Mitogen-activated protein kinases (MAPKs) are rapidly phosphorylated and activated in response to various extracellular stimuli in many different cell types. Such regulation of MAPK results from sequential activation of a series of protein kinases. The kinases that phosphorylate MAPKs, the MAP kinase kinases (MEKs) are also activated by phosphorylation. MEKs are related in sequence to the yeast protein kinases Byr1 (from Schizosaccharomyces pombe) and Ste7 (from Saccharomyces cerevisiae), which function in the pheromone-induced signaling pathway that results in mating. Byr1 and Ste7 are in turn regulated by the protein kinases Byr2 and Ste11. The amino acid sequence of the mouse homolog of Byr2 and Ste11, denoted MEKK (MEK kinase), was elucidated from a complementary DNA sequence encoding a protein of 672 amino acid residues (73 kilodaltons). MEKK was expressed in all mouse tissues tested, and it phosphorylated and activated MEK. Phosphorylation and activation of MEK by MEKK was independent of Raf, a growth factor-regulated protein kinase that also phosphorylates MEK. Thus, MEKK and Raf converge at MEK in the protein kinase network mediating the activation of MAPKs by hormones, growth factors, and neurotransmitters.

ABSTRACT

Nitric oxide (NO) is a poison, and organisms employ diverse systems to protect against its harmful effects. In Escherichia coli, ygaA encodes a transcription regulator (b2709) controlling anaerobic NO reduction and detoxification. Adjacent to ygaA and oppositely transcribed are ygaK (encoding a flavorubredoxin (flavoRb) (b2710) with a NO-binding non-heme diiron center) and ygbD (encoding a NADH:(flavo)Rb oxidoreductase (b2711)), which function in NO reduction and detoxification. Mutation of either ygaA or ygaK eliminated inducible anaerobic NO metabolism, whereas ygbD disruption partly impaired the activity. NO-sensitive [4Fe-4S] (de)hydratases, including the Krebs cycle aconitase and the Entner-Doudoroff pathway 6-phosphogluconate dehydratase, were more susceptible to inactivation in ygaK or ygaA mutants than in the parental strain, and these metabolic poisonings were associated with conditional growth inhibitions. flavoRb (NO reductase) and flavohemoglobin (NO dioxygenase) maximally metabolized and detoxified NO in anaerobic and aerobic E. coli, respectively, whereas both enzymes scavenged NO under microaerobic conditions. We suggest designation of the ygaA-ygaK-ygbD gene cluster as the norRVW modulon for NO reduction and detoxification.Nitric oxide (NO) is present throughout the biosphere (1-3). In humans, tightly regulated NO synthases produce sufficient NO to poison pathogens, opportunistic organisms, and neoplastic tissue (4, 5). Nanomolar NO potently inhibits terminal oxidases and aerobic respiration (6, 7) and alters the amphibolic and regulatory reactions of the citric acid cycle enzyme aconitase by destroying its labile [4Fe-4S] center (7-10). In addition, significant secondary toxicity of NO can occur via reactions of NO 2 , ONOO Ϫ , NO Ϫ , dinitrosyl iron, and nitrosothiols (11-14). It has become increasingly evident that most organisms metabolize and detoxify NO. Enzymes decompose NO in microorganisms (1, 3, 15-18) and humans (7) and prevent the accumulation of toxic NO levels. Nitric-oxide reductases (NORs) 1 metabolize NO to N 2 O in anaerobic denitrifying bacteria and fungi and likely serve an additional role in minimizing NO toxicity (1,3,19). Nitric-oxide dioxygenases (NODs) convert NO to NO 3 Ϫ in organisms as diverse as bacteria and mammals and have been shown to protect aerobic cells from NO damage (7, 20 -26). In microorganisms, (flavo)hemoglobins catalyze NO dioxygenation (20 -24, 27, 28).In the accompanying article (17), we provide evidence for an inducible and robust NO-metabolizing and -detoxifying activity in anaerobic Escherichia coli. Attempts to biochemically identify the NO reduction system have been complicated by its instability. Moreover, the E. coli genome lacks a NOR belonging to either the cytochrome bc complex or cytochrome P450 families (1). The list of proteins displaying a reductase activity for NO in vitro with potential for function in E. coli is long and includes flavohemoglobin (flavoHb) (27, 28), cytochrome c or cЈ (29), multi-heme nitrite reductase (2, 30), copper-n...

