MOLECULAR AND CELLULAR BIOLOGY, Dec. 2004, p. 10757–10765 0270-7306/04/$08.00⫹0 DOI: 10.1128/MCB.24.24.10757–10765.2004 Copyright © 2004, American Society for Microbiology. All Rights Reserved.
Vol. 24, No. 24
Restoration of NF-B Activation by Tumor Necrosis Factor Alpha Receptor Complex-Targeted MEKK3 in Receptor-Interacting Protein-Deficient Cells Marzenna Blonska,1 Yun You,1 Romas Geleziunas,2 and Xin Lin1* Department of Microbiology and Immunology, School of Medicine and Biomedical Sciences, University at Buffalo, State University of New York, Buffalo, New York,1 and Merck Research Laboratories, West Point, Pennsylvania2 Received 26 May 2004/Returned for modification 6 July 2004/Accepted 15 September 2004
Receptor-interacting protein (RIP) plays a critical role in tumor necrosis factor alpha (TNF-␣)-induced NF-B activation. However, the mechanism by which RIP mediates TNF-␣-induced signal transduction is not fully understood. In this study, we reconstituted RIP-deficient Jurkat T cells with a fusion protein composed of full-length MEKK3 and the death domain of RIP (MEKK3-DD). In these cells, MEKK3-DD substitutes for RIP and directly associates with TRADD in TNF receptor complexes following TNF-␣ stimulation. We found that TNF-␣-induced NF-B activation was fully restored by MEKK3-DD in these cells. In contrast, expression of a fusion protein composed of NEMO, a component of the IB kinase complex, and the death domain of RIP (NEMO-DD) cannot restore TNF-␣-induced NF-B activation in RIP-deficient cells. These results indicate that the role of RIP is to specifically recruit MEKK3 to the TNF-␣ receptor complex, whereas the forced recruitment of NEMO to the TNF-␣ receptor complex is insufficient for TNF-␣-induced NF-B activation. Although MEKK2 has a high degree of homology with MEKK3, MEKK2-DD, unlike MEKK3-DD, also fails to restore TNF-␣-induced NF-B activation in RIP-deficient cells, indicating that RIP-dependent recruitment of MEKK3 plays a specific role in TNF-␣ signaling. NF-B is a family of transcription factors involved in inflammation and innate immunity (16). In unstimulated cells, NF-B is sequestered in the cytoplasm through an interaction with a family of inhibitory proteins, known as IB. Following the treatment of cells with various stimuli, IB is phosphorylated by the IB kinase (IKK) complex (10). The IKK complex contains three subunits: IKK␣, IKK, and IKK␥/NEMO. Both IKK␣ and IKK are serine/threonine protein kinases, while NEMO is a regulatory subunit (10). Phosphorylated IB is rapidly ubiquitinated and degraded in the 26S proteasome complex (10), which releases NF-B. NF-B is then translocated into the nucleus, where it regulates the transcription of its target genes (7, 16). One of the most potent NF-B activators is tumor necrosis factor alpha (TNF-␣), a major proinflammatory cytokine. TNF-␣ functions through two distinct surface receptors, 55kDa receptor 1 (TNF-R1) and 75-kDa receptor 2 (TNF-R2). TNF-R1 plays the predominant role in induction of cellular responses by soluble TNF-␣ (3). Treatment of cells with TNF-␣ initiates signal transduction cascades leading to activation of IKK. But the molecular mechanisms that regulate IKK activity are not fully defined. The binding of TNF-␣ to TNF-R1 leads to the recruitment of TNF-R1-associated death domain (TRADD), an adaptor protein, into the receptor complex. TRADD subsequently recruits other effector proteins: TNF receptor-associated factor 2 (TRAF2) (9), Fas-associated
* Corresponding author. Mailing address: Department of Microbiology and Immunology, School of Medicine and Biomedical Sciences, University at Buffalo, SUNY, 138 Farber Hall, 3435 Main St., Buffalo, NY 14214. Phone: (716) 829-3284. Fax: (716) 829-2158. E-mail:
[email protected].
