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(Received for publication, July 31,
1995; and in revised form, October 30, 1995) From the
B-Raf is regulated by Ras protein and acts as a
mitogen-activated protein (MAP) kinase kinase kinase in PC12 cells and
brain. Ras protein undergoes a series of post-translational
modifications on its C-terminal CAAX motif, and the
modifications are critical for its function. To elucidate the role of
the post-translational modifications in interaction with, and
activation of, B-Raf, we have analyzed a direct association between
H-Ras and B-Raf, and constructed an in vitro system for B-Raf
activation by H-Ras. By using methods based on inhibition of yeast
adenylyl cyclase or RasGAP activity and by in vitro binding
assays, we have shown that the segment of B-Raf corresponding to amino
acid 1-326 binds directly to H-Ras with a dissociation constant (K
Ras protein is a plasma membrane-associated guanine
nucleotide-binding protein that cycles between a GTP-bound active form
and a GDP-bound inactive form, and operates in key processes of
intracellular signal transduction systems that are involved in
regulation of cell growth and differentiation. In higher eukaryotes
including Caenorhabditis elegans, Drosophila
melanogaster, and vertebrates, Ras is a key regulator that
mediates signal transduction from cell surface tyrosine kinase
receptors to the nucleus via activation of the MAP ( B-raf gene was discovered as a transforming gene in NIH3T3
cell transfection assays with human Ewing sarcoma DNA(9) , and
its protein product consists of 765 amino acid residues that contain
three distinct regions of conservation with Raf-1; CR1, CR2, and
CR3(2, 10) . In contrast to the ubiquitous
distribution of Raf-1 in a variety of mammalian organs, expression of
B-Raf is confined to brain and testis (11) . Another member of
the Raf family, A-Raf, is expressed most abundantly in ovary and
epididymis(11) . Recent studies have shown that B-Raf, instead
of Raf-1, is responsible for Ras-dependent activation of the MAP kinase
pathway in PC12 cells and mammalian (rat and bovine)
brain(12, 13, 14, 15) . Ras
proteins undergo a series of post-translational modifications on their
unique C-terminal region called a CAAX motif (C, cysteine; A,
aliphatic; and X, any amino acid) (for reviews, see (16, 17, 18) ). The first stage of the
processing consists of three successive modifications of the CAAX motif: (i) farnesylation of the cysteine residue, (ii) proteolytic
cleavage of the amino acids AAX, and (iii) methyl
esterification of the new C-terminal cysteine. This first stage of
modification converts the primary translation product into an
intermediate form. In the case of H-Ras, it is further modified by
acylation with palmitic acid on cysteine residues (Cys-181 and Cys-184)
immediately upstream of the CAAX motif, finally yielding the
post-translationally fully modified form. These modifications are
essential for anchoring Ras proteins to the plasma membrane (19, 20) and for a number of biological activities of
Ras: malignant transformation of NIH3T3
cells(19, 20) , induction of neuronal differentiation
of PC12 cells(21) , and induction of germinal vesicle breakdown
in Xenopus laevis oocytes (22) by activated Ras. The
activity of H-Ras to activate Raf-1 in vivo was also reported
to be dependent on the modifications(23) . However, these in vivo experiments entail an inherent problem in separating
the effect of the modifications on the activity of Ras from that on its
membrane anchoring. Recently an in vitro pure reconstituted
system was used to show that the post-translational modifications,
especially farnesylation, are critical for activation of Saccharomyces cerevisiae adenylyl cyclase which is an
immediate downstream effector of Ras in this organism(24) .
This suggested that the post-translational modifications are required
for activation of Ras effectors. Efficient activation of MAP kinases by
Ras in crude cell-free extracts from X. laevis oocytes was
also reported to depend on the modifications (25, 26) . However, requirement of the modifications
of Ras has not been examined in vitro for the Raf-1 activation
because a cell-free system for the Raf-1 activation has not been
established. To analyze the molecular mechanism underlying the
requirement of the post-translational modifications for the Raf
activation, we have established a cell-free system derived from rat
brain cytosol in which exogenously added H-Ras protein can activate MAP
kinase/ERK2 through activation of B-Raf and MEK. We have also examined
the effect of the modifications on direct association of H-Ras with
B-Raf, and the result is compared with that obtained on the B-Raf
activation.
