Laura A. Solt‡, Lisa A. Madge‡, and Michael J. May‡§1
From the‡Department of Animal Biology and the§Mari Lowe Center for Comparative Oncology, University of Pennsylvania
School of Veterinary Medicine, Philadelphia, Pennsylvania 19104
Proinflammatory NF-?B activation requires the I?B (inhibi-
tor of NF-?B) kinase (IKK) complex that contains two catalytic
subunits named IKK? and IKK? and a regulatory subunit
named NF-?B essential modulator (NEMO). NEMO and IKK?
are essential for tumor necrosis factor (TNF)-induced NF-?B
however, the role of NEMO association with each kinase in
NF-?B signaling and IKK complex formation remains unclear.
To address this question, we stably reconstituted IKK??/?and
IKK??/?murine embryonic fibroblasts (MEFs) with wild-type
(WT) or NBD-deficient (?NBD) versions of IKK? and IKK?,
respectively. TNF-induced classical NF-?B activation in
IKK??/?MEFs was rescued by IKK?WTbut not IKK??NBD,
whereas neither IKK?WTnor IKK??NBDaffected IL-1-induced
scriptional activity was absent in IKK??/?cells. Reconstitution
with either IKK?WTor IKK??NBDrescued both IL-1 and TNF-
ent transcription. Stably expressed IKK?WTor IKK?WTassoci-
IKK?-IKK?-NEMO complex. In contrast, although the
IKK??NBDand IKK??NBDmutants associated with endoge-
nous IKKs containing an NBD, these dimeric endogenous
IKK-IKK?NBDcomplexes did not associate with NEMO.
These findings therefore demonstrate that formation of the
heterotrimeric IKK?-IKK?-NEMO holocomplex absolutely
requires two intact NEMO-binding domains.
NF-?B2describes a family of transcription factors that regu-
late the inducible expression of many genes essential for innate
range of stimuli activates NF-?B, including ligation of innate
immune receptors (e.g. TLRs), antigen receptor engagement (B
cell receptor and T cell receptor), and proinflammatory cyto-
kines (e.g. IL-1 and TNF) (1). NF-?B activation by these stimuli
is normally rapid and transient; however, constitutive NF-?B
activity occurs in some chronic inflammatory diseases, solid
tumors, leukemias, and lymphomas (1, 2). Understanding the
molecular mechanisms that regulate NF-?B activity will there-
The five NF-?B family members are NF-?B1/p105 and
NF-?B2/p100 that are processed to generate p50 and p52,
respectively, p65 (RelA), c-Rel, and RelB (1). These proteins
homo- or heterodimerize to form transcriptionally active (e.g.
the cytosol of resting cells by members of the inhibitory family
of I?B proteins. The I?Bs include I?B?, I?B?, I?B?, and the C
termini of p105 and p100. The prototypic NF-?B-I?B complex
expressed in most cell types is a heterodimer of p50 and p65
associated with I?B? (1). Following cell stimulation, the I?Bs
are rapidly phosphorylated, ubiquitinated, and then degraded
nucleus, where they bind target gene promoters and regulate
The I?B proteins are phosphorylated by the high molecu-
lar weight heterotrimeric I?B (inhibitor of NF-?B) kinase
named IKK? (IKK1) and IKK? (IKK2) and a noncatalytic sub-
unit named NEMO (NF-?B essential modulator) or IKK? (1, 6,
7). NEMO is critical for proinflammatory IKK activation
(8–12), and we previously identified a domain within the C
with NEMO (13, 14). A cell-permeable peptide spanning this
NEMO-binding domain (NBD) disrupts the IKK complex and
blocks proinflammatory NF-?B activation, confirming the cru-
cial role of NEMO association for IKK complex activation (13).
