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B
Kinase ß
Department of Clinical Laboratories,1 Program in Molecular Pharmacology and Chemistry, Memorial Sloan-Kettering Cancer Center,2 Structural Biology Program, Department of Physiology and Biophysics, Mount Sinai School of Medicine, New York, New York3
Received 3 February 2004/ Returned for modification 9 March 2004/ Accepted 10 May 2004
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ABSTRACT |
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B
kinase ß (IKKß) and IKK enzymatic activity in vitro, whereas AA did not have this effect. When cells were loaded with AA and induced to generate DHA by oxidative stress in cells expressing a constitutive active IKKß, NF-B activation was inhibited. Our results identify a dual molecular action of vitamin C in signal transduction and provide a direct linkage between the redox state of vitamin C and NF-B signaling events. AA quenches ROS intermediates involved in the activation of NF-B and is oxidized to DHA, which directly inhibits IKKß and IKK enzymatic activity. These findings define a function for vitamin C in signal transduction other than as an antioxidant and mechanistically illuminate how vitamin C down-modulates NF-B signaling. |
INTRODUCTION |
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-lactone oxidase, the final enzyme in its biosynthetic pathway from glucose (25). Under physiological conditions, vitamin C predominantly exists in its reduced form, ascorbic acid (AA); it also exists in trace quantities in the oxidized form, dehydroascorbic acid (DHA). There are two known mechanisms for transporting vitamin C (21). A universal system, present in all cells, transports vitamin C as DHA via facilitative glucose transporters (34). Once inside the cell, DHA is rapidly reduced and accumulates as AA (34, 35). The second transport system is functional in specialized cells where AA is directly transported into cells via sodium-dependent AA cotransporters (SVCT1 and/or SVCT2) (33). AA functions as a cofactor for enzymes involved in the biosynthesis of collagen (26), carnitine (28), and norepinephrine (18) and in the amidation of hormones (8). In plasma and cells, AA is a powerful antioxidant, quenching reactive oxygen species (ROS) and reactive nitrogen species (10, 14). Intracellular vitamin C can prevent cell death and inhibit mutations induced by oxidative stress (12, 22, 37). During the process of quenching free radicals, ascorbate donates an electron, becoming the unstable intermediate ascorbyl radical that can be reversibly reduced back to ascorbate. Ascorbyl radical can donate a second electron and be converted to DHA (13, 14). DHA may be reduced back to AA or be irreversibly hydrolyzed to 2,3-diketo-gulonic acid, which then is metabolized to threonic and oxalic acid (14). In cells loaded with AA and exposed to hydrogen peroxide, AA is converted to DHA, some of which effluxes from the cells via the glucose transporters, thereby providing a mechanism for recycling vitamin C to the extracellular medium (12). Alternatively, intracellular DHA can be transported to intracellular compartments and organelles (2, 20). DHA functions primarily as a readily transportable form of vitamin C (36).
ROS play a key role in cellular responses as chemical second messenger molecules, and conversely, antioxidants modulate selected signaling responses (24). For example, ROS activate transcription factors, such as NF-B, that are important in host defense, inflammation, and apoptosis (1, 11, 32). Pro-inflammatory cytokines, such as tumor necrosis factor alpha (TNF-), hydrogen peroxide, and ceramide, activate NF-B by inducing the phosphorylation of IB proteins (11, 19). Phosphorylated IB releases NF-B and is itself degraded via proteasomal pathways (17), while unphosphorylated IB associates with NF-B in the cytosol, preventing its nuclear migration. It was initially reported that AA inhibits TNF--induced NF-B activation in endothelial cells via activation of p38 mitogen-activated protein kinase (MAPK) (4); however, we recently showed that AA suppresses TNF--dependent activation of NF-B by inhibiting the activation of kinases involved in the phosphorylation of IB (6).
We investigated the modulation of NF-B activation by vitamin C and found that DHA directly inhibited the kinase activity of IKKß and IKK in vitro and in cellular assays. Thus, our data suggest a dual mechanisms of action of vitamin C in regulating NF-B function. First, as an antioxidant quenching ROS, AA inhibits ROS-mediated signaling events. Second, after oxidization to DHA, vitamin C directly inhibits IKK kinase activity.
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MATERIALS AND METHODS |
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Immunoblotting analysis.
