1. Molecular Cell, Vol. 12, 1287–1300, November, 2003, Copyright 2003 by Cell Press
Mechanisms of Proinflammatory Cytokine-
Induced Biphasic NF-B Activation
Baltimore, 1989). Stimulation of these cells by many
distinct stimuli, including proinflammatory cytokines
such as tumor necrosis factor (TNF)-␣ and interleukin
Christian Schmidt,1,2
Bailu Peng,2
Zhongkui Li,2
Guido M. Sclabas,2
Shuichi Fujioka,2
Jiangong Niu,4
Marc Schmidt-Supprian,5
Douglas B. Evans,2
James L. Abbruzzese,3
and Paul J. Chiao1,2,4,
* (IL)-1, leads to phosphorylation of IB␣, which triggers
rapid degradation of the inhibitors (Ghosh and Karin,1
Department of Molecular and Cellular Oncology
2
Department of Surgical Oncology 2002). Consequently, NF-B proteins are translocated
into the nucleus, where they activate transcription of3
Department of Gastrointestinal Medical Oncology
The University of Texas M.D. Anderson Cancer Center target genes (Karin et al., 2002; Li and Verma, 2002).
One of the key target genes regulated by NF-B is its1515 Holcombe Boulevard
Houston, Texas 77030 inhibitor IB␣. A feedback inhibition pathway for control
of IB␣ gene transcription and downregulation of tran-4
Program in Cancer Biology
The University of Texas Graduate School sient activation of NF-B activity has been described
(Cheng et al., 1994; Chiao et al., 1994; Sun et al., 1993).of Biomedical Sciences at Houston
Houston, Texas 77030 NF-B activation induced by proinflammatory cyto-
kines is biphasic, consisting of a transient phase medi-5
Center for Blood Research
Harvard Medical School ated through IB␣ followed by a persistent phase medi-
ated through IB (Thompson et al., 1995). Recent200 Longwood Avenue
Boston, Massachusetts 02115 studies have shown that biphasic NF-B activation is
induced in response to lipopolysaccharide (LPS) stimu-
lation in an animal model and in various cell types and
is important for the host defense, underlying the pro-Summary
and antiinflammatory function of nitric oxide (Connelly
et al., 2001; Han et al., 2002). Hoffmann and colleaguesThe transcription factor NF-B regulates genes in-
volved in innate and adaptive immune response, in- have shown that IB-NF-B signaling has bimodal char-
acteristics: IB␣ mediates rapid NF-B activation andflammation, apoptosis, and oncogenesis. Proinflam-
matory cytokines induce the activation of NF-B in strong negative feedback regulation, resulting in an os-
cillatory NF-B activation profile, whereas IB and IB⑀both transient and persistent phases. We investigated
the mechanism for this biphasic NF-B activation. Our respond more slowly to IB kinase (IKK) activation and
act to dampen the long-term oscillations of the NF-Bresults show that MEKK3 is essential in the regulation
of rapid activation of NF-B, whereas MEKK2 is impor- response (Hoffmann et al., 2002). However, the mecha-
nisms have not been elucidated.tant in controlling the delayed activation of NF-B in
response to stimulation with the cytokines TNF-␣ and NF-B is activated through complex signaling cas-
cades that are integrated by activation of IKK (Li andIL-1␣. MEKK3 is involved in the formation of the
IB␣:NF-B/IKK complex, whereas MEKK2 partici- Verma, 2002), which phosphorylates IB bound to NF-B
complexes as its substrates (Zandi et al., 1998). Thepates in assembling the IB:NF-B/IKK complex;
these two distinct complexes regulate the proinflam- signaling cascades that regulate IKK activity may be
also cell type dependent (Li and Verma, 2002). Onematory cytokine-induced biphasic NF-B activation.
Thus, our study reveals a novel mechanism in which genetic study revealed that PKC is not required for
IKK activation in embryonic fibroblasts but is requireddifferent MAP3K and IB isoforms are involved in spe-
cific complex formation with IKK and NF-B for regu- for IKK activation in lungs (Leitges et al., 2001). Another
report showed that MEKK3 is necessary in TNF-␣-inducedlating the biphasic NF-B activation. These findings
provide further insight into the regulation of cytokine- NF-B activation in fibroblasts (Yang et al., 2001). How-
ever, the underlying molecular mechanism for the bipha-induced specific and temporal gene expression.
sic NF-B activation was unknown.
In the present study, we sought to elucidate the mech-Introduction
anism by which proinflammatory cytokine-induced bi-
phasic NF-B activation is regulated. Our results showNF-B is a family of pleiotropic transcription factors that
orchestrate the expression of a plethora of genes that that MEKK3 regulates rapid activation of NF-B, whereas
MEKK2 controls a delay in activation of NF-B in re-play key roles in growth, oncogenesis, differentiation,
apoptosis, tumorigenesis, and immune and inflamma- sponse to proinflammatory cytokine stimulation.
tory responses (Karin et al., 2002; Li and Verma, 2002).
In most cell types, NF-B proteins are sequestered in the Results
cytoplasm in an inactive form through their noncovalent
association with the inhibitor IB (Sen and Baltimore, TNF-␣-Induced NF-B Activation Is Delayed
1986). This association masks the nuclear localization in MEKK3Ϫ/Ϫ
and IB␣Ϫ/Ϫ
Cells
signal of NF-B, thereby preventing NF-B nuclear To determine the time course for induction of NF-B
translocation and DNA binding activity (Baeuerle and activation, we stimulated wild-type, MEKK3Ϫ/Ϫ
, and
IB␣Ϫ/Ϫ
cells with TNF-␣ and isolated nuclear extracts
for measuring the NF-B DNA binding activity (Figure*Correspondence: pjchiao@mdanderson.org
2. Molecular Cell
1288
Figure 1. MEKK3 Is Required for Rapid Activation of NF-B and Phosphorylation of IB␣ in Response to TNF-␣
(A) Delayed activation of NF-B in MEKK3Ϫ/Ϫ
and IB␣Ϫ/Ϫ
cells. Electrophoretic mobility shift assay (EMSA) for analysis of B DNA binding
activity. Wild-type (WT) and MEKK3Ϫ/Ϫ
MEFs and IB␣Ϫ/Ϫ
MFCs were stimulated with TNF-␣ for the indicated times (in minutes), followed by
fractionation into cytoplasmic and nuclear extracts. Ten micrograms of nuclear extracts was used in this analysis using HIV B probe. Oct-1
probe was used as a loading control.
(B) Western blot analysis of IB␣ and IB degradation. Fifty micrograms of cytoplasmic protein was probed with anti-phosphorylated IB␣
(p-IB␣), anti-IB␣, and anti-IB antibodies. Relative protein loading was shown by the use of anti--actin antibody.
(C) EMSA was performed to determine the specificity of inducible RelA/p50 NF-B DNA binding activity. Competition and supershift assays
were performed using 15 g of nuclear protein from TNF-␣-stimulated cells as indicated.
