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FLASH Coordinates NF-κB Activity via TRAF2

FLASH Coordinates NF-κB Activity via TRAF2 THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 276, No. 27, Issue of July 6, pp. 25073–25077, 2001 © 2001 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. FLASH Coordinates NF-kB Activity via TRAF2* Received for publication, April 3, 2001, and in revised form, May 4, 2001 Published, JBC Papers in Press, May 4, 2001, DOI 10.1074/jbc.M102941200 Yun-Hee Choi‡, Ki-Bae Kim‡, Hyun-Hee Kim, Gil-Sun Hong, Yun-Kyung Kwon, Chul-Woong Chung, Yang-Mi Park, Zhong-Jian Shen, Byung Ju Kim, Soo-Young Lee§, and Yong-Keun Jung¶ From the Department of Life Science, Kwangju Institute of Science and Technology, Puk-gu, Kwangju 500-712 and the §Division of Molecular Life Science, Ewha University, Seoul 120-750, Korea FLASH is a protein recently shown to interact with caspase cascade (4, 8 –10). Similarly, Fas (CD95/APO-1) re- the death effector domain of caspase-8 and is likely to be cruits FADD to its activated receptor to induce apoptosis (11). a component of the death-inducing signaling complex in In contrast, with respect to stress signaling and immune receptor-mediated apoptosis. Here we show that anti- response, TNF-R interacts with TRAFs and RIP, leading to the sense oligonucleotide-induced inhibition of FLASH ex- activation of NF-kB. Whereas overexpression of the wild type a-induced activation of NF-kB pression abolished TNF- TRAF2, -5, or -6 activates NF-kB, their truncated versions in HEK293 cells, as determined by luciferase reporter lacking zinc-binding domains inhibit NF-kB activation induced kB responsive promoter. gene expression driven by a NF- by various stimuli (12–17). Whereas TRAF2 transduces TNF- Conversely, overexpression of FLASH dose-dependently a-mediated activation of NF-kB, TRAF6 is associated with kB, an effect suppressed by dominant neg- activated NF- interleukin-1 and CARD4 signaling (17, 18), indicating that a, and partially by ative mutants of TRAF2, NIK, and IKK TRAFs are common mediators for NF-kB activation and dis- those of TRAF5 and TRAF6. TRAF2 was co-immunopre- play an ability to stimulate signal-specific NF-kB activation. cipitated with FLASH from the cell extracts of HEK293 Subsequent activation of NIK, a member of the mitogen-acti- cells or HeLa cells stably expressing exogenous FLASH vated protein kinase family (4, 19, 20), and the downstream (HeLa/HA-FLASH). Furthermore, serial deletion map- kinases, IKKa and IKKb, leads to the phosphorylation of IkBs ping demonstrated that a domain spanning the residues for degradation and the activation of NF-kB (20 –25). kB as efficiently as the 856 –1191 of FLASH activated NF- It has recently been reported that FLASH is likely to be a full-length and could directly bind to TRAF2 in vitro and component of DISC involved in Fas- and TNF-mediated apo- in the transfected cells. Taken together, these results kB ac- ptosis (26). FLASH contains a death-effector-domain-recruiting suggest that FLASH coordinates downstream NF- a domain (DRD) in the C-terminal region, which interacts with tivity via a TRAF2-dependent pathway in the TNF- signaling. the death effector domain (DED) of caspase-8 or FADD (26). Still, transient overexpression of FLASH marginally affects apoptosis (26, 27), making its precise function with respect to TNF-a is a pleiotropic cytokine associated with various cel- receptor-mediated signaling (e.g. via TNF-R) unclear. In addi- lular defense responses, with lethal effects such as septic shock tion, the finding that caspase-8 and FADD may be involved in with inflammation, and with apoptosis in susceptible cells (1, the signaling to NF-kB activation as well as to apoptosis (28, 2). TNF-a signaling is transduced through its receptor, TNF-R, 29) suggests that FLASH may function to coordinate stress to simultaneously elicit two opposing effects: apoptosis and responses, a possibility that prompted us to investigate the role activation of an anti-apoptotic transcription factor NF-kB (3– of FLASH in NF-kB activation. In this report, we used an 5). During initiation of apoptosis, FADD is complexed with antisense oligonucleotide (AS) and overexpression analysis to activated TNF-R1 and TRADD via the death domain (4, 6, 7) show that FLASH transduces the TNF-a signal, leading to the and recruits caspase-8 to the resultant death-inducing signal- activation of NF-kB via a TRAF2-NIK-IKK-dependent pathway. ing complex (DISC), which leads to apoptosis via activation of a EXPERIMENTAL PROCEDURES Reagents—Anti-IkBa and anti-tubulin antibodies were purchased * This work was supported by the Molecular Medicine Research from Santa Cruz Biotechnology (Santa Crutz, CA) and Sigma, respec- Group Program (98-MM-01-01-A-03), the KOSEF (Protein Network Research Center), and the National Research Laboratory program (to tively. Anti-HA antibody and anti-TRAF2 antibody (SC-7346) were Y. K. J.). The costs of publication of this article were defrayed in part by from Roche Molecular Biochemicals (Mannheim, Germany) and Santa the payment of page charges. This article must therefore be hereby Cruz Biotechnology, respectively. TNF-a and all other molecular biol- marked “advertisement” in accordance with 18 U.S.C. Section 1734 ogy grade materials were from Sigma or New England Biolabs (Hert- solely to indicate this fact. fordshire, UK). ‡ Supported by the Brain Korea 21 project. These authors contributed Construction of Recombinant Expression Plasmids—pME18S-FLAG equally to this work. and pME18S-FLAG-FLASH were kindly provided by Dr. Yonehara To whom correspondence should be addressed: Department of Life (University of Kyoto, Japan). pHA-FLASH was generated by subcloning Science, Kwangju Institute of Science and Technology, 1 Oryong-dong, the FLASH cDNA into the EcoRI/XbaI sites of pcDNA-HA plasmid. Puk-gu, Kwangju 500-712, Korea. Tel.: 82-62-970-2492; Fax: 82-62-970- FLASH deletion constructs were assembled by polymerase chain reac- 2484; E-mail: [email protected]. tion (PCR) using the following synthetic oligonucleotides as primers: The abbreviations used are: TNF-a, tumor necrosis factor-a; FLASH, 59-CCGGAATTCATGGCAGATGATGACAATGGT-39 and 59-ATAA- FLICE-associated huge protein; AS, antisense oligonucleotide; DED, GAATGCGGCCGCCTAGCTCTCCATGCTAACAACT-39 for pME18S- death effector domain; DRD, DED-recruiting domain; NAD, NF-kB- FLAG-DA-(1– 858) (pFL-DA-FLASH); 59-CCGGAATTCATGGAGAGCT- activating domain; DISC, death-inducing signaling complex; RT-PCR, CATGTGCAATT-39 and 59-ACCGGGCCCCTATCCAGTTCTAGGCAA- reverse transcription-polymerase chain reaction; PAGE, polyacrylamide AGA-39 for pcDNA-HA-DB-(856 –1552) (pHA-DB-FLASH); 59-CCGGA- gel electrophoresis; HA, hemagglutinin; b-gal, b-galactosidase; HEK, hu- ATTCATGGCAGATGATGACAATGGT-39 and 59-ATAAGAATGCGGC- man embryonic kidney cells; GST, glutathione S-transferase; TRAF, TNF receptor-associated factor; NIK, NF-kB-inducing kinase; Ikk, IkB kinase. CGCCTACAGTGAAGATTTAAAATTC-39 for pME18S-FLAG-DC- This paper is available on line at http://www.jbc.org 25073 This is an Open Access article under the CC BY license. 