From the
Osmotic shock induces a variety of biochemical and physiological
responses in vertebrate cells. By analyzing extracts obtained from rat
3Y1 fibroblastic cells exposed to hyper-osmolar media, we have found
that mitogen-activated protein kinases (MAPKs) and stress-activated
protein kinases (SAPKs, also known as JNKs) are both activated in
response to osmotic shock. MAPKK1 (MEK1) was also activated markedly.
Furthermore, Raf-1 and MEKK were activated strikingly by the osmotic
shock. Activation of Raf-1 and MEKK in response to osmotic shock was
detected also in PC12 cells, in which MEKK activation by the osmotic
shock was much stronger than that by epidermal growth factor.
Activation of SAPKs in PC12 cells by the osmotic shock was also more
marked than that by epidermal growth factor. The activated MEKK
phosphorylated not only MAPKKs but also XMEK2, which is distantly
related to MAPKK. Recombinant wild-type XMEK2, but not kinase-negative
XMEK2, was able to phosphorylate and activate recombinant SAPK
Mitogen-activated protein kinase (MAPK)
In yeast a
number of MAPKK/MAPK cascades have been found and each kinase cascade
is functioning in its specific cellular
pathway
(5, 23, 24) . In contrast, in vertebrate
cells the same MAPKK and the same MAPK molecules function commonly in
diverse biological processes as described above. Recent studies,
however, revealed other members of the MAPK superfamily in vertebrate
cells; stress-activated protein kinases (SAPKs, also known as
JNKs)
(25, 26, 27, 28, 29, 30) and p38/MPK2 (HOG1) (31-34). These kinases are
distantly related to classical MAPKs, and may have separate and/or
overlapping functions.
To see possible functional separation or
redundancy of these members of the MAPK superfamily, identification of
extracellular signals that elicit activation of these kinases and
dissection of their signaling pathways are needed. As a first step to
approach these problems, we surveyed conditions that lead to activation
of both classical and novel members of the MAPK superfamily, and found
that osmotic shock induces marked activation of both MAPKs and SAPKs in
fibroblastic cells. Interestingly, both Raf-1 and MEKK have been found
to be activated markedly also by osmotic shock. In addition, we have
found that XMEK2 (35), which is distantly related to MAPKK, can
function as a SAPK kinase and can be activated directly by MEKK.
We surveyed conditions that induce activation of both
classical and novel members of the MAPK superfamily in rat 3Y1
fibroblastic cells by using kinase detection assays within gels
containing substrate proteins (in-gel kinase assays), as the assays are
sensitive and suitable to detect activation of both isotypes of
classical mammalian MAPKs (43-kDa MAPK (ERK1) and 41-kDa MAPK (ERK2))
and have been applied successfully to rat PC12 cells and 3Y1
cells
(38, 43) . Rat 3Y1 cells were exposed to various
extracellular stimuli such as growth factors, phorbol esters, heat
shock, and osmotic shock, and extracts from these cells were analyzed
by in-gel kinase assays containing MBP, microtubule-associated protein
2 (MAP2), c-Jun (Jun), histone, or casein.
Activation of
MAPKK activity also occurred when cells were exposed to hyper-osmolar
media with NaCl. Various times after the exposure of the cells to 0.7
M NaCl, MAPKK1 was immunoprecipitated from the cell extracts
and assayed for MAPKK activity (=MAPK activating activity).
MAPKK1 was activated in a time-dependent manner (Fig. 2). This
time course was similar to that of MAPK activation (Fig. 1A,
MBP).
During this
study we noted that XMEK2 can be phosphorylated by SAPK, as shown in
Fig. 5B in which phosphorylation of kinase-negative
XMEK2 was evident in the presence of SAPK. Then, we carried out the
kinase detection assay within gels containing XMEK2. Extracts from 3Y1
cells exposed to various concentrations of NaCl were analyzed by this
assay. Two kinases with apparent molecular mass of 54 and 46 kDa were
found to be activated by hyper-osmolar media (Fig. 7A).
