Oncogene (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 receptor signaling complexes Danying Chen1, Liang-Guo Xu2, Lei Chen1, Lixia Li1, Zhonghe Zhai1, Hong-Bing Shun,1,2 1Department of Cell 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). Gene knockout study, however, threonine protein 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 proteins 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 growth factor 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 tyrosine kinase activity.Ligand 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 breast cancer 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 proline-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 apoptosis (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 insulin receptor 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 Signal Transduction Antibody ArrayTM antibody, and then detected by ECL.The positive signals system (Hypromatrix, Inc.).