Báo cáo khoa học: TNFR1-induced activation of the classical NF-jB pathway Harald Wajant1 and Peter Scheurich2 pptx

MINIREVIEW
TNFR1-induced activation of the classical NF-jB pathway
Harald Wajant
1
and Peter Scheurich
2
1 Division of Molecular Internal Medicine, Department of Internal Medicine II, University Hospital Wu
¨
rzburg, Germany
2 Institute of Cell Biology and Immunology, University of Stuttgart, Germany
The NF-jB system
The nuclear factor of kappa B (NF-jB) proteins are
dimeric transcription factors composed of five different
subunits, namely p65 (RelA), RelB, cRel, p50 and p52.
In unstimulated cells, the NF-jB transcription dimers
are retained in the cytoplasm in an inactive state by
masking of their nuclear localization sequence [1,2].
Two different structural modes of the blockade of the
nuclear localization sequence can be distinguished
(Fig. 1). In the first case, a NF-jB dimer interacts
intermolecularly with an inhibitor of kappa B (IjB)
protein under the formation of an inactive ternary
complex. In the second case, blockade of the nuclear
localization sequence is achieved by intramolecular
binding of an inhibitory domain. This is possible
because the NF-jB subunits p50 and p52 are initially
produced as large precursor proteins of 105 and
100 kDa, respectively, carrying IjB protein-like inhibi-
tory domains in their C-terminal parts [1,2]. The two
mechanisms of NF-jB dimer inhibition correspond to
the existence of two different NF-jB-activating path-

ways called the classical and the alternative NF-jB
pathway (Fig. 1). The classical NF-jB pathway is initi-
ated by activation of tumor necrosis factor (TNF)
receptor-associated factor (TRAF) adapter proteins
and the subsequent stimulation of the IjB kinase
(IKK) complex, which among others also contains the
related kinases IKK1 and IKK2 and the structural ⁄
regulatory component NF-jB essential modulator
Keywords
apoptosis; caspase 8; IKK necrosis; NF-jB,
NEMO; RIP1; TNF; TRADD; TRAF2
Correspondence
H. Wajant, Division of Molecular Internal
Medicine, Medical Clinic and Polyclinic II,
University Hospital Wu
¨
rzburg, Ro
¨
ntgenring
11, 97070 Wu
¨
rzburg, Germany
Fax: 49 931 201 71070
Tel: 49 931 201 71000
E-mail:
(Received 12 October 2010, revised 9
December 2010, accepted 11 December
2010)
doi:10.1111/j.1742-4658.2011.08015.x
The molecular mechanisms underlying activation of the IjB kinase (IKK)

complex are presumably best understood in the context of tumor necrosis
factor (TNF) receptor-1 (TNFR1) signaling. In fact, it seems that most, if
not all, proteins relevant for this process have been identified and extensive
biochemical and genetic data are available for the role of these factors in
TNF-induced IKK activation. There is evidence that protein modification–
independent assembly of a core TNFR1 signaling complex containing
TNFR1-associated death domain, receptor interacting kinase 1, TNF
receptor-associated factor 2 and cellular inhibitor of apoptosis protein 1
and 2 starts a chain of nondegrading ubiquitination events that culminate
in the recruitment and activation of IKK complex-stimulating kinases and
the IKK complex itself. Here, we sum up the known details of TNFR1-
induced IKK activation, address arising contradictions and discuss possible
explanations resolving the apparent discrepancies.
Abbreviations
cIAP, cellular inhibitor of apoptosis protein; HOIL-1, heme-oxidized IRP1 ubiquitin ligase; IjB, inhibitor of kappa B; IKK, IjB kinase;
IL, interleukin; LUBAC, linear ubiquitin chain assembly complex; MEF, murine embryonal fibroblast; NEMO, NF-jB essential modulator;
NF-jB, nuclear factor of kappa B; PK, protein kinase; RIP1, receptor-interacting kinase 1; RNF11, RING finger protein 11; S1P, sphingosine-
1-phosphate; TAX1BP1, Tax1 binding protein; TNF, tumor necrosis factor; TRADD, TNFR1-associated via death domain; TRAF2, TNF
receptor-associated factor 2.
862 FEBS Journal 278 (2011) 862–876 ª 2011 The Authors Journal compilation ª 2011 FEBS
(NEMO) [1,2]. The activated IKK complex phos-
phorylates IjB proteins, thereby triggering their prote-
asomal degradation. As a consequence, the NF-jB
dimers are released in the cytoplasm and can now
translocate into the nucleus. Activation of the alterna-
tive NF-jB pathway is independent of IKK2 and
NEMO and requires degradation of TRAF proteins
and subsequent activation of IKK1 [1,2]. IKK1 phos-
phorylates p100 and thereby triggers its processing to
p52. This event results in the conversion of p100-inhib-

ited NF-jB complexes into p52-containing NF-jB
dimers capable of translocating into the nucleus [1,2].
Different NF-jB dimers regulate different target genes.
The two NF-jB pathways have, therefore, many non-
redundant functions. Although the classical pathway
mainly acts in innate immunity, the alternative path-
way is of central relevance for the organogenesis of
lymphoid tissue.
The TNF-induced core signaling
complex of TNFR1
Ligand-induced reorganization of preassembled recep-
tor complexes enables TNFR1 to recruit the adapter
protein TNF receptor-associated protein with a death
domain (TRADD) and the serine–threonine kinase
receptor-interacting protein 1 (RIP1) [3]. TRADD and
RIP1 contain a C-terminal death domain which medi-
ates binding to the death domain of TNFR1. There
are contradictory reports claiming competitive, but
also cooperative, effects in the recruitment of TRADD
and RIP1 to TNFR1, but in sum, it is generally
accepted that RIP1 and TRADD are capable of inter-
acting independently with TNFR1 [4–6]. In addition,
TRADD and RIP1 also interact strongly with each
other by virtue of their death domains, which could be
of importance for the assembly ⁄ integrity of a cytosolic,
TRADD and RIP1-containing complex that is formed
upon TNFR1 stimulation (for more details see the
accompanying minireview by O’Donnell and Ting [7]).
Upon association with ligated TNFR1, TRADD
further recruits the adapter protein TRAF2 via its

