©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Purification and Characterization of REKS from Xenopus Eggs
IDENTIFICATION OF REKS AS A Ras-DEPENDENT MITOGEN-ACTIVATED PROTEIN KINASE KINASE KINASE (*)

(Received for publication, October 4, 1994; and in revised form, November 8, 1994)

Shinya Kuroda (1) (2)(§) Kazuya Shimizu (1) (2)(§) Bunpei Yamamori (1) (2)(§) Shuji Matsuda (1) (2)(§) Katsunori Imazumi (1) (2)(§) Kozo Kaibuchi (2) (3)(¶) Yoshimi Takai (1) (2)(§) (3)(**)

From the  (1)Department of Molecular Biology and Biochemistry, Osaka University Medical School, Suita 565, the (2)Department of Biochemistry, Kobe University School of Medicine, Kobe 650, and the (3)Department of Cell Physiology, National Institute of Physiological Sciences, Okazaki 444, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

We have previously identified a protein factor, named REKS (Ras-dependent Extracellular signal-regulated kinase/Mitogen-activated protein kinase kinase (MEK) Stimulator), which is necessary for Ras-dependent MEK activation. In this study, we attempted to highly purify and characterize REKS. We have highly purified REKS by successive column chromatographies using a cell-free assay system in which REKS activates recombinant extracellular signal-regulated kinase 2 through recombinant MEK in a guanosine 5`-O-(thiotriphosphate) (GTPS)-Ki-Ras-dependent manner. REKS formed a stable complex with GTPS-Ras; REKS was coimmunoprecipitated with GTPS-Ki-Ras or GTPS-Ha-Ras, but not with GDP-Ki-Ras or GDP-Ha-Ras by an anti-Ras antibody. REKS was adsorbed to a GTPS-glutathione S-transferase (GST)-Ha-Ras-coupled glutathione-agarose column but not to a GDP-GST-Ha-Ras-coupled glutathione-agarose column and was coeluted with GTPS-GST-Ha-Ras by reduced glutathione. The minimum molecular mass of REKS was estimated to be about 98 kDa on SDS-polyacrylamide gel electrophoresis. REKS phosphorylated this 98-kDa protein as well as recombinant MEK. REKS was not recognized by any of the anti-c-Raf-1, anti-Mos, and anti-mSte11 antibodies. These results indicate that REKS is a Ras-dependent MEK kinase.


INTRODUCTION

Three ras genes encode proteins with M(r) values of about 21,000, named Ha-Ras, Ki-Ras, and N-Ras. Ras exhibits GDP/GTP binding and GTPase activities. They have two interconvertible forms: GDP-bound inactive and GTP-bound active forms. The GDP-bound form is converted to the GTP-bound form by the GDP/GTP exchange reaction, which is regulated by GDP/GTP exchange protein, whereas the GTP-bound form is converted to the GDP-bound form by the GTPase reaction, which is regulated by GTPase-activating protein (for review, see (1) ). Four GDP/GTP exchange proteins, including mCdc25(2, 3) , mSos(4) , C3G(5) , and Smg GDP dissociation stimulator(6) , and two GTPase-activating proteins, Ras GTPase-activating protein (7, 8) and neurofibromin(9) , have thus far been identified. GTP-Ras interacts with its specific target protein. A Ras target molecule has first been identified to be adenylate cyclase in Saccharomyces cerevisiae(10) . However, the target molecule of Ras in higher eukaryotes still remains to be identified. Recent studies indicate that Ras positions upstream of the MEK(^1)/ERK cascade in Xenopus oocytes (11, 12, 13) and mammalian cells(14, 15) . ERK is phosphorylated and activated by MEK in response to many extracellular signals (for a review, see (16) ). In this signal cascade, MEK is phosphorylated and activated by its kinases (MEK kinases)(17, 18, 19, 20, 21, 22, 23, 24) . Raf is one of the MEK kinases(17, 18, 19) . Raf has been positioned downstream of Ras in many signal transduction pathways. Genetic analyses of eye development and embryonic structure formation in Drosophila and of vulval induction in Caenorhabditis elegans have clarified that Raf functions downstream of Ras(25, 26) . Moreover, several groups have reported that c-Raf-1 directly binds to GTP-Ras in a cell-free system (27, 28, 29, 30) and to wild-type Ras and dominant active Ras in a yeast two-hybrid system(31, 32) . Experiments using antisense c-Raf-1 expression constructs have positioned c-Raf-1 downstream of Ras in proliferation and transformation of NIH/3T3 cells(33) . Recently, it has been shown that c-Raf-1 is activated as a result of its recruitment to the plasma membrane(34, 35) . Although these results strongly suggest that Ras, c-Raf-1, MEK, and ERK function in the same signaling pathway, no evidence has so far been obtained that GTP-Ras directly activates c-Raf-1 in a cell-free system. Mos is a germ cell-specific kinase that is synthesized to initiate maturation of Xenopus oocytes(36) . Mos has also been shown to be a MEK kinase(22, 23) . The relationship between Mos and Ras has not yet been clarified. On the other hand, the cDNA of the mammalian Ste11 homologue, termed mSte11, has been isolated from NIH/3T3 cells by use of the reverse-transcriptase polymerase chain reaction, and mSte11 has been shown to phosphorylate and activate MEK (20) . Recently, it has been shown that another MEK kinase, immunoprecipitated by an anti-mSte11 antibody, and B-Raf phosphorylate MEK and that the expression of oncogenic Ras in PC12 cells results in the activation of this MEK kinase and B-Raf(24) . Moreover, it has been shown that phosphatidylinositol 3-kinase directly interacts with GTP-Ras but not with GDP-Ras (37) and that GTP-Ras slightly activates phosphatidylinositol 3-kinase in a cell-free system (38) . Thus, the direct target molecule of Ras in higher eukaryotes still remains to be fully understood.

