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triggers a conformational change, causing CARMA1 to

bind to B cell leukemia/lymphoma (BCL10) and mucosa-

associated lymphoid tissue lymphoma translocation pro-

tein (MALT1), forming the CARMA1-BCL10-MALT1

(CBM) complex. Through a mechanism that may involve

TNF receptor-associated factor (TRAF6), both BCL10 and

MALT1 become polyubiquitinated. The IkB kinase (IKK)

complex is then recruited to the CBM complex via the

IKK g polyubiquitin binding motif. This association leads

to polyubiquitination of IKK g and phosphorylation of

IKK b by TGF-b activated kinase (TAK1), activating

IKK b. IKK b then phosphorylates inhibitor of kB (IkBa),

triggering its proteasomal degradation, enabling nuclear

translocation of canonical NF-kB heterodimers comprised

of p65 reticuloendotheliosis viral oncogene homolog A

(RELA) and p50 proteins. Once in the nucleus, NF-kB

governs the transcription of numerous genes involved in T

cell survival, proliferation, and effector functions.

AcƟvaƟon signal

MALT1-mediated acƟons

Key:

Calcium-mediated signal

Calmodulin

SLP-76

PI3K

PDK1GLK

PKCθ

NF-κ B

CARMA1

TCR CD28

BCL10

MALT1

NF-κ B NF-κ B

Iκ Bα

TCR-dependent PKCθ acƟvaƟon

CD28-dependent PKCθ

acƟvaƟon

ADAP

CK1α

HPK1

CBM complex formaƟonTAK1 recruitment

CBM complex formaƟonCARMA1 phosphorylaƟon

CARMA1 phosphorylaƟon

MIB2Calcineurin

IKK recruitmentand ubiquiƟnaƟon

BCL10 dephosphorylaƟon

BCL10

CYLD

A20

RELB

T cell adhesion

JNK acƟvaƟon

NF-κ B acƟvaƟon

Caspase 8

Gene transcripƟon

cFLIPFLIP

IKK recruitment

cFLIP cleavage

TRAF6

TRAF2 IKK ubiquiƟnaƟon

Homer3AcƟn regulaƟon

TAK1

CaMKIICARMA1 and BCL10phosphorylaƟon

Ca2

IKKα IKKβ

U

P

IKKγ IKKγ U

P AcƟvaƟng phosphorylaƟon

U K63-polyubiquiƟnaƟon

TRENDS in Immunology

Figure1 . Newdevelopments in the T cell receptor (TCR)-to-nuclear factor (NF)-kB signaling pathway. TheTCR transmits signals through the linker for activation of T cells

(LAT)–SH2 domain containing leukocyte protein of 76 kDa (SLP76) complex, and possibly through glucokinase (GCK)-like kinase (GLK) to activate protein kinase (PK)Cu.

CD28 signals through phosphoinositide 3-kinase (PI3K) and phosphoinositide-dependent kinase (PDK1) to activate PKCu.

Activated PKCu

induces formation of the CARD-containing MAGUK protein (CARMA1), B cell leukemia/lymphoma (BCL10),mucosa-associated lymphoid tissue lymphoma translocation protein (MALT1) (CBM) complex.

Newly identified proteins acting on CARMA1 and BCL10 are shown on the left. MALT1 enzymatic protein cleaving functions and caspase-8 recruitment are shown on the

right. The CBM complex transmits activating signals to IkB kinase (IKK) through the ubiquitin ligases TNF receptor-associated factor (TRAF6), TRAF2 and/or mind bomb-2

(MIB2). IKK phosphorylates inhibitor of kB (IkBa) leading to IkBa ubiquitination and degradation, allowing NF-kB nuclear translocation and gene transcription.

Review Trends in Immunology xxx xxxx, Vol. xxx, No. x

TREIMM-1014; No. of Pages 13

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Recent data suggest that aspects of the consensus model

for TCR signaling are overly simplistic, and that additional

molecules play a role in the TCR-to-NF-kB cascade. Here,

we summarize data suggesting that multiple signalosomes

participate in TCR activation of NF-kB, and describe the

negative regulatory mechanisms that control this path-

way. We also discuss evidence for connections between

control of NF-kB activation and other cellular processes,

such as actin remodeling. Overall, the emerging picture is

that the TCR-to-NF-kB signaling cascade is a crucial nexus

that both governs and is regulated by a diverse network of

T cell biological processes.

New developments in the TCR-to-NF-kB pathway

Deletion of the genes encoding PKCu and CBM complex

proteins results in impaired TCR-induced NF-kB activa-

tion. Recent work has also identified several additional

molecules that regulate this pathway (Figure 1).

PKC u

Phosphorylated PKCu connects LAT and SLP76 with the

CBM complex [4,5]. The protein kinase PDK1 is consid-

ered essential for PKCu activation as PDK1-deficient Jur-

kat and primary CD4 T cells show a defect in PKCu

phosphorylation and NF-kB activation [6,7]. However,

there is a lack of in vitro evidence that PDK1 directly

phosphorylates PKCu. Moreover, PDK1 activation is

dependent on CD28 engagement, whereas PKCu IS

translocation andNF-kB activation can occur in a purely

CD3-dependent manner, without participation of CD28

[6,8–10]. These observations suggest that another kinase

links theTCR–CD3complex with PKCu. Indeed,GCK-like

kinase (GLK), a SLP76-regulated kinase, was recently

reported to phosphorylate directly PKCu both in vitro

and in primary T cells and T cell lines in response to

TCRstimulation[11]. Additionally, GLK-deficient murine

lymph node cells exhibit reduced PKCu and IKK phos-

phorylation, correlating with reduced cytokine and anti-

body production. Collectively, these data suggest that

PDK1 and GLK1 might function together to inducePKCu

phosphorylation and activate NF-kB (Figure 1). Alterna-

tively, GLK and/or PDK1 may be utilized in an exclusive

manner to phosphorylate PKCu, depending on the activa-

tion and/or differentiation state of the T cells, the type of

antigen-bearing stimulatory cell, etc. Elucidation of such

details will require a careful comparison of the GLK and

PDK1 knockout models under a variety of T cell activation

paradigms.

Gene transcripƟon

NF-κ B

Iκ Bα

NF-κ B

Iκ Bα

NF-κ B

IKKα IKKβ

BCL10MALT1

P

BCL10MALT1

TRAF6U

P

U

U

P

U

P

TCR CD28

PKCθP

P

L a t e

A20

BCL10P

U

p62

GAKIN

CARMA1

CARMA1

CARMA1

CARMA1

CARMA1

P

CK1α

PP2A

SLP-76

PKCθ

ZAP70P

P

PDK1PI3KL

AT P

SLP-76

P

LCK

PLCγ

ZAP70 LCK

STS

IP3

DAG

P

LAT

U

c-Cbl

SLP-76

P

HPK1

SHP1

NF-κ B

Autophagosome

Proteasome

Endosome-bound

signalosome

p62

Membrane-associated

signalosome

Cytosolic signalosome

AcƟvaƟon signal

Key:

InhibiƟon signal

Inhibitory phosphorylaƟon

AcƟvaƟng phosphorylaƟon

K48-polyubiquiƟnaƟon

P

P

PP4c

IKKγ IKKγ U

U

K63-polyubiquiƟnaƟon

UbiquiƟnaƟon, undetermined form

TAK1

CYLD

?

