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Hydrogen Peroxide Activates NF-κB through Tyrosine Phosphorylation of IκBα and Serine Phosphorylation of p65

Hydrogen Peroxide Activates NF-κB through Tyrosine Phosphorylation of IκBα and Serine... THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 278, No. 26, Issue of June 27, pp. 24233–24241, 2003 © 2003 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Hydrogen Peroxide Activates NF-B through Tyrosine Phosphorylation of IB and Serine Phosphorylation of p65 EVIDENCE FOR THE INVOLVEMENT OF IB KINASE AND Syk PROTEIN-TYROSINE KINASE* Received for publication, December 5, 2002, and in revised form, April 21, 2003 Published, JBC Papers in Press, April 23, 2003, DOI 10.1074/jbc.M212389200 Yasunari Takada‡, Asok Mukhopadhyay‡§, Gopal C. Kundu¶, Ganapati H. Mahabeleshwar ¶, Sujay Singh**, and Bharat B. Aggarwal‡ ‡‡ From the ‡Cytokine Research Laboratory, Department of Bioimmunotherapy, The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030, the ¶National Center for Cell Science (NCCS), NCCS Complex, Pune-411 007, India, and Imgenex, San Diego, California 92121 Although it is well established that reactive oxygen Nuclear factor-B (NF-B) is a transcription factor consist- intermediates mediate the NF-B activation induced ing of a group of five proteins, namely c-Rel, RelA (p65), Rel B, by most agents, how H O activates this transcription NF-B1 (p50 and p105), and NF-B2 (p52) (1). In the resting 2 2 factor is not well understood. We found that treatment state, NF-B is sequestered in the cytoplasm through its tight of human myeloid KBM-5 cells with H O activated 2 2 association with specific inhibitory proteins, called inhibitors of NF-B in a dose- and time-dependent manner much as NF-B(IB), belonging to a gene family consisting of IB, tumor necrosis factor (TNF) did but unlike TNF, H O 2 2 IB,IB,IB, Bcl-3, p100, and p105 (1). On activation by had no effect on IB degradation. Unexpectedly, how- agents such as TNF, IB is phosphorylated at serine residues ever, like TNF-induced activation, H O -induced 2 2 32 and 36, ubiquitinated at lysine residues 21 and 22, and NF-B activation was blocked by the calpain inhibitor degraded through the proteosomal pathway, thus exposing the N-Ac-Leu-Leu-norleucinal, suggesting that a proteoso- nuclear localization signals on the p50-p65 heterodimer. Then mal pathway was involved. Although H O activated 2 2 p65 undergoes phosphorylation, leading to nuclear transloca- IB kinase, it did not induce the serine phosphoryla- tion and binding to a specific sequence in DNA, which in turn tion of IB. Like TNF, H O induced the serine phos- 2 2 results in gene transcription. phorylation of the p65 subunit of NF-B, leading to its The phosphorylation of IB is catalyzed by IB kinase nuclear translocation. We found that H O induced the 2 2 (IKK), which consists of IKK-, IKK-, and IKK- (also called tyrosine phosphorylation of IB, which is needed for NF-B essential modulator (NEMO)) (1). Gene deletion studies NF-B activation. We present several lines of evidence have established that IKK- is essential for NF-B activation to suggest that the Syk protein-tyrosine kinase is in- by TNF (2– 4). IKK- deletion, however, has no effect on NF-B volved in H O -induced NF-B activation. First, H O 2 2 2 2 activation by most agents. Which kinase induces the phospho- activated Syk in KBM-5 cells; second, H O failed to 2 2 activate NF-B in cells that do not express Syk protein; rylation of p65 is controversial, but protein kinase A, casein third, overexpression of Syk increased H O -induced kinase II, IKK-, and IKK- have all been implicated (5–10). 2 2 NF-B activation; and fourth, reduction of Syk tran- The phosphorylation of p65 at serine 529 has been shown to be scription using small interfering RNA inhibited H O - required for the TNF-induced transcriptional activity of NF- 2 2 induced NF-B activation. We also showed that Syk B (11). induced the tyrosine phosphorylation of IB, which NF-B is activated by a wide variety of agents, including all caused the dissociation, phosphorylation, and nuclear 18 members of the TNF superfamily, interleukin-1, interleu- translocation of p65. Thus, overall, our results demon- kin-17, interleukin-18, lipopolysaccharide, H O , ceramide, 2 2 strate that H O induces NF-B activation, not through 2 2 phorbol esters, growth factors, UV, X-rays, and -radiation serine phosphorylation or degradation of IB, but (12). Whether all these agents activate NF-B through the through Syk-mediated tyrosine phosphorylation of same pathway as described above is not clear. Certain agents IB. activate NF-B not through serine phosphorylation but through tyrosine phosphorylation of IB: nerve growth factor, erythropoietin, pervanadate, hypoxia, and silica (13–18). The tyrosine phosphorylation of IB by most agents does not lead * This work was supported in part by the Clayton Foundation for to IB degradation. Pervanadate-induced NF-B activation, Research (to B. B. A.), Department of Defense United States Army however, leads to tyrosine phosphorylation and degradation of Breast Cancer Research Program Grant BC010610 (to B. B. A.), Lung IB (19). Surprisingly, UV-C-induced NF-B activation is me- Chemoprevention PO1 Grant CA91844 from the National Institutes of Health (to B. B. A.), and a P50 Head and Neck Specialized Programs of diated through the degradation of IB that involves phospho- Research Excellence (SPORE) grant from the National Institutes of rylation of neither serine nor the tyrosine residue of IB (20). Health (to B. B. A.). The costs of publication of this article were defrayed Pervanadate-induced tyrosine phosphorylation of IB blocks in part by the payment of page charges. This article must therefore be the TNF-induced serine phosphorylation of IB and NF-B hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. § Present address: National Institute of Immunology, New Delhi, India. The abbreviations used are: NF-B, nuclear factor kappa B; IB, ** Supported by NIGMS, National Institutes of Health Grant inhibitory subunit of NF-B; IKK, IB kinase; TNF, tumor necrosis lck R43 GM68358-01. factor; Syk, spleen tyrosine kinase; p56 , lymphocyte-specific protein- ‡‡ To whom correspondence should be addressed. Tel.: 713-792-3503 tyrosine kinase; EMSA, electrophoretic mobility shift assay; ALLN, or 6459; Fax: 713-794-1613; E-mail: [email protected]. N-acetyl-leucyl-leucyl-norleucinal; siRNA, small interfering RNA. This paper is available on line at http://www.jbc.org 24233 This is an Open Access article under the CC BY license. 24234 Mechanism of H O –induced NF-B Activation 2 2 treated cells were incubated with antibodies against either p50 or p65 activation (16, 21), indicating potential stereochemical of NF-B for 30 min at 37 °C before the complex was analyzed by hindrance. EMSA. Antibodies against cyclin D1 and preimmune serum were in- NF-B activation of most agents has been shown to require cluded as negative controls. The dried gels were visualized, and radio- the generation of reactive oxygen intermediates, in studies that active bands quantitated by a PhosphorImager (Amersham Bio- used either reactive oxygen intermediate quenchers, such as sciences) using ImageQuant software. N-acetylcysteine, or antioxidant enzymes, such as glutathione Western Blot Analysis—To determine the levels of protein expression in cytoplasm or nuclear extracts, we prepared each extract (37) from peroxidase, superoxide dismutase, -glutamylcysteine synthe- TNF-treated cells and fractionated them by SDS-PAGE. After electro- tase, and thioredoxin (22–29). Additionally, there are reports phoresis, the proteins were electrotransferred to nitrocellulose mem- that H O activates NF-B (23, 24, 30). Although it has been 2 2 branes, blotted with each antibody, and detected by ECL regent (Am- shown that H O -induced NF-B is blocked by N-acetylcys- 2 2 ersham Biosciences). The density of the bands was measured using a teine (24), how H O activates NF-B is not fully understood 2 2 personal densitometer scan v1.30 and ImageQuant software version 3.3 (31–33). In the present report, we investigated the mechanism (Amersham Biosciences). IB Kinase Assay—The IKK assay was performed by a method of H O -induced NF-B activation. We found that H O -in- 2 2 2 2 described previously (38). Briefly, IKK complex from cytoplasm was duced NF-B activation occurred without degradation of IB. precipitated with antibody against IKK-, followed by treatment with Instead, H O activated Syk protein-tyrosine kinase, which in 2 2 protein A/G-Sepharose beads (Pierce). After a 2-h incubation, the turn induced tyrosine phosphorylation of IB, leading to beads were washed with lysis buffer and then assayed in kinase assay NF-B activation. mixture containing 50 mM HEPES (pH 7.4), 20 mM MgCl ,2mM dithiothreitol, 20 Ci of [- P]ATP, 10 M unlabeled ATP, and 2 g MATERIALS AND METHODS of substrate GST-IB-(1–54). After the immunocomplex was incu- Reagents—Bacteria-derived human rTNF, purified to homogeneity bated at 30 °C for 30 min, the reaction was terminated by boiling with with a specific activity of 5  10 units/mg, was kindly provided by SDS sample buffer for 5 min. Finally, the protein was resolved on 10% Genentech (South San Francisco, CA). Penicillin, streptomycin, SDS-PAGE, the gel was dried, and the radioactive bands were visu- Iscove’s modified Dulbecco’s medium, RPMI 1640 medium, fetal bo- alized by PhosphorImager. To determine the total amounts of IKK- vine serum, and LipofectAMINE 200 were obtained from Invitrogen. and IKK- in each sample, 30 g of the cytoplasmic protein was H O was obtained from Sigma. Antibodies anti-p65, anti-p50, anti- resolved on 7.5% SDS-PAGE, electrotransferred to a nitrocellulose 2 2 IB, anti-cyclin D1, and anti-phosphotyrosine (PY99) were obtained membrane, and then blotted with either anti-IKK- or anti-IKK- from Santa Cruz Biotechnology (Santa Cruz, CA). Phospho-specific antibodies. anti-IB (Ser-32) antibody was purchased from Cell Signaling (Bev- Syk Kinase Assay—To examine the activity of protein-tyrosine ki- erly, MA). Anti-IKK- and anti-IKK- antibodies were kindly pro- nase Syk induced by H O , we immunoprecipitated with anti-Syk an- 2 2 vided by Imgenex (San Diego, CA). Anti-Syk and anti-phosphoty- tibody, and then performed the in vitro kinase assay using the GST- rosine (4G10) antibodies were purchased from Neo markers IB-(1–54) as the substrate. Briefly, cells were pretreated with 100 (Fremont, CA). Antibody that recognizes the serine 529-phosphoryl- g/ml ALLN (to prevent proteolytic degradation; Ref. 39) for 1 h, then ated form of p65 was obtained from Rockland Laboratories (Gilberts- stimulated with H O for 5 min and whole cell extracts were prepared 2 2 ville, PA). Wild type Syk-cDNA as described previously (34), was in the lysis buffer (1% Triton X-100, 1 mM phenylmethanesulfonyl kindly provided by Dr. Susette C. Mueller (Georgetown University fluoride, 20 g/ml leupeptin, 5 mM sodium orthovanadate, and 2 mM Medical School, Washington, D. C.). EDTA). The kinase protein was immunoprecipitated using anti-Syk Generation of siRNA Plasmid Vector—IMG-800 (pSuppressorNeo, antibody, followed by protein A/G-Sepharose beads. After 2 h, the beads Imgenex, CA) vector was used for construction of 21-bp head-to-head were washed with the lysis buffer and assayed in a kinase assay TM hairpins of human Syk (GenBank accession number NM 003177.2, mixture containing 50 mM HEPES (pH 7.4), 50 mM MgCl ,5mM sodium bp 153–173, 555–575, or 1443–1463). For each construction, two orthovanadate, 10 M unlabeled ATP, and 5 g of substrate GST-IB- complementary oligonucleotides containing human Syk sequences (1–54)) and [- P]ATP. After 30 min incubation at 30 °C, the samples were synthesized (MWG-Biotech, High point, NC) and annealed to were boiled with SDS sample buffer for 5 min, then subjected to poly- generate double stranded DNAs, which were cloned into the SalI and acrylamide gel electrophoresis under denaturing conditions (10% SDS- XbaI cloning sites of IMG-800. The sequence (bp 555–575) used was PAGE), and the dried gels were visualized, and radioactive bands TCGAGCAGACATGGAA CCTGCAGGGGAGTACTGCCCTGCAG- quantitated by a PhosphorImager using ImageQuant software. The GTTCCATGTCTGCTTTTT (sequences are in bold letter, stem loop tyrosine phosphorylation of IB by Syk was also determined by using sequences are in italics, and SalI cloning overhang site is unlabeled ATP in the kinase reaction mixture as described above, and underlined). then performing Western blot analysis using anti-phosphotyrosine Cell Lines—The leukemic cell line KBM-5 is phenotypically myeloid antibody. with monocytic differentiation. The cell lines Jurkat (human T cells), Nuclear Localization of p65 NF-B by Immunocytochemistry—The lck syk JCaM1 (p56 and p72 deficient), human breast cancer cells MCF-7, effect of H O on the nuclear translocation of p65 was examined by 2 2 human lung cancer cells H1299 and mouse macrophage RAW 264.7 the immunocytochemical method as described (40). Briefly, treated cells were obtained from the American Tissue and Cell Culture Collec- cells were plated on a poly-L-lysine-coated glass slide by centrifuga- lck tion (ATCC, Rockville, MD). JCaM1 cells transfected with the p56 tion using a cytospin 4 (Thermoshendon, Pittsburg, PA), air-dried, gene were kindly supplied by Dr. Arthur Weiss (The University of fixed with cold acetone, and permeabilized with 0.2% of Triton X-100. California, San Francisco, CA). The characterization of these cells has After being washed in phosphate-buffered saline, slides were blocked been previously reported (35). KBM-5 cells were cultured in Iscove’s with 5% normal goat serum for 1 h and then incubated with rabbit modified Dulbecco’s medium supplemented with 15% fetal bovine se- polyclonal anti-human p65 or IB antibodies at 1:100 dilutions. rum; Jurkat and JCaM1 cells were cultured in RPMI 1640 medium with After overnight incubation at 4 °C, the slides were washed, incubated 10% fetal bovine serum, 100 units/ml penicillin, and 100 g/ml with goat anti-rabbit IgG-Alexa 594 (Molecular Probes, Eugene, OR) streptomycin. at 1:100 dilutions for 1 h, and counterstained for nuclei with Hoechst Electrophoretic Mobility Shift Assays (EMSA)—To determine NF-B 33342 (50 ng/ml) for 5 min. Stained slides were mounted with mount- activation, we performed EMSA as described (36). Briefly, nuclear ing medium purchased from Sigma and analyzed under a fluores- extracts prepared from TNF-treated cells (2  10 /ml) were incubated cence microscope (Labophot-2, Nikon, Tokyo, Japan). Pictures were with P-end-labeled 45-mer double-stranded NF-B oligonucleotide (10 captured using Photometrics Coolsnap CF color camera (Nikon, g of protein with 16 fmol of DNA) from the human immunodeficiency Lewisville, TX) and MetaMorph version 4.6.5 software (Universal virus long terminal repeat, 5-TTGTTACAAGGGACTTTCCGCTGGG- Imaging, Downingtown, PA). GACTTTCCAGGGAGGCGTGG-3 (boldface indicates NF-B binding Immunoprecipitation Assays—Cells were lysed for 30 min on ice in sites) for 30 min at 37 °C, and the DNA-protein complex formed was whole cell extraction buffer (20 mM HEPES, pH 7.9, 50 mM NaCl, 1% separated from free oligonucleotide on 6.6% native polyacrylamide gels. Nonidet P-40, 2 mM EDTA, 0.5 mM EGTA, 2 g/ml aprotinin, 2 g/ml A double-stranded mutated oligonucleotide, 5-TTGTTACAACT- leupeptin, 0.5 mM phenylmethanesulfonyl fluoride, and 2 mM sodium CACTTTCCGCTGCTCACTTTCCAGGGAGGCGTGG-3, was used to orthovanadate). Lysate containing 500 g of proteins in extraction examine the specificity of binding of NF-B to the DNA. The specificity buffer was incubated with 1 g/ml concentration of antibodies over- of binding was also examined by competition with the unlabeled oligo- night. Immunocomplex was precipitated using protein A/G-Sepharose nucleotide. For supershift assays, nuclear extracts prepared from TNF- beads for1hat4 °C. Beads were washed with extraction buffer and Mechanism of H O –induced NF-B Activation 24235 2 2 resuspended in SDS sample buffer, boiled for 5 min, and fractionated in SDS-PAGE. Syk-siRNA and Syk-cDNA Transfection—To determine the role of Syk protein-tyrosine kinase in the H O -induced NF-B activation, 2 2 2 10 cells were transfected either with small interfering RNA-Syk (siRNA-Syk), or with wild type-Syk cDNA (Syk-WT). Two g of plasmid in each case was diluted in 200 l of Dulbecco’s modified Eagle’s medium (without serum and antibiotics), and then mixed with 200 lof Dulbecco’s modified Eagle’s medium containing 4 l of LipofectAMINE 2000. This mixture was added to the cells and incubated for 4 h. After incubation, medium was changed into RPMI 1640 and maintained for an additional 48 h. These cells were then used to examine the expres- sion of Syk protein by Western blot analysis, NF-B activity by EMSA, and Syk activity by the kinase assay as described above. RESULTS In this report we investigated the effect of H O on NF-B 2 2 activation, IB phosphorylation, IB degradation, p65 phos- phorylation, and nuclear translocation, and the role of protein- tyrosine kinase Syk in H O -induced NF-B activation. Be- 2 2 cause NF-B activation by TNF is well understood, we used TNF as a control for most studies. As determined by trypan blue dye exclusion and Hoechst staining methods, the treat- ment of cells with 500 M H O for 2 h had no significant effect 2 2 on cell viability (viability was greater than 98%). Both H O and TNF Activate DNA Binding Activity of NF-B 2 2 in a Dose- and Time-dependent Manner—To determine the effect of H O on the activation of NF-B, KBM-5 cells were 2 2 treated with different concentrations of H O for 120 min or 2 2 TNF for 30 min. Nuclear extracts were prepared for analysis of NF-B activation by EMSA. Both H O and TNF induced 2 2 NF-B activation in a dose-dependent manner in KBM-5 cells (Fig. 1A); maximum activation with H O occurred at 500 M. 2 2 NF-B was activated by both agents in a time-dependent man- ner (Fig. 1B). TNF induced NF-B activation within 15 min, and activation continued for 240 min, whereas, H O -induced 2 2 NF-B activation started at 60 min, reached maximum at 120 min, and then decreased at 240 min. The maximum TNF- induced NF-B activation was 14-fold; with H O it was 3–7- 2 2 FIG.1. A and B, dose response (panel A) and time course (panel B)of fold. Thus, H O induced NF-B activation more slowly than 2 2 activation of NF-B by TNF (left panel) and H O (right panel) in 2 2 did TNF. The slower kinetics and difference in the optimum KBM-5 cells. A, KBM-5 cells (2  10 /ml) were treated with different concentrations of TNF for 30 min or H O for 120 min. After these level of activation suggest that the mechanism of NF-B acti- 2 2 treatments, nuclear extracts were prepared and then assayed for vation by H O is different from that of TNF. 2 2 NF-B activation by EMSA, as described under “Materials and Meth- Because NF-B is a complex of proteins, various combina- ods.” B, time-dependent activation of NF-BbyH O . Cells (2  10 /ml) 2 2 tions of Rel/NF-B protein can constitute an active NF-B were treated with 0.1 nM TNF or 500 M H O for the indicated times. 2 2 heterodimer that binds to a specific sequence in DNA (1). To After treatment, nuclear extracts were prepared and then assayed for NF-B activation by EMSA. C, NF-B induced by H O is composed of show that the retarded band visualized by EMSA in H O - 2 2 2 2 p65 and p50 subunits. Nuclear extracts were prepared from untreated treated cells was indeed NF-B, we incubated nuclear extracts or 500 M H O -treated cells (2  10 /ml), incubated for 30 min with 2 2 from H O -stimulated cells with antibodies to either the p50 2 2 different antibodies or unlabeled NF-B oligo probes, and then assayed (NF-B1) or the p65 (RelA) subunit of NF-B. Both shifted the for NF-B activation by EMSA. band to a higher molecular mass (Fig. 1C), thus suggesting that the H O -activated complex consisted of p50 and p65 subunits. 2 2 Neither preimmune serum nor anti-cyclin D1 antibody had any IB (for references see Ref. 1). So we investigated whether effect. Excess unlabeled NF-B (100-fold) caused complete dis- H O induces serine phosphorylation of IB. To stabilize the 2 2 appearance of the band, and a mutant oligonucleotide of NF-B phosphorylated IB, we blocked degradation of IB using did not affect NF-B binding activity. the proteosome inhibitor ALLN (39). Western blot analysis H O Induces NF-B Activation without Degrading IB— using phospho-specific anti-IB antibody (Fig. 2B) showed 2 2 TNF-induced NF-B activation requires the degradation of that TNF induced the phosphorylation of IB but H O had no 2 2 IB (for references see Ref. 1). Whether H O -induced NF-B effect on the serine phosphorylation of IB. 2 2 activation is also mediated through IB degradation was in- ALLN Inhibits NF-B Activation Induced by Both TNF and vestigated. To determine this, cells were treated with 0.1 nM H O —ALLN blocked not only TNF-induced NF-B activation, 2 2 TNF or 500 M H O for the indicated times, extracted the but also H O -induced NF-B activation (Fig. 2C). Thus IB 2 2 2 2 cytoplasmic protein, and analyzed for IB on 10% SDS-PAGE phosphorylation and degradation are critical in TNF-induced using anti-IB antibody. TNF induced IB degradation NF-B activation. within 15 min after treatment, and IB was resynthesized at H O Induces Translocation and Phosphorylation of 2 2 60 min (Fig. 2A). However, H O did not induce IB degrada- p65—We analyzed the effect of TNF and H O on translocation 2 2 2 2 tion at any time points. and phosphorylation of p65 by Western blot analysis. Both TNF TNF Induces IB Serine Phosphorylation but H O Does and H O induced nuclear translocation of p65 in a time-de- 2 2 2 2 Not—TNF induces phosphorylation of serines 32 and 36 of pendent manner. On TNF stimulation, p65 nuclear transloca- 24236 Mechanism of H O –induced NF-B Activation 2 2 These results suggest that H O activated IKK but had no 2 2 effect on the serine phosphorylation of IB. H O Does Not Activate NF-B in Syk-deficient Jurkat 2 2 Cells—To investigate further the role of Syk in H O -induced 2 2 NF-B activation, we used JCaM1 cells known to lack both Lck (35) and Syk protein expression (41). Western blot analysis revealed that Jurkat cells expressed Syk protein but JCaM1 cells expressed very little or no Syk (Fig. 5A). The anti-Syk antibody was specific as it did not recognize Lck protein ex- lck pression in p56 -reconstituted JCaM1 cells (Fig. 5A). TNF activated NF-B in both Jurkat and JCaM1 cells and induced IB degradation in both the cell lines (Fig. 5B). The kinetics of TNF-induced NF-B activation, however, was slightly slower in JCaM1 cells than in Jurkat cells (15 versus 30 min) and the overall magnitude of activation was also lower (7.1- versus 5.1-fold). In contrast, H O activated NF-B in Jurkat cells but 2 2 not in JCaM1 cells (Fig. 5C), indicating an essential role of Syk protein expression in H O -induced activation. 