Figure 1:
Measurement of
H-Ras binding to MBP-B-Raf by adenylyl cyclase inhibition assay. A, adenylyl cyclase activity was measured in the presence of 1
pmol of GTP
Figure 3:
In vitro association of the
post-translationally modified and unmodified forms of H-Ras with the
N-terminal segment of B-Raf. A, the post-translationally
modified forms of H-Ras (lanes 1, 2, 6, and 7) and H-Ras
Figure 2:
Inhibition of RasGAP-stimulation of GTPase
activities of the modified and unmodified forms of H-Ras by MBP-B-Raf. A, the post-translationally modified and unmodified forms of
H-Ras loaded with [
Figure 4:
Partial purification and characterization
of Ras-dependent MAP kinase stimulation activity. A, rat brain
cytosol was fractionated by column chromatography on a Mono S column. Solid and broken lines indicate NaCl concentration
and absorbance at 280 nm, respectively. A 15-µl aliquot of each
fraction was assayed for phosphorylation activity of myelin basic
protein in the presence of GST-MEK and GST-ERK2 along with 2 pmol each
of GTP
Figure 5:
Dose-dependent stimulation of MAP kinase
activity by the post-translationally modified and unmodified forms of
H-Ras. A 15-µl aliquot of the fraction 42 was assayed for
phosphorylation activity of myelin basic protein in the presence of
GST-MEK and GST-ERK2 along with varying concentrations of the modified
(
To
examine which step in the process of modifications of H-Ras is critical
for activation of B-Raf, we constructed and purified
H-Ras
Figure 6:
Stimulation of B-Raf and adenylyl cyclase
activities by C-terminal mutants of H-Ras. A, the activity of
B-Raf to induce phosphorylation of myelin basic protein was measured in
the presence of 0.5 pmol each of the various forms of H-Ras protein;
the post-translationally fully modified and unmodified forms of
wild-type H-Ras, H-Ras
We have shown here that post-translational modifications of
H-Ras are not required for association with one of its effector
molecule, B-Raf, but are essential for activation of B-Raf. The
farnesylation step of the modifications, not the palmitoylation, is
shown to be responsible for this effect. B-Raf is a serine/threonine
kinase which is expressed specifically in neuronal tissues and
testis(11) , while Raf-1 is ubiquitously expressed in all cell
types. Although the association of Raf-1 with Ras has been a subject of
extensive investigation, B-Raf is not well analyzed for its interaction
with Ras protein. In this paper we have shown for the first time that
the N-terminal segment of B-Raf makes a direct association with H-Ras
in a GTP-dependent manner. No association is observed with the effector
mutant H-Ras It was shown that B-Raf, not Raf-1,
mediates the nerve growth factor-induced activation of the MAP kinase
cascade through interaction with Ras in PC12 cell(13) . In rat
brain the association of MEK1 with Ras is dependent on B-Raf, not on
Raf-1(14) . These data prompted us to establish a cell-free
system for Ras-dependent stimulation of MAP kinase activity through
B-Raf activation using rat brain cytosol. While this work was in
progress, Yamamori et al.(15) reported establishment
of such a system from bovine brain cytosol and purification of a
Ras-dependent MEK kinase, which turned out to be a complex of B-Raf and
14-3-3 proteins, although the possibility of presence of other
minor components was not excluded completely. Here we also have
constructed a similar in vitro system and employed it to
quantitatively examine the effect of the post-translational
modifications of Ras on its activity. A number of studies showed
that the post-translational modifications are essential for the
biological activities of Ras observed in vivo. However, in
these systems, the effect of the modifications on the activity of Ras
could not be examined separately from the effect on its membrane
anchoring. A recent study using a baculovirus coinfection
assay(23) , which demonstrated that the modifications of Ras
are required for Raf-1 activation, also cannot get rid of this
fundamental problem. Here we have employed the in vitro cell-free system for Ras-dependent B-Raf activation to show that
the post-translational modifications are critical for the ability of
H-Ras to activate B-Raf. To determine which step of modifications is
responsible for the acquisition of the ability to activate B-Raf, we
also examined the B-Raf stimulation activity of H-Ras mutants,
H-Ras The mechanism by
which Raf is activated by Ras is still unclear. It was proposed that a
major consequence of the direct association with Ras is to recruit
Raf-1 to the plasma membrane, where Raf-1 is subject to activation by
an unknown mechanism(36, 37) . This was based on the
finding that attachment of the C-terminal peptide of K-Ras containing
the farnesylation signal and the polybasic region to the Raf-1 C
terminus abrogated the requirement of Ras for the Raf-1
activity(36, 37) . Obviously, the establishment of the
cell-free system from rat brain cytosol strongly suggests that this
membrane translocation model cannot be simply applied to B-Raf. The
observed requirement of the post-translational modifications of H-Ras
(especially farnesylation) for the activation process of B-Raf implies
that the modifications are required for B-Raf to undergo a further
conformational change for assuming its active conformation than that
induced by the association with Ras. Alternatively, if an additional
factor is required for the activation, the modifications may facilitate
the association of Ras or B-Raf with it. This may reflect a difference
in the activation mechanisms of Raf-1 and of B-Raf. Recently, it was
reported that the H-Ras
Volume 271,
Number 9,
Issue of March 1, 1996 pp. 4671-4678
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
) comparable to that of Raf-1 and that
the binding is not significantly affected by the post-translational
modifications. However, when the activity of B-Raf to stimulate MAP
kinase was measured by using a cell-free system derived from rat brain
cytosol, we observed that the unmodified form of H-Ras possesses an
almost negligible activity to activate B-Raf in vitro compared
to the fully modified form. H-Ras
mutant, which
was farnesylated but not palmitoylated, was equally active as the fully
modified form. These results indicate that the post-translational
modifications, especially farnesylation, are required for H-Ras to
activate B-Raf even though they have no apparent effect on the binding
properties of H-Ras to B-Raf.
