Diverse ubiquitin signaling in NF-κB activation

Diverse ubiquitin signaling in NF-kBactivationKazuhiro Iwai

Department of Molecular and Cellular Physiology, Graduate School of Medicine, Kyoto University, Yoshida-konoe-cho, Sakyo-ku,

Kyoto 606-8501, Japan

Review

Nuclear factor-kappa B (NF-kB) is a transcription factorinvolved in a wide variety of phenomena including in-flammation, immune responses, and cell survival. Ab-normal activation of NF-kB occurs in many pathologicalconditions, such as allergic and auto-inflammatory dis-eases and malignancies. As a result, the signal-inducedNF-kB activation pathway has been extensively studiedand revealed to be regulated by ubiquitination. Severaltypes of polyubiquitin chains exist and the type of chainseems to impact how ubiquitinated proteins are regu-lated. Recently, different types of polyubiquitin chains,including linear (M1) and K11 chains, have been impli-cated in NF-kB activation. This review discusses existingevidence of the differential roles played by various ubi-quitin chains, particularly K63, M1, and K11 chains, inNF-kB activation.

The ubiquitin conjugation systemUbiquitin, a globular protein of 76 amino acids, is a pro-tein-based post-translational modifier highly conservedthroughout eukaryotes. The ubiquitin conjugation systemfunctions in most cases by conjugating polyubiquitinchains (ubiquitin polymers) to proteins (Box 1). Thereare several types of polyubiquitin chains, and it has beenhypothesized that the type of polyubiquitin chain deter-mines the mode of protein regulation (Box 1). The ubiquitinsystem was first identified as part of an energy-dependentdegradation system, and the significant roles played by theubiquitin-proteolytic pathway in biology and medicine arenow well known [1,2]. The importance of this pathwaycomes from its timely and selective recognition of specificsubstrates for ubiquitination, which are then marked fordegradation [3]. Timely and selective protein modificationis a crucial feature of many modes of protein regulation, notjust degradation,[4] and, as such, nondegradative roles forubiquitination, including signal transduction, were subse-quently recognized and now widely accepted [5,6]. Thenuclear factor-kappa B (NF-kB) activation pathway isprobably the best-studied signaling cascade in which theubiquitin system is involved [7,8].

The NF-kB activation pathwayNF-kB is a dimeric transcription factor of Rel homologydomain-containing proteins, including p65 (RelA), RelB, c-Rel, p105/p50 (NF-kB1), and p100/p52 (NF-kB2), whichplays a central role in inflammatory responses by inducing

Corresponding author: Iwai, K. ([email protected]).Keywords: ubiquitin; linear ubiquitination; NF-kB; LUBAC.

0962-8924/$ – see front matter � 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.101

the expression of proinflammatory molecules. Besides in-flammation, NF-kB is also involved in many biologicalphenomena including cell survival and innate and ac-quired immune responses [9,10]. NF-kB is inactive inresting cells as it resides in the cytoplasm bound to inhibi-tor proteins called inhibitors of kBs (IkBs) and is inducedby infection, UV or inflammatory cytokines. Because ab-normal activation of NF-kB occurs in many pathologicalconditions such as allergic and auto-inflammatory diseasesand neoplasms, the signal-induced NF-kB activation path-way has been extensively studied [10–12]. NF-kB activa-tion pathways are basically subdivided into two distinctbranches, known as the canonical and non-canonical path-ways, and the ubiquitin system is involved in both [13,14].

In the canonical pathway, NF-kB is rapidly activated byvarious agents such as inflammatory cytokines or Toll-likereceptor (TLR) ligands and is involved in a wide variety ofbiological phenomena including inflammatory and im-mune responses. Upon stimulation with various stimuli,the IKK (IkB kinase) complex, which comprises IKKa,IKKb, and NF-kB essential modulator (NEMO; also calledIKKg), is activated and phosphorylates specific Ser resi-dues within IkBs [15]. Phosphorylated IkBs are degradedafter being conjugated with K48-linked chains, whichreleases NF-kB and allows it to translocate to the nucleusand induce the transcription of target genes (Figure 1)[9,10]. Although activation of canonical IKK complex iscommon to all canonical NF-kB activation stimuli, signal-ing pathways to activate the canonical IKK complex aredifferent among the stimuli. Here, initial activation eventsin tumor necrosis factor (TNF)-a and IL-1b signaling arebriefly introduced. Upon binding to TNF-a, TNF receptor-associated protein with a death domain (TRADD) and thereceptor-interacting protein 1(RIP1) kinase are recruitedto the TNF-receptor 1 (TNFR1). TRADD further recruitsTNF receptor-associated factor 2 (TRAF2) and cellularinhibitor of apoptosis proteins (cIAPs) (Figure 1). In thecase of IL-1b signaling, myeloid differentiation primarygene 88 (MyD88) is recruited to the IL-1 receptor (IL-1R),which recruits IL-1b-associated kinases 1 and 4 (IRAK1and IRAK4) to the receptor (Figure 1). IRAK1 binds toTRAF6. Because TRAF2, TRAF6, and cIAPs exhibit ubi-quitin ligase activity, the ubiquitin system is then involvedin the signaling cascade leading to canonical IKK activa-tion — IKKb phosphorylation [10].

