From the Department of Medicine, Koerner
Pavilion, University of British Columbia, Vancouver, British Columbia
V6T 1Z3 and the § Kinexus Bioinformatics Corporation,
Vancouver, British Columbia V6T 1Z4, Canada
Received for publication, December 26, 2000, and in revised form, January 9, 2001
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ABSTRACT |
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We have identified a direct physical interaction
between the stress signaling p38 Mitogen-activated protein
(MAP)1 kinase modules are
involved in the signal transduction of a wide variety of
cellular responses in all eukaryotic organisms including proliferation,
differentiation, and apoptosis (1). At least four distinct and parallel
MAP kinase cascades have been identified, including extracellular signal-regulated kinases 1 and 2 (ERK1/2), the p38 MAP kinases, c-jun N-terminal or stress-activated protein kinases
(JNK/SAPK), and ERK5/big MAP kinase 1 (BMK1). It is well established
that ERK1/2 are typically stimulated by growth-related stimuli through the Raf1/B-MEK1/2-ERK1/2 protein kinase cascade. The JNK and p38 MAP kinases are primarily activated by stress-related signals such as
heat and osmotic shock, UV irradiation, and proinflammatory cytokines
by means of the MAP kinase kinases, MKK3, -4, -6, and -7 (2-4).
Whereas the selective activation of distinct MAP kinase pathways in
response to different extracellular stimuli has been extensively
documented, there is increasing evidence for cross-talk between
distinct MAP kinase pathways. A p38-dependent ERK1/2
activation was observed in several mammalian cell lines including the
human embryonic kidney cell line HEK293 upon arsenite treatment (5). It
was also found that inactivation of p38 by SB202190 treatment resulted
in a delayed and prolonged activation of ERK1/2 in the human hepatoma
cell line, HepG2 (6). In both cases, MEK1 was implicated in the
activation of ERK1/2. Here we report that in HeLa and HEK293 cells,
stress stimuli lead to an inhibition of ERK1/2 via p38 Construction of pGEX-p38 Expression of GST Fusion Proteins in Bacteria--
GST fusion
protein plasmids were transformed into DH5 GST Fusion Protein Pull-down--
Two mg of rat brain lysate or
1 mg of HeLa cell lysate was mixed with 20 µg of immobilized
GST-ERK1, GST-p38 In Vivo Association of ERK1 and p38--
HEK293 or HeLa cells
cultured in Dulbecco's minimum essential medium (DMEM, Life
Technologies, Inc.) containing 10% fetal bovine serum were grown to
50-60% confluence and transfected with pcDNA3-p38 MBP Kinase Assay--
Anti-ERK1 immunoprecipitates bound to
protein A beads were further washed twice in assay dilution buffer (25 mM Interaction of Bacterially Expressed p38 Evidence for the Inhibition of ERK1/2 by p38--
By using
antibody specific for the phosphorylated (activated) form of ERK1/2 in
Western blotting studies, we observed that anisomycin treatment
resulted in decreased ERK1/2 phosphorylation. By contrast, SB203580, a
specific p38 MAP kinase inhibitor, induced increased ERK1/2
phosphorylation in HeLa cells similar to that observed with epidermal
growth factor (EGF) exposure (Fig.
1A). These findings are
consistent with the results in HepG2 cells (6) and indicate that p38
MAP kinase somehow exerts an inhibitory effect on ERK1/2 activation.
Surprisingly, an increase of myelin basic protein (MBP)
phosphotransferase activity associated with anti-ERK1
immunoprecipitates was detected upon anisomycin treatment, which could
be inhibited by including SB203580 in kinase assay reactions (Fig.
1B). This observation indicated that the increase of MBP
phosphotransferase activity precipitated by anti-ERK1 antibody upon
anisomycin treatment was at least partially due to p38 MAP kinases. The
apparent contradiction between the ERK1/2 phosphorylation state in
Western blotting and the MBP phosphotransferase activity associated
with anti-ERK1 immunoprecipitates could be potentially reconciled if
p38 is coprecipitated with ERK1/2 in response to anisomycin treatment.
Previously, we reported that ERK1 was found in immunoprecipitates of a
p38 homologue in immature sea star oocytes (11). Based on these
observations, we postulated that the direct interaction between p38 and
ERK1/2 MAP kinases might play a role in coordinating the regulation of
these two distinct MAP kinase pathways.
