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IRAK2, a member of the interleukin-1 receptor-associated kinase (IRAK) family, has been implicated in Toll-like receptor (TLR)-mediated signaling. We generated IRAK2-deficient mice to examine its function in detail. These mice are resistant to lipopolysaccharide-induced septic shock, because of impaired TLR4-mediated induction of pro-inflammatory cytokines and chemokines. Although IRAK2 deficiency did not affect TLR4-mediated NFκB activation, a reduction of lipopolysaccharide (LPS)-mediated mRNA stabilization contributed to the reduced cytokine and chemokine production observed in bone marrow-derived macrophages from IRAK2-deficient mice. Furthermore, the ratios of LPS-induced cytokine and chemokine mRNAs in translation-active (polysomal) versus translation-inactive (free ribosomes) pools were reduced in IRAK2-deficient macrophages compared with wild type macrophages. Importantly, LPS-induced phosphorylation of MKK3/6, MNK1, and eIF4E was significantly reduced in IRAK2-deficient macrophages compared with wild type macrophages. Moreover, LPS stimulation induced an interaction of IRAK2 with TRAF6, MKK3/6, and MK2, implicating a critical role for mitogen-activated protein kinase signaling in LPS-induced IRAK2-mediated post-transcriptional control. These results reveal that IRAK2 is required for LPS-mediated post-transcriptional control of cytokine and chemokine expression, which plays an essential role in TLR4-induced septic shock.

The transcription factor STAT1 plays a pivotal role in signal transduction of type I and II interferons (IFNs). STAT1 activation leads to changes in expression of key regulatory genes encoding caspases and cell cycle inhibitors. Deficient STAT1 expression in human cancer cells and virally mediated inhibition of STAT1 function have been associated with cellular resistance to IFNs and mycobacterial infection in humans. Thus, given the relative importance of STAT1, we isolated and characterized a human STAT1 intronic enhancer region displaying IFN-regulated activity. Functional analyses by transient expression identified a repressor region and type I and II IFN-inducible elements within the STAT1 enhancer sequence. A candidate IRF-E/GAS/IRF-E (IGI) sequence containing GAAANN nucleotide repeats was shown by gel shift assay to bind to IFN regulatory factor-1 (IRF-1), but not to IFN-stimulated gene factor-3 (ISGF-3) or STAT1–3. An additional larger IGI-binding complex containing IRF-1 was identified. Mutation of the GAAANN repeats within the IGI DNA element eliminated IRF-1 binding and the IFN-regulated activity of the STAT1 intronic enhancer region. Transfection of the IFN-resistant MM96 cell line to express increased levels of IRF-1 protein also elevated STAT1, STAT2, and p48/IRF-9 expression and enhanced cellular responsiveness to IFN-β. Reciprocating regulation between IRF-1 and STAT1 genes and encoded proteins indicates that an intracellular amplifier circuit exists controlling cellular responsiveness to the IFNs.

The transcription factor NFkappaB is activated by numerous stimuli. Once NFkappaB is fully activated, it participates in the regulation of various target genes in different cells to exert its biological functions. NFkappaB has often been referred to as a central mediator of the immune response, since a large variety of bacteria and viruses can lead to the activation of NFkappaB, which in turn controls the expression of many inflammatory cytokines, chemokines, immune receptors, and cell surface adhesion molecules. Recent studies have shown that NFkappaB may function more generally as a central regulator of stress responses, since different stressful conditions, including physical stress, oxidative stress, and exposure to certain chemicals, also lead to NFkappaB activation. Furthermore, NFkappaB blocks cell apoptosis in several cell types. Taken together, these findings make it clear that NFkappaB plays an important role in cell proliferation and differentiation. It is the intention of this review to cover the various NFkappaB-dependent signaling pathways, thereby to achieve a better understanding of the mechanisms of NFkappaB activation and the physiological functions of activated NFkappaB.

Phosphatidylinositol 3'-kinase (PI3K) and the serine/threonine kinase AKT have critical roles in phosphorylating and transactivating the p65 subunit of nuclear factor kappaB (NF-kappaB) in response to the pro-inflammatory cytokines interleukin-1 (IL-1) and tumor necrosis factor (TNF). Mouse embryo fibroblasts (MEFs) lacking either the alpha or beta subunit of IkappaB kinase (IKK) were deficient in NF-kappaB-dependent transcription following treatment with IL-1 or TNF. However, in contrast to IKKbeta-null MEFs, IKKalpha-null MEFs were not substantially defective in the cytokine-stimulated degradation of Ikappabetaalpha or in the nuclear translocation of NF-kappaB. The IKK complexes from IKKalpha- or IKKbeta-null MEFs were both deficient in PI3K-mediated phosphorylation of the transactivation domain of the p65 subunit of NF-kappaB in response to IL-1 and TNF, and constitutively activated forms of PI3K or AKT did not potentiate cytokine-stimulated activation of NF-kappaB in either IKKalpha- or IKKbeta-null MEFs. Collectively, these data indicate that, in contrast to IKKbeta, which is required for both NF-kappaB liberation and p65 phosphorylation, IKKalpha is required solely for the cytokine-induced phosphorylation and activation of the p65 subunit of NF-kappaB that are mediated by the PI3K/AKT pathway.

Type I interferons (IFNs) are cytokines that play a central role in mediating antiviral, antiproliferative, and immunomodulatory activities in virtually all cells. These activities are entirely dependent on the interaction of IFNs with their particular cell surface receptor. In this report, we identify two specific tyrosine residues located within the cytoplasmic domain of IFNAR2c that are obligatory for IFN-dependent signaling. Various IFNAR2c tyrosine mutants were expressed in a human lung fibroscarcoma cell line lacking IFNAR2c (U5A). Stable clones expressing these mutants were analyzed for their ability to induce STAT1 and STAT2 activation, ISGF3 transcriptional complex formation, gene expression, and cell growth regulation in response to stimulation with type I IFNs. The replacement of all seven cytoplasmic tyrosine residues of IFNAR2c with phenylalanine resulted in a receptor unable to respond to IFN stimulation. Substitution of single tyrosines at amino acid residue 269, 316, 318, 337, or 512 with phenylalanine had no effect on IFN-dependent signaling, suggesting that no single tyrosine is essential for IFN receptor-mediated signaling. In addition, IFNAR2c retaining five proximal tyrosines residues (269, 306, 316, 318, and 337) or either two distal tyrosine residues (411 or 512) continued to be responsive to IFN stimulation. Surprisingly, the presence of only a single tyrosine at either position 337 or 512 was sufficient to restore a complete IFN response. These results indicate that IFN-dependent signaling proceeds through the redundant usage of two tyrosine residues in the cytoplasmic domain of IFNAR2c.

