Upstream Signal Transduction of NF-kB Activation

Abstract

NF-κB is a transcriptionfactor governing the expression of genes involved in the immuneresponse, embryo or cell lineage development, cell apoptosis, cellcycle progression, inflammation, and oncogenesis. During past fewyears, tremendous attention has been focused on the upstream signalingpathways leading to the activation of this transcription factor. Manyof these signaling molecules can serve as potential pharmaceuticaltargets for the specific inhibition of NF-κB activation and the subsequent interference ofdisease processes. However, how these molecules interact with eachother is still a debatable issue. Since many nodal signal moleculesin this pathway relay more than one of the upstream signals to theirdownstream targets, it has been speculated that the transmission ofsignals involves a network, rather than a linear sequence in theactivation of NF-κB. Thus, elucidation ofthe detailed relationships among the upstream signaling molecules ofNF-κB activation will be important indeveloping pharmaceutical inhibitors that specifically inhibit theactivation of NF-κB. Such inhibitors wouldbe predicted to have powerful anti-inflammatory and/or anti-carcinogenic effects.

Key words: IKK, kinases, NF-κB,ROIs, signal transduction, ubiquitination.

The NF-κB transcriptionfactor was first discovered in 1986 . Knowledge of the activation ofthis transcription factor lagged behind the understanding of itsfunction. Nevertheless, tremendous progress has been achieved overthe last two years regarding the signal transduction pathways leadingto the activation of NF-κB including thestructure and function of IκB kinasecomplexes (IKK)¹, the upstream signaling pathways, theinteractions among diverse signaling components, and the extracellularregulators .

At present, five mammalian NF-κB family members have been identified and cloned. These includeNF-κB1 (p50/p105),NF-κB2 (p52/p100),RelA(p65),RelB, andc-Rel .All these NF-κB family members share ahighly conserved Rel homology domain (RHD) responsible for DNAbinding, dimerization, and interaction with IκB, the intracellular inhibitor of NF-κB. The C-terminal regions of RelA, RelB and c-Rel contain a transactivating domain that is important for NF-κB-mediated gene transactivation. The C-terminiof the precursor molecules for p50 and p52, p105 and p100, containmultiple copies of the so-called ankyrin repeat, which is found inIκB family members, including IκBα, IκBβ, IκBε,Bcl3, andDrosophila cactus.

Diverse stimuli, which typically include cytokines,mitogens, environmental and occupational hazards, toxic metals,intracellular stresses, viral or bacterial products, and UV light,induce expression of early response genes through the NF-κB family of transcription factors . In restingcells, NF-κB is sequestered in thecytoplasm in an inactive form through its association with one ofseveral inhibitory molecules, including IκBα, IκBβ, IκBε, p105, and p100.Activation of the NF-κB signaling cascaderesults in complete degradation of IκB orpartial degradation of the C-termini of p105 and p100 precursors,allowing the translocation of NF-κB to thenucleus, where it induces transcription (Fig. 1). Activated NF-κB binds to the enhancer or promoter regions oftarget genes and regulates transcription of genes mediating cell-to-cell interaction, intercellular communication, cell recruitment ortransmigration, amplification or spreading of primary pathogenicsignals, and initiation or acceleration of carcinogenesis. Theconsensus binding site of NF-κB is composedof the GGGRNNYYCC sequence, where R is purine, Y is pyrimidine, and Nis any bases.

Figure 1 :Simplified signal transductionpathways of NF-κB activation. Pro-inflammatory signals, mainly TNFα, IL-1 orToll, bind to their corresponding receptors, leading to a recruitmentof receptor-associated proteins, such as MyD88 and IRAK for IL-1R/TLR,TRADD and RIP1 for TNF receptor. In turn these associated proteinsrecruit TRAF2 or TRAF6, both of which activate TAK1 possibly through anon-destructive G76-K63 polyubiquitin chain-dependent mechanism(Ub63). Activated TAK1 or other MAPKKK family kinases, such as NIKand MEKK1, may phosphorylate and activate IKK complexes that areresponsible for the phosphorylation of the IκB protein. Phosphorylated IκB proteins are recognized and modified by theG76-K48 polyubiquitin chain (Ub48) via the SCF-β-TrCP complex. This process is followed byproteasome-mediated degradation of IκBs.Stress signals resulting in the generation of ROIs contribute to theactivation of NF-κB and may involve thesequential activation of ASK1, SEK1 and JNK. Activated JNK inducesthe accumulation of β-TrCP protein, whichfacilitates the ubiquitination process of IκB proteins.

