(Received for publication, October 4, 1994; and in revised form, November 8, 1994)
From the
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 GTP
S-Ras; REKS was
coimmunoprecipitated with GTP
S-Ki-Ras or GTP
S-Ha-Ras, but not
with GDP-Ki-Ras or GDP-Ha-Ras by an anti-Ras antibody. REKS was
adsorbed to a GTP
S-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
GTP
S-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.
Three ras genes encode proteins with M 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(
)/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.
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.
,
with GTP
S-Ki-Ras;
, with GDP-Ki-Ras;
, without Ki-Ras.
-, NaCl concentration. The results shown are
representative of three independent
experiments.
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 GTP
S-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
GTP
S-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.
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 -galactosidase (M
= 116,000), bovine serum albumin (M
= 66,000), ovalbumin (M
=
45,000), glyceraldehyde-3-phosphate dehydroxygenase (M
= 36,000), carbonic anhydrase (M
= 29,000), and trypsin inhibitor (M
= 20,100). The results shown are representative of three
independent experiments.
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
GTP
S-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
= 205,000) was used. The results shown are representative
of three independent experiments.
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 (-Raf), anti-Mos
(
-Mos), or anti-mSte11 (
-mSte11)
antibody as indicated. Lane 1, the peak fraction of the Mono S
column chromatography (large scale); lane 2, the pass fraction
of the GTP
S-GST-Ha-Ras-coupled glutathione-agarose column
chromatography; lane 3, the wash fraction of the
GTP
S-GST-Ha-Ras-coupled glutathione-agarose column chromatography; lane 4, the eluate fraction of the
GTP
S-GST-Ha-Ras-coupled glutathione-agarose column chromatography
(affinity purified REKS). The results shown are representative of three
independent experiments.
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 GTP
S-Ha-Ras, but not
with GDP-Ki-Ras or GDP-Ha-Ras, by an anti-Ras antibody. Moreover, REKS
is adsorbed to the GTP
S-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 GTP
S-Ki-Ras or GTP
S-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.