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Molecular and Cellular Biology, June 2001, p. 3986-3994, Vol. 21, No. 12
0270-7306/01/$04.00+0   DOI: 10.1128/MCB.21.12.3986-3994.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

The alpha  and beta  Subunits of Ikappa B Kinase (IKK) Mediate TRAF2-Dependent IKK Recruitment to Tumor Necrosis Factor (TNF) Receptor 1 in Response to TNF

Anne Devin,1 Yong Lin,1 Shoji Yamaoka,2 Zhiwei Li,3 Michael Karin,3 and Zheng-gang Liu1,*

Department of Cell and Cancer Biology, Medicine Branch, Division of Clinical Sciences, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 208921; Department of Microbiology, School of Medicine, Tokyo Medical and Dental University, Tokyo, Japan2; and Laboratory of Gene Regulation and Signal Transduction, Department of Pharmacology, University of California, San Diego, La Jolla, California 920933

Received 7 December 2000/Returned for modification 26 January 2001/Accepted 26 March 2001


    ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

The activation of Ikappa B kinase (IKK) is a key step in the nuclear translocation of the transcription factor NF-kappa B. IKK is a complex composed of three subunits: IKKalpha , IKKbeta , and IKKgamma (also called NEMO). In response to the proinflammatory cytokine tumor necrosis factor (TNF), IKK is activated after being recruited to the TNF receptor 1 (TNF-R1) complex via TNF receptor-associated factor 2 (TRAF2). We found that the IKKalpha and IKKbeta catalytic subunits are required for IKK-TRAF2 interaction. This interaction occurs through the leucine zipper motif common to IKKalpha , IKKbeta , and the RING finger domain of TRAF2, and either IKKalpha or IKKbeta alone is sufficient for the recruitment of IKK to TNF-R1. Importantly, IKKgamma is not essential for TNF-induced IKK recruitment to TNF-R1, as this occurs efficiently in IKKgamma -deficient cells. Using TRAF2-/- cells, we demonstrated that the TNF-induced interaction between IKKgamma and the death domain kinase RIP is TRAF2 dependent and that one possible function of this interaction is to stabilize the IKK complex when it interacts with TRAF2.


    INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

The transcription factor NF-kappa B plays a critical role in regulating the expression of many cytokines and immunoregulatory proteins (1, 2, 3). NF-kappa B is composed of homo- or heterodimers of Rel and NF-kappa B proteins (1). The transcription activity of NF-kappa B can be elevated by various stimuli, including the proinflammatory cytokine tumor necrosis factor (TNF) (24). When bound to their specific inhibitors, referred to as Ikappa Bs, NF-kappa B dimers are sequestered in the cytoplasm and are therefore inactive (1, 32). In response to various stimuli, Ikappa Bs are phosphorylated by the Ikappa B kinase complex (IKK) and are then rapidly degraded by the proteasome after their polyubiquitination (1). The degradation of Ikappa Bs allows NF-kappa B to translocate into the nucleus and activate its target genes (1).

The three proteins IKKalpha , IKKbeta , and IKKgamma (also called NEMO) were identified as the components of the IKK complex (6, 23, 26, 29, 36, 37, 39, 40). IKKalpha and IKKbeta are two related catalytic subunits sharing about 52% identity, both containing an N-terminal kinase domain, a leucine zipper, and C-terminal helix-loop-helix motifs (12). IKKalpha and IKKbeta can form homo- or heterodimers via their leucine zipper motif, but the predominant IKK complex appears to contain mostly IKKalpha and IKKbeta heterodimers (29). The recent generation of IKKalpha -/- and IKKbeta -/- mice has established that IKKalpha and IKKbeta are required for the activation of NF-kappa B, although the absence of IKKalpha has a much smaller effect due to a compensatory effect of IKKbeta (11, 15, 17, 18, 34). In IKKalpha and IKKbeta double-knockout cells, TNF-induced NF-kappa B activation is completely abolished (16). Interestingly, however, IKKalpha and IKKbeta knockout mice exhibit completely different phenotypes (11, 15, 18, 34). It has also been suggested that IKKalpha plays a role in the activation of IKKbeta (25). However, IKK activation by TNF or interleukin-1 is barely affected in IKKalpha -/- cells (11). Meanwhile, IKKgamma is the regulatory subunit of the complex, and it binds to the C termini of IKKalpha and IKKbeta (22, 29, 37). Studies with IKKgamma -deficient cells have proven the essential role of IKKgamma in the activation of IKK and NF-kappa B (30, 37). Heterozygous female mice with IKKgamma deficiencies exhibit a dermatopathy similar to the human X-linked disorder incontinentia pigmenti (21, 31).