I. IκB kinasecomplexes (IKK)

The expanding family of IKK, which includes theIKKα, IKKβ,IKKγ, and IKKi/ε, has, over the past three years, beenimplicated in the phosphorylation of several IκB proteins and NF-κBfamily proteins, such as IκBα, IκBβ, IκBIKKα and IKKβ share 50% sequence homology. Both proteinscontain a N-terminal kinase domain, a C-terminal region with a leucinezipper, and a helix-loop-helix domain . An activation loop similarto the one found in the MAP-kinase kinase (MEK) family of proteins hasbeen identified between the kinase subdomains VII and VIII of IKKα and IKKβ. Studiesby in vitro or ex vivo approaches indicate that bothIKKα and IKKβare interchangeable in phosphorylating S32/S36 of IκBα, and S19/S23 ofIκBβ. However,substantial differences in function and regulation of IKKα and IKKβ have beendocumented. First, IKKβ is far more potentthan IKKα in IκBα phosphorylation inresponse to proinflammatory stimuli, such as the signals induced byTNFα, IL-1 and LPS . Second, whereasIKKα seems to be more responsible for NF-κB-inducing kinase (NIK) signals, IKKβ appears more important in mediating MEKK1reactions . Third, gene knockout studies demonstrated that IKKα, but not IKKβ, isphysiologically involved in NIK-mediated carboxyl terminalphosphorylation and subsequent process of NF-κB2 (p100) precursor . Fourth, IKKα controls keratinocyte differentiation by akinase-independent mechanism that affects the production ofkeratinocyte differentiation-inducing factor (kDIF) . IKKβ, in contrast, is not necessary for thisfunction. Finally, although both IKKα andIKKβ can phosphorylate multiple regions ofβ-catenin, an opposite effect of IKKα and IKKβ on thetranscriptional activity and intracellular localization of β-catenin has been observed. Using mouse embryofibroblasts (MEF) lacking IKKα or IKKβ gene, Lamberti et al reported that IKKα increased the nuclear localization andtranscriptional activity of β-catenin,whereas IKKβ decreased the nuclearlocalization and transcriptional activity of β-catenin.

Limited information is available concerning anotherrecently identified IKK complex, IKKi/ε.In contrast to the original IKK complex, this new IKK complex doesnot contain IKKα, β or γ . IKKi/ε shares 27% homology with IKKα and IKKβ andpossibly mediates NF-κB-activating kinase(NAK) signaling, PMA/PKCε-induced S36 phosphorylation of IκBα, and NF-κB activation . In contrast to IKKα and IKKβ, which areconstitutively expressed in most cell types, the expression ofIKKi/ε is inducible. In the mousemacrophage cell line, RAW264.7, lipopolysaccharide (LPS) and someother NF-κB-inducing cytokines, candrastically induce the accumulation of IKKi/ε mRNA . Intriguingly, none of these inducersseem to be able to stimulate the kinase activity of transfectedIKKi/ε. Yeast two-hybrid screeningby Nomura et al. recently showed that the C-terminal portion of IKKi/ε can specificallyassociate with the N-terminal domain of I-TRAF/TANK, aninteraction protein of tumor necrosis factorreceptor-associated factor. Thus, it is possiblethat IKKi/ε either acts further upstream ofIKKα/β or at thesame hierarchical level of IKKα/β after its association with I-TRAF/TANK.

The predominant role of IKK is its activity as aserine/threonine kinase phosphorylating IκBfamily proteins . Most of IκB familyproteins contain a conserved DSGXXS motif, where X isany amino acid . IKK is also able to phosphoyrlate NF-κB p65 protein on a non-consensus site, S536 .The ability of IKK to exert its profound kinase activity has led manyintensive investigations to explore its likely role in other cellularresponses. Homology searches of the gene bank protein sequencedatabase reveal that a number of non-IκB/NF-κB familyproteins also contain this motif. These proteins include β-catenin, HIV vpu protein, phosphoinositide 3-kinase enhancer (centaurin), c-Ski, Rho/Rac guanine nucleotideexchange factor, and a number of other potential substrates listed intable 1. Except for β-catenin, which hasbeen recently demonstrated to be an IKK substrate , no experimentaldata so far suggests that IKK can phosphorylate these non-IκB/NF-κB familyproteins. New evidence suggests that IKKαcontrols keratinocyte differentiation and IKKβ attenuates insulin signaling related to type 2diabetes and obesity . Thus, in addition to NF-κB signaling, IKK might be involved in severalother cellular signal transduction pathways in either a kinaseactivity-dependent or a kinase activity-independent manner.