The non-canonical pathway is activated by some TNF-receptor family proteins including lymphotoxin-b receptor(LT-bR), CD40, and BAFF, and is involved in several

6/j.tcb.2012.04.001 Trends in Cell Biology, July 2012, Vol. 22, No. 7 355

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Box 1. The ubiquitin conjugation system

Ubiquitin conjugation to target proteins is mediated through a

cascade of reactions catalyzed by three enzymes: a ubiquitin-

activating enzyme (E1), a ubiquitin-conjugating enzyme (E2), and a

ubiquitin-protein ligase (E3) (Figure I) [77]. In one class of E3s, called

HECT E3s, ubiquitin is first transferred to a conserved Cys residue on

E3 before being transferred to the target [78]. Most other E3s belong

to the RING family of ligases. E3s in this class seem to function as a

scaffold for binding both an E2 carrying ubiquitin and a substrate

molecule, and facilitates the transfer of ubiquitin from the E2 to the

target [77]. Recently, a subtype of RING E3s with RING between RING

fingers (RBR) was shown to transfer ubiquitin to the Cys residue of E3

before transfer to the substrate, as reported for HECT E3s [79]. Once

ubiquitin is conjugated to the target proteins, other ubiquitin

molecules are successively conjugated to ubiquitin bound to targets

to generate polyubiquitin chains [77]. There are several types of

polyubiquitin chains, and the type of polyubiquitin chain is thought to

determine how the conjugated protein is regulated [5,6]. Polyubiqui-

tin chains have been thought to be formed by the conjugation of the

C-terminal carboxyl group of ubiquitin to the e-amino group of one of

the seven Lys residues in another ubiquitin [77]. Polyubiquitin chains

that function as degradation signals are generated on ubiquitin Lys 48

(K48-linked chains) [2,80]. Lys 63-linked (K63-linked) polyubiquitin

chains are involved in signal transduction and DNA repair, but do not

function as a degradation signal by the proteasome [20,81]. Mass

spectrometry analyses show that inter-ubiquitin linkages via all seven

Lys residues within ubiquitin exist in eukaryotic cells [82]; the

ubiquitin system may play much wider roles than have been

identified to date because roles for K6, K27, K29, and K33 ubiquitin

chains have not been described. Moreover, approximately 100 DUBs,

which disassemble polyubiquitin chains or remove ubiquitin from

substrates, exist in humans [83,84]. Thus, ubiquitination is now

regarded as a reversible post-translational protein modification

system that regulates protein function in a wide variety of ways. In

this context, a new type of polyubiquitin chain, the linear polyubi-

quitin (M1) chain, in which ubiquitin is linked via an N-terminal Met

residue to another ubiquitin molecule, has been identified [27].

Further analysis revealed that linear polyubiquitination is involved in

NF-kB activation [17,18].

(a)

s

ss

ss

AM

P+

PP

iAT

P

AM

P+

PP

i

Polyubiquitin chains

Polyubiquitin chains

ATP

K K

HECT E3

E1 E1

E2

Ub

Ub

Ub

Ub

Ub

Ub

Ub

Ub

Ub

Ub

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Ub

Ub

Ub

Ub

Ub

Ub

Ub

Ub

E2Target

proteinsTarget

proteins

RING E3

(b)

TRENDS in Cell Biology

Figure I. The ubiquitin conjugation system. Ubiquitin is conjugated to target proteins via a cascade of reactions catalyzed by three enzymes: E1 ubiquitin-activating

enzymes, E2 ubiquitin-conjugating enzymes, and E3 ubiquitin-protein ligases. Once ubiquitin is conjugated to the target proteins, other ubiquitin molecules are

successively conjugated to ubiquitin already bound to the target to generate polyubiquitin chains. (a) In one class of E3s, called HECT E3s, ubiquitin is first transferred to

a conserved Cys residue within E3 before being transferred to the target protein. (b) Most other E3s belong to the RING family of ligases. E3s in this class seem to

function as a scaffold for the binding of both E2s carrying ubiquitin and the target molecule, and facilitate the transfer of ubiquitin from E2 to the target protein.

Review Trends in Cell Biology July 2012, Vol. 22, No. 7

biological phenomena including B lymphocyte survival andlymphoid organogenesis. Upon stimulation, NF-kB-induc-ing kinase (NIK), which is degraded via cIAP-mediatedubiquitination in resting cells, is stabilized and phosphor-ylates IKKa. Phosphorylated IKKa induces phosphoryla-tion and ubiquitin-dependent processing (partialdegradation) of NF-kB2/p100 to p52, which allows translo-cation of the RelB/p52 heterodimer to the nucleus (Figure 2)[16]. Although the biological function of canonical and non-canonical NF-kB signaling is different, specific differences intarget genes has not been conclusively shown.

