(Received for publication, October 17, 1996)
From the Laboratory of Cellular Oncology, National
Cancer Institute, Bethesda, Maryland 20892-4040 and
§ Molecular Mechanisms of Carcinogenesis Laboratory,
NCI-Frederick Cancer Research and Development Center, ABL-Basic
Research Program, National Cancer Institute,
Frederick, Maryland 21702-1201
The Raf-1 serine/threonine protein kinase plays a central role in many of the mitogenic signaling pathways regulating cell growth and differentiation. The regulation of Raf-1 is complex, and involves protein-protein interactions as well as changes in the phosphorylation state of Raf-1 that are accompanied by alterations in its electrophoretic mobility. We have previously shown that a 33-kDa COOH-terminal, kinase-inactive fragment of Raf-1 underwent a mobility shift in response to the stimulation of cells with serum or phorbol esters. Here we demonstrate that treatment of NIH 3T3 cells or Sf9 cells with hydrogen peroxide (H2O2) also induces the mobility shift of the kinase-inactive Raf-1 fragment. A series of deletion mutants of the Raf-1 COOH terminus were analyzed, and the region required for the mobility shift was localized to a 78-amino acid fragment (residues 566-643). Metabolic labeling revealed that the slower migrating forms of the 33-kDa and of the smaller fragment contained phosphorus. Mutation of a previously characterized phosphorylation site, serine 621, to alanine prevented the mobility shift as well as phosphate incorporation or Src and Ras-dependent kinase activation in Sf9 cells when this mutation was engineered into the full-length Raf-1. Mutation of 621 to aspartate yielded a protein that existed in both the shifted and unshifted forms, demonstrating that a negative charge at 621 was necessary, but not sufficient, for the mobility shift to occur; however, its full-length form was still resistant to activation in the Sf9 system. Additional mutation of nearby serine 624 to alanine blocked the shift, implicating this residue as the site of the second of a two-step modification process leading to the slower migrating form. Co-expression of the 33-kDa fragment with an activated form of mitogen-activated protein kinase kinase in NIH 3T3 led to the appearance of the shifted form in a serum-independent manner. These results demonstrate that a mitogen-activated protein kinase kinase-induced event involving modification of serines 621 and 624 leads to the mobility shift of Raf-1.
Raf-1, the product of the c-raf-1 protooncogene, is a cytoplasmic serine/threonine protein kinase that is activated in response to various mitogenic signals (1, 2). Activation of Raf-1 results in a cascade of events involving the phosphorylation and subsequent activation of additional proteins, including mitogen-activated protein (MAP)1 kinase kinase (MEK) and MAP kinase, which ultimately lead to transcriptional activation and mitogenesis (3, 4). Thus the Raf-1 protein kinase plays a central role in cellular signal transduction and is a key player in transmitting signals from the plasma membrane to the nucleus (5, 6).
The amino terminus of Raf-1 serves as a regulatory domain, while the catalytic domain resides in the carboxyl terminus, which also may contain regulatory elements (7). Earlier studies showed that the activation of Raf-1 by membrane-associated, protein-tyrosine kinases was dependent on cellular Ras activity (8-10). Subsequently, it was found that Raf-1 binds directly to Ras, and the Ras-binding site was localized to the amino terminus of Raf-1 (11-14). The interaction between Raf-1 and Ras results in the re-localization of Raf-1 to the plasma membrane (15-17), where Raf-1 is converted to a catalytically active form through a still unidentified process. Subsequently, activated Raf-1 kinase activates MEK (18-21), which in turn activates MAP kinase (22). Activated MAP kinase phosphorylates both cytosolic and nuclear proteins (23), and plays a role in subsequent events, which lead to immediate early gene expression (24).
It has been demonstrated previously that Raf-1 exhibits an electrophoretic mobility shift that occurs after mitogenic stimulation of cells and that this event correlates with a change in the state of Raf-1 phosphorylation (25-28). Additionally, we have shown that the region of Raf-1 responsible for the serum and phorbol ester-induced shift in gel mobility is localized to a catalytically inactive COOH-terminal fragment of Raf-1 (29). Since the amino-terminal domain of Raf-1 is responsible for direct binding to Ras (14), these results suggested that the modification of Raf-1 can take place in the absence of direct interaction with Ras.
