(Received for publication, September 5, 1995)
From the
The pleiotropic cytokine tumor necrosis factor- (TNF
)
controls the expression of multiple gene products in macrophages and
plays an important role in host defense. TNF
is recognized by the
receptors, CD120a (p55) and CD120b (p75). Ligation of CD120a (p55) by
TNF
or by anti-receptor agonistic antibodies initiates signal
transduction leading to the activation of mitogen-activated protein
kinases (MAPKs) (p42
and
p44
). Phosphorylation and activation of
MAPK are mediated by MAPK kinase (MEK), a family of Thr/Tyr kinases. In
this study, we investigated the preferential involvement of the MEK
isoforms MEK1 and MEK2 in the activation of p42
in mouse macrophages stimulated with TNF
. Exposure of
macrophages to TNF
stimulated a time-dependent increase in the
activity of MEK1 as measured by an in vitro kinase assay using
kinase-inactive p42
(rMAPK
)
as substrate in the presence of
-[
P]ATP.
Maximal activation of MEK1 was detected at 10 min poststimulation and
coincided with maximal transphosphorylation of Tyr and Thr residues of
rMAPK
. By contrast, there was no evidence of MEK2
activation in macrophages in response to TNF
. These data suggest
that MEK1 is the preferred substrate for MEK kinase, the upstream
kinase implicated in activation of the MAPK pathway in macrophages by
TNF
.
Tumor necrosis factor- (TNF
), (
)a
pleiotropic cytokine produced predominantly by macrophages, stimulates
the expression of multiple gene products that collectively mediate the
role of the macrophage in host
defense(1, 2, 3) . TNF
is recognized by
a binary system of receptors, CD120a (p55) and CD120b (p75), belonging
to the TNF/nerve growth factor receptor family, which initiate signal
transduction following receptor oligomerization in the plane of the
plasma membrane. Although cross-linking of each receptor has been shown
to initiate distinct responses in different cell types(4) , a
major emphasis has been placed on investigating the functional
responses and signaling mechanisms activated by CD120a (p55). Ligation
of TNF
by CD120a (p55) has been shown to stimulate the formation
of several second messengers including, ceramide-1-phosphate (5, 6) and 1, 2-diacylglycerol(7) . However,
emerging studies have shown that an important consequence of ligation
of TNF
is the activation of at least two protein kinase cascades,
which result in the activation of mitogen-activated protein kinases
(p42
and p44
) (6, 8, 9) and c-Jun kinases/stress-activated
protein kinases (JNK/SAPK)(10, 11) .
Ligation of
CD120a (p55) by TNF or by receptor-specific polyclonal agonistic
antibodies results in the transient activation of p42
in mouse macrophages and other cell types with peak tyrosine
phosphorylation and catalytic activation occurring
10-15 min
poststimulation(6, 8, 9) . Cross-linking of
CD120a (p55) is rapidly followed by a transient activation of MEKK, a
serine kinase bearing homology to the yeast kinase Ste11, within 30 s
of stimulation of TNF
in the absence of activation of
c-Raf-1(12) . In addition, activation of MEKK is followed by a
transient increase in total MEK catalytic activity as measured by
fractionation of unstimulated and TNF
-stimulated macrophage
lysates by ion-exchange chromatography over a mono-S column followed by
detection of catalytically active MEK in a coupled assay based on its
ability to phosphorylate and activate purified recombinant
p42
. While these studies have clearly
shown MEK to be activated by TNF
, MEK represents a family of dual
specificity Tyr/Thr kinases that co-elute from mono-S columns, thus
raising the question of the specificity of MEK isoform activation by
TNF
.
At least three MEK isoforms (MEK1, MEK2, and MEK3) have
been described (13, 14, 15, 16) .
MEK1 and MEK2 are highly conserved, and purified recombinant MEK1 and
MEK2 have both been shown to phosphorylate and activate purified
recombinant p42 and
p44
(16) . By contrast, MEK3, an
alternatively spliced variant of MEK1, is catalytically inactive with
respect to these substrates (16) and does not appear to be
important in the activation of the MAPK cascade. The aim of the present
study was to investigate the specificity of MEK isoform involvement in
the activation of p42
in primary cultures
of mouse macrophages stimulated with TNF
. Our results show that
although both MEK1 and MEK2 isoforms are present in mouse macrophages,
TNF
preferentially utilizes MEK1 to stimulate the phosphorylation
and activation of p42
.
Figure 1:
Immunoprecipitation of
autophosphorylated rMEK1 and rMEK2
and
S-labeled macrophages to investigate MEK antibody
specificity. Panel A, autoradiograph of autophosphorylated
rMEK1
immunoprecipitated with 1) monoclonal
anti-MEK1 antibody, 2) monoclonal anti-MEK2 antibody, 3) polyclonal anti-MEK antibody, and 4) monoclonal
IgG antibody. Panel B, autoradiograph of autophosphorylated
rMEK2
immunoprecipitated with 1) monoclonal
anti-MEK1 antibody, 2) monoclonal anti-MEK2 antibody, 3) polyclonal anti-MEK antibody, and 4) monoclonal
IgG antibody. Panel C, autoradiograph of
S-labeled macrophages immunoprecipitated with 1)
monoclonal anti-MEK1 antibody, 2) monoclonal anti-MEK2
antibody, and 3) monoclonal IgG antibody.
Figure 2:
In
vitro kinase time course and immunoblot of MEK1-immunoprecipitated
macrophage lysates. Panel A, autoradiograph of MEK1 activity
time course. MEK1 was immunoprecipitated from unstimulated and
TNF-stimulated (40 ng/ml) murine macrophage lysates at 2, 5, 10,
15, and 30 min, and immunoprecipitated MEK1 was then subjected to in vitro kinase assay using recombinant kinase-inactive
p42
(rMAPK
) as substrate.
