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
Trang 1TNFR1-induced activation of the classical NF-jB pathway Harald Wajant1and Peter Scheurich2
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: harald.wajant@mail.uni-wuerzburg.de
(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.
Trang 2(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.
Trang 3mushroom 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 poly-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, markbind-ing it for K48-ubiquitination and proteasomal degradation.
Trang 4mutant 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
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]
Trang 5domain 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
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
p-Ser 536
NF-jB reporter gene fully blocked
[73] TRAF2) ⁄ )
Bone marrow
macrophages
Normal DNA binding (EMSA)
[74]
TRAF5) ⁄ )
MEFs
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]
Trang 6TNFR1-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 functransloca-tional 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 formaaddi-tion 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 phosphorylainhibi-tion and degradainhibi-tion
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 fracregula-tion 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
–
K-31
cIAP1 –
– K63 K63
[77]
[30]
[29]
–
–
– cIAP1 and cIAP2 TRAF2–S1P
– K48, K63 K63
[38,39]
[19,21]
[36]
to tail
[37]
Trang 7these 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.
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]
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 IjBa, enhanced
production of NF-jB-regulated genes and enhanced RIP1 ubiquitination
[59]
Trang 8corresponding 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
Trang 9MAP3 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
Trang 10with 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 IjB
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 regulatact-ing 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 ubiquitin 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 also includes 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 activity 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