(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) 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.
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 ()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.
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 GTPS-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.
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 (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
GTP
S, 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.
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 [-
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.
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 GTPS-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 GTP
S-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).
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
() 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 GTP
S-bound H-Ras subtracted by that in the
presence of the same concentration of GDP-bound
H-Ras.
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, 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.
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, 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.
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, 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) .
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 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.
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 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.