Review article

CD40 and its viral mimic, LMP1: similar means to different ends

Ngan Lam, Bill Sugden*

McArdle Laboratory for Cancer Research, University of Wisconsin-Madison, 1400 University Avenue, Madison, WI 53713, USA

Received 20 May 2002; accepted 10 July 2002

Abstract

CD40 is an important regulator of diverse aspects of the immune response including the T-cell-dependent humoral immune response, the

development of antigen-presenting cells (APCs) and inflammation. Latent membrane protein 1 (LMP1), a protein encoded by Epstein–Barr

Virus (EBV), appears to mimic CD40 in multiple ways. CD40 and LMP1 bind similar sets of cellular signalling proteins and activate

overlapping signalling pathways. Despite many similarities shared between CD40 and LMP1, they also differ substantively. In this review,

we will compare and contrast the signalling mediated by CD40 and LMP1.

D 2002 Elsevier Science Inc. All rights reserved.

Keywords: CD40; LMP1; EBV; TRAF; NF-kB; JNK; p38/MAPK

1. Introduction

CD40 and Epstein–Barr Virus (EBV) are two means to

affect proliferation of human B-lymphocytes. CD40, a

member of the tumour necrosis factor receptor (TNF-R)

family, was originally identified as a costimulator of B-cell

proliferation in vitro when it is activated by antibodies [1].

After 16 years of intensive study, CD40 is now recognized

as an important regulator of diverse aspects of the immune

response including the T-cell-dependent humoral immune

response, the development of antigen-presenting cells and

inflammation (reviewed in Refs. [2–5]). EBV, a human

lymphotrophic gamma herpesvirus, causes infectious mono-

nucleosis and contributes causally to many malignancies

including Burkitt lymphoma, Hodgkin’s lymphoma and

nasopharyngeal carcinoma (reviewed in Refs. [6–8]). Infec-

tion of human resting B-lymphocytes in vitro with EBV

leads to proliferation of the infected cells, an effect similar

to that achieved by activation of the CD40 and IL4

receptors. One viral protein, latent membrane protein 1

(LMP1), has been shown to be important in EBV-mediated

B-cell proliferation and mimics CD40 in multiple ways [9].

LMP1 and CD40 bind similar sets of cellular signal-

ling proteins and activate overlapping signalling pathways.

Despite many similarities shared between CD40 and LMP1,

they also differ substantively. Understanding the similarities

and differences between CD40’s and LMP1’s signalling

provides insights into both CD40 and EBV. In this review,

we will compare and contrast the signalling mediated by

CD40 and LMP1.

2. CD40 and its function

CD40 is a 45–50 kD glycoprotein of 277 amino acid (aa)

belonging to the TNF-R family. It has a 172-aa extracellular

portion, a transmembrane region and a 61-aa cytoplasmic

tail (Fig. 1). The extracellular portion of CD40 contains four

cysteine-rich domains (CRDs) which are conserved among

members of the TNF-R family and mediate direct ligand

binding [10]. It has been recently shown that CD40 self-

associates through its extracellular domain in the absence of

binding its ligand [11]. Self-association has been observed

in other members of TNF-R family and is mediated, as

demonstrated for TNF-R, through a pre-ligand binding

assembly domain (PLAD) which is required for, but not

directly involved in, ligand binding [11]. The cytoplasmic

domain of CD40 is responsible for signal transduction in

that it associates with other molecules involved in signal-

ling. There are multiple CD40 isoforms produced by alter-

native splicing of RNA. Several isoforms, including one

lacking the cytoplasmic domain, can block CD40’s signal-

0898-6568/02/$ - see front matter D 2002 Elsevier Science Inc. All rights reserved.

PII: S0898 -6568 (02 )00083 -9

* Corresponding author. Tel.: +1-608-262-6697.

E-mail address: sugden@oncology.wisc.edu (B. Sugden).

www.elsevier.com/locate/cellsig

Cellular Signalling 15 (2002) 9–16

ling [12]. CD40 is expressed in a broad range of cell types

including most lineages of B cells, monocytes, dendritic

cells, basophils, eosinophils, some T-lymphocytes, endothe-

lial cells, fibroblasts, epithelial cells and neuronal cells.