25074 NF-kB Activation by FLASH fected plasmid DNA was kept constant within individual experiments by adding appropriate amounts of pcDNA or pME18S. HeLa cells stably expressing HA-FLASH (HeLa/HA-FLASH) were generated as described by Chung et al. (38). Antisense Oligonucleotide Treatment—AS-2 (59-ATTCAGCAACT- TACTTGC-39) is an antisense oligonucleotide complementary to human FLASH mRNA and corresponds to a location around the stop codon, 5942–5959 bp downstream of the translation initiation site. Compari- son of this oligonucleotide sequence with the database detected the only homology to the FLASH sequence. The following scrambled sequence was used as a control: (59-GCTACTAGTAGCAGCTAC-39). Cells (3 3 10 per well) were continuously treated with 5 mM FLASH antisense or the scrambled oligonucleotide for 48 h in culture medium containing LipofectAMINE reagent. RNA Isolation and RT-PCR—Total RNA was isolated from HEK293 cells using TRIzol reagent (Life Technologies, Inc.). RT-PCR was per- formed for quantification of FLASH mRNA using b-actin mRNA as a control. Two sets of oligonucleotides were designed; 59-TAGGTGCTTT- TATTGACTTGACACAA-39 (sense) and 59-CAGGAATTCAGCAACT- TATCTGCAT-39 (antisense) (predicted product length: 725 bp) for FLASH primer 1 and 59-GAAGGTAATCATCCTGCATTAGCTGT-39 (sense) and 59-GAGCTTCATTAGCTGCTGGAATCTT-39 (antisense) (predicted product length: 714 bp) for FLASH primer 2. The nucleotide sequences of the b-actin primers were 59-CAACCGCGAGAAGAT- GACCC-39 (sense) and 59-GAAGGAAGGCTGGAAGAGTG-39 (anti- sense) (predicted product length: 457 base pairs). The PCR products were confirmed by DNA sequencing. Luciferase and b-Galactosidase Assays—Cells were harvested 24 h after transfection, and luciferase activities in the cell extracts were determined using a luciferase assay system (Promega). To measure b-galactosidase activity, the cell extracts were mixed with equal amounts of b-galactosidase assay buffer (23) containing 200 mM so- dium phosphate (pH 7.3), 2 mM MgCl , 100 mM b-mercaptoethanol, and 1.33 mg/ml O-nitrophenyl-b-D-galactopyranoside and incubated at 37 °C for 30 min. The absorbance at 420 nm was then measured using an ELISA reader (Molecular Device, Sunnyvale, CA). FIG.1. Suppression of TNF-a-induced activation of NF-kB us- Generation of Anti-FLASH Antibody and Western Blot Analysis— ing a FLASH antisense oligonucleotide. HEK293 cells were co- GST-DRD-FLASH fusion proteins were expressed in BL21(DE3) by transfected for 36 h with NF-kB-luciferase reporter plasmid (pNF-kB- addition of 0.2 mM isopropyl-b-D-thiogalactoside, purified using gluta- luc), pCMV-b-gal, and either 5 mM FLASH AS-2 or a scrambled thione-Sepharose 4B (Amersham Pharmacia Biotech) and administered oligonucleotide (random), and were then incubated with TNF-a (30 into a rabbit in a series of four injections. Anti-FLASH antibody was ng/ml) for an additional 26 h. A, activity of luciferase reporter genes was purified from the serum by antigen-affinity chromatography. For West- normalized to that of b-galactosidase, which served as an internal ern blot analysis, cell lysates were prepared, and protein concentrations control. Bars represent mean 6 S.D. from at least four independent experiments. B, HEK293 cells were lysed, and Western blotting was were determined using a DC protein assay kit (Bio-Rad). Western performed with anti-IkBa antibody. For an internal control, the same blotting was then carried out as previously described (31); proteins were extracts were probed with antibody to a-tubulin. C, reverse transcrip- visualized using an enhanced chemiluminescence system (ECL, Amer- tion of RNA isolated from cells treated with scrambled (Ran) or AS-2 sham Pharmacia Biotech). (AS) oligonucleotides. The PCR reaction was carried out with two dif- In Vitro Binding Assay—The expression of GST fusion proteins in ferent sets of FLASH primers. PCR of b-actin was performed to nor- BL21 (DE3) harboring pGEX-4T, pGEX-DRD-FLASH, or pGEX-NAD- malize FLASH expression. FLASH was induced with 0.2 mM isopropyl-b-D-thiogalactoside during exponential growth. Harvested cells were resuspended and lysed by (1–1191) (pFL-DC-FLASH); 59-CCGGAATTCATGGAGAGCTGCTCAT- sonication in 50 mM Tris-HCl buffer (pH 7.4) containing 1 mM dithio- GTGCAATT-39 and 59-CATTTAGGTGACACTA-39 for pME18S-FLAG- threitol, 0.5 mM EDTA, and 10% (v/v) glycerol. The supernatant lysates DD-(1553–1962) (pFL-DD-FLASH); 59-CCGGAATTCATGGAGAGCTC- were incubated with glutathione-Sepharose 4B. TRAF2, or caspase-8 ATGTGCAATT-39 and 59-CATTTAGGTGACACTA-39 for pME18S- labeled with [ S]methionine using the TNT system (Promega) were FLAG-DE-(856 –1962) (pFL-DE-FLASH). The PCR products were then then added to GST fusion proteins (20 mg each) coupled to glutathione- inserted into the EcoRI/NotI sites of pME18S-FLAG (DA, DC, DD, and Sepharose 4B in a final volume of 500 ml of binding buffer (50 mM DE), the EcoRI/ApaI sites of pcDNA3-HA (pHA-DB-FLASH), or the Tris-HCl, pH 7.4, 1 mM dithiothreitol, 0.5 mM EDTA, 0.01% Triton EcoRI/NotI sites of pcDNA3-HA (pHA-DD-FLASH). GST-NAD-FLASH X-100, 0.5 mg/ml bovine serum albumin, and 10% (v/v) glycerol). After and GST-DRD-FLASH fusion proteins were generated by subcloning being incubated at 4 °C for 2 h with gentle mixing, the beads were PCR products amplified by 59-CGCGGATCCCCTAGAGTTTCTGCTG- washed three times with the binding buffer, separated by 12% SDS- AA-39 and 59-CCGCTCGAGTTACAGTGAAGATTTAAATT-39 for pGST- PAGE, and detected by autoradiography. NAD-FLASH and 59-CGCGGATCCGATAAGAGTAAACTAACTC-39 Immunoprecipitation—HEK293 cells were transfected with pHA- and 59-CCGCTCGAGTTATTCACAGGAGCCAGGAGA-39 for pGST- FLASH, pHA-DB-FLASH, and pTRAF2 plasmids and lysed in radioim- DRD-FLASH into the BamHI/XhoI sites of pGEX4T-3 (Amersham mune precipitation buffer (50 mM Tris-HCl, pH 7.4, 1% Nonidet P-40, Pharmacia Biotech.). All PCR products were confirmed by DNA se- 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EDTA, 1 mM phenyl- quencing. pTRAF2, pTRAF5, pTRAF6, pFL-NIK, pCR3.1-IKKa, domi- methylsulfonyl fluoride, 1 mg/ml each of aprotinin, leupeptin, and pep- nant negative forms of TRAF2, TRAF5, TRAF6, NIK, and IKKa, pNF- statin, 1 mM Na VO ,and1mM NaF). FLASH was immunoprecipitated 3 4 kB-luc, and pCasp8 were described previously (30, 37). from cell lysates after incubation with anti-FLASH and anti-HA anti- Cell Culture, Stable Cells, and DNA Transfection—HEK293 and bodies and protein-A-coupled-Sepharose CL-4B (Amersham Pharmacia Jurkat cells were cultured in Dulbecco’s modified Eagle’s medium and Biotech.) at 4 °C for 2 h. TRAF2, HA-FLASH, and HA-DB-FLASH were RPMI 1640, respectively, supplemented with 10% fetal bovine serum then detected by Western blot analysis using anti-TRAF2 and anti-HA (BIOFLUIDS). Cells were subcultured to a density of 2 3 10 cells/well monoclonal antibodies, respectively. in 6-well dishes and allowed to stabilize for 1 day. Cells were then typically transfected with 600 ng of NF-kB-luciferase reporter plasmid RESULTS (pNF-kB-luc), 200 ng of pCMV-b-gal, and 1 mg of vector or the indicated Suppression of TNF-a-induced NF-kB Activation by a expression plasmid using LipofectAMINE according to the manufactur- er’s instructions (Life Technologies, Inc.). The total amount of trans- FLASH Antisense Oligonucleotide—To identify a role for NF-kB Activation by FLASH 25075 FIG.2. NF-kB activation induced by FLASH expression. HEK293 cells were transfected for 36 h with pNF-kB-luc, pCMV-b-gal and the indicated amounts of pFLASH, pTRAF2, or pHA-DD-FLASH. A, relative NF-kB-driven luciferase activities in transfectants express- ing FLASH, TRAF2, or DD-FLASH; activity in the control cells was arbitrarily set to a value of 1. B, Western blots showing the expression levels of exogenous FLASH (anti-FLASH antibody), DD-FLASH (anti- FIG.3. Mapping of the FLASH domain responsible for NF-kB HA antibody), and TRAF2 (anti-TRAF2 antibody). activation. A, schematic diagrams of full-length FLASH and its dele- tion (D) constructs. The death-effector domain-recruiting domain (DRD) and a putative NF-kB-activating domain (NAD) are indicated. B, HEK293 cells were co-transfected with pNF-kB-luc and either pcDNA FLASH in TNF-a signaling, we initially examined its contri- (control), full-length FLASH, or deletion constructs. One day later, bution to NF-kB activation by directly targeting FLASH ex- luciferase reporter gene assays were performed and for each construct, pression using an AS. Four different ASs were synthesized luciferase activities were adjusted so that the control was 1. Bars indicate mean 6 S.D. of the induction of NF-kB activity relative to the based on the nucleotide sequence of human FLASH and when control from at least four independent experiments. C, HEK293 cells examined, they showed essentially similar effects on TNF-a- were left untreated, treated with TNF-a for 1 h, or transfected with signaling (data not shown). Treating HEK293 cells with