Previous experiments have suggested that induction of the β-R1 gene by interferon (IFN)-β required transcription factor ISGF-3 (IFN-stimulated gene factor-3) and an additional component. We now provide evidence that nuclear factor-κB (NF-κB) can serve as this component. Site-directed mutagenesis of an NF-κB binding site in the β-R1 promoter or over-expression of an IκBα super-repressor abrogated IFN-β-mediated induction of a β-R1 promoter-reporter. IFN-β treatment did not augment abundance of NF-κB but did lead to phosphorylation of the p65 NF-κB subunit. It is proposed that IFN-β-mediated enhancement of the transactivation competence of NF-κB components is required for inducible transcription of the β-R1 promoter. These results provide a novel insight into the role of NF-κB in the transcriptional response to IFN-β.

p53 represses the transcription of cdc2 and cyclin B1, causing loss of Cdc2 activity and G(2) arrest. Here we show that the region -22 to -2 of the cdc2 promoter called the R box is required for repression by p53 but not for basal promoter activity. The R box confers p53-dependent repression on heterologous promoters and binds to p130/E2F4 in response to overexpression of p53. R box-dependent repression requires p21/waf1, and overexpression of p21/waf1 also represses the cdc2 promoter. These observations suggest that p53 represses the cdc2 promoter by inducing p21/waf1, which inhibits cyclin-dependent kinase activity, enhancing the binding of p130 and E2F4, which together bind to and repress the cdc2 promoter.

The LMP2 gene, which encodes a protein required for efficient presentation of viral antigens, requires both unphosphorylated Stat1 and IRF1 for basal expression. LMP2 expression is down-regulated by the adenovirus protein E1A, which binds to Stat1 and CBP/p300, and by the mutant E1A protein RG2, which binds to Stat1 but not to CBP/p300, but not by the mutant protein Delta2-36, which does not bind to either Stat1 or CBP/p300. Stat1 and IRF1 associate in untreated cells and bind as a complex to the overlapping ICS-2/GAS element of the LMP2 promoter. E1A interferes with the formation of this complex by occupying domains of Stat1 that bind to IRF1. These results reveal how adenovirus infection attenuates LMP2 expression, thereby interfering with the presentation of viral antigens.

Defects in apoptosis have been noted in signal transducer and activator of transcription (Stat) 1-null cells in vitro. The purpose of this study was to analyse the keratocyte apoptosis response that occurs in vivo in response to corneal epithelial injury in Stat 1null compared with control mice and to determine whether Stat 1null corneal fibroblasts have a defective response to death receptor activation in vitro. Corneal epithelial scrape injuries were performed in Stat 1-null and wild-type mice. Keratocyte apoptosis was monitored with the quantitative TUNEL assay and confirmed using transmission electron microscopy. Corneal fibroblast apoptosis in response to tumor necrosis factor (TNF) alpha, with and without inhibitors of nuclear factor kappa B (NF-kappaB) activation, was monitored using DNA laddering and the methylene blue assay. Significantly less keratocyte apoptosis was noted in Stat 1-null mice compared with wild-type controls. TNF alpha-induced apoptosis only occurred in wild-type mice in the presence of inhibitors of NF-kappaB activation. Corneal fibroblast TNF alpha-induced apoptosis was defective in Stat 1null corneal fibroblasts whether NF-kappaB activation was blocked or not. Stat 1 has an important role in the keratocyte apoptosis that occurs in response to corneal epithelial injury. Previous studies suggest that the defect is due to a lack of constitutive expression of caspases. This study demonstrates that this defect in apoptosis in Stat 1-null mice is present in vivo in Stat 1-null mice and suggests that Stat 1 could be a therapeutic target for transient inhibition of keratocyte apoptosis to modulate corneal wound healing.

We report preliminary characterization of a gene designated β-R1, which is selectively expressed in response to interferon β (IFN-β) compared with IFN-α. In human astrocytoma cells, β-R1 was induced to an equivalent extent by 10 IU/mL IFN-β or 2500 IU/mL IFN-α2. To address the mechanism of this differential response, we analyzed induction of the β-R1 gene in fibrosarcoma cells and derivative mutant cells lacking components required for signaling by type I IFNs. β-R1 was readily induced by IFN-β in the parental 2fTGH cell line, but not by recombinant IFN-α2, IFN-α Con1, or a mixture of IFN-α subtypes. IFN-α8 induced β-R1 weakly. β-R1 was not induced by IFN-β in mutant cell lines U2A, U3A, U4A, and U6A, which lack, respectively, p48, STAT1, JAK1, and STAT2. U5A cells, which lack the Ifnar 2.2 component of the IFN-α and -β receptor, also failed to express β-R1. U1A cells are partially responsive to IFN-β and IFN-α8 but lacked β-R1 expression, indicating that TYK2 protein is essential for induction of this gene. Taken together, these results suggest that the expression of β-R1 in response to type I IFN requires IFN-stimulated gene factor 3 plus an additional component, which is more efficiently formed on induction by IFN-β compared with IFN-α.

Treatment of Syrian hamster kidney cells with the demethylating agent 5-aza-2'-deoxycytidine (azadC) increased both the frequency and the rate of gene amplification appreciably. AzadC caused substantial DNA demethylation, which is likely to be responsible. The magnitude of the increases depended on the concentrations of both azadC and the drug used for selection. A transient stress response is not responsible since the increases were not dependent on cytotoxicity and were still apparent after several weeks. We discuss mechanisms by which azadC treatment may induce amplification by rendering DNA more prone to this process or by increasing the transcription of genes whose protein products stimulate amplification.

