Receptor-Specific Signaling for Both the Alternative and the Canonical NF-κB Activation Pathways by NF-κB-Inducing Kinase

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Immunity, Vol. 21, 477489, October, 2004, Copyright 2004 by Cell PressReceptor-Specific Signaling for Both theAlternative and the Canonical NF-B ActivationPathways by NF-B-Inducing Kinaselating protein kinase, IKK2, which occurs within a macro-molecular complex, the IKK signalosome, in associa-tion with a structurally homologous kinase, IKK1, andan adaptor protein, NEMO. IKK2-mediated phosphory-lation of IB leads to its proteasomal degradation and,Parameswaran Ramakrishnan,1 Wangxia Wang,1and David Wallach1,*1Department of Biological ChemistryThe Weizmann Institute of Science76100 Rehovothence, activation of its associated NF-B dimers. Re-Israelcent work has yielded some knowledge of an alterna-tive pathway through which NF-B dimers containingp100 are activated. This activation occurs independentlySummaryof IKK2 or NEMO but is dependent on IKK1. Phosphory-lation of p100 upon activation of this pathway leads toThe NF-B-inducing kinase (NIK) induces proteolyticlimited proteolytic processing in which only the IB-processing of NF-B2/p100 and, hence, the generationhomologous region within p100 is degraded, allowingof NF-B dimers such as p52:RelB but was suggestedthe resulting p52 fragment to translocate to the nucleusnot to signal for the processing of IB. Here, we showin association with some other NF-B proteins (mainlythat although the induction of IB degradation in lym-RelB) (Xiao et al., 2001; Senftleben et al., 2001; Solanphocytes by TNF is independent of NIK, its inductionet al., 2002; Coope et al., 2002; Claudio et al., 2002;by CD70, CD40 ligand, and BLyS/BAFF, which all alsoKayagaki et al., 2002; Dejardin et al., 2002; Yilmaz et al.,induce NF-B2/p100 processing, does depend on NIK2003; Hatada et al., 2003).function. Both CD70 and TNF induce recruitment ofMembers of the tumor necrosis factor/nerve growththe IKK kinase complex to their receptors. In the casefactor (TNF/NGF) receptor family control multiple im-of CD70, but not TNF, this process is associated withmune-defense activities as well as certain develop-NIK recruitment and is followed by prolonged receptormental processes through NF-B activation (Wallach etassociation of just IKK1 and NIK. Recruitment of theal., 1999; Locksley et al., 2001). Most of these receptorsIKK complex to CD27, but not that of NIK, dependsare capable of activating the canonical NF-B pathway.on NIK kinase function. Our findings indicate that NIKIn addition, the lymphotoxin- receptor (LTR), whoseparticipates in a unique set of proximal signalingexpression is restricted to stromal cells, and severalevents initiated by specific inducers, which activatereceptors that occur in lymphocytes, including CD40,both canonical and noncanonical NF-B dimers.the BLyS/BAFF receptor, and (as shown here) CD27,also activate the alternative pathway.IntroductionSignaling for NF-B activation by several receptorsof the family is initiated by their binding to adaptor pro-Inducible activation of the dimeric NF-B/Rel transcrip-teins of the TRAF family. In cells treated with TNF, thetion factors contributes to the regulation of a wide arrayTRAFs have been shown to facilitate, collaborativelyof genes. In mammals, this family of transcription factorswith the adaptor protein RIP, recruitment of the signalo-is comprised of five members: p65 (RelA), RelB, c-Rel,some components to the p55 TNF receptor (Zhang etNF-B1 (which occurs both as a precursor, p105, andal., 2000; Devin et al., 2000, 2001). While searching forin a processed form, p50), and NF-B2 (which occursadditional proteins that participate in NF-B activationboth as a precursor, p100, and as its processed product,by the TNF/NGF receptor family, we identified a TRAFp52). Prior to activation, homo- and heterodimers ofbinding serine/threonine protein kinase related to thethese proteins are retained in the cytoplasm by theirMAP3Ks, which can strongly activate NF-B and wasassociation with inhibitory proteins of the IB family.therefore dubbed NF-B-inducing kinase (NIK) (Mali-The precursor forms of NF-B1 and NF-B2 (p105 and nin et al., 1997).p100, respectively) contain C-terminal IB-homologous Initial studies of the function of NIK in which the conse-inhibitory regions. Dimers containing these NF-B pro- quences of overexpression of this kinase and of its mu-teins are retained in the cytoplasm by virtue of the func- tants in cells were assessed suggested that NIK medi-tion of the IB-homologous regions. Moreover, p105 ates activation of the canonical NF-B pathway and thatand p100 can also associate with dimers of other NF- it does so in response to multiple inducers with manyB proteins and impose cytoplasmic retention on them. different physiological functions. However, later studiesActivation occurs by a variety of different agents, mainly of mice of the aly strain, which express a nonfunctionalthrough induced degradation of the IB proteins or of NIK mutant, as well as of NIK-knockout mice, challengedIB-homologous regions in p105 and p100, and conse- the notion that NIK has a functional role in the activitiesquent translocation of the NF-B dimers to the nucleus of most of these inducers. They suggested, rather, that(Ghosh and Karin, 2002). NIK participates selectively in the activation of NF-BMost of our current knowledge of these processes by a restricted set of ligands that specifically affect theconcerns the mechanisms of activation of a ubiquitous development and function of lymphocytes (Shinkura et al.,NF-B dimer, p65:p50. The critical event in initiating this 1999; Yin et al., 2001). Moreover, based on characterizationcanonical pathway is activation of an IB-phosphory- of cells derived from these mutant mice, it was suggestedthat NIK does not participate at all in the canonical NF-B pathway but, rather, serves exclusively to activate*Correspondence: alternative one (see Pomerantz and