From the
The mitogen-activated protein kinase (MAP kinase) pathway is
thought to play an important role in the actions of neurotrophins. A
small molecule inhibitor of the upstream kinase activator of MAP
kinase, MAP kinase kinase (MEK) was examined for its effect on the
cellular action of nerve growth factor (NGF) in PC-12 pheochromocytoma
cells. PD98059 selectively blocks the activity of MEK, inhibiting both
the phosphorylation and activation of MAP kinases in vitro.
Pretreatment of PC-12 cells with the compound completely blocked the
4-fold increase in MAP kinase activity produced by NGF. Half-maximal
inhibition was observed at 2 µM PD98059, with maximal
effects at 10-100 µM. The tyrosine phosphorylation
of immunoprecipitated MAP kinase was also completely blocked by the
compound. In contrast, the compound was without effect on NGF-dependent
tyrosine phosphorylation of the pp140
In the past few years, the mechanisms by which neurotrophins,
such as nerve growth factor (NGF),
The
precise role of the MAP kinase pathway in growth factor action remains
unclear. This enzyme is activated by a wide variety of hormones,
cytokines, and growth factors and in turn directs the phosphorylation
of transcription factors, such as c-Jun and
p62
Although these data
provide compelling evidence supporting a critical role for the MAP
kinase pathway in neuronal differentiation, the possibility remains
that transfected cells may adapt inappropriately to overexpression of
these mutants. In this report we describe the use of a specific
inhibitor of MEK to completely block the biological effect of NGF in
PC-12 cells, providing additional pharmacological evidence that the MAP
kinase pathway is required for the differentiative effects of
neurotrophins.
PD98059 was discovered by screening a chemical library for
inhibitors of the MAP kinase cascade. Its structure is shown in
Fig. 1
. This compound is a selective inhibitor of MAP kinase
kinase or MEK.
The stimulation by hormones of the MAP kinase pathway is
thought to be involved in a number of cellular changes, including both
growth and differentiation. In PC-12 cells, agents such as NGF that
induce neuronal differentiation lead to the persistent
activation
(5, 33) and subsequent nuclear translocation
(26) of the enzyme. In contrast, receptors for other growth
factors, such as EGF, that only transiently activate MAP kinase and do
not induce its nuclear translocation do not result in neuronal
differentiation
(26) . Such correlative data were significantly
extended in recent experiments showing that expression in PC-12 cells
of a constitutively active MEK mutant can itself induce
differentiation
(24) , suggesting that overactivation of this
pathway is sufficient to induce this morphological phenotype. Moreover,
transfection of PC-12 cells with a mutant MEK in which the two
phosphorylation sites required for activation (serine 218/222) were
substituted with alanine produced a dominant interfering phenotype in
which NGF did not induce differentiation
(24) . Although such
experiments strongly suggest that MEK activation is essential for this
cellular action of NGF, it is important to note that overexpression of
dominant interfering mutants such as these are likely to bind but not
dissociate from upstream activating kinases, preventing these kinases
from phosphorylating their endogenous substrates. Thus, these data
indicate an essential role for MEK kinases in neuronal differentiation,
leaving open the issue of the necessity for MEK itself.
In order to
answer this question, we have utilized a novel, small molecular weight
inhibitor of MEK activity, PD98059. This compound can block the
activity of MEK assayed both in vitro with recombinant enzyme
and in a variety of intact cells.
We thank Drs. David Dudley, Alex Bridges, and Kunliang
Guan for helpful discussions.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
receptor
or its substrate Shc and did not block NGF-dependent activation of
phosphatidylinositol 3`-kinase. However, PD98059completely blocked
NGF-induced neurite formation in these cells without altering cell
viability. These data indicate that the MAP kinase pathway is
absolutely required for NGF-induced neuronal differentiation in PC-12
cells.
(
)
promote the
survival and development of sympathetic and sensory neurons have been
the focus of much inquiry. Although a wide variety of signaling
processes may be involved, protein phosphorylation appears to play a
prominent role
(1) . The NGF receptor,
pp140
, is a tyrosine kinase that
undergoes autophosphorylation in response to
NGF
(2, 3, 4) . Activation of this kinase
initiates a cascade of molecular interactions, ultimately resulting in
stimulation of the activity of a number of serine/threonine kinases,
including the mitogen-activated protein (MAP)
kinase
(5, 6) . The activation of this kinase is
catalyzed by MAP kinase kinase or MEK, which phosphorylates MAP kinase
on threonine and tyrosine residues
(7, 8) . MEK is itself
phosphorylated and activated by an upstream kinase or kinases that
appear to require activation of the ras protooncogene
(9, 10) . One of these MEK kinases may
be the product of the raf protooncogene
(11, 12, 13) , although other
MEK kinases have been
identified
(14, 15, 16, 17) .
