Download E ect of SB 203580 on the activity of c-Raf in vitro and in vivo

Oncogene (1999) 18, 2047 ± 2054
ã 1999 Stockton Press All rights reserved 0950 ± 9232/99 $12.00
http://www.stockton-press.co.uk/onc
E€ect of SB 203580 on the activity of c-Raf in vitro and in vivo
Clare A Hall-Jackson*,1, Michel Goedert2, Philip Hedge3 and Philip Cohen1
1
MRC Protein Phosphorylation Unit, Department of Biochemistry, University of Dundee, MSI/WTB Complex, Dow Street,
Dundee DD1 5EH, Scotland; 2MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, England; 3Zeneca
Pharmaceuticals, Alderley Edge, Maccles®eld, Cheshire SK10 4TG, England
The inhibition of SAPK2a/p38 (a mitogen activated
protein (MAP) kinase family member) by SB 203580
depends on the presence of threonine at residue 106.
Nearly all other protein kinases are insensitive to this
drug because a more bulky residue occupies this site
(Eyers et al., 1998). Raf is one of the few protein kinases
that possesses threonine at this position, and we show
that SB 203580 inhibits c-Raf with an IC50 of 2 mM in
vitro. However, SB 203580 does not suppress either
growth factor or phorbol ester-induced activation of the
classical MAP kinase cascade in mammalian cells. One
of the reasons for this is that SB 203580 also triggers a
remarkable activation of c-Raf in vivo (when measured in
the absence of the drug). The SB 203580-induced
activation of c-Raf occurs without any increase in the
GTP-loading of Ras, is not prevented by inhibitors of the
MAPK cascade, protein kinase C or phosphatidylinositide 3-kinase, and is not triggered by the binding of this
drug to SAPK2a/p38. The paradoxical activation of cRaf by SB 203580 (and by another structurally unrelated
c-Raf inhibitor) suggests that inhibitors of the kinase
activity of c-Raf may not be e€ective as anti-cancer
drugs.
Keywords: c-Raf; MAP kinase; SB203580; EGF;
SAPK2; p38
Introduction
Protein kinases form one of the largest protein families
encoded in the human genome, and many of these
enzymes have become potential targets for drug
therapy because abnormal protein phosphorylation is
a cause or a consequence of many diseases. Several
relatively speci®c inhibitors of particular protein
kinases have been developed that have potential for
the treatment of cancer (Law and Lydon, 1996),
diabetes (Ishii et al., 1996), and hypertension (Uehata
et al., 1997). In addition, a class of pyridinyl imidazoles
has been identi®ed that suppress the synthesis (and
some of the actions) of proin¯ammatory cytokines (Lee
et al., 1994) and show promise for the treatment of
rheumatoid arthritis and other chronic in¯ammatory
conditions (Badger et al., 1996).
The pyridinyl imidazole SB 203580 is an inhibitor of
two stress-activated protein kinases (SAPKs) termed
SAPK2a (or p38an) and SAPK2b (or p38b2) (Cuenda
*Correspondence: CA Hall-Jackson
Received 2 July 1998; revised 5 November 1998; accepted 11
December 1998
et al., 1995; Goedert et al., 1997). These enzymes,
which display 75% amino acid sequence identity, are
members of the mitogen-activated protein (MAP)
kinase family, but other members of this gene family
are insensitive to SB 203580. These include SAPK3
(p38g) and SAPK4 (p38d) whose amino acid sequences
are 60% identical to SAPK2a/p38 or SAPK2b/p38b2
(Goedert et al., 1997). SB 203580 also fails to inhibit
many other protein kinases tested (Cuenda et al., 1995)
and, for this reason, it has been used extensively to
identify substrates and physiological roles of SAPK2/
p38 (reviewed in Cohen, 1997).