2 2 We also investigated the ability of H O to activate NF-Bin 2 2 human lung epithelial (H1299) and breast epithelial (MCF-7) cells, which cannot be activated for Syk (Fig. 5D, lower panel). H1299 and MCF-7 cells were treated with H O for the indi- 2 2 cated times, nuclear extracts were prepared and analyzed for NF-B activation by EMSA. Whole cell extracts were analyzed for Syk activation. We found that H O failed to activate Syk 2 2 and this correlated with the lack of activation of NF-Bin H1299 and MCF-7 cells. FIG.2. A, TNF-induces IB degradation but H O does not. KBM-5 2 2 Whether H O can activate NF-B in murine cells was ex- cells (2  10 cells/ml) were treated with 0.1 nM TNF or 500 M H O for 2 2 2 2 the indicated times, and then cytoplasmic extracts were prepared as amined. For this murine macrophage RAW264.7 cells were described under “Materials and Methods.” Cytoplasmic extracts were treated for the indicated times with 500 M H O and then 2 2 fractionated on 10% SDS-PAGE and electrotransferred to nitrocellulose examined for NF-B activation by EMSA and IB degradation membrane. Western blot analysis was used with anti-IB antibody. by Western blot analysis. As shown in Fig. 5E,H O induced For loading control, we used anti--actin antibody. B, TNF-induces 2 2 IB phosphorylation but H O does not. KBM-5 cells (2  10 cells/ml) NF-B activation in murine cells and this was accompanied 2 2 were preincubated with 100 g/ml proteosome inhibitor, ALLN (39), for with the degradation and resynthesis of IB. These results 1 h, and then cells were treated with either 0.1 nM TNF for 15 min or agree with a previous report (32) but differ from that noted in 500 M H O for 60 min. Cytoplasmic extracts were fractionated on 10% 2 2 human cells. SDS-PAGE, and then Western blot analysis was performed with phos- phospecific anti-IB antibody. C, ALLN inhibits NF-B activation H O Does Not Activate NF-B in Lck-reconstituted JCaM1 2 2 induced by both TNF and H O . KBM-5 cells (2  10 cells/ml) were lck 2 2 Cells—JCaM1 cells have been shown to lack both p56 and incubated with 100 g/ml ALLN for 1 h, and then treated with 0.1 nM Syk protein-tyrosine kinases (35). Previously we have shown TNF or 500 M H O for the indicated periods. Nuclear extracts were 2 2 lck that p56 is required for ceramide-induced and HIV-tat-in- analyzed for NF-B activation by EMSA. duced NF-B activation (42, 43). EMSA of JCaM1 cells whose lck p56 expression had been reconstituted revealed that H O 2 2 tion reached maximum 15 min after TNF treatment and grad- activated NF-B in Jurkat cells but not in JCaM1 cells whose ually declined thereafter. In the case of H O , p65 translocation 2 2 lck p56 expression had been reconstituted (Fig. 5F). No IB was slightly induced after 15 min treatment, peaked at 60 min, phosphorylation was noted in either of the cell lines (see lower and diminished thereafter (Fig. 3A). lck panel). In other words, p56 was not required for H O -in- 2 2 Both TNF and H O induced the phosphorylation of p65 in a 2 2 duced NF-B activation or for IB phosphorylation. time-dependent manner, but the kinetics of H O -induced 2 2 H O Induces Tyrosine Phosphorylation of Syk Protein in phosphorylation of p65 was slower than that for TNF (Fig. 3B). 2 2 Jurkat Cells but Not in JCaM1 Cells—Jurkat and Syk-defi- Immunocytochemistry assay showed that while in untreated cient JCaM1 cells treated with H O for the indicated times cells, p65 was localized primarily in the cytoplasm, TNF and 2 2 were immunoprecipitated with anti-Syk antibody and then H O induced translocation of p65 into the nucleus (Fig. 3C). 2 2 subjected to Western blot analysis using anti-phosphoty- However, H O -induced phosphorylation and translocation of 2 2 rosine antibody (4G10). H O -induced tyrosine phosphoryla- p65 to the nucleus were delayed compared with TNF- 2 2 tion of Syk in Jurkat, but not in Syk-deficient JCaM1 cells induced response. (Fig. 6A). H O Induces IB Kinase Activation—Because our results 2 2 H O Induces Tyrosine Phosphorylation of IB Protein in indicated that H O -induced NF-B activation is not mediated 2 2 2 2 Jurkat Cells—Previously our laboratory and others have through the phosphorylation and degradation of IB, we next shown that certain agents can activate NF-B through tyrosine explored whether H O can activate IKK. It has been shown 2 2 phosphorylation even though it does not lead to the degrada- that IKK is required not only for TNF-induced phosphorylation tion of IB (13–18). Because H O likewise did not induce of IB but also for the phosphorylation of p65 (9, 10). An in 2 2 IB degradation, we next determined whether H O induces vitro immune complex kinase assay using GST-IB-(1–54) as 2 2 the substrate showed that both TNF and H O activated IKK tyrosine phosphorylation of IB. Jurkat cells treated with 2 2 H O exhibited tyrosine phosphorylation of IB (Fig. 6B) (the as early as 5 min after TNF treatment, but then activation 2 2 ceased (Fig. 4). H O -induced IKK activation was, however, tyrosine-phosphorylated molecule is the slower migrating spe- 2 2 cies of IB). Pretreatment of cells with ALLN significantly weaker than that induced by TNF. Neither TNF nor H O had 2 2 any effect on the expression of either IKK- or IKK- proteins. enhanced tyrosine phosphorylation of IB (Fig. 6C). These Mechanism of H O –induced NF-B Activation 24237 2 2 FIG.3. A, both TNF and H O induce 2 2 the translocation of p65. KBM-5 cells (2 10 cells/ml) were treated with 0.1 nM TNF or 500 M H O for the indicated 2 2 times. Nuclear extracts were prepared and fractionated on SDS-PAGE, and then Western blot analysis was performed us- ing anti-p65 antibody. B, both TNF and H O induce the phosphorylation of p65. 2 2 KBM-5 cells (2  10 cells/ml) were treated with 0.1 nM TNF or 500 M H O 2 2 for the indicated times. Nuclear extracts were prepared and fractionated on SDS- PAGE, and then Western blot analysis was performed using anti-p65 antibody. C, both TNF and H O induce the nuclear 2 2 translocation of p65. KBM-5 cells were treated with 1 nM TNF for 30 min or 500 M H O for 120 min. Cells were sub- 2 2 jected immunocytochemistry as described under “Materials and Methods.” sufficient for the release of p65 was explored. Jurkat cells were treated with TNF or with H O and analyzed for IB by 2 2 Western blot analysis. TNF induced the degradation of IB in the cytosol (Fig. 7A, left, middle panel) and the translocation of p65 to the nucleus (right, bottom panel). TNF also induced the dissociation of p65 from IB (right, top panel). In the H O - 2 2 treated cells, p65 was translocated into the nucleus (right, FIG.4. Both TNF and H O activate IKK. Cells (2  10 cells/ml) bottom panel) without degradation of IB (left, middle panel). 2 2 were activated with TNF (0.1 nM)orH O (500 M) for the indicated 2 2 We were surprised to see that H O did not induce dissociation 2 2 time intervals. Cytoplasmic extracts were prepared, and 300 gof of p65 from IB (left, top panel). H O induced a slow migrat- 2 2 cytoplasmic extracts were immunoprecipitated with antibody against ing species of IB (left, top panel, and left, middle panel). No IKK-. Thereafter immune complex kinase assay was performed as described under “Materials and Methods.” To examine the effect of IB could be detected in nucleus, neither free (right, middle H O on the level of expression of IKK proteins, 30 g of cytoplasmic 2 2 panel) nor p65 bound (right, top panel). An immunocytochem- extracts were fractionated on 7.5% SDS-PAGE. Western blot analysis istry assay showed that both p65 and IB were localized in the was performed using with anti-IKK- and anti-IKK- antibodies. cytosol of untreated cells (Fig. 7B). The cells treated with TNF lacked IB in the cytosol, and p65 was translocated to the nucleus in these cells. Although H O induced p65 transloca- 2 2 tion into the nucleus, IB remained in the cytosol (Fig. 7B). results suggest that H O induced tyrosine phosphorylation of 2 2 These results suggest that H O induced translocation of p65 IB by activating of Syk tyrosine kinase. 2 2 without degrading IBa. H O -activated Syk Induces Phosphorylation of IB—To 2 2 Overexpression of Syk Enhances H O -induced NF-B Acti- investigate the mechanism of Syk-mediated phosphorylation of 2 2 vation and Tyrosine Phosphorylation of IB—To further con- IB, we performed the in vitro kinase assay using GST-IB- firm the role of Syk protein-tyrosine kinase in H O -induced (1–54) as the substrate. We found that H O -activated Syk 2 2 2 2 could directly phosphorylate IB, and this phosphorylation NF-B activation, we transiently transfected the Jurkat cells with the wild type Syk cDNA-containing expression plasmid. was enhanced by ALLN treatment (Fig. 6D). To further confirm the Syk-mediated tyrosine phosphorylation of IB, we per- As shown in Fig. 8, these cells showed an increase in expression of Syk protein (panel A), enhanced H O -induced NF-B acti- formed the in vitro kinase assay using GST-IB-(1–54) as the 2 2 substrate and unlabeled ATP. The tyrosine-phosphorylated vation (panel B), and an increase in the IB tyrosine phos- phorylation (panel C), over the non-transfected control. Thus IB was detected by Western blot analysis using anti-phos- photyrosine and anti-IB antibodies (Fig. 6E). These results these results suggest that Syk protein-tyrosine kinase mediate H O -induced NF-B through the tyrosine phosphorylation suggest that Syk protein-tyrosine kinase can directly phospho- 2 2 rylate IB on tyrosine residues. of IB. Reduction of Syk Protein by siRNA-Syk Inhibits H O - H O Induces Dissociation of IB from p65 without Degrad- 2 2 2 2 ing IB—Whereas serine phosphorylation leads to the degra- induced NF-B Activation and Tyrosine Phosphorylation of dation of IB, tyrosine phosphorylation does not (13–18). IB—We also used siRNA to suppress Syk expression (44). Whether H O -induced tyrosine phosphorylation of IB was For this, we transiently transfected siRNA-Syk into Jurkat 2 2 24238 Mechanism of H O –induced NF-B Activation 2 2 lck lck FIG.5. A, Jurkat cells express Syk but JCaM1 or p56 -reconstituted cells do not. Jurkat, JCaM1, and JCaM1/p56  cells were lysed, and 30 g of samples of each were fractionated on 7.5% SDS-PAGE. Western blot analysis was performed using anti-Syk antibody. B, TNF activates NF-B and induces IB degradation in both Jurkat- and Syk-deficient JCaM1 cells. Jurkat and JCaM1 cells were treated with 0.1 nM TNF for the indicated times. Nuclear and cytoplasmic extracts were prepared. Nuclear extracts were analyzed for NF-B activation by EMSA. Cytoplasmic extracts were analyzed for IB expression by Western blot analysis using anti-IB antibody. C,H O activates NF-B in Jurkat and but not in 2 2 Syk-deficient JCaM1 cells. Jurkat and JCaM1 cells were treated with 500 M H O for the indicated times. Nuclear and cytoplasmic extracts were 2 2 prepared. Nuclear extracts were analyzed for NF-B activation by EMSA. Cytoplasmic extracts were analyzed for IB expression by Western blot analysis using anti-IB antibody. D,H O does not activate NF-B in human lung epithelial (H1299) and breast epithelial (MCF-7) cells. H1299 2 2 and MCF-7 cells were treated with 500 M H O for the indicated times. Nuclear extracts were prepared and analyzed for NF-B activation by 2 2 EMSA. Whole cell extracts were analyzed for Syk expression. E,H O activates NF-B in murine macrophages RAW 264.7 cells. Cells were treated 2 2 with 500 M H O for the indicated times. Nuclear and cytoplasmic extracts were prepared. Nuclear extracts were analyzed for NF-B activation 2 2 by EMSA. Cytoplasmic extracts were analyzed for IB expression by Western blot analysis using anti-IB antibody. F,H O does not activate 2 2 lck NF-B in JCaM1 cells reconstituted with p56 . Jurkat and Lck-reconstituted JCaM1 cells were treated with H O for the indicated times. Nuclear 2 2 extracts were analyzed for NF-B activation by EMSA. Cytoplasmic extracts were analyzed for IB expression and for phosphorylated IB by Western blot analysis using anti-IB and phospho-specific IB antibodies. cells, and then examined H O -induced NF-B activation and again suggest that Syk plays an important role in NF-B 2 2 IB tyrosine phosphorylation. As shown in Fig. 9, siRNA- activation induced by H O . 