Baltimore [2002] cess was blocked by the proteasomal inhibitor MG132(Figures 1G and 1H). In the presence of MG132, CD70for review). Since lymphocytes of NIK mutant mice ex-hibit a highly aberrant pattern of differentiation (Miya- induced accumulation of polyubiquitinated p100 mole-cules in the Ramos cells (Figure 1I), testifying to ubiquiti-waki et al., 1994; Shinkura et al., 1999; Matsumoto et al.,1999; Yamada et al., 2000; Karrer et al., 2000; Fagarasan nation followed by proteasomal processing of p100 inthe generation of al., 2000), we questioned the validity of assessing thesignaling role of NIK in them by comparing NIK activityin lymphocytes derived from NIK mutant mice to that in Depletion of NIK in Ramos Lymphoblastoid Cellslymphocytes of wild-type mice. or Its Replacement with Nonfunctional NIKIn this study, we reevaluated the function of NIK in Mutants Blocks CD27-Induced Activationlymphocytes by assessing the effect of its depletion or of Both the Canonical and the Alternativeinhibition in vitro in cultured cells of lymphoblastoid NF-B Pathwayslines. We confirmed that NIK is not required in lympho- To examine the participation of NIK in activation of thecytes for activation of the canonical pathway by TNF. various NF-B forms by CD27, we attempted to arrestHowever, we found that NIK plays a crucial role in these the synthesis of NIK in Ramos cells by infecting themcells in activation of the alternative as well as of the with a lentiviral vector expressing hairpin short interfer-canonical pathway by CD40 ligand (CD40L) and BLyS/ ing RNA (siRNA) of NIK. Western analysis confirmed thatBAFF. Furthermore, we showed that CD27, a receptor both in transient transfection and in stable expression,of the TNF/NGF family that is expressed mainly in T lym- the siRNA vector effectively arrested the synthesis ofphocytes and memory B lymphocytes, also requires NIK NIK (Figure 2A, top and middle). Treatment of the NIK-for the induction of both the canonical and the alterna- deficient (NIK) cells with CD70 failed to induce translo-tive pathways. Analysis of the proximal signaling events cation of p52 or of RelB to their nuclei (Figure 1D, right).initiated by CD27 revealed that NIK and, in a way that Surprisingly, it also resulted in inability of CD70 to inducedepends on NIKs kinase function, the IKK signalosome IB degradation or nuclear p65 translocation, both mani-are recruited to this receptor. Triggering of the p55 TNF festations of the canonical pathway (Figure 1E, right).receptor induces prolonged receptor recruitment of the Control tests confirmed that the NIK Ramos cellswhole signalosome, whereas triggering of CD27 induces express CD27 at levels comparable to those in normaltransient signalosome recruitment followed by selective Ramos cells and manifest a normal extent of proteinmaintenance of just IKK1 together with NIK in the re- kinase C (PKC) activation upon CD27 triggering (Erlich-ceptor complex, a process that might constitute the man and Howard, 1999) (Figure 2B and its inset), exclud-initiating events in the NIK-dependent activation of the ing a general aberration of CD27 function as a cause ofcanonical and, subsequently, of the alternative NF-B the inability of the NIK cells to manifest activation ofpathways by CD27. NF-B by this receptor.To verify that this inability indeed results from theabsence of NIK, we restored NIK expression in the NIKResultscells (Figure 2A, bottom). Cells reconstituted with wild-type NIK regained the ability to respond to CD70 withCD27 Induces Processing of Both IBboth an increase in nuclear p52 and a transient decreaseand NF-B2/p100 in Lymphocytesin IB (Figure 2C). On the other hand, expression ofWe recently found that SIVA, a protein previously shownan enzymatically inactive NIK mutant (KD-NIK) in theto associate with the TNF/NGF receptor family memberNIK cells failed to restore their response to either ofCD27, binds to NIK and controls its function (Ramakrish-the CD70 effects. Expression of NIK with a missensenan et al., submitted). This finding led us to suspectmutation corresponding to that of aly mice (aly-NIK)that NIK plays a role in CD27 signaling. We thereforeprompted spontaneous generation of nuclear p52 butexamined the effect of CD27 on the alternative NF-Bdid not restore the ability of the NIK cells to respondactivation pathway in which NIK function has been impli-to CD70 (Figure 2D).cated.Treatment of human peripheral blood mononuclearcells (PBMC) with CD70 induced rapid decrease of IB, Depletion of NIK in Lymphoblastoid Cells BlocksActivation of Both the Canonical and the Alternativeindicating that CD27 can trigger activation of the canoni-cal NF-B pathway (Figures 1A and 1B). In resting NF-B Pathways by CD40L and BLyS/BAFF,but Not Activation of the Canonical PathwayPBMC, CD70 also induced translocation of p52 as wellas of RelB to the nuclei, indicating that CD27 also stimu- by TNF, PMA, or Endoplasmic Reticulum StressStudies of the effects of various ligands of the TNF familylates the alternative NF-B pathway in these cells (Fig-ure 1A). on NF-B activation in lymphocytes have demonstratedactivation of both the canonical and the alternative NF-CD70 also potently induced IB degradation as wellas nuclear translocation of RelB and p52 in cells of the B pathways by CD40L and BLyS/BAFF. On the otherhand, TNF, though capable of effectively triggering theRamos and (to a lesser extent) of the Raji lymphoblastoidlines (Figure 1C and left panels in Figures 1D and 1E). canonical pathway, appears unable to trigger the alter-native pathway (Matsushima et al., 2001; Yin et al., 2001;This translocation was associated with an increase inthe total cellular content of p52 (Figure 1F). Both in Dejardin et al., 2002; Yilmaz et al., 2003). It induces onlya slight increase in nuclear p52, much less than thatthe Ramos cells and in HEK293T cells transfected withCD27, CD70 also induced generation of p52 from p100 induced by ligands such as CD40L (Yilmaz et al., 2003;Derudder et al., 