From the Laboratory of Signal Transduction, Sloan-Kettering Institute, Memorial Sloan-Kettering Cancer Center, New York, New York 10021
Received for publication, September 5, 2000, and in revised form, November 26, 2000
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ABSTRACT |
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We recently established a two-stage in
vitro assay for KSR kinase activity in which KSR never comes in
contact with any recombinant kinase other than c-Raf-1 and defined the
epidermal growth factor (EGF) as a potent activator of KSR kinase
activity (Xing, H. R., Lozano, J., and Kolesnick, R. (2000)
J. Biol. Chem. 275, 17276-17280). That study,
however, did not address the mechanism of c-Raf-1 stimulation by
activated KSR. Here we show that phosphorylation of c-Raf-1 on
Thr269 by KSR is necessary for optimal activation in
response to EGF stimulation. In vitro, KSR specifically
phosphorylated c-Raf-1 on threonine residues during the first stage of
the two-stage kinase assay. Using purified wild-type and mutant c-Raf-1
proteins, we demonstrate that Thr269 is the major c-Raf-1
site phosphorylated by KSR in vitro and that
phosphorylation of this site is essential for c-Raf-1 activation by
KSR. KSR acts via transphosphorylation, not by increasing c-Raf-1 autophosphorylation, as kinase-inactive c-Raf-1(K375M) served as an
equally effective KSR substrate. In vivo, low physiologic doses of EGF (0.001-0.1 ng/ml) stimulated KSR activation and induced Thr269 phosphorylation and activation of c-Raf-1. Low dose
EGF did not induce serine or tyrosine phosphorylation of c-Raf-1. High
dose EGF (10-100 ng/ml) induced no additional Thr269
phosphorylation, but rather increased c-Raf-1 phosphorylation on serine
residues and Tyr340/Tyr341. A Raf-1 mutant with
valine substituted for Thr269 was unresponsive to low dose
EGF, but was serine- and
Tyr340/Tyr341-phosphorylated and partially
activated at high dose EGF. This study shows that Thr269 is
the major c-Raf-1 site phosphorylated by KSR. Furthermore, phosphorylation of this site is essential for c-Raf-1 activation by KSR
in vitro and for optimal c-Raf-1 activation in response to
physiologic EGF stimulation in vivo.
The mammalian raf-1 gene was first identified as the
normal cellular counterpart of v-raf, the transforming gene
of murine sarcoma virus (1). Two other related members of this family, A-raf and B-raf, were discovered subsequently (2,
3). Raf proteins display three conserved domains: a cysteine-rich
amino-terminal domain (CR1), a serine/threonine-rich domain (CR2), and
a C-terminal kinase domain (CR3) (1, 4-7). The Raf-1 serine/threonine
kinase is a central component in many signaling pathways, functioning as a bridge to connect upstream activated tyrosine kinases and Ras to
downstream serine/threonine kinases (8-10). Upon activation, Raf-1
phosphorylates and activates
MEK1,1 resulting in
propagation of the signal to MAPK. MAPK in turn phosphorylates several
regulatory proteins in the cytoplasm and nucleus to alter the program
of transcription and translation (11, 12).
Activation of Raf-1 in vivo is initiated when cytoplasmic
Raf-1, through the Ras-binding domain within CR1, couples to active GTP-bound Ras and is recruited to the plasma membrane. After binding Ras, c-Raf-1 becomes activated by a complex and still incompletely understood mechanism involving phosphorylation and interaction with
membrane lipids (13-18). Confirmation of an essential role for
phosphorylation in regulating Raf-1 kinase function derives from the
identification of critical in vitro and in vivo
phosphorylation sites and mutational analysis of these sites. In
resting cells, Raf-1 is phosphorylated exclusively on serine
(Ser43, Ser621, and Ser624) and
threonine (Thr268) residues. Serine and threonine
phosphorylation can be further increased at these sites and other
selected sites (Ser259,
Ser388/Ser389,
Ser497/Ser499, and Thr269) upon
stimulation with mitogenic stimuli and cytokines and is often
accompanied by tyrosine phosphorylation at positions 340 and 341 (6,
19-32). The relative contribution of these sites to Raf-1 activation
is cell type- and stimulus-specific.
KSR (kinase suppressor of Ras), a
novel member of the Ras/MAPK pathway, was originally identified and
characterized in Drosophila melanogaster and
Caenorhabditis elegans to function as a positive modulator
of Ras function either upstream of or parallel to Raf-1 (33-36). The
isolation of murine and human KSR homologs with a high level of
sequence identity (33) suggested that KSR signaling is evolutionarily
conserved. Since its discovery as a component of the Ras pathway, much
effort has been invested to elucidate the precise role of this molecule
in the regulation of the MAPK cascade. However, a confusing and often
contradictory picture of the role of this protein in signal
transduction has emerged. The presence of an Arg in kinase subdomain II
in mouse and human KSR instead of the conserved Lys normally involved
in ATP binding in mammalian kinases (33) has led to the suggestion that
mammalian KSR might not even function as a kinase. In fact, based on
the inability of a number of groups to detect a KSR kinase activity in vitro (33, 37, 38) and the capacity of KSR to associate with numerous proteins (38, 39), it has been proposed that the primary
mode of KSR signaling is via protein-protein interaction (the
scaffolding model).
In our experience, however, KSR consistently displays kinase activity
in an in vitro assay in which the entire MAPK cascade is
reconstituted with individual recombinant components (30, 40, 41). To
validate our original observations, we recently established a two-stage
in vitro assay for KSR kinase activity (41). During the
first stage, highly purified KSR is incubated with c-Raf-1 in a
reaction mixture containing ATP. In the second stage of the assay,
activated c-Raf-1 is separated from KSR and incubated with a reaction
mixture containing unactivated MEK, unactivated MAPK, and a human
GST-Elk-1 fusion protein. Elk-1 phosphorylation serves as a specific
readout for MAPK activation. Thus, KSR never comes in contact with any
recombinant kinase other than c-Raf-1 in this assay. Using this
approach, we demonstrated that KSR, purified to homogeneity, retained
its activity to reconstitute c-Raf-1-dependent MAPK
activation in vitro. Furthermore, functional integrity of
the KSR kinase domain appeared necessary since substitution of
conserved aspartates involved in phospho-transfer with alanines to
generate a kinase-inactive KSR (D683A/D700A or Ki-KSR) abrogated MAPK
signaling. Critically, endogenous human KSR from A431 cells was capable
of signaling c-Raf-1-dependent activation of the MAPK pathway in vitro, indicating that KSR kinase activity is not
an artifact of overexpression, but a property intrinsic to this
protein. Moreover, we defined EGF as a potent activator of KSR kinase
activity. This study, however, did not address the mechanisms of
c-Raf-1 stimulation by activated KSR.