There are several good candidates for theinhibition of IKK activity. One group is the nonsteroidal anti-inflammatory drugs (NSAIDs), including aspirin and sodium salicylate.NSAIDs have been previously shown to inhibit activation of NF-κB and cytokine-induced mRNA of cell adhesionmolecules . Later studies suggested that these effects of NSAIDs arethe result of specific inhibition of ATP-binding to IKKβ , which is independent of their cyclooxygenase-2 (COX-2) inhibitory activity. A second candidate is the 15d-PGJ₂, an anti-inflammatory cyclopentenone prostaglandin anda natural ligand for peroxisome proliferator-activated receptor γ (PPARγ) . In JurkatT cells or HeLa cells, 15d-PGJ₂ inhibits TPA- or TNFα-induced NF-κBactivation in a PPARγ-independent manner .A direct modification of cysteine 179 (C179) in the activation loop ofIKKβ by 15d-PGJ₂ was observed inan in vitro IKK kinase activity assay . The IC₅₀for inhibition of IKK activity was around 5 μM. The same concentration of 15d-PGJ₂, in contrast, stimulated JNK activity, indicating that15d-PGJ₂ or its analogs may have therapeutic potential fordiseases in which inhibition of IKK and NF-κB may be desirable. However, it should benoted that the inhibitory effect of 15d-PGJ₂ on IKK and NF-κB might be cell type or stimulusdependent. This becomes evident by the fact that 15d-PGJ₂potentiated LPS-induced gene expression of IL-8, a NF-κB targeting gene . The third group of IKKinhibitors includes several plant extracts that have been shown toreduce IKK activity in some experimental systems. These extractsinclude resveratrol, parthenolide, and green tea polyphenol (-)-epigallocatechin-3-gallate . The specificity and potentialapplication of these natural products in inhibiting IKK activityremain to be investigated. Finally, two relatively specific IKKinhibitors are being developed by Signal Pharmaceuticals and NovartisPharma AG, respectively. Signal Pharmaceuticals developed a selectiveIKKβ inhibitor named SPC839 that inhibitsIKKβ with nanomolar potency and IKKα with micromolar potency.

1. MEKK1

MEKK1 is a mammalian serine/threonine kinase in themitogen-activated protein kinase kinase kinase (MAPKKK) group. It wasfound that MEKK1 was a far more important activator for JNK signalingrather than ERK signaling as was proposed originally . The firstevidence indicating the involvement of MEKK1 in signal-induced IKKactivation was provided by Lee et al. . In their studies, theyreported that the addition of the recombinant catalytic domain ofMEKK1 (MEKK1Δ) to the partially enriched fraction of nonstimulated HeLa cellsstimulated an IKK-like kinase activity that phosphorylated IκBα at S32 and S36 andsubsequent ubiquitination and degradation of IκBα. Follow-upstudies demonstrated that overexpression of MEKK1 stimulated the NF-κB-dependent transcriptional reporter .The activation of NF-κB by HTLV Tax proteinwas shown to require MEKK1 . MEKK1 may also contribute to Toll- andIL-1 receptor-mediated IKK activation, as demonstrated by an adaptorprotein, known as evolutionarily conserved signaling intermediate inToll pathways (ECSIT) that could promote the proteolytic activation ofMEKK1 and subsequent activation of NF-κB .Further studies by Mercurio et al. indicated the presence of a proteinin the IKK complex that is recognized by anti-MEKK1 antiserum .

Although MEKK1 has been shown to contribute to IKKactivation in a number of studies, the precise molecular link betweenthese two kinases remains unclear. One intriguing possibility is thatMEKK1 may directly phosphorylate both IKKαand IKKβ at the MAPKK activation loop,176/177 S-X-X-X-S 180/181, where X is any amino acid. Substitution ofthese serines with alanine residues inactivates both kinases, whereasphosphomimetic glutamic acid substitution at these positions resultsin constitutively active kinases . Nevertheless, it remains to beconfirmed whether MEKK1 is a physiological activator of IKK in cellsin response to various stimuli. Indeed, a recent study by Xia et al.demonstrated that inactivation of MEKK1 did not result in animpairment of NF-κB activation in responseto TNFα, IL-1, LPS, and dsRNA.

2. NF-κB-InducingKinase (NIK)

NIK, a member of the MAPKKK family, wasoriginally identified as a tumor necrosis factor (TNFα) receptor associating factor 2 (TRAF2)-interacting kinase whose overexpression results in potent NF-κB activation without any considerable effect onMAPKs . A study using yeast two-hybrid screens identified aninteraction between NIK and IKK, suggesting that NIK might be a directupstream activator of IKK . Transient transfection of NIK into humanembryonic kidney 293 cells indicated that IKKα was more responsive to NIK, whereas IKKβ was slightly more responsive to MEKK1 . Whenthe abilities of MEKK1 and NIK to activate total IKK kinase activityare compared, most of studies show that NIK is a much strongeractivator of the NF-κB transcriptionalreporter than MEKK1. NIK could preferentially phosphorylate IKKα on Sl76 in the activation loop, leading to theactivation of IKKα kinase activity. Incontrast, MEKK1 was found to preferentially phosphorylate thecorresponding serine residue, Sl77, in the activation loop of IKKβ. A dominant negative mutant of NIK blocked NF-κB activation by TNFα, interleukin-1 (IL-1),Fas, Toll-likereceptors 2 and 4 , LMP1 and CD3/CD28 stimulation . Thus, NIK appearsto be a general kinase mediating IKK activation induced by diversestimuli. However, a recent analysis using the NIK-mutant mouse strainalymphoplasia (aly) contradicted this assumption. TheAlymohoplasia mouse strain failed to develop lymphoid organs,such as lymph nodes and Peyer's patches due to a point mutation in theNIK locus . The mutation of NIK locus results in disruption ofinteractions between NIK and IKKα or TRAFproteins. Further analysis indicated that the aly mutation didnot affect TNFα-induced activation of NF-κB but only blocked lymphotoxin-mediatedactivation of NF-κB. Similarly, studiesusing cells derived from NIK-deficient mice have indicated that NIKappears to be dispensable in IKK activation induced by TNFα or IL-1 . Intriguingly, lymphotoxin β induced a normal NF-κB DNA-binding activity in NIK-deficient cells,whereas the same treatment failed to induce NF-κB reporter gene activity or NF-κB target gene expression . It raises thepossibility that NIK may be specifically involved in IKK activationinduced by lymphotoxin but not others.