CD40-ligand (CD40L, CD154, gp39), the ligand for

CD40, is a member of TNF family. CD40L is a trans-

membrane protein of 261 aa. It has a short 23-aa amino

cytoplasmic domain, a transmembrane region and a 215-aa

extracellular tail. The crystal structure of a soluble fragment

of CD40L encompassing aa 161–261 indicates that CD40L

forms a homotrimer [13]. There are two shorter isoforms of

CD40L generated by post-translational modification, a 31-

kD isoform lacking the cytoplasmic domain and an 18-kD

soluble isoform lacking the cytoplasmic domain, the trans-

membrane domain and part of the extracellular domain [14].

Both isoforms are able to form trimers, activate CD40 and

have been detected in heteromultimeric complexes with the

full-length CD40L [14–16]. Given that CD40 self-associ-

ates through its PLAD domain, it is perplexing how CD40L

initiates CD40’s signalling because the triggering cannot be

simply explained as trimerization of CD40 by CD40L. The

initiation of signalling is probably mediated through

changes in CD40’s conformation. It is worthwhile pointing

out that the outcomes of signalling initiated by CD40L can

differ from that initiated by anti-CD40 antibodies [17].

CD40L is mainly expressed in activated but not resting T-

lymphocytes. Its expression in T cells is mostly restricted to

the CD4+ subset, although it is also detected in a small

fraction of CD8+ cells. Some other cell types including mast

cells, basophils and eosinophils can be readily stained with

CD40L antibodies. CD40L can also be found in intracellular

stores of thrombocytes.

The biological significance of the CD40–CD40L inter-

action was first revealed in studies which showed that

patients with an X-linked immunodeficiency with hyper-

IgM (HIGM1) have defects in the gene encoding CD40L

[18–22]. The hallmark of HIGM1 is that patients have low

levels of IgG, IgA, IgE and normal to high levels of IgM in

their serum. They are prone to opportunistic infections such

as Pneumocystis, carinii-induced pneumonia and Crypto-

sporidium-induced diarrhoea. T cells from HIGM1 patients

fail to express functional CD40L. However, their B cells can

produce normal levels of IgG, IgA and IgE when CD40 is

activated in vitro in combination with appropriate cytokines.

The defects in CD40L of HIGM1 patients indicate that

CD40–CD40L interaction is important in controlling T-cell-

dependent Ig class-switching in B cells. The role of CD40–

CD40L interactions in the immune response has been

further supported by the recent finding that patients with

HIGM3 have homozygous mutations in their CD40 gene

Fig. 1. Shown are diagrams of monomeric CD40 and LMP1 as they might localize in the lipid rafts of a membrane. Both molecules are shown as monomers,

although much evidence indicates that a peptide from CD40’s cytoplasmic tail containing a TRAF binding site binds TRAF molecules in a trimeric complex

[73,83], that its extracellular moiety consisting of four cysteine-rich-domain (CRD) is oligomeric [11], and its ligand, CD40L, is trimeric [13]. How all of these

intact molecules associate before and after ligand binding is yet to be determined at atomic resolution. None of LMP1’s structure has been determined. Both

CD40, when bound by its ligand, and LMP1, which apparently lacks a ligand, can bind various TRAF molecules and JAK3 (see text). The TRAF molecules are

depicted as mushrooms based on structures determined by X-ray crystallographic studies [73,74]. CD40 binds TRAF6 directly [82,83]; LMP1 binds TRADD

[89] which binds TRAF2 [90,92]. CTAR1 and CTAR2 refer to the carboxy-terminal activation regions 1 and 2 of LMP1 which associate with TRAF and

TRADD molecules, respectively.

N. Lam, B. Sugden / Cellular Signalling 15 (2002) 9–1610

[23]. HIGM3 patients have immunological and clinical

phenotypes indistinguishable from those of HIGM1 pa-

tients. The role of CD40 in controlling Ig class-switching

in B cells is also confirmed in mice deficient in CD40 and

CD40L, respectively [24,25]. Both CD40 and CD40L

knockout mice have phenotypes similar to those of HIGM1

and HIGM3 patients. Mice null for CD40 or CD40L not

only have defects in their Ig class-switching, but also in their

formation of germinal centres and establishment of B-cell

memory.

Expression of CD40 in many cell types other than B cells

indicates that CD40 participates in multiple biological

activities. Indeed, CD40 expressed in antigen-presenting

cells (APCs) such as dendritic cells and macrophages has

been shown to play a critical role in activation and develop-

ment of APCs and consequently in the priming of T-cell-

mediated immune responses [4,26–29]. CD40 expressed in

endothelial cells contributes to the inflammatory response

[30]. Finally, CD40 has been shown to be involved in the

development, maintenance and survival of neuronal cells

both in vitro and in vivo [31].

