a protein kinase A inhibitor, did not alter either R1BP-RNA binding or R1 mRNA-stability characteristics. Transfectants containing R1 or CAT/R1 cDNA constructs with a deletion of the 49-nt 3'UTR sequence failed to respond in message-stability studies to the effects of PMA, staurosporine or okadaic acid. These observations indicate that a protein kinase C signal pathway regulates ribonucleotide reductase R1 gene expression post-transcriptionally, through a mechanism involving a specific cis-trans interaction at a 49-nt region within the R1 mRNA 3'UTR.
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PMID:Regulation of mammalian ribonucleotide reductase R1 mRNA stability is mediated by a ribonucleotide reductase R1 mRNA 3'-untranslated region cis-trans interaction through a protein kinase C-controlled pathway. 806 98

12-O-Tetradecanoylphorbol-13-acetate (TPA) stimulation of PU-34 cells, a primate bone marrow stromal cell line, resulted in a prolonged elevation of interleukin-11 (IL-11) mRNA, which can be inhibited by protein synthesis inhibitors. Nuclear run-on assays and actinomycin D experiments demonstrated that the up-regulation of IL-11 gene expression is mainly controlled at the posttranscriptional level through the protein kinase C (PKC) pathway. Inhibition of PKC activity by calphostin C generated an IL-11 mRNA degradation intermediate in TPA-stimulated PU-34 cells. This intermediate retains the 5' untranslated region (5'UTR) and coding region of the IL-11 mRNA but has lost the poly(A) tail and the 3'UTR. The mechanisms underlying IL-11 mRNA stabilization were further investigated by transfections with a variety of chimeric IL-11 constructs and deletion mutants. Two important observations were made from these transient expression experiments: (i) the same 3'UTR of IL-11 mRNA shown to confer instability in one chimeric transcript may not function as a destabilizer in another chimeric RNA, and (ii) the 5'UTR, coding region, and 3'UTR all contribute to IL-11 mRNA decay, and labile IL-11 deletion transcripts are not necessarily stabilized by TPA stimulation. Our study suggests that multiple regions within the IL-11 mRNA are involved in TPA-stimulated IL-11 mRNA stabilization, possibly through a unique RNA folding conformation involving interactions of various RNA sequences within the IL-11 mRNA molecule.
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PMID:Interleukin-11 mRNA stabilization in phorbol ester-stimulated primate bone marrow stromal cells. 866 45

In LLC-PK1 cells, urokinase-type plasminogen activator (uPA) mRNA has a short half-life of 70 min. We have previously demonstrated that most of the regulatory regions responsible for the rapid turnover of uPA mRNA in LLC-PK1 cells reside in its 3' untranslated region (3' UTR), where there are at least three regulatory sites, one of which is A+U-rich. This A+U-rich sequence mediates uPA mRNA stabilization induced by protein kinase C (PKC) down-regulation. In this work, we found that uPA mRNA is rather stable in MDA-MB-231 cells with a half-life of 17 h. We compared the stability of hybrid globin mRNA containing different parts of uPA mRNA in its 3' UTR and found that the A+U-rich sequence of uPA mRNA renders otherwise stable globin mRNA unstable in LLC-PK1 cells but not in MDA-MB-231 cells. We identified a cytoplasmic protein of 40 kDa (p40) which specifically interacts with the A+U-rich sequence. Levels of p40 activity as detected by ultraviolet cross-linking were higher in MDA-MB-231 and PKC-down-regulated LLC-PK1 cells than in untreated LLC-PK1 cells. Prior treatment of the cytoplasm with a specific antibody against heterogeneous nuclear ribonucleoprotein C (hnRNP C) significantly reduced p40 activity. These results suggest a correlation between the A+U-rich sequence-dependent uPA mRNA stabilization in vivo and the binding of hnRNP C to the A+U-rich sequence in vitro.
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PMID:Enhanced stability of urokinase-type plasminogen activator mRNA in metastatic breast cancer MDA-MB-231 cells and LLC-PK1 cells down-regulated for protein kinase C--correlation with cytoplasmic heterogeneous nuclear ribonucleoprotein C. 924 23