)kinase
cascade (for reviews, see Refs. 1 and 2). Recent studies demonstrated
that Ras makes a direct association with a serine/threonine kinase
Raf-1, a product of the c-raf-1 proto-oncogene(3, 4, 5, 6, 7, 8) and that
this association leads to stimulation of the activity of Raf-1 to
phosphorylate MAP kinase kinase (MEK) (for reviews, see (1) and (2) ). However, the precise mechanism of the
Raf activation by active form of Ras remains to be clarified.
Materials
The post-translationally modified and
unmodified forms of human H-Ras protein were purified from the membrane
and cytosol fractions of Sf9 cells, respectively, which had been
infected with a recombinant baculovirus carrying the human H-ras cDNA, as described previously(24) . Two H-ras mutants encoding the proteins defective in the post-translational
modifications, H-ras and
H-ras
, were constructed by site-specific
mutagenesis using appropriate mutagenic oligonucleotides as described
elsewhere(24) . The effector mutant H-ras
cDNA was provided by Dr. S. Yokoyama (University of Tokyo, Tokyo,
Japan). Recombinant baculoviruses carrying the mutant H-ras genes were prepared as described before(24) . The
farnesylated but not palmitoylated H-Ras
and the
fully modified H-Ras
proteins were solubilized and
purified from the membrane fractions of Sf9 cells infected with
recombinant baculoviruses carrying the respective H-ras genes
similarly as described before(24) . The unprocessed
H-Ras
protein was purified from the cytosol fraction
of Sf9 cells infected with the corresponding baculovirus. A plasmid
carrying the full-length human B-raf cDNA, pSL-Braf, was
provided by Dr. W. Kolch (Institute for Clinical Molecular Biology and
Tumor Genetics, Germany). The HindIII-ApaI and HindIII-XhoI fragments of the B-raf cDNA
corresponding to amino acid 1-326 and 1-445, respectively,
were cloned into the matching cleavage sites of pMAL-cRI (New England
Biolabs Inc.) to produce pMAL-B-Raf(1-326) and
pMAL-B-Raf(1-445), respectively. The MBP-B-Raf fusion proteins
were purified from Escherichia coli harboring the
corresponding pMAL-B-Raf plasmids by affinity chromatography on amylose
resin(27) . A segment of the human c-raf-1 cDNA
corresponding to amino acid 1-206 of Raf-1 was amplified by a
polymerase chain reaction with suitable primers and cloned into
pMAL-cRI for expression as an MBP-fusion protein in E. coli.
Plasmids for expressing GST-fusion protein of MEK (GST-MEK) or
GST-fusion protein of a kinase negative mutant of ERK2 (GST-KNERK) in E. coli were obtained from Dr. A. Kikuchi (Hiroshima
University, Hiroshima, Japan). A rat cDNA encoding the full-length ERK2
was cloned from a rat brain cDNA library using a polymerase chain
reaction with suitable primers and cloned into pGEX-2T (Pharmacia
Biotech Inc.) for production of GST-fusion protein of ERK2 (GST-ERK2)
in E. coli. GST-MEK, GST-ERK2, and GST-KNERK proteins were
purified by glutathione-agarose chromatography as described
elsewhere(28) . Purified recombinant RasGAP p120 produced in E. coli was provided by Dr. S. Hattori (National Center of
Neurology and Psychiatry, Tokyo, Japan). The anti-H-Ras monoclonal
antibody F235 was purchased from Oncogene Science Inc. (Manhasset, NY).
The polyclonal antibody raised against the C terminus of B-Raf
(ASPKTPIQAGGYGAFPVH) and that raised against the C terminus of Raf-1
(CTLTTSPRLPVF) were purchased from Santa Cruz Biotech. Inc. (Santa
Cruz, CA). Myelin basic protein was purchased from Sigma.
Adenylyl Cyclase Assay
Yeast adenylyl cyclase,
overproduced in yeast TK35-1 harboring the plasmid YEP24-ADC1-CYR1, was
solubilized from the crude membrane fraction with buffer C (50 mM MES/NaOH, pH 6.2, 0.1 mM MgCl
, 0.1 mM EGTA, 1 mM PMSF, and 1 mM 2-mercaptoethanol)
containing 1.5 M NaCl as described previously(29) .
The supernatant (15 µg of protein) after centrifugation at 100,000
g for 1 h was used for each adenylyl cyclase assay.
Measurements of adenylyl cyclase activity dependent on the
GTPS-bound form of H-Ras and its inhibition by the purified
MBP-B-Raf polypeptides were carried out as described
previously(29, 30) .
