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. Author manuscript; available in PMC: 2007 Sep 23.
Published in final edited form as: Mol Cell. 2007 Jun 8;26(5):689–702. doi: 10.1016/j.molcel.2007.05.006

XIAP induces NF-κB activation via the BIR1/TAB1 interaction and BIR1 dimerization

Miao Lu 1, Su-Chang Lin 1, Yihua Huang 1,*, Young Jun Kang 2, Rebecca Rich 3, Yu-Chih Lo 1, David Myszka 3, Jiahuai Han 2, Hao Wu 1
PMCID: PMC1991276  NIHMSID: NIHMS25551  PMID: 17560374

Summary

In addition to caspase inhibition, X-linked inhibitor of apoptosis (XIAP) induces NF-κB and MAP kinase activation during TGFβ and BMP receptor signaling and upon overexpression. Here we show that the BIR1 domain of XIAP, which has no previously ascribed function, directly interacts with TAB1 to induce NF-κB activation. TAB1 is an upstream adapter for the activation of the kinase TAK1, which in turn couples to the NF-κB pathway. We report the crystal structures of BIR1, TAB1 and the BIR1/TAB1 complex. The BIR1/TAB1 structure reveals a striking butterfly-shaped dimer and the detailed interaction between BIR1 and TAB1. Structure-based mutagenesis and knockdown of TAB1 show unambiguously that the BIR1/TAB1 interaction is crucial for XIAP-induced TAK1 and NF-κB activation. We show that although not interacting with BIR1, Smac, the antagonist for caspase inhibition by XIAP, also inhibits the XIAP/TAB1 interaction. Disruption of BIR1 dimerization abolishes XIAP-mediated NF-κB activation, implicating a proximity-induced mechanism for TAK1 activation.

Introduction

The inhibitor of apoptosis proteins (IAPs) were originally identified in baculoviruses but have been subsequently found in diverse organisms (Crook et al., 1993; Duckett et al., 1996; Hay et al., 1995). They are characterized by the presence of conserved baculoviral IAP repeat (BIR) domains (Eckelman et al., 2006; Schimmer et al., 2006). In humans, the current IAP family members include XIAP, cIAP1, cIAP2, ML-IAP, NAIP, ILP2, Survivin and Bruce (Eckelman et al., 2006). Although the first recognized function of IAPs is anti-apoptosis and caspase inhibition, IAPs are now known as a family of multifunctional proteins that also play critical roles in receptor signaling, cell division, copper metabolism, and ubiquitination of proteins for proteasomal degradation (Eckelman et al., 2006; Mufti et al., 2006; Vaux and Silke, 2005).

XIAP is the best-studied member of the IAP family. It contains three baculoviral IAP repeat (BIR) domains and a RING domain (Deveraux et al., 1997; Duckett et al., 1996; Holcik et al., 2001) (Supplemental Fig. S1). Previous structural and biochemical studies have shown that the linker preceding the BIR2 domain of XIAP directly blocks the active sites of caspase-3 and caspase-7 (Chai et al., 2001; Huang et al., 2001; Riedl et al., 2001), while the BIR3 domain sterically hinders caspase-9 dimerization and its activation (Shiozaki et al., 2003). By doing so, XIAP acts as a brake on caspase-mediated cellular dismantling. Upon apoptosis induction, Smac (also known as DIABLO) gets released from the inter-membrane space of the mitochondria and interacts with the BIR2 and BIR3 domains of XIAP to relieve caspase inhibition (Chai et al., 2000; Du et al., 2000; Huang et al., 2003; Verhagen et al., 2000; Wu et al., 2000). The RING domain of XIAP may act as an E3 in the ubiquitination pathway to promote the turnover of a number of cellular proteins as well as itself (Vaux and Silke, 2005). The function of the BIR1 domain is unknown.

In addition to the well-characterized function of XIAP in caspase inhibition, an important function of XIAP is its role in signaling to NF-κB and MAP kinase activation (Birkey Reffey et al., 2001; Lewis et al., 2004; Sanna et al., 1998; Shibuya et al., 1996; Yamaguchi et al., 1999; Yamaguchi et al., 1995). In fact, while the caspase inhibitory function of XIAP does not appear to be conserved in other IAP family members (Eckelman et al., 2006), this signaling function of XIAP is conserved in at least two other IAP members, NAIP and ML-IAP (Sanna et al., 2002). In addition, two other IAPs, cIAP1 and cIAP2, associate with TRAFs in the TNF signaling pathway (Rothe et al., 1995) and may facilitate or regulate TRAF-mediated NF-κB and MAP kinase activation (Tang et al., 2003).