A CYP11B2 gene encoding cytochrome P450 aldosterone synthase (P450aldo) was isolated from a hamster genomic library. The gene, which contained 9 exons, was composed of 9,045 bp, of which 3,722 bp were located in the 5' untranslated region (5' UTR). A TATA box sequence (gataaa) and other putative cis elements, previously named Ad1 to Ad6, were identified in the 5' UTR of the hamster gene comparable to the CYP11B2 gene of other animal species. Footprint analysis showed protection by nuclear protein extracts from hamster adrenal zona glomerulosa (ZG) in the regions containing the above mentioned cis elements. In addition, a new protected cis element, between -143 and -161 bp, was demonstrated, and gel-shift assays revealed that the sequence of this new cis element was specifically retarded by factors in the nuclear extracts of hamster adrenal ZG. We then examined the transcriptional activity of the 5' UTR of the CYP11B2 gene, using chloramphenicol acyltransferase (CAT) as the reporter gene. Ten deletion plasmids were constructed using a modified pCAT vector. Transient transfections of the chimeric reporter constructs into Y1 cells showed that the highest basal promoter activity was obtained with the construct containing up to -134 bp. Increasing the length of the regulatory region of CYP11B2 gene to -167 bp resulted in less than two-thirds of the maximal activity, indicating the probability of putative inhibitory cis elements in this area of the gene. Forskolin stimulated the expression of the reporter gene of deletion plasmids excepting the construct containing only the TATA box, and the highest activity also occurred with the -134 bp construct. TPA had no stimulatory effects on any of the constructs, and interestingly it slightly inhibited CAT activity. In contrast to TPA, staurosporine, an inhibitor of the PKC pathway, stimulated CAT activity. To conclude, the promoter region of the hamster CYP11B2 gene transfected in Y1 cells is responsive to forskolin, indicating that the gene is controlled by the PKA signaling pathway. Paradoxically, staurosporine, but not TPA, stimulates the promoter activity of the CYP11B2 gene, indicating that PKC might, at least in Y1 cells, act as a negative regulator on the aldosterone synthase promoter. Moreover, a new cis element was shown to exert a negative effect on basal as well as on stimulated activities of the hamster promoter CYP11B2 gene.
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PMID:Characterization of the hamster CYP11B2 gene encoding adrenal cytochrome P450 aldosterone synthase. 930 41

14-3-3 proteins constitute a family of well-conserved eukaryotic proteins that possess diverse biochemical activities such as regulation of gene transcription, cell proliferation and activation of protein kinase C. At least 7 subtypes (alpha to theta) of 14-3-3 protein are known, but the zeta subtype of this protein has been cloned only in mammals. We cloned the zeta subtype of 14-3-3 protein (14-3-3 zeta) from the frog, Rana rugosa. The sequence encoded 245 amino acids that share 92% identity with rat and bovine 14-3-3 zeta s, and 92% with human phospholipase A2 (PLA2; 14-3-3 zeta). Northern blot analysis revealed a single band of about 1.8 kb in tadpoles at stage 25. The 14-3-3 zeta mRNA level was high in the brain, lung, spleen and kidney, and low in the heart and testis, as opposed to the mRNA level, which was only faintly detected in the liver, pancreas, ovary and muscle. Furthermore, high similarity in the 3'-untranslated region (3'-UTR) was observed between frog and human 14-3-3 zeta cDNA. The results suggest that 14-3-3 zeta is highly conserved throughout eukaryotic evolution, and that the homologous sequence in the 3'-UTR of 14-3-3 zeta cDNA may be conserved in frogs and humans.
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PMID:Molecular cloning of cDNA for the zeta isoform of the 14-3-3 protein: homologous sequences in the 3'-untranslated region of frog and human zeta isoforms. 945 Mar 88

The RNA-binding protein HuD binds to a regulatory element in the 3' untranslated region (3' UTR) of the GAP-43 mRNA. To investigate the functional significance of this interaction, we generated PC12 cell lines in which HuD levels were controlled by transfection with either antisense (pDuH) or sense (pcHuD) constructs. pDuH-transfected cells contained reduced amounts of GAP-43 protein and mRNA, and these levels remained low even after nerve growth factor (NGF) stimulation, a treatment that is normally associated with protein kinase C (PKC)-dependent stabilization of the GAP-43 mRNA and neuronal differentiation. Analysis of GAP-43 mRNA stability demonstrated that the mRNA had a shorter half-life in these cells. In agreement with their deficient GAP-43 expression, pDuH cells failed to grow neurites in the presence of NGF or phorbol esters. These cells, however, exhibited normal neurite outgrowth when exposed to dibutyryl-cAMP, an agent that induces outgrowth independently from GAP-43. We observed opposite effects in pcHuD-transfected cells. The GAP-43 mRNA was stabilized in these cells, leading to an increase in the levels of the GAP-43 mRNA and protein. pcHuD cells were also found to grow short spontaneous neurites, a process that required the presence of GAP-43. In conclusion, our results suggest that HuD plays a critical role in PKC-mediated neurite outgrowth in PC12 cells and that this protein does so primarily by promoting the stabilization of the GAP-43 mRNA.
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PMID:The RNA-binding protein HuD is required for GAP-43 mRNA stability, GAP-43 gene expression, and PKC-dependent neurite outgrowth in PC12 cells. 1098 10

