[advances in immunology] volume 124 || the ubiquitin system in immune regulation

CHAPTER TWO

The Ubiquitin System in ImmuneRegulationYoon Park, Hyung-seung Jin, Daisuke Aki, Jeeho Lee, Yun-Cai Liu1La Jolla Institute for Allergy and Immunology, La Jolla, California, USA1Corresponding author: e-mail address: [email protected]

Contents

1. Introduction 181.1 The ubiquitin system 181.2 Deubiquitination 191.3 Protein ubiquitination in the immune responses 20

2. E3 Ligases in T-Cell Activation and Anergy 212.1 Cbl-b 232.2 Itch 242.3 GRAIL 252.4 TRAF6 262.5 Peli1 272.6 Roquin 28

3. E3 Ligases in T-Cell Differentiation 283.1 Tregs 293.2 Th1 cells 323.3 Th2 cells 323.4 Th17 cells 33

4. Ubiquitination in NF-κB Signaling 344.1 TNFR1 signaling 344.2 IL-1R/TLR4 signaling 404.3 T-cell receptor signaling 424.4 Noncanonical NF-κB signaling: CD40 45

5. Ubiquitination in Hematopoiesis 465.1 E2 enzyme 475.2 RING finger E3s 475.3 HECT-type E3 505.4 Deubiquitinating enzymes 51

6. Concluding Remarks 51Acknowledgments 53References 53

Advances in Immunology, Volume 124 # 2014 Elsevier Inc.ISSN 0065-2776 All rights reserved.http://dx.doi.org/10.1016/B978-0-12-800147-9.00002-9

17

http://dx.doi.org/10.1016/B978-0-12-800147-9.00002-9

Abstract

The ubiquitin system plays a pivotal role in the regulation of immune responses. Thissystem includes a large family of E3 ubiquitin ligases of over 700 proteins and about100 deubiquitinating enzymes, with the majority of their biological functions remainingunknown. Over the last decade, through a combination of genetic, biochemical, andmolecular approaches, tremendous progress has been made in our understandingof how the process of protein ubiquitination and its reversal deubiquitination controlsthe basic aspect of the immune system including lymphocyte development, differen-tiation, activation, and tolerance induction and regulates the pathophysiological abnor-malities such as autoimmunity, allergy, and malignant formation. In this review, weselected some of the published literature to discuss the roles of protein–ubiquitin con-jugation and deubiquitination in T-cell activation and anergy, regulatory T-cell andT-helper cell differentiation, regulation of NF-κB signaling, and hematopoiesis in bothnormal and dysregulated conditions. A comprehensive understanding of the relation-ship between the ubiquitin system and immunity will provide insight into themolecularmechanisms of immune regulation and at the same time will advance new therapeuticintervention for human immunological diseases.

1. INTRODUCTION

1.1. The ubiquitin systemUbiquitin conjugation to a protein substrate or protein ubiquitination is a

fundamental regulatory mechanism for various cellular processes including

signal transduction, cell cycle control, transcriptional regulation, antigen

presentation, and apoptosis (Hershko & Ciechanover, 1998). A three-step

enzymatic cascade is initiated by the activation of the 76-amino acid poly-

peptide ubiquitin by forming thioester bond between the C-terminal gly-

cine of ubiquitin and an active cysteine group of ubiquitin-activating

enzyme (E1); the activated ubiquitin is then transferred to a ubiquitin-

conjugating enzyme (E2) via the formation of E2-ubiquitin thioester inter-

mediate; in the final step, a ubiquitin–protein ligase (E3) recruits both

ubiquitin-bound E2 and the target protein and promotes ubiquitin transfer

to the substrate by catalyzing an isopeptide bond formation between the

C-terminal glycine of ubiquitin and the ε-amino group of a lysine residue

on the substrate (Pickart, 2001). Ubiquitin has seven lysine residues (K6,

K11, K27, K29, K33, K48, and K63), and any one of them can be conju-

gated to another ubiquitin, which leads to the formation of polyubiquitin

chain of different linkages. In addition, linear polyubiquitin chains can be

generated by the conjugation between the amino terminal methionine

18 Yoon Park et al.

residue of the proceeding ubiquitin and the C-terminal glycine residue of

the incoming ubiquitin (Komander, 2009). The linkage of polyubiquitin

chains adopts distinct functions in diverse cellular processes. For example,

K48- and K11-linked polyubiquitin chains directly target proteins to

undergo proteasomal degradation, whereas modification of target proteins

by K63-linked polyubiquitin chains leads to the regulation of cellular func-

tions such as signal transduction and DNA repair via proteasome-

independent mechanism (Ikeda & Dikic, 2008; Pickart & Fushman,

2004). E3s, which have a total number of over 700 in human genome,

are mainly responsible for determining a substrate specificity and are gener-

ally subdivided into two classes based on the type of protein domain required

for the substrate recognition: the homologous to the E6-associated protein

C-terminus (HECT) and really interesting new gene (RING) domain-

containing E3s. The HECT domain-containing E3s form a thioester bond

with ubiquitin through a conserved cysteine residue within HECT domain

and directly transfer the ubiquitin to target proteins, which contribute to

determine the specificity of ubiquitin chains. The RING domain-

containing E3s direct the transfer of ubiquitin from the ubiquitin loaded

E2 to the substrate via RING domain (Pickart, 2001). Thus, the RING

E3s often contain ubiquitin-binding domains (UBDs) that bind different

types of ubiquitin linkages and influence the fate of target proteins.

1.2. DeubiquitinationUbiquitination is a reversible process, in which the attached ubiquitin chains

can be removed by protease-termed deubiquitinating enzymes (DUBs).

There are approximately 100 DUBs in the human genome, and they can

be categorized into five subclasses depending on their ubiquitin–protease

domains: ubiquitin-specific protease (USP), ubiquitin C-terminal hydrolase,

otubain protease (OTU), Machado–Joseph disease protease, and JAB1/

MPN/Mov34 metalloenzyme. In addition to protease domain, DUBs also

contain UBDs including the zinc finger USP (ZnF-USP) domain, the

ubiquitin-interacting motif, and the ubiquitin-associated domain (UBA),

which mediate the recognition and interaction to specific substrates and

ubiquitin linkages (Komander, Clague, & Urbe, 2009). Three functional

categories of DUBs have been known in the regulation of ubiquitin-

mediated cellular processes: DUBs cleave ubiquitin precursors, which is a

linear fusion of ubiquitin proteins, to generate free ubiquitin; DUBs can res-

cue proteins from degradation and also reverse signaling and trafficking by

19Ubiquitin System in Immune Regulation

removal of ubiquitin chains from substrates that are posttranslationally mod-

ified; and DUBs can contribute to edit ubiquitin signals by modifying

ubiquitin chains (Komander, Clague, et al., 2009; Reyes-Turcu,

Ventii, & Wilkinson, 2009). Although genetic and biochemical studies

reveal an important role of DUBs such as OTU DUB A20 and USP

DUB CYLD that negatively regulate NF-κB signaling by deubiquitinating

signaling molecules in the immune response (Sun, 2008), physiological

functions and target substrates of most DUBs have not been identified yet.

1.3. Protein ubiquitination in the immune responsesThemulticellular organisms, including plants, invertebrates, and vertebrates,

developed intrinsic immune mechanisms to defend themselves against exog-

enous enemies such as microorganisms (microbes) and parasites, collectively

called infectious pathogens. The innate immune response is triggered by the

encounter of germ line-encoded pattern recognition receptors in the host

with the pathogen-derived substances. These receptors recognize conserved

microbial molecules called pathogen-associated molecular patterns (PAMPs)

that are found only in microbes, which lead to the elimination of the invad-

ing microbes by initiating gene transcription of antimicrobial molecules or

proinflammatory cytokines ( Janeway & Medzhitov, 2002). In vertebrates,

innate immune response mounts the adaptive immune response to effi-

ciently respond to distinct microbes and to provide prolonged protection.

The adaptive immune response triggered by pathogens produces

pathogen-specific receptors such as the T-cell receptor and the B-cell

receptor through somatic DNA rearrangement, or humoral antibodies,

which are exclusively programmed to eradicate infectious pathogens

(Medzhitov & Janeway, 1998).

Ubiquitin system has been known to be intimately associated with both

innate and adaptive immune responses via playing a pivotal role in the reg-

ulation of immune tolerance, immune cell development, T-cell differenti-

ation, antigen- or cytokine-induced intracellular signaling pathways, and

hematopoiesis. Over the last decade, there are increasing amounts of liter-

ature documenting the role of the ubiquitin system in many aspects of the

immune regulation and it would be impossible to cite each of them in this

limited forum. In this chapter, we have selected some of the published works

as examples and discuss the current understanding of how protein

ubiquitination or deubiquitination controls diverse immune responses.

20 Yoon Park et al.

2. E3 LIGASES IN T-CELL ACTIVATION AND ANERGY

Both the engagement of T-cell receptor (TCR) by antigenic peptide in

the context of major histocompatibility complex (MHC) and costimulatory

molecules (i.e., CD28) are needed for the complete activation of T cells

(Smith-Garvin, Koretzky, & Jordan, 2009). Binding of the TCR to antigen

conjugated to MHC leads to recruitment of Lck and ZAP-70 to the cytoplas-

mic tailsof the invariantTCRsubunits.ZAP-70 subsequentlyphosphorylates a

transmembraneprotein,LAT.Uponphosphorylation,LATserves as a docking

site for multiple adaptor proteins, Shc–Grb2–SOS, phosphatidylinositol-3

kinase (PI3K), and phospholipase C-γ1. Phospholipase C-γ1 cleaves the

membrane phospholipid phosphatidylinositol-4,5-biphosphate into inosi-

tol 1,4,5-triphosphate (IP3) and diacylglycerol (DAG). IP3 interactswith its

receptor and triggers the opening of the Ca2+ channel and thus release of

Ca2+ into the cytoplasm. TCR-induced DAG production results in the

activationof PKC/Ras/MAPKsignalingpathway.Costimulation provided

by binding of CD28 to its ligands, CD80 or CD86, induces the expression

and increased stabilization of IL-2 mRNA (Appleman & Boussiotis, 2003).

CD28 costimulation is also important for the clustering of T-cell surface

receptors, leading to the formation of supramolecular activation clusters

(SMACs). SMAC formation is an essential process for optimal IL-2 produc-

tion and cell proliferation in T cells. These signaling pathways eventually

lead to cytokine production, clonal expansion, and increased survival of

naı̈ve T cells (Frauwirth & Thompson, 2002). In the case that only antigen

presentation occurs without costimulation, T cells fail to become fully acti-

vated and enter a state of anergy, which is a long-lived, unresponsive state

that depends on Ca2+-mediated signals (Rao, 2009). NFAT drives the

expression of anergy-associated genes that inhibit T-cell activation and

establish the anergic state at different levels. The ubiquitin ligases, gene

related to anergy in lymphocytes (GRAIL), Itch, and Casitas B-cell

lymphoma-b (Cbl-b), block TCR signaling cascades through targeted deg-

radation of the signaling molecules in the anergic T cells (Heissmeyer et al.,

2004; Jeon et al., 2004). Studies that combine genetic and biochemical

approaches have provided accumulated evidence that protein

ubiquitination is a crucialmechanism that controlsT-cell activation and tol-

erance. Here, we discuss the physiological roles of several E3 ubiquitin

ligases in the regulation of T-cell activation and anergy (Fig. 2.1).


Figure 2.1 Regulation of T-cell activation and anergy by the ubiquitin system.Costimulation of CD28 and the TCR complex triggers different signaling pathways thatresult in the activation of transcription factors NFAT, NF-κB, and AP-1. These transcrip-tion factors translocate to the nucleus and cooperate with each other to induce the tran-scription of several genes such as IL-2. In the absence of costimulatory signals, NFATinduces the expression of numerous anergy-associated genes, including thoseencoding several E3 ubiquitin ligases (Cbl-b, Itch, and GRAIL). These E3 ubiquitin ligasespromote the sequestration or degradation of signaling molecules that are essential forT-cell activation. Cbl-b, Itch, and GRAIL promote the degradation of PLCγ1 and PKCθ,thus terminating the signaling downstream of LAT. Cbl-b is also known to directly targetthe p85 subunit of PI3K (phosphoinositide 3-kinase) and attenuate CD28 costimulatorysignaling cascades. Peli1 mediates the ubiquitination and degradation of c-Rel, leadingto inhibition of cytokine production such as IL-2.

22 Yoon Park et al.

2.1. Cbl-bCbl-b, a RING-type E3 ubiquitin ligase, plays an essential role in the reg-

ulation of T-cell activation, immunotolerance, and autoimmunity (Liu &

Gu, 2002; Paolino & Penninger, 2010). Cbl-b-deficient T cells are hyper-

proliferative and able to be fully activated even in the absence of CD28

costimulation, suggesting that Cbl-b can uncouple T-cell activation from

the requirement for CD28 costimulation (Bachmaier et al., 2000; Chiang

et al., 2000). Accordingly, loss of Cbl-b restores impaired T-cell prolifera-

tion in CD28�/� mice (Bachmaier et al., 2000; Chiang et al., 2000).

Paolino et al. (2011) showed a loss-of-function mutation in the Cbl-b

RING finger domain in mice phenocopies, the Cbl-b-knockout pheno-

type, indicating that Cbl-b physiological functions are mediated by the cat-

alytic E3 ligase activity. At the molecular level, Cbl-b functions as a

gatekeeper to prevent the undesired activation of T cells through the

ubiquitination of its target substrates. Cbl-b directly binds and ubiquitinates

p85, the regulatory subunit of PI3K. Nondegradative ubiquitination of p85

prevents its recruitment to CD28, thus inhibiting CD28-triggered PI3K–

Akt activation (Fang & Liu, 2001). A recent study by Guo et al. (2012)

has reported that Pten activity was decreased and PI3K activity was not

increased in Cbl-b�/� T cells upon TCR stimulation. Given that PI3K–

Akt pathway is negatively regulated by Pten (Manning & Cantley, 2007),

it is possible that the heightened activation of Akt in Cbl-b�/� T cells

may be due to reduced Pten activity. In the study, they showed that Cbl-b

suppresses Nedd4 (neural precursor cell expressed, developmentally down-

regulated 4, a HECT E3 ubiquitin ligase)-mediated Pten ubiquitination by

impeding the binding of Pten to Nedd4. Intriguingly, Nedd4 has been also

reported to target Cbl-b for ubiquitin-mediated degradation upon CD28

costimulation (Yang et al., 2008). Thus, the exact biological function of

the Nedd4 and Cbl-b interaction remains to be established. Additionally,

Cbl-b inhibits Vav1-mediated cytoskeleton rearrangements required for

receptor clustering and synapse formation (Krawczyk et al., 2000). Further-

more, Cbl-b-dependent ubiquitination of the adaptor Crk-L represses

T-cell activation by preventing its association with the guanine exchange

factor C3G, thus inhibiting Crk-L/C3G-mediated Rap1 and LFA-1 activa-

tion (Zhang et al., 2003). Cbl-b itself becomes ubiquitinated and degraded

through the proteasome upon CD28 triggering (Zhang et al., 2002). Gruber

et al. (2009) have shown that the defects in the response of PKCθ�/�T cells to activation in vitro and the resistance of PKCθ�/� mice to


experimental autoimmune encephalomyelitis (EAE) in vivo could be rescued

by the concomitant loss of Cbl-b. Mechanistically, TCR and CD28

costimulation induces PKCθ-mediated phosphorylation of Cbl-b, leading

to ubiquitin-dependent degradation of Cbl-b. Cbl-b is upregulated in

anergized T cells (Heissmeyer et al., 2004; Jeon et al., 2004). Cbl-b-deficient

T cells are resistant to ionomycin-induced anergy. It has been suggested that

PLCγ1 and PKCθ are also relevant substrates for Cbl-b anergic functions

(Heissmeyer et al., 2004; Jeon et al., 2004). In support of in vitro data,

Cbl-b-knockout mice cannot be tolerized in vivo ( Jeon et al., 2004). For

instance, while repeated challenge of P14+ TCR-transgenic mice to the

cognate p33 antigen results in T-cell anergy induction, Cbl-b-deficient

P14+ TCR-transgenic mice challenged with p33 exhibited the massive

activation of CD8+ T cells and significant morality mediated by cytokine

storm. Together with the impaired tolerance phenotype, Cbl-b-knockout

mice develop spontaneous autoimmunity and are highly susceptible to

experimentally induced autoimmune disorders such as encephalomyelitis,

arthritis, and diabetes (Chiang et al., 2000; Gronski et al., 2004; Jeon

et al., 2004). Still, future studies are required to uncover the essential sub-

strates and complex mechanisms underlying the role of Cbl-b in T-cell acti-

vation and tolerance.

2.2. ItchItch is a HECT-type E3 ubiquitin ligase involved in the regulation of

immune responses (Liu, 2007), as Itch-deficient mice develop a skin-

scratching phenotype and immunological disorders, manifested by hyper-

plasia of lymphoid organs, and inflammation in the lungs and digestive tract

(Perry et al., 1998). The inflammatory phenotype is associated with a

T helper cell type 2 (Th2)-biased differentiation and a concomitant increase

in Th2 cytokines like IL-4 and IL-5. Itch�/�mice also have higher levels of

IgG1 and IgE as compared with wild-type mice. Itch controls Th2 differ-

entiation by binding and mediating ubiquitin-dependent degradation of

JunB (Fang et al., 2002; Venuprasad et al., 2006). The results are consistent

with previous studies showing increased Th2 differentiation in JunB

transgenic mice and decreased Th2 immune responses in JunB gene-

targeted mice (Hartenstein et al., 2002; Li, Tournier, Davis, & Flavell,

1999). Another study showed that Itch is able to regulate TCR respon-

siveness through promoting the ubiquitination and degradation of BCL10

(Scharschmidt, Wegener, Heissmeyer, Rao, & Krappmann, 2004).

24 Yoon Park et al.

Recently, it was found that Itch and Cbl-b cooperatively induce K33-linked

polyubiquitination of TCR-ζ in a proteolysis-independent manner. This

modification inhibits TCR-ζ phosphorylation and association with the ζchain-associated protein kinase Zap-70 and thereby prevents the activation

of TCR signaling pathway (Huang et al., 2010). Interestingly, the E3 ligase

activity and function of Itch are regulated by multiple mechanisms.

