Cell type-specific activation of mitogen-activated protein kinases by CpG-DNA controls interleukin-12 release from antigen-presenting cells.

Abstract

Activation of antigen-presenting cells (APCs) by invariant constituents of pathogens such as lipopolysaccharide (LPS) or bacterial DNA (CpG-DNA) initiates immune responses. We have analyzed the mitogen-activated protein kinase (MAPK) pathways triggered by CpG-DNA and their significance for cytokine production in two subsets of APCs, i.e. macrophages and dendritic cells (DCs). We found that CpG-DNA induced extracellular signal-regulated kinase (ERK) activity in macrophages in a classic MEK-dependent way. This pathway up-regulated tumor necrosis factor production but down-regulated interleukin (IL)-12 production. However, in DCs, which produce large amounts of IL-12, CpG-DNA and LPS failed to induce ERK activity. Consistent with a specific negative regulatory role for ERK in macrophages, chemical activation of this pathway in DCs suppressed CpG-DNA-induced IL-12 production. Overall, these results imply that differential activation of MAP kinase pathways is a basic mechanism by which distinct subsets of innate immune cells regulate their effector functions.

Full text

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The EMBO Journal Vol.18 No.24 pp.6973–6982, 1999
Cell type-specific activation of mitogen-activated
protein kinases by CpG-DNA controls interleukin-12
release from antigen-presenting cells
Hans Ha ¨cker, Harald Mischak1,
Georg Ha ¨cker, Sema Eser, Norbert Prenzel2,
Axel Ullrich2and Hermann Wagner3
Institute of Medical Microbiology, Immunology and Hygiene,
Technische Universita ¨t Mu ¨nchen, Trogerstraße 9, D-81675 Munich,
1Department of Nephrology, Medizinische Hochschule Hannover,
D-30625 Hannover and2Department of Molecular Biology, Max
Planck Institut fu ¨r Biochemie, Am Klopferspitz 18A, D-82152
Martinsried, Germany
3Corresponding author
e-mail: h.wagner@lrz.tum.de
Activation of antigen-presenting cells (APCs) by
invariant constituents of pathogens such as lipo-
polysaccharide (LPS) or bacterial DNA (CpG-DNA)
initiates immune responses. We have analyzed the
mitogen-activated protein kinase (MAPK) pathways
triggered by CpG-DNA and their significance for
cytokine production in two subsets of APCs, i.e. macro-
phages and dendritic cells (DCs). We found that
CpG-DNAinducedextracellular
kinase (ERK) activity in macrophages in a classic
MEK-dependent way. This pathway up-regulated
tumor necrosis factor production but down-regulated
interleukin(IL)-12production. However,inDCs,which
produce large amounts of IL-12, CpG-DNA and LPS
failed to induce ERK activity. Consistent with a specific
negative regulatory role for ERK in macrophages,
chemical activation of this pathway in DCs suppressed
CpG-DNA-induced IL-12 production. Overall, these
results imply that differential activation of MAP kinase
pathways is a basic mechanism by which distinct
subsets of innate immune cells regulate their effector
functions.
Keywords: CpG-DNA/IL-12/innate immunity/MAP
kinase/TNF
signal-regulated
Introduction
Antigen-presenting cells (APCs), such as macrophages
and dendritic cells (DCs), carry out at least two important
tasks during immune responses towards pathogens: first,
they represent a primary barrier against pathogens through
their antimicrobial activity, and secondly, they provide the
basis for development of adaptive immune responses. In
contrast to cells of the adaptive immune system, innate
immune cells do not require clonal expansion and receptor
rearrangement of antigen-specific cells but recognize
pathogens through structurally conserved, invariant micro-
bial constituents, so-called pattern recognition factors
(PRFs). Recognition of these factors by APCs activates
the cells to exert various effector functions. In macro-
phages, direct antimicrobial functions seem to dominate
© European Molecular Biology Organization
6973
their anti-infectious repertoire (reviewed by Fearon, 1997).
These functions include the production of cytotoxic sub-
stances as well as the secretion of large amounts of
inflammatory cytokines such as tumor necrosis factor
(TNF), interleukin (IL)-6 and, to a lesser extent, IL-12.
