From the
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 GTP
Recent studies indicate that Ras activates the
MAP(
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 A
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.
Bovine brain cytosol (200 ml,
1.4 g of protein) was thawed on ice, and the supernatant was obtained
by centrifugation at 100,000
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).
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 GTP
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 GTP
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 GTP
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
GTP
S (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, GTP
S-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 GTP
S-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 GTP
S-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.
)
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.
perse, 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).
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.
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). GTP
S- 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
GTP
S-Ki-Ras;
, in the presence of GDP-Ki-Ras;
, in the
absence of Ki-Ras. --, NaCl concentration; - - -,
absorbance at 280 nm. C, GTP
S- or GDP-GST-Ha-Ras-coupled
glutathione-agarose column chromatography. Closed bar,
GTP
S-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 GTP
S-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 GTP
S-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
GTP
S-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 GTP
S-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).
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 GTP
S-Ki-Ras. Closed bar, in the presence of
GTP
S-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 GTP
S-Ki-Ras. Open bar,
without GTP
S-Ki-Ras. Closed bar, with
GTP
S-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 GTP
S-Ki-Ras was nearly inactive (data not shown).
The K
value for lipid-modified
GTP
S-Ki-Ras, giving a half-maximum activation, was about 20
nM. We have reported previously that 14-3-3 proteins and
GTP
S-Ki-Ras synergistically stimulate Xenopus REKS
(35) . 14-3-3 proteins purified from rat brain markedly
stimulated the REKS activity in the absence of GTP
S-Ki-Ras,
whereas 14-3-3 proteins slightly stimulated it in the presence of
GTP
S-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 GTP
S-Ki-Ras. The
K
values for 14-3-3 proteins were also
the same in the presence and absence of GTP
S-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
GTP
S-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.
S-Ki-Ras, whereas they
synergistically activate it in the presence of GTP
S-Ki-Ras (35).
In contrast, 14-3-3 proteins markedly stimulated bovine REKS in the
absence of GTP
S-Ki-Ras and slightly stimulated it in the presence
of GTP
S-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 GTP
S-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.
S-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.
S-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.
S-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.
S, guanosine
5`-(3-O-thio)triphosphate; GST, glutathione
S-transferase; PAGE, polyacrylamide gel electrophoresis.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.