Journal articles: 'Ribonucleotide kinase'

1

Chen, Frank Y., Francis M. Amara, and Jim A. Wright. "Posttranscriptional regulation of ribonucleotide reductase R1 gene expression is linked to a protein kinase C pathway in mammalian cells." Biochemistry and Cell Biology 72, no. 7-8 (July 1, 1994): 251–56. http://dx.doi.org/10.1139/o94-036.

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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.

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Merdanovic, Melisa, Elizabeta Sauer, and Joachim Reidl. "Coupling of NAD+ Biosynthesis and Nicotinamide Ribosyl Transport: Characterization of NadR Ribonucleotide Kinase Mutants of Haemophilus influenzae." Journal of Bacteriology 187, no. 13 (July 1, 2005): 4410–20. http://dx.doi.org/10.1128/jb.187.13.4410-4420.2005.

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ABSTRACT Previously, we characterized a pathway necessary for the processing of NAD+ and for uptake of nicotinamide riboside (NR) in Haemophilus influenzae. Here we report on the role of NadR, which is essential for NAD+ utilization in this organism. Different NadR variants with a deleted ribonucleotide kinase domain or with a single amino acid change were characterized in vitro and in vivo with respect to cell viability, ribonucleotide kinase activity, and NR transport. The ribonucleotide kinase mutants were viable only in a nadV + (nicotinamide phosphoribosyltransferase) background, indicating that the ribonucleotide kinase domain is essential for cell viability in H. influenzae. Mutations located in the Walker A and B motifs and the LID region resulted in deficiencies in both NR phosphorylation and NR uptake. The ribonucleotide kinase function of NadR was found to be feedback controlled by NAD+ under in vitro conditions and by NAD+ utilization in vivo. Taken together, our data demonstrate that the NR phosphorylation step is essential for both NR uptake across the inner membrane and NAD+ synthesis and is also involved in controlling the NAD+ biosynthesis rate.

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Dubacq, Caroline, Anne Chevalier, and Carl Mann. "The Protein Kinase Snf1 Is Required for Tolerance to the Ribonucleotide Reductase Inhibitor Hydroxyurea." Molecular and Cellular Biology 24, no. 6 (March 15, 2004): 2560–72. http://dx.doi.org/10.1128/mcb.24.6.2560-2572.2004.

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ABSTRACT The Snf1/AMP-activated kinases are involved in a wide range of stress responses in eukaryotic cells. We discovered a novel role for the Snf1 kinase in the cellular response to genotoxic stress in yeast. snf1 mutants are hypersensitive to hydroxyurea (HU), methyl-methane sulfonate, and cadmium, but they are not sensitive to several other genotoxic agents. HU inhibits ribonucleotide reductase (RNR), and deletion of SNF1 also increased the growth defects of an rnr4 ribonucleotide reductase mutant. The snf1 mutant has a functional checkpoint response to HU insofar as cells arrest division normally and derepress the transcription of RNR genes. The sensitivity of snf1 to HU or to RNR4 deletion may be due to posttranscriptional defects in RNR function or to defects in the repair of, and recovery from, stalled replication forks. The Mig3 repressor was identified as one target of Snf1 in this pathway. Genetic and biochemical analyses suggest that a weak kinase activity is sufficient to confer resistance to HU, whereas a high level of kinase activity is required for optimal growth on carbon sources other than glucose. Quantitative regulation of Snf1 kinase activity may contribute to the specificity of the effector responses that it controls.

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Klein, Beate, and Hartmut Follmann. "Deoxyribonucleotide Biosynthesis in Green Algae. S Phase-Specific Thymidylate Kinase and Unspecific Nucleoside Diphosphate Kinase in Scenedesmus obliquus." Zeitschrift für Naturforschung C 43, no. 5-6 (June 1, 1988): 377–85. http://dx.doi.org/10.1515/znc-1988-5-610.

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NDP kinase and thymidylate kinase are essential for DNA precursor formation in that they phosphorylate the products of de novo deoxyribonucleotide biosynthesis, deoxyribonucleoside 5′-diphosphates and thymidine 5′-monophosphate to the corresponding triphosphates which then serve as DNA polymerase substrates. The two enzymes have been measured in synchronous cultures of the green algae, S. obliquus. Thymidylate kinase exhibits an activity peak at the 11 -12th hour of the 24-hour cell cycle, coinciding with DNA synthesis. Enzyme activity is markedly stimulated in presence of fluorodeoxyuridine in the culture medium. This behaviour of dTMP kinase is very similar to that of three other S phase-specific peak enzymes previously analyzed in synchronous algae, viz. ribonucleotide reductase, thymidylate synthase, and dihydrofolate reductase. In contrast, NDP kinase exhibits high and constant activity through the entire cell cycle. The two kinases have been isolated from cell-free extracts, and separated from each other by chromatography on Blue Sepharose. The peak enzyme, dTMP kinase, has been purified to near homogeneity and its catalytic properties are described; the molecular weight is 56,000. NDP kinase activity is separable into two enzyme fractions, both of molecular weight 100,000 (or higher), which are unspecific with respect to ribonucleotide and deoxyribonucleotide substrates. Characterization and purification of the whole series of deoxyribonucleotide-synthesizing enzymes from one organism provides a basis for in vitro experiments towards reconstitution of an S phase-specific DNA precursor/DNA replication multienzyme aggregate.

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Hiller, Bjoern, Martin Achleitner, Silke Glage, Ronald Naumann, Rayk Behrendt, and Axel Roers. "Mammalian RNase H2 removes ribonucleotides from DNA to maintain genome integrity." Journal of Experimental Medicine 209, no. 8 (July 16, 2012): 1419–26. http://dx.doi.org/10.1084/jem.20120876.

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Ribonucleases H (RNases H) are endonucleases which cleave the RNA moiety of RNA/DNA hybrids. Their function in mammalian cells is incompletely understood. RNase H2 mutations cause Aicardi-Goutières syndrome, an inflammatory condition clinically overlapping with lupus erythematosus. We show that RNase H2 is essential in mouse embryonic development. RNase H2–deficient cells proliferated slower than control cells and accumulated in G2/M phase due to chronic activation of a DNA damage response associated with an increased frequency of single-strand breaks, increased histone H2AX phosphorylation, and induction of p53 target genes, most prominently the cyclin-dependent kinase inhibitor 1 encoding cell cycle inhibitor p21. RNase H2–deficient cells featured an increased genomic ribonucleotide load, suggesting that unrepaired ribonucleotides trigger the DNA damage response in these cells. Collectively, we show that RNase H2 is essential to remove ribonucleotides from the mammalian genome to prevent DNA damage.

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6

Davies, Lawrence C., John A. Stock, S. Elaine Barrie, Rosanne M. Orr, and Kenneth R. Harrap. "Dinucleotide analogs as inhibitors of thymidine kinase, thymidylate kinase and ribonucleotide reductase." Journal of Medicinal Chemistry 31, no. 7 (July 1988): 1305–8. http://dx.doi.org/10.1021/jm00402a008.

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7

Chen, F. Y., F. M. Amara, and J. A. Wright. "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." Biochemical Journal 302, no. 1 (August 15, 1994): 125–32. http://dx.doi.org/10.1042/bj3020125.

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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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Hurta, Robert A. R., and Jim A. Wright. "Regulation of mammalian ribonucleotide reductase by the tumor promoters and protein phosphatase inhibitors okadaic acid and calyculin A." Biochemistry and Cell Biology 70, no. 10-11 (October 1, 1992): 1081–87. http://dx.doi.org/10.1139/o92-153.