2 2 Syk suppressed the expression of Syk protein (panel A), di- DISCUSSION minished H O -induced NF-B activation (panel B), and sup- 2 2 pressed tyrosine phosphorylation of IB (panel C)as By using metabolic inhibitors and antioxidant enzymes, it compared with the nontransfected control. These results has been well established that oxygen radicals are involved in Mechanism of H O –induced NF-B Activation 24239 2 2 FIG.7. A, colocalization of p65 and IB complex in the cytoplasm. Jurkat cells were treated with either 1 nM TNF for 15 min or 500 M H O for 60 min, and then nuclear and cytoplasmic extracts were 2 2 FIG.6. A,H O activates Syk in Jurkat cells but not in JCaM1 cells. 2 2 prepared. Each extract was incubated with anti-p65 antibody over- Jurkat and JCaM1 cells were treated with 500 M H O for the indi- 2 2 night, precipitated with protein A/G-Sepharose beads, and then frac- cated times. Whole cell extracts were prepared, and 500 g of sample tionated on 10% SDS-PAGE. Western blot analysis was performed was incubated with anti-Syk antibody overnight. Immunocomplex was using anti-IB antibody. Nuclear and cytoplasmic extracts were ana- precipitated using protein A/G-Sepharose beads and then fractionated lyzed for IB and p65 expression by SDS-PAGE following Western blot on 7.5% SDS-PAGE. Western blot analysis was performed using anti- analysis using anti-IB and anti-p65 antibodies. B, effect of H O on 2 2 phosphotyrosine antibody (4G10). B,H O induces tyrosine phospho- 2 2 the immunolocalization of p65 and IB. Cells were treated with either rylation of IB in Jurkat cells. Jurkat cells were treated with 500 M 1nM TNF for 30 min or 500 M H O for 120 min. Cells were then 2 2 H O for the indicated times. Whole cell extracts were prepared, and 2 2 processed for immunocytochemistry using anti-p65 and anti-IB an- 500 g of sample was incubated with anti-IB antibody overnight. tibodies, as described under “Materials and Methods.” Immunocomplex was precipitated with protein A/G-Sepharose beads, and then fractionated on 10% SDS-PAGE. Western blot analysis was performed using anti-phosphotyrosine antibody (PY99). C, ALLN en- tion. Rather, it induced tyrosine phosphorylation of IB.H O 2 2 hances the H O -induced tyrosine phosphorylation of IB. Jurkat cells 2 2 activated both IKK- and Syk kinase, and this activation of were preincubated with 100 g/ml ALLN for 1 h and then treated with Syk kinase proved to be critical for tyrosine phosphorylation of 500 M H O for the indicated times. Whole cell extracts were prepared, 2 2 and 500 g of sample was incubated with anti-IB antibody overnight. IB and for NF-B activation. Immunocomplex was precipitated with protein A/G-Sepharose beads, Both TNF and H O activated NF-B, but the kinetics of 2 2 and then fractionated on 10% SDS-PAGE. Western blot analysis was NF-B activation was faster with TNF than that with H O . 2 2 performed using anti-phosphotyrosine antibody (PY99). D,H O -acti- 2 2 These results in themselves suggested that the mechanism of vated Syk induces phosphorylation of IB. Jurkat cells were treated NF-B activation by TNF are different from that of H O . with 500 M H O for 5 min in the presence or absence of ALLN, 2 2 2 2 prepared whole cell extracts, immunoprecipitated with anti-Syk anti- Although reactive oxygen intermediates has been implicated in body, and then performed kinase reaction using GST-IB-(1–54) as TNF-induced NF-B activation (45, 46), this alone may not be the substrate as described under “Materials and Methods.” Syk protein sufficient for NF-B activation. The difference in kinetics may levels were examined by Western blot analysis using anti-Syk antibody. also be related to the receptor-mediated nature of TNF-induced E,H O -activated Syk induces tyrosine phosphorylation of IB. Jur- 2 2 kat cells were treated with 500 M H O for 5 min in the presence or activation whereas H O activity is receptor-independent. 2 2 2 2 absence of ALLN, prepared whole cell extracts, immunoprecipitated We have in fact demonstrated that the mechanism of NF-B with anti-Syk antibody, and then performed kinase reaction as de- activation by H O is significantly different from that of TNF. 2 2 scribed under “Materials and Methods.” Samples were analyzed for For instance, TNF induced IB degradation but H O did not. tyrosine phosphorylation of I B by Western blot analysis using anti- 2 2 phosphotyrosine (4G10) and anti-IB antibodies. For positive control, Other agents are also known to activate NF-B without IB cells were treated with pervanadate (pV) for 60 min. degradation, including pervanadate, hypoxia, erythropoietin, and nerve growth factor (13–18). Our results agree with Dudek et al. (47) and Milligan et al. (48) but differ from Schoonbroodt NF-B activation by most agents. Additionally, that H O et al. (32), who showed that H O induces IB degradation in 2 2 2 2 alone can activate NF-B has been known for almost a decade mouse lymphoma EL4 cells. Whether these differences are but the mechanism has remained unclear. We demonstrate in related to cell type is not clear. We used various T cell lines and the present report that H O activates NF-B without the myeloid cells and found similar results. Our results also sug- 2 2 degrading IB and without inducing its serine phosphoryla- gest that, unlike TNF, H O does not induce serine phospho- 2 2 24240 Mechanism of H O –induced NF-B Activation 2 2 FIG.8. Overexpression of Syk enhances H O -induced NF-B 2 2 activation and IB phosphorylation. A, Jurkat cells were trans- fected with wild type-Syk cDNA (Syk-WT), prepared whole cell extracts and then subjected to SDS-PAGE for protein expression using anti-Syk antibody. B, Syk-WT-transfected cells were treated with 250 M H O FIG.9. Reduction of Syk protein by siRNA-Syk inhibits H O - 2 2 2 2 for the indicated times, and then analyzed for NF-B activation. C,to induced NF-B activation and IB phosphorylation. A, Jurkat determine the effect of overexpressed Syk on IB, cells were treated cells were transfected with siRNA-Syk, prepared whole cell extracts, with 500 M H O for 5 min, prepared whole cell extracts, immunopre- and then subjected to SDS-PAGE for protein expression using anti-Syk 2 2 cipitated with anti-IB antibody, and then analyzed by Western blot antibody. B, siRNA-Syk-transfected cells were treated with 250 M analysis using anti-phosphotyrosine antibody (PY99). H O for the indicated times, and then analyzed for NF-B activation. 2 2 C, to determine the effect of reduced Syk on IB, siRNA-Syk-trans- fected cells were treated with 500 M H O for 5 min, prepared whole rylation of IB. Schoonbroodt et al. (32) also showed that 2 2 cell extracts, immunoprecipitated with anti-IB antibody, and then phosphorylation of serine 32 and 36 of IB is not required for analyzed by Western blot analysis using anti-phosphotyrosine antibody NF-B activation by H O . 2 2 (PY99). As was the case for TNF, H O -induced NF-B activation 2 2 was completely suppressed by ALLN, a proteosomal inhibitor. How ALLN inhibits H O -induced NF-B activation is not Our results indicate that H O induces tyrosine phosphoryl- 2 2 2 2 clear. The proteosome has been implicated in the degradation ation of IB. These results are in agreement with reports by of IB and in the processing of p105 (NF-B1) and p100 Livolsi et al. (31) and Schoonbroodt et al. (32). What kinase (NF-B2) proteins. It is unlikely that ALLN inhibits H O - induces the tyrosine phosphorylation of IB, however, is not 2 2 induced NF-B activation through the inhibition of degrada- clear. Our results provide evidence that Syk protein kinase tion of IB or processing of NF-B proteins. The proteosome may play a major role in this phosphorylation. First, H O 2 2 inhibitors MG132 and lactacystin have also been shown to activated Syk in KBM-5 cells; second H O failed to activate 2 2 suppress H O -induced NF-B activation without affecting NF-B in cells that do not express Syk protein; third, overex- 2 2 IB,-,or- in lens epithelial cells (49). Our results suggest pression of Syk increased H O -induced NF-B activation; and 2 2 the role of another proteosomal-sensitive factor in H O -in- fourth, reduction of Syk transcription using siRNA inhibited 2 2 duced NF-B activation. H O -induced NF-B activation. We also showed that Syk in- 2 2 Our results also demonstrate that even though H O did not duced the tyrosine phosphorylation of IB, which caused the 2 2 induce serine phosphorylation of IB, it did activate IKK, dissociation, phosphorylation, and nuclear translocation of p65. phosphorylated p65 at serine residue 529, and translocated p65 Howe et al. (50) showed that H O -induced phosphorylation of 2 2 to the nucleus. Recently it was demonstrated that IKK can IB through calcium/calmodulin-dependent kinases in Jurkat phosphorylate p65 (9, 10). Therefore, it is possible that H O - cells. H O has been shown to activate Syk (51), a kinase 2 2 2 2 induced IKK is needed not for the phosphorylation of IB but known to be expressed in thymocytes, B lymphocytes, mast for the phosphorylation of p65. Our results are in agreement cells, monocytes, macrophages, neutrophils and platelets, and with a recent report by Kamata et al. (33), who showed that mature T lymphocytes. We found that cells that do not express H O can activate IKK and this activation is essential for Syk, such as JCaM1 (a genetic isolate of Jurkat cells), failed to 2 2 NF-B activation. How H O -induced IKK activates NF-B, activate NF-B on treatment with H O . Lakshminarayanan et 2 2 2 2 however, was not investigated by these workers. Our results al. (52) showed that in human microvessel endothelial cells or differ from Schoonbroodt et al. (32), who showed lack of activa- human lung epithelial A549 cells, TNF could activate NF-B tion of IKK by H O . but H O could not. The inability of H O to activate NF-Bin 2 2 2 2 2 2 Mechanism of H O –induced NF-B Activation 24241 2 2 19. Mukhopadhyay, A., Manna, S. K., and Aggarwal, B. B. (2000) J. Biol. Chem. these cells could have been because of a lack of expression of 275, 8549 – 8555 Syk kinase in these cells. Previously it has been shown that 20. Li, N., and Karin, M. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 13012–13017 lck H O can activate p56 (53). Although Lck has been impli- 21. Singh, S., and Aggarwal, B. B. (1995) J. Biol. Chem. 270, 10631–10639 2 2 22. Staal, F. J., Roederer, M., Herzenberg, L. A., and Herzenberg, L. A. (1990) cated in the NF-B activation by ceramide and by HIV-tat (42, Proc. Natl. Acad. Sci. U. S. A. 87, 9943–9947 43), our results indicate that H O does not induce NF-B 23. Kretz-Remy, C., Mehlen, P., Mirault, M. E., and Arrigo, A. P. (1996) J. Cell 2 2 lck lck Biol. 133, 1083–1093 activation through activation of p53 , as p53 -reconstituted 24. Schreck, R., Rieber, P., and Baeuerle, P. A. (1991) EMBO J. 10, 2247–2258 JCaM1 cells did not respond to H O for NF- B activation. 2 2 25. Giri, D. K., and Aggarwal, B. B. (1998) J. Biol. Chem. 273, 14008 –14014 We found that in TNF-treated cells, almost all the cytoplas- 26. Manna, S. K., Zhang, H. J., Yan, T., Oberley, L. W., and Aggarwal, B. B. (1998) J. Biol. Chem. 273, 13245–13254 mic pool of IB was degraded and the p65 was translocated to 27. Manna, S. K., Kuo, M. T., and Aggarwal, B. B. (1999) Oncogene 18, 4371– 4382 the nucleus. In contrast in H O -treated cells IB was phos- 2 2 28. Shrivastava, A., and Aggarwal, B. B. (1999) Antioxid Redox Signal. 1, 181–191 phorylated but not degraded, remaining bound to p65 in the 29. Matthews, J. R., Wakasugi, N., Virelizier, J. L., Yodoi, J., and Hay, R. T. (1992) Nucleic Acids Res. 20, 3821–3830 cytosol; only a fraction of the p65 was translocated to the 30. Meyer, M., Schreck, R., and Baeuerle, P. A. (1993) EMBO J. 12, 2005–2015 nucleus. These results thus suggest that H O -induced NF-B 31. Livolsi, A., Busuttil, V., Imbert, V., Abraham, R. T., and Peyron, J. F. (2001) 2 2 Eur. J. Biochem. 268, 1508 –1515 activation occurs through a mechanism significantly different 32. Schoonbroodt, S., Ferreira, V., Best-Belpomme, M., and Boelaert, J. R. (2000) from that of TNF. 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(1995) Proc. Natl. Acad. Sci. U. S. A. 92, 18. Koong, A. C., Chen, E. Y., and Giaccia, A. J. (1994) Cancer Res. 54, 1425–1430 4527– 4531 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Biological Chemistry Unpaywall