SB 203580 binds competitively with ATP (Young et
al., 1997) and the three-dimensional structure of
SAPK2a/p38a in a complex with closely related
pyridinyl imidazoles has established that these drugs
are inserted into the ATP-binding pocket of SAPK2a/
p38a (Tong et al., 1997; Wilson et al., 1997). However,
the 4-¯uorophenyl ring of the drug does not make
contact with residues in the ATP-binding pocket that
interact with ATP. One residue that makes contact
with the 4-¯uorophenyl moiety is Thr 106 and recent
studies have established that this residue plays a critical
role in determining sensitivity to SB 203580. Thus its
mutation to Met or Gln, which are present at the
equivalent position in other MAP kinase family
members, or to other residues with bulky side chains,
makes SAPK2a/p38a and SAPK2b/p38b2 insensitive
to SB 203580 (Wilson et al., 1997; Eyers et al., 1998;
Gum et al., 1998). Conversely, the mutation of this
residue to Thr in other MAP kinase family members
(SAPK1/JNK, SAPK3/p38g and SAPK4/p38d) makes
them sensitive to SB 203580 (Eyers et al., 1998; Gum et
al., 1998). Sensitivity to SB 203580 is greatly enhanced
when the side chain at residue 106 is smaller than Thr
(i.e. Ser, Ala or Gly).
Examination of the sequences of protein kinases in
the data bases reveals that a bulky residue is almost
always found at the position equivalent to Thr 106,
explaining why nearly all protein kinases are insensitive
to SB 203580. However, a small number of protein
kinases do have Thr at this position, and we have
shown that two of these enzymes, the Type-II TGFb
receptor and the protein tyrosine kinase lck, are
inhibited by SB 203580 (Eyers et al., 1998). In our
standard assay, conducted at 0.1 mM ATP, the IC50
values for the Type-II TGFb receptor and lck are 40
and 20 mM, respectively, 400 ± 800-fold higher than the
IC50 for inhibition of SAPK2a/p38a and 40 to 80-fold
higher than the IC50 for inhibition of SAPK2b/p38b2.
Nevertheless, the Type-II TGFb receptor becomes
insensitive to SB 203580 when the Thr is changed to
Met, and is inhibited 10-fold more potently when this
residue is changed to Ala (Eyers et al., 1998). Thus the
sensitivity of the Type-II TGFb receptor to SB 203580
Effect of SB203580 on Raf
CA Hall-Jackson et al
2048
also depends on the presence of Thr at the position
equivalent to Thr 106 of SAPK2a/p38. Two mammalian protein kinases (the Type-I TGFb receptor and an
Activin receptor) have Ser and not Thr at this position.
The Type-I TGFb receptor is inhibited by SB 203580
(IC50=20 mM) and sensitivity is abolished when this
residue is mutated to Met (Eyers et al., 1998). No
protein kinases in the public data bases have Ala or
Gly at this position.
SB 203580 does not a€ect the activation of the
classical MAP kinase cascade by a variety of agonists
(Cuenda et al., 1995; Gould et al., 1995; Hazzalin et
al., 1997). However, the protein kinase that is thought
to lie at the head of this cascade, the proto-oncogene cRaf, contains threonine at the position equivalent to
Thr 106 of SAPK2a/p38a. Here we show that c-Raf is
inhibited by SB 203580 in vitro with an IC50 of 2 mM, a
concentration that is 40-fold higher than the IC50 for
SAPK2a/p38a, but only fourfold higher than the IC50
for SAPK2b/p38b2. The failure of SB 203580 to
suppress activation of the MAP kinase cascade in vivo
may be related to a remarkable activation of c-Raf that
occurs when mammalian cells are incubated with this
drug.
Results
E€ect of SB 203580 on activated c-Raf in vitro
The sensitivity of protein kinases to SB 203580
depends on the presence of threonine, or a smaller
residue, at the position equivalent to Thr 106 of
SAPK2a/p38a (see Introduction). Since c-Raf is one
of a small number of protein kinases that possess
threonine at this position, we initially examined the
e€ect of SB 203580 on human c-Raf that had been
activated in insect Sf9 cells by cotransfection of its
DNA with DNA encoding v-Ras and the protein
tyrosine kinase Lck. c-Raf in Sf9 cell extracts is
inhibited by SB 203580 with an IC50 value of 2 mM
(Figure 1). This is 40-fold higher than the IC50 for
SAPK2a/p38a, but only four-fold higher than the
IC50 for SAPK2b/p38b2 (Eyers et al., 1998).
