Dynein Light Chain LC8 Negatively Regulates NF-κB through the Redox-dependent Interaction with IκBα
2008; Elsevier BV; Volume: 283; Issue: 35 Linguagem: Inglês
10.1074/jbc.m803072200
ISSN1083-351X
AutoresYuyeon Jung, Hojin Kim, Sun Hee Min, Sue Goo Rhee, Woojin Jeong,
Tópico(s)Genomics, phytochemicals, and oxidative stress
ResumoRedox regulation of nuclear factor κB (NF-κB) has been described, but the molecular mechanism underlying such regulation has remained unclear. We recently showed that a novel disulfide reductase, TRP14, inhibits tumor necrosis factor α (TNFα)-induced NF-κB activation, and we identified the dynein light chain LC8, which interacts with the NF-κB inhibitor IκBα, as a potential substrate of TRP14. We now show the molecular mechanism by which NF-κB activation is redox-dependently regulated through LC8. LC8 inhibited TNFα-induced NF-κB activation in HeLa cells by interacting with IκBα and thereby preventing its phosphorylation by IκB kinase (IKK), without affecting the activity of IKK itself. TNFα induced the production of reactive oxygen species, which oxidized LC8 to a homodimer linked by the reversible formation of a disulfide bond between the Cys2 residues of each subunit and thereby resulted in its dissociation from IκBα. Butylated hydroxyanisol, an antioxidant, and diphenyleneiodonium, an inhibitor of NADPH oxidase, attenuated the phosphorylation and degradation of IκBα by TNFα stimulation. In addition LC8 inhibited NF-κB activation by other stimuli including interleukin-1β and lipopolysaccharide, both of which generated reactive oxygen species. Furthermore, TRP14 catalyzed reduction of oxidized LC8. Together, our results indicate that LC8 binds IκBα in a redox-dependent manner and thereby prevents its phosphorylation by IKK. TRP14 contributes to this inhibitory activity by maintaining LC8 in a reduced state. Redox regulation of nuclear factor κB (NF-κB) has been described, but the molecular mechanism underlying such regulation has remained unclear. We recently showed that a novel disulfide reductase, TRP14, inhibits tumor necrosis factor α (TNFα)-induced NF-κB activation, and we identified the dynein light chain LC8, which interacts with the NF-κB inhibitor IκBα, as a potential substrate of TRP14. We now show the molecular mechanism by which NF-κB activation is redox-dependently regulated through LC8. LC8 inhibited TNFα-induced NF-κB activation in HeLa cells by interacting with IκBα and thereby preventing its phosphorylation by IκB kinase (IKK), without affecting the activity of IKK itself. TNFα induced the production of reactive oxygen species, which oxidized LC8 to a homodimer linked by the reversible formation of a disulfide bond between the Cys2 residues of each subunit and thereby resulted in its dissociation from IκBα. Butylated hydroxyanisol, an antioxidant, and diphenyleneiodonium, an inhibitor of NADPH oxidase, attenuated the phosphorylation and degradation of IκBα by TNFα stimulation. In addition LC8 inhibited NF-κB activation by other stimuli including interleukin-1β and lipopolysaccharide, both of which generated reactive oxygen species. Furthermore, TRP14 catalyzed reduction of oxidized LC8. Together, our results indicate that LC8 binds IκBα in a redox-dependent manner and thereby prevents its phosphorylation by IKK. TRP14 contributes to this inhibitory activity by maintaining LC8 in a reduced state. Dyneins are large multi-component complexes that function as microtubule-based molecular motors both in the cytoplasm and in flagella (1King S.M. Biochim. Biophys. Acta. 2000; 