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INTRODUCTION |
The serine/threonine kinase c-Raf1 (hereafter referred to as Raf)
is one of the proteins in the cellular
MAP1 kinase signal
transduction cascade controlling cell proliferation and differentiation
among other biological processes. Activation of Raf appears as a
multistep process, which is still incompletely understood (for review,
see Refs. 1-3). The first step necessary for full activation involves
membrane recruitment of Raf via binding to Ras-GTP (4-6). This step is
thought to be a prerequisite for subsequent activating events that
include phosphorylation on tyrosine residues Tyr-340/Tyr-341 of Raf (7,
8). In addition to Tyr-340/Tyr-341 phosphorylation, several
phospho-transfer reactions on serine-/threonine residues have been
linked to Raf activation (9-11). Protein-protein interactions
e.g. with 14-3-3 proteins and heat shock proteins also
influence kinase activity (12, 13). Once in its active state, Raf
phosphorylates and activates the dual specificity kinase MEK, which in
turn facilitates induction of the MAPK ERK.
Class I PI 3-kinases (PI3K) have been shown to express differential
effects on the signaling activities of Raf and the other members of the
MAPK cascade. In various cell types regulatory effects of PI3K on Ras,
Raf, and MEK have been described. At the level of Ras, PI3K have been
reported to exert an activating effect, possibly of a conditional
nature, on Ras-GTP formation (14-16). On the other hand, PI3K were
identified as Ras effectors, binding to and being activated by Ras-GTP
(17, 18). Moreover, it was recently proposed that PI3K are involved in
phosphorylation of MEK on serine 298 via the Rac/Cdc42 effector PAK.
Ser-298 phosphorylation enhances binding of MEK to Raf and can, thus,
lead to a sustained activation of MEK (19).
Phosphorylation of distinct serine residues on Raf has been implicated
in direct regulation of Raf activity. Two PI3K-sensitive protein
kinases affecting Raf kinase activity have been described. Protein
kinase B (PKB), which depends on PI3K lipid products for its
activation, was recently outlined as a Raf Ser-259 kinase (20, 21).
Phosphorylated Ser-259 provides a docking site for 14-3-3 proteins on
Raf, and this interaction has been reported to down-regulate Raf
activity (22-24). The second published effect is mediated by the
serine/threonine kinases PAK2 and -3 (25). PAK is activated via the
small GTPases Rac and Cdc42 which, at least in some systems, can be
regulated by PI3K-dependent activation of guanine
nucleotide exchange factors for Rho family GTPases. In this regard, PAK
has been shown to phosphorylate Ser-338 in Raf, a phosphorylation site
described to be essential for full activation of the kinase (25-27).
These findings contrast to other data demonstrating that
phosphorylation on Ser-338 does not always correlate with Raf kinase
activity (28, 29).
In the present study we have attempted to study the role of PI3K and
Ser-338 in activation of endogenous Raf in U937 myelomonocytic cells. A
permeabilization strategy has been applied that allows manipulation of
the cellular Ras activation independently on PI3K activity status.
Using this method we identified a stimulatory effect of PI3K on
endogenous Raf, which is not mediated by Ras. Moreover, the system has
been exploited to investigate the role of PAK and Ser-338
phosphorylation in the modulation of Raf activity. Our data challenge a
role for serine 338 phosphorylation in c-Raf1 activation.
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EXPERIMENTAL PROCEDURES |
Reagents and Antibodies--
Streptolysin O and GTP
S were
purchased from Sigma. For Western blot analysis anti-phospho-ERK
(E-10), anti-phosphoserine 473-PKB (Cell Signaling), anti-Raf1,
anti-pan-ERK (Transduction Laboratories), anti-PKB (Pharmingen),
anti-phosphoserine 338-Raf (Upstate Biotechnologies), anti-pan-Ras
clone Ras11 (Calbiochem) were used.
PAK (N-19) and Rac2 (C-11)
antibodies were obtained from Santa Cruz Biotechnology. Y13-259
antibody was purified from cell culture supernatant of the
corresponding hybridoma cell line (ATCC CRL 1742). Western blots were
detected using the enhanced chemiluminescence kit (PerkinElmer Life
Sciences). Wortmannin and Genistein were obtained from Alexis Corp.,
LY297002 was from Sigma Chemicals, and the c-Raf inhibitor ZM336372 was
purchased from Calbiochem.
Plasmids and Protein Purification--
The baculoviruses
encoding His-MEK-K97M (MEK KM) and GST-MEK were kind gifts of U. Rapp.
Proteins were purified from SF9 cells using nickel nitrilotriacetic
acid-Sepharose (Qiagen) and glutathione-Sepharose according to
standard protocols. Bacterial expression vectors encoding GST-Ras
binding domain (RBD) for Raf and GST-PAK RBD were kindly provided by J. Downward and G. Bokoch, respectively. The fusion proteins were
expressed in Escherichia coli and purified via affinity
chromatography using glutathione-Sepharose (Amersham Biosciences) as
described (16).