AS-2, pcDNA-HA, pHA-FLASH, pHA-DB-FLASH, pFL-DD-FLASH, or but not with a scrambled oligonucleotide (as a negative con- pTRAF2. After 1 day, cell extracts were prepared and analyzed by trol), abolished TNF-a-induced activation of NF-kB, as as- Western blotting with anti-IkBa antibody. sessed by luciferase reporter gene expression driven by a NF-kB responsive promoter (Fig. 1A), and also suppressed effects of several FLASH deletion mutants (Fig. 3A, DA–DE) TNF-a-induced degradation of IkBa (Fig. 1B). were examined. Expression of these deletions was confirmed in Because we could detect exogenous FLASH expression (Fig. the transfected HEK293 cells by Western blot analysis using 2B) but failed to detect expression of endogenous FLASH with anti-HA or anti-FLAG antibodies (data not shown). Determi- Western blot analysis using anti-FLASH antibody, the effects nation of NF-kB activity following the respective expression of of AS-2 or the scrambled oligonucleotide on FLASH expression each of these constructs showed that the truncation in the DB-, were assessed by RT-PCR (Fig. 1C). RT-PCR and Northern blot DC-, and DE-FLASH constructs had no effect on the ability of analysis have both been used to examine the effects of anti- FLASH to activate NF-kB (Fig. 3B). The DD-FLASH, by con- sense on gene expression (32). FLASH mRNA was undetectable trast, completely abolished NF-kB activation, whereas the DA- in HEK293 cells treated with AS-2, whereas tubulin expression FLASH partially induced NF-kB activity (Fig. 3B). Because the was unaffected, and readily detectable in cells treated with the DB-, DC-, and DE-FLASH contain a common region including a scrambled oligonucleotide, indicating that FLASH expression putative oligomerization domain, the region responsible for the was reduced by AS-2 treatment. These results suggest that activation of NF-kB apparently spans most of the oligomeriza- FLASH is involved in TNF-a-induced activation of NF-kB. tion domain and part of the DA-FLASH domain (Fig. 3A). We Activation of NF-kB by FLASH Expression and Domain designate the common region in the DB-, DC-, and DE-FLASH Mapping for NF-kB Activation—To more directly assess the spanning residues 856 –1191 as the NF-kB activation domain role of FLASH in the activation of NF-kB signaling, we exam- (NAD) of FLASH. ined an effect of its overexpression on NF-kB activity in Because it is known that TNF-a treatment of cells leads to HEK293 cells. We found that, indeed, NF-kB activity was dose- the activation of NF-kB through the phosphorylation and deg- dependently related to FLASH expression (Fig. 2A). The rela- radation of IkBa (33, 34), exposure of HEK293 cells to TNF-a tive levels of expression of FLASH and TRAF2 were confirmed resulted in the degradation of IkBa (Fig. 3C, left panel). Tran- by Western blot analysis (Fig. 2B). sient expression of FLASH, TRAF2, or DB-FLASH led to the FLASH contains DRD at its C terminus and a putative degradation of IkBa, whereas overexpression of the DD-FLASH oligomerization domain at its N terminus (Fig. 3A) (26). To did not affect the degradation of IkBa (Fig. 3C, right panel), ascertain which of these mediates induction of NF-kB activity, consistent with the results of NF-kB activity assays in Fig. 3B. 25076 NF-kB Activation by FLASH FIG.5. In vitro binding of NAD to TRAF2. GST, GST-NAD, and GST-DRD fusion proteins were purified from Escherichia coli. Proteins bound to their affinity resins (each equivalent to 20 mg of protein) were incubated with TRAF2 or caspase-8 labeled with [ S]methionine as described under “Experimental Procedures.” After separation by 12% SDS-PAGE, the bound proteins were detected by autoradiography (up- per panels), and resin-coupled proteins were visualized by Western blotting with anti-GST polyclonal antibody (lower panels). FIG.4. Effects of various dominant negative mutants on FLASH-induced activation of NF-kB. A, HEK293 cells were left untreated or treated with TNF-a after co-transfection with pNF-kB-luc and either pcDNA (control) or expression plasmids encoding the wild type or dominant negative mutants (D/N) of the indicated mediators in the presence or absence of pHA-FLASH. One day later, luciferase activities were measured and normalized to that of b-galactosidase. B, HEK293 cells were co-transfected with pNF-kB-luc and the indicated dominant negative mutants. The cells were then exposed to TNF-a (30 ng/ml) for 6 h, after which luciferase activities were measured. These results demonstrate and confirm that FLASH expression through NAD in DB-FLASH induces NF-kB activation by the degradation of IkBa. FLASH Signals NF-kB Activation through TRAF2-NIK- IKKs—It has previously been shown that various signaling pro- teins downstream of TNF-R, including TRADD, TRAFs, RIP, NIK, FIG.6. Cellular interaction of FLASH with TRAF2. A, proteins from Jurkat cell extracts were immunoprecipitated (IP), separated by and IKKs, are involved in TNF-a-induced activation of NF-kB. We SDS-PAGE, and immunoblotted with preimmune (pre) or anti-TRAF2 therefore examined the respective roles of these proteins in monoclonal antibody. B, proteins from HeLa cell permanently expressing FLASH-induced activation of NF-kB. Expression of dominant neg- HA-FLASH (HeLa/HA-FLASH) were immunoprecipitated with anti-HA ative mutants of TRAF2-(87–501), NIK (K429A,K430A), or IKKa antibody and probed with anti-TRAF2 antibody (upper panel) or anti-HA antibody (lower panel). C, HEK293 cells were transiently transfected with (K44A) inhibited NF-kB activation induced by FLASH (Fig. 4A)or pHA-FLASH in the presence or absence of pTRAF2. One day later, pro- TNF-a (Fig. 4B). On the other hand, although dominant negative teins were immunoprecipitated with anti-FLASH antibody or anti-HA mutants of TRAF5-(205–558) and TRAF6-(289 –530) partially sup- antibody and immunoblotted with anti-TRAF2 antibody. D, HEK293 cells pressed TNF-a- or FLASH-induced activation of NF-kB (Fig. 4B), were co-transfected with pHA-DB-FLASH and pTRAF2, immunoprecipi- tated with anti-HA antibody, and immunoblotted with anti-TRAF2 (upper they showed little effect on both TNF-a- and FLASH-induced acti- panels) or anti-HA antibody (lower panels). vation of NF-kB (Fig. 4, A and B), which thus appears to be mainly mediated via a TRAF2-NIK-IKK-pathway. FLASH Interacts with TRAF2 in Vitro and in Vivo through TRAF2 in Jurkat, HEK 293, and HeLa cells. Immunoprecipi- NAD—That FLASH interacts with TRAF2 was then examined tation with anti-FLASH antibody and subsequent Western blot with in vitro binding assay and immunoprecipitation. GST analysis using anti-TRAF2 antibody revealed that endogenous pull-down assay showed that GST-NAD fusion protein could TRAF2 was co-precipitated with endogenous FLASH, but not bind to TRAF2, whereas GST-DRD interacted only with by preimmune serum (Fig. 6A). Endogenous FLASH could not caspase-8, indicating that NAD of FLASH specifically interacts be detected with Western blot analysis using anti-FLASH an- with TRAF2 in vitro (Fig. 5). Immunoprecipitation assays were tibody in the immunoprecipitates probably because of its low then performed to examine cellular interaction of FLASH and expression level in Jurkat cells. We have then generated HeLa NF-kB Activation by FLASH 25077 cells stably expressing exogenous FLASH tagged with HA ptosis and also of the protein complex including TRAFs leading to (HeLa/HA-FLASH) and further examined the intracellular in- NF-kB signaling, FLASH needs to be further characterized for the teractions. Immunoprecipitation with anti-HA antibody fol- stoichiometry of protein-protein interactions and for a fine-tuning lowed by Western blotting using anti-TRAF2 antibody or an- activity balancing survival and apoptosis. ti-HA antibody showed the presence of TRAF2 and HA-FLASH Acknowledgments—We thank Dr. S. Yonehara (University of Kyoto, in the immunoprecipitates (Fig. 6B). These results indicate Japan) for FLASH cDNA and Dr. N. Spoerel for critical reading of this that FLASH interacts with TRAF2 in the cells. manuscript. We have then examined cellular interaction of NAD of FLASH with TRAF2. HEK293 cells were transfected with REFERENCES pHA-FLASH (Fig. 6C) or pHA-DB-FLASH (Fig. 6D)inthe 1. Beg, A. A., and Baltimore, D. (1996) Science 274, 782–784 2. Serfas, M. S., Goufman, E., Feuerman, M. H., Gartel, A. L., and Tyner, A. L. presence or absence of TRAF2. Immunoprecipitation and West- (1997) Cell Growth and Differ. 