In response to TNF, IKK is quickly activated, which correlates with IKK recruitment to the TNF receptor complex (5, 42). Two components of the TNF receptor 1 (TNF-R1) signaling complex, TNF receptor-associated factor 2 (TRAF2) and the death domain kinase receptor-interacting protein (RIP), were shown to be required for NF-kappa B and IKK activation (5, 13, 35, 38). Although over expression of either RIP or TRAF2 could lead to robust NF-kappa B and IKK activation, the absence of either protein results in decreased TNF-induced NF-kappa B and IKK activation (5, 13, 35, 38). Recently, it has been found that TRAF2 and RIP play distinct signaling roles: TRAF2 recruits IKK to TNF-R1, whereas RIP mediates IKK activation (5). Interestingly, a TNF-induced interaction between IKKgamma and RIP which has been suggested to play a role in IKK recruitment to the TNF-R1 complex has also been observed (42).

In order to understand the mechanism underlying the interaction between TRAF2 and IKK, we investigated the respective role of each IKK subunit in this process. We also addressed the role of the interaction between RIP and IKKgamma in IKK recruitment. We found that IKKalpha and IKKbeta interact with TRAF2, but IKKgamma does not. This interaction requires the leucine zipper motif of IKKalpha or IKKbeta and the RING finger motif of TRAF2. Using IKKgamma -deficient cells, we found that the regulatory subunit is dispensable for IKKalpha and IKKbeta recruitment to the TNF-R1 complex. Although IKKgamma interacts with RIP in response to TNF, this interaction is TRAF2 dependent.


    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Reagents and plasmids. Anti-RIP antibody was purchased from Transduction Laboratories. Anti-TRAF2, anti-Xpress, anti-IKKalpha , anti-TNF-R1-associated death domain protein (anti-TRADD), and antihemagglutinin (anti-HA) antibodies were purchased from Santa Cruz. Anti-IKKgamma and anti-Myc antibodies were from Pharmingen. The anti-IKKbeta antibody was purchased from Upstate Biotechnology. The anti-Flag antibody was purchased from Sigma. The anti-TNF-R1 antibody was from R&D Systems. Human and mouse TNF-alpha (mTNF-alpha ) were purchased from R&D Systems. The mammalian expression plasmids for Myc-RIP, Flag-TRAF2, HA-IKKalpha , HA-IKKbeta , and IKKgamma have been described previously (10, 20, 39). The constructs for different glutatnione S-transferase (GST)-TRAF2 fusion proteins were previously described (14). The constructs for in vitro-translated HA-IKKalpha , HA-IKKbeta , and HA-IKKgamma were generated by subcloning these genes into the pBluescript vector (Stratagene). The expression plasmids for different domains of IKKalpha and IKKbeta were constructed by subcloning the different fragments (HindIII-XbaI for IKKalpha 1-371, HindIII-EcoRV for IKKalpha 1-500, EcoRV-NotI for IKKalpha 500-745, HindIII-BglII for IKKbeta 1-399, BglII-XhoI for IKKbeta 399-577, and BglII-NotI for IKKbeta 399-756) of the IKKalpha and IKKbeta genes into the pcDNA vector (Invitrogen).

Cell culture and transfection. Wild-type (wt), IKKalpha -/-, and IKKbeta -/- mouse fibroblast and HEK293 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum or 10% calf serum, 2 mM glutamine, 100 U/of penicillin/ml, and 100 µg of streptomycin/ml; wt and (IKKgamma )-deficient (5R) rat fibroblast were also cultured in this medium. RIP-/- and TRAF2-/- cells were cultured in the same medium except that 0.3 mg/of G418/ml was included. Transfection experiments were performed with Lipofectamine PLUS reagent by following the instructions provided by the manufacturer (GIBCO/BRL).

Western blot analysis and coimmunoprecipitation. For Western blotting, cells were treated with mTNF-alpha as described in the figure legends and then collected in M2 lysis buffer (20 mM Tris [pH 7], 0.5% NP-40, 250 mM NaCl, 3 mM EDTA, 3 mM EGTA, 2 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 20 mM beta -glycerol phosphate, 1 mM sodium vanadate, 1 µg of leupeptin/ml; 1 µg of aprotinin/ml, 1 µg of pepstatin/ml, and 10 mM pNpp). Fifty micrograms of the cell lysates were fractionated on sodium dodecyl sulfate (SDS)-4 to 20% polyacrylamide gels, and Western blottings were performed with the desired antibodies. The proteins were visualized by enhanced chemiluminescence according to the manufacturer's instructions (Amersham).

