Activation of IKK by TNFa Requires Site-Specific Ubiquitination of RIP1 and Polyubiquitin Binding by NEMO

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1 Molecular Cell 22, , April 21, 2006 ª2006 Elsevier Inc. DOI /j.molcel Activation of IKK by TNFa Requires Site-Specific Ubiquitination of RIP1 and Polyubiquitin Binding by NEMO Chee-Kwee Ea, 1,2 Li Deng, 1,2 Zong-Ping Xia, 1,2 Gabriel Pineda, 1 and Zhijian J. Chen 1, * 1 Howard Hughes Medical Institute Department of Molecular Biology University of Texas Southwestern Medical Center Dallas, Texas Summary The receptor interacting protein kinase 1 (RIP1) is essential for the activation of nuclear factor kb (NF-kB) by tumor necrosis factor a (TNFa). Here, we present evidence that TNFa induces the polyubiquitination of RIP1 at Lys-377 and that this polyubiquitination is required for the activation of IkB kinase (IKK) and NF-kB. A point mutation of RIP1 at Lys-377 (K377R) abolishes its polyubiquitination as well as its ability to restore IKK activation in a RIP1-deficient cell line. The K377R mutation of RIP1 also prevents the recruitment of TAK1 and IKK complexes to TNF receptor. Interestingly, polyubiquitinated RIP1 recruits IKK through the binding between the polyubiquitin chains and NEMO, a regulatory subunit of the IKK complex. Mutations of NEMO that disrupt its polyubiquitin binding also abolish IKK activation. These results reveal the biochemical mechanism underlying the essential signaling function of NEMO and provide direct evidence that signal-induced site-specific ubiquitination of RIP1 is required for IKK activation. Introduction TNFa regulates many cellular functions, including apoptosis, inflammation, immune response, and cell growth and differentiation (reviewed by Tracey and Cerami [1994]). TNFa-induced cellular responses are mediated by TNF receptor 1 (TNF-R1) and TNF receptor 2 (TNF- R2; reviewed by Chen and Goeddel [2002]). Binding of TNFa to TNF-R1 leads to the recruitment of the TNF-R1- associated death domain (TRADD) protein and subsequent recruitment of the FAS-associated death domain (FADD) protein, the TNF receptor-associated factor 2 (TRAF2), and the receptor-interacting protein kinase RIP1. While association of FADD with TRADD triggers the apoptosis program, binding of TRAF2 and RIP to TRADD activates the IkB kinase (IKK) complex and Jun N-terminal kinase (JNK) (Hsu et al., 1996b). The IKK complex is composed of two catalytic subunits, IKKa and IKKb, and an essential regulatory subunit, NEMO (also known as IKKg). IKK phosphorylates the NF-kB inhibitor IkB and targets this inhibitor for degradation by the ubiquitin-proteasome pathway (reviewed by Chen [2005] and Krappmann and Scheidereit [2005]). NF-kB is then liberated to enter the nucleus to turn on downstream target genes involved in immune and inflammatory responses. *Correspondence: zhijian.chen@utsouthwestern.edu 2 These authors contributed equally to this work. RIP1 was initially identified in a yeast two-hybrid screen for proteins that interact with the death domains of Fas and TRADD (Hsu et al., 1996a; Stanger et al., 1995). Subsequent experiments showed that RIP1 is essential for NF-kB activation by TNFa (Kelliher et al., 1998; Ting et al., 1996). RIP1 contains an N-terminal kinase domain, an intermediate domain, and a C-terminal death domain (DD). The intermediate domain, but not the kinase domain of RIP1, mediates NF-kB activation through an unknown mechanism. The role of RIP1 in JNK activation by TNFa has not been conclusively established, as one study showed that JNK activation is normal in RIP1-deficient MEF cells (Kelliher et al., 1998), whereas another study found that JNK activation is impaired in the same cell line (Devin et al., 2003). TRAF proteins such as TRAF2 and TRAF6 contain an N-terminal RING domain commonly found in ubiquitin ligases (E3). Indeed, recent studies have shown that TRAF proteins catalyze the synthesis of K63-linked polyubiquitin chains in conjunction with the ubiquitinconjugating enzyme (E2) complex consisting of Ubc13 and Uev1A (Deng et al., 2000). Several targets of polyubiquitination in the NF-kB pathway have been identified, and these include RIP1, NEMO, TRAF2, and TRAF6 themselves (reviewed by Chen [2005]). Polyubiquitination of these proteins has been proposed to mediate the activation of the TAK1 kinase complex, which in turn phosphorylates and activates the IKK complex (Wang et al., 2001). The TAK1 kinase complex consists of the TAK1 kinase and its associated proteins, TAB1, TAB2, and TAB3 (Cheung et al., 2004; Ishitani et al., 2003; Kanayama et al., 2004; Shibuya et al., 1996; Takaesu et al., 2000; Yamaguchi et al., 1995). TAB2 and TAB3 are homologous proteins that contain a highly conserved C-terminal novel zinc finger (NZF) domain, which binds preferentially to K63-linked polyubiquitin chains (Kanayama et al., 2004). Thus, polyubiquitination of target proteins such as RIP1 and TRAF6 may facilitate the interaction of these proteins with the TAK1 complex, thereby leading to TAK1 and IKK activation. However, direct evidence that ubiquitination of RIP1 or TRAF is required for TAK1 and IKK activation is still lacking. In addition, although NEMO is essential for IKK activation, the biochemical mechanism by which NEMO regulates IKK is currently unknown. In this report, we show that RIP1 is polyubiquitinated at Lys-377 in response to TNFa stimulation. The substitution of Lys-377 by arginine (K377R) blocked RIP1 ubiquitination and abrogated the ability of RIP1 to rescue IKK activation in a RIP1-deficient cell line. Polyubiquitination of RIP1 is required for the recruitment of TAK1 and IKK complexes to the TNF receptor complex after TNFa stimulation. The recruitment of IKK requires the binding between polyubiquitinated RIP1 and NEMO, which contains a previously unidentified polyubiquitin binding domain. Mutations that impair the ability of NEMO to bind to polyubiquitin chains also abrogated the activation of IKK. Collectively, these results show that site-specific polyubiquitination of RIP1 and polyubiquitin binding by NEMO mediate the activation of IKK in the TNFa pathway.

