©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Purification of a Ras-dependent Mitogen-activated Protein Kinase Kinase Kinase from Bovine Brain Cytosol and Its Identification as a Complex of B-Raf and 14-3-3 Proteins (*)

Bunpei Yamamori (1), Shinya Kuroda (1), Kazuya Shimizu (1), Koji Fukui (1), Toshihisa Ohtsuka (1), Yoshimi Takai (1) (2)(§)

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

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
FOOTNOTES
REFERENCES

ABSTRACT

We previously purified a protein factor, named REKS (Ras-dependent Extracellular Signal-regulated Kinase (ERK)/mitogen-activated protein kinase Kinase (MEK) Stimulator), from Xenopus eggs by use of a cell-free assay system in which recombinant GTPS (guanosine 5`-(3-O-thio)triphosphate)-Ki-Ras activates recombinant MEK. By use of this assay system, we purified here bovine REKS to near homogeneity from the cytosol fraction of bovine brain by successive chromatographies of Mono S, Mono Q, GTPS-glutathione S-transferase-Ha-Ras-coupled glutathione-agarose, and Mono Q columns. It was composed of three proteins with masses of about 95, 32, and 30 kDa as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The 95-, 32-, and 30-kDa proteins were identified by immunoblot analysis to be B-Raf protein kinase, 14-3-3 protein, and 14-3-3 protein, respectively. Moreover, the REKS activity was specifically immunoprecipitated by an anti-B-Raf antibody. Bovine REKS was activated by lipid-modified GTPS-Ki-Ras far more effectively than by a lipid-unmodified one. Lipid-modified GDP-Ki-Ras was inactive. Exogenous addition of 14-3-3 proteins stimulated further the REKS activity both in the presence and absence of GTPS-Ki-Ras. These results indicate that at least one of the direct targets of Ras is B-Raf complexed with 14-3-3 proteins in bovine brain.


INTRODUCTION

Recent studies indicate that Ras activates the MAP() kinase cascade consisting of MAP kinase/ERK, MAP kinase kinase/MEK, and MEK kinase in mammals (for reviews, see Refs. 1-4). MAP kinase is phosphorylated at both serine/threonine and tyrosine residues by MEK, and this phosphorylation causes MAP kinase activation (1, 2, 3) . MEK is also phosphorylated at serine/threonine residues by MEK kinase, and this phosphorylation causes MEK activation (5, 6, 7) . Many MEK kinases have been identified; these include c-Raf-1 (8, 9, 10) , B-Raf (11, 12, 13, 14, 15) , Mos (16, 17) , and mSte11 (11, 18) . GTP-Ras directly interacts with B-Raf (13, 14) and c-Raf-1 (19, 20, 21, 22, 23, 24) . Ras genetically positions upstream of c-Raf-1 in Drosophila(25) and Caenorhabditis elegans(26) . Moreover, MAP kinase is activated by B-Raf, but not by c-Raf-1, in intact PC12 cells in response to nerve growth factor (13). Association of MEK with immobilized Ras is dependent on B-Raf, but not on c-Raf-1, in rat brain (14) . MEK activating activity is accompanied with B-Raf, but not with c-Raf-1, in bovine brain or chromaffin cells (15) . However, it has not been demonstrated that GTP-Ras directly activates these MEK kinases in a cell-free assay system.

To identify the direct target of Ras, we have previously developed a cell-free assay system in which GTP-Ras activates MEK (27) . By use of this assay system, we have identified a protein factor that is essential for Ras-dependent MEK activation and tentatively named it REKS (Ras-dependent ERK Kinase Stimulator) (27) . We have also shown that post-translationally lipid-modified Ras is far more effective on the activation of REKS than lipid-unmodified Ras (28) . Consistently, lipid-modifications of Ras are essential for the Ras-dependent activation of c-Raf-1 in insect cells overexpressing Ras and c-Raf-1 (29) . We have recently highly purified REKS from Xenopus eggs and identified a possible protein of REKS with a mass of 98 kDa, which is immunologically distinct from c-Raf-1, Mos, and mSte11 (30) .

On the other hand, the 14-3-3 protein family directly interacts with c-Raf-1 in in vivo and in vitro systems (31, 32, 33, 34) , and 14-3-3 proteins and Ras synergistically activate REKS (35) . The 14-3-3 protein family, consisting of at least seven members, is known to be an activator protein of tyrosine and tryptophan hydroxylases, to modulate the protein kinase C activity, to stimulate secretion, to be a phospholipase Aperse, to stimulate mitochondrial import of protein, to interact with c-Bcr and Bcr-Abl, and to interact with middle tumor antigen of polyomavirus (36) (for reviews, see Refs. 37 and 38).