Cell Culture--
U937 cells were cultured in RPMI1640 media
supplemented with 10% heat-inactivated fetal calf serum in a
humidified atmosphere at 37 °C. Before experiments cells were
serum-starved for 16-18 h in RPMI media supplemented with 0.1% fatty
acid free bovine serum albumin (BSA) and 50 mM HEPES, pH
7.4.
Permeabilization of U937 Cells--
Permeabilization was carried
out essentially as described by Stephens et al. (30). The
bacterial toxin streptolysin O was used to allow influx of GTP
S as
well as of larger molecules such as antibodies. Before the experiments
the cells were serum-starved for 16-18 h and subsequently washed 3 times with HBBSS (15 mM HEPES, pH 7.4, 140 mM
NaCl, 5 mM KCl, 2.8 mM NaHCO3, 1.5 mM CaCl2, 1 mM MgCl2,
0.06 mM MgSO4, 5.6 mM glucose,
0.1% fatty acid free BSA) and 2 times with intracellular buffer (50 mM HEPES, pH 7.4, 107 mM potassium glutamate, 2 mM MgCl2, 1 mM EGTA) at 37 °C.
Thereafter, cells were resuspended at a density of 108
cells/ml in intracellular buffer and kept on ice for 10 min. To start
the experiment, aliquots of 625 µl of the cell suspension were
incubated at 37 °C for 8 min with or without inhibitor. Afterward, 1.85 ml of permeabilization buffer (50 mM HEPES, pH 7.2, 67 mM potassium glutamate, 1 mM EGTA, 6 mM MgCl2, 0.6 mM CaCl2,
100 µM ATP) containing 3 units of streptolysin O and,
where indicated, the appropriate inhibitor, were added. 750 µl of the
cell suspension were removed within the first minute for assays at time
point zero. All incubations were carried out at 37 °C. Additional
aliquots were taken from the suspension 2 and 4 min thereafter. The
cells were spun down and lysed immediately. Cleared lysates were
subjected to Ras and/or Raf and PAK activity assays.
Ras and Rac Pull-down Assay--
To capture activated GTP-bound
GTPases, GST fusion proteins containing the Ras binding domain of Raf
or PAK were used as specific probes for activated Ras and Rac,
respectively. The assays were carried out essentially as described with
some variations (33, 34). Briefly, U937 cells were lysed in 1 ml Ras
pull-down buffer (50 mM Tris/HCl, pH 7.5, 1 mM
EGTA, 150 mM NaCl, 5 mM MgCl2, 1% Nonidet P-40) containing 100 µM GDP and 20 µg/ml
GST-Raf RBD. For Rac assays, cells were lysed in 1 ml of buffer
containing 50 mM Tris HCl, pH 7.5, 150 mM NaCl,
2 mM MgCl2, 50 mM NaF, 10% glycerol, 1% Nonidet P-40, 100 µM GDP, and 20 µg/ml
GST-PAK RBD. The cleared lysates were incubated for 30 min with
glutathione-Sepharose, and the precipitates were washed three times
with Ras or Rac pull-down buffer, respectively. Associated Ras or Rac
protein was detected by subsequent SDS-PAGE and Western blotting.
Raf and PAK Kinase Assays--
U937 cells were lysed in lysis
buffer containing 1% Triton X-100, 50 mM Tris, pH 7.5, 150 mM NaCl, 5 mM EDTA, 1 mM EGTA.
Raf-kinase activity was immunoprecipitated incubating the cleared
lysates with 0.5 µg of anti-Raf antibody and protein G-Sepharose for
2 h. Immunoprecipitates were washed 3 times with lysis buffer and 2 times with kinase buffer (50 mM Tris/HCl, pH 7.5, 0.1 mM EGTA, 13.3 mM MgCl2, 5%
glycerol). The kinase reaction mix contained 2.5 µCi of
[32P]ATP, 1 µg of kinase inactive His-MEK substrate,
and 15 µM unlabeled ATP in 50 µl of kinase buffer. The
kinase reaction was carried out at 30 °C for 30 min and stopped by
adding Laemmli sample buffer to the reactions. The samples were
subjected to a 10% SDS-PAGE gel to separate phosphorylated MEK-K97M
substrate from the precipitated kinase. [32P]Phosphate
incorporation into the MEK substrate was visualized using a Bio-Rad
phosphorimaging system. The PAK kinase assay was carried out
essentially as described (19). Immunoprecipitated PAK2/3 was incubated
with 5 µg of myelin basic protein as a substrate in 50 µl of kinase
buffer containing 20 mM Tris/HCl, pH 8.0, 10 mM
MgCl2, 1 mM dithiothreitol, 100 µM ATP, and 1 µCi of [32P]ATP at 30 °C
for 30 min. Afterward the samples were subjected to a 12.5% SDS-PAGE
gel to separate the substrate myelin basic protein from the PAK kinase.
Phosphate incorporation into the substrate was visualized using a
Bio-Rad phosphorimaging system.