Under physiological states, XIAP plays a role in development by mediating transforming growth factor β (TGFβ and bone morphogenetic protein (BMP) signaling. It bridges the TGFβ and BMP type I receptors to TAK1 (Birkey Reffey et al., 2001; Yamaguchi et al., 1999). TAK1 is a MAP kinase kinase kinase (MAP3K) that activates MAP kinases and NF-κB transcription factors by directly activating MAP kinase kinase (MKK) and the inhibitor of κB kinase (IKK) (Wang et al., 2001; Yamaguchi et al., 1995). It is essential for mesoderm induction and patterning in early Xenopus development (Shibuya et al., 1998; Shibuya et al., 1996; Yamaguchi et al., 1995), for diverse developmental roles such as control of cell shape and regulation of apoptosis in Drosophila (Takatsu et al., 2000) and for vascular development in mice (Jadrich et al., 2003; Jadrich et al., 2006). Injection of XIAP mRNA into dorsal blastomeres enhanced the ventralization of Xenopus embryos in a TAK1-dependent manner (Yamaguchi et al., 1999), confirming the role of XIAP in development. Moreover, XIAP deficiency in mice exhibits delays in the development of the mammary gland in a manner that correlates with delayed NF-κB activation (Olayioye et al., 2005), suggesting that the role of XIAP in development is related to its TAK1 and NF-κB activation ability.

XIAP is differentially up regulated in many forms of human cancers and confers resistance to chemotherapy-induced cell death (Berezovskaya et al., 2005; Wilkinson et al., 2004). In contrast, down-regulation of XIAP with siRNA or antisense oligonucleotides enhances sensitivity to chemotherapy for a variety of malignant cell lines (Chawla-Sarkar et al., 2004; McManus et al., 2004; Sasaki et al., 2000; Tong et al., 2005). XIAP is heavily pursued as a target for anticancer therapy, both by antisense oligonucleotides that target XIAP expression and by small molecules that disrupt protein-protein interactions in XIAP function (Andersen et al., 2005; Schimmer et al., 2006). The NF-κB activating function of XIAP may be important for cancer cell survival under these pathological conditions. In support of this, it has been shown that the anti-apoptotic activity of XIAP is dependent on TAK1-mediated survival signaling (Lewis et al., 2004; Sanna et al., 2002; Sanna et al., 1998) and that XIAP-mediated TAK1-dependent NF-κB activation is important for endothelial cell survival (Hofer-Warbinek et al., 2000; Levkau et al., 2001).

The molecular mechanism of XIAP-mediated TAK1 activation has been a subject of debate. A yeast two-hybrid screen has shown that the region of XIAP comprising the three BIR domains interact with the N-terminal domain of the TAK1-binding protein TAB1 (Yamaguchi et al., 1999), suggesting that XIAP activates TAK1 via TAB1. However, it has also been suggested that XIAP directly interacts with TAK1 (Sanna et al., 2002) and that the RING domain of XIAP acts as an E3 in non-degradative ubiquitination for NF-κB activation (Lewis et al., 2004). To resolve these conflicting issues, we performed a series of biochemical, structural and cell biological experiments. We showed that the BIR1 domain of XIAP, which has no previously ascribed function, interacts specifically with TAB1. We determined the crystal structure of the BIR1/TAB1 complex, as well as the structures of isolated BIR1 and TAB1. Revelation of the BIR1/TAB1 interface allowed us to rigorously determine whether XIAP-induced TAK1 activation is through TAB1. An XIAP construct that contains the BIR domains only efficiently induced TAK1 and NF-κB activation. Structure-based mutations that disrupt TAB1 interaction and siRNA-mediated knockdown of TAB1 showed that the BIR1/TAB1 interaction is crucial for XIAP-induced TAK1 and NF-κB activation. We further showed that despite its not directly interacting with BIR1, Smac, the antagonist for caspase inhibition by XIAP, also inhibited the XIAP/TAB1 interaction.

What is the molecular mechanism of XIAP-TAB1 induced TAK1 activation? It was proposed recently based on the crystal structure of TAB1 that TAB1 is a pseudo-phosphatase bearing the fold of protein phosphatase type 2C (PP2C) and likely binds to and regulates accessibility of phosphorylated residues on substrates downstream of TAK1 or on the TAK1 complex itself (Conner et al., 2006). Our structure of TAB1 alone is highly similar to the reported structure, but our structural analysis suggests that TAB1 cannot bind phosphates due to the mutation of a crucial Arg to Ser, incomplete metal ion binding and excess negative charge at the active site. Instead, our structure revealed a dimerization tendency of the BIR1 domain of XIAP both in the crystals and in solution. We showed that disruption of XIAP dimerization abolished XIAP-mediated NF-κB activation.

Therefore, our study revealed the molecular mechanism of XIAP-induced TAK1 and NF-κB activation and may provide a new target for anti-cancer therapy. The molecular mechanism identified here may also have general implications for TAK1 activation in other signaling systems.

Results

The BIR1 Domain of XIAP is Sufficient for TAB1 Interaction

To determine which BIR domain of XIAP is responsible for TAB1 interaction, we first used Native PAGE band shift assay to dissect this interaction. Surprisingly, the BIR1 domain, which has no previously ascribed function, is the only BIR domain that interacted with the N-terminal domain of TAB1. This interaction was confirmed by co-migration of BIR1 and TAB1 on gel filtration chromatography (Fig. 1A). We further used surface plasmon resonance to quantitatively determine whether BIR1 is sufficient for TAB1 interaction. The measurements showed that BIR1 and BIR1–3 of XIAP have similar affinity for TAB1 (Table 1, Fig. 1B, 1C), demonstrating that BIR1 is sufficient for TAB1 interaction.

Figure 1. Biochemical analysis of the interaction between the BIR1 domain of XIAP and the N-terminal domain of TAB1.