N-terminal TRAF-binding domain (Fig. 2). TRAF2
consists of an N-terminal RING domain followed by
five zinc fingers and a C-terminal TRAF domain,
which mediates homotrimerization and interaction
with TRADD [8]. TRAF2 forms stable homotrimeric
mushroom-shaped complexes capable of interacting
with one TRADD molecule with each of its protomers.
The interaction of TRAF2 and TRADD is much
stronger compared with TRAF2 binding to those
members of the TNF receptor family that directly
interact with TRAF proteins, such as, for example,
TNFR2. Although a similar surface at the edge of the
-
-
Fig. 1. Activation of classical and alternative NF-jB signaling. The classical NF-jB pathway can be activated by a broad range of stimuli,
including most ligands of the TNF family, IL-1, a variety of pathogen associated molecular patterns (e.g. lipopolysaccharide) and physical
stress (e.g. UV irradiation). Activation of the classical NF-jB pathway involves stimulation of the kinase activity of the IKK complex and prote-
olytic degradation of IjB proteins. The alternative NF-jB pathway is activated by a limited subgroup of TNF ligands and involves activation of
NIK-mediated stimulation of IKK1 and conversion of p100-containing NF-jB complexes into p52-containing NF-jB complexes by proteolytic
processing of p100 to p52.
H. Wajant and P. Scheurich TNFR1 induced NF-jB signaling
FEBS Journal 278 (2011) 862–876 ª 2011 The Authors Journal compilation ª 2011 FEBS 863
mushroom cap of a TRAF2 complex mediates inter-
action with TRADD as well as TRAF2-binding TNF
receptors, the actual molecular contacts enabling these
interactions are entirely different [9]. TNF-induced
recruitment of TRAF2 to TNFR1 is abrogated in
TRADD-deficient murine embryonal fibroblasts
(MEFs) [10–12], but there is still significant TRAF2
recruitment in TRADD-deficient macrophages [12].

Because RIP1 is known to directly interact with
TRAF2 and is highly expressed in macrophages, it was
suggested that RIP1 might contribute to TRAF2
recruitment to TNFR1, thereby compensating to some
extent for TRADD deficiency in this regard [12].
Nevertheless, in the absence of RIP1, TRADD is fully
sufficient to recruit TRAF2 into the TNFR1 signaling
complex. TRAF2 forms complexes with cellular inhibi-
tor of apoptosis protein (cIAP)-1 and cIAP2 with high
efficacy and therefore typically all these proteins are
part of the TNF-induced TNFR1 signaling complex
[13].
TRADD, RIP1, TRAF2 and cIAPs are
crucially involved in TNFR1-induced
NF-jB signaling
It is evident from studies with TRADD-, TRAF2- and
RIP1-deficient mice that all these molecules play
important roles in TNFR1-induced activation of the
classical NFjB pathway, but are not absolutely indis-
pensable. Although TNFR1-induced phosphorylation
and degradation of IjBa are almost completely abol-
ished in TRADD-deficient MEFs, these hallmarks of
classical NF-jB signaling are only attenuated in
TRADD-deficient macrophages [11,12]. These findings
correspond well to the differential capability of
TNFR1 in these cells to recruit TRAF2 in a TRADD-
independent fashion (see above). The available genetic
evidence also argues for a cell type-specific dependency
of TNFR1-induced activation of the classical NF-jB
pathway from RIP1 (Table 1). No significant signs of

TNF-induced NF-jB signaling were found in a human
Fig. 2. Formation of the NF-jB-stimulating TNFR1 signaling complex. In a first step, binding of TNF to TNFR1 triggers recruitment of the
death domain-containing proteins RIP1 and TRADD. In a second step, there is recruitment of TRAF2–cIAP1 ⁄ 2 complexes to TNFR1-bound
TRADD. At this point, there is no evidence that protein modifications, such as phosphorylation or ubiquitination, play a role in the assembly
of the TNFR1 signaling complex. The TRAF2-associated E3 ligases cIAP1 and cIAP2 (and possibly also TRAF2) now modify RIP1, TRAF2 and
themselves with K63-linked ubiquitin chains. This creates docking sites for the LUBAC complex, an E3 ligase capable of forming linear poly-
ubiquitin chains. The LUBAC complex ubiquitinates NEMO, a subunit of the IKK complex, which by help of its IKK2 subunit also interacts
with TRADD-bound TRAF2. In parallel, the TAK1-TAB 2 complex interacts with K63-ubiquitin modified RIP1 by use of the K63-ubiquitin bind-
ing TAB 2 subunit. TAK1 become activated and then phosphorylates and activates IKK2 which in turn now phosphorylates IjBa, marking it
for K48-ubiquitination and proteasomal degradation.
TNFR1 induced NF-jB signaling H. Wajant and P. Scheurich
864 FEBS Journal 278 (2011) 862–876 ª 2011 The Authors Journal compilation ª 2011 FEBS
mutant cell line lacking RIP1 and in Abelson murine
leukemia virus-transfomed pre-B-cell lines derived
from RIP1 knockout mice (Table 1). By contrast, for
MEFs derived from RIP1 knockout mice, almost unaf-
fected TNF-induced NF-jB signaling has been
reported (Table 1). Remarkably, reconstitution experi-
ments with kinase-dead mutants suggest that the
kinase activity of RIP1 is dispensable for its role in
TNFR1-induced NF-jB activation [14].
Reports describing variable contributions of RIP1 to
TNFR1-mediated NF-jB activation correspond well
with the observation that the C-terminal subunit of the
heterodimeric oncoprotein MUC1 can substitute for
RIP1 in TNFR1-induced activation of the classical
NF-jB pathway. MUC1 is initially produced as a
typ I transmembrane polypeptide which is rapidly
autoproteolytically cleaved in its sea urchin sperm
protein, enterokinase and agrin (SEA) module near the

membrane. The resulting N-terminal (MUC1-N) and
C-terminal (MUC1-C) fragments tightly interact by
noncovalent interaction. In MCF-10A cells, MUC1-C
has been shown to be recruited to TNFR1 in a
TRADD- and TRAF2-dependent manner after TNF
stimulation, whereas RIP1 was found to be dispensable
for this interaction [15]. Furthermore, molecular
knockdown of TRADD, TRAF2, TAK1 or TAB 2, all
known to be of relevance in TNFR1-induced and
RIP1-mediated NF-jB activation, also resulted in
downregulation of TNF-induced and MUC1-mediated
NF-jB signaling in MCF-10A cells. These data are in
accordance with the hypothesis that MUC1-C is capa-
ble of substituting for RIP1 in TNFR1-mediated acti-
vation of the NF-jB signaling pathway. As expected,
knockdown of MUC1-C also strongly reduced the
TNF-induced NF-jB response [15].
MEFs derived from TRAF2-deficient mice showed
only a slightly reduced efficiency of TNF-induced
NF-jB activation, whereas MEFs derived from
TRAF2–TRAF5 double-deficient mice were severely
impaired in this regard [16]. Accordingly, TRAF2 and
TRAF5 were proposed to have at least partially redun-
dant functions in TNFR1-induced NF-jB activation,
although recruitment of TRAF5 into the TNFR1 sig-
naling complex has not been demonstarted to date.
Notably, various studies analyzing MEFs deficient for
TRAF2, TRAF5 or both molecules revealed quite con-
tradictory effects on TNF-induced NF-jB signaling
(Table 2). Because disagreements have been also

