COMMUNICATION
Mitogen-activated Protein Kinase/ERK Kinase Kinases 2 and 3 Activate Nuclear Factor-
B through I
B Kinase-
and I
B
Kinase-
*
Quan
Zhao and
Frank S.
Lee
From the Department of Pathology and Laboratory Medicine,
University of Pennsylvania School of Medicine,
Philadelphia, Pennsylvania 19104
 |
ABSTRACT |
Recent evidence indicates that nuclear
factor-
B (NF-
B), a transcription factor critically important for
immune and inflammatory responses, is activated by a protein kinase
cascade. The essential features of this cascade are that a
mitogen-activated protein kinase kinase kinase (MAP3K) activates an
I
B kinase (IKK) that site-specifically phosphorylates I
B. The
I
B protein, which ordinarily sequesters NF-
B in the cytoplasm, is
subsequently degraded by the ubiquitin-proteasome pathway, thereby
allowing the nuclear translocation of NF-
B. Thus far, only two
MAP3Ks, NIK and MEKK1, have been identified that can activate this
pathway. We now show that MEKK2 and MEKK3 can in vivo
activate IKK-
and IKK-
, induce site-specific I
B
phosphorylation, and, relatively modestly, activate an NF-
B reporter
gene. In addition, dominant negative versions of either IKK-
or
IKK-
abolish NF-
B activation induced by MEKK2 or MEKK3, thereby
providing evidence that these IKKs mediate the NF-
B-inducing
activities of these MEKKs. In contrast, other MAP3Ks, including MEKK4,
ASK1, and MLK3, fail to show evidence of activation of the NF-
B
pathway. We conclude that a distinct subset of MAP3Ks can activate
NF-
B.
 |
INTRODUCTION |
The transcription factor nuclear factor-
B
(NF-
B)1 plays a critical
role in immune and inflammatory responses (1, 2). NF-
B,
prototypically a heterodimer of p50 and p65 subunits, is sequestered in
the cytoplasm of most cell types by virtue of its association with a
family of inhibitor molecules, the I
Bs. Upon exposure to a wide
variety of agents, including the proinflammatory cytokine TNF-
,
lipopolysaccharide, oxidative stress, and the HTLV-I Tax protein, the
I
B protein is phosphorylated at its N terminus. In the case of
I
B
, the most extensively studied I
B isoform, this
phosphorylation occurs at Ser-32 and Ser-36 (3, 4). This
phosphorylation event targets I
B for degradation by the
ubiquitin-proteasome pathway (5), allowing the subsequent nuclear
translocation of NF-
B.
An I
B kinase (IKK) complex with a native molecular mass of 700 kDa
was originally identified in cytoplasmic extracts of HeLa cells and
shown to perform the site-specific phosphorylation of I
B
(6, 7).
A significant advance was the subsequent cloning of the cDNAs for
the catalytic, protein kinase subunits of this complex, IKK-
and
IKK-
(8-12). Several lines of evidence now indicate that IKK-
and IKK-
can be regulated by phosphorylation. The initial
indications were that the IKK complex can be activated in
vitro by the MAP3K MEKK1 (MAPK/ERK kinase kinase 1) (7) and that
the complex, activated either in vitro by MEKK1 or in vivo by exposure of cells to TNF-
can be inactivated by
phosphatase treatment (7, 8). Additional work then demonstrated that (i) mutation of potential phosphoacceptor residues to alanine in the
activation loop of IKK-
or IKK-
abrogated activity (12), (ii)
mutation of these same residues in IKK-
to the phosphoresidue mimetic glutamic acid results in its constitutive activation (12), (iii) both IKK-
and IKK-
can be activated in vivo when
overexpressed with MEKK1 or the related MAP3K NF-
B-inducing kinase
(NIK) (10, 11, 13-15), and (iv) immunoprecipitated NIK can
phosphorylate immunoprecipitated IKK-
(16). Therefore, an important
conceptual advance in our understanding of NF-
B regulation is that
it can be activated by protein kinase cascade, the core elements of
this cascade being a MAP3K and an IKK (7, 10, 17).