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A rapid elevation of ribonucleotide reductase activity was observed with BALB c/3T3 fibroblasts treated with 10 nM okadaic acid, a nonphorbol ester tumor promoter and protein phosphatase inhibitor. Northern blot analysis of the two components of ribonucleotide reductase (R1 and R2) showed a marked elevation of R1 and R2 mRNA expression. Western blot analysis with R1 and R2 specific monoclonal antibodies indicated that the increase in ribonucleotide reductase activity was primarily due to the elevation of the R2 rather than the R1 protein during treatment with okadaic acid. The okadaic acid induced elevations in R1 and R2 message levels occurred without a detectable change in the proportion of cells in S phase and were blocked by treatment of cells with actinomycin D, indicating the importance of the reductase transcriptional process in responding to the action of okadaic acid. Furthermore, down-regulation of protein kinase C with 12-O-tetradecanoylphorbol-13-acetate pretreatment abrogated the okadaic acid mediated elevation of ribonucleotide reductase mRNAs, consistent with the involvement of this signal pathway in the regulation of ribonucleotide reductase and the effects of okadaic acid. Treatment of cells with 2.5 nM calyculin A, another non-phorbol ester tumor promoter and protein phosphatase inhibitor, resulted in a rapid elevation of both R1 and R2 mRNA levels within 10 min of treatment. This first demonstration that the non-phorbol ester tumor promoters and protein phosphatase inhibitors can cause rapid alterations in ribonucleotide reductase gene expression suggests that (i) ribonucleotide reductase, particularly the R2 component, plays a fundamental role in the critical early events involved in the process of tumor promotion, and (ii) illustrates a role for cellular protein phosphatases in the regulation of ribonucleotide reductase and, through this process, the regulation of DNA synthesis.Key words: ribonucleotide reductase, DNA synthesis, okadaic acid, calyculin A, tumor promoter, protein phosphatase.

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9

Eaton, Jana S., Z. Ping Lin, Alan C. Sartorelli, Nicholas D. Bonawitz, and Gerald S. Shadel. "Ataxia-telangiectasia mutated kinase regulates ribonucleotide reductase and mitochondrial homeostasis." Journal of Clinical Investigation 117, no. 9 (September 4, 2007): 2723–34. http://dx.doi.org/10.1172/jci31604.

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10

Lin, Fumin, Thomas J. Ribar, and Anthony R. Means. "The Ca2+/Calmodulin-Dependent Protein Kinase Kinase, CaMKK2, Inhibits Preadipocyte Differentiation." Endocrinology 152, no. 10 (August 23, 2011): 3668–79. http://dx.doi.org/10.1210/en.2011-1107.

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When fed a standard chow diet, CaMKK2 null mice have increased adiposity and larger adipocytes than do wild-type mice, whereas energy balance is unchanged. Here, we show that Ca2+/calmodulin-dependent protein kinase kinase 2 (CaMKK2) is expressed in preadipocytes, where it functions as an AMP-activated protein kinase (AMPK)α kinase. Acute inhibition or deletion of CaMKK2 in preadipocytes enhances their differentiation into mature adipocytes, which can be reversed by 5-aminoimidazole-4-carboxamide ribonucleotide-mediated activation of AMPK. During adipogenesis, CaMKK2 expression is markedly decreased and temporally accompanied by increases in mRNA encoding the early adipogenic genes CCAAT/enhancer binding protein (C/EBP) β and C/EBP δ. Preadipocyte factor 1 has been reported to inhibit adipogenesis by up-regulating sex determining region Y-box 9 (Sox9) expression in preadipocytes and Sox9 suppresses C/EBPβ and C/EBPδ transcription. We show that inhibition of the CaMKK2/AMPK signaling cascade in preadipocytes reduces preadipocyte factor 1 and Sox9 mRNA resulting in accelerated adipogenesis. We conclude that CaMKK2 and AMPK function in a signaling pathway that participates in the regulation of adiposity.

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Kramata, Pavel, and Ivan Votruba. "Enzymes of Human Herpesviruses." Collection of Czechoslovak Chemical Communications 57, no. 8 (1992): 1577–612. http://dx.doi.org/10.1135/cccc19921577.

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The properties of human herpesvirus-encoded enzymes are reviewed and the importance of sequence analysis of viral genomes as well as the experiments on characteristics of enzymes isolated from infected cell cultures are emphasized. The following enzymes are described in detail: DNA replication complex consisting of DNA polymerase, DNA helicase-primase, single-stranded DNA binding protein and origin binding protein, further thymidine kinase, ribonucleotide reductase, deoxyuridine triphosphatase as well as uracil-DNA-glycosylase, deoxyribonuclease and protein kinase. The importance of these enzymes from the point of view of antiviral chemotherapy is discussed.

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Paik, Sehmi, Lauren Senty, Sankar Das, Jody C. Noe, Cindy L. Munro, and Todd Kitten. "Identification of Virulence Determinants for Endocarditis in Streptococcus sanguinis by Signature-Tagged Mutagenesis." Infection and Immunity 73, no. 9 (September 2005): 6064–74. http://dx.doi.org/10.1128/iai.73.9.6064-6074.2005.

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ABSTRACT Streptococcus sanguinis is a gram-positive, facultative anaerobe and a normal inhabitant of the human oral cavity. It is also one of the most common agents of infective endocarditis, a serious endovascular infection. To identify virulence factors for infective endocarditis, signature-tagged mutagenesis (STM) was applied to the SK36 strain of S. sanguinis, whose genome is being sequenced. STM allows the large-scale creation, in vivo screening, and recovery of a series of mutants with altered virulence. Screening of 800 mutants by STM identified 38 putative avirulent and 5 putative hypervirulent mutants. Subsequent molecular analysis of a subset of these mutants identified genes encoding undecaprenol kinase, homoserine kinase, anaerobic ribonucleotide reductase, adenylosuccinate lyase, and a hypothetical protein. Virulence reductions ranging from 2-to 150-fold were confirmed by competitive index assays. One putatively hypervirulent strain with a transposon insertion in an intergenic region was identified, though increased virulence was not confirmed in competitive index assays. All mutants grew comparably to SK36 in aerobic broth culture except for the homoserine kinase mutant. Growth of this mutant was restored by the addition of threonine to the medium. Mutants containing an insertion or in-frame deletion in the anaerobic ribonucleotide reductase gene failed to grow under strictly anaerobic conditions. The results suggest that housekeeping functions such as cell wall synthesis, amino acid and nucleic acid synthesis, and the ability to survive under anaerobic conditions are important virulence factors in S. sanguinis endocarditis.

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13

Zhao, Xiaolan, and Rodney Rothstein. "The Dun1 checkpoint kinase phosphorylates and regulates the ribonucleotide reductase inhibitor Sml1." Proceedings of the National Academy of Sciences 99, no. 6 (March 19, 2002): 3746–51. http://dx.doi.org/10.1073/pnas.062502299.

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Chan, Arthur K., Sujata Persad, David W. Litchfield, and Jim A. Wright. "Ribonucleotide reductase R2 protein is phosphorylated at serine-20 by P34cdc2 kinase." Biochimica et Biophysica Acta (BBA) - Molecular Cell Research 1448, no. 3 (January 1999): 363–71. http://dx.doi.org/10.1016/s0167-4889(98)00115-3.

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15

Downes, C. S., C. Z. Bachrati, S. J. Devlin, M. Tommasino, T. J. Cutts, J. V. Watson, I. Rasko, and R. T. Johnson. "Mammalian S-phase checkpoint integrity is dependent on transformation status and purine deoxyribonucleosides." Journal of Cell Science 113, no. 6 (March 15, 2000): 1089–96. http://dx.doi.org/10.1242/jcs.113.6.1089.