Because their apparent molecular mass and the dependence of their
activation on NaCl concentration are identical to those of
p54
Two recent reports demonstrated that hyper-osmolar media
cause a marked increase in JNK (=SAPK) activity in Chinese
hamster ovary cells (47) and a weak, short-term increase in MAPK
activity in Madin-Darby canine kidney epithelial cells
(48) ,
respectively. The results presented in this study have clearly shown
that osmotic shock with hyper-osmolar media induces activation of both
MAPK (p43 and p41) and SAPK (p54 and p46) in mammalian fibroblastic 3Y1
cells. The activations are rapid and marked, persist for more than 1 h,
and are reversible. This study thus demonstrates that both MAPK and
SAPK could function in the osmotic shock-induced signal transduction
pathway.
During the efforts to dissect upstream pathways resulting
in the activation of MAPKs and SAPKs in this system, we have found that
osmotic shock induces marked activation of MEKK as well as Raf-1.
Lange-Carter and Johnson
(22) recently revealed EGF- or nerve
growth factor-induced p21
We hypothesized that
XMEK2
(35) , which has been isolated as a member of the MAPKK
superfamily but is distinct from MAPKK, might be a SAPK activator.
Consistent with this hypothesis, recombinant wild-type XMEK2, but not
kinase-negative XMEK2, could activate recombinant SAPK
Finally, this study has shown that XMEK2 (SEK1) is one of
the best substrates in vitro for SAPK. In this context, it
should be pointed out that MAPKK is phosphorylated by MAPK
(52) .
Physiological meaning of these apparent feedback phosphorylations still
remains unclear.
We thank Dr. Tetsu Akiyama (Osaka University) and Dr.
Kunihiro Matsumoto (Nagoya University) for stimulating discussions. We
also thank K. Takenaka, K. Shirakabe, and F. Itoh for help in some of
the experiments.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
in vitro. In addition, this activity of XMEK2 was activated by
the activated MEKK. These results suggest that the MAPK cascade
consisting of Raf-1, MAPKK, and MAPK and the SAPK cascade consisting of
MEKK, XMEK2, and SAPK are both activated in response to osmotic shock.
Finally, it was found that XMEK2 is a good substrate for SAPK.
(
)
and its direct activator, MAPK kinase (MAPKK), are
activated in a large number of signal transduction pathways in
vertebrate cells (for review, see Refs. 1-6). MAPKK and MAPK are
thought to form a linear pathway called the MAPKK/MAPK cascade, which
has been shown to play a pivotal role in diverse biological processes
including fibroblastic cell proliferation and
transformation
(7, 8, 9) , PC12 cell
differentiation
(8) , Xenopus oocyte
maturation
(10) , and metaphase II arrest of unfertilized
eggs
(11, 12) . Several serine/threonine kinases such as
Raf-1
(13, 14, 15) , Mos
(16, 17) ,
and MEKK
(18) have been identified as a direct activator for
MAPKK. Raf-1 is a proto-oncogene product and is one of targets of
p21
(for review, see Ref. 19 and 20). Mos is
also a proto-oncogene product and is shown to function during oocyte
maturation
(21) . MEKK is found to be activated by epidermal
growth factor (EGF) and nerve growth factor through a
p21
-dependent pathway in PC12 cells
(22) ,
but the activation in this system is not so strong, suggesting that
MEKK may be activated also in other signaling pathways.
Antibodies
Anti-MEKK antibody (=antibody
against both N- and C-terminal fragments of MEKK) was purchased from
UBI. Anti-Raf-1 antibody used here was characterized
previously
(36) . Anti-Xenopus MAPKK antibody we
previously produced
(37) was shown to react specifically with
mammalian MAPKK1 (MEK1),(
)
and thus used for
immunoprecipitation of rat MAPKK1.
In-gel Kinase Assays
These assays were performed
according to the method previously described
(38) with slight
modifications. Briefly, after electrophoresis, SDS was removed by
washing the gel with 20% 2-propanol. Then, after denaturation with 6
M guanidine HCl and renaturation in a 0.04% Tween
40-containing buffer, the gel was incubated at 22 °C for 1 h with
10 ml of a reaction buffer consisting of 40 mM Hepes, pH 8.0,
0.1 mM EGTA, 20 mM MgCl, 2 mM
dithiothreitol, and 25 µM
[
-
P]ATP (25 µCi). Myelin basic protein
(MBP, 0.5 mg/ml), microtubule-associated protein 2 (MAP2, 0.1 mg/ml),
or c-Jun (0.05 mg/ml) was used as a substrate in gels. These substrate
proteins were added to the separation gel prior to polymerization of
acrylamide. MBP was purchased from Sigma. MAP2 was purified from
porcine brain
(39) , and human c-Jun was bacterially expressed as
a histidine-tagged protein and purified by a nickel affinity column. In
some experiments, gels containing recombinant XMEK2 were used.