In the present study, we have attempted to purify REKS from bovine brain cytosol and to identify it. We have found that bovine brain REKS is composed of B-Raf complexed with 14-3-3 proteins.


EXPERIMENTAL PROCEDURES

Purification of REKS

Bovine brains were obtained from the heads of freshly slaughtered cattle. All the purification procedures were performed at 0-4 °C. Gray matter of cerebra (about 1.6 kg, wet weight) were homogenized in a Waring blender with 1.7 volumes of Homogenizing Buffer (20 mM Tris/HCl at pH 7.9, 2 mM EDTA, 5 µM APMSF, 1 mg/l leupeptin, and 1 mg/l pepstatin A). The homogenate was centrifuged at 100,000 g for 1 h. After one-ninth volume of Buffer A (200 mM Tris/HCl at pH 8.0, 10 mM DTT, 100 mM EGTA, and 50 mM MgCl) was added to the supernatant, one-ninth volume of ethylene glycol was added to give the final concentration of 10%. The supernatant was then immediately frozen in liquid nitrogen and stored at -80 °C until use.

Bovine brain cytosol (200 ml, 1.4 g of protein) was thawed on ice, and the supernatant was obtained by centrifugation at 100,000 g for 1 h. The supernatant was sequentially filtrated through 2.0-, 0.8-, 0.45-, and 0.2-µm filters. The filtrated supernatant (160 ml, 1.1 g of protein) was then applied to a Mono S column (1.0 10 cm) equilibrated with 80 ml of Buffer B (20 mM HEPES/NaOH at pH 7.0, 1 mM DTT, 10 mM EGTA, 5 mM MgCl, and 10 µM APMSF). After the column was washed with 80 ml of Buffer B, elution was performed with a 240-ml linear gradient of NaCl (0-1.0 M) in Buffer B and fractions of 4 ml each were collected. REKS appeared in fractions 80-90 (Fig. 1A). After 1.5 M Tris was added to 160 ml of the active fractions of Fig. 1A to adjust pH to 7.5, the active fractions were diluted with an equal volume of Buffer C (20 mM Tris/HCl at pH 7.5, 1 mM DTT, 1 mM EGTA, 5 mM MgCl, and 10 µM APMSF) and applied to a Mono Q column (1.0 10 cm) equilibrated with 80 ml of Buffer C. After the column was washed with 80 ml of Buffer C, elution was performed with a 240-ml linear gradient of NaCl (0.125-0.5 M) in Buffer C and fractions of 4 ml each were collected. REKS appeared in fractions 118-122 (Fig. 1B). GTPS- or GDP-GST-Ha-Ras (15 nmol each) was separately applied to a glutathione-agarose column (0.6 ml) pre-equilibrated with 9 ml of Buffer D (20 mM Tris/HCl at pH 7.5, 1 mM DTT, 5 mM MgCl, and 1 mM EGTA), and the column was washed with 15 ml of Buffer D. Forty ml of the active fractions of Fig. 1B was applied to this column and washed sequentially with 6 ml of Buffer D, 6 ml of Buffer D containing 0.25 M NaCl, and 6 ml of Buffer D. Elution was performed with 1.8 ml of Buffer D containing 20 mM reduced glutathione (Fig. 1C). The active eluate fractions of Fig. 1C were applied to a Mono Q column (0.16 5 cm) equilibrated with 1 ml of Buffer D. After the column was washed with 2 ml of Buffer D, elution was performed with a linear gradient of NaCl (0-0.5 M) in Buffer D. At 190 mM NaCl, however, the gradient was placed on hold for 4 ml to remove REKS-unbound GST-Ha-Ras and then continued. Fractions of 100 µl each were collected (Fig. 2A). REKS appeared in fractions 93-100.


Figure 1: Purification of REKS from bovine brain cytosol. Each fraction of Mono S column chromatography (15 µl) or of Mono Q column chromatography (7.5 µl) was assayed for the REKS activity. A, Mono S column chromatography. B, first Mono Q column chromatography. , in the presence of GTPS-Ki-Ras; , in the presence of GDP-Ki-Ras; , in the absence of Ki-Ras. --, NaCl concentration; - - -, absorbance at 280 nm. C, GTPS- or GDP-GST-Ha-Ras-coupled glutathione-agarose column chromatography. Closed bar, GTPS-GST-Ha-Ras-coupled glutathione-agarose column chromatography; open bar, GDP-GST-Ha-Ras-coupled glutathione-agarose column chromatography. Lanes 1 and 2, the pass fraction; lanes 3 and 4, the wash fraction; lanes 5 and 6, the eluate fraction. The REKS activity purified by the first Mono Q column chromatography is set at 100%. The results shown are representative of three independent experiments.