1496: 60-75Crossref PubMed Scopus (292) Google Scholar). Cytoplasmic dyneins participate in a variety of intracellular motile processes including mitosis and vesicular transport, whereas axonemal dyneins provide motive force for the beating of cilia and flagella. The 8-kDa dynein light chain (LC8, also known as DLC8 or DLC1) was originally identified in flagellar dynein of Chlamydomonas (2King S.M. Patel-King R.S. J. Biol. Chem. 1995; 270: 11445-11452Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar) and was subsequently found to be a component of cytoplasmic dynein motor (3King S.M. Barbarese E. Dillman III, J.F. Patel-King R.S. Carson J.H. Pfister K.K. J. Biol. Chem. 1996; 271: 19358-19366Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar). LC8 is widely expressed and highly conserved among species, with the Chlamydomonas and human proteins sharing 93% sequence identity (2King S.M. Patel-King R.S. J. Biol. Chem. 1995; 270: 11445-11452Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar, 3King S.M. Barbarese E. Dillman III, J.F. Patel-King R.S. Carson J.H. Pfister K.K. J. Biol. Chem. 1996; 271: 19358-19366Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar, 4Wilson M.J. Salata M.W. Susalka S.J. Pfister K.K. Cell Motil. Cytoskeleton. 2001; 49: 229-240Crossref PubMed Scopus (40) Google Scholar). It also serves essential cellular functions. For instance, in Drosophila, a partial loss-of-function mutation in LC8 results in pleiotropic morphogenetic defects in bristle and wing development, female sterility, and disruption of sensory axon projections (5Dick T. Ray K. Salz H.K. Chia W. Mol. Cell Biol. 1996; 16: 1966-1977Crossref PubMed Scopus (150) Google Scholar, 6Phillis R. Statton D. Caruccio P. Murphey R.K. Development. 1996; 122: 2955-2963Crossref PubMed Google Scholar). Furthermore, a null mutation results in massive cell death via the apoptotic pathway and consequent embryonic death. In addition to being an essential component of the dynein motor complex, LC8 binds to a large number of proteins with diverse biological functions (7Fan J. Zhang Q. Tochio H. Li M. Zhang M. J. Mol. Biol. 2001; 306: 97-108Crossref PubMed Scopus (122) Google Scholar). For example, LC8 associates with and inhibits the activity of neuronal nitric-oxide synthase, giving rise to its alternative designation as PIN (protein inhibitor of neuronal nitric-oxide synthase) (8Jaffrey S.R. Snyder S.H. Science. 1996; 274: 774-777Crossref PubMed Scopus (423) Google Scholar). It also binds to IκBα (9Crepieux P. Kwon H. Leclerc N. Spencer W. Richard S. Lin R. Hiscott J. Mol. Cell Biol. 1997; 17: 7375-7385Crossref PubMed Google Scholar), an inhibitor of NF-κB 4The abbreviations used are:NF-κBnuclear factor κBTNFαtumor necrosis factor αIKKIκB kinaseROSreactive oxygen speciesBHAButylated hydroxyanisolDPIdiphenyleneiodoniumILinterleukinLPSlipopolysaccharideDTTdithiothreitolCM-H2DCFDA5-(and-6-)chloromethyl-2′,7′-dichlorodihydrofluorescein diacetateMnSODMn2+-dependent superoxide dismutaseAEBSF4-(2-aminoethyl)benzene-sulfonyl fluoridesiRNAsmall interfering RNAAMS4-acetamido-4′-maleimidylstilbene-2,2′-disulfonateNoxNADPH oxidaseTrxR1thioredoxin reductase 1p-phosphorylatedHAhemagglutinin. 4The abbreviations used are:NF-κBnuclear factor κBTNFαtumor necrosis factor αIKKIκB kinaseROSreactive oxygen speciesBHAButylated hydroxyanisolDPIdiphenyleneiodoniumILinterleukinLPSlipopolysaccharideDTTdithiothreitolCM-H2DCFDA5-(and-6-)chloromethyl-2′,7′-dichlorodihydrofluorescein diacetateMnSODMn2+-dependent superoxide dismutaseAEBSF4-(2-aminoethyl)benzene-sulfonyl fluoridesiRNAsmall interfering RNAAMS4-acetamido-4′-maleimidylstilbene-2,2′-disulfonateNoxNADPH oxidaseTrxR1thioredoxin reductase 1p-phosphorylatedHAhemagglutinin.;to Bim (Bcl-2-interacting mediator of cell death) and Bmf (Bcl-2-modifying factor) (10Puthalakath H. 