3. NF-κB activatingkinase (NAK)

Several groups independently identified a novelserine/threonine kinase, possibly activating IKK through directphosphorylation in cells stimulated with PMA . This novel kinase wasnamed NAK, TANK-binding-kinase 1 (TBK1), or T2K. Pomerantz andBaltimore cloned NAK by a yeast two-hybrid screen using the N-terminal stimulatory domain of TANK 1-190 fused to GAL4 as bait, and ahuman B-cell library fused to the GAL4 activation domain. The samekinase was also identified by PCR using degenerate primers based onsequences common to IKKα and IKKβ. The amino acid sequence analysis indicatedthat the NAK protein contains a kinase domain at its N-terminus thatexhibits about 30% identity to the corresponding kinase domains ofIKKα and IKKβ,and more than 60% identity to the corresponding kinase domain ofIKKi/ε. The report by Pomerantz andBaltimore showed that NAK might form a ternary complex withTANK andTRAF-2, suggesting that NAK functions to the far more upstream of thesignal cascade leading to IKK activation, whereas in vitrokinase activation assay by Tojima and co-workers demonstratedthat NAK was a direct upstream kinase phosphorylating IKKβ. Interestingly, activation of endogenous NAKresulted in only S36, but not S32 phosphorylation of IκBα, a similarphenomenon observed in recombinant IKKi/ε-mediated IκBαphosphorylation . Since both IKKα andIKKβ are able to phosphorylate both S32 andS36 of IκBαprotein, it is unclear whether IKKi/ε or a novel IKK isozyme functions as adownstream kinase of NAK to induce S36 phosphorylation of IκBα. Transienttransfection studies showed that dominant negative NAK inhibited NF-κB transcriptional reporter activityinduced by PMA, PKCε, andPDGF, but not byTNFα, IL-1β,LPS, or ionizing radiation . These results, therefore, suggest thatNAK is likely to be a downstream kinase of PKCε or related isozymes, and an upstream kinase ofIKK in the signaling pathway through which growth factors, such asPDGF, stimulate NF-κB activity.

4. Akt (PKB)

The pro-survival function of Akt has been welldocumented. The kinase activity of Akt is activated via thephosphoinositide-3-OH kinase (Pl3K) and P13K-dependent kinase 1/2(PDKI/2) signaling pathways . Overexpression or constitutiveactivation of Akt has been associated with tumorigenesis in a numberof studies. As a serine/threonine kinase, Akt is able tophosphorylate the pro-apoptotic proteinBad, the anti-apoptoticprotein Bcl-x, the apoptotic protease caspase-9, the Forkheadtranscription factors, and eNOS . However, considerable controversyremains regarding the involvement of Akt in signal-induced IKKactivation. Studies by Ozes et al. and Xie et al. indicated thatAkt was required for TNFα- or G proteinactivator-induced NF-κB activation bydirectly phosphorylating and activating IKKα in 293, HeLa, and ME-180 cells. A putative Aktphosphorylation site at amino acids 18 to 23 in both IKKα and IKKβ wasidentified. Akt induced T23 phosphorylation of IKKα both in vitro and in vivo.Mutation of T23 significantly decreased Akt-induced IKKα phosphorylation and TNFα-induced NF-κBactivation in 293, HeLa, and ME-180 cells . By contrast, Romashkovaet al. demonstrated that Akt was involved in PDGF-mediated,but not TNF α- or PMA-mediated NF-κB activation in human or rat fibroblasts. Inthis study, the authors suggested that upon PDGF stimulation, Aktcould transiently associate with IKK and induce the activation IKK,especially IKKβ. Several other studies,however, contradicting these reports, suggested that the effects ofAkt on NF-κB did not occur at the level ofIKK activation in several cell types. A study by Delhase and co-workers indicated that Akt activation induced by IGF-I failed toactivate IKKα, IκBα phosphorylationand degradation, or NF-κB DNA binding inHeLa cells, the same cell line used by Ozes et al . Similarly,several recent studies showed that Akt was not involved in TNFα-induced NF-κBactivation in human vascular smooth muscle cells, skin fibroblasts, orendothelial cells . Rather, Akt might enhance the ability of the p65(RelA) transactivation to induce transcription . In Jurkat T-cells,Akt alone failed to activate NF-κB, but itwas capable of potentiating NF-κBactivation induced by PMA, partially by enhancing IκBβ degradation .Evidence further supporting this notion is the observation thatexpression of constitutively active Akt upregulates the mRNA level ofβ-TrCP, a subunit of the SCF-β-TrCP complex responsible for the ubiquitinationof IκBα orIκBβ proteins .Thus, it is possible that Akt phosphorylates IKK in a cell context-and stimulation-dependent manner. It is also highly possible thatseveral different mechanisms are involved in Akt-regulated NF-κB activation. One remaining question is whetheror not upstream kinases of Akt, such as PDK1 and PDK2, also activateIKK, since both IKKα and IKKβ contain a putative PDK1 phosphorylation site(S-F-X-G-T-X-X-Y-X-A-P-E) directly juxtaposed to the MAPKKKphosphorylation site .