For immunoprecipitation assays, 3 × 107 mTNF-alpha (40 ng/ml)-treated or untreated fibroblasts were collected in lysis buffer (50 mM HEPES [pH 7.6], 250 mM NaCl, 0.1% NP-40, 5 mM EDTA, 0.5 mM pherylinethylsulfonyl fluoride, 1 µg of leupeptin/ml, 1 µg of aprotinin/ml, and 1 µg of pepstatin/ml). The lysates were mixed and precipitated with the relevant antibody and protein A-Sepharose beads by incubation at 4°C for 4 h to overnight. The beads were washed four times with 1 ml of lysis buffer, and the bound proteins were resolved in SDS-10% polyacrylamide gels and detected by Western blot analysis. For immunoprecipitations with antibodies that were cross-linked to protein A-Sepharose beads as indicated in the figure legends, antibodies (100 µg of antibody/ml of wet beads) were coupled to the beads with dimethylpimelimidate as previously described (7).

For GST pull-down experiments with in vitro translated, 35S-labeled IKK subunits and different GST-TRAF2 proteins (14), 5 µg of each GST protein was combined with the in vitro translation lysate of each IKK subunit in 1 ml of lysis buffer (see above) and incubated at 4°C for 2 h. Glutathione-Sepharose beads were then added, and incubation was performed overnight. The beads were extensively washed with lysis buffer and the bound proteins were resolved in SDS-10% polyacrylamide gels. The GST-TRAF2 proteins were detected by Coomassie blue staining and the coprecipitated IKK proteins were visualized by autoradiography.


    RESULTS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

IKK is recruited to TNF-R1 through an interaction between IKKalpha or IKKbeta and TRAF2. In response to TNF binding to TNF-R1, a signaling complex is rapidly formed that includes TRADD, RIP, and TRAF2 (8, 9, 10, 19, 27, 28, 33). Recently, IKK was found to be recruited to the same TNF-R1 complex (5); moreover, its recruitment was found to be mediated by TRAF2 (5). The recruitment of IKK to the TNF-R1 signaling complex can be detected by immunoprecipitation experiments with anti-TNF-R1 antibody following TNF treatment. As shown in Fig. 1A, three IKK subunits, IKKalpha , IKKbeta , and IKKgamma were recruited to the TNF-R1 in wt mouse fibroblasts but not in TRAF2-/- fibroblasts. In RIP-/- fibroblasts, IKKalpha and IKKbeta recruitment to TNF-R1 was similar to that in wt cells but the recruitment of IKKgamma was notably decreased in comparison to what was observed in wt cells (Fig. 1A). The recruitment of TRAF2, TRADD, and RIP to TNF-R1 in wt, RIP-/-, and TRAF2-/- fibroblasts is shown in Fig. 1B. As was reported previously (5), TRAF2 plays an essential role in recruiting IKK to TNF-R1.


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  		FIG. 1.   Recruitment of IKK to TNF-R1 requires TRAF2. (A) Cell extracts were prepared from wt, RIP-/- and TRAF2-/- fibroblasts either treated for 2 min. with 40 ng of mTNF-alpha /ml or left untreated. After normalization of protein content according to the protein assay, cell extracts were immunoprecipitated with anti-TNF-R1 antibody overnight. Immunoprecipitates were resolved by SDS-polyacrylamide gel electrophoresis (PAGE), and Western blotting was performed with anti-IKKalpha , anti-IKKbeta , and anti-IKKgamma . Cell extract (1%) from each treated sample was used as a control for protein content (input). (B) Immunoprecipitates were also analyzed by Western blotting with anti-TRAF2, anti-TRADD, or anti-RIP. Numbers on the left are molecular masses in kilodaltons.