To determine the site and nature of the modification(s) of Raf-1 that accompany the shift in electrophoretic mobility, additional deletion mutants of the carboxyl terminus and point mutants within this region were created and analyzed. From these experiments, the site of modification(s) was further localized to a region of 78 amino acids, within which was a known serine phosphorylation site (Ser-621). Therefore, we analyzed the role of this serine, as well as a neighboring serine (Ser-624), in the modification of Raf-1, through the creation of site-directed mutants of these residues. Metabolic labeling and immunoprecipitation, as well as immunoblotting of the proteins encoded by these mutants, revealed that the altered electrophoretic mobility of the Raf-1 COOH-terminal fragments occurs following a two-step process that involves the addition of phosphate to two neighboring serine residues.
Deletion mutants of Raf-1 were constructed to
include a COOH-terminal epitope-tagging sequence encoding the last 12 amino acids of the protein kinase C epsilon () gene to provide rapid detection of the expressed protein. Deletion mutants were obtained by
polymerase chain reaction amplification with the Pfu DNA
polymerase (Stratagene), using a COOH-terminal
-tagged fragment of
Raf-1 (RIII-
) encoding amino acids 381-643 (30), as the template. NH2-terminal sense primers containing a BamHI
restriction site were created at regular intervals originating at amino
acid 381, 412, 443, 474, 505, 536, or 566 of Raf-1. A COOH-terminal
antisense primer was generated containing the
-tag, along with
EcoRI and BamHI restriction sites. Using RIII-
as template and the COOH-terminal
-epitope tagging sequence as
primer with various NH2-terminal primers, six additional
NH2-terminally truncated epitope-tagged Raf-1 constructs
were created. Several additional COOH-terminal
-epitope tagged
constructs were generated using COOH-terminal primers, which originated
at amino acids 612, 581, or 550 along with the
-tag, and with the
NH2-terminal primer originating at amino acid 381. The
full-length Raf-1 mutant plasmid pKSRafS621A was described previously
(31). The plasmid encoding activated MEK-1 and a linked
hygromycin resistance gene was provided by James C. Stone, University
of Alberta, Edmonton (Canada) (32).
The RIII- single and double point mutants were generated by
sequential PCR amplifications (33). The point mutations other than
S621A were created using the RIII-
construct as template, and the
following sense primers (for S621D,
5
CCGGAGCGCT
GAGCCATCC3
; for S621D/S624A,
5
GCGCT
GAGCCA
TTGCATCGGGC3
) along with a
COOH-terminal antisense primer containing the
-tag and
EcoRI and BamHI restriction sites. RIII-
S621A
was generated using the pKSRaf1S621A plasmid as template, the sense
primer 5
CCGGAGCGCTGCCGA
ATCC3
, and the same antisense
primer as above. The PCR product from each of these reactions was then
used as the antisense primer to generate RIII-
mutant fragments,
which were then cloned into baculovirus expression vectors for
expression in Sf9 cells. The presence of point mutations was verified
by direct DNA sequence analysis.
NIH 3T3 cells were maintained at
37 °C in Dulbecco's modified Eagle's medium supplemented with 10%
fetal bovine serum, penicillin/streptomycin, and glutamine. The
isolation of NIH 3T3 cell lines expressing epitope-tagged Raf-1 mutants
is described elsewhere (30). To induce maximal expression of the
recombinant proteins, the transfected cells were changed to serum-free
medium supplemented with 20 µM zinc acetate (to
up-regulate expression from the metallothionine promoter-linked genes)
for 48 h prior to stimulation with 10% serum, 100 nM
PMA, or 500 µM H2O2. Stable
transfectants expressing both RIII- and activated MEK-1
were isolated following cotransfection of cells with the expression
plasmids and selection in neomycin and hygromycin. The Sf9 insect cell
line was maintained in spinner flasks in Grace's supplemented medium
containing 10% fetal bovine serum and 0.1% pluronic F-68 (Life
Technologies, Inc.). To express the Raf-1 deletion mutants, Sf9 cells
were co-transfected with the baculovirus transfer vector
pEVMOD-Raf-
-tag and BaculoGold baculovirus DNA (Pharmingen) using
the Lipofectin reagent (Life Technologies, Inc.). Recombinant
baculoviruses encoding the various Raf-1 proteins were isolated and
enriched according to the manufacturer's protocol (Pharmingen). For
Raf-1 point mutants, the Bac-To-Bac Baculovirus expression system for
Sf9 cells was used (Life Technologies, Inc.). For experimental
purposes, Sf9 cells (2.5 × 106) were infected with
virus and harvested after 48 h.