3. LMP1 and its function

EBV, a herpesvirus, is characterized by its ability to

establish latent infections in B-lymphocytes [6]. This latent

infection induces the proliferation of infected cells both in

vivo and in vitro. In vivo, EBV-induced polyclonal B-cell

proliferation causes infectious mononucleosis in young

adults, and a potential malignant lymphoproliferative dis-

order in immune-compromised people [7]. In vitro, EBV-

induced proliferation efficiently leads to immortalized lym-

phoblastic cell lines (LCLs). Although proliferation of

primary B cells can also be induced by CD40L and IL4 in

vitro, only infection with EBV gives rise to sustained

proliferation and, eventually, immortalized cell lines. One

viral membrane protein LMP1 expressed during latent

infection has been shown to be important for EBV-mediated

B-cell proliferation. Using a recombinant mini-EBV in

which expression of LMP1 is controlled by a tetracycline

inducible promoter to infect primary B cells, Kilger et al. [9]

showed that blocking expression of LMP1 inhibits prolifer-

ation of infected cells. Interestingly, the cells again prolif-

erate when they are treated with CD40L [9]. This restoration

indicates that LMP1 functionally mimics CD40. That LMP1

expressed in B cells of CD40 null mice partially rescues

some of the mouse’s defects, including T-cell-dependent

IgG production by B cells, also indicates that LMP1 mimics

CD40. However, these CD40� /� , LMP1+ mice fail to

produce high affinity IgG and fail to form germinal centres

[32], indicating that LMP1 and CD40 also differ in their

functions.

LMP1 is a membrane protein with a 25-aa amino-

terminal cytoplasmic domain, a six-transmembrane region

and a 200-aa carboxy-terminal cytoplasmic tail (Fig. 1). One

striking feature of LMP1 is that it signals in the apparent

absence of a ligand [33,34], whereas CD40 depends on

CD40L for its signalling. It is thought that the ligand

independency of LMP1 is mediated by aggregation, directly

or indirectly, through its transmembrane domain. For exam-

ple, a recombinant protein consisting of LMP1’s amino-

terminal and transmembrane domain and CD40’s cytoplas-

mic domain efficiently activates CD40’s signalling pathway

in the absence of CD40L [34]. LMP1 is expressed in EBV-

transformed LCLs and has been detected in tumour cells of

many EBV-associated malignancies including Hodgkin’s

lymphoma and nasopharyngeal carcinoma [7]. Early studies

with LMP1 showed that its expression in mouse fibroblasts

and endothelial cells leads to transformation of the cells to

anchorage-independent growth [35,36]. These studies, in

combination with the genetic studies showing LMP1’s

critical role in EBV-mediated transformation, define LMP1

as an oncogene. LMP1’s oncogenic property is further

supported by the observation that transgenic mice, which

express LMP1 in their B cells, have higher incidences of

lymphoma in old age [37]. Although CD40 has not been

shown to be associated with any malignancies, it is found to

be constitutively activated in non-Hodgkin’s lymphoma of a

B-cell lineage (NHL-B) by CD40L which is found abnor-

mally coexpressed in the tumour cells. Suppression of

CD40’s signalling inhibits proliferation of these tumour

cells [38].

4. Signalling pathways activated by CD40 and LMP1

Signal transduction by CD40 and LMP1 have both been

studied intensively. Activation of primary B cells by CD40

leads to their increased secretion of Ig; increased expression

of cell surface adhesion molecules (ICAM-1, CD23), cos-

timulatory molecules of T-cell activation (B7-1) and pro-

teins involved in apoptosis such as Fas or anti-apoptosis

such as A20, Bcl-xl, bfl-1; and production of cytokines

[3,5,39,40]. In other cell types, signalling through CD40

increases expression of similar groups of cellular proteins

such as surface adhesion molecules, costimulatory mole-

cules, apoptotic proteins and cytokines, but with some cell-

type-dependent specificities. The consequences of CD40’s

signalling usually foster survival, proliferation and differ-

entiation of cells [3,5]. However, this signalling has been

shown to inhibit proliferation and induce apoptosis in some

carcinoma cells and lymphoma cells [41–43].