However, the IC50 values became 10-fold higher if
the assays were carried out using c-Raf immunoprecipitated from Sf9 cells (Figure 1), EGF-stimulated
mouse Swiss 3T3 cells (Figure 1) or EGF-stimulated
293 cells (data not shown). Similar results were
obtained if the Swiss 3T3 cells or the 293 cells were
stimulated with PMA (data not shown). Possible
reasons for this di€erence in sensitivity to SB 203580
are considered further under Discussion. The human
B-Raf isoform was also expressed in Sf9 cells and
was found to be inhibited with an IC50 of 7 mM
when assayed in the Sf9 cell lysates without
immunoprecipitation (Results not shown). SK&F
105809 (10 mM), a compound that is closely related
in structure to SB 203580 but does not inhibit
SAPK2a/p38a, also did not inhibit c-Raf or B-Raf
activity (data not shown). c-Raf and B-Raf are
assayed in a coupled assay containing MKK1 and
MAPK2. Neither of these coupling enzymes are
inhibited by SB 203580 (Cuenda et al., 1995).
The drug PD 98059 binds to MKK1 preventing its
phosphorylation and activation by c-Raf (Alessi et al.,
Figure 1 Inhibition of c-Raf activity by SB 203580 in vitro.
Human c-Raf was activated in Sf9 cells by cotransfection with
DNA encoding v-Ras and Lck, and activity was either measured
directly in the cell lysates (*) or after immunoprecipitation (*).
c-Raf was also activated by incubation of mouse Swiss 3T3 cells
for 2 min with 100 ng/ml EGF and assayed after immunoprecipitation (~). The results are presented relative to control
incubations in which SB 203580 was omitted (mean+s.e.m. for
three separate experiments)
1995a). Therefore, SB 203580 could inhibit c-Raf
activity either by binding to c-Raf or by binding to
MKK1. We distinguished between these possibilities,
by studying the e€ect of SB 203580 on the autophosphorylation of c-Raf that occurs when immunoprecipitates are incubated with MgATP. The autophosphorylation of c-Raf was prevented by
SB 203580 at concentrations similar to those that
prevented the activation of MKK1 (data not shown),
indicating that the drug exerts its e€ect by interacting
with c-Raf directly.
Failure of SB 203580 to prevent the activation of MKK1
or MAPK2 in mammalian cells
The ®nding that c-Raf is inhibited by SB 203580 at
micromolar concentrations was surprising because we
have reported previously that the activation of
MAPK1 and MAPK2 by NGF in rat PC12 cells
(Cuenda et al., 1995), by EGF or IGF-1 in human KB
cells (Cuenda et al., 1995; Gould et al., 1995), or by
EGF or phorbol esters in mouse C3H 10T1/2 cells
(Hazzalin et al., 1997), is not inhibited by SB 203580.
In the present study we con®rmed this result for EGFstimulated Swiss 3T3 cells (Figure 2) and IGF-1
stimulated rat L6 myotubes (data not shown). We
also showed for the ®rst time that SB 203580 has no
e€ect on the activation of MKK1 by EGF in Swiss 3T3
cells (Figure 2) at a concentration (10 mM) where it
inhibits the activity of c-Raf by 80% in vitro (Figure 1).
Thus the failure of SB 203580 to inhibit the activation
of MAPK2 is not explained by the activation of a
MAPK phosphatase.
Effect of SB203580 on Raf
CA Hall-Jackson et al
2049
Figure 2 E€ect of SB 203580 on the activation of MKK1 and MAPK2 in vivo. Con¯uent Swiss 3T3 cells were incubated for 18 h
in DMEM containing 0.5% FCS and then for 60 min with or without 10 mM SB 203580 or 50 mM PD 98059 followed by
stimulation for 5 min with 100 ng/ml EGF. The cells were lysed and MKK1 and MAPK2 activities measured after
immunoprecipitation. The results are presented as a percentage of the activity obtained after stimulation for 5 min with EGF,
and are presented as the mean+s.e.m. for three separate experiments. The batch of PD 98059 used in these experiments inhibits the
activation of MKK1 more potently that the sample used in our earlier work, which (at 50 mM) did not suppress the EGF-induced
activation of MAPK2 (Alessi et al., 1995a)
Paradoxical activation of c-Raf by SB 203580 in
mammalian cells
In order to investigate why SB 203580 does not prevent
the growth factor or PMA-induced activation of
MKK1 or MAPK2, we exposed Swiss 3T3 cells to
SB 203580 and then measured c-Raf activity in the
absence of SB 203580 after its immunoprecipitation
from the cell lysates. These experiments revealed that
SB 203580 had induced a remarkable increase in c-Raf
activity which was elevated about 30-fold after 60 min
(Figure 3a). The activation was similar to that attained
after stimulation for 2 min with EGF (Figure 3a),
which is the most potent activator of c-Raf in Swiss
3T3 cells (Alessi et al., 1995a). The activation of c-Raf
by EGF was transient, peaking after 2 min and
returning to near basal levels after 20 min (Figure
3a), but the activation of c-Raf by SB 203580 was
sustained for at least 60 min (Figure 3a). Similar results
were obtained in 293 cells, where exposure to
SB 203580 also increased c-Raf activity 30-fold
between 6 and 60 min (results not shown). In L6
myotubes, SB 203580 increased c-Raf activity 15-fold
after 60 min, similar to the activation of c-Raf induced
by stimulation for 5 min with IGF-1 (results not
shown). c-Raf that had been activated by incubation
of Swiss 3T3 cells with 10 mM SB 203580 and then
immunoprecipitated from the lysates, was just as
sensitive to SB 203580 as c-Raf immunoprecipitated
from the lysates of cells that had been stimulated by
EGF (data not shown). SK&F 105809 (10 mM), which
is closely related in structure to SB 203580 but does
not inhibit SAPK2a/p38a, was unable to induce any
activation c-Raf when Swiss 3T3 cells were incubated
for up to 60 min (data not shown).