Figure 1

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(A) Co-migration of BIR1 and TAB1 N-terminal domain on gel filtration chromatography.

(B) Duplicate responses (left) and isotherms (right) for a two-fold dilution series (0.143–18.25 μM) of TAB1 binding tosurface-tethered BIR1 in an SPR Experiment.

(C) Duplicate responses (left) and isotherms (right) for a two-fold dilution series (0.143–18.25 μM) of TAB1 binding tosurface-tethered BIR1–3 in an SPR Experiment.

(D) Structure-based mutagenesis on the BIR1/TAB1 interaction, showing the peak fractions of gel filtration on BIR1 with WT and mutant TAB1.

Table 1.

Surface plasmon resonance (SPR) and crystallographic statistics.

SPR KDα (μM) KDα (μM)
BIR1–3/TAB1 27.7±0.3 BIR1–3/TAB1D* 16.1±0.2
BIR1/TAB1 14.3±0.1 BIR1/TAB1D* 4.2±0.2
Crystallography TAB1 BIR1 BIR1-TAB1D*
Constructs Residues 1–370 Residues 20–99 Residues 10–99 of BIR1; TAB1D*
Structure determination MAD SAD MR
Data collection
Beamlines X4A of NSLS X4A of NSLS X4C of NSLS
Space group P321 I222 P212121
Cell dimensions
 a, b, c (Å) 143.4, 143.4, 66.1 34.9, 73.0, 81.7 61.0, 108.7, 175.7
Resolution 30-2.4 Å 30-1.8 Å 30–3.1 Å
Rsym 6.4% (27.8%) 5.5% (18.3%) 13.0% (47.7%)
I/σI 27.8 (4.2) 11.9 (2.5) 10.0 (3.2)
Completeness 98.6% (95.9%) 92.6% (66.0%) 82.4% (66.0%)
Redundancy 4.5 (3.1) 3.6 (2.1) 4.3 (2.5)
Refinement
Resolution 30-2.5 Å 30-1.8 Å 30–3.1 Å
No. reflections 26,191 16,989 16,920
Rwork/Rfree 21.4%/24.9% 20.3%/21.3% 21.5%/29.3%
No. atoms
 Protein 2733 614 6268
 Water and ion 65 86 2
Average B-factors
 Protein 53.2 Å2 28.3 Å2 77.1 Å2
 Water and ion 42.6 Å2 39.5 Å2 80.6 Å2
RMS deviations
 Bond lengths/angles 0.009 Å/1.47° 0.007 Å/1.23° 0.007 Å/1.33°
Ramachandran Plot
 Most favored/allowed 90.1%/9.9% 90.9%/9.1% 70.1%/26.0%
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*

TAB1D: residues 1–370 with deletion of residues 133–151.

†

Highest resolution shell is shown in parenthesis.

Structure Determination

We determined the crystal structures of BIR1 at 1.8Å resolution using single wavelength anomalous diffraction of its intrinsic zinc, the N-terminal domain of TAB1 at 2.4Å resolution from a three wavelength anomalous diffraction data set of its selenomethionyl crystal and the BIR1/TAB1 complex at 3.1Å resolution by molecular replacement (Table 1). The TAB1 structure has the fold of mammalian PP2Cα, but with a unique helical extension (Fig. 2A) (Conner et al., 2006). To facilitate crystallization of the BIR1/TAB1 complex, we used an internal deletion mutant of TAB1 (TAB1D, Δ133–151) without this unique helical extension domain. This deletion did not affect its ability to interact with BIR1 or BIR1–3 of XIAP (Table 1).

Figure 2. Structural analysis of the interaction between the BIR1 domain of XIAP and the N-terminal domain of TAB1.

Figure 2

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(A) A ribbon diagram of TAB1. Helices, β-strands and loops are shown in yellow, cyan and pink, respectively.

(B) Dimeric BIR1/TAB1 complex. TAB1 is shown with helices in yellow, β-strands in cyan and loops in pink, respectively. BIR1 is shown with helices in green, β-strands in blue and loops in gray.

(C) A stereo diagram for the detailed interaction between BIR1 and TAB1. Interface residues and their hydrogen bonding interactions are labeled.

(D) Electrostatic surfaces of BIR1 and TAB1, showing the charge and shape complementarity.

BIR1 and TAB1 Form an Extensive and Specific Interface

There are two independent copies of the BIR1/TAB1 complex in the asymmetric unit of the crystal. The two complexes are almost identical with a superimposed RMSD of 0.4Å, suggesting a specific interaction between BIR1 and TAB1. No significant structural changes are observed in TAB1 before and after BIR1 binding or as a result of deletion of the helical extension domain.

The two BIR1/TAB1 complexes form an almost perfect non-crystallographic dimer (176° rotation, Fig. 2B). The dimer resembles the shape of a butterfly with the two BIR1 molecules as the body and the two TAB1 molecules as the wings. The interaction between BIR1 and TAB1 is extensive, burying approximately 1,400 Å2 surface areas at each interface (Fig. 2C). There are a total of 121 pairs of Van der Waals interactions, which are mixed with both hydrophobic and hydrophilic contributions.