5. Mixed-Lineage Kinase 3 (MLK3)

MLK3, another member of the MAPKKK family, containsan N-terminal SH3 domain, followed by the catalytic domain and twotandem leucine/isoleucine zippers, a basic region, a Cdc42/Rac bindingmotif, and a proline-rich C terminus . Based on these structuralcharacteristics, MLK3 may associate with a variety of protein modules.Studies by Hehner et al. suggested that MLK3 could directly associatewith IKK complex through its leucine zipper domain and phosphorylateSl76 of IKKα and Sl77 and Sl8l of IKKβ. Transfection of Jurkat T cells with a kinase-mutated form of MLK3 blocked CD3-CD28 signal- and PMA-induced NF-κB transcriptional activity. No significantinfluence of this mutated MLK3 was observed on either TNFα- or IL-1-induced NF-κB activation. These results suggest that MLK3may be important in mediating T-cell co-stimulation-induced activationof IKK and consequent NF-κB-dependenttranscription. MLK3 has also been shown to form a complex with a JNKscaffold protein JIP and stimulate JNK activation . Thus, MLK3 mayfunction as an integral molecule between the signaling pathwaysleading to the activation of NF-κB and JNK,which would provide a molecular explanation why many stimuli induceNF-κB and JNK simultaneously under certaincircumstances.

6. TGFβ-ActivatedKinase 1 (TAK1)

TAK1 is a member of the MAPKKK (MAP3K) family,which was originally identified as a kinase mediating the signalingpathway of TGFβ superfamily members .Transfection of the cells with an activated form of TAK1, in which theN-terminal 22 amino acids are deleted, induces expression of areporter gene governed by TGFβ-responsivepromoter . However, the biochemical link between TGFβ and TAK1 has been elusive. Ironically, thecontributions of TAK1 to signal-induced NF-κB and JNK activation have been studiedintensively. Several new insights into the roles of TAK1 in IKKactivation have emerged in the cellular response to cytokines or Tollsignals . The first evidence to suggest that TAK1 is involved in NF-κB signaling came from studies in whichoverexpression of TAK1 together with its activator protein, TAK1binding protein 1 (TAB1), induced the nuclear translocation of NF-κB in a NIK-independent manner . Furtherstudies indicated a direct physiological interaction between TAK1 andIKK in unstimulated cells . Recruitment of TAB1 and/or TAB2 to TAK1activates the kinase activity of TAK1, resulting in phosphorylation ofthe serine residues in the activation loop of IKK and subsequentdissociation of TAK1 from IKK complex . In Drosophila, a nullmutation in the TAK1 gene produces phenotypes similar to that ofmutations in immune deficiency (Imd), and IKK, suggesting thatTAK1 is a direct kinase mediating Imd signals . Geneticstudies and sequential chromatographic purification by Wang et al.demonstrate that the kinase activity of TAK1 in response to IL-1 isdependent on the TRAF6 protein, which has been modified by a distinctpolyubiquitin chain assembled through the lysine 63 (K63) at eachubiquitin molecules. In contrast to the polyubiquitin chain in whichthe C-terminal glycine 76 (G76) of one ubiquitin is ligated to the K48side chain of the neighboring ubiquitin, the polyubiquitin chainlinked through G76-K63 does not target proteins for proteasomaldegradation, but rather, activates the function of proteins (seebelow) . The major debatable issues in TAK1-induced IKK activationare the involvement and hierarchical position of NIK. Whereas severalreports clearly suggest that NIK is not involved in TAK1-induced IKKactivation and TAK1 is a direct upstream kinase phosphorylating IKK inHeLa cells treated with IL-1 , Ninomiya-Tsuji et al. showed that NIKis a mediator of TAK1-induced IKK activation in IL-1-treated 293cells. It is unclear whether this discrepancy is due to the cell typeor subtle differences in overexpression of the dominant-negativeinactive NIK mutant.

7. Other Kinases

A variety of other kinases have been reported tofunction upstream of IKK. Because of the lack of evidence of directassociation of these kinases with IKK upon activation or specificphosphorylation site(s) of these kinases on IKK, it is unclear whetherthese kinases are direct upstream kinases phosphorylating andactivating of IKK, or far more distal kinases indirectly activatingIKK. These kinases include Cot , PKCζ, PKCα, PKCθ, or PKR etc. In light of the fact that a variety ofkinases can affect IKK, it seems likely that different cell types andstimuli may utilize distinct upstream kinases for the activation ofIKK. An example to support this notion is the observation thatPKCθ and Cot kinase participate in CD3-CD28costimulation signal-induced, but not TNFα-induced, activation of NF-κB .