Cell extracts were prepared as previously described (6). Immunoblot analysis was performed with the following rabbit polyclonal antibodies: anti-phospho-IB, anti-IB, anti-p38 MAPK, anti-phospho-p38 MAPK, anti-phosphorylated p44/42 MAPKs (Cell Signaling Technology, Beverly, Mass.) anti-p44/42 MAPKs (Upstate Biotech, Lake Placid, N.Y.) and anti-FLAG antibody (Sigma). Membranes were incubated with horseradish peroxidase-conjugated anti-rabbit immunoglobulin G antibody, and the proteins were revealed using enhanced chemiluminescence assay (Amersham Pharmacia Biotech, Piscataway, N.J.).
Transfection and luciferase assays.
HeLa cells were transiently transfected with pNFB-luc (Clontech, Palo Alto, Calif.) or cotransfected with plasmids containing IKKß(SS/EE) (a constitutively active IKKß in which serines 177 and 181 had been replaced by glutamic acid) or its mutants by the calcium phosphate method or Superfect (QIAGEN, Valencia, Calif.). Cells were incubated with 30 ng of TNF- per ml in triplicate for the time indicated in the figures. Luciferase activity was determined using the luciferase reporter assay system (Promega).
Kinase assays.
Endogenous IKKß was isolated from TNF--treated cellular extracts by immunoprecipitation with anti-IKKß antibody (Santa Cruz Biotechnology, Santa Cruz, Calif.). IKKß(SS/EE) and its mutants were isolated from transfected HeLa cell extracts with anti-FLAG antibody beads (Sigma). Recombinant IKK and IKKß were isolated from transfected 293T cells. Cells were transiently transfected using Genejammer transfection reagent (Stratagene, La Jolla, Calif.) with plasmids containing IKK or IKKß. After 48 h, the cells were harvested, and the kinases were isolated by immunoprecipitation. The kinase activity of endogenous IKKß, recombinant IKK and IKKß, and IKKß(SS/EE) was determined after 15 min at 30°C in buffer containing 10 µM ATP, 1 µCi of [-32P]ATP and 1 µg of substrate (glutathione S-transferase [GST]-IB). The phosphorylated substrate was detected by autoradiography (6). MAPK (p38) activity in vitro was analyzed using a purified GST fusion enzyme and phosphorylated heat- and acid-stable protein 1 (PHAS-1) as the substrate (Calbiochem), and the kinase reaction was performed as indicated by the manufacturer (Calbiochem).
DHA binding assay in vitro. IKKß(SS/EE) and its mutants were isolated from transfected HeLa cells by immunoprecipitation with anti-FLAG beads. The beads carrying IKKß were incubated in kinase buffer for 15 min at 30°C with 6 mM [14C]DHA or 6 mM [14C]AA. [14C]DHA was generated in incubation buffer (pH 5.5) by incubating [14C]AA with ascorbate oxidase. The kinase was eluted from the beads with FLAG peptide, and the associated radioactivity was counted with a Beckman LS 6000LL counter.
Detection of ROS.
HeLa cells were kept for 16 h in serum-free medium containing 0.2% bovine serum albumin. The cells were then loaded with vitamin C, washed, and treated for 10 min with 50 ng of TNF- per ml. Cells were then incubated with 20 µM 2',7'-dichlorodihydrofluorescein diacetate (DCF) for 10 min at 37°C, and ROS were visualized with fluorescence using an Olympus BX60 microscope (40x, DP40, WIBA) equipped with a Qimaging digital charge-coupled device camera (Retiga 1300C) and QCapture software.
Site-directed mutagenesis. Site-directed mutagenesis was performed using the QuikChange XL site-directed mutagenesis kit from Stratagene per the manufacturer's instructions.
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RESULTS |
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-induced activation of NF-B.
We used HeLa cells as a model system to investigate the molecular mechanisms of inhibition of NF-B activation by vitamin C. Cells were loaded with vitamin C by exposing them to DHA (6). This procedure circumvents artifacts associated with the pro-oxidant effects of AA in tissue culture medium (7). Consistent with our previous findings, HeLa cells transfected with the NF-B-dependent luciferase reporter construct (pNFB-luc) induced a 20-fold increase in luciferase activity after treatment with TNF- (Fig. 1A). Cells loaded with 4.0 mM AA showed a weaker induction of luciferase activity of approximately eightfold, corresponding to a 60% inhibition of luciferase activity. Intracellular concentrations of 2.5 mM AA had no significant effect on TNF--induced luciferase activity (P = 0.21, n = 3, one-tailed t test) (Fig. 1A). TNF- induced phosphorylation of IB and cells loaded with 4 mM AA showed decreased phosphorylation of IB (Fig. 1B). These results suggested that intracellular vitamin C inhibits TNF--induced NF-B transcriptional responses by preventing the phosphorylation of IB.
