From the Department of Physiological Chemistry,
Graduate School of Pharmaceutical Sciences, University of Tokyo,
7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan, the ¶ Department
of Immunology, Medical Institute of Bioregulation, Kyushu University,
Higashi-ku, Fukuoka 812-8582, Japan, the
Department of
Biochemistry, Akita University School of Medicine,
Akita 010-8543, Japan, and the ** University Health
Network, Departments of Medical Biophysics and Immunology, University
of Toronto, Toronto, Ontario M5G 2C1, Canada
Received for publication, December 26, 2002, and in revised form, February 27, 2003
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ABSTRACT |
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Stress-activated protein kinase/c-Jun
NH2-terminal kinase (SAPK/JNK), belonging to the
mitogen-activated protein kinase family, plays an important role in
stress signaling. SAPK/JNK activation requires the phosphorylation of
both Thr and Tyr residues in its Thr-Pro-Tyr motif, and SEK1 and MKK7
have been identified as the dual specificity kinases. In this study, we
generated mkk7 The SAPK/JNK1 is a
member of the family of mitogen-activated protein kinase (MAPK).
This MAPK is activated not only by many types of cellular stresses,
including changes in osmolarity, heat shock, and UV irradiation, but
also by serum, lysophosphatidic acid, and inflammatory cytokines
(interleukin-1 Targeted gene-disruption experiments in mice demonstrate that both SEK1
and MKK7 are required for embryonic development.
Sek1 In this regard, several in vitro experiments have shown that
SAPK/JNK is activated synergistically by SEK1 and MKK7 (7-9). The
synergistic activation may be related to the enzymatic properties of
the two MAPKKs: SEK1 prefers the Tyr residue and MKK7 prefers the Thr
residue of the MAPK. We have also reported that the synergistic activation of SAPK/JNK in response to stress signals is attenuated with
a decreased level of its Tyr phosphorylation in
sek1 To reveal the molecular mechanism of the synergistic activation of
SAPK/JNK by SEK1 and MKK7 in living cells, we generated mkk7 Generation of MKK7-deficient ES Cells--
A 17-kbp DNA fragment
of mkk7 gene was isolated from a genomic 129/J mouse
library. Targeting vector 1 contained a 585-bp short arm, a 6.7-kbp
long arm, three loxP sequences, and a hygromycin resistance cassette
(Hyg) in antisense orientation to mkk7 transcription. Targeting vector 2 contained a 709-bp short arm, a 5.2-kbp long arm,
and a neomycin resistance cassette (Neo) in antisense orientation to
mkk7 transcription. The linearized targeting vector 1 was
electroporated into E14K ES cells. ES cell colonies resistant to
hygromycin (0.2 mg/ml; Invitrogen) were screened for homologous
recombination by PCR (30 s at 94 °C, 30 s at 60 °C, 1 min at
72 °C, for 40 cycles) using primers specific for mkk7
genomic sequences and Hyg as described below. Next, the
mkk7+/hyg ES cells were transfected with Cre
recombinase expression vector, and cell colonies sensitive to
hygromycin were screened for deletion of the region containing exons
4-13 and Hyg by PCR. The linearized targeting vector 2 was
electroporated into mkk7+/del ES cells.
Retargeted ES cell colonies resistant to G418 (0.3 mg/ml; Invitrogen)
were screened for homologous recombination by PCR using primers
specific for mkk7 genomic sequences and Neo as described
below. As a result, two mkk7neo/del clones (001 and 002) were independently obtained. Both clones lack MKK7 completely,
and they are henceforth referred to as mkk7 Plasmids--
Plasmids that express FLAG-tagged SEK1, MKK7 Antibodies--
Antibodies against SAPK/JNK1 (C-17 and FL),
MKK7/MEK7 (T-19), and SEK1/MEK4 (C-20) were purchased from Santa Cruz
Biotechnology, Inc. Anti-phospho-SAPK/JNK (9251), and anti-phospho-SEK1
(9151) Abs were from Cell Signaling Technology. Anti-phospho-Tyr (PY20) and anti-phospho-Thr-Pro (P-Thr-Pro-101) mAbs were from BD Transduction Laboratories and Cell Signaling Technology, respectively. Anti-FLAG (M2) Ab and anti-HA affinity matrix were purchased from Sigma-Aldrich Co. and Roche Diagnostics, respectively. Rat anti-SEK1 (KN-001) and
anti-MKK7 (KN-004) mAbs applicable to immunoprecipitation and
immunoblotting were prepared in our laboratory as described previously
(10).
Transfection--
ES cells were plated at 2 × 106 cells onto a 60-mm dish and transfected 1 day later
with 8 µg of plasmid DNA using LipofectAMINE 2000 (Invitrogen). The
cells, after being cultured for 1 day, were transferred to four 35-mm
dishes and cultured for another day. Human embryonic kidney 293T cells
were plated at 1 × 106 cells onto a 35-mm dish and
transfected 1 day later with 4 µg of plasmid DNA using LipofectAMINE
2000 (Invitrogen). The cells were stimulated and subjected for the
assays of SAPK/JNK activity, immunoprecipitation, and immunoblotting.
