MEK2 regulates ribonucleotide reductase activity through functional interaction with ribonucleotide reductase small subunit p53R2

The p53R2 protein, a newly identified member of the ribonucleotide reductase family that provides nucleotides for DNA damage repair, is directly regulated by p53. We show that p53R2 is also regulated by a MEK2 (ERK kinase 2/MAP kinase kinase 2)-dependent pathway. Increased MEK1/2 phosphorylation by serum stimulation coincided with an increase in the RNR activity in U2OS and H1299 cells. The inhibition of MEK2 activity, either by treatment with a MEK inhibitor or by transfection with MEK2 siRNA, dramatically decreased the serum-stimulated RNR activity. Moreover, p53R2 siRNA, but not R2 siRNA, significantly inhibits serum-stimulated RNR activity, indicating that p53R2 is specifically regulated by a MEK2-dependent pathway. Co-immunoprecipitation analyses revealed that the MEK2 segment comprising amino acids 65–171 is critical for p53R2–MEK2 interaction, and the binding domain of MEK2 is required for MEK2-mediated increased RNR activity. Phosphorylation of MEK1/2 was greatly augmented by ionizing radiation, and RNR activity was concurrently increased. Ionizing radiation-induced RNR activity was markedly attenuated by transfection of MEK2 or p53R2 siRNA, but not R2 siRNA. These data show that MEK2 is an endogenous regulator of p53R2 and suggest that MEK2 may associate with p53R2 and upregulate its activity.     

MEK2 regulates ribonucleotide reductase activity through functional interaction with ribonucleotide reductase small subunit p53R2

The p53R2 protein, a newly identified member of the ribonucleotide reductase family that provides nucleotides for DNA damage repair, is directly regulated by p53. We show that p53R2 is also regulated by a MEK2 (ERK kinase 2/MAP kinase kinase 2)-dependent pathway. Increased MEK1/2 phosphorylation by serum stimulation coincided with an increase in the RNR activity in U2OS and H1299 cells. The inhibition of MEK2 activity, either by treatment with a MEK inhibitor or by transfection with MEK2 siRNA, dramatically decreased the serum-stimulated RNR activity. Moreover, p53R2 siRNA, but not R2 siRNA, significantly inhibits serum-stimulated RNR activity, indicating that p53R2 is specifically regulated by a MEK2-dependent pathway. Co-immunoprecipitation analyses revealed that the MEK2 segment comprising amino acids 65–171 is critical for p53R2–MEK2 interaction, and the binding domain of MEK2 is required for MEK2-mediated increased RNR activity. Phosphorylation of MEK1/2 was greatly augmented by ionizing radiation, and RNR activity was concurrently increased. Ionizing radiation-induced RNR activity was markedly attenuated by transfection of MEK2 or p53R2 siRNA, but not R2 siRNA. These data show that MEK2 is an endogenous regulator of p53R2 and suggest that MEK2 may associate with p53R2 and upregulate its activity.     

Kinetic studies on the reduction of the R2 subunit of mouse ribonucleotide reductase with hydroxyurea, hydrazine, phenylhydrazine and hydroxylamine