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Chem. 2005; 280: 8172-8179Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). nuclear factor κB tumor necrosis factor α IκB kinase reactive oxygen species Butylated hydroxyanisol diphenyleneiodonium interleukin lipopolysaccharide dithiothreitol 5-(and-6-)chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate Mn2+-dependent superoxide dismutase 4-(2-aminoethyl)benzene-sulfonyl fluoride small interfering RNA 4-acetamido-4′-maleimidylstilbene-2,2′-disulfonate NADPH oxidase thioredoxin reductase 1 phosphorylated hemagglutinin. nuclear factor κB tumor necrosis factor α IκB kinase reactive oxygen species Butylated hydroxyanisol diphenyleneiodonium interleukin lipopolysaccharide dithiothreitol 5-(and-6-)chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate Mn2+-dependent superoxide dismutase 4-(2-aminoethyl)benzene-sulfonyl fluoride small interfering RNA 4-acetamido-4′-maleimidylstilbene-2,2′-disulfonate NADPH oxidase thioredoxin reductase 1 phosphorylated hemagglutinin. The transcription factor NF-κB is a key regulator of immune and inflammatory responses and exists as a homo- or heterodimer composed of members of the Rel/NF-κB family of proteins, including RelA (p65), RelB, c-Rel, NF-κB1 (p105/p50), and NF-κB2 (p100/p52) (15Hayden M.S. Ghosh S. Genes Dev. 2004; 18: 2195-2224Crossref PubMed Scopus (3345) Google Scholar). The most common form of NF-κB in mammalian cells is the heterodimer composed of RelA (p65) and p50 (16Baldwin Jr., A.S. Annu. Rev. Immunol. 1996; 14: 649-683Crossref PubMed Scopus (5552) Google Scholar). Under basal conditions, NF-κB is present in the cytoplasm as an inactive complex with the inhibitor protein IκB. However, in response to a variety of stimuli, including TNFα, interleukin (IL)-1β, and lipopolysaccharide (LPS), IκBα is phosphorylated at residues Ser32 and Ser36 by IKK and then ubiquitinated at residues Lys21 and Lys22, resulting in its degradation by the 26 S proteasome (15Hayden M.S. Ghosh S. Genes Dev. 2004; 18: 2195-2224Crossref PubMed Scopus (3345) Google Scholar). The degradation of IκBα exposes the nuclear localization signal of NF-κB, resulting in its translocation to the nucleus, where it regulates the transcription of various target genes that control the immune system, cell growth, and inflammation (17Pahl H.L. Oncogene. 1999; 18: 6853-6866Crossref PubMed Scopus (3427) Google Scholar). Recent findings suggest that ROS such as H2O2 activate NF-κB (18Gloire G. Legrand-Poels S. Piette J. Biochem. Pharmacol. 2006; 72: 1493-1505Crossref PubMed Scopus (1200) Google Scholar). The observations that potent NF-κB activators such as TNFα, IL-1β, and LPS trigger the production of ROS (19Kim Y.S. Morgan M.J. Choksi S. Liu Z.G. Mol. Cell. 2007; 26: 675-687Abstract Full Text Full Text PDF PubMed Scopus (412) Google Scholar, 20Vlahopoulos S. Boldogh I. Casola A. Brasier A.R. Blood. 1999; 94: 1878-1889Crossref PubMed Google Scholar, 21Li Q. Harraz M.M. Zhou W. Zhang L.N. Ding W. 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The molecular mechanisms underlying these observations remain poorly understood, however, and the contribution of redox regulation to NF-κB activation remains unclear because of some conflicting reports (26Bowie A. O'Neill L.A. Biochem. Pharmacol. 