MEKK1–JNK1 signaling induces phosphorylation of Itch and subsequent

activation of its ligase activity (Gao et al., 2004). In contrast, tyrosine phos-

phorylation of Itch induced by Fyn negatively modulates its function by the

inhibition of the association with the substrate JunB (Yang et al., 2006). In

addition, Nedd4 family-interacting protein 1 (Ndfip1) functions as an acti-

vator for Itch (Oliver et al., 2006). Besides its role in T-cell differentiation,

Itch also plays a critical role in the process of T-cell anergy induction. As in

the case of Cbl-b and GRAIL, Itch is upregulated in an anergic T cell.

Upregulation of these E3 ligases induces the proteolysis of critical signal

molecules such as PLCγ1 or PKCθ that blocks T-cell activation even upon

effective stimulation (Heissmeyer et al., 2004). It remains to be defined

whether targets of Itch differ in resting versus anergic T cells. By using a

soluble antigen-induced tolerance induction mouse model, it was found

that Itch-deficient mice are resistant to Th2 tolerance induction, which indi-

cates that Itch is important in the tolerogenic process of Th2 cells

(Venuprasad et al., 2006). The critical role of Itch in the regulation of

autoinflammation/immunity has been highlighted by the finding that

human patients who have a mutation resulting in the deficiency in Itch have

multiple immunological defects, including asthma-like chronic lung disease

and multisystem autoimmunity (Lohr et al., 2010).

2.3. GRAILGRAIL (also known as RNF128) is a transmembrane RING-type E3 ligase

(Whiting, Su, Lin, & Fathman, 2011). GRAIL was identified to be highly

upregulated in anergic CD4+ T cells (Anandasabapathy et al., 2003;

Heissmeyer et al., 2004). Consistent with the notion that GRAIL is involved

in T-cell anergy induction, constitutive expression of GRAIL was sufficient

to render naı̈ve CD4+ T cells into anergic state (Anandasabapathy et al.,

2003). In addition, expression of the E3 ubiquitin ligase-inactive form of

GRAIL blocks the induction of anergy in CD4 T cells in vivo (Seroogy

et al., 2004). The strong evidence for the crucial role of GRAIL in T-cell

tolerance arises from genetic studies. In accordance with in vitro data,


GRAIL-deficient mice cannot be tolerized in vivo (Kriegel, Rathinam, &

Flavell, 2009; Nurieva et al., 2010). Oral tolerance is abolished in vivo using

two different models. Moreover, aged GRAIL-deficient mice display

increased infiltration of inflammatory cells into the lung and kidney and

exacerbation of EAE, suggesting a critical role of GRAIL in preventing lym-

phoproliferative and autoimmune responses (Nurieva et al., 2010). In par-

ticular, Grail�/� CD4+ T cells are hyperproliferative upon TCR

stimulation in vitro and in vivo. WhenGRAIL�/�CD4+ T cells were acti-

vated under Th-cell-polarizing conditions, these cells exhibited enhanced

IFN-γ expression in Th1 cells, lowered IL-4 in Th2 cells, and elevated

IL-17 in Th17 cells. Like many E3 ligases, GRAIL appears to ubiquitinate

a variety of target proteins. It has been reported that CD40 ligand, CD83,

CD151, CD81, RhoGDI, Arp2/3-5, and coronin 1A could be substrates of

GRAIL (Whiting et al., 2011). Genetically, Nurieva et al. (2010) showed

that GRAIL downmodulates the expression of TCR–CD3 complex via

the ubiquitin–proteasome pathway, suggesting that GRAIL controls the

thresholds for TCR responsiveness. Kriegel et al. (2009) showed that loss

of GRAIL increases total levels of ERK. Obviously, further investigation

is required to define the exact molecular mechanisms how GRAIL controls

naı̈ve CD4+ T-cell proliferation and anergy.

2.4. TRAF6Tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6) is a

member of TRAF protein family, which plays a critical role in both innate

and adaptive immune responses by mediating signal transduction from

members of the TNF superfamily, the Toll-like receptor/interleukin-1

receptor (TLR/IL-1R) family, and the TCR (Inoue, Gohda, &

Akiyama, 2007; Kobayashi, Walsh, & Choi, 2004). Although it has been

shown that TRAF6 functions downstream of the TCR to mediate IκBkinase (IKK) activation by binding to mucosa-associated lymphoid tissue

(MALT) 1 in Jurkat T cells (Kanayama et al., 2004), a role for TRAF6 in

TCR signaling-induced NF-κB activation has not been well defined.

A study using the chimeric mice showed that TRAF6 deficiency induces

chronic T-cell activation and progressive inflammatory disease (Chiffoleau

et al., 2003), indicating a critical role for TRAF6 in the regulation of

T-cell homeostasis. Furthermore, T-cell-specific knockout of TRAF6

(Traf6-ΔT) in mice resulted in an inflammatory disorder accompanied

by splenomegaly and lymphadenopathy (Ea, Deng, Xia, Pineda, & Chen,

26 Yoon Park et al.

2006). Traf6-ΔT mice also showed abnormal Th2 cytokine production; ele-

vated serum levels of IgG1, IgE, and IgM; and DNA autoantibodies. In vitro,

TRAF6-deficient T cells hyperproliferate in response to anti-CD3 stimula-

tion independently of CD28 costimulation. Furthermore, the loss of TRAF6

restores the ability of CD28�/� T cells to respond efficiently to CD3 stim-

ulation, indicating that TRAF6 deficiency renders T cells resistant to

anergizing signals (King et al., 2008). At the molecular level, TRAF6 defi-

ciency results in the hyperactivation of PI3K–Akt pathway and the decreased

expression of Cbl-b under anergizing conditions. However, it remains unclear

how TRAF6 regulates Cbl-b expression and whether decreased Cbl-b

expression is responsible for the hyperactivation of PI3K/Akt.

2.5. Peli1Peli (also called Pellino) is a RING-type E3 ubiquitin ligase that was first

identified in Drosophila melanogaster as interacting with Pelle, the Drosophila

orthologue of mammalian IRAKs ( Jin, Chang, & Sun, 2012). A gene-

targeting study has revealed that Peli1 mediates IKK activation by the

TRIF-dependent TLR pathway (Chang, Jin, & Sun, 2009). In addition,

Peli1 function is important for the regulation of T-cell activation and

homeostasis (Chang et al., 2011). Peli1 is highly expressed in T cells and fur-

ther elevated following T-cell activation. Peli1-deficient T cells are hyper-

responsive to TCR and CD28 stimulation in vitro and display an activated

phenotype in vivo. Interestingly, Peli1-deficient T cells are refractive to reg-

ulatory T cell (Treg) and transforming growth factor β (TGFβ) suppression.More profound autoimmune symptoms were revealed in aged Peli1-

deficient mice compared to wild-type littermates. Peli1-deficient mice

develop spontaneous autoimmunity, characterized by enlarged spleens

and peripheral lymph nodes, severe immune cell infiltration in multiple

organs, and elevated antinuclear autoantibodies and immunoglobulin depo-

sition in kidney glomeruli. Peli1 appears to negatively regulate c-Rel by

mediating the K48 ubiquitination and degradation of c-Rel in activated

T cells. c-Rel undergoes ubiquitin-dependent degradation in T cells in

response to TCR/CD28 signals. However, the induction of c-Rel

ubiquitination is largely blocked in the Peli1�/� T cells. These observa-

tions suggest that Peli1 may regulate intrinsic T-cell tolerance through

preventing aberrant accumulation of c-Rel during T-cell activation. In

future, lineage-specific deletion of Peli-1 in mouse would be a useful tool

to illustrate the differing roles of Peli-1 in different cell types.


2.6. RoquinRoquin family proteins, which belong to RING-type E3 ubiquitin ligases

and act as RNA-binding proteins, are involved in regulating the stability and

translation of mRNA (Heissmeyer & Vogel, 2013). Initial characterization

of Roquin-defective mice (referred to as sanroque mice), which have a

single-point mutation (M199R) in the Roquin-1 protein, exhibited

splenomegaly, spontaneous T-cell activation and germinal center formation,

and high levels of autoreactive antibodies (Linterman et al., 2009; Yu et al.,

2007). Surprisingly, mice with a T-cell-specific deletion of the Roquin-1-

encoding gene Rc3h1 failed to exhibit a breach in self-tolerance or have

changes in follicular T-cell (Tfh) differentiation (Bertossi et al., 2011). Sim-

ilar findings were observed for mice with a T-cell-specific deletion of its

paralog Rc3h2, encoding Roquin-2 (Pratama et al., 2013; Vogel et al.,

2013). Instead, combined deletion of both paralogs in the T cell caused accu-

mulation of T cells with an effector and Tfh phenotype, suggesting the

redundant function of Roquin-1 andRoquin-2 in the control of T-cell acti-

vation and Tfh differentiation (Pratama et al., 2013; Vogel et al., 2013). Rec-

ognition of a cis-element in the 30-untranslated region of the ICOS mRNA

by Roquin-1 facilitates degradation of the transcript through recruiting the

mRNA decay machinery by interacting with the decapping enzyme Edc4

and helicase Rck (Glasmacher et al., 2010). In addition, it has been identified

that Ox40 acts as a new target of Roquin-1 and Roquin-2 (Vogel et al.,

2013). Combined ablation of Rc3h1 and Rc3h2 in T cells induced the ele-

vated expression of Ox40 and the activation of the alternative NF-κB path-

way. It remains to be investigated through which molecular network

Roquin regulates T-cell activation and Tfh differentiation and if the E3

ligase activity of Roquin is involved in mRNA regulation. It will be also

important to find out what other mRNA and mRNA-associated substrates

are regulated by the Roquin family proteins.

3. E3 LIGASES IN T-CELL DIFFERENTIATION

Naı̈ve CD4+ T cells can differentiate into various effector or suppres-

sive cell types, depending on the encounters of different pathogens or cyto-

kine milieu, and differentiation of such T-cell subtypes is controlled by

critical and specific transcription factors. Control of T-cell differentiation

is critical to maintain immune responses with their activities against

28 Yoon Park et al.

infection, inflammation, and cancer. Many key molecules are regulated by

ubiquitination/deubiquitination enzymes to influence T-cell development

and differentiation. Here, we discuss recent studies that have been per-

formed to understand the role of the ubiquitination pathway in regulatory

T-cell (Treg) and T-helper cell (Th) development and function (Fig. 2.2).

3.1. TregsTregs play an important role in maintaining immune homeostasis by

suppressing many kinds of immune cells. Most Tregs express Foxp3, a mas-

ter transcription factor, which is crucial for their suppressive activities. It has

been studied that several E3 ubiquitin ligases regulate the differentiation

and/or the function of Treg. One of the E3 ubiquitin ligases, GRAIL, is

responsible for Treg function. GRAIL-deficient mice showed autoimmu-

nity without immune tolerance, suggesting that GRAIL plays in T-cell tol-

erance and Treg functions (Kriegel et al., 2009). GRAIL was also able to

diminish TCR signaling through ubiquitination of CD3ζ, thereby loss of

GRAIL leads to lack of functional Treg and tolerance induction (Nurieva

et al., 2010). Tregs are classified into two distinct populations: one is natu-

rally occurring Treg (nTreg) generated in thymus and another is induced

Treg (iTreg) developed in periphery. Both Treg subtypes shared many com-

mon features in function and expression of Treg markers. However, nTreg

can be distinguished from iTreg by the expression of specific molecules such

as helios and neuropilin-1 (Thornton et al., 2010; Weiss et al., 2012), even

though it has been reported that iTregs were also able to express helios both

in vitro and in vivo (Kim et al., 2012). TGFβ is essential for the development

of iTregs. The E3 ubiquitin ligase Cbl-b is required for TGFβ-mediated

iTreg generation. Cbl-b-deficient T cells were defective in Treg induction

in response to TGFβ because Cbl-b deficiency showed impaired PI3K–

AKT–Foxo signaling (Harada et al., 2010). In addition to Treg generation,

Cbl-b expression in target cells is also required for Treg function because

Cbl-b-deficient T cells were resistant to TGFβ-mediated suppression by

functional Tregs (Adams et al., 2010), indicating that Cbl-b is important

for both Treg itself and target cells. Deubiquitinase CYLD has been exam-

ined for TGFβ-mediated Treg generation. CYLD regulates TGFβ signalingby deubiquitination of Smad7, which is responsible for downstream signal-

ing of TGFβ (Zhao et al., 2011). However, lack of CYLD showed impaired

Treg function with reduced expression of CD25 and CTLA4, which are key


Figure 2.2 The ubiquitin system regulates the differentiation of T helper cells. (A) NaïveT cells differentiate into Foxp3+ regulatory T cells in response to TGFβ and IL-2. BothCbl-b and CYLD regulate TGFβ signaling through Foxo1/3a and Smad7, respectively.The stability of Foxp3 is maintained by deubiquitinase USP7, whereas Foxp3 can bereduced by E3 ubiquitin ligase Stub1. Skp2 promotes the conversion of Foxp3+ cellsinto non-Treg (Foxp3�) cells, while Ubc13 and CYLD prevent this conversion by regu-lation of IL-10, CD25, or CTLA4 expression. (B) IL-12 triggers Th1 cells through STAT4–T-bet pathway. SLIM inhibits Th1 differentiation by inducing degradation of STAT4. ICOSsignaling is required for T-cell activation; however, roquin suppresses ICOS signalingpathway, which drives the reduction of Th1 cells. (C) IL-4 promotes STAT6–GATA3 sig-naling pathway for the induction of Th2 cells. BMI-1 associates with GATA3, which canprevent the degradation of GATA3 by unknown ubiquitinase(s). IL-25 signaling duringTh2 polarization is regulated by E3 ligase Act1. Itch regulates JunB throughubiquitination. However, in the absence of Itch, JunB can escape from degradation

30 Yoon Park et al.

molecules for Treg function (Reissig et al., 2012), indicating that CYLD is

indispensible for Treg generation as well as function.

The maintenance of Treg stability is important for their suppressive

activity. The ubiquitin-conjugating enzyme Ubc13 is known to regulate

Treg conversion. Ubc13�/� Tregs were prone to convert efficiently into

Th1 or Th17 cells together with Foxp3 loss (Chang et al., 2012). Ubc13 is

able to control IKK, which is critical for IL-10 and SOCS1 expression. Even

though E3 ubiquitin ligase Itch is known as a critical regulator in Th2 cells,

Itch ablation in Tregs did not showed impaired Treg function both in vitro

and in vivo and exhibited normal Foxp3 expression and stability; however,

the conditional knockout mice showed excessive inflammation in the lung

and skin ( Jin, Park, Elly, & Liu, 2013). Notably, Itch-deficient Tregs pro-

duce large amounts of Th2 cytokines including IL-4 and IL-5, which are

attributed to the triggering of the inflammatory immune response. Unlikely

Ubc13�/� Tregs, Itch�/� Tregs promote Th2-biased pathology by

gaining the additional ability, such as producing inflammatory cytokines

without losing Foxp3. This conversion is reversible because effector

T cells can undergo the conversion into Treg or vice versa. The F-box pro-

tein S-phase kinase-associated protein 2 (Skp2) is an essential component of

Skp–Cullin–F-box (SCF) ubiquitin ligase complex and is involved in the

conversion between Treg and effector T cells. Downregulation of Skp2

induced the conversion of pathogenic T cells into regulatory T cells

(Wang et al., 2012). Skp2 was able to regulate cell cycle regulators (p21

and p27) and Foxo proteins. In contrast, overexpression of Skp2 in Tregs

caused the loss of Foxp3 and reduced Treg function. In many cases above,

Foxp3 stability is a key limiting factor for Tregmaintenance; however, it was

not clear that these enzymes regulate directly Foxp3 protein level.

Recently, two groups reported the ubiquitination-related enzymes that

are responsible for the regulation of Foxp3 protein. The E3 ubiquitin ligase

Stub1 plays an important role in ubiquitination of Foxp3. Stub1 was able to

negatively regulate Foxp3 because overexpression of Stub1 led to the loss of

Foxp3 and the increase of Th1 cells with autoimmunity (Chen et al., 2013).

and, in turn, accumulated JunB induces IL-4 expression. (D) Ndfip1 deficiency inducesIL-4 production in T cells and then promotes the recruitment of IL-6-producing eosin-ophils into the lung, thus regulating Th17 development indirectly. SLIM controls Th17cells by the inhibition of STAT3 activation. USP18 modulates IL-2 expression, which candampen Th17 polarization. Ro52 blocks IL-23 signaling which is essential for the main-tenance of Th17 cells.


In contrast, the DUB USP7 maintains the stability by Foxp3

deubiquitination (van Loosdregt et al., 2013). USP7 expression is highly

upregulated in Foxp3+ Tregs. Knockdown of USP7 or DUB inhibitor

induced the loss of Foxp3 protein in Tregs. Therefore, Foxp3 stability

may be precisely regulated by genes related in ubiquitination/

deubiquitination.

3.2. Th1 cellsEffector CD4+ T cells such as Th1, Th2, and Th17 play an important role

for protection from infection and cancer. However, excessive effector func-

tions could lead to adverse effects such as inflammation and autoimmunity.

Thus, the development and function of effector T cells are tightly regulated

to maintain a balance between immune responses and tolerance.

IL-12 is known to drive Th1 differentiation through the signal trans-

ducer and activator of transcription 4 (STAT4) activation. A STAT

ubiquitin E3 ligase, SLIM (STAT-interacting LIM protein), has been shown

to regulate Th1 generation. SLIM deficiency results in increased IFNγ pro-duction by Th1 cells (Tanaka, Soriano, & Grusby, 2005). SLIM interacts

with activated STAT4, leading to proteaosome-mediated degradation. In

addition, T-bet is a key transcription factor for Th1 differentiation. Muta-

tion in Lys-313 of T-bet prevented ubiquitination with enhanced stability

( Jang, Park, Hong, & Hwang, 2013), even though it remains to be eluci-

dated which enzyme(s) is/are responsible for T-bet ubiquitination. Another

E3 ligase Roquin may be involved in Th1-dependent autoimmunity

because the sanroque mice have a point mutation of roquin with higher sus-

ceptibility to autoimmunity. Overexpression of roquin in CD4 T cells

showed the increased Th1 cells in a collagen-induced arthritis model by reg-

ulating CD28/ICOS signaling during T-cell activation ( Ji et al., 2012).