8, 951–961 ern blot analysis showed that endogenous (Fig. 6C, middle 3. Hsu, H., Xiong, J., and Goeddel, D. V. (1995) Cell 81, 495–504 panel) or exogenous TRAF2 (Fig. 6C, right panel) was co-pre- 4. Hsu, H., Shu, H. B., Pan, M. G., and Goeddel, D. V. (1996) Cell 84, 299 –308 5. Wang, C. Y., Mayo, M. W., Korneluk, R. G., Goeddel, D. V., and Baldwin Jr., cipitated with FLASH by anti-FLASH antibody or anti-HA A. S. (1998) Science 281, 1680 –1683 antibody. As expected from in vitro binding assays, DB-FLASH 6. Shu, H. B., Takeuchi, M., and Goeddel, D. V. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 13973–13978 was co-immunoprecipitated with TRAF2 by anti-HA antibody 7. Hu, W. H., Johnson, H., and Shu, H. B. (2000) J. Biol. Chem. 275, from lysates of HEK293 cells transfected with both pHA-DB- 10838 –10844 FLASH and pTRAF2 (Fig. 6D). These results suggest that 8. Boldin, M. P., Goncharov, T. M., Goltsev, Y. V., and Wallach, D. (1996) Cell 85, 803– 815 FLASH may directly interact with TRAF2 through its NAD. 9. Muzio, M., Chinnaiyan, A. M., Kischkel, F. C., O’Rourke, K., Shevchenko, A., Ni, J., Scaffidi, C., Bretz, J. D., Zhang, M., Gentz, R., Mann, M., Krammer, DISCUSSION P. H., Peter, M. E., and Dixit, V. M. (1996) Cell 85, 817– 827 10. Yuan, J., Shaham, S., Ledoux, S., Ellis, H. M., and Horvitz, H. R. (1993) Cell Previous studies have suggested that TNF-R-mediated apoptosis 75, 641– 652 and NF-kB activation pathways pass through TRADD, a death 11. Boldin, M. P., Mett, I. L., Varfolomeev, E. E., Chumakov, I., Shemer-Avni, Y., domain-containing adaptor protein interacting with TNF-R in a Camonis, J. H., and Wallach, D. (1995) J. Biol. Chem. 270, 387–391 12. Coa, Z., Xiong, J., Takeuchi, M., Kurama, T., and Goeddel, D. V. (1996) Nature TNF-dependent process; TRADD-FADD-caspase-8 and TRADD- 383, 443– 446 TRAF2-RIP-NIK-IKKs cascades lead to the induction of apoptosis 13. Rothe, M., Sarma, V., Dixit, V. M., and Goeddel, D. V. (1995) Science 269, 1424 –1427 and activation of an anti-apoptotic transcription factor NF-kB, re- 14. Nakano, H., Oshima, H., Chung, W., Williams-Abbott, L., Ware, C. F., Yagita, spectively. Whereas FLASH may be required for the activation of H., and Okumura, K. (1996) J. Biol. Chem. 271, 14661–14664 caspase-8 during Fas- and TNF-R-mediated apoptosis (26, 27), the 15. Ishida, T., Mizushima, S., Azuma, S., Kobayashi, N., Tojo, T., Suzuki, K., Aizawa, S., Watanabe, T., Mosialos, G., Kieff, E., Yamamoto, T., and Inoue, data presented here provide the first evidence for involvement of J. (1996) J. Biol. Chem. 271, 28745–28748 FLASH in NF-kB activation by TNF-R. The physical interaction of 16. Ishida, T., Tojo, T., Aoki, T., Kobayashi, N., Ohishi, T., Watanabe, T., FLASH with TRAF2, demonstrated here in vitro and in vivo (Figs. Yamamoto, T., and Inoue, J. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 9437–9442 5 and 6), lends further support to the idea that FLASH transduces 17. Ling, L., and Goeddel, D. V. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, TNF-a signals via a TRAF2-dependent pathway of NF-kB activa- 9567–9572 18. Bertin, J., Nir, W. J., Fischer, C. M., Tayber, O. V., Errada, P. R., Grant, J. R., tion. Though dominant negative mutants of TRAF5 and 6 also Keilty, J. J., Gosselin, M. L., Robison, K. E., Wong, G. H. W., Glucksmann, partially suppressed FLASH- and TNF-a-mediated activation of M. A., and DiStefano, P. S. (1999) J. Biol. Chem. 274, 12955–12958 NF-kB (Fig. 4, A and B), complex formation of FLASH with addi- 19. Liu, Z. G., Hsu, H., Goeddel, D. V., and Karin, M. (1996) Cell 87, 565–576 20. Malinin, N. L., Boldin, M. P., Kovalenko, A. V., and Wallach, D. (1997) Nature tional signal mediators leading to NF-kB signaling such as other 385, 540 –544 TRAFs, TRADD, or RIP remains to be elucidated. 21. Didonato, J. A., Hayakawa, M., Rothwarf, D. M., Zandi, E., and Karin, M. (1997) Nature 388, 548 –554 FLASH as a component of apoptotic signaling complexes is likely 22. Mercurio, F., Zhu, H., Murray, B. W., Shevchenko, A., Bennett, B. L., Li, J. W., to mediate apoptosis signals probably triggered by cell surface Young, D. B., Barbosa, M., Mann, M., Manning, A., and Rao, A. (1997) receptors. However, the fact that FLASH activates NF-kB may Science 278, 860 – 866 23. Re ´ gnier, C. H., Song, H. Y., Gao, X., Goeddel, D. V., Cao, Z., and Rothe, M. explain the observation that in many cell types, TNF treatment did (1997) Cell 90, 373–383 not induce apoptosis in the absence of gene expression. With re- 24. Woronicz, J. D., Gao, X., Cao, Z., Rothe, M., and Goeddel, D. V. (1997) Science 278, 866 – 870 spect to the role of FLASH as an activator interacting with 25. Zandi, E., Rothwarf, D. M., Delhase, M., Hayakawa, M., and Karin, M. (1997) caspase-8 and FADD, the lack of a significant increase of apoptosis Cell 91, 243–252 following overexpression of both FLASH and caspase-8 or the in- 26. Imai, Y., Kimura, T., Murakami, A., Yajima, N., Sakamaki, K., and Yonehara, S. (1999) Nature 398, 777–785 ability of TNF-a to induce apoptosis in a subset of tumor cells may 27. Medema, J. P. (1999) Nature 398, 756 –757 be attributed to FLASH-mediated activation of NF-kB. NAD-me- 28. Inohara, N., Koseki, T., Lin, J., Peso, L., Lucas, P. C., Chen, F. F., Ogura, Y., diated activation of NF-kB may antagonize DRD-mediated apo- and Nu ´n ˜ ez, G. (2000) J. Biol. Chem. 275, 27823–27831 29. Newton, K., Harris, A. W., Bath, M. L., Smith, K. G. C., and Strasser, A. (1998) ptotic signals by encoding inhibitory proteins such as IAPs and EMBO J. 17, 706 –718 IEX-1L (5, 35, 36). Moreover, recent reports that FADD and 30. Wong, B. R., Josien, R., Lee, S. Y., Vologodskaia, M., Steinman, R. M., and Choi, Y. (1998) J. Biol. Chem. 273, 28355–28359 caspase-8 may be required for cell survival and proliferation during 31. Jung, Y., Miura, M., and Yuan, J. (1996) J. Biol. Chem. 271, 5112–5117 heart and thymus development may be explained by our observa- 32. Vassar, R., Bennett, B. D., Babu-Khan, S., Kahn, S., Mendiaz, E. A., Denis, P., Teplow, D. B., Ross, S., Amarante, P., Loeloff, R., Luo, Y., Fisher, S., Fuller, tions of FLASH-mediated activation of NF-kB. This speculation is J., Edenson, S., Lile, J., Jarosinski, M. A., Biere, A. L., Curran, E., Burgess, reinforced by our observation that FLASH transduced NF-kB sig- T., Louis, J. C., Collins, F., Treanor, J., Rogers, G., and Citron, M. (1999) naling evoked by caspase-8 (data not shown). Science 286, 735–741 33. Baeuerle, P. A., and Henkel, T. (1994) Annu. Rev. Immunol. 12, 141–179 FLASH seems to be an upstream component of various receptor- 34. Reuther, J. Y., and Baldwin, A. S. (1999) J. Biol. Chem. 274, 20664 –20670 mediated signals including TNF-a and most likely has a dual 35. LaCasse, E. C., Baird, S., Korneluk, R. G., and MacKenzie, A. E. (1998) function in apoptosis and NF-kB signaling. We have additional Oncogene 17, 3247–3259 36. Chu, Z. L., McKinsey, T. A., Liu, L., Genty, J. J., Malim, M. H., and Ballard, evidences that FLASH is also an indispensable component in re- D. W. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 10057–10062 ceptor-mediated apoptosis. As a component of DISC during apo- 37. Kim, I. K., Chung, C. W., Woo, H. N., Hong, G. S., Nagata, S. J., and Jung, Y. K. (2000) Biochem. Biophys. Res. Commun. 277, 311–316 38. Chung, C. W., Song, Y. H., Kim, I. K., Yoon, W. J., Ryu, B. R., Jo, D. G., Woo, Y. H. Choi, K. B. Kim, B. J. Kim, and Y. K. Jung, manuscript in H. N., Kwon, Y. K., Kim, H. H., Gwag, B. J., Mook-Jung, I. H., and Jung, preparation. Y. K. (2001) Neurobiol. Disease 8, 162–172 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Biological Chemistry Unpaywall