These immunoprecipitation experiments with cell extracts do not provide information about the biochemical basis for the interaction between IKK and TRAF2, although it has previously been shown that the RING domain of TRAF2 is essential for this interaction (5). It is important to know, for instance, whether IKK binds directly to TRAF2 and, if so, which IKK subunit mediates this interaction. To address these issues, we performed GST pull-down experiments using GST-TRAF2 fusion proteins and different IKK subunits. Since the TRAF domain of TRAF2 is dispensable for downstream signaling as long as the N-terminal domain is oligomerized (4), we used the GST fusion proteins containing the RING finger (amino acids 1 to 105), the zinc finger (76 to 282), and the RING and zinc fingers (1 to 225) of TRAF2, as described before (14). The three 35S-labeled IKK proteins were generated by in vitro translation with wheat germ lysate. In these experiments, GST alone was used as a negative control. As shown in Fig. 2A and B, both IKKalpha and IKKbeta bound to the RING finger domain of TRAF2, whereas they did not interact with the zinc finger region. The presence of both the RING and zinc fingers strengthened the interaction between TRAF2 and IKKalpha or IKKbeta (Fig. 2A and B). The amounts of the different GST-TRAF2 fusion proteins precipitated in these experiments are shown in Fig. 2A and B. In contrast, IKKgamma did not show any considerable interaction with the different GST-TRAF2 proteins (Fig. 2C). These data suggested that either IKKalpha or IKKbeta can bind directly to the RING domain of TRAF2.



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  		FIG. 2.   IKKalpha and IKKbeta interact with the RING domain of TRAF2. In vitro-translated IKKalpha (A), IKKbeta (B), and IKKgamma (C) were mixed with either GST or different GST-TRAF2 proteins (18), and then GST pull-down experiments were performed. Precipitates were resolved by SDS-PAGE (top panels), and the coprecipitation of different IKK subunits was detected by autoradiography. The precipitation of different GST proteins was examined by Coomassie blue staining. The in vitro translation lysate for each subunit was used as a control. Numbers on the left are molecular masses in kilodaltons.

The leucine zipper domain of IKKalpha and IKKbeta is essential for their interaction with TRAF2. IKKalpha and IKKbeta are related catalytic subunits with an overall identity of about 52% (12). Both contain an N-terminal kinase domain, a leucine zipper, and a C-terminal helix-loop-helix motif (12). In order to determine which region of these proteins was involved in their interaction with TRAF2, we generated expression constructs for different truncated IKKalpha and IKKbeta proteins as shown in Fig. 3A and C. These constructs were then used to perform coimmunoprecipitation experiments. In these experiments, the different truncated IKKalpha or IKKbeta proteins were ectopically expressed together with Flag-TRAF2 in HEK293 cells. After Flag-TRAF2 was immunoprecipitated with anti-Flag antibody, the immune complexes were analyzed by Western blotting with anti-HA or anti-Xpress antibody. As shown in Fig. 3B and D, the leucine zipper motif of IKKalpha or IKKbeta is essential for interaction with TRAF2. The kinase domain and helix-loop-helix domain of IKKalpha or IKKbeta failed to interact with TRAF2. These data indicate that IKKalpha and IKKbeta interact with TRAF2 through their leucine zipper motifs. Alternatively, the interaction may require a dimer whose formation depends on the leucine zipper motif.


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  		FIG. 3.   The leucine zipper domain of IKKalpha and IKKbeta interacts with TRAF2. (A) Diagrams of different IKKalpha constructs used for the mapping of IKKalpha interaction with TRAF2. (B) HEK293 cells were cotransfected with 5 µg of Flag-TRAF2 and 5 µg of each of the IKKalpha expression plasmids [HA-IKKalpha , HA-IKKalpha (1-371), HA-IKKalpha (1-500), and Xpress-IKKalpha (500-745)]. Cells were collected 24 h after transfection, and cell extracts were immunoprecipitated with anti-Flag antibody overnight. Immunoprecipitates were resolved by SDS-PAGE, and Western blotting was performed with anti-HA, anti-Xpress, or anti-Flag. Cell extract (4%) from each sample was used as a control (input). (C) Diagrams of different IKKbeta constructs used for mapping IKKbeta interaction with TRAF2. (D) HEK293 cells were cotransfected with 5 µg of Flag-TRAF2 and 5 µg of each of the IKKbeta constructs [HA-IKKbeta , HA-IKKbeta (1-399), Xpress-IKKbeta (399-577), and Xpress-IKKbeta (399-756)]. Twenty four hours after transfection, immunoprecipitation experiments and Western blotting were performed as described for panel B. Numbers on the right are molecular masses in kilodaltons.