However, as described earlier, it has been reported that roquin is also

involved in Tfh development (Pratama et al., 2013; Vogel et al., 2013).

3.3. Th2 cellsFor Th2 differentiation, IL-4 is a critical cytokine responsible for the gen-

eration of Th2 cells via STAT6–GATA3 signaling pathway. Polycomb

group proteins such as BMI-1 and RING1b are related in Th2 differentia-

tion. BMI-1 promotes Th2 polarization by interaction with GATA3 and

this interaction prevents GATA3 from ubiquitin-mediated degradation

(Hosokawa et al., 2006). By the overexpression of BMI-1, the stability of

32 Yoon Park et al.

GATA3 increased. Thus, GATA3 stability by BMI-1 is critical for Th2 dif-

ferentiation. As described earlier, the E3 ligase Itch is also known to play a

critical role in Th2 development because Itch�/� mice showed enhanced

Th2 immune responses. Itch is able to control Th2 response by regulating

JunB, which is critical for target gene expression in Th2 cells (Fang et al.,

2002). Moreover, MEKK1 signaling after TCR stimulation regulates Itch

(Enzler et al., 2009; Venuprasad et al., 2006). Activated MEKK1 becomes

phosphorylated and is able to recruit Itch. Then, Itch is phosphorylated by

JNK; thus activated Itch can negatively regulate JunB, which is responsible

for IL-4 expression. In addition, it has been reported that the U-box-type E3

ubiquitin ligase Act1 is required for Th2 induction. Act1�/� T cells had

impaired Th2 responses with less expression of GATA3 and Gfi1

(Swaidani et al., 2011). This was due to the lack of IL-25 signaling in

Act�/� T cells.

3.4. Th17 cellsIL-6 signaling is required for the induction of Th17 cells, since IL-6 activates

STAT3 phosphorylation through IL-6 receptor, and then the activated

STAT3 induces the transcription of Rorc, which is a key transcription factor

for the expression of IL-17. SLIM was known to play a role in Th1 devel-

opment by regulating STAT4, as described earlier. However, SLIM is also

involved in Th17 differentiation by inducing the degradation of STAT3

through ubiquitination (Tanaka et al., 2011). Therefore, SLIM�/�T cells showed enhanced Th17 differentiation. Ro52 was identified as an

E3 ligase and known to control proinflammatory cytokine production by

regulating interferon regulatory factor. Ro51�/� mice showed increased

Th17 cells and this phenotype was abolished from Ro51�/� IL23p19�/�mice (Espinosa et al., 2009). Thus, Ro52 regulates Th17 differentiation

through IL-23–Th17 pathway. Ndfip1 is an adapter protein that binds to

E3 ligase Itch, which is responsible for Th2 cells. However, Th17 cells were

induced from Ndfip1�/� mice, even though Ndfip1�/� T cells were not

efficient for Th17 differentiation (Ramon, Beal, Liu, Worthen, & Oliver,

2012). The induction of Th17 cells was caused by increasing

IL-6-producing eosinophils in lungs fromNdfip1�/�mice, since enhanced

Th2 response was able to recruit those eosinophils into lungs. Thus, Th17

cells could be regulated indirectly by Ndfip1�/� T cells. Recently,

deubiquitinase USP18 was identified as a regulator in Th17 differentiation.

USP18�/� T cells showed a defect in Th17 polarization (Liu et al., 2013).


USP18 regulates TGFβ-activated kinase-1 (TAK1)–TAB1 complex by

deubiquitination, which inhibits IL-2 expression. Since IL-2 is known to

inhibit Th17 generation, USP18�/� T cells were resistant to Th17

differentiation.

4. UBIQUITINATION IN NF-κB SIGNALING

The nuclear factor kappa B (NF-κB) family of transcription factors,

which consists of p50, p52, p65 (RelA), c-Rel, and RelB, is a key regulator

of various cellular processes including immune response, inflammation, and

cell survival (Hayden & Ghosh, 2008). The NF-κB family is present in the

cytosol bound to inhibitory proteins of κB family (IκB) under nonstimulated

conditions. Upon numerous stimuli such as inflammatory cytokines, antigen

receptors, and microorganisms, IκB is phosphorylated by IKK complex and

subsequently undergoes ubiquitination and proteasomal degradation, which

allows NF-κB to translocate to the nucleus from cytosol and regulates the

transcription of a variety of target genes. Alternatively, specific members

of TNF family including CD40 ligand, BAFF, and lymphotoxin-β induce

noncanonical NF-κB pathway, which is initiated from NF-κB-inducingkinase (NIK) activation. Following NIK activation, IKKα is phosphorylated

and in turn a precursor NF-κB subunit, p100, is polyubiquitinated and sub-

sequently processed by proteasome to p52, which leads to the formation of

p52/RelB complexes (Skaug, Jiang, & Chen, 2009). As such, ubiquitination

is critically involved in the NF-κB activation pathways regulating IκB degra-

dation, NF-κB precursor processing, and protein kinases’ activation via

degradation-dependent and -independent mechanisms. Therefore, a better

knowledge of ubiquitination-mediated regulation of NF-κB activationwould

let us understand and develop therapeutic agents for inflammatory diseases.

4.1. TNFR1 signalingTNF-α is a proinflammatory cytokine that activates multiple cellular responses

including NF-κB and apoptosis. The binding of TNF-α to TNF receptor 1

(TNFR1) induces the trimerization of this receptor and recruitment of

TNFR-associated protein with a death domain (TRADD) and signaling mol-

ecules. TRADD forms a complex 1 with TNF receptor-associated factor 2

(TRAF2), TRAF5, cellular inhibitor of apoptosis protein 1 (cIAP1), cIAP2,

and receptor-interacting protein 1 (RIP1) that activates NF-κB (Chen &

Goeddel, 2002; Micheau & Tschopp, 2003) (Fig. 2.3). RIP1 has been known

as an adaptor protein for TNF-α-induced NF-κB activation by interacting

34 Yoon Park et al.

Figure 2.3 See legend on next page

with and recruiting the signaling molecules. Upon TNF-α stimulation,

TNFR1-boundRIP1 is rapidly polyubiquitinated with K63-linked ubiquitin

chains and it recruits IKK complexes and TAK1 to TNFR1 via binding to

their UBDs. This interaction between K63-linked polyubiquitin chains on

RIP1 and NEMO (a regulatory subunit of the IKK complex) is a critical step

for NF-κB activation (Ea et al., 2006; Kanayama et al., 2004; Li, Kobayashi,

Blonska, You, & Lin, 2006; Wu, Conze, Li, Srinivasula, & Ashwell, 2006).

Mutation on K377 of RIP1 that is the key lysine residue for ubiquitination

prevents the recruitment of IKK complex to TNFR1 and leads to inactivation

of IKK. It is further confirmed that NEMO mutations, which are unable to

bind with polyubiquitin chains, fail to restore IKK activation in NEMO-

deficient cells (Ea et al., 2006; Wu et al., 2006). TAK1 is also recruited to

TNFR1 through the interaction with TAB2/3 and polyubiquitinated

RIP1 (Kanayama et al., 2004) and then activates IKK. Although the require-

ment for TAK1 has been generally believed for IKK phosphorylation, it still

remains unclear whether TAK1 directly phosphorylates IKK or mediates the

activation through MEKK3 (Blonska et al., 2005; Li et al., 2006).

The RING domain-containing ubiquitin E3 ligase TRAF2, which is

one of the components of TNFR1–TRADD signaling complex (Hsu,

Shu, Pan, & Goeddel, 1996), is involved in the formation of K63-linked

ubiquitin chains on RIP1. A previous study reported that TNF-α-inducedRIP1 polyubiquitination is reduced in TRAF2-deficient MEFs (Lee,

Shank, Cusson, & Kelliher, 2004); however, there is no solid evidence that

Figure 2.3—Cont’d NF-κB activation in TNFR1 and IL-1R/TLR4 signaling pathways.Stimulation of TNFR1 by TNF-α (left) induces the formation of a membrane receptorcomplex 1 (complex 1): TRADD, TRAF2, TRAF5, cIAP1, cIAP2, and RIP1. RIP1 ispolyubiquitinated with K63 or K11 or M1 (linear)-linked chains and it results in therecruitment of TAK1 and IKK complexes. K63-linked polyubiquitin chain bound TAK1complex (TAK1, TAB1, and TAB2/3) activates IKK, and it leads to the phosphorylationand degradation of IκBα proteins, which allows p50/p65 heterodimers to enter thenucleus and bind to NF-κB target genes. DUBs including A20, CYLD, Cezanne, andOTULIN can reverse NF-κB activation by removing K63- or M1-linked polyubiquitinchains from RIP1. Upon ligation of IL-1R or TLRs by IL-1β or PAMPs (right), Myd88 is rec-ruited to ligand-bound receptors, which in turn assemble the receptor signaling com-plex with IRAK1, IRAK4, TRAF6, and Ubc13. Activated TRAF6, together with ubc13 andPeli, facilitates the synthesis of K63-linked polyubiquitin chains, which recruits TAK1 andIKK complexes. M1-linked polyubiquitin chains also positively regulate IKK activation bycooperatively modifying the signaling molecules with K63-linked polyubiquitin chains.Engagement of LPS to TLR4 leads to the association with TRIM and TRIF, and their sub-sequent binding to TRAF6 and RIP1 results in TAK1 and IKK activation by promoting theconjugation of K63-linked polyubiquitination to RIP1.

36 Yoon Park et al.

TRAF2 directly catalyzes polyubiquitin chain to RIP1. Furthermore,

TRAF2 and TRAF5 have a functional redundancy on NF-κB activation

(Lee et al., 1997; Tada et al., 2001; Yeh et al., 1997). These accumulated

data obtained with biochemical and genetic studies indicate that TRAF2 is

required for TNF-α-induced NF-κB activation by recruiting another E3

ligase to RIP1.

The cIAP1 and cIAP2 are also RING domain-containing ubiquitin E3

ligases that have been suggested to catalyze RIP1 ubiquitination. Upon

TNF-α stimulation, TRAF2 recruits cIAP1/2 to TNFR1 and in turn

receptor-associated cIAP1/2 promotes K63-linked polyubiquitination of

RIP1 (Bertrand et al., 2008; Shu, Takeuchi, & Goeddel, 1996). A recent

report showed that cIAP1, together with UbcH5, generates K11-linked

polyubiquitin chains on RIP1 in TNFR1 signaling complex, which leads

to the induction of NF-κB activation in a nondegradative manner

(Dynek et al., 2010). Although genetic studies on cIAP1/2 clearly present

a critical role of cIAP1/2 in RIP1 ubiquitination and NF-κB activation

by TNF-α (Mahoney et al., 2008; Varfolomeev et al., 2008), the detailed

working mechanism underlying TRAF2/5 and cIAP1/2 interplay remains

elusive.

The Ubc13/Uev1A is the E2 ubiquitin-conjugating enzyme that specif-

ically catalyzes K63-linked ubiquitin chains. It has been reported that a

dominant-negative mutant of Ubc13 blocks TRAF2-mediated NF-κB acti-

vation in TNF-α signaling (Deng et al., 2000). However, several genetic

studies have shown that Ubc13 has a limited role in TNF-α-inducedNF-κB activation. MEFs derived from Ubc13-deficient mice exhibit no

defect in IKK activation upon TNF-α stimulation, and further, a human cell

line endogenously expressing K63R ubiquitinmutant did not alter IKK acti-

vation by TNF-α (Xu, Skaug, Zeng, & Chen, 2009; Yamamoto, Okamoto,

et al., 2006). These results suggest a possibility that either alternate E2/E3

enzymes or polyubiquitin chains may be involved in regulating TNF-α-induced NF-κB activation.

LUBAC (linear ubiquitin chain assembly complex) that consists of

HOIP (also known as RNF31), HOIL-1 (also known as RBCK1), and

Sharpin is an E3 ligase complex for linear ubiquitin chain formation, which

is known to regulate IKK activation in TNF-α signaling (Iwai, 2012). Over-

expression and genetic deletion studies of HOIL-1 and HOIP showed that

LUBAC catalyzed linear polyubiquitin chains to NEMO in response to

TNF-α, which leads to NF-κB activation. Furthermore, Sharpin-deficient

MEFs, macrophages, and B cells also displayed an impaired IKK and NF-κB


activation upon TNF-α stimulation (Gerlach et al., 2011; Tanaka et al.,

2011; Tokunaga et al., 2011). Linear polyubiquitin chains provide a scaffold

for recruiting IKK complex through the binding to NEMO, which seems to

be a similar mechanism with RIP-mediated IKK activation by K63-linked

polyubiquitin chains. Indeed, both K63-linked and linear ubiquitin exhibit

equivalent open conformations; however, it has been reported that these

two chains are recognized by distinct components of NF-κB signaling

(Komander, Reyes-Turcu, et al., 2009). Structural and in vitro binding stud-

ies suggest that UBAN (ubiquitin binding in ABIN and NEMO) motif of

NEMO preferentially binds to linear diubiquitin chain, whereas K63-linked

ubiquitin chains are specifically recognized by NZF domain of TAB2/3

(Komander, Reyes-Turcu, et al., 2009; Rahighi et al., 2009). Thus, a

NEMO mutant that is unable to bind to linear ubiquitin chains partially

abolishes NF-κB activation (Hadian et al., 2011). These results indicate that

K63-linked and linear ubiquitin chains possibly collaborate to induce IKK

activation through the selective binding to TAB2/3 and NEMO, respec-

tively. However, it is still controversial because K11- and K63-linked poly-

ubiquitin chains, not diubiquitin chain, have been shown to efficiently bind

to full-length NEMO (Dynek et al., 2010; Laplantine et al., 2009). In addi-

tion, the requirement and role of accessory components of LUBAC on

NF-κB activation remain unclear. Both HOIL-1L and Sharpin deficiency

exhibited partial impairment of TNF-α-induced NF-κB activation, which

suggests that LUBACmay have a redundant function with other proteins in

NF-κB signaling.

Sharpin deletion by spontaneous null mutation in cpdm (chronic prolif-

erative dermatitis) mice results in severe inflammation and immune system

malfunction, which is proposed to represent the important role of LUBAC

in TNF-α signaling (Gerlach et al., 2011; Tanaka et al., 2011; Tokunaga

et al., 2011). However, conflicting results were reported that the inflamma-

tory phenotype in cpdmmice is mainly caused by hyperactivation of NF-κBin IL-1 signaling. Crossing cpdmmice with IL-1RAcP (IL-1receptor acces-

sory protein)-knockout mice rescued inflammatory phenotype of cpdm

mice, and further, proteasome inhibitor bortezomib treatment, which is also

known as a NF-κB inhibitor, reduced skin inflammation (Liang,

Seymour, & Sundberg, 2011). In addition, Sharpin-mediated β1-integrininhibition could be another cause of inflammation (Rantala et al., 2011).

Most recently, an important role of lymphocytes on the regulation of sys-

temic inflammation, but not skin inflammation, in cpdm mice is revealed

by genetic studies, which provides another aspect for the understanding

38 Yoon Park et al.

of inflammatory responses in cpdm mice, and thus suggests a possibility that

Sharpin may play a distinct function in regulating lymphocyte function and

control autoimmunity (Potter et al., 2014). Therefore, further studies are

required to clearly understand the role of linear ubiquitin chains/LUBAC

in TNF-α-induced NF-κB activation and control of inflammatory

responses.

Deubiquitination plays a key role in NF-κB signaling pathways by

reversing the effect of ubiquitination. A20 is one of the most well-studied

DUBs that contains OTU DUB domain at the N-terminus and Cys2/

Cys2 ZnF E3 ligase domain at the C-terminus, which plays a dual role

for RIP1 regulation in TNFR1 signaling (Ma &Malynn, 2012). K63-linked

polyubiquitin chains on RIP1 are removed by OTU DUB domain of A20

and then C-terminal E3 ligase domain of A20 conjugates K48-linked poly-

ubiquitin chains to RIP1 for proteasomal degradation (Wertz et al., 2004).

This negative regulation of TNF-α-induced NF-κB signaling by A20 is fur-

ther confirmed by genetic ablation of A20 in mice that exhibit prolonged

NF-κB responses and development of severe multiorgan inflammation

(Lee et al., 2000). A20 assembles a complex with TAXBP1 (binding protein

of the hTLV TAX) and other E3 ligases including Itch and RNF11 to atten-

uate TNFR1 signaling (Shembade, Harhaj, Liebl, & Harhaj, 2007;

Shembade et al., 2008; Shembade, Parvatiyar, Harhaj, & Harhaj, 2009),

although future studies are needed to elucidate the precise mechanism. Most

recent studies propose that A20 also inhibits LUBAC-induced NF-κB acti-

vation in a DUB activity-independent manner by the interaction between

ZnF domain of A20 and linear ubiquitin chains (Tokunaga et al., 2012;

Verhelst et al., 2012). CYLD is another DUB that is known to negatively

regulate TNF-α-induced NF-κB signaling. USP domain of CYLD has a

DUB activity for K63-linked and linear polyubiquitin chains on RIP1,

TRAF2, and NEMO, which leads to suppress NF-κB activation

(Brummelkamp, Nijman, Dirac, & Bernards, 2003; Komander, Reyes-

Turcu, et al., 2009; Kovalenko et al., 2003; Tokunaga et al., 2012;

Trompouki et al., 2003). OTULIN/gumby is most recently characterized

OTU family DUB that exclusively recognizes linear polyubiquitin chains.

Overexpression of OTULIN inhibits TNF-α-induced NF-κB signaling

by preventing the association of NEMO with polyubiquitinated RIP1

(Fiil et al., 2013; Keusekotten et al., 2013). Several other DUBs have been

characterized as regulators of TNF-α-induced NF-κB signaling: Cezanne

(Enesa et al., 2008), USP21 (Xu et al., 2010), USP31 (Tzimas et al.,

2006), USP11 (Sun et al., 2010; Yamaguchi, Kimura, Miki, & Yoshida,


2007), and USP4 (Zhou et al., 2012). These DUBs exhibit DUB activity

toward components of NF-κB signaling for terminating NF-κB activation.

Further biochemical and genetic studies will be required to demonstrate the

roles of these DUBs.