III. Mechanisms of Ubiquitination in NF-κB Activation

As detailed above, the activation of NF-κB by most of the extracellular inducers isdependent on the phosphorylation and subsequent degradation of IκB proteins. A crucial step during this processis the phosphorylation-dependent conjugation of IκB proteins with polyubiquitin chain, a markerrequired for the proteasomal degradation of IκBα. Whereas theubiquitination sites on IκBβ and IκBε have not been definitely identified , lysines21 and 22 (K21 and K22) on the IκBα protein were considered as the major sitesconjugated by the polyubiquitin chain .

Figure 2 :SCF-β-TrCPubiquitin ligase complex-mediated ubiquitination of IκB proteins. The basic components of this E3complex include Skp1, Cul-1 (CDC53), and the F-box protein, β-TrCP. β-TrCPrecognizes and links the phosphorylated IκBproteins to this complex allowing the ubiquitination of IκBs by ubiquitin-conjugating enzyme E2 followingthe C-terminal G residue activation of ubiquitin by ubiquitin-activating enzyme E1.

Ubiquitin is a highly conserved and heat stable 76-amino acid protein found in virtually all types of eukaryotic cells .Ubiquitination of proteins involves three or four sequential steps(Fig. 2). Initially, the C-terminal glycine (G76) of ubiquitin isactivated by ATP to form a high energy thiolester intermediatecatalyzed by the ubiquitin-activating enzyme (Uba or E1). Activatedubiquitin is then transferred from E1 to one of many distinctubiquitin-conjugating enzymes (Ubc or E2), forming a similarthioester-linked complex. Finally, with the aid of ubiquitin ligases(E3), an isopeptide bond is formed between the activated C-terminalG76 of ubiquitin and an ε-NH₂group of a K residue of the substrate. In successive reactions,polyubiquitin chain, is synthesized by progressive transfer ofubiquitin moieties to K48 or K63 of the previously conjugatedubiquitin molecule, forming G76-K48 or G76-K63 isopeptide bonds. Anassembly factor, named Ufd or E4, may be required for this process .The specificity of protein ubiquitination is usually determined by theubiquitin ligase E3 that recognizes specific substrates. At leastthree types of ubiquitin ligase E3 complexes have been welldocumented. These ligase complexes include the Skp1-cullin-F-box(SCF) complex, the VHL protein-elongin B-elongin complex (VBC), andthe anaphase promoting complex (APC). The ubiquitin ligase E3,responsible for the ubiquitination of IκBα, is the SCFcomplex containing a F-box/Trp-Asp repeating (WD) protein named β-TrCP (Fig. 2). Following phosphorylation ofS32 and S36 in the conserved DSGXXS motif of IκBα by IKK, the β-TrCP subunit of the SCF complex recognizes andbinds to the phosphorylated DSGXXS motif of IκBα . Thebinding of SCF to IκBα results in the association of SCF with specificE2s, including Ubc3, Ubc4, Ubc5, and Ubc9 . These E2s are able tocatalyze the ubiquitin conjugation of IκBα and the assemblyof G76-K48 polyubuquitin chain. Consistent with the likely role forSCF-β-TrCP as a ubiquitin ligase complexconjugating polyubiquitin chain to IκBα, is the observation that Slimb protein,a Drosophila homology of mammalian β-TrCP, is required for the ubiquitination ofCactus, an IκB-like proteininhibiting the activation of the Drosophila NF-κB homolog, Dorsal . During dorsoventralpatterning of the early Drosophila embryo, the Dorsalprotein is activated specifically on the ventral side of the embryo bythe Toll receptor-signaling pathway. These findings point to theexistence of an evolutionarily conserved pathway for specificubiquitination of the IκBα protein for the purpose of dynamic signaltransduction from the receptor to NF-κB.

In parallel studies of signal-induced IκBα ubiquitination,several reports indicated that this process could be antagonized bySUMO-1 (small ubiquitin-related modifier-1) modification of IκBα on the sameresidues where the polyubiquitin chain is conjugated , or by unknownproduct(s) of nonpathogenic Salmonella bacteria . SUMO-1 isone of the best-characterized members of ubiquitin-related proteins.Conjugation of SUMO-1 to substrates requires SUMO-1-activating enzymeAos/Uba2, and SUMO-1-conjugating enzyme, Ubc9. Although substratescan be modified by several SUMO-1 at distinct sites, no multi-SUMO-1chains are apparently formed . In contrast to ubiquitination ofIκBα protein,SUMO-1 conjugation does not target IκBα to proteasomal degradation . The inhibitionof NF-κB by certain bacterial pathogens maybe through a mechanism affecting the conjugation of SUMO-1 on IκB proteins. One example is the observation thatYopJ, a protein product encoded by a 70-kB plasmid harbored inthe Yersinia species that caused the Black Death in the MiddleAges, inhibits MEKK1-induced NF-κBactivation. Earlier study by Orth et al. indicated that theinhibition of NF-κB by YopJ isthrough direct interaction of YopJ with IKKβ but not with IKKα.Structural analysis of YopJ protein by the same group latersuggested that YopJ might be a SUMO-protease promoting theconversion of precursor SUMO-1 to mature SUMO-1 . Nevertheless,whether YopJ enhances the conjugation of SUMO-1 on IκBα has not beendemonstrated.

