(2003) 22, 4348–4355 & 2003 Nature Publishing Group All rights reserved 0950-9232/03 $25.00 www.nature.com/onc

NIK is a component of the EGF/heregulin signaling complexes

Danying Chen1, Liang-Guo Xu2, Lei Chen1, Lixia Li1, Zhonghe Zhai1, Hong-Bing Shun,1,2

1Department of Biology and Genetics, College of Life Sciences, Peking University, Beijing 100871, China; 2Department of Immunology, National Jewish Medical and Research Center, University of Colorado Health Sciences Center, 1400 Jackson Street, k516c, Denver, CO 80206, USA

Nuclear factor jB-inducing kinase (NIK) is a member of Introduction the MAP kinase kinase kinase family that was first identified as a component of the TNF-R1-induced NF-jB activation pathway (TNF, tumor necrosis factor; nuclear Nuclear factor kB-inducing kinase (NIK) is a serine/ factor kappaB, NF-jB). knockout study, however, threonine kinase belonging to the MAP kinase suggests that NIK is dispensable for TNF-R1- but kinase kinase family, which also includes MEKK1-4, required for lymphotoxin-b receptor-induced NF-jB ASK1, and Raf, among others (Malinin et al., 1997). activation. A NIK kinase inactive mutant is a potent NIK was first identified as a TRAF2-interacting protein inhibitor of NF-jB activation triggered by various stimuli, by yeast two-hybrid screening and was shown to be suggesting that NIK is involved in a broad range of NF-jB involved in the TNF-R1-induced NF-kB activation activation pathways. To unambiguously identify signaling pathway (Malinin et al., 1997) (TNF, tumor necrosis pathways that NIK participates in, we screened antibody factor; nuclear factor kappaB, NF-kB).Subsequently, it arrays for that are associated with NIK. This has been suggested that NIK interacts with various effort identified ErbB4, one of the EGF/heregulin TRAFs and can activate IKK, a kinase complex that is receptors, and Grb7, an adapter protein associated with critically involved in NF-kB activation triggered by ErbB4 (ErbB, epidermal receptor family divergent stimuli (Lin et al., 1998a, b). However, gene protein; EGF, epidermal growth factor; Grb, growth knockout experiments indicate that NIK is essential for factor receptor bound). Coimmunoprecipitation experi- lymphotoxin-b- but not TNF-R-induced NF-kB activa- ments demonstrated that NIK interacted with Grb7, as tion (Matsushima et al., 2001; Yin et al., 2001). well as Grb10 and Grb14, but not Grb2. Domain mapping Furthermore, it is suggested that NIK functions at a experiments indicated that the central GMdomain of nuclear rather than a cytoplasmic step in the lympho- Grb7 was sufficient for its interaction with NIK. toxin-b-induced NF-kB activation pathway (Yin et al., Coimmunoprecipitation experiments also indicated that 2001).Since a kinase inactive mutant of NIK can block Grb7 and NIK could be simultaneously recruited into NF-kB activation by various stimuli, such as TNF, IL1, signaling complexes of all known EGF/heregulin recep- and LPS, this suggests that NIK may be involved in a tors, including EGFR, ErbB2, ErbB3, and ErbB4. In broad range of NF-kB activation pathways. reporter gene assays, NIK could potentiate Grb7, ErbB2/ Previously, it has been shown that epidermal growth ErbB4, and EGF-induced NF-jB activation. A NIK factor (EGF) can activate NF-kB through undefined kinase inactive mutant could block ErbB2/ErbB4 and pathways (Obata et al., 1996; Sun and Carpenter, 1998; EGF-induced NF-jB activation. Moreover, EGF/here- Biswas et al., 2000; Habib et al., 2001). EGF family gulin receptors activated NF-jB in wild-type, but not members interact with receptors belonging to the EGF NIKÀ/À embryonic fibroblasts. Our findings suggest that receptor family, including epidermal growth factor NIK is a component of the EGF/heregulin receptor receptor (EGFR), epidermal growth factor receptor signaling complexes and involved in NF-jB activation family protein (ErbB)2, ErbB3, and ErbB4 (Pawson, triggered by these receptors. 1995).The cytoplasmic domains of the EGF receptor Oncogene (2003) 22, 4348–4355. doi:10.1038/sj.onc.1206532 family members, except for ErbB2, have intrinsic kinase activity. stimulation leads to Keywords: NIK; EGF; Grb7; NF-kB; signaling homo- or heterodimerization of the EGF receptor family members and their autophosphorylation.This process creates docking sites for downstream signaling molecules that contain the SH2 domain (Pawson, 1995). Interaction of these signaling molecules with the receptors initiates intracellular signaling cascades that *Correspondence: Hong-Bing Shu, Department of Immunology, lead to activation of a number of transcription factors National Jewish Medical and Research Center, University of Colorado such as AP-1 and STATS (Hill and Treisman, 1995). Health Sciences Center, 1400 Jackson Street, k516c, Denver, CO Previous studies also suggest