Assay of SAPK/JNK Activity--
ES cells were plated
at 1.5 × 106 cells per 35-mm dish and cultured
overnight. As chemical and physical stresses, the cells were stimulated
by sorbitol (0.5 M; Wako), anisomycin (3 µg/ml; Sigma),
nocodazole (0.5 µg/ml for 1 h; Sigma), UV light (1 kJ/m2), and heat shock (44 °C for 10 min). SAPK/JNK
proteins were immunoprecipitated at 4 °C for 2 h using the
anti-SAPK/JNK polyclonal Ab (C-17; Santa Cruz Biotechnology, Inc.). The
SAPK/JNK activity in the precipitated fractions was measured with
glutathione S-transferase-c-Jun as an in vitro
substrate in the presence of 60 µM
[ Immunoprecipitarion and Immunoblotting--
To detect the
phosphorylation of Tyr and Thr residues in the Thr-Pro-Tyr motif of
endogenous SAPK/JNK, ES cells were plated at ~2 × 107 cells onto a 150-mm dish and mixed with 2 ml of a lysis
buffer (20 mM HEPES, pH 7.4, 1% Nonidet P-40, 10 mM NaCl, 0.05% 2-mercaptoethanol, 5 mM EDTA,
0.1 mM phenylmethylsulphonyl fluoride, 100 µM
Na3VO4, 20 µg/ml of leupeptin, 50 mM NaF, and 1 mM benzamidine). The cell lysates
were incubated with anti-SAPK/JNK Ab and protein A-Sepharose (Pharmacia) at 4 °C for 2 h, and the immuno-complexes were
washed several times with the lysis buffer. The samples were analyzed by SDS-PAGE and immunoblotting. Proteins were electrophoretically transferred to a polyvinylidene difluoride membrane (Bio-Rad) and
probed with anti-phospho-Tyr, phospho-Thr, and SAPK/JNK Abs. The bands
were visualized by SuperSignal West Pico chemiluminescent substrate for
the development of immunoblots using a horseradish peroxidase-conjugated second Ab according the manufacturer's
instructions (Pierce). Endogenous SEK1 and MKK7 were immunoprecipitated
with anti-SEK1 (KN-001) and anti-MKK7 (KN-004) mAbs and detected with anti-SEK1 (C-20) and anti-MKK7 (T-19) Abs, respectively.
All experiments were repeated at least three times with different
batches of the cell samples, and the results were fully reproducible.
Hence, most of the data shown are representative of several independent experiments.
Generation of MKK7-deficient ES Cells--
To examine the role of
MKK7 in SAPK/JNK regulation, we generated
mkk7 Impaired Stress-induced SAPK/JNK Activation in
MKK7-deficient ES Cells--
To examine the role of MKK7 in
stress-induced activation of SAPK/JNK, mkk7
The impairment of SAPK/JNK activation observed in the two MAPKK mutant
cells was further investigated with the different concentrations of
sorbitol. As shown in Fig. 2E, the
concentration-dependent activation curve of SAPK/JNK had a very
steep upstroke at ~0.15 M sorbitol in wild-type ES cells.
Interestingly, such steep activation of SAPK/JNK was markedly
attenuated in mkk7 SEK1 and MKK7 Are Indispensable for Stress-induced
SAPK/JNK Activation in ES Cells--
The above results in
Fig. 2 suggest that SEK1 and MKK7 synergistically contribute in the
stress-induced stimulation of SAPK/JNK in ES cells. To elucidate the
qualitative difference of the two MAPKKs, SEK1 and MKK7 expression
vectors were transfected into sek1 Properties of SEK1- and MKK7-induced Phosphorylation of
SAPK/JNK--
It has recently been reported that the
phosphorylation of Thr and Tyr residues in the Thr-Pro-Tyr motif of
SAPK/JNK is required for the full activation of the MAPK and that SEK1
and MKK7 preferentially phosphorylate the Tyr and Thr residues,
respectively, in vitro (7-9). Therefore, we examined the
stress-induced phosphorylation state of endogenous SAPK/JNK in
sek1 Loss of Stress-induced Thr Phosphorylation of SAPK/JNK
in Cells Expressing a Kinase-dead Mutant of SEK1--
In ES cells,
SEK1 and MKK7 clearly contributed to the dual phosphorylation of
SAPK/JNK in response to stress stimuli, and the MKK7-induced Thr
phosphorylation seemed to require the prior Tyr phosphorylation by
SEK1. Therefore, we further investigated the action of SEK1, which was
supposed to be involved in the prior Tyr phosphorylation of SAPK/JNK.
For the analysis, we used a human embryonic kidney cell line (293T) for
transient transfection because of low efficiency of transfection into
ES cells. HA-tagged JNK1 was co-expressed with FLAG-dnSEK1, which lacks
kinase activity, in the cells using pCMV5 mammalian vectors. In a
series of the experiments, the expression of HA-JNK1 was almost
constant (Fig. 5C). Thr and
Tyr phosphorylation of exogenous HA-JNK1 in response to 1 kJ/m2 of UV irradiation was measured. UV irradiation
induced the Thr and Tyr phosphorylation of HA-JNK1 (Fig. 5,
A and B, lane 3); however, not only
Tyr but also Thr phosphorylation was lost in the dnSEK1-expressing
cells (Fig. 5, A and B, lane 4). These
results clearly show that the stress-induced Thr phosphorylation of
SAPK/JNK requires the prior Tyr phosphorylation by SEK1 in living
cells.