Monoclonal IgG is used as a negative control. Panel B, anti-MEK1 immunoblot of the samples shown in panel A above. No Subst., no substrate; Autophos.,
autophosphorylation of the rMAPK
substrate in the absence
of cell lysate.
In contrast to these findings, there was no
detectable basal or TNF-stimulated activation of MEK2 (Fig. 3A) at either 5 or 10 min. To verify that the
inability to detect activation of MEK2 was not due to an inability of
the assay procedure to detect activation of the kinase, macrophages
were stimulated with a variety of well characterized stimuli including:
PMA (10 ng/ml), ATP (100 µM), calcium ionophore A23187 (1
µM), platelet-activating factor (1 µM), and
CSF-1 (1000 units/ml). None of these stimuli were capable of activating
MEK2 in this assay although MEK2 protein was detected in immunoblots of
these immunoprecipitates. However, as shown in Fig. 3A and in marked contrast to macrophages, basal MEK2 activity was
detected in unstimulated neutrophils, and a modest increase in activity
associated with a decrease in the electrophoretic mobility of
rMAPK
was detected following stimulation of neutrophil
suspensions with PMA (10 ng/ml) for 10 min. Fig. 3B shows an immunoblot of the anti-MEK2 immunoprecipitates confirming
that equivalent amounts of MEK2 antigen were immunoprecipitated from
unstimulated and stimulated cells. These data thus indicate that
stimulation of mouse macrophages with TNF
resulted in a selective
activation of MEK1 in the absence of a detectable increase in MEK2
catalytic activity. In addition, and in contrast to MEK1, MEK2 appeared
to be catalytically silent in unstimulated mouse macrophages.
Figure 3:
In vitro kinase time course and
immunoblot of MEK2-immunoprecipitated macrophage lysates. Panel
A, autoradiograph of MEK2 activity time course. MEK2 was
immunoprecipitated from unstimulated and TNF-stimulated (40 ng/ml)
murine macrophage lysates at 5 and 10 min and unstimulated (U)
and PMA-stimulated (P) (10 ng/ml) human neutrophil (PMN) lysates; the immunoprecipitated MEK2 was then subjected
to an in vitro kinase assay using rMAPK
as
substrate. Autophosphorylated rMAPK
is used to localize
rMAPK
. Monoclonal IgG is used as a negative control. Panel B, anti-MEK2 immunoblot of the samples shown in panel A above. hc, IgG heavy
chain.
Figure 4:
Phosphoamino acid analysis of
phosphorylated rMAPK acting as substrate for the in
vitro kinase reaction of anti-MEK1 immunoprecipitates of
unstimulated and TNF
-stimulated (40 ng/ml) macrophage lysates at
2-, 5-, 10-, and 15-min time points. Autoradiograph of phosphoamino
acids separated by TLC on cellulose plate is shown. Phosphorylated
bands are compared with phosphotyrosine (P-Tyr),
phosphothreonine (P-Thr), and phosphoserine (P-Ser)
standards visualized on TLC plates by ninhydrin development.
Work reported by Zheng and Guan (16) has shown that
autophosphorylation of GST-MEK1 and GST-MEK2 on Ser and Thr residues is
sufficient to activate MEK activity in a transphosphorylation assay
using ERK1 and ERK2 as substrates although full activation of ERK
required upstream activators present in cytosolic extracts of epidermal
growth factor-stimulated Swiss 3T3 cells. The results of the present
study indicate that while native MEK1 undergoes autophosphorylation in
the presence of -[
P]ATP in vitro,
this was not associated with an increase in the Thr and Tyr
phosphorylation of p42
nor, as we have previously
shown(12) , is there detectable total MEK catalytic activity in
lysates of unstimulated macrophages. In addition, when
p42
was immunoprecipitated from lysates of
unstimulated and TNF
-stimulated
[
P]orthophosphate-labeled mouse macrophages,
radioactivity was detected in p42
following
stimulation with TNF
but not in lysates of unstimulated
macrophages (9) . Similar findings have been reported in human
fibroblasts(8) . These findings thus suggest that the
constitutive autophosphorylation on Ser residues of MEK1 is
insufficient for activation of p42
.
Although
this report has focused on the activation of MEK1 (and thus the
MAPK/ERK pathway) by TNF, recent studies have also shown that
TNF
activates the JNK/SAPK pathway resulting in the
phosphorylation of c-Jun(10, 11) . Indeed, it has been
suggested that the SAPK pathway may be the primary pathway of
activation by TNF
(30) . These reports, however, were
conducted predominantly in transformed fibroblast cell lines and in
PC12 cells. In recent work (
)we have confirmed that JNK is
activated in macrophages in response to TNF
. The significance of
the activation of these different MAPK pathways in the regulation of
macrophage functions is largely unknown. However, unlike the situation
in Swiss 3T3 cells, macrophages do not undergo programmed cell death in
response to TNF
, and thus the activation of other MAPK/ERK
pathways such as p42
may be an important
determinant in cell survival and differentiation in response to
TNF
(2, 22) .
In conclusion, the findings of
this and previous work (9, 12) support the concept of
TNF activation of the MAPK/ERK pathway in macrophages;
specifically, TNF
causes aggregation of CD120a (p55), which
initiates the rapid and transient activation of an MEKK followed by the
selective and sequential activation of MEK1 and p42
in the absence of activation of c-Raf-1 and MEK2. Moreover, the
specificity of MEK1 activation by TNF
suggests a substrate
preference by MEKK for this MEK isoform.