Our prior study provided experimental evidence for KSR phosphorylation
of Thr269, located within the CR2 domain of c-Raf-1, as a
mechanism for c-Raf-1 activation in response to tumor necrosis
factor- Cell Culture and EGF Treatment--
COS-7 cells (American Type
Culture Collection) were maintained in high glucose Dulbecco's
modified Eagle's medium supplemented with 10% fetal bovine serum
(Life Technologies, Inc.), penicillin, and streptomycin at 37 °C in
5% CO2 (41). For EGF stimulation, cells were placed in
serum-free medium for 12 h prior to the treatment.
Expression of KSR and Raf-1 in COS-7 Cells--
Mouse
pcDNA-FLAG-KSR, pcDNA-FLAG-Ki-KSR(D683A/D700A), human
pcDNA-FLAG-c-Raf-1, and the c-Raf-1 mutant pcDNA-FLAG-TV-Raf
substituted with a valine residue at position 269 were generated as
previously described (30). The pUSEamp-FLAG-c-Raf-1(K375M) construct
was purchased from Upstate Biotechnology, Inc. (catalog no. 21-165). pCMV-Myc-KSR and pCMV-Myc-Ki-KSR were generated by subcloning from
pcDNA3 (30) into pCMV-Myc (Invitrogen) using the sites flanking
BamHI and XhoI and sequenced.
For EGF studies, COS-7 cells were plated at a density of 1.5 × 106 cells in 150-mm plates (Corning Inc.) and grown
overnight to ~70% confluence. Culture medium was replenished with
fresh medium 1 h before transfection, and cells were transfected
with 5 µg of DNA/plate using FuGeneTM 6 transfection
reagent (Roche Molecular Biochemicals) according to the manufacturer's
instructions. At 48 h post-transfection, cells were placed in
serum-free medium for 12 h prior to treatment with EGF (0.001-100
ng/ml; Upstate Biotechnology, Inc.) for 3 min. Cells were then
harvested in Nonidet P-40 lysis buffer (25 mM Tris (pH
7.5), 137 mM NaCl, 10% glycerol, 1% Nonidet P-40, 2 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin/soybean trypsin inhibitor, and 5 mM
NaVO4). The homogenate was centrifuged at 10,000 × g for 5 min at 4 °C, the supernatant collected and precleared with protein A/G-agarose (Amersham Pharmacia Biotech), and
protein content was measured using BCA Reagent A (Pierce). Lysates were
divided into aliquots and stored at Immunoprecipitation of KSR and c-Raf-1--
FLAG-tagged proteins
were immunoprecipitated from 500-µg (or as otherwise indicated) COS-7
lysates using 60 µl of agarose-conjugated mouse anti-FLAG Ab M2
(Sigma, catalog no. 1205) as previously described (41). KSR or Raf-1
immune complexes were collected by centrifugation and washed five times
with Nonidet P-40 lysis buffer containing 1.0 M NaCl to
remove contaminating proteins (see below). The beads were subsequently
washed once with reaction buffer (100 mM Tris (pH 7.5), 25 mM Immunoaffinity Purification of Human FLAG-c-Raf-1, FLAG-TV-Raf,
and FLAG-c-Raf-1(K375M)--
Lysates were generated from four 150-mm
plates of COS-7 cells transfected with FLAG-c-Raf-1, FLAG-TV-Raf or
FLAG-c-Raf-1(K375M) and diluted to 1 µg/µl with Nonidet P-40 lysis
buffer. Raf-1 proteins were immunoprecipitated with 1 ml of
agarose-conjugated anti-FLAG Ab M2 at 4 °C for 3 h. c-Raf-1
immune complexes were collected by centrifugation and washed five times
with Nonidet P-40 lysis buffer containing 1.0 M NaCl and
once with ice-cold phosphate-buffered saline. The beads were then
resuspended in 5 ml of phosphate-buffered saline and transferred to an
equilibrated Poly-Prep chromatography column (Bio-Rad), and the gel bed
was washed twice with 5 ml of phosphate-buffered saline. FLAG-Raf
proteins were competitively displaced with 5 volumes of 0.5 ml of
phosphate-buffered saline containing 100 µg/ml FLAG octapeptide
(Sigma). 5 µl of each fraction was resolved by 10% SDS-PAGE, and
proteins were silver-stained using the Bio-Rad silver staining kit. The
identity of eluted proteins was determined by Western blot analysis
using a rabbit anti-human c-Raf-1 Ab (Upstate Biotechnology, Inc.,
catalog no. 06-901) and mouse anti-FLAG Ab M2 (Sigma, catalog no.
F-3165). The Raf-1-containing fractions were pooled, concentrated with Microcon 3 columns (Amicon, Inc.), dialyzed overnight with storage buffer (50 mM Tris-HCl (pH 7.5), 0.1 mM EGTA,
0.1 mM EDTA, 0.1% 2- Two-stage KSR Activity Assay and Raf Activity Assay--
KSR
kinase activity was measured by the two-stage assay as previously
described (41). Briefly, in the first stage of the assay,
immunopurified KSR was incubated at 30 °C in 15 µl of reaction buffer containing 60 µM ATP, 7.5 mM
MgCl2, and the indicated amounts (10 and 100 ng) of either
human His-c-Raf-1 coexpressed with Ras and Lck in Sf9 insect
cells (Upstate Biotechnology, Inc.) or FLAG-c-Raf-1 or FLAG-TV-Raf
prepared as described above. After 10 min, KSR-containing beads were
pelleted (14,000 × g) for 3 min at 4 °C, and 10 µl of supernatant containing activated c-Raf-1 was added in the
second stage of the assay to 20 µl of reaction buffer containing 60 µM ATP, 7.5 mM MgCl2, 0.1 µg of
unactivated murine GST-MEK1 (Upstate Biotechnology, Inc.), 1.0 µg of
unactivated murine GST-ERK2/MAPK (Upstate Biotechnology, Inc.), and 2 µg of human GST-Elk-1 fusion protein (New England Biolabs).
After 20 min, the reaction was stopped by addition of 10 µl of
Laemmli buffer. For some studies, kinase-inactive MEK1(K97M-MKK1) was
used as substrate to assess Raf-1 activity in the second stage of the
assay. Phosphorylated Elk-1 or MEK1 was resolved by 7.5 or 10%
SDS-PAGE as indicated and visualized by Western blot analysis using
rabbit anti-phospho-Elk-1(Ser383) Ab (catalog no. 9181, BioLabs) or anti-phospho-MEK1(Ser217/Ser221)
Abs (catalog no. 9121, BioLabs), respectively. For studies examining the activity of Raf-1 directly immunoprecipitated from COS-7 cells, the
first stage of the assay was omitted, and 30 µl of reaction mixture
was used. It should be noted that a prior study showed that although
triply transfected His-c-Raf-1 is active, it is only partially
activated and can be further activated 6-8-fold by KSR (28).
