MOLECULAR AND CELLULAR BIOLOGY, Nov. 2004, p. 9658–9667
0270-7306/04/$08.00?0 DOI: 10.1128/MCB.24.21.9658–9667.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.
Vol. 24, No. 21
Lipopolysaccharide Activation of the TPL-2/MEK/Extracellular
Signal-Regulated Kinase Mitogen-Activated Protein Kinase
Cascade Is Regulated by I?B Kinase-Induced
Proteolysis of NF-?B1 p105†
S. Beinke,1‡ M. J. Robinson,1‡ M. Hugunin,2and S. C. Ley1*
Division of Immune Cell Biology, National Institute for Medical Research, London, United Kingdom,1
and Molecular and Cellular Biology, Abbott Bioresearch Center, Worcester, Massachusetts2
Received 27 January 2004/Returned for modification 4 March 2004/Accepted 3 August 2004
The MEK kinase TPL-2 (also known as Cot) is required for lipopolysaccharide (LPS) activation of the
extracellular signal-regulated kinase (ERK) mitogen-activated protein (MAP) kinase cascade in macrophages
and consequent upregulation of genes involved in innate immune responses. In resting cells, TPL-2 forms a
stoichiometric complex with NF-?B1 p105, which negatively regulates its MEK kinase activity. Here, it is shown
that lipopolysaccharide (LPS) stimulation of primary macrophages causes the release of both long and short
forms of TPL-2 from p105 and that TPL-2 MEK kinase activity is restricted to this p105-free pool. Activation
of TPL-2, MEK, and ERK by LPS is also demonstrated to require proteasome-mediated proteolysis. p105 is
known to be proteolysed by the proteasome following stimulus-induced phosphorylation of two serines in its
PEST region by the I?B kinase (IKK) complex. Expression of a p105 point mutant, which is not susceptible to
signal-induced proteolysis, in RAW264.7 macrophages impairs LPS-induced release of TPL-2 from p105 and
its subsequent activation of MEK. Furthermore, expression of wild-type but not mutant p105 reconstitutes LPS
stimulation of MEK and ERK phosphorylation in primary NF-?B1-deficient macrophages. Consistently,
pharmacological blockade of IKK inhibits LPS-induced release of TPL-2 from p105 and TPL-2 activation.
These data show that IKK-induced p105 proteolysis is essential for LPS activation of TPL-2, thus revealing a
novel function of IKK in the regulation of the ERK MAP kinase cascade.
Lipopolysaccharide (LPS) stimulation of Toll-like receptor 4
(TLR4) on macrophages leads to the induction of genes that
function in the innate and adaptive immune responses to gram-
negative bacterial infection (32). These include proinflamma-
tory cytokines, chemokines, the major histocompatibility com-
plex, and costimulatory molecules (23). LPS induction of these
genes involves activation of NF-?B transcription factors and
each of the major mitogen-activated protein (MAP) kinase
subtypes (extracellular signal-regulated kinases 1 and 2 [ERK-
1/2], Jun amino-terminal kinases, and p38) (32).
MAP kinases are phosphorylated and activated by MAP
kinase kinases, which in turn are phosphorylated and activated
by MAP kinase kinase kinases in conserved three-tiered kinase
cascades (9). LPS activation of ERK-1/2 MAP kinases in mac-
rophages requires the serine/threonine kinase TPL-2 (12) (also
known as Cot ). TPL-2 functions as a MAP kinase kinase
kinase which phosphorylates and activates the ERK-1/2 ki-
nases, MEK-1/2 (28). LPS induction of tumor necrosis factor
alpha (TNF-?) and cyclooxygenase 2 (COX-2) is dramatically
reduced in TPL-2-deficient macrophages due to defective
ERK-1/2 activation (12, 13). Consequently, TPL-2?/?mice are
resistant to LPS/D-galactosamine-induced endotoxin shock
(12). TPL-2 is also required for TNF-? and CD40 ligand to
stimulate MEK-1/2 activation (14), suggesting an important
role for TPL-2 in both innate and adaptive immune responses.
NF-?B dimers are retained in the cytoplasm of unstimulated
cells through their interaction with a family of inhibitory pro-
teins, termed I?Bs (15). The I?B family includes NF-?B1 p105,
which retains associated NF-?B dimers by virtue of its C-
terminal ankyrin repeats. p105 is also constitutively processed
by the proteasome to produce the NF-?B transcription factor,
p50 (19). Genetic studies with mice have indicated that p105 is
particularly important for the cytoplasmic retention of p50
homodimers (17). Following cellular stimulation with ligands,
such as TNF-?, two serines in the p105 PEST region are
rapidly phosphorylated by the I?B kinase (IKK) complex (20,
29). This creates a binding site for the ubiquitin E3 ligase,
SCF?TrCP, which promotes p105 ubiquitination (16, 20, 26),
leading predominantly to the complete degradation of p105 by
the proteasome. Associated NF-?B (Rel) subunits are thereby
released to translocate into the nucleus and modulate target
Earlier studies from this laboratory demonstrated that the
C-terminal half of NF-?B1 p105 forms a high-affinity, stoichi-
ometric association with TPL-2 (3, 4). This interaction is re-
quired to maintain TPL-2 protein stability. Consequently, the
steady-state levels of TPL-2 are very low in p105-deficient cells
(3, 37), and LPS activation of MEK is severely reduced in bone
marrow-derived macrophages (BMDMs) generated from NF-
?B1?/?mice (37). Interaction of p105 with TPL-2 also nega-
tively regulates its MEK kinase activity by preventing access to
* Corresponding author. Mailing address: National Institute for
Medical Research, Division of Immune Cell Biology, The Ridgeway,
Mill Hill, London NW7 1AA, United Kingdom. Phone: 44-20-8816-
2463. Fax: 44-20-8906-4477. E-mail: firstname.lastname@example.org.
‡ S.B. and M.J.R. contributed equally to this study.
† Supplemental material for this article may be found at http:
MEK (3, 37). In unstimulated BMDMs, therefore, TPL-2
MEK kinase activity is blocked (37), since all detectable TPL-2
is complexed with p105 (21). However, following LPS stimu-
lation, TPL-2 MEK kinase activity increases, indicating that
TPL-2 is released from p105 inhibition (37).