Loss of Stress-induced Thr Phosphorylation of SAPK/JNK
in Cells Expressing the No Tyr-phosphorylated Forms of
SAPK/JNK--
Next, we investigated the contribution of the
Tyr residue in the Thr-Pro-Tyr motif of SAPK/JNK, phosphorylation of
which was supposed to proceed before the Thr modification by MKK7 in
the sequential phosphorylation. For the analysis, we constructed three kinds of HA-JNK1 mutants (APF, APY, and TPF), in which the Thr (T)-Pro
(P)-Tyr (Y) motif was replaced with Ala (A), and Phe (F), respectively.
The HA-JNK mutants and wild type were expressed in 293T cells by
transfection, and the phosphorylation and kinase activity were
measured. In a series of the experiments, the expression of HA-JNK1 was
almost constant (Fig. 6C). The
UV-induced Thr and Tyr phosphorylation and its ability to phosphorylate
glutathione S-transferase-c-Jun as substrate could be
detected in the wild-type HA-JNK1/TPY (Fig. 6, lane 1). Tyr
phosphorylation of the HA-JNK/APY mutant was also detected (Fig.
6B, lane 3), although its kinase activity was
completely lost (Fig. 6D, lane 3). Interestingly, the Thr phosphorylation could not be detected in the HA-JNK1/TPF mutant, in which the Tyr residue was replaced by Phe (Fig.
6A, lane 4). These results clearly show that the
stress-induced Thr phosphorylation of SAPK/JNK requires the
phosphorylated Tyr residue in living cells.
SAPK/JNK Interacts More Preferentially with SEK1 than
with MKK7--
To understand the molecular mechanism of the prior Tyr
phosphorylation of SAPK/JNK by SEK1, we examined the association of SAPK/JNK with SEK1. HA-JNK1 was co-expressed with FLAG-SEK1 and FLAG-MKK7
We also examined whether the above interaction is observable in ES
cells that did not overexpress the HA-JNK1 and FLAG-SEK1/MKK7 It has been reported in in vitro experiments that
synergistic activation of SAPK/JNK requires the phosphorylation of both Thr and Tyr residues within the Thr-Pro-Tyr motif by the two different activators, SEK1 and MKK7 (7-9). Although the two MAPKKs are capable
of catalyzing both phosphorylations, SEK1 prefers the Tyr
phosphorylation and MKK7 prefers the Thr phosphorylation in vitro (Fig. 8A). In a previous study, we reported that
SEK1 is essentially required for synergistic activation of SAPK/JNK in murine ES cells because the activation by various stresses was markedly
attenuated in sek1 First, MKK7 seemed to be a selective MAPKK for the synergistic
activation and the Thr phosphorylation of SAPK/JNK. The mutant mkk7 Tournier et al. have recently reported that the two MAPKKs,
MKK7 and SEK1, differently contribute in various stress-induced activation of SAPK/JNK using primary murine embryo fibroblasts isolated
from sek1 Besides SAPK/JNK, other members of MAPK family, ERK and p38, have also
two MAPKKs. ERK is activated by MKK1 and MKK2, and p38 is stimulated by
MKK3 and MKK6 (15). Therefore, the presence of two MAPKKs is a common
feature of mammalian MAPK-signaling pathways. Fleming et al.
have reported that MKK3 and MKK6 have a strong preference for the
phosphorylation of Tyr residue within the Thr-Gly-Tyr motif of p38 MAPK
(9). Other MAPKKs have also unique biochemical properties. Therefore,
ERK and p38 may be regulated cooperatively by two MAPKKs, as has been
observed in SAPK/JNK. Studies in other MAPKK-deficient cells would be
required for the elucidation of the functional significances of two
activators in ERK and p38 MAPK-signaling pathways.
Recently, Ferrell et al. have proposed the interesting
concept that SAPK/JNK cascade could, in principle, function as a
sensitivity amplifier, which converts graded inputs into more
switch-like outputs, allowing the cascade to filter out noise and yet
still respond decisively to supra-threshold stimuli (16-19). They have shown in Xenopus oocytes that SAPK/JNK responds to
physiological and pathological stimuli, such as progesterone and
sorbitol, in an all-or-none manner (20). The activation of SAPK/JNK by
the stimuli was graded at the level of a population of oocytes;
however, at the level of an individual oocyte, the stimulatory response seemed to be switch-like. In the present study, we have also observed a
very steep concentration-dependent response in the
activation of SAPK/JNK by hyperosmolar stress (i.e.
sorbitol) in murine ES cells (Fig. 2E). Furthermore, as
described in the Introduction, our recent work showed that both SEK1
and MKK7 are required for full SAPK/JNK activation and hepatoblast
proliferation in developing mice (6). This suggests that the
all-or-none type MAPK activation also occurs in mammalian cells at an
individual cell level only when the two MAPKKs are simultaneously
activated. Therefore, this MAPK signaling should strictly proceed
without errors, essentially through the two separated signals, one of
which activates SEK1 and the other activates MKK7. Although the
molecular mechanism whereby the two MAPKKs are simultaneously
stimulated by various stress signals remains to be resolved, it is
tempting to speculate that the existence of the two separated pathways
leading to SAPK/JNK activation may physiologically function as a
fail-safe mechanism as proposed previously (10).