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In eukaryotic cells arrested in S-phase, checkpoint controls normally restrain mitosis until after replication. We have identified an array of previously unsuspected factors that modulate this restraint, using transformed hamster cells in which cycle controls are known to be altered in S-phase arrest. Arrested cells accumulate cyclin B, the regulatory partner of the mitotic p34(cdc2) kinase, which is normally not abundant until late G(2) phase; treatment of arrested cells with caffeine produces rapid S-phase condensation. We show here that such S-phase checkpoint slippage, as visualised through caffeine-dependent S-phase condensation, correlates with rodent origin and transformed status, is opposed by reverse transformation, and is favoured by c-src and opposed by wnt1 overexpression. Slippage is also dependent on a prolonged replicative arrest, and is favoured by arrest with hydroxyurea, which inhibits ribonucleotide reductase. This last is a key enzyme in deoxyribonucleotide synthesis, recently identified as a determinant of malignancy. Addition of deoxyribonucleosides shows that rapid S-phase condensation is suppressed by a novel checkpoint mechanism: purine (but not pyrimidine) deoxyribonucleosides, like reverse transformation, suppress cyclin B/p34(cdc2) activation by caffeine, but not cyclin B accumulation. Thus, ribonucleotide reductase has an unexpectedly complex role in mammalian cell cycle regulation: not only is it regulated in response to cycle progression, but its products can also reciprocally influence cell cycle control kinase activation.

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Nguyen, Alexandra, Melanie Dzulko, Janine Murr, Yun Yen, Günter Schneider, and Oliver H. Krämer. "Class 1 Histone Deacetylases and Ataxia-Telangiectasia Mutated Kinase Control the Survival of Murine Pancreatic Cancer Cells upon dNTP Depletion." Cells 10, no. 10 (September 23, 2021): 2520. http://dx.doi.org/10.3390/cells10102520.

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Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive disease with a dismal prognosis. Here, we show how an inhibition of de novo dNTP synthesis by the ribonucleotide reductase (RNR) inhibitor hydroxyurea and an inhibition of epigenetic modifiers of the histone deacetylase (HDAC) family affect short-term cultured primary murine PDAC cells. We used clinically relevant doses of hydroxyurea and the class 1 HDAC inhibitor entinostat. We analyzed the cells by flow cytometry and immunoblot. Regarding the induction of apoptosis and DNA replication stress, hydroxyurea and the novel RNR inhibitor COH29 are superior to the topoisomerase-1 inhibitor irinotecan which is used to treat PDAC. Entinostat promotes the induction of DNA replication stress by hydroxyurea. This is associated with an increase in the PP2A subunit PR130/PPP2R3A and a reduction of the ribonucleotide reductase subunit RRM2 and the DNA repair protein RAD51. We further show that class 1 HDAC activity promotes the hydroxyurea-induced activation of the checkpoint kinase ataxia-telangiectasia mutated (ATM). Unlike in other cell systems, ATM is pro-apoptotic in hydroxyurea-treated murine PDAC cells. These data reveal novel insights into a cytotoxic, ATM-regulated, and HDAC-dependent replication stress program in PDAC cells.

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Liu, Li, Jiri Veis, Wolfgang Reiter, Edwin Motari, Catherine E. Costello, John C. Samuelson, Gustav Ammerer, and David E. Levin. "Regulation of Pkc1 Hyper-Phosphorylation by Genotoxic Stress." Journal of Fungi 7, no. 10 (October 17, 2021): 874. http://dx.doi.org/10.3390/jof7100874.

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The cell wall integrity (CWI) signaling pathway is best known for its roles in cell wall biogenesis. However, it is also thought to participate in the response to genotoxic stress. The stress-activated protein kinase Mpk1 (Slt2, is activated by DNA damaging agents through an intracellular mechanism that does not involve the activation of upstream components of the CWI pathway. Additional observations suggest that protein kinase C (Pkc1), the top kinase in the CWI signaling cascade, also has a role in the response to genotoxic stress that is independent of its recognized function in the activation of Mpk1. Pkc1 undergoes hyper-phosphorylation specifically in response to genotoxic stress; we have found that this requires the DNA damage checkpoint kinases Mec1 (Mitosis Entry Checkpoint) and Tel1 (TELomere maintenance), but not their effector kinases. We demonstrate that the casein kinase 1 (CK1) ortholog, Hrr25 (HO and Radiation Repair), previously implicated in the DNA damage transcriptional response, associates with Pkc1 under conditions of genotoxic stress. We also found that the induced association of Hrr25 with Pkc1 requires Mec1 and Tel1, and that Hrr25 catalytic activity is required for Pkc1-hyperphosphorylation, thereby delineating a pathway from the checkpoint kinases to Pkc1. We used SILAC mass spectrometry to identify three residues within Pkc1 the phosphorylation of which was stimulated by genotoxic stress. We mutated these residues as well as a collection of 13 phosphorylation sites within the regulatory domain of Pkc1 that fit the consensus for CK1 sites. Mutation of the 13 Pkc1 phosphorylation sites blocked hyper-phosphorylation and diminished RNR3 (RiboNucleotide Reductase) basal expression and induction by genotoxic stress, suggesting that Pkc1 plays a role in the DNA damage transcriptional response.

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18

Sanvisens, N., A. M. Romero, X. An, C. Zhang, R. de Llanos, M. T. Martinez-Pastor, M. C. Bano, M. Huang, and S. Puig. "Yeast Dun1 Kinase Regulates Ribonucleotide Reductase Inhibitor Sml1 in Response to Iron Deficiency." Molecular and Cellular Biology 34, no. 17 (June 23, 2014): 3259–71. http://dx.doi.org/10.1128/mcb.00472-14.

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19

BRACCHI-RICARD, Valerie, Sailen BARIK, Cherie DELVECCHIO, Christian DOERIG, Ratna CHAKRABARTI, and Debopam CHAKRABARTI. "PfPK6, a novel cyclin-dependent kinase/mitogen-activated protein kinase-related protein kinase from Plasmodium falciparum." Biochemical Journal 347, no. 1 (March 27, 2000): 255–63. http://dx.doi.org/10.1042/bj3470255.

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We have isolated a novel protein kinase cDNA, PfPK6, by differential display RT-PCR (DDRT-PCR) of mRNA obtained from different asexual erythrocytic stages of Plasmodium falciparum, which shows sequence similarity to both cyclin-dependent kinase (CDK) and mitogen-activated protein kinase (MAPK) family members. The 915 bp open reading frame (ORF) is interrupted by seven introns and encodes a 305-residue polypeptide with a predicted molecular mass of 35848 Da. Several cDNA clones with some of the intron sequences were isolated, indicating alternate or defective splicing of PfPK6 transcripts because the gene seems to be a single copy located on chromosome 13. The similarity of the catalytic domain of PfPK6 to those of CDK2 and MAPK is 57.3% and 49.6%, respectively. The signature PSTAIRE (single-letter amino acid codes) CDK motif is changed to SKCILRE in PfPK6. The TXY residues that are phosphorylated in MAPKs for their activation are T173PT in PfPK6. Three size classes of PfPK6 transcripts of 6.5, 2.0 and 1.1 kb are up-regulated during the transition of P. falciparum from ring to trophozoite. Western blot analysis suggested the expression of a 35 kDa polypeptide in trophozoites and schizonts. Immunofluorescence studies indicated both nuclear and cytoplasmic localization of PfPK6 in trophozoite, schizont and segmenter stages. In vitro, recombinant PfPK6 phosphorylated itself and also exogenous substrates, histone and the small subunit of the malarial ribonucleotide reductase (R2). The kinase activity of PfPK6 is sensitive to CDK inhibitors such as olomoucine and roscovitine. PfPK6 showed a preference for Mn2+ over Mg2+ ions as a cofactor. The Lys38 → Arg mutant is severely defective in its interaction with ATP and bivalent cations and somewhat defective in catalytic rate for R2 phosphorylation.

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Tsao, Ning, Ya-Chi Yang, Yu-Jyun Deng, and Zee-Fen Chang. "The direct interaction of NME3 with Tip60 in DNA repair." Biochemical Journal 473, no. 9 (April 26, 2016): 1237–45. http://dx.doi.org/10.1042/bcj20160122.

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Cellular supply of dNTPs via RNR (ribonucleotide reductase) is crucial for DNA replication and repair. It has been shown that DNA-damage-site-specific recruitment of RNR is critical for DNA repair efficiency in quiescent cells. The catalytic function of RNR produces dNDPs. The subsequent step of dNTP formation requires the function of NDP kinase. There are ten isoforms of NDP kinase in human cells. In the present study, we identified NME3 as one specific NDP kinase that interacts directly with Tip60, a histone acetyltransferase, to form a complex with RNR. Our data reveal that NME3 recruitment to DNA damage sites depends on this interaction. Disruption of interaction of NME3 with Tip60 suppressed DNA repair in serum-deprived cells. Thus Tip60 interacts with RNR and NME3 to provide site-specific synthesis of dNTP for facilitating DNA repair in serum-deprived cells which contain low levels of dNTPs.