Pretreatment of Swiss 3T3 cells for 60 min with
SB 203580 (in the absence of growth factors or PMA)
failed to induce any activation of MAPK2, but slightly
enhanced EGF-dependent activation of c-Raf (Figure
3a) and MAPK2 (Figure 3b). Pretreatment with
SB 203580 also failed to induce any activation of
MAPK2 in 293 cells or L6 myotubes (results not
shown).
Mechanism of activation of c-Raf by SB 203580
We have reported previously that PD 98059, a drug
that binds to MKK1 and thereby prevents its
activation by c-Raf, also stimulates the basal activity
of c-Raf several-fold as well as the rate and extent of cRaf activation by PDGF (Alessi et al., 1995a). These
®ndings suggest that the activity of c-Raf is suppressed
by an activated `downstream' component of the
classical MAP kinase pathway. However, SB 203580
does not induce any activation of MKK1 or MAPK2
(Figure 2). Moreover, incubation of Swiss 3T3 cells
with PD 98059 (50 mM), which prevents the activation
of MAPK2 by EGF (Figure 2), failed to prevent the
much larger activation of c-Raf induced by SB 203580
(Figure 4). Thus activation of c-Raf by SB 203580
occurs independently of the activation of the MAP
kinase cascade.
The activation of c-Raf by di€erent agonists can
result from the stimulation of several signalling
pathways, including the activation of protein kinase
C (PKC) (Morrison and Cutler, 1997) or phosphatidylinositide (PtdIns) 3-kinase (Cross et al., 1994).
However, c-Raf activation is una€ected by Ro 318220
(Figure 4), an inhibitor of PKC and some other protein
kinases (Alessi, 1997), under conditions where the
PMA-induced activation of MAPK2 measured in the
same cells was decreased by 85%. Similarly wortmannin, an inhibitor of PtdIns 3-kinase, does not a€ect the
activation of c-Raf by SB 203580 under conditions
Effect of SB203580 on Raf
CA Hall-Jackson et al
2050
Figure 4 E€ect of PD 98059, wortmannin and Ro 318220 on the
activation of c-Raf by SB 203580 in Swiss 3T3 cells. Con¯uent
cells were incubated in DMEM containing 0.5% FCS for 18 h
and then pretereated with (+) or without (7) 50 mM PD 98059
(60 min), 100 nM wortmannin (10 min) or 0.3 mM Ro 318220
(60 min) prior to incubation for 60 min with 10 mM SB 203580 in
the continued presence of these compounds. The results are
presented as a percentage of the activity obtained after incubation
for 60 min with SB 203580 in the absence of any other compound
Figure 3 E€ect of SB 203580 on the activation of c-Raf and
MAPK2 in vivo. Con¯uent Swiss 3T3 cells were incubated on 18 h
in DMEM containing 0.5% FCS then incubated for the times
indicated with 10 mM SB 203580 (*) or 100 ng/ml EGF (*).
Stimulation with EGF was also carried our after pretreatment for
60 min with 10 mM SB 203580 (~). The activities of c-Raf (a) and
MAPK (b) were then measured after immunoprecipitation from
the cell lysates. The results are presented as the mean+s.e.m. for
three separate experiments
where wortmannin suppresses IGF-1-induced activation of protein kinase B by 95%. Thus c-Raf activation
induced by SB 203580 does not appear to occur via
either of these pathways.