BIR1 and TAB1 exhibit shape complementarity with the concave surface of BIR1 receiving the convex surface of TAB1 (Fig. 2D). The region of TAB1 involved in BIR1 interaction resides at the back side of the TAB1 structure (Fig. 2A, 2C). These include residues mainly from α2, α3, and β9 (Fig. 2A, 3A). Based on surface area burial, residues D213 and F216 of TAB1 contribute most to the interaction. Residues of BIR1 involved in TAB1 interaction reside at α2, α3, and loops preceding them and after α4 (Fig. 3B). Based on surface area burial, residues Y75, V80 and L98 contribute most to the interaction.

Figure 3. Structure-based sequence alignment.

Figure 3

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(A) Alignment of TAB1, PP2Cα and PstP. Secondary structures of TAB1 are labeled. Residues at the interface with BIR1 are highlighted in green and residues at the active site of PP2C-like domains are highlighted in magenta.

(B) Alignment of BIR1 from different species. Secondary structures of BIR1 are labeled. Residues at the interface with TAB1 are highlighted in cyan and residues at the dimerization interface are highlighted in red.

(C) Structure-based mutagenesis on the BIR1/TAB1 interaction, showing the peak fractions of gel filtration chromatography on TAB1 with WT and mutant BIR1.

A number of hydrogen bonding interactions are observed at the BIR1/TAB1 interface (Fig. 2C). These include an ion pair between E212 of TAB1 and R84 of BIR1, hydrogen bonds between the carboxylate of D213 of TAB1 and the amide nitrogens of A79 and V80 of BIR1, hydrogen bonds between the carboxylate of E271 of TAB1 and the side chain hydroxyls of S43 and T46 of BIR1, and a side chain hydrogen bond between Q276 of TAB1 and Y75 of BIR1. Residues at the BIR1/TAB1 interface are mostly conserved across species (Fig. 3), suggesting that the interaction is preserved through evolution. In contrast, these residues show poor conservation among PP2Cα and PstP, suggesting that BIR1 interaction is a unique property of TAB1.

The BIR2 and BIR3 domains of XIAP have been shown to interact with the Smac dimer (Huang et al., 2003; Wu et al., 2000). The surface of BIR1 equivalent to the Smac interaction surface of BIR2 and BIR3 is opposite to its TAB1-binding surface. In contrast, the dimerization surface of BIR1 overlaps with the equivalent Smac-binding surface of BIR2 and BIR3 (see below).

The BIR1/TAB1 Interaction is Crucial for XIAP-induced NF-κB Activation

We performed extensive structure-based mutagenesis on the BIR1/TAB1 interaction. TAB1 mutants D213A and F216A both completely abolished the ability of TAB1 to interact with BIR1 (Fig. 3C). Single site mutations of BIR1, including Y75G, V80A and L98G, did not completely eliminate the ability of BIR1 to interact with TAB1. In contrast, V80D, which introduces a negative charge to the interface, eliminated the ability of BIR1 to interact with TAB1 (Fig. 3C). Double mutations of any combination on these residues of BIR1 or the triple mutation also completely knocked out the interaction (Fig. 3C).

To determine whether the BIR1/TAB1 interaction is critical for XIAP-induced TAK1 activation, we introduced the TAB1-binding disruptive mutation V80D and the TAB-1 binding reduced mutant V80A into the mammalian expression construct of XIAP containing the BIR1–3 domains. We transfected WT and mutant XIAP into 293T cells and determined their ability to activate NF-κB, the downstream effector of TAK1 activation (Fig. 4A). While WT XIAP efficiently induced NF-κB activation, the V80D and the V80A mutants of XIAP exhibited reduced ability to activate NF-κB. The effect of the V80A mutant is less drastic than the V80D mutant, consistent with the residual ability of V80A to binding TAB1 (Fig. 3C, 4A). In addition, XIAP constructs with and without the RING domain induced similar level of NF-κB activation, suggesting that the RING is not critical for XIAP-induced signaling in these cells.

Figure 4. XIAP/TAB1 interaction is critical for XIAP-induced NF-κB activation.

Figure 4

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(A) Defective and weakened NF-κB activation by the BIR1–3 mutant V80D and V80A.

(B) Defective and weakened TAK1 activation by the BIR1–3 mutant V80D and V80A.

(C) Defective NF-κB activation by BIR1–3 of XIAP in MEFs with SiRNA-mediated TAB1 knockdowns.

(D) Smac inhibits XIAP/TAB1 interaction as shown by gel filtration.

To determine whether the reduced ability of XIAP mutants to activate NF-κB is due to impaired TAK1 activation, we performed in vitro kinase assay on immunoprecipitated TAK1 in XIAP transfected cells (Fig. 4B). While WT full length or BIR1–3 of XIAP efficiently induced TAK1 activation as shown by the phosphorylation of the TAK1 substrate MKK6, the V80D and the V80A mutants exhibited much reduced TAK1 activation. Consistent with the degree of defectiveness in TAB1 interaction, the V80D mutant showed the most drastic impairment in TAK1 activation.

