(Received for publication, May 9, 1995; and in revised form, August 8, 1995)
From the
PD 098059 has been shown previously to inhibit the
dephosphorylated form of mitogen-activated protein kinase kinase-1
(MAPKK1) and a mutant MAPKK1(S217E,S221E), which has low levels of
constitutive activity (Dudley, D. T., Pang, L., Decker, S. J., Bridges,
A. J., and Saltiel, A. R.(1995) Proc. Natl. Acad. Sci. U. S. A. 92, 7686-7689). Here we report that PD 098059 does not
inhibit Raf-activated MAPKK1 but that it prevents the activation of
MAPKK1 by Raf or MEK kinase in vitro at concentrations
(IC = 2-7 µM) similar to those
concentrations that inhibit dephosphorylated MAPKK1 or
MAPKK1(S217E,S221E). PD 098059 inhibited the activation of MAPKK2 by
Raf with a much higher IC
value (50 µM) and
did not inhibit the phosphorylation of other Raf or MEK kinase
substrates, indicating that it exerts its effect by binding to the
inactive form of MAPKK1. PD 098059 also acts as a specific inhibitor of
the activation of MAPKK in Swiss 3T3 cells, suppressing by 80-90%
its activation by a variety of agonists. The high degree of specificity
of PD 098059 in vitro and in vivo is indicated by its
failure to inhibit 18 protein Ser/Thr kinases (including two other
MAPKK homologues) in vitro by its failure to inhibit the in vivo activation of MAPKK and MAP kinase homologues that
participate in stress and interleukin-1-stimulated kinase cascades in
KB and PC12 cells, and by lack of inhibition of the activation of p70
S6 kinase by insulin or epidermal growth factor in Swiss 3T3 cells. PD
098059 (50 µM) inhibited the activation of p42
and isoforms of MAP kinase-activated protein kinase-1 in Swiss
3T3 cells, but the extent of inhibition depended on how potently c-Raf
and MAPKK were activated by any particular agonist and demonstrated the
enormous amplification potential of this kinase cascade. PD 098059 not
only failed to inhibit the activation of Raf by platelet-derived growth
factor, serum, insulin, and phorbol esters in Swiss 3T3 cells but
actually enhanced Raf activity. The rate of activation of Raf by
platelet-derived growth factor was increased 3-fold, and the subsequent
inactivation that occurred after 10 min was prevented. These results
indicate that the activation of Raf is suppressed and that its
inactivation is accelerated by a downstream component(s) of the MAP
kinase pathway.
Stimulation of cells with growth factors and cytokines, or
exposure to cellular stresses, activates several signal transduction
pathways that have specific physiological roles. These include at least
three in which a mitogen-activated protein kinase (MAPK) ()homologue is involved. In one pathway, cell stimulation
leads to the sequential activation of p21
and
the protein kinases c-Raf, MAP kinase kinase-1 and -2 (MAPKK1, MAPKK2),
and p42 and p44 MAP kinases (p42
, p44
).
These MAPKs phosphorylate a variety of proteins in vivo including MAP kinase-activated protein (MAPKAP) kinases 1
and
1
(also known as Rsk-1 and Rsk-2(1) ). The sustained
activation of p42/p44
is not only required, but it is
sufficient to induce the proliferation or differentiation of several
cells(2) .
In order to dissect MAPK pathways and to
elucidate their physiological roles, one approach has been to generate
dominant negative mutants and overexpress them in cells. For example,
dominant negative forms of p21, c-Raf, and
MAPKK1 all inhibit the activation of p42/p44
and the
growth factor-induced proliferation or differentiation of several
cells(3) . However, although dominant-negative mutants are
useful, the generation of cell lines that stably express them is time
consuming, and their expression may lead to erroneous conclusions. For
example, overexpression of an inactive form of MAPKK1 that can be
phosphorylated by Raf may not only prevent the activation of endogenous
wild-type MAPKK1, but also the activation of other cellular substrates
of Raf that might lie in distinct signaling pathways. The need to
express a dominant negative mutant for many hours may also result in
unwanted secondary effects. Similarly, the use of dominant negative
mutants of Raf may affect Ras-dependent processes that are independent
of Raf.