Most phenotypes induced by CD40 investigated in cell

culture can be reproduced by LMP1. The ability of LMP1 to

induce a panel of anti-apoptotic proteins and to protect cells

from apoptosis [44–47] has elicited much attention because

this phenotype of LMP1 could contribute to oncogenesis

mediated by EBV. Nevertheless, LMP1 is cytostatic when

expressed at high levels [48,49]. The cytostatic function of

LMP1 is mediated through its transmembrane domain and

can be separated from its positive signalling [48]. Neither

N. Lam, B. Sugden / Cellular Signalling 15 (2002) 9–16 11

the significance nor the mechanism of this inhibitory effect

of LMP1 has been elucidated.

Both CD40 and LMP1 induce multiple signalling path-

ways yielding activation of NF-kB, JNK, p38/MAPK and

STAT proteins [50–56]. The roles of these signalling path-

ways in CD40’s and LMP1’s functions have been inves-

tigated with genetic studies and specific inhibitors. These

studies have found that the activation of NF-kB and p38/

MAPK both contribute to CD40’s and LMP1’s functions

[56–63]. There is no direct study on roles of JNK kinase.

Activation of STAT proteins is not essential for their

function, at least in B cells [64–66]. Phenotypes induced

by CD40 and LMP1 seem to be regulated either independ-

ently or cooperatively by different pathways. For CD40,

studies from DNA microarrays in combination with specific

inhibitors have shown that NF-kB seems mainly to upregu-

late gene expression, while p38/MAPK largely downregu-

lates gene expression in B cells [60].

5. Mechanism of signalling transduction

CD40’s signalling initiates at the plasma membrane by

binding of CD40 to CD40L or its antibodies. Upon activa-

tion, CD40 redistributes into lipid rafts which are membrane

microdomains enriched in sphingolipids and cholesterol

[67,68]. In contrast, about 30% of LMP1 associates with

the lipid rafts at steady state [69]. It is thought that the

association of CD40 and LMP1 with lipid rafts provides a

platform for assembly of a signalling complex and is thus

important for generating their signals. Three observations

are consistent with this notion. First, activated CD40 and

LMP1 recruit tumour necrosis factor receptor associated

factors (TRAFs) which are their interacting proteins impor-

tant for signalling into the lipid rafts [67,68,70]. Second, a

drug that disrupts the lipid rafts inhibits CD40’s signalling

[68]. Third, targeting the signalling domains of CD40 and

LMP1 to lipid rafts activate their signals [69].

5.1. TRAF- and TRADD-mediated pathways

Both CD40 and LMP1 transduce signals through their

cytoplasmic tails. In common with other members of the

TNF-R family, CD40’s and LMP1’s cytoplasmic tails lack

intrinsic kinase activity and interact with a family of adaptor

proteins TRAFs. TRAFs were first identified as proteins that

interact with TNF-R and are important for its signalling. Six

TRAFs have been identified in human cells designated

TRAF1 through 6 (reviewed in Refs. [71,72]). TRAF

proteins are characterized by their having a conserved

TRAF homology domain which includes a TRAF-C domain

and a coil–coil (C–C) domain at their carboxy-termini.

Solutions of the crystal structures of the TRAF homology

domains of both TRAF2 and TRAF3 have revealed their

trimeric mushroom-like structure with the TRAF-C domain

in the cap and the C–C domain in the stalk [73–74,120].

That TRAFs form a trimeric structure is consistent with the

hypothesis that TNF-R family members, including CD40,

initiate signalling in a trimeric complex. Co-crystal struc-

tures of TRAF domains with small peptides from either

TNF-R or CD40 [73–74,120] indicate that the TRAF-C

domains dictate receptor-binding and specificity. Also, dif-

ferent TRAFs can form heterooligomers through their

TRAF domains [76]. All TRAFs except TRAF1 have a

ring finger domain and multiple zinc finger domains at their

amino termini. Overexpression of TRAF 2, 5 or 6, but not of

TRAF 1 or 3, has been shown to induce activation of NF-kB

and JNK [77]. It is not clear how TRAFs transduce signals

mechanistically, although TRAF1, 2, 3, 5 and 6 have been

shown to interact with NF-kB-inducing kinase (NIK)

through their TRAF domains [77]. Nevertheless, the ring

finger and zinc finger domains appear to be essential for

TRAFs’ functions because derivatives of TRAFs containing

only a TRAF domain act as dominant negative inhibitors.

