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Essential Role of Nuclear Factor (NF)- k B-inducing Kinase and Inhibitor of k B (I k B) Kinase a in NF k B Activation through Lymphotoxin b Receptor, but Not through Tumor Necrosis Factor Receptor I

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Essential Role of Nuclear Factor (NF)- k B-inducing Kinase and Inhibitor of k B (I k B) Kinase a in NF k B Activation through Lymphotoxin b Receptor, but Not through Tumor Necrosis Factor Receptor I
    J. Exp. Med. ©  The Rockefeller University Press • 0022-1007/2001/03/631/06 $5.00Volume 193, Number 5,March 5, 2001631–636  Brief Definitive Report   631   Essential Role of Nuclear Factor (NF)-    B–inducing Kinase and Inhibitor of   B (I    B) Kinase   in NF-    B Activation through Lymphotoxin   Receptor, but Not through Tumor Necrosis Factor Receptor I  By Akemi Matsushima,  *  Tsuneyasu Kaisho,   ‡  Paul D. Rennert,   §  Hiroyasu Nakano,     Kyoko Kurosawa,    Daisuke Uchida,  *  Kiyoshi Takeda,   ‡  Shizuo Akira,   ‡  and Mitsuru Matsumoto  *   From the *   Division of Informative Cytology, Institute for Enzyme Research, University of Tokushima, Tokushima 770-8503, Japan; the ‡   Department of Host Defense, Research Institute for Microbial Diseases, and Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Corporation, Osaka University, Osaka 565-0871, Japan; the §   Department of Immunology and Inflammation, Biogen, Incorporated, Cambridge, Massachusetts 02142; and the    Department of Immunology and CREST, Japan Science and Technology Corporation, Juntendo University School of Medicine, Tokyo 113-8421, Japan  Abstract   Both nuclear factor (NF)-      B–inducing kinase (NIK) and inhibitor of    B (I      B) kinase (IKK)have been implicated as essential components for NF-      B activation in response to many exter-nal stimuli. However, the exact roles of NIK and IKK      in cytokine signaling still remain con-troversial. With the use of in vivo mouse models, rather than with enforced gene-expressionsystems, we have investigated the role of NIK and IKK      in signaling through the type I tumor necrosis factor (TNF) receptor (TNFR-I) and the lymphotoxin    receptor (LT      R), a receptor essential for lymphoid organogenesis. TNF stimulation induced similar levels of phosphoryla-tion and degradation of I      B      in embryonic fibroblasts from either wild-type or NIK-mutantmice. In contrast, LT      R stimulation induced NF-      B activation in wild-type mice, but the re-sponse was impaired in embryonic fibroblasts from NIK-mutant and IKK      -deficient mice.Consistent with the essential role of IKK      in LT      R signaling, we found that development of Peyer’s patches was defective in IKK      -deficient mice. These results demonstrate that both NIKand IKK      are essential for the induction of NF-      B through LT      R, whereas the NIK–IKK      pathway is dispensable in TNFR-I signaling.Key words:alymphoplasia • cytokine signaling • I      B • Akt kinase • Peyer’s patch  Introduction   The transcription factor nuclear factor (NF)-      B plays a piv-otal role in the regulation of innate immunity, stress re-sponses, inflammation, and the inhibition of apoptosis (1,2). The activity of NF-      B is tightly regulated by cytokinesand other external stimuli. In most cell types, NF-      B ispresent as a heterodimer comprising 50-kD (p50) and 65-kD (p65) subunits and is sequestered in the cytoplasm by amember of the inhibitor of    B (I      B) family of inhibitoryproteins. NF-      B activation requires the degradation of I      Bproteins, and the mechanisms of I      B degradation and sub-sequent NF-      B activation have been the subject of intenseinvestigation (3). Those studies have revealed two impor-   tant classes of kinase involved in this pathway: mitogen-activated protein kinase kinase kinase (MAP3K) and itsdownstream target, I      B kinase (IKK) (4, 5). NF-      B–induc-ing kinase (NIK) is structurally related to MAP3K and has   been identified as a TNFR-associated factor (TRAF)2-interacting protein (6). On the basis of the finding that ki-nase-inactive mutants of NIK transfected into 293-EBNAcells abolished NF-      B activation in response to TNF or bycotransfection with type I TNF receptor (TNFR-I), NIKwas considered to be involved in an NF-      B–inducing sig-naling cascade induced by TNF (6). Subsequently, NIK was   Address correspondence to Mitsuru Matsumoto, Division of Informative   Cytology, Institute for Enzyme Research, University of Tokushima,3-18-15 Kuramoto, Tokushima 770-8503, Japan. Phone: 81-88-633-7432; Fax: 81-88-633-7434; E-mail:   on O c  t   o b  er 2  ,2  0 1  5  j   em.r  u pr  e s  s . or  gD  ownl   o a d  e d f  r  om  Published March 5, 2001   632  Role of NIK and IKK    in TNF and Lymphotoxin Signaling   demonstrated to phosphorylate IKK      and IKK      , which di-rectly associate with I      B      and specifically phosphorylate iton serines 32 and 36 (4, 5). These studies suggested that in-teraction of NIK and IKK constitutes an essential step for NF-      B activation. However, in vivo studies with mutantmice have thrown some doubt on the essential roles of NIKand IKK      in NF-      B activation, at least as induced by TNF.The alymphoplasia (   aly   ) mouse is a natural strain with a mu-tated NIK (7). Despite the NIK mutation, upregulation of vascular cell adhesion molecule 1 (VCAM-1) after stimula-tion with TNF was present in aly   mouse embryonic fibro-blasts (EFs) (8). It was also reported that aly   mice exhibitedsimilar TNF-mediated endotoxin shock after generalizedLPS administration (7). These observations suggested thatNIK was not a critical element in the TNF signaling path-way to NF-      B activation. The in vivo role of IKK in NF-      B activation was also examined using gene-targeted mice.Although TNF-induced NF-      B activation was markedlyreduced in IKK      -deficient EFs (9, 10),   NF-      B activationfrom IKK      -deficient EFs was normal (11, 12) or dimin-ished but still present (13) after stimulation with TNF. Sur-prisingly, mice deficient in IKK      showed perinatal deathassociated with limb and skin abnormalities, suggesting thatIKK      plays an essential role in the regulation of gene ex-pression required for the development of limb and skinrather than for TNF signaling (11–13). Thus, the role of NIK–IKK      in NF-      B activation through TNFR signalingrequires further investigation.The lymphotoxin    receptor (LT      R) has emerged as asignaling system required for the development of lymphoidorgans (14, 15). Although LT      R has been shown to bindTRAF2, -3, -4, and -5, but not TRAF6 (16, 17), and toactivate NF-      B after receptor ligation (18), the molecular mechanisms by which LT      R exerts its biological activitiesare still poorly understood. aly   mice and LT ligand– or LT      R-deficient mice share a unique phenotype, which in-cludes the lack of LN and Peyer’s patches (PPs) and a dis-turbed splenic architecture (7, 14, 15). Therefore, we spec-ulated that NIK plays a role in LT      R signaling. Thishypothesis was supported by the demonstration that upreg-ulation of VCAM-1 after stimulation with agonistic anti-LT      R mAb was absent from aly   mouse EFs (8).Phenotypic analyses of mutant and gene-targeted mice,as described above, have unveiled the essential roles of NIKin lymphoid organogenesis and of IKK      in limb and skindevelopment. However, the roles of NIK and IKK      in cy-tokine signaling still remain controversial. We have ap-proached this question with the use of in vivo mouse mod-els. Here, we show that NIK–IKK      constitutes an essentialpathway for the induction of NF-      B through LT      R,whereas this pathway is dispensable in TNFR-I signaling.  