death domain (FADD) (9), and receptor-interacting protein (RIP) (8, 21). RIP interacts directly with TRADD via its death domain (DD) (8). It has been demonstrated that TRAF2 plays an essential role in IKK recruitment to the TNF-R1 complex (4), but IKK activation requires the presence of RIP in the same complex (4, 11). In TRAF2⫺/⫺ fibroblasts, IKK activation is significantly reduced compared to that in wild-type (wt) cells, but the remaining IKK activity is sufficient for NF-B activation (4, 24). In contrast, in RIP⫺/⫺ cells, IKK is recruited to the TNF-R1 complex but its activation is almost completely abolished (4, 11). Furthermore, the kinase activity of RIP is not required for RIP to mediate TNF-␣-induced NF-B activation (21). Kinase-deficient RIP(K45A) restored TNF-␣-induced IKK activation as efficiently as wt RIP in RIP⫺/⫺ fibroblasts (4). Therefore, it has been proposed that IKK activation requires its phosphorylation by an upstream kinase(s) other than RIP. However, the molecular mechanism by which RIP mediates TNF-␣-induced IKK activation remains to be determined. It has been proposed that several kinases, mainly mitogenactivated protein kinase kinase kinase (MAP3K) family members, play an important role in TNF-␣-induced NF-B activation (13, 18, 20, 22, 27). Some of these kinases were coprecipitated with RIP (MEKK1 and MEKK3) (12, 14, 22) or TRAF2 (TAK1) (2, 20). One study has shown that MEKK3 plays a role in assembling the IKK/IB␣/NF-B complex following cytokine stimulation, whereas MEKK2 is associated with the IKK/IB/NF-B complex (18). However, the best genetic evidence supporting the role of these MAP3Ks in TNF-␣-induced NF-B activation is that this activation is significantly impaired in MEKK3-deficient mouse embryonic fibroblasts (22). In the present study, we tested the hypothesis that the role of
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RIP in the TNF-␣ pathway is mainly to recruit a MAP3K to the TNF-R1 complex. To do this, we reconstituted RIP-deficient Jurkat T cells with fusion proteins composed of full-length MEKK3 or MEKK2 and the DD of RIP (M3-DD and M2-DD cell lines, respectively). In these cells, MEKK3-DD or MEKK2-DD proteins would presumably substitute for RIP and directly associate with TRADD in the TNF-R1 complex following TNF-␣ stimulation. We found that TNF-␣-induced NF-B activation was fully restored in M3-DD cells, but not in M2-DD cells. In addition, we found that the kinase activity of MEKK3 was essential in this process. In contrast, expression of a fusion protein composed of NEMO, a component of the IKK complex, and the DD of RIP (NEMO-DD) failed to restore TNF-␣-induced NF-B activation in the RIP-deficient Jurkat T cells. Together, our results demonstrate that the role of RIP in the TNF-␣ signaling pathway is to specifically recruit MEKK3 into the TNF-R1 complex. MATERIALS AND METHODS Reagents and plasmids. Antibodies specific for Myc (A14) or Flag epitope tags and for NEMO, IKK␣ (H-744), p-IKK (Ser 181), IKK (H-470), TNF-R1 (H-5), Bcl10 (H-197), and -tubulin (D-10) were obtained from Santa Cruz Biotechnology (Santa Cruz, Calif.). Anti-RIP monoclonal antibodies were purchased from BD Transduction Laboratories (San Diego, Calif.). Rabbit antiMEKK3 antibodies were kindly provided by B. Su (M. D. Anderson Cancer Center, Houston, Tex.). Recombinant human TNF-␣ was obtained from Endogen (Woburn, Mass.). The coding sequences of MEKK3, MEKK2, NEMO, and the RIP DD were amplified by PCR from previously constructed plasmids (5, 19). Kinase-deficient forms of MEKK3 or MEKK2 were generated by replacing the active-site lysine at position 391 or 395 with alanine or methionine, respectively. The XbaI/EcoRI fragments for MEKK3, MEKK2, and NEMO, followed by a EcoRI/BamHI fragment for the DD of RIP, were inserted into the corresponding sites of the pcDNA3.1(⫺) vector in frame with a C-terminal Flag epitope tag coding sequence. RIP and TRADD cDNA was obtained by reverse transcription-PCR. The plasmids encoding Myc-RIP and Myc-TRADD were generated by inserting RIP cDNA or TRADD cDNA into the BamHI and EcoRI sites of the pRK-Myc vector. All expression plasmids were verified by restriction enzyme digestions and DNA sequencing. The plasmid encoding FlagIKK was described previously (6). Cell cultures and transfection. RIP-deficient (RIP⫺) Jurkat T cells were kindly provided by B. Seed (Massachusetts General Hospital, Boston) (21). wt and RIP⫺ cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), 100 U of penicillin/ml, and 100 g of streptomycin/ml. Human embryo kidney 293 (HEK293) cells were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% FBS and the antibiotics described above. Cells were grown in 5% CO2 at 37°C and passaged every 3 days. Stable transfection of RIP⫺ cells was established by electroporation with a gene pulser (Bio-Rad, Hercules, Calif.) at 250 V and 950 F. Plasmids (10 g) encoding MEKK3-DD, MEKK3(K391A)-DD, MEKK2-DD, or NEMO-DD were transfected into 107 RIP⫺ cells. The cells were incubated at 37°C for 2 days and than treated with G418 (1 mg/ml) for 2 weeks. The clones that were resistant to G418 were collected and examined for the presence of fusion proteins. HEK293 cells were transfected by the calcium phosphate coprecipitation method (1 to 4 g of DNA per 7 ⫻ 105 cells). Western blotting, coimmunoprecipitation, and kinase assay. Cells were transfected with vectors and lysed in a buffer containing 50 mM