The main task of DCs appears to be the control of
the adaptive immune system through appropriate antigen
presentation. Immature DCs process protein antigens from
pathogens and present peptides that serve as a stimulus
for specific T-cell receptors (for a review, see Cella et al.,
1997). Either direct recognition of PRFs or T-helper cell-
mediated CD40 ligation activates APCs to transit to
professional APCs via up-regulation of major histo-
compatibility complex (MHC) class II and co-stimulatory
molecules and secretion of large amounts of IL-12 and
TNF.
Bacterial cell wall components, e.g. lipopolysaccharides
(LPS) and peptidoglycans (Hoffmann et al., 1999) are
well characterized PRFs. Over the last few years, bacterial
DNA has also been recognized as a powerful stimulus of
innate immune cells in vivo and in vitro (for a review, see
Wagner, 1999). Its immune-stimulatory potential is based
on the presence of unmethylated CpG-‘motifs’, which
often contain the nucleotide sequence purine-purine-CG-
pyrimidine-pyrimidine(Krieg
sequences are present in bacterial DNA at almost the
statistically expected frequency but are evolutionarily
suppressed (‘CG suppression’) and methylated in ver-
tebrate DNA (Sved and Bird, 1990). Recognition of this
invariant structural feature by the vertebrates’ innate
immune cells qualifies bacterial CpG-DNA as a PRF. The
stimulatory principle contained in double-stranded DNA
canbetransferredtosingle-stranded
tides (ODNs) that contain the described CpG motif (CpG
ODN) (Yamamoto et al., 1992). Commonly, DNA with
immune-stimulatory capacity based on CpG-‘motifs’ is
referred to as CpG-DNA.
In macrophages and bone marrow-derived dendritic
cells (BMDDCs), CpG-DNA has been shown to induce
release of the cytokines TNF, IL-6 and IL-12 (Stacey
et al., 1996; Sparwasser et al., 1997a, 1998). While in
macrophages the pro-inflammatory cytokines TNF and
IL-6 quantitatively dominate IL-12, DCs secrete large
amounts of IL-12 (Ha ¨cker et al., 1998; Sparwasser et al.,
1998). Additionally, CpG-DNA induces maturation of
DCs, characterized by up-regulation of MHC class II and
co-stimulatory molecules such as CD80 and CD86, which
results in highly efficient antigen presentation (Sparwasser
et al., 1998). Induction of this DC maturation accompanied
by the production of large amounts of IL-12 might explain
the exceptional potency of CpG-DNA as an adjuvant
(Lipford et al., 1997a).
We have investigated signal transduction pathways
activated by CpG-DNA. Recently, we and others found
etal.,1995).These
oligonucleo-

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H.Ha ¨cker et al.
that CpG-DNA signaling involves activation of NF-κB
and induction of the stress kinases c-Jun N-terminal kinase
(JNK) 1/2 and p38 in macrophages, DCs and B cells
(Stacey et al., 1996; Ha ¨cker et al., 1998; Yi and Krieg,
1998; for a review, see Ha ¨cker, 1999).
In this study, we focused on MAPK pathways induced
by CpG-DNA and LPS in APCs displaying a distinct
differentiation status, i.e. macrophages and DCs, with the
emphasis on the role of the ERK pathway for release of
the cytokines IL-12 and TNF.
Results
CpG-DNA induces ERK activity in primary
macrophages and the macrophage cell line
RAW264.7
Previously, we showed that CpG-DNA activates the stress
kinases JNK and p38 in macrophage cell lines and primary
BMDDCs (Ha ¨cker et al., 1998). To decipher further
the signaling events involved in APC activation, we
investigated the role of the classic MAPKs, the ERKs, in
cells stimulated with CpG-DNA. Although this is a path-
way that has been characterized mainly during growth
factor-dependent signaling, it also has been shown to be
used by other PRFs such as LPS and peptidoglycans
(Dziarski et al., 1996; Sanghera et al., 1996). Initially, we
stimulated the macrophage cell line RAW264.7 with CpG
ODN or a control oligonucleotide where the CpG has
been inverted to GpC (GpC ODN). This control ODN
allows differentiation between specific, CpG-dependent
events and non-specific effects of the ODN backbone
(Sparwasser et al., 1997b). As shown in Figure 1A,
stimulation of the cells with CpG ODN led to a rapid
induction of ERK activity that peaked after 30 min to a
?5-fold induction over background and declined slowly
afterwards. In contrast, the control GpC ODN did not
induce significant ERK activity, confirming that ERK
activation depended on the bacterial ‘CpG-motif’.