Interaction of ERK1/2 and p38 in Vitro--
To examine whether a
physical interaction occurs between p38 and ERK1, we expressed
glutathione S-transferase (GST) fusion proteins of the
full-length human ERK1 and p38 Interaction of ERK1/2 and p38 Is p38
Activity-dependent--
We next examined the interaction
between ERK1 and p38
We further monitored the physical interaction of these two MAP kinases
in mammalian cells by coimmunoprecipitation. HeLa cells were
transfected with human p38
Similarly, interaction between ERK1 and p38 Direct Interaction Occurs between Purified ERK1 and p38 in
Vitro--
We further examined whether a direct interaction occurs
between p38 p38 Does Not Suppress MEK1 Phosphorylation to Inhibit
ERK1/2--
To assess the roles of p38-ERK1/2 interaction in
regulating the activation of ERK1/2, we activated endogenous p38
kinases by transfecting HeLa cells with MKK6(E) DNA and then treated
the cells with EGF and monitored the activation of ERK1/2 and their upstream activating kinase, MEK1. In response to EGF treatment, the
phosphorylated forms of ERK1/2 were present at much lower levels in
MKK6(E)-transfected cells than in nontransfected HeLa cells
(Fig. 5A), indicating an
inhibitory effect of active p38 on ERK1/2. Moreover, no apparent
difference in the phosphorylation of MEK1 at its activation sites was
observed under these two circumstances (Fig. 5B). These
results indicate that p38 suppresses ERK1/2 by a mechanism independent
of MEK1 phosphotransferase activity.
In summary, our study provides the first experimental evidence for the
direct interaction between two MAP kinases lying in two distinct
signaling cascades, raising the possibility that the direct interaction
between p38
Combined with previous studies, our results indicate that the
communication between p38 and the ERK1/2 pathways may act through two
distinct modes. Active p38 may suppress ERK1/2 phosphotransferase activity either through inhibition of upstream activating kinases of
ERK1/2 or through direct interaction between p38 and ERK1/2. Based on
our observation of the direct association between p38 MAP kinase and the
mitogen-activated protein kinases ERK1 and ERK2 by affinity
chromatography and coimmunoprecipitation studies. Phosphorylation and
activation of p38
enhanced its interaction with ERK1/2, and this
correlated with inhibition of ERK1/2 phosphotransferase activity. The
loss of epidermal growth factor-induced activation and
phosphorylation of ERK1/2 but not of their direct activator MEK1 in
HeLa cells transfected with the p38
activator MKK6(E) indicated that
activated p38
may sequester ERK1/2 and sterically block their
phosphorylation by MEK1.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
.
Phosphorylated p38
is capable of forming a complex with ERK1/2, and
it prevents their phosphorylation by MEK1/2.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
--
The pGEX-p38
wild-type and
p38
(AF) dominant negative mutant were constructed by subcloning the
respective cDNA sequences into pGEX-4T vector (Amersham Pharmacia
Biotech). The human p38 full-length sequences were obtained by
digesting pcDNA3-flagp38
wild-type and p38
(AF) plasmids, a
generous gift of Dr. J. Han (the Scripps Research Institute, La Jolla,
CA). pGEX-ERK1 was obtained by subcloning human ERK1 cDNA
full-length sequence into pGEX-2T vector (Amersham Pharmacia Biotech).
The resulting GST fusion protein constructs were verified by DNA
sequencing (7).
bacteria. Expression of
GST fusion proteins was induced by 0.5 mM
isopropyl-1-thio-
-D-galactopyranoside at 37 °C
for 3 h. GST fusion proteins were purified as described previously
on glutathione-agarose (Sigma) and eluted from beads with reduced
glutathione; GST tag was cleaved with thrombin when necessary (8).