In the present study, the mechanism by which TPL-2 is
activated after LPS stimulation of BMDMs was investigated.
Evidence is presented that IKK-induced p105 proteolysis is
required to generate a pool of p105-free TPL-2 in LPS-stim-
ulated cells which phosphorylates and activates MEK. Conse-
quently, LPS activation of ERK in macrophages is dependent
on the activity of the IKK complex.
MATERIALS AND METHODS
cDNA constructs, antibodies, and recombinant proteins. Hemagglutinin (HA)
epitope-tagged wild-type NF-?B1 p105 (HA-p105), HA-p105S927A,S932A(HA-
p105SSAA), Myc epitope-tagged wild-type TPL-2 (Myc-TPL-2), and kinase-inac-
tive Myc-TPL-2D270A(Myc-TPL-2KD) cDNAs have been described previously (4,
20). For stable transfection of RAW264.7 cells, these cDNAs were subcloned in
the pMX-1 vector (Ingenius). For retroviral infection of BMDMs, FLAG
epitope-tagged wild-type p105 (FL-p105) and p105S927A,S932A(FL-p105SSAA)
were generated by PCR, subcloned into a pMSCV-based vector, and verified by
The rabbit antibodies used to immunoprecipitate TPL-2 (70-mer) and NF-?B1
p105 (anti-p105N) have previously been described (20). For Western blotting,
anti-p105C antibody was used to detect total p105 (29), and a previously de-
scribed anti-phospho-S927-p105 antibody (29) was used to detect p105 phosphor-
ylated on serine 927. A commercial anti-TPL-2 antibody (Santa Cruz) was used
to detect TPL-2 on Western blots. Antibodies against MEK-1/2, phospho(S217/
S221)-MEK-1/2 (activated MEK-1/2; phospho-MEK), p38, and phospho(T180/
Y182)-p38 (activated p38; phospho-p38) were purchased from Cell Signaling
Technology. Anti-ERK-1b was generously provided by Jeremy Tavare (Univer-
sity of Bristol, Bristol, United Kingdom), and anti-phospho(T185/Y187)-ERK
(activated ERK; phospho-ERK) was purchased from Biosource. Antibodies used
to immunoprecipitate and Western blot HA epitope-tagged proteins have been
described previously (20). Tubulin (detected with TAT-1 anti-?-tubulin mono-
clonal antibody, kindly provided by Keith Gull, University of Manchester, United
Kingdom) was used as a loading control protein on Western blots of total cell
extracts. Glutathione S-transferase (GST)–MEK and GST-ERK proteins were
both kindly provided by Richard Marais (CR-UK, London, United Kingdom).
Mouse strains and cell lines. NF-?B1 knockout mice (30) (from Jackson
Laboratories, Bar Harbor, Maine) and BALB/c mice were bred in a specific-
pathogen-free environment at the National Institute for Medical Research (Lon-
don, United Kingdom).
BMDMs from BALB/c mice were prepared as described previously (36).
Briefly, bone marrow cells were plated in complete BMDM medium (RPMI
1640; Sigma) supplemented with 10% fetal bovine serum, antibiotics, and 20%
L-cell conditioned medium. After 24 h, nonadherent cells were transferred to
90-mm dishes (Nunc; 6 ? 106cells). On day 4 of culture, the cells were further
supplemented with complete BMDM medium. After a total of 7 days of culture,
the adherent macrophages were harvested and plated for experiments. More
than 95% of the resulting cell populations were positive for the macrophage
marker F4/80, as judged by flow cytometric analysis (data not shown).
RAW264.7 cells were kindly provided by Lynn Williams (Kennedy Institute,
London, United Kingdom) and maintained in Dulbecco’s modified Eagle’s me-
dium (Invitrogen) containing 10% fetal bovine serum and antibiotics. For stable
transfection, 5 ? 107RAW264.7 cells were transfected by electroporation with
10 ?g of pMX-1 expression vector containing cDNA inserts encoding HA-p105,
HA-p105SSAA, Myc-TPL-2, or MycTPL-2KD. As a control, cells were transfected
with pMX-1 vector containing no insert (empty vector [EV]). Transfected cells
were cultured for a further 48 h and cloned by limiting dilution under neomycin
selection (complete Dulbecco’s modified Eagle’s medium plus 1 mg of G418/ml;
Invitrogen). After 1 to 3 weeks, clones were expanded and tested for expression
of transfected proteins by Western blotting for HA epitope tag or TPL-2. Pos-
itive clones were maintained in selection medium. Prior to experiments, the
RAW264.7 cell lines were passaged once without G418. All experiments were
performed with at least two independent clones and generated similar results.
Protein analysis. BMDMs (3 ? 106cells) or RAW264.7 cells (4 ? 106) were
plated in 60-mm dishes (Nunc). After 18 h in culture, cells were stimulated with
LPS (1 ?g/ml; Salmonella enterica serovar Minnesota; Alexis Biochemicals) for
the times shown or phorbol myristate acetate (PMA) (100 ng/ml; Sigma) for 7.5
min or left untreated. Where appropriate, cells were preincubated with the
indicated concentrations of MG132 proteasome inhibitor (Biomol), BAY 11-
7082 IKK inhibitor (Calbiochem), or dimethyl sulfoxide (DMSO) vehicle control
for 30 min prior to stimulation. Cells were washed once in phosphate-buffered
saline prior to lysis in 1% NP-40 containing buffer A (50 mM Tris [pH 7.5], 150
mM NaCl, 1 mM EDTA, 1 mM EGTA, 50 mM NaF, 1 mM Na3VO4, 100 nM
okadaic acid [Calbiochem], 2 mM Na4P2O7plus a mixture of protease inhibitors
[Roche Molecular Biochemicals]).