The ubiquitin-proteolytic pathway plays a crucial role inboth activation pathways as mentioned above; however,the involvement of nondegradative ubiquitin modificationshas been described only in the canonical pathway. Becausemost of the recent progress on the involvement of theubiquitin system in NF-kB activation is related to itsnondegradative functions [17], this review will discuss

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recent advances in the involvement of ubiquitination inthe canonical NF-kB activation pathway. To date, severalcontradictory results have been reported regarding thepotentially different roles of the variable polyubiquitinchains in the NF-kB activation pathway, especially theirroles in IKK activation [18,19]. Because the role of differentubiquitin chains in NF-kB activation has not yet beenconclusively revealed, we first summarize the reportedroles for K63, M1, and K11 chain modifications in thecanonical NF-kB activation pathway. Then, we speculateon the potential nondegradative roles of each ubiquitinmodification in the regulation of NF-kB based on currentknowledge.

K63-linked chains in NF-kB activationThe nondegradative role of ubiquitination in signal trans-duction was first identified in the involvement of K63 chainsin NF-kB activation, and this has since been extensively

TRAF2 TRADD

cIA

Ps RIP1

P P

PP

IKKβ

IκBαp65

(Rel A)

p65(Rel A)

NF-κB

p50

p50

MyD88

TRAF6

NEMO(IKKγ)

IKKα

IRAK4

IRAK1

K48-linked polyubiquitinationof lκBα

Degradationof lκBα

Ub Ub Ub Ub

IKKcomplex

Nucleartranslocation

DNA

Nucleus

TN

FR

IL-R

Ub Ub


TNF-α IL-1β

Figure 1. The canonical nuclear factor-kappa B (NF-kB) activation pathway. NF-kB (p65–p50 heterodimer in this figure) resides within the cytoplasm of resting cells in a

form bound to the inhibitor protein, IkBa. Upon binding to tumor necrosis factor (TNF)-a, TNF receptor-associated protein with a death domain (TRADD) and the

receptor-interacting protein 1 (RIP1) kinase are recruited to the TNF-receptor 1 (TNFR1). TRADD further recruits TNF receptor-associated factor 2 (TRAF2) and cellular

inhibitor of apoptosis proteins (cIAPs). In the case of IL-1b signaling, myeloid differentiation primary gene 88 (MyD88) is recruited to the IL-1 receptor (IL-1R), which

recruits IL-1b-associated kinases 1 and 4 (IRAK1 and IRAK4) to the receptor. IRAK1 binds to TRAF6. Because TRAF2, TRAF6, and cIAPs exhibit ubiquitin ligase activity, the

ubiquitin system is then involved in the signaling cascade leading to canonical IkB kinase (IKK) activation, namely IKKb phosphorylation. Activated IKK then

phosphorylates IkBa, which triggers K48-linked polyubiquitination of the protein leading to its degradation. This liberates NF-kB, which translocates to the nucleus and

induces the expression of target genes.


studied [20]. Because excellent reviews on the role of K63chains in NF-kB signaling have been published [7,8], we willonly briefly summarize the current hypothesis in TNF-a-and IL-1b-induced NF-kB activation. In TNF-a signaling,cIAPs or TRAF2 recruited to TNFR1 generates K63 chainson RIP1 [21]. IL-1b induces TRAF6-mediated K63 chaingeneration on TRAF6 itself and on IRAK1 (Figure 3a)[20,22]. Then, K63 chains recruit the TAK1–TAB1–TAB2/3 complex and the IKK complex to the receptor complexesvia K63-selective binding of TAB2/3 or NEMO, respectively[7]. TAK1 then phosphorylates IKKb, which leads to thephosphorylation and degradation of IkBs (Figure 3a) [23].Recently, unanchored K63 chains were proposed to be cru-cial for IKK activation [19]. However, several reports havesuggested that K63-linked chains may not play importantroles in canonical NF-kB activation. Ubc13–Uev1a, an E2complex, specifically generates K63 chains together withvarious E3s, including TRAF6 and TRAF2 [20]. NF-kBactivation mediated by TNF-a, IL-1b, and TLR ligands isnot overtly affected in cells isolated from Ubc13 knockoutmice, although activation of MAPKs by these stimuli isseverely impaired [24]. However, other reports suggestedthat IL-1b-induced IKK activation is heavily attenuated inUbc13 knockout mouse embryonic fibroblasts (MEFs) [25].Using ubiquitin-replacement technology, K63 chains were

shown to be indispensable for IL-1b-induced, but not TNF-a-induced, NF-kB activation [26]. Thus, although K63 chainsplay crucial roles in signaling and possibly in IL-1b-inducedNF-kB activation, they may not be essential for canonicalNF-kB activation, at least in some occasions, such as TNF-astimulation.