4.2. IL-1R/TLR4 signalingIL-1R and TLRs are cytoplasmic TIR (Toll-IL-1 receptor) domain-

containing transmembrane proteins. Upon ligation of IL-1R or TLRs by

IL-1β or PAMPs, respectively, these receptors, except TLR3, recruit

TIR-containing adaptor proteins MyD88, MAL (also known as TIRAP),

TRIF, and TRAM through the interaction between TIR domains

(Verstrepen et al., 2008). Among TLRs, LPS-induced TLR4 activation ini-

tiates two pathways that are controlled by distinct adaptor pairs: the

MyD88–MAL and the TRAM and TRIF. Recruited MyD88 by MAL

assembles the receptor signaling complex with IRAK1, IRAK4, and

TRAF6. IRAK4 phosphorylates and recruits IRAK1, which then binds

to RING E3 ubiquitin ligase TRAF6. The complex-bound TRAF6 subse-

quently catalyzes the formation of K63-linked polyubiquitin chains and

these ubiquitin chains recruit TAK1/TAB and IKK complexes, which lead

to NF-κB activation. Another RING E3 ubiquitin ligase Peli is also known

to regulate signaling components in the IL-1R/TLR4 pathway (Moynagh,

2009). Upon LPS ligation, TLR4 binds to TRIM and subsequently recruits

TRIF, which binds to TRAF6 and RIP1. K63-linked polyubiquitination of

both TRAF6 and RIP1 is known to promote TAK1 and NF-κB activation

(Vallabhapurapu & Karin, 2009).

Despite the importance of K63-linked ubiquitin chains as a key player for

IL-1R/TLR4 signaling, the requirement of major E2 and E3 ubiquitin

enzymes, which are thought to synthesize K63-linked ubiquitin chains,

for NF-κB activation still remains elusive. TRAF6 has been proposed to

cooperate with E2 ubiquitin enzyme Ubc13/Uev1A for facilitating K63-

linked polyubiquitination of various substrates such as IRAK1 and TAK1

in response to IL-1β or LPS (Conze, Wu, Thomas, Landstrom, &

Ashwell, 2008; Wang et al., 2001; Windheim, Stafford, Peggie, &

Cohen, 2008). However, like TRAF2, genetic analyses showed conflicting

results on the role of Ubc13 in NF-κB activation in IL-1R/TLR4 signaling.

Conditional deletion of Ubc13 in BMDMs (bone marrow-derived macro-

phages), B cells, and MEFs did not impair TRAF6-mediated NF-κB acti-

vation upon IL-1β or TLR ligands’ stimulation (Yamamoto, Okamoto,

40 Yoon Park et al.

et al., 2006), whereas NF-κB activation by LPS was reduced in macrophages

and splenocytes derived from Ubc13+/� mice (Fukushima et al., 2007).

Furthermore, there is a discrepancy on the function of RING domain of

TRAF6 in IL-1R/TLR4 signaling, although TRAF6-deficient cells were

reported to display a significant failure of NF-κB activation upon IL-1βor LPS treatment (Lomaga et al., 1999). Reconstitution of TRAF6 mutant

lacking RING domain in TRAF6-deficient MEFs rescued the effect of

TRAF6 deficiency on IL-1β- and LPS-induced NF-κB activation

(Kobayashi et al., 2001). In contrast, RING-domain point mutant

(C70A) of TRAF6 failed to restore NF-κB activation in TRAF6-deficient

cells (Lamothe et al., 2007). Therefore, more studies are needed to define the

precise mechanism of Ubc13/Uev1A–TRAF6-mediated regulation of

IL-1R/TLR4 signaling, and thus, characterization of alternate E2–E3

enzymes, which may play an essential role in this pathway, is also needed.

Recently, LUBAC E3 ligase complex has been suggested to catalyze lin-

ear polyubiquitin chains to NEMO upon IL-1β or TLR agonists, leading to

IKK activation (Tokunaga et al., 2009), although it is not clear whether

there is a functional redundancy between K63-linked and linear poly-

ubiquitin chains. A more recent study by Emmerich et al. (2013) proposes

that K63-linked and linear ubiquitin chains cooperatively work together to

modify the signaling molecules in IL-1R/TLR4 pathway includingMyD88

and IRAK4 through assembling either K63-linked/linear hybrid ubiquitin

chains or interaction between same proteins. However, positive regulation

of IKK activation by linear ubiquitin chains/LUBAC is still controversial,

since Sharpin-deficient macrophages exhibit no alteration of IKK activation

upon TLR stimulation (Zak et al., 2011). Another alternate ubiquitin chain-

mediated regulation of IKK activation in IL-1R signaling has been revealed

by recent biochemical studies. Unanchored K63-linked polyubiquitin

chains, which are not conjugated to any proteins, are polymerized by

TRAF6 and Ubc13/Uev1A and then activate TAK1 through the binding

to TAB2 in response to IL-1β (Xia et al., 2009). Future study will be needed

to demonstrate the precise working mechanism and in vivo role of unan-

chored polyubiquitin chains in IKK–NF-κB activation pathway.

IL-1R/TLR4 signaling is attenuated by DUBs. A20 has been appeared

to cleave K63-linked polyubiquitin chains from TRAF6. A20 deficiency in

macrophages results in the prolonged TRAF6 ubiquitination upon LPS

stimulation (Boone et al., 2004). Both biochemical and genetic studies reveal

that A20 inhibits IL-1β-induced interaction between TRAF6 and UBc13/

Uev1A or UbcH5 and then triggers their ubiquitination and proteasomal


degradation (Shembade, Ma, & Harhaj, 2010). CYLD also negatively reg-

ulates NF-κB activation in IL-1R/TLR4 signaling through its DUB activity

(Sun, 2010b). In addition, a recent study reported that USP7 has a DUB

activity for TRAF6 and NEMO in TLR pathway (Daubeuf et al., 2009).

4.3. T-cell receptor signalingBinding of an antigenic peptide–MHC complex to TCR and coreceptor

CD28 triggers the initiation of TCR signaling through activation of tyrosine

kinase cascade including Src/Syk family kinases, PI3K, and PDK1.

A subsequent phosphorylation of serine/threonine kinase PKCθ by

PDK1 phosphorylates CARMA1 and it leads to assembly and recruitment

of CBM (CARMA1–BCL10–MALT1) complex to membrane. CBM

complex activates IKK via promoting formation of K63-linked poly-

ubiquitin chains, leading to NF-κB activation (Schulze-Luehrmann &

Ghosh, 2006; Thome, Charton, Pelzer, & Hailfinger, 2010) (Fig. 2.4).

Modification of TCR/CD28 signaling components by K63-linked

polyubiquitin chains is a crucial process for TCR-induced NF-κB activa-

tion. TRAF2 and TRAF6 have been suggested to conjugate K63-linked

polyubiquitin chains onto TCR downstream molecules. In a cell-free sys-

tem, oligomerization of TRAF6 is induced by oligomeric forms of BCL10

and MALT1, which activates E3 ligase activity of TRAF6 for NEMO

ubiquitination (Sun, Deng, Ea, Xia, & Chen, 2004). TRAF6 also catalyzes

the polymerization of K63-linked ubiquitin chains on both BCL10 and

MALT1 that contributes to the recruitment of IKK complex and probably

promotes further TRAF6-mediated activation of IKK (Oeckinghaus et al.,

2007; Wu & Ashwell, 2008). Despite the clear role of TRAF2 and TRAF6

in NF-κB activation in TCR/CD28 signaling, genetic deletion of TRAF2

or TRAF6 in mice did not affect NF-κB activation by TCR stimulation,

which indicates the functional redundancy between TRAF2 and TRAF6

in T cells (King et al., 2006). Indeed, RNAi-mediated knockdown exper-

iment shows that expression of both TRAF2 and TRAF6 RNAi severely

abrogates NF-κB activation upon TCR ligation (Sun et al., 2004).

Ubc13/Uev1A has been thought to play an important role for NF-κB acti-

vation in TCR signaling by catalyzing K63-linked polyubiquitin chains

with TRAF6, which in turn activates TAK1 and IKK. Thus, Ubc13/

Uev1A, together with MALT1, promotes BCL10-induced NEMO modi-

fication by K63-linked polyubiquitin chains in TCR signaling (Zhou et al.,

2004). Although Ubc13 deficiency in thymocytes resulted in the significant

42 Yoon Park et al.

Figure 2.4 See legend on next page

impairment of TAK1 activation, PMA/ionophore-induced NF-κB activa-

tion exhibited a moderate decrease (Yamamoto, Sato, et al., 2006). These

results suggest a possibility that other E2 or E3 enzymes, which have either

cooperative or alternative roles, may be involved in NF-κB activation in

TCR signaling.

Modulating CBM complex activity is also a critical part of TCR-

induced NF-κB signaling. Genetic deletion of each component of the

CBM complex in mice displayed significant impairment of TCR-induced

IKK activation, which represents an indispensable role of CBM complex on

NF-κB activation in TCR signaling (Egawa et al., 2003; Ruland et al., 2001;

Ruland, Duncan, Wakeham, & Mak, 2003). BCL10 has been thought to

undergo ubiquitin-mediated downregulation by several E3 ligases such as

Itch, NEDD4, and β-TrCP for restraining TCR signaling, although it is

controversial whether it is autophagy-mediated lysosomal degradation

(Paul, Kashyap, Jia, He, & Schaefer, 2012; Scharschmidt et al., 2004) or

proteasomal degradation (Lobry, Lopez, Israel, & Weil, 2007). CARMA1

is also negatively regulated by ubiquitin modification. Monoubiquitination

of CARMA1 by E3 ubiquitin ligase Cbl-b disrupts the interaction with

BCL10, which results in the inhibition of NF-κB activation (Kojo et al.,

2009). MALT1 has a protease domain at C-terminus that is ubiquitinated

by TRAF6 for recruiting IKK complex (Oeckinghaus et al., 2007) and thus

Figure 2.4—Cont’d NF-κB activation in TCR signaling and noncanonical pathways.Upon TCR stimulation (left), a kinase cascade including Src/Syk family kinases, PI3Kand PDK1, is activated, which in turn phosphorylates serine/threonine kinase PKCθ.Subsequently, CARMA1 is phosphorylated by PKCθ, which promotes the associationof CARMA1 with BCL10–MALT1 and recruits CBM (CARMA1–BCL10–MALT1) complextomembrane. MALT1-meidated recruitment of TRAF2 and TRAF6 to CBM complex leadsto IKK activation via the conjugation of K63-linked polyubiquitin chains to BCL10,MALT1, and NEMO. Modification of CBM complex with other ubiquitin linkage types,such as K48-linked or mono-ubiquitin chains, also contributes to the regulation ofNF-κB activation. While A20 and CYLD play a negative role for TCR-induced NF-κBactivation by the cleavage of K63-linked polyubiquitin chains, USP9X positively regu-lates NF-κB activation via promoting CBM complex formation. In noncanonical NF-κBactivation (right), ligation of CD40L to CD40 in B cells induces the recruitment of TRAF2and cIAP1/2 to TRAF3, and it leads to the proteasomal degradation of TRAF3 by mod-ified cIAP1/2 with K63-linked polyubiquitin chains. TRAF3 degradation allows NIK to bedisassociated from cIAP1/2 and then accumulated, which results in IKKα-mediated p100phosphorylation and its processing to p52. In steady state, NIK constitutively undergoesproteasomal degradation by E3 complex (TRAF3, TRAF2, and cIAP1/2)-mediatedubiquitination. A20 binds and inhibits cIAP1-mediated degradation of NIK for promot-ing noncanonical NF-κB activation.

44 Yoon Park et al.

required for CARMA1 binding for the formation or stabilization of CBM

complex (Che et al., 2004). However, protease activity of MALT1 has been

reported to have a limited effect on TCR-induced NF-κB activation.

BCL10 is one of the known MALT1 targets that is processed by MALT1

protease activity. BCL10 cleavage is required for T-cell activation by regu-

lating T-cell adhesion (Rebeaud et al., 2008), but is not involved in NF-κBactivation. Another known target of MALT1 is A20, which is a critical neg-

ative regulator of NF-κB signaling. Interestingly, A20 also targets MALT1

for disrupting the sustained interaction between MALT1 and IKK complex

upon TCR stimulation by removing K63-linked polyubiquitin chains, lead-

ing to the termination of IKK activity (Duwel et al., 2009). Although

MALT1 inactivates A20 by protease activity-mediated cleavage, it seems

to be dispensable for initial IKK/NF-κB activation by TCR stimulation

(Coornaert et al., 2008). A more recent study reported that MALT1 is mod-

ified by monoubiquitination, which enhances its protease activity and leads

to enhancement of lymphocyte activation and survival (Pelzer et al., 2013).

CYLD has been known to negatively regulate NF-κB activation in TCR

signaling by removing K63-linked polyubiquitin chains from TAK1.

CYLD-deficient T cells exhibit a hyperresponsive phenotype and constitu-

tively active NF-κB (Reiley et al., 2007). We recently reported that USP9X

DUB plays a positive role for TCR-induced NF-κB activation via facilitat-

ing CBM complex formation (Park, Jin, & Liu, 2013). As there is still a lack

of knowledge of DUBs that possibly play a crucial role in TCR-induced

NF-κB signaling, future studies will be required to characterize and establish

the role of DUBs.

4.4. Noncanonical NF-κB signaling: CD40Engagement of a subset of TNFR family member, CD40 with its ligand

CD40L on B cells activates noncanonical NF-κB signaling pathway in a

ubiquitin–proteasome-dependent manner. The proteasomal processing of

p100 to p52 is the key process of the noncanonical NF-κB signaling, which

is mediated by NIK and IKKα. CD40 ligation-induced activation of NIK

phosphorylates IKKα, which in turn leads to phosphorylation and subse-

quent processing of p100 (Sun, 2010a). In steady state, NIK protein level

is constitutively regulated by TRAF3-mediated ubiquitination and

proteasomal degradation. Although TRAF3 plays an indispensable role in

NIK regulation as proved by genetic studies (Wallach & Kovalenko,

2008), TRAF3 has been thought to have a role as an adaptor that recruits


cIAP1/2, which assemble degradative polyubiquitin chains to NIK (Vince

et al., 2007). Upon CD40 stimulation, TRAF3 recruits both TRAF2 and

the associated cIAP1/2 to the receptor and then TRAF2 catalyzes K63-

linked polyubiquitin chains on cIAP1/2. Modified cIAP1/2 with K63-

linked ubiquitin facilitates K48-linked polyubiquitination of TRAF3,

which results in proteasomal degradation. TRAF3 degradation causes disas-

sociation of NIK from cIAP1/2, thereby stabilizingNIK for p100 processing

and NF-κB activation (Vallabhapurapu et al., 2008; Zarnegar et al., 2008).

A recent study proposes an “allosteric regulation model” for the mechanism

of NIK stabilization in LTβR signaling that activates noncanonical NF-κBsignaling in stromal cells, which suggests that LTβRplays as an allosteric reg-

ulator between NIK and TRAFs by competitive binding (Sanjo, Zajonc,

Braden, Norris, & Ware, 2010). More structural and biochemical studies

are required to elucidate dynamics in the receptor–TRAFs–cIAPs–NIK

complex.

Unlike canonical NF-κB signaling, deubiquitination is thought to have a

positive role in noncanonical NF-κB signaling, although related DUBs and

their role are largely unknown. A recent study reported that A20 promotes

the activation of noncanonical NF-κB pathway in a catalytic activity-

independent manner through binding to cIAP1, which results in NIK sta-

bilization (Yamaguchi, Oyama, Kozuka-Hata, & Inoue, 2013). Apparently,

more studies are needed to understand the mechanism of ubiquitin-

mediated regulation of noncanonical NF-κB signaling.

5. UBIQUITINATION IN HEMATOPOIESIS

Throughout the life-span, adult hematopoiesis continuously provides

specific subsets of immune cells, which function in innate and adaptive

immunity. Hematopoietic stem cells (HSCs) that are rare population in

the bone marrow and defined by Lin�Sca-1+c-Kit+ (LSK) can be function-

ally characterized with self-renewal capacity and multiple lineage differen-

tiation (Orkin & Zon, 2008). Because most HSCs are kept in a quiescent

state in order to prevent from HSC exhaustion, HSC fate decisions such as

quiescence, self-renew, and differentiation into mature lineage populations

are closely associated with the regulation of cell cycle. Therefore, previous

studies have intensively focused on the regulatory mechanisms how the

quiescence of HSCs is coordinated by cell-intrinsic or -extrinsic regulators

using genetically altered mice models (Orford & Scadden, 2008; Pietras,

Warr, & Passegue, 2011). However, it is only recently emerged that

46 Yoon Park et al.

posttranslational modification of proteins by ubiquitination plays an im-

portant role in HSC homeostasis as described later.

5.1. E2 enzymeUbc13 is an ubiquitin-conjugating (E2) enzyme that specifically catalyzes

K63-linked ubiquitin chains. In order to clarify the function of Ubc13 on

hematopoiesis, Wu, Yamamoto, Akira, and Sun (2009) crossed Ubc13 floxed

mice crossed with Mx-Cre mice and demonstrate that these mutant mice die

of hematopoietic failure with pancytopenia within 2weeks after the induction

ofUbc13 deletion. Ubc13 deficiency leads to the reduction of LSK and hema-

topoietic progenitor LK population due to increased apoptotic cells. More-

over, Ubc13�/� LSK cells fail to compete against Ubc13+/+ cells in

mixed bone marrow transplantation model. Mechanistically, β-catenin, acomponent of Wnt signal transduction, which contributes to maintenance

of HSC function (Kirstetter, Anderson, Porse, Jacobsen, & Nerlov, 2006),

is significantly elevated in Ubc13�/� hematopoietic cells including HSCs

(Wu et al., 2009). Although it still remains unclear whether Ubc13 directly

targets β-catenin for ubiquitin–proteasome degradation or indirectly regulates

β-catenin expression level through ubiquitination for another target protein,

these findings clearly demonstrate that K63-linked protein ubiquitination

could control adult hematopoiesis at HSC level.