The activation of c-Raf by growth factors, such as
EGF, is dependent on the activation of Ras. In
unstimulated Swiss 3T3 cells the % of GTP-Ras
relative to GDP-Ras+GTP-Ras is 10.7+2.6%, which
increases to 44+2.4% after stimulation for 2 min with
EGF (Figure 5). However, after exposure to SB 203580
for 60 min, at which time c-Raf activation is maximal
(Figure 2), the proportion of GTP-Ras does not
increase at all (10.3+0.5%) (Figure 5). There was
also no increase in GTP-Ras after exposure of the cells
to SB 203580 for 2 or 10 min (data not shown). Thus
the activation of c-Raf by SB 203580 does not result
from an increase in the level of GTP-Ras.
Prolonged mitogenic stimulation leads to the
hyperphosphorylation of c-Raf, which can be visua-
Figure 5 Lack of e€ect of SB 203580 on GTP-loading of Ras in
Swiss 3T3 cells. Cells were 32p-labelled as described in Materials
and methods before stimulation with 100 ng/ml EGF (2 min) or
10 mM SB 203580 (60 min). Ras was immunoprecipitated from the
cell lysates and the bound guanine nucleotides eluted and
separated by thin layer chromatography. The ®gure shows an
autoradiograph of a representative experiment. Quantitation
(+s.e.m. for three separate experiments) is presented in the text
lised by a reduction in its electrophoretic mobility.
However, pretreatment of Swiss 3T3 cells with
SB 203580 for 6 ± 200 min does not induce any change
in the electrophoretic mobility of c-Raf (results not
shown).
Effect of SB203580 on Raf
CA Hall-Jackson et al
2051
Figure 6 E€ect of SB 203580 on the activities of MAPKAP
kinase-2 and c-Raf in 293 cells. Cells were incubated for 18 h in
DMEM containing 0.5% FCS prior to treatment for 60 min with
the indicated concentrations of SB 203580. (a) e€ect of SB 203580
on MAPKAP kinase-2 measured as a percentage of the activity
observed in the absence of the drug; (b) e€ect of SB 203580 on cRaf activity measured as fold activation relative to that obtained
in the absence of the drug. MAPKAP kinase-2 and c-Raf were
assayed after immunoprecipitation from cell lysates and the
results are presented as mean+s.e.m for three separate
experiments
The activation of c-Raf by SB 203580 is not caused by
interaction of the drug with SAPK2/p38a
Since SB 203580 inhibits SAPK2a/p38a and SAPK2b/
p38b (collectively termed SAPK2/p38), as well as cRaf, the activation of c-Raf by SB 203580 could have
resulted from the binding of this drug to SAPK2/p38,
rather than c-Raf. If SB 203580 exerted its e€ects via
interaction with SAPK2/p38, then signals that activate
SAPK2/p38 might have been expected to suppress the
activation of c-Raf by growth factors. However,
neither osmotic shock, UV-C irradiation or the
protein synthesis inhibitor anisomycin, which are all
potent activators of SAPK2/p38 in Swiss 3T3 and
other cells, have any e€ect on the EGF-induced
activation of MAPK2 (Doza et al., 1998).
Figure 7 E€ect of transfection with an SB 203580-insensitive
mutant of SAPK2a/p38 mutant on the activation of MAPKAP
kinase-2 and c-Raf by SB 203580. 293 cells were transfected with
Thr 106Met SAPK2a/p38a (hatched bars) or mock-transfected
(®lled bars). The cells were then incubated for 18 h in DMEM
containing 0.5% FCS prior to treatment for 60 min with (+) or
without (7) 1 mM SB 203580. (a) E€ect of SB 203580 on
MAPKAP kinase-2 activity; (b) e€ect of SB 203580 on c-Raf
activity. The protein kinases were assayed after their immunoprecipitation from the cell lysates and the results are presented as
mean+s.d. for a representative experiment. Similar results were
obtained in one other experiment
MAPKAP kinase-2, an immediate downstream
target of SAPK2/p38, is partially active in unstimulated 293 cells, and maximal suppression of this basal
level of activation is observed when the cells are ®rst
preincubated with 1 mM SB 203580 (Figure 6a). In
contrast, 1 mM SB 203580 usually induces about a
threefold activation of c-Raf and activation rises to
16-fold as the SB 203580 concentration is increased
to 10 mM (Figure 6b). These results are consistent
with the activation of MAPKAP kinase-2 being
blocked by the interaction of SB 203580 with
SAPK2/p38 and with c-Raf activation being triggered by the interaction of SB 203580 with another
Effect of SB203580 on Raf
CA Hall-Jackson et al
2052
protein that binds to the drug with lower anity
(presumably c-Raf itself).