Materials and Methods   Mice.aly   /      , aly   /   aly   , and C57BL/6J mice were purchasedfrom CLEA Japan. The mice were maintained under pathogen-free conditions and were handled in accordance with the Guide-lines for Animal Experimentation of Tokushima University   School of Medicine. The experiments were initiated at 8–12 wkof age. IKK      -deficient mice were generated by gene targeting asdescribed previously and maintained at Osaka University (11).   Use of EF to Assess Signaling through TNFR-I and LT        R.   EFswere established as described previously (8, 11). EFs from aly   /   aly   mice, IKK      -deficient mice, and C57BL/6J wild-type mice werecultured in DMEM (GIBCO BRL) supplemented with 10%heat-inactivated FCS (GIBCO BRL), 2 mM l   -glutamine, 100U/ml penicillin, and 100    g/ml streptomycin at a density of 7  10 5  cells per 60-mm culture dish. After incubation with controlmAb Ha4/8 (2  g/ml), agonistic anti-LT  R mAb AC.H6 (2  g/ml; reference 19), or recombinant human TNF (Genzyme,Inc.), whole cell lysates were harvested from the dish with a lysisbuffer containing 1% NP-40 (Sigma-Aldrich) and subjected toWestern blot analysis as described previously (20). The followingAbs were used: rabbit antipeptide Ab directed against I  B   ( sc-371; Santa Cruz Biotechnology), phospho-specific I  B  (cat. no. 9241; New England Biolabs), and polyclonal rabbit Abagainst actin (Biomedical Technologies). For blockade of thephosphatidylinositol-3  -OH kinase (PI[3]K)–Akt pathway, EFswere treated with 1  M wortmannin (Calbiochem) for 30 minbefore stimulation with TNF. For blockade of proteasome activ-ity, EFs were treated with 100  M N  -acetyl-Leu-Leu-norleuci-nal (ALLN; Nacalai Tesque) for 1 h. Use of NF-   B Reporter Assay to Assess Signaling through TNFR-I and LT    R. EFs cultured in a 35-mm culture dish (2   10 5 cells) were transfected with 2  g of a reporter plasmid comprisingthree repeats of the NF-  B site upstream of a minimal thymi-dine kinase promoter and a luciferase gene in the pGL-2 vec-tor (Promega), together with 2  g of  -actin promoter–driven  -galactosidase expression plasmid. Transfected cells were incu-bated in the presence of recombinant human TNF (100 U/ml) or agonistic anti-LT  R mAb AC.H6 for 8 h. After 24 h, the cellswere harvested in PBS and lysed in a luciferase lysis buffer, LC-  (Piccagene). Luciferase assays were performed with a luminome-ter (Lumat LB 9507; Berthold). Activity was normalized to  -galactosidase activity, and data were expressed as the fold acti-vation compared with stimulation by control anti-KLH hamster mAb Ha4/8.  Assessment of LT    R Expression on EFs with Flow-cytometric Anal-ysis. EFs were incubated with anti-LT  R mAb AF.H6 (19) or control mAb Ha4/8. After washing twice with PBS, cells wereincubated with FITC-conjugated anti–hamster IgG mAb (cloneG94-56; PharMingen). Cells were analyzed with a FACSCali-ber™ flow cytometer (Becton Dickinson) with CELLQuest™software. Mouse mAb–producing hybridoma cells were used asnegative control.  Assessment of PP Formation with Whole-Mount Immunohistochem-istry. Whole-mount immunohistochemistry for the detection of PP was performed as described (21). In brief, 2% paraformalde-hyde (pH 7.4)-fixed intestines from 18.5 days postcoitus (d.p.c.)embryos were incubated with mAb against VCAM-1 (PharMin-gen) and then with horseradish peroxidase (HRP)-conjugatedanti–rat Ig (Tago Immunologicals). Color development for bound HRP was done with diaminobenzidine.  