Figure 2: Second Mono Q column chromatography of the affinity-purified REKS. Each fraction (1 µl) was assayed for the REKS activity. Another aliquot (60 µl) of each fraction was subjected to SDS-PAGE followed by silver staining and immunoblot analysis. A, REKS activity. B, silver staining. C, immunoblot analysis with anti-B-Raf and anti-14-3-3 protein antibodies. Lanes 1 and 3, bovine brain cytosol (100 µg of protein); lanes 2 and 4, 60 µl of fraction 94. An arrowhead and arrows indicate the positions of the 95-kDa protein, and both the 32- and 30-kDa proteins, respectively. The protein markers used were myosin (M = 200,000), -galactosidase (M = 116,000), bovine serum albumin (M = 66,000), ovalbumin (M = 45,000), glyceraldehyde-3-phosphate dehydrogenase (M = 36,000), carbonic anhydrase (M = 29,000), and trypsin inhibitor (M = 20,100). The results shown are representative of three independent experiments.



Materials and Other Procedures

Post-translationally lipid-modified and -unmodified Ki-Ras, GST-MEK, GST-ERK2, and GST-Ha-Ras were prepared as described (35, 39) . The same sample of an anti-14-3-3 protein polyclonal antibody as used previously (35) was kindly provided by T. Isobe (Tokyo Metropolitan University, Tokyo, Japan). Anti-c-Raf-1, anti-A-Raf, and anti-B-Raf polyclonal antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The same sample of rat brain 14-3-3 proteins as used previously (35) was kindly provided from T. Yamauchi (Tokushima University, Tokushima, Japan). 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 (35) . SDS-PAGE was performed by the method of Laemmli (40) . Protein concentrations were determined with bovine serum albumin as a standard protein by the method of Bradford (41) . Immunoblot was carried out as described (35) .

RESULTS

When filtrated bovine brain cytosol was subjected to a Mono S column chromatography and each fraction was assayed for the REKS activity, two peaks appeared (Fig. 1A). The second peak was enhanced by GTPS-Ki-Ras, but not by GDP-Ki-Ras, whereas the first peak was not affected by GTPS-Ki-Ras. Moreover, the second peak was dependent on MEK, whereas the first peak was not (data not shown). Therefore, the second peak was a bovine counterpart of Xenopus REKS. When the active fractions of Fig. 1A were subjected to a Mono Q column chromatography and each fraction was assayed for the REKS activity, three peaks appeared (Fig. 1B). The first peak was enhanced by GTPS-Ki-Ras, whereas the second and third peaks were not. All peaks were dependent on recombinant MEK (data not shown). Therefore, the first peak was REKS and the other peaks were unknown MEK kinases. The active fractions of Fig. 1B were applied to a GTPS-GST-Ha-Ras-coupled glutathione-agarose column chromatography. About 45% of the total REKS activity was adsorbed to the column and was eluted by reduced glutathione (Fig. 1C). About 70% of GTPS-GST-Ha-Ras was also eluted by reduced glutathione (data not shown). Any REKS activity was not adsorbed to the GDP-GST-Ha-Ras-coupled glutathione-agarose column. When the active eluate fractions of Fig. 1C were subjected to a Mono Q column chromatography, the REKS activity was detected as a single peak (Fig. 2A). When each fraction was subjected to SDS-PAGE followed by protein staining, the peak of REKS was accompanied with four proteins and most GST-Ha-Ras was recovered in other fractions (Fig. 2B). The masses of these proteins were about 95, 49, 32, and 30 kDa. The protein with a mass of 49 kDa was GST-Ha-Ras itself. The proteins with masses of 95, 32, and 30 kDa were identified by immunoblot analysis to be B-Raf, 14-3-3 protein, and 14-3-3 protein, respectively (Fig. 2C).