2 Molecular Cell 246 Figure 1. TNFa Induces K63-Linked Polyubiquitination of RIP1 at K377 (A) Jurkat cells were stimulated with GST-TNFa for the indicated lengths of time and then the receptor-associated signaling complex was isolated by using glutathione Sepharose. The bound proteins were immunoblotted with an antibody against RIP1 (top) or GST (middle). Cell lysates were also immunoblotted with an antibody against RIP1 or phospho-ikba (bottom two panels). On the right, the signaling complex bound to the glutathione Sepharose beads was disrupted by 1% SDS, and then RIP1 was immunoprecipitated after dilution of SDS. Ubiquitinated RIP1 was detected with a ubiquitin antibody. (B) HEK293 cells were transfected with expression vectors encoding HA-Ub or its lysine mutants as illustrated (left). The transfected cells were stimulated with TNFa for 2 min and then RIP1 was immunoprecipitated. Ubiquitination of RIP1 was detected with an HA antibody (middle). On the right, cell lysates were immunoblotted with an HA antibody that detects the transfected ubiquitin and its mutants.

3 Mechanisms of IKK Activation by Ubiquitination 247 Results RIP1 Is Ubiquitinated at Lys-377 in Response to TNFa Stimulation Stimulation of cells with TNFa leads to the recruitment of RIP1 to TNF-R1, and the receptor bound RIP1 undergoes a form of covalent modification that resembles polyubiquitination (Chen et al., 2002; Zhang et al., 2000). To establish a system for the study of RIP1 polyubiquitination, we stimulated Jurkat cells with GST-TNFa and then isolated the TNF-R1 complex by using glutathione Sepharose. GST-TNFa treatment led to the activation of IKK and the recruitment of RIP1 to the TNF-R1 complex (Figure 1A, top). A fraction of RIP1 recruited to the TNF- R1 complex formed high molecular weight species. To verify that these species are polyubiquitinated RIP1, we dissociated TNF-R1 bound proteins with 1% SDS and then immunoprecipitated RIP1 with a RIP1-specific antibody after dilution of SDS. The precipitated proteins were then analyzed by immunoblotting with a ubiquitinspecific antibody. The high molecular weight species were detected with the ubiquitin antibody, confirming that RIP1 was polyubiquitinated in response to stimulation with GST-TNFa (Figure 1A, right). To determine the ubiquitin linkage of polyubiquitin chains on RIP1, we transfected HEK293 cells with HA-tagged ubiquitin mutants containing only one lysine at position 63 (K63) or 48 (K48) or those with a single point mutation at position 63 (R63) or 48 (R48; Figure 1B, left). The cells were stimulated with GST-TNFa for 2 min, and then RIP1 was immunoprecipitated and immunoblotted with an HA antibody. As shown in Figure 1B, RIP1 polyubiquitination was observed with ubiquitin mutants containing a lysine at position 63 (K63 and R48), but not with those lacking a lysine at position 63 (R63 and K48). Thus, the polyubiquitin chains on RIP1 were linked primarily through K63 of ubiquitin. To map the polyubiquitination site(s) of RIP1, we focused on the intermediate domain of RIP1 because overexpression of this domain is sufficient to activate NF-kB (Hsu et al., 1996a; Ting et al., 1996). As shown in Figure 1C, there are five conserved lysine residues in the intermediate domain of RIP1 from human, mouse, and rat. We mutated each of these lysine residues to arginine in human RIP1 and then introduced them back into RIP1 2/2 Jurkat cells by using retroviral infection. Stable RIP1 mutant cell lines were established, and the expression of RIP1 was verified by immunoblotting (Figure 1D). We chose those stable cell lines expressing the exogenous RIP1 at a level similar to that of the endogenous protein. Strikingly, a point mutation at Lys- 377 (K377R) abolished TNFa-induced polyubiquitination of RIP1, whereas mutations at other lysine residues had little effect (Figure 1E). These results indicate that K377 is the primary polyubiquitination site of RIP1 after TNFa stimulation. Polyubiquitination of RIP1 Is Essential for TNFa-Mediated IKK and NF-kB Activation To determine if the ubiquitination of RIP1 is required for IKK activation, we stimulated the stable cell lines harboring different mutations of RIP1 with TNFa, immunoprecipitated the endogenous IKK complex, and then measured IKK activity by using an N-terminal fragment of IkBa (GST-IkBa-NT) as a substrate. As shown in Figure 2A, IKK activity was rapidly induced by TNFa in cells containing wild-type RIP1, but not in cells lacking RIP1. Importantly, a point mutation at K377, but not at other lysines, abolished the ability of RIP1 to rescue IKK activation in the RIP1-deficient cells. Furthermore, RT-PCR analysis showed that in RIP1-K377R cells, TNFa failed to induce the NF-kB target genes intercellular adhesion molecule 1 (ICAM-1) and monocyte chemoattractant protein 1 (MCP-1; Figure 2B). Control experiments showed that the constitutive expression of GAPDH was normal in the RIP1 2/2 and RIP1-K377R cells. These results indicate that polyubiquitination of RIP1 at K377 is required for the activation of IKK and NF-kB in response to TNFa stimulation. Defective Polyubiquitination of RIP1 Sensitizes Cells to TNFa-Induced Apoptosis It has been shown that RIP1-deficient cells are sensitive to TNFa-induced cell death, owing to the defect of NFkB activation, which prevents cell death (Ting et al., 1996). To test the susceptibility of RIP1-K377R cells to TNFa treatment, we performed the TNFa cytotoxicity assay that measures the reduction of a tetrazolium compound (MTT) by the mitochondria of viable cells. In the absence of the protein synthesis inhibitor cycloheximide (CHX), cells containing wild-type RIP1 were resistant to TNFa treatment (Figure 3A). In contrast, the viability of RIP1 2/2 and RIP1-K377R cells decreased by 40% after TNFa treatment for 24 hr. The cytotoxicity of TNFa was further enhanced in the presence of both TNFa and CHX. To determine if TNFa-induced cell death was due to apoptosis, we examined the cleavage of poly-adpribose polymerase (PARP), a biochemical marker for apoptosis. Consistent with the cytotoxicity assay, PARP cleavage was detected in cells lacking RIP1 or containing RIP1-K377R, but not in cells containing wild-type RIP1 (Figure 3B). Thus, the absence of RIP1 polyubiquitination sensitizes cells to TNFa-induced apoptosis. Polyubiquitination of RIP1 Is Required for the Recruitment of TAK1 and IKK Complexes to TNF-R1 The binding of trimeric TNFa to its receptor leads to the rapid recruitment of several signaling proteins to the receptor complex, including TRADD, TRAF2, RIP1, TAK1, and IKK complexes (Hsu et al., 1996a; Lee et al., 2004). To determine if polyubiquitination of RIP1 is required for the recruitment of downstream signaling molecules to the receptor complex, we stimulated the RIP1 mutant (C) Sequence alignment of the intermediate domain of RIP1 from human, mouse, and rat. The conserved lysine residues are numbered and indicated by arrow. (D) Expression of RIP1 mutants in stable Jurkat cell clones lacking endogenous RIP1. Tubulin serves as a loading control. (E) Jurkat cell clones expressing various RIP1 mutants were stimulated with GST-TNFa and then the receptor complex was isolated and analyzed by immunoblotting as described in (A).