? Unknown mediator

CRADD

BCL10

U

E ar l y

TRENDS in Immunology

LAT

P

P

U

U

U

Figure 2. Negative regulation of T cell receptor (TCR)-to-nuclear factor (NF)-kB signaling. TheTCR/CD28activation signal, shown by green arrows, passes throughmultiple

intermediate signaling proteins, ultimately causingNF-kB activation. Negative modulation activities are depicted by red arrows. Yellow ovals indicatemembraneproximaland cytosolic signalosomes described in detail in the text.



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CARMA1

CARMA1, a critical target of PKCu phosphorylation,

resides in lymphocytes in an inactive state. Extensive

CARMA1 mutagenesis data suggest that this inactive

state is maintained by intramolecular interactions that

prevent the CARMA1 CARD from interacting with the

CARD of BCL10 [12,13]. PKCu phosphorylates human

CARMA1 at three serine

residues, S552, S645 and S637(S564, S657, S649 in mice) [1]. Phosphorylation at S552

and S645 is critical for the CARMA1 conformational

change that enables binding to BCL10–MALT1, leading

to further signal transmission [14,15] (CBM complex

post-translational modifications are reviewed in [1]). By

contrast, S649 phosphorylation suppresses CARMA1-

mediated NF-kB and C-jun N-terminal kinase (JNK)

activation [16]. Although PKCu induces CARMA1 S649

phosphorylation in vitro and PKC inhibitors block the

phosphorylation event in vivo, there is to date little evi-

dence that PKCu directly phosphorylates CARMA1 at

S649 in living cells. Also, the phosphorylation events of

opposing function do not occur simultaneously; the acti-

vating phosphorylation at S552/S645 is early and tran-sient, whereas the inhibitory phosphorylation at S649 is

delayed, but sustained for a longer duration. Considering

both the absence of evidence of direct PKCu phosphory-

lation of CARMA1 S649 and the rapid PKCu activation

kinetics post-TCR activation, it remains a strong possi-

bility that the delayed CARMA1S649 phosphorylation is

mediated by anunidentified PKCu-dependent kinase that

is activated and/or interacts with CARMA1 in a delayed

manner (Figure 2).

Apart from PKCu, three additional protein kinases,

hematopoietic progenitor kinase (HPK-1), casein kinase

(CK1a) and calcium/calmodulin-dependent protein kinase

(CaMKII)

are

capable

of

phosphorylating

CARMA1

andregulating CBM complex activity (Figure 1). In Jurkat T

cells, siRNA knockdown of HPK-1 reduces IKK enzymatic

activity, NF-kB nuclear translocation, and T cell survival

[17]. HPK-1 overexpression produces the opposite effect

[18]. Demonstration of TCR-regulated interaction between

HPK-1 and CARMA1 explains the ability of HPK-1 to

stimulate NF-kB activation. These authors also provided

evidence that HPK-1 phosphorylates CARMA1 at S551, a

site distinct from the residues modified by PKCu [18].

However, the overall role of HPK-1 in lymphocyte function

remains a hotly debated issue, because mouse knockout

data have demonstrated that HPK-1 suppresses lympho-

cyte activation, due to its inhibitory effect on the proximal

signaling protein SLP76 [19,20] (see Negative regulation of

TCR signaling to NF-kB, below).

CK1a, another kinase that acts via modification of

CARMA1, has been identified by mass spectrometry as a

CARMA1 binding partner. CK1a induces TCR-mediated

NF-kB activation in Jurkat cells and interleukin (IL)-2

secretion in primary human T cells [21]; probably by

recruiting activated IKK b to the CBM complex. Although

CK1a phosphorylates CARMA1 at S608, limiting NF-kB

activation, the positive CK1a function supercedes its

negative role. [21]. Thus CK1a enzymatic activity opposes

its IKK recruiting function, indicating that CK1a is also

a bifunctional regulator of the TCR-to-NF-kB pathway.

Accumulating data suggest that the TCR-to-NF-kB

pathway is also regulated by the protein kinase, CaMKII.

TCR stimulation results in a rapid increase in T cell

cytosolic calcium, which binds CaM, triggering activation

of CaMKII and the phosphatase, calcineurin (see also

BCL10 section below) (Figure 1). Both enzymes influence

the TCR-to-NF-kB pathway. In Jurkat cells, following

APC

+

antigen

or

CD3

+

CD28

stimulation

[22,23], CaMKIIis recruited to the IS. CaMKII inhibition by siRNA knock-

down (g or d isoform) or by the pharmacological inhibitor,

KN-93, reduces NF-kB activation [23]. However, the mech-

anism of CaMKII action is unclear. In a cell-free system,

CaMKII can phosphorylate CARMA1 at S109 [23] and

BCL10 at S138 [24] or at S48 and T91 [22]. Studies in

Jurkat cells have shown CARMA1 S109 phosphorylation

assists in CARMA–BCL10 binding [23], and BCL10 T91

phosphorylation promotes K63-polyubiquitination of

BCL10 and signaling to IKK [22]. By contrast, there is

evidence that BCL10 phosphorylation at S138 might have

a negative effect on NF-kB activation [24]. Considering the

above data, the mechanism by which CaMKII connects

calcium–CaM signals to NF-kB remains largely unclear.Future studies using genetic deletion or RNAi silencing of

CaMKII in primary T cells will be required to clarify the

role of CaMKII in TCR activation of NF-kB.

Interestingly, CARMA1 phosphorylation is necessary

but not sufficient for CBM complex formation. An addi-

tional essential step involves interaction with the integrin

receptor regulatory protein adhesion and degranulation-

promoting adapter protein (ADAP) (Figure 1). ADAP-defi-

cient T cells show impaired TCR- and CD28-dependent

proliferation and reduced cytokine secretion. ADAP binds

both CARMA1, facilitating CBM complex assembly [25],

and TAK1, promoting IKK activation [26], and these

actions

might

account

for

the

effect

of

ADAP

on

NF-kBactivation and cytokine secretion. The molecular mecha-

nism by which ADAP enhances CBM complex assembly

remains unclear. The ADAP–CARMA1 interaction is also

critical for cyclin-dependent kinase (CDK)2 and cyclin E

expression [27]. As cyclins regulate progression through

the cell cycle, this observation suggests a molecular basis

for the impaired T cell proliferation observed in ADAP

deficiency.