(D) Northern blot analysis for the expression of the NF-B-inducible gene IB␣. Twenty-five micrograms of total RNA from wild-type and
MEKK3Ϫ/Ϫ
MEFs stimulated with TNF-␣ for the indicated times (in minutes) was analyzed using a murine IB␣ cDNA probe; glyceraldehyde-
3. Regulation of Biphasic NF-B Activation
1289
1A) and cytoplasmic fractions for Western blot analysis MEKK3 and IB␣ Are Crucial in Assembling the
Signal-Dependent Complex Formation with IKK(Figure 1B). Within 15 min of TNF-␣ stimulation of wild-
type cells, the NF-B DNA binding activity was maxi- To test our hypothesis, we performed coimmunoprecipi-
tation assays to determine whether MEKK3 plays a rolemally induced and lasted for 60–120 min (Figure 1A). In
contrast, at 15 and 60 min after TNF-␣ stimulation of in assembling the IKK:IB␣:NF-B complex following
cytokine stimulation. Both MEKK3 and IKK2 were de-MEKK3Ϫ/Ϫ
and IB␣Ϫ/Ϫ
cells, the NF-B DNA binding
activity was only marginally induced, and the maximum tected in anti-IB␣ immunoprecipitates isolated from
wild-type cells stimulated with either TNF-␣ or IL-1␣,activity was not reached until 120 min (Figure 1A). Cyto-
kine-induced p50/p50 homodimer accumulation was whereas no MEKK3 or IKK2 was found in unstimulated
wild-type and cytokine-stimulated MEKK3Ϫ/Ϫ
or IB␣Ϫ/Ϫ
also delayed in MEKK3 null cells, suggesting that the
IKK-mediated p105 degradation was also affected (Heiss- cells (Figure 2A). In additional coimmunoprecipitation
experiments, MEKK3 was detected to be associatedmeyer et al., 1999, 2001).
Using the cytoplasmic extracts from wild-type, with RelA in TNF-␣- and in IL-1␣-stimulated wild-type
cells but not in IB␣Ϫ/Ϫ
cells (Figure 2B). IKK2 was de-MEKK3Ϫ/Ϫ
, and IB␣Ϫ/Ϫ
cells, we found that IB␣ is phos-
phorylated and degraded in wild-type cells after 15 min tected in RelA immunoprecipitates from TNF-␣- and
IL-1␣-stimulated cells, but reduced levels of IKK2 wereof stimulation by TNF-␣, whereas in MEKK3Ϫ/Ϫ
cells,
IB␣ phosphorylation and degradation could not be de- coimmunoprecipitated by anti-RelA antibody in MEKK3-
and IB␣ null cells (Figure 2B). Taken together, thesetected (Figure 1B). Degradation of IB occurred at 60
min after TNF-␣ stimulation in wild-type, MEKK3Ϫ/Ϫ
, and results show that in the absence of IB␣ or MEKK3, the
signal-induced MEKK3:IKK:IB␣:NF-B complex is notIB␣Ϫ/Ϫ
cells (Figure 1B), findings that are consistent
with those previously reported (Thompson et al., 1995) assembled, suggesting that MEKK3 and IB␣ are crucial
components in assembling the signal-dependent forma-and with the delayed and decreased NF-B activation
in TNF-␣-stimulated MEKK3Ϫ/Ϫ
and IB␣Ϫ/Ϫ
cells (Figure tion of this complex.
To determine whether IKK1, IKK2, and NEMO are re-1A). Competition and supershift assays showed the
presence of RelA and p50 in the NF-B DNA binding quired in MEKK3-mediated formation of the IKK:IB␣:
NF-B complex, we investigated the signal-dependentactivity in wild-type, MEKK3Ϫ/Ϫ
, and IB␣Ϫ/Ϫ
cells (Figure
1C). These results suggest that lack of either IB␣ formation of the MEKK3:IKK:IB␣:NF-B complex in
IKK2Ϫ/Ϫ
, IKK1Ϫ/Ϫ
, and NEMOϪ/Ϫ
cells (Li et al., 1999a,or IB␣ phosphorylation results in a similar delay of
NF-B activation. 1999b; Schmidt-Supprian et al., 2000). Our results show
that NEMO and IKK2 are essential for assembling ofWe next measured the time course for expression of
the NF-B downstream target genes IB␣ and IEX-1 the MEKK3:IKK:IB␣:NF-B complex, whereas IKK1 is
dispensable (Figures 2C and 2D). A higher level of IKK2(Chiao et al., 1994; Wu et al., 1998) (Figures 1D and 1E).
In wild-type cells, the IB␣ mRNA was induced by TNF-␣ coimmunoprecipitable RelA was detected in wild-type
cells than in MEKK3- and IB␣ null cells after cytokineat 15 min, and its level peaked after 60 min (Figure
1D); in MEKK3Ϫ/Ϫ
cells, the TNF-␣ induction of IB␣ was stimulation (Figures 2E and 2F). This result is consistent
with the findings shown in Figure 2B and suggests thatsignificantly delayed and the expression level greatly
decreased (Figure 1D). The expression of IEX-1 was also IKK2 is partitioned between different IB:NF-B com-
plexes (Figures 2E and 2F). Both MEKK3 and IB␣ werepostponed and reduced in both MEKK3Ϫ/Ϫ
and IB␣Ϫ/Ϫ
cells (Figure 1E). To confirm that IB␣ is essential for detected in IKK2 and IKK1 immunoprecipitates from
TNF-␣- or IL-1␣-stimulated wild-type, but not in unstim-rapid induction of NF-B activation, we analyzed the
TNF-␣-induced IB␣ promoter activity in the IB␣LacZ/LacZ
ulated, cells (Figures 2E and 2F). We could not distin-
guish whether IKK1:IKK2 or IKK2:IKK2 dimers wereand IB␣ϩ/LacZ
cells, which were newly cultured from
mouse embryos in which the IB␣ gene was homozy- present in these complexes because of the heterodimer
formation between IKK1 and IKK2 (Figures 2E and 2F);gously or heterozygously replaced with a lacZ gene (Beg
et al., 1995a). The TNF-␣-induced and IB␣ promoter- these results were consistent with previously reported
findings (Zandi et al., 1997). The IB␣:NF-B complexmediated transcription of lacZ was reduced and post-
poned in IB␣LacZ/LacZ
cells but not in IB␣ϩ/LacZ
cells (Fig- was not detected in association with IKK2 and IKK1 in
the TNF-␣- or IL-1␣-stimulated MEKK3 null cells (Figuresure 1F). These results further demonstrate that in the
absence of either MEKK3 or IB␣, rapid and transient 2E and 2F). MEKK3 was not detected in the IKK2 and
IKK1 immunoprecipitates from IB␣Ϫ/Ϫ
cells after stimu-NF-B activation is inhibited. We speculated that the
signal-dependent formation of MEKK3 with the IKK: lation (Figures 2E and 2F). The IB␣:NF-B complex was
not detected in association with IKK2, but MEKK2 andIB␣:NF-B complex is required for rapid and transient
activation of NF-B and that other members of MAP3K the IB:NF-B complex were associated with IKK2 in
the TNF-␣- or IL-1␣-stimulated MEKK3 null cells (Figureregulate the interaction between the NF-B:IB com-
plexes and IKK. 2G). These results suggested that in response to cyto-
3-phosphate dehydrogenase (GAPDH) probe was used as a relative RNA loading control.
(E) Northern blot analysis was performed to determine the expression of the NF-B inducible gene IEX-1 and control GAPDH.