Despite their significant structural similarities, genetic anal-
yses of IKK? and IKK? revealed distinct roles for the kinases
during NF-?B activation (1, 6, 7, 15). In this regard, TNF-in-
duced I?B? degradation is dependent upon IKK? and also
sical NF-?B pathway and is defined as NEMO- and IKK?-de-
pendent I?B phosphorylation and degradation releasing
?B-inducing kinase)-dependent processing of NF-?B2/p100 to
generate p52 (18–22). This mechanism is activated in the
Grants 1RO1-HL-080612 and T32-AI-055428-06. This work was also sup-
ported by W. W. Smith Charitable Trust Grant H0703.
1To whom correspondence should be addressed: Dept. of Animal Biology,
University of Pennsylvania School of Veterinary Medicine, 3800 Spruce St.
2The abbreviations used are: NF-?B, nuclear factor-?B; IKK, I?B kinase; MEF,
murine embryonic fibroblast; NBD, NEMO-binding domain; IL, interleukin;
sorting; EMSA, electrophoretic mobility shift assay.
THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 284, NO. 40, pp. 27596–27608, October 2, 2009
© 2009 by The American Society for Biochemistry and Molecular Biology, Inc.Printed in the U.S.A.
27596 JOURNAL OF BIOLOGICAL CHEMISTRYVOLUME 284•NUMBER 40•OCTOBER 2, 2009
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cal NF-?B pathway (15). Ligation of only a subset of receptors,
including the lymphotoxin-? receptor, CD40, and BAFF-R,
activates the IKK?-dependent noncanonical pathway, and the
resulting p52, together with RelB, regulates a panel of chemo-
kine and cytokine genes required for lymphoid organogenesis
and B-cell maturation (18–23). IKK? has also been shown to
play several roles in regulating the transcriptional activity of
classical NF-?B that are separate from its upstream signaling
function as an I?B kinase (24–29).
Since TNF-induced I?B? degradation and classical NF-?B
classical pathway activation has emerged in which IKK? is
redundant (15). It remains, however, that IKK? associates via
its NBD with NEMO (14), and we previously questioned
whether this association plays a functional role in classical
NF-?B signaling (30). Surprisingly, we found that although
TNF-induced I?B? degradation requires NEMO and IKK?,
IL-1-induced classical pathway activation is intact in cells lack-
ing IKK?. Furthermore, IL-1-induced NF-?B activation in
IKK?-deficient cells was blocked by the NBD peptide, demon-
IL-1- but not TNF-induced classical NF-?B activation (30).
Intriguingly, Lam et al. (31) recently demonstrated that IKK?
plays a crucial compensatory role in regulating constitutive
classical NF-?B pathway activation in a subset of diffuse large
B-cell lymphoma cells in which IKK? has been pharmacologi-
cally inhibited. These findings therefore identify differences in
the absolute requirements for the separate IKK subunits acti-
vated in a NEMO-dependent manner by distinct stimuli and
suggest that targeting only IKK? may not effectively block dys-
regulated classical NF-?B activation. Consequently, determin-
ing the role of the interaction of each IKK subunit with NEMO
will provide novel insight into the mechanisms that regulate
NEMO-dependent classical NF-?B activation.
To address this question, we determined the effects of indi-
signaling and IKK complex formation. We stably reconstituted
IKK??/?and IKK??/?murine embryonic fibroblasts (MEFs)
with wild-type (WT) or NBD-deficient (?NBD) versions of
IKK? and IKK?, respectively. Reconstitution of IKK??/?
MEFs with IKK?WTbut not IKK??NBDrescued TNF-induced
classical NF-?B activation, confirming the requirement for the
of either IKK?WTor IKK??NBD. Classical NF-?B transcrip-
tional activity was absent in IKK??/?cells, and reconstitution
induced transcription. This therefore demonstrates that asso-
ciation with NEMO is not necessary for IKK?-dependent
noprecipitation analysis and size exclusion chromatography
revealed that stably expressed IKK?WTor IKK?WTassociated
with endogenous IKKs and NEMO in IKK??/?or IKK??/?