An alternative strategy is to identify small cell-permeant molecules that are specific inhibitors of particular protein kinases. An advantage of this approach is that the effects of these inhibitors can be investigated in any cell in vivo. Moreover, these inhibitors may have therapeutic potential as anti-cancer, or anti-inflammatory agents, or as immunosuppressants. Several such inhibitors have recently been described, including an inhibitor of the epidermal growth factor (EGF) receptor tyrosine kinase(4) , which may be useful for treating human tumors that overexpress this receptor, and a specific inhibitor of the MAP kinase homologue termed reactivating kinase (RK) or p38(5) . The latter inhibitor prevents the synthesis of interleukin-1 (IL-1) and tumor necrosis factor in monocytes induced by bacterial endotoxins and also blocks several actions of these cytokines on other cells(6) . For these reasons, it may be efficaceous in the treatment of inflammatory diseases, such as rheumatoid arthritis. Another compound, rapamycin, prevents the IL-2-induced activation of p70 S6 kinase and the proliferation of T-cells (7, 8) and may therefore be useful as an immunosuppressant.
One of our laboratories recently described the first synthetic inhibitor of the MAP kinase pathway. This substance, PD 098059 (Fig. 1) was identified as a noncompetitive inhibitor of MAP kinase kinase (MAPKK) by screening a compound library using a MAP kinase cascade assay comprising unphosphorylated MAPKK1 (which possesses low basal activity) and unphosphorylated MAP kinase and monitoring phosphorylation of myelin basic protein. PD 098059 was subsequently demonstrated to inhibit the constitutively active mutant (MAPKK1(S217E,S221E)) in which the serine residues phosphorylated by c-Raf had been mutated to glutamic acid(9) . PD 098059 inhibited the activation of MAP kinase by growth factors in vivo and, consistent with a key role for this pathway in the proliferation of some cells and the differentiation of others, it reversed the transformed phenotype induced by Ras overexpression in KRNK and K-balb cells (9) or the nerve growth factor (NGF)-induced differentiation of PC12 cells(10) . We now report that although PD 098059 inhibits MAPKK1(S217E, S221E), it surprisingly has no effect on MAPKK1 or MAPKK2 that have been activated by Raf. This remarkable finding has led us to demonstrate that PD 098059 blocks the activation of MAPKK1 by Raf or MEK kinase in vitro and that it acts in vivo as a highly specific inhibitor of the activation of MAPKK.
Figure 1: Structure of PD 098059.
p42 was assayed
by its ability to phosphorylate myelin basic protein (16) and
MAPKAP kinase 1
/
by the phosphorylation of a peptide related
to the C terminus of ribosomal protein S6
[Gly-245,Gly-246]S6-(218-249)(1, 17) .
One unit of p42
or MAPKAP kinase-1
/
was that
amount which catalyzed the phosphorylation of 1 nmol of substrate
peptide in 1 min. Protein kinase activities in immunoprecipitates were
measured by adding the other assay components to the tubes containing
the immunoprecipitated enzyme.
RK kinase from KB cells and MKK4 from
PC12 cells were assayed by the activation of RK(18) . RK
itself(18) , MAPKAP kinase-2(18) , p70 S6
kinase(19) , protein kinase A(20) , protein kinase
C(21) , 5`-AMP-activated protein kinase(22) ,
cyclin A cyclin-dependent protein kinase-2(23) , phosphorylase
kinase(24) , glycogen synthase kinase-3
/
(17) , and myosin light chain kinase (25) were assayed as described previously. c-Jun kinase (JNK)
from IL-1-stimulated KB cells was assayed using a
GST-c-Jun-(1-191) fusion protein(26) , a gift from Dr. R.
Treisman (Imperial Cancer Research Fund, London).
PD 098059 was identified during
a screen to identify inhibitors of dephospho-MAPKK and was subsequently
found to inhibit MAPKK(S217E,S221E). PD 098059 inhibited both of these
enzymes with an IC of 2 µM. Inhibition was
essentially complete at 50 µM ((9) ; see Fig. 2) and unaffected by prior incubation of bacterially
expressed dephospho-MAPKK1 with PP2A under conditions that completely
inactivated the phosphorylated form of MAPKK1 (data not shown).
However, to our surprise, PD 098059 did not inhibit MAPKK1 that had
been phosphorylated by c-Raf in vitro or MAPKK activity in
cell lysates prepared from EGF-stimulated Swiss 3T3 cells (Fig. 2).