 Four reductions of the R2 subunit of mouse ribonucleotide reductase have been studied and found to exhibit different behaviour from that of Escherichia coli R2. An important difference is that there is no stable met-R2 (Fe₂ ^II I) form of mouse R2. With hydroxyurea, hydrazine and hydroxylamine uniphasic kinetics are observed for the combined reduction of radical Tyr ^˙ and Fe₂ ^II I components to tyrosine and Fe₂ ^II respectively. The rate constants, determined at 370 nm (emphasising Fe^III decay) and 417 nm (emphasising Tyr ^˙ decay), differ by factors of 2–3, allowing some mechanistic features to be defined. The studies with hydrazine are particularly important. In the case of E. coli R2, a first phase corresponding to two-equivalent reduction of the met-R2 component has been observed [18]. It is likely that the four times slower second phase reaction of active E. coli R2 also corresponds to the Fe₂ ^II I → Fe₂ ^II change and is followed by fast intramolecular Fe₂ ^II reduction of the higher potential Tyr ^˙. The latter changes are believed to hold also for (active) mouse R2. The Fe^IIFe^III semi forms have been detected at low levels by EPR for mouse R2 (9%) and E. coli (∼5%) in previous studies. Further substrate reduction of Fe^IIFe^III occurs at a comparable rate to account for the transient behaviour of Fe^IIFe^III. For mouse R2 the combined Fe^III decay processes (which we are unable to separate) give smaller uniphasic rate constants at 370 nm than at 417 nm. A fitted-base-line (FBL) treatment of absorbance changes at 417 nm targets more closely the Tyr ^˙ decay as a means of monitoring the rate-determining step. The FBL method gives rate constants k (M^–1 s^–1) at 25  °C and pH 7.5 for hydroxyurea (1.46), hydrazine (0.163) and hydroxylamine (4.4). Surprisingly, phenylhydrazine, with a less strong reduction potential (0.25 V), gives a substantially faster reduction of the Tyr ^˙ as the only redox step (rate constant 27 M^–1 s^–1). In this case a slower second phase at 370 nm is independent of reductant and corresponds to rate-controlling release of Fe^III. Overall the results indicate a more reactive redox centre for mouse R2 and help develop further an understanding of factors affecting the reactivity of R2. Received: 11 October 1996 / Accepted: 11 February 1997     

Mutations within the membrane domain of HMG-CoA reductase confer resistance to sterol-accelerated degradation

The pivotal event for sterol-induced degradation of the cholesterol biosynthetic enzyme HMG-CoA reductase is binding of its membrane domain to Insig proteins in the endoplasmic reticulum. Insigs are carriers for gp78, an E3 ubiquitin ligase that marks reductase for proteasomal degradation. We report here the isolation of mutant Chinese hamster ovary cell lines, designated SRD-16, -17, and -18, in which sterol-induced ubiquitination and degradation of reductase are severely impaired. These cells were produced by chemical mutagenesis and selection with SR-12813, a compound that mimics sterols in stimulating ubiquitination and degradation of reductase. Each SRD cell line was found to contain a point mutation in one reductase allele, resulting in substitutions of aspartate for serine-60 (SRD-16), arginine for glycine-87 (SRD-17), and proline for alanine-333 (SRD-18). Sterols failed to promote ubiquitination and degradation of these reductase mutants, owing to their decreased affinity for Insigs. Thus, three different point mutations in reductase, all of which localize to the membrane domain, disrupt Insig binding and abolish sterol-accelerated degradation of the enzyme.     

Mutations in the IGF-II pathway that confer resistance to lytic reovirus infection

Background  

Viruses are obligate intracellular parasites and rely upon the host cell for different steps in their life cycles. The characterization of cellular genes required for virus infection and/or cell killing will be essential for understanding viral life cycles, and may provide cellular targets for new antiviral therapies.     

Mutations in the human cytomegalovirus UL27 gene that confer resistance to maribavir

Previous drug selection experiments resulted in the isolation of a human cytomegalovirus (CMV) UL97 phosphotransferase mutant resistant to the benzimidazole compound maribavir (1263W94), reflecting the anti-UL97 effect of this drug. Three other CMV strains were plaque purified during these experiments. These strains showed lower-grade resistance to maribavir than the UL97 mutant. Extensive DNA sequence analyses showed no changes from the baseline strain AD169 in UL97, the genes involved in DNA replication, and most structural proteins. However, changes were identified in UL27 where each strain contained a different mutation (R233S, W362R, or a combination of A406V and a stop at codon 415). The mutation at codon 415 is predicted to truncate the expressed UL27 protein by 193 codons (32% of UL27) with a loss of nuclear localization. The expression of full-length UL27 as a green fluorescent fusion protein in uninfected fibroblasts resulted in nuclear and nucleolar fluorescence, whereas cytoplasmic localization was observed when codons 1 to 415 were similarly expressed. Viable UL27 deletion mutants were created by recombination and showed slight growth attenuation and maribavir resistance in cell culture. Marker transfer experiments confirmed that UL27 mutations conferred maribavir resistance. The UL27 sequence was well conserved in a sample of 16 diverse clinical isolates. Mutation in UL27, a betaherpesvirus-specific early gene of unknown biological function, may adapt the virus for growth in the absence of UL97 activity.     