Assay for GTPase Activity
The post-translationally
modified and unmodified forms of H-Ras (2 pmol) were first loaded with
[-
P]GTP by incubation in a mixture
containing 20 mM Tris/HCl, pH 7.4, 5 mM MgCl
, 10 mM EDTA, 1 mM DTT, and 1.7
µM [-
P]GTP (3,000 cpm/pmol) at
30 °C for 25 min, followed by addition of MgCl
to 15
mM. The GTPase reactions were initiated by addition of a fixed
amount of the [-
P]GTP-bound H-Ras into a
reaction mixture (100 µl) containing 20 mM Tris/HCl, pH
7.4, 10 mM MgCl
, 5 mM EDTA, 1 mM DTT, and 1 mM GTP with or without 400 pM recombinant RasGAP p120. After incubation at 25 °C for various
periods, the reaction was stopped by rapid filtration through a
nitrocellulose filter, and the GTP-hydrolyzing activity was examined by
measuring the residual amount of [-
P]GTP
retained on the filter as described previously(30) . For
measuring the inhibition of GAP activity, similar incubations were
carried out for 5 min in the presence of various amounts of
MBP-B-Raf(1-326), and the amounts of
[
-
P]GTP remaining bound to H-Ras were
measured as described above and compared with those without the
MBP-B-Raf polypeptide.
In Vitro Binding Assay
MBP-B-Raf and MBP-Raf-1
polypeptides were attached to amylose resin in the column buffer (20
mM Tris/HCl, pH 7.4, 0.2 M NaCl, 1 mM EDTA,
10 mM 2-mercaptoethanol, and 1 mM NaN
) by
continuous mixing for 2 h on ice, and the resin was washed twice with
the column buffer. Fifteen-µl aliquots of the resin with
MBP-B-Raf(1-326), MBP-B-Raf(1-445), or
MBP-Raf-1(1-206) attached were incubated with various forms of
H-Ras (10 pmol), which had been loaded with GDP
S or GTPS, for
30 min at 25 °C and for 30 min at 4 °C in a total volume of 100
µl of the Ras-binding buffer (20 mM Tris/HCl, pH 7.4, 5
mM MgCl
, 1 mM EDTA, 1 mM DTT,
and 0.1% Lubrol PX). To evaluate the binding affinity between B-Raf and
H-Ras, increasing concentrations of H-Ras were added. After washing
three times with the Ras-binding buffer, MBP-fusion proteins with the
bound H-Ras were eluted from the resin by 10 mM maltose and
separated by SDS-PAGE (12% gel). Bound H-Ras was detected by Western
immunoblotting with the anti-H-Ras monoclonal antibody F235. The upper
half of the gel was stained with Coomassie Brilliant Blue to detect the
MBP-fusion proteins.Preparation and Fractionation of the Rat Brain
Cytosol
All manipulations were carried out at 0-4 °C.
A rat (Wister, 300 g of body weight, the Center of Japan Biological
Chemistry Co. Ltd.) brain was homogenized in 7 ml of buffer A (20
mM Tris/HCl, pH 8.0, 2 mM EDTA, 1 mM DTT,
2.5 mM MgCl, 0.3 M sucrose, 10 µg/ml
leupeptin, 20 µg/ml aprotinin, and 1 mM PMSF) with a
Potter-Elvehjem homogenizer. The homogenate was centrifuged at 100,000
g for 1 h, and the resulting supernatant (20 mg of
protein) was applied to a Mono S HR5/5 column (0.5 5 cm)
(Pharmacia) equilibrated with buffer B (20 mM HEPES/NaOH, pH
6.8, 2 mM EDTA, 1 mM DTT, 2.5 mM MgCl, 10 µg/ml leupeptin, and 10 µM (p-amidinophenyl)methanesulfonyl fluoride). A linear
gradient elution was performed between 15 ml each of 0 and 1 M NaCl in buffer B, and 1-ml fractions were collected.Assay of Ras-dependent MAP Kinase Stimulation
Activity
Ras-dependent MAP kinase stimulation activity was
measured by phosphorylation of myelin basic protein in the presence of
GTPS-bound or GDP-bound H-Ras. The sample (15 µl) to be tested
was preincubated for 10 min at 30 °C in a final volume of 50 µl
containing 20 mM Tris/HCl, pH 8.0, 6 mM HEPES/NaOH,
pH 6.8, 1.5 mM EGTA, 1 mM EDTA, 1 mM DTT, 5
mM MgCl
, 120 µM ATP, 80 nM GST-MEK, and varying amounts of GTPS- or GDP-bound H-Ras,
followed by addition of 10 µl of 3 µM recombinant
GST-ERK2 and another incubation for 10 min. The final phosphorylation
reaction was initiated by addition of 20 µl of reaction mixture
containing 20 mM Tris/HCl, pH 8.0, 1.5 mM EGTA, 1
mM EDTA, 1 mM DTT, 5 mM MgCl
, 50
µM [-
P]ATP (1,000 cpm/pmol),
and 50 µM myelin basic protein. After 20-min incubation at
30 °C, a 30-µl aliquot of the reaction mixture was spotted onto
a piece of phosphocellulose paper (Whatman P81). After washing with 75
mM phosphoric acid, the radioactivity on the paper was
measured by liquid scintillation spectrometry. To measure Ras-dependent
phosphorylation of ERK2, the assay was performed similarly except that
the reaction mixture contained purified GST-KNERK in place of GST-ERK2
and myelin basic protein and that GST-KNERK was absorbed onto
glutathione-agarose resin after the final phosphorylation reaction. The
resin was briefly washed with 20 mM Tris/HCl, pH 8.0, 1 mM EDTA, 1 mM DTT, and 5 mM MgCl
, and
subjected to SDS-PAGE (10% gel) and autoradiography.Immunodepletion Study
A 150-µl aliquot of the
active fraction (fraction 42) of the Mono S column chromatography was
incubated with continuous mixing for 1 h at 4 °C with 30 µl of
protein A-Sepharose resin alone or of the resin to which 1.5 µg of
either the anti-Raf-1 polyclonal antibody or the anti-B-Raf polyclonal
antibody was attached. After a brief centrifugation, 15 µl of the
supernatant were assayed for the Ras-dependent MAP kinase stimulation
activity as described above.