For experiments in which total p105 levels were quantified, washed BMDMs
were extracted directly into sodium dodecyl sulfate-polyacrylamide gel electro-
phoresis (SDS-PAGE) sample buffer. Extracts were passed through a 23-gauge
needle to shear DNA and boiled prior to SDS-PAGE. p105 levels were detected
on Western blots by enhanced chemiluminescence (Amersham Biosciences) and
quantified using a Fuji image reader (LAS-3000). p105 levels were normalized
Covalent coupling of antibodies to protein A-Sepharose (Amersham Bio-
sciences), immunoprecipitation, and Western blotting of proteins were carried
out as described previously (18). p105 was precleared from BMDM lysates by
immunodepletion twice with anti-p105N antibody coupled to protein A-Sepha-
rose. p105 was similarly immunodepleted from lysates of stably transfected
RAW264.7 cells but with a mixture of anti-p105N and anti-HA (12CA5) anti-
bodies coupled to protein A Sepharose. Complete depletion of p105 was con-
firmed by Western blotting. The percentage of TPL-2 released from p105 after
LPS stimulation of BMDMs was determined by scanning Western blots using a
Bio-Rad calibrated imaging densitometer (GS710). TPL-2 levels were normal-
ized against ?-tubulin.
Assay of TPL-2 phosphorylation and MEK kinase activity. To analyze TPL-2
phosphorylation, LPS-stimulated BMDMs (8 ? 106cells) were lysed in kinase
lysis buffer (buffer A containing 0.5% NP-40, 5 mM Na ?-glycerophosphate, and
0.1% 2-mercaptoethanol,) and TPL-2 was immunoprecipitated for 4 h with
anti-TPL-2 antibody coupled to protein A-Sepharose. Beads were washed twice
in kinase lysis buffer and twice in protein phosphatase 2A (PP2A) buffer (Up-
state) and then resuspended in 50 ?l of PP2A buffer. PP2A (0.5 U; Upstate) was
added to the appropriate tubes with or without the phosphatase inhibitors, NaF
(1 mM) and okadaic acid (100 nM). Control tubes had no addition. Samples were
incubated for 30 min at 30°C. Bound TPL-2 was eluted from beads by boiling in
SDS-PAGE sample buffer and then subjected to Western blotting after 10%-
To assay TPL-2 MEK kinase activity, BMDMs (8 ? 106cells) or RAW264.7
cells (10 ? 106cells) were plated in 90-mm dishes (Nunc). After 18 h in culture,
cells were stimulated with LPS for the indicated times and then extracted with
kinase lysis buffer. Lysates were immunoprecipitated for 4 h with anti-TPL-2
antibody coupled to protein A-Sepharose. To analyze p105-free TPL-2, lysates
were first immunodepleted twice for 2 h with p105N antibody or control immu-
noglobulin coupled to protein A-Sepharose. Subsequently, these cleared lysates
were immunoprecipitated with anti-TPL-2 antibody, anti-p105N antibody, or
control immunoglobulin (Ig). Specific antibody-coupled beads were washed four
times in kinase lysis buffer and twice with kinase buffer (50 mM Tris [pH 7.5], 5
mM ?-glycerophosphate, 0.1 mM sodium vanadate, 100 nM okadaic acid, 10 mM
MgCl2, 0.1 mM EGTA, 0.03% Brj35, 0.1% 2-mercaptoethanol). The beads were
resuspended in 25 ?l of kinase buffer supplemented with 1 mM ATP, 6.5 ?g of
GST-MEK/ml, and 100 ?g of GST-ERK/ml and incubated for 30 min at room
temperature on a shaker. After beads were pelleted by centrifugation, 2 ?l of the
supernatant was added to 48 ?l of kinase buffer containing 0.33 mg of myelin
basic protein (MBP) (Sigma)/ml, 0.1 mM ATP, and 2.5 ?Ci of [?-32P]ATP
(Amersham Biosciences) and incubated at room temperature for 10 min. The
assay was terminated by adding 50 ?l of 2? SDS sample buffer, and labeled MBP
was revealed by autoradiography after 12.5% acrylamide SDS-PAGE. Immuno-
precipitated protein was eluted from the antibody-coupled beads with 0.2 M
glycine (pH 2.5), resolved by 10% acrylamide SDS-PAGE, and subjected to
Retrovirus infection of BMDMs. Amphoteric recombinant retrovirus were
produced by transfecting wild-type FL-p105 or FL-p105SSAA, subcloned in the
pMSCV-based vector, into the Plat-E packaging cell line (25). These cells were
transfected using GeneJuice transfection reagent (Merck Biosciences) according
to the manufacturer’s instructions. Transfected cells were cultured at 37°C for 48
to 72 h, at which time culture supernatants were removed and filtered (pore size,
0.4 ?m). Virus was concentrated 10-fold by centrifugation prior to BMDM
BMDMs from NF-?B1?/?mice were prepared essentially as described above,
with minor modifications. Bone marrow cells were plated in complete BMDM
VOL. 24, 2004 ROLE FOR IKK IN REGULATION OF ERK MAP KINASE CASCADE9659
medium at 106cells/well (culture volume, 2 ml) of a six-well plate (Sarstedt).
Following 48 h of incubation, 200 ?l of concentrated virus was added per well,
and the plates were centrifuged at 2,000 ? g for 1 h at room temperature. Each
well was further supplemented with complete BMDM medium (2 ml) 2 and 48 h
after infection. On day 7, cells were replated in fresh BMDM medium (1.5 ? 106
cells/well; Nunc six-well plates). On the following day, BMDMs were stimulated
with LPS (10 ng/ml) and analyzed as described above. Flow cytometric analysis
confirmed that ?95% of cells prepared in this way were F4/80 positive.
LPS stimulation of primary macrophages induces release of
TPL-2 from p105. The MEK kinase activity of TPL-2 is inhib-
ited by its interaction with NF-?B1 p105 (3, 37). In unstimu-
lated BMDMs, the majority of TPL-2 is associated with p105
(Fig. 1D) (21), and consequently, TPL-2 MEK kinase activity
cannot be detected in anti-TPL-2 immunoprecipitates (37)
(Fig. 1A). However, LPS stimulation of BMDMs causes a
marked increase in TPL-2 MEK kinase activity with kinetics
similar to phosphorylation of endogenous MEK on its activa-
tion loop (Fig. 1A) (37). In the following sections, the physi-
ological mechanism by which TPL-2 is released from p105
inhibition after LPS stimulation is investigated.