(18, 19) , other kinases such as
pp90
(20) and MAPKAP
(21) , and
intermediate regulators of protein synthesis, such as
PHAS-1
(22, 23) . Studies using constitutively active
mutants of MEK (24) have shown that persistent activation of this
pathway may be transforming in some fibroblast cell lines. Moreover,
transfection of PC-12 cells with such constitutively active mutants
induced neuronal differentiation, whereas dominant negative interfering
mutations blocked differentiation
(24) .
Materials
I-Labeled anti-mouse IgG
(3000 Ci/mmol) was from Amersham Corp.,
[
-
P]ATP (3000 Ci/mmol) was from DuPont NEN,
Dulbecco's modified Eagle's medium was from ICN (Irvine,
CA), NGF 2.5 S was from Bioproducts for Science (Indianapolis, IN), and
anti-phosphotyrosine and anti-Shc antibodies were from Upstate
Biotechnology (Lake Placid, NY). All other reagents were from Sigma and
were the highest quality available. Anti-pp44
(16) and anti-trk(4) antisera were developed as
described previously. Anti-MEK antisera was the generous gift of Dr.
Kunliang Guan (University of Michigan).
Cell Culture
PC-12 cells were grown on
collagen-coated plastic culture dishes in Dulbecco's modified
Eagle's medium with 10% fetal bovine serum and 5% horse serum, as
described
(5, 6) . Prior to treatment with NGF, medium
was replaced for 1 h with serum-free medium.
Assay of MAP Kinase Activity
This activity was
assayed as described
(5) . Briefly, 10-µl aliquots of cell
lysates were incubated with pig brain microtubule-associated protein-2
(MAP-2) or myelin basic protein at 0.2 mg/ml for 10 min at 30 °C in
a final volume of 25 µl containing 50 mM Tris-HCl (pH
7.4), 2 mM EGTA, 10 mM MgCl, and 40
µM [
-
P]ATP (1 µCi). The
reaction was stopped by the addition of Laemmli SDS sample buffer, and
phosphorylated MAP-2 was resolved by 7.2% SDS-PAGE. Coomassie
Blue-stained phospho-MAP-2 or myelin basic protein was excised from the
gels, and incorporated radioactivity was measured by Cerenkov counting.
Anti-phosphotyrosine Immunoblots
For analysis of
protein tyrosine phosphorylation in whole cell lysates, PC-12 cells
were grown in 60-mm dishes. After treatment, cells were washed once
with ice-cold phosphate-buffered saline, followed by the addition of
100 µl of Laemmli SDS sample buffer. Samples were heated at 95
°C for 5 min, and 30-µg protein aliquots were loaded onto 8%
SDS-polyacrylamide gels. Proteins were transferred to nitrocellulose
paper and immunoblotted with anti-phosphotyrosine antiserum. In some
experiments, lysates were subject to immunoprecipitation. PC-12 cells
were grown in 150-mm dishes prior to treatment. Cells were washed once
with ice-cold phosphate-buffered saline and lysed in 1 ml of 50
mM HEPES, pH 7.5, 150 mM NaCl, 10% glycerol, 1%
Triton X-100, 1.5 mM MgCl, 1 mM EDTA, 10
mM sodium pyrophosphate, 100 µM sodium
orthovanadate, 100 mM NaF, 30 mMp-nitrophenyl phosphate, 10 µg/ml aprotinin, 10 µg/ml
leupeptin, and 1 mM phenylmethanesulfonyl fluoride (HNTG
buffer) according to Margolis et al.(25) . Lysates were
centrifuged for 10 min at 10,000
g, and supernatants
were incubated for 60 min with antiserum. Immunoprecipitates were mixed
for 30 min with protein A-Sepharose beads, and immune complexes bound
to the beads were washed 3 times with 1 ml of the same lysis buffer.
Immune complexes were solubilized in 25 µl of Laemmli sample buffer
and loaded onto 8% polyacrylamide gels.
Assay of MEK
Cells (150-mm dishes) were serum
deprived for 16 h. After growth factor treatments, cells were lysed in
HNTG buffer, and lysates were immunoprecipitated with anti-MEK antisera
(1:200 dilution) for 2 h at 4 °C. This antiserum precipitates both
MEK1 and MEK2. Immunoprecipitates were mixed with Protein A-Sepharose
beads for 30 min, and beads were washed twice with 1 ml of HNTG buffer.
The sample was then resuspended in 100 µl of reaction buffer
containing 20 mM Tris-HCl, pH 7.4, 10 mM
MgCl, 1 mM MnCl
, 1 mM EGTA.