These findings have already begun to provide a framework for
understanding how NF-
B can be activated by diverse stimuli. For
example, compelling evidence has been presented to show that NIK
mediates the NF-
B-inducing activity of TNF-
(17, 18), whereas
MEKK1 mediates the NF-
B-inducing activity of Tax (15). Thus,
different stimuli can activate NF-
B by targeting different MAP3Ks.
These findings moreover raise the possibility that yet other MAP3Ks
might activate NF-
B. MAP3Ks were originally identified as components
of signaling cascades in which a MAP3K phosphorylates and activates a
MAP2K, which in turn phosphorylates and activates a MAPK; the latter
include the mitogen-activated ERK and the stress-activated c-Jun
N-terminal kinase (JNK, also known as stress-activated protein kinase)
and p38 families (19). Here we show that MEKK2 and MEKK3, but not
certain other MAP3Ks, can activate NF-
B, and show that this
activation occurs by their activation of IKK-
and IKK-
.
 |
EXPERIMENTAL PROCEDURES |
Plasmids--
pCMV5-HA-MEKK2 (20), pCMV5-HA-MEKK3 (20), and
pCMV5-
MEKK4 (21) were gifts of Dr. Gary Johnson (National Jewish
Medical and Research Center). pcDNA3-ASK1 (22) was a gift of Dr.
Hidenori Ichijo (The Cancer Institute, Tokyo). pcDNA3-MLK3 was
constructed by subcloning into the BamHI
(blunt)/EcoRI site of pcDNA3 the 2.7-kilobase pair
NcoI (blunt)/EcoRI coding sequence fragment of
pPTK1-3.2 (23); the latter was a gift of Dr. Richard Spritz (University of Wisconsin-Madison). (PRDII)3E1bCAT, a
reporter gene that contains three copies of the NF-
B binding site
from the interferon-
enhancer, an E1b promoter, and the CAT gene, was a gift of Dr. Tom Maniatis (Harvard University). The sources of
(PRDII)2CAT, which contains two copies of the NF-
B
binding site from the interferon-
enhancer, pCMV5-MEKK1, which
encodes for a C-terminal 672-residue fragment of MEKK1 (24), and all other plasmids have been described (7, 14).
Tissue Culture and Transfection--
HeLa cells were maintained
as described (7). Transfections performed in 3.5-cm-diameter wells were
conducted by calcium phosphate precipitation (25) or by using Fugene 6 according to the manufacturer's instructions (Boehringer Mannheim).
CAT and protein measurements were performed as described (7, 26).
Immunoprecipitations--
Cells were washed once with
Dulbecco's phosphate-buffered saline containing 1 mM EDTA
and then lysed by the addition of 1 ml of buffer B (14) containing 10 µg/ml leupeptin and 1 mM phenylmethylsulfonyl fluoride.
After centrifugation of the whole cell lysate at 16,000 × g for 10 min at 4 °C, the supernatant was incubated with
10 µl of M2-agarose with end over end rotation for 1 h at
4 °C. The resin was then washed three times with buffer B and eluted
by the addition of 20 µl of 2× SDS-PAGE loading buffer.
Western Blotting--
Immunoprecipates were subjected to
SDS-PAGE and then transferred to Immobilon-P membranes (Millipore).
Membranes were blocked and then incubated with anti-I
B
(C-21,
Santa Cruz Biotechnology), anti-phospho-Ser-32 I
B
(New England
Biolabs), anti-Flag (D-8, Santa Cruz Biotechnology), or anti-JNK1
(C-17, Santa Cruz Biotechnology) polyclonal rabbit antibodies. After
washing, the membranes were incubated with anti-rabbit IgG-horseradish
peroxidase conjugates, washed, and then developed using SuperSignal
substrate (Pierce).