In initial experiments, the effect of LPS on TPL-2 was de-
termined by Western blotting. Alternative translational initia-
tion on a second methionine (at residue 30) results in the
expression of two TPL-2 isoforms (1), M1-TPL-2 and M30-
TPL-2 (Fig. 1A). Similar to published experiments (37), LPS
induced proteolysis of the longer isoform, M1-TPL-2, at 30
min, whereas the shorter isoform, M30-TPL-2, was stable (Fig.
1A and C). Prior to inducing its degradation, LPS stimulation
induced a marked decrease in mobility of essentially all of
M1-TPL-2 in SDS-PAGE (Fig. 1A and C). In some experi-
ments, a very small fraction of M30-TPL-2 also underwent an
LPS-induced mobility shift. Treatment of anti-TPL-2 immuno-
precipitates with PP2A demonstrated that these mobility shifts
were due to phosphorylation of TPL-2 (Fig. 1B).
Western blotting of anti-p105N immunoprecipitates re-
vealed a decrease in copurifying M1-TPL-2 after LPS stimu-
lation (Fig. 1C). Previously this LPS-induced decrease in p105-
FIG. 1. LPS stimulation induces a p105-free pool of TPL-2 which activates MEK. BMDMs (BALB/c) were stimulated with LPS for the
indicated times. (A) TPL-2 was immunoprecipitated from cell lysates with anti-TPL-2 antibody, and its MEK kinase activity was determined by
coupled MEK/ERK kinase assay. Labeled MBP substrate was visualized by autoradiography after SDS-PAGE, and the levels of immunoprecipi-
tated TPL-2 were determined by Western blotting. Lysates were also subjected to Western blotting with an anti-phospho-(S217/S221)-MEK-1/2
antibody (phospho-MEK) to determine activation of endogenous MEK by LPS. (B) TPL-2 was immunoprecipitated from cell lysates. Beads were
incubated with 0.5 U of PP2A, 0.5 U of PP2A plus the phosphatase inhibitors NaF and okadaic acid (PP2A ? Inhibitors), or control buffer.
Immunoprecipitated TPL-2 was revealed by Western blotting. (C) Cell lysates and anti-p105 immunoprecipitates were subjected to Western
blotting. (D) Total cell lysates (lysates) and p105 cleared lysates were subjected to Western blotting for TPL-2 and p105. Equal protein loading
was confirmed by probing for ?-tubulin. (E) Cell lysates were immunoprecipitated with anti-TPL-2 antibody, anti-p105N antibody, or control Ig
(control) after first immunodepleting with either anti-p105N antibody (p105) or control Ig (control) as indicated. Immunoprecipitates were assayed
for associated MEK kinase activity and TPL-2 levels as for panel A. Cell lysates were subjected to Western blotting to confirm depletion of p105
and activation of MEK phosphorylation by LPS.
9660 BEINKE ET AL.MOL. CELL. BIOL.
associated TPL-2 had been suggested to result from the
selective dissociation of M1-TPL-2 from p105 (37). However,
this was not clear from the present analyses due to proteolysis
of both M1-TPL-2 and p105 after LPS stimulation (Fig. 1A and
C). Therefore, to determine conclusively whether LPS induces
the dissociation of TPL-2 from its inhibitor p105, an assay was
developed which could directly detect low levels of p105-free
TPL-2. To do this, lysates of BMDMs were precleared of p105
by immunodepletion and then subjected to Western blotting
for TPL-2. Similar to previous results (21), no p105-free TPL-2
was detected in unstimulated cells (Fig. 1D). However, LPS
stimulation induced the appearance of both M1-TPL-2 and
M30-TPL-2 in the p105-depleted lysate, peaking at 15 min, at
which time TPL-2 MEK kinase activity was also maximal (Fig.
1A). By densitometric scanning of Western blots, it was deter-
mined that 21% (standard error of the mean [SEM], ?4.6%;
n ? 4) of total TPL-2 present in unstimulated cells was re-
leased from p105 after 15 min of LPS stimulation.
To establish whether activated TPL-2 resides in the p105-
free pool, TPL-2 was immunoprecipitated from lysates of LPS-
stimulated BMDMs which had been precleared with either
anti-p105 antibody or control Ig. Similar levels of TPL-2 MEK
kinase activity were isolated from p105-free and control ly-
sates, despite the amount of TPL-2 immunoprecipitated from
p105-free lysates being only a fraction of that isolated from
control lysates (Fig. 1E). Consistent with published data (37),
no MEK kinase activity was detected when p105 was isolated
directly with anti-p105N antibody, although large amounts of
TPL-2 were copurified (Fig. 1E).
Together these data show that LPS activation of TPL-2 in
BMDMs involves the release of both M1 and M30 TPL-2 from
p105 and that TPL-2 MEK kinase activity is restricted to this
LPS activation of TPL-2 and MEK requires proteasome
activity. LPS stimulates p105 proteolysis by the proteasome in
THP-1 monocytes and 70Z/3 pre-B cells (11, 16). Therefore,
one possible mechanism by which LPS might promote the
release of TPL-2 from p105 in BMDMs is to stimulate protea-
some-mediated proteolysis of p105.
To determine whether LPS induces p105 proteolysis in BM-
DMs rapidly enough to account for the kinetics of TPL-2
release from p105 and TPL-2 activation, p105 levels were
quantified by Western blotting in replicate experiments. To
ensure that total p105 was assayed, cells were extracted directly
into SDS-PAGE sample buffer. After 15 min of LPS stimula-
tion, when LPS-induced release of TPL-2 from p105 and
TPL-2 MEK kinase activity were maximal (Fig. 1A and D),
p105 levels decreased by 28% (standard error of the mean,
?4%; n ? 3; P ? 0.02) compared with unstimulated control
(Fig. 2A). Treatment of BMDMs with the proteasome inhibi-
tor MG132 prior to stimulation blocked the LPS-induced de-
creases in the levels of both p105 and I?B? (Fig. 2B). Prote-
olysis of M1-TPL-2 induced by LPS stimulation was also
inhibited by MG132 (Fig. 2B). These data indicate that LPS
stimulation of BMDMs induces proteasome-mediated prote-
olysis of p105 with kinetics that correlate with TPL-2 release
from p105 and TPL-2 activation.
