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(Received for publication, September 5, 1995) From the
The pleiotropic cytokine tumor necrosis factor-
Tumor necrosis factor- Ligation of
CD120a (p55) 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
Figure 1:
Immunoprecipitation of
autophosphorylated rMEK1
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
In contrast to these findings, there was no
detectable basal or TNF
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
Figure 4:
Phosphoamino acid analysis of
phosphorylated rMAPK
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 Although
this report has focused on the activation of MEK1 (and thus the
MAPK/ERK pathway) by TNF In conclusion, the findings of
this and previous work (9, 12) support the concept of
TNF
Volume 270,
Number 46,
Issue of November 17, 1995 pp. 27391-27394
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
-induced
Activation of p42
in Mouse Macrophages (*)
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(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.
(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) .
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. 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.
Materials
C3H/HeJ mice were bred at the National
Jewish Center Biological Resource Center and were used throughout the
study to avoid the possibility of stimulation by trace amounts of
endotoxin contaminants(17) . Anti-MEK1 and anti-MEK2 monoclonal
antibodies and rabbit anti-MEK antibody were purchased from
Transduction Laboratories (Lexington, KY). Monoclonal rat anti-mouse
IgG directed against an epitope of the T-cell receptor chain was
a kind gift from Dr. John Kappler, National Jewish Center, Denver,
CO(18) . Anti-rabbit and anti-mouse IgG
F(ab`)
-horseradish peroxidase-conjugated antibodies and
-[
P]ATP (Redivue,
3000 Ci/mmol) were
purchased from Amersham Life Sciences, Arlington Heights, IL.
Histidine-tagged recombinant kinase-inactive p42
(rMAPK
), wild type p42
(rMAPK
), wild type mouse MEK1 (rMEK1
),
and wild type MEK2 (rMEK2
) were expressed in Escherichia coli and purified as described
previously(19, 20) . The plasmid constructs containing
these cDNAs were provided by Dr. Gary Johnson, National Jewish Center,
Denver, CO. An additional purified kinase-inactive p42
(K52R), previously described(21) , was a kind gift from
Dr. Michael Weber, University of Virginia School of Medicine,
Charlottesville, VA. Recombinant mouse TNF
was generously provided
by Genentech Inc., San Francisco, CA.Macrophage Isolation and Culture
Bone
marrow-derived macrophages were cultured from femoral and tibial bone
marrow as described previously (22, 23) at a density
of 2.4 
10
cells/cm at 37 °C for
5-6 days. Eighteen hours prior to stimulation, the growth medium
was changed to one containing 0.1% (v/v) heat-inactivated fetal bovine
serum and no L929 cell-conditioned medium since CSF-1 present in
L-cell-conditioned medium has been found to activate the MAPK
pathway(9) .Neutrophil Isolation and Preparation
Neutrophils
(PMNs) were isolated by the plasma-Percoll method (24) and were
resuspended at 25 10
cells/ml in KRPD containing
0.25% human serum albumin, 1 mM phenylmethylsulfonyl fluoride,
10 µg/ml leupeptin, and 10 µg/ml aprotinin. 25 10 PMNs were preincubated for 30 min at 37 °C, stimulated with
10 ng/ml PMA for 10 min, and centrifuged at 14,000 g for 20 s to terminate the reaction. Cell pellets were lysed at 4
°C in lysis buffer as described below.Biosynthetic Labeling with