reported in cells of the same genetic background, they
might reflect differences in the cultivation conditions
or cell culture-related adaptation ⁄ selection processes.
In any case, these discrepancies illustrate the problems
and pitfalls in making generalizations based only on
studies of MEFs. In line with this, initial analyses of
cIAP1- and cIAP2-deficient MEFs gave no evidence
for a role of these molecules in TNFR1 signaling
[17,18]. However, follow-up studies demonstrated
impaired TNF-induced IjBa degradation in MEFs
when expression of both cIAP proteins had been
downregulated by RNA interference [19–21]. At a first
glance, the NF-jB activation-promoting function of
cIAP1 and -2 observed in the context of TNFR1 sig-
naling dissents from other reports showing NF-jB
activation after depletion of cIAP1 and -2 with second
mitochondria-derived activator of caspase (SMAC)
mimetics [22,23]. However, cIAP1 and -2 are not only
involved in TNFR1-induced activation of the IKK
complex, but are also responsible for the constitutive
degradation of the MAP3K NF-jB-inducing kinase
(NIK) [24,25], which phosphorylates and thereby acti-
vates IKK1 to trigger limited processing of p100.
Accordingly, cIAP1 and -2 are negative regulators of
the alternative NF-jB pathway and SMAC mimetics
have consequently been identified as strong inducers of
p100 processing. The still puzzling observation that
SMAC mimetics also activate the classical NF-jB
pathway might be related to the fact that the IjB
Table 1. Effect of receptor-interacting kinase 1 (RIP1) deficiency

on tumor necrosis factor (TNF)-induced activation of the classical
NF-jB pathway. IKK, IjB kinase; IL, interleukin; MEF, murine
embryonal fibroblast; NF-jB, nuclear factor of kappa B
Model
Observed effects on TNF-induced
activation of the classical
NF-jB pathway Ref.
RIP1 def.
Jurkat
No TNF-induced activation of a
NF-jB-regulated reporter gene
[68]
RIP1 def.
Jurkat
Complete inhibition of TNF-induced
IjBa phosphorylation (western
blot) and DNA-binding of NF-jB
(EMSA)
[39]
Abelson-transformed
B-cells
Completely inhibited TNF-induced
DNA-binding of NF-jB (EMSA)
[69]
RIP1 def. MEFs No or at best traces (< 10% of
wild-type) of TNF-induced IKK
activation and IL-6 induction
[14]
RIP1 def. MEFs Minor (10–30% of wild-type) but
significant TNF-induced IKK

activation
[4]
RIP1 def. MEFs Moderately attenuated
TNF-stimulated degradation of
IjBa and only modestly reduced
induction (residual induction
70–80% of wild-type) of
NF-jB-regulated target genes in
primary and SV40 transformed
MEFs
[70]
H. Wajant and P. Scheurich TNFR1 induced NF-jB signaling
FEBS Journal 278 (2011) 862–876 ª 2011 The Authors Journal compilation ª 2011 FEBS 865
domain of one of the subunits of a p100 homodimer
can inhibit p65 ⁄ p50 NF-jB dimers which are typically
regulated by the classical pathway [26]. Together, in
this special case activation of the alternative NF-jB
pathway and p100 processing result in the release of a
‘classical’ NF- jB dimer in a fashion independent of
IKK complex activity and IjB degradation. Further-
more, TNF production itself is regulated by NF-jBs
and SMAC mimetics thus induce production of TNF
and TNF-mediated apoptosis in some cell types but
upregulation of TNF may also lead to enhanced
NF-jB activation [22,23].
Table 2. Effect of TNF receptor-associated (TRAF)2 and TRAF5 deficiency on tumor necrosis factor (TNF)-induced activation of the classical
NF-jB pathway. cIAP, cellular inhibitor of apoptosis protein; IKK, IjB kinase; IL, interleukin; MEF, murine embryonal fibroblast; NF-jB,
nuclear factor of kappa B. FLIP, FLICE-inhibitory protein.
Cell type
Effect on TNF-induced activation of the classical NF-jB pathway

Ref.IKK activity
IjBa
phosphorylation
IjBa
degradation
Nuclear
translocation and
phosphorylation of
p65
NF-jB target
Genes
TRAF2
) ⁄ )
MEFs
Attenuated, basal
activity
enhanced
Minor
inhibition
[71]
TRAF2
) ⁄ )
MEFs
Normal DNA
binding (EMSA)
[16]
TRAF2
) ⁄ )
MEFs
Delayed, basal

activity normal
Attenuated Strongly
reduced
A20 and IL-6 strongly
reduced
[29]
TRAF2
) ⁄ )
MEFs
Enhanced and
prolonged
Poorly
affected
Higher basal P-p65,
but poorly
inducible
[72]
TRAF2
) ⁄ )
MEFs
Unchanged Reduced Strongly reduced
p-Ser 536
NF-jB reporter gene
fully blocked
[73]
TRAF2
) ⁄ )
Bone marrow
macrophages
Normal DNA

binding (EMSA)
[74]
TRAF5
) ⁄ )
MEFs
Normal [16]
TRAF5
) ⁄ )
Bone marrow
macrophages
Normal DNA
binding (EMSA)
[75]
TRAF2
) ⁄ )
TRAF5
) ⁄ )
MEFs
Basally increased,
but TNF-induced
maximal effect
comparable with
wild-type
Strong, but
not complete
inhibition
FLIP and cIAP1
un-changed
But IL-6, IP10 and
ICAM-1 reduced