/
mouse embryonic
stem (ES) cells in addition to
sek1
/
cells and compared the
two kinases in terms of the activation and phosphorylation of JNK.
Although SAPK/JNK activation by various stress signals was markedly
impaired in both sek1
/
and
mkk7
/
ES cells, there were
striking differences in the dual phosphorylation profile. The severe
impairment observed in mkk7
/
cells was
accompanied by a loss of the Thr phosphorylation of JNK without marked
reduction in its Tyr-phosphorylated level. On the other hand, Thr
phosphorylation of JNK in sek1
/
cells was also attenuated in addition to a decreased level of its Tyr
phosphorylation. Analysis in human embryonic kidney 293T cells
transfected with a kinase-dead SEK1 or a Thr-Pro-Phe mutant of JNK1
revealed that SEK1-induced Tyr phosphorylation of JNK1 was followed by
additional Thr phosphorylation by MKK7. Furthermore, SEK1 but not MKK7
was capable of binding to JNK1 in 293T cells. These results indicate
that the Tyr and Thr residues of SAPK/JNK are sequentially
phosphorylated by SEK1 and MKK7, respectively, in the stress-stimulated
ES cells.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and tumor necrosis factor-
). The activated
SAPK/JNK phosphorylates transcription factors c-Jun, Jun D, and
activating transcription factor-2 to regulate gene expression for the
stress response. Activation of SAPK/JNK requires the phosphorylation of
Tyr and Thr residues located in a Thr-Pro-Tyr motif in the activation
loop between VII and VIII of the kinase domain. The phosphorylation is
catalyzed by the dual specificity kinases SEK1 (also known as MKK4) and MKK7 (SEK2), which are capable of catalyzing the phosphorylation of
both Thr and Tyr residues in vitro (1, 2).
/
embryos die between embryonic day 10.5 (E10.5) and E12.5 with impaired liver formation (3-5). Furthermore, we
have recently reported that SEK1 is crucial for hepatocyte growth
factor-induced activation of SAPK/JNK in developing hepatoblasts of
mouse embryos. On the other hand, mkk7
/
embryos die between E11.5 and E12.5 with similar impairment of liver
formation and SAPK/JNK activation (6). These results clearly show that
both SEK1 and MKK7 play indispensable roles in hepatoblast
proliferation during mouse embryogenesis. Distinct biochemical
properties between SEK1 and MKK7 may be critical for the indispensable
roles of the two activators of SAPK/JNK in vivo.
/
mouse ES cells that retain MKK7 at the
same level as the wild-type cells (10). SAPK/JNK activation by UV
irradiation and Fc
-receptor stimulation was also attenuated in the
mkk7
/
mast cells. Despite the impaired
SAPK/JNK activation in the mkk7
/
cells, the
expression of SEK1 was strongly up-regulated, and SEK1 protein was
phosphorylated upon stimulation (11). Thus, both SEK1 and MKK7 seem to
be required for the synergistic and functional activation of SAPK/JNK
in a variety of mammalian cells.
/
mouse ES cells by gene
targeting, in addition to sek1
/
ES cells,
and further investigated the contribution of SEK1 and MKK7 to the
activation and phosphorylation of the MAPK. Our present results clearly
show that both SEK1 and MKK7 are required for the synergistic
activation of SAPK/JNK in response to various stimuli in ES cells.
Furthermore, we propose a sequential phosphorylation mechanism of
SAPK/JNK by the two activators, SEK1 and MKK7, in the stress-stimulated
living cells.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
ES cells in this manuscript. Specific primer sets used were 5'-GCC AAA
ACA CGG AGT GCT GG-3' and 5'-ATG TGA CCA GGC AGG AGT GG-3' for
wild-type (+) allele, 5'-TTA AGG CAA CTG GCA GAG-3' and 5'-AGC TGA CTC
TAG AGC TTG-3' for hyg allele, 5'-ATC TGC CTG TAG CAT GCC-3' and 5'-ACT
CCA AAC ACC TCC CAC-3' for del allele, 5'-GGA TGT GGA ATG TGT GCG AG-3'
and 5'-AGC TGG AAC CAC GCG CAA TGT GAG-3' for neo allele. Recombinant
ES cell clones were confirmed by Southern blotting of
XbaI-digested genomic DNA hybridized to a 530-bp 3'-flanking probe.