[
Macrophage monolayers were
[S]Methionine
S]methionine-labeled as previously described (2) . The cells were lysed on ice in 1 ml of modified RIPA
buffer, pelleted by centrifugation at 14,000
g for 10
min at 4 °C, and the supernatants were precleared with 15 µl of
protein A-Sepharose. Five µl (1.25 µg) of monoclonal anti-MEK1,
anti-MEK2 antibody, or irrelevant monoclonal IgG were added to the
precleared lysates and rotated overnight at 4 °C. Thirty µl of
protein A-Sepharose were then added to each tube and rotated for 1 h at
4 °C. The immunoprecipitates were washed five times with lysis
buffer and boiled in an equal volume of 2 Laemmli before
separating by 10% SDS-PAGE under reducing conditions. The gel was dried
and the radioactive bands were localized by fluorography.In Vitro Kinase Assay and
Immunolocalization
Macrophage monolayers were lysed on ice in 1
ml of ice-cold lysis buffer composed of 10 mM Tris/HCl, pH
7.4, containing 1% (v/v) Triton X-100, 5 mM EDTA, 50 mM NaCl, 5 mM NaF, 0.1% (w/v) bovine serum albumin, 20
µg/ml aprotinin, 1 mM phenylmethylsulfonyl fluoride, and 2
mM Na
VO
. Insoluble nuclear material
was pelleted by centrifugation at 14,000 g for 10 min
at 4 °C, and the supernatants were precleared with 15 µl of
protein A-Sepharose. 5 µl of anti-MEK antibody (1.25 µg) were
then added to the precleared lysates along with 12 µl of protein
A-Sepharose and rotated for 2 h at 4 °C. The immunoprecipitates
were washed twice with lysis buffer and twice with PAN buffer (10
mM PIPES, pH 7.0, containing 100 mM NaCl and 21
µg/ml aprotinin) and resuspended in kinase buffer (20 mM PIPES, pH 7.2, containing 10 mM MnCl and 20
µg of aprotinin) containing 20 µCi of
-[
P]ATP and
500 ng of rMAPK
as substrate in a final volume of 80 µl. The reactions were
incubated at 30 °C for 20 min and were terminated by the addition
of 20 µl of 5
Laemmli sample buffer containing 100 mM dithiothreitol, boiled for 5 min, and separated by SDS-PAGE
through 12% gels under reducing conditions and transferred to PVDF
membranes for autoradiography and Western analysis. P-Labeled phosphoproteins were detected by autoradiography
using Kodak X-Omat AR5 film. In some experiments, rMEK1
and rMEK2
were autophosphorylated in vitro by incubating
600 ng of purified recombinant protein in 17.5
µl of PAN buffer (10 mM PIPES, pH 7.0, containing 100
mM NaCl and 21 µg/ml aprotinin), 2.5 µl of 10
kinase buffer (200 mM PIPES, pH 7.2, containing 100
mM MnCl and 200 mg/ml aprotinin), and 20 µCi
of -[
P]ATP per 25-µl reaction mixture
at 30 °C for 4 h. Immunolocalization of MEK isoforms by Western
blotting was as described by Towbin et al.(25) . Bound
antibody was detected with anti-rabbit or anti-mouse IgG F(ab`)
horseradish peroxidase-conjugated antibody as the secondary
antibody. The enhanced chemiluminescence method was used to detect
bound conjugated secondary antibody. All experiments were conducted a
minimum of three times.Phosphoamino Acid Analysis of in Vitro Phosphorylated
rMAPK
Phosphoproteins were excised from PVDF
membranes and subjected to acid hydrolysis in 6 N HCl at 110
°C for 1 h, lyophilized(26, 27) , and
reconstituted in 15 µl of pH 1.9 buffer (2.5% v/v concentrated
formic acid, 7.8% v/v glacial acetic acid) containing phosphotyrosine,
phosphothreonine, and phosphoserine standards at a final concentration
of 5 mg/ml. 15 µl of each sample were loaded on a cellulose TLC
plate and chromatographed in a solvent system composed of
ethanol:butanol:glacial acetic acid:water (1:1:1:1)(28) .