2003), probably through stimulating themolecules encoded by transfected cDNA, and this pro-NIK-Mediated Canonical NF-B Pathway Activation479Figure 1. CD27 Induces Both the Canonical and the Alternative Pathways in Lymphocytes, and Their Induction Is Prevented by Arrest ofNIK SynthesisEffects of CD70 on the cytoplasmic levels of IB and on the cytoplasmic and nuclear levels of NF-B proteins in (A) resting PBMC, (B) PBMCstimulated for 48 hr with PHA (1 g/ml) followed by a 12 hr resting period without PHA, (C) Raji cells, and (D) and (E) Ramos cells (normaland NIK). Effects of CD70 (F) on the total endogenous cellular levels of p100 and p52 in Ramos cells and on the total levels of p100 andp52 encoded by transiently expressed cDNA for p100 fused at its N terminus to the myc tag (myc-p100) in (G) Ramos cells and in (H) HEK293Tcells that transiently express CD27. The effect of transiently expressed myc-NIK on p100 processing in the HEK293T cells served as a positivecontrol. The effect of the proteasomal inhibitor MG132 on p52 generation in the myc-p100-transfected cells was assessed in the Ramos cellsby applying it for the last 2 hr of a 4 hr period of stimulation with CD70 and in the HEK293T cells by applying it throughout the 4 hr period ofincubation with CD70. (I) Effect of CD70 on p100 ubiquitination in Ramos cells, assessed in the presence and in the absence of the proteasomalinhibitor MG132. (NS, nonspecific)synthesis of p100 (de Wit et al., 1998) and releasing Supplemental Figure S1 at with no visible changep52:p65 dimers held latent in the cytoplasm by IB(Coope et al., 2002). TNF also induces synthesis of RelB in the cellular levels of IB (see Hatada et al. [2003] for asimilar effect of BLyS/BAFF on primary B lymphocytes).(Bren et al., 2001), part of which accumulates in thenucleus, apparently through induced nuclear transloca- CD40L and BLyS/BAFF also induced marked increasesin nuclear p52 as well as in RelB (left panels of Figurestion of p100:RelB dimers (Yilmaz et al., 2003).The responses of the Ramos cells to CD40L, BLyS/ 3A and 3C). TNF induced nuclear translocation of RelB,but only a slight increase in nuclear p52 (Figure 3E, left).BAFF, and TNF in this study were consistent with thosereports. All three ligands induced activation of the ca- Assessment of coimmunoprecipitation of various NF-B proteins from nuclear extracts of the Ramos cellsnonical pathway, as reflected in rapid nuclear transloca-tion of p65 (left panels of Figures 3B, 3D, and 3F). This confirmed that whereas CD70 enhances nuclear accu-mulation mainly of RelB:p52 (and of RelB:p100), TNFtranslocation was associated with a decrease in IB(left panels of Figures 3B and 3F) or, in the case of BLyS/ induces increased nuclear levels of RelB:p100 withoutincreasing RelB:p52 (Figure 3G).BAFF, phosphorylation of IB (Figure 3D, left) as wellas increased DNA binding of the p65:p50 dimer (see On further examining the impact of the arrest of NIKImmunity480Figure 2. siRNA-Mediated Ablation of NIK Synthesis in Ramos Cells and Its Restoration by Transfection of Wild-Type or Mutant NIK cDNA(A) Suppression of NIK synthesis by expression of NIK siRNA. Top: suppression of the synthesis of transiently expressed myc-tagged NIK inHEK293T cells by cotransfection of pSUPER-NIK at ratios of 1:1, 1:2, 1:3, and 1:5. Middle: analysis of NIK (and, for comparison, of IKK1) inRamos cells constitutively expressing lentiviral-pSUPER-NIK (NIK cells) in comparison to Ramos cells transduced with lentiviral-GFP ascontrol. Bottom: reconstitution of NIK expression in the NIK Ramos cells by constitutive expression of myc-tagged NIK.(B) CD70-induced PKC activation in normal (black bars) and NIK (white bars) Ramos cells. Bars represent the means of triplicate tests. CD27levels in normal and NIK Ramos cells are shown in the inset.(C and D) CD70-induced degradation of IB and nuclear accumulation of p52 in (C) NIK cells reconstituted with wild-type NIK and (D)normal Ramos cells (control), NIK cells, and NIK cells reconstituted with KD-NIK and with aly-NIK. Western blot analysis of the expressionof the myc-tagged NIK mutants is shown at the bottom of (D). WB, Western blot; IP, immunoprecipitation.expression on the response of the Ramos cells to the NIK depletion also had no effect on IB degradationin response to thapsigargin, an inhibitor of the sarco-three ligands, we found that as with the effects of CD70,all effects of CD40L and BLyS/BAFF on NF-B activa- endoplasmic reticulum Ca2-adenosine triphosphatasethat triggers activation of NF-B through induction oftionthose ascribed to the canonical pathway as wellas those related to the alternative pathwaywere ar- endoplasmic reticulum stress (Pahl and Baeuerle, 1996),or to 4-phorbol-12-myristate-13-acetate, an agent acti-rested in the NIK cells (right panels of Figures 3A3D).Similarly, transfection of cells of the Raji lymphoblastoid vating NF-B through stimulation of PKC (Sen and Balti-more, 1986) (Figure 3I).line with NIK siRNA resulted in a marked decrease inactivation of both the canonical and the alternative NF-B pathways by CD40L (Figure 3H). In contrast, the NIK-Dependent Activation of the Canonical PathwayCan Be Blocked by Introduction of an Anti-NIKinduction of IB degradation by TNF and the resultingnuclear translocation of p65, as well as the induction of Antibody into CellsBesides the loss of responsiveness to the effects ofnuclear translocation of p100 and RelB by TNF, occurredin the NIK-deficient cells just as effectively as in the CD70, CD40, and BLyS/BAFF on NF-B, the NIK-defi-cient Ramos cells also displayed some constitutive al-cells expressing NIK (right panels of Figures 3E and 3F).NIK-Mediated Canonical NF-B Pathway Activation481Figure 3. Ablation of NIK Synthesis in Lymphoblastoid Cells Compromises Activation of Both the Canonical and the Alternative NF-B Pathwaysby CD40L and BLyS/BAFF but Does Not Affect Activation of the Canonical Pathway by TNF, Thapsigargin, or PMA(AF; I) Kinetic analysis of the responses of normal and NIK Ramos cells to the indicated ligands. Thapsigargin was applied at 5 M asdescribed (Leonardi et al., 2002) and PMA at 100 ng/ml. For detection of phosphorylated IB, cells were pretreated for 2 hr with theproteasomal inhibitor MG132.