Assessment of Association between NIK and IKK   . Proteinswere expressed by transfecting expression constructs with the in-dicated cDNAs into COS-7 cells. Extracts were prepared 30 h af-ter transfection. Immunoprecipitation and Western blot analysiswas performed as described previously (20, 22). Full-sized, Flag-tagged wild-type NIK (6), Flag-tagged aly -type NIK, and Myc-tagged IKK   (22) were expressed in pCR3 vectors (Invitrogen); aly -type NIK cDNA was generated by the introduction of an   on O c  t   o b  er 2  ,2  0 1  5  j   em.r  u pr  e s  s . or  gD  ownl   o a d  e d f  r  om  Published March 5, 2001  633 Matsushima et al.Brief Definitive Report amino acid substitution (G860R) into the COOH-terminal re-gion of human NIK (7) by site-directed mutagenesis. Anti-FlagmAb (clone M2) and anti-Myc mAb (clone 9E10) were fromSigma-Aldrich and Santa Cruz Biotechnology, respectively. Results and Discussion Retained TNFR-I Signaling in NIK-mutant EFs. NIKwas srcinally identified as a kinase that participates inan NF-  B–inducing signaling cascade induced by TNF,CD95, and IL-1 (6). To assess the impact of NIK mutationin TNF responsiveness, we treated EFs from both wild-type and aly  mice with human TNF, which signals onlythrough mouse TNFR-I (23), and assessed NF-  B activa-tion by Western blot analysis. Rapid I  B   degradationconcomitant with the appearance of phosphorylated I  B  was observed with similar kinetics in EFs from both wild-type and aly  mice (Fig. 1 A). 30 min after stimulation withTNF, I  B   started to recover similarly in both wild-typeand aly  mice (Fig. 1 B). I  B   degradation in response toTNF was also indistinguishable between wild-type and aly mice (data not shown). We also tested TNF responsivenessby titrating the TNF concentration between 0.1 and 100U/ml. In this range, TNF sensitivity assessed by I  B   deg-radation, and I  B   phosphorylation was indistinguishablebetween wild-type and aly  mice (Fig. 1 C). Using an NF-  B–binding oligonucleotide probe in an electrophoreticmobility shift assay (EMSA), we also observed a very simi-lar level of NF-  B activation between wild-type and aly mice in TNF-stimulated EFs or TNF-stimulated thy-mocytes (data not shown). Furthermore, IL-1 and IL-6production from TNF-stimulated EFs was indistinguishablebetween wild-type and aly  mice (data not shown). Theseresults demonstrate that NF-  B activation through TNFR-Iis not affected by the NIK mutation.Recently, it was demonstrated that Akt serine–threoninekinase is involved in the activation of NF-  B by TNF (24).Although the results described above do not suggest a rolefor NIK in TNFR-I signaling, it is possible that NIK playsan important role in NF-  B activation in combinationwith Akt. We therefore tested the combined effect of NIKand Akt in the NF-  B–inducing pathway downstream of the TNFR-I. I  B   degradation occurred 10 min after TNF stimulation in wild-type EFs, even in the presence of 1  M wortmannin, a sufficient concentration for blockadeof the PI(3)K–Akt pathway (references 24 and 25; Fig. 1D). This suggests that Akt, the downstream target of PI(3)K, by itself has no major role in NF-  B activation byTNF. Additionally, no obvious effect of wortmannin onI  B   degradation was observed in aly  mouse EFs, indicat-ing that NF-  B activation by TNF can occur even whenthe functions of both NIK and Akt are inhibited. We failedto observe phosphorylation of Akt in response to TNFwhen we probed the same blot with phospho-specific anti-Akt Ab in this experimental setting (data not shown).These results suggest that neither NIK nor Akt is essentialfor NF-  B activation by TNF and that other IKK kinase(s)can substitute for NIK and Akt in NF-  B activation byTNF, at least in EFs. Elucidation of the IKK kinase(s) thatactivates IKK in response to TNF awaits further study. Indispensable Role of NIK and IKK    in NF-   B Activationthrough LT    R. We intended to evaluate the role of NIKin TNFR-I and LT  R signaling with the use of in vivomouse models, rather than introducing enforced gene-expression systems. To this end, we transfected EFs (whichexpress both TNFR-I and LT  R) only with a reporter plasmid that has three repeats of the NF-  