To identify a direct target molecule of Ras, we have established a cell-free assay system using Xenopus oocyte extract in which Ras activates ERK through MEK(39) . By use of this assay system, we have identified a protein factor, tentatively named REKS (Ras-dependent ERK Kinase Stimulator), for the Ras-dependent MEK activation(39) . Recently, we have modified this cell-free assay system by use of recombinant MEK and recombinant ERK(40) . We have, moreover, shown that posttranslationally lipid-modified Ras is far more effective on the activation of REKS (41) and yeast adenylate cyclase (42) than lipid-unmodified Ras in cell-free assay systems. Kataoka's group (43) has also reported the similar results for the Ras-dependent activation of yeast adenylate cyclase. It has also been reported that lipid modification of Ras is necessary for the activation of c-Raf-1 in insect cells overexpressing Ras and c-Raf-1(44) .

In these earlier reports, it has not been examined, however, whether GTP-Ki-Ras or GTP-Ha-Ras directly interacts with REKS, whether REKS is a protein kinase, or whether REKS is the same as or different from other MEK kinases including c-Raf-1, Mos, and mSte11. In the present study, we have first attempted to highly purify REKS and have addressed these important issues by use of the purified sample.


EXPERIMENTAL PROCEDURES

Materials and Chemicals

Post-translationally lipid-modified Ki-Ras and Ha-Ras were purified from the membrane fraction of insect cells, which were infected with baculovirus carrying the cDNAs of Ki-Ras and Ha-Ras, respectively(6) . GTPS-Ki-Ras, GTPS-Ha-Ras, GDP-Ki-Ras, and GDP-Ha-Ras were prepared as described previously(6) . The cDNA of mouse MEK was cloned as described(40) . The kinase-negative MEK was generated by the site-directed mutagenesis of Lys to Trp(45) . Recombinant wild-type MEK, kinase-negative MEK, ERK2, and Ha-Ras were purified from overexpressing Escherichia coli as GST fusion proteins using a glutathione-Sepharose 4B column as described(46) . An anti-Ras monoclonal antibody (RASK-4) was kindly provided by H. Shiku (Nagasaki University, Nagasaki, Japan). An anti-Xenopus c-Raf-1 monoclonal antibody was kindly provided by L.T. Williams (University of California, San Francisco, CA). An anti-Xenopus Mos polyclonal antibody was kindly provided by N. Sagata (Kurume University, Fukuoka, Japan). An anti-Xenopus ERK polyclonal antibody was kindly provided by E. Nishida (University of Kyoto, Kyoto, Japan). An anti-MEK polyclonal antibody was generated as described(40) . An anti-mSte11 polyclonal antibody was purchased from Transduction Laboratories (Lexington, KY). Anti-A-Raf and anti-B-Raf polyclonal antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Myelin basic protein was purchased from Sigma.