To ensure that the observation made in a cell line was
representative of primary macrophages, we next examined
ERK activation in peritoneal washout cells (PWCs). PWCs
were collected and cultured overnight. The cells were then
stimulated with CpG ODN, GpC ODN or, as a positive
control, with LPS, a known inducer of ERK activity
in macrophages (Sanghera et al., 1996). Again, strong
activation of ERK was observed upon CpG ODN or LPS
stimulation, while GpC ODN failed to induce ERK activity
(Figure 1B). Activation of ERK as measured by this
in vitro kinase assay (Figure 1A and B) correlated directly
with ERK phosphorylation at their activation site, Thr202/
Tyr204, as determined by Western blotting (data not
shown).
The upstream activating kinases of ERK are the
MAPK/ERK kinases (MEK) 1/2, which are activated by
phosphorylation at Ser217/221 (Alessi et al., 1994). Using
antibodies that recognize specifically the Ser217/221-
phosphorylated form of MEK1/2, we examined activation
of MEK1/2 by CpG ODN. As shown in Figure 1C, clear
induction of phosphorylation and thus activation of
MEK1/2 could be demonstrated in CpG ODN-activated
RAW264.7 cells. LPS was again used as a positive control.
To show that the ERK activity induced by CpG ODN in
macrophages is dependent on MEK1/2, we transfected
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Fig. 1. CpG ODN induce ERK activity in RAW264.7 cells and
primary macrophages in a MEK-dependent way. (A) A total of
2 ? 106RAW264.7 cells were stimulated in complete culture medium
for the indicated times with CpG ODN or GpC ODN (1 µM). Then,
cells were lysed and kinase activity was determined by immune
complex kinase assay with antibodies to ERK-2 and myelin basic
protein (MBP) as substrate. The radioactivity incorporated into MBP
was quantitated after SDS–PAGE by PhosphorImager analysis. The
values obtained were normalized against the activity in non-stimulated
cells and are presented as fold induction. (B) PWCs were prepared as
described in Materials and methods. Stimulation was performed in
complete culture medium. Cells were stimulated with CpG ODN or
GpC ODN (1 µM) or LPS (10 ng/ml) for the time indicated. After
stimulation, cells were lysed and kinase activity was determined by
immune complex kinase assay with antibodies to ERK-2 and MBP as
substrate. Quantification was performed as described in (A).
(C) RAW264.7 cells were stimulated with 1 µM CpG ODN or 10 ng/
ml LPS for the times indicated. Cell lysates were prepared and
analyzed by Western blotting with antibodies against total MEK1/2
and against the Ser217/221 phosphorylated forms of MEK1/2.
(D) RAW264.7 cells were transiently transfected with an expression
vector for HA-tagged ERK-2 or HA-tagged JNK-1, together with
either a control vector or an expression vector for MEKdn as
indicated. At 24 h post-transfection, cells were left untreated or were
stimulated with 1 µM CpG ODN for 20 min. After stimulation, cells
were lysed and kinases were immunoprecipitated with antibodies to
the HA tag. In vitro kinase assays were performed with MBP or
GST–Jun(79) as substrate.
RAW264.7 cells with a dominant-negative form of MEK
(MEKdn) and activated these cells with CpG ODN. As
shown in Figure 1D, MEKdn inhibited activation of ERK.
This inhibition was specific as activation of JNK was not
reduced in the presence of MEKdn (Figure 1D). Taken

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CpG-DNA cell type-specific activation of MAP kinases
together, these results demonstrate that CpG-DNA induces
ERK activity via a classic MEK-dependent pathway.
CpG-DNA-induced MEK/ERK activity regulates
expression of TNF and IL-12
As a next step, we addressed the question of the biological
significance of this ERK activation. Recently, we and
others have noted that CpG-DNA induces the production
of large amounts of TNF but small amounts of IL-12 in
macrophage cell lines and primary peritoneal macrophages
(Sparwasser et al., 1997b; Sweet et al., 1998). Secretion
of both cytokines upon CpG ODN stimulation requires
p38 activity (Ha ¨cker et al., 1998). While no selective
inhibitor of ERK is available at present, the MEK inhibitor
PD98059 can be used to inhibit MEK and subsequent
MEK-dependent ERK activation (Dudley et al., 1995).