5.2. RING finger E3sIt has been demonstrated that multisubunit E3s such as the cullin RING

ligase (CRL) superfamily and the Fanconi anemia (FANC) E3 complex

are known to control HSC self-renewal and differentiation. Heterozygosity

of Cul4A E3 ligase causes functional defects of HSCs with impaired engraft-

ment capacity and differentiation into multiple lineages after serial transplan-

tation (Li et al., 2007). In addition, either Cul4A or Cul4B targets

homeobox protein, HoxB4, for ubiquitin–proteasomal degradation (Lee

et al., 2013). HoxB4 has been found to positively regulate HSC proliferation

ex vivo (Amsellem et al., 2003; Antonchuk, Sauvageau, & Humphries, 2002;

Krosl et al., 2003). Indeed, transduction of degradation-resistant HoxB4

protein into human HSCs confers augmented self-renewal capacity (Lee

et al., 2013). As shown in these studies, although it still remains elusive

regarding the function of Cul4 in HSCs, these indicate that Cul4-based

E3 complex might target the other substrates as well as HoxB4.


One of F-box proteins, Skp2, forms SCF complex and function in sub-

strate recognition. Genetic deletion of Skp2 increased frequencies and abso-

lute cell numbers of LSK cells in cell-intrinsic manner (Rodriguez et al.,

2011; Wang et al., 2011). The report from Rodriguez et al. (2011) demon-

strates that Skp2 deficiency results in the elevated protein levels of CDK

inhibitors (CKIs) such as p27 and p130 and diminished cell cycle entry in

LSK cells, leading to reduced short-term engraftment. In contrast, Wang

et al. (2011) reveal that Skp2�/� LSK cells display promoted cell cycle

entry due to the increasedCyclin D1 gene expression. This study also reports

that these defects found in Skp2�/� HSCs eventually lead to the aug-

mented repopulation potential, although there was no significant difference

in the first three-round transplantation between Skp2+/+ and Skp2�/�cells. These observations are further supported with greater repopulation

capacity in Skp2�/� cells under competitive condition (Wang et al.,

2011). Although there are some discrepancies between the two studies, it

suggests that Skp2 SCF complex might contribute to adult hematopoiesis

via regulating HSC homeostasis.

The F-box and WD repeat-containing protein Fbw-7, which is known

as E3 component for ubiquitin–proteasomal degradation of c-Myc, also

plays an important role in maintaining HSC quiescence. Enforced expres-

sion of Fbw-7α in LSK cells by retrovirus transduction decreases cycling cells

with the reduction of c-Myc protein and augmented repopulation capacity

after ex vivo culture (Iriuchishima et al., 2011). On the other hand, inducible

deletion of Fbw-7 from hematopoietic cells leads to reduction of LSK and

LK cells without affecting apoptosis. Fbw-7�/� LSK cells exhibit promoted

cell cycle progression, especially characterized with decreased cells residing

in G0 phase, eventually leading to exhaustion of HSCs. Functionally, Fbw-7

deficiency causes loss of long-term repopulation capability. These defects are

able to explain by the accumulated c-Myc protein in Fbw-7�/� HSCs

(Matsuoka et al., 2008; Thompson et al., 2008). Consistent with these obser-

vations, Reavie et al. (2010) demonstrate that highly c-Myc-expressing LSK

cells exhibited accelerated cell cycle progression and less engraftment. This

study also shows that deregulated cell cycle progression found in Fbw-7-

deficient HSCs could be rescued by compound with the lack of single copy

of c-Myc.

One of the 13 FANC proteins, FANCL, contains a RING finger-like

PHD domain and catalyzes monoubiquitination of FANCD2 and FANCI,

which are required for cellular resistance in response to DNA damage

(Moldovan & D’Andrea, 2009). Lentiviral knockdown of FANCL

48 Yoon Park et al.

expression in human CD34+ cord blood stem and progenitor cells leads to

reduced expansion of multilineage progenitor cells in colony-forming assay

and reduced c-Myc expression due to attenuated Wnt/β-catenin signal

transduction (Dao et al., 2012). It is also demonstrated that ubiquitin-specific

peptidase 1 (USP1), which functions in deubiquitination of FANCD2,

implicates in regulating HSC self-renewal. FANCD2�/�mice show mar-

ked disturbed HSC function with decreased progenitor activity and

impaired engraftment, whereas USP-1 deficiency leads to augmented

HSC pool but eventually loss of reconstitution ability (Parmar et al.,

2010). Taken together, these studies suggest that the DNA repair system

mediated by FANCE3 complex is crucial for maintenance of HSC function.

With regard to cellular response to genomic damage, murine double

minute 2 (Mdm2) has been identified as an E3 to negatively regulate p53

through ubiquitin–proteasome pathway. Although Mdm2�/� mice die

at embryo, genetic compound of hypomorphilic allele of p53 (p53515C),

which lacks its apoptotic activity, is able to rescue from lethality found

in Mdm2�/� background (Liu et al., 2007). These mice exhibit hemato-

poietic failure due to the loss of LSK cells with cell cycle arrest. Notably,

reactive oxygen species (ROS), which induce DNA damage, are significantly

elevated inMdm2�/�p53515C/515C bone marrow cells compared to those of

Mdm2+/�p53515C/515C, and treatment with antioxidant N-acetyl cysteine

partially rescued dysfunction in Mdm2�/�p53515C/515C cells (Abbas et al.,

2010). Thus, oxygenation status in HSCs could impact on properly retaining

HSC function. Indeed, it is also reported that hypoxic microenvironment in

the bone marrow is associated with HSC quiescence. A component of CRL

E3 complex, von Hippel–Lindau (VHL), targets hypoxia-inducible factor-1α(HIF-1α), which is known as an essential transcription factor for cellular

response to hypoxia. Loss of VHL in hematopoietic cells causes impaired

repopulation capacity with decreased cell cycling, defective homing, and aug-

mented cell death (Takubo et al., 2010). And defects found in VHL-deficient

HSCs are HIF-1α dependent.

Epigenetic modification via chromatin-remodeling proteins is required

for transcriptional regulation of genes in eukaryotic cells. Polycomb group

proteins function as transcriptional repressors by modifying chromatin struc-

tures and can be divided into two complexes known as Polycomb repressive

complex (PRC) 1 and 2. PRC1 complex catalyzes lysine 119 of histone

H2A monoubiquitination through RING finger domain (Morey &

Helin, 2010). It has been shown that RING1B, a catalytic subunit of

PRC1, negatively regulates HSC proliferation via controlling cyclin D2


protein level (Cales et al., 2008). Moreover, BMI-1, which is a component

of PRC1 and stimulates RING1B E3 activity, has been found to regulate

self-renewal of HSCs. BMI-1�/� HSCs are hypoproliferative and show

impaired repopulation capacity (Iwama et al., 2004; Park et al., 2003;

Sauvageau et al., 1995). In contrast, retroviral introduction of BMI-1 in

mouse CD34�LSK cells and human cord blood CD34+ cells exhibits aug-

mented proliferation and long-term repopulation ability (Iwama et al., 2004;

Rizo, Dontje, Vellenga, de Haan, & Schuringa, 2008). BMI-1 appears to

negatively regulate expression of some CKIs, such as p16Ink4a and p19Arf

in HSCs (Iwama et al., 2004; Park et al., 2003).

Intracellular signal transduction via protein tyrosine kinases is implicated

in a variety of cellular processes. The RING finger E3, c-Cbl, which targets

activated receptor tyrosine kinases, negatively regulates HSC proliferation.

Although c-Cbl�/�mice are viable and fertile, impaired HSC quiescence is

found in those mice. Genetic deletion of c-Cbl leads to the augmented HSC

proliferation in cell-intrinsic manner and enhanced reconstitution capacity

under competitive conditions. Mechanistically, loss of c-Cbl induces hyper-

activated thrombopoietin-mediated signal transduction due to accumulated

STAT5, eventually leading to increased c-Myc expression (Rathinam,

Thien, Langdon, Gu, & Flavell, 2008).

5.3. HECT-type E3In comparison to the contribution of RING-type E3s in HSC homeostasis,

it still remains largely unknown how HECT-type E3s can regulate HSCs.

One of HECT-type E3, Itch, which belongs to NEDD4 family, is highly

expressing in LSK compartment of mouse bone marrow. Particularly

long-term HSCs, which are most primitive HSC population defined by

CD150+CD48� expression, contain more Itch mRNA. Itch�/� mice

show expanded HSC pool due to augmented cell cycle entry. Competitive

repopulation assay reveals that HSCs from Itch�/� mice had repopulating

advantage over Itch+/+ cells. Furthermore, deletion of Itch cell intrinsically

leads to hematopoietic recovery after myeloablation by 5-FU administra-

tion. Consistent with these observations, Itch�/� LT-HSCs still retain

more progenitor properties than Itch+/+ cells even after 20 days in vitro cul-

ture in the presence of cytokines. These findings show that Itch deficiency

confers greater capability of self-renewal in HSCs. Notably, although Itch

deficiency leads to Notch1 accumulation in Lin� bone marrow cells,

Itch�/� mice do not show any hematological malignancy, unlike other

50 Yoon Park et al.

dysregulated Notch1 signaling animal models (Aifantis, Raetz, &

Buonamici, 2008; Rathinam,Matesic, & Flavell, 2011). These findings indi-

cate that Itch could have other substrates for ubiquitination in HSCs and it

might contribute to protect against leukemogenesis in vivo.

5.4. Deubiquitinating enzymesIn addition to USP1 as described earlier, deubiquitination of H2A by a

DUB, Myb-like SWIRM and MPN domains containing protein 1

(Mysm1), is important for HSC homeostasis. Although inactivation of

Mysm1 results in expanded LSK pool, repopulation capacity of HSCs

derived from mutant mice is significantly attenuated. It is also shown that

these phenotypes were associated with increased ROS production and

p53 expression (Nijnik et al., 2012). The host cell factor 1 (HCF-1) func-

tions transcriptional regulation of cell cycle at G1/S-phase through the asso-

ciation with E2F1 and E2F4 (Tyagi, Chabes, Wysocka, & Herr, 2007).

HCF-1 is modified with K48-linked polyubiquitin chains, whereas it is

reversed by a DUB, BRCA1-associated protein 1 (BAP1) (Machida,

Machida, Vashisht, Wohlschlegel, & Dutta, 2009; Misaghi et al., 2009).

Dey et al. (2012) showed that BAP1-deficient LSK cells exhibited less col-

ony formation capability in methylcellulose culture and failure of engraft-

ment, although BAP1-deficient mice had more LSK cells in the spleen

and bone marrow. They also found marked reduction of HCF-1 levels in

BAP1-deficient splenocytes, indicating that BAP1 might stabilize HCF-1

in vivo (Dey et al., 2012). However, it still remains elusive whether the loss

of BAP1 could affect cell cycle progression of HSCs in HCF-1-

dependent manner.

6. CONCLUDING REMARKS

There has been significant progress made over the past decade in

understanding the ubiquitin system-mediated regulation of immune

responses. The components of the ubiquitin system like E2s, E3s, and DUBs

have been implicated in many aspects of both innate and adaptive immune

responses, such as modulation of inflammatory and antigen receptor signal-

ing, determination of T cell fate, andmaintenance of HSC homeostasis. Fur-

thermore, it has been identified that ubiquitin presents itself as an important

regulator of immune responses through generating unanchored poly-

ubiquitin chains. However, we are still in an early stage with numerous

questions that need to be answered. Indeed, human genome encodes


approximately over 700 E3s and only some of them have been functionally

characterized (Deshaies & Joazeiro, 2009). In addition, identification and

functional analysis of many alternative substrates and ubiquitin recognition

domains, such as F-box-, VHL box-, and ZNF-containing proteins (�300),

are also required. Therefore, quantitative proteomics and genomic high-

throughput screenings in targeted immune responses (e.g., T helper cells

under each subset polarizing conditions) will be needed for gaining a deeper

understanding the role of ubiquitin system in immune regulation.

Since an immune response is differentially regulated depending on the

type of polyubiquitin linkages, which can propagate or terminate the

immune response by the degradation of regulatory proteins or activation

of protein kinases, detecting physiologically functional polyubiquitin chains

in immune cells is an important future direction. Although genetic studies of

ubiquitin in the germ line may not be easily achieved, recently advanced

mass spectroscopic techniques and ubiquitin linkage-specific antibodies will

allow us to distinguish ubiquitin chain topology in immune cells

(Kirkpatrick, Denison, & Gygi, 2005; Matsumoto et al., 2010; Newton

et al., 2008; Wang et al., 2008). Together with structural and biochemical

studies, these approaches may provide insight into understanding the regu-

latory mechanism of immune signaling molecules by ubiquitin

modification.

The therapeutic exploitations of the ubiquitin system have been

achieved in the treatment of malignant diseases such as multiple myeloma

by inducing malignant cell death or immune cell activation. Proteasome

inhibitor, Bortezomib (Velcade), is approved by the US FDA as an antican-

cer drug for multiple myeloma and mantle cell lymphoma (Chen, Frezza,

Schmitt, Kanwar, & Dou, 2011). Although a precise working mechanism

of Bortezomib is not fully unveiled, it has clearly shown that proteasome

inhibition by Bortezomib leads to apoptosis in malignant cells by

upregulating proapoptotic proteins and downregulating antiapoptotic pro-

teins. Recent reports revealed that the modulation of not only proteasome

but also E3 ligase activity is critical for treating multiple myeloma. Thalid-

omide and its analogs, lenalidomide and pomalidomide, induce the degra-

dation of the transcription factors, Ikaros and Aiolos, via enhancing the

activity of the CRBN E3 ligase complex, which leads to the cytotoxic effect

on myeloma cells and additional increase of T-cell activation (Kronke et al.,

2014; Lu et al., 2014). Similarly, SMAC mimetics, which promotes

autoubiquitination and proteasomal degradation of cIAPs (Chen &

Huerta, 2009), have recently proved to work in cancer patients through

52 Yoon Park et al.

phase 1 clinical trials (Beug et al., 2014). As seen in the development of small

molecules as an anticancer drug, a detailed mechanistic understanding of the

ubiquitin pathway is essential for the design of new therapeutic approaches.

Therefore, further determining the basic mechanism of ubiquitination,

including how to assemble specific types of ubiquitin chains, how to select

substrates, and how to coordinate E3s and DUBs, will be ultimately required

to develop therapeutic treatments for human diseases such as autoimmune

diseases, infectious diseases, allergic diseases, and cancer.

ACKNOWLEDGMENTSWe apologize for omitting many other important publications in this limited review. This

work is supported by the National Institutes of Health (RO1AI78272, RO1AI62969, and

PO1AI089624).

REFERENCESAbbas, H. A., Maccio, D. R., Coskun, S., Jackson, J. G., Hazen, A. L., Sills, T. M., et al.

(2010). Mdm2 is required for survival of hematopoietic stem cells/progenitors via damp-ening of ROS-induced p53 activity. Cell Stem Cell, 7(5), 606–617.

Adams, C. O., Housley, W. J., Bhowmick, S., Cone, R. E., Rajan, T. V., Forouhar, F., et al.(2010). Cbl-b(�/�) T cells demonstrate in vivo resistance to regulatory T cells but acontext-dependent resistance to TGF-beta. Journal of Immunology, 185(4), 2051–2058.

Aifantis, I., Raetz, E., & Buonamici, S. (2008). Molecular pathogenesis of T-cell leukaemiaand lymphoma. Nature Reviews Immunology, 8(5), 380–390.

Amsellem, S., Pflumio, F., Bardinet, D., Izac, B., Charneau, P., Romeo, P. H., et al. (2003).Ex vivo expansion of human hematopoietic stem cells by direct delivery of the HOXB4homeoprotein. Nature Medicine, 9(11), 1423–1427.

Anandasabapathy, N., Ford, G. S., Bloom, D., Holness, C., Paragas, V., Seroogy, C., et al.(2003). GRAIL: An E3 ubiquitin ligase that inhibits cytokine gene transcription isexpressed in anergic CD4+ T cells. Immunity, 18(4), 535–547.

Antonchuk, J., Sauvageau, G., & Humphries, R. K. (2002). HOXB4-induced expansion ofadult hematopoietic stem cells ex vivo. Cell, 109(1), 39–45.

Appleman, L. J., & Boussiotis, V. A. (2003). T cell anergy and costimulation. ImmunologicalReviews, 192, 161–180.

Bachmaier, K., Krawczyk, C., Kozieradzki, I., Kong, Y. Y., Sasaki, T., Oliveira-dos-Santos, A., et al. (2000). Negative regulation of lymphocyte activation and autoimmunityby the molecular adaptor Cbl-b. Nature, 403(6766), 211–216.

Bertossi, A., Aichinger, M., Sansonetti, P., Lech, M., Neff, F., Pal, M., et al. (2011). Loss ofRoquin induces early death and immune deregulation but not autoimmunity. Journal ofExperimental Medicine, 208(9), 1749–1756.

Bertrand, M. J., Milutinovic, S., Dickson, K. M., Ho, W. C., Boudreault, A., Durkin, J.,et al. (2008). cIAP1 and cIAP2 facilitate cancer cell survival by functioning as E3 ligasesthat promote RIP1 ubiquitination. Molecular Cell, 30(6), 689–700.

Beug, S. T., Tang, V. A., Lacasse, E. C., Cheung, H. H., Beauregard, C. E., Brun, J., et al.(2014). Smac mimetics and innate immune stimuli synergize to promote tumor death.Nature Biotechnology, 32(2), 182–190.

Blonska, M., Shambharkar, P. B., Kobayashi, M., Zhang, D., Sakurai, H., Su, B., et al.(2005). TAK1 is recruited to the tumor necrosis factor-alpha (TNF-alpha) receptor 1


http://refhub.elsevier.com/B978-0-12-800147-9.00002-9/rf0005



































complex in a receptor-interacting protein (RIP)-dependent manner and cooperates withMEKK3 leading to NF-kappaB activation. Journal of Biological Chemistry, 280(52),43056–43063.

Boone, D. L., Turer, E. E., Lee, E. G., Ahmad, R. C.,Wheeler, M. T., Tsui, C., et al. (2004).The ubiquitin-modifying enzyme A20 is required for termination of Toll-like receptorresponses. Nature Immunology, 5(10), 1052–1060.

Brummelkamp, T. R., Nijman, S. M., Dirac, A. M., & Bernards, R. (2003). Loss of thecylindromatosis tumour suppressor inhibits apoptosis by activating NF-kappaB.Nature, 424(6950), 797–801.

Cales, C., Roman-Trufero, M., Pavon, L., Serrano, I., Melgar, T., Endoh, M., et al. (2008).Inactivation of the polycomb group protein Ring1b unveils an antiproliferative role inhematopoietic cell expansion and cooperation with tumorigenesis associated with Ink4adeletion. Molecular and Cellular Biology, 28(3), 1018–1028.