HEPES (pH 7.4), 250 mM NaCl, 1% Nonidet P-40, 1 mM EDTA, 1 mM Na3VO4, 1 mM NaF, 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM dithiothreitol, and a protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany). The cell lysates were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting or immunoprecipitated with anti-Flag M2 (or anti-c-Myc)–agarose affinity gel (Sigma-Aldrich, St. Louis, Mo.). The immunoprecipitates were washed with lysis buffer five times and eluted with 2⫻ SDS loading buffer. After being boiled (4 min), the samples were fractionated on SDS–10% PAGE gel and transferred to nitrocellulose membranes. Immunoblots were incubated with specific primary antibodies, followed by horseradish peroxidase-conjugated secondary antibodies and were developed by the enhanced chemiluminescence method according to the manufacturer’s protocol (Pierce,
MOL. CELL. BIOL. Rockford, Ill.). For the kinase assay the immunoprecipitates were washed three times with lysis buffer and once with kinase buffer containing 10 mM HEPES (pH 7.4), 1 mM MnCl2, 5 mM MgCl2, 12.5 mM glycerol-2-phosphate, 0.1 mM Na3VO4, 4 mM NaF, and 1 mM dithiothreitol. The reactions were performed in the kinase buffer with 20 M cold ATP and 10 Ci of [␥-32P]ATP at 30°C for 30 min. The reactions were stopped by adding 2⫻ SDS loading buffer, and the samples were boiled for 4 min. The eluted proteins were fractionated on SDS– 10% PAGE gel and transferred to nitrocellulose membranes, followed by autoradiography. Electrophoretic mobility shift assay (EMSA). Nuclear extracts were prepared from Jurkat wt or mutant cells after various stimulations. Cells (2 ⫻ 107) were resuspended in 400 l of lysis buffer (10 mM HEPES [pH 7.9], 10 mM KCl, 0.1 mM EDTA, 1 mM dithiothreitol, 0.5 mM PMSF, 0.4% Nonidet P-40, 1% protease inhibitor cocktail) and incubated on ice for 15 min. The nuclei were pelleted, and the cytoplasmic proteins were carefully removed. The nuclear pellets were then resuspended in 100 l of extraction buffer (20 mM HEPES [pH 7.9], 0.4 M NaCl, 1 mM EDTA, 1 mM dithiothreitol, 0.5 mM PMSF, 1% protease inhibitor cocktail). After vortexing for 30 min at 4°C, the samples were centrifuged (13,000 ⫻ g, 10 min) and the nuclear proteins in the supernatant were collected. Protein concentrations of nuclear extracts were determined by the Bio-Rad protein assay using bovine serum albumin as the standard. Nuclear extract (10 g) was incubated with a 32P-labeled, double-stranded, NF-B-specific oligonucleotide probe or an Oct-1 probe as a control (Promega, Madison, Wis.) for 15 min at room temperature. After incubation, samples were fractionated on a 5% polyacrylamide gel and visualized by autoradiography.
RESULTS MEKK3-DD fusion protein restores TNF-␣-induced NF-B activation in RIP-deficient cells. TNF-␣-induced NF-B activation is mediated by RIP and a downstream kinase, which is necessary for IKK activation (11, 21, 22). Previous studies suggest that MEKK3 connects RIP to the IKK complex (22). To further confirm that RIP is associated with MEKK3 in a signaling complex, we expressed MEKK3 and RIP in HEK293 cells. Consistent with previous findings, we found that expression of RIP was specifically associated with MEKK3 (Fig. 1A). To examine whether the role of RIP is to recruit MEKK3 to the TNF-R1 complex, we constructed an expression vector encoding a fusion protein composed of full-length MEKK3 and the DD of RIP (MEKK3-DD) (Fig. 1B). RIP-deficient Jurkat cells were stably transfected with Flag epitope-tagged MEKK3-DD or a kinase-defective mutant form of it [MEKK3(K391A)-DD] (Fig. 2A, lanes 2 and 3). Expression of these fusion proteins and TNF-␣ stimulation did not alter the expression levels of endogenous MEKK3 in these cells (Fig. 2B). TNF-␣-induced NF-B activation in these cells was examined by EMSA (Fig. 2C). The expression of MEKK3-DD fully restored TNF-␣-induced NF-B activation in the absence of RIP, whereas MEKK3(K391A)-DD failed to restore this activity (Fig. 2C). Consistent with these results, IB␣ was degraded in the TNF-␣-treated M3-DD stable cell line (Fig. 2D). These data suggest that RIP-dependent recruitment of MEKK3 is required for TNF-␣-induced NF-B activation. Expression of the MEKK2-DD fusion protein does not restore TNF-␣-induced NF-B activation in the absence of RIP. MEKK2 is the most closely related homolog of MEKK3 (1), and it has been shown that both MEKK2 and -3 activate IKK␣ and IKK when overexpressed (27). We next examined whether the MEKK2-DD fusion protein (Fig. 1B) was able to restore NF-B activation in the absence of RIP. Similarly, RIP⫺ cells stably expressing the MEKK2-DD fusion protein (M2-DD cell line) were stimulated with TNF-␣ for 30 min and NF-B binding activity was examined by EMSA. The expres-
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FIG. 1. MEKK3 physically associates with RIP. (A) HEK293 cells (7 ⫻ 105) were transiently transfected with 1 g of Flag-tagged MEKK3 and cotransfected with 1.5 g of Myc-tagged RIP. The cells were lysed 24 h later, and the lysates were precipitated with anti-Flag M2–agarose affinity gel. Immunocomplexes (top and middle) and whole-cell lysates (bottom) were subjected to SDS-PAGE and analyzed by Western blotting (WB) with the indicated antibodies. IP, immunoprecipitation. (B) Schematic diagram of MEKK3-DD, MEKK2-DD, and NEMO-DD fusion proteins. The RIP DD was fused to full-length MEKK3, MEKK2, or NEMO. RD, regulatory domain.