GST-MEK Pull-down Assay--
Immobilized GST-MEK fusion proteins
(10 µg) or GST alone (20 µg) were incubated with cell lysate
corresponding to 2 × 107 U937 cells, treated as
indicated. After 2 h at 4 °C, complexes were washed 4 times
with lysis buffer, solubilized in SDS-PAGE sample buffer, and resolved
by SDS-PAGE. Raf bound to MEK was analyzed by Western blotting.
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RESULTS |
Ras and Raf Activation in U937 Cells Are Sensitive to Wortmannin
and Genistein--
Fetal calf serum and the protein kinase
C-activating agent TPA have been initially used to characterize Ras and
Raf activation in U937 cells. In addition, we investigated the effects
of insulin and ATP, which are known to stimulate a tyrosine kinase and
G-protein-coupled receptor respectively. All four agonists induced Ras
and Raf activation (Fig. 1), and in all
cases activation was blunted by the PI3K inhibitor wortmannin and the
broad-specificity tyrosine kinase blocker genistein (Fig. 1). Use of a
second, structurally unrelated PI3K inhibitor (LY294002) yielded
similar results (data not shown). These data implicate tyrosine
kinase(s) and PI3K in the activation process of Ras, confirming and
extending data from a recent study using the same cell type (16). In
that study PI3K were found to modulate Ras activity via down-regulation
of Ras-GAP proteins in U937 cells. To achieve Ras-GTP loading in a
background of PI3K inhibition we introduced non-hydrolyzable GTP
analogues into permeabilized cells. In the present study we have used
this approach as a way to dissect PI3K effects on Ras and Raf
activity.

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Fig. 1.
Activation of Ras and Raf in U937 cells is
sensitive to wortmannin and genistein. U937 cells were starved for
16 h in RPMI medium containing 0.1% BSA and 25 mM
HEPES, pH 7.4. Before the experiment, the cells were washed,
resuspended in HBBSS buffer at 107 cells/ml, and incubated
at 37 °C for 5 min. The cells then were preincubated with 100 nM wortmannin, 100 µM genistein, or
Me2SO carrier for 10 min and subsequently stimulated with
10% fetal calf serum, 100 nM TPA, 10 µg/ml insulin, or
100 µM MgATP as indicated. The cells were lysed, and the
cleared lysates were used for the Ras-GTP pull-down assay (upper
panel). Supernatants of the lysates were used for Raf
immunoprecipitation and subsequent kinase assay. Phosphorylation of the
Raf substrate MEK KM was analyzed by SDS-page and phosphorimaging
(lower panel). WB, Western blot.
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Differential Regulation of Ras and Raf by PI3K and Tyrosine
Kinases--
Permeabilization of U937 cells with streptolysin O in the
presence of GTP
S led to a time-dependent activation of
the Raf kinase (Fig. 2). Inclusion of the
Raf inhibitor ZM336372 in the kinase reaction abrogated phosphorylation
of the substrate MEK KM, demonstrating the specificity of the
immunoprecipitation protocol (Fig. 2). We next tested the effect of the
PI3K inhibitor wortmannin and the tyrosine kinase inhibitor genistein
on GTP
S-induced responses. Accumulation of active Ras in response to
GTP
S was abolished by genistein and only slightly decreased by
wortmannin (Fig. 3A), in line
with previous observations which indicated that tyrosine kinases but
not PI3K do regulate basal exchange factor systems for Ras in these
cells (16). Again, the structurally unrelated PI3K inhibitor LY294002
yielded analogous results to wortmannin. The slight reduction in active
Ras accumulation by wortmannin could be due to the fact that PI3K
blockade abrogates basal Ras-GTP levels in U937 cells such that
formation of active Ras via GTP
S uptake adds upon a decreased basal
level of Ras-GTP in wortmannin-treated cells (16). Irrespective of
these considerations, the data in Fig. 3A demonstrate that
GTP
S perfusion of permeabilized U937 cells can be used to activate
Ras in a background of PI3K inhibition, which provides the adequate
conditions to investigate the input of PI3K at the level of Raf apart
from PI3K effects on Ras activity.

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Fig. 2.
Suitability of the
GTP S approach for measuring Raf kinase
activity in U937 cells. U937 cells were starved overnight and
prepared for permeabilization as described under "Experimental
Procedures." Samples were taken within the first minute after the
addition of the permeabilization mix, before pore formation started
(time = 0). One minute after the addition of the permeabilization
mix the time course was started, and samples were taken after an
additional 2 and 4 min. The cells were lysed, and a Raf kinase assay
was performed. Where indicated 25 µM ZM336372 were added
to the kinase reaction to test for specificity of the reaction.
Phosphorylation of the Raf substrate MEK KM was analyzed.
WB, Western blot.
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Fig. 3.
Ras-GTP loading and Raf activation in
GTP S treated U937 cells. The
permeabilization experiment was carried out as described for Fig. 2.