Either IKKalpha or IKKbeta alone is sufficient to mediate the recruitment of IKK to the TNF-R1 complex in response to TNF. Since both IKKalpha and IKKbeta can interact with TRAF2 efficiently, we next investigated which one of them is responsible for physiological IKK recruitment following TNF treatment. We addressed this question by performing TNF-R1 immunoprecipitation experiments with IKKalpha -/- and IKKbeta -/- mouse fibroblasts (11, 18). As before, the immune complexes were analyzed by Western blotting sequentially with anti-IKKbeta , anti-IKKgamma , and anti-TRADD antibodies (Fig. 4A) or with anti-IKKalpha , anti-IKKgamma , and anti-RIP (Fig. 4B). In the absence of either IKKalpha or IKKbeta , IKK complexes containing IKKbeta and IKKgamma or IKKalpha and IKKgamma , respectively, were still recruited to TNF-R1 upon TNF treatment with an efficiency similar to that in wt fibroblasts (Fig. 4A and B). As controls, the TNF-induced recruitment of TRADD and/or RIP to TNF-R1 in IKKalpha -/- and IKKbeta -/- cells was also examined, and TRADD and RIP were found to be recruited to TNF-R1 normally (Fig. 4A and B). The protein expression levels of IKKalpha , IKKbeta , and IKKgamma in wt, IKKalpha -/-, and IKKbeta -/- cells were measured by Western blotting. As shown in Fig. 4C, the expression levels of each subunit, when present, are similar in all three types of cells.


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  		FIG. 4.   IKKalpha or IKKbeta alone is sufficient to mediate the interaction between IKK and TRAF2. (A) Cell extracts were prepared from wt and IKKalpha -/- fibroblasts either left untreated or treated with 40 ng of mTNF/ml. After normalization of protein content according to the protein assay, cell extracts were immunoprecipitated with anti-TNF-R1 antibody overnight. Immunoprecipitates were resolved by SDS-PAGE, and Western blotting was performed sequentially with anti-IKKalpha , anti-IKKbeta , anti-IKKgamma , anti-TRADD, and anti-RIP antibodies. Cell extract (1%) from each treated sample was used as a control for protein content (input). (B) Immunoprecipitation experiments were performed as described for panel A except that IKKbeta -/-, instead of IKKalpha -/-, fibroblasts were used. Western blotting was performed sequentially with anti-IKKalpha , anti-IKKbeta , anti-IKKgamma , and anti-RIP. (C) The same amount of cell extract from wt, IKKalpha -/-, or IKKbeta -/- cells was used for measuring the expression of IKKalpha , IKKbeta , and IKKgamma by Western blotting. (D) Cell extracts were prepared from IKKalpha -/- and IKKbeta -/- fibroblasts either treated with 40 ng of mTNF/ml or left untreated. After normalization of protein content according to the protein assay, cell extracts were immunoprecipitated with anti-IKKgamma antibody overnight. Immunoprecipitates were resolved by SDS-PAGE, and Western blotting was performed with anti-IKKbeta in IKKalpha -/- cells and anti-IKKalpha in IKKbeta -/- cells. Cell extract (1%) from each treated sample was used as a control for protein content (input). Numbers on the left are molecular masses in kilodaltons.

Since IKKgamma is normally complexed with both IKKalpha and IKKbeta in wt cells (18), we wanted to confirm that IKKgamma still forms a complex with IKKbeta or IKKalpha in IKKalpha -/- or IKKbeta -/- cells, respectively. To accomplish this, we performed immunoprecipitation experiments with anti-IKKgamma antibody in IKKalpha -/- and IKKbeta -/- cells. As shown in Fig. 4D, IKKgamma efficiently interacts with IKKbeta in the absence of IKKalpha and with IKKalpha in the absence of IKKbeta . TNF treatment had no effect on these interactions. These results suggest that either IKKalpha or IKKbeta alone together with IKKgamma is sufficient for recruitment to TNF-R1.

The TNF-induced interaction between RIP and IKKgamma requires TRAF2. Recently, IKKgamma has been found to interact with RIP in response to TNF, therefore, it has been proposed that IKKgamma mediates IKK recruitment to TNF-R1 (42). However, the results shown in Fig. 1 and previous studies (5) indicated that TRAF2, not RIP, is essential for bringing IKK to TNF-R1. Since the interaction between RIP and IKKgamma was observed in the presence of TRAF2 (42), we investigated whether TRAF2 is required for this interaction. Consistent with a previous report (42), when RIP was overexpressed with either IKKalpha , IKKbeta , or IKKgamma it was coprecipitated only with IKKgamma (Fig. 5A). To test whether RIP interacts with IKKgamma in the absence of TRAF2 in response to TNF treatment, we performed coimmunoprecipitation experiments in wt and TRAF2-/- cells. As shown in Fig. 5B, RIP was coprecipitated with IKKgamma in TNF-treated wt cells but not in TNF-treated TRAF2-/- cells. These results suggest that TRAF2 is necessary for the interaction of RIP with IKKgamma under physiological conditions.