that EGF can activate NF- 80206, USA; E-mail: [email protected] Received 3 January 2003; revised 20 February 2003; accepted 26 kB in several types of cells, including smooth muscle, February 2003 A431, fibroblasts, and EGF receptor-overexpressing NIK is a component of the EGF/heregulin D Chen et al 4349 cells (Obata et al., 1996; Sun and proteins.The antibodies are immobilized on a nitrocel- Carpenter, 1998; Biswas et al., 2000; Habib et al., 2001). lulose membrane, each at a predetermined position, and The growth factor receptor bound (Grb)7 family they retain their capabilities of recognizing and captur- members are some of the adapter molecules that interact ing antigens as well as antigen-associated proteins.We with activated receptor tyrosine kinases, including the transfected 293 cells with an expression plasmid for EGF receptor family members.The Grb7 family Flag-NIK and the cell lysate was used to incubate with consists of Grb7, Grb10, and Grb14, which do not the antibody array membrane.Using anti-Flag anti- have intrinsic enzymatic activity and signal through body, we detected that 21 antibodies could recruit Flag- their interaction with downstream proteins (Daly, 1998; NIK, probably directly or indirectly through their Han et al., 2001). Unlike Grb2, which contains an SH2 antigens.These include antibodies against NBK, Mad- domain flanked by two SH3 domains, the Grb7 family 1, p38, CD3-e, eNOS, HSP-70, ANT, DAXX, SODD, members contain an N-terminal -rich (PR) thyroid receptor a1, Sp1, ErbB4, TNF-R1, B7-1, region, a central GM region that is homologous with DMBT1, GATA-1, IRF2, TRADD, BOK, caspase-2, the C. elegans protein Mig10, and a C-terminal SH2 and Grb7.Results for part of the membrane are shown domain (Daly, 1998; Han et al., 2001). The SH2 domain in Figure 1.Previously, it was shown that Grb7 is of the Grb7 family is responsible for interaction with the associated with EGF receptor family members, includ- majority of identified Grb7-interacting proteins, such as ing EGFR, ErbB2, ErbB3, and ErbB4 (Margolis et al., the EGF receptor family members, Shc, Raf, Tek/Tie2, 1992; Stein et al., 1994; Janes et al., 1997; Daly, 1998; and FAK, among others (Han et al., 2001). It has been Fiddes et al., 1998; Han et al., 2001). Thus, recruitment shown that the PR domain of Grb7 family members of NIK to two independent antibodies against ErbB4 interacts with c-Abl and Tankyrase (Frantz et al., 1997; and Grb7, respectively, suggests a high possibility that Han et al., 2001; Lyons et al., 2001). Proteins that NIK is a true component of the ErbB4/Grb7 signaling interact with the large central GM domain of the Grb7 complex. family members are not known. In addition to their roles in EGF receptor signaling, NIK interacts with Grb7, Grb10, and Grb14, but not Grb2 the Grb7 family members play divergent roles in various signaling pathways and pathophysiological processes. To determine whether NIK interacts with Grb7, we Grb7 is overexpressed in some human primary cancers transfected 293 cells with expression plasmids for HA- and tumor cell lines, suggesting a possible role in tagged NIK and Flag-tagged Grb7 and performed tumorigenesis (Stein et al., 1994). Various studies have immunoprecipitation experiments.These experiments also suggested a role for Grb7 in the regulation of cell suggest that NIK interacts with Grb7 in 293 cells migration (Tanaka et al., 1998; Han and Guan, 1999; (Figure 2).The Grb7 adapter family contains three Han et al., 2000; Lee et al., 2000; Vaysssiere et al., 2000). members, including Grb7, Grb10, and Grb14 (Daly, Grb10 has been found to be associated with mitochon- 1998; Han et al., 2001). Coimmunoprecipitation experi- dria, where it interacts with Raf1 and is likely to be ments indicate that NIK also interacts with Grb10 and involved in the regulation of (Nantel et al., Grb14 (Figure 2).These interactions are specific because 1998, 1999).It was also reported that Grb10 is recruited Grb2, an adapter protein not belonging to the Grb7 to the c-Kit receptor in the course of SCF-mediated family, does not interact with NIK under the same activation of c-Kit and forms a complex with Akt (Jahn condition (Figure 2). et al., 2002). Grb14 was recently shown to be a specific inhibitor of catalytic activity (Berezia Domain mapping of interaction between Grb7 and NIK et al., 2002). To unambiguously identify the signaling pathways It has been shown that the SH2 domain of Grb7 family that NIK participates in, we searched NIK-associated members is responsible for interaction with most proteins by screening antibody arrays.This effort identified ErbB4 and Grb7 as two proteins that are associated with NIK.Our findings suggest that NIK is a component of the EGF/heregulin receptor signaling complexes and involved in NF-kB activation by these receptors.