Expression of Recombinant XMEK2 and SAPK
The
XMEK2 coding region
(35) was amplified by using reverse
transcriptase-polymerase chain reaction and inserted into
BamHI site of the pGEX-2T (Pharmacia Biotech Inc.) and pMAL-c2
to construct glutathione S-transferase fusion protein and
maltose-binding protein fusion protein, respectively. The proteins were
purified by glutathione-Sepharose affinity chromatography (for
glutathione S-transferase fusion protein) or by amylose resin
(for maltose-binding protein fusion protein) followed by
phenyl-Sepharose column chromatography. The purified proteins were
dialyzed against a buffer consisting of 20 mM Tris-HCl (pH
7.5), 2 mM EGTA, 2 mM dithiothreitol, 1 mM
phenylmethylsulfonyl fluoride, and 1% aprotinin and stored at -80
°C. No difference was observed between both types of recombinant
XMEK2 in any of the experiments presented here. The kinase-negative
XMEK2 was obtained by a lysine-to-arginine mutation in the presumptive
ATP-binding site as described
(35) , and then glutathione
S-transferase and maltose-binding protein fusion proteins were
prepared as above. The SAPK coding region
(29) was
amplified by using reverse transcriptase-polymerase chain reaction and
inserted into the BamHI site of the pET16b. The
histidine-tagged protein was purified from bacterial lysates by a
nickel affinity column.
Cell Cultures and Preparation of Cell Extracts
Rat
3Y1 cells were maintained in Dulbecco's modified Eagle's
medium (DMEM) supplemented with 10% fetal calf serum. Rat PC12 cells
were maintained in Dulbecco's modified Eagle's medium
supplemented with 0.35% glucose, 5% heat-inactivated horse serum, and
10% fetal calf serum. Confluent cultures of rat 3Y1 cells were exposed
to various concentrations of NaCl. Semiconfluent cultures of rat PC12
cells were exposed to various concentrations of NaCl or 30 nM
EGF. Various times after the exposure, cells were lysed in a buffer
consisting of 20 mM Tris-HCl (pH 7.5), 10 mM EGTA, 60
mM -glycerophosphate, 10 mM MgCl
, 1%
Triton X-100, 2 mM dithiothreitol, 1 mM vanadate, 1
mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 10
µg/ml pepstatin, and 1% aprotinin and centrifuged at 16,000
g for 30 min at 2 °C. The supernatant (approximately 0.1
mg/ml cellular proteins) was subjected to in-gel kinase assays or
immune complex kinase assays.
Immunoprecipitation and Immune Complex Kinase
Assays
For immunoprecipitation, 5 µg of the appropriate
antibody was added to 400-µl aliquots of lysate (approximately 40
µg of cellular proteins) and incubated for 4 h at 4 °C. 20
µl of protein A-Sepharose was added to the tubes, and incubation
continued for a further 1 h. The precipitates were washed three times
with the buffer containing 0.5 M NaCl. To assay for MAPKK1,
the immunocomplex with anti-MAPKK1 antibody, bound to 20 µl of
protein A-Sepharose, was suspended in 15 µl of a buffer consisting
of 20 mM Tris-HCl (pH 7.5), 2 mM EGTA, 10 mM
MgCl, and 100 µM ATP (referred to as kinase
buffer) containing 5 µg of Xenopus recombinant
MAPK
(40, 41) , and incubated for 20 min at 30 °C.