sion of MEKK2-DD failed to rescue NF-B activation in TNF␣-treated RIP⫺ cells (Fig. 3A). Consistent with this observation IB␣ degradation was not observed in stimulated M2-DD cells (Fig. 3B). Recently, it has been suggested that MEKK3 regulates the initial induction of NF-B, whereas MEKK2 controls a delayed induction of NF-B in response to TNF-␣ stimulation (18). To test this possibility, we analyzed TNF-␣-induced NF-B activation at various time points in Jurkat, RIP⫺, M3-DD, and M2-DD cells. The cells were either untreated or stimulated with TNF-␣ for 30, 60, and 120 min. NF-B binding activity was maximally induced after 30 min of stimulation in Jurkat and M3-DD cells; this was followed by a slight decrease at 60 and 120 min in Jurkat cells and a significant reduction at these time points in M3-DD cells. In RIP⫺ and M2-DD cells only marginal NF-B activation was observed following TNF-␣
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treatment (Fig. 4A). The inability of MEKK2-DD to restore TNF-␣-induced NF-B activation could not be attributed to lower MEKK2-DD expression levels, since the relative expression of MEKK2-DD in M2-DD cells was higher than that of MEKK3-DD in M3-DD cells (Fig. 4B). Taken together, these results suggest that MEKK2 is not involved in RIP-dependent NF-B activation. Recruitment of the NEMO-DD fusion protein to the TNF-R1 complex is not sufficient to activate NF-B. Our next question was whether a direct recruitment of the IKK complex to TNF-R1 was sufficient to restore NF-B activation in the absence of RIP. RIP⫺ cells were stably transfected with a plasmid encoding a fusion protein containing full-length NEMO and the DD of RIP (Fig. 1B). The expression level of this fusion protein in the transfected cells (NE-DD cells) was comparable to the level of endogenous RIP in Jurkat cells (Fig. 5A). Since the IKK complex is composed of a NEMO homodimer bound to either an IKK␣/IKK heterodimer or an IKK homodimer (17), it is not surprising that we succeeded in coprecipitating Flag-tagged NEMO-DD with endogenous NEMO. Moreover, NEMO-DD was constitutively incorporated into endogenous IKK complexes in a TNF-␣-independent manner (Fig. 5B). Next, NE-DD cells were stimulated with TNF-␣ or phorbol myristate acetate plus CD28 antibodies for 30 min and NF-B binding activity was examined by EMSA. NEMO-DD could not rescue TNF-␣-induced NF-B activation in the absence of RIP (Fig. 5C), and TNF-␣ stimulation failed to induce IB␣ degradation in NE-DD cells (Fig. 5D). Taken together, these results show that the recruitment of the IKK complex directly to the TNF-R1 complex is not sufficient to restore TNF-␣-induced NF-B activation in the absence of RIP, and the complete activation of IKK requires a RIP-dependent recruitment of a signaling intermediate, such as a MAP3K. MEKK3-DD, MEKK2-DD, and NEMO-DD fusion proteins associate with TRADD and are recruited into the TNF-R1 complex. RIP and TRADD interact via their DDs (8). We next examined whether MEKK3-DD, MEKK2-DD, and NEMO-DD fusion proteins were able to associate with TRADD due to the presence of the DD of RIP. HEK293 cells were cotransfected with the Flag-tagged fusion proteins or proteins without the DD and Myc-tagged TRADD. As shown in Fig. 6, TRADD was effectively coprecipitated with MEKK3-DD (Fig. 6A, lane 6), MEKK2-DD (Fig. 6B, lane 4), and NEMO-DD (Fig. 6C, lane 4). These interactions were specific because no association between MEKK3, MEKK2, or NEMO and TRADD was observed (Fig. 6). These results demonstrate that, even though all three proteins are capable of interacting with TRADD, only the MEKK3-TRADD interaction leads to restoration of TNF-␣-mediated activation of NFB. To examine whether MEKK3-DD, MEKK2-DD, or NEMO-DD could be recruited into the TNF-R1 complex in a signal-dependent manner, TNF-R1 complexes were immunoprecipitated from NE-DD cells (Fig. 7A) or M3-DD and M2-DD cells (data not shown) following TNF-␣ stimulation. Since the expression level of NEMO-DD in NE-DD cells is higher than those of MEKK3-DD and MEKK2-DD in M3-DD and M2-DD cells, respectively, we could detect only the association of NEMO-DD (Fig. 7A), not that of MEKK3-DD and
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FIG. 3. The MEKK2-DD fusion protein does not rescue TNF-␣mediated NF-B activation in RIP-deficient cells. (A) Jurkat and RIP⫺ cells and cells expressing MEKK2-DD (M2-DD) were stimulated as described for Fig. 2C, and NF-B binding activity was analyzed by EMSA. ns, nonspecific. (B) The expression of IB␣ in TNF-␣ (10 ng/ml)-stimulated cells was determined by Western blotting with antiIB␣ antibodies. The membrane was reprobed with anti--tubulin antibodies (a loading control).