[71]
TRAF2
) ⁄ )
TRAF5
) ⁄ )
MEFs
Delayed and
attenuated,
basal activity
normal
Delayed and
weak
Reduced DNA
binding (EMSA)
[16]
TRAF2
) ⁄ )
TRAF5
) ⁄ )
MEFs
Residual IjBa
phosphorylation
[35]
TRAF2
) ⁄ )
TRAF5
) ⁄ )
MEFs
Basally increased,
but TNF-induced

maximal effect
comparable with
wild-type
Higher basal P-p65
but poor increase
by TNF
[76]
TRAF2
) ⁄ )
TRAF5
) ⁄ )
MEFs
Strongly reduced
P-Ser 536
[51]
TNFR1 induced NF-jB signaling H. Wajant and P. Scheurich
866 FEBS Journal 278 (2011) 862–876 ª 2011 The Authors Journal compilation ª 2011 FEBS
TNFR1-induced activation of classical
NF-jB signaling is associated with
recruitment of MAP3K and the IKK
complex to ubiquitinated components
of the TNFR1 core signaling complex
Based on co-immunoprecipitation experiments showing
TNF-induced recruitment of the IKK complex to
TNFR1 in wild-type and RIP1-deficient cells, but
defective recruitment in TRAF2-deficient cells, an ini-
tial simple model of TNFR1-induced NF-jB activation
was proposed [4]. According to this model, TRAF2 is
responsible for the recruitment of the IKK complex to
the TNFR1 core signaling complex where RIP1 acti-

vates the kinase subunits of the IKK complex. Early
on, it became evident that TNFR1, TRAF2 and
particularly RIP1 undergo ubiquitination in the
TNFR1 core signaling complex (Table 3). Ubiquitina-
tion of TNFR1 occurs after TNF-induced transloca-
tion into lipid rafts, but the functional consequences
and the E3 ligases involved are still obscure [27]. The
multiubiquitin chains found in TNFR1-associated
TRAF2 and RIP1 are not linked via K48, typically
marking the modified proteins for proteasomal degra-
dation, but are linked via K63 or linearly by head-
to-tail conjugation. These latter two ubiquitination
forms are now known to represent recognition sites for
ubiquitin binding proteins in the absence of degrada-
tion [28–30]. In fact, the IKK complex and the
IKK-activating TAK1–TAB 2–TAB 3 complex recruit
to the ubiquitinated TNFR1 core signaling complex by
means of their respective ubiquitin-binding subunits
NEMO and TAB 2. NEMO also becomes multiubiqui-
tinated in course of TNFR1-induced NF-jB activation
in a TRAF2- and RIP1-dependent manner (Table 3).
Ubiquitination of ubiquitin binding proteins ⁄ protein
complexes is not unusual and may serve to facilitate
formation of stable supramolecular protein complexes
of ubiquitinated proteins and ubiquitin binding pro-
teins. There is further evidence that TAK1 is activated
by K63 multiubiquitination and then phosphorylates
IKK1 and IKK2 in their activation loops to trigger
IjBa phosphorylation and proteasomal degradation of
IjBa [31]. An important role of TAK1, TAB 2 and

TAB 3 in TNFR1-induced activation of the classical
NF-jB is evident from analyses of knockout mice and
RNA interference experiments (Table 4). As observed
for other components of the TNFR1 signaling com-
plex, however, these molecules are not absolutely
obligate for TNF-induced NF-jB signaling. MEKK3
is another MAP3K family that has been implicated in
TNFR1-mediated IKK activation. MEKK3
) ⁄ )
MEFs
display strongly impaired TNF-induced NF-jB activa-
tion and biochemical studies further showed that this
kinase interacts with RIP1 and TRAF2 [32]. MEKK3
can phosphorylate IKKs, but also stimulates TAK1.
Whether TAK1 and MEKK3 act in a redundant man-
ner in TNFR1-induced activation of the IKK complex
or whether these two molecules cooperate in this
regard is currently unclear. There is further evidence
that MEKK2 is also involved in TNFR1-induced
IKK activation. Notably, MEKK2 and MEKK3 target
different NF-jB complexes after TNF stimulation [33].
TNF induces formation of an early complex composed
of MEKK3, IjBa and the IKK complex, but in addi-
tion also the independent and delayed formation of
a complex consisting of MEKK2, IjBb and again
the IKK complex [33]. Correspondingly, MEKK3
deficiency primarily reduces early NF-jB activation
by TNF, whereas MEKK2 downregulation by RNA
interference results in downregulation of delayed
NF-jB activity. Combined inhibition of MEKK2 and

MEKK3 in turn results in an almost complete inhibi-
tion of TNF-induced phosphorylation and degradation
of IjBa ⁄ b [33]. Nevertheless, there is no detailed
knowledge of the interplay of MEKK2, MEKK3
and TAK1 in TNFR1-induced NF-jB signaling and,
in particular, it is unclear whether ubiquitination of
MEKK3 or MEKK2 plays a role.
E3 ligases involved in TNFR1-induced
activation of the classical NF-jB
pathway
Several E3 ligases have been implicated in the regula-
tion of TNF-induced NF-jB signaling. A fraction of
Table 3. Ubiquitination of components of the NF-jB-stimulating
TNF receptor-associated factor 1 (TNFR1) signaling complex.
LUBAC, linear ubiquitin chain assembly complex; NEMO, NF-jB
essential modulator; NF-jB, nuclear factor of kappa B; RIP1, recep-
tor-interacting kinase 1; S1P, shingosine-1-phosphate; TRAF2, TNF
receptor-associated factor 2.
Modified
component
Modified
residue(s) E3 ligase
Ubiquitin
linkage type Ref
TRAF2 –
–
K-31
cIAP1
–
–

K63
K63
[77]
[30]
[29]
cIAP1 – – – [19,22]
cIAP2 – – – [19,22]
RIP1 K-377
–
–
–
cIAP1 and
cIAP2
TRAF2–S1P
–
K48, K63
K63
[38,39]
[19,21]
[36]
TAK1 K-158 – K63 [31]
NEMO K-285, K309 LUBAC Linear head
to tail
[37]
H. Wajant and P. Scheurich TNFR1 induced NF-jB signaling
FEBS Journal 278 (2011) 862–876 ª 2011 The Authors Journal compilation ª 2011 FEBS 867
these ligases modifies components of the TNFR1 sig-
naling complex by K48-linked multiubiquitin chains
and thus prompt their proteasomal degradation. These
E3 ligases are discussed in the paragraph dedicated to