After addition of 10 µg of MBP and
[
-
P]ATP (1 µCi), the reaction mixture
was incubated for a further 15 min. The reaction was stopped by
addition of Laemmli's sample buffer. Phosphorylated MBP was
resolved by SDS-PAGE and quantified by an image analyzer (FUJIX BAS
2000). To assay for MAPKK activating activity (=MAPKK-K
activity), the immunocomplex with anti-MEKK antibody or anti-Raf-1
antibody, bound to 20 µl of protein A-Sepharose, was suspended in
15 µl of the kinase buffer containing 5 µg of Xenopus recombinant MAPKK
(42) , 20 µg of recombinant
kinase-negative MAPK (glutathione S-transferase fusion
protein, see Ref. 42), and [
-
P]ATP (5
µCi). The reaction mixture was incubated for 1 h at 30 °C. The
reaction was stopped by addition of Laemmli's sample buffer.
Phosphorylated glutathione S-transferase kinase-negative MAPK
was resolved by SDS-PAGE and quantified by an image analyzer. To assay
for XMEK2 phosphorylating activity, the MEKK immunoprecipitate was
suspended in 15 µl of the kinase buffer containing 5 µg of
recombinant kinase-negative XMEK2 and
[
-
P]ATP (10 µCi). The reaction was
stopped by addition of Laemmli's sample buffer. Phosphorylated
XMEK2 was resolved by SDS-PAGE and autoradiographed. To assay for XMEK2
activating activity, the MEKK immunoprecipitate was suspended in 15
µl of the kinase buffer containing 5 µg of recombinant
wild-type XMEK2 and 10 µg of recombinant SAPK
, and incubated
for 1 h at 30 °C. The reaction mixture was then subjected to Jun
in-gel kinase assay to detect enhanced activity of recombinant
SAPK
.
Measurement of Protein Kinases in Vitro
To measure
the activity of XMEK2 to activate SAPK, either 2 or 4 µg of
recombinant SAPK
was mixed with 5 µg of recombinant XMEK2 in a
final volume of 15 µl of kinase buffer containing 10 µg of Jun
and [
-
P]ATP (1 µCi). The reaction
mixture was incubated for the indicated times at 20 °C. The
reaction was stopped by addition of Laemmli's sample buffer.
Phosphorylated Jun was resolved by SDS-PAGE and quantified by an image
analyzer. To assay for SAPK
phosphorylating activity, 5 µg of
either kinase-negative XMEK2 or wild-type XMEK2 was mixed with 5 µg
of recombinant SAPK
in a final volume of 15 µl of the kinase
buffer containing [
-
P]ATP (1 µCi). The
reaction mixture was incubated at 30 °C for 10 min. The reaction
was stopped by addition of Laemmli's sample buffer. To assay for
MAPK activating activity, 5 µg of recombinant MAPK was mixed with
either 2 µg of recombinant MAPKK or 5 µg of recombinant XMEK2
in a final volume of 15 µl of kinase buffer. The reaction mixture
was incubated at 30 °C for 30 min. Then, 10 µg of MBP and
[
-
P]ATP (1 µCi) were added to the
reaction mixture and incubated for a further 30 min. The reaction was
stopped by addition of Laemmli's sample buffer. Phosphorylated
MBP was resolved by SDS-PAGE and quantified by an image analyzer.
(
)
Among these stimuli, hyper-osmolar media (with NaCl) were
found to induce activation of both MAPKs and SAPKs markedly (see
Fig. 1
). MBP is a good substrate for
MAPKs
(38, 43, 44, 45, 46) , but
not for SAPKs
(25, 28) , and Jun is a good substrate for
SAPKs
(25, 28) , but not for MAPKs. MAP2 can serve as a
good substrate for both SAPKs and
MAPKs
(25, 38, 43, 44, 45, 46) ,
but under the conditions used here activation of SAPKs was more clearly
visible than that of MAPKs in in-gel (MAP2) assays. Histone and casein
are very poor substrates for both MAPKs and
SAPKs
(25, 38, 43, 44, 45, 46) .