In order to obtain further evidence that the
activation of c-Raf by SB 203580 results from its
interaction with c-Raf, and not with SAPK2/p38, we
transfected 293 cells with a SAPK2a/p38 mutant in
which Thr 106 had been changed to Met to make it
insensitive to low concentrations of SB 203580 (Wilson
et al., 1997; Eyers et al., 1998). In cells transfected with
this mutant, MAPKAP kinase-2 activity is elevated,
but can no longer be suppressed by 1 mM SB 203580 in
contrast to untransfected cells (Figure 7a). In contrast,
basal c-Raf activity increased several-fold after
transfection with the Thr 106Met mutant, but c-Raf
could still be activated further by incubating the cells
with SB 203580 (Figure 7b).
Discussion
In this paper we have shown that SB 203580 inhibits cRaf (Figure 1) and B-Raf (results not shown) at
micromolar concentrations, consistent with the sensitivity to this drug being determined by the presence of
threonine (or a smaller residue) at the position
equivalent to Thr 106 of SAPK2a/p38. The sensitivity
of c-Raf to SB 203580 was 10-fold greater if the
enzyme was assayed directly in Sf9 cell extracts than
when assayed after immunoprecipitation (Figure 1).
This suggests that the interaction of c-Raf with the
anti-c-Raf antibody decreases the anity of c-Raf for
the drug. An alternative explanation is that the
sensitivity of c-Raf to the drug is enhanced by its
interaction with a protein(s) that is present in the
extracts but removed by immunoprecipitation. The
latter explanation seems less likely because the c-Raf
assay employed is extremely sensitive and the Sf9 cell
extracts have to be diluted 30 000-fold to ensure that
initial rate conditions are met. However, whichever of
the two explanations is correct, the IC50 value measured
without immunoprecipitation is more likely to re¯ect
the situation in intact cells.
SB 203580 does not prevent the activation of MKK1
or MAPK2 by a variety of growth factors or phorbol
esters in many cells (e.g. Figure 2). One possible
explanation for these ®ndings is that the activity of
Raf isoforms are not rate-limiting for agonist-induced
activation of the MAP kinase cascade in the cells that
we have studied, and that, in the presence of SB 203580,
another growth factor/phorbol ester-stimulated protein
kinase(s) mediates the activation of MKK1. However,
another explanation is suggested by the observation that
incubation of mammalian cells with SB 203580 induces
a remarkable activation of c-Raf (when measured in the
absence of the drug). Thus Raf inhibition may be
counterbalanced by its reactivation. This e€ect is not
mediated by the interaction of SB 203580 with
SAPK2a/p38a (Figure 7b), but with another protein
that binds to SB 203580 with lower anity (Figure 6b).
This protein is likely to be Raf itself, because exactly the
same phenomenon is observed with another inhibitor of
c-Raf that is structurally unrelated to SB203580 (Clare
Hall-Jackson, Philip Cohen and Philip Hedge, unpublished work).
The activation of c-Raf by two structurally unrelated
c-Raf inhibitors suggests that, in the absence of growth
factors/phorbol esters, a mechanism may exist by
which c-Raf suppresses its own activation. This
putative feedback loop would have to involve a novel
pathway because it is una€ected by inhibitors of
protein kinase C, PtdIns 3-kinase or the MAP kinase
cascade (Figure 4), and does not involve changes in the
GTP loading of Ras (Figure 5). However, other
interpretations are possible. For example, the interaction of these drugs with c-Raf may cause c-Raf to
oligomerise (Farrar et al., 1996; Luo et al., 1996) or to
be targetted to the plasma membrane (Leevers et al.,
1994; Stokoe et al., 1994), which are well-established
mechanisms for inducing the activation of c-Raf.