REKS Assay

REKS activity was assayed by measuring the phosphorylation of myelin basic protein by recombinant GST-ERK2 in the presence of recombinant GST-MEK as described(40) . GTPS-Ki-Ras, GTPS-Ha-Ras, GDP-Ki-Ras, or GDP-Ha-Ras was added as indicated. Namely, a REKS sample to be assayed was incubated for 10 min at 30 °C in a final volume of 50 µl containing 20 mM Tris/HCl, pH 8.0, 120 µM ATP, 10 mM MgCl(2), 6 mM EGTA, 80 nM recombinant GST-MEK, and 100 nM GTPS-Ki-Ras, GTPS-Ha-Ras, GDP-Ki-Ras, or GDP-Ha-Ras. After the 10-min incubation, 10 µl of 3 µM recombinant GST-ERK2 was added. The reaction mixture was incubated for additional 20 min at 30 °C. Then, 20 µl of a reaction mixture containing 20 mM Tris/HCl, pH 8.0, 100 µM [-P]ATP (600 cpm/pmol), 220 µM myelin basic protein, 10 mM MgCl(2), and 6 mM EGTA was added. Incubation was continued for another 10 min at 30 °C, after which 30 µl of the reaction mixture was spotted onto a phosphocellulose paper sheet. The sheet was washed with 75 mM phosphoric acid, and the radioactivity was measured by liquid scintillation spectrometry.

Preparation of the Cytosol of Xenopus Eggs

Eggs were obtained from fully mature Xenopus laevis females as described(39, 47) . Eggs, dejellied with cysteine and washed with modified modified Ringer's solution containing 5.0 mM HEPES/NaOH, pH 7.8, 0.1 M NaCl, 2.0 mM KCl, 1.0 mM MgSO(4), 2.0 mM CaCl(2), and 0.1 mM EDTA were activated by electric shock as described (48) to make them enter into interphase and inactivate endogenous ERK and MEK activities(49, 50) . The cytosol of activated eggs was obtained by centrifugation as described previously(40) .

Partial Purification of REKS from the Cytosol by Mono Q Column Chromatography

The cytosol of activated eggs (24 mg of protein, 6 ml) was applied to a Mono Q column (0.5 times 5 cm) equilibrated with Buffer A containing 20 mM Tris/HCl, pH 8.0, 1 mM dithiothreitol, 5 mM MgCl(2), 10 mM EGTA, and 10 µM (p-amidinophenyl)methanesulfonyl fluoride. After the column was washed with 50 ml of Buffer A, elution was performed with a 15-ml linear gradient of NaCl (0-0.5 M) in Buffer A, and fractions of 1 ml each were collected. An aliquot of each fraction (15 µl) was assayed for the REKS activity.

Coimmunoprecipitation of REKS with GTPS-Ras by an Anti-Ras Antibody

The peak 2 (67.5 µl) of the Mono Q column chromatography was incubated with an anti-Ras antibody coupled to Protein A-Sepharose beads (Pharmacia Biotech Inc.) in the presence of [S]GTPS-Ki-Ras, [S]GTPS-Ha-Ras, [^3H]GDP-Ki-Ras, or [^3H]GDP-Ha-Ras (22.5 µl) at the final concentration of 500 nM. After the incubation for 30 min at 4 °C, the immunocomplex was precipitated and washed 3 times with Buffer A containing 0.5% Nonidet P-40. The precipitate and the supernatant were assayed for the REKS activity. The amounts of Ki-Ras or Ha-Ras in the precipitate and the supernatant were determined by measuring the radioactivity of [S]GTPS or [^3H]GDP bound to Ki-Ras or Ha-Ras.