BCL10

As a result of the above modifications and interactions of

CARMA1, the constitutively associated BCL10–MALT1

complex associates with CARMA1, forming the CBM com-

plex [3,4]. Immediately following TCR stimulation, BCL10

is phosphorylated and ubiquitinated. BCL10 post-transla-

tional modification is complex, with many reported sites of

modification. The regulation and significance of many

modifications remain poorly understood (see [1,5] for re-

view). To date, evidence suggests that IKK b is one of the

principal kinases responsible for BCL10 phosphorylation

(Figure 2) (see above for discussion of BCL10 phosphory-

lation by CaMKII). Initially, IKK b-phosphorylation of

BCL10 stabilizes the CBM complex, but subsequent IKK b

phosphorylation of BCL10 triggers BCL10 dissociation

from MALT1 [28] and/or BCL10 degradation [29]. Thus

BCL10 phosphorylation may be an important step for



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terminating TCR signals to NF-kB. Interestingly, calci-

neurin, a calcium-dependent phosphatase, has been

reported to dephosphorylate BCL10 [30,31], stabilizing

the CBM complex and enhancing NF-kB activation

(Figure 1). Calcineurin, primarily known for its role in

TCR-induced activation of the nuclear factor of activated T

cells (NFAT) transcription factor, is also involved in lyso-

some-dependent

degradation

of

PLCg1 and

PKCu

upon

Tcell restimulation, resulting in T cell anergy [32]. As PKCu

is essential for NF-kB activation, these seemingly conflict-

ing roles of calcineurin in TCR activation of NF-kB require

further examination.

An area of intense recent investigation involves eluci-

dation of the mechanism and purpose of BCL10 ubiquiti-

nation. Reports indicate that BCL10 is modified by

K63-polyubiquitination [33,34] or both K48- and K63-

polyubiquitination with K63- preceding K48-polyubiquiti-

nation [35]. Mutagenesis data suggest that both K48- and

K63-polyubiquitin chains are conjugated exclusively to

lysines 31 and 63 of BCL10 [35]. K63-polyubiquitination

of Bcl10 is triggered by interaction with CARMA1 [12,13].

The identity of the BCL10 ubiquitin ligase is highly con-troversial, with neural precursor cell expressed, develop-

mentally downregulated (NEDD4) and ITCH [36], cellular

inhibitor of apoptosis (cIAP2) [37], b-transducin repeats-

containing proteins bTrCP [29], and TRAF6 [35] all impli-

cated as E3 ligases targeting BCL10. These uncertainties

aside, the emerging consensus is that BCL10 ubiquitina-

tion serves two important purposes: initial transmission of

the NF-kB activation signal [33,35], followed by BCL10

degradation and signal termination [29,33,35,36] (see Neg-

ative regulation of TCR signaling to NF-kB, below). BCL10

ubiquitination is important for signal transmission,

because ubiquitin chains create binding sites for IKK g

[35] and p62 [33] (Figure 2); two molecules critical for

NF-kB activation. Interestingly, the requirement for p62

and certain other signaling molecules seems to depend on

the stage of T cell differentiation (Box 1).

Another emerging area of interest regarding the biology

of BCL10 involves the relation between TCR-dependent

post-translational

modification

of

BCL10

and regulationof the T cell actin cytoskeleton [38]. Although current evi-

dence suggests that actin cytoskeletal dynamics do not

directly influence NF-kB activation, actin polymerization

has a profound influenceon the immunological synapseand

TCR microclusters (see Cell membrane and cytosolic com-

plexes in TCR signaling to NF-kB, below), indirectly linking

regulation of the actin cytoskeleton to TCR signaling to

downstream transcription factors [39,40]. Also, there are

multiple points of intersection between TCR-to-NF-kB sig-

naling and regulation of the actin cytoskeleton, suggesting

an as-yet poorly understood mechanistic coupling of the

regulation of these pathways. TCR stimulation alters the

T cell actin cytoskeleton, enabling T cell spreading and

conjugation with APCs. In Jurkat cells, successful actinremodeling post-TCR stimulation requires BCL10 S138

phosphorylation [38] and IKK b–Homer3 association at

the IS [41] (Figure 1). Interestingly,BCL10 S138 phosphor-

ylation is alsorequired for macrophage Fc receptor-induced

actin polymerization, leading to phagosome formation [42].

Phosphorylation of S138 is unrelated to CARMA1–BCL10–

MALT1 mediated NF-kB activation in both T cells and

macrophages [38,42]. Similarly, the IKK b–Homer3 mediat-

edactin regulation in T cells is also NF-kB independent [41].

The identity of the precise mechanisms that link phospho-

BCL10 and Homer3 to actin remains to be determined.

Box 1. Differences in TCR-to-NF-kB signaling between naı̈ve and effector/memory T cells

Compared to naı ¨ve T cells, effector and memory cells demonstrate a

faster response to antigen stimulation. This accelerated response of

effector/memory T cells may partly reflect differentiation-dependent

changes in NF-kB signaling mechanisms. One proposed mechanism

is that crucial signaling prote ins in dif ferent iated T cells are

constitutively phosphorylated, allowing more rapid activation. How-

ever, investigations have failed to reveal differences in phosphoryla-

tion states of ZAP70 and PLC-g between naı ¨ve andmemory cells [111].

A second possible mechanism is that signaling proteins in effector/

memory T cells may be constitutively associated with lipid rafts;

cholesterol-rich regions in the cell membrane that are sites of

signaling molecule aggregation. Studies have indeed demonstrated

increased interaction of signaling proteins with lipid rafts in memoryCD4 and CD8 T cells [112,113], but the functional consequences have

not been established.

A third potential mechanism is that key signaling proteins may be

more highly expressed in effector/memory T cells. Single cell

experimental studies and computer modeling suggest a correlation

between increased signaling protein expression and T cell functional

capacity [114]. Multiple studies have shown higher total ZAP70

protein expression in CD4 effector and memory T cells, compared

to naı ¨ve T cells [115,116]. Furthermore, data show that IFN-g

production occurs selectively from effector T cells expressing the

highest levels of ZAP70, and that IFN-g expression can be reduced by

silencingZAP70 using siRNA. Another study hasdemonstratedhigher

levels of the adaptor protein BCL10 in both effector CD4 and memory

CD8 T cells, compared to naı ¨ve T cells [33], although the functional

importance of this difference remains unexplored. Importantly,

elevations of signaling protein levels do not universally accompany

T cell differentiation. For example, relative to naı ¨ve T cells, SLP-76

levelsare increased in effector T cells butdecreased inmemoryT cells

[117].