2,
and HA-tagged JNK1 were constructed as described previously (10). The
cDNA encoding FLAG-tagged SEK1 kinase dominant-negative mutant
(dnSEK1) by substituting Lys-129 with Arg (K129R) was cloned into
mammalian expression vector pCMV5. The cDNAs encoding SAPK/JNK1
mutants Ala-Pro-Phe (APF), Ala-Pro-Tyr (APY), and Thr-Pro-Phe (TPF),
were constructed by Kunkel method using the following three primers: 5'-GGA ACG AGT TTT ATG ATG GCG CCT TTT GTA GTG
ACT CGC TAC TAC AGA GCA CC-3' for APF mutant, 5'-GGA ACG AGT TTT ATG
ATG GCG CCT TAT GTA GTG ACT CGC TAC TAC AGA GCA CC-3' for
APY mutant, and 5'-GGA ACG AGT TTT ATG ATG ACG CCT TTT GTA GTG
ACT CGC TAC TAC AGA GCA CC-3' for TPF mutant.
-32P]ATP as described previously (12, 13).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
ES cells using two targeting vectors
in the process of mkk7+/+,
mkk7+/hyg, mkk7+/del, and
mkk7neo/del construction as follows (Fig.
1). First, the exons 4-13 region of
mkk7 gene was deleted from one allele using targeting vector 1 and Cre recombinase (Fig. 1A). Second, another allele of
mkk7 was disrupted by replacing the site of activating
phosphorylation in exon 9 with neomycin resistance cassette using
targeting vector 2 (Fig. 1B). As a result, two clones (001 and 002) of mkk7neo/del ES cells that completely
lack 48-kDa MKK7 were independently obtained (Fig. 1, C and
D). Thus, the null-mutant mkk7neo/del
clones are used as mkk7
/
ES cells in this
study.
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Fig. 1.
Targeted gene disruption of murine mkk7.
A and B, partial restriction map of murine
genomic mkk7 sequences and construction of targeting vectors
1 and 2. Targeting vector 1 (A) and 2 (B) contain
a hygromycin resistance gene (Hyg) plus three loxP sequences
(triangles) and a neomycin resistance gene (Neo),
respectively. The 14 exons of mkk7 are shown as
boxes, and MKK7-coding regions as filled boxes.
The genomic mkk7 3'-flanking probe used for Southern
blotting is indicated as gray boxes. The predicted
structures of targeted alleles are shown as hyg,
del, and neo, respectively. Restriction enzymes
used for the vector construction (H, HindIII, and
HincII) and genomic Southern blotting (XbaI) are
indicated by arrows. C, genomic analysis of ES
cells. Genomic DNAs isolated from wild-type
mkk7+/+, mkk7+/hyg,
mkk7+/del, mkk7+/neo, and
mkk7neo/del E14K ES cells were digested with
XbaI and analyzed by Southern blotting using the 3'-flanking
probe. Molecular weight markers and mkk7 alleles
(wt, 13 kb; hyg, 5 kb; del, 7.3 kb;
neo, 7.8 kb) are indicated by bars and
arrows, respectively. D, Western blot analysis of
48-kDa MKK7 in ES cells. MKK7 was immunoprecipitated and immunoblotted
with anti-MKK7 mAb and polyclonal Ab, respectively. The position of
48-kDa MKK7 is indicated by an arrow. No MKK7 was detected
in the two independent clones of mkk7neo/del ES
cells (001 and 002). The amount of MKK7 in
sek1 /
ES cells was comparable with that in
wild-type cells.
/
ES cells were incubated under various conditions, together with sek1
/
and wild-type cells. Fig.
2 shows the time courses of SAPK/JNK activity in response to a protein synthesis inhibitor (A, 3 µg/ml of anisomycin), heat shock (B, 44 °C for 10 min),
UV irradiation (C, 1 kJ/m2), and hyperosmolar
stress (D, 0.5 M sorbitol) in the two MAPKK mutant and wild-type ES cells. These stresses markedly stimulated SAPK/JNK activity in wild-type ES cells. Such stimulation, however, was
severely impaired in both MAPKK mutant cells, although the cells
contained SAPK/JNK at the same level as wild-type cells (see Fig.
4C). Thus, the stress-induced full activation of SAPK/JNK seems to require both SEK1 and MKK7 in ES cells.
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Fig. 2.
Time courses of stress-induced activation of
SAPK/JNK in wild-type, sek1 /
, and
mkk7
/
ES cells. Wild-type,
sek1
/
, and mkk7
/
ES cells were stimulated with anisomycin (A, 3 µg/ml),
heat shock (B, 44 °C for 10 min), UV (C, 1 kJ/m2) or sorbitol (D, 0.5 M and
E, the indicated concentrations) for the indicated times or
30 min (E). Cell lysates were prepared from the stimulated
cells and immunoprecipitated with an anti-SAPK/JNK (C-17) Ab. SAPK/JNK
activity in the precipitated fractions was measured with glutathione
S-transferase-c-Jun as a substrate in the presence of
[
-32P]ATP as described under "Experimental
Procedures." The insets of A-D show the
32P-phosphorylated glutathione
S-transferase-c-Jun, and the activity is expressed as the
-fold stimulation compared with the control level observed in wild-type
ES cells without the stress. The data shown are representative of three
independent experiments.