To further demonstrate that TAB1 is responsible for XIAP-mediated NF-κB activation, we transfected the BIR1–3 domain of XIAP into two independent cultures of primary MEFs in which TAB1 has been knocked down using two different SiRNA constructs (Kang et al., 2006). Western blots and semi-quantitative PCR confirmed the much reduced expression of TAB1 for both SiRNA constructs (Fig. 4C). XIAP exhibited much reduced ability to activate NF-κB in both these cells in comparison with its effect in control MEFs (Fig. 4C). This demonstrates that the BIR1/TAB1 interaction is crucial for XIAP-induced TAK1 activation and argues against direct association of XIAP with TAK1 as the means of TAK1 activation.

Although Smac Does Not Interact with BIR1, It Antagonizes the XIAP/TAB1 Interaction via Steric Exclusion

Because the caspase inhibitory function of XIAP is antagonized by Smac, a mitochondrial protein that gets released during apoptosis, we wondered if Smac may also inhibit the XIAP/TAB1 interaction and therefore the signaling function of XIAP. We have shown previously that the Smac dimer interacts simultaneously with BIR2 and BIR3 domains of XIAP (Huang et al., 2003). This interaction excludes the interaction of the linker of XIAP, which resides before the BIR2 domain, with caspase-7 or caspase-3. To determine whether Smac also antagonizes the XIAP/TAB1 interaction, we used gel filtration to determine complex formation. While XIAP BIR1–3 co-migrated with TAB1, this interaction was abolished in the presence of Smac (Fig. 4D), demonstrating that Smac inhibits the interaction between TAB1 and XIAP. Therefore, Smac also antagonizes the ability of XIAP to activate TAK1 and regulates XIAP via several different mechanisms.

Since Smac does not interact with BIR1 (Liu et al., 2000; Wu et al., 2000), antagonizing XIAP/TAB1 interaction by Smac is likely a result of steric exclusion, rather than direct competition. In this steric exclusion, simultaneous binding of the BIR2 and BIR3 domains of XIAP with the Smac dimer sterically prevents the binding of the BIR1 domain of the same XIAP molecule to TAB1. In support of this, a synthetic dimeric Smac peptide dAVPI did not inhibit the XIAP/TAB1 interaction (data not shown).

TAB1 Can Neither Catalyze De-phosphorylation Nor Bind Phosphates

Like human PP2Cα (Das et al., 1996) and the bacterial phosphatase PstP (Pullen et al., 2004), the structure of TAB1 has the fold of PP2C-like domains with a central β-sheet flanked by α-helices at either side (Fig. 2A, 5A). Catalysis by PP2Cs requires a two-metal binding center (Mg2+ or Mn2+) at the active site, localized within the top channel of the β-sandwich (Das et al., 1996) (Fig. 2A, 5A, 5B). For human PP2Cα, Mn2+ stimulates its activity with a lower Km than Mg2+, although the physiological metal ion for catalysis is likely to be Mg2+ (Pato and Kerc, 1991). In the crystal structure of PP2Cα, the Mn2+ at site 1 directly contacts three Asp residues, D60, D239 and D282. The Mn2+ at site 2 only directly interacts with one Asp residue, D60. Other ligands for this metal include the carbonyl oxygen of G61 and three water molecules, two of which are stabilized by interactions with E37 and D38. In TAB1, D60, D239 and D282 of PP2Cα are changed to N69, E290, and E356, respectively while E37, D38 and G61 of PP2Cα are conserved (Fig. 3A, 5B). Another conserved Asp, D146 of PP2Cα, which coordinates a third metal ion near the active site in the bacterial phosphatase PstP and may enhance catalysis (Pullen et al., 2004), is changed to Thr in TAB1 (Fig. 3A, 5B). Lack of a complete active site suggests that TAB1 does not have phosphatase activity, a conclusion that is also supported experimentally using artificial substrates (Conner et al., 2006) (and data not shown).

Figure 5. TAB1 does not have phosphatase activity.

Figure 5

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(A) Superposition of human TAB1 (yellow), human phosphatase PP2Cα (green) and bacterial phosphatase PstP (pink).

(B) Superposition of the active sites of TAB1 (yellow) and PP2Cα and PstP. Two metal ions and one phosphate ion are bound at the PP2Cα active site. Residues important for metal ion coordination and catalysis are labeled in black for TAB1 and green for PP2Cα.

(C) Only one Mn2+ ion is bound at the TAB1 active site when soaked with MnCl2. The Fo-Fc map is shown at 10σ level.

(D) Electrostatic surface of the PP2Ca active site. The location of the bound phosphate ion is shown.

(E) Electrostatic surface of the same region in TAB1.

It has been proposed that TAB1 may be a pseudo-phosphatase, possibly binding to phosphorylated residues on TAK1 or other molecules in the pathway and regulating their activity (Conner et al., 2006). However, phosphate binding, as shown in the PP2Cα structure, requires water molecules bound to both metal ions and a crucial Arg residue R33 (Das et al., 1996). In TAB1, R33 is changed to a Ser residue, S46 (Fig. 3A, 5B). In addition, soaking of MnCl2 into the crystals showed that only one Mn2+ is bound (Fig. 5C). This Mn2+ corresponds to the PP2Cα ion 2. Furthermore, the two mutated metal coordinating negatively charged surface residues, E290 and E356 of TAB1, are not coordinating any metals. Electrostatic surface calculations showed while PP2Cα active site is highly positively charged, the same region of TAB1 is largely negatively charged with the exception of the single metal ion surface (Fig. 5D, 5E). Variation in the phosphate binding residue, lack of a metal ion, and excess negative charges strongly suggest that TAB1 can not bind phosphates or phosphorylated residues.