(F) Colorimetric assay was performed to determine the -galacosidase (-gal) activity induced by TNF-␣ in IB␣ϩ/LacZ
and IB␣LacZ/LacZ
MEFs
established from the 15-day-old embryos from one pregnancy. The genotype of these MEFs was confirmed by PCR and Western blot analysis
using the antibodies indicated; ϩ indicates wild-type IB␣, and L denotes LacZ. The time course stimulation of TNF-␣ is indicated: white, 0
min; yellow, 30 min; red, 60 min; and green, 120 min.
4. Molecular Cell
1290
Figure 2. MEKK3 Is Required for Signal-Dependent Interaction of NF-B:IB␣ with the IKK Complex
Immunoprecipitation assays, from the cytoplasmic extracts of wild-type, MEKK3Ϫ/Ϫ
, IKK2Ϫ/Ϫ
, RelAϪ/Ϫ
, and IB␣Ϫ/Ϫ
cells with or without TNF-␣
or IL-1␣ stimulation as indicated, were performed with (A) anti-IB␣ antibody and (B) anti-RelA antibodies, followed by Western blot analysis
using the antibodies indicated. As shown in (C) and (D), association of MEKK3 with the IKK complex requires NEMO. Immunoprecipitation
assays using the cytoplasmic extracts of wild-type, IKK2Ϫ/Ϫ
, NEMOϪ/Ϫ
, IKK1Ϫ/Ϫ
, and MEKK3Ϫ/Ϫ
MEFs stimulated with TNF-␣ or IL-1␣ as indicated
were performed with anti-MEKK3 (C) and anti-IKK2 (D), followed by Western blot analysis using the antibodies indicated. (E), (F), and (G) show
the signal-dependent formation of two different complexes: MEKK3:IKK:IB␣:NF-B and MEKK2:IKK:IB:NF-B. Immunoprecipitation (IP)
assays using the cytoplasmic extracts of wild-type, MEKK3Ϫ/Ϫ
, and IKK2Ϫ/Ϫ
cells and IB␣Ϫ/Ϫ
cells with or without TNF-␣ or IL-1␣ stimulation
were performed with anti-IKK2 and anti-IKK1 antibodies followed by Western blot (WB) analysis using the antibodies indicated.
5. Regulation of Biphasic NF-B Activation
1291
kine stimulation, two inducible complexes, MEKK3:
IB␣:NF-B:IKK and MEKK2: IB:NF-B:IKK, are inde-
pendently formed.
Formation of the MEKK3:IB␣:NF-B:IKK Complex
Is Induced Rapidly and Transiently, but Formation
of the MEKK2:IB:NF-B:IKK Complex Is More Stable
To further demonstrate the cytokine-induced formation
of the MEKK3:IB␣:NF-B:IKK and MEKK2:IB:NF-
B:IKK complexes, we show that TNF-␣ and IL-1␣ in-
duce transient formation of the MEKK3:IB␣:NF-B:IKK
complex and more stable formation of the MEKK2:
IB:NF-B:IKK complex in wild-type cells, whereas
only the MEKK2:IB:NF-B:IKK complex is induced in
MEKK3 null cells (Figures 3A and 3B). We also deter-
mined the time course of this complex formation in wild-
type MEFs stimulated with TNF-␣ for 90 s, 5 min, and
15 min. IKK2-precipitable MEKK3 and IB␣ were de-
tected at 90 s after TNF-␣ stimulation, and both levels
were decreased at 5 min (Figure 3C). At 15 min after
TNF-␣ stimulation, neither MEKK3 nor IB␣ was de-
tected in the anti-IKK2 immunoprecipitates (Figure 3C).
Our results suggest that the formation of the MEKK3:
IKK:IB␣:NF-B complex is both signal dependent and
transient. Consistent with previous reports (DiDonato et
al., 1997; Mercurio et al., 1997; Regnier et al., 1997;
Zandi et al., 1997), we also found that the interaction
between IKK2 and IB␣ in E1A-transformed human em-
bryonic kidney (HEK)-293 cells was preexisting and sig-
nificantly increased after stimulation with either TNF-␣
or IL-1␣ (data not shown). This observation is consistent
with a previous report that IKK is directly or indirectly
associated with NF-B and IB proteins in HeLa cells,
and many tumor cell lines exhibit constitutive or high
basal NF-B activity (Gilmore et al., 1996; Heilker et
al., 1999). Our results also show that IKK:IB␣:NF-B
complex formation induced by PMA is independent of
MEKK3 (Figure 3D).
MEKK3 Kinase Activity Is Required
for MEKK3:IKK:IB␣:NF-B Complex Formation
To determine whether the kinase activity of MEKK3 is
required for the formation of the MEKK3:IKK:IB␣:NF-B
complex, we transfected plasmids encoding HA-tagged
wild-type MEKK3 and mutant MEKK3 (K391M) into
MEKK3Ϫ/Ϫ
cells and HA-tagged wild-type IKK2 and mu-
tant IKK2 (K44M) into IKK2Ϫ/Ϫ
cells, followed by immuno-
precipitation using anti-HA antibody (Figures 4A and
4B). We found that IKK2 and IB␣ were coprecipitated
only with HA-tagged wild-type MEKK3 in TNF-␣-stimu-
lated cells but not with mutant MEKK3 (K391M) (Figure
4A). These findings indicated that the MEKK3 kinase
activity was required for signal-dependent formation of
the MEKK3:IKK:IB␣:NF-B complex. Either the HA-
tagged wild-type IKK2 or the HA-tagged mutant IKK2
Figure 3. Time Course of the Complex Formation in Wild-Type (WT), (K44M) coprecipitated with MEKK3 and IB␣ proteins in
MEKK3Ϫ/Ϫ
, and IKK2Ϫ/Ϫ
MEFs Stimulated with TNF-␣ or IL-1
Immunoprecipitation (IP) assays using the cytoplasmic extracts of
wild-type, MEKK3Ϫ/Ϫ
, and IKK2Ϫ/Ϫ
cells with TNF-␣ (A) or IL-1␣ (B)
(D) PMA-induced NF-B activation is independent of MEKK3. Immu-stimulation at 0, 5, and 60 min were performed with anti-IKK2 anti-
noprecipitation assays from cytoplasmic extracts of wild-type,body, followed by Western blot (WB) analysis using the antibodies
MEKK3Ϫ/Ϫ
, and IKK2Ϫ/Ϫ
and IB␣Ϫ/Ϫ
cells, with or without PMA stimu-indicated. (C) Wild-type MEFs were stimulated with TNF-␣ as indi-
lation, were performed with anti-IKK2 antibody, followed by Westerncated; IKK2 was precipitated from these cytoplasmic extracts, fol-
blot analysis using the antibodies indicated.lowed by Western blot analysis using the antibodies as indicated.