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induces the MAPK pathway activating p44/42 MAPKs and p38 MAPK (16). Therefore, we investigated whether intracellular AA could also inhibit the activation of these kinases. TNF- induced the phosphorylation of p44/42 MAPK in HeLa cells as assayed by immunoblotting (Fig. 1C). Loading cells with AA inhibited TNF--induced phosphorylation of p44/42 MAPK (Fig. 1C). Cells treated with TNF- showed a modest increase in phosphorylated p38 MAPK, but loading cells with AA resulted in a substantial decrease in p38 MAPK phosphorylation (Fig. 1D). Since the activation of NF-B by TNF- involves ROS (11), we measured the relative levels of ROS induced by TNF- in cells loaded with vitamin C. Intracellular ROS was detected using DCF and fluorescence microscopy. TNF- treatment induced an increase in intracellular ROS; however, the levels of ROS in cells loaded with 4 mM vitamin C were barely detected (Fig. 1D). These results suggest that vitamin C prevents TNF--dependent activation of NF-B via inhibition of the phosphorylation of IB by quenching ROS involved in the activation of IKK and also decreases the accumulation of ROS that is a result of signaling.
Intracellular vitamin C inhibits a constitutively active IKKß.
IKKß is activated by phosphorylation at serines 177 and 181 and phosphorylates IB. The replacement of these serines with glutamic acids yields a constitutively active IKKß, IKKß(SS/EE) (38). To investigate the possibility that vitamin C has another mode of action beyond inhibiting the activation of IKKß, we analyzed the activity of IKKß(SS/EE) in cells loaded with vitamin C. Cotransfection of pNFB-luc and a plasmid expressing IKKß(SS/EE) induced a 12-fold increase in luciferase activity (Fig. 2A). However, cells loaded with 4 mM vitamin C inhibited IKKß(SS/EE)-dependent luciferase activity. Lower concentrations of AA (2.5 mM) had no significant effect (P = 0.11, n = 3, one-tailed t test) (Fig. 2A). To determine whether vitamin C inhibits the activity of IKKß(SS/EE), we measured the levels of phosphorylated IB in cells transfected with IKKß(SS/EE). IKKß(SS/EE)-dependent phosphorylation of IB was inhibited in cells loaded with vitamin C (Fig. 2B). Analysis of IKKß(SS/EE) showed that intracellular vitamin C had no effect on its expression (data not shown). Thus, the inhibition of IB phosphorylation induced by the expression of IKKß(SS/EE) in cells loaded with the vitamin points to direct inhibition of IKKß(SS/EE) activity by vitamin C.
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in vitro.
Since intracellular vitamin C inhibited IKKß(SS/EE)-induced phosphorylation of IB, we examined whether AA would directly inhibit the kinase activity of IKKß in a cell-free system. Activated IKKß isolated by immunoprecipitation from TNF--treated HeLa cells was immobilized on protein G-agarose beads and then assayed for in vitro kinase activity (6). The addition of 1 or 10 mM AA to the kinase reaction mixture had no effect on IKKß activity (Fig. 3A). Under similar assay conditions, 100 mM AA inhibited IKKß activity, but this inhibition was abolished when the kinase reaction mixture included dithiothreitol (DTT), suggesting that the inhibitory activity was associated with oxidation of AA (Fig. 3A). Under nonreducing conditions, AA oxidizes to DHA, which can then be hydrolyzed to oxalic and threonic acid (13, 14).
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and p38 MAPK. The kinase activities of IKK and IKKß isolated from transfected cells were inhibited by DHA. At 500 µM, DHA inhibited almost all kinase activity (Fig. 3E). p38 MAPK was also inhibited by DHA, but 500 µM DHA inhibited approximately 50% of the kinase activity (Fig. 3E). To further investigate the mechanism of kinase inhibition by DHA, endogenous IKKß immobilized to protein G was incubated with DHA or AA, and beads were washed to remove AA and DHA prior to the kinase reaction (Fig. 4A). IKKß preincubated with 1 mM DHA was completely inhibited, whereas preincubation with 0.1 mM DHA caused retention of approximately 40% activity. Preincubation with AA resulted in no decrease in IKKß activity (Fig. 4A). These findings suggest that DHA inhibits IKKß by altering its enzymatic properties, likely by binding directly to the kinase and interfering with ATP or substrate binding or catalysis. DHA inhibition was maintained after the beads were washed, indicating that DHA did not bind to ATP or the substrate. Interference with ATP binding was investigated by analyzing whether DHA would inhibit IKKß activity when incubated in the presence of ATP. ATP partially competed the inhibitory activity of DHA (Fig. 4B), implying that DHA could be interfering with ATP binding to IKKß.