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Sanvisens, Nerea, Antonia M. Romero, Caiguo Zhang, Xiaorong Wu, Xiuxiang An, Mingxia Huang, and Sergi Puig. "Yeast Dun1 Kinase Regulates Ribonucleotide Reductase Small Subunit Localization in Response to Iron Deficiency." Journal of Biological Chemistry 291, no. 18 (March 12, 2016): 9807–17. http://dx.doi.org/10.1074/jbc.m116.720862.

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Albert, Daniel A., and Edwardine Nodzenski. "M2 subunit of ribonucleotide reductase is a target of cyclic AMP-dependent protein kinase." Journal of Cellular Physiology 138, no. 1 (January 1989): 129–36. http://dx.doi.org/10.1002/jcp.1041380118.

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Boccalatte, Francesco E., Claudia Voena, Chiara Riganti, Amalia Bosia, Lucia D'Amico, Ludovica Riera, Mangeng Cheng, et al. "The enzymatic activity of 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/IMP cyclohydrolase is enhanced by NPM-ALK: new insights in ALK-mediated pathogenesis and the treatment of ALCL." Blood 113, no. 12 (March 19, 2009): 2776–90. http://dx.doi.org/10.1182/blood-2008-06-161018.

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AbstractAnaplastic large cell lymphoma represents a subset of neoplasms caused by translocations that juxtapose the anaplastic lymphoma kinase (ALK) to dimerization partners. The constitutive activation of ALK fusion proteins leads to cellular transformation through a complex signaling network. To elucidate the ALK pathways sustaining lymphomagenesis and tumor maintenance, we analyzed the tyrosine-kinase protein profiles of ALK-positive cell lines using 2 complementary proteomic-based approaches, taking advantage of a specific ALK RNA interference (RNAi) or cell-permeable inhibitors. A well-defined set of ALK-associated tyrosine phosphopeptides, including metabolic enzymes, kinases, ribosomal and cytoskeletal proteins, was identified. Validation studies confirmed that vasodilator-stimulated phosphoprotein and 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/inosine monophosphate cyclohydrolase (ATIC) associated with nucleophosmin (NPM)–ALK, and their phosphorylation required ALK activity. ATIC phosphorylation was documented in cell lines and primary tumors carrying ALK proteins and other tyrosine kinases, including TPR-Met and wild type c-Met. Functional analyses revealed that ALK-mediated ATIC phosphorylation enhanced its enzymatic activity, dampening the methotrexate-mediated transformylase activity inhibition. These findings demonstrate that proteomic approaches in well-controlled experimental settings allow the definition of informative proteomic profiles and the discovery of novel ALK downstream players that contribute to the maintenance of the neoplastic phenotype. Prediction of tumor responses to methotrexate may justify specific molecular-based chemotherapy.

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Guetsova, Maria L., Karine Lecoq, and Bertrand Daignan-Fornier. "The Isolation and Characterization of Saccharomyces cerevisiae Mutants That Constitutively Express Purine Biosynthetic Genes." Genetics 147, no. 2 (October 1, 1997): 383–97. http://dx.doi.org/10.1093/genetics/147.2.383.

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In response to an external source of adenine, yeast cells repress the expression of purine biosynthesis pathway genes. To identify necessary components of this signalling mechanism, we have isolated mutants that are constitutively active for expression. These mutants were named bra (for bypass of repression by adenine). BRA7 is allelic to FCY2, the gene encoding the purine cytosine permease and BRA9 is ADE12, the gene encoding adenylosuccinate synthetase. BRA6 and BRA1 are new genes encoding, respectively, hypoxanthine guanine phosphoribosyl transferase and adenylosuccinate lyase. These results indicate that uptake and salvage of adenine are important steps in regulating expression of purine biosynthetic genes. We have also shown that two other salvage enzymes, adenine phosphoribosyl transferase and adenine deaminase, are involved in activating the pathway. Finally, using mutant strains affected in AMP kinase or ribonucleotide reductase activities, we have shown that AMP needs to be phosphorylated to ADP to exert its regulatory role while reduction of ADP into dADP by ribonucleotide reductase is not required for adenine repression. Together these data suggest that ADP or a derivative of ADP is the effector molecule in the signal transduction pathway.

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Piao, C., M. Jin, H. B. Kim, S. M. Lee, P. N. Amatya, J. W. Hyun, I. Y. Chang, and H. J. You. "Ribonucleotide reductase small subunit p53R2 suppresses MEK–ERK activity by binding to ERK kinase 2." Oncogene 28, no. 21 (April 27, 2009): 2173–84. http://dx.doi.org/10.1038/onc.2009.84.

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Zhang, Yong-Wei, Tamara L. Jones, Scott E. Martin, Natasha J. Caplen, and Yves Pommier. "Implication of Checkpoint Kinase-dependent Up-regulation of Ribonucleotide Reductase R2 in DNA Damage Response." Journal of Biological Chemistry 284, no. 27 (May 5, 2009): 18085–95. http://dx.doi.org/10.1074/jbc.m109.003020.

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Milho, Ricardo, Michael B. Gill, Janet S. May, Susanna Colaco, and Philip G. Stevenson. "In vivo function of the murid herpesvirus-4 ribonucleotide reductase small subunit." Journal of General Virology 92, no. 7 (July 1, 2011): 1550–60. http://dx.doi.org/10.1099/vir.0.031542-0.

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The difficulty of eliminating herpesvirus carriage makes host entry a key target for infection control. However, its viral requirements are poorly defined. Murid herpesvirus-4 (MuHV-4) can potentially provide insights into gammaherpesvirus host entry. Upper respiratory tract infection requires the MuHV-4 thymidine kinase (TK) and ribonucleotide reductase large subunit (RNR-L), suggesting a need for increased nucleotide production. However, both TK and RNR-L are likely to be multifunctional. We therefore tested further the importance of nucleotide production by disrupting the MuHV-4 ribonucleotide reductase small subunit (RNR-S). This caused a similar attenuation to RNR-L disruption: despite reduced intra-host spread, invasive inoculations still established infection, whereas a non-invasive upper respiratory tract inoculation did so only at high dose. Histological analysis showed that RNR-S−, RNR-L− and TK− viruses all infected cells in the olfactory neuroepithelium but unlike wild-type virus then failed to spread. Thus captured host nucleotide metabolism enzymes, up to now defined mainly as important for alphaherpesvirus reactivation in neurons, also have a key role in gammaherpesvirus host entry. This seemed to reflect a requirement for lytic replication to occur in a terminally differentiated cell before a viable pool of latent genomes could be established.

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Corda, Yves, Sang Eun Lee, Sylvine Guillot, André Walther, Julie Sollier, Ayelet Arbel-Eden, James E. Haber, and Vincent Géli. "Inactivation of Ku-Mediated End Joining Suppresses mec1Δ Lethality by Depleting the Ribonucleotide Reductase Inhibitor Sml1 through a Pathway Controlled by Tel1 Kinase and the Mre11 Complex." Molecular and Cellular Biology 25, no. 23 (December 1, 2005): 10652–64. http://dx.doi.org/10.1128/mcb.25.23.10652-10664.2005.