Measurement of Direct Association between B-Raf and
H-Ras by Adenylyl Cyclase Inhibition Assay
Although a previous
study showed that B-Raf forms a complex with Ras and exists in an
activated state in the complex(14) , their direct association
has not been rigorously demonstrated. We examined whether B-Raf could
bind directly to H-Ras protein and thereby compete for it with yeast
adenylyl cyclase in vitro as observed for Raf-1 and Schizosaccharomyces pombe Byr2
proteins(29, 30) . Because production of the
full-length B-Raf protein in E. coli turned out to be
impossible, we chose to express its N-terminal segments of 326 and 445
amino acid residues as MBP-fusion proteins. B-Raf(1-326)
contained CR1 only, while B-Raf(1-445) encompassed both CR1 and
CR2. Because the Ras-binding activity of Raf-1 had been shown to reside
solely in the CR1(6, 31) , we expected that the
N-terminal segments represent the Ras-binding activity of B-Raf. As
shown in Fig. 1A, both the purified
MBP-B-Raf(1-326) and MBP-B-Raf(1-445) polypeptides
efficiently inhibited H-Ras-dependent adenylyl cyclase activity in a
dose-dependent manner when added to the assay mixture containing
Mg, the GTP
S-bound form of the
post-translationally modified H-Ras, and crude extract of yeast
TK35-1 cells harboring YEP24-ADC1-CYR1. No inhibition was
observed when MBP only was added to the reaction mixture instead of
MBP-B-Raf or when MBP-B-Raf(1-445) was added to a reaction
mixture for measuring adenylyl cyclase activity dependent on
Mn
instead of Mg
and H-Ras (Fig. 1A). Dissociation constants (K
) of Raf-1 and Byr2 for Ras had been determined
in solution by kinetic analyses of their inhibition patterns of
Ras-dependent adenylyl cyclase activity(29, 30) . We
employed this method to estimate the K value of
B-Raf N terminus for H-Ras. Adenylyl cyclase activities dependent on
various concentrations of H-Ras were measured with the addition of
varying concentrations of the purified MBP-B-Raf(1-326) (data not
shown) or MBP-B-Raf(1-445) to the reaction mixtures (Fig. 1B). At each point of H-Ras concentration in the
presence of the competitor, we obtained free H-Ras concentration
available for adenylyl cyclase activation as that required for giving
the same adenylyl cyclase activity in the absence of the competitor. A
difference between the original and the free concentrations of H-Ras
was regarded as that bound to the competitor, and a reciprocal of this
value was plotted against a reciprocal of the free H-Ras concentration (Fig. 1C). This gave a series of straight lines for
each value of the competitor, which converged on the horizontal axis.
The data indicated that the competitor polypeptide bound directly to
H-Ras protein and competitively sequestered it from adenylyl cyclase.
The K values of B-Raf(1-326) and
B-Raf(1-445) for H-Ras were calculated from the points of
intersection with the horizontal axis and determined to be about 7
nM (data not shown) and 5 nM (Fig. 1C), respectively. These values are
comparable to those of Raf-1 for H-Ras, 3.5 nM, of yeast
adenylyl cyclase for yeast Ras2, 7 nM, or of Byr2 for Ras2, 1
nM (see (29) for the data and a detailed description
on the kinetic analysis).
S-bound form of H-Ras with the addition of various
amounts of MBP-B-Raf(1-326) (
), MBP-B-Raf(1-445)
(
), and MBP (
). Essentially similar inhibition assay by
MBP-B-Raf(1-445) was carried out in the presence of 2.5 mM Mn instead of Mg
and H-Ras
(
). Values on the vertical axis represent percentages of the
activities obtained in the presence of the MBP-fusion proteins compared
with those obtained in their absence. B, adenylyl cyclase
activities dependent on various concentrations of H-Ras were measured
in the presence of various amounts of MBP-B-Raf(1-445) as
follows: 0 (), 2 (), 4 (
), and 8 pmol (
). One
unit of activity is defined as 1 pmol of cAMP formed in 1 min of
incubation with 1 mg of protein at 30 °C under standard assay
conditions. C, double-reciprocal plot analysis of the binding
reaction between MBP-B-Raf(1-445) and H-Ras. The amounts of free
and B-Raf-bound H-Ras were calculated as described in the text. The
symbols correspond to those used in B.
Measurement of B-Raf Association with H-Ras by Inhibition
of RasGAP Activity
The affinity of B-Raf could not be determined
for the post-translationally unmodified form of H-Ras by the adenylyl
cyclase inhibition assay because the unmodified form of H-Ras possesses
a negligible activity to stimulate yeast adenylyl cyclase(32) .