A final possible mechanism is that differentiated T cells might

utilize signaling molecules that are distinct from those involved in

naı ¨ve cell signaling. Two studies have demonstrated that the

scaffolding protein p62 is required for TCR-to-NF-kB signaling in

effector T cells, but not in naı ¨ve T cells [33,118]. Consistent with these

findings, p62 expression is dramatically increased concomitant with

TCR stimulation and effector differentiation [119]. These studies have

indicated that differentiation-dependent increases in expression of

certain intermediate signaling proteins and utilization of distinctsignaling proteins may enhance TCR signaling in effector/memory T

cells, versus naı ¨ve T cells.

Apart from changes in signal transduction, alterations in transcrip-

tion factor utilization and chromatin modification contribute to the

rapid responses of memory T cells to antigen stimulation. The

increase in IFN-g production by memory CD4 T cells compared to

naı ¨ve cells is associatedwith a switch fromT-bet- to NF-kB-dependent

transcription. Additionally,memory cells exhibit increasedexpression

and promoter binding of the NF-kB subunit p50 [120]. Also, the

kinetics of gene transcription maybe accelerated inmemoryT cells by

histone acetylation of the promoter regions of specif ic genes,

including perforin and Eomes, facilitating transcription factor binding

[121]. Thus, epigenetic modifications and differential transcription

factor usage may contribute to the rapid transcriptional response of

memory T cells.



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MALT1

MALT1, the third member of the CBM complex, plays a

more nebulous role in TCR activation of NF-kB. Initial

studies suggested that MALT1 functions as a scaffolding

protein that participates in NF-kB activation by connect-

ing the CBM complex with TRAF6. Later studies showed

that MALT1 contributes less to TCR activation of NF-kB

than

BCL10

or

PKCu

in

primary

mouse

T

cells

[43]

andthat silencing MALT1 expression in Jurkat cells reduces

but does not completely block IkBa phosphorylation [44].

Additionally, in B cells, BCL10 is essential for RELA

activation, whereas MALT1 is dispensable [45]. Thus,

there is an emerging understanding of MALT1 as a protein

which modifies antigen receptor-mediated signaling to NF-

kB, without being strictly required for NF-kB activation.

Moreover, the protease activity of MALT1 is associated

with regulation of diverse cellular functions, most of which

are distinct from NF-kB signal transduction. MALT1

cleaves target proteins after arginine residues preceded

by a serine [44,46]. To date, four MALT1 substrates,

BCL10, A20, cylindromatosis (CYLD) and reticuloendothe-

liosis viral oncogene homolog B (RELB), have been identi-fied (Figure 1). MALT1 cleavage of BCL10 and CYLD

regulates T cell adhesion [44] and JNK activation [47]

respectively, with no detected effect on NF-kB signal

transduction. Another target of MALT1 cleavage is A20,

a known inhibitor of NF-kB activation [46]. However,

MALT1 cleaves only a small fraction of the total cellular

A20 pool [46], and blockade of MALT1 protease activity

fails to limit IKK activation [48]. Thus, the significance of

A20 cleavage as a mechanism regulating TCR signaling to

NF-kB remains uncertain. Among known MALT1 sub-

strates, RELB is most compelling as an NF-kB signal

mediator that is cleaved to regulate NF-kB activation.

MALT1-dependent

RELB

cleavage

and

concomitant

deg-radation leads to increased RELA and c-REL DNA binding

[49,50]. How RELB degradation affects RELA or c-REL

activity is not well understood, but it might reflect compe-

tition between different NF-kB heterodimers for DNA

binding sites, or inhibition of canonical activation by

gene products of the noncanonical (i.e., RELB-dependent)

pathway.

Caspase-8

The protease caspase-8 is well known as a mediator of

apoptosis. Impaired lymphocyte activation in caspase-8-

deficient mice has also suggested a role of caspase-8 in the

TCR-to-NF-kB pathway [51]; a finding now confirmed and

extended by several additional studies (Figure 1). Caspase-

8 recruits IKK to activated BCL10–MALT1 by a TRAF6-

dependent mechanism [51,52], and MALT1 activates cas-

pase-8, causing caspase-8-mediated cleavage of the NF-kB

inducer cellular FLICE (Caspase-8)-inhibitory protein,

long form (c-FLIPL) [53] (reviewed in [54]). Additionally,

caspase-8 proteolytic activity is required for CD3/CD28-

dependent NF-kB activation, because T cells treated with

the caspase inhibitor Z-VAD-FMK or expressing the cas-

pase-8 catalytically inactive mutant, C360S, fail to induce

NF-kB [51]. Interestingly, in HEK293 cells, neither Z-

VAD-FMK nor caspase-8 C360S blocks caspase-8-induced

NF-kB activation [55], suggesting that the mechanistic

link between caspase-8 and NF-kB activation might be

cell-type specific. Thus, additional mechanistic data are

required to yield a clear understanding of how caspase-8

participates in this signaling pathway.

IKK complex

A key step in TCR activation of NF-kB is CBM complex-

dependent

K63-polyubiquitination

of

IKK g. TRAF6

hasbeen proposed as the ubiquitin ligase that performs this

key function [1,3,54] (Figure1). However, TRAF6-deficient

T cells do not show impaired NF-kB activation [56], sug-

gesting either that TRAF6 does not play such a role, or that

there are redundant ubiquitin ligases that compensate for

loss of TRAF6. For example, TRAF2, an ubiquitin ligase

recruited by the caspase-8–FLIP complex is also capable of

polyubiquitinating IKK g [53]. Another possible candidate

is the ubiquitin ligase mind bomb-2 (MIB2), which binds to

BCL10 and promotes IKK g ubiquitination and NF-kB

activation in transfection–overexpression experiments

[57] (Figure 1). Thus, it is possible that several ubiquitin

ligases contribute to K63-polyubiquitination of IKK g. It is

also possible that there is a key role for modification of IKK g by M1 (linear head-to-tail linked) polyubiquitin

chains. M1 polyubiquitination plays a key role in TNF

receptor activation of NF-kB [58], but involvement in

TCR-mediated activation of IKK is undetermined. Resolu-

tion of these lingering mechanistic questions will require

compelling genetic and biochemical evidence using prima-

ry T cells to definitively demonstrate which ligases and

which ubiquitin modifications are essential.

Activation of IKK requires a combination of IKK gubqui-

tination and IKK b phosphorylation. The latter process is

mediated by the protein kinase TAK1 [59] (Figure1). TAK1

activation seems to be dependent on PKCu, but not on the

CBM

complex

members

CARMA1

and

BCL10

[60]. Theadapter molecule ADAP is required for TAK1 recruitment

to PKCu [26]. However, the precise mechanism of PKCu-

mediated TAK1 activation is not well defined. The IKK b

phosphorylation events can be countered by the protein

phosphatase 4 regulatory subunit 1-protein phosphatase 4

regulatory subunit (PP4R1–PP4c) phosphatase complex,

limiting IKK activation and T cell function [61] (Figure 2).