Involvement of linear (M1) chains in NF-kB activationRecently, involvement of a new type of ubiquitin modifica-tion, namely linear (M1, summarized in Box 2) polyubi-quitination, in NF-kB activation has emerged [18]. In thecase of linear chains, the linear ubiquitin chain assemblycomplex (LUBAC), composed of HOIP, HOIL-1L, andSHARPIN, determines the type of chain generated [27],although in the case of Lys-linked chains, E2s are believedto determine the type of ubiquitin chains [6,28]. TNF-a-induced nuclear localization of p65, a subunit of NF-kB, isheavily impaired in primary hepatocytes isolated fromHOIL-1L knockout mice [29]. Moreover, TNF-a-inducedIKK activation and expression of NF-kB target genes areseverely attenuated in MEFs from these knockout mice[29]. In mice, null mutation of SHARPIN causes chronicproliferative dermatitis (cpdm) [30]. MEFs from cpdm miceexhibit attenuated TNF-a-induced nuclear localizationof p65 and IKK activation. In addition, CD40- and

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Rel B

Rel B p52

Rel B

Nucleus

DNA

p52

Proteasomalprocessing

of p100/NF-κB2

Nucleartranslocation

p100

IKKα

(NF-κB2)

Rel B p100(NF-κB2)

IKKα

P P

PP

cIA

Ps

TRAF2 TRAF3

NIK

LT-β

R

LT-β

K48-linkedpolyubiquitinationof p100/NF-κB2

Ub Ub Ub UbUb Ub


Figure 2. The non-canonical NF-kB activation pathway. Upon stimulation with lymphotoxin-b receptor (LT-bR), or other receptors including CD40, NF-kB-inducing kinase

(NIK), which is degraded via cIAP-mediated ubiquitination in resting cells, is stabilized and phosphorylates IKKa. Phosphorylated IKKa induces phosphorylation and

ubiquitin-dependent processing (partial degradation) of NF-kB2/p100 to p52, which allows translocation of the RelB/p52 heterodimer to the nucleus.


LT-bR-induced canonical NF-kB activation is attenuatedin cells from cpdm and HOIL-1L knockout mice; non-canonical signaling is unimpaired [29,31,32]. One recentreport showed that TLR2 ligand-mediated IkBa degrada-tion is not heavily attenuated in bone marrow macro-phages from cpdm mice [33]. Biochemical and massspectrometry analyses revealed that NEMO was the sub-strate for linear polyubiquitination [29,31,32,34]. Indeed,linear polyubiquitination of NEMO is attenuated in cpdmMEFs [31]. The precise mechanism by which linear poly-ubiquitination of NEMO induces IKK activation has notyet been identified. However, because introduction ofNEMO conjugated to noncleavable linear polyubiquitincan activate NF-kB, but introduction of GFP conjugatedto linear polyubiquitin cannot, linearly polyubiquitinatedNEMO seems critical for activation of IKK [31]. Thus, thecurrent concept for LUBAC-mediated NF-kB activation isas follows: upon stimulation with agents such as TNF-a,IL-1b, or TLR ligands, LUBAC recognizes and linearlypolyubiquitinates NEMO, which induces IKK activationand subsequent degradation of IkBa (Figure 3b). Becauseloss of SHARPIN reduces signal-induced NF-kB activationand induces the phenotype of cpdm mice, M1 chains playcrucial roles in maintenance of cell health, possibly bybeing involved in NF-kB activation [17]. However, NF-kB activation induced by several stimuli is not completelyabolished in HOIL-1L knockout or cpdm mice[29,31,32,34]. Therefore, it is not yet clear whether linear

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polyubiquitination is indispensable for TNF-a- or IL-1b-mediated canonical IKK activation. Deleting the linearpolyubiquitination activity of LUBAC in mice, for exampleby knocking out HOIP (the catalytic subunit of LUBAC), isneeded to examine the precise role played by M1 chains inNF-kB activation.

K11 chains in NF-kB activationK11 chains are conjugated to the substrates of a specificubiquitin ligase, anaphase promoting complex (APC), andmark the conjugated proteins for degradation by the pro-teasome [35]. The specificity of K11 chain elongation isdetermined by an E2 called Ube2S [28]. However, K11chains can also be generated by cIAP ligases, together withUbcH5 s, and then conjugated to RIP1 [36,37]. In the caseof RIP1, K11 chains do not function as a degradationsignal, but are involved in NF-kB activation. Thus, cur-rently at least four different ubiquitin chains — K11, K48,K63, and M1 chains — are known to be involved in canoni-cal NF-kB activation. Of these, K63, K11, and M1 areinvolved in a nondegradative signaling process leadingto IKK activation (Figure 3c).