Hydrogen Peroxide Activates NF-κB through Tyrosine Phosphorylation of IκBα and Serine Phosphorylation of p65

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THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 278, No. 26, Issue of June 27, pp. 24233–24241, 2003 © 2003 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Hydrogen Peroxide Activates NF-B through Tyrosine Phosphorylation of IB and Serine Phosphorylation of p65 EVIDENCE FOR THE INVOLVEMENT OF IB KINASE AND Syk PROTEIN-TYROSINE KINASE* Received for publication, December 5, 2002, and in revised form, April 21, 2003 Published, JBC Papers in Press, April 23, 2003, DOI 10.1074/jbc.M212389200 Yasunari Takada‡, Asok Mukhopadhyay‡§, Gopal C. Kundu¶, Ganapati H. Mahabeleshwar ¶, Sujay Singh**, and Bharat B. Aggarwal‡ ‡‡ From the ‡Cytokine Research Laboratory, Department of Bioimmunotherapy, The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030, the ¶National Center for Cell Science (NCCS), NCCS Complex, Pune-411 007, India, and Imgenex, San Diego, California 92121 Although it is well established that reactive oxygen Nuclear factor-B (NF-B) is a transcription factor consist- intermediates mediate the NF-B activation induced ing of a group of five proteins, namely c-Rel, RelA (p65), Rel B, by most agents, how H O activates this transcription NF-B1 (p50 and p105), and NF-B2 (p52) (1). In the resting 2 2 factor is not well understood. We found that treatment state, NF-B is sequestered in the cytoplasm through its tight of human myeloid KBM-5 cells with H O activated 2 2 association with specific inhibitory proteins, called inhibitors of NF-B in a dose- and time-dependent manner much as NF-B(IB), belonging to a gene family consisting of IB, tumor necrosis factor (TNF) did but unlike TNF, H O 2 2 IB,IB,IB, Bcl-3, p100, and p105 (1). On activation by had no effect on IB degradation. Unexpectedly, how- agents such as TNF, IB is phosphorylated at serine residues ever, like TNF-induced activation, H O -induced 2 2 32 and 36, ubiquitinated at lysine residues 21 and 22, and NF-B activation was blocked by the calpain inhibitor degraded through the proteosomal pathway, thus exposing the N-Ac-Leu-Leu-norleucinal, suggesting that a proteoso- nuclear localization signals on the p50-p65 heterodimer. Then mal pathway was involved. Although H O activated 2 2 p65 undergoes phosphorylation, leading to nuclear transloca- IB kinase, it did not induce the serine phosphoryla- tion and binding to a specific sequence in DNA, which in turn tion of IB. Like TNF, H O induced the serine phos- 2 2 results in gene transcription. phorylation of the p65 subunit of NF-B, leading to its The phosphorylation of IB is catalyzed by IB kinase nuclear translocation. We found that H O induced the 2 2 (IKK), which consists of IKK-, IKK-, and IKK- (also called tyrosine phosphorylation of IB, which is needed for NF-B essential modulator (NEMO)) (1). Gene deletion studies NF-B activation. We present several lines of evidence have established that IKK- is essential for NF-B activation to suggest that the Syk protein-tyrosine kinase is in- by TNF (2– 4). IKK- deletion, however, has no effect on NF-B volved in H O -induced NF-B activation. First, H O 2 2 2 2 activation by most agents. Which kinase induces the phospho- activated Syk in KBM-5 cells; second, H O failed to 2 2 activate NF-B in cells that do not express Syk protein; rylation of p65 is controversial, but protein kinase A, casein third, overexpression of Syk increased H O -induced kinase II, IKK-, and IKK- have all been implicated (5–10). 2 2 NF-B activation; and fourth, reduction of Syk tran- The phosphorylation of p65 at serine 529 has been shown to be scription using small interfering RNA inhibited H O - required for the TNF-induced transcriptional activity of NF- 2 2 induced NF-B activation. We also showed that Syk B (11). induced the tyrosine phosphorylation of IB, which NF-B is activated by a wide variety of agents, including all caused the dissociation, phosphorylation, and nuclear 18 members of the TNF superfamily, interleukin-1, interleu- translocation of p65. Thus, overall, our results demon- kin-17, interleukin-18, lipopolysaccharide, H O , ceramide, 2 2 strate that H O induces NF-B activation, not through 2 2 phorbol esters, growth factors, UV, X-rays, and -radiation serine phosphorylation or degradation of IB, but (12). Whether all these agents activate NF-B through the through Syk-mediated tyrosine phosphorylation of same pathway as described above is not clear. Certain agents IB. activate NF-B not through serine phosphorylation but through tyrosine phosphorylation of IB: nerve growth factor, erythropoietin, pervanadate, hypoxia, and silica (13–18). The tyrosine phosphorylation of IB by most agents does not lead * This work was supported in part by the Clayton Foundation for to IB degradation. Pervanadate-induced NF-B activation, Research (to B. B. A.), Department of Defense United States Army however, leads to tyrosine phosphorylation and degradation of Breast Cancer Research Program Grant BC010610 (to B. B. A.), Lung IB (19). Surprisingly, UV-C-induced NF-B activation is me- Chemoprevention PO1 Grant CA91844 from the National Institutes of Health (to B. B. A.), and a P50 Head and Neck Specialized Programs of diated through the degradation of IB that involves phospho- Research Excellence (SPORE) grant from the National Institutes of rylation of neither serine nor the tyrosine residue of IB (20). Health (to B. B. A.). The costs of publication of this article were defrayed Pervanadate-induced tyrosine phosphorylation of IB blocks in part by the payment of page charges. This article must therefore be the TNF-induced serine phosphorylation of IB and NF-B hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. § Present address: National Institute of Immunology, New Delhi, India. The abbreviations used are: NF-B, nuclear factor kappa B; IB, ** Supported by NIGMS, National Institutes of Health Grant inhibitory subunit of NF-B; IKK, IB kinase; TNF, tumor necrosis lck R43 GM68358-01. factor; Syk, spleen tyrosine kinase; p56 , lymphocyte-specific protein- ‡‡ To whom correspondence should be addressed. Tel.: 713-792-3503 tyrosine kinase; EMSA, electrophoretic mobility shift assay; ALLN, or 6459; Fax: 713-794-1613; E-mail: [email protected]. N-acetyl-leucyl-leucyl-norleucinal; siRNA, small interfering RNA. This paper is available on line at http://www.jbc.org 24233 This is an Open Access article under the CC BY license. 24234 Mechanism of H O –induced NF-B Activation 2 2 treated cells were incubated with antibodies against either p50 or p65 activation (16, 21), indicating potential stereochemical of NF-B for 30 min at 37 °C before the complex was analyzed by hindrance. EMSA. Antibodies against cyclin D1 and preimmune serum were in- NF-B activation of most agents has been shown to require cluded as negative controls. The dried gels were visualized, and radio- the generation of reactive oxygen intermediates, in studies that active bands quantitated by a PhosphorImager (Amersham Bio- used either reactive oxygen intermediate quenchers, such as sciences) using ImageQuant software. N-acetylcysteine, or antioxidant enzymes, such as glutathione Western Blot Analysis—To determine the levels of protein expression in cytoplasm or nuclear extracts, we prepared each extract (37) from peroxidase, superoxide dismutase, -glutamylcysteine synthe- TNF-treated cells and fractionated them by SDS-PAGE. After electro- tase, and thioredoxin (22–29). Additionally, there are reports phoresis, the proteins were electrotransferred to nitrocellulose mem- that H O activates NF-B (23, 24, 30). Although it has been 2 2 branes, blotted with each antibody, and detected by ECL regent (Am- shown that H O -induced NF-B is blocked by N-acetylcys- 2 2 ersham Biosciences). The density of the bands was measured using a teine (24), how H O activates NF-B is not fully understood 2 2 personal densitometer scan v1.30 and ImageQuant software version 3.3 (31–33). In the present report, we investigated the mechanism (Amersham Biosciences). IB Kinase Assay—The IKK assay was performed by a method of H O -induced NF-B activation. We found that H O -in- 2 2 2 2 described previously (38). Briefly, IKK complex from cytoplasm was duced NF-B activation occurred without degradation of IB. precipitated with antibody against IKK-, followed by treatment with Instead, H O activated Syk protein-tyrosine kinase, which in 2 2 protein A/G-Sepharose beads (Pierce). After a 2-h incubation, the turn induced tyrosine phosphorylation of IB, leading to beads were washed with lysis buffer and then assayed in kinase assay NF-B activation. mixture containing 50 mM HEPES (pH 7.4), 20 mM MgCl ,2mM dithiothreitol, 20 Ci of [- P]ATP, 10 M unlabeled ATP, and 2 g MATERIALS AND METHODS of substrate GST-IB-(1–54). After the immunocomplex was incu- Reagents—Bacteria-derived human rTNF, purified to homogeneity bated at 30 °C for 30 min, the reaction was terminated by boiling with with a specific activity of 5  10 units/mg, was kindly provided by SDS sample buffer for 5 min. Finally, the protein was resolved on 10% Genentech (South San Francisco, CA). Penicillin, streptomycin, SDS-PAGE, the gel was dried, and the radioactive bands were visu- Iscove’s modified Dulbecco’s medium, RPMI 1640 medium, fetal bo- alized by PhosphorImager. To determine the total amounts of IKK- vine serum, and LipofectAMINE 200 were obtained from Invitrogen. and IKK- in each sample, 30 g of the cytoplasmic protein was H O was obtained from Sigma. Antibodies anti-p65, anti-p50, anti- resolved on 7.5% SDS-PAGE, electrotransferred to a nitrocellulose 2 2 IB, anti-cyclin D1, and anti-phosphotyrosine (PY99) were obtained membrane, and then blotted with either anti-IKK- or anti-IKK- from Santa Cruz Biotechnology (Santa Cruz, CA). Phospho-specific antibodies. anti-IB (Ser-32) antibody was purchased from Cell Signaling (Bev- Syk Kinase Assay—To examine the activity of protein-tyrosine ki- erly, MA). Anti-IKK- and anti-IKK- antibodies were kindly pro- nase Syk induced by H O , we immunoprecipitated with anti-Syk an- 2 2 vided by Imgenex (San Diego, CA). Anti-Syk and anti-phosphoty- tibody, and then performed the in vitro kinase assay using the GST- rosine (4G10) antibodies were purchased from Neo markers IB-(1–54) as the substrate. Briefly, cells were pretreated with 100 (Fremont, CA). Antibody that recognizes the serine 529-phosphoryl- g/ml ALLN (to prevent proteolytic degradation; Ref. 39) for 1 h, then ated form of p65 was obtained from Rockland Laboratories (Gilberts- stimulated with H O for 5 min and whole cell extracts were prepared 2 2 ville, PA). Wild type Syk-cDNA as described previously (34), was in the lysis buffer (1% Triton X-100, 1 mM phenylmethanesulfonyl kindly provided by Dr. Susette C. Mueller (Georgetown University fluoride, 20 g/ml leupeptin, 5 mM sodium orthovanadate, and 2 mM Medical School, Washington, D. C.). EDTA). The kinase protein was immunoprecipitated using anti-Syk Generation of siRNA Plasmid Vector—IMG-800 (pSuppressorNeo, antibody, followed by protein A/G-Sepharose beads. After 2 