Next, pharmacological blockade of the proteasome with
MG132 was used to investigate initially whether p105 proteol-
ysis might be an important step in the generation of p105-free
active TPL-2 in BMDMs. Western blotting with phospho-an-
tibodies revealed that MG132 inhibited LPS stimulation of
MEK and ERK phosphorylation (Fig. 2C) in a dose-dependent
fashion (supplemental figure). However, LPS activation of p38
phosphorylation (Fig. 2C) and phorbol ester stimulation of
MEK phosphorylation (Fig. 2D), which are both independent
of TPL-2 (12), were unaffected by MG132 treatment. Two
other inhibitors of proteasome function, clasto-lactacystin
?-lactone and ALLN, also inhibited LPS-induced phosphory-
lation of MEK and ERK in a dose-dependent fashion (supple-
mental figure). Each of the proteasome inhibitors blocked LPS
stimulation of MEK and ERK phosphorylation, p105 proteol-
ysis, and I?B? degradation with similar potency. ALLM, an
ALLN congener that is not active on the proteasome, did not
affect LPS induction of MEK and ERK phosphorylation. Sim-
ilar to MG132, clasto-lactacystin ?-lactone and ALLN had no
effect on LPS stimulation of p38 phosphorylation.
Western blotting of p105-depleted lysates revealed that LPS-
induced release of TPL-2 from p105 was prevented by MG132
treatment (Fig. 2E). Furthermore, MG132 was also found to
significantly reduce LPS stimulation of TPL-2 MEK kinase
activity detected in coupled MEK/ERK kinase assays (Fig. 2F).
The experiments described in this section demonstrate that
there is an essential proteasome-mediated proteolytic step in
the TLR4 signaling pathway that activates the ERK MAP
kinase cascade and suggest that this regulates the generation of
the pool of p105-free TPL-2 which can phosphorylate MEK.
Signal-induced proteolysis of NF-?B1 p105 is essential for
LPS activation of MEK phosphorylation. Stimulation of p105
proteolysis by LPS is mediated via the IKK complex (16).
TNF-?-induced proteolysis of p105 is induced by IKK phos-
phorylation of p105 serines 927 and 932 (20, 29). A phos-
phopeptide-specific antibody which recognizes p105 phosphor-
ylated on serine 927 (29) was used to confirm that LPS-induced
proteolysis of p105 also involves phosphorylation of the p105
PEST region. To do this, p105 was immunoprecipitated from
lysates of BMDMs, which had been pretreated with MG132
prior to LPS stimulation, and subjected to Western blotting
with an anti-phospho-S927-p105 antibody (29). LPS stimula-
tion induced the rapid phosphorylation of p105 on serine 927
(Fig. 2G). Significantly, LPS stimulation induced maximal p105
serine 927 phosphorylation at 7.5 min. Thus, LPS-induced
phosphorylation of p105 is very rapid and precedes MEK phos-
phorylation, which peaks at 15 min (Fig. 1A). In the following
experiments, a genetic approach was taken to determine the
importance of IKK-induced phosphorylation of p105 in LPS-
induced TPL-2 activation.
TPL-2 is the major MEK kinase activated by LPS in the
murine macrophage cell line RAW264.7 (8). Stable expression
of kinase-inactive TPL-2 in these cells blocked LPS induction
of MEK phosphorylation, while induction of p38 phosphory-
lation was not affected (Fig. 3A). Thus, LPS activation of
MEK is dependent on TPL-2 kinase activity in RAW264.7
cells. In addition, MG132 pretreatment of RAW264.7 cells
inhibited LPS stimulation of MEK phosphorylation (Fig. 3B),
TPL-2 release from p105 (Fig. 3C), and TPL-2 activation (Fig.
3D). Therefore, similar to primary macrophages, LPS activa-
tion of TPL-2 and MEK requires proteasome activity in
RAW264.7 cells. In the following experiments, RAW264.7
cells were used as a model system to determine whether the
VOL. 24, 2004ROLE FOR IKK IN REGULATION OF ERK MAP KINASE CASCADE 9661
inhibitory effect of MG132 reflects a requirement for signal-
induced p105 proteolysis in LPS activation of TPL-2 and MEK.
RAW264.7 cells were stably transfected with vectors encod-
ing wild-type HA-p105 (HA-p105WT) or a mutant with inacti-
vating mutations at the IKK phosphorylation sites, HA-
p105SSAA(20, 29), which is not susceptible to signal-induced
proteolysis. Clones were selected which expressed similar lev-
els of transfected protein. LPS stimulation of MEK phosphor-
ylation was very similar in cells transfected with HA-p105WT
compared with empty vector control (Fig. 4A). However, LPS-
induced MEK phosphorylation was significantly reduced (60%
? 10% reduction; n ? 3; P ? 0.01) in HA-p105SSAA-trans-
fected cells from levels in HA-p105WTcells (Fig. 4A). LPS-
induced p38 phosphorylation (Fig. 4A) was similar in the two
cell lines (98% ? 12%; n ? 3). There was also no difference in
phorbol ester activation of MEK phosphorylation (Fig. 4B).
Analysis of p105-depleted lysates indicated that LPS induc-
tion of TPL-2 release from p105 was markedly reduced by
expression of HA-p105SSAAcompared with results for HA-
p105WT(Fig. 4C). Additionally, coupled MEK/ERK kinase
assays demonstrated that LPS activation of TPL-2 MEK kinase
activity was dramatically reduced in p105SSAA-transfected cells
relative to that in HA-p105WTtransfected cells (Fig. 4D).