4 Molecular Cell 248 Figure 2. Ubiquitination of RIP1 at K377 Is Required for IKK Activation (A) Jurkat cells stably expressing RIP1 mutants were stimulated with TNFa for the indicated time, and then anti-nemo antibody was used to immunoprecipitate the IKK complex. The IKK assay was carried out by using the N terminus of IkBa (GST-IkBa-NT) and g- 32 P-ATP as substrates (top). The amounts of IKKb in the immunoprecipitates (middle) and RIP1 in the cell lysates were determined by immunoblotting (bottom). Abbreviation: IP / KA, immunoprecipitation followed by IKK kinase assay. (B) Jurkat cell clones as indicated were stimulated with or without TNFa for 20 hr, and the induction of human ICAM-1 and MCP-1 was measured by RT-PCR. The constitutive expression of GAPDH RNA was also measured as a control. cells with GST-TNFa and then isolated the TNF-R1 complex by using glutathione Sepharose. As shown in Figure 4A, GST-TNFa treatment led to the recruitment of TRADD, TRAF2, TAK1, TAB2, IKKb, and NEMO to TNF- R1 in cells containing wild-type RIP1. Interestingly, slower-migrating forms of TAB2 were also observed after TNFa treatment. These modified forms of TAB2 likely resulted from phosphorylation because they were sensitive to l phosphatase treatment (Figure 4A, top right). Significantly, TAK1, TAB2, IKKb, and NEMO were not recruited to TNF-R1 in RIP1 2/2 and RIP1-K377R cells while the recruitment of TRADD and TRAF2 was normal (Figure 4A, lanes 10 12). RIP1-K377R cells remained capable of binding to TRAF2 and TRADD (Figure 4B), suggesting that the conservative substitution at K377 does not alter the overall structure of RIP1. Thus, the failure to recruit the TAK1 and IKK complexes to the receptor in RIP1-K377R cells is likely due to defective polyubiquitination of RIP1. Figure 3. Ubiquitination of RIP1 at K377 Is Required to Protect Cells from TNFa-Induced Cell Death (A) Jurkat cells expressing RIP1 mutants were treated with TNFa or cyclohexamide (CHX), or both, for 24 hr and then the cell viability was measured by MTT assay. (B) Jurkat cells were treated with or without TNFa for 24 hr and then the cleavage of PARP was analyzed by immunoblotting. NEMO Binds to Polyubiquitinated RIP1 in Response to TNFa Treatment We have previously shown that TAB2 contains a C-terminal NZF domain that binds preferentially to K63 polyubiquitin chains (Kanayama et al., 2004). This polyubiquitin binding likely contributes to the binding of TAB2 to polyubiquitinated RIP1 in TNFa-stimulated cells. To determine if polyubiquitination of RIP1 at K377 mediates the binding between RIP1 and TAB2, we stimulated cells containing the wild-type or K377R mutant of RIP1 with TNFa and then immunoprecipitated TAB2 with a specific antibody. As shown in Figure 5A, the TNFa-dependent binding between TAB2 and RIP1 was observed only in cells expressing the wild-type RIP1, but not the K377R mutant. Moreover, the predominant form of RIP1 associated with TAB2 was polyubiquitinated. Parallel experiments showed that similar amounts of TAK1 associated with TAB2 in the wild-type and RIP1-K377R mutant cells. We also carried out similar sets of experiments

5 Mechanisms of IKK Activation by Ubiquitination 249 Figure 4. Ubiquitination of RIP1 Mediates the Recruitment of Signaling Complexes to the TNF Receptor (A) Jurkat stable cell lines were stimulated with GST-TNFa for the indicated time. The receptor-associated signaling complex was isolated by using glutathione Sepharose and then analyzed by immunoblotting with the indicated antibodies. In the top right panel, the receptor complex was treated with l phosphatase or buffer alone and then analyzed by immunoblotting with a TAB2 antibody. (B) The K377R mutation of RIP1 does not affect its association with TRAF2 or TRADD. Flag-tagged TRAF2 or TRADD was coexpressed with Flag-RIP1 or K377R in HEK293 cells. The association between RIP1 and TRAF2 or TRADD was analyzed by immunoprecipitation with an antibody against TRAF2 (top left) or TRADD (top right) followed by immunoblotting with the Flag antibody. IgG was used as a control antibody for immunoprecipitation. The bottom panel is an immunoblot of the cell lysates using the Flag antibody. to examine whether NEMO interacts with RIP1 in a ubiquitination-dependent manner. Remarkably, TNFa stimulation led to the binding between NEMO and polyubiquitinated RIP1 in the wild-type, but not K377R mutant cells, whereas similar amounts of IKKb associated with NEMO in both cell types (Figure 5B). We also found that IKKb was not recruited to the TNF-R1 in NEMO-deficient Jurkat cells (Figure 5C). The recruitment of TAB2 and ubiquitinated RIP1 to the receptor was normal in the absence of NEMO (Figure 5C), consistent with NEMO functioning downstream of TAB2 and RIP1. To determine if the TAK1 kinase complex is required for the recruitment of IKKb to TNF-R1, we used RNAi to knock down the expression of TAK1 or both TAB2 and TAB3. In the absence of components of the TAK1 complex, TNFa-induced activation of IKK activation was defective (Figure 5D, middle panels). However, in contrast to the requirement of NEMO for the receptor recruitment of IKKb, RNAi of TAK1, TAB2, and TAB3 had no effect on IKKb recruitment (Figure 5D, top panels). Thus, although the TAK1 complex is required for IKK activation, it is not required for the recruitment of IKK/NEMO complex to