Phosphorylation of c-Raf-1 by KSR in Vitro--
c-Raf-1
phosphorylation was determined by 32P labeling and by
Western blot analysis of threonine, serine, and tyrosine
phosphorylation. For radiolabeling, immunoprecipitated KSR was washed
and incubated for 30 min at 30 °C in 30 µl of reaction buffer
containing 60 µM ATP, 7.5 mM
MgCl2, 30 µCi of [ Phosphatase assay--
For these studies, 300 ng of purified
human FLAG-c-Raf-1 was treated with 4 µg of Type III potato acid
phosphatase (Sigma) in the presence of 20 µM each
aprotinin and leupeptin for 30 min prior to or after KSR
phosphorylation. Phosphatase treatment was terminated with 500 µl of
cold Nonidet P-40 lysis buffer containing 10 mM sodium
vanadate. Thereafter, FLAG-c-Raf-1 was quantitatively immunoprecipitated with 40 µl of anti-FLAG beads at 4 °C for
2 h. Dephosphorylated c-Raf-1 was then used in either the first or
second stage of the KSR activity assay as described above. For studies
employing pre-dephosphorylated c-Raf-1, KSR remained throughout both
stages of the assay, which had no discernible effect on reconstitution
of MAPK signaling.
KSR Activates c-Raf-1 in Vitro by Phosphorylation--
Our
previous investigation established a two-stage in vitro
assay for KSR kinase activity (41). During the first stage, highly
purified KSR is incubated with c-Raf-1 in a reaction mixture containing
ATP. In the second stage of the assay, activated c-Raf-1 is separated
from KSR and incubated with a reaction mixture containing unactivated
GST-MEK, unactivated GST-MAPK, and a human GST-Elk-1 fusion protein.
Thus, KSR never comes in contact with any recombinant kinase other than
c-Raf-1 in this assay. Using this procedure, we demonstrated that KSR,
washed to homogeneity with high salt, retained its activity toward
c-Raf-1, indicating that the kinase activity was intrinsic to KSR.
Furthermore, we showed that KSR kinase activity was enhanced by EGF stimulation.
Our previous study, however, did not address the mechanism of c-Raf-1
stimulation by activated KSR (41). To investigate whether
activated KSR stimulates c-Raf-1 by direct phosphorylation, COS-7 cells
transiently transfected with FLAG-KSR were treated for 3 min with 50 ng/ml EGF. We showed previously that this EGF dose and duration of
treatment induce maximal KSR activation (41). Thereafter, KSR was
immunoprecipitated, washed with high salt, and incubated with
recombinant human His-c-Raf-1 substrate that had been coexpressed with
Ras and Lck in Sf9 insect cells in a reaction mixture containing
[
Activation of c-Raf-1 kinase is tightly regulated by multiple
phosphorylation events. In resting cells, c-Raf-1 is phosphorylated exclusively on several serine and threonine residues. Serine and threonine phosphorylation can be further increased at selected sites
upon stimulation with mitogenic stimuli and cytokines and is often
accompanied by tyrosine phosphorylation at positions 340 and 341 (6,
20-32). To investigate the contribution of threonine, serine, and
tyrosine phosphorylation to the overall increased c-Raf-1
phosphorylation by KSR in vitro, Western blot analysis was
performed using anti-phosphothreonine, anti-phosphoserine, and
anti-phosphotyrosine antibodies. Treatment of COS-7 cells with 50 ng/ml
EGF for 3 min markedly increased the capacity of immunoprecipitated KSR
to increase c-Raf-1 threonine phosphorylation when reconstituted
in vitro (Fig. 1C, lane 6). However,
serine and tyrosine phosphorylation of c-Raf-1 by EGF-activated KSR was not detected (data not shown). Furthermore, Ki-KSR, whether isolated from untreated or EGF-treated COS-7 cells, failed to support threonine phosphorylation of recombinant His-c-Raf-1 (lanes 3 and
4), indicating that the kinase activity of KSR was necessary
for c-Raf-1 phosphorylation. It should be noted that the level of
c-Raf-1 was similar in all incubations (Fig. 1C, lower
panel). These results suggest that threonine phosphorylation may
be involved in c-Raf-1 activation by activated KSR and that the kinase
activity of KSR is obligatory.
Immunoaffinity-purified Human FLAG-c-Raf-1 Can Support KSR-mediated
MAPK Activation in Vitro, and Thr269 of c-Raf-1 Is
Required--
Thr269 is located within the CR2 domain of
c-Raf-1, a region documented to critically regulate c-Raf-1
phosphorylation and activation (6). We previously proposed that this
threonine might be important for phosphorylation and activation of
c-Raf-1 by KSR in response to tumor necrosis factor-
To investigate the contribution of Thr269 to threonine
phosphorylation of c-Raf-1 by KSR in vitro, the capacity of
our highly purified recombinant FLAG-c-Raf-1 and FLAG-TV-Raf to support
c-Raf-1 threonine phosphorylation by KSR was determined by Western
analysis using the anti-phosphothreonine antibody, as in Fig.
1C. Fig. 2B (lane 1) shows that
autophosphorylation of recombinant human FLAG-c-Raf-1 on threonine
residues was very low. Immunoprecipitates from COS-7 cells transfected
with vector alone or from KSR-overexpressing cells left untreated
failed to confer c-Raf-1 threonine phosphorylation (lanes
2-5). However, EGF treatment of COS-7 cells significantly increased the capacity of immunoprecipitated KSR to increase c-Raf-1 threonine phosphorylation in vitro (lane 6).
These results are consistent with prior studies showing that
recombinant human FLAG-c-Raf-1, purified to homogeneity, could be
activated in vitro (26, 42-45). Homogeneous FLAG-TV-Raf,
however, was not phosphorylated by KSR preparations from EGF-treated or
untreated cells (lanes 7 and 8). Furthermore, an
equivalent amount of Ki-KSR (data not shown), whether isolated from
untreated or EGF-treated COS-7 cells, failed to support threonine
phosphorylation of recombinant FLAG-c-Raf-1 (lanes 9 and
10), indicating the necessity of kinase activity of KSR for
c-Raf-1 phosphorylation. These studies indicate that the kinase
activity of KSR and Thr269 of c-Raf-1 are required for
Raf-1 phosphorylation by KSR in vitro.
