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)

Ribonucleotide reductase is responsible for the production of the deoxyribonucleotides required for DNA synthesis. The enzyme is composed of two dissociable subunits, proteins M1 and M2, which are inactive alone, but are fully active when combined. From mouse S49 T lymphoma cells we have isolated and separated the two subunits and used each for determining the activity of the complementary subunit in extracts from cells of different phases in the cell cycle. Treatment of S49 cells with cAMP analogs (e.g. Bt2cAMP) results in the protein kinase-dependent arrest of the cells in the G1 phase of the cell cycle. Ribonucleotide reductase (holoenzyme) activity fell in S49 cells treated for more than 16 h with Bt2cAMP but was unchanged during short term treatments. The activity of protein M2 was decreased in parallel to the overall activity of ribonucleotide reductase, while protein M1 activity changed less. Removal of bt2cAMP after 24 h exposure resulted in increased holoenzyme and protein M2 activities. Centrifugal elutriation of exponentially growing S49 cells separated cells into a 90% pure G1 cell population a mixture of G1 and early S phase cells and a 95% pure S phase/G2 cell population. The specific catalytic activity of protein M1 was the same in all these fractions while that of protein M2 was decreased 60% in the G1 cell population. These results demonstrate that the ribonucleotide reduction necessary for DNA synthesis is regulated in a cell cycle-dependent fashion by the activity of the protein M2 subunit of ribonucleotide reductase.
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PMID:Ribonucleotide reductase in cultured mouse lymphoma cells. Cell cycle-dependent variation in the activity of subunit protein M2. 627 86

We have analysed the intracellular localisation of herpes simplex virus type 1 ribonucleotide reductase during infection of cultured cells by indirect immunofluorescence using polyclonal and monoclonal antibodies specific for the R1 and R2 subunits. Three different viruses were used to infect cells, wild-type strain 17+ and two temperature-sensitive mutants, ts 1222, which produces R1 only, and ts 1207, which expresses a normal R2 and an altered R1 that fails to interact with R2 at the nonpermissive temperature because of an amino acid substitution in R1. R1 was detected 2 hr postinfection with all three viruses and remained evenly distributed throughout the cytoplasm. R2 was not observed until 4 hr postinfection and, in contrast to the even distribution of R1, was localised in discrete cytoplasmic foci close to the nucleus. In double-labelling experiments both R1 and R2 were found in these foci where they presumably associate to form the active enzyme. As expected R2 was not detectable in cells infected with ts 1222. In ts 1207-infected cells it formed wild-type-like foci, indicating that interaction with R1 is not required for R2 focus formation. R1 was present in a twofold excess over R2 in wild-type-infected cells. We suggest that the uncomplexed R1 could perform a role associated with the protein kinase present in the N-terminal domain.
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PMID:Intracellular localisation of herpes simplex virus type 1 ribonucleotide reductase subunits during infection of cultured cells. 749 85

Ribonucleotide reductase is a highly regulated activity responsible for reducing ribonucleotides to deoxyribonucleotides, which are required for DNA synthesis and DNA repair. We have tested the hypothesis that malignant cell populations contain alterations in signal pathways important in controlling the expression of the two genes that code for ribonucleotide reductase, R1 and R2. A series of radiation and H-ras transformed mouse 10T1/2 cell lines with increasing malignant potential were exposed to stimulators of cAMP synthesis (forskolin and cholera toxin), an inhibitor of cAMP degradation (3-isobutyl-1-methylxanthine) and a biologically stable analogue of cAMP (8-bromo-cAMP). Dramatic elevations in the expression of the R1 and R2 genes at the message and protein levels were observed in malignant metastatic populations, which were not detected in the normal parental cell line or in cells capable of benign tumor formation. These changes in ribonucleotide reductase gene expression occurred without any detectable modifications in the rates of DNA synthesis, showing that they were regulated by a novel mechanism independent of the S phase of the cell cycle. Furthermore, studies with forskolin (a stimulator of the protein kinase A signal pathway) and the tumor promoter 12-0-tetradecanoylphorbol-13-acetate (a stimulator of the protein kinase C signal pathway), alone or in combination, indicated that their effects on R1 and R2 gene expression in a highly malignant cell line were greater than when they were tested individually, suggesting that the two pathways modulating R1 and R2 gene expression can cooperate to regulate ribonucleotide reduction, and interestingly this can occur in a synergistic fashion. Also, a direct relationship between H-ras expression and ribonucleotide reductase gene expression was observed; analysis of forskolin mediated elevations in R1 and R2 message levels closely correlated with the levels of H-ras expression in the various cell lines. In total, these studies demonstrate that ribonucleotide reductase expression is controlled by a complex process, and malignant ras transformed cells contain alterations in the regulation of signal transduction pathways that lead to novel modifications in ribonucleotide reductase gene expression. This signal mechanism, which is aberrantly regulated in malignant cells, may be related to regulatory pathways involved in determining ribonucleotide reductase expression in a S phase independent manner during periods of DNA repair.
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PMID:Alterations in the cyclic AMP signal transduction pathway regulating ribonucleotide reductase gene expression in malignant H-ras transformed cell lines. 750 77