BIR1 Dimerization is Crucial for XIAP-induced NF-κB Activation

The lack of phosphatase activity of TAB1 makes it unlikely that TAB1 directly regulates the phosphorylation state of TAK1 and thereby activates TAK1. In contrast, the dimeric assembly of the BIR1/TAB1 structure in the crystal prompted us to speculate that dimerization of BIR1 may lead to proximity-induced TAK1 activation. Interestingly, the same BIR1 dimer in the dimeric BIR1/TAB1 complex is also observed in the BIR1 crystal lattice in the absence of TAB1 (Fig. 6A). This dimerization is mediated by mutual interactions among R62, D71, R72, D77, R82, K85 and V86, in which V86 is completely buried at the interface (Fig. 6B). This dimerization interface of BIR1 is equivalent to the Smac interacting surfaces of BIR2 and BIR3.

Figure 6. BIR1 dimerization is critical for XIAP-mediated NF-κB activation.

Figure 6

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(A) Superposition of the BIR1 dimer in the crystal of BIR1 alone (red) and in complex with TAB1 (green).

(B) Dimerization interface of BIR1, showing important residues and their hydrogen bonding interactions.

(C) V86E mutant of BIR1 still interacts with TAB1 as shown by gel filtration.

(D) Apparent molecular mass of BIR1 WT and V86E mutant as a function of concentration as measured by dynamic light scattering.

(E) Defective NF-κB activation by the V86E mutant. The TAB1-binding defective mutant V80D was used as a control.

(F) A schematic model for XIAP-mediated TAK1 and NF-κB activation.

To determine whether BIR1 has a tendency to dimerize in solution, we performed dynamic light scattering measurements at BIR1 concentrations from 10 mg/ml and up (Fig. 6D). The calculated molecular weight of monomeric BIR1 is 10.5 kD. The apparent molecular mass of BIR1 increased significantly as a function of concentration, to approximately dimeric at 30 mg/ml, suggesting that BIR1 does have a tendency to dimerize in solution. Similar dynamic light scattering measurements have also been used previously to assess dimerization tendency of human CD4 (Wu et al., 1997).

To determine whether this dimerization is important for XIAP-induced TAK1 activation, we mutated V86 to Glu to introduce a buried negative charge at the dimerization interface. While the V86E mutant retained its ability to interact with TAB1 (Fig. 6C), its dimerization tendency was abrogated as shown by dynamic light scattering measurements (Fig. 6D). Remarkably, upon transfection, the mutant XIAP also had much reduced ability to activate NF-κB (Fig. 6E), demonstrating that XIAP dimerization is important for TAK1 activation and its associated biological effects. As a control, the TAB1-binding defective XIAP mutant V80D also showed decreased NF-κB activation.

Discussion

Establishment of the XIAP-TAB1-TAK1 Pathway for XIAP-mediated NF-κB Activation

The multifunctional IAP family member XIAP participates in diverse cellular functions including caspase inhibition, signal transduction, copper metabolism and ubiquitination. For the signaling function, XIAP bridges TGFβ and BMP type I receptors to TAK1 and NF-κB activation under physiological conditions and induces survival signaling in cancer cells under pathological conditions. Here we establish that XIAP activates TAK1 via interaction with the TAK1-binding protein TAB1. While the RING domain of XIAP has been shown to interact with the receptors, we mapped the TAB1-interacting region of XIAP to the BIR1 domain. The affinity of XIAP or BIR1 with TAB1 is similar to several bona fide interactions involved in signal transduction such as the SH3-peptide and SH2-peptide interactions (Ferreon and Hilser, 2004).

The N-terminal domain of TAB1 interacts with BIR1 of XIAP and the C-terminal tail of TAB1 interacts with the kinase domain of TAK1 (Fig. 6F). Under endogenous conditions, TAB1 and TAK1 are constitutively associated with each other without TAK1 activation (Wang et al., 2001). In the crystal structure of a fusion protein containing the TAK1 kinase domain and the C-terminal tail of TAB1, TAB1 interacts with the C-terminal lobe of TAK1 (Brown et al., 2005). This interaction is similar to the binding of the myristoylated N-terminus with the c-Abl kinase domain and presumably induces conformational changes in the kinase domain (Nagar et al., 2003). However, this interaction does not appear to directly lead to kinase activation as the fusion kinase is not phosphorylated at the crucial residues of the active site loop (Brown et al., 2005). Consistent with this hypothesis, the myristate-bound c-Abl kinase is also not active (Nagar et al., 2003).

The current study suggests that during TGFβ and BMP signaling or upon XIAP upregulation, XIAP interacts with TAB1 and brings the associated TAK1 into proximity for trans-phosphorylation and activation. Previous studies have suggested the involvement of XIAP RING domain in XIAP oligomerization and a residual self-association ability of RING-deleted XIAP (Silke et al., 2002; Silke et al., 2005). The ability of the RING domain to mediate XIAP oligomerization may be the reason for its requirement in signaling in some situations (Lewis et al., 2004). Therefore, it appears that while the RING domain may make XIAP a constitutive oligomer, the BIR1 domain mediates a specific and dynamic dimerization for TAK1 activation (Fig. 6F).