6 Molecular Cell 250 Figure 5. Ubiquitination of RIP1 at K377 Mediates Its Association with TAB2 and NEMO (A) Jurkat cells stably expressing RIP1 or RIP1(K377R) were stimulated with TNFa. The TAB2 complex was immunoprecipitated and then analyzed by immunoblotting with an antibody against RIP1 or TAK1. (B) Similar to (A) except that NEMO antibody was used in lieu of TAB2 antibody for immunoprecipitation. (C) NEMO is required for the receptor recruitment of IKKb. Wild-type and NEMO 2/2 Jurkat cells were treated with GST-TNFa, and the receptorassociated complex was isolated by using glutathione Sepharose beads. Proteins within the complex were detected by immunoblotting with the indicated antibodies. (D) The TAK1 complex is not required for the receptor recruitment of IKKb. HEK293 cells were transfected with sirna oligos corresponding to GFP (control), TAK1, or TAB2 and TAB3. The cells were stimulated with GST-TNFa, and the receptor-associated complex was isolated by using glutathione Sepharose. The presence of IKKb in the receptor complex was determined by immunoblotting (top two panels). IKK assays were also performed after immunoprecipitation with a NEMO antibody (middle two panels). The efficiency of RNAi was evaluated by immunoblotting of cell lysates with an antibody against TAK1, TAB2, or TAB3 (bottom three panels). the receptor. Taken together, these results indicate that polyubiquitination of RIP1 mediates the independent recruitment of TAB2 and NEMO, which in turn recruits TAK1 and IKK, respectively, to TNF-R1. NEMO Binds to Polyubiquitin Chains The preferential binding of NEMO to polyubiquitinated RIP1 suggests that NEMO may be a polyubiquitin binding protein. To test this possibility, we synthesized free polyubiquitin chains by using in vitro enzymatic reactions. For the synthesis of K48-linked polyubiquitin chains, a reaction containing E1, Ubc3 (E2), Skp1- Cul1-Roc1-bTrCP (E3), and ubiquitin was carried out (Kanayama et al., 2004; Spencer et al., 1999; Zheng et al., 2002). A similar reaction was used to synthesize K63-linked polyubiquitin chains, except that Ubc13/ Uev1A (E2) and TRAF6 (E3) were used (Deng et al., 2000). These polyubiquitin chains were conjugated to Sepharose beads (Kanayama et al., 2004), which were then used to pull down NEMO. As shown in Figure 6A, K63 polyubiquitin chains pulled down significantly more NEMO than did K48 chains, whereas mono-ub had little binding to NEMO (Figure 6A, top). In contrast, the S5a (Rpn10) subunit of the proteasome bound to K48 and K63 chains with similar affinity (Figure 6A, bottom). These results indicate that NEMO binds preferentially to K63 polyubiquitin chains. Polyubiquitin Binding by NEMO Is Required for IKK Activation To determine if polyubiquitin binding by NEMO is required for IKK activation, we attempted to map the polyubiquitin binding domain of NEMO (Figure 6B). NEMO contains two coil-coiled domains, CC1 (residues ) and CC2 ( ), a leucine zipper (LZ) ( ), and a zinc finger motif ( ; [Rothwarf et al., 1998; Yamaoka et al., 1998]). In addition, an N-terminal region of NEMO (50 93) mediates its binding to IKKa and IKKb (May et al., 2000). We expressed various truncation mutants of NEMO in HEK293 cells as Flag-tagged

7 Mechanisms of IKK Activation by Ubiquitination 251 Figure 6. NEMO Binds to Polyubiquitin Chains (A) Monoubiquitin (Ub), K48-, or K63-linked polyubiquitin chains (Ub n ) were covalently conjugated to Sepharose beads, which were then incubated with purified Flag- NEMO (top) or His 10 -S5a (bottom) proteins. Proteins bound to the beads were detected with an antibody against NEMO or Penta- His. Lane 1 shows 20% of NEMO or S5a protein used for the binding experiments. (B) Diagram of the structural domains of NEMO and different deletion mutants. The results of ubiquitin binding and IKK activation experiments are summarized on the right. (C) Different deletion mutants of Flag-tagged NEMO were incubated with K63-linked polyubiquitin chains and then NEMO was immunoprecipitated (IP) with a Flag antibody. The precipitated proteins were analyzed by immunoblotting with an antibody against ubiquitin or Flag. (D) Different amounts of NEMO deletion mutants were added to 1.3E2 (NEMO 2/2 ) extracts. The activation of IKK was initiated by adding recombinant TRAF6 and ATP to the extracts. After incubation for 1 hr, phosphorylation of IkBa was determined by immunoblotting with an IkBa antibody. fusion proteins, purified these proteins with the Flag antibody, and tested their ability to bind to polyubiquitin chains by coimmunoprecipitation followed by immunoblotting with a ubiquitin antibody. As shown in Figure 6C, deletion of the N-terminal 86 residues encompassing the IKK binding sites (86 419) or the C-terminal 54 residues including the zinc finger motif (1 365) had no effect on polyubiquitin binding by NEMO. However, further deletion into the LZ region (1 196 and 1 302) abolished the binding of NEMO to polyubiquitin. We also examined the ability of NEMO deletion mutants to restore IKK activation by TRAF6 in cell extracts derived from a NEMOdeficient cell line, 1.3E2. The wild-type and NEMO mutant lacking the zinc finger domain (1 365) were capable of restoring IKK activation when each was added back to the NEMO-deficient extracts, consistent with the previous results that deletion of the zinc finger domain did not affect IKK activation by IL-1b or LPS (Huang et al., 2002; Makris et al., 2002). In contrast, the NEMO mutant lacking the IKK binding region (86 419) or failing to bind polyubiquitin (1 302) was also defective in restoring IKK activation (Figure 6D), suggesting that polyubiquitin binding of NEMO may be linked to its ability to mediate IKK activation. Recent structural studies have shown that most, if not all, of known ubiquitin binding domains bind to a hydrophobic patch on the surface of ubiquitin that surrounds an invariant Ile-44 (Hicke et al., 2005). Therefore, to identify potential residues that might mediate the binding of NEMO to polyubiquitin, we searched for conserved hydrophobic residues within the ubiquitin binding domain ( ) of NEMO. This analysis identified several hydrophobic residues that are conserved among human, bovine, mouse, and Drosophila, including I307, Y308, and F312 (Figure 7A). We mutated I307 and F312 to alanine, and Y308 to serine, and then examined the effect on the ability of NEMO to restore IKK activation in NEMO-deficient extracts. Strikingly, a point mutation at Y308 abolished the ability of NEMO to restore IKK activation (Figure 7B, lane 4). The F312A mutation also partially inhibited IKK activation, whereas the I307A mutation had little effect (Figure 7B, lanes 3 and 5). These