Functional differences between distinct ubiquitin chainsin NF-kB activationThe importance of, and the precise roles for, K63 andlinear chains in canonical NF-kB activation are currentlyunder extensive debate. In the canonical NF-kB activation

(a)

IRAK4

IRAK1

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IKKβ

TRADDTRAF2

TRAF6

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IKKcomplex

PP

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K11 chain

cIA

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M1 chain

K63 chain

UbUb

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TAB2

IL-R IL-R

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P

HOIL-1L

Ub Ub Ub Ub Ub

UbUb

UbUb

UbUb

UbUb

UbUb

HOIPNEMO(IKKγ)

NEM

O

(IKKγ)

NE

MO

(IKK

γ)

SHARPIN

P

PP


IL-1β IL-1β TNF-α TNF- α

Figure 3. Current hypothetical roles of K63, M1, and K11 chains in NF-kB activation. (a) K63-linked chains generated upon stimulation by molecules such as IL-1b or TNF-a

recruit the TAK1–TAB1–TAB2/3 complex and the IKK complex through the K63-selective binding of TAB2/3 or NEMO (NF-kB essential modulator), respectively. Then, TAK1

may phosphorylate IKKb, which leads to the phosphorylation and degradation of IkBs. Although TAB2/3 is selectively bound to K63 chains, NEMO is shown to bind to M1

chains more efficiently than to K63 chains. Also, the structural analysis of IKKb suggests that IKKb is activated by trans-autophosphorylation. (b) Upon stimulation by

various agents, including TNF-a and IL-1b, linear ubiquitin chain assembly complex (LUBAC), which comprises HOIL-1L, HOIP, and SHARPIN, recognizes and linearly

polyubiquitinates NEMO, which may induce IKK activation leading to the degradation of IkBa. However, the mechanism underlying IKK activation by linear ubiquitination of

NEMO has not been described, and there is evidence that TLR2 ligand-mediated IkBa degradation is not heavily attenuated in bone marrow macrophages in SHARPIN-

deficient cpdm mice. (c) Upon stimulation by TNF-a, c-IAPs conjugates K11 chains to RIP1, which might induce IKK activation and lead to the degradation of IkBa. Because

involvement of K11 chains in IKK activation is not yet well established, the precise role of K11 chains has not been elucidated.


pathway, IKK is activated by phosphorylation of IKKb

[10,15]. Here, I discuss the roles of different ubiquitinchains in the phosphorylation of IKKb. Most kinases,including IKKb, are activated by phosphorylation, whichis achieved either by trans-autophosphorylation or byupstream kinases [38]. It is hypothesized that TAK1 isan upstream kinase that phosphorylates IKKb [14]. In-deed, TAK1 phosphorylates IKKb in vitro, and TNF-a-and IL-1b-induced NF-kB activation is almost completelyabolished in TAK1-deficient MEFs [20,39,40]. However,canonical NF-kB activation is somewhat augmented inTAK1-null neutrophils [41], which suggests that TAK1 isnot the sole upstream kinase for IKKb or that IKKb mightbe activated also by trans-autophosphorylation. The crys-tal structure of IKKb was identified recently and showsthat IKKb possesses a dimerization domain. Dimeriza-tion-defective IKKb mutants fail to be activated [42].Therefore, IKK might be phosphorylated by trans-autop-hosphorylation, and upstream kinases for IKKb may notbe necessary. TAK1 forms a complex with TAB1 andTAB2/TAB3 [40]. Because TAK1 is recruited to the stim-ulated receptor complex by K63 chains, owing to the K63-specific binding activity of TAB2 and TAB3 [7], K63chains may not be always indispensable for IKKb phos-phorylation. The finding that K63-linked chains are dis-pensable for TNF-a-mediated NF-kB activation, butindispensable for activation via IL-1b, may supportthis notion [26]. However, K63 chains may play crucialroles in canonical NF-kB activation in some settings asdiscussed below.

NEMO, which is a regulatory subunit of IKK, is anindispensable component of canonical IKK activation[10,15]. The ubiquitin binding activity of NEMO is aprerequisite for IKK activation [43,44]. NEMO possessestwo ubiquitin binding domains: one is located in themiddle of the protein, referred to as the UBAN, NOA,NUB or CoZi domain [45]; the other is in the C-terminalzinc finger (ZF) domain [46]. NEMO constructs with muta-tions in the UBAN domain, or deletion of the ZF, cannotrestore the signal-dependent activation of NF-kB inNEMO-deficient cells [46,47]. Incubation of polyubiquitinchains with IKK complexes containing wild type (WT)NEMO, but not a NEMO mutant defective for ubiquitinbinding, activates IKK and IkBa phosphorylation [19].Mutation of NEMO is causative for an inherited diseasecalled anhidrotic ectodermal dysplasia with immunodefi-ciency (EDA-ID) [48]. Mutations in the UBAN and ZFdomains are found in EDA-ID, which further supportsthe importance of the ubiquitin-binding properties ofNEMO in IKK activation [47,49]. The binding specificityof NEMO to ubiquitin chains remains controversial. TheUBAN domain preferably binds to linear di-ubiquitin (Kd