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and IKKß (Fig. 7). These processes link oxidative stress to kinase inhibition via the antioxidant, vitamin C.
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DISCUSSION |
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B responses are also known to utilize ROS in signaling, and it has been shown that activation of NF-B is inhibited by overexpression of antioxidant enzymes and antioxidants, such as pyrrolidine dithiocarbamate, N-acetyl-L-cysteine, thioredoxin, glutathione, and vitamin C (4, 6, 9, 11, 23). In this study, we sought to define the role of vitamin C in inhibiting the phosphorylation of IB in a mechanistic manner. The importance of vitamin C as an intracellular antioxidant is well documented and inhibits cell death induced by hydrogen peroxide (12, 37), apoptosis induced by FAS binding to CD95 (27), and DNA damage induced by oxidative stress (22). In addition to these functions, vitamin C is a cofactor for various enzymatic hydroxylation reactions (13, 14). All of these functions of vitamin C, including its role as a cofactor, rely on its ability to donate electrons, becoming oxidized to DHA in the process. DHA functions mainly in the transport of vitamin C (36) but has also been reported to inhibit hexokinase activity (8a).
We discovered that DHA can function as a kinase inhibitor and directly inhibit the activity of IKKß, IKK, and p38 MAPK, expanding our knowledge of how vitamin C modulates ROS-dependent NF-B signaling (4, 6). The initial evidence pointing to vitamin C inhibition of IKKß enzymatic activity was the down-modulation of responses induced by the constitutively active IKKß [IKKß(SS/EE)]. Studies in vitro indicated that the oxidized form of vitamin C inhibited immunopurified IKKß and IKKß(SS/EE). Using radioactively labeled [14C]DHA, we found that DHA binds to IKKß, whereas AA showed neither inhibitory activity nor binding activity to IKKß in vitro.
The inhibitory effect of DHA on IKK and ß enzymatic activities defines two mechanisms for the negative regulation of NF-B responses by vitamin C. First, AA quenches signaling ROS induced by the cytokine-receptor interaction, preventing the activation of ROS-mediated responses, and also quenches ROS that result from signaling. Second, as a consequence of AA oxidation, DHA is generated and inhibits the kinase(s) activated by ROS. This point was experimentally demonstrated in cells loaded with AA and induced to generate DHA by stressing them with H2O2 or DMNQ. These findings suggest an anti-inflammatory role for vitamin C in inhibiting TNF- signal initiation and transduction via IKKß.
The precise mechanism of kinase inhibition by DHA is not certain, but we propose that DHA binds to a pocket near the active site of the kinase. The results of the initial competition experiments with ATP suggested that it could interact with the ATP binding site, but the results of binding assays indicated that an intact ATP site was not required for DHA binding. These apparent conflicting results can be reconciled by the presence of an independent binding site for DHA. Docking simulation and computer modeling indicated that DHA could bind near the catalytic site and not in the ATP binding site (data not shown).
It has been reported that vitamin C inhibits the enzymatic activity of rabbit muscle adenylyl kinase; however, the inhibition was overcome by first incubating AA with DTT (29). These results suggest that AA was converted to DHA and that DHA could be the molecule inhibiting adenylyl kinase. The soluble guanylyl cyclase was also reported to be repressed by AA (31). Cells loaded with vitamin C and under oxidative stress induced a transient cell cycle arrest at the G2/M checkpoint, and it was postulated that DHA induced cell cycle arrest by delaying the activation of cyclin B-cdc2 (3). These results can be rationalized by hypothesizing that DHA may be affecting kinase reaction(s) important in cell cycle control. Our data indicate that DHA can inhibit IKKß, IKK, and p38 MAPK, while T4 polynucleotide kinase was resistant to DHA inhibition (data not shown).
Inhibitors of NF-B activation, such as salicylate and aspirin, play important roles in decreasing inflammation and perhaps oncogenesis. These agents inhibit the kinase activity of IKKß by competing with ATP binding (38). Our findings with vitamin C as an NF-B inhibitor point to a role for the vitamin in inflammation and apoptosis. Vitamin C can inhibit the activation of NF-B by two distinct but related mechanisms: down-regulating ROS induced activation and directly inhibiting IKK and IKKß.
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ACKNOWLEDGMENTS |
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, and pFLAG-CMV2-IKK2(SS/EE) plasmids. This work was supported in part by grants from the National Institutes of Health (CA30388), New York State Department of Health, and Lebensfeld Foundation.
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FOOTNOTES |
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