Results

Identification of NIK-associated proteins by screening of antibody arrays Figure 1 Identification of NIK-associated proteins by screening of To search for potential NIK-associated proteins, we antibody arrays.The antibody array membranes were incubated with cell lysate containing Flag-NIK, probed with anti-Flag used the Antibody ArrayTM antibody, and then detected by ECL.The positive signals system (Hypromatrix, Inc.). Each Antibody ArrayTM correspond to: ErbB4 (A5), B7-1 (D1), DMBT1 (D4), GATA-1 membrane contains 400 antibodies against well-studied (D6), Grb7 (J6)

Oncogene NIK is a component of the EGF/heregulin D Chen et al 4350 Grb7 Grb10 Grb14 Grb2 a IP Ab: CCαF αF CαF CαF kDa: 1 102 418 533 Pro GM SH2 NIK 97- b ) 533 418) 68- - - -102) -418) (1 rb7(103 IgG Grb7 Grb7(1 Grb7 Grb7(103 G α α α α α 43- IP Ab: C HA C HA C HA C HA C HA kDa Western blot: αHA NIK 97-

Lysate 68- kDa NIK IgG 97- 68- Grb10 43- Grb14 43- Grb7 Western blot: αFlag

Lysate kDa 31- NIK Grb2 97- Grb7 68- Grb7(103-533) Western blot: αHA+ αFlag 43- Grb7(1-418) Grb7(103-418) Figure 2 NIK interacts with Grb7, Grb10, and Grb14, but not 31- Grb2.293 cells ( B5 Â 106) were transfected with 10 mgofan expression plasmid for HA-tagged NIK, together with 10 mgofan 19- expression plasmid for Flag-tagged Grb7, Grb10, Grb14, or Grb2. Grb7(1-102) Coimmunoprecipitation was performed with anti-Flag antibody (aF) or control IgG (C), and Western blot analysis was performed Western blot: αFlag+ αHA with anti-HA antibody (upper panel).Expression levels of NIK, Grb7, Grb10, Grb14, and Grb2 were confirmed by Western blot Figure 3 Domain mapping of Grb7 interaction with NIK. analysis of the lysates with anti-Flag and anti-HA antibodies (a) Schematic presentation of Grb7 deletion mutants.( b) Coim- (lower panel) munoprecipitations between Grb7 mutants and NIK.293 cells (B5 Â 106) were transfected with 10 mg of an expression plasmid for Flag-tagged NIK, together with 10 mg of an expression plasmid for HA-tagged Grb7 or its various mutants.Coimmunoprecipitation identified binding partners (Daly, 1998; Han et al., was performed with anti-HA antibody (aHA) or control IgG (C), 2001).To determine which domain of Grb7 interacts and Western blot analysis was performed with anti-Flag antibody (upper panel).Expression levels of NIK, Grb7, and its mutants with NIK, we constructed a series of HA epitope-tagged were confirmed by Western blot analysis of the lysates with anti- deletion mutants of Grb7, including Grb7(1–102) (the Flag and anti-HA antibodies (lower panel) PR domain), Grb7(1–418) (the PR and GM domains), Grb7(103–533) (the GM and SH2 domains), and Grb7(103–418) (the GM domain).We cotransfected these deletion mutants with Flag-tagged NIK into 293 including EGFR, ErbB2, ErbB3, and ErbB4 cells and performed coimmunoprecipitation experi- (Figure 4a).We also found that EGFR/ErbB2 could ments.These experiments suggest that the central GM form a complex with Grb7, Grb10, Grb14, and NIK, domain of Grb7, aa103–418, is sufficient for its respectively (Figure 4b). interaction with NIK (Figure 3). We next determined whether NIK is associated with Grb7 and the EGF receptors in untransfected cells. Previously, it was shown that Grb7 is coamplified, NIK is recruited to EGF/heregulin receptor signaling overexpressed, and in a tight complex with ErbB2 (Stein complexes et al., 1994; Akiyama et al., 1997; Tanaka et al., 1997). To determine whether NIK is recruited to EGF/ Therefore, we have used a breast cancer cell line, MCF7, heregulin receptor signaling complexes, we transfected for endogenous coimmunoprecipitation experiments. 293 cells with expression plasmids for HA-tagged Grb7 The results suggest that NIK is constitutively associated and NIK, together with Flag-tagged ErbBs, and with Grb7 and the EGFR/ErbB2 complex (Figure 4b). performed coimmunoprecipitation experiments.The It is possible that the EGFR/ErbB2 complex is results suggest that Grb7 and NIK can be simulta- constitutive active in MCF7 cells and thus results in a neously recruited to all EGF/heregulin receptors, constitutive recruitment of NIK to the complex.