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  		FIG. 5.   TRAF2 is required for the TNF-induced interaction between IKKgamma and RIP. (A) HEK293 cells were cotransfected with 5 µg of Myc-RIP and 5 µg of each of the HA-tagged IKK subunits. Cells were collected 24 hours after transfection, and cell extracts were used for immunoprecipitation experiments with anti-HA antibody. Immunoprecipitates were resolved by SDS-PAGE, and Western blotting was performed with anti-Myc and anti-HA. (B) Immunoprecipitation experiments were performed with cell extracts prepared from wt and TRAF2-/- fibroblasts with or-without mTNF (40 ng/ml) treatment. After normalization of protein content according to the protein assay, cell extracts were immunoprecipitated with anti-IKKgamma antibody overnight. Immunoprecipitates were resolved by SDS-PAGE, and Western blotting was performed with anti-RIP or anti-IKKbeta . Cell extract (2%) from each treated sample was used as a control for protein content (input). Numbers on the left are molecular masses in kilodaltons.

IKKgamma is not essential for TNF-induced IKK recruitment to TNF-R1. To further understand the role of IKKgamma in IKK recruitment, we tested whether IKK can be recruited to TNF-R1 in the absence of IKKgamma . To do so, we performed immunoprecipitation experiments with anti-TNF-R1 antibody in Rat-1 and 5R fibroblasts, the latter being IKKgamma deficient (37). In these experiments TNF-R1 complexes were immunoprecipitated from either untreated or TNF-treated Rat-1 and 5R cells and the immunoprecipitates were analyzed by Western blotting sequentially with anti-IKKalpha , anti-IKKbeta , and anti-RIP antibodies. As shown in Fig. 6A, both IKKalpha and IKKbeta were efficiently recruited to TNF-R1 in 5R cells following TNF treatment. However, the levels of IKKalpha and IKKbeta recruitment in 5R cells were slightly decreased compared to that in Rat-1 cells. As a control, the TNF-induced recruitment of RIP was examined and was found to be similar in both types of cells (Fig. 6A). These results indicated that IKKgamma was dispensable for the TNF-induced recruitment of IKK to TNF-R1 although its presence enhances the efficiency of IKK recruitment. This conclusion was further confirmed by the immunoprecipitation experiments with anti-TRAF2 antibody (Fig. 6B). The expression levels of IKKalpha , IKKbeta , and IKKgamma in Rat-1 and 5R cells were examined by Western blotting. As shown in Fig. 6C, IKKalpha and IKKbeta were expressed similarly in both cell types. Thus, it is IKKalpha or IKKbeta but not IKKgamma that plays an essential role in the recruitment of IKK to the TNF-R1 signaling complex.


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  		FIG. 6.   IKKgamma is not essential for TNF-induced IKK recruitment to TNF-R1. (A) Immunoprecipitation experiments were performed with cell extracts prepared from wt Rat-1 and 5R cells with or without mTNF (40 ng/ml) treatment. After normalization of protein content according to the protein assay, cell extracts were immunoprecipitated with anti-TNF-R1 antibody overnight. Immunoprecipitants were resolved by SDS-PAGE, and Western blotting was performed sequentially with anti-IKKalpha , anti-IKKbeta , and anti-RIP antibodies. Cell extract (1%) from each treated sample was used as a control for protein content (input). (B) Similar experiments were performed as described for panel A except that anti-TRAF2 antibody was used for immunoprecipitation. Western blotting was performed with anti-IKKbeta and anti-RIP antibodies. (C) The same amount of cell extract from Rat-1 or 5R cells was applied to SDS-PAGE for Western blotting with anti-IKKalpha , anti-IKKbeta , and anti-IKKgamma antibodies. Numbers on the left are molecular masses in kilodaltons.