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ABSTRACT RAD53 and MEC1 are essential Saccharomyces cerevisiae genes required for the DNA replication and DNA damage checkpoint responses. Their lethality can be suppressed by increasing the intracellular pool of deoxynucleotide triphosphates. We report that deletion of YKU70 or YKU80 suppresses mec1Δ, but not rad53Δ, lethality. We show that suppression of mec1Δ lethality is not due to Ku−-associated telomeric defects but rather results from the inability of Ku− cells to efficiently repair DNA double strand breaks by nonhomologous end joining. Consistent with these results, mec1Δ lethality is also suppressed by lif1Δ, which like yku70Δ and yku80Δ, prevents nonhomologous end joining. The viability of yku70Δ mec1Δ and yku80Δ mec1Δ cells depends on the ATM-related Tel1 kinase, the Mre11-Rad50-Xrs2 complex, and the DNA damage checkpoint protein Rad9. We further report that this Mec1-independent pathway converges with the Rad53/Dun1-regulated checkpoint kinase cascade and leads to the degradation of the ribonucleotide reductase inhibitor Sml1.

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Ahluwalia, Amrita, Neil Hoa, Michael K. Jones, and Andrzej S. Tarnawski. "NSAID-induced injury of gastric epithelial cells is reversible: roles of mitochondria, AMP kinase, NGF, and PGE2." American Journal of Physiology-Gastrointestinal and Liver Physiology 317, no. 6 (December 1, 2019): G862—G871. http://dx.doi.org/10.1152/ajpgi.00192.2019.

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Nonsteroidal anti-inflammatory drugs (NSAIDs) such as diclofenac (DFN) and indomethacin (INDO) are extensively used worldwide. Their main side effects are injury of the gastrointestinal tract, including erosions, ulcers, and bleeding. Since gastric epithelial cells (GEPCs) are crucial for mucosal defense and are the major target of injury, we examined the extent to which DFN- and INDO-induced GEPC injury can be reversed by nerve growth factor (NGF), 16,16 dimethyl prostaglandin E2 (dmPGE2), and 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR), the pharmacological activator of the metabolic sensor AMP kinase (AMPK). Cultured normal rat gastric mucosal epithelial (RGM1) cells were treated with PBS (control), NGF, dmPGE2, AICAR, and/or NSAID (DFN or INDO) for 1–4 h. We examined cell injury by confocal microscopy, cell death/survival using calcein AM, mitochondrial membrane potential using MitoTracker, and phosphorylation of AMPK by Western blotting. DFN and INDO treatment of RGM1 cells for 2 h decreased mitochondrial membrane potential and cell viability. NGF posttreatment (initiated 1 or 2 h after DFN or INDO) reversed the dissipation of mitochondrial membrane potential and cell injury caused by DFN and INDO and increased cell viability versus cells treated for 4 h with NSAID alone. Pretreatment with dmPGE2 and AICAR significantly protected these cells from DFN- and INDO-induced injury, whereas dmPGE2 and AICAR posttreatment (initiated 1 h after NSAID treatment) reversed cell injury and significantly increased cell viability and rescued the cells from NSAID-induced mitochondrial membrane potential reduction. DFN and INDO induce extensive mitochondrial injury and GEPC death, which can be significantly reversed by NGF, dmPGE2, and AICAR. NEW & NOTEWORTHY This study demonstrated that mitochondria are key targets of diclofenac- and indomethacin-induced injury of gastric epithelial cells and that diclofenac and indomethacin injury can be prevented and, importantly, also reversed by treatment with nerve growth factor, 16,16 dimethyl prostaglandin E2, and 5-aminoimidazole-4-carboxamide ribonucleotide.

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Yue, Yingying, Chang Zhang, Xuejiao Zhang, Shitian Zhang, Qian Liu, Fang Hu, Xiaoting Lv, et al. "An AMPK/Axin1-Rac1 signaling pathway mediates contraction-regulated glucose uptake in skeletal muscle cells." American Journal of Physiology-Endocrinology and Metabolism 318, no. 3 (March 1, 2020): E330—E342. http://dx.doi.org/10.1152/ajpendo.00272.2019.

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Contraction stimulates skeletal muscle glucose uptake predominantly through activation of AMP-activated protein kinase (AMPK) and Rac1. However, the molecular details of how contraction activates these signaling proteins are not clear. Recently, Axin1 has been shown to form a complex with AMPK and liver kinase B1 during glucose starvation-dependent activation of AMPK. Here, we demonstrate that electrical pulse-stimulated (EPS) contraction of C2C12 myotubes or treadmill exercise of C57BL/6 mice enhanced reciprocal coimmunoprecipitation of Axin1 and AMPK from myotube lysates or gastrocnemius muscle tissue. Interestingly, EPS or exercise upregulated total cellular Axin1 levels in an AMPK-dependent manner in C2C12 myotubes and gastrocnemius mouse muscle, respectively. Also, direct activation of AMPK with 5-aminoimidazole-4-carboxamide ribonucleotide treatment of C2C12 myotubes or gastrocnemius muscle elevated Axin1 protein levels. On the other hand, siRNA-mediated Axin1 knockdown lessened activation of AMPK in contracted myotubes. Further, AMPK inhibition with compound C or siRNA-mediated knockdown of AMPK or Axin1 blocked contraction-induced GTP loading of Rac1, p21-activated kinase phosphorylation, and contraction-stimulated glucose uptake. In summary, our results suggest that an AMPK/Axin1-Rac1 signaling pathway mediates contraction-stimulated skeletal muscle glucose uptake.

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Sigmond, Jennifer, Andries M. Bergman, Leticia G. Leon, Willem J. P. Loves, Eveline K. Hoebe, and Godefridus J. Peters. "Staurosporine increases toxicity of gemcitabine in non-small cell lung cancer cells: role of protein kinase C, deoxycytidine kinase and ribonucleotide reductase." Anti-Cancer Drugs 21, no. 6 (July 2010): 591–99. http://dx.doi.org/10.1097/cad.0b013e32833a3543.

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Theodoropoulou, Sofia, Paraskevi E. Kolovou, Yuki Morizane, Maki Kayama, Fotini Nicolaou, Joan W. Miller, Evangelos Gragoudas, Bruce R. Ksander, and Demetrios G. Vavvas. "Retinoblastoma cells are inhibited by aminoimidazole carboxamide ribonucleotide (AICAR) partially through activation of AMP‐dependent kinase." FASEB Journal 24, no. 8 (April 6, 2010): 2620–30. http://dx.doi.org/10.1096/fj.09-152546.

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Boxem, M., D. G. Srinivasan, and S. van den Heuvel. "The Caenorhabditis elegans gene ncc-1 encodes a cdc2-related kinase required for M phase in meiotic and mitotic cell divisions, but not for S phase." Development 126, no. 10 (May 15, 1999): 2227–39. http://dx.doi.org/10.1242/dev.126.10.2227.

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We have identified six protein kinases that belong to the family of cdc2-related kinases in Caenorhabditis elegans. Results from RNA interference experiments indicate that at least one of these kinases is required for cell-cycle progression during meiosis and mitosis. This kinase, encoded by the ncc-1 gene, is closely related to human Cdk1/Cdc2, Cdk2 and Cdk3 and yeast CDC28/cdc2(+). We addressed whether ncc-1 acts to promote passage through a single transition or multiple transitions in the cell cycle, analogous to Cdks in vertebrates or yeasts, respectively. We isolated five recessive ncc-1 mutations in a genetic screen for mutants that resemble larval arrested ncc-1(RNAi) animals. Our results indicate that maternal ncc-1 product is sufficient for embryogenesis, and that zygotic expression is required for cell divisions during larval development. Cells that form the postembryonic lineages in wild-type animals do not enter mitosis in ncc-1 mutants, as indicated by lack of chromosome condensation and nuclear envelope breakdown. However, progression through G1 and S phase appears unaffected, as revealed by expression of ribonucleotide reductase, incorporation of BrdU and DNA quantitation. Our results indicate that C. elegans uses multiple Cdks to regulate cell-cycle transitions and that ncc-1 is the C. elegans ortholog of Cdk1/Cdc2 in other metazoans, required for M phase in meiotic as well as mitotic cell cycles.

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Yang, Pei-Ming, Li-Shan Lin, and Tsang-Pai Liu. "Sorafenib Inhibits Ribonucleotide Reductase Regulatory Subunit M2 (RRM2) in Hepatocellular Carcinoma Cells." Biomolecules 10, no. 1 (January 9, 2020): 117. http://dx.doi.org/10.3390/biom10010117.