For the
experiments shown in Figs. 1 and 2, cells were washed twice with
ice-cold phosphate-buffered saline, harvested by resuspension of the
cell pellet in buffer A (20 mM Tris, pH 7.5, 2 mM EDTA, 2 mM EGTA, containing 1 mM
phenylmethylsulfonyl fluoride, 20 µg/ml aprotinin, 10 µg/ml
leupeptin, and 5 mM sodium orthovanadate), and disrupted by
Dounce homogenization (100 strokes). Lysates were adjusted to 1 × Laemmli sample buffer concentration and stored at 70 °C. For the
experiments shown in Table I and Figs. 3, 4, 5, 6, cytosolic extracts of
insect cells and mammalian cells were prepared as follows. The cells
were washed twice with ice-cold phosphate-buffered saline, harvested by
resuspension of the cell pellet in buffer B (0.05% SDS, 20 mM Tris, pH 8.0, 137 mM NaCl, 10% glycerol,
1% Nonidet P-40, 0.5% sodium deoxycholate, 2 mM EDTA,
containing 1 mM phenylmethylsulfonyl fluoride, 20 µg/ml aprotinin, 10 µg/ml leupeptin, and 5 mM sodium
orthovanadate) for insect cells and buffer C (same as B except 0.1%
SDS) for mammalian cells. Cytosolic fractions were isolated by
centrifugation of the cell homogenates at 15,000 × g
for 10 min at 4 °C. Lysates for use in immunoblotting were adjusted
to 1 × Laemmli sample buffer concentration and stored at
70 °C.
Immunoprecipitation and Immunoblot Analysis
Immunoprecipitation was performed on cytosolic extracts using
anti-PKC (5 µg/ml; Life Technologies, Inc.) and protein
A-Sepharose beads (Calbiochem). After incubation for 4-24 h at
4 °C, immunoprecipitates were washed as described previously (34)
and processed for SDS-PAGE. Immunoprecipitates were prepared for
electrophoresis by the addition of 2 × Laemmli sample buffer with
-mercaptoethanol and boiled prior to electrophoresis on an
SDS-polyacrylamide gel (12%). The proteins were transferred to
nitrocellulose, and immunoblot analysis was performed using 10 µg/ml
anti-PKC-
antibody as described previously (30).
Cell labeling experiments were performed by
first rinsing infected cells once with either methionine- or
phosphate-deficient medium. The cells then were incubated either in
methionine-deficient medium containing 2.5% dialyzed fetal calf serum
and 250 µCi/ml [35S]methionine
(Expre35S35S Label; DuPont NEN), or in
phosphate-deficient medium with 2.5% dialyzed fetal calf serum and 1 mCi/ml [32P]orthophosphate (Amersham). After 3 h of
labeling, the cells were stimulated by the addition of 500 µM hydrogen peroxide (H2O2) for
the final hour of labeling. The labeled cells were harvested and
disrupted, and immunoprecipitation of the -tagged Raf-1 proteins was
carried out as described above.
Recombinant human
-epitope-tagged Raf-1 proteins were specifically immunoprecipitated
as described above from lysates of singly infected Sf9 cells, or from
cells coexpressing Raf-1, p21ras, and activated
pp60src. The immunoprecipitates were washed three times with
lysis buffer, and once with kinase buffer (20 mM Tris, pH
7.4, 20 mM NaCl, 1 mM dithiothreitol, 10 mM MgCl2). Raf kinase activity was assayed by
the phosphorylation of kinase-negative MEK, and the autophosphorylation of Raf-1 was assayed by omitting the MEK from the reactions (35). Briefly, the washed immunoprecipitates were incubated in 40 µl of
kinase buffer containing 100 ng of kinase-negative MEK, 10 µM ATP, and 5 µCi of [
-32P]ATP at
30 °C for 30 min. The reactions were terminated by addition of
Laemmli sample buffer and boiling. The samples were separated by
SDS-PAGE, and phosphorylation of the kinase-inactive MEK was determined
by autoradiography. Alternatively, Raf-1 protein kinase activity was
assayed using a coupled assay of extracellular-regulated kinase and MAP
kinase activation (21), and the activity was determined by the
phosphorylation of myelin basic protein by the activated MAP kinase.