GTPS-GST-Ha-Ras-coupled Glutathione-Agarose Column Affinity Chromatography of REKS

To make GTPS-GST-Ha-Ras-coupled or GDP-GST-Ha-Ras-coupled glutathione-agarose column, GTPS-GST-Ha-Ras or GDP-GST-Ha-Ras was prepared as described(6) . GTPS-GST-Ha-Ras or GDP-GST-Ha-Ras (5 nmol each) was separately applied to a glutathione-agarose column (200 µl) preequilibrated with 3.6 ml of Buffer B containing 20 mM Tris/HCl, pH 8.0, 1 mM dithiothreitol, 5 mM MgCl(2), and 1 mM EGTA(46) , and the column was washed with 5.0 ml of Buffer B. About 95% of GTPS-GST-Ha-Ras or GDP-GST-Ha-Ras was adsorbed to the column. The REKS sample applied to this column was purified by the Mono S column chromatography as described (40) except that the cytosol of activated eggs (160 mg of protein, 40 ml) was applied and that the elution was performed with a 15-ml linear gradient of NaCl (0-1.0 M). After MgCl(2) and GTPS or GDP were added to this REKS sample (3 ml) to give the final concentrations of 15 mM and 10 µM, respectively, in order to prevent the dissociation of guanine nucleotides from GST-Ha-Ras during the application of the REKS sample, the REKS sample was applied to the GTPS-GST-Ha-Ras-coupled glutathione-agarose column or the GDP-GST-Ha-Ras-coupled glutathione-agarose column. After the column was washed with 4 ml of Buffer B, elution was performed with 600 µl of Buffer B containing 20 mM reduced glutathione. The eluate fraction of the GTPS-GST-Ha-Ras-coupled glutathione-agarose column chromatography was used for the experiments as affinity-purified REKS.

Phosphorylation of MEK by REKS

Affinity-purified REKS (5 µl) was supplemented with 200 ng of each of GST alone, wild-type GST-MEK, or kinase-negative GST-MEK (40 µl each). The reaction was started by adding the mixture (5 µl) containing 20 mM Tris/HCl, pH 8.0, 200 µM [-P]ATP (5,000 cpm/pmol), and 15 mM MgCl(2) and continued for 10 min at 30 °C. The reaction was terminated by the addition of Laemmli's sample buffer (25 µl) and subjected to SDS-PAGE. The radioactivity of P incorporated into MEK was detected by bioimaging analyzer BAS2000 (Fujix, Tokyo) as described previously (39) .

Phosphorylation of Affinity-purified REKS

Affinity-purified REKS (50 µl) was incubated for 2 min at 30 °C with the reaction mixture (5 µl) containing 20 mM Tris/HCl, pH 8.0, 110 µM [-P]ATP (50,000 cpm/pmol), 1 mM dithiothreitol, 5 mM MgCl(2), and 1 mM EGTA. The reaction was stopped by the addition of Laemmli's sample buffer (27.5 µl) and was subjected to SDS-PAGE followed by autoradiography.

Other Procedures

SDS-PAGE was performed by the method of Laemmli(51) . Protein concentrations were determined with bovine serum albumin as a standard protein by the method of Bradford(52) . Immunoblot was carried out as described(40) .


RESULTS

Partial Purification of REKS by Mono Q Column Chromatography

The cytosol of activated eggs was subjected to a Mono Q column chromatography. When each fraction was assayed for the REKS activity, three peaks (peaks 1-3) were detected in the presence of both recombinant MEK and recombinant ERK2 (Fig. 1A). The activity of only peak 2 was enhanced by GTPS-Ki-Ras but not by GDP-Ki-Ras. In the presence of recombinant ERK2 alone, two peaks were detected in the same fractions as those of peaks 1 and 3 of Fig. 1A ( Fig. 1B ). The activities of both peaks were independent of GTPS-Ki-Ras. In the absence of recombinant MEK and recombinant ERK2, only one peak was detected at the same position as that of peak 3 of Fig. 1A (Fig. 1C). This peak was also independent of GTPS-Ki-Ras. The similar results were obtained when Ha-Ras was used instead of Ki-Ras (data not shown). Immunoblot analysis of MEK and ERK revealed that the immunoreactivities of MEK and ERK were detected in fractions 4-6 and 10-12, respectively. These results indicate that peaks 1 and 2 are MEK and REKS, respectively, and that peak 3 contains ERK and an unknown myelin basic protein kinase. These results are essentially consistent with our earlier observations that REKS is required for the Ras-dependent activation of ERK2 through MEK(39, 40) .


