Mammalian ribonucleotide reductase, which occupies a key position in the synthesis of DNA, is a highly controlled enzyme activity, because it is solely responsible for the de novo reduction of ribonucleoside diphosphates to their corresponding deoxyribonucleoside diphosphate forms, required for DNA synthesis. Ribonucleotide reductase consists of two dissimilar protein components often called M1 and M2, which are independently regulated during cell proliferation. The M1 component contains multiple effector binding sites and is responsible for the complex allosteric regulation of the enzyme, whereas the M2 protein contains nonheme iron and a unique tyrosyl-free radical required for ribonucleotide reduction. Since the reaction is rate limiting for DNA synthesis, ribonucleotide reductase plays an important role in regulating cell division, and hence, cell proliferation. There are many inhibitors of ribonucleotide reductase and perhaps the most valuable one from a cell biology, biochemistry, and clinical point of view is the hydroxamic acid, hydroxyurea. This drug has also been very useful as a selective agent for isolating a variety of mammalian mutant cell lines altered in ribonucleotide reductase gene expression. Regulatory, structural, and biological characteristics of ribonucleotide reductase are reviewed, including evidence that ribonucleotide reductase, particularly the M2 protein, has an important early role to play in tumor promotion. In addition, modifications in the expressions of genes altered in hydroxyurea-resistant mutants and cultured in the absence or presence of hydroxyurea are discussed, with emphasis on changes in M2 protein, M1 protein, and the iron-storage protein ferritin.(ABSTRACT TRUNCATED AT 250 WORDS)
Ribonucleotide reductase, a rate-limiting enzyme in the synthesis of DNA, consists of two nonidentical subunits, proteins M1 and M2. Hydroxyurea, a specific inhibitor of DNA synthesis, acts by destroying the unique tyrosyl free radical of protein M2. In the past, we have described a mouse L cell line which exhibited a stable resistance to high concentrations of hydroxyurea [McClarty, G. A., Chan, A.,& Wright, J.A. (1986) Somat. Cell Mol. Genet. 12, 121-131]. When this line was grown in the absence of hydroxyurea, the cells contained a modest but stable elevation in ribonucleotide reductase activity. However, the activity was further increased on the addition of drug to the culture medium. This was accompanied by an increase in protein M2 activity as shown by activity titration experiments. Likewise, removal of hydroxyurea resulted in a decrease in M2 activity. In the present study, we make use of recently isolated cDNAs and monoclonal antibodies for both the M1 and M2 proteins to further our understanding of the mechanism of hydroxyurea resistance at the molecular level in a subclone of this cell line. Our results indicated that protein M1 levels were elevated 2-3-fold and protein M2 levels were increased about 50-fold in the mutant cells when they were grown in the absence of hydroxyurea, compared to wild-type cells. These protein increases were accompanied by corresponding elevations in the levels of mRNAs for both subunits and increased rates of transcription of both genes. There was a 6-fold amplification in the gene copy number for protein M2.(ABSTRACT TRUNCATED AT 250 WORDS)
Hydroxyurea was used as a selective agent in culture, to isolate by a stepwise procedure, a unique mouse L cell line called LHF which exhibited a stable resistance to high concentrations of drug (5 mM). LHF cells contained an elevation in ribonucleotide reductase activity which depended upon whether cells were previously cultured in the presence or absence of hydroxyurea. M1 immunoprecipitation and M2 titration experiments indicated that both ribonucleotide reductase subunits were elevated in drug-resistant cells. Interestingly, a very large drug-dependent change in the M2 activity (about a 100-fold) was observed. Studies on enzyme activity with cycloheximide and actinomycin D indicated that the hydroxyurea-dependent increase in activity required de novo protein synthesis and transcriptional activity. These results are different from other ribonucleotide reductase overproducing cell lines previously described, and indicate that hydroxyurea modulates enzyme activity by an interesting mechanism.
Ribonucleotide reductase is responsible for supplying the deoxyribonucleotides required for DNA synthesis and repair. The active enzyme consists of two dissimilar protein components called R1 and R2. Immunoprecipitation of R1 and R2 proteins from [32P]orthophosphate-labeled exponentially growing mouse L cells showed that the R2 protein but not the R1 protein of ribonucleotide reductase could be phosphorylated in vivo. Two-dimensional phosphopeptide mapping experiments of trypsin-digested R2 protein showed a major spot containing more than 90% of the total radioactivity and a minor spot with the remaining radioactivity. Phosphoamino acid analysis of R2 phosphorylated protein indicated that phosphorylation occurred exclusively on serine. Protein kinase C, cAMP-dependent protein kinase, p34cdc2, and CDK2 were capable of phosphorylating the R2 protein in vitro, whereas casein kinase II was not. To determine whether any of these enzymes could phosphorylate peptides observed to be phosphorylated in actively growing cells, tryptic phosphopeptide maps of R2 that had been phosphorylated in vitro were compared with maps of R2 that had been isolated from [32P]-labeled cells. Only the phosphopeptide maps obtained with p34cdc2 and CDK2 matched the pattern found in [32P]-labeled cells. Experiments in which tryptic digests from different samples were mixed prior to two-dimensional separation demonstrated comigration of phosphopeptides obtained by in vivo phosphorylation with phosphopeptides derived from p34cdc2 or CDK2 obtained by in vitro phosphorylations. These studies indicate that protein R2 phosphorylation may play an important role in the regulation of ribonucleotide reduction, DNA synthesis, and cell cycle progression, and suggest a potentially important p34cdc2 and/or CDK2 regulation point in DNA replication.
A human Myc epitope is frequently used to tag proteins for expression experiments in nonhuman cells. We used the monoclonal 9E10 antibody specific for this epitope to analyse the expression of four proteins carrying the Myc tag in cells transfected with expression vectors. While all four proteins can be detected by immunofluorescence and immunoprecipitation assays, surprisingly, only two proteins could be detected in Western blot analysis, indicating that epitope recognition by the monoclonal antibody can be blocked in some membrane-retained ectopic proteins. Other techniques such as immunofluorescence and immunoprecipitation assays can be successfully used with the 9E10 antibody to determine potential expression of Myc-tagged proteins.
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