Therefore, we used a method based on inhibition of the
GTPase-stimulating activity of RasGAP p120, whose interaction with
H-Ras had been shown to be unaffected by the
modifications(32) . We purified the unmodified form of H-Ras
from the cytosol fraction of Sf9 cells infected with the baculovirus
carrying the H-ras cDNA. The purified protein migrated slower
than the fully modified form upon SDS-PAGE (see Fig. 3A). Previous studies employing
[H]mevalonate labeling confirmed that H-Ras
purified from the cytosol fraction of Sf9 cells was not
farnesylated(33) . We also characterized the proteins by
reversed phase chromatography, and found that H-Ras from the cytosol
fraction eluted earlier than the modified form at the position expected
for the unmodified form from the previous report (34) (data not
shown). In the absence of RasGAP p120,
[-
P]GTP bound to Ras decreased at the same
rate in the unmodified and modified forms of H-Ras. Also, no difference
was observed in GTP-hydrolyzing activity stimulated by RasGAP p120 (Fig. 2A). H-Ras interacts directly with RasGAP p120 at
its effector region(35) , and the interaction is subject to
competitive inhibition by the Ras effectors, Raf-1 and
Byr2(4, 5, 30) . We examined whether B-Raf
N-terminal polypeptides interfered with the activity of RasGAP to
stimulate GTP-hydrolysis of H-Ras. As shown in Fig. 2B,
we observed a dose-dependent inhibition of the RasGAP activity by
MBP-B-Raf(1-326), and the extent of inhibition was not
significantly affected by the post-translational modifications of
H-Ras. The result suggested that the B-Raf N-terminal polypeptide has
an almost equal activity to bind the unmodified and modified forms of
H-Ras and thereby competitively sequester them from RasGAP p120.
(lanes 5 and 10), and the unmodified form of H-Ras (lanes 3, 4, 8, and 9) (10 pmol each) were loaded with
GTP
S (T) or GDP
S (D), and incubated with
MBP-B-Raf(1-326) (0.2 µg) or MBP-Raf-1(1-206) (0.5
µg) immobilized on amylose resin as described under
``Experimental Procedures.'' MBP-fusion proteins with the
bound H-Ras were eluted by 10 mM maltose and separated by
SDS-PAGE (12% gel). MBP-B-Raf(1-326) and MBP-Raf-1(1-206)
were detected by staining with Coomassie Brilliant Blue (shown by the arrows in the upper panel). H-Ras proteins were
detected by immunoblotting with the anti-H-Ras monoclonal antibody F235 (middle panel). Similarly, 0.1 aliquot of H-Ras put into each
of the binding reactions was detected by immunoblotting with the
anti-H-Ras antibody. B, various concentrations; 25 nM (lanes 1 and 5), 50 nM (lanes 2 and 6), 100 nM (lanes 3 and 7), and 200 nM (lanes 4 and 8), of
the post-translationally modified (lanes 1-5) and
unmodified (lanes 6-10) forms of H-Ras were loaded with
GTPS, and incubated with the fixed amount (0.2 µg) of
immobilized MBP-B-Raf(1-326). MBP-B-Raf(1-326) and the
bound H-Ras was detected as described in A. The result shown
is a representative of three independent experiments, which gave
equivalent results.
-
P]GTP were incubated in
the presence or absence of 40 fmol of RasGAP p120 for the indicated
periods, and the radioactivities remaining bound to H-Ras were measured
as described under ``Experimental Procedures.'' The modified
H-Ras incubated with (
) or without () RasGAP. The unmodified
H-Ras incubated with () or without (
) RasGAP. B,
increasing concentrations of MBP-B-Raf(1-326) were added to the
RasGAP assay reaction mixture containing the modified () or
unmodified () form of H-Ras. Percentages of inhibition of RasGAP
activity are plotted against the added MBP-B-Raf(1-326)
concentrations.