To confirm that the effect of KSR to induce c-Raf-1 phosphorylation
in vitro results from transphosphorylation, studies were performed using kinase-defective FLAG-c-Raf-1(K375M). Kinase-inactive FLAG-c-Raf-1(K375M), which autophosphorylates very weakly (6), was
purified to homogeneity from COS-7 cells by immunoaffinity chromatography. Incubation of purified wild-type c-Raf-1 or
FLAG-c-Raf-1(K375M) with immunoprecipitates from EGF-treated COS-7
cells transfected with vector alone failed to confer threonine
phosphorylation onto c-Raf-1 (Fig. 2C, lanes 1 and 2). However, FLAG-KSR immunoprecipitated from
EGF-treated COS-7 cells phosphorylated FLAG-c-Raf-1(K375M) and
wild-type c-Raf-1 on threonine residues to the same extent (lanes
3 and 4). These results collectively indicate that
c-Raf-1 phosphorylation is not a result of c-Raf-1 autophosphorylation, but rather via transphosphorylation by activated KSR.
To determine whether Thr269 phosphorylation regulates
c-Raf-1 activity, we examined the capacity of purified FLAG-c-Raf-1 or FLAG-TV-Raf to support KSR-mediated activation of MAPK using the two-stage kinase assay as described above (41). A high (100 ng) and a
low (10 ng) dose of purified recombinant human FLAG-c-Raf-1 or
FLAG-TV-Raf was tested using KSR isolated from lysates of EGF-treated COS-7 cells (Fig. 3A).
Immunoprecipitates from COS-7 cells transfected with vector alone
failed to confer activity onto recombinant FLAG-c-Raf-1, as measured by
reconstitution of the MAPK cascade using Elk-1 phosphorylation as
readout (lanes 1-3). KSR immunoprecipitated from
EGF-treated COS-7 cells, when incubated in the first stage of the assay
without Raf-1, also yielded no effect (lane 4). However, KSR
immunoprecipitated from EGF-treated COS-7 cells activated 10 ng of
FLAG-c-Raf-1 about as effectively as it activated an equal amount of
triply transfected commercial His-c-Raf-1 (lane 5 versus
lane 7). Increasing the amount of recombinant human
FLAG-c-Raf-1 in this assay to 100 ng yielded no further increase in
KSR-initiated reconstitution of the MAPK cascade in vitro
(lane 6), indicating that 10 ng of FLAG-c-Raf-1 was optimal
under our assay conditions. These results show that
immunoaffinity-purified recombinant human FLAG-c-Raf-1, although free
of contamination, is replete in mediating KSR signaling. In contrast,
substitution of threonine with valine at amino acid 269 of c-Raf-1
(lane 8) abrogated KSR-mediated Raf-1-dependent MAPK activation. The lack of signaling by 10 ng of TV-Raf was not due
to an insufficient quantity of TV-Raf in the assay, as 100 ng of
FLAG-TV-Raf also failed to elicit MAPK activation (lane 9).
To provide evidence that it is the threonine phosphorylation of c-Raf-1
by KSR that dictates activation, we dephosphorylated c-Raf-1 before or
after KSR treatment using potato acid phosphatase, prior to
reconstitution of the MAPK cascade. As shown in Fig. 3B,
although potato acid phosphatase treatment abrogated KSR activation of
c-Raf-1 (lane 6 versus lane 5), c-Raf-1
dephosphorylated prior to KSR treatment could still be partially
activated (lane 7). These data show that KSR phosphorylates
c-Raf-1 on Thr269 and that this phosphorylation site is
essential for c-Raf-1 activation by KSR.
In a prior study, we showed that endogenous KSR from A431 cells, which
signal constitutively through the EGF receptor, was constitutively
active (41). Endogenous KSR purified from unstimulated A431 cells, like
overexpressed KSR from EGF-treated COS-7 cells, phosphorylated c-Raf-1
on threonine residues and increased c-Raf-1 activity (data not shown).
(In fact, endogenous KSR isolated from A431 cells is ~1.6-fold more
active than FLAG-tagged KSR isolated from EGF-treated COS-7 cells.)
Endogenous KSR from A431 cells, however, neither phosphorylated nor
activated TV-Raf (data not shown). Thus, the phosphorylation of
Thr269 and the activation of c-Raf-1 in vitro by
KSR are not an artifact of KSR transfection, but a property intrinsic
to this protein.
Physiologic Doses of EGF Activate KSR in Vivo--
Recently,
Wennstrom and Downward (46) proposed that low dose EGF activated
the Ras/MAPK signaling pathway by a different mechanism than high dose
EGF in COS-7 cells. To better understand the involvement of KSR in
intact cells, we reevaluated the dose dependence of EGF to enhance KSR
kinase activity using the two-stage assay. For these studies, we
treated COS-7 cells, transiently transfected with FLAG-KSR or vector,
with varying doses of EGF (0.001-100 ng/ml for 3 min), isolated KSR
from lysates, and reconstituted the MAPK cascade using our homogeneous
preparation of human c-FLAG-Raf-1 (41). As anticipated,
immunoprecipitates that lack KSR, from cells transfected with vector
only, did not activate the MAPK cascade (Fig.
4A, lanes 1 and
2). However, EGF conferred dose-dependent activation of KSR kinase activity (lanes 4-8). The maximal
KSR activity was achieved with 0.1 ng/ml EGF (lane 6). This
dose is within the physiologic range for EGF-stimulated mitogenesis
(47). (Note that this and subsequent studies resolved phosphorylated Elk-1 by 10% SDS-PAGE to separate multiply phosphorylated Elk-1 forms
(see below).)
To determine whether KSR activation corresponded to c-Raf-1 threonine
phosphorylation in vivo, the phosphorylation pattern after
low and high dose EGF was determined in cells transiently transfected
with FLAG-c-Raf-1 in addition to Myc-KSR. Myc-tagged KSR constructs
were employed to avoid co-immunoprecipitation of KSR with c-Raf-1. Fig.
4B shows that cells cotransfected with Myc-tagged KSR and
FLAG-c-Raf-1 displayed minimal threonine phosphorylation (upper
panel, lane 3), which was markedly increased upon
treatment with 0.1 ng/ml EGF, a dose that maximally activated KSR
(upper panel, lane 4). (Note that cells
transfected with c-Raf-1 alone displayed no threonine phosphorylation
(Fig. 5B, lane 3);
the modest threonine phosphorylation observed in Fig. 4B due
to KSR coexpression is examined in detail in Fig. 5B.) High
dose EGF (100 ng/ml), which induced no further increase in KSR kinase
activity, also induced no further increase in threonine phosphorylation (Fig. 4B, upper panel, lane 8 versus
lane 4).
