©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Activation of Protein Kinase Cascades by Osmotic Shock (*)

Satoshi Matsuda (§) , Hiroshi Kawasaki (§) , Tetsuo Moriguchi (§) , 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
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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 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.


INTRODUCTION

Mitogen-activated protein kinase (MAPK)() 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.

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.


MATERIALS AND METHODS

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.


RESULTS

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.() 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).

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).


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.

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 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).




DISCUSSION

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 -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.

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 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.

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.


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.

§
S. M., H. K., and T. M. contributed equally to this work.

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

The abbreviation used are: MAPK, mitogen-activated protein kinase; MAPKK, MAPK kinase; MAPKK-K, MAPKK kinase; SAPK, stress-activated protein kinase; JNK, jun N-terminal kinase; EGF, epidermal growth factor; MBP, myelin basic protein; MAP2, microtubule-associated protein 2; PAGE, polyacrylamide gel electrophoresis.

T. Moriguchi, Y. Gotoh, and E. Nishida, manuscript in preparation.

H. Kawasaki, Y. Gotoh, and E. Nishida, unpublished results.

T. Moriguchi, H. Kawasaki, S. Matsuda, Y. Gotoh, and E. Nishida, unpublished observations.


ACKNOWLEDGEMENTS

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.


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