Hence we examined secretion of TNF and IL-12 upon
CpG ODN stimulation in RAW264.7 cells in the presence
of PD98059. As shown in Figure 2A, inhibition of ERK
activation resulted in a significant suppression of TNF
release. However, unexpectedly, inhibition of the ERK
pathway resulted in a dose-dependent increase of IL-12
secretion, up to ?10-fold (Figure 2A). Comparable results,
i.e. strong up-regulation of IL-12 in the presence of
PD98059, were obtained with PWCs (data not shown).
Recently, another potent MEK inhibitor, UO126, has
become available. In contrast to PD98059, which primarily
seems to inhibit MEK1/2 activation by blockade of the
access of activating enzymes, UO126 is able to inhibit
the activated, phosphorylated form of MEK1/2 (Favata
et al., 1998). Qualitatively, the same results were obtained
with UO126 as with PD98059, although its effects on
cytokine production were more pronounced with respect
to both TNF suppression and up-regulation of IL-12
(Figure 2B).
Significance of the CpG-DNA-induced ERK activity
for regulation of TNF and IL-12 p40 mRNA levels
and promoter activity
IL-12 in its active form, IL-12 p70, consists of a hetero-
dimer of the proteins p40 and p35 (Gubler et al., 1991;
Wolf et al., 1991). While IL-12 p35 is expressed more
ubiquitously, IL-12 p40 expression appears to be restricted
to cell types that express biologically active IL-12 and is
strongly inducible by LPS and CD40 ligation in certain
cell types (D’Andrea et al., 1992; Shu et al., 1995). The
data available suggest that IL-12-inducing stimuli act via
enhancement of IL-12 p40 promoter activity (Murphy
et al., 1995; Ma et al., 1996; Plevy et al., 1997). TNF
production is regulated at both the transcriptional and
post-transcriptional level (for a review, see Beutler and
Kruys, 1995). To define further the level of cytokine
regulation by CpG-DNA-induced MEK/ERK activity, we
measured mRNA levels by RNase protection assays and
promoteractivityusingRAW264.7cells,stablytransfected
with a luciferase gene under the control of either the
TNF or the IL-12 p40 promoter. RAW264.7 cells were
stimulated with CpG ODN in either the absence or
presence of the MEK inhibitors PD98059 or UO126, and
RNA was prepared and subjected to RNase protection
assays using TNF or IL-12 p40 sequences as probes.
As shown in Figure 2C, CpG ODN induces rapid up-
regulation of TNF mRNA. Both PD98059 and UO126 led
6975
to a slight decrease in constitutive mRNA levels, but no
substantial effect on CpG ODN-induced up-regulation of
TNF mRNA was observed (Figure 2C). These results
correlate well with experiments using the RAW264.7 TNF
promoter transfectants: even at the highest concentration
of PD98059 (50 µM, as used in the RNase protection
assay), no significant effect on CpG ODN-induced TNF
promoter activity was observed (Figure 2D). We conclude
from these experiments that CpG-DNA-induced ERK
activity up-regulates TNF primarily at a post-transcrip-
tional level. This result is in accordance with published
observations on LPS-induced TNF secretion, where ERK
activation acted primarily at a post-transcriptional level
(Bhat et al., 1998).
In contrast to the regulation of TNF, CpG ODN-induced
up-regulation of both IL-12 p40 mRNA and IL-12 p40
promoter activity is controlled by ERK signaling: inhib-
ition of MEK by PD98059 or UO126 led to a strong
increase in CpG ODN-induced IL-12 p40 mRNA levels
(Figure 2C). In direct correlation with this up-regulation,
PD98059 strongly enhanced the CpG ODN-induced activ-
ity of the IL-12 p40 promoter in stably transfected
RAW264.7 cells (Figure 2D).
To control further for the possibility that the observed
effects of the MEK inhibitors were due to the inhibition
of other kinases, we also examined the effect of dominant-
negative MEK-1 and of a constitutively active Raf mutant
on the IL-12 p40 promoter. RAW264.7 cells were transi-
ently transfected with the IL-12-p40 promoter–luciferase
construct and either dominant-negative MEK-1 or Raf-
BXB, the constitutively active kinase domain of RAF.
RAF BxB on its own can activate MEK1/2 and sub-
sequently ERK1/2 (Kyriakis et al., 1992). The results,
shown in Figure 2E and F, confirm the findings obtained
with the MEK inhibitor: inhibition of MEK activity using
the dominant-negative construct also increased the CpG
ODN-induced IL-12 p40 promoter activity in a dose-
dependent way (Figure 2E). Furthermore, activation of
MEK by the upstream kinase Raf-1 decreased the capacity
ofCpGODNtoinducetheIL-12p40promoter(Figure2F).