In contrast to enhanced threonine phosphorylation, at low dose EGF,
there was no effect on serine or tyrosine phosphorylation. Neither
cells expressing c-Raf-1 alone (data not shown) nor cells coexpressing
Myc-KSR displayed c-Raf-1 tyrosine phosphorylation (Fig. 4B,
middle panel, lane 3). Furthermore, low dose EGF
had no obvious effect on tyrosine phosphorylation (middle
panel, lane 4), whereas high dose EGF, as often
reported (21, 23, 48, 49), effected substantive
Tyr340/Tyr341 phosphorylation (lane
8). As described by others (6, 22, 25, 29, 50), c-Raf-1 was
phosphorylated on serine residues in resting cells whether expressed
alone (data not shown) or in cells coexpressing Myc-KSR (lower
panel, lane 3). As with tyrosine phosphorylation, low
dose EGF had no effect on serine phosphorylation, but increased serine
phosphorylation at high doses (lower panel, lane 4 versus lane 8). These studies indicate a close
association of EGF-induced KSR activation with threonine
phosphorylation of c-Raf-1 in vivo.
Substitution of Thr269 of c-Raf-1 Blocks Raf-1
Activation by KSR in Vivo--
We next assessed the requirement of
Thr269 for c-Raf-1 activation by KSR in vivo
using the 0.1 ng/ml EGF dose. Fig. 5A (lanes 1 and 2) shows that anti-FLAG-Raf-1 immunoprecipitates from
COS-7 cells transfected with vector alone displayed no significant
effect to reconstitute MAPK activation. As reported by others (23, 26,
42, 44), immunoprecipitated FLAG-c-Raf-1 alone, in the absence of EGF
treatment, also exhibited no apparent kinase activity (lane
3). Upon stimulation with 0.1 ng/ml EGF, FLAG-c-Raf-1 activity was
significantly increased (lane 4). Multiple supershifted
forms of phosphorylated Elk-1 were observed, reflecting different
degrees of Elk-1 phosphorylation. Yang et al. (51) similarly
observed multiply phosphorylated forms of Elk-1 in an in
vitro kinase assay employing MAPK isolated from EGF-treated COS-1
cells. Increased c-Raf-1 activity was also detected after EGF
stimulation by direct phosphorylation of Ki-MEK1(K97M-MKK1) (data not
shown). In contrast, substitution of Thr269 with Val
abrogated the enhanced kinase function of FLAG-c-Raf-1, rendering
TV-Raf incapable of transducing an EGF signal (lane 6).
These data support a potentially important role of Thr269
in c-Raf-1 function under physiologic conditions in
vivo.
The capacity of KSR to activate c-Raf-1 in vivo was further
evaluated by coexpression of Myc-KSR and FLAG-c-Raf-1. Myc-tagged KSR
coexpressed with FLAG-c-Raf-1, in the absence of EGF, substantially enhanced the ability of immunoprecipitated FLAG-c-Raf-1 to activate the
MAPK cascade in vitro (Fig. 5A, lane
7), comparable to that of FLAG-c-Raf-1 when expressed alone and
immunoprecipitated from EGF-stimulated COS-7 cells (lane 4).
Moreover, EGF treatment markedly augmented the capacity of KSR to
activate c-Raf-1, producing multiple supershifted Elk-1 bands of high
intensity (lane 8). In contrast to wild-type KSR, Ki-KSR
failed to support c-Raf-1 activation with or without EGF treatment
(lanes 9 and 10). In fact, Ki-KSR appeared to
serve a dominant-negative function, blocking EGF-induced c-Raf-1
activation (compare lanes 4 and 10). These
results are consistent with our published study that showed that KSR
mediated and Ki-KSR prevented ceramide-induced apoptosis through Ras
and c-Raf-1 in BAD-overexpressing COS-7 cells (40). These results clearly indicate that the kinase function of KSR is required for it to
act as a c-Raf-1 activator in vivo as well as in
vitro (Figs. 1 and 2). The significance of Thr269 in
c-Raf-1 activation by KSR in response to EGF stimulation was further
investigated in COS-7 cells cotransfected with wild-type KSR and
TV-Raf. As observed in the in vitro studies, this single amino acid substitution abrogated c-Raf-1 activation by KSR in vivo (Fig. 5A, lanes 11 and 12).
It should be noted that cotransfection of Myc-tagged KSR and
FLAG-tagged Raf-1 constructs did not affect the level of their
respective expression in these studies (Fig. 5A,
lower panel).
If phosphorylation of Thr269 is necessary for c-Raf-1
activation by KSR, it is anticipated that TV-Raf would be defective in this event. Although c-Raf-1 overexpressed in COS-7 cells was not
phosphorylated on threonine residues (Fig. 5B, lane
3), KSR coexpression, which resulted in c-Raf-1 activation,
conferred threonine phosphorylation (lane 7). Low dose EGF
treatment, which enhanced c-Raf-1 activation in the presence of KSR,
further increased threonine phosphorylation (lane 8).
However, Ki-KSR failed to support c-Raf-1 threonine phosphorylation
with or without EGF treatment (lanes 9 and 10).
Moreover, as in Fig. 5A, Ki-KSR appeared to serve a
dominant-negative function, abrogating c-Raf-1 phosphorylation (Fig.
5B, lane 10 versus lane 4). In
contrast, TV-Raf, which could not be activated by KSR with or without
EGF, failed to display threonine phosphorylation (lanes 6 and 12). TV-Raf is not globally inactive however, as high
dose EGF (100 ng/ml) induced serine and
Tyr340/Tyr341 phosphorylation of TV-Raf and
stimulated partial activation (data not shown). Collectively, these
results strongly argue that KSR signals through Thr269 to
activate c-Raf-1 in response to physiologic EGF stimulation in
vivo.
This investigation extends our studies on the role of KSR in
c-Raf-1 activation using highly purified reagents. In our prior investigation, we showed, using a two-stage assay in which KSR comes in
contact only with commercially available His-c-Raf-1, that purified KSR
from EGF-treated cells activates c-Raf-1 (41). Here, we show that
EGF-activated KSR phosphorylated c-Raf-1 during the first stage of this
assay, specifically on threonine residues. The mechanism of
KSR-mediated c-Raf-1 phosphorylation appears to be via
transphosphorylation rather than increased c-Raf-1 autophosphorylation, as Ki-c-Raf-1(K375M) effectively served as a KSR substrate.