Chromosome-mediated gene transfer of hydroxyurea resistance and amplification of ribonucleotide reductase activity.

Metaphase chromosomes purified from a hydroxyurea-resistant Chinese hamster cell line were able to transform recipient wild-type cells to hydroxyurea resistance at a frequency of 10(-6). Approximately 60% of the resulting transformant clones gradually lost hydroxyurea resistance when cultivated for prolonged periods in the absence of drug. One transformant was subjected to serial selection in higher concentrations of hydroxyurea. The five cell lines generated exhibited increasing relative plating efficiency in the presence of the drug and a corresponding elevation in their cellular content of ribonucleotide reductase. The most resistant cell line had a 163-fold increase in relative plating efficiency and a 120-fold increase in enzyme activity when compared with the wild-type cell line. The highly hydroxyurea-resistant cell lines had strong electron paramagnetic resonance signals characteristic of an elevated level of the free radical present in the M2 subunit of ribonucleotide reductase. Two-dimensional electrophoresis of cell-free extracts from one of the resistant cell lines indicated that a 53,000-dalton protein was present in greatly elevated quantities when compared with the wild-type cell line. These data suggest that the hydroxyurea-resistant cell lines may contain an amplification of the gene for the M2 subunit of ribonucleotide reductase.     

Mutations that confer resistance to 2-deoxyglucose reduce the specific activity of hexokinase from Myxococcus xanthus

The glucose analog 2-deoxyglucose (2dGlc) inhibits the growth and multicellular development of Myxococcus xanthus. Mutants of M. xanthus resistant to 2dGlc, designated hex mutants, arise at a low spontaneous frequency. Expression of the Escherichia coli glk (glucokinase) gene in M. xanthus hex mutants restores 2dGlc sensitivity, suggesting that these mutants arise upon the loss of a soluble hexokinase function that phosphorylates 2dGlc to form the toxic intermediate, 2-deoxyglucose-6-phosphate. Enzyme assays of M. xanthus extracts reveal a soluble hexokinase (ATP:D-hexose-6-phosphotransferase; EC 2.7.1.1) activity but no phosphotransferase system activities. The hex mutants have lower levels of hexokinase activities than the wild type, and the levels of hexokinase activity exhibited by the hex mutants are inversely correlated with the ability of 2dGlc to inhibit their growth and sporulation. Both 2dGlc and N-acetylglucosamine act as inhibitors of glucose turnover by the M. xanthus hexokinase in vitro, consistent with the finding that glucose and N-acetylglucosamine can antagonize the toxic effects of 2dGlc in vivo.     

Reversion of hydroxyurea resistance, decline in ribonucleotide reductase activity, and loss of M2 gene amplification

A key rate-limiting reaction in the synthesis of DNA is catalyzed by ribonucleotide reductase, the enzyme which reduces ribonucleotides to provide the deoxyribonucleotide precursors of DNA. The antitumor agent, hydroxyurea, is a specific inhibitor of this enzyme and has been used in the selection of drug resistant mammalian cell lines altered in ribonucleotide reductase activity. An unstable hydroxyurea resistant population of mammalian cells with elevated ribonucleotide reductase activity has been used to isolate three stable subclones with varying sensitivities to hydroxyurea cytotoxicity and levels of ribonucleotide reductase activities. These subclones have been analyzed at the molecular level with cDNA probes encoding the two nonidentical subunits of ribonucleotide reductase (M1 and M2). Although no significant differences in M1 mRNA levels or gene copy numbers were detected between the three cell lines, a strong correlation between cellular resistance, enzyme activity, M2 mRNA and M2 gene copies was observed. This is the first demonstration that reversion of hydroxyurea resistance is directly linked to a decrease in M2 mRNA levels and M2 gene copy number, and strongly supports the concept that M2 gene amplification is an important mechanism for achieving resistance to this antitumor agent through elevations in ribonucleotide reductase.     