Oncogene NIK is a component of the EGF/heregulin D Chen et al 4351 a Grb7 collaborates with NIK to activate NF-kB EGFR ErbB2 ErbB3 ErbB4 It has been shown that NIK is involved in NF-kB IP Ab: αF CCCCαF αF αF activation (Malinin et al., 1997; Lin et al., 1998a, b; Yin kDa et al., 2001). To determine whether Grb7 has a similar function, we performed NF-kB luciferase reporter ErbBs gene assays.These experiments indicated that over- 97- NIK expression of Grb7 could weakly activate NF-kBina dose-dependent manner (Figure 5a).In addition, Grb7 68- could collaborate with NIK to activate NF-kB Grb7 (Figure 5a).To exclude the possibility that Grb7 affects NIK expression but not NIK signaling, we examined 43- IgG NIK levels in all transfections by Western blot analysis. Western blot: αFlag+ αHA As shown in Figure 5a, NIK levels were not significantly changed with the increased expression of Grb7. b

αErbB2 αErbB2 αErbB2 + FR+ R FR+ EG EGF EG IP Ab: α C α C α C kDa ErbB2 EGFR NIK 97-

68- Grb10 Grb14 Grb7

43- Western blot: αFlag+ αHA

c ErbB2 α IgG + b7 EGFR Gr IP Ab: controlα α kDa

97- NIK 68-

IgG 43- Western blot: αNIK

Figure 4 NIK is recruited to EGF/heregulin receptor signaling complexes.( a) NIK and Grb7 are associated with ErbBs.293 cells (B5 Â 106) were transfected with an expression plasmid for Flag- tagged EGFR, ErbB2, ErbB3, or ErbB4 (10 mg), together with expression plasmids for HA-tagged Grb7 and NIK (each 10 mg). Coimmunoprecipitation was performed with anti-Flag antibody (aF) or control IgG (C), and Western blot analysis was performed with anti-HA and anti-Flag antibodies.( b) NIK and Grb7, Grb10, or Grb14 are recruited to EGFR/ErbB2 complex.293 cells Figure 5 Interaction of Grb7 with NIK in NF-kB activation.( a) (B5 Â 106) were transfected with expression plasmids for Flag- Grb7 collaborates with NIK to activate NF-kB.293 cells tagged EGFR and ErbB2 (each 10 mg), together with expression (B1 Â 105) were transfected with 0.2 kg of NF-kB-luciferase plasmids for HA-tagged NIK and Flag-tagged Grb7, Grb10, or reporter plasmid, 0.2 mg of pRL-TK Renilla luciferase reporter Grb14 (each 10 mg).Coimmunoprecipitation was performed with plasmid, and the indicated amounts (mg) of expression plasmids.At anti-EGFR and anti-ErbB2 antibodies (aEGFR þ aErbB2) 16 h after transfection, luciferase activities were measured and or control IgG (C), and Western blot was performed with anti- normalized based on Renilla luciferase levels.The levels of NIK HA and anti-Flag antibody mixture.( c) NIK is associated with expression in the transfected cells are shown under the graph.( b) Grb7 and EGF receptors in untransfected cells.MCF7 cells Effects of Grb7 mutants on NIK-induced NF-kB activation.293 (B2 Â 107) were lysed and the lysate was immunoprecipitated with cells (B1 Â 105) were transfected with reporter plasmids and the a mixture of rabbit anti-EGFR (1 mg) þ anti-ErbB2 (1 mg) anti- indicated expression plasmids, and luciferase assays were per- bodies, rabbit anti-Grb7 antibody (2 mg), or rabbit control IgG formed as above.Data shown are averages and standard deviations (2 mg).Western blot analysis was performed with rabbit anti-NIK of relative luciferase activities from three independent experiments antibody (transfection was performed in triplicate in each experiment)

Oncogene NIK is a component of the EGF/heregulin D Chen et al 4352 We also determined the effects of different Grb7 NIK is involved in NF-kB activation mediated by EGF/ domains on NIK-induced NF-kB activation.These heregulin receptors experiments suggest that the N-terminal PR domain To determine the role of Grb7 in the NF-kB activation (aa1–102) can inhibit NIK-induced NF-kB activation, while the central GM domain is sufficient for collabora- pathways mediated by ErbBs, we performed NF-kB luciferase reporter gene assays.The results indicated tion with NIK to activate NF-kB (Figure 5b).More- that overexpression of ErbB2/ErbB4 could activate NF- over, the SH2 domain of Grb7 can enhance its collaborative ability in NIK-induced NF-kB activation kB and this was enhanced by EGF stimulation (Figure 6a).In these assays, NIK could collaborate (Figure 5b).