the termination of TNF-induced NF-jB signaling (see
below). However, the RING finger domain-containing
molecules TRAF2, cIAP1 and cIAP2 themselves pos-
ses E3 ubiquitin ligase activity. Together with the lin-
ear ubiquitin chain assembly complex (LUBAC),
consisting of the heme-oxidized IRP1 ubiquitin ligase
(HOIL-1) and HOIL-1-interacting protein HOIP, there
are four E3 ligases capable of modifying components
of the TNFR1 signaling complex with ‘nondegrading’
regulatory multiubiquitin chains, either linearly linked
or via lysine residues distinct from K48 of ubiquitin,
especially K63. TRAF2 tightly interacts constitutively
by use of the N-terminal part of the TRAF domain
with the BIR domains of cIAP1 and cIAP2 [13].
As discussed above, upon TNF stimulation, TRAF2
and the associated cIAPs are recruited to TNFR1-
bound TRADD with the help of the C-terminal half
of the TRAF domain of TRAF2. Thus, the E3 ligases
TRAF2, cIAP1 and cIAP2 enter the TNFR1 signaling
complex essentially independent of the presence of
ubiquitin chains. By contrast, reconstitution experi-
ments of TRAF2 or cIAP knockout cells with the
Table 4. Effects of knockout ⁄ knockdown of components of the TNF receptor-associated factor 1 (TNFR1) signaling complex on TNFR1-
induced activation of the classical NF-jB pathway. cIAP, cellular inhibitor of apoptosis protein; HOIL-1, heme-oxidized IRP1 ubiquitin ligase;
IKK, IjB kinase; LUBAC, linear ubiquitin chain assembly complex; MEF, murine embryonal fibroblast; NEMO, NF-jB essential modulator;
NF-jB, nuclear factor of kappa B; PKC, protein kinase C; RIP1, receptor-interacting kinase 1; TRADD, TNFR1-associated death domain.
Target Effect of knockout ⁄ knockdown on TNF-induced NF-jB signaling Ref.
TRADD MEFs: strongly reduced phosphorylation and degradation of IjBa; no TNFR1-associated RIP1 polyubiquitination
in MEFs
Macrophages: only partial reduction of phosphorylation and degradation of IjBa

[10–12]
TRAFs See Table 2
RIP1 See Table 1
cIAPs cIAP1 KO MEFs: normal TNF-induced NF-jB signaling and constitutive enhanced cIAP2 expression
cIAP2 KO MEFs: normal TNF-induced NF-jB signaling
cIAP2 KO MEFs + cIAP1 siRNA: no IjBa degradation
cIAP1 siRNA in C2C12 cells: no IjBa degradation
cIAP1 or cIAP2 siRNA in MEFs or hepatocytes: normal IjBa degradation
cIAP1 and cIAP2 siRNA in MEFs or hepatocytes: no IjBa degradation
[17–20]
LUBAC HOIL-1 KO MEFs: Reduced phosphorylation and degradation of IjBa and reduced gene induction
HOIL-1 and ⁄ or HOIP siRNA in HeLa: Reduced phosphorylation and degradation of IjBa and reduced gene
induction; normal recruitment of TRADD, but reduced recruitment of TRAF2, RIP1, TAK1 and NEMO
[34,37]
TAB 2 MEFs: delayed TAK1 activation, but reduced NF-jB–DNA binding and attenuated phosphorylation and
degradation of IjBa
[66]
TAK1 MEFs: almost complete inhibition of phosphorylation and degradation of IjBa and reporter gene activity;
reduced p65 phosphorylation on S536
[31]
MEKKs MEKK2 KO MEFs: Delayed phase of NF-jB -DNA binding is blocked
MEKK3 KO MEFs: inhibited interaction of IjBa, but not IjBb containing NF-
jB complexes to the IKK complex;
NF-jB -DNA binding, phosphorylation and degradation of IjBa are delayed and almost completely reduced
MEKK2-MEKK3 DKO MEFs: complete inhibition of NF-jB -DNA binding, phosphorylation and degradation
of IjBa
[32,33]
PKCf Primary embryonal fibroblasts: Reduced reporter gene synthesis and DNA binding activity but grossly normal
IKK activation
Lung cells: Strongly reduced IKK activation and DNA binding activity

[48]
PKCe MEFs: TRAF2 phophorylation, IKK activation, interaction of TRAF2 with the IKK complex and expression of
NF-jB -regulated gene are reduced
MEFs + PKCd siRNA: phosphorylation, IKK activation, interaction of TRAF2 with the IKK complex and
expression of NF-jB -regulated gene almost completely blocked
[29]
A20 MEFs: Sustained phosphorylation and degradation of IjBa and enhanced production of NF-jB -regulated genes [78]
TAX1BP1 MEFs: Prolonged NF-jB -DNA binding, sustained phosphorylation and degradation of IjBa and enhanced
production of NF-jB -regulated genes
[57]
Itch MEFs: Prolonged NF-jB -DNA binding, sustained phosphorylation and degradation of IjBa and enhanced
production of NF-jB -regulated genes; enhanced K63 ubiquitination and reduced K63 ubiquitination of RIP1
[58]
RNF11 siRNA: Prolonged NF-jB -DNA binding, sustained phosphorylation and degradation of Ij Ba, enhanced
production of NF-jB-regulated genes and enhanced RIP1 ubiquitination
[59]
TNFR1 induced NF-jB signaling H. Wajant and P. Scheurich
868 FEBS Journal 278 (2011) 862–876 ª 2011 The Authors Journal compilation ª 2011 FEBS
corresponding E3 ligase activity-defective mutants
revealed that recruitment of LUBAC is indirect and
dependent on cIAP- but not TRAF2-mediated ubiqui-
tination events [34]. Indeed, the HOIP subunit of
LUBAC in particular interacts with K48-, K63- and
linearly linked ubiquitin chains [34]. The hypothesis
that cIAP1 ⁄ 2-mediated ubiquitination of one or more
components of the TNFR1 core signaling complex is
crucially involved in recruitment and activation of the
IKK complex is in good agreement with functional
and biochemical data. Cells with absent or downregu-
lated cIAP1 and -2 showed no RIP1 ubiquitination, no

recruitment of LUBAC and no significant IjBa degra-
dation [34].
TRAF2 itself is also a reasonable substrate for
ubiquitination in vitro and becomes modified with
K63-linked polyubiquitin chains within the TNFR1
signaling complex [29]. K63-ubiquitination of TRAF2
primarily occurs at lysine 31 and is dependent on
TNF-induced phosphorylation of TRAF2 on T117 by
protein kinase (PK)Cd and PKCe [29]. Reconstitution
experiments with TRAF2 knockout MEFs and
TRAF2 mutants that are defective in T117 phospory-
lation and ubiquitination of K31, together with analy-
sis of PKCe knockout MEFs with knockdown of
PKCd, revealed further evidence of a crucial role of
K63-ubiquitination of the RING domain of TRAF2
for recruitment of the IKK complex into the TNFR1-
signaling complex and activation of the NF-jB path-
way [29]. However, there is also strong evidence that
K63-ubiquitination of TRAF2, and therefore its RING
domain, is not involved in TNFR1-mediated NF-jB
activation. Together, reconstitution experiments in
MEFs derived from TRAF2 ⁄ 5 double-deficient mice
with TRAF2 mutants revealed that the capability of
this molecule to interact with cIAPs is necessary to
restore TNF-induced NF-jB activation, whereas its
RING ⁄ E3 ligase domain is dispensable. The function
of TRAF2 as a bona fide E3 ligase is also controver-
sial. Some studies making use of transient expression
experiments reported TRAF2 autoubiquitination that
was dependent on the RING domain of TRAF2 and