2000; 59: 13-23Crossref PubMed Scopus (818) Google Scholar, 27Hayakawa M. Miyashita H. Sakamoto I. Kitagawa M. Tanaka H. Yasuda H. Karin M. Kikugawa K. EMBO J. 2003; 22: 3356-3366Crossref PubMed Scopus (363) Google Scholar). We recently showed that a novel disulfide reductase, TRP14, inhibits TNFα-induced NF-κB activation by suppressing the phosphorylation of IκBα, and we identified LC8 as a potential substrate of TRP14 (28Jeong W. Chang T.S. Boja E.S. Fales H.M. Rhee S.G. J. Biol. Chem. 2004; 279: 3151-3159Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). The proteins β-arrestin and κB-Ras inhibit NF-κB by interacting with IκBα and IκBβ, respectively (29Witherow D.S. Garrison T.R. Miller W.E. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 8603-8607Crossref PubMed Scopus (216) Google Scholar, 30Fenwick C. Na S.Y. Voll R.E. Zhong H. Im S.Y. Lee J.W. Ghosh S. Science. 2000; 287: 869-873Crossref PubMed Scopus (88) Google Scholar, 31Huxford T. Ghosh G. Methods Enzymol. 2005; 407: 527-534Crossref Scopus (6) Google Scholar), indicating that IκB-binding proteins are potentially important regulators of NF-κB function. We have therefore investigated whether LC8 might serve as a molecular intermediary that links the disulfide reductase activity of TRP14 to NF-κB regulation. We now show that LC8 inhibits IκBα phosphorylation by IKK through its redox-dependent interaction with IκBα and that TRP14 regulates this inhibitory activity by maintaining LC8 in a reduced state. Reagents and Antibodies—LPS, butylated hydroxyanisol (BHA), diphenyleneiodonium (DPI), NADPH, and dithiothreitol (DTT) were obtained from Sigma; 4′,6-diamidino-2-phenylindole and 5-(and-6-)chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate (CM-H2DCFDA), acetyl ester were from Molecular Probes; MG132 was from Calbiochem; glutathione-Sepharose and nickel-chelating Sepharose were from Amersham Biosciences; and TNFα and IL-1β were from R & D Systems. Normal rabbit IgG was from Caltag Laboratories; mouse monoclonal and rabbit polyclonal antibodies to LC8 were from BD Biosciences and Phoenix Pharmaceuticals, respectively; rabbit antibodies to phosphorylated (p-) IκBα, to p-IKKα/β, and to IKKβ were from Cell Signaling Technology; a monoclonal antibody to p65 and rabbit polyclonal antibodies to IκBα and to IKKγ were from Santa Cruz Biotechnology; a monoclonal antibody to β-actin was from Abcam; rabbit polyclonal antibody to Mn2+-dependent superoxide dismutase (MnSOD) was from Upstate Biotechnology; a monoclonal antibody to the FLAG epitope was from Sigma; and a rat antibody to the hemagglutinin (HA) epitope was from Roche Applied Science. Horseradish peroxidase-conjugated goat antibodies to rabbit or mouse IgG were from Amersham Biosciences Bioscience, and Alexa Fluor 488-conjugated monoclonal IgG was from Molecular Probes. Cloning and Mutagenesis of Human LC8 cDNA—A mammalian expression vector for LC8, pFLAG-LC8, was described previously (28Jeong W. Chang T.S. Boja E.S. Fales H.M. Rhee S.G. J. Biol. Chem. 2004; 279: 3151-3159Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). Cysteine mutants of LC8 (C2S, C24S, and C56A, in which Cys2, Cys24, and Cys56 are individually replaced by serine or alanine) were generated with the use of a site-directed mutagenesis kit (Stratagene) and complementary primers containing a 1-bp mismatch that converts the codon for cysteine to one for serine or alanine. To express HA-LC8 or LC8-FLAG proteins, cDNAs for wild-type LC8 or cysteine mutants of LC8 were cloned into the XbaI and BamHI sites of