Posttranscriptional regulation of ribonucleotide reductase R1 gene expression is linked to a protein kinase C pathway in mammalian cells

1994 ◽  
Vol 72 (7-8) ◽  
pp. 251-256 ◽  
Author(s):  
Frank Y. Chen ◽  
Francis M. Amara ◽  
Jim A. Wright
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Ribonucleotide Reductase ◽  
Mrna Stability ◽  
Posttranscriptional Regulation ◽  
Signal Pathway ◽  
Kinase C ◽  
Gf 109203X ◽  
Protein Kinase C Inhibitor ◽  
R1 Gene

A rate-limiting reaction in DNA synthesis is catalyzed by ribonucleotide reductase, the enzyme responsible for reducing ribonucleotides to provide the deoxyribonucleotide precursors of DNA. In this study, we have tested the hypothesis that posttranscriptional regulation of ribonucleotide reductase R1 gene expression is controlled by a protein kinase C signal pathway. We show that mouse BALB/c 3T3 fibroblasts treated with the potent and highly specific protein kinase C inhibitor bisindolylmaleimide GF 109203X contain significantly reduced steady-state levels of R1 mRNA and protein. Message half-life studies demonstrate that this is due, at least in part, to a marked decrease in R1 message stability in cells treated with the protein kinase C inhibitor. Furthermore, the protein kinase C signal pathway appears to be specifically involved in this regulation since 8-bromo-cAMP, a modulator of the protein kinase A pathway, had no effect on R1 mRNA levels or stability properties. Cross-linking assays revealed that the binding activity of a R1 mRNA 3′-untranslated region binding protein (R1BP), which was previously shown to be involved in the regulation of R1 mRNA stability, was significantly elevated after treatment of the cells with GF 109203X, in a dose-dependent manner. However, treatment with 8-bromo-cAMP at concentrations up to 2.5 mM did not obviously affect the basic level of the R1BP–RNA interaction. These observations provide a better understanding of the biochemical signals that are critical for the cis–trans interaction-mediated posttranscriptional regulation of ribonucleotide reductase R1 gene expression.Key words: protein kinase C, ribonucleotide reductase, mRNA stability, RNA–protein interaction, bisindolylmaleimide GF 109203X, 8-bromo-cAMP.

scholarly journals 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

1994 ◽  
Vol 302 (1) ◽  
pp. 125-132 ◽  
Author(s):  
F Y Chen ◽  
F M Amara ◽  
J A Wright
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Okadaic Acid ◽  
Ribonucleotide Reductase ◽  
Mrna Stability ◽  
Untranslated Region ◽  
Rna Binding ◽  
Kinase C ◽  
The Stability ◽  
Dose Dependent

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.

scholarly journals Protein Kinase C Inhibitor Generates Stable Human Tolerogenic Dendritic Cells

2013 ◽  
Vol 191 (5) ◽  
pp. 2247-2257 ◽  
Author(s):  
Takuya Matsumoto ◽  
Hitoshi Hasegawa ◽  
Sachiko Onishi ◽  
Jun Ishizaki ◽  
Koichiro Suemori ◽  
...  
Keyword(s):  
Dendritic Cells ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Kinase C ◽  
Tolerogenic Dendritic Cells ◽  
Protein Kinase C Inhibitor

The protein kinase C inhibitor H-7 activates human neutrophils: effect on shape, actin polymerization, fluid pinocytosis and locomotion

1990 ◽  
Vol 96 (1) ◽  
pp. 99-106
Author(s):  
H.U. Keller ◽  
V. Niggli ◽  
A. Zimmermann ◽  
R. Portmann
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Actin Polymerization ◽  
Kinase Inhibitor ◽  
Human Neutrophils ◽  
Kinase C ◽  
Chemotactic Peptides ◽  
Protein Kinase C Inhibitor ◽  
Shape Changes ◽  
Stimulation Of

The present study demonstrates new properties of H-7. The protein kinase inhibitor H-7 is a potent activator of several neutrophil functions. Stimulation of initially spherical nonmotile neutrophils elicits vigorous shape changes within a few seconds, increases in cytoskeletal actin, altered F-actin distribution, increased adhesiveness and a relatively small increase in pinocytic activity. H-7 has also chemokinetic activities. Depending on the experimental condition, H-7 may elicit or inhibit neutrophil locomotion. It failed to induce chemotaxis. Thus, the response pattern elicited by H-7 is different from that of other leukocyte activators such as chemotactic peptides, PMA or diacylglycerols. The finding that H-7 can elicit shape changes, actin polymerization and pinocytosis suggests that these events can occur without activation of protein kinase C (PKC). PMA-induced shape changes and stimulation of pinocytosis were not inhibited by H-7.

scholarly journals Protein kinase C isoenzymes in airway smooth muscle

1997 ◽  
Vol 324 (1) ◽  
pp. 167-175 ◽  
Author(s):  
Benjamin L. J. WEBB ◽  
Mark A. LINDSAY ◽  
Peter J. BARNES ◽  
Mark A. GIEMBYCZ
Keyword(s):  
Smooth Muscle ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Oligonucleotide Primer ◽  
Mrna Levels ◽  
Kinase C ◽  
Gf 109203X ◽  
Bona Fide

The protein kinase C (PKC) isoenzymes expressed by bovine tracheal smooth muscle (BTSM) were identified at the protein and mRNA levels. Western immunoblot analyses reliably identified PKCα, PKCβI and PKCβII. In some experiments immunoreactive bands corresponding to PKCδ, PKCϵ and PKCθ were also labelled, whereas the γ, η and ζ isoforms of PKC were never detected. Reverse transcriptase PCR of RNA extracted from BTSM using oligonucleotide primer pairs designed to recognize unique sequences in the PKC genes for which protein was absent or not reproducibly identified by immunoblotting, amplified cDNA fragments that corresponded to the predicted sizes of PKCδ, PKCϵ and PKCζ, which was confirmed by Southern blotting. Anion-exchange chromatography of the soluble fraction of BTSM following homogenization in Ca2+-free buffer resolved two major peaks of activity. Using ϵ-peptide as the substrate, the first peak of activity was dependent upon Ca2+ and 4β-PDBu (PDBu = phorbol 12,13-dibutyrate), and represented a mixture of PKCs α, βI and βII. In contrast, the second peak of activity, which eluted at much higher ionic strength, also appeared to comprise a combination of conventional PKCs that were arbitrarily denoted PKCα′, PKCβI′ and PKCβII′. However, these novel enzymes were cofactor-independent and did not bind [3H]PDBu, but were equally sensitive to the PKC inhibitor GF 109203X compared with bona fide conventional PKCs, and migrated on SDS/polyacrylamide gels as 81 kDa polypeptides. Taken together, these data suggest that PKCs α′, βI′ and βII′ represent modified, but not proteolysed, forms of their respective native enzymes that retain antibody immunoreactivity and sensitivity to PKC inhibitors, but have lost their sensitivity to Ca2+ and PDBu when ϵ-peptide is used as the substrate.

Sign in / Sign up

Export Citation Format

Share Document