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The main curative treatments for hepatocellular carcinoma (HCC) are surgical resection and liver transplantation, which only benefits 15% to 25% of patients. In addition, HCC is highly refractory and resistant to cytotoxic chemotherapy. Although several multi-kinase inhibitors, such as sorafenib, regorafenib, and lenvatinib, have been approved for treating advanced HCC, only a short increase of median overall survival in HCC patients was achieved. Therefore, there is an urgent need to design more effective strategies for advanced HCC patients. Human ribonucleotide reductase is responsible for the conversion of ribonucleoside diphosphate to 2′-deoxyribonucleoside diphosphate to maintain the homeostasis of nucleotide pools. In this study, mining the cancer genomics and proteomics data revealed that ribonucleotide reductase regulatory subunit M2 (RRM2) serves as a prognosis biomarker and a therapeutic target for HCC. The RNA sequencing (RNA-Seq) analysis and public microarray data mining found that RRM2 was a novel molecular target of sorafenib in HCC cells. In vitro experiments validated that sorafenib inhibits RRM2 expression in HCC cells, which is positively associated with the anticancer activity of sorafenib. Although both RRM2 knockdown and sorafenib induced autophagy in HCC cells, restoration of RRM2 expression did not rescue HCC cells from sorafenib-induced autophagy and growth inhibition. However, long-term colony formation assay indicated that RRM2 overexpression partially rescues HCC cells from the cytotoxicity of sorafenib. Therefore, this study identifies that RRM2 is a novel target of sorafenib, partially contributing to its anticancer activity in HCC cells.

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Marsolier, Marie-Claude, Pascal Roussel, Christophe Leroy, and Carl Mann. "Involvement of the PP2C-Like Phosphatase Ptc2p in the DNA Checkpoint Pathways of Saccharomyces cerevisiae." Genetics 154, no. 4 (April 1, 2000): 1523–32. http://dx.doi.org/10.1093/genetics/154.4.1523.

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Abstract RAD53 encodes a conserved protein kinase that acts as a central transducer in the DNA damage and the DNA replication checkpoint pathways in Saccharomyces cerevisiae. To identify new elements of these pathways acting with or downstream of RAD53, we searched for genes whose overexpression suppressed the toxicity of a dominant-lethal form of RAD53 and identified PTC2, which encodes a protein phosphatase of the PP2C family. PTC2 overexpression induces hypersensitivity to genotoxic agents in wild-type cells and is lethal to rad53, mec1, and dun1 mutants with low ribonucleotide reductase activity. Deleting PTC2 specifically suppresses the hydroxyurea hypersensitivity of mec1 mutants and the lethality of mec1Δ. PTC2 is thus implicated in one or several functions related to RAD53, MEC1, and the DNA checkpoint pathways.

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Bhalla, Amit. "Clofarabine: a next-generation deoxyadenosine analogue." International Journal of Basic & Clinical Pharmacology 7, no. 5 (April 23, 2018): 1048. http://dx.doi.org/10.18203/2319-2003.ijbcp20181660.

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Acute lymphoblastic leukaemia (ALL) is the most common of the paediatric leukaemias. It is estimated that the use of modern combination chemotherapy results in long-term remission in nearly 80% of children diagnosed with ALL. Despite therapy advances, approximately 20% of children with ALL, experience leukaemia relapse. Clofarabine (2-chloro-2’-fluoro-2’-deoxy-9-β-D-arabinofuranosyladenine) is a second-generation nucleoside analogue and is structurally related to fludarabine and cladribine which are widely used in the treatment of lymphoproliferative disorders. Clofarabine exhibits greater affinity to deoxycytidine kinase (dCyd kinase) and prolonged retention in leukaemic blasts compared to fludarabine and cladribine. Clofarabine inhibits both DNA polymerases and ribonucleotide reductase (RNR). This results in impaired DNA synthesis through inhibition of DNA elongation as well as depletion of deoxyribonucleotides. Accumulation of clofarabine triphosphate, in the blasts of patients with refractory leukemia has been demonstrated. Prolonged intracellular half-life of 24 hours for clofarabine triphosphate. Clofarabine is indicated for the treatment of pediatric patients 1 to 21 years old with relapsed or refractory acute lymphoblastic leukemia after at least two prior regimens.

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OLIVER, F. Javier, Mary K. L. COLLINS, and Abelardo LÓPEZ-RIVAS. "Regulation of the salvage pathway of deoxynucleotides synthesis in apoptosis induced by growth factor deprivation." Biochemical Journal 316, no. 2 (June 1, 1996): 421–25. http://dx.doi.org/10.1042/bj3160421.

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Here we describe changes in dNTP metabolism that precede DNA fragmentation in a model of apoptosis driven by deprivation of the cytokine interleukin 3 (IL-3). In haemopoietic BAF3 cells, IL-3 withdrawal leads to a rapid decrease in the size of dATP, dTTP and dGTP pools without affecting dCTP levels. This imbalance in dNTP pools precedes DNA fragmentation and is accompanied by down-regulation of enzymes controlling the de novo and salvage pathways of dNTP synthesis, ribonucleotide reductase and thymidine kinase (TK) respectively. Readdition of IL-3 results in a rapid, protein synthesis-independent restoration of normal dNTP pools, enhanced TK activity and increased precursor incorporation through the salvage pathway. Up-regulation of TK activity after IL-3 readdition is prevented by the protein kinase C (PKC) inhibitor staurosporin, but not by tyrosine kinase inhibitors. Furthermore activation of PKC by phorbol esters mimics the stimulatory effect of IL-3 on TK activity, suggesting that PKC might be involved in regulating this effect. These results indicate that regulation by IL-3 of the salvage pathway of dNTP synthesis plays a role in the maintenance of cellular dNTP pool balance and suggests that alterations in dNTP metabolism after IL-3 deprivation could be a relevant event in the commitment of haemopoietic cells to apoptosis.

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Gao, Jin, and Des R. Richardson. "The potential of iron chelators of the pyridoxal isonicotinoyl hydrazone class as effective antiproliferative agents, IV: the mechanisms involved in inhibiting cell-cycle progression." Blood 98, no. 3 (August 1, 2001): 842–50. http://dx.doi.org/10.1182/blood.v98.3.842.

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Abstract Some chelators of the pyridoxal isonicotinoyl hydrazone class have antiproliferative activity that is far greater than desferrioxamine (DFO). In this study, DFO was compared with one of the most active chelators (311) on the expression of molecules that play key roles in cell-cycle control. This was vital for understanding the role of iron (Fe) in cell-cycle progression and for designing chelators to treat cancer. Incubating cells with DFO, and especially 311, resulted in a decrease in the hyperphosphorylated form of the retinoblastoma susceptibility gene product (pRb). Chelators also decreased cyclins D1, D2, and D3, which bind with cyclin-dependent kinase 4 (cdk4) to phosphorylate pRb. The levels of cdk2 also decreased after incubation with DFO, and especially 311, which may be important for explaining the decrease in hyperphosphorylated pRb. Cyclins A and B1 were also decreased after incubation with 311 and, to a lesser extent, DFO. In contrast, cyclin E levels increased. These effects were prevented by presaturating the chelators with Fe. In contrast to DFO and 311, the ribonucleotide reductase inhibitor hydroxyurea increased the expression of all cyclins. Hence, the effect of chelators on cyclin expression was not due to their ability to inhibit ribonucleotide reductase. Although chelators induced a marked increase in WAF1 and GADD45 mRNA transcripts, there was no appreciable increase in their protein levels. Failure to translate these cell-cycle inhibitors may contribute to dysregulation of the cell cycle after exposure to chelators.