The ‘all-or-none’ NF- kB response

At a single cell level, signaling pathways may be broadly

classified as digital or analog. Whereas analog signaling is

a graded response that is proportional to stimulus intensi-

ty, digital signaling produces an all-or-none response,

irrespective of intensity of the activation stimulus [62].

In T cells, increasing TCR stimulation strength leads to an

increasing percentage of cells with IkBa phosphorylation

and RELA activation, without altering per cell level of the

two proteins [63]. This observation suggests a digital sig-

naling mechanism similar to findings regarding tumor

necrosis factor (TNF)a-induced NF-kB signaling [64],

and TCR-induced extracellular signal-regulated kinase

(ERK) phosphorylation [65,66] or NFATc2 activation

[67]. By contrast, bypassing the TCR via stimulation with

phorbol 12-myristate 13-acetate (PMA+ionomycin) leads

to a graded (analog) NF-kB response in T cells [63,67],

indicating that signal digitization occurs at an early



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TCR-proximal step. The molecular switch enabling digiti-

zation of TCR signals remains to be determined.

The above studies have demonstrated that, rather than

the early understanding of TCR activation of NF-kB as a

simple linear cascade (PKCu!CBMcomplex!IKK), there

is a complex web of interconnected signaling molecules

that link the TCR to NF-kB. In particular, there is accu-

mulating

evidence

of

multiple

signaling

inputs

converging on key proteins, particularly CARMA1 and BCL10. Addi-

tionally, the mechanistic understanding of the crucial

functions of certain mediators in transmitting the activat-

ing signal to NF-kB, for example, MALT1, is incomplete.

Also, the precise roles and relative importance of kinases

recently implicated as regulators of this pathway, includ-

ing PDK1, GLK, HPK-1, and CK1a remain to be well

defined. Crosstalk between the TCR-to-NF-kB pathway

andother cellular processes, such as regulation of the actin

cytoskeleton and T cell–APC interactions, is an emerging

area of interest for which there is currently very limited

mechanistic understanding.

Cell surface and cytosolic complexes in TCR signaling toNF-kB

Cell surface TCR microclusters and the IS

The concept of the IS developed in the late 1990s when

imaging revealed micrometer-sized clusters, composed of

surface receptors and signaling proteins, at the T cell–APC

intercellular contact site [68,69]. Initially, the IS was

proposed as the site at which intracellular signaling from

the TCR is initiated and sustained. Later studies showed

that initiation of TCR signaling and tyrosine kinase acti-

vation can be detected within seconds following stimula-

tion and before IS formation, in peripheral TCR-rich

regions termed microclusters [70,71]. Current data suggest

that

the

earliest

signaling

events

occur

in

these

peripheralmicroclusters, which move centripetally and eventually

fuse to form the mature IS.

Recent data also cast doubt on the model that the IS

represents the primary site of sustained signaling to down-

streammediators. Specifically, TCR signaling to NF-kB can

occurin theabsence of an IS [72,73]. Also, as detailedbelow,

accumulating data point towards the existence of cytosolic

signaling clusters/signalosomes thatmay sustain andregu-

late TCR signal transduction. Although the IS might not be

involved in signal initiation or maintenance, the IS has

certain other critical functions. Experimental studies and

computer modeling haverevealed that at low antigen–MHC

concentrations, the IS amplifies TCR activationby grouping

together antigens, TCR, and signaling proteins. This allows

the TCR to engage rapidly the smallnumber of stimulatory

ligands, resulting in efficient signal transduction [74]. Also,

the IS may serve as a site of TCR internalization and

degradation, serving to limit signal strength and/or dura-

tion [75]. Interestingly, ubiquitination of as-yet-undefined

central supramolecular activation cluster (c-SMAC) pro-

teins leads to interactionwith theubiquitin-binding protein,

tumor susceptibility gene (TSG101), a component of

the endosomal sorting complex required for transport

(ESCRT). This interaction is requiredfor c-SMAC formation

and organization, as well as for termination of signaling

by microclusters and TCR degradation [76]. These

observations suggest that TCR signaling and signal termi-

nation by TCR degradation are intimately connected.

Cytosolic T cell signalosomes

Emerging data suggest that several distinct cellular sites

may together coordinate TCR signaling to NF-kB. Imme-

diately following TCR engagment, the integral membrane

protein

LAT

and

the

cytosolic

protein

SLP76

are

recruitedto the region immediately below the TCR microclusters [3].

ZAP70-mediated phosphorylation of LAT and SLP76

enables these adaptor proteins to bind downstream med-

iators, transmitting the activation signal. Data suggest

that following initial recruitment, LAT and SLP76 disso-

ciate from TCR microclusters in endosomes [77] (Figure 2).

SLP76 remains on the the outer cytosolic side of the

endosome, and its attachment to endosomes is mediated

indirectly through LAT. Endosome-associated LAT and

SLP76 remain phosphorylated, indicating that these adap-

tors are capable of binding effector molecules and continu-

ing the signaling cascade. LAT–SLP76 endosomes might

represent sites of continous signaling to downstream med-

iators, similar to the signaling complexes on intracellularendosomal membranes observed downstream of the epi-

dermal growth factor receptor (EGFR) [78,79]. LAT–

SLP76 endosomes might also have a role in termination

of TCR signaling, as inhibition of microcluster movement

results in enhanced TCR signaling [80,81].

There is also evidence for cytosolic signalosomes con-

taining BCL10 and MALT1 that specifically sustain NF-

kB-activation. Upon PKCu activation, CARMA1 is

recruited to the plasma membrane, apparently undergoing

a conformational change that opens access to binding sites

for BCL10 and MALT1, leading to activation of IKK [3,4]

(Figure 2). However, the mechanism that links the mem-

brane

bound

CBM

complex

to

IKK

activation

is

not

welldefined. One possibility is that BCL10–MALT1 clusters

interact transiently with membrane bound CARMA1, fol-

lowed by redistribution to a cytosolic site at which these

proteins transmit the signal to IkBa–NF-kB. Biochemical

analysis of Jurkat cells has revealed the existence of two

complexes [34]: an early membrane-bound CBM complex

(Figure 2), and a late BCL10–MALT–IKK complex, and

this late complex has been shown to interact inducibly with

IkBa. Additionally, in a cell-free system, purified recombi-

nant BCL10 and MALT1 proteins along with TRAF6,

ubiquitin-conjugating enzyme (UBC13), and TAK1 were

sufficient for IKK activation, demonstrating that the

BCL10–MALT1 complex can activate IKK in a manner

that is independent of concurrent physical association with

CARMA1 [48] (i.e., although CARMA1 is required for

transmitting the activating signal to BCL10, it does not

need to remain associated with BCL10–MALT1). Impor-

tantly, IKK activation is mediated by a small fraction of the

total BCL10 and MALT1 pool that exists as high molecular

weight oligomers, providing biochemical evidence of

BCL10–MALT1 signalosomes. However, it is also impor-

tant to note that there are as yet no published biochemical

data showing that similar signaling-competent oligomers

of BCL10–MALT1 exist in intact cells or cellular lysates.