MEKK2-DD (data not shown), with the TNF-R1 complex following TNF-␣ stimulation. To confirm that MEKK3-DD can be recruited into TNF-R1 complexes, we transiently transfected MEKK3-DD into
FIG. 2. MEKK3-DD, but not the kinase-deficient mutant version of it, restores TNF-␣-induced NF-B activation in RIP-deficient cells. (A) Levels of MEKK3-DD and MEKK3(K391A)-DD fusion protein expression. RIP-deficient Jurkat cells (2 ⫻ 107) stably transfected with Flag-tagged MEKK3-DD (M3-DD cell line) or MEKK3(K391A)-DD
[M3(K391A)-DD cell line] were lysed and immunoprecipitated (IP) with anti-Flag M2–agarose affinity gel. Immunoprecipitates (top) and whole-cell lysates (middle and bottom) were subjected to SDS-PAGE and probed with anti-Flag antibodies or anti-RIP and anti--tubulin antibodies, respectively. WB, Western blotting. (B) Levels of endogenous MEKK3 in Jurkat, M3-DD, and M3(K391A)-DD cells. The cells were either untreated or stimulated with TNF-␣ (20 ng/ml) for 5 min. Lysates were subjected to SDS-PAGE and probed with anti-MEKK3 or anti--tubulin antibodies. (C) NF-B binding activity was analyzed by EMSA. Jurkat, RIP-deficient [RIP(⫺)], M3-DD, and M3(K391A)-DD cells were either untreated or stimulated with TNF-␣ (10 ng/ml) or phorbol myristate acetate (PMA; 10 ng/ml) plus a CD28 monoclonal antibody (CD28Ab; 1 g/ml) for 30 min. Nuclear extracts (10 g) from these cells were incubated with 32P-labeled probes containing NF-B binding sites for 15 min and then subjected to electrophoresis in 5% polyacrylamide gel and autoradiography. ns, nonspecific. (D) Time course of IB␣ degradation in TNF-␣ (10 ng/ml)stimulated cells. The level of IB␣ expression was determined by Western blotting with anti-IB␣ antibodies. The membrane was reprobed with anti--tubulin antibodies (a loading control).
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FIG. 4. Differential effects of MEKK2-DD and MEKK3-DD expression on TNF-␣-induced NF-B activation in RIP-deficient cells. (A) Jurkat, RIP⫺, M2-DD, and M3-DD cells were stimulated with TNF-␣ (10 ng/ml) for the indicated times, and EMSA was performed using 32P-labeled probes containing NF-B or Oct-1 binding sites. ns, nonspecific. (B) Levels of MEKK2-DD and MEKK3-DD fusion protein expression. Cells (2 ⫻ 107) were lysed and immunoprecipitated (IP) with anti-Flag M2–agarose affinity gel. Immunoprecipitates (top) and whole-cell lysates (bottom) were subjected to SDS-PAGE and probed with anti-Flag, anti-RIP, or anti--tubulin antibodies. WB, Western blotting.