To further confirm that REKS includes B-Raf, we examined the immunoprecipitation of REKS with an anti-B-Raf antibody. After the REKS sample of Fig. 1B was incubated with or without an anti-B-Raf antibody, its activity was measured in the supernatant and precipitate (Fig. 3A). In the presence of the antibody, the REKS activity was recovered in the precipitate, whereas, in the absence of the antibody, the REKS activity was recovered in the supernatant. Moreover, the REKS activity was not immunoprecipitated by any of the antibodies against A-Raf and c-Raf-1 and no immunoreactivities against A-Raf and c-Raf-1 were detected in the REKS sample of Fig. 1B (data not shown). It may be noted that the recovery of the REKS activity in the precipitate was about 500% (Fig. 3A). To clarify the reason for this increase in the recovery, the REKS activity was measured in the presence or absence of the anti-B-Raf antibody. The REKS activity increased about 5-fold in the presence of the antibody compared to that in the absence of the antibody (Fig. 3B).


Figure 3: Immunoprecipitation of REKS by an anti-B-Raf antibody and effect of an anti-B-Raf antibody on the REKS activity. A, immunoprecipitation of REKS by an anti-B-Raf antibody. The first peak of Fig. 1B (100 µl) was incubated with or without an anti-B-Raf antibody (40 µl) for 1.5 h at 4 °C. After Protein A-Sepharose beads (20 µl) were added, the immunocomplex was precipitated and washed sequentially with Buffer C containing 0.5% Nonidet P-40 and with Buffer C. The precipitate (1 µl) or supernatant (1 µl) was assayed for the REKS activity in the presence or absence of GTPS-Ki-Ras. Open bar, in the absence of GTPS-Ki-Ras. Closed bar, in the presence of GTPS-Ki-Ras. Lanes 1, 2, 5, and 6, without an anti-B-Raf antibody; lanes 3, 4, 7, and 8, with an anti-B-Raf antibody. Lanes 1-4, supernatant; lanes 5-8, precipitate. B, effect of an anti-B-Raf antibody on the REKS activity. After the REKS sample of Fig. 1B (100 µl) was incubated with or without an anti-B-Raf antibody (5 µl) for 1.5 h at 4 °C, the mixture (1 µl) was assayed for the REKS activity in the presence or absence of GTPS-Ki-Ras. Open bar, without GTPS-Ki-Ras. Closed bar, with GTPS-Ki-Ras. Lanes 1 and 2, without an anti-B-Raf antibody; lanes 3 and 4, with an anti-B-Raf antibody. The results shown are representative of three independent experiments.



We have shown previously that lipid-modified Ras is far more effective than lipid-unmodified Ras on the activation of Xenopus REKS (28) . Similarly, lipid-modified GTPS-Ki-Ras activated bovine REKS, whereas lipid-unmodified GTPS-Ki-Ras was nearly inactive (data not shown). The Kvalue for lipid-modified GTPS-Ki-Ras, giving a half-maximum activation, was about 20 nM. We have reported previously that 14-3-3 proteins and GTPS-Ki-Ras synergistically stimulate Xenopus REKS (35) . 14-3-3 proteins purified from rat brain markedly stimulated the REKS activity in the absence of GTPS-Ki-Ras, whereas 14-3-3 proteins slightly stimulated it in the presence of GTPS-Ki-Ras (data not shown). However, the maximal levels activated by the addition of 14-3-3 proteins were almost the same in the presence and absence of GTPS-Ki-Ras. The Kvalues for 14-3-3 proteins were also the same in the presence and absence of GTPS-Ki-Ras and were about 100 nM.

DISCUSSION

We have purified here REKS from bovine brain cytosol and shown that bovine brain REKS is composed of at least three proteins with masses of about 95, 32, and 30 kDa. The 95-, 32-, and 30-kDa proteins have been identified to be B-Raf, 14-3-3 protein, and 14-3-3 protein, respectively. These results are consistent with the earlier observations that MAP kinase is activated by B-Raf in response to nerve growth factor in PC12 cells (13) , that association of MEK with immobilized Ras is dependent on B-Raf in rat brain (14) , and that MEK activating activity is accompanied with B-Raf in bovine brain or chromaffin cells (15) . Because we have purified here REKS by the GTPS-GST-Ha-Ras-coupled glutathione-agarose column chromatography, the purified REKS is likely to be complexed with GTPS-GST-Ha-Ras and to be converted to an active form. Therefore, the possibility cannot be excluded that an inactive form of REKS contains a component other than B-Raf and 14-3-3 proteins.