6. Molecular Cell
1292
and transfected plasmids encoding an HA-tagged wild-
type IKK2, an HA-tagged kinase-defective IKK2 mutant
(K44M), an HA-tagged triple mutant (K44M/S177, S181A)
(Wang et al., 2001), and an HA-tagged constitutive acti-
vated IKK2 kinase mutant (S177, 181E) into IKK2Ϫ/Ϫ
cells,
followed by immunoprecipitation using anti-HA antibody
(Figure 4C). Our results show that IB␣, IB, RelA,
MEKK2, and MEKK3 were coprecipitated with HA-
tagged wild-type and K44M mutant IKK2 in TNF-␣ and
IL-1␣-stimulated cells (Figure 4C). MEKK2 and MEKK3
were, but IB␣, IB, and RelA were not, coprecipitated
with HA-tagged IKK2 mutant (K44M/S177, 181A) (Figure
4C). The coimmunoprecipitation of HA-tagged constitu-
tive activated IKK2 kinase mutant (S177, 181E) revealed
the presence of IB␣, IB, and RelA with or without
cytokine stimulations; however, only MEKK2 and MEKK3
were coprecipitated from the cells stimulated with
TNF-␣ and IL-1␣. These findings suggest the following:
(1) IKK2 kinase activity, which is defective in IKK2 K44M
mutant, is not required for signal-dependent formation
of the MEKK3:IKK:IB␣:NF-B complex; (2) activation
of IKK2, with phosphorylation of S177 and S181 on its
activation loop, is essential in the interaction with
IB:NF-B complexes but is not required for association
with MEKK2 and MEKK3; and (3) constitutively activated
IKK2, the phosphorylation mimetic IKK2 kinase mutant
(S177, 181E), binds to IB:NF-B complexes indepen-
dent of TNF-␣ and IL-1␣ stimulation, but association of
the constitutive active IKK2 with MEKK2 and MEKK3 is
still regulated by cytokine stimulation.
MEKK3 Phosphorylates S177 and S181
in the Activation Loop of IKK2
Because our data show that MEKK3 organizes signal-
dependent formation of the MEKK3:IKK:IB␣:NF-B
complex and that the catalytic activity of MEKK3 is es-
sential for the formation of this complex, we sought to
determine whether MEKK3 is required for IKK2 phos-
phorylation after TNF-␣ stimulation. We transfected
wild-type and MEKK3Ϫ/Ϫ
cells with Flag-tagged catalyti-
cally inactive IKK2 (K44M), which lacks autophosphory-
lation activity, to unveil phosphorylation at the serines
(S177 and S181) in the activation loop of IKK2 and a
triple-point mutant in which the two serine residues in
Figure 4. Catalytic Activity of MEKK3 Is Essential for Complex For- the activation loop were also mutated (K44M/S177,
mation with IB␣:NF-B and IKK2 181A) (Lee et al., 1997; Wang et al., 2001). Our results
MEKK3Ϫ/Ϫ
MEFs were transfected with HA-tagged MEKK3 and show that TNF-␣-activated MEKK3 is required for phos-
MEKK3 (K391M) (A), or IKK2Ϫ/Ϫ
MEFs were transfected with HA-
phorylation of the IKK2 activation loop (Figures 5A and
tagged IKK2 and IKK2 (K44M) (B), and IKK2Ϫ/Ϫ
MEFs were trans-
5B). This finding is consistent with the reduced andfected with HA-tagged IKK2 expression constructs encoding wild-
delayed NF-B activation in MEKK3 null cells, as showntype (WT) IKK2, IKK2 (K44M), IKK2 (K44M/S177, 181A), and IKK2
in Figure 1A, and the delayed phosphorylation of IKK2,(S177, S181E) (C). These transfectants were stimulated with TNF-␣
or IL-1␣ as indicated. From these cytoplasmic extracts, immunopre- as detected in MEKK3 null cells at 60 min TNF-␣ stimula-
cipitation (IP) using anti-HA antibodies was performed by Western tion (Figures 5A and 5B). Taken together, these results
blot (WB) analyses, as indicated.
suggest that MEKK3 selectively activates IKK:IB␣:
NF-B complex in a rapid and transient mode, whereas
MEKK2 may selectively activate IKK:IB:NF-B com-TNF-␣-stimulated IKK2Ϫ/Ϫ
cells (Figure 4B), suggesting
that the IKK2 kinase activity was not required for signal- plex in a delay phase, in response to stimulation with
proinflammatory cytokines.dependent formation of this complex. Consistent with
these results, phosphorylation of IB␣ was detected in We next sought to determine whether MEKK3 directly
phosphorylates S177 and S181 in the activation loopthe TNF-␣-stimulated cells transfected either with HA-
tagged wild-type MEKK3 or IKK2 (Figures 4A and 4B). of IKK2. MEKK3 is biochemically purified to apparent
homogeneity from the cytoplasmic fraction using affinityTo further determine the role of IKK2 in the formation
of the MEKK3:IKK:IB␣:NF-B complex, we generated a chromatography; a single band is detected in the
MEKK3 fraction by silver staining (Figure 5C) and identi-constitutively activated IKK2 kinase mutant (S177, 181E)
7. Regulation of Biphasic NF-B Activation
1293
Figure 5. MEKK3 Phosphorylates S177 and S181 in the Activation Loop of IKK2.
(A) MEKK3 induces phosphorylation of IKK2 and (B) delayed phosphorylation of IKK2 in MEKK3 null MEFs. Wild-type (WT) and MEKK3Ϫ/Ϫ
MEFs transfected with Flag-tagged mutant IKK2 (K44M) and Flag-tagged triple-point mutant IKK2 (K44M/S177A/S181A) stimulated with TNF-␣
for 5 min in (A) and 60 min in (B) were coimmunoprecipitated using anti-Flag antibody and reprobed at the membrane with anti-Flag antibody,
which served as a loading control, and assayed for their ability to be phosphorylated in the presence of [␥-32
P]ATP. Silver staining (C),
immunoblotting of purified MEKK3 (D), and kinase assay (E). In an in vitro system, MEKK3 phosphorylates the polypeptide mapping to the
regulatory loop of IKK2 (indicated as “wt peptide”) but not the polypeptides mapping an S→A mutated regulatory loop of IKK2 (indicated as
“mt peptides”). The migration of molecular weight markers (in kilodaltons) is also indicated.
fied by anti-MEKK3 antibodies in immunoblotting (Fig- that MEKK3 directly phosphorylates IKK2 at S177 and
S181 in the activation loop.ure 5D). The in vitro kinase assay was performed with
purified MEKK3 using wild-type IKK2 peptide (wt IKK2
peptide, 170-KELDQQSLCTSFVGTLQYLA-190) corre- TNF-␣- and IL-1␣-Induced Delayed NF-B Activation
Is Inhibited in MEFs with siRNA-Mediatedsponding to the phosphorylation site of the IKK2 activa-
tion loop, and mutant peptides with alanine substitution Knockdown Levels of MEKK2
Since our results suggest that MEKK2 is involved in(mt IKK2 peptide, 170-KELDQQALCTAFVGTLQYLA-190)
served as a control. Autophosphorylation of MEKK3 and formation of the IB:NF-B:IKK complex and delayed
NF-B activation, RNA interference approaches werephosphorylation of the wild-type IKK2 peptides but not
the mutant peptide by the purified MEKK3 is demon- carried out to knock down the level of MEKK2 expres-
sion. As shown in Figures 6A and 6B, both TNF-␣- andstrated (Figure 5E). Taken together, these results show
8. Molecular Cell
1294
Figure 6. MEKK2 Regulates the Delayed NF-B Activation in Response to Cytokine Stimulation Using siRNA Directed Against MEKK2.