Figure 1: Partial purification of REKS from the cytosol of activated Xenopus eggs by Mono Q column chromatography. A 15-µl aliquot of each fraction of the Mono Q column chromatography was assayed for the REKS activity in the presence of various combinations of GST-MEK, GST-ERK2, and GTPS-Ki-Ras or GDP-Ki-Ras. A, with recombinant MEK and recombinant ERK2. B, with ERK2 alone. C, without MEK and ERK2. bullet, with GTPS-Ki-Ras; circle, with GDP-Ki-Ras; up triangle, without Ki-Ras. -, NaCl concentration. The results shown are representative of three independent experiments.



Coimmunoprecipitation of REKS with GTPS-Ras by an Anti-Ras Antibody

To show interaction of REKS with GTPS-Ras, we examined whether REKS is coimmunoprecipitated with GTPS-Ki-Ras or GTPS-Ha-Ras by an anti-Ras antibody. GTPS-Ki-Ras was incubated with the peak 2 of the Mono Q column chromatography and anti-Ras antibody-coupled Protein A-agarose beads. After the incubation for 30 min at 4 °C, the mixture was centrifuged. The REKS activity was recovered in the precipitate but not in the supernatant (Table 1). GTPS-Ki-Ras was also mostly recovered in the precipitate. In contrast, when the similar experiment was done with GDP-Ki-Ras, GDP-Ki-Ras was mostly recovered in the precipitate, whereas the REKS activity was recovered in the supernatant but not in the precipitate. The similar results were obtained when Ha-Ras was used instead of Ki-Ras (data not shown). We have purified REKS by the Mono S column chromatography as described previously(40) . The similar results were obtained when REKS purified by the Mono S column chromatography was used instead of that purified by the Mono Q column chromatography (data not shown).



GTPS-GST-Ha-Ras-coupled Glutathione-Agarose Column Affinity Chromatography of REKS

To obtain another line of evidence for interaction of REKS with GTPS-Ras, we first prepared a large amount of REKS by a large scale of a Mono S column chromatography. By use of this sample, we examined whether REKS interacts with GTPS-GST-Ha-Ras coupled to a glutathione-agarose column. In this large scale experiment, 40 ml of the cytosol of activated eggs was subjected to a Mono S column chromatography under the same conditions as described (40) , except that the elution was performed with a 15-ml linear gradient of NaCl (0-1.0 M). In the large scale of the Mono S column chromatography, the REKS activity became mostly Ras-independent for an unknown reason. However, the activity still absolutely required both recombinant MEK and recombinant ERK2. This REKS sample was collected and subjected to the GTPS-GST-Ha-Ras-coupled glutathione-agarose column. The column was washed with Buffer B and was eluted by reduced glutathione. About 60% of the REKS activity was adsorbed to the column and was eluted by reduced glutathione (Fig. 2). This eluate fraction was used for the experiments as affinity-purified REKS. About 80% of GTPS-GST-Ha-Ras was also eluted by reduced glutathione (data not shown). About 40% of the REKS activity was detected in the pass fraction. This reason was not known, but REKS might be degraded by proteolysis in its Ras-binding domain, or this activity might be derived from another contaminating MEK kinase(s), such as c-Raf-1, Mos, and mSte11. The relationship between REKS and other MEK kinases is described below. In contrast, REKS was not adsorbed to the GDP-GST-Ha-Ras-coupled glutathione-agarose column, and the most REKS activity was recovered in the pass fraction.