pCGN or into the EcoRI and BamHI sites of pFLAG-CMV5.1, respectively. For expression of LC8 in bacteria, the human LC8 gene was cloned into the NdeI and EcoRI sites of pET15b or pET17b. Preparation of Recombinant Proteins—Escherichia coli BL21(DE3) transformed with pET17b-LC8 was cultured at 37 °C in LB medium supplemented with ampicillin (100 μg/ml). Isopropyl-β-d-thiogalactopyranoside was added to the culture at a final concentration of 0.4 mm when the optical density at 600 nm had reached 0.5. After incubation for an additional 3 h, the cells were harvested by centrifugation and stored at -70 °C until use. The frozen cells were suspended in a solution containing 20 mm Tris-HCl (pH 8.0), 1 mm EDTA, and 1 mm 4-(2-aminoethyl)benzene-sulfonyl fluoride (AEBSF) and were disrupted by sonication. After the removal of debris by centrifugation, the remaining soluble fraction was applied at a flow rate of 2 ml/min to a DEAE-Sepharose column that had been equilibrated with a solution containing 20 mm Tris-HCl (pH 8.0) and 1 mm EDTA. The flow-through fraction was collected and then applied to a gel filtration column (G3000SW; Tosoh Bioscience) that had been equilibrated with a solution containing 50 mm HEPES-NaOH (pH 7.0) and 0.1 m NaCl. The fractions containing LC8 were pooled and dialyzed against 10 mm HEPES-NaOH (pH 7.0). For the bacterial expression of IκBα, a DNA fragment encoding human IκBα was amplified by PCR from HeLa cell cDNA and cloned into the NdeI and BamHI sites of pET14b. His6-tagged IκBα was purified from lysates of the transformed E. coli cells by affinity chromatography on nickel-chelating Sepharose. In Vitro Kinase Assay—The phosphorylation of IκBα (1 μg) was performed for 30 min at 30 °C with an IKK immune complex in a final volume of 40 μl containing 50 mm Tris-HCl (pH 7.5), 1 mm EDTA, 5 mm DTT, 1 mm MgCl2, 1 mm Na3VO4, 5 mm glycerophosphate, 100 μm ATP, and 10 μCi of [γ-32P]ATP. IκBα and LC8 were incubated for 30 min at 30 °C before the addition of the IKK complex. The IKK complex was isolated by immunoprecipitation with antibody to IKKγ and protein A-Sepharose from HeLa cells that had been exposed to TNFα (20 ng/ml) for 10 min. The kinase reaction mixtures were fractionated by SDS-PAGE on a 4–20% gradient gel, and the radioactivity associated with IκBα band was quantified with the use of a phosphorimaging device (Molecular Dynamics). NF-κB Reporter Assay—NF-κB activity was measured with the use of a dual luciferase reporter assay system. The cells were transfected for 24 h with 0.25 μg of pNF-κB-Luc (NF-κB reporter plasmid; Stratagene), 0.25 μg of pRL-SV40 (internal control), and either pFLAG-CMV2 or pFLAG-LC8. The total amount of plasmid DNA was adjusted with pFLAG-CMV2. A dual luciferase assay was subsequently performed with a kit (Promega). The activity of firefly luciferase was normalized by that of the Renilla enzyme and was then expressed as fold increase relative to the normalized value for cells transfected with pFLAG-CMV2. Depletion of LC8 by RNA Interference—A small interfering RNA (siRNA) corresponding to nucleotides 315–333 (relative to the translation initiation site) of human LC8 cDNA (5′-GGACTGCAGCCTAAATTCC-3′) was synthesized with T7 RNA polymerase (32Donze O. Picard D. Nucleic Acids Res. 2002; 30: e46Crossref PubMed Scopus (240) Google Scholar) and was introduced into HeLa cells with the use of Oligofectamine (Invitrogen) as described (28Jeong W. Chang T.S. Boja E.S. Fales H.M. Rhee S.G. J. Biol. Chem. 2004; 279: 3151-3159Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). RNA