Briefly, Raf-1 immunoprecipitates were incubated with 2 µg of
recombinant MAP kinase (Santa Cruz Biotechnology) in reaction mixtures
(40 µl) of kinase buffer containing 200 µM ATP along
with 1 µg of recombinant active MEK (Santa Cruz Biotechnology). After
30 min at 30 °C, 10 µl of the reaction mixtures were added to 40 µl of ice-cold kinase buffer containing 100 µM
Na3VO4. Ten microliters of the diluted reaction
mixture was combined with 30 µl of kinase buffer containing 20 µg
of myelin basic protein and 5 µCi of [
-32P]ATP.
After incubation for 20 min at 30 °C, assays were terminated by the
addition of SDS-PAGE sample buffer, the samples were resolved by
SDS-PAGE, and phosphoproteins were visualized by autoradiography.
Previously, we identified a 33-kDa catalytically
inactive COOH-terminal fragment of Raf-1 (amino acids 381-643;
designated RIII-), which exhibited a Ras-independent serum- and
phorbol ester-induced shift in gel mobility that mimicked the shift
observed with full-length Raf-1 (29). Recent interest has focused on the possible role(s) of oxygen free radicals, including hydrogen peroxide (H2O2), in the regulation of various
signal transduction processes. Oxygen free radicals have been suggested
to play a role in regulating several enzymes involved in transmembrane
signaling pathways, including protein kinase C, tyrosine-specific
protein kinases, and MAPK in certain cell systems (36-38). Moreover,
in one recent report the normal responses of vascular smooth muscle cells to platelet-derived growth factor, including MAPK activation and
DNA synthesis, were shown to require H2O2
generation (39).
Since Raf-1 functions as an upstream activator in the MAPK kinase
cascade, we investigated whether Raf-1 might also be affected by
treatment of cells with H2O2. To examine the
effect of various agents on Raf-1 mobility, quiescent NIH 3T3 cells
expressing RIII- were treated with 10% serum, 100 nM
PMA, or 500 µM H2O2. Cytosolic extracts were isolated and separated by SDS-PAGE, then subjected to
immunoblot analysis (Fig. 1). In quiescent cells, only a
single electrophoretic form of the RIII-
protein was observed (Fig. 1, lane 1). In contrast, two forms of RIII-
were observed
following treatment with serum or phorbol ester, as shown previously,
or hydrogen peroxide (Fig. 1, lanes 2-4). The similar
migration rates of the upper band induced by each treatment suggested
that this form of RIII-
resulted from a similar modification(s) in
each instance.
Experiments performed using the Sf9 insect cell system to analyze the
activation of Raf-1 have revealed that co-expression of Src, Ras, or
PKC with Raf-1 led to its activation (9, 10, 20, 31). To develop a
simplified system for the analysis of Raf-1 COOH-terminal
modifications, we examined whether treatment of Sf9 cells expressing
only RIII- with H2O2 would lead to a shift
in electrophoretic mobility similar to that observed in mammalian
cells. After 48 h of infection with an RIII-
expressing baculovirus, readily detectable levels of RIII-
protein were expressed (Fig. 2). Unexpectedly, a doublet
corresponding to RIII-
was observed in the untreated cells (Fig. 2,
lane 1). The doublet is probably a result of the presence of
serum in the infected Sf9 culture. However, the more rapidly migrating
form, corresponding to the unmodified form of RIII-
, was found in
greater amount in the untreated cells. As early as 30 min after
H2O2 treatment, a greater amount of the more
slowly migrating form of Raf-1 was observed (Fig. 2, lane
2), and after 60 min, almost all of the RIII-
was found in the
modified form (Fig. 2, lane 3). Thus, a similar shift to a
more slowly migrating form of RIII-
was noted both in insect cells
and in mammalian cells following H2O2 treatment.