The cells were pretreated with inhibitor (wortmannin, 100 nM; genistein, 100 µM) or Me2SO
for 10 min as indicated. The cells were lysed in the appropriate lysis
buffers, and the lysates were used for either Ras pull-down experiments
(A) or Raf kinase assay (B). For the Ras
pull-down assay, lysates were incubated with GSH-Sepharose and GST-Raf
RBD. After washing, the precipitates were subjected to SDS-PAGE, and
Ras was detected. Samples for the Raf kinase assay were treated as
described under "Experimental Procedures." WB, Western
blot.
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We, thus, proceeded to test the effect of PI3K inhibition on
GTP
S-induced Raf activation. In marked contrast to its effects on
Ras activation pattern, wortmannin caused a strong decrease in
GTP
S-stimulated Raf kinase activity (Fig. 3B), indicating that PI3Ks regulate Raf activity via a mechanism independent of its
action on Ras. Contrary to the effects of wortmannin, preincubation of
the cells with the tyrosine kinase inhibitor genistein, which effectively suppressed Ras-GTP accumulation, only minimally affected Raf activation by GTP
S (Fig. 3B). However, genistein
blocked ERK activation in permeabilized U937 cells subjected to the
streptolysin O/GTP
S protocol (Fig. 3B). From these data
we conclude (i) the existence of an additional genistein-sensitive
step(s) downstream or parallel to Raf needed for MAPK activation by
GTP
S and (ii) the presence of a Ras-independent, GTP
S-driven
pathway for Raf activation that is active in a background of tyrosine
kinase inhibition. Indeed, the Ras-neutralizing antibody Y13-259, which
interferes with Ras/effector interactions (31), completely abolished
accumulation of active Ras as well as Raf activation in permeabilized
GTP
S-loaded U937 cells (Fig. 4) but
was unable to block the GTP
S-dependent appearance of
active Raf in cells pretreated with genistein. The data point to a
Y13-259-insensitive G-protein that can induce Raf activation in a
background of tyrosine kinase inhibition.

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Fig. 4.
The Ras antibody Y13-259 blocks
GTP S induced Raf activation but does not block
GTP S/genistein elicited Raf activation.
U937 cells were washed and resuspended in intracellular buffer
and incubated at 37 °C for 5 min. Before permeabilization the cells
were incubated with 100 nM wortmannin, 100 µM
genistein, or Me2SO carrier for 10 min as indicated. Where
indicated 10 µg of Y13-259 antibody were added to the
permeabilization mix. After 1 min 1 mM GTP S was added to
the cells, and the first sample was taken immediately. Additional
samples were taken after 2 and 4 min as indicated. The upper
panel shows a phosphorimaging scan of the phosphorylated MEK KM
substrate. The lower panel shows a -Raf Western blot
(WB) to analyze the amount of immunoprecipitated Raf. The
activity of Ras was determined using the Ras pull-down method
(middle panel).
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The lack of correlation of cRaf1 and ERK activation observed in
genistein-treated U937 cells suggests the existence of alternative pathways linking Ras to ERK activation. One obvious candidate in this
regard is B-Raf. Several recent reports make a strong case for B-Raf
being the major transducer of Ras-signals to ERK (2, 32). Indeed we
have detected wortmannin-sensitive activation of endogenous U937 B-Raf
in response to TPA, serum, and insulin (data not shown). However,
investigation of B-Raf action in our system is rendered arduous by the
fact that at least five splicing variants of B-Raf are expressed in
U937 cells (data not shown). A detailed study aimed at deciphering the
role of B-Raf in Ras-dependent ERK activation is currently
under way in our laboratory.
Ser-338 Phosphorylation and Raf Activity Do Not Correlate in Their
Sensitivities to Wortmannin and Genistein--
PI3Ks have been
recently proposed to contribute to full activation of Raf by inducing
phosphorylation of Ser-338 in Raf via a Rac/PAK-dependent
pathway (27). To test if the requirement for PI3K in GTP
S-induced
Raf activation in U937 cells correlates with Ser-338 phosphorylation,
we performed Western blots using a well characterized phosphoserine
338-specific antibody (28). Fig. 5 shows
that Raf became phosphorylated on Ser-338 upon GTP
S loading of U937
cells. Treatment with wortmannin did not significantly alter
GTP
S-dependent Ser-338 phosphorylation, although a
slight decrease was observed in some experiments. Moreover, the
previously observed Ras-independent activation of Raf by GTP
S in
genistein-treated cells was not accompanied by Ser-338 phosphorylation.
These data indicate that Ser-338 phosphorylation does not correlate
with Raf activity. Moreover PI3K seem to be dispensable for Ser-338 phosphorylation on Raf in U937 cells loaded with GTP
S.

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Fig. 5.
PAK and Rac activities in
GTP S-stimulated U937 cells do not correlate
with activity and serine 338 phosphorylation of Raf. After
incubation of the cells with 100 nM wortmannin, 100 µM genistein, or carrier, the cells were permeabilized
and stimulated with 1 mM GTP S. The lysates were
subjected to a Raf kinase assay (A, top panel) or
a PAK kinase assay (B, top panel) and a Rac
pull-down assay (B, lower panel). For the PAK
assay PAK was immunoprecipitated with an antibody specific for the PAK2
and 3 isoforms. Myelin basic protein (MBP) was used as a
substrate to measure kinase activity. Rac-GTP was precipitated from the
lysates using a GST-PAK RBD probe, and Rac was visualized in a Western
blot (WB) with a Rac2 antibody. 50 µg of total cell lysate
of each sample was subjected to SDS-PAGE, and the activity of ERK was
assessed in a Western blot using phospho-specific antibodies directed
against the active forms of the kinases. The -pan ERK Western blot
was performed as a loading control (A).