= 1.4 mM) rather than K63 linked di-ubiquitin (Kd =131 mM) [50]. Structural analyses confirm high-affinitybinding of the UBAN domain to linear di-ubiquitin[47,50]. However, in the case of longer ubiquitin chains,full-length NEMO also bound to other types of ubiquitinchains, including K11 and K63 linkages [36,46].

NEMO itself is also ubiquitinated. K63-linked chainsare conjugated onto NEMO upon stimulation of NOD2 or

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Box 2. The unique features of linear (M1) polyubiquitin

chains

As described (Box 1), M1 chains are generated by conjugating the

carboxyl group of a ubiquitin monomer to the a-amino group of the

first Met residue of another ubiquitin monomer. Although it has

been suggested that E2 enzymes determine the type of polyubiqui-

tin chain in the case of Lys-linked chains [85] [e.g. E2 complexes

containing Ubc13 (Ubc13-Uev1a and Ubc13-MMS2) only generate

K63-linked chains], the specificity of M1 chains is determined by the

E3 ligase [27]. To date, LUBAC, composed of HOIL-1L, HOIP, and

SHARPIN, is the only reported E3 known to specifically generate M1

chains [17]. This is advantageous for probing the roles of specific

ubiquitin chains within an organism, because E2 enzymes can bind

to several E3 enzymes and conjugate polyubiquitin chains to

substrates specifically recognized by those E3s. For example, the

E2 complex containing Ubc13 can conjugate K63-linked chains to

numerous substrates [86], which suggests that K63 chains play a

wide variety of roles. Moreover, K63 chains can be generated by

other E2 enzymes, such as UbcH5 s [87]. Until now, genetic analysis

could not readily be applied to probe the function of specific Lys-

linked chains within organisms. However, linear polyubiquitination

as discussed above enables the use of genetic analysis techniques

to examine the function of linear chains.


TCRs [51–53]. NEMO is also the major target for linearpolyubiquitination by LUBAC, which is suspected to playcrucial roles in IKK activation [29,31,32,34].

Considering these observations, several possible differ-ential roles of variable ubiquitin chains exist. So far,genetic evidence suggests that M1 chains are involved inNF-kB activation. siRNA-mediated knockdown of HOIL-1L in SHARPIN-deficient cpdm MEFs attenuates IL-1b-and TNF-a-mediated IkBa degradation almost completely.Therefore, it might be the case that M1 chains play acentral role in IKK activation upon stimulation with IL-1b or TNF-a [31]. Because linear polyubiquitin chains areconjugated and recognized by NEMO, it may be possiblethat M1 chains conjugated to NEMO are recognized byNEMO in trans in another IKK complex, thereby inducingmultimerization of the IKK complex. Upon multimeriza-tion, IKKb could dimerize and be phosphorylated by trans-autophosphorylation (Figure 4a) [18]. Alternatively, it maybe plausible that binding of ubiquitin to the UBAN domaincould induce conformational changes in NEMO [47], there-by changing the positions of IKKa and IKKb and leading tophosphorylation of IKKb (Figure 4b) [18]. Considering theresults of structural analyses of IKKb [42], the formerseems the more likely scenario. Although it might bepossible that LUBAC-mediated linear polyubiquitinationplays a central role in IKKb phosphorylation in someoccasions, including TNF-a stimulation, other mecha-nism(s) could also collaterally exist in the canonical path-way for NF-kB activation. M1 chains might be dispensablefor canonical NF-kB activation in some instances becauseit has not been shown whether LUBAC is recruited to anyreceptor complex that induces canonical NF-kB activation.K63 and K11 chains are conjugated to components of thereceptor complex upon stimulation with various agents.Thus, it might be plausible that, in cases when a longpolyubiquitin chain is generated upon stimulationwith various stimuli, including TNF-a or IL-1b, multipleNEMO proteins could recognize one K63- or K11-linkedpolyubiquitin chain, and induce multimerization of the

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IKK complex leading to phosphorylation of IKKb

(Figure 4c,d) [36,46]. The possibility that upstreamkinases, including TAK1, are recruited to the activatedreceptor complex together with the IKK complex by K63chains to phosphorylate IKKb cannot be ruled out(Figure 3a) [18].