We have compared the protein kinase activities of the R1 subunits from herpes simplex virus types 1 (HSV-1) and 2 (HSV-2) ribonucleotide reductase following expression in Escherichia coli. Autophosphorylation activity was observed when kinase assays were performed with immunoprecipitated R1 or proteins purified to homogeneity, and the activity was stimulated by the basic protein protamine. Transphosphorylation of histones or calmodulin by purified or immunoprecipitated HSV-1 and HSV-2 R1 was not observed, and our results suggest that the activities of these two proteins are similar. We further characterized the protein kinase activity of HSV-1 R1 by producing insertion and deletion mutants constructed with a plasmid expressing R1 amino acids 1 to 449. C-terminal deletion analysis identified the catalytic core of the enzyme as comprising residues 1 to 292, and this polypeptide will be useful for structural determinations by X-ray crystallography. Insertion of a 4-amino-acid sequence at sites within the protein kinase domain identified regions essential for activity; insertions at residues 22 and 112 completely inactivated activity, and an insertion at residue 136 reduced activity sixfold. Similar insertions at residues 257, 262, 292, and 343 had no effect on activity. The ATP analog 5'-fluorosulfonylbenzoyladenosine, which covalently modifies conventional eukaryotic kinases at an essential lysine residue within the active site, did label HSV R1, but this labelling occurred outside the N-terminal domain. These data indicate that the HSV R1 kinase is novel and distinct from other eukaryotic protein kinases.
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PMID:Characterization of the novel protein kinase activity present in the R1 subunit of herpes simplex virus ribonucleotide reductase. 760 68

The large subunit of the HSV-2 ribonucleotide reductase (RR) (ICP10) is a chimera consisting of a serine threonine (Ser/Thr) protein kinase domain at the amino terminus and the RR domain at the carboxy terminus. Transformed human cells that constitutively express ICP10 (JHLa1) were stained with anti-LA-1 antibody (recognizes ICP10 amino acids 13-26) and immunogold-conjugated goat anti-rabbit IgG and were examined by electron microscopy. ICP10-associated gold particles were observed on the cell surface and in structures with ultrastructural characteristics of endocytic vesicles, multivesicular bodies, and lysosomes, consistent with endocytic internalization. ICP10 was also associated with the cytoskeleton fraction of JHLa1 cells and, at least in part, it colocalized with actin filaments. This was evidenced by immunoprecipitation of [35S]methionine-labeled cell fractions and immunofluorescent staining of Triton-treated cells with anti-LA-1 antibody and phalloidin. Endocytic localization of gold particles was not seen in cells that constitutively express the ICP10 transmembrane (TM)-deleted mutant p139TM (JHL15). p139TM did not associate with the cytoskeleton and was almost entirely localized within the cytoplasm. raf and Erk evidenced decreased mobility consistent with an activated state in JHLa1, but not JHL15, cells, and chloramphenicol acetyl transferase (CAT) expression from a c-fos/cat hybrid construct was significantly increased in JHLa1 but not JHL15 cells. The data indicate that effector molecules downstream of ras are activated in JHLa1 cells and the ICP10 TM segment plays a critical role in ICP10 intracellular localization and its ability to activate signaling pathways. This behavior is analogous to that of an activated growth factor receptor kinase.
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PMID:Intracellular internalization and signaling pathways triggered by the large subunit of HSV-2 ribonucleotide reductase (ICP10). 761 72