(G) An immunoprecipitation analysis of the NF-B complexes that occur in the nuclei of Ramos cells 15 min and 4 hr after application of TNFor CD70 to the cells.(H) The response of Raji cells to CD40 ligand and its modulation by siRNA-mediated suppression of NIK synthesis.(J) A comparison of basal levels of the various NF-B proteins in control and NIK Ramos cells.terations of their basal NF-B protein levels (Figure 3J). peptide corresponding to the phosphorylated NIK acti-vation loop (-pNIK) effectively blocked the in vitro ki-Expression of those NF-B proteins whose levels werefound to decrease is known to depend in part on NF- nase function of NIK (Figure 4A). We therefore attemptedto introduce these antibodies into the Ramos cells as aB activation. This decrease is reminiscent of that ob-served in lymphocytes of aly mice (Yamada et al., 2000). means of blocking NIK function. As shown in Figure 4B,treatment of the Ramos cells with a protein transfectionIt probably reflects arrest of the effects of some auto-crine mediators that continuously cause mild activation kit allowed effective, though transient, introduction ofimmunoglobulins into the cells. Introduction of theof NF-B in a NIK-dependent manner in the Ramos cells.To exclude the possibility that the differences in ligand -pNIK antibodies into the Ramos cells had no effecton the induction of IB degradation by TNF. However,effects observed between the NIK-deficient and the nor-mal cells are secondary to such constitutive alterations the antibodies effectively blocked the induction of IBdegradation by CD70 or CD40L (Figure 4C). On examin-occurring in the cells as a consequence of prolongedNIK deficiency, we sought a way to instantaneously ab- ing cells of several other lymphoblastoid lines, we foundthose of the BJAB line to be just as amenable to transfec-late NIK function in cells. NIK activation requires phos-phorylation of its activation loop (Lin et al., 1998). A tion with the antibodies as the Ramos cells. CD40L (butnot CD70) induced IB degradation in these cells, andmonoclonal antibody that we raised against a phospho-Immunity482as well as p65 nuclear translocation in these cells inresponse to either TNF or CD70 but had no effect on theCD70-induced nuclear accumulation of p52 (Figure 5A).The two NF-B activation pathways also differ withregard to the IKK species that mediate them. IB phos-phorylation is mediated by IKK2 and was reported tooccur also in cells devoid of IKK1 (Ghosh and Karin,2002). In contrast, the phosphorylation that targets p100for processing depends on IKK1. Consistently, siRNA-mediated suppression of the synthesis of this kinasein Ramos cells decreased the CD70-induced nuclearaccumulation of p52. Surprisingly, IKK1 depletion alsocompromised the induction of IB degradation andof p65 nuclear translocation by CD70, suggesting thatsome unique, IKK1-dependent mechanism participatesin activation of the canonical pathway by CD70 (Fig-ure 5B).Both TNF and CD70 were found to enhance the in vitrokinase function of the IKK signalosome as manifestedby the effectiveness of the self-phosphorylation of theIKKs and phosphorylation of their associated NEMO(Figure 5C) as well as by the effectiveness of the in vitroIKK-mediated phosphorylation of GST-IB (Figure 5D).However, whereas activation of the signalosome by TNFwas not affected by NIK deficiency, the effect of CD70Figure 4. Introduction of Antibodies against the Phosphorylated Ac- on the signalosome was aborted in NIK-deficient cellstivation-Loop of NIK (p-NIK) into Ramos and BJAB Cells Blocks (Figures 5C and 5D).the Induction of IB Degradation by CD70 and CD40L, but NotThese findings suggest that both TNF and CD70 medi-by TNFate phosphorylation of IB by activation of the IKK(A) In vitro inhibitory effect of p-NIK on NIK kinase function. Self-signalosome complex, but that the activation in re-phosphorylation of myc-NIK immunoprecipitated from transientlysponse to the two ligands occurs via different mecha-transfected HEK293T cells was determined in the presence of in-creasing amounts of p-NIK (0.5 g, 1.0 g, and 2 g/25 l kinase nisms, with only the CD70 effect being dependent onreaction volume) or with control IgG (2 g). Autoradiogram of the NIK and on IKK1 function.phosphorylated protein is compared to Western blot analysis of NIKlevels in the same samples.NF-B Activation by CD27 Is Associated with Receptor(B) Introduction of antibodies into Ramos cells with a protein-trans-Recruitment of the Signalosome and of Just IKK1fection reagent. Uptake of FITC-tagged immunoglobulin by theRamos cells was assessed by fluorescence microscopy at the indi- In a Manner Dependent on NIK Kinase Function.cated times after transfection. It Is Also Associated with Recruitment of NIK(C) Assessment of the effect of introduction of p-NIK antibody (or, Itself to the Receptor Independentlyfor comparison, of mouse IgG or of -NIK81 that had no effect on of Its Kinase FunctionNIK kinase activity in vitro) on the induction of IB degradation byThe earliest known event in activation of the signalo-CD70, CD40L, and TNF in Ramos cells and (D) by CD40L in BJABsome by TNF is its recruitment to the p55 TNF receptor,cells. The ligands were applied 4 hr after transfection of the antibody(10 g/ml of transfection volume) was started. Results are represen- a process facilitated by recruitment of the adaptor pro-tative of three experiments, each performed in duplicate. teins RIP and TRAF2 to that receptor. CD70 does notinduce recruitment of RIP to CD27. However, as shownin Figure 6A, it does induce recruitment of TRAF2. Inter-this induction was significantly reduced when the estingly, the TRAF2 molecules recruited to CD27 dis--pNIK antibodies were introduced (Figure 4D). These played extensive electrophoretic