Phosphorylation appears to be required for c-Raf-1 activation, as
subsequent dephosphorylation with potato acid phosphatase reversed the
KSR effect. Consistent with a requirement for threonine phosphorylation
in c-Raf-1 activation by KSR, c-Raf-1 in which Thr269, the
putative KSR phosphorylation site, has been substituted with valine
could be neither phosphorylated nor activated by KSR in
vitro. In these latter studies, a great deal of attention was paid
to utilizing c-Raf-1 reagents purified to homogeneity, as the
commercially available material was contaminated with numerous unknown
proteins. However, Thr269 phosphorylation alone may be
insufficient for activation, as bacterially expressed c-Raf-1 cannot be
activated by KSR,2 suggesting
that an unknown post-translational modification of c-Raf-1 primes it
for KSR activation. Consistent with this notion, c-Raf-1,
dephosphorylated prior to KSR treatment, could still be partially
activated by KSR-dependent phosphorylation. Whether priming
represents an irreversible post-phosphorylation conformational change
in c-Raf-1 by kinases other than KSR or an unknown post-translational modification will require further investigation. That
Thr269 was also required for c-Raf-1 activation in
vivo was determined by overexpression of wild-type or TV-Raf-1
protein with or without kinase-active or -inactive KSR and then
stimulation of cells with EGF. In contrast to wild-type c-Raf-1,
TV-Raf-1 could not be activated by physiologic concentrations of EGF or
by concomitant overexpression of kinase-active KSR. Furthermore, the
combination that delivered the largest activation, coexpression of
wild-type KSR with c-Raf-1 and then treatment with physiologic doses of
EGF, was ineffective in the absence of Thr269. However,
TV-Raf is not globally inactive, as it can be fully activated in
vivo by 12-O-tetradecanoylphorbol-13-acetate (30) or
partially activated by super-physiologic doses of EGF. These studies
provide evidence that Thr269 is required for
KSR-dependent c-Raf-1 activation in vivo.
Evidence that the kinase activity of KSR is required for c-Raf-1
activation in vivo as well as in vitro was
established. Ki-KSR, in which two conserved aspartates involved in
phospho-transfer were substituted with alanine residues, did not
increase c-Raf-1 phosphorylation or activation in vitro,
indicating that simple contact of c-Raf-1 with KSR was insufficient for
either event. However, this investigation does not rule out a direct
interaction between these proteins, as both proteins used in the first
stage of the reconstitution assay were purified to homogeneity.
Similarly, Ki-KSR neither activated c-Raf-1 in vivo nor
supported EGF-dependent c-Raf-1 activation. In fact, Ki-KSR
blocked EGF-induced c-Raf-1 activation, consistent with prior studies
in COS-7 cells that showed that Ki-KSR served a dominant-negative
function, antagonizing ceramide-induced apoptosis signaled through
c-Raf-1 (40).
Activated KSR, immunoprecipitated from COS-7 cells treated with EGF,
phosphorylated c-Raf-1 in vitro exclusively on threonine residues (Figs. 1 and 2). Under our assay conditions, increases in
serine and tyrosine phosphorylation of Raf-1 by activated KSR were not
observed. This is consistent with the recognition that mammalian KSRs
contain the motif YI(L)APE in subdomain VIII, which is conserved among
serine/threonine kinases (33). Direct tyrosine phosphorylation of Raf-1
by KSR was not expected since mammalian KSR does not contain the
conserved sequence HKDLR required for tyrosine kinase activity. This
latter sequence is present in both C. elegans and D. melanogaster KSR (33), suggesting that KSR activation of C. elegans and Drosophila Raf might occur through a
fundamentally different mechanism than in the mammalian systems.
It is noteworthy that maximal activation of KSR as measured by
reconstitution of its activity in vitro occurred with
physiologic doses of EGF (0.1 ng/ml). Recently, it has become clear
that these low doses activate a specific high affinity subset of EGF
receptors to signal Ras/MAPK-mediated mitogenesis in vivo
(46). Moreover, the high EGF doses commonly used to stimulate cells in
culture often yield antiproliferative signals (52-57). Strength of
stimulation as a determinant in the assembly of different downstream
signaling complexes has been reported during platelet-derived growth
factor- and EGF-induced MAPK activation in COS-7 cells (58). For EGF, Wennstrom and Downward (46) recently suggested that low dose EGF
may activate an Shc-Grb2-Sos complex that is not EGF
receptor-associated to induce Ras activation, whereas high dose EGF
stably recruits Shc and Grb2 to the EGF receptor. Furthermore, a
protein kinase C-mediated pathway through c-Raf-1 to ERK (46, 59, 60)
may be initiated only at high EGF concentrations sufficient for
phosphorylation of phospholipase C In this study, we further define the mechanism of KSR activation of
c-Raf-1. In vitro, KSR from EGF-stimulated cells
phosphorylates and transactivates human c-Raf-1. The kinase activity of
KSR and Thr269 of c-Raf-1 are the minimal requirements for
this event. Similarly, these properties are required for optimal
c-Raf-1 activation in vivo. That physiologic EGF is
sufficient for maximal KSR signaling implies that this mechanism is
involved in proliferative responses upon signaling through the EGF
receptor. A better understanding of the mechanism of KSR as a Ras
effector and Raf-1 activator in vitro should help elucidate
the biological function of this protein in vivo.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
and ceramide (30). KSR failed to phosphorylate or activate a
Raf-1 mutant in which Thr268, a putative
autophosphorylation site, and Thr269 were both substituted
with valine resides (Raf-1(T268V/T269V)). To examine the mechanism of
c-Raf-1 activation by KSR in response to EGF stimulation and the
significance of Thr269 in c-Raf-1 activation, we generated
recombinant human FLAG-c-Raf-1 and FLAG-Raf-1(T269V) (FLAG-TV-Raf) and
purified them to homogeneity. Using our newly defined two-stage
in vitro KSR activity assay, we show that Thr269
is the major c-Raf-1 site phosphorylated by KSR and that
transphosphorylation of this site is essential for c-Raf-1 activation
by KSR in vitro. Furthermore, Thr269 appears to
be required for optimal activation of c-Raf-1 by KSR in response to
physiologic EGF stimulation in vivo.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
80 °C for subsequent use. KSR
and c-Raf-1 expression were determined by Western blot analysis as
described (41).
-glycerophosphate, 5 mM EGTA, 1 mM dithiothreitol, and 1 mM NaVO4)
before measuring kinase activity.
-mercaptoethanol, 0.1 mM phenylmethylsulfonyl fluoride, and 50% glycerol), and
protein content was measured using BCA Reagent A.