Chang, M., Jin, W., Chang, J. H., Xiao, Y., Brittain, G. C., Yu, J., et al. (2011). Theubiquitin ligase Peli1 negatively regulates T cell activation and prevents autoimmunity.Nature Immunology, 12(10), 1002–1009.

Chang, M., Jin,W., & Sun, S. C. (2009). Peli1 facilitates TRIF-dependent Toll-like receptorsignaling and proinflammatory cytokine production. Nature Immunology, 10(10),1089–1095.

Chang, J. H., Xiao, Y., Hu, H., Jin, J., Yu, J., Zhou, X., et al. (2012). Ubc13 maintains thesuppressive function of regulatory T cells and prevents their conversion into effector-likeT cells. Nature Immunology, 13(5), 481–490.

Che, T., You, Y., Wang, D., Tanner, M. J., Dixit, V. M., & Lin, X. (2004). MALT1/par-acaspase is a signaling component downstream of CARMA1 and mediates T cellreceptor-induced NF-kappaB activation. Journal of Biological Chemistry, 279(16),15870–15876.

Chen, Z., Barbi, J., Bu, S., Yang, H. Y., Li, Z., Gao, Y., et al. (2013). The ubiquitin ligaseStub1 negatively modulates regulatory T cell suppressive activity by promoting degra-dation of the transcription factor Foxp3. Immunity, 39(2), 272–285.

Chen, D., Frezza, M., Schmitt, S., Kanwar, J., & Dou, Q. P. (2011). Bortezomib as the firstproteasome inhibitor anticancer drug: Current status and future perspectives. CurrentCancer Drug Targets, 11(3), 239–253.

Chen, G., & Goeddel, D. V. (2002). TNF-R1 signaling: A beautiful pathway. Science,296(5573), 1634–1635.

Chen, D. J., & Huerta, S. (2009). Smac mimetics as new cancer therapeutics. Anti-CancerDrugs, 20(8), 646–658.

Chiang, Y. J., Kole, H. K., Brown, K., Naramura, M., Fukuhara, S., Hu, R. J., et al. (2000).Cbl-b regulates the CD28 dependence of T-cell activation. Nature, 403(6766),216–220.

Chiffoleau, E., Kobayashi, T., Walsh, M. C., King, C. G., Walsh, P. T., Hancock, W. W.,et al. (2003). TNF receptor-associated factor 6 deficiency during hemopoiesis inducesTh2-polarized inflammatory disease. Journal of Immunology, 171(11), 5751–5759.

Conze, D. B., Wu, C. J., Thomas, J. A., Landstrom, A., & Ashwell, J. D. (2008). Lys63-linked polyubiquitination of IRAK-1 is required for interleukin-1 receptor- andToll-like receptor-mediated NF-kappaB activation. Molecular and Cellular Biology,28(10), 3538–3547.

Coornaert, B., Baens, M., Heyninck, K., Bekaert, T., Haegman, M., Staal, J., et al. (2008).T cell antigen receptor stimulation induces MALT1 paracaspase-mediated cleavage ofthe NF-kappaB inhibitor A20. Nature Immunology, 9(3), 263–271.

Dao, K. H., Rotelli, M. D., Petersen, C. L., Kaech, S., Nelson, W. D., Yates, J. E., et al.(2012). FANCL ubiquitinates beta-catenin and enhances its nuclear function. Blood,120(2), 323–334.

54 Yoon Park et al.





















































Daubeuf, S., Singh, D., Tan, Y., Liu, H., Federoff, H. J., Bowers, W. J., et al. (2009). HSVICP0 recruits USP7 to modulate TLR-mediated innate response. Blood, 113(14),3264–3275.

Deng, L., Wang, C., Spencer, E., Yang, L., Braun, A., You, J., et al. (2000). Activation of theIkappaB kinase complex by TRAF6 requires a dimeric ubiquitin-conjugating enzymecomplex and a unique polyubiquitin chain. Cell, 103(2), 351–361.

Deshaies, R. J., & Joazeiro, C. A. (2009). RING domain E3 ubiquitin ligases. Annual Reviewof Biochemistry, 78, 399–434.

Dey, A., Seshasayee, D., Noubade, R., French, D. M., Liu, J., Chaurushiya, M. S., et al.(2012). Loss of the tumor suppressor BAP1 causes myeloid transformation. Science,337(6101), 1541–1546.

Duwel, M.,Welteke, V., Oeckinghaus, A., Baens, M., Kloo, B., Ferch, U., et al. (2009). A20negatively regulates T cell receptor signaling to NF-kappaB by cleaving Malt1 ubiquitinchains. Journal of Immunology, 182(12), 7718–7728.

Dynek, J. N., Goncharov, T., Dueber, E. C., Fedorova, A. V., Izrael-Tomasevic, A., Phu, L.,et al. (2010). c-IAP1 and UbcH5 promote K11-linked polyubiquitination of RIP1 inTNF signalling. The EMBO Journal, 29(24), 4198–4209.

Ea, C. K., Deng, L., Xia, Z. P., Pineda, G., & Chen, Z. J. (2006). Activation of IKK byTNFalpha requires site-specific ubiquitination of RIP1 and polyubiquitin binding byNEMO. Molecular Cell, 22(2), 245–257.

Egawa, T., Albrecht, B., Favier, B., Sunshine, M. J., Mirchandani, K., O’Brien, W., et al.(2003). Requirement for CARMA1 in antigen receptor-induced NF-kappaB activation and lymphocyte proliferation. Current Biology, 13(14), 1252–1258.

Emmerich, C. H., Ordureau, A., Strickson, S., Arthur, J. S., Pedrioli, P. G., Komander, D.,et al. (2013). Activation of the canonical IKK complex by K63/M1-linked hybridubiquitin chains. Proceedings of the National Academy of Sciences of the United States ofAmerica, 110(38), 15247–15252.

Enesa, K., Zakkar, M., Chaudhury, H., Luong le, A., Rawlinson, L., Mason, J. C., et al.(2008). NF-kappaB suppression by the deubiquitinating enzyme Cezanne: A novel neg-ative feedback loop in pro-inflammatory signaling. Journal of Biological Chemistry,283(11), 7036–7045.

Enzler, T., Chang, X., Facchinetti, V., Melino, G., Karin, M., Su, B., et al. (2009). MEKK1binds HECT E3 ligase Itch by its amino-terminal RING motif to regulate Th2 cytokinegene expression. Journal of Immunology, 183(6), 3831–3838.

Espinosa, A., Dardalhon, V., Brauner, S., Ambrosi, A., Higgs, R., Quintana, F. J., et al.(2009). Loss of the lupus autoantigenRo52/Trim21 induces tissue inflammation and sys-temic autoimmunity by disregulating the IL-23–Th17 pathway. Journal of ExperimentalMedicine, 206(8), 1661–1671.

Fang, D., Elly, C., Gao, B., Fang, N., Altman, Y., Joazeiro, C., et al. (2002). Dysregulation ofT lymphocyte function in itchy mice: A role for Itch in TH2 differentiation. NatureImmunology, 3(3), 281–287.

Fang, D., & Liu, Y. C. (2001). Proteolysis-independent regulation of PI3K by Cbl-b-mediated ubiquitination in T cells. Nature Immunology, 2(9), 870–875.

Fiil, B. K., Damgaard, R. B.,Wagner, S. A., Keusekotten, K., Fritsch,M., Bekker-Jensen, S.,et al. (2013). OTULIN restricts Met1-linked ubiquitination to control innate immunesignaling. Molecular Cell, 50(6), 818–830.

Frauwirth, K. A., & Thompson, C. B. (2002). Activation and inhibition of lymphocytes bycostimulation. Journal of Clinical Investigation, 109(3), 295–299.

Fukushima, T., Matsuzawa, S., Kress, C. L., Bruey, J. M., Krajewska, M., Lefebvre, S., et al.(2007). Ubiquitin-conjugating enzyme Ubc13 is a critical component of TNF receptor-associated factor (TRAF)-mediated inflammatory responses. Proceedings of the NationalAcademy of Sciences of the United States of America, 104(15), 6371–6376.






















































Gao, M., Labuda, T., Xia, Y., Gallagher, E., Fang, D., Liu, Y. C., et al. (2004). Jun turnoveris controlled through JNK-dependent phosphorylation of the E3 ligase Itch. Science,306(5694), 271–275.

Gerlach, B., Cordier, S.M., Schmukle, A. C., Emmerich, C. H., Rieser, E., Haas, T. L., et al.(2011). Linear ubiquitination prevents inflammation and regulates immune signalling.Nature, 471(7340), 591–596.

Glasmacher, E., Hoefig, K. P., Vogel, K. U., Rath, N., Du, L., Wolf, C., et al. (2010).Roquin binds inducible costimulator mRNA and effectors of mRNA decay to inducemicroRNA-independent post-transcriptional repression. Nature Immunology, 11(8),725–733.

Gronski, M. A., Boulter, J. M., Moskophidis, D., Nguyen, L. T., Holmberg, K.,Elford, A. R., et al. (2004). TCR affinity and negative regulation limit autoimmunity.Nature Medicine, 10(11), 1234–1239.

Gruber, T., Hermann-Kleiter, N., Hinterleitner, R., Fresser, F., Schneider, R., Gastl, G., et al.(2009). PKC-theta modulates the strength of T cell responses by targeting Cbl-b forubiquitination and degradation. Science Signaling, 2(76), ra30.

Guo, H., Qiao, G., Ying, H., Li, Z., Zhao, Y., Liang, Y., et al. (2012). E3 ubiquitin ligaseCbl-b regulates Pten via Nedd4 in T cells independently of its ubiquitin ligase activity.Cell Reports, 1(5), 472–482.

Hadian, K., Griesbach, R. A., Dornauer, S., Wanger, T. M., Nagel, D., Metlitzky, M., et al.(2011). NF-kappaB essential modulator (NEMO) interaction with linear and lys-63ubiquitin chains contributes to NF-kappaB activation. Journal of Biological Chemistry,286(29), 26107–26117.

Harada, Y., Harada, Y., Elly, C., Ying, G., Paik, J. H., DePinho, R. A., et al. (2010). Tran-scription factors Foxo3a and Foxo1 couple the E3 ligase Cbl-b to the induction of Foxp3expression in induced regulatory T cells. Journal of Experimental Medicine, 207(7),1381–1391.

Hartenstein, B., Teurich, S., Hess, J., Schenkel, J., Schorpp-Kistner, M., & Angel, P. (2002).Th2 cell-specific cytokine expression and allergen-induced airway inflammation dependon JunB. The EMBO Journal, 21(23), 6321–6329.

Hayden, M. S., & Ghosh, S. (2008). Shared principles in NF-kappaB signaling. Cell, 132(3),344–362.

Heissmeyer, V., Macian, F., Im, S. H., Varma, R., Feske, S., Venuprasad, K., et al. (2004).Calcineurin imposes T cell unresponsiveness through targeted proteolysis of signalingproteins. Nature Immunology, 5(3), 255–265.

Heissmeyer, V., & Vogel, K. U. (2013). Molecular control of Tfh-cell differentiation byRoquin family proteins. Immunological Reviews, 253(1), 273–289.

Hershko, A., & Ciechanover, A. (1998). The ubiquitin system.Annual Review of Biochemistry,67, 425–479.

Hosokawa, H., Kimura, M. Y., Shinnakasu, R., Suzuki, A., Miki, T., Koseki, H., et al.(2006). Regulation of Th2 cell development by Polycomb group gene Bmi-1 throughthe stabilization of GATA3. Journal of Immunology, 177(11), 7656–7664.

Hsu, H., Shu, H. B., Pan, M. G., & Goeddel, D. V. (1996). TRADD–TRAF2 andTRADD–FADD interactions define two distinct TNF receptor 1 signal transductionpathways. Cell, 84(2), 299–308.

Huang, H., Jeon, M. S., Liao, L., Yang, C., Elly, C., Yates, J. R., 3rd., et al. (2010). K33-linked polyubiquitination of T cell receptor-zeta regulates proteolysis-independentT cell signaling. Immunity, 33(1), 60–70.

Ikeda, F., & Dikic, I. (2008). Atypical ubiquitin chains: New molecular signals. ‘ProteinModifications: Beyond the Usual Suspects’ review series. EMBOReports, 9(6), 536–542.

Inoue, J., Gohda, J., & Akiyama, T. (2007). Characteristics and biological functions ofTRAF6. Advances in Experimental Medicine & Biology, 597, 72–79.

56 Yoon Park et al.





















































Iriuchishima, H., Takubo, K., Matsuoka, S., Onoyama, I., Nakayama, K. I., Nojima, Y.,et al. (2011). Ex vivo maintenance of hematopoietic stem cells by quiescence inductionthrough Fbxw7α overexpression. Blood, 117(8), 2373–2377.

Iwai, K. (2012). Diverse ubiquitin signaling in NF-kappaB activation. Trends in Cell Biology,22(7), 355–364.

Iwama, A., Oguro, H., Negishi, M., Kato, Y., Morita, Y., Tsukui, H., et al. (2004).Enhanced self-renewal of hematopoietic stem cells mediated by the polycomb geneproduct Bmi-1. Immunity, 21(6), 843–851.

Janeway, C. A., Jr., & Medzhitov, R. (2002). Innate immune recognition. Annual Review ofImmunology, 20, 197–216.

Jang, E. J., Park, H. R., Hong, J. H., & Hwang, E. S. (2013). Lysine 313 of T-box is crucialfor modulation of protein stability, DNA binding, and threonine phosphorylation ofT-bet. Journal of Immunology, 190(11), 5764–5770.

Jeon,M.S.,Atfield,A.,Venuprasad,K.,Krawczyk,C., Sarao,R.,Elly,C., et al. (2004).Essentialrole of the E3 ubiquitin ligase Cbl-b in T cell anergy induction. Immunity, 21(2), 167–177.

Ji, Y. R., Kim, H. J., Yu, D. H., Bae, K. B., Park, S. J., Yi, J. K., et al. (2012). Enforcedexpression of roquin protein in T cells exacerbates the incidence and severity of exper-imental arthritis. Journal of Biological Chemistry, 287(50), 42269–42277.

Jin, W., Chang, M., & Sun, S. C. (2012). Peli: A family of signal-responsive E3 ubiquitinligases mediating TLR signaling and T-cell tolerance. Cellular & Molecular Immunology,9(2), 113–122.

Jin, H. S., Park, Y., Elly, C., & Liu, Y. C. (2013). Itch expression by Treg cells controls Th2inflammatory responses. Journal of Clinical Investigation, 123(11), 4923–4934.

Kanayama, A., Seth, R. B., Sun, L., Ea, C. K., Hong, M., Shaito, A., et al. (2004). TAB2 andTAB3 activate the NF-kappaB pathway through binding to polyubiquitin chains.Molec-ular Cell, 15(4), 535–548.

Keusekotten, K., Elliott, P. R., Glockner, L., Fiil, B. K., Damgaard, R. B., Kulathu, Y., et al.(2013). OTULIN antagonizes LUBAC signaling by specifically hydrolyzing Met1-linked polyubiquitin. Cell, 153(6), 1312–1326.

Kim, Y. C., Bhairavabhotla, R., Yoon, J., Golding, A., Thornton, A. M., Tran, D. Q., et al.(2012). Oligodeoxynucleotides stabilize Helios-expressing Foxp3+ human T regulatorycells during in vitro expansion. Blood, 119(12), 2810–2818.

King, C. G., Buckler, J. L., Kobayashi, T., Hannah, J. R., Bassett, G., Kim, T., et al. (2008).Cutting edge: Requirement for TRAF6 in the induction of T cell anergy. Journal ofImmunology, 180(1), 34–38.

King, C. G., Kobayashi, T., Cejas, P. J., Kim, T., Yoon, K., Kim, G. K., et al. (2006).TRAF6 is a T cell-intrinsic negative regulator required for the maintenance of immunehomeostasis. Nature Medicine, 12(9), 1088–1092.

Kirkpatrick, D. S., Denison, C., & Gygi, S. P. (2005). Weighing in on ubiquitin: The expan-ding role of mass-spectrometry-based proteomics. Nature Cell Biology, 7(8), 750–757.

Kirstetter, P., Anderson, K., Porse, B. T., Jacobsen, S. E., & Nerlov, C. (2006). Activation ofthe canonical Wnt pathway leads to loss of hematopoietic stem cell repopulation andmultilineage differentiation block. Nature Immunology, 7(10), 1048–1056.

Kobayashi, N., Kadono, Y., Naito, A., Matsumoto, K., Yamamoto, T., Tanaka, S., et al.(2001). Segregation of TRAF6-mediated signaling pathways clarifies its role inosteoclastogenesis. The EMBO Journal, 20(6), 1271–1280.

Kobayashi, T., Walsh, M. C., & Choi, Y. (2004). The role of TRAF6 in signal transductionand the immune response. Microbes and Infection, 6(14), 1333–1338.

Kojo, S., Elly, C., Harada, Y., Langdon,W. Y., Kronenberg,M., & Liu, Y. C. (2009).Mech-anisms of NKT cell anergy induction involve Cbl-b-promoted monoubiquitination ofCARMA1. Proceedings of the National Academy of Sciences of the United States of America,106(42), 17847–17851.























































Komander, D. (2009). The emerging complexity of protein ubiquitination. Biochemical Soci-ety Transactions, 37(Pt 5), 937–953.

Komander, D., Clague, M. J., &Urbe, S. (2009). Breaking the chains: Structure and functionof the deubiquitinases. Nature Reviews Molecular Cell Biology, 10(8), 550–563.

Komander, D., Reyes-Turcu, F., Licchesi, J. D., Odenwaelder, P., Wilkinson, K. D., &Barford, D. (2009). Molecular discrimination of structurally equivalent Lys 63-linkedand linear polyubiquitin chains. EMBO Reports, 10(5), 466–473.

Kovalenko, A., Chable-Bessia, C., Cantarella, G., Israel, A., Wallach, D., & Courtois, G.(2003). The tumour suppressor CYLD negatively regulates NF-kappaB signalling bydeubiquitination. Nature, 424(6950), 801–805.

Krawczyk, C., Bachmaier, K., Sasaki, T., Jones, R. G., Snapper, S. B., Bouchard, D., et al.(2000). Cbl-b is a negative regulator of receptor clustering and raft aggregation in T cells.Immunity, 13(4), 463–473.