Figure 1:
Activation of SAPK and MAPK in response
to osmotic shock. A, 3Y1 cells were exposed to 0.5 M
NaCl and cell lysates were prepared at the indicated times. An aliquot
of each extract was subjected to the kinase detection assay within a
polyacrylamide gel (in-gel assay) containing MBP (top panel),
Jun (middle panel), or MAP2 (bottom panel). The
autoradiographs were shown. The positions of p54 and
p46
are indicated by closed and open
arrowheads, respectively. p43
(ERK1) and
p41
(ERK2) are shown by arrows in a top
panel.B, 3Y1 cells were exposed to indicated
concentrations of NaCl for 60 min. Then, cell lysates were prepared and
an aliquot of each extract was subjected to in-gel assay containing
MAP2 or Jun. The positions of p54
(closed
arrowhead) and p46
(open arrowhead) are
shown in each panel. C, 3Y1 cells were stimulated with 0.75
M NaCl for 5 min and then medium was changed to 0 M
NaCl. Cell lysates were obtained at the indicated times after the
medium was changed to 0 M NaCl. An aliquot of cell lysate was
subjected to in-gel assay containing MAP2. The positions of
p54
(closed arrowhead) and p46
(open arrowhead) are shown.
Both p43 (ERK1) and p41
(ERK2) were
activated in a time-dependent manner when 3Y1 cells were exposed to
hyper-osmolar media (Fig. 1A, MBP,
arrows). The activation was detected within 3 min of exposure
of the cells to 0.5 M NaCl and peaked about 30-60 min
after the exposure (Fig. 1A, MBP). At the maximum,
almost full activation occurred, as almost full conversion to the
electrophoretically retarded band was observed in the immunoblotting
with anti-MAPK antibody (data not shown). Concomitant with the
activation of MAPKs, p54
and p46
were
activated by the osmotic shock; the time course of their activation was
similar to that of MAPK activation (Fig. 1A, Jun and
MAP2, arrowheads). Identification of these two polypeptides as
SAPK was shown by their substrate specificity in in-gel kinase assays;
their ability to phosphorylate Jun and MAP2 and their inability to
phosphorylate MBP, histone, or casein (Fig. 1A and data
not shown) and reactivity to anti-SAPK antibody that had been produced
by immunizing rabbits with recombinant rat SAPK
(data not shown).
Activation of about half of the molecules was achieved at the maximum,
as revealed by the mobility shift in immunoblotting (data not shown).
The activation was dependent on the concentration of NaCl; the maximum
activation was observed at 0.7 M NaCl
(Fig. 1B). The activation was reversible. When the cells
were exposed to 0.7 M NaCl for 5 min and then returned to
normal medium, inactivation of SAPKs occurred rapidly
(Fig. 1C), suggesting that mechanisms inactivating SAPKs
may be operating constitutively under normal conditions. Activation of
MAPKs and SAPKs in 3Y1 cells could be induced by hyper-osmolar media
with sorbitol as well as with NaCl (data not shown).
Figure 2:
Activation of MAPKK1 in response to
osmotic shock. 3Y1 cells were exposed to 0.7 M NaCl and cell
lysates were prepared at the indicated times. A 400-µl aliquot of
cell lysates was subjected to immunoprecipitation with anti-MAPKK1
antibody and the immunoprecipitate was assayed for MAPKK1 activity as
described under ``Materials and
Methods.''
Since previous studies have shown that Raf-1 and MEKK can
work as a MAPKK kinase that phosphorylates and activates MAPKK, we
followed their activity in rat 3Y1 cells after osmotic shock. Extracts
were obtained from the cells that had been exposed to 0.7 M
NaCl for 0, 20, and 50 min, and were subjected to immunoprecipitation
with anti-Raf-1 antibody or anti-MEKK antibody, and each
immunoprecipitate was assayed for the MAPKK activating activity. Both
Raf-1 and MEKK were found to be activated by the exposure of the cells
to hyper-osmolar media (Fig. 3). These activations were quite
striking and reproducibly observed.
Figure 3:
Activation of MEKK and Raf-1 in response
to osmotic shock. Rat 3Y1 cells were exposed to 0.7 M NaCl and
cell lysates were prepared at 0, 20, and 50 min after the exposure. A
400-µl aliquot of cell lysates was subjected to immunoprecipitation
with anti-MEKK antibody (left) or anti-Raf-1 antibody
(right) immobilized on protein A-Sepharose, and assayed for
MAPKK activating activity as described under ``Materials and
Methods.''