The ring finger domains have been identified as encoding

ubiquitin ligase activities [78]. It has recently been shown

that the ubiquitin ligase activity of TRAF6 acts in concert

with an ubiquitin-activating enzyme and an ubiquitin-con-

jugating enzyme to activate the TAK1 kinase, which can

phosphorylate IKK and MKK to induce NF-kB and p38/

MAPK pathways [79,80].

CD40 has been shown to interact with TRAF1, TRAF2,

TRAF3 and TRAF6 through two different motifs in its

cytoplasmic tail [76,81,82] (Fig. 1). A PXQXT consensus

TRAF-binding motif from aa 250 to 254 binds TRAF1, 2

and 3. Another distinct motif in the cytoplasmic tail closer to

the membrane interacts with TRAF6. The different TRAFs

bind CD40 with different affinities measured in vitro; the

order of binding is TRAF2>TRAF3HTRAF1 and TRAF6

[83]. Although TRAF5 cannot bind CD40 directly, it can be

recruited to CD40 by TRAF3 in a heterooligomeric complex

[76,81].

The two motifs involved in binding to TRAFs have been

shown to be indispensable for CD40’s signalling and

function in a cooperative manner. Functional examinations

of mutants of CD40 in cell culture demonstrate that its

binding sites for TRAF2, 3 and 6 are required for full

activation of both the NF-kB and JNK signalling pathways

[75,84], while the TRAF6-binding site is solely responsible

for activation of the p38/MAPK pathway [75,81]. Mutants

of CD40, which have defective binding sites for TRAF2 and

3 but an intact site for TRAF6, can stimulate 70% of the

level of NF-kB’s activity and close to 100% of that of JNK’s

activity as does wild-type CD40. The results from experi-

ments in cell culture have been confirmed in mice null for

CD40 and transgenic for wild-type or different mutants of

human CD40 in their B cells [85]. Wild-type human CD40

fully rescues the deficiencies of the T-dependent humoral

immune response of the CD40-null mice. The mice trans-

genic for CD40 mutants with only TRAF2 and 3 or only

TRAF6-binding sites still show immunological defects but

are restored for some phenotypes. Those having only the

N. Lam, B. Sugden / Cellular Signalling 15 (2002) 9–1612

TRAF2- and 3-binding site partially rescue Ig class-switch-

ing; those having only the TRAF6-binding site rescue Ig

class-switching and extrafollicular B-cell differentiation.

The formation of germinal centres requires the presence of

both binding sites [85].

LMP1, like CD40, has two motifs in its carboxy-terminal

cytoplasmic domain, designated CTAR1 and CTAR2,

respectively, that are required for LMP1’s signalling via

TRAF molecules (Fig. 1). CTAR1 contains a consensus

TRAF binding motif PXQXT and has been shown to bind

TRAF1, 2 and 3 [86]; their affinities rank in the order of

TRAF3>TRAF1>TRAF2 [87]. Unlike CD40, the second

activation domain CTAR2 of LMP1 does not bind TRAF6,

rather it binds TNF-R associated death domain protein

(TRADD) which is another adaptor protein involved in

TNF-R mediated signalling [88,89]. The amino-terminus

of TRADD has been co-crystallized in a trimeric complex

with the TRAF domain of TRAF2 [90]. The carboxy-

terminus of TRADD contains a death domain which inter-

acts with TNF-R and mediates the induction of apoptosis

and the activation of NF-kB [88,91]. TRADD interacts

through its amino-terminus with LMP1 differently than it

does with TNF-R [92]. TRADD-mediated activation of NF-

kB has been shown to be dependent on TRAF2 [92,93].

The requirement for CTAR1 and CTAR2 for LMP1’s

function has been explored genetically in cell culture and in

the context of EBV. LMP1’s two domains, CTAR1 and

CTAR2, as with CD40, are required for efficient activation

of NF-kB and p38/MAPK [54,56,62,94,95]. CTAR2 of

LMP1 accounts for about 60–70% of activation of NF-kB

[54,62]. CTAR2 has also been shown to be fully responsible

for the activation of JNK [55,96], required for transforma-

tion of mouse fibroblasts [97], but not for murine endothe-

lial cells [35]. Viral genetic studies indicate that CTAR1 and

CTAR2 are both required for efficient proliferation of

infected primary B cells [64,98,99]. Viruses harbouring

mutants of LMP1 having only functional CTAR1 or CTAR2

have less than 10% of wild-type virus’s activity to stimulate

cell proliferation [64].