Protein Kinase Assays--
Immunocomplex kinase assays for IKK
and JNK were performed essentially as described (14), except that 10 µCi of [
-32P]ATP was employed per assay, 1 µg of
GST-I
B
(5-55) was used in the IKK assays, and the ATP
concentration employed to initiate the IKK reactions was 50 µM instead of 200 µM. Kinase activities were quantitated using a Molecular Dynamics Storm 860 PhosphorImager.
 |
RESULTS |
MEKK2 and MEKK3 Induce NF-
B Activity and Site-specific
Phosphorylation of I
B
--
To examine the possibility that
MAP3Ks other than MEKK1 or NIK might activate NF-
B HeLa cells were
cotransfected with a reporter gene that contains two NF-
B binding
sites and expression constructs for a series of MAP3Ks, including MEKK2
(20), MEKK3 (20), the catalytic domain of MEKK4 (
MEKK4) (21),
apoptosis signal-regulating kinase 1 (ASK1) (22), and mixed-lineage
kinase 3 (MLK3, also known as protein-tyrosine kinase 1 or SH3
domain-containing proline-rich kinase (23, 27, 28). All can activate
the JNK pathway (20-22, 28). In addition, MEKK3, MEKK4, and ASK1 can
activate the p38 pathway (22, 29, 30), whereas MEKK2 and MEKK3 can
activate the ERK pathway (20). As shown in Fig.
1A, under conditions where
overexpression of the positive controls MEKK1 and NIK induces activation of the NF-
B reporter gene, overexpression of MEKK3 (as
reported previously; Ref. 31) and, to a lesser extent, MEKK2 induces
activation as well.
MEKK4, ASK1, and MLK3 did not induce activation
in either these cells (Fig. 1A) or the murine fibroblast cell line L929 (data not shown) but as expected did induce robust activation of coexpressed JNK1 in HeLa cells (data not shown).

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Fig. 1.
Activation of NF- B
and site-specific phosphorylation of
I B induced by MEKK2
and MEKK3. A, HeLa cells were cotransfected by calcium
phosphate precipitation with 3 µg of (PRDII)2CAT and 6 µg of pCMV5-MEKK1, pCMV5-HA-MEKK2, pCMV5-HA-MEKK3, pCMV5- MEKK4,
pcDNA3-NIK, pcDNA3-ASK1, pcDNA3-MLK3, or pCMV5. Cells were
harvested 41 h post-transfection. CAT activities were normalized
to protein concentrations of extracts. Shown is a representative
result, performed in duplicate with standard deviations, from three
independent experiments. B, HeLa cells were cotransfected
using Fugene 6 with 0.5 µg of expression vectors for wild-type
(WT) (pCMV4-FlagI B ) or mutant (M)
(pCMV4-FlagI B (S32A/S36A)) I B , and 4 µg of
pCMV5-HA-MEKK2, pCMV5-HA-MEKK3, or pCMV5. 24 h post-transfection,
the epitope-tagged I B was immunoprecipitated with M2-agarose and
subjected to 12% SDS-PAGE. Top, immunoblotting
(IB) was first performed with anti-I B antibodies
(C-21, Santa Cruz Biotechnology). The positions of unphosphorylated
(I B ) and phosphorylated (P-I B ) are indicated to the
right, and those of molecular mass markers (in kDa) are
shown on the left. Bottom, the immunoblot was
then stripped and reprobed with anti-phospho-Ser-32 I B antibodies
(New England Biolabs). Shown are representative results from three
independent experiments.
|
|
NIK and MEKK1 activate NF-
B by inducing the site-specific
phosphorylation of I
B. In the case of I
B
, this
phosphorylation, which occurs at Ser-32 and Ser-36, is manifested by
slower mobility when I
B
is examined by SDS-PAGE (3, 4). To
examine whether MEKK2 and MEKK3 might act through the same mechanism,
HeLa cells were cotransfected with expression vectors for Flag-tagged
wild-type or phosphorylation-defective (S32A/S36A) I
B
and
expression vectors for MEKK2, MEKK3, or empty expression vector. The
Flag-tagged I
B
was then immunoprecipitated with anti-Flag
antibodies and examined by Western blotting with anti-I
B
antibodies. As shown in Fig. 1B, MEKK2 and MEKK3 both induce
the appearance of a more slowly migrating I
B
species (top
panel, lanes 3 and 5, upper bands) that is abolished when an S32A/S36A I
B
mutant is
examined (lanes 4 and 6), consistent with this
species being N-terminally phosphorylated I
B
.