The vast majority of ubiquitination reactions inwhich the proteins are ubiquitinated via G76-K48 assembly of thepolyubiquitin chain target protein for proteasomal degradation.Examples include the ubiquitination of IκBα, p53, cyclins, c-Jun, and others. This is not, however, the case of ubiquitination ofproteins via G76-K63 assembly of polyubiquitin chain. The biochemicalevidence of G76-K63 assembly of the polyubiquitin chain remainselusive, but it appears to be independent of proteasomal degradation .Recent studies by Wang et al. suggest that linkage of the G76-K63polyubiquitin chain with TRAF6 protein plays an important role inmediating TLR/IL-1R signal-induced activation of TAK1, an upstreamkinase of IKK. TRAF6 itself exhibits the ubiquitin ligase E3 activityby the structural characteristic of RING fingers in its C-terminus.

The nature of the upstream regulators that promoteG76-K63 ubiquitination of TRAF6 is less clear. One good candidate,however, is the Ubc complex composed of Ubc13, a member of theubiquitin-conjugating enzyme E2 family, and Uev1A, a ubiquitin-conjugating E2 enzyme variant . In yeast and mammalian cells, bothUbc13 and Uev1A are considered the major enzymes required for thesynthesis of G76-K63 polyubiquitin . In chromatographic purificationof HeLa cell cytoplasmic extracts, the Ubc13/Uev1A complex was foundto be co-eluted with TRAF6 and appeared to be essential in TRAF6-induced TAK1 and subsequent IKK activation . However, inDrosophila, this Ubc13/Uev1A-induced K63 polyubiquitination ofTRAF6 has yet to be established, despite the identification of theDrosophila homologs of Ubc13 and Uev1A, bendless anddUev1A, respectively . Interestingly, Ubc13/Uev1A was alsofound to interact genetically with a DNA repair protein, Rad5 ,indicating that it is likely to be coupled to a number of cellularprocesses. Such a finding supports the likelihood that the activationof the Ubc complex provides a mechanism by which IKK signals can beselectively activated during cellular damage response in vivo.

IV. ROIs: Critical Mediators or bystanders inNF-κB Activation?

Oxidative stress is a hallmark ofpathophysiological response resulting from the alterations of cellularredox homoeostasis due to either an over-production of reactive oxygenintermediates (ROIs) or a deficiency in buffering or scavenging systemfor ROIs . Typically, the oxidatively stressed cells exhibit damageof their macromolecules leading to lipid peroxidation, oxidation ofamino acid side chains (especially cysteine), DNA damage, stressresponse kinase activation and gene expression associated with cellcycle arrest and/or cell apoptosis. Moderate oxidative stress withoutsevere damage of structural and functional macromolecules can berecovered due to the activation of cellular defense systems includingnonenzymatic and enzymatic antioxidants. A number of stress responsegenes are induced to protect cells from the oxidative stress or torepair the ROI-mediated damages. A sustained oxidative stressproduced during chronic or acute inflammatory response and/onenvironmental toxicant exposure, however, will be cytotoxic.

Among all the known oxidative stress inducers,H₂O₂ and some environmental toxic metals orparticles are perhaps the most potent and well studied . Many otheragents, such as TNFα, IL-1 and bacterial orviral proteins, also induce oxidative stress . Since the discovery ofNF-κB, hundreds of reports have indicatedthat some extracellular stimuli that induce oxidative stress alsoactivate NF-κB . Thus, it is not toosurprising that many researchers attributed a role for ROIs in signal-induced NF-κB activation . Some evenproposed that ROIs might be universal molecules mediating theactivation of NF-κB in response to a broadrange of stimuli . However, the conclusion that ROIs mediate NF-κB activation has been strongly challenged .First, correlations between ROI generation and NF-κB activation do not necessarily mean ROIs areessential mediators linking upstream signals to NF-κB activation. Under certain circumstances, ROIgeneration may be simply a bystander signal or a secondary response toNF-κB activation. Second, caution shouldbe exercised in interpreting the inhibitory effects of a variousantioxidants on signal-induced NF-κB. Manyantioxidants can disturb the normal cellular redox status thatmaintains the basal signaling potential required for the activation ofNF-κB or other intracellular biochemicalevents even under the non-oxidative stress conditions. In addition,many low-molecular-weight antioxidants may inhibit NF-κB by non-antioxidant actions . Third, it shouldbe noted that several studies show that ROIs fail to activate NF-κB in many experimental systems . Finally,emerging evidence suggests that the DNA binding activity of activatedand nuclear translocated NF-κB requiresreducing conditions . Oxidation or nitrosylation of the cysteineresidue in the DNA binding domain of the NF-κB p50 subunit suppresses the DNA binding andtranscriptional activity of NF-κB .

