From the
Stress-activated protein kinases (SAPKs) or c-Jun amino-terminal
kinases (JNKs), which belong to a subgroup of the mitogen-activated
protein kinase (MAPK) superfamily, are activated in response to a
variety of stresses in mammalian cells. An activity to activate a
recombinant rat SAPK
Recent studies identified two novel subgroups of the
mitogen-activated protein kinase (MAPK)
A direct
activator for classical MAPKs, MAPK kinase (MAPKK, also called MEK) was
identified by fractionating extracts obtained from stimulated cells and
turned out to be a dual specificity kinase (for reviews, see Refs.
13-17). Inhibition of MAPKK activity by an anti-MAPKK
neutralizing antibody inhibited MAPK activation during oocyte
maturation
(18) , and immunodepletion of MAPKK prevented the
v-Ras p21-induced activation of MAPK in a cell-free system
(19) .
Furthermore, the dominant-negative form of MAPKK suppressed the
functions of the MAPK pathway
(20, 21, 22) .
These results suggest that MAPKK and MAPK form a linear pathway (the
MAPKK/MAPK cascade), which defines one of the central signal
transduction pathways.
As for p38/MPK2, efforts have been directed
at dissecting upstream pathways resulting in p38/MPK2 activation in the
heat shock- or arsenite-induced signaling pathway
(8) . As for
SAPK/JNK, however, dissection of the upstream activating pathways by
fractionating cell extracts has not been carried out. Most recently,
two mammalian cDNAs, SEK1
(23) /MKK4
(24) and
MKK3
(24) , encoding protein kinases distantly related to MAPKK
were isolated. SEK1/MKK4 can act as a direct activator for SAPK/JNK
when expressed in cells
(23, 24) . MKK4 was shown to
function also as an activator for p38/MPK2
(24) . On the other
hand, MKK3 can act solely as an activator for p38/MPK2
(24) . In
this study, we fractionated extracts obtained from fibroblastic cells
exposed to hyperosmolar media to identify an activity to activate
SAPK/JNK. Multiple activator fractions have been obtained. One is
identified as XMEK2/SEK1/MKK4, and the others are previously
unidentified factors.
Extracts obtained from rat fibroblastic 3Y1 cells that had
been exposed to hyperosmolar media (0.7 M NaCl) for 60 min or
left untreated, respectively, were subjected to Q-Sepharose
chromatography, and each fraction was assayed for both the c-Jun
phosphorylating activity (SAPK/JNK activity) and the SAPK/JNK
activating activity. The latter activity was measured by using
recombinant SAPK
A recent report has shown that recombinant MKK4
(XMEK2/SEK1) can work not only as an activator for SAPK/JNK but also as
an activator for p38/MPK2 in vitro(24) . Immune complex
kinase assay with anti-XMEK2 antibodies revealed that the
immunoprecipitate from the total lysate or the partially purified
fractions was able to phosphorylate efficiently a kinase-negative
mutant of a bacterially produced recombinant MPK2
(Fig. 3B, lower,
In this study, the SAPK
We thank F. Itoh for help in some of the experiments.
We also thank Dr. Tetsu Akiyama (Osaka University) and Dr. Kunihiro
Matsumoto (Nagoya University) for stimulating discussion.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
was detected in extracts obtained from rat
fibroblastic 3Y1 cells exposed to hyperosmolar media and was resolved
into unadsorbed and adsorbed fractions on Q-Sepharose chromatography.
The adsorbed activity was identified as XMEK2/SEK1/MKK4 by using
several anti-XMEK2 antibodies. Thus, a 45-kDa protein that was
recognized specifically by these anti-XMEK2 antibodies co-eluted with
the SAPK
activating activity during chromatography on Q-Sepharose
and Superose 6, and the activity could be immunoprecipitated by the
antibodies from these fractions. The unadsorbed activity, whose level
was much greater than that of the adsorbed activity, did not contain
XMEK2/SEK1/MKK4 and was also activated in a time-dependent manner by
osmotic shock. This activity was further resolved into several peaks
during chromatography on heparin-Sepharose and hydroxylapatite. Most of
these peaks eluted separately from major peaks of a kinase activity
toward p38/MPK2, another subgroup of the MAPK superfamily, whereas the
activated XMEK2/SEK1/MKK4 could phosphorylate p38/MPK2 efficiently.