pattern modification,findings further confirm that although NIK does not par- probably corresponding to ubiquitination. CD70 also in-ticipate in activation of the canonical pathway by TNF, duced recruitment of the signalosome. Recruitment ofits function in lymphocytes is crucial for activation of the signalosome to the TNF receptor was prolonged,the canonical pathway by some other ligands. whereas association of the three components of thesignalosome with CD27 could be observed only for afew minutes. At later time points, the amounts of IKK2NIK Is Required for Activation of the IKK KinaseComplex by CD70 but Not for Its Activation by TNF and NEMO in the CD27 complex sharply decreased.Surprisingly, however, the amounts of IKK1 associatedThe critical event in the canonical pathway is stimulationof the IKK signalosome, a complex formed between with the receptor remained high for a long time (Figure6A, left). Similar selective maintenance of IKK1 in associ-the IB-kinases and NEMO. On the other hand, sinceactivation of the alternative pathway is independent of ation with CD27 after CD70 treatment was also observedin PBMC (Figure 6B).NEMO, it is believed to be mediated through activationof kinase complex(es) distinct from the signalosome. Both TNF and CD70 also induced recruitment of allthree components of the canonical NF-B complexConsistently, siRNA-mediated depletion of NEMO fromthe Ramos cells greatly decreased IB degradation (IB, p65, and p50). Although p100 processing is in-NIK-Mediated Canonical NF-B Pathway Activation483Figure 5. CD70 Induces Stimulation of the IKK Signalosome and NF-B-Activation in a NIK- and IKK1-Dependent Manner(A and B) Effects of siRNA-mediated ablation of synthesis of (A) NEMO and (B) IKK1 on the responses of Ramos cells to CD70 and TNF.(C) Self-phosphorylation of the IKKs and phosphorylation of NEMO in an in vitro kinase test of the IKK signalosome 15 min after applicationof CD70 or TNF to normal and to NIK Ramos cells.(D) In vitro IB phosphorylation activity of the IKK signalosome in Ramos cells, isolated both in (C) and in (D) by immunoprecipitation withantibodies to IKK1, compared to cellular IB levels at various times after application of CD70 or TNF to normal and to NIK Ramos cells.duced by CD70 and not by TNF, receptor recruitment to CD27 depends on the NIK kinase function, recruit-ment of NIK itself to the receptor seems to occur inde-of p100 was induced by TNF, but not by CD70 (Figure6A, right). This recruitment was previously suggested to pendently of its enzymatic activity.occur through binding of the death domain in p100 tothat in the p55 TNF receptor-associated adaptor protein DiscussionTRADD and appears to serve not to activate NF-Bbut to amplify death induction by this receptor through The restricted pattern of biological functions in whichNIK participates appears to be at variance with whatcaspase-8 activation (Wang et al., 2002).In NIK cells, recruitment to the p55 TNF receptor might be anticipated from the wide-ranging impact thatthis enzyme can have on NF-B activation. NIK can bindoccurred just as effectively as in the wild-type cells (Fig-ure 6C, right). In contrast, recruitment of the signalo- IKK1 and activate it (Regnier et al., 1997; Ling et al.,1998). It can also bind p100 and phosphorylate it (Xiaosome components to CD27 was completely abolished(Figures 6C and 6D, left panels). Introduction of wild- et al., 2001), and, by activating IKK1, it facilitates thephosphorylation of p100 by the latter kinase (Senftlebentype NIK, but not of kinase-dead NIK, to the NIK cellsreinstated the recruitment in response to CD70 (Figure et al., 2001). Activation of IKK1 by NIK can also lead tophosphorylation and activation of IKK2 by IKK1 and,6D, middle and right).CD70, but not TNF, also induced recruitment of NIK consequently, to IKK2-mediated phosphorylation of IB(OMahony et al., 2000). NIK thus seems to be capableto its receptor. This recruitment could be observed bothin cells expressing the wild-type enzyme and in those of activating both the alternative and the canonical NF-B activation pathways. The present study shows thatthat expressed its kinase-dead mutant (Figure 6D). Thus,while the recruitment of the signalosome components NIK indeed participates in the activation of both path-Immunity484Figure 6. CD70 Induces Recruitment of the IKK Signalosome Followed by Selective Recruitment of IKK1 to CD27 in a Way that Depends onNIK Kinase Function as Well as Recruitment of NIK Independently of Its Kinase Function(A) Kinetic analysis of recruitment of TRAF2 and RIP, the components of the IKK signalosome (IKK1, IKK2, and NEMO), the components ofthe canonical NF-B complex (IB, p65, and p50), and p100 to CD27 and the p55 TNF receptor complexes in Ramos cells at various timepoints after CD70 or TNF application, compared to composition of the cytoplasmic IKK signalosome (isolated, prior to stimulation, by the useof an antibody to NEMO; right) and to the cellular levels of IB (bottom).(B and C) In vitro IB phosphorylation activity and the presence of IKK signalosome components in receptor complexes and cytoplasmicsignalosomes. (B) CD27 complexes and signalosome preparations isolated from resting PBMC before stimulation and after stimulation withCD70 for 20 min. (C) Receptor complexes associated with CD27 (left) and the p55 TNF receptor (right) isolated from control and NIK Ramoscells before stimulation and after stimulation with CD70 or TNF for 20 min.