Wild-type (WT), wild-type [WT (MEKK2)] and MEKK3 null MEFs [MEKK3Ϫ/Ϫ
(MEKK2)] transfected with siRNA oligonucleotides against MEKK2
followed by stimulation with TNF-␣ (A) and IL-1␣ (B) as indicated. Ten micrograms of nuclear extracts was used in electrophoretic mobility
shift assays for determining the NF-B activities in these cells using HIV B probe. Oct-1 probe was used as a loading control. The cytoplasmic
extracts were used in Western blot analysis to determine the degradation of IB␣ and IB induced by TNF-␣ (C) and IL-1␣ (D), with -actin
as a loading control. (E and F) MEKK2 associates with IB. Wild-type fibroblasts were transfected with siRNA oligonucleotides targeted
against MEKK2 and stimulated as indicated. Cytoplasmic extracts were coimmunoprecipitated with MEKK2 and IB antibodies, respectively.
The precipitates were analyzed with the antibodies indicated. (G) Overexpression of MEKK2⌬3UT rescued MEKK2 siRNA-mediated inhibition
of NF-B activation. Wild-type fibroblasts were transfected with and without siRNA oligonucleotides against MEKK2 and MEKK2⌬3UT expres-
sion vector as indicated. Whole-cell lysate was used for Western blot analysis with anti-Flag, MEKK2, and MEKK3 antibodies. Anti-RelA
antibody served as a loading control. The NF-B DNA binding activities were determined using the nuclear extracts by eletrophoretic mobility
shift assay. Oct-1 probe was used as a loading control. (H) MEKK3 siRNA-mediated inhibition of MEKK3 expression. Wild-type fibroblasts
were transfected with and without siRNA oligonucleotides against MEKK3 and expression vector encoding HA and His-tagged MEKK3, as
indicated. Whole-cell lysate was used for Western blot analysis with anti-HA, MEKK2, and MEKK3 antibodies. Anti-RelA antibody served as
a loading control.
9. Regulation of Biphasic NF-B Activation
1295
IL-1␣-induced delayed activation of NF-B were inhib- than IKK2 (Burke et al., 1999). The role of IB␣ and IB
ited in wild-type and MEKK3 null fibroblasts in which C termini in interactions with MEKK2 and MEKK3 is
expression of MEKK2 was knocked down by siRNA di- further supported by this finding.
rected against the 3Ј untranslated region of MEKK2. To further demonstrate that MEKK2 specifies IB:
Consistent with the inhibition of NF-B DNA binding NF-B and that MEKK3 specifies IB␣:NF-B com-
activity, the degradation of IB was also blocked by plexes as substrates for IKK in regulation of proinflam-
siRNA against MEKK2 (Figures 6C and 6D). The phos- matory cytokine-induced biphasic NF-B activation, we
phorylation and degradation of IB␣ was inhibited in performed the bimolecular fluorescence complementa-
MEKK3 null cells but not in wild-type cells in which tion assay (BiFC) to visualize the interaction between
expression of MEKK2 was knocked down by siRNA di- MEKK2 and IB:NF-B:IKK and between MEKK3 and
rected against the 3Ј untranslated region of MEKK2 (Fig- IB␣:NF-B:IKK. The bimolecular fluorescence comple-
ures 6C and 6D). As shown in Figures 6E and F, IB mentation approach and the use of IB␣YN with p65YC
but not IB␣ was coimmunoprecipitated with MEKK2. were previously described (Hu et al., 2002). Cotransfec-
MEKK2 but not MEKK3 was coimmunoprecipitated with tion experiments were performed with MEKKYC and
IB. These results suggest that MEKK2 interacts with IBYN expression vectors as well as positive and nega-
the IKK:IB:NF-B complex. The specificity of siRNA tive controls as indicated in Figure 7C. As the results of
targeted at MEKK2 and MEKK3 was demonstrated in BiFC, the fluorescent signals were observed in the cells
Figures 6G and 6H. The siRNA-mediated reduction of expressing both MEKK3YC and IB␣YN and MEKK2YC
MEKK2 protein was overcome by overexpression of a and IBßYN (Figure 7C). The fluorescent signals reached
plasmid encoding a Flag-tagged MEKK2 lacking the 3Ј peak intensity at 2 min after TNF-␣ stimulation in the
untranslated region (MEKK2⌬3UT) (Figure 6G). Further- cells expressing both MEKK3YC and IB␣YN and MEK-
more, overexpression of MEKK2⌬3UT in MEKK2-knock- K2YC and IBßYN. The fluorescent signals decayed
down cells induced NF-B, suggesting overcompensa-
quickly and were barely detectable in the cells express-
tion for the reduced level of MEKK2 protein (Figure 6G).
ing both MEKK3YC and IB␣YN after 15 min of TNF-␣
This finding is consistent with a previously published
stimulation. However, the fluorescent signals were per-
report (Zhao and Lee, 1999). Taken together, these re-
sistent in the cells expressing both MEKK2YC and I-
sults suggest that MEKK3 plays an essential role in the
BßYN after 60 min of TNF-␣ stimulation. Overexpression
regulation of rapid and transient activation of NF-B,
of MEKK3 and MEKK2 has been shown to induce NF-
whereas MEKK2 regulates the delayed activation of
B activation (Zhao and Lee, 1999) and may cause the
NF-B in response to cytokine stimulation.
appearance of fluorescence signals without TNF-␣ stim-
ulation in these transfected cells. In the same assay, no
Proinflammatory Cytokine Induces Formation
fluorescence signals were observed in the cells cotrans-
of MEKK3:IB␣:NF-B:IKK and MEKK2:IB:NF-
fected with both MEKK3YC and IBßYN or with both
B:IKK Complexes in Biphasic NF-B Activation
MEKK2YC and IB␣YN expression vectors, suggesting
Because our results show that MEKK3 is associated
that the complex formation did not occur between MEK-
with the IB␣:NF-B:IKK complex and that MEKK2 is
K3YC and IBßYN, or between MEKK2YC and IB␣YN.