Isolation, Reverse Transcription, and Real Time PCR Analysis—Cells that had been stimulated with TNFα (20 ng/ml) for 1 h were harvested, and total RNA was isolated1 with the use of the TRIzol reagent (Invitrogen) and quantified by measurement of absorbance at 260 nm. Reverse transcription was performed with 2 μg of total RNA and Moloney murine leukemia virus reverse transcriptase (Promega) for 1 h at 42 °C followed by 10 min at 70 °C, and the resulting cDNAs were subjected to real time PCR analysis with primers (sense and antisense, respectively) for human IκBα (5′-GCTGAAGAAGGAGCGGCTACT-3′ and 5′-TCGTACTCCTCGTCTTTCATGGAC-3′), human IL-8 (5′-ATGACTTCCAAGCTGGCCGT-3′ and 5′-TTACATAATTTCTGTGTTGGC-3′), human cyclooxygenase-2 (5′-CCTTCCTCCTGTGCCTGATG-3′ and 5′-ACAATCTCATTTGAATCAGGAAGCT-3′), and human β-actin (5′-ATGAGCTGCGTGTGGCTC-3′ and 5′-GGCGTACAGGGATAGCAC-3′). PCRs were performed with an ABI Prism 7300 sequence detection system and SYBRGreen PCR Master Mix (Applied Biosystems). Determination of Redox Status of LC8—HeLa cells in six-well plates were washed with ice-cold phosphate-buffered saline and exposed directly to 10% trichloroacetic acid to prevent further oxidation. The precipitates were washed with 10% trichloroacetic acid and with acetone and were then suspended in 100 μl of reaction buffer (100 mm Tris-HCl, pH 8.8, 1 mm EDTA, 1.5% SDS, 1 mm AEBSF, leupeptin (10 μg/ml), aprotinin (10 μg/ml)) containing 20 mm 4-acetamino-4′-maleimidylstilbene-2,2′-disulfonate (AMS) or 40 mm N-ethylmaleimide. The reaction mixtures were incubated for 90 min at 30 °C, after which the reaction was stopped by the addition of 25 μl of 5× nonreducing SDS-PAGE sample buffer. AMS and N-ethylmaleimide were used to mask free thiol groups. LC8 Interacts with IκBα and Thereby Inhibits Its Phosphorylation by IKK—Although LC8 was previously shown to interact physically with the regulatory domain of IκBα (9Crepieux P. Kwon H. Leclerc N. Spencer W. Richard S. Lin R. Hiscott J. Mol. Cell Biol. 1997; 17: 7375-7385Crossref PubMed Google Scholar), the physiological function of this association has remained unknown. We first confirmed the interaction between LC8 and IκBα in HeLa cells by immunoprecipitation with antibody to IκBα. Not only transiently expressed FLAG-LC8 but also endogenous LC8 was immunoprecipitated together with IκBα (Fig. 1A). To examine whether LC8 inhibits IκBα phosphorylation by IKK, we assayed the kinase activity of the immunoprecipitated IKK complex with recombinant IκBα as the substrate in the presence of LC8. LC8 indeed inhibited the phosphorylation of IκBα by the IKK complex (Fig. 1B). LC8 Expression Inhibits TNFα-induced NF-κB Activation— Given that LC8 inhibited IKK-mediated phosphorylation of IκBα in vitro, we next investigated the effect of forced expression of LC8 on the NF-κB signaling pathway in HeLa cells. Expression of FLAG-LC8 inhibited NF-κB activation by TNFα in a concentration-dependent manner (Fig. 2A) as well as attenuated the TNFα-induced nuclear translocation of RelA (p65) (Fig. 2B). To determine which step of the NF-κB signaling pathway is influenced by LC8, we transfected cells with an expression vector for LC8 together with those for IKKβ or p65. LC8 inhibited the increase in NF-κB activity induced by IKKβ overexpression but not that attributable to overproduction of p65 (Fig. 2C), indicating that LC8 inhibits the NF-κB signaling pathway at a step between IKK activation and p65 nuclear translocation. However, the IKK complexes immunoprecipitated from cells transfected with the LC8 expression vector