Both K63 and M1 chains are necessary for IL-1b-medi-ated NF-kB activation [26,31]. K11 chains are generatedby cIAPs, which are shown to be indispensable for TNF-a-mediated NF-kB activation by generating variable chainsincluding K63 and K48 chains [36,54,55]. Therefore, K11,M1, and possibly K63 chains seem necessary for TNF-a-mediated NF-kB activation [31,36]. It has been reportedthat loss of cIAPs, which generate both K63 and K11chains, heavily attenuated recruitment of LUBAC toTNFR1 receptor complex in TNF-a-signaling [56], sug-gesting that LUBAC is involved in downstream signalingevents in TNF-a-mediated NF-kB activation. Moreover,profound suppression of LUBAC expression suppressesNF-kB activation induced by both IL-1b and TNF-a al-most completely [31]. Considering that M1 chains areconjugated to and recognized by NEMO, a crucial regula-tor of the canonical IKK complex [29,47], M1 chains mightplay a role in direct activation of IKK, via IKKb phos-phorylation, upon stimulation with IL-1b or TNF-a. If so,what are the roles of K63 and K11 chains in IL-1b- or TNF-a-mediated NF-kB activation? K63 chains are shown to beindispensable for IL-1b-induced NF-kB activation [26].The ligase activity of cIAPs is necessary for recruitmentof LUBAC to the TNF receptor complex as mentionedabove, and LUBAC possesses ubiquitin binding activity[56,57]. Indeed, HOIP, a component of LUBAC, has affini-ty for K63 chains [56], although it has not been shownwhether LUBAC can bind to K11 chains. Therefore, it istempting to speculate that K63 and K11 chains function torecruit LUBAC to the IL-1 receptor or TNFR complex andinduce NF-kB activation (Figure 5). In addition to NEMO,RIP1 is linearly ubiquitinated by LUBAC upon TNF-astimulation [32]. It has been suggested that the ligaseactivity of LUBAC is necessary for stabilizing the TNFreceptor signaling complex [56]; therefore, linear chainsmight be involved in stabilization of the TNF signalingcomplex by being conjugated to RIP1 and lead to NF-kBactivation. However, further work will be needed to clarifythe differential roles of distinct ubiquitin chains in NF-kBactivation.

Role of deubiquitinating enzymes in NF-kB activationThe roles played by deubiquitinating enzymes (DUBs) in thecanonical NF-kB activation pathway are briefly discussedhere. In the canonical pathway, DUBs, which disassemblepolyubiquitin chains or remove ubiquitin from a substrate,are involved in downregulating NF-kB [58,59]. A20, whichpossesses deubiquitinating activity, is induced by NF-kB[60]. Deletion of A20 in mice results in premature lethalitywith massive systemic inflammation and cachexia as aresult of deregulated NF-kB activation [61]. A20 has ubi-quitin ligase activity in addition to its deubiquitinatingactivity; A20 suppresses NF-kB by deubiquitinating K63chains on RIP1, a crucial molecule in TNF-a-mediated NF-kB activation, and marking the protein for degradation by

IKKα

IKKα

IKKβIKKβ

IKKβ

IKKcomplex

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IL-R

TN

FR

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FR

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Conformationalchange

(a) (b)

PP PPP

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Ub Ub Ub Ub UbHOIP

NEMO(IKKγ)

NEMO(IKKγ)

SHARPIN

IKKαIKKαIKKβ

IKKβ

IKKcomplex

M1 chain

HOIL-1L

Ub Ub Ub Ub UbHOIP

NEMO(IKKγ)

NEMO(IKKγ)

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P

(c) (d)

IRAK4 TRADD

clA

PsIRAK1

MyD88

RIP1TRAF2

IKKβIKKβ

IKK

βIK

Kβ

IKKα

IKKβIKKβ

IKKα

IKKα

IKKα IKKα

IKKα

TRAF6

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P

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P

K63 chain K63 chainK11

chain

UbUb

UbUb

UbUb

UbUb

UbUb

UbUb

Ub

Ub

UbUb

UbUb

UbUb

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IL-R

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(IKKγ)

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TNF-α IL-1 β TNF- αIL-1β

TNF-αIL-1β

Figure 4. Possible roles of the different polyubiquitin chains in IKK activation. (a) M1 chains conjugated to NEMO could be recognized by NEMO within another IKK complex

and induce multimerization of the IKK complex. Upon multimerization, IKKb dimerizes and is phosphorylated by trans-autophosphorylation. (b) M1 chains conjugated to

NEMO could be recognized by the UBAN motif of another NEMO molecule. Binding of M1 chains to the UBAN domain would induce conformational changes in NEMO,

which affects the position of IKKa and IKKb leading to the phosphorylation of IKKb. (c) Long K63 chains that are conjugated to components of the IL-1 signaling complex

upon stimulation could be recognized by multiple NEMO proteins, inducing multimerization of the IKK complex and leading to phosphorylation of IKKb. (d) Long K11 or K63

chains conjugated to RIP1 upon stimulation with TNF-a could be recognized by multiple NEMO proteins, inducing multimerization of the IKK complex and leading to

phosphorylation of IKKb.