Imaging studies in primary mouse T cells and D10 T

cells have demonstrated the presence of TCR-induced



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cytosolic BCL10–MALT1 clusters, termed POLKADOTS

[9,82], at locations that are distinct from membrane bound

PKCu, a marker for the IS. Furthermore, these BCL10–

MALT1 clusters are also enriched in the downstream

signaling protein TRAF6 [82]. Formation of the POLKA-

DOTS clusters is dependent both on expression of the

autophagy adaptor protein p62 and K63-polyubiquitina-

tion

of

BCL10

[33]. Interaction

between

BCL10–MALT1and p62 and the resultant signal transmission to IKK is

driven by BCL10 K63-polyubiquitination (Figure 2). The

formation of the proposed POLKADOTS signalosome is

strongly correlated with IKK a / b phosphorylation [33] and

RelA nuclear translocation [63], supporting a direct role in

NF-kB activation.

The above studies provide compelling evidence that

TCR signaling to NF-kB involves several organized signa-

losomes that concentrate groups of interacting signaling

proteins. Interestingly, these signalosomes are not univer-

sally required for TCR signaling to NF-kB. Specifically,

NF-kBactivation can occur in the absence of the IS, and the

POLKADOTS signalosome apparently plays no role in

naı̈ve T cells (Box 1). Although early data suggest thatsuch signalosomes represent cellular platforms for coordi-

nating and precisely regulating positive and negative sig-

nals to NF-kB,much further work is needed to define fully

the components and functions of these intriguing macro-

molecular structures. Areas for future focus include: de-

termining how TSG101 and the ESCRT complex interact

with microclusters and c-SMAC constitutents to regulate

signal transmission by the TCR; better establishing the

role of endosomal LAT–SLP76 in activation of downstream

transcription factors; establishing the mechanistic connec-

tion between the IS and POLKADOTS signalosome; and

defining inmolecular detail how mechanistic requirements

for

NF-kB activation

change

with

T

cell

differentiation(Box 1).

Negative regulation of TCR signaling to NF-kB

TCR activation of NF-kB is critical for T cell proliferation

and differentiation. However, unrestricted and persistent

NF-kB activation can lead to development of autoimmune

diseases and neoplasms [83], cellular senescence [84], or

apoptosis [85]. In order to strike a balance between pro-

ductive T cell activation and deleterious consequences of

excessive NF-kB activation, TCR signaling has to be pre-

cisely regulated. After T cell activation, negative regula-

tionstarts at the level of cell surface receptors (e.g., byTCR

endocytosis and degradation) and continues at multiple

steps of cytoplasmic and nuclear signaling. In this review,

we focus on cytoplasmic mechanisms that limit TCR-to-

NF-kB signaling (cell surface receptor and nuclear regula-

tionmechanisms are reviewed in [2,3] and [86], respective-

ly). Cytosolic negative regulators and mechanisms of

action are listed in Table 1 and illustrated in Figure 2.

TCR-proximal regulatory mechanisms

Successful transmission of the activation signal from the

TCR to NF-kB requires dynamic regulation at multiple

intermediate steps. TCR engagement results in the phos-

phorylation and/or ubiquitination of many intermediates,

and these post-translational modifications activate or

repress signal transmission. The TCR-proximal signaling

events involving ZAP70, LCK, LAT, and SLP-76 are dy-

namically regulated by multiple protein phosphatases and

kinases (Figure 2). Data suggest that Src homology region

2 domain-containing phosphatase (SHP-1), well known for

its ability to regulate T cell signaling, dephosphorylates

LCK [87], ZAP70 [88], and SLP-76 [89]. Mice with T cell-

specific

deletion

of

SHP-1

exhibit

increased

expansion

of CD8 effector (but not memory) T cells in response to viral

infection [90]. In addition to SHP-1, ZAP70 can also be

dephosphorylated by suppressor of T cell receptor signal-

ing Sts-1 and Sts-2, limiting TCR stimulation. Deletion of

both Sts-1 and Sts-2 causes T cell hyper-responsiveness,

with augmented cytokine secretion in response to antigen

stimulation [91]. In stimulated T cells, Sts-1 and -2 dele-

tion results in accumulation of hyper-phosphorylated and

ubiquitinated proteins [92]. Deletion of either Sts-1 or Sts-

2 alone does not produce any detectable phenotype, thus,

there is likely a wide overlap in the function of these

related molecules [93].

LCK activity is controlled by two opposing enzymes:

c-src tyrosine kinase (CSK), a protein kinase, and CD45, aprotein phosphatase. Data show that CSK-mediated in-

hibitory phosphorylation of LCK diminishes TCR activa-

tion (Figure 2), whereas CSK silencing amplifies TCR-

induced IL-2 production [94]. CD45 removes this inhibitory

phosphate group from LCK, allowing LCK to participate in

TCR signaling [95]. SLP-76 activity is also limited by

phosphorylation. After the initial transient ZAP70-medi-

ated activating phosphorylation of SLP-76 (at Y112, Y128

and Y145; reviewed in [96]), HPK-1 adds a phosphate

group to SLP-76 at S376 (Figure 2), causing increased

binding to the inhibitory protein 14-3-3 [19,20]. T cells

lacking HPK-1 demonstrate CD3-induced hyperprolifera-

tion

and

increased

cytokine

secretion,

although

it

is

un-clear whether this effect is mediated by the effect of HPK-1

on NF-kB or on other T cell transcription factors (e.g.,

NFAT and activator protein AP-1).

TCR-distal regulatory mechanisms

In the case of the CBM complex and other TCR-distal

mediators of NF-kB activation, these proteins were

identified relatively recently, and regulatory mechanisms

are therefore only now being recognized. One protein newly

identified as a CARMA1 regulator is the serine–threonine

phosphatase, PP2A (Figure 2). As discussed above,

PKCu phosphorylation of CARMA1 at Ser645 is critical

for assembly of the CBM complex [1]. PP2A removes the

phosphate group from Ser645, destabilizing the CARMA1–

BCL10 interaction and reducing NF-kB activation.

Further, siRNA silencing of PP2A results in higher

TCR-induced IL-2 and interferon (IFN)-g production

[97]. However, it is also important to note that PP2A

directly deactivates the IKK complex [98], making it un-

clear whether inhibition of NF-kB activation is primarily

due to PP2A effects on CARMA1 or IKK.