Figure 6 NIK is required for NF-kB activation mediated by EGF/heregulin receptors.( a) Effects of NIK and its kinase inactive mutant on ErbB2/ErbB4- and Grb7-mediated NF-kB activation.293 cells ( B1 Â 105) were transfacted with 0.3 mg of NF-kB-luciferase reporter plasmid, 0.3 mg of RSV-b-galactosidase plasmid, and the indicated expression plasmids (each 1 mg).At 16 h after transfection, luciferase activities were measured and normalized based on b-galactosidase levels.Data shown are averages and standard deviations of relative luciferase activities from one representative experiment in which each transfection was performed in triplicate.( b) EGFR/ ErbB2-mediated NF-kB activation in wild-type and NIKÀ/À embryonic fibroblasts.Wild-type or NIK À/À embryonic fibroblasts (B1 Â 105) were transfected with expression plasmids for EGFR (1 mg) and ErbB2 (1 mg) or empty control plasmid (2 mg), together with NF-kB-luciferase (0.5 mg) and RSV-b-galactosidase reporter plasmids.At 8 h after transfection, cells were treated with EGF (10 ng/ml) or left untreated for 6 h.Reporter gene assays were performed as in ( a).( c) ErbB2/ErbB4-mediated NF-kB activation in wild-type and NIKÀ/À embryonic fibroblasts.Wild-type or NIK À/À embryonic fibroblasts (B1 Â 105) were transfected with expression plasimids for ErbB2 (1 mg) and ErbB4 (1 mg) or empty control plasmid (2 mg), together with NF-kB-luciferase (0.5 mg) and RSV-b-galactosidase reporter plasmids.At 8 h after transfection, cells were treated with heregulin (10 ng/ml) or left untreated for 6 h.Reporter gene assays were performed as in (a)