The transmembrane region of LMP1 in addition to

CTAR1 and CTAR2 is essential for LMP1’s signalling.

Aggregation of LMP1 through its transmembrane domain

is pivotal for the assembly of a signalling complex. It is not

known what structure the transmembrane region imposes on

the cytoplasmic tail, although it is likely to be trimeric given

TRAFs and TRADD are all trimeric. Interestingly, fusion of

the trimeric protein chloramphenicol acetyltransferase

(CAT) to the cytoplasmic tail of CD40 when properly

targeted to membrane restores CD40’s wild-type activity,

while a parallel fusion of LMP1 does not [69]. The trans-

membrane region of LMP1 assembles a distinct complex

from that of CD40. In fact, a fusion of LMP1’s amino-

terminus and transmembrane domain with CD40’s cytoplas-

mic domain not only signals in the absence of CD40L, but

also activates NF-kB’s activities to a greater extent than

does CD40 plus its ligand [69]. The ability of LMP1’s

transmembrane region to assemble a more efficient complex

may be attributed to its ability to form higher ordered

aggregates. Consistent with this notion is the finding that

a dodecameric form of CD40L is more effective than a

trimeric CD40L in activating B cells in vitro [100]. Fur-

thermore, TRAF6 binds to CD40 more tightly when CD40

is in high density as measured in surface plasmon resonance

studies [83]. Interestingly, formation of higher ordered

clustering has been observed in Tall-1 which is another

member of TNF family [101]. The functional unit of Tall-1

is a 200-A diameter virus-like assembly consisting of 20

Tall-1 trimers. A mutant of Tall-1 that can form trimers but

not the virus-like complex binds to its receptor with similar

affinity as does wild-type Tall-1 but is defective in initiating

signalling [101]. All of these observations are consistent

with a model in which LMP1 signals efficiently because it

can form some higher order complex than a trimer.

Although some of the requirements for CD40 and

LMP1 to signal have been elucidated, the roles of different

adaptor proteins in their signalling are far from clear.

Because both CD40 and LMP1 interact with multiple

adaptor proteins, it is important to understand how each

adaptor protein contributes to signalling. The overexpres-

sion of TRAF2, 5 or 6 in cell culture, respectively, is

sufficient to induce activation of NF-kB [82,102,103]. Ad

ditionally, expression in cells of dominant negative forms

of TRAF2, 5 or 6 inhibits CD40’s signalling [82,102–

104]. The cell culture experiments indicate that TRAF2,

TRAF5 and TRAF6 may be involved in the activation of

CD40’s signalling. One caveat of these cell culture experi-

ments is that some derivatives of TRAF proteins that can

act as dominant negative mutants also can heterooligomer-

ize [76]. Thus, dominant negative derivative of TRAFs,

when expressed at high levels, could potentially inhibit

other TRAFs nonspecifically and yield misleading results.

Genetic studies using knockout mice have therefore been

especially valuable in confirming experiments in cell

culture. B cells from TRAF2� /� TNFR1� /� double-

knockout [105] and TRAF6� /� knockout mice [106] fail

to proliferate in response to activation of CD40 in vitro.

Activation of NF-kB cannot be detected in those cells.

Furthermore, TRAF2� /� TNFR1� /� double-knockout

mice are defective in T-cell-dependent Ig isotype-switching

[105]. On the other hand, B cells from TRAF5 knockout

mice [107] show only a reduced response to CD40-

activation in vitro. These mice have a normal T-cell-

dependent humoral immune response to high concentra-

tions of antigen, but a reduced ability to produce high

affinity IgG to low concentrations of antigen. These results

from knockout mice indicate that TRAF2 and TRAF6, but

not TRAF5, are essential for CD40’s signalling in B cells.

Although both TRAF1 and TRAF3 bind CD40, and a

dominant negative derivative of TRAF3 inhibits CD40’s

signalling [108,109], B cells from both TRAF1� /� and

TRAF3� /� knockout mice are normal in their response to

CD40-activation in vitro [110,111]. However, TRAF1 and

N. Lam, B. Sugden / Cellular Signalling 15 (2002) 9–16 13

TRAF3 may contribute to other aspects of CD40’s signal-

ling. For example, TRAF3 may be a negative regulator of

CD40’s signalling. The overexpression of TRAF3 inhibits

CD40’s signalling in B cells in culture [112]. Also, it has

been found that in endothelial cells, shear stress upregulates

TRAF3 both in vitro and in vivo and inhibits CD40-

dependent functions [113].