Effect of hydroxyurea on T4 ribonucleotide reductase.

Phage T4-induced ribonucleotide reductase, purified to homogeneity, catalyzes the reduction of the four ribonucleotides CDP, UDP, ADP, and GDP to the corresponding deoxyribonucleotides. The enzyme is an order of magnitude more sensitive to hydroxyurea than the corresponding Escherichia coli enzyme. Fifty per cent inhibition occurs at 10 micrometer hydroxyurea. Inhibition is complete at a high concentration of the drug, and there is no differential effect on the four substrates. Treatment of T4 ribonucleotide reductase or its isolated subunits with hydroxyurea does not lead to their irreversible inactivation.     

Altered ribonucleotide reductase activity in mammalian tissue culture cells resistant to hydroxyurea

Four Chinese hamster ovary cell lines and one mouse L cell line have been isolated which are resistant to the cytotoxic effects of hydroxyurea and guanazole. These five cell lines contain an altered ribonucleotide reductase activity as judged by a decreased sensitivity to the inhibitory action of both drugs. This is strong evidence that ribonucleotide reductase is one of the lethal sites of action for these two antitumour agents. The results are also consistent with the view that mammalian cell variants can arise from structural gene mutations.     

EPR stopped-flow studies of the reaction of the tyrosyl radical of protein R2 from ribonucleotide reductase with hydroxyurea.

The reaction of the functional tyrosyl radical in protein R2 of ribonucleotide reductase from E. coli and mouse with the enzyme inhibitor hydroxyurea has been studied by EPR stopped-flow techniques at room temperature. The rate of disappearance of the tyrosyl radical in E. coli protein R2 is k2 = 0.43 M-1 s-1 at 25 degrees C. The reaction follows pseudo-first-order kinetics up to 450 mM hydroxyurea indicating that no saturation by hydroxyurea takes place even at this high concentration. Transient nitroxide-like radicals from hydroxyurea have been detected for the first time in the reaction of hydroxyurea with protein R2 from E. coli and mouse, indicating that 1-electron transfer from hydroxyurea to the tyrosyl radical is the dominating mechanism in the inhibitor reaction. The hydroxyurea radicals appear in low steady-state concentrations during 2-3 half-decay times of the tyrosyl radical and disappear thereafter.     

Purification and characterization of recombinant mouse and herpes simplex virus ribonucleotide reductase R2 subunit.

Overexpression of recombinant mouse and herpes simplex virus ribonucleotide reductase small subunit (protein R2) has been obtained by using the T7 RNA polymerase expression system. Both proteins, which constitute about 30% of the soluble Escherichia coli proteins, have been purified to homogeneity by a rapid and simple procedure. At this stage, few of the molecules contain the iron-tyrosyl free-radical center necessary for activity; however, addition of ferrous iron and oxygen under controlled conditions resulted in a mouse R2 protein containing 0.8 radical and 2 irons per polypeptide chain. In this reaction, one oxygen molecule was needed to generate each tyrosyl radical. Both proteins had full enzymatic activity. EPR spectroscopy showed that iron-center/radical interactions are considerably stronger in both mouse and viral proteins than in E. coli protein R2. CD spectra showed that the bacterial protein contains 70% alpha-helical structure compared to only about 50% in the mouse and viral proteins. Light absorption spectra between 310 and 600 nm indicate close similarity of the mu-oxo-bridged binuclear iron centers in all three R2 proteins. Furthermore, the paramagnetically shifted iron ligand proton NMR resonances show that the antiferromagnetic coupling and ligand arrangement in the iron center are nearly identical in all three species.     