Infection of macrophages with the bacterium Listeria monocytogenes (Lm) stimulates NFkB signalling and the synthesis of type I Interferons (IFN-I). As a secondary response IFN-I signal through the type I IFN receptor (IFNAR) to activate the ISGF3 complex containing STAT1, STAT2 and IRF9 subunits. Using ChIP-Seq we provide a genome-wide analysis of genes with binding sites for both NFkB and the IFN-I activated STATs. In particular we define the group of genes displaying synergistic activation by STAT and NFkB pathways. This group includes the Nos2 gene encoding inducible nitric oxide synthase. Detailed analysis of this gene provides a mechanistic explanation how STAT and NFkB pathways cooperate in promoter activation, i.e. the establishment of activating histone marks and the recruitment and phosphorylation of RNA polymerase II. In particular we demonstrate a signal integrator function for the mediator complex. In summary our data explain the functional interplay between two major antimicrobial pathways.

A single high dose of IFN β activates powerful cellular responses in which many antiviral, pro-apoptotic, and anti-proliferative proteins are highly expressed. The expression of IFN-stimulated genes (ISGs) is mediated by ISGF3, consisting of IRF9 and tyrosine phosphorylated STATs 1 and 2. This initial response to IFN β is rapid and transient, so that the expression of most ISGs returns to basal levels within a day. However, the expression of some ISGs is prolonged for many days. We found that high levels of STAT1, STAT2, and IRF9 proteins that lack tyrosine phosphorylation are critical for the prolonged second phase response to IFN β . The expression of the STAT1, STAT2, and IRF9 proteins is increased by IFN β at late times (>24 h), and these proteins form a complex resembling ISGF3 (named unphosphorylated ISGF3, U-ISGF3), and bind to ISREs of target genes independently of tyrosine phosphorylation of STATs 1 and 2. U-ISGF3 induces only a subset of ISGs (20–30% of the total), which are mostly known to be antiviral genes and are also highly expressed in therapy-resistant cancer cells, but not cytotoxic ISGs. We observed that, in the absence of interferon treatment, high levels of U-ISGF3 significantly reduced the replication of many RNA viruses. High U-ISGF3 levels are correlated with resistance of cancer cells to DNA damage induced by radiation, etoposide, or doxorubicin. Knocking STAT1 and IRF9 down to decrease U-ISGF3 levels sensitized cancer cells to doxorubicin-induced DNA damage. Continuous exposure of cells to a low level of IFN β , often seen in cancers, leads to steady-state increases in the expression of only the U-ISGF3-dependent genes, with no sustained increases of IFN β -induced cytotoxic genes. Our study has elucidated that the U-ISGF3-dependent response to IFN β prolongs the antiviral effects, but leads to DNA damage resistance, which inhibits responses to cancer therapy.

The ubiquitous inducible transcription factor NF-κB plays central roles in immune and inflammatory responses and in tumorigenesis. Complex posttranslational modifications of the p65 subunit (RelA) are a major aspect of the extremely flexible regulation of NF-κB activity. Although phosphorylation, acetylation, ubiquitination, and lysine methylation of NF-κB have been well described, arginine methylation has not yet been found. We now report that, in response to IL-1β, the p65 subunit of NF-κB is dimethylated on arginine 30 (R30) by protein-arginine methyltransferase 5 (PRMT5). Expression of the R30A and R30K mutants of p65 substantially decreased the ability of NF-κB to bind to κB elements and to drive gene expression. A model in which dimethyl R30 is placed into the crystal structure of p65 predicts new van der Waals contacts that stabilize intraprotein interactions and indirectly increase the affinity of p65 for DNA. PRMT5 was the only arginine methyltransferase that coprecipitated with p65, and its overexpression increased NF-κB activity, whereas PRMT5 knockdown had the opposite effect. Microarray analysis revealed that ∼85% of the NF-κB-inducible genes that are down-regulated by the R30A mutation are similarly down-regulated by knocking PRMT5 down. Many cytokine and chemokine genes are among these, and conditioned media from cells expressing the R30A mutant of p65 had much less NF-κB-inducing activity than media from cells expressing the wild-type protein. PRMT5 is overexpressed in many types of cancer, often to a striking degree, indicating that high levels of this enzyme may promote tumorigenesis, at least in part by facilitating NF-κB-induced gene expression.

NF-κB, a central coordinator of immune and inflammatory responses, must be tightly regulated. We describe a NF-κB regulatory pathway that is driven by reversible lysine methylation of the p65 subunit, carried out by a lysine methylase, the nuclear receptor–binding SET domain-containing protein 1 (NSD1), and a lysine demethylase, F-box and leucine-rich repeat protein 11 (FBXL11). Overexpression of FBXL11 inhibits NF-κB activity, and a high level of NSD1 activates NF-κB and reverses the inhibitory effect of FBXL11, whereas reduced expression of NSD1 decreases NF-κB activation. The targets are K218 and K221 of p65, which are methylated in cells with activated NF-κB. Overexpression of FBXL11 slowed the growth of HT29 cancer cells, whereas shRNA-mediated knockdown had the opposite effect, and these phenotypes were dependent on K218/K221 methylation. In mouse embryo fibroblasts, the activation of most p65-dependent genes relied on K218/K221 methylation. Importantly, expression of the FBXL11 gene is driven by NF-κB, revealing a negative regulatory feedback loop. We conclude that reversible lysine methylation of NF-κB is an important element in the complex regulation of this key transcription factor.

Type I and II IFNs both play an important role in the induction of inflammation and the clearance of microbial infections. The order that leukocytes are exposed to such IFNs seems to matter for the subsequently induced signaling pathways and genes. Previously, it was shown that priming of mouse macrophages with IFN-gamma enhances IFN-alpha-induced activation of STAT3, and particularly the activation of STAT1. Moreover, in mouse macrophages, the induction of inflammatory STAT1 target genes by IFN-alpha is strongly enhanced after pre-exposure to IFN-gamma. Although we could confirm the enhanced induction of PY-STAT1 by IFN-beta after IFN-gamma priming of primary human monocytes, the formation of PY-STAT3 remained the same. In contrast, we observed strongly increased IFN-beta-induced activation of STAT5 after pre-exposure of primary human monocytes and THP-1, a human monocytic cell line, to IFN-gamma. Mechanistically, we could confirm in human monocytes the published data in mice, which showed that the enhanced type I IFN-induced activation of STAT1 after priming with IFN-gamma is dependent on the increased activity of the tyrosine kinase Syk. However, our data showed that the enhanced induction of PY-STAT5 in human monocytes did not depend on Syk, suggesting that the enhancement of the activation of STAT5 occurs via a different mechanism than the augmentation of PY-STAT1. We are currently investigating which tyrosine kinase is responsible for the enhanced induction of PY-STAT5 by IFN-beta after IFN-gamma priming. Once identified, this will provide us a tool to study which IFN-beta-induced genes depend on either STAT1 or STAT5 activation and insights into the altered biological response in human monocytes after pre-exposure to IFN-gamma. Finally, because activation of STAT5 is generally associated with increased cell survival and proliferation, we will study whether the increased activation of STAT5 opposes the IFN-beta-induced apoptosis in human monocytes that normally occurs via the activation of STAT1.