/
and
sek1
/
ES cells without significant change in
the half-maximum effective concentration of sorbitol. Impairment of
SAPK/JNK activation in the two MAPKK mutant cells was also observed in
other stress signals, including a microtubule-disruptive reagent,
nocodazole, and genotoxic stresses, such as etoposide and
arabinofuranosyl-cytosine (data not shown).
/
or
mkk7
/
ES cells (Fig.
3). SEK1 could rescue the impaired
SAPK/JNK activation in response to UV irradiation and heat shock in
sek1
/
ES cells (Fig. 3A).
However, MKK7 isoforms
1,
1, and
2, could not restore the
SAPK/JNK activation in sek1
/
ES cells. On
the other hand, MKK7
1 and
1, but not SEK1, could rescue the
impaired SAPK/JNK activation in response to heat shock, anisomycin, and
nocodazole in mkk7
/
ES cells (Fig.
3B). These results clearly show that SEK1 and MKK7 serve
different functions in the stress-induced SAPK/JNK activation in ES
cells.
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Fig. 3.
Effects of SEK1 or MKK7 expression on
stress-induced SAPK/JNK activation in sek1 /
and mkk7
/
ES cells. The two mutant
sek1
/
(A) and
mkk7
/
ES cells (B) were
transfected with MKK7
1,
1,
2, or SEK1 expression vector. The
transfected ES cells, after being cultured for 24 h, were
stimulated with UV (1 kJ/m2 and further incubation for 25 min), heat shock (HS, 43 °C for 30 min), anisomycin
(Ani, 10 µg/ml for 30 min), or nocodazole (Noc,
0.5 µg/ml for 1 h). Cell lysates were prepared and
immunoprecipitated with an anti-SAPK/JNK (C-17) Ab. SAPK/JNK activity
in the precipitated fractions was measured as described in Fig.
2.
/
and mkk7
/
ES cells together with wild-type cells. The three cell types were
stimulated with 0.5 M of sorbitol (Fig. 4, lanes
2, 6, and 10), 1 kJ/m2 of UV
irradiation (lanes 3, 7, and 11), and
3 µg/ml of anisomycin (lanes 4, 8, and
12). The endogenous SAPK/JNK was immunoprecipitated and
analyzed by immunoblotting with phospho-specific Abs. In a series of
the present experiments, these ES cells expressed almost the same
amounts of SAPK/JNK (Fig. 4C). The existence of
stress-induced Thr and Tyr phosphorylation within the Thr-Pro-Tyr motif
of SAPK/JNK in wild-type cells could be detected with
anti-phospho-Thr-Pro and anti-phospho-Tyr Abs, respectively (Fig. 4,
A and B, lanes 10-12).
Interestingly, Thr but not Tyr phosphorylation was almost completely
abolished in mkk7
/
cells (Fig.
4, A and B,
lanes 2-4) in which stress-induced
phosphorylation of SEK1 was retained at the same level as wild-type
cells (Fig. 4, D and E, lanes 5 and
6). On the other hand, the Tyr phosphorylation of SAPK/JNK
was greatly impaired in sek1
/
cells (Fig.
4B, lanes 6-8) in accordance with our
previous report (10). Surprisingly, the Thr phosphorylation was also
markedly attenuated in sek1
/
cells (Fig.
4A, lanes 6-8), although this mutant
contained the same amount of 48-kDa MKK7 as wild-type cells (Fig.
1D). These results indicate that SEK1 phosphorylates the Tyr
residue of SAPK/JNK and that MKK7 preferentially phosphorylates the Thr
residue of Tyr-phosphorylated SAPK/JNK rather than its
non-phosphorylated form. In other words, MKK7-induced Thr
phosphorylation requires the prior phosphorylation of SAPK/JNK at the
Tyr residue by SEK1 in the stress-stimulated ES cells (see Fig.
8C).
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Fig. 4.
Stress-induced Thr and Tyr phosphorylation of
endogenous SAPK/JNK in wild-type, sek1 /
,
and mkk7
/
ES cells. ES cells were
stimulated with 0.5 M sorbitol (lanes 2,
6, 10), 1 kJ/m2 of UV irradiation
(lanes 3, 7, 11), and 3 µg/ml of
anisomycin (lanes 4, 8, 12) and
further incubated at 37 °C for 25 min. Cell lysates were prepared,
and endogenous SAPK/JNK and SEK1 were immunoprecipitated
(IP) with anti-SAPK/JNK (C-17) polyclonal Abs
(A-C) and anti-SEK1 (KN-001) mAb (D and
E), respectively. The Thr phosphorylation (A) and
Tyr phosphorylation (B) of SAPK/JNK, together with
phosphorylated SEK1 (D) were determined using anti-phospho
Abs as described under "Experimental Procedures." IB,
immunoblots.
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Fig. 5.