, or GST alone on glutathione-agarose beads at
4 °C with rotation. After a 2-h incubation, beads were washed three
times with 50 mM Tris (pH 8.0), 150 mM NaCl,
and 1% Nonidet P-40 followed by separation of bound proteins by
SDS-polyacrylamide gel electrophoresis (PAGE). Proteins were
transferred to nitrocellulose membrane, and immunoblotting was
performed with either anti-p38
or anti-ERK1-CT antibody
(StressGen, Victoria, British Columbia, Canada).
using
SuperFect reagent (Qiagen, Mississauga, Ontario, Canada) as per the
manufacturer's instructions. For each 100-mm dish, 5 µg of plasmid
DNA were introduced into cells using 30 µl of SuperFect reagent in
serum-free medium. After a 3-h incubation, cells were starved in fresh
serum-free DMEM. 24 h after transfection, cells were stimulated
with anisomycin (Sigma) or arsenite (Sigma) alone or in combination
with SB203580 (Calbiochem) in serum-free DMEM as indicated in the
figure legends. Cells were lysed in 500 µl of lysis buffer (150 mM NaCl, 20 mM Tris pH 8.0, 0.5% (w/v) Nonidet
P-40, 1 mM dithiothreitol (DTT), 20 mM
-glycerophosphate, 1 mM
Na3VO4, 1 mM phenylmethylsulfonyl
fluoride, 10 µg/ml aprotinin, and 10 µg/ml leupeptin) and sonicated
for 30 s. Cell debris was removed by centrifugation at 13,000 rpm
for 15 min at 4 °C. Protein concentration was determined by the
Bradford assay (9). Cell lysates were precleared with 40 µl of
protein A-Sepharose (Amersham Pharmacia Biotech) beads and then
incubated with anti-ERK1-CT antibody (1 µg/ml of lysate) at 4 °C
overnight. 40 µl of protein A-Sepharose beads were then added to
precipitate immunocomplexes and washed with lysis buffer three times;
immunoprecipitates were analyzed by immunoblotting with anti-p38
antibody.
-glycerophosphate, 20 mM MOPS, pH
7.2, 5 mM EGTA, 2 mM EDTA, 20 mM
MgCl2, 1 mM Na3VO4, 0.25 mM DTT) and then incubated with 20 µg of MBP (Sigma)
in the same buffer supplemented with 50 µM ATP and 10 µCi of [
-32P]ATP (Amersham Pharmacia Biotech) in a
volume of 20 µl at 30 °C for 15 min. Reactions were stopped by
spotting onto P81 paper. After three washes in 1%
H3PO4, phosphotransferase activity was quantitated by liquid scintillation counting.
and ERK1 in
Vitro--
Eluted GST-p38
wild-type or GST-p38
(AF) mutant
fusion protein was first incubated with bacterially expressed MKK6(E),
a constitutively active form of MKK6, at 30 °C for 2 h in 1×
kinase buffer (20 mM Hepes, pH 7.4, 10 mM
MgCl2, 1 mM DTT, 150 µM
phenylmethylsulfonyl fluoride, 0.4 mM ATP). GST-p38
proteins were then absorbed onto to glutathione-agarose beads and
incubated with bacterially expressed ERK1 at 4 °C for 2 h.
Following three washes in lysis buffer, the beads were boiled in 2×
Laemmli sample buffer (10), and the bound proteins were separated on
SDS-PAGE. Conversely, p38
was first incubated with GST-MKK6(E) beads
for activation under the conditions described above. GST-MKK6(E) beads
were pelleted and discarded, and GST-ERK1 beads were added to the
supernatants. After a 2-h incubation, the bound proteins were resolved
by SDS-PAGE.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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Fig. 1.
Effects of anisomycin and SB203580 on the
ERK1/2 phosphorylation and MBP kinase activity associated with
anti-ERK1 immunoprecipitates. A, HeLa cells were
treated with anisomycin (10 µg/ml, 30 min), SB203580 (5 µM, 20 min), and EGF (100 ng/ml, 15 min; Calbiochem),
respectively, and the phosphorylation of ERK1/2 was determined by
Western blot analysis with anti-active ERK1/2 antibody (Santa Cruz
Biotechology). B, MBP kinase activity was determined
in anti-ERK1 immunoprecipitates from anisomycin-, SB203580-, or
EGF-treated HeLa cells. The MBP kinase activity associated with
anti-ERK1 immunoprecipitates from anisomycin-treated cells could be
inhibited by including 5 µM SB203580 in the kinase assay
reaction. The data shown are the means +/ S.E. of 4 experiments.
in bacteria and used them to affinity
purify proteins from a rat brain lysate. Immunoblotting with
anti-p38
antibody revealed the presence of p38
protein on the
GST-ERK1 beads at a level well above that bound to GST alone (Fig.