(D) Comparison of the kinetics of recruitment of NIK and IKK1 to CD27 and to the p55 TNF receptor complexes at various times after applicationof CD70 or TNF to NIK Ramos cells and to NIK cells replenished with wild-type or enzymatically inactive NIK mutant (KD-NIK).NIK-Mediated Canonical NF-B Pathway Activation485Figure 7. Speculative Model of the Mechanisms Initiating NF-B Activation by TNF and CD70The figure presents an outline of the molecular events leading from activation of the p55 TNF receptor by TNF (left) and of the CD27 receptorby CD70 (right) to NF-B activation. TNF induces NIK-independent recruitment of all three core components of the signalosome to its receptorin a way that depends on interaction of these components with TRAFs and RIP. This recruitment initiates the canonical pathway only. CD70induces recruitment and massive ubiquitination of TRAF2, but not of RIP. It also induces the recruitment of NIK and, in a way that dependson the kinase function of NIK, induces also the recruitment first of the whole signalosome and then of only IKK1 to CD27. Recruitment of thewhole signalosome to this receptor and the consequent activation of IKK1 in it by NIK might be the mechanism for initiation by this receptorof the canonical pathway, and the subsequent recruitment of IKK1 might be the mechanism for initiation by this receptor of the alternativepathway. Broken lines pointing at p100 or RelB represent their induction upon activation of the canonical pathway by TNF and CD70 and theconsequent translocation of the p100:RelB complex to the nucleus.ways, but that this participation is inducer specific. It dependent activation by CD70 might provide a clue asto how the participation of NIK in activation of the canon-was detectable only in response to ligands capable ofstimulating both the canonical and the alternative NF- ical pathway is restricted to the effect of specific in-ducers. Activation of the canonical pathway by CD70,B activation pathways and not in response to an in-ducer such as TNF, which (at least in the cells studied like its activation by TNF, is associated with recruitmentof the signalosome components IKK1, IKK2, and NEMOhere) can stimulate only the canonical pathway. Someof the receptors that signal for NF-B activation can do to the receptor complex. Unlike TNF, however, CD70also induces recruitment of NIK to its receptor. More-so by more than one mechanism, with each mechanismoperating in a different region of the receptors intracel- over, NIK deficiency, while not affecting recruitment ofthe signalosome to the TNF receptor, prevented its re-lular domain and leading to activation of a different setof NF-B dimers (e.g., Coope et al. [2002] and Saito et cruitment to CD27.Expression of kinase-dead NIK or the NIK aly mutantal. [2003]). NIK might thus turn out to function in a waythat is even more restricted by participating in one of failed to restore the response to CD70. This failure isprobably due in part to the inability of these mutantsthe mechanisms of signaling by a receptor, and not inthe others, and thus contributing to the function of this to phosphorylate IKK1. The finding that IKK1 depletioncompromised the induction not only of the alternativereceptor only in particular cell types and differentiationstates. Some of the data obtained while assessing the pathway but also of the canonical pathway by CD27 isindeed consistent with the suggestion that in the caseimpact of the NIK aly mutation or of overexpression ofits kinase-dead mutant on NF-B activation are indeed of this receptor, NIK-mediated IKK1 phosphorylation iscrucial for activation of both pathways. Our findingsconsistent with the notion that this kinase participatesin activation of the canonical pathway in a way that is indicate, however, that the kinase function of NIK con-tributes not only to phosphorylation of IKK1 but alsospecific both to certain cell types and to a particularreceptor (Garceau et al., 2000; Smith et al., 2001). to recruitment of the signalosome components to theCD27 complex.Our comparison of the initiating events in the NIK-independent activation of NF-B by TNF and in the NIK- CD27 associates with members of the TRAF adaptorImmunity486relevant expression constructs (see below). TNF, a gift from Dr. The ability of NIK to bind to these adaptor pro-Adolf, Boehringer Institute, Vienna, Austria, was applied to cells atteins might assist its recruitment to CD27. However,a concentration of 100 ng/ml. MG132 was purchased from Calbio-TRAFs also associate with the TNF receptor, to whichchem, and thapsigargin, 4-phorbol-12-myristate-13-acetate, andNIK is not recruited. In fact TRAFs associate with all phytohemagglutinin (PHA) from Sigma. [32P]ATP and Ficoll-Paquemembers of the TNF/NGF family, and yet only some were from Amersham Biosciences, G418 from Life Technologies,of these receptors are known to involve NIK in NF-B and the MACSelect Kk.II transfected cell selection kit was from Mil-tenyi Biotec.activation. Thus, there must be other determinants, inaddition to the association of TRAFs with a receptor, thatAntibodiesdictate NIK recruitment to this receptor and activation ofAnti-p52 antibody was purchased from Upstate Biotechnologies; anti-NF-B by the receptor in a NIK-dependent manner.bodies against p65, p52, p50, RelB, c-Rel, CD27, TRAF2, Lamin A/C,Recruitment of the signalosome along with NIK to Oct-1, NEMO, and IKK1 (M280 and H744) from Santa Cruz Biotechnol-CD27 is followed shortly by a sharp decrease in both ogy; anti-RIP, anti-IKK2, and anti-NEMO antibodies from BD-Phar-IKK2 and NEMO in the receptor complex. Both IKK1 mingen; anti-FLAG, anti-FLAG M2-beads, and anti- actin from Sigma;anti-ubiquitin from Covance, anti-IB from Transduction Labora-and NIK, however, remain associated with the receptortories; and anti-phospho-IB from Cell Signaling Technology. Afor a long time. The latter form of the CD27 complexmonoclonal antibody against the phosphorylated NIK activation loopprobably serves to initiate the alternative pathway pre-(-pNIK) was raised by immunizing mice with a KLH-coupled peptideviously shown to depend on the function of IKK1 andcorresponding to the NIK activation loop in which Thr559 was phos-NIK but not of IKK2 or NEMO (see the hypothetical model phorylated. The anti-NIK monoclonal antibody NIK-81 was raisedin Figure 7). by immunizing mice with a KLH-coupled peptide corresponding toLike the recruitment of the whole signalosome to a sequence within the NIK kinase domain (RLGRGSFGEVHRMEDK;amino acids 405420). Both anti-NIK and anti-myc (clone-9E10)CD27, the subsequent preferential association of IKK1monoclonal antibodies were purified from mouse ascitic fluids onwith the receptor cannot be observed in cells devoid ofaffinity columns to which their corresponding peptides wereNIK or in cells expressing nonfunctional NIK mutants. tempting to speculate that these two stages in therecruitment are mechanistically linked, thereby ensuringCellsthat initiation of the NIK-dependent alternative activa- PBMC were isolated from buffy coat samples by Ficoll-Paque gradi-tion pathway is coupled to that of the canonical pathway. ent centrifugation at 450 g. The PBMC, as well as cells of theIndeed, whereas there are many stimuli that activate the human B lymphoblastoid lines of Burkitt lymphoma origin, Ramos,Raji, and BJAB, were cultured in RPMI medium, and HEK293T cellscanonical pathway alone, all agents found to date toin Dulbeccos modified Eagles medium supplemented with 10%activate the alternative pathway are also known to acti-fetal calf serum, 100 U/ml penicillin, and 100 g/ml streptomycin.vate the canonical one. This dual ability has clear physio-logical significance. The NF-B dimers generated by theExpression Vectorstwo pathways recognize different DNA sequence motifsThe cDNAs for the extracellular domains of mCD70, hCD40L, andand, thus, by affecting different promoters, can control hBLyS/BAFF were PCR-amplified from ESTs and cloned in fusionthe expression of different genes (Perkins et al., 1992; with a leader sequence, a modified leucine zipper, and FLAG tagLin et al., 1995; Dejardin et al., 2002; Hoffmann et al., (Fanslow et al., 1994) into pcDNA3 (Invitrogen). pCS3MTNIK andpCS3MT-NIK KK429430AA, expression vectors for wild-type and2003). However, activation of both pathways by thekinase-dead NIK fused N-terminally to the myc tag, were obtainedsame inducers allows for functional interactions be-from Dr. Michael Kracht, Germany. N-terminally myc-tagged p100tween the genes regulated by the two pathways. More-(myc-p100) was constructed by PCR with pCMVSPORT6-p100 (In-over, it allows the two signaling pathways to affect eachvitrogen) as the template and then cloned into pcDNA3. The expres-others activation. Thus, by triggering the synthesis of sion vector encoding human CD27 (pcDNAGS-hCD27) was pur-p100 as well as of RelB (de Wit et al., 1998; Bren et al., chased from Invitrogen. pEGFP was purchased from Clontech.2001), which together form the precursor dimer affected Human NIK with a mutation (G860R) corresponding to that of themouse aly mutation (G855R) (Shinkura et al., 1999) was generatedby the alternative pathway, the canonical pathway po-with a site-directed mutagenesis kit (Stratagene). (The sequencestentiates the alternative pathway. Conversely, sinceinof the oligonucleotides used for site-directed mutagenesis, PCR,addition to binding to RelBp100 also associates withand suppression of protein synthesis by RNA interference are listedthe dimers controlled by the canonical pathway and thusin the Supplemental Data.)blocks their function, its processing by the alternativepathway helps to perpetuate the activation of the canon- Plasmid Transfections, Immunoblotting,ical pathway. and ImmunoprecipitationsTo allow for coordination of the activation mecha- For detection of endogenous NIK, Ramos cells (24 108; 1 108cells/ml) were lysed and NIK was immunoprecipitated from the ly-nisms for the two functionally distinct yet interactingsate with affinity-purified mouse anti-NIK antiserum coupled to pro-sets of NF-B dimers by the same inducer, they needtein G-Sepharose beads. The precipitated protein was detected byto be controlled by both common and distinct regulatoryWestern blotting with the NIK-81 antibody and the SuperSignal Westelements. Previous studies have disclosed several com-Femto Chemiluminescent Detection Kit (Pierce).ponents unique to the alternative or to the canonical For immunoprecipitation of the CD27-receptor complexes, ly-pathway. Our study shows that NIK can serve as a com- sates of CD70-treated cells, prepared as described for the kinasemon proximal signaling molecule in situations where tests below, were incubated for 4 hr at 4C with 25 l of 50% M2-FLAG agarose beads per ml of lysate. The TNF-receptor complexboth mechanisms of NF-B activation are triggered.was precipitated as described (Zhang et al., 2000). The signalosomefrom cell lysates was immunoprecipitated through IKK1 or NEMOExperimental Proceduresas described (Dejardin et al., 2002).For immunoprecipitation of the NF-B proteins with anti-RelBReagentsantibody, nuclear extracts from 1020 106 cells were diluted tomCD70, hCD40L, and hBLyS/BAFF were produced by large-scaletransfection of human embryonic kidney (HEK) 293T cells with the achieve the following composition: 0.5% NP-40, 10 mM HEPES (pHNIK-Mediated Canonical NF-B Pathway Activation4877.9), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol For exploration of the effectiveness of the pSUPER-NIK, HEK293Tcells were transfected by the calcium phosphate precipitation(DTT), 1 mM PMSF, and 1 complete protease inhibitor cocktail.The immunoprecipitated proteins were resolved on 4%12% Bis- method (Sambrook et al., 1989) with this plasmid together withpCS3MT-NIK. To express the pSUPER-NIK constitutively in theTris NuPAGE gels (Invitrogen), transferred to a nitrocellulose mem-brane, and analyzed with the enhanced chemiluminescence (ECL) Ramos cells, we used a lentiviral vector as described (Lois et al.,2002). The cassette of H1 promoter NIK RNAi was transferredWestern blotting detection system (Amersham).For ligand activation of lymphoid cell lines and PBMC, typically from the pSUPER vector to the GFP-expressing FUGW lentiviralvector. Transduced cells were sorted by FACS for GFP expression1 106 cells were stimulated for the indicated time periods withthe relevant ligands, and nuclear and cytoplasmic extracts were (FACS Vantage, Becton-Dickinson). The sorted cells continued tomanifest expression of GFP and deficiency of NIK for