Effects of SEK1 inhibition on stress-induced
Thr and Tyr phosphorylation of SAPK/JNK in 293T cells. 293T cells
were transfected with 1 µg of pCMV5/HA-JNK1, together with
(lanes 2 and 4) or without (lanes 1 and 3) 1 µg of pCMV5/FLAG-dnSEK1. The transfected
293T cells, after being cultured for 24 h, were stimulated with 1 kJ/m2 of UV irradiation and incubated for 25 min
(lanes 3 and 4). Cell lysates were prepared, and
HA-JNK1 was immunoprecipitated (IP) with anti-HA
affinity matrix. The Thr phosphorylation (A) and Tyr
phosphorylation (B) of HA-JNK1 were determined using
anti-phospho Abs. HA-JNK1 (C) and FLAG-dnSEK1
(D) were determined using anti-SAPK/JNK (FL) and anti-FLAG
(M2) Abs, respectively. IB, immunoblots.
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Fig. 6.
Effects of amino acid replacement of the TPY
motif on the stress-induced Thr and Tyr phosphorylation of SAPK/JNK in
293T cells. 293T cells were transfected with 1 µg of TPY-wild
type (lane 1), APF- (lane 2), APY- (lane
3), and TPF- (lane 4) mutant forms of HA-JNK1
expression vectors. The transfected 293T cells, after being cultured
for 24 h, were stimulated with 1 kJ/m2 of UV
irradiation and further incubated for 25 min. Cell lysates were
prepared and immunoprecipitated (IP) with anti-HA affinity
matrix. The Thr phosphorylation (A) and Tyr phosphorylation
(B) of HA-JNK1 were determined using anti-phospho Abs.
HA-JNK1 (C) was determined using anti-SAPK/JNK (FL)
polyclonal Abs. The SAPK/JNK activity (D) was measured as
described under "Experimental Procedures." IB,
immunoblots.
2 in 293T cells using pCMV5 mammalian vectors (10). The
transfected cells were stimulated with the protein synthesis inhibitor
anisomycin. The cell lysates were immunoprecipitated with anti-HA
affinity matrix and analyzed for the phosphorylation of SEK1 using
anti-phospho-SEK1 and anti-FLAG Abs. The expression of HA-JNK1 was
almost constant (Fig. 7C). The
different ratios of FLAG-SEK1 and FLAG-MKK7
2 expression vectors
induced varied expression levels of FLAG-SEK1 and FLAG-MKK7
2
proteins, but the sum of the expressed proteins was almost constant in
each of the experiments (Fig. 7A). FLAG-SEK1 was
phosphorylated in response to anisomycin in the presence and absence of
HA-JNK1 (Fig. 7B). Interestingly, both phosphorylated and
non-phosphorylated forms of FLAG-SEK1 could be coimmunoprecipitated
with HA-JNK1; however, FLAG-MKK7
2 was not (Fig. 7, D and
E). The interaction between SEK1 and SAPK/JNK seemed to be
comparable with that observed between JIP-1 and SAPK/JNK (data not
shown). These results clearly show that SAPK/JNK interacts more
preferentially with SEK1 than with MKK7 and suggest that the
interaction might be responsible for the prior Tyr phosphorylation of
SAPK/JNK by SEK1 (see Fig.
8C).
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Fig. 7.
Preferential association of SAPK/JNK with
SEK1. 293T cells were transfected with different amounts (0, 0.5, or 1 µg) of pCMV5/FLAG-tagged SEK1 and/or MKK7 2 expression
vectors, together with (lanes 4-6 and 10-12) or
without (lanes 1-3 and 7-9) 1 µg of
pCMV5/HA-JNK1. The transfected 293T cells, after being cultured for
24 h, were stimulated with 3 µg/ml of anisomycin (lanes
1-6) or not (lanes 7-12). Cell lysates were prepared
(A-C) and immunoprecipitated (IP) with anti-HA
affinity matrix (D and E). Expression of SEK1
plus MKK7
2 (A), phosphorylated SEK1
(B), and HA-JNK1 (C) were determined using
anti-FLAG (M2), anti-phospho-SEK1, and anti-SAPK/JNK (FL) Abs,
respectively. Co-immunoprecipitated SEK1 plus MKK7
2
(D) and phosphorylated SEK1 (E) were determined
using anti-FLAG (M2) and anti-phospho-SEK1, respectively.
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Fig. 8.
Schematic description of SAPK/JNK
phosphorylation by SEK1 and MKK7 under various conditions.
A, synergistic activation of SAPK/JNK by the dual
specificity kinase SEK1 or MKK7, which has been reported in in
vitro conditions (7-9). B, activation of SAPK/JNK by
SEK1 or MKK7 associated with their scaffold proteins, JIP-1 and MEKK1
(1, 2). C, synergistic activation of SAPK/JNK through
sequential phosphorylation by SEK1 plus MKK7 in murine ES cells
observed in the present study. See "Discussion" for further
explanation. TPY, Thr-Pro-Tyr motif.
2. However, we could not detect their direct interaction in the native ES
cells or in mouse tissues, including brain and liver (data not shown).
This may be because of low expression levels of the endogenous
proteins. Alternatively, endogenous SEK1 may localize near or exist as
a non-associated form with SAPK/JNK in native cells, and there may be a
molecular mechanism for releasing SEK1 rapidly after the
phosphorylation of SAPK/JNK.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
murine ES cells (10).