MEKK4, ASK1, and
MLK3 did not induce the appearance of this more slowly migrating
species (data not shown). This I
B
species was examined further by
reprobing this blot with antibodies specific for phospho-Ser-32
I
B
. As shown in Fig. 1B (bottom panel,
lanes 3 and 5), the slower migrating I
B
species induced by MEKK2 or MEKK3 is immunoreactive with these antibodies. We conclude that MEKK2 and MEKK3 can induce site-specific, N-terminal phosphorylation of I
B
in vivo.
MEKK2 and MEKK3 Activate Both IKK-
and IKK-
--
Both MEKK1
and NIK induce site-specific phosphorylation of I
B by activating
IKK-
and IKK-
. To examine whether MEKK2 or MEKK3 acts by the same
mechanism, HeLa cells were cotransfected with expression constructs for
Flag-tagged IKK-
, IKK-
, or JNK1 and expression constructs for
MEKK1, MEKK2, MEKK3, NIK, or MLK3. The IKK or JNK was then
immunoprecipitated with anti-Flag antibodies, and the kinase activities
of the immunoprecipitated proteins were measured by using as substrates
GST fused to the N terminii of I
B
(residues 5-55) or c-Jun
(residues 1-79), respectively, in the presence of
[
-32P]ATP.
As shown in Fig. 2, (A and
B, top panels), not only do MEKK1 (lane
2) and NIK (lane 5) activate IKK-
and IKK-
activity, as reported previously (10, 11, 13-15), but MEKK2 and MEKK3 do so as well (lanes 3 and 4). Immunoblot
analysis reveals comparable IKK expression levels (Fig. 2, A
and B, lower panels). Furthermore, the MEKK2- or
MEKK3-activated IKK-
and IKK-
display the expected substrate
specificity, because a S32A/S36A double mutation in the I
B
substrate abolishes phosphorylation of the latter (data not shown). As
a negative control, MLK3 does not significantly activate either IKK but
does activate JNK (Fig. 2, A, B, and
C, upper panels, lane 6) as expected
(28). ASK1 and
MEKK4 likewise activate JNK but neither IKK-
nor
IKK-
(data not shown). NIK, in contrast, activates both IKKs but not
JNK (Fig. 2, A, B, and C, upper
panels, lane 5) as reported previously (14, 18).

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Fig. 2.
Activation of both IKK-
and IKK- by MEKK2 and MEKK3. HeLa
cells were cotransfected using Fugene 6 with 3 µg of pCMV5-MEKK1,
pCMV5-HA-MEKK2, pCMV5-HA-MEKK3, pcDNA3-NIK, pcDNA3-MLK3, or pCMV5,
and 0.5 µg of pRK-FlagIKK- (A), pRK-FlagIKK-
(B), or pcDNA3-FlagJNK1 (C). Whole cell
extracts were prepared 24 h post-transfection and divided into two
equal aliquots, and IKK or JNK from each aliquot was then
immunoprecipitated with M2-agarose. One set of immunoprecipitates was
then assayed for kinase activity (KA) toward GST-I B
(5-55) (A and B, top) or GST-cJun (1-79)
(C, top) in the presence of
[ -32P]ATP and then analyzed by 12% SDS-PAGE and
autoradiography. The positions of the substrates are indicated to the
right. The relative degrees of substrate 32P
incorporation are indicated below the gels. The other set of
immunoprecipitates was subjected to SDS-PAGE and analyzed by
immunoblotting (IB) using anti-Flag (D-8, Santa Cruz
Biotechnology) (A and B, bottom) or
anti-JNK1 (C-17, Santa Cruz Biotechnology) (C,
bottom) antibodies. The positions of IKK or JNK are
indicated to the right. Shown are representative results
from three to four independent experiments.
|
|
The potencies of MEKK1, MEKK2, MEKK3, and NIK in activating IKK-
,
IKK-
, or an NF-
B reporter gene were analyzed in more detail (Fig.
3). All four MAP3Ks induce
dose-dependent increases in the activities of coexpressed
IKK-
or IKK-
. In the case of coexpressed IKK-
, the dose
response curves are roughly comparable (Fig. 3A; see also
Fig. 2A). In the case of coexpressed IKK-
, NIK is a
somewhat less potent activator than the other MAP3Ks (Fig.