months.prepared as described (Schreiber et al., 1989) and analyzed by West-ern blotting. Raji cells were depleted of NIK by nucleofection (solution V, pro-gram S18) with a mixture of 4 g of NIK siRNAs (3 g of siRNAmyc-p100 was transiently expressed in Ramos cells by nucleofec-tion with the nucleofector kit T and program G10 (Amaxa biosys- #1511 and 1 g of #NC3; Ambion) and 1 g of the pMACS Kk.IIplasmid (for selection of the transfected cells). Cells nucleofectedtems). Cells were stimulated with CD70 16 hr after nucleofection forthe indicated time periods, and this was followed by Western analy- with lamin A siRNA served as a control. Cells were selected bymagnetic sorting 36 hr after transfection, according to the manufac-sis of the whole-cell lysates.For assessment of CD27-induced processing of ectopically ex- turers instructions (Miltenyi Biotec). Sorted cells were allowed togrow for a further 36 hr and were then stimulated with CD40L forpressed myc-p100 in HEK293T cells, the cells were cotransfectedwith myc-p100 and human CD27 expression vectors (or, as a posi- the indicated times.Ramos cells were depleted of NEMO by nucleofection (solutiontive control, with myc-NIK vector). Twenty-four hours after CD27transfection CD70 was applied for 4 hr with or without MG132 (25 V, program G16) with a mixture of the relevant pSuper construct(1.5 g) and NEMO-specific Smartpool siRNA (3.5 g; Dharmacon).M in all experiments), and this was followed by lysis and West-ern blotting. They were depleted of IKK1 by nucleofection (solution V, programG10) with a mixture of three pSUPER-RNAi constructs for IKK1Ubiquitination of p100 was assessed as described (Mordmuller etal., 2003). The p100 was immunoprecipitated from CD70-stimulated (1 g each) and a synthetic siRNA (2 g). As controls, Ramos cellswere nucleofected with pSUPER containing a scrambled siRNA se-cells with a mixture of two anti-p100 antibodies (C5, Santa Cruz andanti-NFB2, Upstate). MG132 was applied for the last hour of the 4 quence plus lamin A siRNA. Stimulation with CD70 or TNF was done30 hr after nucleofection in NEMO-depleted cells and 72 hr afterhr stimulation. For Western analysis of the whole-cell lysate, cellswere boiled in 1 LDS sample buffer (Invitrogen). nucleofection in IKK1-depleted cells.For reintroduction of NIK and its mutants into the NIK cells, thecells were nucleotransfected with the NIK-expressing vector with Protein Kinase C AssayPKC activity in control and NIK-deficient Ramos cells after CD70the Nucleofector Kit V, according to the manufacturers instructions(Amaxa Biosystems). Briefly, 2 106 NIK cells were nucleofected stimulation was measured by the Signatect Protein Kinase C AssaySystem (Promega). The enzymatic activity of PKC was determinedwith 4 g of NIK plasmid and 1 g of pEGFP in solution V in theNucleofector device with program S18 or G10. Cells stably express- by subtracting the values obtained when assaying in the absenceof phospholipids from those obtained in their the transfected proteins were selected for growth in G418(1 mg/ml).AcknowledgmentsAntibody TransfectionAntibodies were transfected into cells in serum-free medium with We are grateful to Dr. Reuven Agami for the pSUPER vector, Dr.Yuri Goltsev for the construct for expression of lucine-zipper fusionthe Pro-ject Protein Transfection Reagent Kit (Pierce), according tothe manufacturers instructions. Ligands were applied in regular proteins, and Drs. David Baltimore, Eric Brown, Carlos Lois, and(serum-containing) medium 34 hr after antibody transfection. Didier Trono for the lentiviral vector and for their advice on its use.We thank Ms. Inna Kolesnik, Ms. Tatiana Shalevich, Dr. ShaolingKinase Tests Ma, and Mr. Sergei Viukov for technical assistance; Dr. Ayala SharpThe in vitro IKK kinase activity of the receptor complexes and cyto- and Mr. Eitan Ariel for assistance in cell sorting; Ms. Taisia Shmush-plasmic signalosome complex, with a bacterially expressed GST- kovich for assistance in generation of the expression vector forIB(154) as substrate, was assessed as described (Uhlik et al., CD40L; and Dr. Orith Leitner, Ms. Anat Bromberg, and Mr. Alon Levy1998; Dejardin et al., 2002). Briefly, 24 108 Ramos cells were for assistance in generation of monoclonal antibodies. Our thankslysed by rotation for 30 min at 4C in lysis buffer (20 mM HEPES are also due to Drs. Elena Appel, Chaya Brodie, Dorit Landstein,[pH 7.6], 250 mM NaCl, 0.5% NP-40, 20 mM -glycerophosphate, Judith Gan, Tania Goncharov, Andrew Kovalenko, Yael Pewzner-1 mM EDTA, 20 mM p-nitrophenyl phosphate, 0.1 mM sodium vana- Jung, and Stefan Leu for useful advice and discussions and to, 2 mM sodium fluoride, 1 mM DTT, 1 mM PMSF, and 1 com- Shirley Smith for scientific editing. This work was supported in partplete protease inhibitor cocktail). Cellular debris was removed by by a grant from Ares Trading S.A., Switzerland.centrifugation at 10,000 g, and the lysate was precleared withprotein A/G beads to which rabbit/mouse preimmune serum was Received: November 7, 2003adsorbed and then subjected to immunoprecipitation for 2 hr at Revised: August 5, 20044C. The immunoprecipitates were washed four times with lysis Accepted: August 6, 2004buffer and twice with kinase buffer (20 mM HEPES [pH 7.6], 20 mM Published: October 19, 2004MgCl2, 20 mM -glycerophosphate, 1 mM EDTA, 2 mM p-nitrophenylphosphate, and 2 mM DTT), and the kinase reaction was performed Referencesin kinase buffer (40 l) containing 1 g GST-IB(154) and[32P]ATP at 30C for 30 min. The kinase activity of NIK was assessed Bren, G.D., Solan, N.J., Miyoshi, H., Pennington, K.N., Pobst, L.J.,under the same conditions with myc-tagged NIK that had been and Paya, C.V. (2001). Transcription of the RelB gene is regulatedoverexpressed in transfected HEK293T cells and immunoprecipi- by NF-kappaB. Oncogene 20, 77227733.tated with anti-myc antibody. The kinase test was carried out inBrummelkamp, T.R., Bernards, R., and Agami, R. (2002). 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