Also discussed above, CARMA1 phosphorylation at

S649 and S608 by PKCu (directly or indirectly) [16] and

CK1a [21], respectively, impairs NF-kB signaling. A phos-

phorylation-independent mechanism of CARMA1 regula-

tion involves binding of CARMA1 to the kinesin motor



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protein Guanylate kinase-associated kinesin (GAKIN)

[99]. This interaction results in CARMA1 movement away

from PKCu at the IS and reduces CARMA1 interaction

with BCL10 (Figure 2). Post-TCR stimulation CARMA1–

BCL10 binding is also interrupted by caspase and receptor

interacting protein adaptor with death domain (CRADD),

which competes with CARMA1 to bind BCL10 (Figure 2).

Accordingly, CRADD-deficient murine T cells show in-

creased CARMA1–BCL10 binding and RELA activation.

CRADD deletion results in increased T cell cytokine pro-

duction with concanavalin A stimulation, but does not

yield any identified autoimmune phenotype [100].

Regulation of TCR signaling by ubiquitin-modifying

enzymes

The ubiquitin-editing enzyme, A20, has been long known

as an inhibitor of TNF-dependent NF-kB activation. A20

can both remove K63-polyubiquitin chains from and add

K48-polyubiquitin chains to target proteins [101]. A20 has

a high basal expression in T cells, and it can limit TCR-

induced NF-kB activity by removing K63-polyubiquitin

chains from the CBM complex protein, MALT1 [102]

(Figure 2). Interestingly, MALT1 can cleave A20 in a

TCR-dependent manner, and this proteolytic cleavage

has been suggested to disrupt the ability of A20 to limit

TCR activation of NF-kB [46]. A20 is also capable of

cleaving K63-polyubiquitin chains from receptor-interact-

ing serine/threonine-protein kinase (RIP1), TRAF6, and

IKK g, limiting TNF-, IL-1-, and lipopolysaccharide (LPS)-

dependent NF-kB activation [103]. However, there is so far

no evidence that these specific mechanisms regulate the

TCR-to-NF-kB pathway. A20 activity is critical for down-

regulating NF-kB activity in numerous cell types, there-

fore, A20-deficient mice (Tnfaip3À/À) die shortly after birth

due to multiorgan inflammation [101]. Although condition-

al inactivation of A20 in B cells results in autoantibody

production and lupus-like disease [101], the in vivo role of

A20 in T cells remains uncertain.

A second ubiquitin-modifying enzyme, CYLD, has been

reported as a modulator of JNK and NF-kB activation

[101]. CyldÀ/À mice develop spontaneous autoimmune

colitis. Biochemical studies of CyldÀ/À peripheral T cells

Table 1. Cytosolic proteins involved in negative regulation of TCR-to-NF-kB signaling.

Protein mediator Proposed mechanism Gene deletion or silencing phenotype Refs

SHP-1 (phosphatase) Dephosphorylates LCK,

ZAP70, SLP-76

me/me ‘moth-eaten’ mouse

TCR-induced thymocyte hyperproliferation, increases IL-2

production

Increased CD8 effector cell expansion with viral infection

[87–90,122]

STS-1, STS-2 (phosphatase) Dephosphorylates ZAP-70 Sts1À / À Sts2 À / À double knockout

T cell hyperproliferation

Increased IL-2, -4, -5, -10, IFN-g secretionIncreased susceptibility in murine multiple scelerosis model

[91]

CSK (protein kinase) LCK inhibitory phosphorylation siRNA silencing: increased TCR-induced IL-2 secretion [94]

CBL-B, C-CBL (E3 ubiquitin

ligase)

C-CBL: LAT trafficking to

endosomes

Cbl-b À / À: increased lymphocyte proliferation and secretion

of IL-2 and antibody

c-Cbl À / À: lymphoid hyperplasia

[77,81,107]

HPK-1 (protein kinase) SLP-76 inhibitory

phosphorylation increases

14-3-3 binding

Map4k1À / À: T cell hyperproliferation, increased T cell

cytokines and B cell antibody production

Increased susceptibility to autoimmune encephalitis

[19,20]

PP2A (phosphatase) Dephosphorylates CARMA1

and IKK

siRNA silencing: increased IL-2 and IFN-g secretion

TAXmediated PP2A inhibition: constitutive IKK activation

[97,98]

CK1a (protein kinase)a Phosphorylates CARMA1 CK1a inactive kinase mutant: increased NF-kB activity

CK1a silencing: reduced NF-kB activity, T cell IL-2 secretion

and proliferation (contradictory results suggest CK1a is a

bifunctional regulator; dominant effect is positive regulation)

[21]

PKCu (protein kinase)a

(direct or indirect effect)

Late CARMA1phosphorylation

at S649

Mutation of S649 phosphorylation site increases IKK and

NF-kB activation in Jurkat cells(PKCu knockout blocks NF-kB activation;dominant effect is

positive regulation)

[16]

GAKIN (kinesin 13B) Competes with BCL10 for

CARMA1 binding

shRNA silencing: increased IKK activation and IL-2 secretion [99]

CRADD (adaptor protein) Competes with CARMA1 to

bind BCL10

Cradd À / À: increased T cell CARMA1–BCL10 binding, RELA

activation and cytokine secretion

[100]

Autophagy pathway,

proteasomes

BCL10 degradation Autophagy-deficient T cells: increased IL-2 secretion, CD25

expression; decreased survival

Inhibition of BCL10 phosphorylation: increased secretion of

IL-2 and TNFa

[28,29,33,36,108]

A20 (deubiquitinase and

ubiquitin ligase)

Removal of K63-polyubiquitin

from MALT1

Tnfaip3 À / À: multiorgan inflammation, leading to death

B cell conditional knock out: lupus-like disease, increased

antibody secretion

[101,102]

CYLD (deubiquitinase) Removal of K63-polyubiquitin

from TAK1

Cyld À / À: increased IKKb phosphorylation

Hyper-responsive T and B cellsSpontaneous colonic infiltration

[104,105]

PP4R1–PP4c (phosphatase) IKKa / b dephosphorylation PP4R1-silenced T cells demonstrate enhanced IKK

phosphorylation and NF-kB activation

[61]

aBifunctional NF-kB regulator with stimulatory effect over-riding negative function.



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and B cells showed constitutively phosphorylated IKK b

and activated NF-kB [104,105]. This is explained by loss of

CYLD-mediated TAK1 K63-deubiquitination [106], which

suppresses TAK1 activation in wild type lymphocytes

(Figure 2). Curiously, in CD3+CD28-stimulated Jurkat T

cells, MALT1 cleaves CYLD increasing activation of JNK,

but not NF-kB [47]. Further studies of A20À/À and CyldÀ/À

T

cells

will

be

required

to

determine

the

relative

impor-tance of these regulators of protein ubiquitination in TCR-

to-NF-kB signaling, and to assess whether there is any

redundancy in their activities.

Regulation of TCR signaling by molecular sequestration

Emerging data suggest a novel mechanism of TCR signal

transduction regulation, whereby cytosolic mediators are

selectively sequestered in cytosolic vesicular compart-

ments, making them unavailable for signal transmission.