These results indicate the existence of multiple activators for
SAPK/JNK; one is XMEK2/SEK1/MKK4, and the others are previously
undescribed factors.
(
)
superfamily in addition to classical MAPKs in vertebrate
cells. One subgroup is called stress-activated protein kinase
(SAPK)
(1, 2, 3) or c-Jun amino-terminal kinase
(JNK)
(4, 5, 6) , and the other is
p38/MPK2
(7, 8, 9, 10) . The classical
MAPKs are characterized by having the Thr-Glu-Tyr (TEY) sequence as the
dual phosphorylation motif that is required for their activation
(11-14), whereas SAPK/JNK and p38/MPK2 are characterized by a TPY
sequence
(3, 5, 6) and a TGY
sequence
(7, 8, 10) , respectively.
Preparation of Cell Extracts
Confluently grown
3Y1 cells, which were exposed to 0.7 M NaCl for indicated
times or left untreated, were washed once with ice-cold Hepes-buffered
saline, scraped into buffer A consisting of 20 mM Tris, pH
7.5, 2 mM EGTA, 25 mM -glycerophosphate, 2
mM DTT, 1 mM vanadate, 1 mM
phenylmethylsulfonyl fluoride, and 1% aprotinin (300 µl of
buffer/100-mm dish), and were homogenized. The homogenate was
centrifuged first at 1000
g for 3 min, then at 400,000
g for 20 min, and the supernatant was used as the cell
extracts.
Preparation of Recombinant Proteins
Rat
SAPK
(3) , Xenopus MPK2
(8) , Xenopus XMEK2
(25) , and human c-Jun
(26) coding regions were
amplified by reverse transcriptase polymerase chain reaction. A
kinase-negative mutant of MPK2 (KN-MPK2) was produced by mutagenesis of
Lys-54 to Arg by the method of Kunkel et al.(27) using
a mutagenic primer 5`-CTGAAACGGCCTCGAGAGTTTTCTTACAGCAATAC-3`. c-Jun
cDNA was inserted into pET3. SAPK
and XMEK2 cDNAs were subcloned
into pET16b. KN-MPK2 cDNA was subcloned into pET28a. These genes were
expressed in a bacteria strain BL21(DE3)pLysS as His-tagged proteins
and purified according to manufacturer's instructions (Novagen).
A kinase-negative MAPK (KN-MAPK) was expressed as a glutathione
S-transferase fusion protein and purified as
described
(28) .
Preparation of Anti-XMEK2
Antibodies
Anti-COOH-terminal XMEK2 antiserum was raised in
rabbits against a bovine serum albumin-coupled peptide
(KILEQMPVSPSSPMYVD) corresponding to the extreme COOH-terminal sequence
of XMEK2
(25) . Anti-recombinant XMEK2 antisera were raised in
both rabbits and mice by immunizing them with His-tagged XMEK2.
Assay of Protein Kinase Activities
To measure the
activity to phosphorylate c-Jun, MPK2, and MAPK, samples were incubated
for 30 min at 30 °C with 3 µg of His-tagged c-Jun, KN-MPK2, or
KN-MAPK in a final volume of 15 µl of a solution containing 20
mM Tris, pH 7.5, 10 mM MgCl, and 50
µM [
-
P]ATP (1 µCi). To
measure the activity to activate SAPK
, samples were first
incubated for 30 min at 30 °C with 0.2 µg of wild-type
His-tagged SAPK
in a solution containing 20 mM Tris-Cl,
pH 7.5, 10 mM MgCl
, and 100 µM ATP
and subsequently for 20 min at 20 °C with 3 µg of His-tagged
c-Jun and 1 µCi [
-
P]ATP in the same
solution (final volume, 15 µl). The reaction was stopped by
addition of Laemmli's sample buffer and boiling. After SDS-PAGE,
phosphorylation of these proteins was quantified by an image analyzer
(Fujix BAS2000).