Reactions were initiated by the addition of 10 µCi of
-
P]ATP (50 µM) and 10 µg of a
glutathione S-transferase-fusion protein containing
p44
with a Lys
Arg substitution
(MAPK/KA).
(
)
This protein can be
phosphorylated by MEK but is catalytically inactive and does not
autophosphorylate. After a 15-min incubation at 25 °C, reactions
were stopped with 20 µl of Laemmli sample buffer, and
phospho-MAPK/KA was detected by SDS-PAGE followed by
autoradiography.
Assay of Phosphatidylinositol (PI) 3-Kinase
This
was assayed essentially as described previously
(27) with the
following modifications. Lysates were prepared from confluent cells
(100-mm plates) and treated with the indicated concentrations of NGF
for 5 min. Cells were lysed in 500 µl of Nonidet P-40 lysis buffer,
and lysates were precleared with rabbit IgG and Pansorbin, 5 µl of
4G10 were added, and the samples were incubated overnight. Immune
complexes were isolated using 50 µl of protein-G/A agarose. PI
3-kinase was assayed by adding to the immunoprecipitate 50 µl of
reaction buffer (10 mM Tris (pH 7.5), 0.2 mM EGTA,
100 mM NaCl, and 20 mM MgCl), 0.4 mg of
phosphatidylinositol, and 10 µM
[
-
P]ATP (10 µCi), followed by
incubation for 20 min at room temperature. The reaction was stopped by
extraction with organic solvents; the organic phase was dried down and
chromatographed on silica gel TLC. Incorporated radioactivity was
determined by autoradiography.
To evaluate its ability to block the
stimulation of MAP kinase, PC-12 cells were preincubated with
0-100 µM PD98059 for 10 min prior to the addition of
100 nM NGF for 2 min. Following the treatment, lysates were
assayed for MAP kinase activity by evaluating the phosphorylation of
myelin basic protein (Fig. 2). As described
previously
(5) , NGF produced almost a 4-fold increase in MAP
kinase activity. Pretreatment of cells with PD98059 alone was without
effect, but the compound completely blocked stimulation of the enzyme
by NGF, returning the enzyme activity to basal levels. Half-maximal
inhibition was observed at approximately 2 µM, and
complete inhibition was observed at 10-100 µM. This
blockade of MAP kinase activation by NGF was also observed in 3T3 cells
expressing the trk protooncogene. Direct addition of PD98059
to the assay incubation had no effect on MAP kinase activity (data not
shown).
Figure 1:
Structure of
PD98059.
Figure 2:
PD98059 blocks the activation of
MAP kinase by NGF. Serum-deprived PC-12 cells in 100-mm dishes were
treated with an indicated concentration of PD98059 for 30 min prior to
the addition of 50 ng/ml NGF for 5 min. Cells were lysed and MAP kinase
activity was assayed as described under ``Experimental
Procedures,'' using myelin basic protein (MBP) as
substrate. Following SDS-PAGE, the phosphorylated substrate was excised
from the gel and quantitated by Cerenkov counting. Results are
expressed as the means of triplicate determinations ± S.D. and
are representative of five separate experiments. Activity assayed in
the absence of NGF was 1.2 10
cpm.
MAP kinase is thought to be activated by the dual
specificity kinase, MEK. To confirm that PD98059 blocks MAP kinase
activation via inhibition of MEK, we examined the activity of MEK in an
immune complex assay (Fig. 3). PC-12 cells were preincubated with
or without 20 µM PD98059 and then exposed to NGF for 5
min. Cells were lysed, and lysates were immunoprecipitated with an
anti-MEK antisera that recognizes both MEK1 and MEK2. MEK activity was
assayed by evaluating the phosphorylation of a glutathione
S-transferase-fusion protein containing a catalytically
inactive MAP kinase. Treatment of cells with NGF caused a
4-fold increase in MEK activity. This stimulation was inhibited
approximately 70% by preincubation of cells with PD98059.
Interestingly, direct addition of PD98059 to the assay inhibits MEK
activity by over 90%, although this inhibition can be reversed by
extensive washing.
It is likely that the reduced inhibition
described here results from some dissociation of PD98059 from the
enzyme complex during immunoprecipitation and washing.
Figure 3:
PD98059 inhibits the activation
of MEK by NGF. Serum-deprived PC-12 cells were grown in 150-mm dishes.
Cells were preincubated for 30 min with 20 µM PD98059 or
dimethyl sulfoxide, followed by a 5-min exposure to NGF. Cell lysates
were immunoprecipitated with anti-MEK antisera, and the phosphorylation
of glutathione S-transferase-MAPK/KA was assayed as
described under ``Experimental Procedures.'' The migration of
glutathione S-transferase-MAPK/KA is shown in the
autoradiograph. GST-ERK, glutathione
S-transferase-extracellular regulated kinase. In B,
regions of the gel corresponding to the phosphorylated band were
excised and subject to Cerenkov counting. Identical results were
obtained in two separate experiments.