Figure 2: Effect of PD 098059 on the activity of different forms of MAPKK1. MAPKK activity was assayed after preincubation for 10 min with the indicated concentrations of PD 098059, and the results are presented relative to control incubations in which the inhibitor was omitted. The concentrations of MAPKK1 in the assays were as follows: dephospho-MAPKK1, 0.3 µM; MAPKK1(S217E,S217E), 10 nM; Raf-activated MAPKK1, 0.2 nM. Closed circles, MAPKK assayed in lysates from Swiss 3T3 cells stimulated for 2 min with EGF; open circles, MAPKK1 maximally phosphorylated at Ser-217 and Ser-221 by c-Raf; closed triangles, dephosphorylated MAPKK1; open triangles, MAPKK1(S217E,S221E). MAPKK1 maximally phosphorylated at Ser-217 and Ser-221 has a 7000-fold higher activity than the dephosphorylated enzyme and 180-fold higher activity than MAPKK1(S217E,S221E). Each MAPKK preparation was diluted to give similar MAPKK activity in the assay in the absence of PD 098059. Similar results were obtained in three experiments.
Since the phosphorylated form of MAPKK1 has a far
higher activity than either MAPKK1(S217E,S221E) or dephospho-MAPKK1,
these results suggested that PD 098059 might interact specifically with
the inactive conformation of MAPKK and prompted us to investigate its
effect on the activation of MAPKK1 in vitro. PD 098059
inhibited both the activation (Fig. 3A) and phosphorylation (Fig. 3B) of MAPKK1 in vitro by either c-Raf or
MEK kinase with IC values of 4 ± 2 µM,
similar to the IC
for inhibition of dephospho-MAPKK1 or
MAPKK1(S217E,S221E) (Fig. 2). Identical results were obtained
using c-Raf expressed in insect Sf9 cells (Fig. 3) or
immunoprecipitated from EGF-stimulated Swiss 3T3 cells (not shown). The
activation of MAPKK2 by c-Raf was also inhibited by PD 098059 but over
10 times less potently (IC
= 50 µM)
than MAPKK1. Like Raf-activated MAPKK1, Raf-activated MAPKK2 was not
inhibited by 50 µM PD 098059 (see Table 2).
Figure 3: Effect of PD 098059 on the activation and phosphorylation of MAPKK1 by c-Raf and MEK kinase in vitro. A, activation by c-Raf and MEK kinase. c-Raf and MEK kinase were diluted to achieve an equivalent reactivation of MAPKK1 in the absence of any inhibitor (100%). The MAPKK1 concentration in the assays was 0.3 µM. After a 10-min preincubation with the indicated concentration of PD 098059, c-Raf (open circles) and MEK kinase (closed circles) were assayed by the activation of MAPKK1. Similar results were obtained in three experiments. B, same as A, except that c-Raf or MEK kinase were assayed by the phosphorylation of MAPKK1 (see ``Experimental Procedures''). Similar results were obtained in two experiments.
In contrast to the inhibition of MAPKK1 phosphorylation by c-Raf and MEK kinase, PD 098059 did not inhibit the (weak) phosphorylation of myelin basic protein and casein (29) by c-Raf, the autophosphorylation of c-Raf, or the activation of MKK4 by MEK kinase (data not shown).
The
effect of 50 µM PD 098059 on activation of the downstream
targets of MAPKK, namely p42 and MAPKAP kinases
1
/
varied from agonist to agonist (Table 1). PD 098059
(50 µM) prevented the activation of p42
and
MAPKAP kinase-1
/
by insulin (the weakest activator of MAPKK)
almost completely, but with PDGF, TPA, and serum (which are stronger
activators of MAPKK) the activation of p42
and MAPKAP
kinases-1
/
was only inhibited by 70-80% and
26-52%, respectively. After stimulation for 5 min with EGF (the
strongest activator of MAPKK) the activation of p42
and
MAPKAP kinase-1
/
was only inhibited by 33 and 13%,
respectively (Table 1). However, as the concentration of EGF was
reduced, the extent of inhibition of p42
activation by
50 µM PD 098059 increased progressively. After stimulation
for 10 min with 100 ng/ml EGF, the inhibition of p42
activation was only 8%, but at 0.1 ng/ml and 0.01 ng/ml EGF it
was 89 and 100%, respectively (Fig. 4). PD 098059 cannot be used
at higher concentrations because of its low solubility in aqueous
solution. These results are considered further under
``Discussion.''
Figure 4:
Effect of PD 098059 on the activation of
p42 in Swiss 3T3 cells at different concentrations of
EGF. Cells were incubated for 90 min in the absence (open
circles) or presence (closed circles) of 50 µM PD 098059 and stimulated for 10 min at the indicated
concentrations of EGF. The cells were lysed, and p42
was
immunoprecipitated and assayed as described under ``Experimental
Procedures.'' The p42
activity is plotted relative
to the specific activity of p42
in the lysate after
stimulation with 100 ng/ml EGF in the absence of PD 098059, which was
0.45 ± 0.02 units/mg.