8 Molecular Cell 252 Figure 7. Polyubiquitin Binding by NEMO Is Required for IKK Activation (A) Sequence alignment of the coiled-coil and LZ domains of NEMO from human, mouse, bovine, and Drosophila. The asterisk (*) indicates invariant residues, whereas the dot (.) indicates highly conserved residues. The leucine residues postulated to form an LZ are underlined, and the invariant hydrophobic residues within the coiled-coil domain are numbered and shown in bold. (B) Wild type and mutants of NEMO as indicated were added to 1.3E2 extracts, and the activation of IKK by TRAF6 was determined by immunoblotting with an IkBa antibody. (C) Wild type and mutants of NEMO were incubated with Sepharose beads conjugated with monoubiquitin (Ub), K48-, or K63-linked polyubiquitin chains (Ub n ). Proteins bound to the beads were immunoblotted with a NEMO-specific antibody. In lanes 9 and 10, purified endogenous IKK

9 Mechanisms of IKK Activation by Ubiquitination 253 NEMO mutants were examined for their ability to bind to K48- or K63-linked polyubiquitin chains (Figure 7C). Consistent with the IKK activation assay, the Y308S and F312A mutations drastically reduced the binding of NEMO to polyubiquitin (Figure 7C, lanes 5 8). Although the I307A mutation decreased the binding of NEMO to K48 chains, this mutation did not affect the binding of NEMO to K63 chains (Figure 7C, lane 4) or the activation of IKK (Figure 7B, lane 3). The aforementioned experiments showed that the LZ ( ) region of NEMO including Y308 and F312 is required for polyubiquitin binding as well as IKK activation. To determine if the LZ region of NEMO is sufficient to bind to polyubiquitin, we expressed this fragment as a GST fusion protein in E. coli and tested its ability to bind K63 polyubiquitin chains (Figure 7D). The LZ fragment alone ( ) did not bind polyubiquitin nor did a fragment containing the second coiled-coil (CC2) region of NEMO ( ; Figure 7D, lanes 3 and 4). However, a longer fragment ( ) containing both CC2 and LZ of NEMO was capable of binding to polyubiquitin (Figure 7D, lane 5). Further deletion experiments showed that a 66 residue fragment ( ) of NEMO containing the C-terminal portion of CC2 domain and the N-terminal portion of LZ domain (Figure 7D, lane 9) was sufficient to bind polyubiquitin chains (Figure 7D, lane 9). Importantly, the Y308S and F312A mutations markedly reduced the binding of this NEMO fragment to polyubiquitin chains (Figure 7D, lanes 6, 10, and 11). To determine if Y308S disrupts the overall structure of NEMO, we carried out two sets of experiments. In the first set, we analyzed the elution profile of the NEMO fragment containing CC2 and LZ ( ) on a gel filtration column. Previous studies using a shorter NEMO fragment containing CC2 and LZ (residues in mouse NEMO) showed that this fragment forms a trimer (Agou et al., 2004). Gel filtration (Superdex-200) analysis of NEMO ( ) showed that this protein fragment, which has a calculated molecular weight of about 19 kda, eluted at a position corresponding to more than 100 kda (Figure 7E). Although this apparent molecular size is larger than the predicted size of a trimer of NEMO (residues ), it is possible that the apparent large size is due to the coiled-coil structure, which causes the aberrant elution of proteins from gel filtration columns. In any case, the elution profile of the NEMO- Y308S mutant protein was indistinguishable from that of the wild-type NEMO, suggesting that the mutation at Y308 does not cause a global structural alteration of NEMO that disrupts its oligomerization. In the second set of experiments, we tested whether Y308S mutation of NEMO affects its ability to form an IKK complex (Figure 7F). When the NEMO-deficient extracts (from 1.3E2 cells) were applied to the gel filtration column (Superdex-200), IKKb eluted at a position corresponding to w500 kda. The addition of wild-type NEMO or Y308S mutant to the 1.3E2 extracts caused IKKb to elute at a position corresponding to w700 kda, which is the size of an intact IKK complex. Thus, the Y308S mutation does not affect the ability of NEMO to form an intact IKK complex. Collectively, these results indicate that the Y308S mutation of NEMO specifically impairs its ability to bind to polyubiquitin chains without altering its overall structure. If the inhibition of IKK by the Y308S mutation of NEMO is due to the inability of this mutant to bind to polyubiquitin chains, it may be possible to rescue IKK activation if the ubiquitin binding activity of the NEMO mutant is restored. To test this possibility, we fused NEMO-Y308S to the NZF domain of Npl4, which is known to bind to polyubiquitin chains (Kanayama et al., 2004; Meyer et al., 2002). However, the NEMO fusion protein (Y308S-NZF) did not bind to polyubiquitin chains as strongly as did the wild-type protein (Figure S1A available in the Supplemental Data with this article online). Nevertheless, this fusion protein partially activated IKK in the presence of TRAF6 (Figure S1B), further supporting the importance of ubiquitin binding in the regulation of IKK by NEMO. To determine if ubiquitin binding of NEMO is required for IKK activation in vivo, we expressed the wild-type and Y308S mutant of NEMO in the NEMO 2/2 Jurkat cell line by retroviral infection. These cells were stimulated with GST-TNFa and then the IKK complex was isolated by immunoprecipitation to measure its kinase activity. As shown in Figure 7G, the activation of IKK was markedly reduced in cells containing the Y308S mutant of NEMO (top four panels). To determine if NEMO-Y308S complex was used for the binding assay. The bottom panel shows the immunoblotting of 20% of NEMO or IKK complex used in the binding experiments. (D) Purified GST-NEMO proteins as indicated were incubated with K63-linked polyubiquitin chains and then absorbed to glutathione Sepharose