To
further localize the region within the Raf-1 COOH terminus required for
modification(s) to occur, which result in a shift in gel mobility, a
series of deletion mutants of the RIII- (amino acids 381-643)
protein were created and expressed in Sf9 cells. Six
NH2-terminal and three COOH-terminal deletion mutants were constructed in 31 amino acid intervals encompassing residues 381-643 of Raf-1 (Table I). After 48 h of infection with
the various virus stocks, Sf9 cells were treated with
H2O2 for 60 min to fully induce the
characteristic shift in electrophoretic mobility. All of the
NH2-terminal deletion mutants exhibited a mobility shift after treatment (Table I), including the smallest polypeptide (RIX-
), which encompasses only amino acids 566-643 of Raf-1. In
contrast, all deletions from the COOH terminus resulted in proteins
that exhibited only a single band in the absence or presence of
H2O2. Further efforts to define the minimal
fragment (RIX-
), which still exhibited a band shift, were
unsuccessful, probably as a result of instability, since no expression
of the smaller proteins was detected (data not shown). We conclude that
a region of Raf-1 comprising only amino acids 566-643 is capable of
undergoing modification(s) resulting in a shift in gel mobility upon
stimulation of cells with H2O2.
A change in the
phosphorylation state of Raf-1 after mitogenic stimulation has been
suggested as the explanation for the shift in migration rate of Raf-1
on SDS-polyacrylamide gels (26). To test this possibility, in
vivo labeling experiments were performed with Sf9 cells expressing
RIII- and RIX-
, to examine the phosphorylation state of these
COOH-terminal Raf-1 fragments. After 48 h of viral infection,
cells were labeled continuously with either
[35S]methionine or [32P]orthophosphate for
4 h, with 500 µM H2O2
treatment for the final 60 min. Immunoprecipitation analysis of
[35S]methionine-labeled cell extracts revealed that
RIII-
is expressed as a doublet under these conditions (Fig.
3, lane 2). Following 32Pi labeling, however, the major band observed
was the upper band of the RIII-
doublet (Fig. 3, lane 5).
These results link the modification responsible for the migration shift
of RIII-
to the presence of phosphate in the protein. Only a low
level of 35S label was incorporated into RIX-
, likely
due to the low number of methionine and cysteine residues present in
this fragment (Fig. 3, lane 3). However, a doublet was
visible with much longer exposures of the gel (data not shown).
Nonetheless, RIX-
did exhibit significant 32Pi incorporation (Fig. 3, lane 6),
and once again, only a single band was observed in the
32Pi-labeled lysates. These results suggested
that the change in the rate of migration of the Raf-1 COOH-terminal
fragments may be associated with a phosphorylation event occurring
between residues 566 and 643.
One site of modification
that lies within the 566-643 region is serine 621, which was
previously shown to be constitutively phosphorylated in fibroblast
cells (31). Additionally, mutation of this site to an alanine resulted
in the absence of phosphorylation of Raf-1, and these mutants exhibited
a dominant-negative phenotype (7, 40). To determine what role serine
621 might play in the mobility shift observed in RIII- upon
mitogenic stimulation, point mutations resulting in a change of this
residue to either an alanine (RIII-
S621A) or aspartate (RIII-
S621D) were constructed and expressed in Sf9 cells. Although RIX-
is
the smallest fragment that exhibits a mobility shift, the resolution
between the two forms is more evident using RIII-
. The S621A
mutation prevents phosphorylation at site 621, while the negatively
charged residue in S621D should mimic the presence of a phosphorylated
serine. As expected, mutation of serine 621 to alanine prevented
further modification of RIII-
, resulting in the presence of only the faster migrating form of the protein (Fig. 4, lane
2). Surprisingly, however, mutation of serine 621 to aspartate
resulted in the presence of a doublet (Fig. 4, lane 3),
comparable to the doublet observed for wild-type RIII-
(Fig. 4,
lanes 1 and 5). If phosphorylation of this site
alone was responsible for the induced mobility shift, we would have
expected to observe the presence of a single, slowly migrating band.