In Vitro Binding of B-Raf N Terminus to the Modified and
Unmodified Forms of H-Ras
The post-translationally modified and
unmodified forms of H-Ras were loaded with GTPS or GDP
S, and
examined for binding to MBP-B-Raf(1-326) and
MBP-Raf-1(1-206), which had been immobilized on amylose resin as
described under ``Experimental Procedures.'' Both of the
MBP-fusion proteins bound efficiently to the GTPS form but not to
the GDP
S form of both post-translationally modified and unmodified
forms of H-Ras (Fig. 3A). The binding was abolished by
a substitution of asparagine for aspartic acid at position 38 in the
effector loop of H-Ras. This mutation had been shown to abolish the
Raf-1 binding(4) . We next examined whether the
post-translational modifications affected the binding affinity of H-Ras
to B-Raf (Fig. 3B). When increasing concentrations of
the modified and unmodified forms of H-Ras were used for binding
reactions with MBP-B-Raf(1-326), the amounts of bound H-Ras
increased and finally reached a plateau level. No difference in the
binding pattern was observed between the modified and unmodified forms (Fig. 3B). From the data, we estimated an apparent K value of B-Raf to both forms of H-Ras as the
concentration of H-Ras exhibiting a half maximal binding, and obtained
a value of around 50 nM. This is a bit higher than that
obtained by the adenylyl cyclase inhibition assay. These results
indicated that H-Ras binds to the B-Raf N terminus in a GTP-dependent
manner presumably at its effector domain and that the
post-translational modifications of H-Ras have no effect on the
binding.Establishment of a Cell-free System for Ras-dependent
B-Raf Activation
It was shown that activation of the MAP kinase
cascade by Ras protein is mediated by B-Raf, not by Raf-1, in a crude
extract from rat brain(14) . We established here a cell-free
system derived from rat brain cytosol in which exogenously added H-Ras
could activate protein kinases MEK and ERK2 in vitro. The
cytosolic extract (20 mg of protein) was applied to a Mono S HR5/5
column equilibrated with buffer B, and washed with 20 ml of the same
buffer. Subsequently a linear gradient elution was performed between 0
and 1 M NaCl in a total volume of 30 ml. One-ml fractions were
collected and assayed for stimulation of the activity of MAP kinase to
phosphorylate myelin basic protein in the presence and absence of the
GTPS-bound H-Ras as described under ``Experimental
Procedures.'' The MAP kinase activity stimulated by H-Ras was
eluted as a single peak around fractions 41-44 (Fig. 4A). The peak fraction 42 was used for subsequent
studies. The Ras-dependent activation was not observed by either the
GDP-bound H-Ras or the GTP
S-bound H-Ras
(Fig. 4B). The Ras-dependent phosphorylation of
myelin basic protein also depended on the inclusion of recombinant
GST-ERK2 in the reaction mixture (Fig. 4B), indicating
that H-Ras induced the enhanced phosphorylation of myelin basic protein
through the activation of ERK2. When GST-fusion protein of a kinase
negative mutant of ERK2, GST-KNERK, was used as a substrate, the same
fraction exhibited H-Ras-dependent stimulation of its phosphorylation,
and this activity was dependent on the presence of recombinant MEK (Fig. 4C). Both the Ras-dependent phosphorylations of
myelin basic protein and GST-KNERK were efficiently inhibited by the
addition of MBP-B-Raf(1-326) (Fig. 4, B and C). This may be caused by competitive sequestration of Ras by
the B-Raf N-terminal polypeptide from the endogenous Ras effector
molecule. B-Raf, the major Raf family protein in brain, was detected in
the peak fraction of the Ras-dependent MAP kinase stimulation activity
when examined by immunoblotting with the anti-B-Raf antibody (Fig. 4D). Next, we performed an immunodepletion study
to examine whether B-Raf is responsible for the Ras-dependent
stimulation of MAP kinase activity. The Ras-dependent activity was
almost completely depleted by incubation with the anti-B-Raf antibody,
but not with the anti-Raf-1 antibody (Fig. 4E). Thus,
we concluded that H-Ras stimulated the MEK-MAP kinase pathway via
activation of B-Raf in this soluble cell-free system.
S-bound H-Ras (
) or GDP-bound H-Ras () as
described under ``Experimental Procedures.'' B, a
15-µl aliquot of the fraction 42 was assayed for the
phosphorylation activity of myelin basic protein as described under
``Experimental Procedures'' with the addition or omission of
following ingredients; 2 pmol each GDP- or GTPS-bound H-Ras (columns 1 and 2), 2 pmol each GDP- or
GTP
S-bound H-Ras
(columns 3 and 4), 2 pmol each GDP- or GTP
S-bound H-Ras with omission of
GST-ERK2 (columns 5 and 6), and 2 pmol each GDP- or
GTP
S-bound H-Ras with the addition of 5 pmol (columns 7 and 8) or 20 pmol (columns 9 and 10) of
MBP-B-Raf(1-326). C, a 15-µl aliquot of the fraction
42 was assayed for phosphorylation of GST-KNERK as described under
``Experimental Procedures'' without H-Ras (lane 1),
or with the addition of 2 pmol each of GDP-bound (lane 2) or
GTP
S-bound H-Ras (lanes 3-5) except that
recombinant MEK was omitted in lane 4 and that 20 pmol of
MBP-B-Raf(1-326) was added in lane 5. The arrowhead indicates the position of phosphorylated GST-KNERK. D,
the rat brain cytosol (3.5 µg of protein) (lane 1) and the
fraction 42 (1.5 µg of protein) (lane 2) were separated by
SDS-PAGE (10% gel), and immunoblotted with the anti-B-Raf antibody. The arrowhead indicates the position of the 95-kDa B-Raf. E, the fraction 42 was preincubated with protein A-Sepharose
alone (columns 1 and 2) or that attached with 1.5
µg each of the anti-Raf-1 antibody (column 3) or
anti-B-Raf antibody (column 4). After a brief centrifugation,
15 µl of the supernatant were assayed for phosphorylation of myelin
basic protein in the presence of 2 pmol each of GDP- or GTP
S-bound
H-Ras as described under ``Experimental Procedures'' except
that 20 pmol of MBP-B-Raf(1-326) were added to the reaction
mixture in column 2. Ras-dependent stimulation of the
phosphorylation was calculated by subtracting the radioactivity
incorporated into myelin basic protein in the presence of GDP-bound
H-Ras from that in the presence of GTP
S-bound H-Ras. The values
were presented as percentages of the activities obtained under
preincubation with protein A-Sepharose only (column
1).