Column Chromatography
Cell extracts (50 ml, 60 mg
of protein) were loaded onto a Q-Sepharose column (12 ml, Pharmacia
Biotech Inc.) equilibrated with buffer A. The flow through fractions
were pooled as ``unadsorbed'' fractions, and adsorbed
proteins were eluted with a 200-ml linear gradient of 0-0.5
M NaCl. The fractions were assayed for the SAPK
activating activity as described above. The adsorbed active fractions
were pooled and concentrated by Centricon-30 (Amicon) and then loaded
onto a Superose 6 HR 10/30 column (Pharmacia) equilibrated with buffer
B (20 mM Tris-Cl, pH 7.5, 2 mM EGTA, 25 mM
-glycerophosphate, 100 mM NaCl, 2 mM DTT, 0.01%
Brij-35). The unadsorbed fractions of the Q-Sepharose column
chromatography were pooled and then loaded onto a HiTrap heparin column
(5 ml, Pharmacia) equilibrated with buffer C (20 mM Tris, pH
7.5, 2 mM EGTA, 25 mM
-glycerophosphate, 2
mM DTT, 1 mM vanadate, 0.2% aprotinin, 0.01% Brij-35)
and proteins were eluted with a 90-ml gradient of 0-0.4
M NaCl. Fractions that eluted at 0.12-0.16 M
NaCl (fractions 5-11 in Fig. 4A) and at
0.2-0.24 M NaCl (fractions 16-21) were collected
and pooled as the first peak and the second peak, respectively. The
first peak was diluted 3-fold with buffer C, loaded again onto a HiTrap
heparin column, and eluted with a 85-ml gradient of 0-0.4
M NaCl. The second peak was loaded onto a hydroxylapatite
column (1 ml, Bio-Rad) equilibrated with buffer D (10 mM
potassium phosphate, pH 7.0, 25 mM
-glycerophosphate, 0.2
mM EGTA, 100 mM NaCl, 2 mM DTT, 1
mM vanadate), and proteins were eluted with a 22-ml gradient
of 0.01-0.4 M potassium phosphate.
Figure 4:
Column chromatography of the
Q-Sepharose-unadsorbed SAPK activating activity. The unadsorbed
fractions of the Q-Sepharose chromatography (see Fig. 1A) were
fractionated by heparin-Sepharose chromatography (A). The
KN-MAPK phosphorylating activity (), KN-MPK2 phosphorylating
activity (
), and the SAPK activating activity (
) were
measured as described under ``Materials and Methods.'' The
first (fractions 5-11) and the second (fractions 16-21)
peaks of the SAPK activating activity were subjected to chromatography
on heparin-Sepharose (B) and hydroxylapatite (C),
respectively. Each fraction was assayed for the SAPK activating
activity (
) and the KN-MAPK phosphorylating activity (
) or
KN-MPK2 phosphorylating activity (
) as described under
``Materials and Methods.''
Immunoprecipitation
3Y1 cell extracts (100 µl)
were incubated with 3 µl of anti-XMEK2 antibody for 1 h at 4 °C
and further with 30 µl of 1:1 slurry of protein A-Sepharose beads
(Pharmacia) for 1 h at 4 °C. The immune complex on beads was washed
three times with a solution containing 20 mM Tris-Cl, pH 7.5,
500 mM NaCl, 2 mM DTT, and 0.05% Tween 20 and then
used as the anti-XMEK2 immunoprecipitate. To detect SAPK activating
activity or MPK2 phosphorylating activity of the anti-XMEK2
immunoprecipitate, the immune complex was washed once with buffer A and
incubated for 30 min at 30 °C either with 0.5 µg of wild-type
His-tagged SAPK and 3 µg of His-tagged c-Jun or with 3 µg
of KN-MPK2 in a solution (final volume, 15 µl) containing 20
mM Tris-Cl, pH 7.5, 10 mM MgCl
, and 100
µM [
-
P]ATP (3 µCi). After
SDS-PAGE, the radioactivity was detected by autoradiography and
quantified by using an image analyzer (Fujix BAS2000). The anti-XMEK2
immunoprecipitate alone had no kinase activity toward c-Jun.