Epidermal Growth Factor Receptor (EGFR) and Nuclear Factor kappa B (NF- κ B) both play key roles in promoting tumorigenesis in a variety of cancers but their connection is not yet well understood. We have used Western assays, transcription analysis of known NF- κ B target genes, and chromatin immunopreciptiation (ChIP) assays to show that EGF activates NF- κ B-dependent signaling robustly, through I kappa B (I κ B) and I κ B kinase (IKK) phosphorylation. We then studied how NF- κ B-dependent transcription is regulated differentially by EGFR, compared to the Interleukin-1 receptor (IL-1R), a classical NF- κ B activator. Our preliminary global analysis of NF- κ B promoter enrichment of EGF- and IL-1-activated genes leads us to propose that EGF induces distinct NF- κ B targets that are not induced by IL-1. We are currently testing this hypothesis by global ChIP-seq and RNA-seq analyses, to identify target genes that are differentially activated by the two signals and have identified a novel set of NF- κ B-dependent genes that are induced by EGF but not by IL-1. We will investigate the underlying mechanisms of differential NF- κ B gene regulation by analyzing post-translational modifications of NF- κ B and induction of specific transcriptional co-regulators that are activated differentially by the two signals. We expect that this work will lead to a better understanding of how EGF-dependent pathways contribute to NF- κ B-dependent gene expression in normal and cancer cells, and we hope that it leads to improved treatment of EGFR-driven cancers, in which NF- κ B is known to play a key role in mediating resistance to therapy.

Carbamyl-P synthetase (EC 2.7.2.9), aspartate transcarbamylase (EC 2.1.3.2), and dihydro-orotase (EC 3.5.2.3), the first three enzymes of the de novo pathway for synthesis of pyrimidine nucleotides, have been co-purified as a single oligomeric protein from a mutant line of hamster cells selected for its ability to resist N-(phosphonacetyl)-L-aspartate (PALA), a potent and specific inhibitor of aspartate transcarbamylase. All three enzymes overaccum,late in the mutant cells (Kempe, T.D., Swyryd, E.A., Bruist, M., and Stark, G.R. (1976) Cell 9, 541-550) and the oligomer represents nearly 10% of the total cellular protein. Tens of milligrams of oligomer have been purified to homogeneity by a simple and rapid procedure, with recovery of about 50% of all three activities. The pure protein contains only one size of polypeptide, Mr approximately 200,000, as revealed by electrophoresis in danaturing gels. All three enzyme activities are associated with this polypeptide, indicating that it is multifunctional. Further evidence for a multifunctional protein is provided by titration of the oligomer with radioactive PALA, which reveals that the number of PALA binding sites approximately equals the number of polypeptide chains. The isolated multifunctional protein is a mixture of trimers and hexamers.

N-(phosphonacetyl)-l-aspartate (PALA) is an analog of the activated complex for the reaction catalyzed by aspartate transcarbamylase and has previously been shown to be a potent and specific inhibitor of the enzymes from Escherichia coli and mouse spleen. PALA is now shown to be a potent inhibitor of de novo pyrimidine nucleotide biosynthesis in five lines of mammalian cells in culture. When the concentration of PALA in the medium is 10-4 m, simian virus 40-transformed hamster cells do not divide and, within about 2 days, have detached from the culture dishes and appear dead. The toxicity of 10-4 m PALA is prevented completely if 10-4 m uridine is present in the medium, showing that PALA inhibits uridylic acid biosynthesis specifically. The same effect can be observed with the other cell lines. When the external concentration of radioactive PALA is in the range 10-4 to 10-5 m, its concentration in the cytosol is approximately equal to the external concentration. Most of the PALA recovered from cells remains undegraded. In crude cell extracts, the Ki for PALA is about 1 x 10-9 m at 37° and pH 7.5. Addition of 1 x 10-7 m PALA in vitro to an extract of transformed hamster cells inhibits by 60% the incorporation of radioactivity from CO2 into UMP and CMP; approximately 10-6 m PALA is required to give comparable inhibition of incorporation from carbamyl-P. Higher concentrations give virtually complete inhibition in both cases. These results suggest that PALA may find clinical use in suppressing cellular proliferation. A small number of the virus-transformed hamster cells become resistant to 10-4 m PALA in culture and retain resistance after they are grown for many generations in the absence of the drug. Resistance is accompanied by a large increase in aspartate transcarbamylase activity.

Specific chemical cleavage of proteins at S-cyanocysteine residues leads to formation of iminothiazolidinecarboxylyl peptides which, because their amino termini are blocked, are not susceptible to Edman degradation. A catalyst prepared from NiCl2 and NaBH4 converts 2-iminothiazolidine-4-carboxylate to alanine and iminothiazolidine carboxylylglycine to Ala-Gly in good yield. Unmodified proteins treated with this catalyst in 8M guanidinium chloride are recovered in good yield, with quantitative conversion of methionine to amino-butyrate and half-cystine to alanine by desulfuration. The catalyst also induces cleavage of a small subclass of peptide bonds, probably Phe-Thr and Phe-Ser sequences, producing discrete fragments.

We describe a new, rapid, and convenient procedure for purifying 2',5'-oligoadenylate synthetases, employing precipitation with ammonium sulfate, fractionation by gel filtration, rapid binding to poly(I) X poly(C) cellulose, and elution with 0.35 M KCl. Unlike previously published methods, the procedure does not require sedimentation of the enzyme at 200,000 X g. Therefore, it is more general and more likely to succeed with synthetases extracted from a variety of cells or tissues, or from different subcellular fractions. We have purified the enzymes from two sources to apparent homogeneity, about 2500-fold from the cytoplasm of HeLa cells in 40% yield and more than 400,000-fold from the cytoplasm of rabbit reticulocytes in 25% yield. The specific activity of the HeLa enzyme is about 4 times higher than reported previously. The physical and functional properties of the pure enzymes are very similar to those reported by others for preparations of 2',5'-oligoadenylate synthetase from rabbit reticulocytes, mouse L cells, and human HeLa cells. A new affinity matrix was prepared by linking periodate-oxidized poly(I) X poly(C) to a hydrazide derivative of finely divided cellulose. Poly(I) X poly(C) cellulose binds about twice as much synthetase as the corresponding amount of poly(I) X poly(C) paper and activates the bound enzyme about three times better.