B site upstreamof a minimal thymidine kinase promoter and a luciferasegene, and then stimulated the transfected EFs with TNF or agonistic anti-LT  R mAb (AC.H6). EFs from both wild-type and aly  mice showed upregulation of luciferase activityin response to human TNF (Fig. 2 A). The normal level of TNF responsiveness in aly  mice is consistent with the re-sults shown above (Fig. 1). In contrast to TNF stimulation,NF-  B activation in response to agonistic anti-LT  R mAbwas significantly reduced in aly  mouse EFs, indicating thatNIK is involved in LT  R signaling (Fig. 2 A).With the combination of EFs and agonistic anti-LT  RmAb, signals for NF-  B activation assessed by EMSA werenot strong enough to evaluate the role of NIK in NF-  B Figure 1. NF-  B activation inresponse to TNF is retained inEFs from NIK-mutant mice. EFsfrom wild-type mice (WT) and aly  mice (aly) were stimulatedwith TNF (100 U/ml), and cellswere harvested at the indicatedtime points (A and B). I  B   deg-radation (detected by anti-I  B  Ab) and I  B   phosphorylation(detected by phospho-specificanti-I  B   Ab) was assessed byWestern blot analysis. Arrow in-dicates phosphorylated I  B   (A).EFs were stimulated with differ-ent concentrations (Conc.) of TNF ranging from 0.1 to 100U/ml as indicated, and cells wereharvested 7 min after stimulation (C). EFs were stimulated with TNF (100 U/ml) with or without prior treatment with 1  M wortmannin (D). The sameblot was probed with anti-actin Ab (bottom).   on O c  t   o b  er 2  ,2  0 1  5  j   em.r  u pr  e s  s . or  gD  ownl   o a d  e d f  r  om  Published March 5, 2001  634 Role of NIK and IKK   in TNF and Lymphotoxin Signaling activation through the LT  R (our unpublished observa-tion). Involvement of NIK in LT  R signaling was there-fore examined by detection of I  B   phosphorylation in re-sponse to agonistic anti-LT  R mAb. In wild-type EFsstimulated with agonistic anti-LT  R mAb for 1 h, I  B  phosphorylation was easily detected by Western blot analy-sis (Fig. 2 B). In contrast, aly  mouse EFs showed mini-mal, if any, phosphorylated I  B   after LT  R stimulation.Taken together, these results demonstrate that NIK is es-sential for NF-  B activation in LT  R signaling, which ac-counts for the abnormal lymphoid organogenesis in aly mice.We have previously demonstrated that EFs isolated fromIKK  -deficient mice can activate NF-  B in response toTNF and IL-1, suggesting that IKK   is not essential for ei-ther the TNF or IL-1 signaling pathways (11). The dis-pensable role of IKK   in NF-  B activation throughTNFR-I was also confirmed by the detection of phosphor- ylated I  B   in IKK  -deficient EFs after TNF stimulation(Fig. 2 C). Because NIK is essential for LT  R signaling, asdemonstrated above, and NIK has been shown to phos-phorylate IKK   (26), it is important to determine whether IKK   is also essential for LT  R signaling. We thereforetreated EFs from IKK  -deficient mice with agonistic anti-LT  R mAb and assessed NF-  B activation by the detec-tion of phosphorylated I  B  . We found that IKK  -defi-cient EFs showed no I  B   phosphorylation after LT  Rstimulation, suggesting that NIK–IKK   constitutes an im-portant pathway in LT  R signaling (Fig. 2 B). LT  R ex-pression assessed by flow-cytometric analysis with anti-LT  R mAb (AF.H6) was similar among wild-type, aly ,and IKK  -deficient EFs (Fig. 2 D). The basal level of NF-  B activation, assessed by the treatment of EFs with ALLNalone, which blocks the degradation of phosphorylatedI  B   by proteasomes (3), was reduced in aly  mice and re-duced more profoundly in IKK  -deficient mice (Fig. 2 B).The essential role of IKK   in LT  R signaling was ex-amined by investigation of lymphoid organogenesis in Figure 3. Lack of PP de-velopment in IKK  -deficientmice. Embryonic intestines iso-lated from control littermates(A) and IKK  -deficient mice(B) were