HEK293 cells. TNF-R1 complexes in the transfected cells were immunoprecipitated with anti-TNF-R1 antibody-conjugated beads. We found that MEKK3-DD was recruited into TNF-R1 complexes following TNF-␣ stimulation (Fig. 7B). Together, these results indicate that MEKK3-DD restoration of TNF-␣induced NF-B activation is due to the recruitment of MEKK3-DD into TNF-R1 complexes via its association with TRADD. MEKK3 associates with and phosphorylates IKK. To further confirm that MEKK3 is a kinase that links RIP to the IKK complex, we performed coimmunoprecipitation experiments using vectors encoding two IKK subunits: IKK and NEMO. HEK293 cells were cotransfected with Myc-tagged MEKK3, MEKK3(K391A), MEKK2, or MEKK2(K395M) and Flagtagged IKK or NEMO. After 24 h, cells were harvested and
FIG. 5. The NEMO-DD fusion protein is incorporated into the IKK complex, but its recruitment to the TNF-R1 complex is not sufficient for NF-B activation in the absence of RIP. (A) Jurkat, RIP⫺, and RIP⫺ cells expressing NEMO-DD (NE-DD) were either untreated or stimulated with TNF-␣ (10 ng/ml). The cells were lysed and subjected to SDS-PAGE and Western blotting (WB) with anti-RIP antibodies that recognize the DD of RIP. (B) Jurkat, RIP⫺, and NE-DD cells were either untreated or stimulated with TNF-␣ (20 ng/ml) for 5 min. The cells were lysed and precipitated with anti-Flag M2–agarose affinity gel. Immunocomplexes (left) and whole-cell lysates (right) were subjected to SDS-PAGE and analyzed by Western blotting with antibodies against IKK␣, IKK, NEMO, Flag, or Bcl10 as a loading control. IP, immunoprecipitation. (C) Jurkat, RIP⫺, and NE-DD cells were stimulated as described for Fig. 2C, and NF-B binding activity was analyzed by EMSA. PMA, phorbol myristate acetate; ns, nonspecific. (D) The expression of IB␣ in TNF-␣ (10 ng/ml)-stimulated cells was determined by Western blotting with antiIB␣ antibodies. The membrane was reprobed with anti--tubulin antibodies (a loading control).
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FIG. 7. NEMO-DD and MEKK3-DD fusion proteins are recruited to the TNF-R1 complex in TNF-␣-treated cells. (A) Jurkat or NE-DD cells (2 ⫻ 107) were either untreated or stimulated with TNF-␣ (20 ng/ml) for the indicated times. These cells were lysed and precipitated with mouse monoclonal anti-TNF-R1 antibodies. The immunocomplexes (top) and whole-cell lysates (middle and bottom) were subjected to SDS-PAGE and analyzed by Western blotting (WB) with anti-Flag or anti-TNF-R1 antibodies. IP, immunoprecipitation; Ig, immunoglobulin. (B) HEK293 cells (7 ⫻ 105) were transiently transfected with 1 g of empty vector (pcDNA3) or Flag-tagged MEKK3-DD. Twenty-four hours later the cells were stimulated with TNF-␣ (20 ng/ml) for 10 min and lysates were tested as described for panel A.
FIG. 6. MEKK3-DD, MEKK2-DD, and NEMO-DD fusion proteins associate with TRADD. HEK293 cells (7 ⫻ 105) were transiently transfected with 1.3 g of expression plasmids encoding several constructs: Myc-tagged TRADD, Flag-tagged MEKK3, or MEKK3-DD (A), MEKK2 or MEKK2-DD (B), or NEMO or NEMO-DD (C) or with control vectors pRK6 and pcDNA3. Cells were lysed and precipitated with anti-Flag M2–agarose affinity gel. The immunocomplexes were subjected to SDS-PAGE and analyzed by Western blotting (WB) with anti-Myc or anti-Flag antibodies. The levels of expression of TRADD were assessed with anti-Myc immunoblotting of total cell lysates. IP, immunoprecipitation.
the lysates were immunoprecipitated with anti-Flag affinity gel. Immunoblotting analysis revealed that MEKK3 and the kinase-defective mutant form of it were specifically associated with IKK (Fig. 8A, lanes 3 and 6). Moreover, MEKK3-DD was also coprecipitated with IKK (Fig. 8B, lane 5). Interestingly, MEKK3 and MEKK3-DD failed to associate with NEMO (Fig. 8A, lanes 4 and 7). These results demonstrate that MEKK3 may physically interact with the IKK complex through IKK. The interaction of MEKK3, but not the kinasedeficient form of MEKK3 (Fig. 9, top), with IKK could effectively induce a direct or indirect phosphorylation of IKK (Fig. 9, bottom). On the other hand, we were unable to coprecipitate MEKK3 or MEKK3-DD with endogenous IKK (Fig. 8C), suggesting that the interaction between MEKK3 and IKK may be transient or require additional components, such as adaptor proteins. Surprisingly, although it has been shown that the overex-
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FIG. 9. Kinase-active form of MEKK3 phosphorylates IKK. HEK293 cells (7 ⫻ 105) were transiently transfected with 1.3 g of empty vector (pcDNA3) (lane 1) or expression plasmids encoding Myc-MEKK3 (lane 2) and Myc-MEKK3(K391A) (lane 3). The cells were lysed 24 h later and precipitated with anti-c-Myc–agarose affinity gel. The immunocomplexes were subjected to an in vitro kinase assay. Whole-cell lysates were analyzed by Western blotting (WB) with antiphospho-IKK and anti-IKK antibodies. IP, immunoprecipitation.