conjugating K48 chains [62]. An alternative mechanism forNF-kB suppression by A20 has also been suggested, where-by A20 disassembles binding between TRAF6, TRAF2 orcIAPs, and E2s, induces degradation of the E2s (UbcH5 sand Ubc13) by the proteasome, and inhibits ubiquination byother E3s. The deubiquitinating activity and, possibly, E3activity of A20 appears indispensable for this function [63],whereas A20 binding to the ubiquitin ligases RNF11 andItch is shown to be required [64,65]. Recently, a deubiqui-

tination-independent mechanism for suppression of NF-kBby A20 has also emerged. In this mechanism, A20 sup-presses NF-kB activation by binding to NEMO in a ubiquitinchain-dependent manner and suppresses IKK activation ina process that does not involve the deubiquitination activityof A20 [66]. A cylindromatosis gene product, CYLD, func-tions as a DUB, and the cyld gene predisposes to tumors ofskin appendages [67–69]. CYLD mutants lacking deubiqui-tination are unable to suppress NF-kB activation [59].

361

TRADD IRAK4

TN

FR

IL-R

IRAK1

TRAF2

MyD88

Ub

UbUb

Ub

UbUb

Ub

Ub

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Ub HO

IP

HO

IP

HO

IL-1L

IKKα

IKKα

IKKβ

IKK

βH

OIL-1L

SH

AR

PIN

NE

MO

(IKKγ)

NE

MO

(IKKγ)

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AR

PIN

TRAF6

RIP1

K11 chain K63 chain

cIA

Ps Ub

Ub

UbUb

UbUb

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Ub

UbUbUb

UbUb

UbUb

Ub

Ub


TNF-α IL-1β

Figure 5. Possible roles of K63 and K11 chains in NF-kB activation besides IKK

activation. K63-linked chains that are conjugated to components of the IL-1b- or

TNF-a-signaling complex upon stimulation, and K11-linked chains conjugated to

RIP1 by cIAP ligases in TNF-a-signaling complex, may recruit LUBAC to the IL-1

receptor or the TNF receptor, respectively, and induce linear polyubiquitination of

NEMO. Linearly polyubiquitinated NEMO could induce IKKb phosphorylation.


Because CYLD cleaves not only K63 but also M1 chains [70],CYLD suppresses NF-kB by cleaving these chains. In addi-tion to A20 and CYLD, the DUBs Cezanne and USP21 arealso involved in suppression of NF-kB activation by remov-ing ubiquitin chains from RIP1 [71,72]. However, the precisemechanism underlying the action of these DUBs in NF-kBsignaling has not been clarified.

Concluding remarksThis short article discussed several mechanisms involvedin the ubiquitin conjugation system in the canonical NF-kBactivation pathway that activate the IKK complex com-posed of IKKa, IKKb, and NEMO [10,15]. It might be thecase that M1 chains play a major role in IKKb phosphor-ylation upon TNF-a or IL-1b stimulation. However, otherubiquitin chains also may play a major role upon stimula-tion with other molecules, such as the TCR [73]. In addi-tion, even after stimulation with TNF-a or IL-1b, otherubiquitin chains may play a role in activation of IKK insome cases. A specific ubiquitin chain exerts its distinctfunctions via recognition by ubiquitin binding domainsthat display preferential binding activity for that specificchain [44,74]. M1 chains expressed in Saccharomyces cer-evisiae, in which M1 chains do not exist endogenously,function as a degradation signal, albeit weakly [75].

362

Because specific binding domains for M1 chains may notexist in yeast owing to the lack of M1 chains, M1 chainsmight function as a weak degradation signal by bindingwith weak affinity to ubiquitin-binding domains on theproteasome that recognize K48 chains [76]. Thus, canoni-cal IKK activation induced by stimuli such as TNF-a- or IL-1b might not be completely abolished in cells lackingsynthesis of M1 chains because long K63 or K11 chainsmay be recognized by NEMO, which activates IKK(Figure 5c,d). The ubiquitin conjugation system is a regu-latory mechanism that modulates protein function [77].Although the structures of polyubiquitin chains differ fromeach other, there are many similarities, and the ubiquitinbinding domains that preferentially bind to one chain mayrecognize other chains, albeit with lower affinity. There-fore, in the canonical NF-kB activation pathway, severalchains might be redundantly involved in phosphorylationof IKKb or the signaling cascades leading from stimulationto IKKb phosphorylation, although M1 chains might playpredominant roles in phosphorylation of IKKb possibly bytrans-autophosphorylation in the case of TNF-a signaling.

AcknowledgmentsThe work in my laboratory was supported by Grants-in-Aid from theMinistry of Education, Culture, Sports, Science and Technology of Japan,and the CREST Japan Science Technology Corporation.

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Diverse ubiquitin signaling in NF-κB activation

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