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PAK Activation Correlates with Ser-338 Phosphorylation on Raf but
Not with Raf Activity--
The small GTPase Rac and its effector PAK
have been described as mediators of PI3K-dependent Ser-338
phosphorylation in other systems. To collect further evidence for the
role of this presumptive pathway in U937 cells, we investigated the
activation status of both proteins in our experimental setting.
Permeabilization of U937 cells in the presence of GTP
S led to a
time-dependent activation of PAK (Fig. 5). GTP
S-induced
PAK activation was not abolished by preincubation of the cells with
wortmannin at the same concentration which efficiently blocked Raf, ERK
(Fig. 3B), and PKB activation (data not shown) but was
completely blunted by pretreating the cells with genistein (Fig. 5). To
complement these results, we also assessed the activation status of the
GTPase Rac2 as the upstream activator of PAK. GTP
S-stimulated Rac2
activation showed an analogous activation pattern to PAK, with
wortmannin only causing a minor reduction in active Rac2 formation and
genistein having a more severe effect. Both Rac2 and PAK activation
correlate with Ser-338 phosphorylation, in agreement with the notion
that Rac-activated PAK is a/the Raf Ser-338 kinase (25). In our hands
all three parameters did not correlate with Raf activity induced by
GTP
S, indicating that they may not contribute to Raf activation.
Ser-338 Phosphorylation and Raf Activity Do Not Correlate in
Space--
The results presented above raised the possibility that
Ser-338 phosphorylation is not essential for Raf activation in U937 cells. Alternatively, Ser-338 phosphorylation and the unidentified PI3K
input at the level of Raf may represent two independent sequential or
parallel steps, both co-operatively contributing to the Raf activation
process. To collect further evidence as to the role of Ser-338
phosphorylation in the Raf activation process, we analyzed the
subcellular distribution of Raf after GTP
S loading of permeabilized U937 cells. GTP
S-stimulated U937 cells were lysed in a hypotonic, detergent-free buffer, and cytosolic (S100) and membrane fractions (P100) were separated and tested for Raf activity and
Ser-338-phosphorylated Raf (Fig. 6).
Although the bulk of Raf protein and Raf activity was present in S100,
it was the particulate fraction that exhibited GTP
S and
time-dependent appearance of Raf with high specific activity. This is in agreement with the current view on the importance of membrane localization for full Raf activation. In marked contrast, no Ser-338 phosphorylated Raf was detectable in the particulate fraction, whereas a strong signal for phosphoserine 338 was observed in
the soluble fraction. These data show that the majority of Ser-338-phosphorylated Raf did not co-localize with the active kinase.
Membrane-bound Ras was used as a marker for the efficiency of the
fractionation procedure (Fig. 6) in this experiment. Together these
data are in line with the lack of correlation between Ser-338 phosphorylation and Raf kinase activity observed above.

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Fig. 6.
Ser-338 phosphorylated Raf cannot be detected
in the membrane fraction after GTP S
stimulation. U937 cells were washed and resuspended in
intracellular buffer at 1 × 108 cells/ml and
incubated at 37 °C for 5 min. The cells were permeabilized and
stimulated with 1 mM GTP S permeabilization. Samples were
lysed in a hypotonic buffer after 0, 2, and 4 min as indicated and
homogenized by passing the suspension through a 27-gauge needle. The
cytosolic and membrane fractions were separated by differential
centrifugation, as described under "Experimental Procedures." The
lysates of the membrane fraction (P100) and the cytosolic fraction
(S100) were subjected to Raf immunoprecipitation. The
immunoprecipitates were used for an in vitro Raf kinase
assay (top panel), and the precipitates were analyzed for
Raf and Ser-338 phosphorylated Raf in Western blots. Purity of the
fractionation was determined by a pan-Ras Western blot (WB)
performed with 50 µg of lysates from each fraction.
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Ser-338 Phosphorylation Does Not Correlate with Raf Activity and
MEK Binding Activity of Raf--
Next we tried to assess the physical
interaction between Raf and MEK after stimulation of intact U937 cells
to determine the contribution of Ser-338-phosphorylated Raf to the
observed Raf activation. Cell extracts of TPA-stimulated U937 cells
were depleted of MEK-binding proteins by incubation with immobilized
GST-MEK or GST as a control, and Raf activity assays were performed on the various fractions (Fig. 7). GST-MEK
protein efficiently precipitated Raf kinase activity, whereas GST
affinity precipitates contained no detectable MEK-phosphorylating
activity. The amount of GST-MEK-bound Raf increased with stimulation in
a time-dependent fashion, and the binding decreased after
10 min, correlating with the observed kinetics of Raf stimulation.