FLASH Coordinates NF-κB Activity via TRAF2

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THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 276, No. 27, Issue of July 6, pp. 25073–25077, 2001 © 2001 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. FLASH Coordinates NF-kB Activity via TRAF2* Received for publication, April 3, 2001, and in revised form, May 4, 2001 Published, JBC Papers in Press, May 4, 2001, DOI 10.1074/jbc.M102941200 Yun-Hee Choi‡, Ki-Bae Kim‡, Hyun-Hee Kim, Gil-Sun Hong, Yun-Kyung Kwon, Chul-Woong Chung, Yang-Mi Park, Zhong-Jian Shen, Byung Ju Kim, Soo-Young Lee§, and Yong-Keun Jung¶ From the Department of Life Science, Kwangju Institute of Science and Technology, Puk-gu, Kwangju 500-712 and the §Division of Molecular Life Science, Ewha University, Seoul 120-750, Korea FLASH is a protein recently shown to interact with caspase cascade (4, 8 –10). Similarly, Fas (CD95/APO-1) re- the death effector domain of caspase-8 and is likely to be cruits FADD to its activated receptor to induce apoptosis (11). a component of the death-inducing signaling complex in In contrast, with respect to stress signaling and immune receptor-mediated apoptosis. Here we show that anti- response, TNF-R interacts with TRAFs and RIP, leading to the sense oligonucleotide-induced inhibition of FLASH ex- activation of NF-kB. Whereas overexpression of the wild type a-induced activation of NF-kB pression abolished TNF- TRAF2, -5, or -6 activates NF-kB, their truncated versions in HEK293 cells, as determined by luciferase reporter lacking zinc-binding domains inhibit NF-kB activation induced kB responsive promoter. gene expression driven by a NF- by various stimuli (12–17). Whereas TRAF2 transduces TNF- Conversely, overexpression of FLASH dose-dependently a-mediated activation of NF-kB, TRAF6 is associated with kB, an effect suppressed by dominant neg- activated NF- interleukin-1 and CARD4 signaling (17, 18), indicating that a, and partially by ative mutants of TRAF2, NIK, and IKK TRAFs are common mediators for NF-kB activation and dis- those of TRAF5 and TRAF6. TRAF2 was co-immunopre- play an ability to stimulate signal-specific NF-kB activation. cipitated with FLASH from the cell extracts of HEK293 Subsequent activation of NIK, a member of the mitogen-acti- cells or HeLa cells stably expressing exogenous FLASH vated protein kinase family (4, 19, 20), and the downstream (HeLa/HA-FLASH). Furthermore, serial deletion map- kinases, IKKa and IKKb, leads to the phosphorylation of IkBs ping demonstrated that a domain spanning the residues for degradation and the activation of NF-kB (20 –25). kB as efficiently as the 856 –1191 of FLASH activated NF- It has recently been reported that FLASH is likely to be a full-length and could directly bind to TRAF2 in vitro and component of DISC involved in Fas- and TNF-mediated apo- in the transfected cells. Taken together, these results kB ac- ptosis (26). FLASH contains a death-effector-domain-recruiting suggest that FLASH coordinates downstream NF- a domain (DRD) in the C-terminal region, which interacts with tivity via a TRAF2-dependent pathway in the TNF- signaling. the death effector domain (DED) of caspase-8 or FADD (26). Still, transient overexpression of FLASH marginally affects apoptosis (26, 27), making its precise function with respect to TNF-a is a pleiotropic cytokine associated with various cel- receptor-mediated signaling (e.g. via TNF-R) unclear. In addi- lular defense responses, with lethal effects such as septic shock tion, the finding that caspase-8 and FADD may be involved in with inflammation, and with apoptosis in susceptible cells (1, the signaling to NF-kB activation as well as to apoptosis (28, 2). TNF-a signaling is transduced through its receptor, TNF-R, 29) suggests that FLASH may function to coordinate stress to simultaneously elicit two opposing effects: apoptosis and responses, a possibility that prompted us to investigate the role activation of an anti-apoptotic transcription factor NF-kB (3– of FLASH in NF-kB activation. In this report, we used an 5). During initiation of apoptosis, FADD is complexed with antisense oligonucleotide (AS) and overexpression analysis to activated TNF-R1 and TRADD via the death domain (4, 6, 7) show that FLASH transduces the TNF-a signal, leading to the and recruits caspase-8 to the resultant death-inducing signal- activation of NF-kB via a TRAF2-NIK-IKK-dependent pathway. ing complex (DISC), which leads to apoptosis via activation of a EXPERIMENTAL PROCEDURES Reagents—Anti-IkBa and anti-tubulin antibodies were purchased * This work was supported by the Molecular Medicine Research from Santa Cruz Biotechnology (Santa Crutz, CA) and Sigma, respec- Group Program (98-MM-01-01-A-03), the KOSEF (Protein Network Research Center), and the National Research Laboratory program (to tively. Anti-HA antibody and anti-TRAF2 antibody (SC-7346) were Y. K. J.). The costs of publication of this article were defrayed in part by from Roche Molecular Biochemicals (Mannheim, Germany) and Santa the payment of page charges. This article must therefore be hereby Cruz Biotechnology, respectively. TNF-a and all other molecular biol- marked “advertisement” in accordance with 18 U.S.C. Section 1734 ogy grade materials were from Sigma or New England Biolabs (Hert- solely to indicate this fact. fordshire, UK). ‡ Supported by the Brain Korea 21 project. These authors contributed Construction of Recombinant Expression Plasmids—pME18S-FLAG equally to this work. and pME18S-FLAG-FLASH were kindly provided by Dr. Yonehara To whom correspondence should be addressed: Department of Life (University of Kyoto, Japan). pHA-FLASH was generated by subcloning Science, Kwangju Institute of Science and Technology, 1 Oryong-dong, the FLASH cDNA into the EcoRI/XbaI sites of pcDNA-HA plasmid. Puk-gu, Kwangju 500-712, Korea. Tel.: 82-62-970-2492; Fax: 82-62-970- FLASH deletion constructs were assembled by polymerase chain reac- 2484; E-mail: [email protected]. tion (PCR) using the following synthetic oligonucleotides as primers: The abbreviations used are: TNF-a, tumor necrosis factor-a; FLASH, 59-CCGGAATTCATGGCAGATGATGACAATGGT-39 and 59-ATAA- FLICE-associated huge protein; AS, antisense oligonucleotide; DED, GAATGCGGCCGCCTAGCTCTCCATGCTAACAACT-39 for pME18S- death effector domain; DRD, DED-recruiting domain; NAD, NF-kB- FLAG-DA-(1– 858) (pFL-DA-FLASH); 59-CCGGAATTCATGGAGAGCT- activating domain; DISC, death-inducing signaling complex; RT-PCR, CATGTGCAATT-39 and 59-ACCGGGCCCCTATCCAGTTCTAGGCAA- reverse transcription-polymerase chain reaction; PAGE, polyacrylamide AGA-39 for pcDNA-HA-DB-(856 –1552) (pHA-DB-FLASH); 59-CCGGA- gel electrophoresis; HA, hemagglutinin; b-gal, b-galactosidase; HEK, hu- ATTCATGGCAGATGATGACAATGGT-39 and 59-ATAAGAATGCGGC- man embryonic kidney cells; GST, glutathione S-transferase; TRAF, TNF receptor-associated factor; NIK, NF-kB-inducing kinase; Ikk, IkB kinase. CGCCTACAGTGAAGATTTAAAATTC-39 for pME18S-FLAG-DC- This paper is available on line at http://www.jbc.org 25073 This is an Open Access article under the CC BY license. 