Distribution of MEK Isoforms in Mouse
Macrophages
The distribution of MEK1 and MEK2 in mouse
macrophages was investigated by immunoprecipitation of S-labeled macrophage lysates with monoclonal anti-MEK1 and
anti-MEK2 antibodies. The specificity of the monoclonal antibodies was
determined by immunoprecipitating purified
P-autophosphorylated rMEK1
and rMEK2
with each antibody as well as with a rabbit polyclonal anti-MEK
antibody that reportedly recognizes shared epitopes on both proteins
and an irrelevant monoclonal antibody as a negative control. As can be
seen in Fig. 1A,
P-labeled rMEK1
was immunoprecipitated with anti-MEK1 antibody but not by
anti-MEK2 antibody. Conversely, rMEK2
was
immunoprecipitated by anti-MEK2 antibody but not by anti-MEK1 antibody (Fig. 1B). Thus, the anti-MEK1 and anti-MEK2 monoclonal
antibodies recognized their respective antigens but failed to
cross-react with one another to any significant degree. To determine if
both MEK1 and MEK2 were present in mouse macrophages MEK1 and MEK2 were
immunoprecipitated from [
S]methionine-labeled
macrophages and analyzed by SDS-PAGE through 10% polyacrylamide gels
followed by fluorography. As can be seen in Fig. 1C,
S-labeled MEK1 and MEK2, with respective molecular masses
of
45 and
46 kDa, were immunoprecipitated from lysates of
mouse macrophages confirming the presence of both MEK isoforms in these
cells.
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.
TNF
To determine
which MEK isoform was activated by TNF-induced Activation of MEK1, macrophage monolayers were
stimulated with an optimal concentration of TNF (40 ng/ml) for
time intervals up to 30 min. The cells were then lysed and
immunoprecipitated with monoclonal anti-MEK1 or anti-MEK2 antibodies,
and the immunoprecipitates were subjected to an in vitro kinase assay using purified rMAPK as substrate in the
presence of
-[
P]ATP. The reaction mixtures
were separated by SDS-PAGE, transferred to PVDF membranes, and analyzed
by autoradiography and immunoblotting. As shown in Fig. 2A, there was a marked increase in phosphorylation
of rMAPK
in response to stimulation with TNF
. The
TNF-stimulated increase in activity of MEK1 was initially detected
at 5 min, peaked at 10 min, and diminished to basal levels by 30 min (Fig. 2A). A maximal 5-6-fold increase in
phosphorylation of the rMAPK was seen following
stimulation with TNF
for 10 min. Western blotting of the
immunoprecipitates with anti-MEK1 antibody revealed that equivalent
amounts of MEK1 were immunoprecipitated at each time point both in the
presence and absence of TNF (Fig. 2B). In addition
to its ability to transphosphorylate rMAPK, basal
autophosphorylation of MEK1 was detected (i) in unstimulated
macrophages and (ii) appeared to increase in response to both a medium
change and, additionally, in response to TNF
(Fig. 2A). Autophosphorylation of MEK1 has also been
observed by Gardner and colleagues(29) . We also observed a
mobility shift in MEK1 to a higher apparent molecular weight at 10 and
15 min (Fig. 2B). However, by 30 min poststimulation,
the mobility shift in MEK1 was no longer apparent. Thus, exposure of
macrophages to TNF was associated with the activation of MEK1 with
a time course that paralleled the previously reported total catalytic
activity of MEK measured by a coupled peptide phosphorylation
assay(12) .
-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.
-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.
-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.
MEK1 Activation Parallels the Tyr and Thr Phosphorylation
of rMAPK
In previously reported work(12) ,
minimal catalytic activity of MEK was detected in lysates of
unstimulated macrophages, and thus the finding of autophosphorylation
of MEK1 in anti-MEK1 immunoprecipitates of unstimulated macrophages was
somewhat unexpected. To further investigate this finding, we analyzed
the phosphoamino acid composition of the MEK1 in vitro kinase
reaction mixtures under unstimulated conditions and in response to
TNF. Macrophage monolayers were stimulated with TNF (40
ng/ml) for 2-30 min or were incubated in medium alone, lysed, and
immunoprecipitated with anti-MEK1, and the immunoprecipitates were
subjected to an in vitro kinase assay in the presence of
rMAPK and
-[
P]ATP. The
reaction mixtures were then separated by SDS-PAGE, transferred to PVDF
membranes, and localized by autoradiography, and the
P-labeled rMAPK
bands and MEK1 bands were
excised and subjected to phosphoamino acid analysis. As can be seen in Fig. 4, in the absence of stimulation, there was minimal
phosphorylation of rMAPK
on tyrosine and threonine
residues. However, in response to stimulation with TNF
an increase
in phosphorylation of threonine and tyrosine residues was detected that
peaked at 10 min. Of note, there was also an absence of phosphorylation
of serine residues at each time point in the excised rMAPK bands. However, when the MEK1 band was excised and subjected to
phosphoamino acid analysis, there was detectable phosphorylation on
serine residues (data not shown). These findings are compatible with
the known ability of MEK1 to undergo autophosphorylation on serine
residues in vitro(16, 29) . Collectively,
these findings indicate that (i) peak activation of MEK1 detected by in vitro phosphorylation of rMAPK
coincided with
the peak level of Thr/Tyr phosphorylation of rMAPK
and
(ii) the basal autophosphorylation of MEK1 detected in the absence (or
presence) of stimulation was due to phosphorylation on serine residues.
