FAF1 Suppresses IκB Kinase (IKK) Activation by Disrupting the IKK Complex Assembly*

This study presents a molecular inhibitory mechanism by Fas-associated factor 1 (FAF1) on IκB kinase (IKK) activation, where divergent NF-κB-activating stimuli converge. FAF1 interacts with IKKβ in response to proinflammatory stimuli (such as tumor necrosis factor-α, interleukin-1β, and lipopolysaccharide) and suppresses IKK activation. Interaction of the leucine-zipper domain of IKKβ with FAF1 affected the IKK heterocomplex (IKKα/β) and homocomplex (IKKα/α, IKKβ/β) formations and attenuated IKKγ recruitment to IKKβ. Overexpression of FAF1 reduced the level of IKKβ activity, whereas FAF1 depletion increased the activity. These results indicate that FAF1 inhibits IKK activation and its downstream signaling by interrupting the IKK complex assembly through physical interaction with IKKβ. Taken together, FAF1 robustly suppresses NF-κB activation through the inhibition of IKK activation in combination with previously reported cytoplasmic retention of NF-κB p65 (Park, M. Y., Jang, H. D., Lee, S. Y., Lee, K. J., and Kim, E. (2004) J. Biol. Chem. 279, 2544–2549). Such redundant suppression would prevent inadvertent activation of the NF-κB pathway.

Fas-associated factor 1 (FAF1) 3 is evolutionarily conserved from flies to mammals (1)(2)(3)(4) and is involved in various key biological processes. FAF1 potentiates the Fas pathway as a member of the Fas death-inducing signaling complex and mediates chemotherapeutic-induced cell death (5,6). FAF1 also functions as an integral regulatory component of the transient receptor potential vanilloid type 1 (TRPV1) signaling pathway (7). FAF1 is involved in the ubiquitination pathway and interacts with ubiquitin and valosin-containing protein, which is a multiubiquitin chain-targeting factor (8). In addition, FAF1 inhibits the chaperone activities of the heat-shock proteins Hsc70 and Hsp70 (9).
FAF1 is also involved in the nuclear factor-B (NF-B) signaling pathway. FAF1 inhibits NF-B activation in HEK 293 cells by binding to NF-B p65 (10). NF-B inhibition by FAF1 has also been reported in Drosophila (4). Caspar, a fly homolog of human FAF1, selectively suppresses the immune deficiency (Imd) pathway. Loss-of-function caspar mutants constitutively expressed antibacterial genes in the absence of bacterial infections, indicating that caspar is an endogenous suppressor of the Imd pathway. Selective involvement of IB kinase (IKK) complex in the fly Imd pathway led us to examine the regulatory mechanism, if any, between FAF1 and the IKK complex.
The IKK complex is mainly composed of two catalytic subunits, IKK␣/IKK1 and IKK␤/IKK2, and a regulatory subunit, IKK␥/NF-B essential modulator (NEMO)/IKKAP1. Knockout mouse studies have demonstrated that IKK␤ has a dominant role in NF-B activation induced by proinflammatory cytokines, whereas IKK␣ is essential for morphogenic signaling (11). Although IKK␥ lacks the catalytic function, IKK␥ is essential for activation of the IKK complex. Both IKK␣ and IKK␤ contain an N-terminal kinase domain, a central leucine-zipper (LZ) domain, and a C-terminal helix-loop-helix (HLH) domain (12)(13)(14). Homo-and hetero-oligomerizations between IKK␣ and IKK␤ occur through their LZ domains, and the HLH domains mediate recruitment of IKK␥ to the IKK complex.
This study reveals a novel function of FAF1: as an endogenous suppressor of IKK activation. FAF1 disrupts IKK complex assembly through physical interaction with the LZ domain of IKK␤. Association between FAF1 and IKK␤ was induced by proinflammatory stimuli. Such an induced interaction indicates that FAF1 is an NF-B pathway suppressor with a unique mode of action.

EXPERIMENTAL PROCEDURES
Reagents and Constructs-Recombinant tumor necrosis factor-␣ (TNF␣) and interleukin-1␤ (IL-1␤) (R&D Systems Inc., * This research was supported by Grant CBM31-A2300-01-00-00 from the Center for Biological Modulators of the 21st Century Frontier R&D Program, MOST, Korea. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Transfection and RNA Interference-Cell transfection experiments were performed using the calcium phosphate precipitation method with 1 g of plasmid (15). Duplex siRNAs with two thymidine residues (dTdT) at the 3Ј-end of the sequence were provided by Qiagen Inc. (Valencia, CA) and transfected according to the manufacturer's instructions (Biontex, Munich, Germany). The target sequences were: IKK␣ (sense, 5Ј-GCAGGCUCUU-UCAGGGACA-3Ј) (16) and FAF1 (sense, 5Ј-CCACCUUCAUCAUC-UAGUC-3Ј). The siRNA-resistant FAF1 and FAF1-FID (Fas-interacting domain) plasmids were constructed using site-directed mutagenesis of GFP-FAF1 and GFP-FAF1-FID using the following oligonucleotide: 5Ј-CCGCCGUCGUCGUCUAGUC-3Ј (changed nucleotides are underlined).
Immunoprecipitation-The HEK 293 cells were lysed in lysis buffer (10), and the lysates were subject to centrifugation. A total of 200 g of protein from the supernatant was incubated with corresponding antibodies for 2 h at 4°C. Immunoprecipitates were washed and immunoblotted with the indicated antibodies.