stained with anti– VCAM-1 mAb. Arrows indicatethe sites of PPs. Original magni-fication,  10. Figure 2. Impaired NF-  Bactivation in response to LT  Rstimulation in NIK-mutant andIKK  -deficient EFs. Wild-typeEFs and aly  mouse EFs weretransfected with NF-  B re-porter plasmids and stimulatedwith TNF or agonistic anti-LT  R mAb. 8 h later, NF-  Bactivation was assessed by themeasurement of luciferase activi-ties. Data were expressed as foldactivation compared with stimu-lation by control mAb, and the re-sults were plotted as the mean  SEM for a total of four indepen-dent experiments. White andblack bars represent wild-typeEFs and aly  mouse EFs, respec-tively (A). EFs from wild-type, aly , and IKK  -deficient mice(IKK   /  ) were stimulated withagonistic anti-LT  R mAb for 1 h, and I  B   phosphorylationwas assessed by Western blotanalysis (B, top). For the assessment of the basal level of NF-  B activation, EFs were treated with ALLN alone. Phosphorylated I  B   is indicated as P.NS, nonspecific bands. The same blot was probed with anti-actin Ab (B, bottom). TNF stimulation induced I  B   phosphorylation in IKK  -deficientEFs as in wild-type and aly  EFs (C). LT  R expression assessed by flow-cytometric analysis with anti-LT  R mAb (D, thick line) was similar among wild-type, aly ,   and IKK  -deficient EFs (D). Anti-KLH mAb Ha4/8 (D, thin line) and mouse hybridoma cells (top left) were used as negative control.   on O c  t   o b  er 2  ,2  0 1  5  j   em.r  u pr  e s  s . or  gD  ownl   o a d  e d f  r  om  Published March 5, 2001  635 Matsushima et al.Brief Definitive Report IKK  -deficient mice. Because IKK  -deficient mice showperinatal death associated with abnormal limb and skindevelopment, lymphoid organogenesis in IKK  -deficientmice was assessed by the development of PP from 18.5d.p.c. embryos. PP formation in control embryos ( n     8)was easily detected by whole-mount immunohistochemis-try with mAb against VCAM-1 (Fig. 3 A); VCAM-1   cellsaccumulate at the site of PP development starting from15.5 d.p.c. and can be used as a stromal marker for PP for-mation (21). In contrast, no PP formation was detected inintestines isolated from IKK  -deficient embryos ( n     7;Fig. 3 B). A similar lack of VCAM-1   cell accumulation inembryonic intestines has been demonstrated in aly  mice(21). This result shows that LT  R signaling is fundamen-tally impaired in IKK  -deficient mice. Together, thesefindings are important in providing clear evidence thatIKK   is involved in cytokine receptor signaling in vivo.It is important to note that abnormal lymphoid organo-genesis in IKK  -deficient mice is not due to defective re-ceptor activator of NF-  B (RANK) signaling, becausemice deficient in RANK have PPs despite their lack of pe-ripheral LNs (27); RANK activates NF-  B by recruitingTRAF6, which has not been observed to associate withLT  R (16, 17). It remains possible, however, that thereexist other undefined NIK–IKK  -activating receptor path-ways involved in lymphoid organogenesis beyond LT  R.The above data strongly suggest that NIK and IKK   to-gether control LT  R signaling with a close mechanistic re-lationship in their pathway. We have therefore reasonedthat impaired LT  R signaling in aly  mice may be due todefective interaction between mutated NIK and IKK  . Toinvestigate this, NIK and IKK   were coexpressed in COS-7cells, and protein interactions were assessed by immuno-precipitation. Association of wild-type NIK with IKK  was easily detected (Fig. 4). In contrast, association of aly type NIK, which corresponds to a G855R substitution inmice, with IKK   was disrupted by the mutation, providingfurther support for the role of NIK–IKK   as an essentialpathway for NF-  B activation in LT  R signaling. Despitethis finding, the possibility remains that aly  mice mighthave a different phenotype from NIK-null mutation