Effect of Post-translational Modifications of H-Ras on
Its Ability to Stimulate B-Raf Activity in Vitro
We used the in vitro cell-free system for examination of the effect of the
post-translational modifications of H-Ras on its ability to activate
B-Raf. The GTPS-bound forms of post-translationally modified and
unmodified H-Ras were added to the reaction mixture containing the Mono
S peak fraction for measuring stimulation of MAP kinase activity. As
shown in Fig. 5, the modified H-Ras efficiently activated
phosphorylation of myelin basic protein. A half maximal activation was
observed at the H-Ras concentration of about 2 nM, which was
in good coincidence with the K
value of B-Raf N
terminus for the modified H-Ras determined by the adenylyl cyclase
inhibition assay. On the other hand, the post-translationally
unmodified H-Ras exhibited an almost undetectable activity of
stimulating the phosphorylation compared to the modified form.
) or unmodified () form of H-Ras as described under
``Experimental Procedures.'' The results were expressed as
the radioactivity incorporated into myelin basic protein obtained in
the presence of GTPS-bound H-Ras subtracted by that in the
presence of the same concentration of GDP-bound
H-Ras.
, which was farnesylated but lacked the two
cysteine residues to be palmitoylated, and H-Ras
,
which lacked the cysteine residue to be farnesylated and, therefore,
was not modified at all. The activities of these mutants to stimulate
B-Raf were examined similarly by using the in vitro system. As
shown in Fig. 6A, H-Ras
activated
phosphorylation of myelin basic protein as efficiently as the fully
modified form of H-Ras at the concentration of 5 nM. In
contrast, H-Ras
had an almost negligible activity at
the same concentration (Fig. 1A) or even at 20 nM (data not shown). Essentially similar result was obtained when the
activities of H-Ras
and H-Ras
to stimulate S. cerevisiae adenylyl cyclase were
examined (Fig. 6B). This result is consistent with our
previous observation that farnesylation, not palmitoylation, of yeast
Ras2 is essential for its ability to activate adenylyl cyclase in
vitro(24) . These results indicated that the
post-translational modifications of H-Ras, especially the farnesylation
step, are critical for the activation of B-Raf as well as of yeast
adenylyl cyclase.
, and
H-Ras
. The results were shown similarly as
described in Fig. 5. B, adenylyl cyclase activity was
measured in the presence of 10 pmol of the various forms of H-Ras as
described in A.
, suggesting that B-Raf binds to the
effector domain of Ras. Further, we have determined the K
value of B-Raf N terminus for the
post-translationally modified H-Ras by the yeast adenylyl cyclase
inhibition assay. The value is comparable to those of other
Ras-effector molecules for their homologous Ras proteins. There exists
little difference in the estimated K values of
B-Raf segments containing CR1 only and both CR1 and CR2, suggesting
that CR1 contains a major Ras-binding site(s) as observed for
Raf-1(6, 31) . which is farnesylated but not
palmitoylated, and H-Ras
which is neither
farnesylated nor palmitoylated. The result clearly indicated that
farnesylation but not palmitoylation is the critical step for the B-Raf
activation by H-Ras. The effect of the modifications on the ability of
H-Ras to bind to B-Raf was examined by both the RasGAP inhibition assay
and the in vitro binding assay, and the result clearly
indicated that the modifications are not required for efficient
association between H-Ras and B-Raf N terminus.
mutant has the ability to
activate the MAP kinase cascade in vivo possibly through
activation of Raf-1 as efficiently as the wild-type even though it is
localized in the cytosol but not on the plasma membrane(38) .
The unmodified mutant H-Ras
, also located in the
cytosol, failed to activate the MAP kinase cascade. This is similar to
our observations about B-Raf and adenylyl cyclase in this study and in
a previous report (24) . Another report (39) has
appeared showing that the Ras2
mutant, which is
farnesylated but not palmitoylated, is located in the cytosol and still
maintains its full biological activity, whereas the unmodified mutant
Ras2
completely lost the activity. This also suggests
that the farnesylation of Ras protein, but not its membrane targeting,
is required for yeast Ras to fulfill its function in the yeast cells.
These results plus our observations suggest that the post-translational
modifications, especially farnesylation, of Ras protein is generally
essential for its ability to activate its effectors.
)S,
guanosine 5`-O-(2-thiotriphosphate); GTPS, guanosine
5`-O-(3-thiotriphosphate); PAGE, polyacrylamide gel
electrophoresis.
We thank Dr. S. Yokoyama for H-ras cDNA, Dr. W. Kolch for pSL-Braf, Dr. A. Kikuchi for E. coli expressing GST-MEK and GST-KNERK, and Dr. S. Hattori for purified
recombinant RasGAP p120. We also thank A. Seki and Y. Kawabe for help
in preparation of this manuscript.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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