It is also curious that incubation of Swiss 3T3 cells
for 60 min with SB 203580 does not trigger any
activation of MAPK2 and yet subsequent exposure to
EGF (in the continued presence of SB 203580), which
only increases c-Raf activity a further 3 ± 4-fold (Figure
3a), triggers essentially complete activation of MAPK2
(Figure 3b, Alessi et al., 1995a). This observation could
be explained if Raf activity was not rate-limiting for
the activation of MKK1 in these cells or if, in addition
to activating c-Raf per se EGF makes c-Raf competent
to activate MKK1 in vivo by facilitating the interaction
of these protein kinases in an unknown way.
Many oncogenes and tumour promoters are thought
to induce cell transformation, at least in part, by
causing inappropriate activation of the MAP kinase
cascade. Consistent with this view, the drug PD 98059
which interacts with MKK1 and prevents its activation
in vivo, reverses the transformed phenotype of several
Ras-transformed cell lines. However, SB 203580, as
well as another c-Raf inhibitor that bears no structural
relationship to SB 203580, fails to prevent growth
factor or phorbol ester-induced activation of MAP
kinase. The latter inhibitor also fails to reverse the
phenotype of Ras-transformed cell lines (P Hedge,
unpublished work). These observations suggest that
inhibitors of the kinase activity of Raf may not be
e€ective as anti-cancer drugs.
Materials and methods
Materials
Ro 318220 was a kind gift from Dr D Bradshaw (Roche
Pharmaceutical Company, Welwyn Garden City, UK),
PD 98059 and SB 203580 were purchased from Calbiochem and wortmannin and phorbol 12, 13 myristate
acetate (PMA) from Sigma. These drugs were dissolved
in dimethyl sulphoxide (DMSO) at concentrations of 10 ±
50 mM. They were either diluted appropriately in aqueous
bu€ers just prior to use, or added directly to the cell
culture media to achieve ®nal concentrations of 100 nM ±
50 mM. Control experiments contained the equivalent
amounts of DMSO which did not exceed 0.2% (v/v) in
any experiment. This concentration did not a€ect the
activity or activation of any protein kinase examined.
Epidermal growth factor (EGF) was purchased from
Boehringer Mannheim and the monoclonal anti-H-Ras
antibody (Y13 259) from Oncogene Science Products. The
antibodies against c-Raf (Alessi et al., 1995b), MAPKAP
kinase-2 (Clifton et al., 1996), MAP kinase kinase-1
(MKK1) and MAPK2 (also called ERK2) were raised in
sheep at the Scottish Antibody Production Unit (Carluke,
Lanarkshire, UK). The anti-MKK1 antibodies were raised
against the peptide PKKKPTPIQLNPAPDG correspond-
Effect of SB203580 on Raf
CA Hall-Jackson et al
ing to residues 2 ± 17 and the anti-MAPK2 antibodies
against the C-terminal peptide ± EETARFQPGYRS. All
peptides were conjugated to both bovine serum albumin
and keyhole limpet haemocyanin before injection into the
sheep.
Cell culture and stimulation
Con¯uent mouse Swiss 3T3 ®broblasts or human 293 cells
were incubated for 18 h in Dulbecco's modi®ed Eagles
medium (DMEM) containing 0.5% FCS. After treatment
with growth factors or inhibitors as indicated, each 10-cm
dish of cells was lysed in 0.4 ml of ice-cold Bu€er A
(20 mM Tris acetate, pH 7.5, 0.27 M sucrose, 1% (by mass)
Triton X-100, 1 mM EDTA, 1 mM EGTA, 50 mM sodium
¯uoride, 10 mM sodium b-glycerophosphate, 5 mM sodium
pyrophosphate, 1 mM sodium orthovanadate, 0.1% (by
vol) 2-mercaptoethanol, 0.1 mM phenylmethylsulphonyl
¯uoride, 1 mM benzamidine, 5 mg/ml leupeptin), and the
lysates frozen immediately in liquid nitrogen and stored at
7808C until use. Protein concentrations were determined
by the method of Bradford (1976).
Immunoprecipitation of protein kinases c-Raf was immunoprecipitated from 400 mg cell lysate protein, MKK1 from
100 mg of cell lysate and MAPK2 and MAPKAP kinase-2
from 50 mg cell lysate protein using 5 ml of protein GSepharose conjugated to 4 mg of the appropriate antibody
(Alessi et al., 1995b; Clifton et al., 1996).