Baby hamster kidney (BHK) cells selected simultaneously with N-phosphonacetyl-L-aspartate (PALA) and methotrexate (MTX) gave rise to doubly resistant colonies at frequencies 20 to 260 times greater than the product of the independent frequencies found with PALA or MTX alone. Double resistance was due to amplification of both target genes, CAD and DHFR. Four independent doubly resistant "MP" lines were selected and characterized. Cells resistant to coformycin, pyrazofurin, or ouabain were generated from all four MP lines at rates up to 25 times greater than the rates for BHK cells. These three drugs select cells that have amplified the genes for their target enzymes. Therefore, we conclude that the four MP lines have an amplificator phenotype. All four grew much more slowly than BHK cells, indicating that the amplificator phenotype may be linked to significant defects in metabolism or cell division.

A quick, accurate assay for specific DNA sequences is described in which whole cells are treated with 0.4 m sodium hydroxide at 80°C. DNA is relatively resistant to alkaline hydrolysis, whereas proteins and RNA are degraded rapidly. The DNA in NaOH is then transferred through a slot directly onto a nylon membrane and hybridized with a probe. Since the procedure is so simple, many samples can be analyzed in a short time. A single-copy gene can be detected in as few as 1000 cells and, since the DNA from 105 cells can be loaded through a single slot, the sensitivity is sufficient to detect one specific DNA sequence per 100 cells. Accurate quantitative analysis can be achieved by normalizing the amount of DNA available for hybridization in each slot, using a probe derived from total DNA.

Complex mixtures of 2′,5′-oligoadenylates are formed in cells and tissues under several different circumstances, and methods for analyzing such mixtures are reviewed. Separation is achieved by high-performance liquid chromatography and quantitation by competition-binding assays, using three different types of antibodies or a specific binding protein, or by functional assay, using preparations of an endonuclease specifically activated by some of the 2′,5′-oligoadenylates. Representative results from three different biological systems are presented. The function of 2′,5′-oligoadenylates as activators of intracellular RNA degradation is discussed, along with the possibility that these compounds may serve as signals for other intracellular regulatory processes.

N-(phosphonacetyl)-l-aspartate (PALA), a transition state analogue for aspartate transcarbamylase, is an effective inhibitor of the enzyme in a number of tissues when administered to mice. After a single injection of 1 mg of [3H]PALA (1 µCi per mg), PALA concentrations reach a peak of 1.5 x 10-6 m within 2 hours in spleen, and remain at a constant level of 5 x 10-7 m for at least 72 hours in both spleen and liver. PALA concentrations in jejunum are maintained at 10-7 m for at least 72 hours following a single subcutaneous injection. More than 90% of the tissue radioactivity can be recovered as unchanged PALA. The drug is equally effective when given either orally or by subcutaneous injection. Using the activity of aspartate transcarbamylase as a measure of the effective concentration of PALA in the tissue, a single oral dose of as little as 100 µg gives maximum inhibition of the enzyme. Aspartate transcarbamylase activity is maximally inhibited within 15 min of administration and 72 hours later the enzyme activity in spleen is still substantially inhibited. Addition of 1 x 10-7 m PALA in vitro to an extract of spleen inhibits by 80% the incorporation of radioactivity from CO2 into UMP and CMP; approximately 10-6 m PALA is required to give comparable inhibition of incorporation from carbamyl-P. Mice injected with 1 mg of PALA 1 hour before injection with isoproterenol incorporated [14C]thymidine into the DNA of their submaxillary glands at a rate no greater than that of control animals, whereas incorporation by mice injected with isoproterenol alone was markedly stimulated, indicating that PALA effectively blocks the supply of pyrimidine nucleotides required for DNA synthesis in this instance of drug-stimulated proliferation.

Some preparations of both native aspartate transcarbamylase from Escherichia coli and catalytic subunit have fewer tight binding sites per oligomer for carbamyl-P than the number of catalytic peptide chains. In contrast, the number of sites for the tight-binding inhibitor N-(phosphonacetyl)-L-aspartate does equal the number of catalytic chains in each case. Binding of the labile carbamyl-P was determined using rapid gel filtration, with conversion to stable carbamyl-L-aspartate during collection. Native enzyme (six catalytic chains) obtained from cells grown under the conditions of J.C. Gerhart and H. Holoubek (J. Biol. Chem. (1967) 242, 2886-2892) has 5.4 tight sites for carbamyl-P at pH 8.0 (KD = 9.9 muM), whereas native enzyme from cells grown with higher concentrations of glucose, uracil, and histidine (to yield more enzyme per unit volume of culture) has only 1.9 tight sites at pH 8.0 (KD = 4.6 muM) and only 2.3 tight sites at pH 7.0 (KD = 2.6 muM). At pH 8.0, catalytic subunit (three catalytic chains) obtained from the former native enzyme has 2.2 tight sites for carbamyl-P (KD = 2.4 muM) and the number of sites is 2.3 in the presence of 35 mM succinate, whereas catalytic subunit obtained from the latter native enzyme has 1.8 tight sites (KD = 3.6 muM) in the absence of succinate and 2.3 tight sites in its presence. The number of tight binding sites is also less than the number of subunit peptide chains in 19F nuclear magnetic resonance experiments performed with catalytic subunit and two fluorinated analogs of carbamyl-P at comparable concentrations of analogs and active sites. A model is proposed in which incomplete removal of formylmethionine from the NH2 termini of the enzyme under conditions of extreme depression affects affinity for ligands.