25074 NF-kB Activation by FLASH fected plasmid DNA was kept constant within individual experiments by adding appropriate amounts of pcDNA or pME18S. HeLa cells stably expressing HA-FLASH (HeLa/HA-FLASH) were generated as described by Chung et al. (38). Antisense Oligonucleotide Treatment—AS-2 (59-ATTCAGCAACT- TACTTGC-39) is an antisense oligonucleotide complementary to human FLASH mRNA and corresponds to a location around the stop codon, 5942–5959 bp downstream of the translation initiation site. Compari- son of this oligonucleotide sequence with the database detected the only homology to the FLASH sequence. The following scrambled sequence was used as a control: (59-GCTACTAGTAGCAGCTAC-39). Cells (3 3 10 per well) were continuously treated with 5 mM FLASH antisense or the scrambled oligonucleotide for 48 h in culture medium containing LipofectAMINE reagent. RNA Isolation and RT-PCR—Total RNA was isolated from HEK293 cells using TRIzol reagent (Life Technologies, Inc.). RT-PCR was per- formed for quantification of FLASH mRNA using b-actin mRNA as a control. Two sets of oligonucleotides were designed; 59-TAGGTGCTTT- TATTGACTTGACACAA-39 (sense) and 59-CAGGAATTCAGCAACT- TATCTGCAT-39 (antisense) (predicted product length: 725 bp) for FLASH primer 1 and 59-GAAGGTAATCATCCTGCATTAGCTGT-39 (sense) and 59-GAGCTTCATTAGCTGCTGGAATCTT-39 (antisense) (predicted product length: 714 bp) for FLASH primer 2. The nucleotide sequences of the b-actin primers were 59-CAACCGCGAGAAGAT- GACCC-39 (sense) and 59-GAAGGAAGGCTGGAAGAGTG-39 (anti- sense) (predicted product length: 457 base pairs). The PCR products were confirmed by DNA sequencing. Luciferase and b-Galactosidase Assays—Cells were harvested 24 h after transfection, and luciferase activities in the cell extracts were determined using a luciferase assay system (Promega). To measure b-galactosidase activity, the cell extracts were mixed with equal amounts of b-galactosidase assay buffer (23) containing 200 mM so- dium phosphate (pH 7.3), 2 mM MgCl , 100 mM b-mercaptoethanol, and 1.33 mg/ml O-nitrophenyl-b-D-galactopyranoside and incubated at 37 °C for 30 min. The absorbance at 420 nm was then measured using an ELISA reader (Molecular Device, Sunnyvale, CA). FIG.1. Suppression of TNF-a-induced activation of NF-kB us- Generation of Anti-FLASH Antibody and Western Blot Analysis— ing a FLASH antisense oligonucleotide. HEK293 cells were co- GST-DRD-FLASH fusion proteins were expressed in BL21(DE3) by transfected for 36 h with NF-kB-luciferase reporter plasmid (pNF-kB- addition of 0.2 mM isopropyl-b-D-thiogalactoside, purified using gluta- luc), pCMV-b-gal, and either 5 mM FLASH AS-2 or a scrambled thione-Sepharose 4B (Amersham Pharmacia Biotech) and administered oligonucleotide (random), and were then incubated with TNF-a (30 into a rabbit in a series of four injections. Anti-FLASH antibody was ng/ml) for an additional 26 h. A, activity of luciferase reporter genes was purified from the serum by antigen-affinity chromatography. For West- normalized to that of b-galactosidase, which served as an internal ern blot analysis, cell lysates were prepared, and protein concentrations control. Bars represent mean 6 S.D. from at least four independent experiments. B, HEK293 cells were lysed, and Western blotting was were determined using a DC protein assay kit (Bio-Rad). Western performed with anti-IkBa antibody. For an internal control, the same blotting was then carried out as previously described (31); proteins were extracts were probed with antibody to a-tubulin. C, reverse transcrip- visualized using an enhanced chemiluminescence system (ECL, Amer- tion of RNA isolated from cells treated with scrambled (Ran) or AS-2 sham Pharmacia Biotech). (AS) oligonucleotides. The PCR reaction was carried out with two dif- In Vitro Binding Assay—The expression of GST fusion proteins in ferent sets of FLASH primers. PCR of b-actin was performed to nor- BL21 (DE3) harboring pGEX-4T, pGEX-DRD-FLASH, or pGEX-NAD- malize FLASH expression. FLASH was induced with 0.2 mM isopropyl-b-D-thiogalactoside during exponential growth. Harvested cells were resuspended and lysed by (1–1191) (pFL-DC-FLASH); 59-CCGGAATTCATGGAGAGCTGCTCAT- sonication in 50 mM Tris-HCl buffer (pH 7.4) containing 1 mM dithio- GTGCAATT-39 and 59-CATTTAGGTGACACTA-39 for pME18S-FLAG- threitol, 0.5 mM EDTA, and 10% (v/v) glycerol. The supernatant lysates DD-(1553–1962) (pFL-DD-FLASH); 59-CCGGAATTCATGGAGAGCTC- were incubated with glutathione-Sepharose 4B. TRAF2, or caspase-8 ATGTGCAATT-39 and 59-CATTTAGGTGACACTA-39 for pME18S- labeled with [ S]methionine using the TNT system (Promega) were FLAG-DE-(856 –1962) (pFL-DE-FLASH). The PCR products were then then added to GST fusion proteins (20 mg each) coupled to glutathione- inserted into the EcoRI/NotI sites of pME18S-FLAG (DA, DC, DD, and Sepharose 4B in a final volume of 500 ml of binding buffer (50 mM DE), the EcoRI/ApaI sites of pcDNA3-HA (pHA-DB-FLASH), or the Tris-HCl, pH 7.4, 1 mM dithiothreitol, 0.5 mM EDTA, 0.01% Triton EcoRI/NotI sites of pcDNA3-HA (pHA-DD-FLASH). GST-NAD-FLASH X-100, 0.5 mg/ml bovine serum albumin, and 10% (v/v) glycerol). After and GST-DRD-FLASH fusion proteins were generated by subcloning being incubated at 4 °C for 2 h with gentle mixing, the beads were PCR products amplified by 59-CGCGGATCCCCTAGAGTTTCTGCTG- washed three times with the binding buffer, separated by 12% SDS- AA-39 and 59-CCGCTCGAGTTACAGTGAAGATTTAAATT-39 for pGST- PAGE, and detected by autoradiography. NAD-FLASH and 59-CGCGGATCCGATAAGAGTAAACTAACTC-39 Immunoprecipitation—HEK293 cells were transfected with pHA- and 59-CCGCTCGAGTTATTCACAGGAGCCAGGAGA-39 for pGST- FLASH, pHA-DB-FLASH, and pTRAF2 plasmids and lysed in radioim- DRD-FLASH into the BamHI/XhoI sites of pGEX4T-3 (Amersham mune precipitation buffer (50 mM Tris-HCl, pH 7.4, 1% Nonidet P-40, Pharmacia Biotech.). All PCR products were confirmed by DNA se- 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EDTA, 1 mM phenyl- quencing. pTRAF2, pTRAF5, pTRAF6, pFL-NIK, pCR3.1-IKKa, domi- methylsulfonyl fluoride, 1 mg/ml each of aprotinin, leupeptin, and pep- nant negative forms of TRAF2, TRAF5, TRAF6, NIK, and IKKa, pNF- statin, 1 mM Na VO ,and1mM NaF). FLASH was immunoprecipitated 3 4 kB-luc, and pCasp8 were described previously (30, 37). from cell lysates after incubation with anti-FLASH and anti-HA anti- Cell Culture, Stable Cells, and DNA Transfection—HEK293 and bodies and protein-A-coupled-Sepharose CL-4B (Amersham Pharmacia Jurkat cells were cultured in Dulbecco’s modified Eagle’s medium and Biotech.) at 4 °C for 2 h. TRAF2, HA-FLASH, and HA-DB-FLASH were RPMI 1640, respectively, supplemented with 10% fetal bovine serum then detected by Western blot analysis using anti-TRAF2 and anti-HA (BIOFLUIDS). Cells were subcultured to a density of 2 3 10 cells/well monoclonal antibodies, respectively. in 6-well dishes and allowed to stabilize for 1 day. Cells were then typically transfected with 600 ng of NF-kB-luciferase reporter plasmid RESULTS (pNF-kB-luc), 200 ng of pCMV-b-gal, and 1 mg of vector or the indicated Suppression of TNF-a-induced NF-kB Activation by a expression plasmid using LipofectAMINE according to the manufactur- er’s instructions (Life Technologies, Inc.). The total amount of trans- FLASH Antisense Oligonucleotide—To identify a role for NF-kB Activation by FLASH 25075 FIG.2. NF-kB activation induced by FLASH expression. HEK293 cells were transfected for 36 h with pNF-kB-luc, pCMV-b-gal and the indicated amounts of pFLASH, pTRAF2, or pHA-DD-FLASH. A, relative NF-kB-driven luciferase activities in transfectants express- ing FLASH, TRAF2, or DD-FLASH; activity in the control cells was arbitrarily set to a value of 1. B, Western blots showing the expression levels of exogenous FLASH (anti-FLASH antibody), DD-FLASH (anti- FIG.3. Mapping of the FLASH domain responsible for NF-kB HA antibody), and TRAF2 (anti-TRAF2 antibody). activation. A, schematic diagrams of full-length FLASH and its dele- tion (D) constructs. The death-effector domain-recruiting domain (DRD) and a putative NF-kB-activating domain (NAD) are indicated. B, HEK293 cells were co-transfected with pNF-kB-luc and either pcDNA FLASH in TNF-a signaling, we initially examined its contri- (control), full-length FLASH, or deletion constructs. One day later, bution to NF-kB activation by directly targeting FLASH ex- luciferase reporter gene assays were performed and for each construct, pression using an AS. Four different ASs were synthesized luciferase activities were adjusted so that the control was 1. Bars indicate mean 6 S.D. of the induction of NF-kB activity relative to the based on the nucleotide sequence of human FLASH and when control from at least four independent experiments. C, HEK293 cells examined, they showed essentially similar effects on TNF-a- were left untreated, treated with TNF-a for 1 h, or transfected with signaling (data not shown). Treating HEK293 cells with AS-2, pcDNA-HA, pHA-FLASH, pHA-DB-FLASH, pFL-DD-FLASH, or