Because XIAP is highly up-regulated in many cancer cells (Schimmer et al., 2006), the high expression level in these cells may facilitate TAK1 activation by XIAP and contribute to the effect of XIAP in promoting cancer cell survival. The current study may provide a new target for suppressing XIAP function in cancer cells.

BIR1 as a General Mediator of Signaling and Dimerization

This is the first time that any function of XIAP has been ascribed to the BIR1 domain, which is highly conserved across species. One surface of BIR1 is used for TAB1 interaction while the opposite surface, which is equivalent to the Smac binding surface of BIR2 and BIR3, is used for BIR1 dimerization.

In this context, it is interesting to note that the BIR1 domain of two other IAP family members, cIAP1 and cIAP2, has also been shown recently to mediate signaling and dimerization (Samuel et al., 2006; Varfolomeev et al., 2006; Zhou et al., 2005). The BIR1 domain of cIAP1 and cIAP2 mediates the interaction with TRAF1 and TRAF2, the major signaling proteins in intracellular signal transduction of TNF receptors (Samuel et al., 2006; Varfolomeev et al., 2006). In the most frequent chromosomal translocation associated with MALT lymphomas, the c-IAP2 BIR domains are fused with the paracaspase MALT1, resulting in constitutive cIAP2-MALT1 oligomerization and NF-κB activation. The BIR1 domain, but not BIR2 or BIR3 of cIAP2, mediates the oligomerization of the cIAP2-MALT1 fusion protein and is required for NF-κB activation (Zhou et al., 2005). Oligomerization and TRAF interaction of cIAPs are apparently mediated by different surfaces of BIR1 as the TRAF-binding mutants of cIAP2-MALT1 still oligomerize and activate NF-κB (Varfolomeev et al., 2006). In the cIAP2-MALT1 fusion, cIAP2 appears to provide the oligomerization ability of the protein while MALT1 recruits other proteins for NF-κB activation (Zhou et al., 2005).

Broad Biological and Mechanistic Implications

Unlike the caspase inhibitory function, the signaling function of XIAP is conserved in several other IAPs such as NAIP and ML-IAP. These IAPs may also induce TAK1 activation via a similar TAB1-mediated mechanism.

TAK1 also plays a much broader role in survival signaling by acting as the upstream kinase for NF-κB and MAP kinase activation in multiple receptor signaling pathways including IL-1 receptor, Toll-like receptors, some TNF receptors and antigen receptors (Chen, 2005; Deng et al., 2000; Wang et al., 2001). In these pathways, the signaling protein TRAF6 acts as an E3 to mediate non-degradative K63-linked polyubiquitination of itself and of downstream targets. Instead of TAB1, the TAK1-binding adapter protein TAB2 (or its related protein TAB3) links TRAF6 to TAK1 (Kanayama et al., 2004; Takaesu et al., 2000). In this TRAF6-TAB2-TAK1 pathway, ubiquitination of TRAF6 promotes TAB2 recruitment and TAK1 activation. Because TRAF6 is induced to trimerize or form other oligomers upon receptor stimulation (Park et al., 1999; Ye et al., 2002), the current study implicates a general oligomerization-dependent mechanism for TAK1 activation in these diverse TAK1 and NF-κB activation pathways.

METHODS

Protein preparation and mapping of the XIAP/TAB1 interaction

Human XIAP deletion mutants (BIR1: residues 20–99; BIR2: residues 124–256; BIR3: residues 257–336; BIR1–3: residues 1–336), human TAB1 N-terminal domain (residues 1–370), and TAB1D (residues 1–370 with deletion of 133–151) were expressed as GST-fusion proteins in bacteria, purified using glutathione beads and cleaved with thrombin to remove the GST tag. The cleaved proteins contain two additional residues GS at the beginning of the proteins. Human BIR1 (residues 10–99) was expressed as an N-terminally His-tagged protein and purified by Ni-affinity chromatography. The tag was cleaved off by thrombin treatment. Smac was expressed and purified as described previously (Chai et al., 2000; Huang et al., 2003). For mapping studies, purified XIAP and TAB1 constructs were mixed, incubated and subject to Native PAGE using the PhastSystem (GE Biosciences) and gel filtration using Superdex 200 HR 10/30 (GE Biosciences).

Surface plasmon resonance (SPR) measurements

Binding studies were performed at 20°C using a Biacore 2000 optical biosensor equipped with a streptavidin-coated CM4 research-grade sensor chip and equilibrated with running buffer (20 mM Tris, 75 mM NaCl, 0.005% P20, 0.1 mg/ml BSA, pH 8.0). BIR1 and BIR1–3 of XIAP were incubated with EZ-link sulfo-NHS-LC-LC-biotin for one hour, passed over a fast desalting column to remove free biotin, and captured at densities of 250 and 515 response units (RU), respectively, on the surfaces of two flow cells. Two-fold dilution series of TAB1 (0, 0.143, 0.285, 0.570, 1.14, 2.28, 4.56, 9.13, and 18.3 μM) and TAB1D (0, 0.203, 0.406, 0.813, 1.63, 3.25, 6.50, 13.0, and 26.0 μM) were tested in duplicate for binding to the surface-tethered XIAP proteins. The binding responses were double referenced (Myszka, 1999) and fit to a simple binding isotherm to determine affinity using Scrubber 2 (BioLogic Software Ltd., Campbell, Australia).