MEFs, respectively. This association resulted in the formation
of the heterotrimeric IKK?-IKK?-NEMO complex. In con-
trast, despite the ability of the IKK??NBDand IKK??NBD
mutants to associate with endogenous IKKs containing an
NBD, these mutants formed dimeric endogenous IKK-
IKK?NBDcomplexes that did not associate with NEMO. These
meric IKK?-IKK?-NEMO holocomplex absolutely requires
the presence of two intact NEMO-binding domains.
Reagents and Cell Culture—Recombinant human IL-1? was
obtained from Peprotech (Rocky Hill, NJ). Recombinant
human TNF and recombinant mouse LT?1?2 were from R&D
Systems (Minneapolis, MN). Polyclonal rabbit anti-IKK? (sc-
7218), rabbit anti-NEMO (sc-8330), goat anti-NEMO (sc-
8256), rabbit anti-p100/p52 (sc-298), and rabbit anti-IKK?
(sc-8014) antisera were from Santa Cruz Biotechnology, Inc.
(Santa Cruz, CA). Monoclonal anti-?-tubulin (T5168) was
anti-phospho-IKK?/? (Ser176/180) (catalog number 2694), and
anti-histone 3 (catalog number 9715) were from Cell Signaling
mal goat IgG (sc-2028), and donkey anti-mouse IgG (sc-2518),
used as nonspecific antibodies in immunoprecipitations, were
from Santa Cruz Biotechnology. Immobilized Protein A/G
beads were from Pierce, and Protein G-Sepharose beads were
from Amersham Biosciences. Horseradish peroxidase-conju-
gated secondary antibodies against either rabbit or mouse IgG,
AffiniPure goat anti-mouse IgG light chain-specific, and IgG
secondary antibodies were from Jackson ImmunoResearch
Laboratories (West Grove, PA).
WT, IKK??/?, and IKK??/?MEFs were generously pro-
vided by Dr. Inder Verma (Salk Institute for Biological Studies,
La Jolla, CA). Plat-E cells were kindly provided by Dr. Tadaichi
Kitamura (Institute of Medical Science, University of Tokyo,
Japan), and Phoenix cells were provided by Dr. Garry Nolan
(Stanford University, Stanford, CA). All cells were maintained
in Dulbecco’s modified Eagle’s medium (Invitrogen) supple-
mented with 10% fetal calf serum, 2 mM L-glutamine, penicillin
(50 units/ml), and streptomycin (50 ?g/ml). For all experi-
ments, unless otherwise indicated, cells were cultured in either
6-well tissue culture trays or 100-mm dishes and were stimu-
lated with IL-1? (10 ng/ml) or TNF (10 ng/ml) when they
reached 80% confluence.
performed by PCR using cloned Pfu DNA polymerase (Strat-
agene, La Jolla, CA). Complementary DNA encoding full-
length IKK? or IKK? 1–733 (IKK??NBD) were subcloned into
the HindIII and NotI sites of the LZRS-pBMN-lacZ retroviral
vector (kindly provided by Dr. Garry Nolan). Resulting LZRS-
IKK?WTand LZRS-IKK??NBDwere transiently transfected
using Fugene6 into Phoenix cells and selected for gene expres-
sion 24 h after transfection using puromycin (1 ?g/ml). Puro-
mycin-resistant cells were used to derive conditioned medium
to provide a retroviral stock for MEF transduction. For cell
transduction, IKK??/?MEFs were washed and incubated for
8 h with retrovirus-conditioned medium containing Polybrene
replaced with normal growth medium. The transduction proc-
ess was repeated a further three times until cells became posi-
tive for IKK? when visualized by immunoblotting.
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J. Biol. Chem.
Laura A. Solt, Lisa A. Madge and Michael J.
Assembly and Classical NF-
B Kinase Complex
NEMO-binding Domains of Both IKK
Mechanisms of Signal Transduction:
doi: 10.1074/jbc.M109.047563 originally published online August 7, 2009
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