Oncogene NIK is a component of the EGF/heregulin D Chen et al 4353 with ErbB2/ErbB4 and Grb7 to activate NF-kB.On the effecter of EGF/heregulin receptor-mediated NF-kB other hand, a kinase inactive mutant of NIK, but not activation pathways. that of MEKK3, inhibited ErbB2/ErbB4-mediated NF- Previously, it has been shown that the Grb7 family kB activation (Figure 6a). members are phosphorylated on serine and threonine To determine whether NIK is involved in EGF/ residues (Stein et al., 1994; Ooi et al., 1995; Daly et al., heregulin receptor-induced NF-kB activation under 1996; Dong et al., 1997; Reilly et al., 2000). The physiological conditions, we examined whether EGF/ is either constitutive or can be induced heregulin receptors can activate NF-kB in NIK-deficient by stimulation with growth factors, such as EGF, embryonic fibroblasts by reporter gene assays.As shown heregulin, and PDGF.Since NIK is a serine/threonine in Figure 6b, c, we found that EGF/heregulin receptors protein kinase, it would be interesting to examine could activate NF-kB in wild-type, but not NIK- whether NIK is responsible for the serine/threonine deficient cells.Taken together, these data suggest that phosphorylation of the Grb7 family members.It is also NIK is a component of EGF/heregulin receptor- possible that NIK functions as a kinase for other mediated NF-kB activation pathways. downstream signaling proteins, for example, c-Abl and Tankyrase, two proteins that are associated with the PR domain of the Grb7 family members. Grb7 has been shown to be amplified and over- Discussion expressed in concert with ErbB2 in several breast cancer cell lines and other cancer cell types (Margolis et al., NIK is a serine/threonine protein kinase that has been 1992; Akiyama et al., 1997; Tanaka et al., 1997). It has implicated in NF-kB activation pathways.In this study, also been shown recently that EGF-induced NF-kB we found that NIK interacts with Grb7 family members activation was a major pathway of cell-cycle progression and is a component of the EGF/heregulin receptor- in estrogen-receptor negative breast cancer cells (Biswas mediated NF-kB activation pathways.Our studies et al., 2000). In addition, human tumor cell lines support previous reports that EGF/heregulin can resistant to TNF express high levels of EGFR, ErbB2, activate the transcriptional factor NF-kB (Obata et al., or ErbB3 (Hoffmann et al., 1998). Since NF-kB 1996; Sun and Carpenter, 1998; Biswas et al., 2000; activation can prevent cells from apoptosis induced by Habib et al., 2001) and further provide a mechanistic divergent stimuli (Van Antwerp et al., 1996; Beg and explanation for this observation. Baltimore, 1996; Liu et al., 1996; Wang et al., 1998), it is Grb7 was initially identified as an EGF receptor possible that NF-kB activation is involved in cell binding protein (Margolis et al., 1992); thereafter many proliferation and malignancy triggered by overexpres- other binding partners have been reported for Grb7. sion of EGF receptor family members.The identifica- These include HER2/Shc, SHPTP2, PDGFR, ErbB2, tion of NIK as a component of EGF/heregulin receptor- ErbB3, ErbB4, c-Kit, FAK, Tek/Tie2, c-Kit/SCFR, mediated NF-kB activation pathways may therefore , IR, and (Han et al., 2001). Most of these provide a molecular target for drug development against binding partners are upstream regulators of Grb7 family cancers caused by overexpression of EGF/heregulin proteins.Little is known about potential downstream receptor family members. effectors of the Grb7 family proteins.Previous studies have shown that the PR domain of Grb10 interacts with c-Abl in vitro (Frantz et al., 1997). It has also been suggested that the PR domain of Grb14 interacts with Materials and methods Tankyrase in the mammalian overexpression system and Reagents this interaction is involved in vesicle trafficking (Lyons et al., 2001). However, it is unknown whether these Recombinant EGF and heregulin (R&D Systems), monoclo- proteins are associated with the Grb7 family members in nal antibodies against FLAG and the HA (Sigma) epitopes, untransfected cells. rabbit polyclonal antibodies against EGFR, ErbB2, Grb7 and In this study, we identified NIK as a protein NIK (Santa Cruz Biotechnology), and 293 cells (ATCC) were purchased from the indicated resources.Wild-type and NIK- interacting with the GM domain of Grb7.NIK also deficient mouse embryonic fibroblasts (Dr Robert Schreiber, interacts with Grb10 and Grb14, two other members of Washington University at St Louis), and MCF7 cells (Dr Gary the Grb7 family.As far as we know, NIK is the first Johnson, University of Colorado Health Sciences Center) were protein that has been shown to interact with the large provided by the indicated investigators. central GM domain of the Grb7 family members. Endogenous coimmunoprecipitation experiments have Constructs suggested that NIK is associated with Grb7 and is a component of the EGF receptor signaling complex.A NF-kB luciferase reporter construct (Dr Gary Johnson, kinase inactive mutant of NIK, but not that of MEKK3, University of Colorado Health Sciences Center), mammalian inhibits Grb7 and ErbB2/ErbB4-mediated NF-kB acti- expression plasmids for Flag-NIK (Dr David Goeddel, vation.Moreover, experiments with NIK-deficient Tularik Inc.), EGFR, ErbB2-4 (Dr Gibbes Johnson, Center for Biologics Evaluation and Research) were provided by the embryonic fibroblasts indicate that NIK is required for indicated investigators.Mammalian expression plasmids for EGF/heregulin receptor-mediated NF-kB activation. HA-NIK, HA- or FLAG-tagged Grb2, Grb10, Grb14, Grb7 Therefore, we propose that NIK is a downstream and its deletion mutants were constructed by PCR amplifica-

Oncogene NIK is a component of the EGF/heregulin D Chen et al 4354 tion of the corresponding cDNA fragments and subsequently in 1 ml of lysis buffer (15 mm Tris, 120 mm NaCl, 1% Triton, cloning into a CMV promoter-based vector containing a 50 or 25 mm KCl, 2 mm EGTA, 2 mm EDTA, 0.1 mm DTT, 0.5% 30 HA or FLAG tag. Triton X-100, 10 mg/ml leupeptin, 0.5 mm phenylmethylsulfo- nyl fluoride, pH 7.5). For each immunoprecipitation, a 0.4-ml Antibody array screening aliquot of lysate was incubated with 0.5 mg of the indicated monoclonal antibody or control mouse IgG and 25 mlof1:1 The antibody arrays that contain 400 antibodies were slurry of GammaBind A Plus-Sepharose (Amersham Pharma- purchased from Hypromatrix, Inc.To screen for NIK- cia) for at least 2 h.The sepharose beads were washed three 6 associated proteins, 293 cells (5 Â 10 ) were transfected with times with 1 ml of lysis buffer.The precipitates were 10 mg of expression plasmid for Flag-NIK.At 24 h after fractionated on SDS–PAGE and subsequent Western blot transfection, cells were lysed in 1 ml of Triton Extraction analysis was performed as described (Chen et al., 2002). m m m Solution (15 m Tris, pH 7.5, 120 m NaCl, 1% Triton, 25 m For endogenous coimmunoprecipitation, MCF cells m m m KCl, 2 m EGTA, 2 m EDTA, 0.1 m DTT, 0.5% Triton X- (B6 Â 107) were lysed in 3 ml of lysis buffer.A 1 ml aliquot m 100, 10 mg/ml leupeptin, 0.5 m phenylmethylsulfonyl fluor- of lysate was incubated with a mixture of rabbit anti-EGFR ide).The cell lysate was collected and applied to preblocked (1 mg) and anti-ErbB2 (1 mg) antibodies, a rabbit anti-Grb7 Antibody ArrayTM membranes for 2 h.The membranes were antibody (2 mg), or control rabbit IgG (2 mg).The subsequent m m then washed with TBS-T buffer (150 m NaCl, 25 m Tris, immunoprecipitation and Western blot (with a rabbit anti- 0.05% Tween-20, pH 7.5) and incubated with HRP-con- NIK antibody) experiments were performed as described jugated anti-Flag antibody for 2 h at room temperature.The above. membranes were washed again and the signals were detected All immunoprecipitation experiments were performed at by ECL. least two times and similar results were obtained.