pression of MEKK2 also activates NF-B (27), we did not observe an association of MEKK2 with IKK or NEMO when MEKK2 was expressed at a level similar to that of MEKK3 (Fig. 8A, lanes 9, 10, 12, and 13). The interaction between MEKK2 and IKK was detectable only when we significantly increased the amount of the transfected MEKK2 (data not shown). Together, these results indicate that MEKK3 has a higher affinity for association with IKK than MEKK2. DISCUSSION Upon stimulation, TNF-R1 recruits TRADD and possibly other known or yet-unidentified proteins to form a signaling complex. TRADD further recruits RIP, FADD, and TRAF2 to the signaling complex (8, 9). This complex triggers the NF-B signaling pathway via recruitment of the IKK complex (Fig. 10) (15, 26). Earlier studies indicate that FADD and TRAF2 are required for TNF-␣-induced apoptosis and JNK activation (23, 25), respectively, whereas RIP is required for TNF-␣-induced IKK activation (11, 21). RIP is a serine/threonine kinase containing an N-terminal kinase domain (KD), which is followed by an intermediate domain (ID), and a C-terminal DD. It has been shown that the DD of RIP is required for linking RIP to
FIG. 8. MEKK3 and MEKK3-DD associate with IKK. (A) HEK293 cells (7 ⫻ 105) were transiently transfected with 1.3 g of empty vector (pcDNA3) (lane 1) or expression plasmids encoding Myc-tagged MEKK3 (lanes 2 to 4), MEKK3(K391A) (lanes 5 to 7), MEKK2 (lanes 8 to 10), or MEKK2(K395M) (lanes 11 to 13) and cotransfected with 1 g of Flag-tagged IKK (lanes 3, 6, 9, and 12) or NEMO (lanes 4, 7, 10, and 13). The cells were lysed and precipitated with anti-Flag M2–agarose affinity gel. The immunocomplexes were subjected to SDS-PAGE and analyzed by Western blotting (WB) with
anti-Myc antibodies. The levels of expression of the transfected products were assessed with anti-Myc and anti-Flag immunoblots of total cell lysates. IP, immunoprecipitation. (B) HEK293 cells (7 ⫻ 105) were transiently transfected with 1.3 g of Myc-tagged MEKK3 (lanes 2, 5, and 6) and cotransfected with Flag-tagged IKK or NEMO. The cells were lysed and tested as described for panel A. (C) HEK293 cells (7 ⫻ 105) were transiently transfected with Flag-tagged MEKK3, MEKK3DD, NEMO, or NEMO-DD expression vectors, and the cells were lysed 24 h later. The lysates were precipitated with anti-Flag M2– agarose affinity gel. Immunocomplexes (top and middle) and wholecell lysates (bottom) were subjected to SDS-PAGE and analyzed by Western blotting with antibodies against IKK or Flag.
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FIG. 10. Working model of TNF-␣-induced NF-B activation in RIP-deficient cells expressing MEKK3-DD or NEMO-DD fusion proteins.
the upstream signaling component TRADD, whereas the KD of RIP is dispensable for TNF-␣-induced NF-B activation. In contrast, the ID is required for TNF-␣-induced IKK activation (8, 21). However, how the ID of RIP induces IKK activation is not fully understood. In the present study, we found that expression of a fusion protein composed of MEKK3 and the DD of RIP (MEKK3-DD) effectively restored TNF-␣-induced NF-B activation in RIP-deficient cells. The kinase activity of MEKK3 is required for the restoration of NF-B activation, since the kinase-deficient version of MEKK3-DD fails to do so (Fig. 2). Since MEKK3-DD associates with TRADD through the DD of RIP, MEKK3-DD is presumably recruited into the TNF-R1–TRADD complex following TNF-␣ stimulation in a RIP-independent manner (Fig. 10). Thus, these results indicate that the main function of the KD and ID of RIP is to recruit a MAP3K into the TNF-R1 complex. Although the expression of MEKK3-DD in RIP⫺ cells (M3-DD cells) effectively restores TNF-␣-induced NF-B activation, the kinetics of these activations for wt Jurkat and M3-DD cells are different. TNF-␣-induced NF-B activation was maximally induced after 30 min of stimulation in both Jurkat and M3-DD cells. This activation was slightly decreased at 120 min in Jurkat cells but rapidly diminished in M3-DD cells (Fig. 4). This result suggests that the KD and ID of RIP may be important for sustained NF-B activation. However, the kinetics of TNF-␣-induced NF-B activation in RIP⫺ cells restored with either wt or kinase-deficient versions of RIP are almost identical (data not shown), indicating that the kinase activity of RIP is not involved in controlling the down-regulation of TNF-␣-induced NF-B activation. Previous studies suggest that both MEKK2 and MEKK3 can activate NF-B and are involved in TNF-␣-induced NF-B activation (18, 22, 27). Although MEKK2 and MEKK3 have a high degree of homology, MEKK2-DD, unlike MEKK3-DD, failed to restore TNF-␣-induced NF-B activation in RIP⫺ cells (Fig. 3 and 4), indicating that the RIP-dependent recruitment of a MAP3K is specific. Since the N-terminal regulatory domains of MEKK3 and MEKK2 are approximately 65% ho-
MOL. CELL. BIOL.