h, the beads Imgenex, CA) vector was used for construction of 21-bp head-to-head were washed with the lysis buffer and assayed in a kinase assay TM hairpins of human Syk (GenBank accession number NM 003177.2, mixture containing 50 mM HEPES (pH 7.4), 50 mM MgCl ,5mM sodium bp 153–173, 555–575, or 1443–1463). For each construction, two orthovanadate, 10 M unlabeled ATP, and 5 g of substrate GST-IB- complementary oligonucleotides containing human Syk sequences (1–54)) and [- P]ATP. After 30 min incubation at 30 °C, the samples were synthesized (MWG-Biotech, High point, NC) and annealed to were boiled with SDS sample buffer for 5 min, then subjected to poly- generate double stranded DNAs, which were cloned into the SalI and acrylamide gel electrophoresis under denaturing conditions (10% SDS- XbaI cloning sites of IMG-800. The sequence (bp 555–575) used was PAGE), and the dried gels were visualized, and radioactive bands TCGAGCAGACATGGAA CCTGCAGGGGAGTACTGCCCTGCAG- quantitated by a PhosphorImager using ImageQuant software. The GTTCCATGTCTGCTTTTT (sequences are in bold letter, stem loop tyrosine phosphorylation of IB by Syk was also determined by using sequences are in italics, and SalI cloning overhang site is unlabeled ATP in the kinase reaction mixture as described above, and underlined). then performing Western blot analysis using anti-phosphotyrosine Cell Lines—The leukemic cell line KBM-5 is phenotypically myeloid antibody. with monocytic differentiation. The cell lines Jurkat (human T cells), Nuclear Localization of p65 NF-B by Immunocytochemistry—The lck syk JCaM1 (p56 and p72 deficient), human breast cancer cells MCF-7, effect of H O on the nuclear translocation of p65 was examined by 2 2 human lung cancer cells H1299 and mouse macrophage RAW 264.7 the immunocytochemical method as described (40). Briefly, treated cells were obtained from the American Tissue and Cell Culture Collec- cells were plated on a poly-L-lysine-coated glass slide by centrifuga- lck tion (ATCC, Rockville, MD). JCaM1 cells transfected with the p56 tion using a cytospin 4 (Thermoshendon, Pittsburg, PA), air-dried, gene were kindly supplied by Dr. Arthur Weiss (The University of fixed with cold acetone, and permeabilized with 0.2% of Triton X-100. California, San Francisco, CA). The characterization of these cells has After being washed in phosphate-buffered saline, slides were blocked been previously reported (35). KBM-5 cells were cultured in Iscove’s with 5% normal goat serum for 1 h and then incubated with rabbit modified Dulbecco’s medium supplemented with 15% fetal bovine se- polyclonal anti-human p65 or IB antibodies at 1:100 dilutions. rum; Jurkat and JCaM1 cells were cultured in RPMI 1640 medium with After overnight incubation at 4 °C, the slides were washed, incubated 10% fetal bovine serum, 100 units/ml penicillin, and 100 g/ml with goat anti-rabbit IgG-Alexa 594 (Molecular Probes, Eugene, OR) streptomycin. at 1:100 dilutions for 1 h, and counterstained for nuclei with Hoechst Electrophoretic Mobility Shift Assays (EMSA)—To determine NF-B 33342 (50 ng/ml) for 5 min. Stained slides were mounted with mount- activation, we performed EMSA as described (36). Briefly, nuclear ing medium purchased from Sigma and analyzed under a fluores- extracts prepared from TNF-treated cells (2  10 /ml) were incubated cence microscope (Labophot-2, Nikon, Tokyo, Japan). Pictures were with P-end-labeled 45-mer double-stranded NF-B oligonucleotide (10 captured using Photometrics Coolsnap CF color camera (Nikon, g of protein with 16 fmol of DNA) from the human immunodeficiency Lewisville, TX) and MetaMorph version 4.6.5 software (Universal virus long terminal repeat, 5-TTGTTACAAGGGACTTTCCGCTGGG- Imaging, Downingtown, PA). GACTTTCCAGGGAGGCGTGG-3 (boldface indicates NF-B binding Immunoprecipitation Assays—Cells were lysed for 30 min on ice in sites) for 30 min at 37 °C, and the DNA-protein complex formed was whole cell extraction buffer (20 mM HEPES, pH 7.9, 50 mM NaCl, 1% separated from free oligonucleotide on 6.6% native polyacrylamide gels. Nonidet P-40, 2 mM EDTA, 0.5 mM EGTA, 2 g/ml aprotinin, 2 g/ml A double-stranded mutated oligonucleotide, 5-TTGTTACAACT- leupeptin, 0.5 mM phenylmethanesulfonyl fluoride, and 2 mM sodium CACTTTCCGCTGCTCACTTTCCAGGGAGGCGTGG-3, was used to orthovanadate). Lysate containing 500 g of proteins in extraction examine the specificity of binding of NF-B to the DNA. The specificity buffer was incubated with 1 g/ml concentration of antibodies over- of binding was also examined by competition with the unlabeled oligo- night. Immunocomplex was precipitated using protein A/G-Sepharose nucleotide. For supershift assays, nuclear extracts prepared from TNF- beads for1hat4 °C. Beads were washed with extraction buffer and Mechanism of H O –induced NF-B Activation 24235 2 2 resuspended in SDS sample buffer, boiled for 5 min, and fractionated in SDS-PAGE. Syk-siRNA and Syk-cDNA Transfection—To determine the role of Syk protein-tyrosine kinase in the H O -induced NF-B activation, 2 2 2 10 cells were transfected either with small interfering RNA-Syk (siRNA-Syk), or with wild type-Syk cDNA (Syk-WT). Two g of plasmid in each case was diluted in 200 l of Dulbecco’s modified Eagle’s medium (without serum and antibiotics), and then mixed with 200 lof Dulbecco’s modified Eagle’s medium containing 4 l of LipofectAMINE 2000. This mixture was added to the cells and incubated for 4 h. After incubation, medium was changed into RPMI 1640 and maintained for an additional 48 h. These cells were then used to examine the expres- sion of Syk protein by Western blot analysis, NF-B activity by EMSA, and Syk activity by the kinase assay as described above. RESULTS In this report we investigated the effect of H O on NF-B 2 2 activation, IB phosphorylation, IB degradation, p65 phos- phorylation, and nuclear translocation, and the role of protein- tyrosine kinase Syk in H O -induced NF-B activation. Be- 2 2 cause NF-B activation by TNF is well understood, we used TNF as a control for most studies. As determined by trypan blue dye exclusion and Hoechst staining methods, the treat- ment of cells with 500 M H O for 2 h had no significant effect 2 2 on cell viability (viability was greater than 98%). Both H O and TNF Activate DNA Binding Activity of NF-B 2 2 in a Dose- and Time-dependent Manner—To determine the effect of H O on the activation of NF-B, KBM-5 cells were 2 2 treated with different concentrations of H O for 120 min or 2 2 TNF for 30 min. Nuclear extracts were prepared for analysis of NF-B activation by EMSA. Both H O and TNF induced 2 2 NF-B activation in a dose-dependent manner in KBM-5 cells (Fig. 1A); maximum activation with H O occurred at 500 M. 2 2 NF-B was activated by both agents in a time-dependent man- ner (Fig. 1B). TNF induced NF-B activation within 15 min, and activation continued for 240 min, whereas, H O -induced 2 2 NF-B activation started at 60 min, reached maximum at 120 min, and then decreased at 240 min. The maximum TNF- induced NF-B activation was 14-fold; with H O it was 3–7- 2 2 FIG.1. A and B, dose response (panel A) and time course (panel B)of fold. Thus, H O induced NF-B activation more slowly than 2 2 activation of NF-B by TNF (left panel) and H O (right panel) in 2 2 did TNF. The slower kinetics and difference in the optimum KBM-5 cells. A, KBM-5 cells (2  10 /ml) were treated with different concentrations of TNF for 30 min or H O for 120 min. After these level of activation suggest that the mechanism of NF-B acti- 2 2 treatments, nuclear extracts were prepared and then assayed for vation by H O is different from that of TNF. 2 2 NF-B activation by EMSA, as described under “Materials and Meth- Because NF-B is a complex of proteins, various combina- ods.” B, time-dependent activation of NF-BbyH O . Cells (2  10 /ml) 2 2 tions of Rel/NF-B protein can constitute an active NF-B were treated with 0.1 nM TNF or 500 M H O for the indicated times. 2 2 heterodimer that binds to a specific sequence in DNA (1). To After treatment, nuclear extracts were prepared and then assayed for NF-B activation by EMSA. C, NF-B induced by H O is composed of show that the retarded band visualized by EMSA in H O - 2 2 2 2 p65 and p50 subunits. Nuclear extracts were prepared from untreated treated cells was indeed NF-B, we incubated nuclear extracts or 500 M H O -treated cells (2  10 /ml), incubated for 30 min with 2 2 from H O -stimulated cells with antibodies to either the p50 2 2 different antibodies or unlabeled NF-B oligo probes, and then assayed (NF-B1) or the p65 (RelA) subunit of NF-B. Both shifted the for NF-B activation by EMSA. band to a higher molecular mass (Fig. 1C), thus suggesting that the H O -activated complex consisted of p50 and p65 subunits. 2 2 Neither preimmune serum nor anti-cyclin D1 antibody had any IB (for references see Ref. 1). So we investigated whether effect. Excess unlabeled NF-B (100-fold) caused complete dis- H O induces serine phosphorylation of IB. To stabilize the 2 2 appearance of the band, and a mutant oligonucleotide of NF-B phosphorylated IB, we blocked degradation of IB using did not affect NF-B binding activity. the proteosome inhibitor ALLN (39). Western blot analysis H O Induces NF-B Activation without Degrading IB— using phospho-specific anti-IB antibody (Fig. 2B) showed 2 2 TNF-induced NF-B activation requires the degradation of that TNF induced the phosphorylation of IB but H O had no 2 2 IB (for references see Ref. 1). Whether H O -induced NF-B effect on the serine phosphorylation of IB. 2 2 activation is also mediated through IB degradation was in- ALLN Inhibits NF-B Activation Induced by Both TNF and vestigated. To determine this, cells were treated with 0.1 nM H O —ALLN blocked not only TNF-induced NF-B activation, 2 2 TNF or 500 M H O for the indicated times, extracted the but also H O -induced NF-B activation (Fig. 2C). Thus IB 2 2 2 2 cytoplasmic protein, and analyzed for IB on 10% SDS-PAGE phosphorylation and degradation are critical in TNF-induced using anti-IB antibody. TNF induced IB degradation NF-B activation. within 15 min after treatment, and IB was resynthesized at H O Induces Translocation and Phosphorylation of 2 2 60 min (Fig. 2A). However, H O did not induce IB degrada- p65—We analyzed the effect of TNF and H O on translocation 2 2 2 2 tion at any time points. and phosphorylation of p65 by Western blot analysis. Both TNF TNF Induces IB Serine Phosphorylation but H O Does and H O induced nuclear translocation of p65 in a time-de- 2 2 2 2 Not—TNF induces phosphorylation of serines 32 and 36 of pendent manner. On TNF stimulation, p65 nuclear transloca- 24236 Mechanism of H O –induced NF-B Activation 2 2 These results suggest that H O activated IKK but had no 2 2 effect on the serine phosphorylation of IB. H O Does Not Activate NF-B in Syk-deficient Jurkat 2 2 Cells—To investigate further the role of Syk in H O -induced 2 2 NF-B activation, we used JCaM1 cells known to lack both Lck (35) and Syk protein expression (41). Western blot analysis revealed that Jurkat cells expressed Syk protein but JCaM1 cells expressed very little or no Syk (Fig. 5A). The anti-Syk antibody was specific as it did not recognize Lck protein ex- lck pression in p56 -reconstituted JCaM1 cells (Fig. 5A). TNF activated NF-B in both Jurkat and JCaM1 cells and induced IB degradation in both the cell lines (Fig. 5B). The kinetics of TNF-induced NF-B activation, however, was slightly slower in JCaM1 cells than in Jurkat cells (15 versus 30 min) and the overall magnitude of activation was also lower (7.1- versus 5.1-fold). In contrast, H O activated NF-B in Jurkat cells but 2 2 not in JCaM1 cells (Fig. 5C), indicating an essential role of Syk protein expression in H O -induced activation. 