We examined the capability of pseudorabies virus (PRV) to replicate in vitro in porcine peripheral blood mononuclear cells (PBMC) and characterized the phenotype of infected cells. In addition, we investigated whether inactivation of various PRV proteins or the expression of a foreign gene affected this replication. Finally, we studied the replication of PRV strains in concanavalin A (Con A)-stimulated lymphocytes. The replication of PRV mutants with inactivated glycoproteins gE or gG, thymidine kinase (TK), ribonucleotide reductase (RR) or US3-encoded protein kinase (PK), and the replication of PRV vector strains expressing the envelope glycoprotein E1 of hog cholera virus (HCV) were studied. By adherence of PBMC to plastic, monocytes and lymphocytes were largely separated. Infected monocytes were analysed with an immunostaining monolayer assay and infected lymphocytes were analysed with immunofluorescence staining and flow cytometry. We found that the wild-type NIA-3 virus replicated in both lymphocyte and monocyte cultures. NIA-3 infected relatively more monocytes (> 90%) than non-adherent B cells (46-65%) and T cells (17-28%); approximately equal numbers of CD4+ and CD8+ T cells were infected. Although E1 is probably involved in adsorption of HCV to host cells, the expression of E1 by PRV vector strains did not change the level of replication. Inactivation of TK and RR, but not inactivation of gE, gG or PK, severely affected the replication in both monocytes and lymphocytes. Con A stimulation of lymphocytes restored the reduced replication of the TK mutant, but not of the RR mutant. Moreover, Con A stimulation of lymphocytes reduced the replication of the wild-type NIA-3 virus. We concluded that both viral TK and RR activity are important for efficient replication of PRV in resting lymphocytes. Furthermore, Con A-stimulated lymphocytes can restore the viral TK defect and PRV replication can also be influenced by cellular metabolism.
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PMID:Role of viral proteins and concanavalin A in in vitro replication of pseudorabies virus in porcine peripheral blood mononuclear cells. 778 71

The 140-kDa RR1 protein from both herpes simplex virus type 1 and type 2 (HSV-1 and HSV-2) functions as the large subunit of viral ribonucleotide reductase (RR) and contains an intrinsic serine/threonine protein kinase (PK) activity at the unique NH2-terminal domain. This PK activity is capable of autophosphorylation and reported to transphosphorylate histone and calmodulin by some groups but not by others. It has been suggested that the lack of consensus in the finding of transphosphorylation activity with RR1 protein may be due to HSV strain variations used in different laboratories. In the present study, we have attempted to resolve this issue by immunospecifically isolating the 140-kDa RR1 protein from four different strains of HSV-1 including F, KOS, HF, and MP and three different strains of HSV-2 including G, 333, and MS and subjecting them to immunocomplex kinase assays. In PK assays autophosphorylation of 140-kDa RR1 was readily observed in protein immunopurified from all HSV-1 and HSV-2 strains used in this study. However, using the same assay no transphosphorylation activity was observed with either the 38-kDa RR2 protein present in immunocomplex along with the RR1, or with histone, when added as an exogenous PK substrate. This conclusion is further supported by the evidence that a commercial preparation of PK (protein kinase catalytic subunit from bovine heart) readily phosphorylated histone under the conditions used for RR1 immunocomplex kinase assay. These results show that the 140-kDa RR1 protein contains an autokinase activity but it is incapable of transphosphorylating heterologous substrates such as histone. In addition, we show that the RR enzyme complex (140 and 38-kDa proteins) is associated with purified HSV-2 virions.
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PMID:The 140-kDa RR1 protein from both HSV-1 and HSV-2 contains an intrinsic protein kinase activity capable of autophosphorylation but it is transphosphorylation defective. 788 45

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.
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PMID:Posttranscriptional regulation of ribonucleotide reductase R1 gene expression is linked to a protein kinase C pathway in mammalian cells. 789 63

The large subunit (R1) of herpes simplex virus (HSV) ribonucleotide reductase is a bifunctional protein consisting of a unique N-terminal protein kinase domain and a ribonucleotide reductase domain. Previous studies showed that the two functional domains are linked by a protease sensitive site. Here we provide evidence for two subdomains, of 30K and 53K, within the reductase domain. The two fragments, which were produced by limited proteolysis and were resistant to further degradation, remained tightly associated in a complex containing two molecules of each. They were capable of binding the R2 subunit of HSV ribonucleotide reductase with approximately the same affinity as the intact protein but the complex did not complement the small subunit (R2) to give an active enzyme. At low concentrations (0.4 micrograms/ml) of trypsin or V8 protease, cleavage between the subdomains was prevented by the presence of the N-terminal protein kinase domain. At higher protease concentrations (1 micrograms/ml) the N-terminal domain is extensively proteolysed and the 30K and 53K domains were generated. Identical results were obtained using purified R1 isolated from infected cell extracts or following expression in Escherichia coli. The origin of the two domains was investigated by N-terminal sequencing of the 53K fragment and by examining their reactivity with a panel of R1-specific monoclonal antibodies which we isolated and epitope mapped for that purpose. The trypsin cleavage site was found to lie between arginine 575 and asparagine 576, and proteolysis in this region was not prevented by the presence of R2 or the nonapeptide YAGAVVNDL. We propose that the ribonucleotide reductase region of HSV R1 exists in a two domain structure, and that the interdomain linking region is protected by the unique N terminus.
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PMID:Identification of structural domains within the large subunit of herpes simplex virus ribonucleotide reductase. 799 27

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


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