Cell transfection and reporter gene assays 293 cells (1 Â 105) were seeded in 12-well dishes and transfected the following day by the standard calcium phosphate Abbreviations precipitation (Sambrook et al., 1989). Mouse embryonic NIK, nuclear factor kB-inducing kinase; Grb, growth factor fibroblasts (1 Â 105) were seeded in 12-well dishes and receptor bound; EGFR, epidermal growth factor receptor; transfected the following day with Lipofectamine 2000 reagent ErbB, epidermal growth factor receptor family protein; EGF, (Invitrogen) by following procedures suggested by the epidermal growth factor; TRADD, tumor necrosis factor manufacturer.Within the same experiment, each transfection associated death domain protein; RIP, receptor interacting was performed in triplicate and, where necessary, empty protein; TNF, tumor necrosis factor; NF-kB, nuclear factor control plasmid was added to ensure that each transfection kappaB; STAT, signal transducers and activators of transcrip- receives the same amount of total DNA.To normalize for tion. transfection efficiency, 0.1 mg of pRL-TK Renilla luciferase or RSV-b-galactosidase reporter plasmid was added to each Acknowledgements transfection.Luciferase and b-galactosidase assays were We thank Xiaoyan Li for technical resistance, Dr Robert performed as previously described (Chen et al., 2002). Schreiber for NIK-/- EF cells, and members of the Shu Luciferase activities were normalized on the basis of Renilla laboratories for discussion.This work was supported in part luciferase or b-galactosidase expression levels. by the National Natural Science Foundation of China (39925016 and 30100097), the Chinese High-Technology Coimmunoprecipitation and Western blot analysis program (2001AA221281), the Special Funds for Major State For transient transfection and coimmunoprecipitation experi- Basic Research of China (G19990539), the Ellison Medical ments, transfected 293 cells from each 100-mm dish were lysed Foundation, and the NIH (R01 AI49992-01).

References

Akiyama N, Sasaki H, Ishizuka T, Kishi T, Sakamoto H, Fiddes RJ, Campbell DH, Janes PW, Sivertsen SP, Sasaki H, Onda M, Hirai H, Yazaki Y, Sugimura T and Terada M. Wallasch C and Daly RJ.(1998). J. Biol. Chem., 273, 7717– (1997). Cancer Res., 57, 3548–3553. 7724. Antwerp DJ, Martin SJ, Kafri T, Green DR and Verma IM. Frantz JD, Giorgetti-Peraldi S, Ottinger EA and Shoelson SE. (1996). Science, 274, 787–789. (1997). J. Biol. Chem., 272, 2659–2667. Beg AA and Baltimore D.(1996). Science, 274, 782–784. Habib AA, Chatterjee S, Park SK, Ratan RR, Lefebvre S and Berezia V, Kasus-Jacobi A, Perdereau D, Cariou B, Girard J Vartanian T.(2001). J. Biol. Chem., 276, 8865–8874. and Burnol AF.(2002). J. Biol. Chem., 277, 4845–4852. Han DC and Guan JL.(1999). J. Biol. Chem., 274, 24425– Biswas DK, Cruz AP, Gansberger E and Pardee AB.(2000). 24430. Proc. Natl. Acad. Sci. USA, 97, 8542–8547. Han DC, Shen TL and Guan JL.(2000). J. Biol. Chem., 275, Chen D, Li X, Zhai Z and Shu HB.(2002). J. Biol. Chem., 277, 28911–28917. 15985–15991. Han DC, Shen TL and Guan JL.(2001). Oncogene, 20, 6315– Daly RJ.(1998). Cell. Signal., 10, 613–618. 6321. Daly RJ, Sanderson GM, Janes PW and Sutherland RL. Hill CS and Treisman K.(1995). Cell, 80, 199–211. (1996). J. Biol. Chem., 271, 12502–12510. Hoffmann M, Schmidt M and Wels W.(1998). Cancer Dong LQ, Du H, Porter SG, Kolakowski Jr LF, Lee AV, Immunol. Immunother., 47, 167–175. Mandarino LJ, Fan J, Yee D, Liu F and Mandarino J. Jahn T, Seipel P, Urschel S, Peschel C and Duyster J.(2002). (1997). J. Biol. Chem., 272, 29104–29112. Mol. Cell. Biol., 22, 979–991.