2A). Under similar conditions,
GST-p38
was able to pull down both ERK1 and ERK2 proteins (Fig.
2B). These results indicated a specific, direct interaction
between ERK1/2 and p38
.
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Fig. 2.
Interaction of ERK1 and
p38 in rat brain lysate revealed by GST fusion
protein pull-down experiments. A, after incubation with
rat brain lysate, GST-ERK1 and GST beads were washed and immunoblotted
with anti-p38
antibody. B, ERK1/2 were found on
GST-p38
beads after incubation with rat brain lysate.
in HeLa cells treated with stimuli that
specifically activate either the ERK1/2 or p38 MAP kinase pathways. The
treatment of anisomycin resulted in a significant increase of p38
that was bound by GST-ERK1 (data not shown). No apparent difference of
the amount of p38
protein purified by GST-ERK1 fusion protein
between EGF-treated and untreated control HeLa cells was observed.
Treating HeLa cells with SB203580 prior to anisomycin
stimulation diminished the association of ERK1 with p38
. The
correlation of the enhancement of the binding of ERK1 and p38
with
p38 kinase activation indicated that the interaction between these two
MAP kinases was dependent upon the p38 but not the ERK1 activity status.
full-length DNA and then treated with
anisomycin 24 h later. Cell lysate was prepared and precipitated with anti-ERK1-CT antibody. Anti-ERK1 immunoprecipitates were probed
with anti-p38
antibody in Western blot analysis for coprecipitated p38
. As shown in Fig. 3A, a
much higher level of p38
protein was detected in the anti-ERK1
precipitates from anisomycin-treated HeLa cells than in those from
untreated control cells. This finding was consistent with the GST
fusion protein pull-down results and further confirmed the requirement
of p38 kinase activity for the interaction between ERK1 and p38
.
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Fig. 3.
Coimmunoprecipitation of ERK1 and
p38 in human cell lines in a p38 kinase
activity-dependent manner. A, after
transfection with pcDNA3-p38
and treatment with anisomycin (10 µg/ml, 30 min), HeLa cells were lysed, and the lysate was
precipitated by anti-ERK1-CT antibody. Anti-ERK1 precipitates were
immunoblotted with anti-p38
antibody. B, HEK293 cells
were transfected with pcDNA3-p38
24 h prior to treatment
with arsenite (0.5 mM, 4 h) or a combination of
SB203580 (5 µM, 60 min prior to arsenite) and arsenite
(SB+Arsenite). Cell lysates were prepared and
immunoprecipitation was performed with anti-ERK1-CT antibody.
Immunoprecipitates were probed with anti-p38
antibody. p38
was
present in higher levels in arsenite-treated cells than found in
SB203580-treated and untreated control cells.
in HEK293 cells
transfected with p38
was observed in a p38
activity-dependent manner (Fig. 3B). Compared
with untreated control cells, an increasing level of p38
protein
precipitated by anti-ERK1-CT antibody was found in arsenite-treated
HEK293 cells. Pretreating HEK293 cells with SB203580 prior to arsenite
reduced the level of p38
precipitated by anti-ERK1-CT to that of
untreated control. These results confirmed a p38
activity-dependent association of p38
with endogenous
ERK1 in mammalian cells.
and ERK1 using recombinant forms of these MAP kinases. Bacterially expressed ERK1 protein was mixed with GST-p38
wild-type or dominant negative mutant p38
(AF) that was preincubated with MKK6(E), a p38-specific upstream activating kinase. p38
(AF) is a kinase-inactive mutant with the substitution of two activating phosphorylation sites by alanine and phenylalanine (5), whereas MKK6 is
constitutively activated by replacement of two activating phosphorylation sites with glutamic acid (12). The ERK1 protein was
affinity-purified by p38
wild-type protein after its activation by
MKK6(E) (Fig. 4A). No MKK6
protein was detected in the p38
/ERK1 complexes (data not shown),
indicating that p38
and ERK1 can interact with each other without
involvement of other proteins. Consistent with the requirement of p38
kinase activity in the formation of ERK1/p38 complexes, ERK1
precipitated by p38
wild-type protein was present at a higher level
than that obtained with the p38
(AF) mutant. Similar observations
were made in the converse experiment, where GST-ERK1 bound active
wild-type but not inactive wild-type and dominant negative forms of
p38
(Fig. 4B). Furthermore, Western blotting analysis
with anti-phospho-p38
antibody revealed the p38
protein
coprecipitated with ERK1 was in its phosphorylated form (Fig.