The activation of both Raf-1 and
MEKK in response to high NaCl concentrations was observed also in rat
PC12 cells (Fig. 4A). It was reproducibly observed that
MEKK activation by osmotic shock (NaCl) was much stronger than that by
EGF while Raf-1 activation by both stimuli was comparable
(Fig. 4A). Both p54 and p46
were activated markedly by the osmotic shock in PC12 cells
(Fig. 4B), while they were activated very weakly by EGF
(Fig. 4B). Thus, SAPK activation correlated apparently
with MEKK activation and MEKK might function upstream of SAPK. If so,
it is reasonable to assume that MEKK may phosphorylate and activate a
presumptive SAPK-activating kinase which should be distantly related to
MAPKK, because MAPKK is shown to be incapable of activating SAPK in
vitro (Ref. 28, data not shown). Then, we hypothesized that
XMEK2
(35) , which encodes a member of the MAPKK superfamily and
is distantly related to MAPKK in structure, might be a SAPK kinase and
serve as a substrate for MEKK. Consistent with this hypothesis,
bacterially-produced XMEK2 was phosphorylated by the anti-MEKK
immunoprecipitate obtained from rat 3Y1 cells (see
Fig. 6A). In addition, the ability of MEKK to
phosphorylate XMEK2 was activated by the osmotic shock and EGF, like
the MAPKK activating activity (data not shown), supporting that MEKK
phosphorylates XMEK2.
Figure 4:
Activation of MEKK, Raf-1, and SAPK in
PC12 cells. A, cell lysates were prepared from PC12 cells
treated with none, 0.7 M NaCl for 15 min, or 30 nM
EGF for 3 min. A 400-µl aliquot of cell lysates was subjected to
immunoprecipitation with anti-MEKK antibody (left) or
anti-Raf-1 antibody (right) immobilized on protein
A-Sepharose, and the immunoprecipitate was assayed for the activity as
in Fig. 3. B, PC12 cells were exposed to 0.4 or 0.8 M
NaCl for 60 min or 30 nM EGF for 3 min. Each lysate was
subjected to in-gel kinase assay containing Jun. The positions of
p54 (closed arrowhead) and p46
(open arrowhead) are
indicated.
Figure 6:
Phosphorylation and activation of XMEK2 by
MEKK. 3Y1 cells were exposed to 0.7 M NaCl. Cell lysates were
prepared at 0, 20, and 50 min after exposure. A 400-µl aliquot of
cell lysates was subjected to immunoprecipitation with anti-MEKK
antibody immobilized on protein A-Sepharose beads. The
immunoprecipitate was assayed for XMEK2 phosphorylating activity as
described under ``Materials and Methods.'' The
autoradiography of phosphorylated XMEK2 is shown (A). The same
immunoprecipitate was assayed for XMEK2 activating activity
(B). XMEK2 activity was determined by the SAPK activating
activity, and the SAPK
activity was determined by in-gel assay
containing Jun.
Fig. 5A shows that recombinant
wild-type XMEK2, but not kinase-negative XMEK2, could activate
recombinant, bacterially expressed SAPK in vitro (Fig. 5A) and that XMEK2 phosphorylated SAPK
in vitro (Fig. 5B). However, XMEK2 could not
activate recombinant MAPK (Fig. 5C). These results
suggest that XMEK2 can work as a SAPK kinase.
Figure 5:
Activation and phosphorylation of
SAPK by XMEK2. A, after incubation of recombinant
SAPK
(2 µg (
) or 4 µg (
,
)) with either
recombinant wild-type XMEK2 (WT-XMEK2,
,
) or
kinase-negative XMEK2 (KN-XMEK2,
) for the indicated times, the
SAPK
was assayed for the activity to phosphorylate Jun as
described under ``Materials and Methods.'' The basal activity
of SAPK
was subtracted in each time point. B, either
WT-XMEK2 or KN-XMEK2 was incubated with recombinant SAPK
in the
presence of [
-
P]ATP for 30 min at 30
°C. The phosphorylated proteins were resolved by SDS-PAGE and
autoradiographed. The positions of recombinant XMEK2 and recombinant
SAPK
are indicated as an arrow and arrowhead,
respectively. C, recombinant MAPK was incubated with either
recombinant wild-type MAPKK (MAPKK) or recombinant wild-type XMEK2
(XMEK2) for 30 min at 30 °C. Then, the MAPK activity toward MBP was
quantified as described under ``Materials and
Methods.''