mologous whereas their C-terminal kinase domains are 94% conserved, it is conceivable that the N-terminal regulatory domain confers differential regulation on these two kinases (1). This difference could be due to the fact that MEKK3 has a higher affinity for binding to IKK than MEKK2 (Fig. 8A). Together, these results suggest that TNF-␣-induced NF-B activation is mediated by RIP-dependent recruitment of MEKK3 into the TNF-R1 complex (Fig. 10). NEMO/IKK␥ is an essential subunit of the IKK complex that is critical for TNF-␣-induced NF-B activation. NEMO constitutively associates with IKK and IKK␣ (17). Although NEMO-DD also assembles into a complex with IKK␣ and IKK (Fig. 5B), associates with the upstream adaptor TRADD through the DD (Fig. 6C), and is recruited into the TNF-R1 complex following TNF-␣ stimulation (Fig. 7A), the expression of NEMO-DD failed to restore TNF-␣-induced NF-B activation in RIP⫺ cells (Fig. 5C and D). This result suggests that the recruitment of the IKK complex into the TNF-R1 complex alone is not sufficient for TNF-␣-induced NF-B activation. Our studies now reveal that it is essential to recruit a MAP3K, most likely MEKK3, into the TNF-R1 complex to activate NF-B. The recruitment of MEKK3 to the TNF-R1 complex may activate its kinase activity by an unknown mechanism. The activated MEKK3 is likely involved in the phosphorylation of IKK of the IKK complex (Fig. 9), as previously proposed. Consistent with this model, our results indicate that MEKK3 and MEKK3-DD associate with IKK but not with NEMO (Fig. 8A and B). In summary, our data demonstrate that MEKK3 is a critical and specific signaling molecule in RIP-dependent NF-B activation. The role of RIP in the TNF-␣ pathway is to recruit MEKK3 to the TNF-R1 complex (Fig. 10). In addition, we found that MEKK3 physically interacted with IKK. However, how MEKK3 is activated after being recruited to the TNF-R1 complex and whether MEKK3 is able to phosphorylate IKK directly remain to be determined. ACKNOWLEDGMENTS We thank B. Seed for providing the RIP-deficient Jurkat T cells and B. Su for providing anti-MEKK3 antibodies. This work was supported by a Public Health Service grant to X.L. (AI50848) from the National Institutes of Health. Y.Y. is supported by a Buswell Fellowship from the University at Buffalo. REFERENCES 1. Blank, J. L., P. Gerwins, E. M. Elliott, S. Sather, and G. L. Johnson. 1996. Molecular cloning of mitogen-activated protein/ERK kinase kinases (MEKK) 2 and 3. Regulation of sequential phosphorylation pathways involving mitogen-activated protein kinase and c-Jun kinase. J. Biol. Chem. 271:5361–5368. 2. Bouwmeester, T., A. Bauch, H. Ruffner, P. O. Angrand, G. Bergamini, K. Croughton, C. Cruciat, D. Eberhard, J. Gagneur, S. Ghidelli, C. Hopf, B. Huhse, R. Mangano, A. M. Michon, M. Schirle, J. Schlegl, M. Schwab, M. A. Stein, A. Bauer, G. Casari, G. Drewes, A. C. Gavin, D. B. Jackson, G. Joberty, G. Neubauer, J. Rick, B. Kuster, and G. Superti-Furga. 2004. A physical and functional map of the human TNF-alpha/NF-kappa B signal transduction pathway. Nat. Cell Biol. 6:97–105. 3. Chen, G., and D. V. Goeddel. 2002. TNF-R1 signaling: a beautiful pathway. Science 296:1634–1635. 4. Devin, A., A. Cook, Y. Lin, Y. Rodriguez, M. Kelliher, and Z. Liu. 2000. The distinct roles of TRAF2 and RIP in IKK activation by TNF-R1: TRAF2 recruits IKK to TNF-R1 while RIP mediates IKK activation. Immunity 12:419–429. 5. Ellinger-Ziegelbauer, H., K. Brown, K. Kelly, and U. Siebenlist. 1997. Direct activation of the stress-activated protein kinase (SAPK) and extracellular signal-regulated protein kinase (ERK) pathways by an inducible mitogen-
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