Oncogene NIK is a component of the EGF/heregulin D Chen et al 4355 Janes PW, Lackmann M, Church WB, Sanderson GM, Obata H, Biro S, Arima N, Kaieda T, Eto H, Miyata M and Sutherland RL and Daly RJ.(1997). J. Biol. Chem., 274, Tanaka H.(1996). Biochem. Biophys. Res. Commun., 224, 30896–30905. 27–32. Lee H, Volonte D, Galbiati F, Lyengar P, Lublin DM, Ooi J, Yajnik V, Immanuel D, Gordon M, Moskow JJ, Bregman DB, Milson MT, Campos-Gonzalez R, Bouzahzah Buchberg AM and Margolis B.(1995). Oncogene, 10, 1621– B, Pestell RG, Scherer PE and Lisanti MP.(2000). Mol. 1630. Endocrinol., 14, 1750–1755. Pawson T.(1995). Nature, 373, 573–580. Lin L, Cao Z and Goeddel DV.(1998a). Proc. Natl. Acad. Sci. Reilly JF, Mickey G and Maher PA.(2000). J. Biol. Chem., USA, 95, 3792–3379. 275, 7771–7778. Lin X, Mu Y, Cunningham ET, Marcu KB, Geleziunas R and Sambrook J, Fritch EF and Maniatis T.(1989). Molecular Greene WC.(1998b). Mol. Cell. Biol., 18, 5899–5907. Cloning: A Laboratory Manual.2nd edn.Cold Spring Liu Z, Hsu H, Goeddel DV and Karin M.(1996). Cell, 87, Harbor Laboratory Press: Cold Spring Harbor, NY. 565–576. Stein D, Wu J, Fuqua S, Roonprapunt C, Yajnik V, Lyons RJ, Deane R, Lynch DK, Ye ZS, Sanderson GM, Eyre D’Eustachio P, Moskow J, Buchberg A, Osborne C and HJ, Sutherland GR and Daly RJ.(2001). J. Biol. Chem., 276, Margolis B.(1994). EMBO J., 13, 1331–1340. 17172–17180. Sun L and Carpenter G.(1998). Oncogene, 16, 2095–2102. Malinin NL, Boldin MP, Kovalenko AV and Wallach D. Tanaka S, Mori M, Akiyoshi T, Tanaka Y, Mafune K, Wands (1997). Nature, 385, 540–544. J and Sugimachi K.(1997). Cancer Res., 57, 28–31. Margolis B, Silvennoinen O, Comoglio F, Roonprapunt C, Tanaka S, Mori M, Akiyoshi T, Tanaka Y, Mafune KI, Wants Skolnik E, Ullrich A and Schlessinger J.(1992). Proc. Natl. JR and Sugimachi K.(1998). J. Clin. Invest., 102, 821–827. Acad. Sci. USA, 89, 8894–8898. Vaysssiere B, Zalcman G, Mache Y, Mirey G, Ligensa T, Matsushima A, Kaisho T, Rennert PD, Nakano H, Kurosawa Weidner KM, Chardin P and Camonis J.(2000). FEBS K, Uchida D, Takeda K, Akira S and Matsumoto M. Lett., 467, 91–96. (2001). J. Exp. Med., 193, 631–636. Wang CY, Mayo MW, Korneluk RG, Goeddel DV and Nantel A, Huber M and Thomas DY.(1999). J. Biol. Chem., Baldwin Jr AS.(1998). Science, 281, 1680–1683. 274, 35719–35724. Yin L, Wu L, Wesche H, Arthur CD, White JM, Goeddel DV Nantel A, Mohammad-Ali K, Sherk J, Posner BI and Thomas and Schreiber RD.(2001). Science, 291, 2162–2165. DY.(1998). J. Biol. Chem., 273, 10475–10484.

Oncogene