2 2 We also investigated the ability of H O to activate NF-Bin 2 2 human lung epithelial (H1299) and breast epithelial (MCF-7) cells, which cannot be activated for Syk (Fig. 5D, lower panel). H1299 and MCF-7 cells were treated with H O for the indi- 2 2 cated times, nuclear extracts were prepared and analyzed for NF-B activation by EMSA. Whole cell extracts were analyzed for Syk activation. We found that H O failed to activate Syk 2 2 and this correlated with the lack of activation of NF-Bin H1299 and MCF-7 cells. FIG.2. A, TNF-induces IB degradation but H O does not. KBM-5 2 2 Whether H O can activate NF-B in murine cells was ex- cells (2  10 cells/ml) were treated with 0.1 nM TNF or 500 M H O for 2 2 2 2 the indicated times, and then cytoplasmic extracts were prepared as amined. For this murine macrophage RAW264.7 cells were described under “Materials and Methods.” Cytoplasmic extracts were treated for the indicated times with 500 M H O and then 2 2 fractionated on 10% SDS-PAGE and electrotransferred to nitrocellulose examined for NF-B activation by EMSA and IB degradation membrane. Western blot analysis was used with anti-IB antibody. by Western blot analysis. As shown in Fig. 5E,H O induced For loading control, we used anti--actin antibody. B, TNF-induces 2 2 IB phosphorylation but H O does not. KBM-5 cells (2  10 cells/ml) NF-B activation in murine cells and this was accompanied 2 2 were preincubated with 100 g/ml proteosome inhibitor, ALLN (39), for with the degradation and resynthesis of IB. These results 1 h, and then cells were treated with either 0.1 nM TNF for 15 min or agree with a previous report (32) but differ from that noted in 500 M H O for 60 min. Cytoplasmic extracts were fractionated on 10% 2 2 human cells. SDS-PAGE, and then Western blot analysis was performed with phos- phospecific anti-IB antibody. C, ALLN inhibits NF-B activation H O Does Not Activate NF-B in Lck-reconstituted JCaM1 2 2 induced by both TNF and H O . KBM-5 cells (2  10 cells/ml) were lck 2 2 Cells—JCaM1 cells have been shown to lack both p56 and incubated with 100 g/ml ALLN for 1 h, and then treated with 0.1 nM Syk protein-tyrosine kinases (35). Previously we have shown TNF or 500 M H O for the indicated periods. Nuclear extracts were 2 2 lck that p56 is required for ceramide-induced and HIV-tat-in- analyzed for NF-B activation by EMSA. duced NF-B activation (42, 43). EMSA of JCaM1 cells whose lck p56 expression had been reconstituted revealed that H O 2 2 tion reached maximum 15 min after TNF treatment and grad- activated NF-B in Jurkat cells but not in JCaM1 cells whose ually declined thereafter. In the case of H O , p65 translocation 2 2 lck p56 expression had been reconstituted (Fig. 5F). No IB was slightly induced after 15 min treatment, peaked at 60 min, phosphorylation was noted in either of the cell lines (see lower and diminished thereafter (Fig. 3A). lck panel). In other words, p56 was not required for H O -in- 2 2 Both TNF and H O induced the phosphorylation of p65 in a 2 2 duced NF-B activation or for IB phosphorylation. time-dependent manner, but the kinetics of H O -induced 2 2 H O Induces Tyrosine Phosphorylation of Syk Protein in phosphorylation of p65 was slower than that for TNF (Fig. 3B). 2 2 Jurkat Cells but Not in JCaM1 Cells—Jurkat and Syk-defi- Immunocytochemistry assay showed that while in untreated cient JCaM1 cells treated with H O for the indicated times cells, p65 was localized primarily in the cytoplasm, TNF and 2 2 were immunoprecipitated with anti-Syk antibody and then H O induced translocation of p65 into the nucleus (Fig. 3C). 2 2 subjected to Western blot analysis using anti-phosphoty- However, H O -induced phosphorylation and translocation of 2 2 rosine antibody (4G10). H O -induced tyrosine phosphoryla- p65 to the nucleus were delayed compared with TNF- 2 2 tion of Syk in Jurkat, but not in Syk-deficient JCaM1 cells induced response. (Fig. 6A). H O Induces IB Kinase Activation—Because our results 2 2 H O Induces Tyrosine Phosphorylation of IB Protein in indicated that H O -induced NF-B activation is not mediated 2 2 2 2 Jurkat Cells—Previously our laboratory and others have through the phosphorylation and degradation of IB, we next shown that certain agents can activate NF-B through tyrosine explored whether H O can activate IKK. It has been shown 2 2 phosphorylation even though it does not lead to the degrada- that IKK is required not only for TNF-induced phosphorylation tion of IB (13–18). Because H O likewise did not induce of IB but also for the phosphorylation of p65 (9, 10). An in 2 2 IB degradation, we next determined whether H O induces vitro immune complex kinase assay using GST-IB-(1–54) as 2 2 the substrate showed that both TNF and H O activated IKK tyrosine phosphorylation of IB. Jurkat cells treated with 2 2 H O exhibited tyrosine phosphorylation of IB (Fig. 6B) (the as early as 5 min after TNF treatment, but then activation 2 2 ceased (Fig. 4). H O -induced IKK activation was, however, tyrosine-phosphorylated molecule is the slower migrating spe- 2 2 cies of IB). Pretreatment of cells with ALLN significantly weaker than that induced by TNF. Neither TNF nor H O had 2 2 any effect on the expression of either IKK- or IKK- proteins. enhanced tyrosine phosphorylation of IB (Fig. 6C). These Mechanism of H O –induced NF-B Activation 24237 2 2 FIG.3. A, both TNF and H O induce 2 2 the translocation of p65. KBM-5 cells (2 10 cells/ml) were treated with 0.1 nM TNF or 500 M H O for the indicated 2 2 times. Nuclear extracts were prepared and fractionated on SDS-PAGE, and then Western blot analysis was performed us- ing anti-p65 antibody. B, both TNF and H O induce the phosphorylation of p65. 2 2 KBM-5 cells (2  10 cells/ml) were treated with 0.1 nM TNF or 500 M H O 2 2 for the indicated times. Nuclear extracts were prepared and fractionated on SDS- PAGE, and then Western blot analysis was performed using anti-p65 antibody. C, both TNF and H O induce the nuclear 2 2 translocation of p65. KBM-5 cells were treated with 1 nM TNF for 30 min or 500 M H O for 120 min. Cells were sub- 2 2 jected immunocytochemistry as described under “Materials and Methods.” sufficient for the release of p65 was explored. Jurkat cells were treated with TNF or with H O and analyzed for IB by 2 2 Western blot analysis. TNF induced the degradation of IB in the cytosol (Fig. 7A, left, middle panel) and the translocation of p65 to the nucleus (right, bottom panel). TNF also induced the dissociation of p65 from IB (right, top panel). In the H O - 2 2 treated cells, p65 was translocated into the nucleus (right, FIG.4. Both TNF and H O activate IKK. Cells (2  10 cells/ml) bottom panel) without degradation of IB (left, middle panel). 2 2 were activated with TNF (0.1 nM)orH O (500 M) for the indicated 2 2 We were surprised to see that H O did not induce dissociation 2 2 time intervals. Cytoplasmic extracts were prepared, and 300 gof of p65 from IB (left, top panel). H O induced a slow migrat- 2 2 cytoplasmic extracts were immunoprecipitated with antibody against ing species of IB (left, top panel, and left, middle panel). No IKK-. Thereafter immune complex kinase assay was performed as described under “Materials and Methods.” To examine the effect of IB could be detected in nucleus, neither free (right, middle H O on the level of expression of IKK proteins, 30 g of cytoplasmic 2 2 panel) nor p65 bound (right, top panel). An immunocytochem- extracts were fractionated on 7.5% SDS-PAGE. Western blot analysis istry assay showed that both p65 and IB were localized in the was performed using with anti-IKK- and anti-IKK- antibodies. cytosol of untreated cells (Fig. 7B). The cells treated with TNF lacked IB in the cytosol, and p65 was translocated to the nucleus in these cells. Although H O induced p65 transloca- 2 2 tion into the nucleus, IB remained in the cytosol (Fig. 7B). results suggest that H O induced tyrosine phosphorylation of 2 2 These results suggest that H O induced translocation of p65 IB by activating of Syk tyrosine kinase. 2 2 without degrading IBa. H O -activated Syk Induces Phosphorylation of IB—To 2 2 Overexpression of Syk Enhances H O -induced NF-B Acti- investigate the mechanism of Syk-mediated phosphorylation of 2 2 vation and Tyrosine Phosphorylation of IB—To further con- IB, we performed the in vitro kinase assay using GST-IB- firm the role of Syk protein-tyrosine kinase in H O -induced (1–54) as the substrate. We found that H O -activated Syk 2 2 2 2 could directly phosphorylate IB, and this phosphorylation NF-B activation, we transiently transfected the Jurkat cells with the wild type Syk cDNA-containing expression plasmid. was enhanced by ALLN treatment (Fig. 6D). To further confirm the Syk-mediated tyrosine phosphorylation of IB, we per- As shown in Fig. 8, these cells showed an increase in expression of Syk protein (panel A), enhanced H O -induced NF-B acti- formed the in vitro kinase assay using GST-IB-(1–54) as the 2 2 substrate and unlabeled ATP. The tyrosine-phosphorylated vation (panel B), and an increase in the IB tyrosine phos- phorylation (panel C), over the non-transfected control. Thus IB was detected by Western blot analysis using anti-phos- photyrosine and anti-IB antibodies (Fig. 6E). These results these results suggest that Syk protein-tyrosine kinase mediate H O -induced NF-B through the tyrosine phosphorylation suggest that Syk protein-tyrosine kinase can directly phospho- 2 2 rylate IB on tyrosine residues. of IB. Reduction of Syk Protein by siRNA-Syk Inhibits H O - H O Induces Dissociation of IB from p65 without Degrad- 2 2 2 2 ing IB—Whereas serine phosphorylation leads to the degra- induced NF-B Activation and Tyrosine Phosphorylation of dation of IB, tyrosine phosphorylation does not (13–18). IB—We also used siRNA to suppress Syk expression (44). Whether H O -induced tyrosine phosphorylation of IB was For this, we transiently transfected siRNA-Syk into Jurkat 2 2 24238 Mechanism of H O –induced NF-B Activation 2 2 lck lck FIG.5. A, Jurkat cells express Syk but JCaM1 or p56 -reconstituted cells do not. Jurkat, JCaM1, and JCaM1/p56  cells were lysed, and 30 g of samples of each were fractionated on 7.5% SDS-PAGE. Western blot analysis was performed using anti-Syk antibody. B, TNF activates NF-B and induces IB degradation in both Jurkat- and Syk-deficient JCaM1 cells. Jurkat and JCaM1 cells were treated with 0.1 nM TNF for the indicated times. Nuclear and cytoplasmic extracts were prepared. Nuclear extracts were analyzed for NF-B activation by EMSA. Cytoplasmic extracts were analyzed for IB expression by Western blot analysis using anti-IB antibody. C,H O activates NF-B in Jurkat and but not in 2 2 Syk-deficient JCaM1 cells. Jurkat and JCaM1 cells were treated with 500 M H O for the indicated times. Nuclear and cytoplasmic extracts were 2 2 prepared. Nuclear extracts were analyzed for NF-B activation by EMSA. Cytoplasmic extracts were analyzed for IB expression by Western blot analysis using anti-IB antibody. D,H O does not activate NF-B in human lung epithelial (H1299) and breast epithelial (MCF-7) cells. H1299 2 2 and MCF-7 cells were treated with 500 M H O for the indicated times. Nuclear extracts were prepared and analyzed for NF-B activation by 2 2 EMSA. Whole cell extracts were analyzed for Syk expression. E,H O activates NF-B in murine macrophages RAW 264.7 cells. Cells were treated 2 2 with 500 M H O for the indicated times. Nuclear and cytoplasmic extracts were prepared. Nuclear extracts were analyzed for NF-B activation 2 2 by EMSA. Cytoplasmic extracts were analyzed for IB expression by Western blot analysis using anti-IB antibody. F,H O does not activate 2 2 lck NF-B in JCaM1 cells reconstituted with p56 . Jurkat and Lck-reconstituted JCaM1 cells were treated with H O for the indicated times. Nuclear 2 2 extracts were analyzed for NF-B activation by EMSA. Cytoplasmic extracts were analyzed for IB expression and for phosphorylated IB by Western blot analysis using anti-IB and phospho-specific IB antibodies. cells, and then examined H O -induced NF-B activation and again suggest that Syk plays an important role in NF-B 2 2 IB tyrosine phosphorylation. As shown in Fig. 9, siRNA- activation induced by H O . 