Figure 2: GTPS-GST-Ha-Ras-coupled glutathione-agarose column affinity chromatography of REKS. The peak fraction of the Mono S column chromatography (large scale) was applied to a GTPS-GST-Ha-Ras-coupled glutathione-agarose column or a GDP-GST-Ha-Ras-coupled glutathione-agarose column. After the column was washed with 4 ml of Buffer B, GST-Ha-Ras was eluted by reduced glutathione. Each fraction was assayed for the REKS activity. The REKS activity of the peak fraction of the Mono S column chromatography is set at 100%. Closed bar, the affinity purification with GTPS-GST-Ha-Ras; openbar, the affinity purification with GDP-GST-Ha-Ras; lanes1 and 2, the pass fraction; lanes 3 and 4, the wash fraction; lanes 5 and 6, the eluate fraction. The results shown are representative of three independent experiments.



Phosphorylation of MEK by REKS

To examine whether REKS is a MEK kinase, we explored the activity of REKS to phosphorylate MEK using recombinant wild-type GST-MEK and recombinant kinase-negative mutant GST-MEK. In the latter, a lysine 97 at the ATP binding site of MEK was changed to tryptophan so that it showed no kinase activity. The wild-type GST-MEK underwent autophosphorylation, whereas the kinase-negative mutant did not (Fig. 3, lanes2 and 3). In the presence of affinity-purified REKS, both the wild-type and mutant kinases, but not GST, were phosphorylated (Fig. 3, lanes4-6). Therefore, REKS is a MEK kinase and activates MEK presumably by this phosphorylation.


Figure 3: Phosphorylation of MEK by REKS. Affinity-purified REKS was incubated with recombinant wild-type GST-MEK or recombinant kinase-negative GST-MEK. After the incubation, the reaction was stopped by the addition of Laemmli's buffer, and the sample was subjected to SDS-PAGE (10% polyacrylamide gel) followed by quantification by an image analyzer. Lanes 1-3, without affinity-purified REKS; lanes 4-6, with affinity purified REKS. Lanes 1 and 4, with GST; lanes 2 and 5, with kinase-negative GST-MEK; lanes 3 and 6, with wild-type GST-MEK. Arrowhead and arrow indicate the positions of GST-MEK and GST, respectively. The protein markers used were beta-galactosidase (M(r) = 116,000), bovine serum albumin (M(r) = 66,000), ovalbumin (M(r) = 45,000), glyceraldehyde-3-phosphate dehydroxygenase (M(r) = 36,000), carbonic anhydrase (M(r) = 29,000), and trypsin inhibitor (M(r) = 20,100). The results shown are representative of three independent experiments.



Phosphorylation of Affinity-purified REKS

In the next set of experiments, we examined the phosphorylation of affinity-purified REKS. Affinity-purified REKS was incubated in the presence of [-P]ATP and subjected to SDS-PAGE followed by autoradiography. A phosphorylated protein with a molecular mass of 98 kDa was detected (Fig. 4A). It is noteworthy that this protein was detected as a doublet. This protein with a molecular mass of 98 kDa was also observed when the gel was stained with silver (Fig. 4B). This protein was not observed in the eluate fraction of the GDP-GST-Ha-Ras-coupled glutathione-agarose column chromatography. Moreover, this protein was coimmunoprecipitated with GTPS-GST-Ha-Ras by an anti-Ras antibody (data not shown). Judging from these observations, a 98-kDa protein shown with an arrowhead in Fig. 4is most likely to be REKS.


Figure 4: Phosphorylation of affinity-purified REKS. The eluate fraction of the GTPS-GST-Ha-Ras-coupled glutathione-agarose column chromatography or of the GDP-GST-Ha-Ras-coupled glutathione-agarose column chromatography was incubated with 10 µM [-P]ATP (50,000 cpm/pmol) for 30 °C for 2 min, and the reaction was stopped by the addition of Laemmli's sample buffer. The sample was then subjected to SDS-PAGE (10% polyacrylamide gel) followed by autoradiography. A, autoradiography; B, silver staining. Lane 1, the eluate fraction of the GDP-GST-Ha-Ras-coupled glutathione-agarose column chromatography; lane 2, the eluate fraction of the GTPS-GST-Ha-Ras-coupled glutathione-agarose column chromatography (affinity purified REKS). An arrowhead indicates the 98-kDa protein. The protein markers used were the same as those shown in the legend to Fig. 3except that myosin (M(r) = 205,000) was used. The results shown are representative of three independent experiments.