The ubiquitin ligases of the Casitas B cell lymphoma (CBL)

family, CBL-B and C-CBL, negatively regulate T cell acti-

vation. Both CBL-B and C-CBL knockout results in higher

NF-kBactivation, lymphoid hyperplasia, and increased IL-

2 secretion from T cells [107]. Reports indicate that CBLactivity might depend on promotion of TCR trafficking to

lysosomes [107] and/or on ubiquitination of the adaptor

protein LAT, targeting LAT for endosomal sequestration

[81] (Figure 2). A similar mechanism involving the distal

TCR signaling adaptor BCL10 has also recently been

reported. Data suggest that BCL10 is selectively targeted

for sequestration within autophagosomes [33], followed by

lysosomal degradation [33,36]. The proteasome also con-

tributes to TCR-dependent BCL10 degradation via an

unclear mechanism [29,33]. The role of autophagy in lim-

iting TCR activation of NF-kB is further supported by the

observation that T cell-specific inactivation of the autop-

hagy

genes

ATG7

[108]

or

ATG3

[33]

results

in

increasedIL-2 secretion and CD25 expression. BCL10 autophagy is

highly selective, because the BCL10 binding partner

MALT1 is neither detected inside autophagosomes nor

degraded [33]. The process whereby MALT1 is separated

from BCL10, protecting MALT1 from degradation, might

depend on autophagosome formation [33] and/or IKK b-

mediated BCL10 phosphorylation [28,29]. Data showing

that phosphorylation of BCL10 by IKK b causes BCL10 to

dissociate from the CBM complex [28] suggest that IKK b

has a dual function, both positively and negatively regu-

lating the TCR-to-NF-kB pathway.

Interestingly, the process of BCL10 autophagy occurs at

the POLKADOTS signalosome, requiring the same molec-

ular interactions that play a central role in TCR activation

ofNF-kB. Specifically, autophagy of BCL10 requires inter-

action between p62 and K63-polyubiquitinated BCL10

[33,35,36] (Figure 2). Thus, p62 is an additional bifunc-

tional regulator of TCR signaling to NF-kB, controlling

both assembly of the POLKADOTS signalosome and deg-

radation of its key signaling component, BCL10. How NF-

kB activation and BCL10 autophagy (a signal limiting

mechanism) are coordinately regulated at the POLKA-

DOTS signalosome will require further investigation.

The above studies illustrate that negative regulatory

mechanisms act at multiple points in the TCR-to-NF-kB

signaling cascade. Interestingly, diverse independent

mechanisms including phosphorylation, dephosphoryla-

tion, ubiquitination, deubiquitination, protein trafficking,

andprotein degradation contribute to limiting activation of

TCR signaling. These data suggest that negative regula-

tion of this pathway is critical for T cell function and/or

survival, in line with emerging data that unrestrained

NF-kB activation has a spectrum of possible deleterious

outcomes.

Concluding remarks

Recent findings have significantly altered our views of TCR

signaling to NF-kB. Certain mediators, such as PKCu and

CARMA1, are phosphorylated by multiple kinases, dispel-

ling early models that postulated a simple linear cascade

by which the TCR transmits activating signals to NF-kB.

Studies have also revealed that cytosolic mediators in this

pathway do not all coalesce in a single TCR-proximal

signalosome at the IS. Rather, cytosolic signalosomes far

removed from the TCR are also integral to NF-kB activa-

tion. Despite these new complexities, precise molecular

mechanisms of signal transmission are gradually being

revealed, with several essential post-translational modifi-cations and their downstream effects now identified. For

example, BCL10, once thought to be a simple adaptor

connecting CARMA1 to MALT1, is now known to undergo

K63-polyubiquitination, triggering association with p62

and activation of IKK. Additionally, numerous mecha-

nisms of negative regulation have been recognized,

indicating that the TCR-to-NF-kB pathway is tightly mod-

ulated. Four kinases (PKCu, HPK-1, CK1a, and IKK b) and

two ubiquitin-binding proteins (TSG101 and p62) play a

dual role in signaling to NF-kB, with both activating and

signal limiting activities. Precise control of NF-kB activa-

tion may guarantee production of exact levels of cell cycle

Box 2. Directions for future research

Elucidation of differencesbetween naı ¨ve and effector T cells in the

TCR-to-NF-kB pathway. In particular, it is unclear why effector/

memory T cells require a cytosolic signalosome to successfully

activate NF-kB (Box 1), and why levels of signaling proteins differ

between naı ¨ve and effector/memory cells.

Defining how the CBM complex and the POLKADOTS signalo-

some are mechanistically connected to IKK activation. Related

questions include how BCL10 is physically separated from

MALT1, allowing selective BCL10 degradation, and whether

MALT1 function changes after its signaling partner BCL10 is

degraded.

Revealing the mechanism and purpose of coupling NF-kB

activation with other T cell signaling cascades. In particular, it iscurrently unclear why JNKactivation andactin polymerization are

closely networked to the TCR-to-NF-kB cascade.

Exploring the role of miRNAs in TCR-to-NF-kB signaling. miRNAs

are emerging as key regulators of T cell activation, with likely

effects on signaling to NF-kB. Indeed, recent data show mir-146

andmir-155 affectmediators utilized by theTCR-to-NF-kB cascade

and alter T cell behavior [123–126].

Determining the role of atypical ubiquitination in TCR-to-NF-kB

signaling. To date, most research in this pathway has focused on

typical K48- and K63-polyubiquitination, due to the existence of

highly specific tools that enable detailed study of these modifica-

tions. The six atypical ubiquitins have demonstrated function in

NF-kB activation [58] but there are few data to date regarding how

these under-studied ubiquitin polymers modulate TCR signaling

to NF-kB.



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proteins, thereby avoiding failed cell division and apopto-

sis. Emerging data also suggest that signaling to NF-kB is

coupled to seemingly unrelated biological processes such as

regulation of the actin cytoskeleton. Additionally, there

may be considerable differences in mechanisms of TCR

activation of NF-kB utilized by T cells at distinct stages of

differentiation. Despite these recent advances in our un-

derstanding

of

this

pathway,

there

remain

many

mecha-nistic details that are poorly understood. Important

questions for the field are outlined in Box 2.

AcknowledgmentsThe authors thank A. Snow, C. Gray, K. McCorkell, M. May, and C-Z.

Giam for critical reading of themanuscript. Supported by grants from the

US National Institutes of Health (AI057481 to B.C.S.), the Center for

Neuroscience and Regenerative Medicine (CNRM) (to B.C.S.), and pre-

doctoral fellowships (to S.P.) from the American Heart Association

(10PRE3150039) and the Henry M. Jackson Foundation. The views

expressed are those of the authors and do not necessarily reflect those of

the Uniformed Services University or the Department of Defense. The

authors declare no competing financial interests.

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