The roles of different adaptor proteins in LMP1’s signal-

ling have been tested only in cell culture. Several lines of

evidence indicate that TRADD, TRAF2 and TRAF6 maybe

essential for LMP1 to signal. Expression of a dominant

negative mutant of TRADD containing only the amino-

terminus of the protein and thus lacking its death domain

inhibits the activation of NF-kB and p38/MAPK but not that

of JNK by LMP1 [92]. Expression of a dominant negative

derivative of TRAF2 blocks the activation of NF-kB and

p38/MAPK but not that of the JNK pathway by LMP1

[56,92,114]. This inhibitory effect is different for CD40; the

dominant negative form of TRAF2 blocks both NF-kB’s

and JNK’s activation by CD40 in cell culture [92,103].

Finally, LMP1 fails to activate p38/MAPK in mouse cells

null for TRAF6 or in the presence of a dominant negative

mutant of TRAF6 [115], although TRAF6 appears to bind

LMP1 indirectly. As with CD40, LMP1’s signalling is

inhibited by overexpression of TRAF3 [62].

5.2. JAK–STAT pathway

In addition to interacting with TRAFs or TRADD,

both CD40 and LMP1 have been shown to have a third

motif in their cytoplasmic domains which associates with

JAK3 [116,117]. Interactions between JAK3, CD40 and

LMP1 activate members of STAT family of transcription

factors. However, so far, these interactions appear non-

essential for both CD40 and LMP1’s functions in B cells.

B cells from JAK3 deficient patients respond to CD40-

activating antibodies normally [65]. Additionally, EBV

can induce and maintain proliferation of B cells from

JAK3-deficient patients [66]. Furthermore, a recombinant

EBV harbouring a mutant of LMP1 lacking its JAK3-

binding site induces and maintains proliferation in B cells

as efficiently as does wild-type virus [64,118]. Although

apparently unimportant in B cells, activation of JAK3-

STAT pathway by CD40 and LMP1 is likely to be

influential in other cell types, as indicated by experiments

showing that STAT5a is activated by CD40 in monocytes

but not in B cells [119].

6. Conclusion

Genetic and biochemical studies have demonstrated that

both CD40 and LMP1 contain multiple domains acting

cooperatively to promote signalling. Both CD40 and

LMP1 activate NF-kB, JNK, p38/MAPK and JAK-STAT

pathways, and both require the same proteins such as

TRAF2 and TRAF6 for their functions. However, it is clear

that CD40 and LMP1 assemble different signalling com-

plexes. Although several members of the TRAF family

interact with both LMP1 and CD40, they interact with

different affinities. CD40 but not LMP1 directly interacts

with TRAF6. Furthermore, LMP1 but not CD40 interacts

with TRADD. Finally, the transmembrane domain of LMP1

allows the assembly of a complex which signals through

NF-kB peculiarly efficiently. These different signalling

complexes are likely to dictate the differences in the out-

comes of their signalling through their specificity, strength

and duration of that signalling. One insight into LMP1’s

signalling is likely to be provided by elucidating the

structure of LMP1’s signalling complex. It will be fascinat-

ing to learn how this viral mimic of a cellular receptor sig-

nals so efficiently in the absence of a ligand.

Both CD40 and LMP1 appear to regulate specific pro-

cesses, only some of which are shared. The outcomes of

these differences are obvious in vivo as demonstrated by

LMP1 transgenic/CD40� /� mice which still exhibit a

subset of the defects characteristic of CD40� /� mice.

However, the differences are difficult to discern in cell

culture on examining a limited set of cellular effectors.

Experiments using cDNA microarrays should provide mech-

anistic insights into the different outcomes of CD40’s and

LMP1’s signalling [60]. We envisage that, by comparing and

contrasting the profiles of expression in cells stimulated via

CD40 or LMP1, we shall, for example, identify the genes

pivotal in the formation of germinal centres mediated by

CD40 and pivotal for the cellular transformation mediated

by LMP1.

Acknowledgements

We are grateful to Ulrike Dirmeier, Ajamete Kaykas and

Mark Sandberg for their critical reviews of the manuscript.

This work is supported by grants from the National

Institutes of Health: CA22443, CA70723 and CA14520.

Bill Sugden is an American Cancer Society Research

Professor.

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