©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Evidence for Multiple Activators for Stress-activated Protein Kinases/c-Jun Amino-terminal Kinases
EXISTENCE OF NOVEL ACTIVATORS (*)

Tetsuo Moriguchi (§) , Hiroshi Kawasaki (§) , Satoshi Matsuda (§) , Yukiko Gotoh (¶) , Eisuke Nishida (¶)

From the (1) Department of Genetics and Molecular Biology, Institute for Virus Research, Kyoto University, Sakyo-ku, Kyoto 606-01, Japan

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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 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.


INTRODUCTION

Recent studies identified two novel subgroups of the mitogen-activated protein kinase (MAPK)() 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.

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.


MATERIALS AND METHODS

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.


RESULTS AND DISCUSSION

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. 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.

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, ); 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.

In this study, the SAPK 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.


FOOTNOTES

*
This work was supported in part by grants-in-aid from the Ministry of Education, Science and Culture of Japan, the Toray Science Foundation, and the Mitsubishi Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
These authors contributed equally to this work.

To whom correspondence should be addressed. Fax: 81-75-751-3992.

The abbreviations used are: MAPK, mitogen-activated protein kinase; MAPKK, MAPK kinase; SAPK, stress-activated protein kinase; JNK, c-Jun amino-terminal kinase; DTT, dithiothreitol; PAGE, polyacrylamide gel electrophoresis.


ACKNOWLEDGEMENTS

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.


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