Therefore, the modification at Ser-621 appears to be necessary, but not
sufficient, for the induced mobility shift of RIII-
.
To identify a possible second site of modification, the sequences of
different Raf proteins in the region from amino acids 566-643 were
examined for the most evolutionarily conserved residues. One site,
serine 624, was of particular interest because of its conservation
between the three isoforms of Raf and its proximity to serine 621. To
examine what effect this residue might have on the mobility shift of
the Raf-1 COOH terminus, we constructed a double point mutant in the
RIII- construct, which changed serine 624 to alanine and serine 621 to aspartate (RIII-
S621D/S624A). Immunoblot analysis of cytosolic
extracts from Sf9 cells expressing this double mutant after
H2O2 treatment revealed a band pattern that
differed from the one that was observed with the single mutant (RIII-
S621D). Only the more rapidly migrating band was observed in
lysates containing the RIII-
S621D/S624A double mutant (Fig. 4,
lane 4). These data suggest that two modifications occur in the COOH terminus of Raf-1 to result in the electrophoretic mobility shift of RIII-
. Apparently, an initial modification occurs at serine
621, and this is required to allow a second modification to occur at
serine 624.
To determine whether the electrophoretic mobility
changes reflected alterations in the phosphate content of Raf-1
proteins, in vivo labeling experiments were performed using
the RIII- single point mutants, RIII-
S621A and RIII-
S621D.
Infected Sf9 cells were labeled with either
[35S]methionine or [32P]orthophosphate,
exposed to H2O2 for 60 min, then lysed, and the
lysates were analyzed as for Fig. 3. Metabolic labeling experiments with [35S]methionine showed that adequate levels of
protein were expressed for wild-type RIII-
and the two point mutants
of RIII-
(Fig. 5, lanes 1-3). A doublet
was evident for both wild-type RIII-
and RIII-
S621D, while only
a single, more rapidly migrating band was observed for RIII-
S621A
(consistent with the immunoblot analysis in Fig. 4). In contrast, when
cells were labeled with 32Pi, phosphate was
incorporated into both the faster and more slowly migrating forms of
wild-type RIII-
protein (Fig. 5, lane 4), while no
phosphate was incorporated into the RIII-
S621A protein (Fig. 5,
lane 5). Although the RIII-
S621D mutant expressed both forms when [35S]methionine-labeled lysates were analyzed
(Fig. 5, lane 3), only the upper band was
32P-labeled (Fig. 5, lane 6). This indicates
that an alteration requiring a negative charge at site 621 occurred in
the RIII-
S621D protein. This modification, which produced the
migration shift, also involves the addition of phosphate to the
RIII-
protein.
Since the activation of Raf-1 appears to involve
phosphorylation, we were interested in examining potential protein
kinases that might induce the observed mobility shift of RIII-. It
has been proposed that the shift in Raf-1 electrophoretic mobility may
result from a feedback loop following the activation of the MEK/MAPK
pathway (41-43). To examine the effect MEK-1 might have on the
migration of RIII-
, we introduced an activated form of MEK-1 (32),
which has been shown to induce focus formation in mammalian cells (44,
45), into a NIH 3T3-derived cell line expressing RIII-
. Immunoblot
analysis was performed with proteins extracted from stable
transfectants after serum deprivation for 48 h. In the absence of
exogenous activated MEK-1, only a single, faster migrating form of
RIII-
was observed in serum-deprived cells (Fig. 6,
lane 2). However, in serum-deprived transfectants expressing
activated MEK-1 (lane 3), a doublet was observed that was
comparable to the doublet observed in exponentially growing RIII-
cells (lane 1). Thus, the presence of activated MEK-1 is capable of inducing the mobility shift of RIII-
in mammalian cells.