Mutations in ftsZ that confer resistance to SulA affect the interaction of FtsZ with GTP.

Mutations in the essential cell division gene ftsZ confer resistance to SulA, a cell division inhibitor that is induced as part of the SOS response. In this study we have purified and characterized the gene products of six of these mutant ftsZ alleles, ftsZ1, ftsZ2, ftsZ3, ftsZ9, ftsZ100, and ftsZ114, and compared their properties to those of the wild-type gene product. The binding of GTP was differentially affected by these mutations. FtsZ3 exhibited no detectable GTP binding, and FtsZ9 and FtsZ100 exhibited markedly reduced GTP binding. In contrast, FtsZ1 and FtsZ2 bound GTP almost as well as the wild type, and FtsZ114 displayed increased GTP binding. Furthermore, we observed that all mutant FtsZ proteins exhibited markedly reduced intrinsic GTPase activity. It is likely that mutations in ftsZ that confer sulA resistance alter the conformation of the protein such that it assumes the active form.     

Differential effect of hydroxyurea on a ribonucleotide reductase system.

Infection of Escherichia coli with phage T4 induces a large increase in ribonucleotide reductase activity. We show that hydroxyurea inhibits T4-induced CDP, ADP, UDP, and GDP reductase activities in vitro. Moreover, there are significant differences in the degree of inhibition of each ribonucleotide reductase activity. The reductase activities for CDP and ADP are more sensitive to hydroxyurea than those for UDP and GDP, particularly at high hydroxyurea molarities. As little as 5 x 10(-4)M hydroxyurea lowers CDP and ADP reductase activities to 25 to 30% whereas as much as 0.5 M hydroxyurea is needed to lower UDP and GDP reductase activities to 50%.     

Stable suppression of the R2 subunit of ribonucleotide reductase by R2-targeted short interference RNA sensitizes p53(-/-) HCT-116 colon cancer cells to DNA-damaging agents and ribonucleotide reductase inhibitors

Ribonucleotide reductase catalyzes the production of deoxyribonucleoside diphosphates, the precursors of deoxyribonucleoside triphosphates for DNA synthesis. Mammalian ribonucleotide reductase (RNR) is a tetramer consisting of two non-identical homodimers, R1 and either R2 or p53R2, which are considered to be involved in DNA replication and repair, respectively. We have demonstrated that DNA damage by doxorubicin and cisplatin caused a steady elevation of the R2 protein in p53(-/-) HCT-116 human colon carcinoma cells but induced degradation of the protein in p53(+/+) cells. To evaluate the involvement of R2 in response to DNA damage, p53(-/-) HCT-116 cells were stably transfected with an expression vector transcribing short hairpin/short interference RNA directed against R2 mRNA. Stably transfected clones exhibited a pronounced reduction of the R2 protein with no change in the cellular growth rate. Furthermore, short interference RNA-mediated reduction of the R2 protein caused a marked increase in sensitivity to the DNA-damaging agent cisplatin as well as to the RNR inhibitors Triapine and hydroxyurea. Ectopic expression of p53R2 partially reversed the cytotoxicity of cisplatin but not that of RNR inhibitors to R2 knockdown cells. The increase in sensitivity to cisplatin and RNR inhibitors was correlated with the suppression of dATP and dGTP levels caused by stable expression of R2-targeted short interference RNA. These results indicated that DNA damage resulted in elevated levels of the R2 protein and dNTPs and, consequently, enhanced the survival of p53(-/-) HCT-116 cells. The findings provide evidence that R2-RNR can be employed to supply dNTPs for the repair of DNA damage in cells with an impaired p53-dependent induction of p53R2.