the dimeric Ubc13–Uev1A conjugating enzyme com-
plex [28,30]. By contrast, elucidation of the structure
of the RING domain and the first zinc finger of
TRAF2 revealed an unfavorable interface for interac-
tion with Ubc13 and Ubc-related E2 proteins, which in
this report, also correlated with a lack of TRAF2
autoubiquitination activity [35]. However, these dis-
crepancies may become resolved in view of a recent
study by Alvarez et al. [36] identifying sphingosine-1-
phosphate (S1P) as a cofactor for the E3 ligase activity
of TRAF2 using in vitro RIP1 ubiquitinations assays
with Ubc13–Uev1a as E2 component. Future studies
must now clarify whether binding of S1P to TRAF2
induces, for example, a structural change enabling
TRAF2–Ubc13 interaction. In conclusion, TRAF2
likely represents an authentic E3 ligase, but the rele-
vance of this activity for the role of TRAF2 in
TNFR1-induced NF-jB signaling remains unresolved.
Although recruitment of LUBAC is dependent on ini-
tial cIAP1 ⁄ 2-mediated ubiquitination of components
of the TNFR1 core signaling complex, there is evi-
dence that LUBAC increases overall ubiquitination,
thereby improving recruitment of the IKK complex.
In line with these arguments, there is reduced inter-
action of NEMO with the TNFR1 signaling complex
in cells with LUBAC knockdown [34]. Additional con-
sequences are reduced recruitment of TRAF2, RIP1
and TAK1, despite normal TNF–TNFR1 interaction.
Conversely, in cells with ectopic expression of
LUBAC, a prolonged and increased formation of the

TNFR1 core signaling complex was observed. Thus,
the role of LUBAC in TNF-induced NF-jB activation
seems not to be restricted to recruitment of NEMO
and the IKK complex, but may also involve stabiliza-
tion of the TNFR1 signaling complex as a whole. In
accordance with the proposed supporting and stabi-
lizing nature of the LUBAC, NF-jB activation was
significantly reduced but not fully absent in HOIL-1-
deficient MEFs and LUBAC knockdown cells [34–37].
Notably, ubiquitination of RIP1 in the TNFR1 signal-
ing complex seems to be independent of LUBAC
activity, emphasizing results from other studies which
have identified cIAP1 ⁄ 2 as the major E3 ligases of
RIP1 in TNFR1 signaling [19].
RIP1 ubiquitination and its relevance
for TNFR1-induced NF-jB activation
RIP1 is the most strongly ubiquitinated component of
the TNFR1 signaling complex. RIP1 can be modified
with K63-linked multiubiquitin chains, mediating the
recruitment of various ubiquitin-binding proteins
involved in TNF signaling, but also with K48-linked
ubiquitin chains that prompt proteasomal degradation.
TNF-induced K63 ubiquitination of RIP1 occurs pref-
erentially at lysine 377 [38–40] and is dependent on
TRAF2 and cIAPs, whereby the latter seems to be the
essential E3 ligases. Reconstitution experiments in
RIP1-deficient cells with a RIP1 mutant carrying a
defective K63 ubiquitination acceptor site (RIP1–
K377R), suggest that TNFR1-associated ubiquitinated
RIP1 serves as a recruitment platform for the binding

of a complex containing the ubiquitin-binding proteins
TAB 2 and TAB 3, and the TAB 2 ⁄ 3-interacting
H. Wajant and P. Scheurich TNFR1 induced NF-jB signaling
FEBS Journal 278 (2011) 862–876 ª 2011 The Authors Journal compilation ª 2011 FEBS 869
MAP3 kinase, TAK1 [14,38]. Remarkbly, the affinity
of TAB 2 for K63-linked ubiquitin chains is much
higher than for linear ubiquitin chains [41]. TNF-
induced recruitment of the TAB 2 ⁄ 3–TAK1 complex is
followed by K63-linked polyubiquitination of TAK1
at K158 [31]. The latter has been be achieved with
purified TRAF2 in in vitro assays and reconstitution
experiments with TAK1
) ⁄ )
MEFs and a TAK1–
K158R mutant, further arguing for a crucial role of
this event in TNFR1-induced activation of the classical
NF-jB pathway [31]. Ubiquitinated TNFR1-bound
RIP1 can also interact with the IKK complex [38,40].
Whereas TRAF2 binding to the IKK complex relies
on interaction with the leucin zipper motif of IKK1 or
IKK2, ubiquitinated RIP1 interacts with the NEMO
subunit of the IKK complex [38,40,42]. The relative
contribution of these two interactions to recruitment
of the IKK complex within the TNFR1 signaling core
complex is currently unclear. One study reported an
only slight reduction in TNF-induced recruitment of
IKK1 and IKK2 to TNFR1 in NEMO-deficient MEFs
[42], whereas others found no recruitment of these kin-
ases in NEMO-deficient MEFs [40] or NEMO-deficient
Jurkat cells [38]. In addition, the unexpected observa-

tion has been reported that NEMO interacts better
with LUBAC-generated head-to-tail linked linear
ubiquitin chains than with K63-multiubiquitin chains
[34,37]. It is tempting to speculate that interaction of
nonubiquitinated NEMO with K63-polyubiquitinated
RIP1 initially stabilizes the interaction of the IKK
complex with TRAF2, whereas after LUBAC-cata-
lyzed modification of NEMO with linearly linked
ubiquitin chains, NEMO and the IKK complex are
stabilized in the TNFR1 signaling complex via interac-
tion with the ubiquitin-binding domains of LUBAC.
Despite the convincing biochemical evidence for an
important role of K63 ubiquitination of RIP1, TAK1
and NEMO in TNF-induced NF-jB activation, this
concept is challenged by recent findings. First, TNF-
induced IjBa degradation and p65 nuclear transloca-
tion have been reported to be unchanged in MEFs
deficient for Ubc13 [43], a part of the Ubc13–Uev1A
heteromeric E2 complex catalyzing the K63 ubiquitina-
tion of RIP1 on K377 [38]. Second, inducible replace-
ment of endogenous ubiquitin with K63R mutants in
U2SO cells showed no effect on TNF-induced IKK
activation and IjBa degradation, but abrogated inter-
leukin (IL)-1-induced NF-jB signaling, underscoring
the feasibility of this approach [44]. However, the lat-
ter study is based on a knockdown strategy of endoge-
nous ubiquitin and concomitant expression of the
K63R ubiquitin mutant. Because inhibition of endoge-
nous ubiquitin expression by knockdown approaches is
unavoidably incomplete, it can not entirely be ruled