RIP plays a role in stabilizing IKK. Since the RIP-IKKgamma interaction is not essential for TNF-induced IKK recruitment, we next investigated the possible function of the RIP-IKKgamma interaction in TNF-induced IKK activation. According to the results shown in Fig. 1A, the amount of recruited IKKgamma , but not of IKKalpha or IKKbeta , was decreased in RIP-/- cells in comparison with amounts in wt cells. Because IKKgamma normally forms a complex with IKKalpha and IKKbeta in RIP-/- cells (data not shown), one explanation for this observation is that the TNF-induced TRAF2-IKK interaction interfered with the binding of IKKgamma to IKKalpha and IKKbeta . To test this possibility, we examined whether the presence of TRAF2 disrupts the IKKalpha -IKKgamma interaction. In these experiments, Flag-IKKalpha and HA-IKKgamma were ectopically coexpressed with increasing amounts of Flag-TRAF2. After HA-IKKgamma was immunoprecipitated, the precipitates were analyzed by Western blotting for Flag-IKKalpha , Flag-TRAF2, and HA-IKKgamma . As shown in Fig. 7A, in the absence of TRAF2, IKKalpha and IKKgamma interacted nicely, and this interaction was gradually disrupted as the expression level of TRAF2 was increased. Similar amounts of HA-IKKgamma were immunoprecipitated in these experiments and some of the coexpressed Flag-TRAF2 was also detected. The expression levels of Flag-IKKalpha , Flag-TRAF2, and HA-IKKgamma are shown in Fig. 7A. When Flag-TRAF2(87-501), which lacks the RING finger domain and is thus incapable of recruiting IKK to the TNF-R1 (5), was used in a similar experiment, it had no effect on the interaction between IKKalpha and IKKgamma (Fig. 7B). The expression levels of Flag-IKKalpha , Flag-TRAF2(87-501), and IKKgamma were examined as shown in Fig. 7B. These data indicate that the interaction of TRAF2 and IKKalpha had some interfering effect on the interaction between IKKalpha and IKKgamma .


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  		FIG. 7.   RIP is required to stabilize the interaction between IKKalpha and IKKgamma when IKKalpha binds to TRAF2. (A) HEK293 cells were cotransfected with 3 µg of Flag-IKKalpha , 3 µg of HA-IKKgamma , and increasing amounts of Flag-TRAF2 as shown. After 24 h, cell extracts were collected and used for immunoprecipitation with anti-HA antibody. Immunoprecipitates were resolved by SDS-PAGE, and Western blotting was performed with anti-Flag antibody and anti-HA. (B) HEK293 cells were cotransfected with 3 µg of Flag-IKKalpha , 3 µg of HA-IKKgamma , and 0 or 4 µg of Flag-TRAF2(87-501) as shown. Then immunoprecipitation and Western blotting were performed as described for panel A. (C) HEK293 cells were cotransfected with 3 µg of Flag-IKKalpha , 2 µg of HA-IKKgamma , 2 µg of Myc-RIP, and increasing amounts of Flag-TRAF2 as shown. Immunoprecipitation and Western blotting were performed as described for panel A except that anti-Myc antibody was used to detect Myc-RIP expression.

Because RIP can also interact with TRAF2 (10), when RIP is recruited to the TNF-R1 complex, RIP may stabilize the IKK complex by simultaneously interacting with both TRAF2 and IKKgamma . If this is true, the presence of RIP will counteract the interfering effect of TRAF2 on the interaction between IKKalpha and IKKgamma . To test this hypothesis, we performed the coimmunoprecipitation experiments as described in the legend to Fig. 7A except with the addition of RIP. As shown in Fig. 7C, the expression of RIP in these experiments completely prevented the disruptive effect of TRAF2 and restored the interaction of IKKalpha and IKKgamma . The presence of Flag-TRAF2, Myc-RIP, and HA-IKKgamma was also examined (Fig. 7C). The expression of IKKalpha , IKKgamma , TRAF2, and RIP was detected by Western blotting (Fig. 7C). These results implied that one possible function of RIP in TNF-induced IKK activation is to stabilize IKK after its recruitment to the TNF-R1 complex.


    DISCUSSION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

The regulation of TNF-induced NF-kappa B activation is complex, and one of the key steps in this process is the activation of IKK (12). To be activated by TNF, IKK needs to be quickly recruited to the TNF-R1 complex following TNF treatment (5, 42). Recently it was reported that TRAF2 is essential for TNF-induced IKK recruitment (5). However, because RIP has been found to interact with IKKgamma in response to TNF, it has been suggested that the RIP-IKKgamma interaction is accountable for bringing IKK to TNF-R1 (42). In this study, we demonstrated that the two catalytic subunits of IKK, IKKalpha and IKKbeta , interact with TRAF2 to mediate the TNF-induced IKK recruitment to TNF-R1 and that the regulatory subunit of IKK, IKKgamma , is not essential for this recruiting process. Using TRAF2-/- fibroblasts, we also showed that the RIP-IKKgamma interaction is TRAF2 dependent. Moreover, we proposed that one possible function of RIP in TNF-induced IKK activation is to stabilize the IKKgamma subunit in the IKK complex.