To determine whether the Raf-1 modifications involving
residues 621 and 624 might alter the kinase activity of the native Raf-1 protein, full-length wild-type Raf-1, or mutant proteins bearing
amino acid changes at these residues were expressed in Sf9 cells, and
the kinase activity of the immunoprecipitated Raf-1 proteins was
assayed (Fig. 7). As the in vitro kinase
activity of Raf-1 has been shown to be greatly stimulated when the
protein is isolated from Sf9 cells co-expressing pp60src and
p21ras (9, 31), we analyzed the activity of Raf-1 in triply
infected Sf9 cells (Fig. 7, lanes 2-6), as well as in cells
expressing Raf-1 alone (lanes 7-11). The phosphorylation of
kinase-inactive MEK by Raf-1 (Fig. 7B), as well as Raf-1
autophosphorylation (Fig. 7A) was assayed (35). In addition,
we employed a coupled assay (21) in which Raf-1 immunoprecipitates were
incubated with wild-type MEK and wild-type MAP kinase, and the
activation of MAP kinase was determined by the phosphorylation of
myelin basic protein (Fig. 7C, MBP).
In each type of assay, the activity of wild-type Raf-1 was readily
detected when the protein was isolated from triply infected cells
(lane 2), while only a low level of activity was observed in
precipitates from cells infected singly with Raf-1 (lane 7). As previously reported (31), mutation of serine 621 to alanine (S621A)
abolished the in vitro kinase activity of Raf-1 (lanes 3 and 8). Unexpectedly, Raf-1 protein with serine 621 changed to aspartate (S621D; lanes 4 and 9) was
also defective in its kinase activity. Thus, replacement of the normal
serine with a neutral amino acid (alanine) or a negatively charged
amino acid (aspartate) at this site has a similar effect on Raf-1
activity. As the modification of serine 624 requires a negative charge
at site 621 (see above), we also analyzed the double point mutant in
which the S621D mutation was combined with a serine-to-alanine change
at amino acid 624 (S621D/S624A; lanes 5 and 6 and
lanes 10 and 11). The double mutant protein,
which in the RIII- form is unable to undergo the modifications
required to cause a shift in electrophoretic mobility, was also found
to be kinase-inactive. In all cases, the wild-type and mutant Raf-1
proteins were expressed and were present in the immunoprecipitates, as
detected by immunoblotting (Fig. 7D). We conclude that the
presence of a negative charge at amino acid 621 does not yield an
activated, or even a kinase-competent Raf-1 protein. Furthermore,
preventing the modification of serine 624 by replacing it with alanine
does not restore kinase activity to the protein in which serine 621 has
been changed to aspartate.
Several laboratories have presented data suggesting a correlation
between the phosphorylation state of Raf-1 and activation of its kinase
activity (31, 46, 47). While hyperphosphorylation also has been
suggested to play a role in the shift in electrophoretic mobility of
activated Raf-1 on SDS-polyacrylamide gels (48-50), the identity of
specific site(s) responsible for this shift in gel mobility has
remained elusive. Here we present evidence that modification of serine
624, along with phosphorylation of serine 621, is required for the
electrophoretic mobility shift of a COOH-terminal fragment (RIII-)
comprising residues 566-643 of Raf-1. The band shift noted with the
RIII-
fragment resembles the serum- and PMA-induced shift observed
with full-length Raf-1. It is likely that the same modifications we
have described here in the context of Raf-1 COOH-terminal fragments
also occur with full-length Raf-1, since this protein displays an
analogous electrophoretic mobility shift in response to treatment of
cells with the same agents.
The regulation of serine 624 appears to be complex; it is a two-step
process requiring the initial, obligatory phosphorylation of a
neighboring site (serine 621) prior to the modification of serine 624 to produce the characteristic gel mobility shift. While mutation of
serine 621 to alanine abolished the mobility shift of RIII-,
mutation of serine 621 to aspartate did allow the shift to occur,
although only a portion of the S621D protein was in the more slowly
migrating form. Mutation of serine 624 to alanine, in the context of
S621D, prevented the mobility shift. Thus, a negative charge at residue
621 is necessary for the shift to occur, but it is not sufficient to
produce the shift. In vivo phosphate labeling experiments
revealed that phosphate was incorporated into both the upper and lower
bands of the wild-type RIII-
protein, as well as into the slowly
migrating form of the S621D protein, but not into the S621A mutant.
These results suggest that a second modification, involving serine 624, is required for the mobility shift, and that this change occurs only
after the modification of serine 621.