Kriegel, M. A., Rathinam, C., & Flavell, R. A. (2009). E3 ubiquitin ligase GRAIL controlsprimary T cell activation and oral tolerance. Proceedings of the National Academy of Sciencesof the United States of America, 106(39), 16770–16775.

Kronke, J., Udeshi, N. D., Narla, A., Grauman, P., Hurst, S. N., McConkey, M., et al.(2014). Lenalidomide causes selective degradation of IKZF1 and IKZF3 in multiplemyeloma cells. Science, 343(6168), 301–305.

Krosl, J., Austin, P., Beslu, N., Kroon, E., Humphries, R. K., & Sauvageau, G. (2003).In vitro expansion of hematopoietic stem cells by recombinant TAT-HOXB4 protein.Nature Medicine, 9(11), 1428–1432.

Lamothe, B., Besse, A., Campos, A. D., Webster, W. K., Wu, H., & Darnay, B. G. (2007).Site-specific Lys-63-linked tumor necrosis factor receptor-associated factor 6 auto-ubiquitination is a critical determinant of I kappa B kinase activation. Journal of BiologicalChemistry, 282(6), 4102–4112.

Laplantine, E., Fontan, E., Chiaravalli, J., Lopez, T., Lakisic, G., Veron, M., et al. (2009).NEMO specifically recognizes K63-linked poly-ubiquitin chains through a new bipar-tite ubiquitin-binding domain. The EMBO Journal, 28(19), 2885–2895.

Lee, E. G., Boone, D. L., Chai, S., Libby, S. L., Chien, M., Lodolce, J. P., et al. (2000).Failure to regulate TNF-induced NF-kappaB and cell death responses in A20-deficientmice. Science, 289(5488), 2350–2354.

Lee, S. Y., Reichlin, A., Santana, A., Sokol, K. A., Nussenzweig, M. C., & Choi, Y. (1997).TRAF2 is essential for JNK but not NF-kappaB activation and regulates lymphocyteproliferation and survival. Immunity, 7(5), 703–713.

Lee, T. H., Shank, J., Cusson, N., & Kelliher, M. A. (2004). The kinase activity of Rip1 is notrequired for tumor necrosis factor-alpha-induced IkappaB kinase or p38 MAP kinaseactivation or for the ubiquitination of Rip1 by Traf2. Journal of Biological Chemistry,279(32), 33185–33191.

Lee, J., Shieh, J. H., Zhang, J., Liu, L., Zhang, Y., Eom, J. Y., et al. (2013). Improved ex vivoexpansion of adult hematopoietic stem cells by overcoming Cul4-mediated degradationof HOXB4. Blood, 121(20), 4082–4089.

Li, B., Jia, N., Waning, D. L., Yang, F. C., Haneline, L. S., & Chun, K. T. (2007). Cul4A isrequired for hematopoietic stem-cell engraftment and self-renewal. Blood, 110(7),2704–2707.

Li, H., Kobayashi, M., Blonska, M., You, Y., & Lin, X. (2006). Ubiquitination of RIP isrequired for tumor necrosis factor alpha-induced NF-kappaB activation. Journal of Bio-logical Chemistry, 281(19), 13636–13643.

Li, B., Tournier, C., Davis, R. J., & Flavell, R. A. (1999). Regulation of IL-4 expression bythe transcription factor JunB during T helper cell differentiation. The EMBO Journal,18(2), 420–432.

58 Yoon Park et al.




















































Liang, Y., Seymour, R. E., & Sundberg, J. P. (2011). Inhibition of NF-kappaB signalingretards eosinophilic dermatitis in SHARPIN-deficient mice. Journal of Investigative Der-matology, 131(1), 141–149.

Linterman, M. A., Rigby, R. J., Wong, R. K., Yu, D., Brink, R., Cannons, J. L., et al.(2009). Follicular helper T cells are required for systemic autoimmunity. Journal of Exper-imental Medicine, 206(3), 561–576.

Liu, Y. C. (2007). The E3 ubiquitin ligase Itch in T cell activation, differentiation, and tol-erance. Seminars in Immunology, 19(3), 197–205.

Liu, Y. C., & Gu, H. (2002). Cbl and Cbl-b in T-cell regulation. Trends in Immunology, 23(3),140–143.

Liu, X., Li, H., Zhong, B., Blonska, M., Gorjestani, S., Yan,M., et al. (2013). USP18 inhibitsNF-kappaB and NFAT activation during Th17 differentiation by deubiquitinating theTAK1–TAB1 complex. Journal of Experimental Medicine, 210(8), 1575–1590.

Liu, G., Terzian, T., Xiong, S., Van Pelt, C. S., Audiffred, A., Box, N. F., et al. (2007). Thep53-Mdm2 network in progenitor cell expansion during mouse postnatal development.Journal of Pathology, 213(4), 360–368.

Lobry, C., Lopez, T., Israel, A., & Weil, R. (2007). Negative feedback loop in T cell acti-vation through IkappaB kinase-induced phosphorylation and degradation of BCL10.Proceedings of the National Academy of Sciences of the United States of America, 104(3),908–913.

Lohr, N. J., Molleston, J. P., Strauss, K. A., Torres-Martinez, W., Sherman, E. A.,Squires, R. H., et al. (2010). Human ITCH E3 ubiquitin ligase deficiency causes syn-dromic multisystem autoimmune disease. American Journal of Human Genetics, 86(3),447–453.

Lomaga, M. A., Yeh, W. C., Sarosi, I., Duncan, G. S., Furlonger, C., Ho, A., et al. (1999).TRAF6 deficiency results in osteopetrosis and defective interleukin-1, CD40, and LPSsignaling. Genes & Development, 13(8), 1015–1024.

Lu, G., Middleton, R. E., Sun, H., Naniong, M., Ott, C. J., Mitsiades, C. S., et al. (2014).Themyeloma drug lenalidomide promotes the cereblon-dependent destruction of Ikarosproteins. Science, 343(6168), 305–309.

Ma, A., & Malynn, B. A. (2012). A20: Linking a complex regulator of ubiquitylation toimmunity and human disease. Nature Reviews Immunology, 12(11), 774–785.

Machida, Y. J., Machida, Y., Vashisht, A. A., Wohlschlegel, J. A., & Dutta, A. (2009). Thedeubiquitinating enzyme BAP1 regulates cell growth via interaction with HCF-1. Journalof Biological Chemistry, 284(49), 34179–34188.

Mahoney, D. J., Cheung, H. H., Mrad, R. L., Plenchette, S., Simard, C., Enwere, E., et al.(2008). Both cIAP1 and cIAP2 regulate TNFalpha-mediated NF-kappaB activation.Proceedings of the National Academy of Sciences of the United States of America, 105(33),11778–11783.

Manning, B. D., & Cantley, L. C. (2007). AKT/PKB signaling: Navigating downstream.Cell, 129(7), 1261–1274.

Matsumoto, M. L., Wickliffe, K. E., Dong, K. C., Yu, C., Bosanac, I., Bustos, D., et al.(2010). K11-linked polyubiquitination in cell cycle control revealed by a K11linkage-specific antibody. Molecular Cell, 39(3), 477–484.

Matsuoka, S., Oike, Y., Onoyama, I., Iwama, A., Arai, F., Takubo, K., et al. (2008). Fbxw7acts as a critical fail-safe against premature loss of hematopoietic stem cells and develop-ment of T-ALL. Genes & Development, 22(8), 986–991.

Medzhitov, R., & Janeway, C. A., Jr. (1998). Innate immune recognition and control ofadaptive immune responses. Seminars in Immunology, 10(5), 351–353.

Micheau, O., & Tschopp, J. (2003). Induction of TNF receptor I-mediated apoptosis via twosequential signaling complexes. Cell, 114(2), 181–190.





















































Misaghi, S., Ottosen, S., Izrael-Tomasevic, A., Arnott, D., Lamkanfi, M., Lee, J., et al.(2009). Association of C-terminal ubiquitin hydrolase BRCA1-associated protein 1 withcell cycle regulator host cell factor 1. Molecular and Cellular Biology, 29(8), 2181–2192.

Moldovan, G. L., & D’Andrea, A. D. (2009). How the fanconi anemia pathway guards thegenome. Annual Review of Genetics, 43, 223–249.

Morey, L., & Helin, K. (2010). Polycomb group protein-mediated repression of transcrip-tion. Trends in Biochemical Sciences, 35(6), 323–332.

Moynagh, P. N. (2009). The Pellino family: IRAK E3 ligases with emerging roles in innateimmune signalling. Trends in Immunology, 30(1), 33–42.

Newton, K., Matsumoto, M. L., Wertz, I. E., Kirkpatrick, D. S., Lill, J. R., Tan, J., et al.(2008). Ubiquitin chain editing revealed by polyubiquitin linkage-specific antibodies.Cell, 134(4), 668–678.

Nijnik, A., Clare, S., Hale, C., Raisen, C., McIntyre, R. E., Yusa, K., et al. (2012). Thecritical role of histone H2A-deubiquitinase Mysm1 in hematopoiesis and lymphocytedifferentiation. Blood, 119(6), 1370–1379.

Nurieva, R. I., Zheng, S., Jin, W., Chung, Y., Zhang, Y., Martinez, G. J., et al. (2010). TheE3 ubiquitin ligase GRAIL regulates T cell tolerance and regulatory T cell function bymediating T cell receptor-CD3 degradation. Immunity, 32(5), 670–680.

Oeckinghaus, A., Wegener, E., Welteke, V., Ferch, U., Arslan, S. C., Ruland, J., et al.(2007). Malt1 ubiquitination triggers NF-kappaB signaling upon T-cell activation.The EMBO Journal, 26(22), 4634–4645.

Oliver, P. M., Cao, X., Worthen, G. S., Shi, P., Briones, N., MacLeod, M., et al. (2006).Ndfip1 protein promotes the function of itch ubiquitin ligase to prevent T cell activationand T helper 2 cell-mediated inflammation. Immunity, 25(6), 929–940.

Orford, K. W., & Scadden, D. T. (2008). Deconstructing stem cell self-renewal: Geneticinsights into cell-cycle regulation. Nature Reviews Genetics, 9(2), 115–128.

Orkin, S. H., & Zon, L. I. (2008). Hematopoiesis: An evolving paradigm for stem cell biol-ogy. Cell, 132(4), 631–644.

Paolino, M., & Penninger, J. M. (2010). Cbl-b in T-cell activation. Seminars in Immunopa-thology, 32(2), 137–148.

Paolino, M., Thien, C. B., Gruber, T., Hinterleitner, R., Baier, G., Langdon, W. Y., et al.(2011). Essential role of E3 ubiquitin ligase activity in Cbl-b-regulated T cell functions.Journal of Immunology, 186(4), 2138–2147.

Park, Y., Jin, H. S., & Liu, Y. C. (2013). Regulation of T cell function by the ubiquitin-specific protease USP9X via modulating the CARMA1–BCL10–MALT1 complex.Proceedings of the National Academy of Sciences of the United States of America, 110(23),9433–9438.

Park, I. K., Qian, D., Kiel, M., Becker, M. W., Pihalja, M., Weissman, I. L., et al. (2003).Bmi-1 is required for maintenance of adult self-renewing haematopoietic stem cells.Nature, 423(6937), 302–305.

Parmar, K., Kim, J., Sykes, S. M., Shimamura, A., Stuckert, P., Zhu, K., et al. (2010). Hema-topoietic stem cell defects in mice with deficiency of Fancd2 or Usp1. Stem Cells, 28(7),1186–1195.

Paul, S., Kashyap, A. K., Jia,W., He, Y.W., & Schaefer, B. C. (2012). Selective autophagy ofthe adaptor protein BCL10 modulates T cell receptor activation of NF-kappaB.Immunity, 36(6), 947–958.

Pelzer, C., Cabalzar, K., Wolf, A., Gonzalez, M., Lenz, G., & Thome, M. (2013). The pro-tease activity of the paracaspase MALT1 is controlled by monoubiquitination. NatureImmunology, 14(4), 337–345.

Perry, W. L., Hustad, C. M., Swing, D. A., O’Sullivan, T. N., Jenkins, N. A., &Copeland, N. G. (1998). The itchy locus encodes a novel ubiquitin protein ligase thatis disrupted in a18H mice. Nature Genetics, 18(2), 143–146.

60 Yoon Park et al.





















































Pickart, C. M. (2001). Mechanisms underlying ubiquitination. Annual Review of Biochemistry,70, 503–533.

Pickart, C. M., & Fushman, D. (2004). Polyubiquitin chains: Polymeric protein signals. Cur-rent Opinion in Chemical Biology, 8(6), 610–616.

Pietras, E. M., Warr, M. R., & Passegue, E. (2011). Cell cycle regulation in hematopoieticstem cells. Journal of Cell Biology, 195(5), 709–720.

Potter, C. S., Wang, Z., Silva, K. A., Kennedy, V. E., Stearns, T. M., Burzenski, L., et al.(2014). Chronic proliferative dermatitis in sharpin null mice: Development of anautoinflammatory disease in the absence of B and T lymphocytes and IL4/IL13 signaling.PLoS One, 9(1), e85666.

Pratama, A., Ramiscal, R. R., Silva, D. G., Das, S. K., Athanasopoulos, V., Fitch, J., et al.(2013). Roquin-2 shares functions with its paralog Roquin-1 in the repression ofmRNAs controlling T follicular helper cells and systemic inflammation. Immunity,38(4), 669–680.

Rahighi, S., Ikeda, F., Kawasaki, M., Akutsu, M., Suzuki, N., Kato, R., et al. (2009). Specificrecognition of linear ubiquitin chains by NEMO is important for NF-kappaB activation.Cell, 136(6), 1098–1109.

Ramon, H. E., Beal, A. M., Liu, Y., Worthen, G. S., & Oliver, P. M. (2012). The E3ubiquitin ligase adaptor Ndfip1 regulates Th17 differentiation by limiting the productionof proinflammatory cytokines. Journal of Immunology, 188(8), 4023–4031.

Rantala, J. K., Pouwels, J., Pellinen, T., Veltel, S., Laasola, P., Mattila, E., et al. (2011).SHARPIN is an endogenous inhibitor of beta1-integrin activation. Nature Cell Biology,13(11), 1315–1324.

Rao, A. (2009). Signaling to gene expression: Calcium, calcineurin andNFAT.Nature Immu-nology, 10(1), 3–5.

Rathinam, C., Matesic, L. E., & Flavell, R. A. (2011). The E3 ligase Itch is a negative reg-ulator of the homeostasis and function of hematopoietic stem cells. Nature Immunology,12(5), 399–407.

Rathinam, C., Thien, C. B., Langdon, W. Y., Gu, H., & Flavell, R. A. (2008). The E3ubiquitin ligase c-Cbl restricts development and functions of hematopoietic stem cells.Genes & Development, 22(8), 992–997.

Reavie, L., Della Gatta, G., Crusio, K., Aranda-Orgilles, B., Buckley, S. M., Thompson, B.,et al. (2010). Regulation of hematopoietic stem cell differentiation by a single ubiquitinligase–substrate complex. Nature Immunology, 11(3), 207–215.

Rebeaud, F., Hailfinger, S., Posevitz-Fejfar, A., Tapernoux,M., Moser, R., Rueda, D., et al.(2008). The proteolytic activity of the paracaspase MALT1 is key in T cell activation.Nature Immunology, 9(3), 272–281.

Reiley, W. W., Jin, W., Lee, A. J., Wright, A., Wu, X., Tewalt, E. F., et al. (2007).Deubiquitinating enzyme CYLD negatively regulates the ubiquitin-dependent kinaseTak1 and prevents abnormal T cell responses. Journal of Experimental Medicine, 204(6),1475–1485.

Reissig, S., Hovelmeyer, N.,Weigmann, B., Nikolaev, A., Kalt, B., Wunderlich, T. F., et al.(2012). The tumor suppressor CYLD controls the function of murine regulatory T cells.Journal of Immunology, 189(10), 4770–4776.

Reyes-Turcu, F. E., Ventii, K. H., &Wilkinson, K. D. (2009). Regulation and cellular roles ofubiquitin-specific deubiquitinating enzymes. Annual Review of Biochemistry, 78, 363–397.

Rizo, A., Dontje, B., Vellenga, E., de Haan, G., & Schuringa, J. J. (2008). Long-term main-tenance of human hematopoietic stem/progenitor cells by expression of BMI1. Blood,111(5), 2621–2630.

Rodriguez, S., Wang, L., Mumaw, C., Srour, E. F., Lo Celso, C., Nakayama, K., et al.(2011). The SKP2 E3 ligase regulates basal homeostasis and stress-induced regenerationof HSCs. Blood, 117(24), 6509–6519.






















































Ruland, J., Duncan, G. S., Elia, A., del Barco Barrantes, I., Nguyen, L., Plyte, S., et al.(2001). BCL10 is a positive regulator of antigen receptor-induced activation ofNF-kappaB and neural tube closure. Cell, 104(1), 33–42.

Ruland, J., Duncan, G. S., Wakeham, A., &Mak, T.W. (2003). Differential requirement forMalt1 in T and B cell antigen receptor signaling. Immunity, 19(5), 749–758.

Sanjo, H., Zajonc, D. M., Braden, R., Norris, P. S., & Ware, C. F. (2010). Allosteric reg-ulation of the ubiquitin:NIK and ubiquitin:TRAF3 E3 ligases by the lymphotoxin-betareceptor. Journal of Biological Chemistry, 285(22), 17148–17155.

Sauvageau, G., Thorsteinsdottir, U., Eaves, C. J., Lawrence, H. J., Largman, C.,Lansdorp, P. M., et al. (1995). Overexpression of HOXB4 in hematopoietic cells causesthe selective expansion of more primitive populations in vitro and in vivo.Genes &Devel-opment, 9(14), 1753–1765.

Scharschmidt, E., Wegener, E., Heissmeyer, V., Rao, A., & Krappmann, D. (2004). Deg-radation of BCL10 induced by T-cell activation negatively regulates NF-kappaB signaling. Molecular and Cellular Biology, 24(9), 3860–3873.

Schulze-Luehrmann, J., & Ghosh, S. (2006). Antigen-receptor signaling to nuclear factorkappa B. Immunity, 25(5), 701–715.