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Pons, Miriam, Yanira Zeyn, Stella Zahn, Nisintha Mahendrarajah, Brent D. G. Page, Patrick T. Gunning, Richard Moriggl, Walburgis Brenner, Falk Butter, and Oliver H. Krämer. "Oncogenic Kinase Cascades Induce Molecular Mechanisms That Protect Leukemic Cell Models from Lethal Effects of De Novo dNTP Synthesis Inhibition." Cancers 13, no. 14 (July 10, 2021): 3464. http://dx.doi.org/10.3390/cancers13143464.

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The ribonucleotide reductase inhibitor hydroxyurea suppresses de novo dNTP synthesis and attenuates the hyperproliferation of leukemic blasts. Mechanisms that determine whether cells undergo apoptosis in response to hydroxyurea are ill-defined. We used unbiased proteomics to uncover which pathways control the transition of the hydroxyurea-induced replication stress into an apoptotic program in chronic and acute myeloid leukemia cells. We noted a decrease in the serine/threonine kinase RAF1/c-RAF in cells that undergo apoptosis in response to clinically relevant doses of hydroxyurea. Using the RAF inhibitor LY3009120, we show that RAF activity determines the sensitivity of leukemic cells toward hydroxyurea. We further disclose that pharmacological inhibition of the RAF downstream target BCL-XL with the drug navitoclax and RNAi combine favorably with hydroxyurea against leukemic cells. BCR-ABL1 and hyperactive FLT3 are tyrosine kinases that causally contribute to the development of leukemia and induce RAF1 and BCL-XL. Accordingly, the ABL inhibitor imatinib and the FLT3 inhibitor quizartinib sensitize leukemic cells to pro-apoptotic effects of hydroxyurea. Moreover, hydroxyurea and navitoclax kill leukemic cells with mutant FLT3 that are resistant to quizartinib. These data reveal cellular susceptibility factors toward hydroxyurea and how they can be exploited to eliminate difficult-to-treat leukemic cells with clinically relevant drug combinations.

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Mulherin, Andrew J., Amy H. Oh, Helena Kim, Anthony Grieco, Lina M. Lauffer, and Patricia L. Brubaker. "Mechanisms Underlying Metformin-Induced Secretion of Glucagon-Like Peptide-1 from the Intestinal L Cell." Endocrinology 152, no. 12 (October 4, 2011): 4610–19. http://dx.doi.org/10.1210/en.2011-1485.

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Glucagon-like peptide-17-36NH2 (GLP-1) is secreted by the intestinal L cell in response to both nutrient and neural stimulation, resulting in enhanced glucose-dependent insulin secretion. GLP-1 is therefore an attractive therapeutic for the treatment of type 2 diabetes. The antidiabetic drug, metformin, is known to increase circulating GLP-1 levels, although its mechanism of action is unknown. Direct effects of metformin (5–2000 μm) or another AMP kinase activator, aminoimidazole carboxamide ribonucleotide (100–1000 μm) on GLP-1 secretion were assessed in murine human NCI-H716, and rat FRIC L cells. Neither agent stimulated GLP-1 secretion in any model, despite increasing AMP kinase phosphorylation (P < 0.05–0.01). Treatment of rats with metformin (300 mg/kg, per os) or aminoimidazole carboxamide ribonucleotide (250 mg/kg, sc) increased plasma total GLP-1 over 2 h, reaching 37 ± 9 and 29 ± 9 pg/ml (P < 0.001), respectively, compared with basal (7 ± 1 pg/ml). Plasma activity of the GLP-1-degrading enzyme, dipeptidylpeptidase-IV, was not affected by metformin treatment. Pretreatment with the nonspecific muscarinic antagonist, atropine (1 mg/kg, iv), decreased metformin-induced GLP-1 secretion by 55 ± 11% (P < 0.05). Pretreatment with the muscarinic (M) 3 receptor antagonist, 1-1-dimethyl-4-diphenylacetoxypiperidinium iodide (500 μg/kg, iv), also decreased the GLP-1 area under curve, by 48 ± 8% (P < 0.05), whereas the antagonists pirenzepine (M1) and gallamine (M2) had no effect. Furthermore, chronic bilateral subdiaphragmatic vagotomy decreased basal secretion compared with sham-operated animals (7 ± 1 vs. 13 ± 1 pg/ml, P < 0.001) but did not alter the GLP-1 response to metformin. In contrast, pretreatment with the gastrin-releasing peptide antagonist, RC-3095 (100 μg/kg, sc), reduced the GLP-1 response to metformin, by 55 ± 6% (P < 0.01) at 30 min. These studies elucidate the mechanism underlying metformin-induced GLP-1 secretion and highlight the benefits of using metformin with dipeptidylpeptidase-IV inhibitors in patients with type 2 diabetes.

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Jian, Ming-Yuan, Yanping Liu, Qian Li, Paul Wolkowicz, Mikhail Alexeyev, Jaroslaw Zmijewski, and Judy Creighton. "N-cadherin coordinates AMP kinase-mediated lung vascular repair." American Journal of Physiology-Lung Cellular and Molecular Physiology 310, no. 1 (January 1, 2016): L71—L85. http://dx.doi.org/10.1152/ajplung.00227.2015.

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Injury to the pulmonary circulation compromises endothelial barrier function and increases lung edema. Resolution of lung damage involves restoring barrier integrity, a process requiring reestablishment of endothelial cell-cell adhesions. However, mechanisms underlying repair in lung endothelium are poorly understood. In pulmonary microvascular endothelium, AMP kinase α1 (AMPKα1) stimulation enhances recovery of the endothelial barrier after LPS-induced vascular damage. AMPKα1 colocalizes to a discrete membrane compartment with the adhesion protein neuronal cadherin (N-cadherin). This study sought to determine N-cadherin's role in the repair process. Short-hairpin RNA against full-length N-cadherin or a C-terminally truncated N-cadherin, designed to disrupt the cadherin's interactions with intracellular proteins, were expressed in lung endothelium. Disruption of N-cadherin's intracellular domain caused translocation of AMPK away from the membrane and attenuated AMPK-mediated restoration of barrier function in LPS-treated endothelium. AMPK activity measurements indicated that lower basal AMPK activity in cells expressing the truncated N-cadherin compared with controls. Moreover, the AMPK stimulator 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) failed to increase AMPK activity in cells expressing the modified N-cadherin, indicating uncoupling of a functional association between AMPK and the cadherin. Isolated lung studies confirmed a physiologic role for this pathway in vivo. AMPK activation reversed LPS-induced increase in permeability, whereas N-cadherin inhibition hindered AMPK-mediated repair. Thus N-cadherin coordinates the vascular protective actions of AMPK through a functional link with the kinase. This study provides insight into intrinsic repair mechanisms in the lung and supports AMPK stimulation as a modality for treating vascular disease.

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Chung, T. D., J. P. Wymer, C. C. Smith, M. Kulka, and L. Aurelian. "Protein kinase activity associated with the large subunit of herpes simplex virus type 2 ribonucleotide reductase (ICP10)." Journal of Virology 63, no. 8 (1989): 3389–98. http://dx.doi.org/10.1128/jvi.63.8.3389-3398.1989.

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Mazumder, Aprotim, Katja Tummler, Mark Bathe, and Leona D. Samson. "Single-Cell Analysis of Ribonucleotide Reductase Transcriptional and Translational Response to DNA Damage." Molecular and Cellular Biology 33, no. 3 (November 26, 2012): 635–42. http://dx.doi.org/10.1128/mcb.01020-12.

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ABSTRACTThe ribonucleotide reductase (RNR) enzyme catalyzes an essential step in the production of deoxyribonucleotide triphosphates (dNTPs) in cells. Bulk biochemical measurements in synchronizedSaccharomyces cerevisiaecells suggest thatRNRmRNA production is maximal in late G1and S phases; however, damaged DNA inducesRNRtranscription throughout the cell cycle. But suchen massemeasurements reveal neither cell-to-cell heterogeneity in responses nor direct correlations between transcript and protein expression or localization in single cells which may be central to function. We overcame these limitations by simultaneous detection of singleRNRtranscripts and also Rnr proteins in the same individual asynchronousS. cerevisiaecells, with and without DNA damage by methyl methanesulfonate (MMS). Surprisingly, RNR subunit mRNA levels were comparably low in both damaged and undamaged G1cells and highly induced in damaged S/G2cells. Transcript numbers became correlated with both protein levels and localization only upon DNA damage in a cell cycle-dependent manner. Further, we showed that the differentialRNRresponse to DNA damage correlated with variable Mec1 kinase activity in the cell cycle in single cells. The transcription ofRNRgenes was found to be noisy and non-Poissonian in nature. Our results provide vital insight into cell cycle-dependent RNR regulation under conditions of genotoxic stress.