At least three forms of Raf-1 have been observed when quiescent cells are mitogenically stimulated (31),2 suggesting successive post-translational processing of the protein. Similarly, PKC undergoes several phosphorylation events to generate a mature, catalytically active form (51-53). The first modification of PKC is a transphosphorylation event that activates the kinase, while the second event is an autophosphorylation process that appears to play a role in the subcellular localization of PKC (54). Additionally, an acidic residue must be present at the site of the first modification in order for the second event to occur (55). This requirement for a negative charge in order for a second phosphorylation to occur also has been reported for the regulation of glycogen synthase, CDK2, and MAP kinase (56-58). Similarly, based on our results, the addition of phosphate moieties on Raf-1 in a successive manner appears to regulate the electrophoretic mobility, and possibly the activity, of this protein.
Serine 621 of Raf-1 has previously been shown to be phosphorylated in quiescent as well as in platelet-derived growth factor-stimulated fibroblasts (31). The importance of this residue for Raf-1 function is suggested by the lack of kinase activity exhibited by Raf-1(S621A) in vitro and in vivo (31, 40). Here we have made the unexpected observation that substitution of serine 621 with aspartate (S621D) also yields an inactive kinase. Using a constitutively activated COOH-terminal fragment of Raf-1, other investigators have independently reached the same conclusion (59). The results suggest that either Raf-1 proteins with a negative charge (e.g. a phosphorylated serine) at this site are inactive, or that the aspartate substitution does not completely mimic the phosphorylated serine. Our finding that mutation of serine 621 to aspartate does permit the electrophoretic mobility shift to occur, while the alanine 621 mutation does not, suggests that aspartate at this residue does substitute for at least this aspect of serine 621 function.
Our results raise several questions regarding the regulation of Raf-1
function. One is the nature of the modifications themselves. While the
metabolic labeling experiments clearly demonstrated the incorporation
of phosphate into RIII- as a correlate of the mobility shift, the
phosphate group could comprise part of a more complex modification
rather than phosphorylation per se. Indeed, some
investigators have found no effect by either phosphoserine- or
phosphotyrosine-specific phosphatases on Raf-1 enzymatic activity (17,
29, 60). In contrast, more recent studies have shown that treatment of
Raf-1 with membrane-associated protein phosphatases can inactivate its
kinase activity (47). Thus, while the simplest explanation is that both
serine 621 and serine 624 undergo phosphorylation, we cannot exclude
the possibility that other modifications occur at these sites. A second
and related question is the identity of the enzymes responsible for the
modifications of the Raf-1 COOH terminus. There could be a single
enzyme that modulates both events, or there might be two distinct
enzymes involved. The occurrence of phosphorylation at serine 621 in
both quiescent and stimulated cells makes it difficult to speculate
about the identity of the kinase(s) responsible. However, the ability
of MEK-1, which has a very strict substrate specificity for MAPK (45,
61), to induce the Raf-1 shift implies that MAPK, or another kinase
activated by MAPK, may be responsible for the modification of serine
624.
A final critical issue is the role of these residues and their
modifications in the regulation of Raf-1 activity. Initially, it
appeared that the mobility shift of Raf-1 correlated with increased kinase activity of the protein, especially since agents that induced the shift (e.g. serum, PMA) also activated Raf-1 activity.
Later reports, however, suggested that Raf-1 activation might actually occur prior to the mobility shift, raising the possibility that the
mobility shift may in fact reflect a feedback mechanism to turn off
Raf-1 (42, 43). Thus, it also is possible that the more highly modified
(slower migrating) form of Raf-1 may actually be inactive. The
experiments described here do not definitively distinguish between
these possibilities. Indeed, the lack of kinase activity by
Raf-1(S621A) would seem to support the former model, while the lack of
activity of Raf-1(S621D) would seem to support the latter. However, the
fact that MEK-1 can induce the band shift in the RIII- fragment when
co-expressed in intact NIH 3T3 cells is most easily interpreted in
light of the feedback model. Further experiments are required to
determine the precise manner in which Raf-1 enzymatic activity is
affected by these modifications.
We thank William Vass for expertise with cell culture and transfections. We gratefully acknowledge the invaluable advice and technical assistance of Maureen Johnson, Richard Roden, and Sylvia Felzmann. We thank Dr. Sheldon Steiner for the support and encouragement of his laboratory.