beads. The binding of polyubiquitin chains to the beads was analyzed by immunoblotting with a ubiquitin antibody (top). The GST-NEMO protein fragments used in the binding assays were stained with Coomassie blue (bottom). (E) GST-NEMO proteins were cleaved into GST and NEMO fragments with thrombin and then subjected to gel filtration chromatography on Superdex-200. Fractions containing the NEMO fragment were resolved by SDS-PAGE and stained with Coomassie blue. (F) Wild type (WT) or the Y308S mutant of NEMO was added to 1.3E2 extracts, which were then subjected to gel filtration chromatography by using Superdex-200. IKKb in the fractions was detected by immunoblotting. (G) Jurkat cells stably expressing wild-type NEMO or NEMO-Y308S were stimulated with GST-TNFa for the indicated times and then the IKK complex was immunoprecipitated with a NEMO antibody to measure its kinase activity with GST-IkBa(NT) and g- 32 P-ATP as substrates (top). An aliquot of the IKK complex was immunoblotted with an antibody against IKKb (second panel from the top). Aliquots of the cell lysates were also immunoblotted with an antibody against NEMO or phospho-ikba (third and fourth panels from the top). To measure the recruitment of NEMO or RIP1 to the TNF receptor, glutathione Sepharose beads were used to isolate the receptor complexes, which were then immunoblotted with an antibody against NEMO or RIP1 (bottom two panels). (H) A model of IKK activation by TNFa. Stimulation of cells with the trimeric TNFa leads to the trimerization of TNF receptor (TNF-R1) and subsequent recruitment of signaling proteins, including TRADD, TRAF2, TRAF5, and RIP1. The TRAF proteins recruit Ubc13/Uev1A to polyubiquitinate RIP1 at K377. Polyubiquitinated RIP1 then recruits the TAK1 kinase complex through the interaction between the polyubiquitin chains and the NZF domain of TAB2. The polyubiquitin chains on RIP1 can also bind to NEMO, resulting in recruitment of the IKK complex. IKKb in the complex is then phosphorylated and activated by TAK1, leading to the phosphorylation, ubiquitination, and degradation of IkB. NF-kB subsequently translocates into the nucleus to activate gene expression.

10 Molecular Cell 254 could be recruited to the TNF receptor, the receptor complex was isolated by using glutathione Sepharose after cells were stimulated with GST-TNFa (Figure 7G, bottom two panels). Immunoblotting experiments showed that although RIP1 was still polyubiquitinated and recruited to the receptor, only wild-type NEMO, but not the Y308S mutant, was recruited to the receptor. These experiments demonstrate that polyubiquitin binding of NEMO is required for IKK activation by TNFa. Discussion In this report, we show that RIP1 is polyubiquitinated at K377 after TNFa stimulation and that polyubiquitinated RIP1 binds to TAB2 and NEMO, resulting in the receptor recruitment of the TAK1 and IKK complexes, respectively. Importantly, a point mutation at K377 of RIP1 blocks its polyubiquitination and prevents the receptor recruitment of TAK1 and IKK as well as the activation of these kinases. Furthermore, a point mutation at Y308 of NEMO abrogates its binding to polyubiquitin chains as well as its ability to mediate IKK activation. Collectively, our results show that polyubiquitination of RIP1 and polyubiquitin binding by NEMO are essential for the activation of IKK by TNFa. We propose that the polyubiquitin chains on RIP1 serve as a platform to recruit both TAK1 and IKK complexes, allowing TAK1 to phosphorylate and activate IKK (Figure 7H). It is rather unusual that a point mutation at K377 is sufficient to prevent the polyubiquitination of RIP1. In most ubiquitination substrates, the ubiquitination sites are quite promiscuous, as mutations of the primary ubiquitination sites can cause cryptic ubiquitination at secondary sites on the molecule. In fact, when we first tried to map the ubiquitination site of RIP1 by overexpression in HEK293 cells, we found that none of the lysine mutations prevented RIP1 ubiquitination or NF-kB activation. Only when we established stable cell lines expressing different lysine mutants of RIP1 at a level comparable to that of the endogenous protein did we observe the effect of K377 mutation on RIP1 ubiquitination. Thus, endogenous RIP1 may form a complex with other proteins, which covers the surface of RIP1 and prevents nonspecific ubiquitination. The difficulty in mapping the specific ubiquitination sites of other targets such as TRAF2 and TRAF6 has been a hurdle in proving the physiological significance of ubiquitination of these proteins. At least in the case of RIP1, our results show that signal-induced site-specific ubiquitination of RIP1 is essential for TNFa signaling. In contrast to our finding that RIP1 is required for the recruitment of the IKK complex after TNFa stimulation (Figure 4A), a previous report showed that IKKa and IKKb coprecipitated with TNF-R1 in RIP1-deficient cells (Devin et al., 2000). The reasons for these discrepant results are not clear but could be due to the different conditions employed for the receptor pull-down experiments. While Devin et al. used an antibody against TNF-R1 to immunoprecipitate the receptor complex in the presence of 0.1% NP40, our current study used glutathione Sepharose to pull down GST-TNFa and its receptor complex in the presence of 1% Triton X-100. It is possible that IKKa and IKKb can be recruited to the receptor through TRAF2 or TRAF2 autoubiquitination, but this association is relatively weak and can be disrupted under the more stringent conditions used in our study. Our study shows that TNFa stimulation leads to polyubiquitination of RIP1, thus allowing RIP1 to bind to NEMO in a ubiquitination-dependent manner (Figure 5B). However, it has been shown previously that RIP1 can bind to NEMO in yeast two-hybrid and coimmunoprecipitation experiments (Zhang et al., 2000). These discrepant results can be best explained by the fact that in the latter