This attenuation was accompanied with a decreased level of the Tyr
phosphorylation. In the present study, we also generated
mkk7
/
ES cells and compared the two mutant
ES cells in terms of the activation and phosphorylation of SAPK/JNK.
Our present results not only confirm the synergistic activation of
SAPK/JNK reported previously but also indicate the unique properties of
SEK1 and MKK7 in the stress-induced phosphorylation of the MAPK as follows.
/
ES cells had a defect in synergistic
SAPK/JNK activation in response to a variety of stimuli (Fig. 2). This
defect could be selectively rescued by the introduction of MKK7
isoforms (
1 and
1) but not by SEK1 (Fig. 3B). Thr
phosphorylation of SAPK/JNK observed in wild-type cells was almost
completely abolished in mkk7
/
ES cells (Fig.
4A, lanes 2-4; see also Fig.
8C). Second, the properties of MKK7, which preferentially
catalyzes Thr phosphorylation of SAPK/JNK, seemed to be dependent on
another MAPKK, SEK1, because the Thr phosphorylation was greatly
impaired in sek1
/
ES cells, which retain
MKK7 expression (Fig. 4A, lanes 6-8). This idea was supported by the additional results as follows. 1)
Inhibition of SEK1 by the expression of its dominant-negative form
(dnSEK1) blocked Thr phosphorylation of SAPK/JNK in addition to Tyr
modification. 2) The SAPK/JNK mutant (TPF), which lacks phosphorylatable Tyr residue, could not be phosphorylated at the Thr
residue. 3) SEK1 could associate SAPK/JNK more preferentially than MKK7
and make a complex of SEK1 and SAPK/JNK without MKK7. Thus, we present
a novel activation mechanism that SEK1-induced Tyr phosphorylation of
SAPK/JNK is followed by additional Thr phosphorylation by MKK7 in
stress-stimulated ES cells (Fig. 8C). In other words, MKK7
preferentially phosphorylates the Thr of Tyr-phosphorylated SAPK/JNK.
On the other hand, SEK1 catalyzes Tyr phosphorylation of the MAPK in a
manner independent on MKK7-induced Thr phosphorylation.
/
,
mkk7
/
, and dual deficient mice (14). Their
report shows that MKK7 is more important than SEK1 in the activation of
SAPK/JNK by proinflammatory cytokines in murine embryo fibroblasts.
SAPK/JNK activation in response to UV and anisomycin was almost
completely lost in sek1
/
mkk7
/
murine embryo fibroblasts, but
approximately half stimulation of SAPK/JNK was retained in
sek1
/
or mkk7
/
murine embryo fibroblasts. In contrast, SAPK/JNK activation in response
to TNF
and IL-1
was almost completely lost in
mkk7
/
cells, but 50% stimulation was
observed in sek1
/
cells. Thus, their results
are somewhat different from ours observed in the single mutant of
sek1
/
or mkk7
/
ES
cells, where SAPK/JNK activation by various stimuli was greatly reduced. These differences may be caused by the specificity of cell
types used. ES cells were derived directly from preimplantation embryos
and maintained in vitro under undifferentiated conditions. Thus, the molecular mechanism of SAPK/JNK activation observable in ES
cells may be considered a prototype in mammalian cells. In more
differentiated cells, other cellular proteins, such as the JIP group of
scaffold proteins, may regulate the protein interaction among SEK1,
MKK7, and SAPK/JNK to alter the properties of the MAPKKs, resulting in
the gain of dual-specificity kinase activity (Fig. 8B).
However, overexpression of JIP1, -2, or -3 by cDNA transfection did
not support this possibility (data not shown). Therefore, it will be
critical to investigate the two phosphorylated states of the
Thr-Pro-Tyr motif in endogenous SAPK/JNK and associated proteins to
understand the molecular mechanism of synergistic activation of the
MAPK in each type of cells.
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FOOTNOTES |
---|
* This work was supported in part by research grants from the "Research for the Future" Program of the Japan Society for the Promotion of Science (JSPS-RFTF 96L00505), the Mitsubishi Foundation, and the Scientific Research Funds of the Ministry of Education, Culture, Sports, Science and Technology of the Japanese Government.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.
§ Both authors contributed equally to this work.
To whom correspondence should be addressed. Tel.:
81-3-5841-4754; Fax: 81-3-5841-4751; E-mail:
nishina@mol.f.u-tokyo.ac.jp.
Published, JBC Papers in Press, March 6, 2003, DOI 10.1074/jbc.M213182200
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ABBREVIATIONS |
---|
The abbreviations used are: SAPK, stress-activated protein kinase; JNK, c-Jun N-terminal kinase; MAPK, mitogen-activated protein kinase; E, embryonic day; ERK, extracellular signal-regulated kinase; MKK, mitogen-activated protein kinase kinase; MAPKK, mitogen-activated protein kinase kinase; SEK, stress-activated protein kinase/extracellular signal-regulated kinase kinase; ES, embryonic stem; Ab, antibody; mAb, monoclonal antibody; HA, hemagglutinin; Hyg, hygromycin resistance cassette; Neo, neomycin resistance cassette; dnSEK1, dominant-negative SEK1 mutant.
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