The signal transduction pathway, such as theupstream and proximal kinases, leading to the activation of NF-κB by TNF, IL-1, Toll, LPS, and CD28, has beenclearly identified. However, only limited information is available tosuggest the responsiveness of these kinases to ROIs . The evidence toimplicate ROIs as stimulators of IKK is based on the elevated IKKactivity in human epithelial cells or mouse fibroblast cells by theH₂O₂ treatment . In our own studies, we found amodest induction of IKK activity in cellular response to chromium(VI),a potent intracellular H₂O₂ inducer (Chen etal., unpublished). Nevertheless, studies challenging this observationexist. Li and Karin could not detect IKK kinase activity in HeLacells stimulated with UV-C, another intracellularH₂O₂ inducer , despite the fact that UV-Cinduced IκBαdegradation and NF-κB DNA binding.Similarly, Korn et al. described that H₂O₂itself failed to stimulate IKK, but rather, inhibited TNFα-induced IKK activity. It is highly likely thatH₂O₂ inactivates IKK through direct oxidation ofa conserved cysteine 179 (C179) in the kinase domain of IKKβ, a mechanism similar to the inactivation ofIKKβ by 15d-PGJ2 and a high concentrationof arsenic .

Figure 3 : Model for the ROI-induced oxidation of Trxand the subsequent ubiquitination of IκBs.Oxidation of the C-X-X-C motif of Trx induces its dissociation fromASK1, thereby allowing the dimerization and activation of ASK1. JNKis activated by SEK1 that has been phosphorylated by ASK1, leading tothe accumulation of β-TrCP which isrequired for the processes of IκBubiquitination.

Whereas IKK seems to be a less favorable target-point in ROI-modulated NF-κB activation,kinases other than IKK may serve as bridge molecules linking ROIs tothe activating signals of NF-κB. One suchkinase, JNK, merits special attention, not only because of itsunequivocal activation in response to ROIs, but also because of itspotential link to the ubiquitination and subsequent degradation ofIκBα . Theactivation of JNK by ROIs appears to be mediated by the activation ofASK1, a member of the MAPKKK family that phosphorylates and activatesSEK1 (MKK4), an upstream kinase of JNK (Fig. 3). In resting cells,ASK1 binds with high affinity to the reduced form of thioredoxin (Trx)which serves as an inhibitor of ASK1 by preventing the dimerization ofASK1 . Oxidation of the C-X-X-C motif of Trx by ROIs induces thedissociation of Trx from ASK1, thereby allowing the dimerization ofASK1 and the consequent activation of JNK. We have previouslyreported that inhibition of JNK by overexpression of a dominantnegative SEK1 impaired the degradation of IκBα and the activationof NF-κB induced by vanadate . Thisobservation was further substantiated by Spiegelman et al. whoprovided convincing evidence indicating the contribution of JNK to thesignal-induced ubiquitination of IκBα protein. Activation of JNK resulted inaccumulation of β-TrCP, a subunit of theSCF-β-TrCP complex that recognizes thephosphorylates DSGXXS motif within the IκBα protein and causesthe subsequent ubiquitination . While JNK has been implicated in thestabilization of a number of short-lived mRNAs in response to stress ,it is plausible to speculate that the JNK-mediated accumulation ofβ-TrCP is through stabilization of theβ-TrCP mRNA. Indeed, analysis of the β-TrCP mRNA sequence by Spiegelman et al.revealed a closely resembled JNK response element in addition to twoAU-rich elements in the 3’-UTR region of β-TrCP mRNA. Since ubiquitination of the IκBα protein ispotentially a rate-limiting step, the abundance of β-TrCP regulated by JNK may serve as an importantpoint of regulation in ROI-induced NF-κBactivation.

Table 1: Confirmed and putative IKK substrates inmammalian cells.

Summary

Increasing evidence implicates dysregulation ofsignaling pathways leading to the activation of the NF-κB transcription factor in the pathology ofvarious diseases, including autoimmune diseases, neurodegenerativediseases, inflammation, and cancers . Moreover, several humandiseases due to the inherited mutations in the genes encoding NF-κB signaling molecules have been recentlydescribed . The signal transduction pathways of NF-κB activation therefore represent potentialtargets for therapeutic intervention. As discussed above, tremendousadvances have been made in our understanding of the upstream signalingpathways of NF-κB activation. Yet thisunderstanding is not complete. A fundamental goal for future studiesis to focus on the structural and functional aspects of theparticipating components in these pathways. It seems likely that newaspects of NF-κB signaling will bediscovered through such studies.

¹Abbreviations:

IKK, IκB kinasecomplex; JNK, c-Jun N-terminal kinase; MAPK, mitogen-activated proteinkinases; ROIs, reactive oxygen intermediates; NIK, NF-κB inducing kinase; NAK, NF-κB activating kinase; TAK1, TGF-β activating kinase 1; MLK, mixed lineage kinase.

Acknowledgments

We thank our colleague Drs Murali Rao and VinceCastranova for helpful suggestions and critique of the manuscript. Weapologize to those whose primary valuable work was not cited becauseof space limitation. Dr. Fei Chen thanks the Health EffectsLaboratory Division/National Institute for Occupational Safety andHealth for its continuing supporting.