Thus, p105 proteolysis is required for LPS stimulation to in-
FIG. 2. Proteasome activity is required for LPS activation of TPL-2. (A) BMDMs were simulated with LPS for the indicated times. Total p105
protein levels in cell lysates were determined by Western blotting and quantified using a Fuji Image reader. Data are presented as means (? SEM;
n ? 3), normalized against ?-tubulin. (B to E) BMDMs were preincubated with the proteasome inhibitor MG132 (40 ?M) or DMSO vehicle
control for 30 min and then stimulated with LPS or PMA for the indicated times. Total cell lysates were subjected to Western blotting for the
indicated proteins. LPS activation of endogenous MEK, ERK, and p38 was monitored with the appropriate phospho-specific antibodies. (E) Cell
lysates were immunodepleted of p105 prior to Western blotting for TPL-2 and ?-tubulin. (F) BMDMs were treated with MG132 (40 ?M) prior
to stimulation with LPS for 15 min. TPL-2 was immunoprecipitated from total cell lysates and then assayed for MEK kinase activity as for Fig.
1A. (G) BMDMs, pretreated with 40 ?M MG132, were simulated with LPS for the indicated times. p105 was immunoprecipitated from total cell
lysates, subjected to Western blotting, and probed with a specific anti-phospho-peptide antibody to monitor p105 serine 927 phosphorylation.
9662 BEINKE ET AL.MOL. CELL. BIOL.
duce efficiently release of TPL-2 from p105, TPL-2 MEK ki-
nase activity, and MEK phosphorylation in RAW264.7 cells.
Expression of HA-p105SSAAin RAW264.7 cells reduced but
did not eliminate LPS activation of the TPL-2/MEK/ERK
pathway. This was presumably due to LPS-induced proteolysis
of endogenous wild-type p105, which was expressed at approx-
imately 20 to 40% of the level of transfected HA-p105SSAAin
the clones analyzed (data not shown). LPS stimulation would
therefore be expected to still liberate some TPL-2 from p105 in
these cells due to signal-induced proteolysis of wild-type en-
dogenous p105, resulting in the residual levels of induced
MEK and ERK phosphorylation detected (Fig. 4A).
To determine whether there is an absolute requirement for
signal-induced p105 proteolysis for LPS activation of the ERK
MAP kinase cascade in primary macrophages, BMDMs were
generated from NF-?B1?/?mice (30). These cells lack any
p105 protein and are also deficient in TPL-2 due to its meta-
bolic instability in the absence of p105 (3, 37). FL-p105WTand
FL-p105SSAAwere expressed in NF-?B1?/?BMDMs using
recombinant retroviruses. Similar to reported results (37), LPS
stimulation of NF-?B1?/?BMDMs infected with control EV
retrovirus did not induce MEK and ERK phosphorylation due
to TPL-2 deficiency (Fig. 4E). Expression of wild-type FL-p105
dramatically increased steady-state levels of TPL-2 and recon-
stituted the ability of LPS to stimulate both MEK and ERK
phosphorylation, as expected (37). Steady-state levels of TPL-2
protein were increased to a similar degree by expression of
FL-p105SSAA(Fig. 4E). However, LPS stimulation completely
failed to induce MEK and ERK phosphorylation in cells ex-
pressing FL-p105SSAA, although LPS-induced p38 phosphory-
lation was similar to control EV (Fig. 4E). These data, which
are consistent with the results of the RAW264.7 cell experi-
ments, demonstrate that LPS-induced p105 proteolysis is an
essential step in the TLR4 signaling pathway that activates the
ERK MAP kinase pathway in primary macrophages.
IKK complex activity is required for LPS activation of
TPL-2. The experiments in the previous section demonstrated
that expression of a p105 mutant that is insensitive to IKK-
induced proteolysis (20) blocked LPS stimulation of the TPL-
2/ERK signaling pathway in macrophages (Fig. 4). Since LPS
stimulation of p105 proteolysis is mediated via the IKK com-
plex (16), these data suggest that IKK activity is required for
LPS activation of this MAP kinase pathway.
To investigate this possibility, BMDMs were pretreated with
the IKK inhibitor BAY 11-7082 (27). Western blotting of cell
lysates demonstrated that this inhibitor blocked LPS-induced
p105 proteolysis, as expected (Fig. 5A, bottom panel). LPS
induction of MEK and ERK phosphorylation was also dramat-
ically reduced by BAY 11-7082 treatment (Fig. 5A). A titration
experiment revealed that BAY 11-7082 inhibition of LPS-in-
FIG. 3. LPS activation of MEK in RAW264.7 cells is dependent on TPL-2 and proteasome activity. (A) RAW 264.7 cells stably transfected with
expression vectors encoding wild-type Myc-TPL-2 (WT), kinase-inactive Myc-TPL-2 (KD), or no insert control (empty vector [EV]) were
stimulated with LPS, and cell lysates were subjected to Western blotting. LPS activation of endogenous MEK and p38 activation were assayed using
phospho-specific antibodies. (B to D) RAW264.7 cells were preincubated with MG132 (40 ?M) or DMSO vehicle control for 30 min and then
stimulated with LPS for the times indicated. (B) Total cell lysates were subjected to Western blotting for the indicated proteins. LPS activation
of MEK and p38 activation were monitored with the phospho-specific antibodies. (C) Cell lysates were immunodepleted of p105 and Western
blotted for TPL-2 and ?-tubulin. (D) TPL-2 was immunoprecipitated from total cell lysates and then assayed for MEK kinase activity as for Fig.
VOL. 24, 2004 ROLE FOR IKK IN REGULATION OF ERK MAP KINASE CASCADE9663
duced MEK phosphorylation occurred over a concentration
range similar to that associated with its inhibitory effects on
LPS-induced I?B? degradation (Fig. 5B). Since I?B? degra-
dation is triggered by its direct phosphorylation by the IKK
complex (19), these data are consistent with the effect of BAY
11-7082 on LPS-induced MEK phosphorylation being medi-
ated by inhibition of the IKK complex. LPS stimulation of p38
phosphorylation (Fig. 5A) or phorbol ester stimulation of
MEK phosphorylation (Fig. 5C) were unaffected by BAY 11-
7082, confirming that its effect on LPS activation of MEK was
As expected from its ability to inhibit IKK-triggered p105
proteolysis, BAY 11-7082 blocked both LPS induction of
TPL-2 release from p105 (Fig. 5D) and TPL-2 MEK kinase
activity (Fig. 5E). Control experiments demonstrated that
BAY 11-7082 did not inhibit TPL-2 MEK kinase activity mea-
sured in a coupled kinase assay in vitro (data not shown).