2 2 Syk suppressed the expression of Syk protein (panel A), di- DISCUSSION minished H O -induced NF-B activation (panel B), and sup- 2 2 pressed tyrosine phosphorylation of IB (panel C)as By using metabolic inhibitors and antioxidant enzymes, it compared with the nontransfected control. These results has been well established that oxygen radicals are involved in Mechanism of H O –induced NF-B Activation 24239 2 2 FIG.7. A, colocalization of p65 and IB complex in the cytoplasm. Jurkat cells were treated with either 1 nM TNF for 15 min or 500 M H O for 60 min, and then nuclear and cytoplasmic extracts were 2 2 FIG.6. A,H O activates Syk in Jurkat cells but not in JCaM1 cells. 2 2 prepared. Each extract was incubated with anti-p65 antibody over- Jurkat and JCaM1 cells were treated with 500 M H O for the indi- 2 2 night, precipitated with protein A/G-Sepharose beads, and then frac- cated times. Whole cell extracts were prepared, and 500 g of sample tionated on 10% SDS-PAGE. Western blot analysis was performed was incubated with anti-Syk antibody overnight. Immunocomplex was using anti-IB antibody. Nuclear and cytoplasmic extracts were ana- precipitated using protein A/G-Sepharose beads and then fractionated lyzed for IB and p65 expression by SDS-PAGE following Western blot on 7.5% SDS-PAGE. Western blot analysis was performed using anti- analysis using anti-IB and anti-p65 antibodies. B, effect of H O on 2 2 phosphotyrosine antibody (4G10). B,H O induces tyrosine phospho- 2 2 the immunolocalization of p65 and IB. Cells were treated with either rylation of IB in Jurkat cells. Jurkat cells were treated with 500 M 1nM TNF for 30 min or 500 M H O for 120 min. Cells were then 2 2 H O for the indicated times. Whole cell extracts were prepared, and 2 2 processed for immunocytochemistry using anti-p65 and anti-IB an- 500 g of sample was incubated with anti-IB antibody overnight. tibodies, as described under “Materials and Methods.” Immunocomplex was precipitated with protein A/G-Sepharose beads, and then fractionated on 10% SDS-PAGE. Western blot analysis was performed using anti-phosphotyrosine antibody (PY99). C, ALLN en- tion. Rather, it induced tyrosine phosphorylation of IB.H O 2 2 hances the H O -induced tyrosine phosphorylation of IB. Jurkat cells 2 2 activated both IKK- and Syk kinase, and this activation of were preincubated with 100 g/ml ALLN for 1 h and then treated with Syk kinase proved to be critical for tyrosine phosphorylation of 500 M H O for the indicated times. Whole cell extracts were prepared, 2 2 and 500 g of sample was incubated with anti-IB antibody overnight. IB and for NF-B activation. Immunocomplex was precipitated with protein A/G-Sepharose beads, Both TNF and H O activated NF-B, but the kinetics of 2 2 and then fractionated on 10% SDS-PAGE. Western blot analysis was NF-B activation was faster with TNF than that with H O . 2 2 performed using anti-phosphotyrosine antibody (PY99). D,H O -acti- 2 2 These results in themselves suggested that the mechanism of vated Syk induces phosphorylation of IB. Jurkat cells were treated NF-B activation by TNF are different from that of H O . with 500 M H O for 5 min in the presence or absence of ALLN, 2 2 2 2 prepared whole cell extracts, immunoprecipitated with anti-Syk anti- Although reactive oxygen intermediates has been implicated in body, and then performed kinase reaction using GST-IB-(1–54) as TNF-induced NF-B activation (45, 46), this alone may not be the substrate as described under “Materials and Methods.” Syk protein sufficient for NF-B activation. The difference in kinetics may levels were examined by Western blot analysis using anti-Syk antibody. also be related to the receptor-mediated nature of TNF-induced E,H O -activated Syk induces tyrosine phosphorylation of IB. Jur- 2 2 kat cells were treated with 500 M H O for 5 min in the presence or activation whereas H O activity is receptor-independent. 2 2 2 2 absence of ALLN, prepared whole cell extracts, immunoprecipitated We have in fact demonstrated that the mechanism of NF-B with anti-Syk antibody, and then performed kinase reaction as de- activation by H O is significantly different from that of TNF. 2 2 scribed under “Materials and Methods.” Samples were analyzed for For instance, TNF induced IB degradation but H O did not. tyrosine phosphorylation of I B by Western blot analysis using anti- 2 2 phosphotyrosine (4G10) and anti-IB antibodies. For positive control, Other agents are also known to activate NF-B without IB cells were treated with pervanadate (pV) for 60 min. degradation, including pervanadate, hypoxia, erythropoietin, and nerve growth factor (13–18). Our results agree with Dudek et al. (47) and Milligan et al. (48) but differ from Schoonbroodt NF-B activation by most agents. Additionally, that H O et al. (32), who showed that H O induces IB degradation in 2 2 2 2 alone can activate NF-B has been known for almost a decade mouse lymphoma EL4 cells. Whether these differences are but the mechanism has remained unclear. We demonstrate in related to cell type is not clear. We used various T cell lines and the present report that H O activates NF-B without the myeloid cells and found similar results. Our results also sug- 2 2 degrading IB and without inducing its serine phosphoryla- gest that, unlike TNF, H O does not induce serine phospho- 2 2 24240 Mechanism of H O –induced NF-B Activation 2 2 FIG.8. Overexpression of Syk enhances H O -induced NF-B 2 2 activation and IB phosphorylation. A, Jurkat cells were trans- fected with wild type-Syk cDNA (Syk-WT), prepared whole cell extracts and then subjected to SDS-PAGE for protein expression using anti-Syk antibody. B, Syk-WT-transfected cells were treated with 250 M H O FIG.9. Reduction of Syk protein by siRNA-Syk inhibits H O - 2 2 2 2 for the indicated times, and then analyzed for NF-B activation. C,to induced NF-B activation and IB phosphorylation. A, Jurkat determine the effect of overexpressed Syk on IB, cells were treated cells were transfected with siRNA-Syk, prepared whole cell extracts, with 500 M H O for 5 min, prepared whole cell extracts, immunopre- and then subjected to SDS-PAGE for protein expression using anti-Syk 2 2 cipitated with anti-IB antibody, and then analyzed by Western blot antibody. B, siRNA-Syk-transfected cells were treated with 250 M analysis using anti-phosphotyrosine antibody (PY99). H O for the indicated times, and then analyzed for NF-B activation. 2 2 C, to determine the effect of reduced Syk on IB, siRNA-Syk-trans- fected cells were treated with 500 M H O for 5 min, prepared whole rylation of IB. Schoonbroodt et al. (32) also showed that 2 2 cell extracts, immunoprecipitated with anti-IB antibody, and then phosphorylation of serine 32 and 36 of IB is not required for analyzed by Western blot analysis using anti-phosphotyrosine antibody NF-B activation by H O . 2 2 (PY99). As was the case for TNF, H O -induced NF-B activation 2 2 was completely suppressed by ALLN, a proteosomal inhibitor. How ALLN inhibits H O -induced NF-B activation is not Our results indicate that H O induces tyrosine phosphoryl- 2 2 2 2 clear. The proteosome has been implicated in the degradation ation of IB. These results are in agreement with reports by of IB and in the processing of p105 (NF-B1) and p100 Livolsi et al. (31) and Schoonbroodt et al. (32). What kinase (NF-B2) proteins. It is unlikely that ALLN inhibits H O - induces the tyrosine phosphorylation of IB, however, is not 2 2 induced NF-B activation through the inhibition of degrada- clear. Our results provide evidence that Syk protein kinase tion of IB or processing of NF-B proteins. The proteosome may play a major role in this phosphorylation. First, H O 2 2 inhibitors MG132 and lactacystin have also been shown to activated Syk in KBM-5 cells; second H O failed to activate 2 2 suppress H O -induced NF-B activation without affecting NF-B in cells that do not express Syk protein; third, overex- 2 2 IB,-,or- in lens epithelial cells (49). Our results suggest pression of Syk increased H O -induced NF-B activation; and 2 2 the role of another proteosomal-sensitive factor in H O -in- fourth, reduction of Syk transcription using siRNA inhibited 2 2 duced NF-B activation. H O -induced NF-B activation. We also showed that Syk in- 2 2 Our results also demonstrate that even though H O did not duced the tyrosine phosphorylation of IB, which caused the 2 2 induce serine phosphorylation of IB, it did activate IKK, dissociation, phosphorylation, and nuclear translocation of p65. phosphorylated p65 at serine residue 529, and translocated p65 Howe et al. (50) showed that H O -induced phosphorylation of 2 2 to the nucleus. Recently it was demonstrated that IKK can IB through calcium/calmodulin-dependent kinases in Jurkat phosphorylate p65 (9, 10). Therefore, it is possible that H O - cells. H O has been shown to activate Syk (51), a kinase 2 2 2 2 induced IKK is needed not for the phosphorylation of IB but known to be expressed in thymocytes, B lymphocytes, mast for the phosphorylation of p65. Our results are in agreement cells, monocytes, macrophages, neutrophils and platelets, and with a recent report by Kamata et al. (33), who showed that mature T lymphocytes. We found that cells that do not express H O can activate IKK and this activation is essential for Syk, such as JCaM1 (a genetic isolate of Jurkat cells), failed to 2 2 NF-B activation. How H O -induced IKK activates NF-B, activate NF-B on treatment with H O . Lakshminarayanan et 2 2 2 2 however, was not investigated by these workers. Our results al. (52) showed that in human microvessel endothelial cells or differ from Schoonbroodt et al. (32), who showed lack of activa- human lung epithelial A549 cells, TNF could activate NF-B tion of IKK by H O . but H O could not. The inability of H O to activate NF-Bin 2 2 2 2 2 2 Mechanism of H O –induced NF-B Activation 24241 2 2 19. Mukhopadhyay, A., Manna, S. K., and Aggarwal, B. B. (2000) J. Biol. Chem. these cells could have been because of a lack of expression of 275, 8549 – 8555 Syk kinase in these cells. Previously it has been shown that 20. Li, N., and Karin, M. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 13012–13017 lck H O can activate p56 (53). Although Lck has been impli- 21. Singh, S., and Aggarwal, B. B. (1995) J. Biol. Chem. 270, 10631–10639 2 2 22. Staal, F. J., Roederer, M., Herzenberg, L. A., and Herzenberg, L. A. (1990) cated in the NF-B activation by ceramide and by HIV-tat (42, Proc. Natl. Acad. Sci. U. S. A. 87, 9943–9947 43), our results indicate that H O does not induce NF-B 23. Kretz-Remy, C., Mehlen, P., Mirault, M. E., and Arrigo, A. P. (1996) J. Cell 2 2 lck lck Biol. 133, 1083–1093 activation through activation of p53 , as p53 -reconstituted 24. Schreck, R., Rieber, P., and Baeuerle, P. A. (1991) EMBO J. 10, 2247–2258 JCaM1 cells did not respond to H O for NF- B activation. 2 2 25. Giri, D. K., and Aggarwal, B. B. (1998) J. Biol. Chem. 273, 14008 –14014 We found that in TNF-treated cells, almost all the cytoplas- 26. Manna, S. K., Zhang, H. J., Yan, T., Oberley, L. W., and Aggarwal, B. B. (1998) J. Biol. 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Published: Jun 1, 2003

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