3B). In contrast, NIK is a substantially more potent
activator of an NF-
B reporter gene than the other three MAP3Ks (Fig.
3C). For example, the NF-
B reporter gene activity induced
by 40 ng of NIK expression vector is comparable or even greater than
that induced by 4000 ng of expression vector for either MEKK1, MEKK2, or MEKK3.

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Fig. 3.
Dose response experiments examining IKK and
NF- B activation by MAP3Ks. HeLa cells
were cotransfected using Fugene 6 with 1 µg of pRK-FlagIKK-
(A), 0.5 µg of pRK-FlagIKK- (B), or 2 µg
of (PRDII)3E1bCAT (C); and 40, 400, or 4000 ng
of pCMV5-MEKK1, pCMV5-HA-MEKK2, pCMV5-HA-MEKK3, or pcDNA3-NIK. The
total DNA dose was brought up to 5 (A), 4.5 (B),
or 6 µg (C) with pCMV5. A and B, IKK
was immunoprecipitated with M2-agarose from whole cell extracts
prepared 23-24 h post-transfection and assayed for activity toward
GST-I B (5-55) in the presence of [ -32P]ATP. IKK
expression levels were analyzed by immunoblots of aliquots of the whole
cell extracts using anti-Flag antibodies. C, cell extracts
prepared 25 h post-transfection were assayed for CAT activity and
normalized to the protein concentrations of the extracts. Shown are
representative results from two to three independent experiments.
|
|
Dominant Negative IKK-
and Dominant Negative IKK-
Inhibit
MEKK2- and MEKK3-induced NF-
B Activation--
The experiments
described above indicate that MEKK2 and MEKK3 can activate both IKK-
and IKK-
in vivo. To examine whether this activation is
functionally significant, HeLa cells were cotransfected with expression
constructs for MEKK1, MEKK2, MEKK3, or empty expression vector,
expression constructs for dominant negative, catalytically inactive
IKK-
(K44A), IKK-
(K44A), or empty expression vector, and an
NF-
B reporter gene. As shown in Fig.
4, under conditions where activation of
the NF-
B reporter gene induced by MEKK1 is almost completely
inhibited by dominant negative IKK-
or dominant negative IKK-
(13, 14, 32), that induced by either MEKK2 and MEKK3 is completely
abolished. This therefore provides evidence that IKK-
and IKK-
mediate the NF-
B inducing activity of MEKK2 and MEKK3.

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Fig. 4.
Inhibition of the
NF- B-inducing activity of MEKK2 and MEKK3 by
dominant negative IKK- and dominant negative
IKK- . HeLa cells were cotransfected using
Fugene 6 with 1.5 µg of (PRDII)3E1bCAT, 3 µg of
pCMV5-MEKK1, pCMV5-HA-MEKK2, pCMV5-HA-MEKK3, or pCMV5, and 1.5 µg of
pRK-FlagIKK- (K44A), pRK-FlagIKK- (K44A), or pCMV5. Cells were
harvested 25 h post-transfection. CAT activities were normalized
to protein concentrations of extracts. Shown is a representative
result, performed in duplicate with standard deviations, from two
independent experiments.
|
|
 |
DISCUSSION |
Here we identify two additional, previously cloned MAP3Ks: MEKK2
and MEKK3, which now join NIK and MEKK1 as activators of IKK and
NF-
B, thereby enlarging our framework for understanding NF-
B
activation. Such knowledge is essential to providing a foundation for
understanding how NF-
B can be activated by such a wide variety of
stimuli. That a distinct subset of NF-
B-inducing MAP3Ks exists is
highlighted by the fact that other MAP3Ks identified as activators of
the JNK and/or p38 pathways, such as MEKK4, ASK1, and MLK3, fail to
activate NF-
B.