Treatment of BMDMs with another IKK2 inhibitor, BMS-
345541 (5), also blocked LPS induction of MEK phosphoryla-
tion and TPL-2 release from p105, similar to results with BAY
11-7082 (data not shown). Thus, in primary macrophages, IKK
activity is required for LPS stimulation of the TPL-2/ERK
Preferential serine 927 phosphorylation of M1-TPL-2-asso-
ciated p105. Densitometric scanning of Western blots of p105-
depleted lysates revealed that LPS stimulation more efficiently
induced the release of M1-TPL-2 from p105 than M30-TPL-2
at early time points (Fig. 6A). With more-prolonged LPS stim-
ulation, M1-TPL-2 levels were reduced due to proteolysis,
whereas the amount of M30-TPL-2 reached a plateau. Since
both TPL-2 isoforms associate with p105 at similar levels (Fig.
1A and 6B, right panels), these data suggest that the pool of
FIG. 4. LPS activation of TPL-2 is dependent on signal-induced p105 proteolysis. (A to D) RAW 264.7 cells stably transfected with vectors
encoding wild-type HA-p105 (WT), HA-p105S927A,S932A(SSAA), or with no insert (EV) were stimulated with LPS or PMA for the indicated times.
(A and B) Total cell lysates were subjected to Western blotting, and phospho-specific antibodies were used to monitor MEK, ERK, and p38
activation. (C) Lysates were immunodepleted of p105 and subjected to Western blotting for TPL-2 and ?-tubulin. (D) TPL-2 was immunopre-
cipitated from lysates of RAW264.7 cells expressing HA-p105 (WT) or HA-p105S927A,S932A(SSAA), and MEK kinase activity was determined by
a coupled MEK/ERK kinase assay as for Fig. 1A. (E) BMDMs generated from NF-?B1?/?mice were infected with recombinant retroviruses
encoding wild-type FL-p105 (WT), FL-p105S927A,S932A(SSAA), or with no insert (EV). Cells were stimulated with LPS (10 ng/ml) for 15 min or
left untreated, and total cell lysates were subjected to Western blotting for the indicated proteins. Endogenous MEK, ERK, and p38 activation was
assayed using phospho-specific antibodies.
9664 BEINKE ET AL.MOL. CELL. BIOL.
p105 that is associated with M1-TPL-2 is more efficiently de-
graded than that which is associated with M30-TPL-2.
Signal-induced p105 proteolysis is triggered by IKK-medi-
ated phosphorylation of the p105 PEST region on serines 927
and 932 (20, 29). It was therefore interesting to determine
whether M1-TPL-2-associated p105 is differentially phosphor-
ylated by the IKK complex relative to M30-TPL-2-associated
p105. To do this, p105 phosphorylated on serine 927 was im-
munoprecipitated from lysates of LPS-stimulated BMDMs us-
ing a phospho-specific antibody (29) and subjected to Western
blotting for associated TPL-2. M1-TPL2 coimmunoprecipi-
tated with phospho-S927-p105 at much higher levels than M30-
TPL-2 from LPS-stimulated cell lysates (Fig. 6B, left panels),
although both TPL-2 isoforms copurified in approximately
equal amounts with anti-p105N antibody (Fig. 6B, right pan-
els). This suggests that M1-TPL-2-associated p105 is phosphor-
ylated by IKK more strongly than M30-TPL-2-associated p105.
Since IKK-induced phosphorylation of p105 induces its degra-
dation (16, 20, 29), this differential phosphorylation provides
an explanation for why M1-TPL-2 is released more efficiently
from p105 than M30-TPL-2 in LPS-stimulated cells.
This study identifies a key step in the activation of the ERK
MAP kinase cascade by LPS in macrophages. LPS stimulation
activates the IKK complex to phosphorylate the p105 PEST
region, which triggers p105 proteolysis by the proteasome.
Consequently, TPL-2 is liberated from its inhibitor, p105 (3,
37). This p105-free active pool of TPL-2 then triggers activa-
tion of the MEK/ERK MAP kinase cascade.
It has previously been suggested that LPS stimulation of
BMDMs induces the release of only M1-TPL-2 from p105
(37). However, using an assay which directly detects p105-free
TPL-2 (Fig. 1D), it is evident that both M1-TPL-2 and M30-
TPL-2 are actually released from p105 following LPS stimula-
tion of BMDMs. Therefore, both TPL-2 isoforms may contrib-
ute to activation of MEK.
LPS stimulation of BMDMs or RAW264.7 macrophages
induces a change in the mobility of the majority of M1-
TPL-2 in SDS-PAGE that is due to phosphorylation (Fig. 1B
and data not shown). A phosphorylation-induced mobility
shift is also evident for the M30-TPL-2 band (Fig. 1B), but
only a very small fraction is modified. Western blotting of
FIG. 5. IKK is required for LPS activation of TPL-2. BMDMs (BALB/c) were preincubated with the IKK inhibitor BAY11-7082 or DMSO
vehicle control and stimulated with LPS or PMA for the indicted times. BAY11-7082 was used at a concentration of 7.5 ?M unless otherwise
indicated. (A to C) Total cell lysates were subjected to Western blotting for the indicated proteins. (D) Lysates were immunodepleted of p105 and
subjected to Western blotting for TPL-2 and ?-tubulin. (E) TPL-2 was immunoprecipitated from total cell lysates and assayed for MEK kinase
activity in a coupled MEK/ERK kinase assay as for Fig. 1A.