or the corresponding empty vector showed no difference in kinase activity measured in vitro with recombinant IκBα as the substrate (Fig. 2D). These results thus suggested that the binding of LC8 to IκBα inhibits its phosphorylation by IKK, without perturbing the activity of IKK itself, in TNFα-treated cells. Depletion of LC8 Promotes TNFα-induced NF-κB Activation— To investigate the role of endogenous LC8 in TNFα-induced NF-κB activation, we depleted HeLa cells of LC8 by RNA interference and then monitored the expression of endogenous MnSOD, which is induced in response to NF-κB activation (33Xu Y. Kiningham K.K. Devalaraja M.N. Yeh C.C. Majima H. Kasarskis E.J. St Clair D.K. DNA Cell Biol. 1999; 18: 709-722Crossref PubMed Scopus (203) Google Scholar). The amount of LC8 was reduced by >90% after transfection of cells for 60 h with an siRNA specific for LC8 mRNA, compared with that apparent in cells transfected with a control RNA. Such depletion of LC8 resulted in a ∼2-fold increase in the abundance of MnSOD in unstimulated cells and a 1.8-fold increase in TNFα-stimulated cells (Fig. 3A). We also determined the effect of LC8 depletion on the transcriptional induction of the NF-κB target genes for IκBα, cyclooxygenase-2, and IL-8 by reverse transcription and real time PCR analysis. Depletion of LC8 markedly increased the amounts of the target gene mRNAs under both basal and TNFα-stimulated conditions (Fig. 3B). In addition, depletion of LC8 promoted the nuclear translocation of p65 stimulated by TNFα (Fig. 3C). These data indicated that depletion of LC8 augments TNFα-induced NF-κB activation. In response to activation signals such as TNFα, IκBα is phosphorylated by IKK and subsequently degraded by the ubiquitin-proteasome system. We therefore examined the effect of LC8 depletion on IκBα phosphorylation and degradation in HeLa cells stimulated with TNFα. Transfection of cells with LC8 siRNA substantially increased the extent of serine phosphorylation of IκBα compared with that apparent in cells transfected with a control RNA, and this effect was accompanied by an increase in the rate of IκBα degradation (Fig. 3, D and E). In contrast, depletion of LC8 had little effect on the phosphorylation status of IKK. These results suggested that LC8 blocks IκBα phosphorylation by IKK but does not inhibit the activity of IKK itself, consistent with the results shown in Fig. 2D. LC8 Inhibits NF-κB Activation by Other Stimuli That Activate IKK—Our results suggested that LC8 inhibits NF-κB activation by interacting with IκBα and thereby preventing its phosphorylation by IKK. Given that IL-1β and LPS also induce IκBα phosphorylation by IKK during activation of NF-κB (15Hayden M.S. Ghosh S. Genes Dev. 2004; 18: 2195-2224Crossref PubMed Scopus (3345) Google Scholar, 34Li N. Karin M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13012-13017Crossref PubMed Scopus (400) Google Scholar), we examined whether LC8 inhibits NF-κB activation by these stimuli. The NF-κB reporter assay showed that forced expression of LC8 inhibited NK-κB activation induced by IL-1β or LPS (Fig. 4A). We also examined the effects of endogenous LC8 on NF-κB activation by LPS or IL-1β. Depletion of LC8 by RNA interference increased MnSOD expression in unstimulated or LPS-stimulated HeLa cells (Fig. 4B), as well as in unstimulated or IL-1β-stimulated HEK293 cells (Fig. 4C). These results thus suggested that LC8 blocks IκBα phosphorylation by IKK in the canonical NF-κB activation pathway. LC8 Forms a Reversible Intermolecular Disulfide Bond between Cys2 Residues