Mutant Syrian hamster cells resistant to N-(phosphonacetyl)-L-aspartate (PALA), a transition state analog inhibitor of aspartate transcarbamylase, overproduce CAD, a multifunctional protein which catalyzes the first three reactions of de novo UMP biosynthesis. Increased levels of a single mRNA cause the overproduction of CAD in all PALA-resistant mutants examined thus far. A recombinant plasmid containing a 2,3-kilobase insert complementary to the 3'-proximal region of this 7.9-kilobase mRNA has been prepared and used to show that the CAD gene is amplified in each of the 10 PALA-resistant mutants examined. Rates of association of CAD sequences in DNA isolated from PALA-sensitive and PALA-resistant cells with labeled plasmid DNA indicated that the degree of amplification is approximately equal to the degree of overproduction of protein and mRNA in each mutant. The patterns of digestion of these DNAs with restriction enzymes confirmed this result and showed that the lower limit for the size of the amplified unit is 19 kilobases, much larger than the mRNA. A comparison of restriction endonuclease digests of the cloned cDNA with digests of genomic DNA indicated that part of this difference is attributable to intervening sequences in the CAD gene. A 10.2-kilobase RNA which contains CAD sequences is found in cytoplasmic fractions from some PALA-resistant mutants but not in wild type cells. Restriction patterns were analyzed by a new method in which fragments of DNA are transferred from agarose gels to diazo paper with a high efficiency which is independent of size.

T antigen in extracts of cells infected with tsA mutants is 2 to 6 times more labile at 32°C or 41°C than the antigen in extracts of cells infected with wild-type SV40, as assayed by complement fixation. The stabilities of wild-type and mutant antigens are not altered by mixing the extracts, and thus the stability is an intrinsic property of each antigen and is not determined by another component of the extract. This observation indicates that T antigen is probably the virus-coded product of the A gene. In cells infected at the permissive temperature of 32°C with a high multiplicity of either wild-type or tsA mutant virus, the amounts of T antigen are approximately equivalent and increase logarithmically during the entire period of infection, up to 96 hr. Cells infected at 32°C for 96 hr with mixtures of wild-type and tsA virus produce T antigen with the stability of wild-type, even when the infection is carried out with up to a 5 fold excess of the mutant. The more stable wild-type antigen may repress, directly or indirectly, the synthesis of the more labile mutant antigen.

Pyridoxal-P reacts specifically with a single lysine residue at the active site of Escherichia coli aspartate transcarbamylase (Greenwell, P., Jewett, S. L., and Stark, G. R. (1973) J. Biol. Chem. 248, 5994-6001). Reduction of the Schiff base with sodium borohydride, succinylation of the remaining lysine residues, and digestion with trypsin result in formation of a single pyridoxyl peptide, which was purified to homogeneity after chromatography on DEAE-cellulose, treatment with alkaline phosphatase, and rechromatography. Amino acid composition and the results of limited sequential degradation showed that this peptide corresponds to residues 62 to 98 in the sequence of Konigsberg and co-workers, and contains 2 residues of lysine (Henderson, L., Roy, D., Martin, D., and Konigsberg, W., personal communication). By similar isolation, a second peptide was obtained from unsuccinylated catalytic subunit, containing only the pyridoxylated lysine, which corresponds to Lys-80. Derivatives of catalytic subunit containing an average of either one, two, or three pyridoxamine-P moieties per trimer have been prepared by reduction. These species, which retain catalytic activity in proportion to their unmodified active sites, were recombined with regulatory subunit to prepare partially modified derivatives of native aspartate transcarbamylase. At pH 8, fluorescence emission bands were observed at 340 nm, due to aromatic amino acids in the protein, and at 395 nm, due to the pyridoxamine-P moiety. Upon excitation at 280 nm energy transfer from protein to pyridoxamine-P was approximately 15%. The properties of the probe were used to study changes accompanying the binding of substrates and inhibitors. The effects of CTP and ATP were small. With the transition state analog N-(phosphonacetyl)-L-aspartate (PALA) or the substrate carbamyl-P, two types of response were observed. Derivatives of catalytic subunit and native enzyme which contain some unmodified sites and hence retain partial catalytic activity gave large increases in fluorescence at 395 nm. However, fully modified inactive derivatives gave much smaller increases. A derivative of native enzyme containing one triply modified and one unmodified catalytic subunit behaved like the other partially modified species. These results indicate that there is communication among the active sites of different catalytic trimers in modified native enzyme, as well as among active sites within the same modified catalytic trimer. The increases in fluorescence result from a red shift of the absorption maximum of the pyridoxamine-P moiety from 315 to 325 nm, which increases the absorbance at the excitation wavelength for fluorescence. At pH 7, the absorption spectrum is already shifted and, consequently, the binding of PALA and carbamyl-P has little effect on the fluorescence. Therefore, the binding of these compounds at pH 8.0 must cause a structural change in the protein, which in turn causes protonation of a group in the modified active sites, altering the spectral properties.

2',5'-oligoadenylates can be assayed sensitively in cell extracts by use of an antiserum having maximum specificity for any compound containing the moiety -pA2'pA2'pA-. These compounds reached high concentrations (25-2000 nM) in monkey CV-1 cells after infection with simian virus 40 (SV40) and treatment with human leukocyte interferon. The levels were highest late in infection and increased in parallel with the accumulation of SV40 late messenger RNAs. Alone, neither interferon nor SV40 caused the 2',5'-oligoadenylate concentrations to increase above the levels present in untreated CV-1 cells, 3 nM or less. Analyses by high performance liquid chromatography revealed little or no (p)pp(A2'p)2A or (p)pp(A2'p)3A, and the extracts showed only very low activity in functional assays with ppp(A2'p)nA-dependent nucleases, equivalent to 3 nM ppp(A2'p)3A or less. Some of the 2',5'-oligoadenylates eluted in the positions of the nonphosphorylated "cores," (A2'p)nA, and a substantial fraction was found in several peaks intermediate between ppp(A2'p)3A and cores. The positions of most of these peaks did not change when digestion with alkaline phosphatase was performed before chromatography, indicating that most of the 2',5'-oligoadenylates lack exposed phosphate groups. In contrast to the effects of infection with SV40, addition of poly(I) X poly(C) to interferon-treated CV-1 cells led to accumulation of high levels (up to 3000 nM) of 2',5'-oligoadenylate-5'-di- or triphosphates capable of activating the ppp(A2'p)nA-dependent ribonuclease.