VOL. 24, 2004 ROLE FOR IKK IN REGULATION OF ERK MAP KINASE CASCADE9665
anti-p105N immunoprecipitates suggests that M1-TPL-2 is
phosphorylated when it is still bound to p105 (Fig. 1C). It
has recently been reported that PP2A phosphatase treat-
ment has no inhibitory effect on the MEK kinase activity
detected in anti-TPL-2 immunoprecipitates from LPS-stim-
ulated RAW264.7 cells (8). Thus, phosphorylation of TPL-2
may not regulate its catalytic activity. Rather, the correla-
tion between the quantitative phosphorylation of M1-TPL-2
and its susceptibility to proteolysis after LPS stimulation
(Fig. 1A and C) suggests that phosphorylation of M1-TPL-2
may play a role in promoting its proteolysis.
MG132 pretreatment of BMDMs blocked LPS-induced pro-
teolysis of M1-TPL-2 (Fig. 2B), showing that this process re-
quires proteasome activity. Analysis of p105-depleted lysates
clearly indicates that the p105-free pool of TPL-2 is degraded
(Fig. 1D). However, since only a fraction of total TPL-2 (ap-
proximately 20%) is released from p105, it is possible that
M1-TPL-2 proteolysis can occur when it is still bound to p105.
Interestingly, expression of HA-p105SSAAin RAW264.7 cells
prevented LPS-induced degradation of M1-TPL-2 (Fig. 4A).
Thus, p105 proteolysis is required for LPS-induced degrada-
tion of M1-TPL-2.
At early time points, M1-TPL-2 is preferentially released
from p105 compared with M30-TPL-2. However, since M1-
TPL-2 is degraded on prolonged LPS stimulation (30 min),
only p105-free M30-TPL-2 accumulates at later time points
(Fig. 6A). Kinetic experiments suggest that following release
from p105, both TPL-2 isoforms may contribute to MEK phos-
phorylation which peaks at 15 min (Fig. 1A and D). However,
since p105-free M30-TPL-2 persists after MEK phosphoryla-
tion has substantially declined (Fig. 1A and D), this isoform
may have a lower specific activity for MEK than M1-TPL-2. In
the future, it will be interesting to determine whether the
kinetic dissimilarities in their LPS-induced release from p105
and degradation translate into functional differences between
the TPL-2 isoforms.
The increased release of M1-TPL-2 from p105 at early time
points relative to M30-TPL-2 (Fig. 6A) appears to occur as a
consequence of preferential IKK-mediated phosphorylation of
M1-TPL-2-associated p105 (Fig. 6B). This is expected to trig-
ger more-pronounced proteolysis of this pool of p105 relative
to M30-TPL-2-associated p105 (Fig. 5) (20, 29). These data
raise the question of why associated TPL-2 affects IKK phos-
phorylation of the p105 PEST region. Both TPL-2 and the IKK
complex bind to p105 via its death domain (2, 3). It is therefore
possible that the M1-TPL-2/p105 complex binds to the IKK
complex more strongly than the M30-TPL-2/p105 complex,
facilitating increased IKK-mediated phosphorylation of M1-
TPL-2-associated p105. An alternative possibility is that M1-
TPL-2 and M30-TPL-2 differentially regulate activation of IKK
associated with p105, since overexpressed TPL-2 activates the
IKK complex and induces the proteolysis of cotransfected p105
(4, 22). However, this model is not supported by the absence of
a global defect in LPS-induced NF-?B activation in TPL-2-
deficient macrophages (12) and the fact that TPL-2 would be
expected to be inactive when it is bound to p105 (37; this
study). Further experiments will clearly be necessary to deter-
mine the mechanism underlying the preferential phosphoryla-
tion by IKK of the pool of p105 that is associated with M1-
The IKK-dependent mechanism of TPL-2 activation raises
the question of the physiological advantage of linking LPS
activation of ERK and NF-?B via p105. LPS upregulation of
both TNF-? and COX-2 is blocked in TPL-2-deficient
BMDMs due to defective activation of the ERK MAP kinase
cascade (12, 13). The blockade in LPS induction of TNF-? is
due to defective transport of TNF-? mRNA from the nucleus
to the cytoplasm (12), whereas COX-2 upregulation is inhib-
ited as a consequence of reduced CREB phosphorylation (13),
a critical regulator of COX-2 transcription (6, 35). Transcrip-
tional induction of both TNF-? (31, 33, 34) and COX-2 (7, 10)
by LPS requires NF-?B binding to sites in their respective
promoters. Thus, LPS upregulation of TNF-? and COX-2 pro-
duction involves coordinated activation of ERK and NF-?B.
The mechanism of TPL-2 activation, which is dependent on
phosphorylation of p105 by the IKK complex, ensures that
ERK is only activated simultaneously with NF-?B activation,
facilitating activation of these genes.
FIG. 6. IKK preferentially phosphorylates p105 complexed with M1-TPL-2. BMDMs (BALB/c) were simulated with LPS for the indicated
times. (A) Total cell lysates or cell lysates depleted of p105 were subjected to Western blotting for TPL-2. Bands were quantified by densitometry,
and data are presented as a mean (? SEM; n ? 4) of the fraction of M1- or M30-TPL-2 that is released from p105 normalized against ?-tubulin
levels. (B) Total p105 or p105 phosphorylated on S927 was immunoprecipitated from total cell lysates by using anti-p105N or anti-phospho-S927-
p105 antibodies, respectively. Associated TPL-2 was detected by Western blotting after SDS-PAGE.
9666 BEINKE ET AL.MOL. CELL. BIOL.
In conclusion, this study demonstrates that LPS activation of
TPL-2 in macrophages requires release from its inhibitor, NF-
?B1 p105. TPL-2 release is shown to occur as a consequence of
IKK-triggered p105 proteolysis by the proteasome. In future
studies, it will be important to determine whether a similar
mechanism to activate MEK is utilized by TNF-? and CD40
ligand, which also require TPL-2 MEK kinase to activate the
ERK MAPK cascade (14).
We thank Alain Israel, Toschio Kitamura, and Phillip Tsichlis for
reagents used in this study. We are also grateful to Lee Johnston and
Hamish Allen for critical reading of the manuscript, to NIMR Biolog-
ical Services, and to other members of the Ley laboratory for their
support during the course of this work.
This study was supported by the United Kingdom Medical Research
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