Taken together, these data show that CpG ODN-driven
activation of the ERK pathway triggers TNF secretion at
a post-transcriptional level but negatively regulates IL-12
p40 production at the promoter level.
Priming of macrophages with interferon-γ does not
affect CpG ODN-induced ERK activity
The data presented here clearly indicate that IL-12 produc-
tion is negatively regulated by CpG-DNA-activated ERK.
Earlierdata,ontheotherhand,haddemonstratedthatCpG-
DNA also activates NF-κB and stress kinase pathways, i.e.
signalingpathwaysthatstimulateIL-12production(Stacey
et al., 1996; Ha ¨cker et al., 1998). This constellation
suggests that IL-12 secretion depends on the balance of
positive and negative stimuli. While in macrophages CpG
ODN appear to activate both stimulatory and inhibitory
pathways, there may exist situations, or cell types, where
other external stimuli shift the balance and prime the
cells towards selective IL-12 production. For example, in
primary macrophages and macrophage cell lines, efficient
induction of IL-12 p40 promoter activity and protein
secretion by LPS is dependent on interferon-γ (IFN-γ)
priming (Ma et al., 1996). We therefore speculated that

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H.Ha ¨cker et al.
Fig. 2. IL-12 and TNF expression is regulated by the MAPK/ERK pathway. (A) A total of 2 ? 105RAW264.7 cells were seeded in 0.5 ml of
complete culture medium and stimulated with CpG ODN (1 µM) for 8 h. Release of TNF (left) and IL-12 (right) was measured by ELISA. Where
PD98059 was used, cells were pre-incubated for 30 min before stimulation with the concentrations of PD98059 indicated. (B) RAW264.7 cells were
treated as described in (A). Instead of PD98059, the MEK inhibitor UO126 was used at the concentrations indicated. Release of TNF (left) and
IL-12 (right) was measured by ELISA. (C) A total of 2 ? 106RAW264.7 cells were seeded in 5 ml of complete culture medium and incubated for
16 h. Then cells were stimulated in the presence or absence of the MEK inhibitors PD98059 (50 µM) or UO126 (5 µM) with CpG ODN (1 µM) for
3, 6 or 9 h. After stimulation, total RNA was prepared and mRNA levels were analyzed by RNase protection analysis using TNF, IL-12 p40 and
GAPDH as probes. Where PD98059 or UO126 was used, cells were pre-treated for 30 min before stimulation. Where PD98059 or UO126 was used
without subsequent stimulation by CpG ODN, cells were incubated for 9.5 h in the presence of the inhibitor before RNA preparation.
(D) RAW264.7 cells with a stably integrated TNF promoter–luciferase reporter (left) or an IL-12 p40 promoter–luciferase reporter (right) were
stimulated for 8 h with CpG ODN (1 µM). Where PD98059 was used, cells were pre-incubated for 30 min before stimulation with the
concentrations of PD98059 indicated. After stimulation, luciferase activity was measured. Each column represents the mean of two independent
stimulations. (E and F) RAW264.7 cells were transiently transfected with the IL-12 p40 promoter–luciferase reporter and increasing amounts of
either an expression vector for MEKdn (E) or an expression vector for RAF BxB (F). The overall amount of DNA per transfection was held
constant by adding the appropriate control vectors. At 18 h post-transfection, cells were left untreated or were stimulated with 1 µM CpG ODN for
8 h and luciferase activity was determined. Each column represents the mean of three independent stimulations; error bars are standard deviation.
IFN-γ priming might inhibit ERK activation and thus lead
to secretion of high levels of IL-12. To this end, we
examined the effect of IFN-γ priming on IL-12 production
by CpG ODN-stimulated macrophages. Figure 3A shows
that IFN-γ priming also strongly enhances the CpG ODN-
induced IL-12 production. This effect is accompanied
by a strong increase in promoter activity (not shown).
However, as in unprimed cells, inhibition of MEK by
PD98059inIFN-γ-primedcellsledtoasubstantialincrease
in IL-12 secretion (Figure 3A). These results suggested
that IFN-γ treatment would not change ERK activity in
response to CpG ODN. Indeed, as shown in Figure 3B,
no difference in ERK activity could be detected in IFN-γ-
treated versus untreated cells. Hence, the IFN-γ priming
6976
for IL-12 secretion by macrophages does not seem to
involve ERK.