Crystallization and structure determination

The crystallization and structure determination of BIR1 will be described elsewhere. Briefly, BIR1 (residues 20–99) was crystallized in 1.5M NaCl, 10% methanol and 0.1M Hepes at pH 7.5. The structure was determined at 1.8Å resolution by single wavelength anomalous diffraction from the intrinsic Zn using Solve/Resolve (Terwilliger, 2004), auto-traced using ARP/wARP (Perrakis et al., 2001) and refined using CNS (Brunger et al., 1998) (Table 1). Human native and selenomethionyl TAB1 (residues 1–370) was crystallized in 1.5M Li2SO4 and 0.1M Hepes at pH 7.5. Multi-wavelength anomalous diffraction data were collected at three wavelengths. The structure was determined at 2.6Å resolution using Solve/Resolve (Terwilliger, 2004) and refined at 2.4Å resolution against a native data set using CNS (Brunger et al., 1998). The final atomic model contains residues 18–370 (Table 1). TAB1D (residues 1–370 with deletion of 133–151) was mixed with excess BIR1 (residues 10–99) and subjected to gel filtration chromatography with Superdex 200 HR 26/60 (GE Biosciences). The BIR1/TAB1D complex peak was collected and concentrated to 20 mg/ml for crystallization under 8.5% PEG8000, 4% Ethylene Glycol and 100 mM Na-HEPES at pH 7.6. The structure was determined at 3.1Å resolution by molecular replacement (Tong, 1993) using the BIR1 and TAB1 structures (Table 1). Structural analysis was performed using O (Jones et al., 1991) and Pymol (DeLano Scientific).

Knockdown of TAB1

Knockdown of TAB1 in MEFs was performed as previously described (Kang et al., 2006) Briefly, oligonucleotides were cloned into the vector pSuper to express short hairpin RNA; the targeting sequences are 5′-AGCAGTCCTTCTCAACAGCAAG-3′(#1) and 5′-AGGCCCTTCTGTGCAAATCTAC-3′ (#2). Cell lysates were prepared and subjected to Western blot using anti-TAB1 or GAPDH antibodies. Total RNA was isolated and semi-quantitative PCR was performed.

NF-κB activation by XIAP

293T cells, MEFs or TAB1-knockdown MEFs (Kang et al., 2006) were transfected using Lipofectamine 2000 (Invitrogen) with NF-κB-luciferase, Renilla-luciferase (in pRT-TK using the herpes simplex virus thymidine kinase promoter, Promega), and control, full-length XIAP, or the following BIR1–3 domain expression vectors of XIAP: WT, V80D, or V86E. After 24 hours, cell lysates were prepared and luciferase activity was measured by luminometer. Fold activation of NF-κB was calculated by dividing measured luciferease activity of NF-κB by internal Renilla luciferase activity.

TAK1 kinase activity assay

293 cells were transfected with 1μg of HA-TAK1, and 3 μg of control, wildtype XIAP, wildtype or mutant forms of XIAP BIR1–3 expression vectors. After 24 hours, cell lysates were prepared in lysis buffer (20 mM HEPES, pH 7.4, 150 mM NaCl, 12.5 mM β-glycerophosphate, 1.5 mM MgCl2, 2 mM EGTA, 10 mM NaF, 1 mM DTT, 1 mM Na3VO4, 1 mM PMSF, and 0.5 % Triton X-100) and immunoprecipitated using anti-HA antibody for 3 hours. The immunoprecipitates were washed three times with lysis buffer and twice with kinase buffer (50 mM HEPES (pH 7.4), 1 mM dithiothreitol, 5 mM MgCl2, and 50 μM ATP). They were incubated with 3 μg of kinase-dead MKK6 in kinase buffer containing 10 μCi of [μ-32P] ATP at 37°C for 30 min. Two third of the immunoprecipitates was subjected to SDS-PAGE and visualized by autoradiography, and the rest was subjected to Western blot analysis to confirm the immuoprecipitation of HA-TAK1.

Dynamic light scattering

Hydrodynamic radius and estimated molecular mass of BIR1 at different concentrations were derived from dynamic light scattering measurements (Harding, 1994) on a DynoPro Molecular Sizing Instrument (Protein Solutions, Inc.).

Supplementary Material

01

Acknowledgments

We thank Dr. Colin Duckett and Arjmand Mufti for help with the project and providing the mammalian expression construct for WT BIR1–3 of XIAP, Dr. Yueming Li for providing the dimeric Smac peptide dAVPI, Jin Wu for maintaining X-ray and computer equipment, and Randy Abramowitz and John Schwanof for data collection at X4A and X4C of NSLS. This work was supported by National Institute of Health (RO1 AI045937 to HW, AI041637 and GM037696 to JH). S.C.L is a postdoctoral fellow of the Cancer Research Institute.

Footnotes

Accession Numbers

The coordinates have been deposited in the RCSB Protein Data Bank with the PDB codes of 2POP for the BIR1/TAB1 structure, 2POI for the BIR1 structure and 2POM for the structure of Mn2+-soaked TAB1.

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