4C).
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Fig. 4.
Direct interaction between bacterially
expressed ERK1 and p38 in
vitro. A, following incubating with 20 µg
of MKK6(E), 50 µg of eluted GST-p38
wild-type (WT) or
GST-p38
(AF) was precipitated with glutathione-agarose beads. 50 µg of thrombin-digested GST-ERK1 was added to GST-p38
beads, and
ERK1 that bound to p38 was detected by Western blotting with
anti-ERK1-CT antibody. B, 50 µg of thrombin-digested
p38
wild-type or p38
(AF) was first incubated with MKK6(E) and
then incubated with 50 µg of GST-ERK1 immobilized on
glutathione-agarose beads. GST-ERK1-bound p38
was detected by
Western blotting with anti-p38
antibody. C, a similar
blot to that shown in B was probed with anti-active p38
antibody (New England Biolabs, Beverly, MA).
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Fig. 5.
p38 suppresses ERK1
activation by EGF without affecting MEK1 phosphorylation. HeLa
cells were transfected either with pcDNA3-MKK6(E) (lane
MKK6) or with pcDNA3-CAT (Control). 24 h
later, both MKK6- and CAT-transfected cells were treated with EGF (100 ng/ml, 15 min). A, activation of ERK1/2 was determined by
Western blotting with anti-active ERK1/2 antibody. B,
phosphorylation of MEK1 was monitored by Western blotting with
anti-active MEK1 antibody (Santa Cruz Biotechnology, Santa Cruz,
CA).
and ERK1 may play a role in coordinating activation of
these two distinct MAP kinase pathways. These findings differ from
those revealed by previous studies in that the cross-talk between the
p38 and ERK1/2 signaling pathways was believed to mediate through
upstream activating kinases of the ERK1/2 cascade. Moreover, the
necessity of p38, but not ERK1, phosphotransferase activity for
the interaction between ERK1 and p38
indicates the cross-talk
between these two MAP kinase pathways is a one-way process.
and ERK1 and
the inhibitory effect of p38 on ERK1/2 phosphotransferase activities
independent of MEK1 phosphorylation, we hypothesize that activated p38
may sequester ERK1/2 and sterically block phosphorylation of these MAP
kinases by MEK1/2. The ability of activated p38 to regulate another
protein kinase allosterically is not restricted to ERK1/2. We have
recently reported that activated p38 can also form a complex with
casein kinase CK2 in HeLa cells, but in that instance p38
activates CK2 (13).
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ACKNOWLEDGEMENTS |
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We thank Dr. J. Han, the Scripps Research
Institute (La Jolla, CA) for pcDNA3-flagp38 wild-type and p38
(AF) dominant negative mutant. Constructs of constitutively active
MKK6(E) mutants, pGEX-MKK6(E) and pcDNA3-MKK6(E), were provided by
Dr. C. Glembotski, San Diego State University and Dr. R. Davis,
University of Massachusetts, respectively.
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FOOTNOTES |
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* This work was supported by operating grants from the Medical Research Council of Canada and the Heart and Stroke Foundation of British Columbia and Yukon, Canada (to S. P.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed: Dept. of Medicine, Koerner Pavilion, 2211 Wesbrook Mall, University of British Columbia, Vancouver, B. C. V6T 1Z3 Canada. Tel.: 604-822-9963; Fax: 604-822-8693; E-mail: spelech@home.com.
Published, JBC Papers in Press, January 18, 2001, DOI 10.1074/jbc.C000917200
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ABBREVIATIONS |
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The abbreviations used are: MAP, mitogen-activated protein; ERK, extracellular signal-regulated kinase; MEK, MAP/ERK kinase; MKK, MAP kinase kinase; GST, glutathione S-transferase; PAGE, polyacrylamide gel electrophoresis; DMEM, Dulbecco's minimum essential medium; DTT, dithiothreitol; MBP, myelin basic protein; MOPS, 3-(N-morpholino)propanesulfonic acid; EGF, epidermal growth factor.
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