Cells resistant to pyrazofurin and 6-azauridine have been selected from a simian virus 40-transformed Syrian hamster line and from a Chinese hamster lung line. By increasing the concentrations of inhibitors in several steps, mutant cells from both lines have been obtained which resist high concentrations (1 to 5 mM) of the two inhibitors separately or together. Orotidine-5'-phosphate decarboxylase (EC 4.1.1.23), the sixth and last enzyme in UMP biosynthesis, is inhibited by the nucleoside monophosphates derived from pyrazofurin or 6-azauridine. The activity of this enzyme is increased in each resistant cell line tested. Furthermore, there is a parallel increase in each case in the activity of the fifth enzyme of the pathway, orotate phosphoribosyltransferase (EC 2.4.2.10), which is not inhibited by pyrazofurin or 6-azauridine monophosphates, and the amount of increase is up to 67 times the level found in wild type cells. In contrast, the activities of the first three enzymes of UMP biosynthesis remain essentially unchanged in the mutants. Resistant Chinese hamster cells remain sensitive to 5-fluorouridine; this indicates that uridine kinase, the enzyme necessary to convert 6-azauridine to the monophosphate, is still functional.

The interactions of several dicarboxylic acids and monoanions with Escherichia coli aspartate transcarbamylase and with its catalytic subunit have been studied by ultraviolet difference spectroscopy and steady state kinetics, with the following major findings. 1. A variety of dicarboxylic acids compete with carbamyl-P for the active sites of unliganded catalytic subunit, with steric requirements very different from those important for competition with L-aspartate for the subunit/carbamyl-P complex. Competition with carbamyl-P is much reduced if the dicarboxylic acid has a positively charged amino group. Acetate and chloride also compete. 2. At pH 7, equal concentrations of lysine acetate and L-aspartate are equally effective in displacing the transition state analog N-(phosphonacetyl)-L-aspartate (PALA) from the active sites of the concentrations of L-aspartate and lysine acetate is constant, increasing the concentration of L-aspartate does not relieve inhibition of the enzyme by PALA (Collins, K.C., and Stark, G. R. (1971) J. Biol. Chem. 246, 6599-6605). Therefore, the L-aspartate/subunit complex, like the acetate/subunit complex, must be incapable of participating in the catalytic reaction. We conclude that the kinetic mechanism is ordered, in agreement with the recent findings of Wedler and Gasser (Wedler, F.C., and Gasser, F.J. (1974), Arch. Biochem. Biophys. 163, 57-68) and in disagreement with the interpretation of Heyde et al. (Heyde, E., Nagabhushanam, A., And Morrison, J.F. (1973) Biochemistry 12, 4718-4726)...

We have investigated the mechanism of overproduction of the multifunctional protein catalyzing the first three steps of UMP biosynthesis in stable mutants of Syrian hamster cells in culture. The rate of degradation of this protein is unaltered in one mutant cell line which overproduces it by 118-fold. In all mutants tested, the increase in the rate of synthesis of this protein is equal to the increase in its steady state concentration. There is a similar correlation between steady state levels of this protein in vivo and the capacity of polysomal RNA isolated from these cells to direct the synthesis of the protein in vitro. The one mutant cell line studied contains large amounts of a polysomal poly(A)-containing RNA (Mr = 2.7 +/- 0.2 times 10(6)) that is not detected in wild type cells. This large RNA co-sediments in sucrose gradients with the capacity to direct the synthesis of the multifunctional protein in vitro.

We have studied expression of the carbamoyl-P synthetase-aspartate transcarbamoylase-dihydroorotase (CAD) gene in growing or confluent Syrian hamster cells, and also in cells arrested by depriving them of serum or restimulated by adding fresh serum. In contrast to other biosynthetic enzymes such as dihydrofolate reductase, the amount of the CAD enzyme decreased very little when growth was arrested, as judged by the small change in aspartate transcarbamoylase activity. However, the level of CAD mRNA was about 10-fold lower in arrested cells than in growing cells. The rate of transcription of CAD, measured by the nuclear run-off technique, decreased roughly in parallel with the decrease in the steady-state level of the mRNA, suggesting that control of transcription is probably an important element in the overall regulation of CAD gene expression. Expression of a different gene which lies near the 5' end of CAD changed in parallel with that of CAD, but expression of two other genes in the same region was insensitive to the growth state of the cells. Therefore, control of gene expression as a function of growth was specific rather than a reflection of more general changes in chromatin structure in the vicinity of CAD.

N-(Phosphonacetyl)-l-aspartate (PALA) is a stable analogue of the transition state in the reaction catalyzed by aspartate transcarbamylase from Escherichia coli; it combines in 1 molecule most of the structural features of the two natural substrates, carbamyl phosphate and l-aspartate. The inhibition constant (Ki) for PALA with the catalytic subunit is 2.7 x 10-8m at 28° and pH 7.0 in 0.2 m imidazole acetate; the value of Ki is approximately the same with the native enzyme. PALA binds to aspartate transcarbamylase about 1000 times more tightly than carbamyl phosphate, the substrate bound most tightly. In addition to tight binding, other data indicate that PALA interacts with the enzyme at both the carbamyl phosphate and l-aspartate sites simultaneously and puts the enzyme into the same contracted conformation as do carbamyl phosphate and succinate (an analogue of l-aspartate) acting together. PALA alone or carbamyl phosphate and succinate together produce the same ultraviolet difference spectrum with the catalytic subunit; the binding of PALA alone or carbamyl phosphate and succinate together at some of the catalytic sites in the native enzyme activates the remaining catalytic sites to the same extent. PALA gives competitive inhibition with carbamyl phosphate but not with l-aspartate, indicating an obligatory binding order for the substrates: carbamyl phosphate must bind before l-aspartate for catalysis to occur. Titration of the catalytic subunit with PALA, monitored by ultraviolet difference spectroscopy, indicates that there are 3.07 active sites per 100,000 daltons. The interaction of PALA with the enzyme is consistent with the "compression" model for aspartate transcarbamylase previously proposed (Collins, K.D., and Stark, G.R., J. Biol. Chem., 244, 1869 (1969)).