associated with the IB:NF-B:IKK complex in re-
Taken together, our results suggest that the formation
sponse to TNF-␣ and IL-1␣ stimulation, we determined
of the MEKK3:IB␣:NF-B:IKK and MEKK2:IB:
whether the N- and C-domains of IB␣ and IB are
NF-B:IKK complexes in biphasic NF-B activation is
required for complex formation. Both Flag-tagged N ter-
induced by cytokines.minus deletion mutants of IB␣ (IB␣⌬N, aa 72–317) and
IB (IB⌬N, aa 58–359) were coimmunoprecipitated
with RelA but not with IKK and MEKK3 or MEKK2 (Figure
Discussion7A). In the coimmunoprecipitation experiments with
Flag-tagged C terminus deletion mutants of IB␣ (I-
Figure 7D summarizes our results and illustrates ourB␣⌬C, aa 1–248) and IB (IB⌬C, aa 1–305), RelA,
working model for the regulation of biphasic NF-B acti-IKK, MEKK3, and MEKK2 were not detected. Figure 7B
vation. Genetic analysis showed that MEKK3, which isillustrates the various IB␣ and IB deletion mutants
thought to activate IKK2 through phosphorylation (Yangand summarizes the findings. These results suggest that
et al., 2001), is necessary for TNF-␣-induced NF-B acti-the N terminus of IB␣ (aa 1–72) and IB (aa 1–58) is
vation. However, how the biphasic NF-B activation isessential for the interaction with IKK and MEKK2 or
regulated, how MEKK3 and MEKK2 regulate IKK2 activ-MEKK3 but not for the association with RelA. The C
ity and subsequent rapid NF-B activation, and howtermini of IB␣ (aa 248–317) and IB (aa 305–359) are
various IB:NF-B complexes are differentially regu-necessary for the formation of the MEKK3:IB␣:NF-
lated through phosphorylation by IKK2 were unclear.B:IKK and MEKK2:IB:NF-B:IKK complexes, re-
Our data demonstrate that the formation of the MEKK3:spectively. These results are consistent with previous
IKK:IB␣:NF-B and MEKK2:IKK:IB:NF-B complexesfindings that an N terminus deletion mutant of IB␣ (aa
is signal dependent. Analysis of the time course for76–318) associated with Rel, but that a C terminus dele-
NF-B activation in wild-type and MEKK3Ϫ/Ϫ
cells re-tion mutant of IB␣ (aa 1–266) did not interact with Rel in
vealed that MEKK3 is essential in the regulation of rapidcoimmunoprecipitation assays (Beauparlant et al., 1996;
activation of NF-B. However, TNF-␣- and IL-1␣-inducedLuque and Gelinas, 1998; Luque et al., 2000). It has also
phosphorylation and degradation of IB are indepen-been shown that the C terminus of IB␣ is important in
activating IKK through interactions with subunits other dent of MEKK3. Using an RNAi approach, we inhibited
11. Regulation of Biphasic NF-B Activation
1297
the expression of MEKK2 and showed that the delay NF-B activation. B-Ras proteins that are associated
only with the NF-B:IB complex may provide an ex-NF-B activation is reduced significantly. Thus, these
results suggest that MEKK2 plays a key role in activation planation for the slower phosphorylation and degrada-
tion of IB compared with IB␣ (Fenwick et al., 2000).of the IB:NF-B complex by mediating the formation
of IKK2 with IB:NF-B complexes in response to vari- Our results obtained from IB␣Ϫ/Ϫ
cells show that in
the absence of IB␣, cytokine-induced NF-B activationous stimuli. In summary, our results show that MEKK3
and MEKK2 regulate the formation of IB␣:NF-B and is delayed and reduced, and no signal-dependent for-
mation of the MEKK3:IKK:IB␣:NF-B complex occurs.IB:NF-B complexes, respectively, as substrates for
IKK in the regulation of proinflammatory cytokine- These results suggest that IB binds to NF-B to func-
tion not only as an inhibitor but also as an anchor forinduced biphasic NF-B activation. Our results also sug-
gest that IKK2 is phosphorylated on S177 and S181 on MEKK3 or MEKK2 and IKK2 complex formation for acti-
vation. The expression of IB␣ induced by NF-B isits activation loop by MEKK3. It is possible that IKK2 is
also phosphorylated by MEKK2 given the 94% similarity not only important for terminating the NF-B response
(Cheng et al., 1994; Chiao et al., 1994; Sun et al., 1993)between the amino acid sequences of MEKK3 and
MEKK2 and the signal-dependent complex formation but also for reinitiating activation of NF-B. Our results
shown in Figure 1 are consistent with those in a recentwith IKK2:IB:NF-B.
To date, much effort has focused on the identification report in which cytokine-induced NF-B activation was
analyzed using IBϪ/Ϫ
IB⑀Ϫ/Ϫ
double knockout MEFsof MAPK cascades that are activated by MAP3K. How-
ever, the upstream regulators of MEKK2/3 have not been (Hoffmann et al., 2002). The authors conclude that IB␣
mediates rapid NF-B activation and strong negativeidentified, and some of them are likely to be activators of
MEKK2/3 in regulation of the biphasic NF-B activation. feedback control, resulting in an oscillatory NF-B acti-
vation profile, whereas IB and IB⑀ respond moreOur data suggest that one of the possible steps by which
MEKK2/3 proteins organize the complex formation is the slowly to IKK activation and act to dampen the long-
term oscillations of the NF-B response (Hoffmann etphosphorylation of Ser177 and Ser181 of the activation
loop in IKK2. Phosphorylation of Ser177 and Ser181 of al., 2002). The bimodal characteristics of IB-NF-B sig-
naling support the key role of IB inhibitors in the regula-the activation loop in IKK2 moves the activation loop
away from the catalytic pocket to allow its interaction tion of the differential NF-B activation (Hoffmann et al.,
2002). Furthermore, the X-ray crystal structure of thewith ATP and polypeptide substrates (IB:NF-B) (Del-
hase et al., 1999), thus allowing the transient formation IB:NF-B complex suggests that the unique structural
features of IB contribute to its ability to mediate per-of MEKK2:IKK:IB:NF-B and MEKK3:IKK:IB␣:NF-B
complexes at similar time points after cytokine stimu- sistent NF-B activation (Malek et al., 2003).
A recent report by Wang and colleagues suggestedlation. This may explain why the kinase activity of
MEKK2/3 is required for the inducible complex forma- that TAK1 is an IKKK that phosphorylates IKK in the
IL-1/TRAF6 pathway in a biochemically reconstitutedtion between MEKK2/3:IKK:IB:NF-B whereas the ki-
nase activity of IKK is dispensable. However, our data system (Wang et al., 2001). Another recent study using
siRNA directed against TAK1 in HeLa cells showed thatdo not exclude that other, as yet unidentified, proteins
are also involved in this complex formation. The mecha- TAK1 was important for NF-B activation in the IL-1
and TNF-␣ signaling pathway (Takaesu et al., 2003).nisms regulating the specific interactions between
IKK:IB:NF-B and MEKK2 and between IKK:IB␣: However, siRNA directed against TAK1 only partially
inhibits IL-1 or TNF-␣-induced NF-B activation (Ta-NF-B and MEKK3 are unclear. It has been shown that
143-3-3⑀ and 14-3-3 bind to MEKK2/3 as scaffold for kaesu et al., 2003). Thus, other MAP3Ks may also partici-
pate in regulation of IKK activation in response toprotein-protein interactions (Adams et al., 2002; Fanger
et al., 1998). Although they do not appear of direct influ- these cytokines.
In summary, our results suggest that the IKK kinasesence on the kinase activity of MEKK3 in vitro, one may
speculate that these scaffold proteins might directly are involved in the specific interaction of NF-B-IB
complexes with IKK, thus providing a possible mecha-or indirectly influence the formation of MEKK2/3:IKK:
IB:NF-B complexes in regulation of the biphasic nism for the biphasic NF-B activation.
Figure 7. Proinflammatory Cytokine Induces Formation of MEKK3:IB␣:NF-B:IKK and MEKK2:IB:NF-B:IKK Complexes in Biphasic
NF-B Activation
(A) Determining the role of N- and C termini of IB␣ and IB in signal-induced formation of MEKK:IB:NF-B:IKK complexes. HEK-293 cells
were transfected with HA-tagged wild-type IB␣, HA-tagged IB␣⌬N (aa 72–317), HA-tagged IB␣ IB␣⌬C (aa 1–248), HA-tagged wild-type
IB, HA-tagged IB⌬N (aa 58–359), and HA-tagged IB⌬C (aa 1–305). These transfectants were stimulated with TNF-␣ or IL-1␣ as indicated.