Whereas the majority of Raf kinase activity was present in pull-down
assays produced with GST-MEK, only a minor amount of total
Ser-338-phosphorylated Raf was bound to MEK, the bulk of Ser-338
phosphorylation being recovered from the post-GST-MEK pull-down
fraction (Fig. 7). This experiment demonstrates that a large portion of
Raf protein did not bind MEK and was not activated in response to TPA.
The majority of Raf phosphorylated on Ser-338 co-fractionated with
inactive Raf, which was unable to bind to MEK, suggesting that Ser-338
phosphorylation is not important for Raf activity and Raf/MEK
interaction.

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Fig. 7.
The majority of Ser-338-phosphorylated Raf is
inactive and does not bind MEK. U937 cells were starved for
16 h in RPMI medium containing 0.1% BSA and 25 mM
HEPES, pH 7.4. Before the experiment the cells were washed, resuspended
in HBBSS at a density of 8 × 106 cells/ml, and
incubated at 37 °C for 10 min. Afterward, the cells were stimulated
with 100 nM TPA for the indicated times and lysed in 750 µl 1% Triton X-100 buffer. The lysates were first subjected
to a GST-MEK or GST-only pull-down using GSH-Sepharose as indicated
(GSH precipitation (GSH-precip.)). The supernatants of these
pull-down experiments were subjected to Raf immunoprecipitation to
assess the amount of remaining Raf and kinase activity (Raf
immunoprecipitation after GSH precipitation). Immunoprecipitates as
well as GSH precipitates were subjected to a Raf kinase assay. The
amount of precipitated kinase was visualized with a Raf antibody.
Ser-338 phosphorylation was tested using a Ser-338 phospho-specific
antibody. WB, Western blot.
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Raf Activity and Ser-338 Phosphorylation Do Not Correlate in
Time--
To further investigate the relationship between Ser-338
phosphorylation and Raf activation, kinetic experiments have been performed using different agonists to challenge intact U937 cells (Fig.
8). TPA stimulated Raf activation with a
peak at 1 min after the addition of the agonist. ERK activation
correlated well with the Raf activity under these conditions and was
detectable up to 10 min after stimulation. Raf activity decreased
faster than ERK activity, indicating that there must be strong negative
regulatory events suppressing Raf activity at later time points.
Ser-338 phosphorylation on Raf was strongly induced by TPA, but the
phosphorylation did not decrease in parallel to the kinase activity.
Phosphorylation of Ser-338 was still detectable 30 min after
stimulation, a time point at which the Raf kinase activity was back to
basal levels (Fig. 8). ATP induced a transient and weaker activation of
Raf kinase with a maximum at 1 min. The activity was rapidly
down-regulated and no longer detectable 5 min after stimulation (Fig.
8). This property correlates well with the ERK phosphorylation observed in response to ATP. The peak of ERK activation was detected 2 min after
stimulation, sharply following the peak of Raf activity. Strong Ser-338
phosphorylation 2 min after stimulation has been observed at a time
point when Raf activity was already heavily down-regulated. Similar
results were obtained using fetal calf serum and insulin as stimuli
(Fig. 8). Therefore, in all cases, regulation of Ser-338
phosphorylation lagged behind Raf kinase activity, providing further
evidence against a role of Ser-338 phosphorylation in the Raf
activation process.

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Fig. 8.
Ser-338 phosphorylation on Raf does not
correlate with Raf activity in agonist stimulated cells. U937
cells were starved for 16 h in RPMI medium containing 0.1% BSA
and 25 mM HEPES, pH 7.4. Before the experiment the cells
were washed and resuspended in HBBSS buffer at 107
cells/ml. The cells were incubated at 37 °C for 5 min and
subsequently stimulated with 100 nM TPA, 100 µM MgATP, 10% fetal calf serum, or 10 µg/ml insulin
for the indicated times. The cells were lysed, and the cleared lysates
were used for Raf immunoprecipitation and subsequent kinase assay.
Phosphorylation of the Raf substrate MEK KM was analyzed by SDS-PAGE
and phosphorimaging. Immunoprecipitated kinase was tested for Ser-338
phosphorylation using a Ser-338 phospho-specific antibody (top
panel). The cell lysate was used for detection of active ERK
(lower panel). Equal loading of lysates was confirmed by
probing with a pan-ERK-antibody.
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Ser-338 Phosphorylation Is Dispensable for Hyper Activation of
Raf--
ZM336372, a potent inhibitor of Raf kinase activity,
specifically inhibits Raf kinase activity versus B-Raf and
other Ser-/Thr kinases in vitro. However, treatment of cells
with ZM336372 has been shown to activate Raf, as assayed on
immunoprecipitated Raf kinase activity in vitro. It has been
postulated that active Raf accumulates in ZM336372-treated cells due to
the shutdown of a negative feedback mechanism sparked by Raf itself
(33).