Relationship between REKS and Other MEK Kinases

c-Raf-1, Mos, and mSte11 kinases are known to phosphorylate and activate MEK(17, 18, 19, 20, 22, 23, 24) . Therefore, in the last set of experiments, we examined whether REKS is one of these kinases. Immunoblot analysis revealed that the immunoreactivities of c-Raf-1, Mos, and mSte11 were detected in the peak fraction of the Mono S column chromatography (Fig. 5, lane1). When REKS was purified by the GTPS-GST-Ha-Ras-coupled glutathione-agarose column, the immunoreactivities of c-Raf-1, Mos, and mSte11 were detected in the pass fraction of the GTPS-GST-Ha-Ras-coupled glutathione-agarose column chromatography (Fig. 5, lane2), whereas any immunoreactivity of c-Raf-1, Mos, and mSte11 was not detected in the wash fraction of the GTPS-GST-Ha-Ras-coupled glutathione-agarose column chromatography (Fig. 5, lane3). Any immunoreactivity of c-Raf-1, Mos, and mSte11 was not detected in affinity-purified REKS (Fig. 5, lane4). Moreover, the 98-kDa protein was not recognized by any of the anti-c-Raf-1, anti-Mos, and anti-mSte11 antibodies (Fig. 5, lanes1-4). It is not known whether an anti-mSte11 antibody cross-reacts with the Xenopus homologue of mSte11, but this antibody cross-reacted with the protein with a molecular mass of 80 kDa, which is similar to 78 kDa, the molecular mass of mSte11(20) . Therefore, this protein is most likely to be the Xenopus homologue of mSte11. Any immunoreactivity of MEK, ERK, A-Raf, or B-Raf was not detected in affinity-purified REKS (data not shown), although the Xenopus homologues of A-Raf and B-Raf have not yet been identified. These results suggest that REKS is a MEK kinase distinct from c-Raf-1, Mos, and mSte11 kinases.


Figure 5: Immunoblot analysis of REKS with anti-c-Raf-1, anti-Mos, and anti-mSte11 antibodies. A 45-µl aliquot of each REKS samples at various purification steps was subjected to SDS-PAGE (10, 12, and 10% polyacrylamide gels for c-Raf-1, Mos, and mSte11, respectively) followed by immunoblotting using an anti-c-Raf-1 (alpha-Raf), anti-Mos (alpha-Mos), or anti-mSte11 (alpha-mSte11) antibody as indicated. Lane 1, the peak fraction of the Mono S column chromatography (large scale); lane 2, the pass fraction of the GTPS-GST-Ha-Ras-coupled glutathione-agarose column chromatography; lane 3, the wash fraction of the GTPS-GST-Ha-Ras-coupled glutathione-agarose column chromatography; lane 4, the eluate fraction of the GTPS-GST-Ha-Ras-coupled glutathione-agarose column chromatography (affinity purified REKS). The results shown are representative of three independent experiments.




DISCUSSION

We have previously developed a cell-free assay system in which mammalian GTP-Ki-Ras or GTP-Ha-Ras activates ERK through MEK activation (39, 40, 41) . By use of this system, we have already identified a protein factor named REKS, which is necessary for the Ras-dependent MEK activation. In this study, we have highly purified REKS from Xenopus eggs by successive column chromatographies. On the other hand, it has been shown that c-Raf-1 directly interacts with GTP-Ras(27, 28, 29, 30, 31, 32) . Therefore, we have examined here whether REKS directly interacts with GTP-Ki-Ras or GTP-Ha-Ras. We have shown here that REKS is coimmunoprecipitated with GTPS-Ki-Ras or GTPS-Ha-Ras, but not with GDP-Ki-Ras or GDP-Ha-Ras, by an anti-Ras antibody. Moreover, REKS is adsorbed to the GTPS-GST-Ha-Ras-coupled glutathione-agarose column but not to the GDP-GST-Ha-Ras-coupled glutathione-agarose column, although REKS purified by large scale of Mono S column chromatography became mostly Ras-independent. The reason why Ras-independent REKS is still adsorbed to the Ras affinity column is not known, but it is possible that REKS purified by large scale of Mono S column chromatography is degraded in the regulatory domain and still conserves its Ras-binding domain. These results indicate that REKS forms a stable complex with GTPS-Ki-Ras or GTPS-Ha-Ras but not with GDP-Ki-Ras or GDP-Ha-Ras. It is likely that Ras forms a complex with REKS resulting in the activation of MEK.