studies both RIP1 and NEMO were overexpressed, whereas in our studies, the interactions of the endogenous proteins were examined. Indeed, when we overexpressed NEMO and RIP1 or RIP1(K377R) in HEK293 cells, we found that NEMO bound to both wild-type and K377R RIP1 in coimmunoprecipitation experiments, indicating that NEMO can bind to RIP1 in a ubiquitination-independent manner when both proteins are overexpressed (data not shown). Thus, the interaction between endogenous NEMO and RIP1 in TNFa-stimulated cells is likely to be bipartite: NEMO could interact with RIP1 weakly and directly, but this interaction is facilitated by the additional binding between NEMO and the polyubiquitin chains on RIP1. This makes sense as only such a bipartite interaction can bring specificity in signaling. If NEMO bound only to polyubiquitin chains, it would have been sequestered by the abundant polyubiquitinated proteins in cells. Similarly, if endogenous NEMO bound to RIP1 tightly and constitutively, the interaction between NEMO and RIP1 would not be regulated by TNFa signaling. Thus, the bipartite interaction between polyubiquitinated RIP1 and NEMO provides a regulatory mechanism analogous to the interaction between a phosphorylated motif (e.g., phosphotyrosine motif) and a domain (e.g., SH2) that recognizes not only the phosphate group but also the surrounding amino acid sequence (reviewed by Sun and Chen [2004]). RIP1 is required for IKK activation by multiple pathways, including the Toll-like receptor (TLR) and DNA damage pathways (Devin et al., 2003; Meylan et al., 2004). TLR3 and TLR4 are known to bind TRIF, an adaptor that contains binding sites for both TRAF6 and RIP1 (Akira and Takeda, 2004). Recent studies have shown that RIP1 is polyubiquitinated in response to stimulation of TLR3, suggesting that ubiquitination of RIP1 may also be involved in the TLR pathways (Cusson-Hermance et al., 2005). It will be interesting to determine whether ubiquitination of RIP1 also occurs at K377 and whether RIP1 ubiquitination is important in TLR signaling. RIP1 is also required for NF-kB activation by DNA damage; however, it is not known whether RIP1 is ubiquitinated in this pathway. A key role of RIP1 polyubiquitination is to recruit the TAK1 kinase complex through the NZF ubiquitin binding domain of TAB2 and TAB3. Recent studies have provided several lines of evidence that firmly establish the central role of TAK1 in NF-kB activation by multiple pathways, including those emanating from IL-1R/TLR, TNFR, and T cell receptors (TCRs). First, TAK1 phosphorylates IKKb at key serine residues in the activation loop, thereby activating IKK (Wang et al., 2001). Second, TAK1 is rapidly activated after stimulation of cells with IL-1b, TNFa, and agonists of TLR and TCR (Kanayama et al., 2004; Lee et al., 2000; Ninomiya-Tsuji et al., 1999; Sakurai et al., 2005). Third, Drosophila harboring

11 Mechanisms of IKK Activation by Ubiquitination 255 mutations in TAK1 are severely defective in antibacterial innate immune responses (Vidal et al., 2001). Finally, TAK1-deficient cells generated by homologous recombination or RNAi are defective in NF-kB activation by multiple pathways (Kanayama et al., 2004; Sato et al., 2005; Shim et al., 2005; Shinohara et al., 2005; Takaesu et al., 2003). However, there is still residual IKK activation in TAK1-deficient cells, suggesting that there may be a TAK1-independent pathway that contributes to the partial activation of IKK. MEKK3 may be a part of this TAK1-independent pathway, as it has been shown that MEKK3-deficient cells are partially defective in NF-kB activation in response to stimulation by multiple agents, including TNFa, IL-1b, and LPS (Huang et al., 2004; Yang et al., 2001). However, we found that although RNAi of TAK1 in HeLa and HEK293 cells led to a severe defect in IKK activation by TNFa and IL-1b, efficient silencing of MEKK3 had little effect on IKK activation in the same experiment (data not shown). Furthermore, simultaneous silencing of TAK1 and MEKK3 did not result in further inhibition of IKK beyond what was achieved with TAK1 RNAi alone. Thus, further research is required to delineate the TAK1-independent pathway of IKK activation. It is interesting to note that the majority of RIP1 recruited to the stimulated TNF-R1 is the polyubiquitinated form, whereas only a very small fraction of total cellular RIP1 is polyubiquitinated (Figure 1A; Zhang et al., 2000). These results suggest that polyubiquitinated RIP1 is preferentially recruited to the TNF-R1 complex, implying that polyubiquitination of RIP1 may facilitate its recruitment to and/or stable association with the receptor. Consistent with this notion, the receptor recruitment of RIP1-K377R, which cannot be polyubiquitinated but is still competent in binding to TRADD and TRAF2, was significantly reduced (Figures 1E and 4B). Further research is required to delineate the mechanism by which polyubiquitinated RIP1 is preferentially recruited to the TNF-R1 receptor complex. Ubiquitination of NEMO has been suggested to play an important role in IKK activation by T cell receptors and DNA damaging agents. However, these studies have led to the identification of different ubiquitination sites on NEMO (reviewed by Chen [2005] and Krappmann and Scheidereit [2005]). For example, K399 appears to be the major ubiquitination site on NEMO during T cell activation (Zhou et al., 2004), whereas K277 and K309 serve as the ubiquitination sites after DNA damage (Huang et al., 2003). In addition, NOD2, a cytosolic protein that activates NF-kB in response to intracellular bacteria, has been shown to promote the ubiquitination of NEMO at K285 (Abbott et al., 2004). It is not clear how ubiquitination of NEMO at different sites could mediate IKK activation. Although our results show that NEMO can bind to polyubiquitin chains and that polyubiquitin binding by NEMO is important for IKK activation, they do not rule out the possibility that