out that residual expression of minute amounts of
endogenous ubiquitin remain present, being sufficient
to allow K63-linked ubiquitination of RIP1 in the
TNFR1 signaling complex. In fact, the strongly
reduced expression of endogenous ubiquitin shown by
Xu et al. [44] is demonstrated using total cell lysates,
but RIP1 ubiquitination is at best detectable in immu-
noprecipitates of TNFR1. Furthermore, Ubc13 could
be substituted in its E2 ligase activity of RIP1 by
UbcH5. Thus, the controversial data regarding the role
of RIP1 in general, and RIP1 ubiquitination in partic-
ular, in TNR1-mediated NF-jB activation is likely to
be related to the use of different cell types, insufficient
sensitivity of functional analyses and underestimated
experimental pitfalls. In any case, additional studies
are required to resolve the contradictions arising from
the available literature.
Regulation of TNF-induced NF-jB
activity by modification of NF-jB
subunits
IKK-induced IjBa degradation and translocation of
the NF-jB subunits into the nucleus is not sufficient
to ensure full transcriptional activity. In addition, the
latter requires post-translational modifications of the
NF-jB subunits themselfes to achieve high DNA-bind-
ing capacity and strong transcriptional activity [1,2].
The regulatory mechanisms directly targeting the
NF-jB subunits are typically of relevance for various
NF-jB inducers and not specific for TNFR1 signaling.
We therefore address only briefly some aspects

involved in the regulation of the activity of p65-con-
taining NF-jB dimers, representing the major NF-jB
targets of TNFR1-induced signaling. Phosphorylation
of p65 on serine residues 276, 311, 529, 536 and 576
has been implicated in the regulation of TNF-induced
NF-jB signaling. The catalytic subunit of PKA
(PKAc) was the first kinase identified as a regulator of
p65 activity by serine 276 phosphorylation [45]. PKAc
is associated with NF-jB–IjBa ⁄ b complexes and
concomitantly released upon degradation of IjBa ⁄ b.
Serine 276 phosphorylation can also be stimulated by
TNF-induced activation of MSK1 (mitogen- and
stress-activated kinase-1) via the p38 and ERK path-
ways [46]. MEFs derived from MSK1–MSK2 DKO
mice showed normal DNA-binding of p65 in response
to TNF, but impaired transcription of a subset of
NF-jB-regulated genes [46]. Phosphorylation of ser-
ine 276 enables recruitment of the cAMP-responsive
element-binding protein (CREB)-binding protein (CBP)
and p300. These transcriptional coactivators interact
TNFR1 induced NF-jB signaling H. Wajant and P. Scheurich
870 FEBS Journal 278 (2011) 862–876 ª 2011 The Authors Journal compilation ª 2011 FEBS
with histone acetyltransfereases and mediate acetyl-
ation of p65 leading to enhanced transcriptional
activity [1].
TNF-induced phosphorylation of serine 311 of p65
has been assigned to the activation of PKCf [47]. Simi-
lar to serine 276, this phosphorylation is not required
for IKK activation, IjBa degradation and DNA-bind-
ing of p65 in embryonal fibroblasts, but has been

rather implicated in CREB-binding protein recruitment
and transactivation [47,48]. Noteworthy, in the lung of
PKCf-knockout mice there was in addition a defect in
TNF-stimulated IKK activation and IjBa degradation
[48]. Phosphorylation of serine 529 of p65 is mediated
by casein kinase II, but is prevented in nonstimulated
cells by the interaction with IjBa [49]. Thus, similar to
PKAc-mediated phosphorylation of serine 276 of p65,
phosphorylation on serine 529 by casein kinase II
occurs in the cytoplasm after TNF-induced degra-
dation of IjBa. Again, p65 phosphorylation on
serine 529 increases the transcriptional activity of
p65-containing NF-jB dimers, but is not required for
their nuclear translocation. p65 can also be phosphory-
lated by IKKs in response to TNF on serine 536 and
on S468 by IKK2 [50–52]. TNF-induced IKK-medi-
ated phosphorylation of p65 is strongly reduced in the
absence of TRAF2 ⁄ 5 or TAK1 [51], suggesting that
p65 is an additional substrate for the IKK complex.
Noteworthy, in detail there are differences in the mech-
anisms of IKK-mediated phosphorylation of p65 and
IjBa. First, the protein Rap1 has been identified
recently as a cofactor improving association of the
IKK complex with p65 and was shown to facilitate
phosphorylation of the latter, but appeared irrelevant
for IjBa phosphorylation [53]. Second, IKK1 seems
dispensable for TNF-induced phosphorylation and
degradation of IjBa in MEFs, whereas both I jB kin-
ases were required for p65 transactivation. Further-
more, IKK1, but not IKK2 or NEMO, corecruit with

p65 and CREB-binding protein to promoters of
NF-jB-regulated genes in TNF stimulated cells [54,55].
Besides acting as a p65 kinase, promoter-bound IKK1
also phosphorylates histone H3 on serine 10 to trigger
its subsequent acetylation on lysine 14 [54,55]. Chro-
matin-bound IKK1 further phosphorylates silencing
mediator of retinoid and thyoid hormone action,
inducing the redistribution of histone deactylase-3-con-
taining silencing mediator of retinoid and thyoid hor-
mone action repressor complexes into the cytosol [56].
Whereas phosphorylation of S536 contributes to TNF-
induced NF-jB activation via the aforementioned
mechanisms, phosphorylation of S468 rather elicits
attenuating effects [52].
Termination of TNFR1-induced NF-jB
activation
The mechanisms by which TNFR1-mediated activation
of the classical NF-jB pathway is shut down are less
well understood than the initiating events, but it is evi-
dent that a variety of mechanisms contribute to this
task. There exist general mechanisms targeting steps in
the pathway downstream of IKK activation which will
not be addressed here, but there are also upstream act-
ing mechanisms regulating the activity of the NF-jB-
stimulating TNFR1 signaling complex.
Multiubiquitination of RIP1 on K63 in the course
of TNFR1 signaling can be antagonized by A20,
a protein containing two ubiquitin-editing domains
with different specificities. An N-terminal de-ubiquiti-
nation domain is capable removing K63-linked u biquitin