Assay of protein kinases c-Raf, MKK1 and MAPK2
(Alessi et al., 1995b) and MAPKAP kinase-2 (Clifton et
al., 1996) were assayed as described previously. c-Raf
activity was measured in a coupled assay containing
MKK1, MAPK2 and its substrate myelin basic protein
(MBP). One unit of c-Raf activity was that amount which
increased the activity of MAPK2 by 1 unit/min. MKK1
was assayed by the activation of bacterially expressed
MAPK2 (Alessi et al., 1995b). One unit of MKK1 activity
was that amount which increased the activity of MAPK2
by 1 unit/min. One unit of MAPK activity was that
amount which catalysed the phosphorylation of 1 nmol of
MBP in one min.
Measurement of guanine nucleotides bound to Ras Confluent
Swiss 3T3 cells were incubated for 18 h in DMEM
containing 0.5% FCS, then for 1 h in phosphate-free
DMEM before labelling for 4 h with 32P-orthophosphate
(5 mCi per 10-cm dish). After stimulation, cells were
washed with ice-cold 20 mM Tris/HCl pH 7.5, 150 mM
NaCl and lysed in Bu€er B (50 mM HEPES, pH 7.4,
100 mM NaCl, 5 mM MgCl2, 1 mg/ml Bovine Serum
Albumin (BSA), 1 mM sodium phosphate, complete
proteinase inhibitor cocktail (Boehringer Mannheim),
100 mM GTP, 100 mM GDP, 1 mM ATP) containing 1%
(by mass) Triton X-114 (Boehringer Mannheim). Nuclei
were removed by centrifugation for 5 min at 13 000 g, and
the supernatant incubated for 3 min at 378C and
centrifuged for 2 min at 13 000 g to separate the Triton
X-114 and aqueous phases. The detergent phase was
diluted 10-fold with Bu€er B plus 0.5 M NaCl. The lysate
was precleared with protein G-Sepharose coupled to rabbit
pre-immune serum (15 min) and Ras immunoprecipitated
using 25 ml protein G-Sepharose beads coupled to 10 mg
anti-H-Ras antibody (60 min). The immunoprecipitates
were washed eight times with Bu€er C (50 mM HEPES,
pH 7.4, 500 mM NaCl, 5 mM MgCl2, 0.1% Triton X-100,
0.005% SDS). GTP and GDP were eluted by incubation
for 2 min at 708C in 2 mM EDTA, 2 mM DTT, 0.2% (by
mass) SDS, 0.5 mM GDP, 0.5 mM GTP and separated on
polyethyleneimine (PEI) cellulose plates (Merck) developed
in 1.2 M ammonium formate, 0.8 M HCl. Plates were
autoradiographed and the radioactive spots cut out and
analysed by Cerenkov counting (Burgering et al., 1991).
Immunoblot analysis Cell lysates (7.5 mg protein) were
separated on 10% polyacrylamide gels and immunoblotted
using 2 mg/ml anti-c-Raf antibody.
SAPK2a/p38a expression vectors Human SAPK2a/p38a
(CSBP2 isoform) and Thr106Met SAPK2a/p38a with a
haemaglutinnin (HA)-tag (YPYDVPDYA) at the amino
terminus were produced by PCR ampli®cation, using the
previously described constructs (Eyers et al., 1998) as
templates. Following DNA sequencing, the ampli®ed
DNAs were subcloned into the eukaryotic vector pCMV5
(Invitrogen).
Transfection of HA-Thr106Met SAPK2a/p38 293 cells
were transfected with the pCMV5 HA-Thr106Met
SAPK2a/p38a construct using a modi®ed calcium phosphate method (Alessi et al., 1996). At 24 h after
transfection, the cells were incubated for a further 18 h
in DMEM plus 0.5% FCS, treated for 1 h with 1 mM
SB 203580 and lysed as for Swiss 3T3 cells (see `Cell
Culture and Stimulation'). `Mock' transfected cells (i.e.
without the addition of DNA) were subjected to the same
transfection procedure in parallel experiments.
Acknowledgements
We thank Ulf Rapp for baculovirus vectors expressing Raf,
Ras and Lck and Andrew Patterson for Sf9 cells expressing
activated Raf. This work was supported by the UK
Medical Research Council (PC and MG), the Royal
Society (PC) and the Louis Jeantet Foundation (PC).
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