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Vakana, Eliza, Jessica K. Altman, Heather Glaser, Nicholas J. Donato, and Leonidas C. Platanias. "Antileukemic effects of AMPK activators on BCR-ABL–expressing cells." Blood 118, no. 24 (December 8, 2011): 6399–402. http://dx.doi.org/10.1182/blood-2011-01-332783.

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Abstract The mammalian target of rapamycin (mTOR) signaling pathway plays a critical role in growth and survival of BCR-ABL transformed cells. AMPK kinase is a metabolic sensor that exhibits suppressive effects on the mTOR pathway and negatively regulates mTOR activity. We report that AMPK activators, such as metformin and 5-aminoimidazole-4-carboxamide ribonucleotide, suppress activation of the mTOR pathway in BCR-ABL–expressing cells. Treatment with these inhibitors results in potent suppression of chronic myeloid leukemia leukemic precursors and Ph+ acute lymphoblastic leukemia cells, including cells expressing the T315I-BCR-ABL mutation. Altogether, our data suggest that AMPK is an attractive target for the treatment of BCR-ABL–expressing malignancies and raise the potential for use of AMPK activators in the treatment of refractory chronic myeloid leukemia and Ph+ acute lymphoblastic leukemia.

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Lu, Jun, Qinghua Wang, Lianghu Huang, Huiyue Dong, Lingjing Lin, Na Lin, Feng Zheng, and Jianming Tan. "Palmitate Causes Endoplasmic Reticulum Stress and Apoptosis in Human Mesenchymal Stem Cells: Prevention by AMPK Activator." Endocrinology 153, no. 11 (November 1, 2012): 5275–84. http://dx.doi.org/10.1210/en.2012-1418.

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Abstract Elevated circulating saturated fatty acids concentration is commonly associated with poorly controlled diabetes. The highly prevalent free fatty acid palmitate could induce apoptosis in various cell types, but little is known about its effects on human mesenchymal stem cells (MSCs). Here, we report that prolonged exposure to palmitate induces human bone marrow-derived MSC (hBM-MSC) and human umbilical cord-derived MSC apoptosis. We investigated the role of endoplasmic reticulum (ER) stress, which is known to promote cell apoptosis. Palmitate activated XBP1 splicing, elF2α (eukaryotic translation initiation factor 2α) phosphorylation, and CHOP, ATF4, BiP, and GRP94 transcription in hBM-MSCs. ERK1/2 and p38 MAPK phosphorylation were also induced by palmitate in hBM-MSCs. A selective p38 inhibitor inhibited palmitate activation of the ER stress, whereas the ERK1/2 inhibitors had no effect. The AMP-activated protein kinase activator aminoimidazole carboxamide ribonucleotide blocked palmitate-induced ER stress and apoptosis. These findings suggest that palmitate induces ER stress and ERK1/2 and p38 activation in hBM-MSCs, and AMP-activated protein kinase activator prevents the deleterious effects of palmitate by inhibiting ER stress and apoptosis.

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Cooper, J., J. Conner, and J. B. Clements. "Characterization of the novel protein kinase activity present in the R1 subunit of herpes simplex virus ribonucleotide reductase." Journal of virology 69, no. 8 (1995): 4979–85. http://dx.doi.org/10.1128/jvi.69.8.4979-4985.1995.

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Al-Moujahed, Ahmad, Fotini Nicolaou, Katarzyna Brodowska, Thanos D. Papakostas, Anna Marmalidou, Bruce R. Ksander, Joan W. Miller, Evangelos Gragoudas, and Demetrios G. Vavvas. "Uveal Melanoma Cell Growth Is Inhibited by Aminoimidazole Carboxamide Ribonucleotide (AICAR) Partially Through Activation of AMP-Dependent Kinase." Investigative Opthalmology & Visual Science 55, no. 7 (July 8, 2014): 4175. http://dx.doi.org/10.1167/iovs.13-12856.

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Ferrandina, Gabriella, Valentina Mey, Sara Nannizzi, Simona Ricciardi, Marco Petrillo, Cristiano Ferlini, Romano Danesi, Giovanni Scambia, and Mario Del Tacca. "Expression of nucleoside transporters, deoxycitidine kinase, ribonucleotide reductase regulatory subunits, and gemcitabine catabolic enzymes in primary ovarian cancer." Cancer Chemotherapy and Pharmacology 65, no. 4 (July 29, 2009): 679–86. http://dx.doi.org/10.1007/s00280-009-1073-y.

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Holmes, B. F., E. J. Kurth-Kraczek, and W. W. Winder. "Chronic activation of 5′-AMP-activated protein kinase increases GLUT-4, hexokinase, and glycogen in muscle." Journal of Applied Physiology 87, no. 5 (November 1, 1999): 1990–95. http://dx.doi.org/10.1152/jappl.1999.87.5.1990.

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Abstract:

This study was designed to determine whether chronic chemical activation of AMP-activated protein kinase (AMPK) would increase glucose transporter GLUT-4 and hexokinase in muscles similarly to periodic elevation of AMPK that accompanies endurance exercise training. The adenosine analog, 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR), has previously been shown to be taken up by cells and phosphorylated to form a compound (5-aminoimidazole-4-carboxamide ribonucleotide) that mimics the effect of AMP on AMPK. A single injection of AICAR resulted in a marked increase in AMPK in epitrochlearis and gastrocnemius/plantaris muscles 60 min later. When rats were injected with AICAR (1 mg/g body wt) for 5 days in succession and were killed 1 day after the last injection, GLUT-4 was increased by 100% in epitrochlearis muscle and by 60% in gastrocnemius muscle in response to AICAR. Hexokinase was also increased ∼2.5-fold in the gastrocnemius/plantaris. Gastrocnemius glycogen content was twofold higher in AICAR-treated rats than in controls. Chronic chemical activation of AMPK, therefore, results in increases in GLUT-4 protein, hexokinase activity, and glycogen, similarly to those induced by endurance training.

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Huang, M., and S. J. Elledge. "Identification of RNR4, encoding a second essential small subunit of ribonucleotide reductase in Saccharomyces cerevisiae." Molecular and Cellular Biology 17, no. 10 (October 1997): 6105–13. http://dx.doi.org/10.1128/mcb.17.10.6105.

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Abstract:

Ribonucleotide reductase (RNR), which catalyzes the rate-limiting step for deoxyribonucleotide production required for DNA synthesis, is an alpha2beta2 tetramer consisting of two large and two small subunits. RNR2 encodes a small subunit and is essential for mitotic viability in Saccharomyces cerevisiae. We have cloned a second essential gene encoding a homologous small subunit, RNR4. RNR4 and RNR2 appear to have nonoverlapping functions and cannot substitute for each other even when overproduced. The lethality of RNR4 deletion mutations can be suppressed by overexpression of RNR1 and RNR3, two genes encoding the large subunit of the RNR enzyme, indicating genetic interactions among the RNR genes. RNR2 and RNR4 may be present in the same reductase complex in vivo, since they coimmunoprecipitate from cell extracts. Like the other RNR genes, RNR4 is inducible by DNA-damaging agents through the same signal transduction pathway involving MEC1, RAD53, and DUN1 kinase genes. Analysis of DNA damage inducibility of RNR2 and RNR4 revealed partial inducibility in dun1 mutants, indicating a DUN1-independent branch of the transcriptional response to DNA damage.

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Journal articles on the topic 'Ribonucleotide kinase'

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