14-3-3 proteins interact directly with c-Raf-1 in in vivo and in vitro systems and enhance its activity (31, 32, 33, 34) . The present result that REKS is B-Raf complexed with 14-3-3 proteins is consistent with these earlier observations (31, 32, 33, 34) . We have described previously that 14-3-3 proteins slightly activate Xenopus REKS in the absence of GTPS-Ki-Ras, whereas they synergistically activate it in the presence of GTPS-Ki-Ras (35). In contrast, 14-3-3 proteins markedly stimulated bovine REKS in the absence of GTPS-Ki-Ras and slightly stimulated it in the presence of GTPS-Ki-Ras, although the maximal levels activated by the addition of 14-3-3 proteins were almost the same in the presence and absence of GTPS-Ki-Ras. This present result is not consistent with our earlier observation for Xenopus REKS, but this discrepancy may be due to the difference of the properties between Xenopus REKS and bovine REKS. Furthermore, to examine whether 14-3-3 proteins are essential for the Ras-dependent activation of bovine REKS, we have attempted to separate 14-3-3 proteins from B-Raf but have not yet succeeded. It remains to be clarified whether 14-3-3 proteins are essential for the Ras-dependent activation of B-Raf.

We have found here that the REKS activity immunoprecipitated by the anti-B-Raf antibody is enhanced by the antibody irrespective of the presence or absence of GTPS-Ki-Ras. The N-terminal domain of c-Raf-1 inhibits its kinase activity presumably by masking its kinase domain (42, 43, 44) . It is possible that the association of an anti-B-Raf antibody with B-Raf relieves it from the inhibitory action of the N-terminal domain, resulting in the activation of REKS. This result has raised a possibility that there may be another factor that mimics the action of the antibody.

We have previously identified a possible protein of Xenopus REKS with a mass of 98 kDa, immunologically distinct from B-Raf (30) . However, Xenopus REKS and bovine REKS share the similar properties. They are adsorbed to the GTPS-GST-Ha-Ras-coupled glutathione-agarose column. They have similar minimum molecular sizes on SDS-PAGE. Lipid modifications of Ras are critical for their activation. These observations suggest that the 98-kDa protein of Xenopus REKS is the counterpart of the 95-kDa protein of bovine REKS. The reason why the 98-kDa protein of Xenopus REKS was not recognized by an anti-B-Raf antibody is not known, but it is possible that the amino acids of the C terminus of the Xenopus 98-kDa protein are different from those of the bovine 95-kDa protein, namely B-Raf, against which the anti-B-Raf antibody was generated.

A-Raf, B-Raf, and c-Raf-1 are expressed in brain (45) . We have identified here REKS as B-Raf, but not A-Raf or c-Raf-1, in brain. On the other hand, c-Raf-1 directly interacts with GTP-Ras in cell-free assay systems (19, 20, 21, 24) and in yeast two hybrid systems (22, 23), and c-Raf-1 preactivated in intact cells activates MEK in cell-free systems (8, 9, 10) . Although these earlier observations suggest that c-Raf-1 activated by GTP-Ras activates MEK, no direct evidence has thus far been obtained that GTP-Ras activates c-Raf-1 or A-Raf in cell-free assay systems. The failure that bovine brain A-Raf or c-Raf-1 is not activated by GTPS-Ki-Ras in our system may be due to the contamination of some interfering materials in the A-Raf or c-Raf-1 fractions, or to the absence of a certain factor necessary for the Ras-dependent MEK activation. B-Raf is expressed only in cerebrum, spinal cord, and testis (45) . If A-Raf or c-Raf-1 is not responsible for the Ras-dependent MEK activation, another B-Raf isoform or another MEK kinase may be present and responsible for the Ras-dependent MEK activation in other tissues.


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 (1994), by grants-in-aid for Abnormalities in Hormone Receptor Mechanisms and for Aging and Health from the Ministry of Health and Welfare, Japan (1994), and by grants from the Yamanouchi Foundation for Research on Metabolic Disease (1994), 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.

§
To whom correspondence should be addressed. Tel.: 81-6-879-3410; Fax: 81-6-879-3419; E-mail: ytakai@molbio.med.osaka-u.ac.jp.

The abbreviations used are: MAP, mitogen-activated protein; ERK, extracellular signal-regulated kinase; MEK, MAP kinase kinase/ERK kinase; REKS, Ras-dependent ERK Kinase Stimulator APMSF, (p-amidinophenyl)methanesulfonyl fluoride; DTT, dithiothreitol; GTPS, guanosine 5`-(3-O-thio)triphosphate; GST, glutathione S-transferase; PAGE, polyacrylamide gel electrophoresis.


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