. The c-Jun phosphorylating activity, which was
stimulated by the exposure of the cells to hyperosmolarity, eluted in
the adsorbed fractions (Fig. 1A,
). The SAPK
activating activity was also greatly stimulated by the osmotic shock,
and the enhanced activity eluted largely in unadsorbed fractions
(fractions 2-8) and slightly in adsorbed fractions (fractions
16-20) (Fig. 1A,
, and inset (
)).
Figure 1:
Activation and fractionation of the
SAPK activating activities. A, rat fibroblastic 3Y1 cells were
exposed to 0.7 M NaCl for 60 min (,
) or left
untreated (
,
). Soluble extracts obtained from these cells
were subjected to chromatography on Q-Sepharose, and each fraction was
assayed for c-Jun phosphorylating activity in the absence (
,
) or presence (
,
) of recombinant rat SAPK
. The
SAPK
activating activity is defined as subtracting the Jun
phosphorylating activity in the absence of SAPK
from that in the
presence of SAPK
, i.e.
minus
, or
minus
. The stimulated SAPK
activating activity (
minus
) that eluted in the adsorbed fractions (fractions
15-21) is shown in the inset (
). The data are
shown in arbitrary units. B, rat 3Y1 cells were exposed to 0.7
M NaCl for indicated times and cell extracts were prepared.
The cell extracts were mixed with 0.5 volume of Q-Sepharose beads
equilibrated with buffer A (see ``Materials and Methods'')
and incubated for 30 min at 4 °C and then centrifuged. The
supernatant was saved, and buffer A containing 0.4 M NaCl was
added to the Q-Sepharose beads. The unadsorbed (the first supernatant)
fraction (
) and the 0.4 M NaCl eluted fraction (
)
were assayed for SAPK activating activity. An aliquot of the cell
extracts was subjected to immune complex kinase assay with anti-XMEK2
antibodies as described under ``Materials and Methods,'' and
the results are shown in the inset.
To identify these SAPK activating activities, we
produced a number of anti-XMEK2 antibodies by using a bacterially
produced recombinant XMEK2 protein and a synthetic peptide
corresponding to a COOH-terminal sequence of XMEK2
(25) as
antigens. XMEK2 cDNA was isolated previously from a Xenopus cDNA library and shown to be distantly related to
MAPKK
(25) . Rabbit anti-COOH-terminal peptide antiserum, and
rabbit and mouse anti-recombinant XMEK2 antisera were obtained, and
subjected to affinity purification on each antigen-immobilized resin or
membrane. All the purified antibodies reacted strongly with recombinant
XMEK2 (data not shown) and recognized mainly a 45-kDa protein in a
variety of mammalian cultured cells (Fig. 2, A and
C). Rabbit and mouse anti-recombinant XMEK2 antibodies were
able to immunoprecipitate this 45-kDa protein (Fig. 2B).
Thus, the 45-kDa protein may be a protein product of a mammalian
homolog of XMEK2, SEK1
(23) or MKK4
(24) , which were
recently cloned from murine and human cDNA libraries, respectively.
This XMEK2/SEK1/MKK4 protein (45-kDa) was detected in various tissues
and cells (Fig. 2, C and D).
Figure 2:
Reactivity of anti-XMEK2 antibodies.