ubiquitination of NEMO is also involved in IKK activation. In fact, we found that mutations that impair polyubiquitin binding by NEMO also abrogate its ubiquitination (Figure 7B and data not shown), suggesting that polyubiquitin binding and ubiquitination of NEMO are coupled. Such a coupling between ubiquitin binding and ubiquitination has been commonly observed in several other proteins; for example, several proteins in the endocytic pathways contain the ubiquitin-interaction motif (UIM) that is required for ubiquitin binding as well as ubiquitination (Hicke et al., 2005). Thus, ubiquitin binding by NEMO may be required for both the recruitment of IKK to polyubiquitinated RIP1 and the ubiquitination of NEMO, and the latter may facilitate the interaction between IKK and TAK1 through the binding between ubiquitinated NEMO and TAB2. In any case, polyubiquitin binding by NEMO is an important mechanism that mediates IKK activation. Several ubiquitin binding motifs have been discovered in the past few years (Hicke et al., 2005). These include UIM, UBA (Ub association), UEV (Ub E2 variant), CUE (Cue1-homologous), PAZ (polyubiquitin-associated zinc finger), and NZF. These motifs have been found in a large variety of proteins, and their ability to bind ubiquitin has been shown to be biologically important in some cases. However, NEMO does not contain any of these motifs but instead uses a domain consisting of portions of the coiled-coil and LZ regions (residues ) to bind to polyubiquitin. This domain is both necessary and sufficient for NEMO to bind to polyubiquitin, and a point mutation at Y308 within this domain abolishes the ability of NEMO to bind polyubiquitin as well as its ability to activate IKK. Thus, NEMO appears to contain a novel ubiquitin binding domain herein referred to as NEMO Ub binding (NUB). Previous studies have shown that this domain forms a trimer (Agou et al., 2004), so it is possible that the formation of this oligomeric structure is required for preferential binding to K63 polyubiquitin chains. Structural studies of the NEMO-ubiquitin complex should provide important insights into the mechanism of IKK activation by ubiquitin. Experimental Procedures TNFa-Induced Recruitment of Signaling Proteins to TNF Receptor Jurkat cells (10 7 cells) were treated with 1 mg/ml GST-TNFa for different lengths of time (for t = 0, GST-TNFa was added after the cells were lysed). Cells were lysed in buffer A (20 mm Tris-HCl [ph 7.5], 150 mm NaCl, 25 mm b-glycerophosphate, 1 mm sodium orthovanadate, 10% glycerol, 0.5 mm DTT, 10 mg/ml leupeptin, and 1 mm PMSF) plus 1% Triton X-100. The cell lysates (500 mg) were incubated with 10 ml glutathione Sepharose beads and mixed endover-end at 4ºC for 1 hr. The beads were then washed once with phosphate-buffered saline (PBS) plus 1% Triton X-100 and twice with PBS plus 0.5% Triton X-100. The bound proteins were analyzed by immunoblotting with various antibodies as indicated. For l phosphatase treatment, receptor bound TAB2 was incubated with 40 U of l phosphatase (New England Biolabs) in a 50 ml reaction at 30ºC for 1 hr. The beads were then washed with PBS and analyzed by immunoblotting with a TAB2-specific antibody. TNFa-Induced Polyubiquitination of RIP1 and Its Association with TAB2 and NEMO Jurkat cells were stimulated with GST-TNFa and then the signaling complexes isolated by using glutathione Sepharose as described above. The complexes were disrupted by the addition of 1% SDS in 50 mm Tris-HCl (ph 7.5) and 1 mm DTT. After removal of the beads by centrifugation, the supernatant was diluted 10-fold in PBS before immunoprecipitation with a RIP1 antibody. Ubiquitination of RIP1 was then analyzed by immunoblotting with a ubiquitin-specific antibody. To examine the association between ubiquitinated RIP1 and TAB2 or NEMO, extracts from TNFa-stimulated cells were immunoprecipitated with an antibody against TAB2 or NEMO, and the precipitated proteins were analyzed by immunoblotting with a RIP1 antibody.

12 Molecular Cell 256 Polyubiquitin Binding by NEMO K48- and K63-linked polyubiquitin chains were synthesized in vitro and then conjugated to Sepharose beads as described previously (Kanayama et al., 2004). The ubiquitin Sepharose beads (2 ml at 1 mg/ml) were then incubated with Flag-NEMO isolated from HEK293 cells (50 ml at 2 ng/ml) or His 10 -S5a (50 ml at 2 ng/ml) at room temperature for 20 min. After washing the beads with buffer B (20 mm Tris-HCl [ph 7.5], 150 mm NaCl, and 0.5 mm DTT), NEMO bound to the beads was analyzed by immunoblotting. The polyubiquitin binding assays were also carried out by incubating polyubiquitin chains with NEMO immobilized on Flag antibody (M2) Sepharose or glutathione Sepharose. In these assays, the high molecular weight K63-linked polyubiquitin chains were partially purified by gel filtration chromatography (Superdex-200). These chains (50 ml at 2 ng/ml) were incubated with the NEMO Sepharose beads at room temperature for 20 min. The beads were washed as described above, and then the bound ubiquitin chains were detected with a ubiquitin antibody. Supplemental Data Supplemental Data include Supplemental Experimental Procedures, Supplemental References, and one figure and can be found with this article online at 245/DC1/. Acknowledgments We thank Dr. Adrian Ting (Mount Sinai School of Medicine) for RIP1 2/2 Jurkat cells, Dr. Shao-Cong Sun (Penn State University) for NEMO 2/2 Jurkat cells, Dr. Gary Nolan (Stanford University) for the Phoenix retroviral packaging cell line, and Dr. Cecile Pickart (Johns Hopkins University) for the pet16b-s5a construct. We also thank Dr. Lijun Sun for critically reading the manuscript. This work was supported by grants from the National Institutes of Health (RO1-GM63692), the Welch Foundation (I-1389), and American Cancer Society (RSG TBE). Z.J.C is a Burroughs Wellcome Fund Investigator in Pathogenesis of Infectious Diseases. 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