IKKalpha and IKKbeta are highly homologous and have the same structural features, including kinase, leucine zipper, and helix-loop-helix motifs (12). The helix-loop-helix motif of IKKalpha and IKKbeta is thought to be involved in regulating their kinase activity, while the leucine zipper motif is essential for the dimerization of IKKalpha and IKKbeta and their kinase activity (12). Although both IKKalpha and IKKbeta are capable of phosphorylating Ikappa B, IKKbeta apparently plays a major role in TNF-induced NF-kappa B activation (11, 18, 26, 34, 36). In this study we identified another function of IKKalpha and IKKbeta , the mediation of the interaction between IKK and TRAF2 in response to TNF. We found that IKKalpha and IKKbeta bind to TRAF2 equally well. It appears that the interaction between IKK and TRAF2 requires the leucine zipper motif of IKKalpha or IKKbeta and the RING finger domain of TRAF2. Therefore, besides being required for the dimerization of IKKalpha and IKKbeta and for IKK kinase activity, the leucine zipper motif of IKKalpha and IKKbeta is also essential for IKK to interact with its upstream effector TRAF2 in response to TNF. The studies with IKKalpha and IKKbeta knockout mice indicated that IKKbeta is the major kinase in TNF-induced NF-kappa B activation, since the deletion of IKKalpha had only a minor effect on this process (11, 15, 16, 18, 34). According to our results, either IKKalpha or IKKbeta alone was capable of mediating TNF-induced IKK recruitment to TNF-R1 (Fig. 4). Because effector molecules, including TRADD, RIP, and TRAF2, were recruited to TNF-R1 normally in IKKalpha -/- and IKKbeta -/- cells, it seems that the varied effects of the deletion of IKKalpha or IKKbeta on TNF-induced NF-kappa B activation are due solely to the difference in the kinase activity of IKKalpha and IKKbeta in terms of Ikappa B phosphorylation.

The third component of IKK is IKKgamma , a regulatory subunit (29, 37). It is known that IKKgamma is required for elevating IKK activity by a variety of stimuli and that it binds to the C termini of IKKalpha and IKKbeta to form the IKK complex (12). In response to TNF treatment, IKKgamma interacts with RIP (42). In our study, we found that the interaction between IKKgamma and RIP is not essential for IKK recruitment to TNF-R1, because IKKalpha and IKKbeta were still recruited efficiently in RIP-/- cells. More importantly, the interaction between IKKgamma and RIP is dependent on the recruitment of the IKK complex to TRAF2. Therefore, the critical role of IKKgamma in TNF-induced IKK activation is not to mediate IKK recruitment. We also found that when IKK bound to TRAF2 in response to TNF, the interaction between IKK and TRAF2 destabilized the IKK complex by weakening the binding of IKKgamma to the other two subunits. Although IKKgamma and TRAF2 bind to different regions of IKKalpha and IKKbeta , it appears that the binding of TRAF2 to IKKalpha and IKKbeta interferes with the interaction between IKKgamma and the two catalytic subunits. To completely retain IKKgamma in the IKK complex after it is recruited to TNF-R1, IKKgamma needs to interact wit RIP. The presence of RIP and IKKgamma may enhance the recruitment of IKK to TNF-R1. But since both RIP and IKKgamma are essential for TNF-induced IKK activation, their major function in this process must be to activate IKK, although the mechanism is still not clear. It is possible that the RIP-IKKgamma interaction results in conformational changes in IKK and, in turn, leads to the autophosphorylation and subsequent activation of IKK. Another possibility is that RIP is required for recruiting the IKK kinase, most likely a mitogen-activated protein kinase kinase kinase, and then the interaction between RIP and IKKgamma primes the IKK kinase to activate IKK. Although the study of the kinetics of TNF-induced IKK activation favors the latter possibility (5), the identification of the putative IKK kinase is a critical step in fully understanding the mechanism of TNF-induced IKK activation.


    ACKNOWLEDGMENTS

We thank W.-C. Yeh and T. W. Mak for TRAF2-/- fibroblasts, M. Kelliher for RIP-/- fibroblasts, and U. Siebenlist for GST-TRAF2 constructs. We are grateful to Joseph Lewis for his assistance in manuscript preparation.


    FOOTNOTES

* Corresponding author. Mailing address: Medicine Branch, NCI, NIH, Bldg. 10, Rm. 6N105, 9000 Rockville Pike, Bethesda, MD 20892. Phone: (301) 435-6351. Fax: (301) 402-1997. E-mail: zgliu{at}helix.nih.gov.


    REFERENCES
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

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Molecular and Cellular Biology, June 2001, p. 3986-3994, Vol. 21, No. 12
0270-7306/01/$04.00+0   DOI: 10.1128/MCB.21.12.3986-3994.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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