-32P]ATP (3000 Ci/mmol), and 300 ng of commercially available His-c-Raf-1, or
FLAG-c-Raf-1 or FLAG-TV-Raf prepared in our laboratory. After 30 min,
KSR-containing beads were pelleted as described above, and Raf-1 was
quantitatively immunoprecipitated from 25 µl of supernatant with 2 µg of rabbit anti-human c-Raf-1 Ab. Raf-1 immune complexes were
captured by 40 µl of protein A/G beads (Amersham Pharmacia Biotech),
resolved by 7.5% SDS-PAGE, and autoradiographed. For threonine,
serine, and tyrosine phosphorylation studies, preparation of KSR immune
complexes and Raf-1 phosphorylation reactions were carried out as
described above, however, [
-32P]ATP was omitted from
the reaction mixture. Raf-1 immunoprecipitated from the reaction
mixture was resolved by 7.5% SDS-PAGE and transferred onto a
polyvinylidene difluoride membrane. Raf-1, phosphorylated on threonine,
serine, or tyrosine residues, was visualized by Western blot analysis
using a mouse anti-phosphothreonine Ab (Zymed Laboratories
Inc., catalog no. 13-9200), a rabbit anti-phosphoserine Ab
(Zymed Laboratories Inc., catalog no. 61-8100), or a
rabbit anti-c-Raf-1(Tyr(P)340/Tyr(P)341) Ab
(BIOSOURCE, catalog no. 44-506), respectively,
according to manufacturers' instructions.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-32P]ATP as described (28, 30). After the
phosphorylation reaction, KSR-containing beads were pelleted, and
His-c-Raf-1 was quantitatively immunoprecipitated from the supernatant
to separate it from potential contaminating proteins in the commercial
His-c-Raf-1 preparation (see Fig. 2A).
32P-Labeled c-Raf-1 was resolved by 7.5% SDS-PAGE and
autoradiographed (Fig. 1B).
The amount of c-Raf-1 used in the assay was titrated so that in the
absence of KSR, Raf-1 autophosphorylation was barely detectable
(lanes 2 and 3). Under these conditions, a slight
increase in c-Raf-1 phosphorylation measured by incorporation of
radioactive phosphate was visualized after a 30-min incubation with KSR
immunoprecipitated from untreated cells (lane 4). However,
KSR immunoprecipitated from EGF-treated COS-7 cells markedly increased
the incorporation of radioactive phosphate into c-Raf-1 (lane
5). Immunoprecipitation from the reaction mixture with normal
rabbit IgG rather than the anti-c-Raf-1 Ab confirmed the specificity of
the anti-c-Raf-1 Ab (lanes 1 and 6).
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Fig. 1.
EGF treatment enhances the capacity of KSR to
phosphorylate recombinant human c-Raf-1 in vitro.
In vitro phosphorylation of c-Raf-1 by KSR
immunoprecipitated (IP) from EGF-treated COS-7 cells was
determined by 32P labeling (B) and by Western
analysis of threonine (C) and serine and tyrosine (not
shown) phosphorylation. For these studies, COS-7 cells, transfected
with mouse FLAG-KSR (B and C) and FLAG-Ki-KSR
(C), were treated with 50 ng/ml EGF for 3 min. KSR was then
immunoprecipitated from 500 µg of lysate, purified to homogeneity,
and used to phosphorylate human His-c-Raf-1 as described under
"Materials and Methods" and summarized in A. Briefly,
immunopurified KSR was incubated in a reaction mixture containing
commercially available human His-c-Raf-1 with (B) or without
(C) [ -32P]ATP. After 30 min, KSR-containing
beads were pelleted by centrifugation; 25 µl of supernatant
(sup) containing c-Raf-1 was collected; and His-c-Raf-1 was
precipitated with 2 µg of rabbit anti-human c-Raf-1 Ab. In
B, 32P-labeled c-Raf-1 immune complexes were
resolved by 7.5% SDS-PAGE and autoradiographed (lanes
2-5). Immunoprecipitation with normal rabbit IgG served as a
control for the specificity of anti-c-Raf-1 Ab (lanes 1 and
6). In C, threonine phosphorylation was detected
by Western blot (WB) analysis using a mouse
anti-phosphothreonine Ab. The lower panel shows that c-Raf-1
levels in each lane were similar by Western analysis. These data
represent one of three similar experiments each.
and ceramide,
as KSR failed to phosphorylate or activate a Raf-1 mutant in which
Thr268, a putative autophosphorylation site, and
Thr269 were both substituted with valine residues
(Raf-1(T268V/T269V)) (30). To further explore the possible regulation
of c-Raf-1 by threonine phosphorylation and the significance of
Thr269 to c-Raf-1 activation, we generated a FLAG-tagged
valine-substituted Thr269 mutant (FLAG-Raf-1(T269V) or
FLAG-TV-Raf). We expressed and isolated this mutant and wild-type
recombinant human FLAG-c-Raf-1 from COS-7 cells by immunoaffinity
chromatography. To purify these proteins to homogeneity, FLAG-c-Raf-1
and FLAG-TV-Raf immune complexes were subjected to five washes with
Nonidet P-40 lysis buffer containing 1.0 M NaCl, loaded
onto a polypropylene column, and then eluted using the FLAG
octapeptide. The purity of our c-Raf-1 preparations was compared with
that of commercial recombinant human His-c-Raf-1 from Sf9 cells
after resolution by 10% SDS-PAGE and silver staining. As shown in Fig.
2A, ~60% of the commercial
His-c-Raf-1 preparation consisted of impurities as assessed by
densitometric analysis. In contrast, high salt-washed and
immunoaffinity-purified human FLAG-c-Raf-1 and FLAG-TV-Raf were
homogeneous and free of contaminants (second and third
lanes). Prolonged staining failed to detect any visible
contaminants (data not shown). The availability of highly purified KSR
immune complexes and recombinant Raf-1 proteins allowed us to directly
assess the requirement of Thr269 for KSR-mediated
activation of MAPK in vitro.
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Fig. 2.
EGF-activated KSR phosphorylates
purified human FLAG-c-Raf-1 in vitro,
and Thr269 of c-Raf-1 is required.
A, silver-stained 10% SDS-polyacrylamide gel of 0.5 µg of
the commercial preparation of His-c-Raf-1 and of human FLAG-c-Raf-1 and
FLAG-TV-Raf purified from COS-7 cells by extensive high salt washing
and immunoaffinity chromatography as described under "Materials and
Methods." The identities of the purified bands as His-c-Raf-1,
FLAG-c-Raf-1, and FLAG-TV-Raf were confirmed by Western blot
(WB) analysis with a rabbit anti-human c-Raf-1 Ab and, where
applicable, also with anti-FLAG monoclonal antibody M2 (not shown).