RESULTS
Interaction between FAF1 and IKK␤ Is Induced by Proinflammatory Stimuli-Endogenous association of FAF1 and IKK␤ was induced in response to proinflammatory stimuli such as TNF␣, IL-1␤, and LPS (Fig. 1A). GST pulldown assays using various GST fusion proteins of the truncated FAF1 showed that the N-terminal half of FAF1 (amino acids 1-381) interact with IKK␤ (Fig. 1B). Similarly, GST pulldown assays between various domains of IKK␤ and FAF1 demonstrated that the LZ domain of IKK␤ strongly binds to FAF1 (Fig. 1C). The kinase domain of IKK␤ also showed weak binding to FAF1.
We asked whether FAF1 affected IKK␤ recruitment to the tetrameric IKK␥, which is the essential regulatory component that facilitates the trans-autophosphorylation of IKK␤ neces- sary for IKK complex activation (21). In vitro-translated 35 Slabeled IKK␥ proteins were treated with ethylene glycol-bissuccinimidyl succinate (EGS), a cross-linking agent, to induce IKK␥ tetramerization. Residual amounts of monomeric IKK␥ were still detected after EGS treatment, as has been previously reported. The addition of FAF1 reduced IKK␤ recruitment to the tetrameric and monomeric forms of IKK␥ in a dose-dependent manner (Fig. 2B), whereas unrelated proteins, such as ␤-tubulin and glyceraldehyde-3-phosphate dehydrogenase, did not (supplemental Fig. S2). Consistent with the above results, binding between the IKK␤-HLH domain and IKK␥ was strongly inhibited by the addition of FAF1 protein (Fig. 2C). Furthermore, IKK phosphorylation was blocked by the addition of FAF1 in a dose-dependent manner, whereas the total amount of exogenous IKK␤ remained unchanged (Fig. 2D). These results demonstrate that FAF1 selectively inhibits IKK activation by disrupting assembly of the fully competent IKK complex.
FAF1 Inhibits the Catalytic Activity of IKK␤-Next, the catalytic activity of IKK␤ in the disrupted IKK complex by FAF1 was measured. FAF1 overexpression reduced the TNF␣-induced catalytic activities of IKK␤ in a dose-dependent manner (Fig. 3A, upper right panel). However, introduction of FAF1specific siRNA increased the level of IKK␤ activity (Fig. 3A, upper left  panel). Similarly, FAF1 overexpression attenuated IL-1␤or LPS-induced IKK␤ activations (Fig. 3A, lower panels). Consistent with these in vivo results, the addition of GST-FAF1 also inhibited the IKK catalytic activity in vitro in a dose-dependent manner (Fig. 3B, right  panel). To further confirm the inhibitory function of FAF1, the level of IB␣, which is an endogenous marker of IKK activity, was examined. After TNF␣ treatment, the level of IB␣ in mock-transfected cells was more rapidly reduced than the level in FAF1transfected cells, indicating that FAF1 inhibits IB␣ phosphorylation (Fig. 3C). The level of IKK␤ activity was inversely correlated with the FAF1 expression level, indicating that FAF1 negatively regulates the IKK␤ activity. When IKK␣-specific siRNA was transfected, FAF1 inhibited IKK␤ activation (supplemental Fig. S3). This indicates that FAF1-mediated IKK␤ activity suppression occurred independently of IKK␣. Considering that fly homologs of mammalian IKK␤ and IKK␥, but not IKK␣, participate in the fly Imd pathway (22), inhibition of the fly Imd pathway by FAF1 correlates well with the IKK␣-independent IKK␤ suppression in mammalian cells shown here.
FAF1-FID Is Crucial for Suppression of IKK␤ Activation -Both the FID and the DEDID of FAF1 interact with IKK␤ (Fig. 1B); therefore, we investigated which domain is responsible for suppression of IKK␤ activation. Transfection of fulllength FAF1 and FAF1-FID led to significant suppression of IKK␤ catalytic activity, whereas FAF1-DEDID and FAF1-⌬FID⅐DEDID did not (Fig. 4A, left panel). Consistent with these cellular results, GST-FAF1-FID successfully inhibited the activity of IKK␤ kinase to its substrate GST-IB␣ in in vitro kinase assays (Fig.  4A, right panel). These data indicate that FAF1-FID is responsible and sufficient for the suppression of IKK␤ activation. This result provides a molecular explanation of the suppression of IKK complex formation by FAF1-FID ( Fig. 2A). We then tested whether FAF1-FID inhibits NF-B activation. FAF1-FID overexpression strongly inhibited NF-B activation by proinflammatory stimuli in HEK 293 cells (Fig.  4B, upper panel). The FAF1-FIDmediated suppression of IKK activation was also shown by reduced transcription of the NF-B target gene cIAP2 (Fig. 4B, lower panel).
ChIP results also showed that FAF1-FID significantly inhibited, whereas siFAF1 increased, recruitment of NF-B to the IB␣ promoter region (Fig. 4C).

DISCUSSION
This study demonstrates that FAF1 has a novel function as a suppressor of IKK complex activation. The FAF1-FID domain selectively inhibits IKK activation by disrupting IKK complex assembly. Together, previous reports of cytoplasmic retention of NF-B p65 and the Imd pathway suppression, and this study on suppression of IKK␤ activation, show that FAF1 is a genuine suppressor of the NF-B pathway.
The detailed mechanism underlying induced binding of FAF1- IKK␤ is yet to be determined. Phosphorylation of FAF1 has been reported (31); however, the phosphorylation status of FAF1 remained unchanged despite stimulation (data not shown). In addition, in vitro binding studies have shown that phosphorylation is not a prerequisite for the binding, indicating that phosphorylation of FAF1 might not be the determining factor. Other types of modification in FAF1 or in FAF1-interacting partners that occur in response to stimuli might facilitate the FAF1-IKK␤ interaction. Proteomic analysis of FAF1-interacting proteins in response to NF-B-activating stimuli would help us to investigate this issue.