Erythro-β-hydroxy-l-aspartate is a good substrate for the catalytic subunit of the aspartate transcarbamylase of Escherichia coli, with Km = 24 mm (approximately the same as the Km for l-aspartate) and Vmax = 0.18 mole per min per g (22% of the Vmax for l-aspartate) at pH 8 and 28°. Aminomalonate is also carbamylated by the enzyme (Km approximately 770 mm and Vmax = 0.014 mole per min per g under the same conditions), but threo-β-hydroxy-dl-aspartate, erythro, threo-β-methyl-dl-aspartate, α-methyl-dl-aspartate, l-glutamate, and O-phospho-dl-serine are all inactive, even at high concentrations of enzyme. dl-α-Methylsuccinate, d- and l-tartrate, d-aspartate, the α- and β-methylaspartates, and threo-β-hydroxy-dl-aspartate are all poor inhibitors (Ki greater than 100 mm), whereas mesotartrate (Ki = 2.7 mm at pH 7 and 28°) and d- and l-malate are good competitive inhibitors for l-aspartate. Since maleate is a much better inhibitor than fumarate, we concluded previously that l-aspartate is bound to the aspartate transcarbamylase-carbamyl phosphate complex with its carboxylates oriented approximately cis to one another. Using erythro-β-hydroxy-l-aspartate as an example, four loci can be designated on the enzyme-carbamyl phosphate complex, corresponding to the positions of the α-amino group (A), the β-hydroxyl group (B), the β-hydrogen (C), and the α-hydrogen (D). When the carboxylates are cis, A is cis to B and C is cis to D. A single set of restrictions on these four loci can be used to rationalize the specificity of the enzyme for analogues of l-aspartate as substrates or inhibitors and the previous observation that l-aspartate binds very poorly to the enzyme-carbamyl phosphate complex. The restrictions are (a) that Loci A and B can accept hydroxyl groups without substantial impairment of binding but Loci C and D cannot, and (b) that an amino or methyl group in any of the four loci undergoes an interaction with the enzyme-carbamyl phosphate complex that prevents tight binding. These restrictions lead to the conclusions that mesotartrate binds with the hydroxyl groups in Loci A and B but not in C and D, that l-malate binds with the hydroxyl group in Locus A, and that d-malate binds with the hydroxyl group in Locus B.

A transient nuclear magnetic resonance method has been used to measure the rate at which succinate, a competitive inhibitor of l-aspartate, binds to the aspartate transcarbamylase of Escherichia coli and to its catalytic subunit. In the presence of catalytic subunit and carbamyl phosphate, the relaxation time T2 for succinate is greatly shortened, while the relaxation time T1 is not affected much. In an analogous experiment with inorganic phosphate in place of carbamyl phosphate, T2 is also greatly shortened and, in both instances, the relaxation times are independent of the nuclear magnetic resonance frequency. The expressions for relaxation in the presence of chemical exchange were examined for limiting cases of frequency independence. Two limiting cases could be distinguished by the opposite dependences of T2 on temperature, and it was demonstrated clearly that succinate exchanges slowly with the enzyme-carbamyl phosphate complex, but rapidly with the enzyme phosphate complex. For exchange of succinate with the catalytic subunit-carbamyl phosphate complex, a bimolecular rate constant of 2.3 x 105 m-1 sec-1 at 33° for binding of succinate and an activation energy of 12 kcal per mole for dissociation of succinate from the complex were found. The exchange of succinate with the catalytic subunit-phosphate complex was too rapid to permit measurement of the rate constant by nuclear magnetic resonance methods, but a rotational correlation time of 0.73 x 10-8 sec at 33° was determined for the ternary complex. The results indicate that succinate has relatively free access to the active site of the subunit-phosphate complex and is bound rigidly. The unusually slow bimolecular rate constant for binding of succinate to the subunit-carbamyl phosphate complex may indicate that a conformational change is concerted with binding. A fast bimolecular step, followed by a slow step, cannot be ruled out entirely, but the concentration of the intermediate complex is probably low (less than 2%). For the native enzyme, the rate constant for binding of the last molecule of succinate to the complex with carbamyl phosphate is 1.9 x 105 m-1 sec-1 at 33°, with an activation energy of 8 kcal per mole for dissociation of succinate from the complex. The rotational correlation time for the native enzyme-phosphate-succinate complex is 2.2 x 10-8 sec at 33°. These results, very similar to those obtained with the catalytic subunit, indicate that the regulatory subunits have little influence on the properties of the active sites when the native enzyme is saturated with succinate.

Spectrophotometric titration studies at pH 8.0 have revealed that pyridoxal 5'-phosphate binds as a Schiff base to the catalytic subunit of Escherichia coli aspartate transcarbamylase with a stoichiometry of 3 pyridoxal phosphates per trimer and a dissociation constant of 0.9 µm. The ultraviolet absorption of the Schiff base near 430 nm is lost upon addition of the inhibitor N-(phosphonacetyl)-l-aspartate (PALA) or the substrate carbamyl-P. In steady state kinetic studies, pyridoxal-P was found to be a competitive inhibitor of carbamyl-P, with an inhibition constant of 4 ± 2 µm. Pyridoxal does not bind to or inhibit the catalytic subunit. These results imply that pyridoxal-P binds at the active site not only through a Schiff base but also through noncovalent, ionic interactions with the phosphate binding region. Reduction of the Schiff base with sodium borohydride yields an N-e-pyridoxyl-l-lysine derivative of the enzyme with 2.9 to 3.0 pyridoxal-P incorporated per catalytic trimer. This reduced derivative is inactive but still binds 3 molecules of PALA per catalytic trimer at pH 8.0. However, the binding of PALA is characterized by at least two dissociation constants in the range 100 to 10 µm. PALA binds much more tightly to unmodified catalytic subunit at pH 8 with a single dissociation constant of about 0.1 µm. Photo-oxidation of the unreduced catalytic subunit-pyridoxal-P Schiff base causes loss of 90% to 95% of the activity and loss of 2.0 ± 0.5 histidine residues per chain. No other amino acid is found to be modified by amino acid analysis. However, 0.5 ± 0.05 molecules per chain of an unknown derivative of pyridoxal-P derivative are covalently bound. Covalently linked dimers and trimers of the catalytic chains are also formed, especially at high protein concentrations. At low concentrations at which dimer and trimer formation is negligible, the covalent incorporation of 0.5 pyridoxal-P per chain does not account for the almost complete loss of enzyme activity. Therefore, the loss of histidine is correlated with loss of activity. These studies indicate that 1 lysine residue and 2 histidine residues are probably near the active site of aspartate transcarbamylase.