B, phosphorylation of c-Raf-1 in vitro by
EGF-activated KSR requires Thr269. COS-7 cells transfected
with mouse FLAG-KSR or FLAG-Ki-KSR were treated with 50 ng/ml EGF for 3 min. Raf-1 phosphorylation by immunopurified FLAG-KSR was as described
in the legend to Fig. 1. Raf-1 alone (lane 1) represents
direct analysis of 300 ng of purified FLAG-c-Raf-1. These data
represent one of three similar experiments. C, threonine
phosphorylation of FLAG-c-Raf-1 by KSR is not due to c-Raf-1 autokinase
activity. FLAG-c-Raf-1 and kinase-inactive FLAG-c-Raf-1(K375M),
purified to homogeneity, were phosphorylated by FLAG-KSR immunopurified
from EGF-treated COS-7 cells as described in the legend to Fig. 1.
These data represent one of three similar experiments. IP,
immunoprecipitate.
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Fig. 3.
Purified human FLAG-c-Raf-1 supports
KSR-mediated MAPK activation in vitro,
and Thr269 of c-Raf-1 is required.
A, the capability of 10 or 100 ng of purified FLAG-c-Raf-1
or FLAG-TV-Raf to support KSR signaling of MAPK activation in
vitro was assessed by the two-stage KSR activity assay as
previously described (41). Briefly, in the first stage of the assay,
FLAG-tagged KSR immunopurified from EGF-treated COS-7 cells was
incubated in 15 µl of reaction buffer containing 60 µM
ATP, 7.5 mM MgCl2, and the indicated amounts
(10 and 100 ng) of either His-tagged or triply transfected human
c-Raf-1 or FLAG-tagged Raf-1 prepared as described under "Materials
and Methods." After 10 min, KSR-containing beads were
pelleted, and 10 µl of supernatant containing activated c-Raf-1 was
added in the second stage of the assay to 20 µl of reaction buffer
containing ATP, unactivated murine GST-MEK1, unactivated murine
GST-ERK2/MAPK, and human GST-Elk-1 fusion protein. After 20 min,
phosphorylated Elk-1 was resolved by 7.5% SDS-PAGE and visualized by
Western blot (WB) analysis using a rabbit
anti-phospho-Elk-1(Ser383) Ab. Complete and even transfer
of the resolved proteins to the polyvinylidene difluoride
membrane was monitored by PhaseGel Blue R (Life Technologies Inc.).
These data represent one of three similar experiments. B,
phosphatase treatment abolishes KSR-stimulated c-Raf-1 activity.
FLAG-c-Raf-1 was treated with 4 µg of potato acid phosphatase
(PAP) for 30 min prior to (PAP-pretreated
Flag-c-Raf-1; lane 7) or after (PAP-treated
Flag-c-Raf-1; lane 6) phosphorylation by KSR in the
first stage of the assay. Thereafter, c-Raf-1 was quantitatively
immunoprecipitated (IP) with anti-FLAG Ab M2.
Dephosphorylated c-Raf-1 was then used in either the first or second
stage of the KSR activity assay as described under "Materials and
Methods." These data represent one of three similar
experiments.
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Fig. 4.
Effect of low and high dose EGF treatment on
the kinase activity of KSR and on the phosphorylation of c-Raf-1
in vivo. A, KSR kinase activity in
response to different doses of EGF. For these studies, cells were
treated with the indicated doses of EGF for 3 min, and KSR kinase
activity was determined by the two-stage in vitro kinase
assay as described in the legend to Fig. 3A. Phosphorylated
Elk-1, resolved by 10% SDS-PAGE to separate multiple supershifted
bands, was visualized by Western blot (WB) analysis as
described under "Materials and Methods." These data represent one
of four similar experiments. B, FLAG-c-Raf-1 was
immunoprecipitated (IP) from EGF-treated COS-7 cell lysates
and resolved by 7.5% SDS-PAGE. In vivo c-Raf-1 threonine,
tyrosine, and serine phosphorylation was determined by Western blotting
as described under "Materials and Methods." These data represent
one of three similar experiments.
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Fig. 5.
EGF-stimulated c-Raf-1 activation by KSR
in vivo requires Thr269.
A: upper panel, COS-7 cells, transfected with
mouse Myc-KSR or Myc-Ki-KSR and coexpressing human FLAG-c-Raf-1 or
FLAG-TV-Raf, were stimulated where indicated with 0.1 ng/ml EGF. The
activity of immunoprecipitated (IP) Raf-1 was measured by
in vitro reconstitution of the MAPK cascade as described
under "Materials and Methods." Phosphorylated Elk-1, resolved by
10% SDS-PAGE, was visualized by Western blot (WB) analysis
as described in the legend to Fig. 2C. Prolonged exposure of
films also allowed detection of trace amounts of supershifted Elk-1
measured in Raf-1 immunoprecipitates from EGF-treated COS-7 cells
transfected with Ki-KSR and c-Raf-1 (lane 10), KSR and
TV-Raf (lane 12), and Ki-KSR and TV-Raf (lane
14), representing <5% of the activity of wild-type c-Raf-1 from
EGF-treated KSR-coexpressing cells (lane 8). Lower
panel, the expression levels of Myc-KSR, Myc-Ki-KSR, FLAG-c-Raf-1,
and FLAG-TV-Raf were determined by Western blot analysis of 20 µg of
total cell lysate using mouse anti-Myc and anti-FLAG Abs. These data
represent one of six similar experiments. B: shown is the
in vivo threonine phosphorylation of FLAG-Raf-1 in COS-7
cells treated with 0.1 ng/ml EGF. FLAG-c-Raf-1 and FLAG-TV-Raf were
immunoprecipitated from 300 µg of EGF-treated COS-7 cell lysates
transfected as described for A, and threonine
phosphorylation was determined by Western analysis as described in the
legend to Fig. 4B. These data represent one of three similar
experiments.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
. Whether receptor-associated
adaptor complexes and protein kinase C activation are involved in the
Tyr and Ser phosphorylation, respectively, observed at high EGF doses
requires further investigation.
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ACKNOWLEDGEMENT |
---|
We thank Dr. J. Lozano for kindly providing pCMV-Myc-KSR and pCMV-Myc-Ki-KSR constructs and for critical reading of the manuscript.
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FOOTNOTES |
---|
* This work was supported by Grant CA42385 from the National Institutes of Health (to R. K.) and by a fellowship from the Medical Research Council of Canada (to H. R. X.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed to: Lab. of Signal
Transduction, Memorial Sloan-Kettering Cancer Center, 1275 York Ave.,
New York, NY 10021. Tel.: 212-639-7558; Fax: 212-639-2767; E-mail:
r-kolesnick@ski.mskcc.org.
Published, JBC Papers in Press, December 27, 2000, DOI 10.1074/jbc.M008096200
2 Y. Zhang and R. Kolesnick, unpublished observation.
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ABBREVIATIONS |
---|
The abbreviations used are: MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; GST, glutathione S-transferase; EGF, epidermal growth factor; Ab, antibody; PAGE, polyacrylamide gel electrophoresis.
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