From these cytoplasmic extracts, immunoprecipitations (IP) using anti-HA antibodies and HEK-293 whole-cell extract (WCE) as a control were
followed by Western blot (WB) analysis with the various antibodies indicated.
(B) Schematic representation of the N- and C-terminal deletion mutants of IB␣ and IB and their interactions with RelA, IKK, and MEKKs.
(C) Bimolecular fluorescence complementation assay for visualization of the specific interaction between MEKK2 and IB:NF-B:IKK, and
MEKK3 and IB␣:NF-B:IKK. HEK-293 cells were transfected with bFosYC and bJunYN plasmids as positive control, EYFP and IB␣YN or
IBßYN as negative control, and four different combinations of MEKK3YC, MEKK2YC, IB␣YN, and IB␣YN expression vectors as indicated.
Forty-eight hours after transfection, these cells were stimulated with TNF-␣ (10 ng/ml) for various times as indicated. The fluorescence images
of HEK-293 cells expressing the proteins indicated at the top of each panel were acquired at the time points specified.
(D) Working model for regulation of cytokine-induced NF-B activation shows that MEKK3 assembles the IB␣:NF-B complex and MEKK2
organizes the IB:NF-B complex as substrates for IKK in regulation of proinflammatory cytokine-induced biphasic NF-B activation.
12. Molecular Cell
1298
Experimental Procedures used B probe (5Ј-CTCAACAGAGGGGACTTTCCGAGAGGCCAT-3Ј)
that contained the B site (underlined) found in the HIV long terminal
Reagents repeat. The competitive binding experiment was performed with
We obtained HEK-293 cells from ATCC. Wild-type, IKK1Ϫ/Ϫ
, and a 50-fold excess of wild-type or mutant B oligonucleotides (5Ј-
IKK2Ϫ/Ϫ
MEFs, and RelAϪ/Ϫ
and IB␣Ϫ/Ϫ
MFCs were kindly provided CTCAACAGAGTTGACTTTTCGAGAGGCCAT-3Ј). Oct-1 probe was
by Dr. Inder M. Verma (The Salk Institute for Biological Studies, La used as a loading control. The supershift experiments were per-
Jolla, CA). IB␣ϩ/LacZ
mice were kindly provided by Dr. Amer A. Beg formed with anti-RelA, anti-p50, and anti-p52 antibodies in the ab-
(Department of Biological Sciences, Columbia University, New York, sence or presence of the control peptides (Santa Cruz Biotech-
NY). Antibody against NEMO was kindly provided by Dr. Alain Israe¨ l nology).
(Institute Pasteur, Paris, France). Antibodies against MEKK3 were
obtained from Transduction Laboratories. Antibodies against IKK1
Immunoprecipitation(H-744), IKK1/2 (H-470), IKK2, RelA, p50, p52, IB␣, and IB were
For immunoprecipitation, cells were lyzed in buffer A on ice for 15obtained from Santa Cruz Biotechnology. Antibody against phos-
min, vortexed for 30 s, and subjected to centrifugation for 20 minpho-IB␣ was obtained from Cell Signaling, anti--actin and Flag
at 14,000 rpm in a precooled centrifuge. The resulting cytoplasmicantibodies were obtained from Sigma, and anti-rabbit horseradish
extract (1 mg) was subjected to immunoprecipitation with 200 ngperoxidase (HRP) and anti-mouse HRP antibodies were obtained
of anti-MEKK3, anti-IKK2, anti-IB␣, or anti-RelA antibodies at 4ЊCfrom Pharmingen. The expression vectors encoding bFosYC,
overnight, followed by Western blotting or kinase assay.bJunYN, IB␣YN, BiFC-YC, and BiFC-YN were kindly provided by
Dr. Tom K. Kerppola (Howard Hughes Medical Institute and Depart-
ment of Biological Chemistry, University of Michigan Medical
Western Blot AnalysisSchool). MEKK2YC and MEKK3YC were constructed by fusion of
The immunporecipitates and cell extracts were resolved on 8%the PCR products of MEKK2 and MEKK3 to the N terminus of BiFC-
Laemmli (SDS) polyacrylamide gels and transferred to PVDF mem-YC (EYFP, aa 1–155–238). IBßYN was generated by fusion of IB
branes (Osmonic) incubated in blocking buffer (10% nonfat milk into the N terminus of BiFC-YN (EYEP, aa 1–154). The expression
PBS) for 4 hr at room temperature and then, to block the heavy andvectors encoding HA-tagged wild-type IKK2, HA-tagged kinase-
light chains of IgG used in immunoprecipitation, incubated withdefective IKK2 mutant (K44M), and HA-tagged triple mutant (K44M/
blocking antibody (porcine multilink anti-mouse, anti-rabbit, anti-S177A/S181A) were kindly provided by Dr. James Chen (Department
goat Ig; DAKO) at a dilution of 1/250 for 16 hr at 4ЊC. The blots wereof Molecular Biology, University of Texas Southwestern Medical
washed twice with PBS for 5 min and incubated with the primaryCenter). An HA-tagged, constitutively activated IKK2 kinase mutant
antibody in PBS containing 0.1% nonfat milk for 16 hr at 4ЊC. For(S177E/S181E) was generated by using a PCR-mediated double-
detection, the blots were washed twice with PBS for 5 min andstranded site-directed mutagenesis kit from Stratagene, and se-
incubated at room temperature with HRP-labeled secondary anti-quences of the mutated sites were confirmed by DNA sequencing.
body for 3 hr in PBS containing 0.1% nonfat milk. An enhancedThe oligonucleotides encoding the sequence of MEKK2 (2397–2416
chemiluminescence kit (Amersham) was used for detection.bp) (5Ј-TATTTCTCTTGATTCTTGG-3Ј), MEKK3 (2760–2779) (5Ј-CAC
AAGTCAGGGCACCTGG-3Ј), and (2071–2090) (5Ј-AGCCAGGATGG
GATAGCTC-3Ј) for RNAi experiments were synthesized by Dhar-
Kinase Assaymacon.