We have previously shown that lipid-modified Ras is more effective for the activation of REKS than lipid-unmodified Ras(41) . Namely, the doses of lipid-modified Ras necessary for REKS activation are far lower than those of lipid-unmodified Ras. We have purified here REKS by the affinity column chromatography using lipid-unmodified GST-Ha-Ras. The reason for REKS to be adsorbed to this column might be due to the large amount of GST-Ha-Ras used for the affinity column chromatography.

We have also examined here whether REKS is a MEK kinase. We have shown that both wild-type MEK and kinase-negative MEK are phosphorylated in the presence of affinity-purified REKS, indicating that REKS is a MEK kinase and activates MEK presumably by this phosphorylation. Furthermore, we have identified here a possible protein of REKS with a molecular mass of 98 kDa that is phosphorylated. This 98-kDa protein is specifically observed in affinity-purified REKS. When GDP-GST-Ha-Ras is used instead of GTPS-GST-Ha-Ras for the affinity purification, no REKS activity is detected, and the 98-kDa protein is not observed in the eluate fraction. Therefore, the 98-kDa protein is most likely to be REKS, but further purification is necessary to examine whether the 98-kDa protein is REKS.

We have examined the relationship between REKS and other MEK kinases thus far reported in the literature(17, 18, 19, 20, 21, 22, 23, 24) . We have shown here that REKS is distinct from c-Raf-1, Mos, and mSte11 kinases, all of which are known to phosphorylate and activate MEK(17, 18, 19, 20, 21, 22, 23, 24) . Moodie et al.(27) has reported that the MEK kinase activity associates with immobilized Ras in rat brain cytosol, where c-Raf-1 is immunodepleted by an anti-c-Raf-1 antibody. This is consistent with our observation that c-Raf-1 is not required for the Ras-dependent activation of MEK. It has recently been shown that a novel MEK kinase with a molecular mass of 98 kDa is activated by Ras in PC12 cells(24) . It has not been shown, however, that this MEK kinase directly interacts with GTP-Ras and is activated by GTP-Ras in a cell-free system. The relationship between REKS and this MEK kinase is currently unknown. We cannot exclude the possibility that REKS is one of the homologues of Raf or mSte11. Cloning of REKS is necessary to address this question.


FOOTNOTES

*
This investigation was supported by grants-in-aid for scientific research and for cancer research from the Ministry of Education, Science, and Culture, Japan(1993, 1994), by grants-in-aid for abnormalities in hormone receptor mechanisms and for aging and health from the Ministry of Health and Welfare, Japan(1993, 1994), and by grants from the Yamanouchi Foundation for Research on Metabolic Disease(1993, 1994), the Research Program on Cell Calcium Signal in the Cardiovascular System(1993), Setsuro Fujii Memorial, the Osaka Foundation for Promotion of Fundamental Medical Research(1993), Uehara Memorial Foundation(1994), and Nissan Science Foundation(1994). 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.

§
Present address: Dept. of Molecular Biology and Biochemistry, Osaka University Medical School, Suita 565, Japan.

Present address: Div. of Signal Transduction, Nara Institute of Science and Technology, Ikoma 630-01, Japan.

**
To whom correspondence should be addressed: Dept. of Molecular Biology and Biochemistry, Osaka University Medical School, 2-2 Yamada-oka, Suita 565, Japan. Tel.: 81-6-879-3410; Fax: 81-6-879-3419; ytakai{at}molbio.med.osaka-u.ac.jp.

(^1)
The abbreviations used are: MEK, mitogen-activated protein kinase kinase/ERK kinase; ERK, extracellular signal-regulated kinase; GTPS, guanosine 5`-O-(thiotriphosphate); GST, glutathione S-transferase; PAGE, polyacrylamide gel electrophoresis.


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