mice.It is unclear whether TRAFs mediate all of the signalingactivities of LT  R. In fact, mice deficient in TRAF2, -3,or -5 show LN development (28, 29). In support of thedispensable role of TRAFs in LT  R signaling, recent mu-tational analyses of the cytoplasmic region of LT  R havedemonstrated a TRAF-independent mechanism of NF-  Bactivation through LT  R (30). Consistent with this idea,TRAF–NIK interaction in COS-7 cells as assessed by im-munoprecipitation was not affected by the aly  mutation(our unpublished observation), although the aly  mutationresides in a putative TRAF-binding domain of NIK (31).Elucidation of the molecular mechanisms by which NIKbecomes activated after LT  R stimulation will be criticalfor understanding the biological nature of LT  R signaling. We thank Drs. J.L. Browning, D. Wallach, T. Takemori, and K.Miyazono for providing us with reagents. We thank Drs. K. Honda,M. Shono, S. Noji, H. Ohuchi, T. Matsuzaki, K. Tsuchida, Z.-g.Liu, and C.F. Ware for valuable suggestions. We also thank Ms. M.Kimura for technical assistance.This work was supported in part by Special Coordination Fundsand Grant-in-Aid for Scientific Research of the Ministry of Educa-tion, Culture, Sports, Science, and Technology, the Japanese Gov-ernment, and by the Japan Research Foundation for Clinical Phar-macology. Submitted: 2 November 2000 Revised: 18 January 2001 Accepted: 24 January 2001 References 1.Ghosh, S., M.J. May, and E.B. Kopp. 1998. NF-  B and Relproteins: evolutionarily conserved mediators of immune re-sponses.  Annu. Rev. Immunol.  16:225–260.2.Van Antwerp, D.J., S.J. Martin, I.M. Verma, and D.R.Green. 1998. Inhibition of TNF-induced apoptosis by NF-  B. Trends Cell Biol.  8:107–111.3.Baldwin, A.S. 1996. The NF-  B and I  B proteins: new dis-coveries and insights.  Annu. Rev. Immunol.  14:649–681.4.Maniatis, T. 1997. Catalysis by a multiprotein I  B kinasecomplex. Science. 278:818–819.5.Stancovski, I., and D. Baltimore. 1997. NF-  B activation:the I  B kinase revealed? Cell. 91:299–302.6.Malinin, N.L., M.P. Boldin, A.V. Kovalenko, and D.Wallach. 1997. MAP3K-related kinase involved in NF-  Binduction by TNF, CD95 and IL-1. Nature. 385:540–544.7.Shinkura, R., K. Kitada, F. Matsuda, K. Tashiro, K. Ikuta,M. Suzuki, K. Kogishi, T. Serikawa, and T. Honjo. 1999.Alymphoplasia is caused by a point mutation in the mousegene encoding Nf-  b-inducing kinase. Nat. Genet.  22:74– 77.8.Matsumoto, M., K. Iwamasa, P.D. Rennert, T. Yamada, R.Suzuki, A. Matsushima, M. Okabe, S. Fujita, and M. Yokoyama. 1999. Involvement of distinct cellular compart-ments in the abnormal lymphoid organogenesis in lympho-toxin-  -deficient mice and alymphoplasia ( aly ) mice definedby the chimeric analysis.  J. Immunol.  163:1584–1591.9.Li, Q., D. Van Antwerp, F. Mercurio, K.F. Lee, and I.M.Verma. 1999. Severe liver degeneration in mice lacking theI  B kinase 2 gene. Science.  284:321–325.10.Tanaka, M., M.E. Fuentes, K. Yamaguchi, M.H. Durnin,S.A. Dalrymple, K.L. Hardy, and D.V. Goeddel. 1999. Em-bryonic lethality, liver degeneration, and impaired NF-  Bactivation in IKK-  -deficient mice. Immunity. 10:421–429.11.Takeda, K., O. Takeuchi, T. Tsujimura, S. Itami, O. Adachi, Figure 4. Disruption of inter-action with IKK   by the aly -typeNIK mutation. Protein extractsfrom COS-7 cells transfectedwith the indicated cDNAs wereimmunoprecipitated with anti-Myc mAb and detected with ei-ther anti-Flag mAb (top) or anti-Myc mAb (center). Expression of NIK was verified by Westernblot analysis of total cell lysateswith anti-Flag mAb (bottom). aly -type NIK (aly) has an amino acid substi-tution (G860R) in the COOH-terminal region. Minus (  ) indicatestransfection with empty vectors.   on O c  t   o b  er 2  ,2  0 1  5  j   em.r  u pr  e s  s . or  gD  ownl   o a d  e d f  r  om  Published March 5, 2001
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