Titration experiments reveal that MEKK2 and MEKK3 are comparable in
potency to NIK in activating coexpressed IKK-
or IKK-
but are
substantially less potent than NIK in activating an NF-
B reporter
gene (Fig. 3). Possible factors that might contribute to this apparent
discrepancy include the following: (i) MEKK2 and MEKK3 might activate
intracellular pathways that inhibit the NF-
B pathway and therefore
could be relatively less effective in activating an NF-
B reporter
gene; (ii) NIK might activate other components of the NF-
B pathway
besides IKK and thus could be relatively more potent in activating an
NF-
B reporter gene; and (iii) overexpressed IKK might respond less
sensitively than endogenous IKK to coexpressed MAP3K and therefore
might not accurately reflect activation of the NF-
B pathway (33). In
any case, relative potencies in transient overexpression assays cannot
be used as the sole criterion for assessing physiologic significance. A
particularly pertinent example is provided by the fact the HTLV-I
protein Tax activates NF-
B through MEKK1 (15) despite the fact that
this MAP3K, like MEKK2 or MEKK3, is substantially less potent
than NIK in activating an NF-
B reporter gene (14, 18).
MEKK2 and MEKK3, like other MAP3Ks, contain both catalytic and
noncatalytic domains. The catalytic domains of MEKK2 and MEKK3 share
96% homology, consistent with the fact that both can activate NF-
B,
whereas their noncatalytic domains are 65% homologous (20). MEKK2 is
activated by treatment of cells by epidermal growth factor (34). In
addition, MEKK2 and MEKK3 bind 14-3-3 proteins, an interaction that is
mediated at least in part through their catalytic domains but that does
not modulate their JNK-inducing activities (35).
Further experimentation will be required to determine the detailed
mechanism by which MEKK2 and MEKK3 activate IKK-
and IKK-
. NIK
activates IKK-
by inducing phosphorylation of Ser-176 in the
activation loop of the latter (16). Phosphorylation of Ser-177 and/or
Ser-181 in IKK-
is essential for its activity, because a double
S176A/S181A mutation abolishes activity (12). Therefore, MEKK2 and
MEKK3 might directly phosphorylate these IKK residues, particularly
because these residues are components of canonical MAP2K activation
loop motifs (SXXXS) (12) that might be predicted to be
substrates for MAP3Ks. It is worth noting, however, that definitive
experimental evidence that either NIK or MEKK1 directly induce IKK-
or IKK-
catalytic activity has yet to be reported.
The fact that many MAP3Ks have the capacity to activate distinct
pathways now raises the problem of how specificity in signaling pathways is achieved. One example of this is provided by the
observation that Tax activates MEKK1 and induces potent NF-
B
activity (15) but only modest JNK activity (15, 36), despite the fact
that MEKK1 overexpression coordinately activates both (7). Thus, the
relative capacities of a MAP3K to activate distinct signaling pathways
may be modulated in a manner that is stimulus-specific.
 |
ACKNOWLEDGEMENTS |
We are grateful to Drs. Gary Johnson,
Hidenori Ichijo, Richard Spritz, Roger Davis, David Wallach, David
Goeddel, Dean Ballard, and Tom Maniatis for gifts of plasmids. We thank
Drs. Mark Tykocinski and Leonard Jarett for support and encouragement.
 |
FOOTNOTES |
*
This work was supported in part by a Pilot Project Award
from the Thomas B. McCabe and Jeannette E. Laws McCabe Fund.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Pathology and
Laboratory Medicine, University of Pennsylvania School of Medicine, 218 John Morgan Bldg., Philadelphia, PA 19104. Tel.: 215-898-4701; Fax:
215-573-2272; E-mail: franklee{at}mail.med.upenn.edu.
 |
ABBREVIATIONS |
The abbreviations used are:
NF-
B, nuclear
factor-
B;
ASK1, apoptosis signal-regulating kinase 1;
CAT, chloramphenicol acetyl transferase;
GST, glutathione
S-transferase;
HTLV-I, human T-cell leukemia virus, type I;
IKK, I
B kinase;
JNK, c-Jun N-terminal kinase;
MAPK, mitogen-activated protein kinase;
MAP2K, mitogen-activated protein
kinase kinase;
MAP3K, mitogen-activated protein kinase kinase
kinase;
MEKK, mitogen-activated protein kinase/ERK kinase kinase;
MLK3, mixed-lineage kinase 3;
NIK, NF-
B inducing kinase;
PAGE, polyacrylamide gel electrophoresis;
TNF-
, tumor necrosis factor
.
 |
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