Figure 5:
PD 098059 has no effect on the activation
of JNK, RK kinase, RK, MAPKAP kinase-2, or p70in
vivo. Panel A, KB cells were incubated for 90 min in the
absence(-) or presence (+) of 50 µM PD 098059
and then stimulated for 15 min with 20 ng/ml IL-1 or 0.5 mM sodium arsenite (A). The cells were lysed and assayed for
JNK activity using GST-Jun-(1-191) as substrate. After
phosphorylation, reactions were denatured in SDS, electrophoresed on a
10% polyacrylamide gel, and autoradiographed. Similar results were
obtained in two experiments. Panel B, KB cells were stimulated
in the absence (C) or presence (A) of arsenite and in
the absence(-) and presence (+) of PD 098059 as in panel
A. The cell lysates were chromatographed on Mono Q and assayed for
RK activity or chromatographed on Mono S and assayed for RK kinase and
MAPKAP kinase-2. The results are given ± S.E. for the number of
experiments shown, as a percentage of the arsenite-stimulated activity
in the absence of PD 098059. Panel C, Swiss 3T3 cells were
incubated in the absence or presence of 50 µM PD 098059 as
in panel A and then for 5 min in the presence (+) or
absence(-) of 100 nM rapamycin prior to stimulation for
15 min with 100 ng/ml EGF (E), 100 ng/ml, insulin (I), or buffer (C, control). The cells were lysed and
assayed for p70
activity after immunoprecipitation. The
results are given ± S.E. for three
experiments.
Figure 6: Effect of PD 098059 on the activation and inactivation of c-Raf by PDGF in Swiss 3T3 cells. Cells were incubated for 90 min in the presence (closed circles) or absence (open circles) of PD 098059 and then stimulated for the times indicated with 50 ng/ml PDGF. The cells were lysed and assayed in triplicate for c-Raf activity after immunoprecipitating the enzyme from the lysates. The results are given ± S.E. for three experiments.
Although PD 098059 inhibits forms of MAPKK1 with a low level of activity (dephospho-MAPKK1 andMAPKK1(S217E,S221E)), we demonstrate in this paper that it does not inhibit the phosphorylated forms of MAPKK1 but instead prevents the activation and phosphorylation of MAPKK1 in vitro (Fig. 3) and in vivo (Table 1). These findings, coupled with the failure to inhibit the phosphorylation of other substrates of c-Raf and MEK kinase, and lack of competition with ATP(9) , indicate that PD 098059 does not bind to the active site of MAPKK1 but instead interacts at another site, thereby blocking access to activating enzymes. It will be interesting to find out whether PD 098059 binds to the activation loop of MAPKK1 in the vicinity of the phosphorylation sites, and our results raise the possibility that PD 098059 may mimic or displace an endogenous allosteric effector of these enzymes. Our observations explain the high degree of specificity of PD 098059, which only inhibits the activation of MAPKK2 weakly and does not affect the activities of 18 protein Ser/Thr kinases (Table 2), four protein Tyr kinases, and phosphatidylinositol 3-kinase (9) that have so far been tested. Moreover PD 098059 does not prevent the in vivo activation of Raf (Table 1, Fig. 6) or the activation of other MAPKK or MAPK homologues (Fig. 5, A and B), which lie in stress- and cytokine-activated signaling pathways, or the insulin-induced or EGF-induced activation of p70 S6 kinase (Fig. 5C).
Our results also demonstrate that inhibitors can be identified that differentiate between the active and inactive conformations of protein kinases. These observations could be of general significance and suggest that when screening for inhibitors of protein kinases, weakly active dephosphoenzymes or mutant enzymes should always be examined in parallel with the fully active phosphoenzyme. Indeed, inhibitors that interact with dephosphoenzymes and prevent their activation by upstream kinases may frequently turn out to be more specific inhibitors than compounds that block catalytic activity per se.