A, extracts obtained from rat 3Y1 cells were immunoblotted
with affinity-purified rabbit anti-COOH-terminal XMEK2 antibody
(lane1), mouse anti-recombinant XMEK2 antibody
(lane2), or rabbit anti-recombinant XMEK2 antibody
(lane3). B, extracts from rat 3Y1 cells
were subjected to immunoprecipitation with rabbit anti-recombinant
XMEK2 antibody (left) or mouse anti-recombinant XMEK2 antibody
(right). Immunoprecipitates (ppt), supernatants
(sup), or total extracts (totalextract)
were electrophoresed and immunoblotted with mouse (left) or
rabbit (right) anti-recombinant XMEK2 antibody. C,
extracts obtained from several cells (each 10 µg of protein) were
subjected to immunoblotting with rabbit anti-COOH-terminal XMEK2
antibody. D, extracts (30 µg) from various mouse tissues
were subjected to immunoblotting with rabbit anti-recombinant XMEK2
antibody. An immunoreactive band (42 kDa) was detected in some
cases below a major 45-kDa band in panelsC and
D.
In our
preliminary experiments, the anti-XMEK2 immunoprecipitate obtained from
the osmotically shocked cells could activate a recombinant SAPK.
Then, in order to assign the activity peak for which the activated
XMEK2/SEK1/MKK4 protein is responsible, each fraction of the
Q-Sepharose chromatography (see Fig. 1A) was subjected
to immunoblotting and immunoprecipitation with anti-XMEK2 antibodies.
The elution of the minor SAPK
activating activity (which equals
the adsorbed activity) (fractions 16-20 in
Fig. 1A) coincided with the elution of the 45-kDa
XMEK2/SEK1/MKK4 protein, and the major activity peak, unadsorbed
fractions (fractions 2-8 in Fig. 1A), did not
contain any reactive proteins (Fig. 3A). The immune
complex kinase assay of each fraction revealed clearly that anti-XMEK2
antibodies could immunoprecipitate the SAPK
activating activity as
well as the SAPK
phosphorylating activity from the adsorbed
fractions, and not from the unadsorbed fractions
(Fig. 3A, lower, +NaCl); the
elution of the activity of the immunoprecipitate was superimposed on
the elution of the 45-kDa protein detected by immunoblotting
(Fig. 3A). The anti-XMEK2 immunoprecipitate from each
fraction obtained from control (untreated) cells showed neither kinase
activity toward SAPK
nor SAPK
activating activity
(Fig. 3A, lower, Cont.). When the
adsorbed activity from osmotically shocked cells was further subjected
to gel filtration chromatography on Superose 6, the SAPK activating
activity in each fraction (Fig. 3B, upper) and
in the immune complex (Fig. 3B, lower,
)
co-eluted completely with the 45-kDa immunoreactive protein
(Fig. 3B, middle) as a single peak with an
apparent molecular mass of
50 kDa for globular proteins
(Fig. 3B). These results demonstrate that
XMEK2/SEK1/MKK4 protein (45 kDa) is responsible for part of the
SAPK/JNK activating activity in osmotically shocked cells, and its
active form may exist largely as a monomer.
Figure 3:
Identification of the minor SAPK
activating activity as XMEK2/SEK1/MKK4. A, Q-Sepharose
fractions (Fig. 1A) were subjected to immunoblotting with
anti-recombinant XMEK2 antibody (upper) or to immune complex
kinase assay for SAPK activating activity (lower; the data
from osmotically shocked cells (+NaCl) and from control
untreated cells (Cont.)) as described under ``Materials
and Methods.'' The radioactivity incorporated into His-tagged
SAPK and c-Jun was detected by autoradiography. Essentially the
same immunoblotting data (upper) were obtained with other two
antibodies. B, active fractions of the Q-Sepharose adsorbed
fractions were subjected to Superose 6 gel filtration chromatography
and each fraction was assayed for SAPK activating activity
(upper), immunoblotted with rabbit anti-recombinant XMEK2
antibody (middle), or immune complex kinase assay for SAPK
activating activity (lower,
) or for KN-MPK2
phosphorylating activity (lower,
). The data are shown in
arbitrary units.