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.
-[
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.
, 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) . 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.
), tumor necrosis factor-; JNK, c-Jun kinase; SAPK,
stress-activated protein kinase; MAPK, mitogen-activated protein
kinase; p42, p42 mitogen-activated protein kinase; rMAPK,
recombinant wild type or kinase-active MAPK; rMAPK
,
recombinant kinase dead MAPK; MEK, MAPK kinase; MEKK, MEK kinase or
MAPK kinase kinase; CSF-1, colony-stimulating factor-1; PMA, phorbol
myristate acetate; PVDF, polyvinyldifluoride; PAGE, polyacrylamide gel
electrophoresis; PIPES, 1,4-piperazinediethanesulfonic acid; PMN,
neutrophil; rMEK1
, recombinant wild type MEK1;
rMEK2
, recombinant wild type MEK2.
)
We are indebted to Dr. Gary Johnson for providing the
kinase or plasmid constructs and for constructive discussion. We also
thank Dr. Michael Weber, University of Virginia School of Medicine,
Charlottesville, VA for generously providing the kinase-inactive
p42 protein K52R.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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L.-F. Lee, G. Li, D. J. Templeton, and J. P.-Y. Ting Paclitaxel (Taxol)-induced Gene Expression and Cell Death Are Both Mediated by the Activation of c-Jun NH2-terminal Kinase (JNK/SAPK) J. Biol. Chem., October 23, 1998; 273(43): 28253 - 28260. [Abstract] [Full Text] [PDF]
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A. Schmid-Alliana, L. Menou, S. Manie, H. Schmid-Antomarchi, M.-A. Millet, S. Giuriato, B. Ferrua, and B. Rossi Microtubule Integrity Regulates Src-like and Extracellular Signal-regulated Kinase Activities in Human Pro-monocytic Cells. IMPORTANCE FOR INTERLEUKIN-1 PRODUCTION J. Biol. Chem., February 6, 1998; 273(6): 3394 - 3400. [Abstract] [Full Text] [PDF]
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L. M. Varela, K. M. Darcy, and M. M. Ip The Epidermal Growth Factor Receptor Is Not Required for Tumor Necrosis Factor-{alpha} Action in Normal Mammary Epithelial Cells Endocrinology, September 1, 1997; 138(9): 3891 - 3900. [Abstract] [Full Text] [PDF]
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M. Yin, S. Q. Yang, H. Z. Lin, M. D. Lane, S. Chatterjee, and A. M. Diehl Tumor Necrosis Factor alpha Promotes Nuclear Localization of Cytokine-inducible CCAAT/Enhancer Binding Protein Isoforms in Hepatocytes J. Biol. Chem., July 26, 1996; 271(30): 17974 - 17978. [Abstract] [Full Text] [PDF]
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A. K. Bhunia, H. Han, A. Snowden, and S. Chatterjee Lactosylceramide Stimulates Ras-GTP Loading, Kinases (MEK, Raf), p44 Mitogen-activated Protein Kinase, and c-fos Expression in Human Aortic Smooth Muscle Cells J. Biol. Chem., May 3, 1996; 271(18): 10660 - 10666. [Abstract] [Full Text] [PDF]
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