In dendritic cells, CpG ODN activates stress
kinases JNK and p38 but fails to induce ERK
activity
There is evidence that in vivo the main source of IL-12
is not macrophages but DCs (De Smedt et al., 1996;
Sousa et al., 1997). In vitro, DCs secrete TNF in amounts
comparable with macrophages, but up to two orders of
magnitude more IL-12 (compare Figures 3A and 5A).
In order to see whether the balance of positive and
negative regulatory signals is different in DCs, we
prepared primary DCs from bone marrow cells (Inaba

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CpG-DNA cell type-specific activation of MAP kinases
Fig. 3. IFN-γ priming effect on CpG ODN-stimulated RAW264.7
cells. (A) A total of 2 ? 105RAW264.7 cells were seeded in 0.5 ml of
complete culture medium with or without 10 ng/ml IFN-γ as indicated.
After 12 h, cells were stimulated with CpG ODN (1 µM) for 8 h in
the presence or absence of PD98059. Where PD98059 was used, cells
were pre-incubated for 30 min before stimulation. The concentration
of IL-12 was measured by ELISA. (B) A total of 1.5 ? 106
RAW264.7 cells were seeded in 2 ml of complete culture medium
with or without 10 ng/ml IFN-γ as indicated. After 12 h, cells were
stimulated with CpG ODN (1 µM) for 15 or 60 min and ERK-2
kinase activity determined in an immune complex kinase assay with
antibodies to ERK-2 and MBP as substrate. The radioactivity
incorporated into MBP was quantitated after SDS–PAGE by
PhosphorImager analysis. The values obtained were normalized against
the radioactivity obtained in non-stimulated cells and are presented as
fold induction.
et al., 1992; Lutz et al., 1999). After 8 days of in vitro
cultureinthepresenceofgranulocyte–macrophagecolony-
stimulating factor (GM-CSF), these cells displayed a
phenotype characteristic for this type of DC culture: the
majority of cells were positive for CD11c (Figure 4A)
and responded to CpG ODN or LPS by up-regulating
MHC class II expression (Figure 4A) and expression of
CD80, CD40 and CD86 (not shown; Sparwasser et al.,
1998).
Stimulation of DCs with CpG ODN and LPS induced
robust activation of JNK and p38 activity (Figure 4B) as
suggested by earlier results (Ha ¨cker et al., 1998). This
activity peaked after 15–30 min (Figure 4C), a similar
time frame as observed in macrophages (Ha ¨cker et al.,
1998). In contrast to the macrophages, however, ERK
activation could not be detected in DCs. As a control,
DCs were stimulated with phorbol ester [phorbol 12-
myristate 13-acetate (PMA)]. Figure 4B and C shows that
PMA did induce rapid and strong activation of ERK but
no significant JNK or p38 activity. Because DCs were
generated in GM-CSF-conditioned medium, we also per-
formed experiments to analyze whether GM-CSF would
6977
Fig. 4. FACS analysis and PRF-induced kinase activity of BMDDCs.
(A) BMDDCs were prepared as described in Materials and methods.
After 9 days of in vitro culture, cells were washed and either left
untreated (upper panel) or stimulated with CpG ODN (1 µM, middle
panel) or LPS (10 ng/ml, lower panel) for 20 h. Cells were stained
with antibodies to CD11c (left, bold) or MHC class II (right, bold) and
the appropriate isotype controls (fine), and analyzed by flow
cytometry. (B) BMDDCs were used after 9 days of in vitro culture. A
total of 5 ? 106cells each were stimulated in complete culture
medium with CpG ODN (1 µM), LPS (10 ng/ml) or PMA (10 ng/ml)
for the times indicated. Then, lysates were prepared and split, and
immune complex assays were performed on different aliquots. As
substrates, MBP was used for ERK-2, GST–Jun(79) for JNK1/2 and
GST–MAPKAP kinase II for p38. (C) The radioactivity incorporated
into MBP (ERK-2), GST–Jun (JNK1/2) and GST–MAPKAP kinase II
(p38) obtained in (B) was quantitated by PhosphorImager analysis.
The values obtained were normalized to the radioactivity in non-
stimulated cells and are presented as fold activation.