Phosphorylation of IKK2 (K44M) and IKK2 (K44M/S177A/S181A) was
measured in an in vitro system (20 l) that contained an ATP bufferCell Culture and Transfection
(50 mM HEPES [pH 7.5], 5 mM MgCl2, and 2 mM ATP) and 10 l ofThe wild-type, MEKK3Ϫ/Ϫ
, NEMOϪ/Ϫ
, IKK1Ϫ/Ϫ
, IKK2Ϫ/Ϫ
, RelAϪ/Ϫ
, IB␣Ϫ/Ϫ
immunoprecipitates from wild-type and MEKK3Ϫ/Ϫ
cells (Wang et(Beg et al., 1995a, 1995b; Li et al., 1999a, 2000; Schmidt-Supprian
al., 2001).et al., 2000; Yang et al., 2001), and HEK-293 cells were grown on
For the in vitro kinase assay, we cloned HA-MEKK3 into pcDNA3.1gelatin-coated tissue culture dishes in DMEM that contained 4.5 g/l
(Not1 and Xho1). MEKK3 was purified by use of sequential anti-HAglucose, glutamin, sodium pyruvate, and nonessential amino acids
and Ni-NTA affinity chromatography and subjected to silver stainingand were supplemented with 12% heat-inactivated fetal bovine se-
and Western blotting (Wang et al., 2001). We synthesized peptidesrum (FBS). TNF-␣ and IL-1␣, obtained from R&D Systems, were
mapping the regulatory loop of IKK2 (170-KELDQQSLCTSFVGTLreconstituted to 10 ng/ml in phosphate-buffered saline (PBS) that
QYLA-190), and peptides with S→A mutations (170-KELDQQALCcontained 130 nM bovine serum albumin (BSA; fraction IV [pH 7.0])
and stored in 25 l aliquots at Ϫ25ЊC before use. For the cytokine TAFVGTLQYLA-190) served as a control. The peptides were purified
stimulation experiments, 10 ng/ml TNF-␣ or IL-1␣ was used. Cells by HPLC. For the kinase reaction, we used 1 l of purified MEKK3
were grown to 75% confluence in 10 cm dishes and transfected and 200 ng of peptide in an in vitro system (20 l) that contained
using Fugene-6 or calcium phosphate with 2–20 g of plasmids an ATP buffer (50 mM HEPES [pH 7.5], 5 mM MgCl2, and 2 mM ATP)
that encoded HA-MEKK3, HA-MEKK3 (K391M), HA-IKK2, HA-IKK2 (Wang et al., 2001). After incubation at 37ЊC for 20 min, the reaction
(K44M), Flag-IKK2 (K44M), or Flag-IKK2 (K44M/S177A/S181A). products were resolved by SDS-polyacrylamide gel electrophoresis,
Forty-eight hours after transfection, cells were stimulated with 10 dried, and exposed to X-ray films (Wang et al., 2001).
ng/ml TNF-␣ or IL-1␣ for various periods. The transfection of MEKK2
siRNA oligonucleotides was performed using TransIT-TKO transfec-
Northern Blot Analysistion reagent (Mirus) according to the manufacturer’s protocol.
Total RNA was extracted using 1 ml of Trizol reagent (Life Science)
per 100 mm dish according to the manufacturer’s instructions. TheColorimetric Assay for -Galactosidase Activity
filters were prehybridized and hybridized at 65ЊC using a 32
P-labeledFor assay of -galactosidase activity, cell extracts from 30,000
MEFs/well were added to 100 l of buffer Z (10 mM KCl, 1 mM 1.1 kb mouse IB␣ cDNA (EcoRI-EcoRI) probe, exposed, and rehy-
MgSO4, 50 mM -mercaptoethanol, 100 mM sodium phosphate [pH bridized with the cDNA probe for glyceraldehyde-3-phosphate dehy-
7.5]) to total volume and preincubated at 37ЊC for 5 min. Then, drogenase (GAPDH) (Chiao et al., 1994).
20 l of o-nitrophenyl -D-galactopyranoside (4 mg/ml in 100 mM
sodium phosphate [pH 7.5]) was added, and incubation was contin-
Bimolecular Fluorescence Complementation Assayued at 37ЊC until a detectable yellow color had formed. The reaction
HEK-293 cells cotransfected with various expression vectors usingwas terminated by addition of 50 l of 1 M Na2CO3, and absorbance
Fugene 6 (Roche, Indianapolis, IN) were maintained in Dulbecco’sat 420 nm was measured. -glactosidase activity was expressed
modified Eagle medium (DMEM) supplemented with 10% FBS andas units (nmol of 0-nitrophenol formed per minute) per milligram
antibiotics at 37ЊC in 5% CO2. The fluorescence emissions wereof protein.
observed in living cells 24–48 hr after transfection with or without
TNF-␣ stimulation using a Nikon TE300 inverted fluorescence micro-Electrophoretic Mobility Shift Assay
scope with a cooled CCD camera. YFP fluorescence was measuredCells were fractionated into cytoplasmic and nuclear fractions ac-
cording to a method previously described (Chiao et al., 1994). We by excitation at 500 nm and emission at 535 nm (Hu et al., 2002).
13. Regulation of Biphasic NF-B Activation
1299
Acknowledgments Gilmore, T.D., Koedood, M., Piffat, K.A., and White, D.W. (1996).
Rel/NF-B/IB proteins and cancer. Oncogene 13, 1367–1378.
We are grateful to Dr. Inder M. Verma for the generous gift of IKK1Ϫ/Ϫ
, Han, S.-J., Ko, H.-M., Choi, J.-H., Seo, K.H., Lee, H.-S., Choi, E.-K.,
IKK2Ϫ/Ϫ
, RelAϪ/Ϫ
, and IB␣Ϫ/Ϫ
cells; Dr. Amer A. Beg and Dr. David Choi, I.-W., Lee, H.-K., and Im, S.-Y. (2002). Molecular mechanisms
Baltimore for IB␣ϩ/LacZ
mice; Alain Israe¨ l for anti-NEMO antibody; for lipopolysaccharide-induced biphasic activation of nuclear fac-
Bing Su for the MEKK3Ϫ/Ϫ
cells; Dr. James Chen for the expression tor-B (NF-B). J. Biol. Chem. 277, 44715–44721.
vectors encoding HA-tagged wild-type IKK2, HA-tagged kinase-
Heilker, R., Freuler, F., Pulfer, R., Di Padova, F., and Eder, J. (1999).
defective IKK2 mutant (K44M), and HA-tagged triple mutant (K44M/
All three IB isoforms and most Rel family members are stably
S177A/S181A); and Dr. Tom K. Kerppola for the expression vectors
associated with the IB kinase 1/2 complex. Eur. J. Biochem.
encoding bFosYC, bJunYN, IB␣YN, BiFC-YC, and BiFC-YN. We
259, 253–261.
are also grateful to Dr. Amer A. Beg for critically reading the manu-
Heissmeyer, V., Krappmann, D., Wulczyn, F.G., and Scheidereit, C.script and M.P. Kracklauer for helpful suggestions. We thank Dr.
(1999). NF-B p105 is a target of IB kinases and controls signalPeng Huang for his assistance in fluorescence microscopy. We also
induction of Bcl-3-p50 complexes. EMBO J. 18, 4766–4778.thank Kate O´ Su´ illeabha´ in for her editorial assistance. The work was
Heissmeyer, V., Krappmann, D., Hatada, E.N., and Scheidereit, C.supported by grants from National Cancer Institute (CA73675-01,
(2001). Shared pathways of IB kinase-induced SCF(TrCP)-medi-R21PA-98-029, and CA78778-01) and a grant from the Lockton Fund
ated ubiquitination and degradation for the NF-B precursor p105for Pancreatic Cancer Research. G.M.S. is a recipient of a Fellowship
and IB␣. Mol. Cell. Biol. 21, 1024–1035.of the Cancer League of Bern, Switzerland.
Hoffmann, A., Levchenko, A., Scott, M.L., and Baltimore, D. (2002).
The IB-NF-B signaling module: temporal control and selectiveReceived: January 15, 2003
gene activation. Science 298, 1241–1245.Revised: September 3, 2003
Hu, C.D., Chinenov, Y., and Kerppola, T.K. (2002). Visualization ofAccepted: September 15, 2003
interactions among bZIP and Rel family proteins in living cells usingPublished: November 20, 2003
bimolecular fluorescence complementation. Mol. Cell 9, 789–798.
Karin, M., Cao, Y., Greten, F.R., and Li, Z.W. (2002). NF-B in cancer:References
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