on Oxidation—LC8 was identified as a potential substrate for the novel disulfide reductase TRP14 in a substrate-trapping experiment on the basis of its formation of a mixed disulfide linkage with TRP14 (28Jeong W. Chang T.S. Boja E.S. Fales H.M. Rhee S.G. J. Biol. Chem. 2004; 279: 3151-3159Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar), suggesting that LC8 likely forms a disulfide bond on oxidation. LC8 exists as a dimer under physiological conditions (35Benashski S.E. Harrison A. Patel-King R.S. King S.M. J. Biol. Chem. 1997; 272: 20929-20935Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar), and the human protein contains three cysteine residues (Cys2, Cys24, and Cys56). To determine whether LC8 forms a disulfide bond on exposure of cells to H2O2, we analyzed its redox status by nonreducing SDS-PAGE after modification of free thiol groups with AMS. Alkylation of thiol groups with AMS increases the molecular mass of the host protein by 490 Da/thiol, resulting in a mobility shift on SDS-PAGE that allows determination of the number of oxidized cysteine residues (36Mezghrani A. Fassio A. Benham A. Simmen T. Braakman I. Sitia R. EMBO J. 2001; 20: 6288-6296Crossref PubMed Scopus (215) Google Scholar). AMS modification of LC8 in unstimulated HeLa cells gave rise to two LC8 bands with markedly retarded electrophoretic mobility in nonreducing SDS-PAGE gels (Fig. 5A); the lower of these two bands appeared to correspond to a fully reduced form of LC8 (LC8-AMS3), whereas the upper band seemed to represent an intermolecular disulfide-linked form (AMS2-LC8-S-S-LC8-AMS2). The intensity of the upper band was increased by exposure of the cells to H2O2 in a concentration-dependent manner. Under reducing conditions, the upper band was shifted to a position below the lower band (LC8-AMS3) that appeared to correspond to AMS2-LC8-SH (Fig. 5A). These results indicated that oxidation of LC8 results in the reversible formation of an intermolecular disulfide bond. We next investigated which of the three cysteine residues (Cys2, Cys24, or Cys56) of LC8 contribute to formation of the intermolecular disulfide bond. We substituted each cysteine residue with either serine or alanine by site-directed mutagenesis and then analyzed the oxidation status of the cysteine mutants in cells exposed to H2O2. Given that LC8 forms an intermolecular disulfide bond, we monitored the levels of disulfide-linked dimer by nonreducing SDS-PAGE after masking of free sulfhydryl groups with N-ethylmaleimide to prevent random disulfide bond formation. The C24S and C56A mutants formed the intermolecular disulfide bond, as did wild-type LC8, whereas the C2S mutant did not (Fig. 5B), suggesting that two Cys2 residues form the disulfide linkage on oxidation of LC8 to the homodimer. It was possible that the environment surrounding the Cys2 residue of recombinant LC8 was affected by the NH2-terminal HA tag, which may have led to artifactual results. To exclude this possibility, we examined the oxidation status of COOH-terminally tagged LC8 (LC8-FLAG). Substitution of Cys2 with serine also abolished intermolecular disulfide bond formation by LC8-FLAG (Fig. 5C). Interaction between LC8 and IκBα Is Redox-dependent—In addition to LC8 being identified as a potential substrate of TRP14, this disulfide reductase was shown to inhibit the TNFα-induced activation of NF-κB (28Jeong W. Chang T.S. Boja E.S. Fales H.M. Rhee S.G. J. Biol. Chem. 2004; 279: 3151-3159Abstract Full Text Full Text PDF PubMed Scopus (6
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