Seroogy, C. M., Soares, L., Ranheim, E. A., Su, L., Holness, C., Bloom, D., et al. (2004).The gene related to anergy in lymphocytes, an E3 ubiquitin ligase, is necessary for anergyinduction in CD4 T cells. Journal of Immunology, 173(1), 79–85.

Shembade, N., Harhaj, N. S., Liebl, D. J., & Harhaj, E. W. (2007). Essential role forTAX1BP1 in the termination of TNF-alpha-, IL-1- and LPS-mediated NF-kappaBand JNK signaling. The EMBO Journal, 26(17), 3910–3922.

Shembade, N., Harhaj, N. S., Parvatiyar, K., Copeland, N. G., Jenkins, N. A., Matesic, L. E.,et al. (2008). The E3 ligase Itch negatively regulates inflammatory signaling pathways bycontrolling the function of the ubiquitin-editing enzyme A20. Nature Immunology, 9(3),254–262.

Shembade, N., Ma, A., & Harhaj, E. W. (2010). Inhibition of NF-kappaB signaling by A20through disruption of ubiquitin enzyme complexes. Science, 327(5969), 1135–1139.

Shembade, N., Parvatiyar, K., Harhaj, N. S., & Harhaj, E. W. (2009). The ubiquitin-editingenzyme A20 requires RNF11 to downregulate NF-kappaB signalling. The EMBOJournal, 28(5), 513–522.

Shu, H. B., Takeuchi, M., & Goeddel, D. V. (1996). The tumor necrosis factor receptor 2signal transducers TRAF2 and c-IAP1 are components of the tumor necrosis factorreceptor 1 signaling complex. Proceedings of the National Academy of Sciences of the UnitedStates of America, 93(24), 13973–13978.

Skaug, B., Jiang, X., & Chen, Z. J. (2009). The role of ubiquitin in NF-kappaB regulatorypathways. Annual Review of Biochemistry, 78, 769–796.

Smith-Garvin, J. E., Koretzky, G. A., & Jordan, M. S. (2009). T cell activation. AnnualReview of Immunology, 27, 591–619.

Sun, S. C. (2008). Deubiquitylation and regulation of the immune response. Nature ReviewsImmunology, 8(7), 501–511.

Sun, S. C. (2010a). Controlling the fate of NIK: A central stage in noncanonical NF-kappaBsignaling. Science Signaling, 3(123), pe18.

Sun, S. C. (2010b). CYLD: A tumor suppressor deubiquitinase regulating NF-kappaB acti-vation and diverse biological processes. Cell Death and Differentiation, 17(1), 25–34.

Sun, L., Deng, L., Ea, C. K., Xia, Z. P., & Chen, Z. J. (2004). The TRAF6 ubiquitin ligaseand TAK1 kinase mediate IKK activation by BCL10 and MALT1 in T lymphocytes.Molecular Cell, 14(3), 289–301.

Sun,W., Tan, X., Shi, Y., Xu, G., Mao, R., Gu, X., et al. (2010). USP11 negatively regulatesTNFalpha-induced NF-kappaB activation by targeting on IkappaBalpha.Cellular Signal-ling, 22(3), 386–394.

62 Yoon Park et al.





















































Swaidani, S., Bulek, K., Kang, Z., Gulen, M. F., Liu, C., Yin, W., et al. (2011). T cell-derived Act1 is necessary for IL-25-mediated Th2 responses and allergic airway inflam-mation. Journal of Immunology, 187(6), 3155–3164.

Tada, K., Okazaki, T., Sakon, S., Kobarai, T., Kurosawa, K., Yamaoka, S., et al. (2001). Crit-ical roles of TRAF2 and TRAF5 in tumor necrosis factor-induced NF-kappaB activation and protection from cell death. Journal of Biological Chemistry, 276(39),36530–36534.

Takubo, K., Goda, N., Yamada, W., Iriuchishima, H., Ikeda, E., Kubota, Y., et al. (2010).Regulation of the HIF-1alpha level is essential for hematopoietic stem cells. Cell StemCell, 7(3), 391–402.

Tanaka, T., Soriano,M. A., &Grusby,M. J. (2005). SLIM is a nuclear ubiquitin E3 ligase thatnegatively regulates STAT signaling. Immunity, 22(6), 729–736.

Tanaka, T., Yamamoto, Y., Muromoto, R., Ikeda, O., Sekine, Y., Grusby, M. J., et al.(2011). PDLIM2 inhibits T helper 17 cell development and granulomatous inflammationthrough degradation of STAT3. Science Signaling, 4(202), ra85.

Thome, M., Charton, J. E., Pelzer, C., & Hailfinger, S. (2010). Antigen receptor signaling toNF-kappaB via CARMA1, BCL10, and MALT1. Cold Spring Harbor Perspectives in Biol-ogy, 2(9), a003004.

Thompson, B. J., Jankovic, V., Gao, J., Buonamici, S., Vest, A., Lee, J. M., et al. (2008).Control of hematopoietic stem cell quiescence by the E3 ubiquitin ligase Fbw7. Journalof Experimental Medicine, 205(6), 1395–1408.

Thornton, A. M., Korty, P. E., Tran, D. Q., Wohlfert, E. A., Murray, P. E., Belkaid, Y.,et al. (2010). Expression of Helios, an Ikaros transcription factor family member, differ-entiates thymic-derived from peripherally induced Foxp3+ T regulatory cells. Journal ofImmunology, 184(7), 3433–3441.

Tokunaga, F., Nakagawa, T., Nakahara, M., Saeki, Y., Taniguchi, M., Sakata, S., et al.(2011). SHARPIN is a component of the NF-kappaB-activating linear ubiquitin chainassembly complex. Nature, 471(7340), 633–636.

Tokunaga, F., Nishimasu, H., Ishitani, R., Goto, E., Noguchi, T., Mio, K., et al. (2012).Specific recognition of linear polyubiquitin by A20 zinc finger 7 is involved inNF-kappaB regulation. The EMBO Journal, 31(19), 3856–3870.

Tokunaga, F., Sakata, S., Saeki, Y., Satomi, Y., Kirisako, T., Kamei, K., et al. (2009).Involvement of linear polyubiquitylation of NEMO in NF-kappaB activation. NatureCell Biology, 11(2), 123–132.

Trompouki, E., Hatzivassiliou, E., Tsichritzis, T., Farmer, H., Ashworth, A., &Mosialos, G.(2003). CYLD is a deubiquitinating enzyme that negatively regulates NF-kappaB acti-vation by TNFR family members. Nature, 424(6950), 793–796.

Tyagi, S., Chabes, A. L., Wysocka, J., & Herr, W. (2007). E2F activation of S phase pro-moters via association with HCF-1 and the MLL family of histone H3K4 met-hyltransferases. Molecular Cell, 27(1), 107–119.

Tzimas, C., Michailidou, G., Arsenakis, M., Kieff, E., Mosialos, G., & Hatzivassiliou, E. G.(2006). Human ubiquitin specific protease 31 is a deubiquitinating enzyme implicated inactivation of nuclear factor-kappaB. Cellular Signalling, 18(1), 83–92.

Vallabhapurapu, S., & Karin, M. (2009). Regulation and function of NF-kappaB transcrip-tion factors in the immune system. Annual Review of Immunology, 27, 693–733.

Vallabhapurapu, S., Matsuzawa, A., Zhang, W., Tseng, P. H., Keats, J. J., Wang, H., et al.(2008). Nonredundant and complementary functions of TRAF2 and TRAF3 in aubiquitination cascade that activates NIK-dependent alternative NF-kappaB signaling.Nature Immunology, 9(12), 1364–1370.

van Loosdregt, J., Fleskens, V., Fu, J., Brenkman, A. B., Bekker, C. P., Pals, C. E., et al.(2013). Stabilization of the transcription factor Foxp3 by the deubiquitinase USP7increases Treg-cell-suppressive capacity. Immunity, 39(2), 259–271.























































Varfolomeev, E., Goncharov, T., Fedorova, A. V., Dynek, J. N., Zobel, K., Deshayes, K.,et al. (2008). c-IAP1 and c-IAP2 are critical mediators of tumor necrosis factor alpha(TNFalpha)-induced NF-kappaB activation. Journal of Biological Chemistry, 283(36),24295–24299.

Venuprasad, K., Elly, C., Gao, M., Salek-Ardakani, S., Harada, Y., Luo, J. L., et al. (2006).Convergence of Itch-induced ubiquitination with MEKK1–JNK signaling in Th2 tol-erance and airway inflammation. Journal of Clinical Investigation, 116(4), 1117–1126.

Verhelst, K., Carpentier, I., Kreike, M., Meloni, L., Verstrepen, L., Kensche, T., et al.(2012). A20 inhibits LUBAC-mediated NF-kappaB activation by binding linear poly-ubiquitin chains via its zinc finger 7. The EMBO Journal, 31(19), 3845–3855.

Verstrepen, L., Bekaert, T., Chau, T. L., Tavernier, J., Chariot, A., & Beyaert, R. (2008).TLR-4, IL-1R and TNF-R signaling to NF-kappaB: Variations on a common theme.Cellular and Molecular Life Sciences, 65(19), 2964–2978.

Vince, J. E., Wong, W. W., Khan, N., Feltham, R., Chau, D., Ahmed, A. U., et al. (2007).IAP antagonists target cIAP1 to induce TNFalpha-dependent apoptosis. Cell, 131(4),682–693.

Vogel, K. U., Edelmann, S. L., Jeltsch, K. M., Bertossi, A., Heger, K., Heinz, G. A., et al.(2013). Roquin paralogs 1 and 2 redundantly repress the ICOS and Ox40 costimulatormRNAs and control follicular helper T cell differentiation. Immunity, 38(4), 655–668.

Wallach, D., &Kovalenko, A. (2008). Self-termination of the terminator.Nature Immunology,9(12), 1325–1327.

Wang, C., Deng, L., Hong, M., Akkaraju, G. R., Inoue, J., & Chen, Z. J. (2001). TAK1 is aubiquitin-dependent kinase of MKK and IKK. Nature, 412(6844), 346–351.

Wang, J., Han, F.,Wu, J., Lee, S.W., Chan, C. H.,Wu, C. Y., et al. (2011). The role of Skp2in hematopoietic stem cell quiescence, pool size, and self-renewal. Blood, 118(20),5429–5438.

Wang, H., Matsuzawa, A., Brown, S. A., Zhou, J., Guy, C. S., Tseng, P. H., et al. (2008).Analysis of nondegradative protein ubiquitylation with a monoclonal antibody specificfor lysine-63-linked polyubiquitin. Proceedings of the National Academy of Sciences of theUnited States of America, 105(51), 20197–20202.

Wang, D., Qin, H., Du,W., Shen, Y.W., Lee, W. H., Riggs, A. D., et al. (2012). Inhibitionof S-phase kinase-associated protein 2 (Skp2) reprograms and converts diabetogenicT cells to Foxp3+ regulatory T cells. Proceedings of the National Academy of Sciences ofthe United States of America, 109(24), 9493–9498.

Weiss, J. M., Bilate, A. M., Gobert, M., Ding, Y., Parkhurst, C. N., Curotto deLafaille, M. A., et al. (2012). Neuropilin 1 is expressed on thymus-derived natural reg-ulatory T cells, but not mucosa-generated induced Foxp3+ T reg cells. The Journal ofExperimental Medicine, 209(10), 1723–1742, S1721.

Wertz, I. E., O’Rourke, K. M., Zhou, H., Eby, M., Aravind, L., Seshagiri, S., et al. (2004).De-ubiquitination and ubiquitin ligase domains of A20 downregulate NF-kappaB sig-nalling. Nature, 430(7000), 694–699.

Whiting, C. C., Su, L. L., Lin, J. T., & Fathman, C. G. (2011). GRAIL: A uniquemediator ofCD4 T-lymphocyte unresponsiveness. FEBS Journal, 278(1), 47–58.

Windheim, M., Stafford, M., Peggie, M., & Cohen, P. (2008). Interleukin-1 (IL-1) inducesthe Lys63-linked polyubiquitination of IL-1 receptor-associated kinase 1 to facilitateNEMO binding and the activation of IkappaBalpha kinase.Molecular and Cellular Biology,28(5), 1783–1791.

Wu, C. J., & Ashwell, J. D. (2008). NEMO recognition of ubiquitinated BCL10 is requiredfor T cell receptor-mediated NF-kappaB activation. Proceedings of the National Academy ofSciences of the United States of America, 105(8), 3023–3028.

64 Yoon Park et al.





















































Wu, C. J., Conze, D. B., Li, T., Srinivasula, S. M., & Ashwell, J. D. (2006). Sensing of Lys63-linked polyubiquitination by NEMO is a key event in NF-kappaB activation[corrected]. Nature Cell Biology, 8(4), 398–406.

Wu, X., Yamamoto, M., Akira, S., & Sun, S. C. (2009). Regulation of hematopoiesis by theK63-specific ubiquitin-conjugating enzyme Ubc13. Proceedings of the National Academy ofSciences of the United States of America, 106(49), 20836–20841.

Xia, Z. P., Sun, L., Chen, X., Pineda, G., Jiang, X., Adhikari, A., et al. (2009). Direct acti-vation of protein kinases by unanchored polyubiquitin chains. Nature, 461(7260),114–119.

Xu, M., Skaug, B., Zeng, W., & Chen, Z. J. (2009). A ubiquitin replacement strategy inhuman cells reveals distinct mechanisms of IKK activation by TNFalpha andIL-1beta. Molecular Cell, 36(2), 302–314.

Xu, G., Tan, X., Wang, H., Sun, W., Shi, Y., Burlingame, S., et al. (2010). Ubiquitin-specific peptidase 21 inhibits tumor necrosis factor alpha-induced nuclear factor kappaBactivation via binding to and deubiquitinating receptor-interacting protein 1. Journal ofBiological Chemistry, 285(2), 969–978.

Yamaguchi, T., Kimura, J., Miki, Y., & Yoshida, K. (2007). The deubiquitinating enzymeUSP11 controls an IkappaB kinase alpha (IKKalpha)-p53 signaling pathway in responseto tumor necrosis factor alpha (TNFalpha). Journal of Biological Chemistry, 282(47),33943–33948.

Yamaguchi, N., Oyama, M., Kozuka-Hata, H., & Inoue, J. (2013). Involvement of A20 inthe molecular switch that activates the non-canonical NF-small ka, CyrillicB pathway.Scientific Reports, 3, 2568.

Yamamoto, M., Okamoto, T., Takeda, K., Sato, S., Sanjo, H., Uematsu, S., et al. (2006).Key function for the Ubc13 E2 ubiquitin-conjugating enzyme in immune receptor sig-naling. Nature Immunology, 7(9), 962–970.

Yamamoto, M., Sato, S., Saitoh, T., Sakurai, H., Uematsu, S., Kawai, T., et al. (2006). Cut-ting edge: Pivotal function of Ubc13 in thymocyte TCR signaling. Journal of Immunology,177(11), 7520–7524.

Yang, B., Gay, D. L., MacLeod, M. K., Cao, X., Hala, T., Sweezer, E. M., et al. (2008).Nedd4 augments the adaptive immune response by promoting ubiquitin-mediated deg-radation of Cbl-b in activated T cells. Nature Immunology, 9(12), 1356–1363.

Yang, C., Zhou,W., Jeon,M. S., Demydenko, D., Harada, Y., Zhou, H., et al. (2006). Neg-ative regulation of the E3 ubiquitin ligase itch via Fyn-mediated tyrosine phosphoryla-tion. Molecular Cell, 21(1), 135–141.

Yeh, W. C., Shahinian, A., Speiser, D., Kraunus, J., Billia, F., Wakeham, A., et al. (1997).Early lethality, functional NF-kappaB activation, and increased sensitivity to TNF-induced cell death in TRAF2-deficient mice. Immunity, 7(5), 715–725.

Yu, D., Tan, A. H., Hu, X., Athanasopoulos, V., Simpson, N., Silva, D. G., et al. (2007).Roquin represses autoimmunity by limiting inducible T-cell co-stimulator messengerRNA. Nature, 450(7167), 299–303.

Zak, D. E., Schmitz, F., Gold, E. S., Diercks, A. H., Peschon, J. J., Valvo, J. S., et al. (2011).Systems analysis identifies an essential role for SHANK-associated RH domain-interacting protein (SHARPIN) in macrophage Toll-like receptor 2 (TLR2) responses.Proceedings of the National Academy of Sciences of the United States of America, 108(28),11536–11541.

Zarnegar, B. J., Wang, Y., Mahoney, D. J., Dempsey, P. W., Cheung, H. H., He, J., et al.(2008). Noncanonical NF-kappaB activation requires coordinated assembly of a regula-tory complex of the adaptors cIAP1, cIAP2, TRAF2 and TRAF3 and the kinase NIK.Nature Immunology, 9(12), 1371–1378.




















































Zhang, J., Bardos, T., Li, D., Gal, I., Vermes, C., Xu, J., et al. (2002). Cutting edge: Reg-ulation of T cell activation threshold by CD28 costimulation through targeting Cbl-b forubiquitination. Journal of Immunology, 169(5), 2236–2240.

Zhang, W., Shao, Y., Fang, D., Huang, J., Jeon, M. S., & Liu, Y. C. (2003). Negative reg-ulation of T cell antigen receptor-mediated Crk-L–C3G signaling and cell adhesion byCbl-b. Journal of Biological Chemistry, 278(26), 23978–23983.

Zhao, Y., Thornton, A. M., Kinney, M. C., Ma, C. A., Spinner, J. J., Fuss, I. J., et al. (2011).The deubiquitinase CYLD targets Smad7 protein to regulate transforming growth factorbeta (TGF-beta) signaling and the development of regulatory T cells. Journal of BiologicalChemistry, 286(47), 40520–40530.

Zhou, H., Wertz, I., O’Rourke, K., Ultsch, M., Seshagiri, S., Eby, M., et al. (2004). BCL10activates the NF-kappaB pathway through ubiquitination of NEMO.Nature, 427(6970),167–171.

Zhou, F., Zhang, X., van Dam, H., Ten Dijke, P., Huang, H., & Zhang, L. (2012).Ubiquitin-specific protease 4 mitigates Toll-like/interleukin-1 receptor signaling andregulates innate immune activation. Journal of Biological Chemistry, 287(14),11002–11010.

66 Yoon Park et al.


















[advances in immunology] volume 124 || the ubiquitin system in immune regulation

Documents