We tested the involvement of Ser-338 phosphorylation in Raf activation
after the addition of ZM336372. Incubation of U937 cells with the
inhibitor ZM336372 led to the recovery of high MEK-phosphorylating
activity in Raf immunoprecipitates (Fig.
9). This activity was much higher than in
Raf immunoprecipitates from TPA-stimulated cells (Fig. 9). In line with
a previous report (33), ZM336372-mediated activation of Raf was not
mirrored by ERK activation in inhibitor-treated cells, suggesting that
ZM336372 induced the formation of active Raf while at the same time
effectively inhibiting Raf enzymatic activity in the cells.
Importantly, ZM336372 did not induce Ser-338 phosphorylation of Raf
(Fig. 9), indicating that this phosphorylation event is not a
constitutive part of ZM336372-induced processes leading to accumulation
of active Raf in U937 cells.

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Fig. 9.
The Raf inhibitor ZM336372 induces Ser-338
phosphorylation-independent Raf activity. U937 cells were starved
for 16 h in RPMI medium containing 0.1% BSA and 25 mM
HEPES, pH 7.4. Before the experiment the cells were washed and
resuspended in fresh media at a density of 106 cells/ml.
Where indicated, 1 µM ZM336372 was added, and the cells
were incubated for 1 h. Afterward, the cells were washed 2 times
with HBBSS and resuspended in the same buffer at 107
cells/ml. The cells were incubated at 37 °C for 5 min and
subsequently stimulated with 100 nM TPA or carrier as
indicated. The cells were lysed, and the cleared lysates were used for
Raf immunoprecipitation and subsequent kinase assay. Phosphorylation of
the Raf substrate MEK KM was analyzed by SDS-PAGE and phosphorimaging
(top panel). Immunoprecipitated kinase was tested for
Ser-338 phosphorylation using a Ser-338 phospho-specific antibody
(second panel). The cell lysate was used for detection of
active ERK. Equal loading of lysates was confirmed by probing with an
ERK antibody. WB, Western blot.
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DISCUSSION |
Ras and numerous other signaling proteins have been shown to
affect c-Raf1 activity, but the mechanisms involved in the activation of the kinase in the cellular context are far from being understood. We
employed GTP
S to stimulate endogenous Ras and Raf in permeabilized U937 cells. Activation of Raf by GTP
S was fully dependent on Ras-GTP
formation, as evidenced by the block induced by the Ras-specific neutralizing antibody Y13-259 (Fig. 4). However, the GTP
S-induced Ras-mediated activation of Raf required the additional input of PI3K
since PI3K inhibitors were able to abolish Raf activity under these
conditions. Thus, two independent signals from Ras and PI3K appear to
be essential for full Raf activation.
Significantly, use of GTP
S as a stimulus throughout our experiments
allowed the exclusion of an effect of PI3K inhibition at the level of
Ras because PI3K controls Ras activation in U937 cells via GAP rather
than guanine nucleotide exchange factor (GEF) activities (16).
PI3K inhibition was reported to blunt basal and agonist-induced Ras
activation in intact U937 cells (16). Wennstrom and Downward (34)
describe similar effects in COS-7 fibroblast-like cells. The authors
demonstrated differential dose-dependencies of Ras-GTP formation and
ERK activation toward PI3K inhibition that is indicative of the
existence of a PI3K-dependent step between Ras and ERK
(34). Here we identified Raf as a target for multiple effects of PI3K
on the MAPK cascade. In addition to its effect on Ras-GTP accumulation
(16), a PI3K signal feeds into the MAPK cascade at the level of Raf.
Both inputs are evidently essential for the stimulation of the MAPK
cascade. Our data substantiate the highly integrative signaling
capacity of Raf.
In cells stimulated with GTP
S, treatment with genistein revealed
another c-Raf1 activation mechanism independent of Ras. We have
previously shown that basal guanine nucleotide exchange by Ras in U937
cells is completely inhibited by high micromolar concentrations of
genistein (16). Indeed, GTP
S did not induce accumulation of active
Ras in the genistein background (Fig. 3), but surprisingly, Raf
activation was unaltered. Furthermore the Ras-neutralizing antibody
Y13-259 did not block GTP
S-induced Raf activation in the
genistein-treated cells, corroborating a radically distinct,
Ras-independent Raf activation process. There are few reports in the
literature showing Ras-independent activation of Raf, and the
underlying mechanisms for these processes are so far poorly defined
(35-38). The data imply that G-protein(s) other than a
Y13-259-sensitive Ras species can activate Raf in a background of
tyrosine kinase inhibition. We have not been able to collect direct
evidence as to the nature of the relevant GTPase.