but not with a scrambled oligonucleotide (as a negative con- pTRAF2. After 1 day, cell extracts were prepared and analyzed by trol), abolished TNF-a-induced activation of NF-kB, as as- Western blotting with anti-IkBa antibody. sessed by luciferase reporter gene expression driven by a NF-kB responsive promoter (Fig. 1A), and also suppressed effects of several FLASH deletion mutants (Fig. 3A, DA–DE) TNF-a-induced degradation of IkBa (Fig. 1B). were examined. Expression of these deletions was confirmed in Because we could detect exogenous FLASH expression (Fig. the transfected HEK293 cells by Western blot analysis using 2B) but failed to detect expression of endogenous FLASH with anti-HA or anti-FLAG antibodies (data not shown). Determi- Western blot analysis using anti-FLASH antibody, the effects nation of NF-kB activity following the respective expression of of AS-2 or the scrambled oligonucleotide on FLASH expression each of these constructs showed that the truncation in the DB-, were assessed by RT-PCR (Fig. 1C). RT-PCR and Northern blot DC-, and DE-FLASH constructs had no effect on the ability of analysis have both been used to examine the effects of anti- FLASH to activate NF-kB (Fig. 3B). The DD-FLASH, by con- sense on gene expression (32). FLASH mRNA was undetectable trast, completely abolished NF-kB activation, whereas the DA- in HEK293 cells treated with AS-2, whereas tubulin expression FLASH partially induced NF-kB activity (Fig. 3B). Because the was unaffected, and readily detectable in cells treated with the DB-, DC-, and DE-FLASH contain a common region including a scrambled oligonucleotide, indicating that FLASH expression putative oligomerization domain, the region responsible for the was reduced by AS-2 treatment. These results suggest that activation of NF-kB apparently spans most of the oligomeriza- FLASH is involved in TNF-a-induced activation of NF-kB. tion domain and part of the DA-FLASH domain (Fig. 3A). We Activation of NF-kB by FLASH Expression and Domain designate the common region in the DB-, DC-, and DE-FLASH Mapping for NF-kB Activation—To more directly assess the spanning residues 856 –1191 as the NF-kB activation domain role of FLASH in the activation of NF-kB signaling, we exam- (NAD) of FLASH. ined an effect of its overexpression on NF-kB activity in Because it is known that TNF-a treatment of cells leads to HEK293 cells. We found that, indeed, NF-kB activity was dose- the activation of NF-kB through the phosphorylation and deg- dependently related to FLASH expression (Fig. 2A). The rela- radation of IkBa (33, 34), exposure of HEK293 cells to TNF-a tive levels of expression of FLASH and TRAF2 were confirmed resulted in the degradation of IkBa (Fig. 3C, left panel). Tran- by Western blot analysis (Fig. 2B). sient expression of FLASH, TRAF2, or DB-FLASH led to the FLASH contains DRD at its C terminus and a putative degradation of IkBa, whereas overexpression of the DD-FLASH oligomerization domain at its N terminus (Fig. 3A) (26). To did not affect the degradation of IkBa (Fig. 3C, right panel), ascertain which of these mediates induction of NF-kB activity, consistent with the results of NF-kB activity assays in Fig. 3B. 25076 NF-kB Activation by FLASH FIG.5. In vitro binding of NAD to TRAF2. GST, GST-NAD, and GST-DRD fusion proteins were purified from Escherichia coli. Proteins bound to their affinity resins (each equivalent to 20 mg of protein) were incubated with TRAF2 or caspase-8 labeled with [ S]methionine as described under “Experimental Procedures.” After separation by 12% SDS-PAGE, the bound proteins were detected by autoradiography (up- per panels), and resin-coupled proteins were visualized by Western blotting with anti-GST polyclonal antibody (lower panels). FIG.4. Effects of various dominant negative mutants on FLASH-induced activation of NF-kB. A, HEK293 cells were left untreated or treated with TNF-a after co-transfection with pNF-kB-luc and either pcDNA (control) or expression plasmids encoding the wild type or dominant negative mutants (D/N) of the indicated mediators in the presence or absence of pHA-FLASH. One day later, luciferase activities were measured and normalized to that of b-galactosidase. B, HEK293 cells were co-transfected with pNF-kB-luc and the indicated dominant negative mutants. The cells were then exposed to TNF-a (30 ng/ml) for 6 h, after which luciferase activities were measured. These results demonstrate and confirm that FLASH expression through NAD in DB-FLASH induces NF-kB activation by the degradation of IkBa. FLASH Signals NF-kB Activation through TRAF2-NIK- IKKs—It has previously been shown that various signaling pro- teins downstream of TNF-R, including TRADD, TRAFs, RIP, NIK, FIG.6. Cellular interaction of FLASH with TRAF2. A, proteins from Jurkat cell extracts were immunoprecipitated (IP), separated by and IKKs, are involved in TNF-a-induced activation of NF-kB. We SDS-PAGE, and immunoblotted with preimmune (pre) or anti-TRAF2 therefore examined the respective roles of these proteins in monoclonal antibody. B, proteins from HeLa cell permanently expressing FLASH-induced activation of NF-kB. Expression of dominant neg- HA-FLASH (HeLa/HA-FLASH) were immunoprecipitated with anti-HA ative mutants of TRAF2-(87–501), NIK (K429A,K430A), or IKKa antibody and probed with anti-TRAF2 antibody (upper panel) or anti-HA antibody (lower panel). C, HEK293 cells were transiently transfected with (K44A) inhibited NF-kB activation induced by FLASH (Fig. 4A)or pHA-FLASH in the presence or absence of pTRAF2. One day later, pro- TNF-a (Fig. 4B). On the other hand, although dominant negative teins were immunoprecipitated with anti-FLASH antibody or anti-HA mutants of TRAF5-(205–558) and TRAF6-(289 –530) partially sup- antibody and immunoblotted with anti-TRAF2 antibody. D, HEK293 cells pressed TNF-a- or FLASH-induced activation of NF-kB (Fig. 4B), were co-transfected with pHA-DB-FLASH and pTRAF2, immunoprecipi- tated with anti-HA antibody, and immunoblotted with anti-TRAF2 (upper they showed little effect on both TNF-a- and FLASH-induced acti- panels) or anti-HA antibody (lower panels). vation of NF-kB (Fig. 4, A and B), which thus appears to be mainly mediated via a TRAF2-NIK-IKK-pathway. FLASH Interacts with TRAF2 in Vitro and in Vivo through TRAF2 in Jurkat, HEK 293, and HeLa cells. Immunoprecipi- NAD—That FLASH interacts with TRAF2 was then examined tation with anti-FLASH antibody and subsequent Western blot with in vitro binding assay and immunoprecipitation. GST analysis using anti-TRAF2 antibody revealed that endogenous pull-down assay showed that GST-NAD fusion protein could TRAF2 was co-precipitated with endogenous FLASH, but not bind to TRAF2, whereas GST-DRD interacted only with by preimmune serum (Fig. 6A). Endogenous FLASH could not caspase-8, indicating that NAD of FLASH specifically interacts be detected with Western blot analysis using anti-FLASH an- with TRAF2 in vitro (Fig. 5). Immunoprecipitation assays were tibody in the immunoprecipitates probably because of its low then performed to examine cellular interaction of FLASH and expression level in Jurkat cells. We have then generated HeLa NF-kB Activation by FLASH 25077 cells stably expressing exogenous FLASH tagged with HA ptosis and also of the protein complex including TRAFs leading to (HeLa/HA-FLASH) and further examined the intracellular in- NF-kB signaling, FLASH needs to be further characterized for the teractions. Immunoprecipitation with anti-HA antibody fol- stoichiometry of protein-protein interactions and for a fine-tuning lowed by Western blotting using anti-TRAF2 antibody or an- activity balancing survival and apoptosis. ti-HA antibody showed the presence of TRAF2 and HA-FLASH Acknowledgments—We thank Dr. S. Yonehara (University of Kyoto, in the immunoprecipitates (Fig. 6B). These results indicate Japan) for FLASH cDNA and Dr. N. Spoerel for critical reading of this that FLASH interacts with TRAF2 in the cells. manuscript. We have then examined cellular interaction of NAD of FLASH with TRAF2. HEK293 cells were transfected with REFERENCES pHA-FLASH (Fig. 6C) or pHA-DB-FLASH (Fig. 6D)inthe 1. Beg, A. A., and Baltimore, D. (1996) Science 274, 782–784 2. Serfas, M. S., Goufman, E., Feuerman, M. H., Gartel, A. L., and Tyner, A. L. presence or absence of TRAF2. Immunoprecipitation and West- (1997) Cell Growth and Differ. 