Moreover, the activity
of XMEK2, i.e. the SAPK activating activity, was activated by
the anti-MEKK immunoprecipitate. This XMEK2 activating activity of MEKK
was activated by the exposure of the cells to hyper-osmolar media
(Fig. 6B). The activation of XMEK2 correlated well with
MEKK-catalyzed phosphorylation of XMEK2 (Fig. 6, A and
B). All these results are consistent with the above hypothesis
that XMEK2 is a SAPK kinase which is activated by MEKK.
and p46
(Fig. 7B),
these two kinases may be p54
and p46
themselves. Thus, XMEK2, an upstream activator for SAPK, is a
substrate, at least in vitro, for SAPK.
Figure 7:
Phosphorylation of XMEK2 by SAPK.
A, 3Y1 cells were exposed to 0, 0.2, 0.7, and 1.0 M
NaCl for 60 min. Cell lysates were then prepared and subjected to
in-gel kinase assay containing 80 µg/ml recombinant wild-type
XMEK2. The autoradiography is shown. Molecular weight standards
(prestained SDS-PAGE standards, Bio-Rad) are indicated on the
left. The positions corresponding to 54 and 46 kDa are
indicated by closed and open arrowheads,
respectively. B, the data shown in A were quantified
by an image analyzer for p54 and p46 kinases as % activity relative to
the maximum activity (0.7 M NaCl) (closed bars). The
same cell lysates were subjected to in-gel kinase assay containing Jun,
and the activities of p54 and p46
were
quantified as above (hatched bars).
-dependent activation
of MEKK in PC12 cells. Their paper was the first that identified
physiological extracellular stimuli inducing activation of MEKK. Our
results indicated that MEKK is activated more markedly by osmotic shock
than by EGF in PC12 cells. Furthermore, in PC12 cells SAPKs were found
to be activated more markedly by osmotic shock than by EGF. Thus,
activation of MEKK correlates with activation of SAPKs rather than
MAPKs. A recent report of Minden et al.(49) has shown
that transfection of MEKK or truncated (activated) MEKK can induce
activation of JNKs (=SAPKs) rather than MAPKs in HeLa cells. In
any case, our data have identified one of the strongest stimuli that
induce activation of endogenous MEKK in cells. This osmotic
shock-induced pathway will be useful for analysis of activation
mechanisms and function of MEKK in cells.
in
vitro. Furthermore, the SAPK activating activity of XMEK2 was
activated by MEKK. In addition, the XMEK2 activating activity of MEKK
was found to be activated by osmotic shock in 3Y1 cells. After
completion of these studies, two papers appeared. One demonstrated that
a mammalian homolog of XMEK2 (named SEK1) works as a SAPK
kinase
(50) , and another showed that MEKK can activate
SEK1
(51) . Thus our present results are completely consistent
with their results, and further suggest that MEKK indeed functions as
an upstream activator for the SEK1/SAPK cascade in cells in an
osmo-sensing signal transduction pathway. In our preliminary
experiments, endogenous XMEK2 (SEK1) protein in 3Y1 cells was activated
in response to hyper-osmolar media.
(
)
Thus it is
reasonable to assume, although not proven, that osmotic shock activates
a kinase cascade consisting of MEKK, XMEK2 (SEK1), and SAPK. Moreover,
as it is well established that Raf-1 works as a MAPKK
kinase
(13, 14, 15) and activation of Raf-1 and
MAPKK in addition to activation of MAPK was shown, here, to occur in
cells exposed to high NaCl concentrations, it is likely that osmotic
shock induces activation of the kinase cascade consisting of Raf-1,
MAPKK, and MAPK. Further studies are needed, however, to answer the
following questions. Is Raf-1 a sole MAPKK kinase in this pathway? Is
MEKK also acting as a MAPKK kinase in addition to a SEK1-activating
kinase? How are Raf-1 and MEKK activated? Is SEK1 a sole SAPK kinase in
this pathway, etc? Detailed biochemical analyses concerning this
osmotic shock-induced pathway and other signaling pathways are in
progress.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.