To further investigate the influence of PI3K on Raf activity, we
assessed phosphorylation of Ser-338 on Raf, which has been proposed to
play an important role in Raf activation (28, 39, 40). Recently, PI3Ks
have been implicated as mediators of the Ser-338 phospho-transfer
reaction in a pathway involving Rac and its effector PAK in
PAK-overexpressing COS7 cells (26, 27). In U937 cells challenged with
GTP
S, Ser-338 phosphorylation can proceed in the presence of PI3K
inhibitors. Moreover, TPA induces strong Ser-338 phosphorylation in
these cells, although it is well established that TPA does not activate
PI3K in U937 cells and related myeloid-derived cells as assessed at the
level of phosphatidylinositol 3,4,5-trisphosphate production (30) and PKB phosphorylation (data not shown). However, GTP
S induces
activation of Rac2, the predominant Rac isoform in leukocytes (41), and PAK to a similar extent in untreated and wortmannin-treated cells, showing that GTP
S-driven Rac/PAK activation does not rely on PI3K
activity. Experimental evidence suggests that PI3K can activate Rac via
phosphatidylinositol 3,4,5-trisphosphate-dependent
regulation of Rho-family guanine nucleotide exchange factors
(42), but also PI3K-independent mechanisms for Rac activation have been reported (43). Our findings show that the accumulation of active Rac in
response to GTP
S is PI3K-independent. The data point to an
involvement of tyrosine kinase(s) in the control of Rac activation since genistein treatment severely reduces GTP
S-driven activation of
Rac. The inhibition of Rac correlated well with a block of PAK
activation, providing indirect evidence for the existence of a Rac/PAK
axis in U937 cells.
GTP
S-dependent Ser-338 phosphorylation on Raf correlates
with Rac and PAK activity under all conditions tested (Fig. 5), providing circumstantial evidence for a causal link between PAK and
Raf-Ser-338 phosphorylation in U937 cells. However, PAK activity does
not correlate with Raf activity, and in particular, Raf activated as a
consequence of Ras-dependent and Ras-independent processes in U937 cells does not exhibit phosphorylation of Ser-338. In line with
these findings, Ser-338-phosphorylated Raf is located in the soluble
fraction of GTP
S-challenged U937 cells, whereas Raf with high
specific activity accumulates in the particulate fraction and is devoid
of Ser-338 phosphorylation. These findings are in agreement with the
study of Chiloeches et al. (44), which reports that in cells
overexpressing PAK, Ser-338-phosphorylated Raf was inactive and located
in the cytosol (44). In fact, it had been noted earlier also by others
that phosphorylation on Ser-338 does not always correlate with Raf
kinase activity (28, 29). Furthermore, we find that the majority of
Ser-338 phosphorylation does not interact with its downstream substrate
MEK and seems to be inactive as a MEK kinase (Fig. 7). Therefore, these
data suggest that Ser-338 phosphorylation is not necessary for Raf activation in U937 cells.
Our results point to the existence of several Raf activation mechanisms
in U937 cells that do not seem to include Ser-338 phosphorylation.
These findings contradict previous work that had made a strong case for
Ser-338 phosphorylation as a crucial step in Raf activation (28, 39,
40). All of these studies relied on transient transfection experiments
with activated versions of Ras and Src and/or serine 338 to alanine Raf
mutants. A caveat inherent to these approaches lies in the prolonged
expression of e.g. oncogenic versions of Ras or S338A Raf
mutants before the experiment. This may alter the regulatory mechanisms
under investigation, e.g. by activation of
feedback-mechanisms, affecting upstream regulators of Raf (45-47). Of
relevance to the present discussion, a feedback loop negatively
regulating Raf kinase activity has emerged in experiments utilizing the
novel Raf selective inhibitor ZM336372 (33). Hall-Jackson et
al. (33) obtain the seemingly paradoxical result that ZM336372
treatment of cells leads to the activation rather than inhibition of
Raf as assayed on immunoisolated Raf in vitro. In line with
these findings, we were able to demonstrate that ZM336372 treatment of
U937 cells led to strong activation of Raf in vitro kinase
activity but did not activate Raf in situ as judged by the
absence of phosphorylated ERK. Remarkably, ZM336372 did not induce
Ser-338 phosphorylation on Raf, indicating that Ser-338 phosphorylation
is not required for Raf activity in this feedback mechanism.
In our hands Ser-338 phosphorylation does not correlate with Raf
activation under all the conditions described above. Nevertheless these
findings cannot exclude that a punctual phosphorylation of Ser-338
might be needed transiently as an initial step in the activation of Raf
kinase after Ras binding, e.g. for recruiting binding
partners or facilitating a conformational change necessary for further
steps in the activation process. However, in our experiments the
majority of Ser-338-phosphorylated Raf is inactive, whereas Raf
activated under different conditions lacks Ser-338 phosphorylation.
In conclusion, our data demonstrate the existence of a
PI3K-dependent step in Raf activation that is distinct from
Ras-GTP or Rac-GTP formation and Ser-338 phosphorylation. Although many molecular details about the mechanisms of MAPK regulation by PI3K are
emerging, the general mode of the regulatory function of these enzymes
is still under discussion (34, 48). The contradictory data in the
literature might indicate that the phosphorylation event as well as the
activation of Raf in general are regulated in a complex cell type
and/or stimulus-dependent manner. Further efforts will be
necessary to clarify the pathways from PI3K leading to Raf and the
specific function of Ser-338 in the activation process of Raf.