8, 951–961 ern blot analysis showed that endogenous (Fig. 6C, middle 3. Hsu, H., Xiong, J., and Goeddel, D. V. (1995) Cell 81, 495–504 panel) or exogenous TRAF2 (Fig. 6C, right panel) was co-pre- 4. Hsu, H., Shu, H. B., Pan, M. G., and Goeddel, D. V. (1996) Cell 84, 299 –308 5. Wang, C. Y., Mayo, M. W., Korneluk, R. G., Goeddel, D. V., and Baldwin Jr., cipitated with FLASH by anti-FLASH antibody or anti-HA A. S. (1998) Science 281, 1680 –1683 antibody. As expected from in vitro binding assays, DB-FLASH 6. Shu, H. B., Takeuchi, M., and Goeddel, D. V. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 13973–13978 was co-immunoprecipitated with TRAF2 by anti-HA antibody 7. Hu, W. H., Johnson, H., and Shu, H. B. (2000) J. Biol. Chem. 275, from lysates of HEK293 cells transfected with both pHA-DB- 10838 –10844 FLASH and pTRAF2 (Fig. 6D). These results suggest that 8. Boldin, M. P., Goncharov, T. M., Goltsev, Y. V., and Wallach, D. (1996) Cell 85, 803– 815 FLASH may directly interact with TRAF2 through its NAD. 9. Muzio, M., Chinnaiyan, A. M., Kischkel, F. C., O’Rourke, K., Shevchenko, A., Ni, J., Scaffidi, C., Bretz, J. D., Zhang, M., Gentz, R., Mann, M., Krammer, DISCUSSION P. H., Peter, M. E., and Dixit, V. M. (1996) Cell 85, 817– 827 10. Yuan, J., Shaham, S., Ledoux, S., Ellis, H. M., and Horvitz, H. R. (1993) Cell Previous studies have suggested that TNF-R-mediated apoptosis 75, 641– 652 and NF-kB activation pathways pass through TRADD, a death 11. Boldin, M. P., Mett, I. L., Varfolomeev, E. E., Chumakov, I., Shemer-Avni, Y., domain-containing adaptor protein interacting with TNF-R in a Camonis, J. H., and Wallach, D. (1995) J. Biol. Chem. 270, 387–391 12. Coa, Z., Xiong, J., Takeuchi, M., Kurama, T., and Goeddel, D. V. (1996) Nature TNF-dependent process; TRADD-FADD-caspase-8 and TRADD- 383, 443– 446 TRAF2-RIP-NIK-IKKs cascades lead to the induction of apoptosis 13. Rothe, M., Sarma, V., Dixit, V. M., and Goeddel, D. V. (1995) Science 269, 1424 –1427 and activation of an anti-apoptotic transcription factor NF-kB, re- 14. Nakano, H., Oshima, H., Chung, W., Williams-Abbott, L., Ware, C. F., Yagita, spectively. Whereas FLASH may be required for the activation of H., and Okumura, K. (1996) J. Biol. Chem. 271, 14661–14664 caspase-8 during Fas- and TNF-R-mediated apoptosis (26, 27), the 15. Ishida, T., Mizushima, S., Azuma, S., Kobayashi, N., Tojo, T., Suzuki, K., Aizawa, S., Watanabe, T., Mosialos, G., Kieff, E., Yamamoto, T., and Inoue, data presented here provide the first evidence for involvement of J. (1996) J. Biol. Chem. 271, 28745–28748 FLASH in NF-kB activation by TNF-R. The physical interaction of 16. Ishida, T., Tojo, T., Aoki, T., Kobayashi, N., Ohishi, T., Watanabe, T., FLASH with TRAF2, demonstrated here in vitro and in vivo (Figs. Yamamoto, T., and Inoue, J. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 9437–9442 5 and 6), lends further support to the idea that FLASH transduces 17. Ling, L., and Goeddel, D. V. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, TNF-a signals via a TRAF2-dependent pathway of NF-kB activa- 9567–9572 18. Bertin, J., Nir, W. J., Fischer, C. M., Tayber, O. V., Errada, P. R., Grant, J. R., tion. Though dominant negative mutants of TRAF5 and 6 also Keilty, J. J., Gosselin, M. L., Robison, K. E., Wong, G. H. W., Glucksmann, partially suppressed FLASH- and TNF-a-mediated activation of M. A., and DiStefano, P. S. (1999) J. Biol. Chem. 274, 12955–12958 NF-kB (Fig. 4, A and B), complex formation of FLASH with addi- 19. Liu, Z. G., Hsu, H., Goeddel, D. V., and Karin, M. (1996) Cell 87, 565–576 20. Malinin, N. L., Boldin, M. P., Kovalenko, A. V., and Wallach, D. (1997) Nature tional signal mediators leading to NF-kB signaling such as other 385, 540 –544 TRAFs, TRADD, or RIP remains to be elucidated. 21. Didonato, J. A., Hayakawa, M., Rothwarf, D. M., Zandi, E., and Karin, M. (1997) Nature 388, 548 –554 FLASH as a component of apoptotic signaling complexes is likely 22. Mercurio, F., Zhu, H., Murray, B. W., Shevchenko, A., Bennett, B. L., Li, J. W., to mediate apoptosis signals probably triggered by cell surface Young, D. B., Barbosa, M., Mann, M., Manning, A., and Rao, A. (1997) receptors. However, the fact that FLASH activates NF-kB may Science 278, 860 – 866 23. Re ´ gnier, C. H., Song, H. Y., Gao, X., Goeddel, D. V., Cao, Z., and Rothe, M. explain the observation that in many cell types, TNF treatment did (1997) Cell 90, 373–383 not induce apoptosis in the absence of gene expression. With re- 24. Woronicz, J. D., Gao, X., Cao, Z., Rothe, M., and Goeddel, D. V. (1997) Science 278, 866 – 870 spect to the role of FLASH as an activator interacting with 25. Zandi, E., Rothwarf, D. M., Delhase, M., Hayakawa, M., and Karin, M. (1997) caspase-8 and FADD, the lack of a significant increase of apoptosis Cell 91, 243–252 following overexpression of both FLASH and caspase-8 or the in- 26. Imai, Y., Kimura, T., Murakami, A., Yajima, N., Sakamaki, K., and Yonehara, S. (1999) Nature 398, 777–785 ability of TNF-a to induce apoptosis in a subset of tumor cells may 27. Medema, J. P. (1999) Nature 398, 756 –757 be attributed to FLASH-mediated activation of NF-kB. NAD-me- 28. Inohara, N., Koseki, T., Lin, J., Peso, L., Lucas, P. C., Chen, F. F., Ogura, Y., diated activation of NF-kB may antagonize DRD-mediated apo- and Nu ´n ˜ ez, G. (2000) J. Biol. Chem. 275, 27823–27831 29. Newton, K., Harris, A. W., Bath, M. L., Smith, K. G. C., and Strasser, A. (1998) ptotic signals by encoding inhibitory proteins such as IAPs and EMBO J. 17, 706 –718 IEX-1L (5, 35, 36). Moreover, recent reports that FADD and 30. Wong, B. R., Josien, R., Lee, S. Y., Vologodskaia, M., Steinman, R. M., and Choi, Y. (1998) J. Biol. Chem. 273, 28355–28359 caspase-8 may be required for cell survival and proliferation during 31. Jung, Y., Miura, M., and Yuan, J. (1996) J. Biol. Chem. 271, 5112–5117 heart and thymus development may be explained by our observa- 32. Vassar, R., Bennett, B. D., Babu-Khan, S., Kahn, S., Mendiaz, E. A., Denis, P., Teplow, D. B., Ross, S., Amarante, P., Loeloff, R., Luo, Y., Fisher, S., Fuller, tions of FLASH-mediated activation of NF-kB. This speculation is J., Edenson, S., Lile, J., Jarosinski, M. A., Biere, A. L., Curran, E., Burgess, reinforced by our observation that FLASH transduced NF-kB sig- T., Louis, J. C., Collins, F., Treanor, J., Rogers, G., and Citron, M. (1999) naling evoked by caspase-8 (data not shown). Science 286, 735–741 33. Baeuerle, P. A., and Henkel, T. (1994) Annu. Rev. Immunol. 12, 141–179 FLASH seems to be an upstream component of various receptor- 34. Reuther, J. Y., and Baldwin, A. S. (1999) J. Biol. Chem. 274, 20664 –20670 mediated signals including TNF-a and most likely has a dual 35. LaCasse, E. C., Baird, S., Korneluk, R. G., and MacKenzie, A. E. (1998) function in apoptosis and NF-kB signaling. We have additional Oncogene 17, 3247–3259 36. Chu, Z. L., McKinsey, T. A., Liu, L., Genty, J. J., Malim, M. H., and Ballard, evidences that FLASH is also an indispensable component in re- D. W. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 10057–10062 ceptor-mediated apoptosis. As a component of DISC during apo- 37. Kim, I. K., Chung, C. W., Woo, H. N., Hong, G. S., Nagata, S. J., and Jung, Y. K. (2000) Biochem. Biophys. Res. Commun. 277, 311–316 38. Chung, C. W., Song, Y. H., Kim, I. K., Yoon, W. J., Ryu, B. R., Jo, D. G., Woo, Y. H. Choi, K. B. Kim, B. J. Kim, and Y. K. Jung, manuscript in H. N., Kwon, Y. K., Kim, H. H., Gwag, B. J., Mook-Jung, I. H., and Jung, preparation. Y. K. (2001) Neurobiol. Disease 8, 162–172

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Published: Jan 1, 2001

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