The immune complex
kinase assay showed that XMEK2/SEK1/MKK4 was activated in a
time-dependent manner when the 3Y1 cells were exposed to hyperosmolar
media with NaCl (Fig. 1B, inset). The major
SAPK activating activity that eluted unadsorbed in Q-Sepharose
chromatography was also activated in response to osmotic shock
(Fig. 1B,
). When the unadsorbed fractions from the
Q-Sepharose chromatography of the osmotically shocked cells (see
Fig. 1A) were subjected to Superose 6 chromatography,
the activity eluted as a broad peak with an apparent molecular mass of
50 kDa for globular proteins (data not shown). However, the
activity was resolved into two peaks on heparin-Sepharose
chromatography (Fig. 4A,
). Thus, there might be
at least three activating factors for SAPK/JNK in the osmotically
shocked cells.
); the elution of
p38/MPK2 phosphorylating activity coincided completely with that of the
SAPK activating activity on Superose 6 chromatography
(Fig. 3B, lower,
and
). Thus, the
activated form of endogenous XMEK2/SEK1/MKK4 may work as an activator
for both SAPK/JNK and p38/MPK2. Next, major SAPK activating activities
(unadsorbed fractions in the Q-Sepharose chromatography) were assayed
for their ability to phosphorylate p38/MPK2 after chromatography on the
heparin-Sepharose. The elution of the major p38/MPK2 phosphorylating
activity (Fig. 4A,
) coincided apparently with the
second SAPK activating activity peak (Fig. 4A,
),
whereas little p38/MPK2 phosphorylating activity was detected in the
first SAPK activating activity peak (Fig. 4A). Although
the kinase activity toward classical MAPK (which equals MAPKK activity;
Fig. 4A,
) was detected in the first SAPK
activating activity peak, this MAPKK activity eluted separately from
the SAPK activating activities in rechromatography on heparin-Sepharose
under different elution conditions (Fig. 4B,
and
). Interestingly, in this rechromatography the SAPK activating
activities eluted broadly with several peaks (Fig. 4B,
). When the second SAPK activating activity peak in the first
heparin-Sepharose chromatography (Fig. 4A) was subjected
to chromatography on hydroxylapatite, the SAPK activating activities
eluted with two peaks (Fig. 4C,
), separately from
the major p38/MPK2 phosphorylating activity (Fig. 4C,
), although the first SAPK activating activity peak on
hydroxylapatite had significant p38/MPK2 phosphorylating activity
(Fig. 4C). These results taken together suggest the
existence of multiple SAPK/JNK activators with different substrate
specificity.
activating activity was
first resolved into two fractions on Q-Sepharose chromatography: the
major unadsorbed peak and the minor adsorbed peak. Because a mammalian
homolog of XMEK2, SEK1, or MKK4, has been shown to work as a SAPK/JNK
activator when expressed in cells or in
vitro(23, 24) , we first thought that the major
peak might correspond to XMEK2/SEK1/MKK4. However, rather surprisingly,
the XMEK2/SEK1/MKK4 protein coincided with the adsorbed, minor peak,
and the major, unadsorbed peak did not contain any proteins reactive to
several anti-XMEK2 antibodies produced here. The major, unadsorbed
fractions were further resolved into several peaks during subsequent
chromatography on heparin-Sepharose and hydroxylapatite. Consistent
with the recent report that MKK4 can act as an activator for p38/MPK2
as well as SAPK/JNK
(24) , the endogenous activated
XMEK2/SEK1/MKK4 was here shown to be able to phosphorylate p38/MPK2.
Some of non-XMEK2/SEK1/MKK4 factors phosphorylated p38/MPK2 and some
did not. Therefore, these results suggest the existence of multiple
activating factors for SAPK/JNK. One of them has been identified as
XMEK2/SEK1/MKK4. The others are supposed to be previously unidentified
factors, as XMEK2/SEK1/MKK4 is the only molecule previously identified
as an activator for SAPK/JNK. Furthermore, as the recently identified
activator for p38/MPK2, MKK3, has been shown to be incapable of
phosphorylating SAPK/JNK
(24) , these non-XMEK2/SEK1/MKK4 factors
described here should not be MKK3. It should be pointed out that
XMEK2/SEK1/MKK4 is responsible for only part of the SAPK/JNK activating
activities and the newly described factors appear to play a major role
in the osmotically shock-induced activation of SAPK/JNK.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.