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The flavoHbs 1 belong to a 1.8 billion-year-old family of globin molecules that includes O 2 -binding Hbs and Mbs isolated from animals, plants, fungi, protozoa, bacteria, and worms (1-6). FlavoHbs have a unique two-domain structure containing linked Hb and reductase domains with extensive homology to the mammalian Hbs and metHb reductases (1, 7). Other Hbs appear to be co-expressed with associated metHb reductases (8). An O 2 transport or storage function, like that of the erythrocytic Hbs and muscle Mbs, has been suggested for some microbial and plant (flavo)Hbs (4, 9); however, other functions including the catalysis of oxidations have long seemed more likely (10 -12).Recently, we described an NO dioxygenase (NOD) produced by Escherichia coli that utilizes O 2 and NAD(P)H to convert NO to nitrate (Equation 1) and identified it as a flavoHb (13,14). Subsequent studies have shown that related bacterial and yeast flavoHbs display a similar NOD activity.A role for flavoHbs in NO detoxification is supported by the ability of flavoHbs to protect bacteria against NO or nitroso compounds (13, 14, 16 -18) and by their induction in bacteria exposed to NO, nitrate, nitrite, or nitroso compounds (13, 14, 17, 19 -22). However, the mechanism of NO detoxification, and thus the function of the flavoHbs, is obscured by the possibility of multiple reaction mechanisms involving NO. Other NO detoxifying activities for flavoHbs, including denitrosylation of nitrosothiols (17), NO reduction (17, 23), and NO sequestration (16, 23), have been offered to explain the protection flavoHbs afford to bacteria against NO and nitrosoglutathione. Thus, an understanding of the biological function(s) of the flavoHbs demands a greater knowledge of their various activities.In this report, steady-state, reduction, and ligand binding kinetics of the E. coli NOD (flavoHb) were measured in order to define its function and the mechanism of NO dioxygenation. We also examined the effects of amino acid substitutions at the highly conserved Tyr(B10) position on NOD activity, reduction, and ligand binding kinetics (7,24). Key differences between flavoHb and other Hbs are discussed in light of this specialized but perhaps ancient NO dioxygenation and detoxification function of hemoglobin. MATERIALS AND METHODSCells and Reagents-The flavoHb-deficient E. coli strain RB9060 (25) was generously provided by Alex Ninfa (University of Michigan). Plasmid pAlter containing the E. coli hmp gene was prepared as described previously (13). FAD, NADPH, and bovine hemin were purchased from Sigma. NADH, bovine liver catalase (260,000 units/ml), and deoxyribonuclease were obtained from Roche Molecular Biochemicals. Saturated NO was prepared as described previously (26). Saturated O 2 (1.14 mM) was prepared by vigorously scrubbing a solution of 50 mM potassium phosphate, pH 7.8, containing 0.1 mM EDTA (buffer A) at 25°C and atmospheric pressure with 99.993% O 2 (Praxair, Bethlehem, PA) in a rubber septum-sealed glass vial vented with a syringe needle. Manganese-containing...

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Treatment of L929 cells with tumor necrosis factor alpha (TNFalpha) activates a programmed cell death pathway resulting in apoptosis. We investigated the intracellular signaling pathways activated in L929 cells by TNFalpha. TNFalpha robustly activates Jun kinase (JNK), a member of the mitogen-activated protein kinase (MAPK) family. In addition, p42(MAPK) is activated, but a 10-fold greater concentration of TNFalpha was required for substantial MAPK activation than was needed for maximal JNK stimulation. Simultaneous treatment of L929 cells with fibroblast growth factor (FGF-2) significantly reduced the apoptotic response to TNFalpha. FGF-2 substantially activated the Raf/MEK/MAPK (where MEK is mitogen-activated protein kinase kinase) pathway but did not affect TNFalpha activation of JNK. These results indicate that although JNK may play an important role in transmitting the TNFalpha signal from the cell surface to the nucleus, activation of the JNK pathway is not sufficient to induce apoptosis. Expression of dominant-negative Asn-17 Ras in L929 cells diminished the FGF-2 stimulation of p42(MAPK) and eliminated the protective effect of FGF-2. Asn-17 Ras expression did not affect JNK activity and had no effect on TNFalpha activation of JNK. Pharmacological inhibition of MEK-1 activity by incubation of cells with the compound PD 098059 blocked p42(MAPK) activation and FGF-2 protection against apoptosis. Interestingly, activated Val-12 Ras expression substantially enhanced TNFalpha-mediated apoptosis in L929 cells, but Val-12 Ras did not constitutively activate MAPK in L929 cells and FGF-2 partially protected Val-12 Ras-expressing cells from TNFalpha-mediated apoptosis. Our data indicate that activation of the MAPK pathway mediates an FGF-2 protective effect against apoptosis and highlights the important role that integration of multiple intracellular signaling pathways plays in the regulation of cell growth and death.