The precise signaling pathways which contribute to amyloid precursor protein (APP) gene expression remain incompletely characterized. We evaluated the role of protein kinases, calcium and phospholipase C (PLC) in modulating APP mRNA levels. There was a rapid 35-40% reduction in the steady state level of APP mRNA upon stimulation of peripheral blood mononuclear cells (PBMC) with phorbol 12-myristate 13-acetate (PMA), A23187 or ionomycin. However the protein kinase C (PKC), protein kinase A (PKA) or PLC pathways did not mediate these changes in APP mRNA levels. Rather, PMA or ionophore caused a rapid activation of extracellular-regulated kinase (ERK). This effect was independent of PKC and sensitive to U0126. After 4 h of PMA treatment, the remaining APP mRNA became indefinitely stable. We propose a model for the biphasic decay of APP mRNA in which ERK activation by PMA causes sequential upregulation of two APP mRNA binding proteins, nucleolin and hnRNP C. We attribute the initial rapid loss of APP mRNA to the helicase activity associated with nucleolin and later stabilization to hnRNP C binding to the 29 base instability element in the 3'-UTR of APP mRNA.
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PMID:Extracellular-regulated kinase controls beta-amyloid precursor protein mRNA decay. 1140 97

Overexpression of protein kinase C-zeta (PKC-zeta) in the leukemic myeloid cell line U937 (U937-PKC-zeta cells), previously shown to induce leukemic cell differentiation, resulted in nearly complete downregulation of leukocyte integrins CD11a, CD11b, CD11d, and CD18, but not CD11c from the cell surface. The steady-state level of mRNAs for the downregulated leukocyte integrins was not detectable by Northern analysis. Nuclear run-on analysis revealed that transcription of all the leukocyte integrin genes except CD11c was reduced 70-90% as compared to control U937-Vector cells [U937 cells transfected with the empty vector pSV2M(2)6]. Transfection analysis of CD11-promoter-luciferase constructs confirmed that transcription of the leukocyte integrin genes was drastically downregulated in U937-PKC-zeta cells. The two c-jun binding sites in the CD11c promoter were essential for continued expression of CD11c in U937-PKC-zeta cells. Additionally, the 3' untranslated region (3' UTR) from CD11b, when fused to the luciferase gene, lead to the destabilization of this chimeric mRNA in U937-PKC-zeta cells. This indicates that downregulation of CD11b expression in U937-PKC-zeta cells is also the result of reduced stability of CD11b mRNA. Thus, overexpression of PKC-zeta in U937 cells leads not only to leukemic cell differentiation, but also to differential regulation of the leukocyte integrins.
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PMID:The leukocyte integrins are regulated by transcriptional and post-transcriptional mechanisms in a leukemic cell that overexpresses protein kinase C-zeta. 1171 5

We have cloned a full-length 2874-bp cDNA coding for tobacco topoisomerase I, with an ORF of 2559 bp encoding a protein of 852 amino acids with a calculated molecular mass of 95 kDa and an estimated pI of 9.51. The deduced amino acid sequence shows homology to other eukaryotic topoisomerases I. Tobacco topoisomerase I was over-expressed in Escherichia coli, and the purified recombinant protein was found to relax both positively and negatively super-coiled DNA in the absence of the divalent cation Mg(2+)and ATP. These characteristic features indicate that the tobacco enzyme is a type I topoisomerase. The recombinant protein could be phosphorylated at (a) threonine residue(s) by protein kinase C. However, phosphorylation did not cause any change in its enzymatic activity. The genomic organization of the topoisomerase I gene revealed the presence of 8 exons and 7 introns in the region corresponding to the ORF and one intron in the 3' UTR region. Transcript analysis using RT-PCR showed basal constitutive expression in all organs examined, and the gene was expressed at all stages of the cell cycle--but the level of expression increased during the G1-S phase. The transcript level also increased following exposure to light, low-temperature stress and abscisic acid, a stress hormone.
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PMID:Cloning and characterization of a cell cycle-regulated gene encoding topoisomerase I from Nicotiana tabacum that is inducible by light, low temperature and abscisic acid. 1207 40

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