chains from RIP1, whereas a C-terminal ubiquitin ligase
domain polyubiquitinates RIP1 with K48-linked ubiqu-
itin chains to trigger its proteasomal degradation. There
is increasing evidence that A20 acts as part of a multi-
protein complex in RIP1 deubiquitination that is formed
15–30 min post TNFR1 stimulation. In addition to A20
and its substrate RIP1 (or TRAF6 in LPS signaling),
this ubiquitin-editing A20 complex al so i ncludes
Tax1-binding protein (TAX1BP1), RING finger protein
11 (RNF11) and Itch [57–59]. Accordingly,
TNF-induced interaction of RIP1 with A20 is inhibited
in MEFs deficient for TAX1BP1 or Itch and in RNF11
knockdown cells [57–59]. Moreover, TNF-induced
NF-jB ac tivity and multiubiquitination of RIP1,
detected in RIP1 immunoprecipitates after 15–30 min
(TAX1BP1, Itch11, RNF11) or in TNFR1 immunopre-
cipitates after 5–25 min (A20), were enhanced in MEFs
deficient for A20, TAX1BP1 or Itch and in RNF11
knockdown cells [57–60]. In accordance with the initial
idea that the ubiquitin-editing A20 complex removes
K63-linked ubiquitin from RIP1 to subsequently mark
it by K48 ubiquitination for proteasomal degradation,
RIP1 is predominantly K48 ubiquitin linked in RIP1
immunoprecipitates of TNF-stimulated wild-type
MEFs, but K63 multiubiquitinated in Itch-deficient
MEFs [58]. Moreover, upon inhibition of protein syn-
thesis, TNF triggers degradation of RIP1 in wild-type,
but not Itch- and TAX1BP1-deficient MEFs [58]. The
gene encoding A20 is regulated by NF-jB and thus
not or only poorly expressed in most cells, but readily

inducible by TNFR1. It is therefore tempting to specu-
late that A20 is particularly important for desensitiza-
tion of cellular NF-jB responsiveness towards
persistent TNF stimulation. In fact, there is experimen-
tal evidence that inducible, but also constitutively
H. Wajant and P. Scheurich TNFR1 induced NF-jB signaling
FEBS Journal 278 (2011) 862–876 ª 2011 The Authors Journal compilation ª 2011 FEBS 871
expressed, A20 has only minor effects on early TNF-
induced activation of NF-jB (< 0.5 h), whereas the
later phase (> 1 h) of NF-jB activity is significantly
attenuated [61]. Furthermore, MEFs derived from
A20-deficient mice show prolonged and enhanced TNF
signaling, which is in good accordance with the deadly
chronic inflammation observed in A20-deficient mice.
A20 can also interact with ABIN proteins, a class of
NF-jB-inhibitory proteins containing a special type of
ubiquitin-binding domain also found in NEMO. The
relation ⁄ relevance of A20 interaction with ABIN
proteins regarding the inhibitory effect of the ubiquin
editing complex of A20, Itch, RNF11 and TAX1BP
on TNF-induced NF-jB activation, however, is
unclear. Analysis of ABIN1-deficient MEFs revealed
an, at best, moderate inhibitory effect on TNF-induced
NF-jB activation [62], but it can not be ruled out
that other A20-interacting ABIN proteins, like ABIN-2
or -3, compensate for ABIN-1 deficiency. The
A20-containing complex is presumably not the only
regulatory factor targeting quality and quantity of
RIP1 modification in the context of TNFR1 signaling.
There is evidence that deubiquitination by USP21

and stimulation of proteasomal degradation by the
TRIAD3a E3 ligase and the endosome associated E3
ligase CARP-2 limit the amplitude of TNF-induced
NF-jB signaling [63–65]. Together, K63-polyubiquin-
ation not only paves the road for the recruitment of
stimulating factors to the TNF1 signaling complex,
but also allows recruitment of inhibitory proteins.
Thus, TAB 2-deficient MEFs show the expected
reduced TNF-induced activation of IKKs, but also
revealed prolonged activation of TAK1 which could be
traced back to inhibition of recruitment of the TAK1
inactivating serine ⁄ threonine protein phosphatase-6
[66]. Furthermore, the NEMO homolog optineurin,
interacting with K63-polyubiquitinated RIP1, also neg-
atively regulates TNFR1-induced NF-jB signaling by
competition with NEMO for RIP1 binding [67].
Conclusions and perspectives
The genetic and biochemical findings highlighted in
this review definitely teach us that there is not simply
a linear chain of events connecting TNFR1 with stim-
ulation of the IKK complex and activation of the clas-
sical NF-jB pathway. Instead, we gain more and
more evidence that recruitment, modification and acti-
vation and inactivation of the E3 ligases and kinases
involved in this process are reciprocally and intimately
linked. As a consequence, it is difficult to experimen-
tally separate an individual aspect of TNFR1-induced
NF-jB signaling even when knockout (knockdown)
cells are used which are reconstituted with point muta-
tions of the deleted protein lacking only one or a sub-

set of its properties. A comprehensive understanding
of the molecular mechanisms involved in TNFR1-
induced activation of the IKK complex and the classi-
cal NF-jB pathway might thus become possible only
with the help of mathematical modeling. The develop-
ment of a detailed and quantitative model of TNFR1-
induced NF-jB activation, however, is complicated by
the high degree of functional redundancy of the signal-
ing molecules involved. Thus, the lack of a particular
signaling protein (including its replacement by a func-
tional defective mutant) often affects a biochemically
defined step in TNFR1 signaling, but not in an all or
nothing way. Moreover, TNFR1-induced activation of
the classical NF-jB pathway, in general, and activa-
tion and inactivation of the IKK complex, in particu-
lar, is highly dynamic. Therefore, the time point
considered, as well as the expression levels of the
involved signaling proteins, can also have a tremen-
dous impact on the quantitative read out of a particu-
lar signaling step. As these parameters may vary from
group to group, between cell types and may differ
slightly from experiment to experiment, it is not really
possible to join data reported from different groups
into one coherent model. To reach the goal of a com-
prehensive model with predictive power of TNFR1-
induced NF-jB signaling, it is necessary to obtain
more accurate and time-resolved data of the biochemi-
cally definable steps involved. In particular, there is a
need for quantitative information regarding the cell
type-specifc expression levels of the most relevant sig-

naling molecules that take part in TNFR1-induced
NF-jB signaling.
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