Although PD 098059 suppressed the
activation of MAPKK in Swiss 3T3 cells by 80-90%, its effect on
the activation of p42 and MAPKAP kinase 1
/
in vivo depended on the strength of activation of c-Raf and
MAPKK by any agonist. PD 098059 (50 µM) prevented the
activation of p42
and MAPKAP kinase-1
/
by
insulin (Table 1) or by low levels (0.01-0.1 ng/ml) of EGF (Fig. 4) almost completely, which were the weakest activators of
these enzymes. PD 098059 (50 µM) partially suppressed the
activation of p42
and MAPKAP kinase-1
/
by
PDGF, TPA, or serum but had little effect on the activation of these
enzymes at high concentrations of EGF (100 ng/ml), the most potent
activator of c-Raf and MAPKK (Table 1). Although PD 098059 (50
µM) suppressed by 85-90% the activation of MAPKK at
high EGF, the activity remaining was still 50% of that observed after
stimulation with PDGF, TPA, or serum and much higher than that observed
after stimulation with insulin. This explains why the activation of
p42
and MAPKAP kinase-1
/
at high EGF is hardly
affected by 50 µM PD 098059 (Table 1).
PD 098059
(50 µM) does not inhibit the in vivo activation
of MAPK and MAPKAP kinase-1 completely when cells are stimulated with
high concentrations of agonists that are potent activators of MAPKK,
and its low solubility in aqueous solution precludes its use at higher
concentrations. An effect of PD 098059 on some biological processes may
therefore only be revealed at low agonist concentrations or by using
cell lines with low numbers of growth factor receptors. For example, we
have noticed that PD 098059 inhibits the NGF-induced differentiation of
some PC12 cells (10) but not others, ()and this may
reflect the different numbers of NGF receptors (and hence the strength
of activation of MAPKK by NGF) in these cell lines. Indeed, in the PC12
cell line where PD 098059 failed to inhibit differentiation, the
activation of MAPK by NGF was only suppressed by 50% after 15 min,
despite an 80% inhibition of MAPKK by PD 098059 after 5 min.
It has been established that the sustained activation of MAPK is
not only required, but is sufficient to induce the differentiation of
PC12 cells(2, 3) .
The results presented in Table 1emphasize how little activation of MAPKK is needed to
produce significant activation of p42 and especially of
MAPKAP kinase 1
/
. For example, either insulin in the absence
of PD 098059, or PDGF, TPA, and serum in the presence of PD 098059,
caused only a 0.3-0.9% conversion of MAPKK to the activated form,
yet this was sufficient to cause 13-21% conversion of
p42
to the activated form and 34-77% conversion of
MAPKAP kinase 1
to the activated form. PD 098059 would appear to
be particularly useful for studying the role of the MAP kinase pathway
in the biological actions of insulin, since it essentially abolished
the activation of MAPK and MAPKAP kinase-1 by insulin in Swiss 3T3 (Table 1) and L6 cells (30) .
An unexpected observation was that PD 098059 enhanced the basal activity of c-Raf and its activation by growth factors in Swiss 3T3 (Table 1) and L6 cells (data not shown) and prevented the inactivation of c-Raf in Swiss 3T3 cells that occurred after stimulation with PDGF for 10 min (Fig. 6). This result suggests that the rate of activation of c-Raf is suppressed and that its rate of inactivation is enhanced by a component of the kinase cascade downstream of Raf. We have also observed that PD 098059 blocks the hyperphosphorylation of c-Raf induced by PDGF in Swiss 3T3 cells and by IGF-1 in L6 cells (data not shown), suggesting that a kinase downstream of c-Raf may be responsible for hyperphosphorylation. These results are also consistent with the increasing evidence that growth factor-induced hyperphosphorylation of c-Raf does not correlate with activation(31, 32) . Furthermore, Ueki et al.(33) reported that the overexpression of MAPK in Chinese hamster ovary cells attenuated the activation of c-Raf and enhanced its hyperphosphorylation by insulin. Although the hyperphosphorylation of c-Raf might contribute to its inactivation, other explanations are possible. For example, activation of MAPK by a variety of growth factors causes hyperphosphorylation of the GTP/GDP exchange factor (Sos), which catalyzes the activation of Ras(34, 35) . Moreover, hyperphosphorylation of Sos in stimulated cells results in its dissociation from GRB2 and hence to the inactivation of Ras(34, 35) . Treatment of L6 cells with PD 098059 inhibits the hyperphosphorylation of Sos following insulin stimulation(30) . Therefore, PD 098059 by inhibiting the activation of MAPK and hence the hyperphosphorylation of Sos in growth factor-stimulated cells may prevent the dissociation of Sos from GRB2 and thus block the inactivation of Ras that would result in a sustained activation of c-Raf (Fig. 6).