To further
confirm that PD98059 specifically blocks the activation of MAP kinase,
we examined the tyrosine phosphorylation of the enzyme. PC-12 cells
were treated with or without PD98059 as described above, followed by
exposure to NGF for 2 min. Cells were lysed, and lysates were
precipitated with an anti-MAP kinase antisera. Immunoprecipitates were
subject to SDS-polyacrylamide gel electrophoresis, followed by
immunoblotting with anti-phosphotyrosine antibodies
(Fig. 4A). As described previously
(6) , NGF
caused the tyrosine phosphorylation of MAP kinase, due to the
activation of its upstream kinase, MEK. Preincubation with PD98059
completely blocked this effect. To confirm the specificity of this
agent, whole cell lysates from the same cells were immunoprecipitated
with anti-trk and anti-Shc antibodies, followed by
anti-phosphotyrosine Western blotting (Fig. 4B). Neither
autophosphorylation of the receptor nor the phosphorylation of its
substrate Shc
(28) was affected by PD98059.
Figure 4:
PD98059 blocks the tyrosine
phosphorylation of MAP kinase but not early NGF-receptor tyrosine
phosphorylations. PC-12 cells were cultured as described in Fig. 2.
Cells were treated with or without 20 µM PD98059 for 30
min, followed by a 5-min exposure to NGF, as indicated. In A,
cell lysates were immunoprecipitated with anti-MAP kinase antisera,
followed by SDS-PAGE. The resulting gels were Western blotted with
anti-phosphotyrosine antibody. In B, lysates were precipitated
with anti-trk or anti-shc antisera, and precipitates
were subject to SDS-PAGE and anti-phosphotyrosine immunoblotting as
described above. IP,
immunoprecipitate.
The binding of
NGF to its receptor and the subsequent stimulation of its tyrosine
kinase activity lead to an increase in PI 3-kinase activity in PC-12
cells
(27) , by virtue of the association of the p85 regulatory
subunit of the enzyme with a 100-kDa substrate of the
receptor
(27) . Recent studies with wortmannin
(29) suggest that the activation of PI 3-kinase may be necessary
for the biological effects of NGF. To evaluate whether PD98059 had any
effect on the stimulation of this enzyme, cells were pretreated with
the compound as described above, followed by exposure to NGF
(Fig. 5). As described previously
(27) , NGF caused the
appearance of PI 3-kinase activity in anti-phosphotyrosine
immunoprecipitates, correlating with activation of the enzyme and
generation of PI-3,4,5-P in PC-12 cells
(30) .
Treatment of cells with PD98059 was without effect on this stimulation.
Moreover, direct addition of the compound to the assay incubation did
not affect enzyme activity (data not shown).
Figure 5:
PD98059 does not block the
activation of PI 3-kinase by NGF. PC-12 cells were preincubated with 20
µM PD98059 and treated with NGF for 5 min as described
above. Cell lysates were immunoprecipitated with anti-phosphotyrosine
antisera, and PI 3-kinase activity was assayed in the
immunoprecipitate. An autoradiograph of the thin layer chromatograph is
shown. Identical results were obtained in three separate
experiments.
Inhibition of the
tyrosine kinase activity of the NGF receptor by staurosporine and
related compounds
(31) or by tyrphostins
(32) can block
NGF-dependent neurite outgrowth. To evaluate the specific role of the
MEK-MAP kinase pathway in the differentiative action of NGF, PC-12
cells were treated with 10 µM PD98059 for 30 min before
the addition of NGF for 2 days (Fig. 6). The compound completely
blocked NGF-induced neurite formation, without significantly altering
cell viability.
Figure 6:
PD98059 inhibits NGF-induced
neurite outgrowth. PC-12 cells were treated with 10 µM
PD98059 for 30 min prior to addition of 50 nM NGF for 3 days.
Cells were photographed using a phase-contrast light
microscope.
This inhibition appears
to be noncompetitive with respect to ATP.
Moreover, the
compound has not been found to block a number of other protein
serine/threonine or tyrosine kinases, even at higher
concentrations.
We show here that PD98059 can completely
inhibit the activation of MAP kinase by NGF in PC-12 cells, presumably
due to the blockade of its upstream activator. In contrast, the
compound has no effect on early phosphorylation events catalyzed by the
pp140
NGF receptor. Interestingly, at these
concentrations, this inhibition is accompanied by the complete blockade
of NGF-induced neuronal differentiation, without impairing cell
viability. In contrast, the compound does not block differentiation of
other tissue culture cells, including 3T3-L1 adipocytes or L-6 myocytes
(data not shown). These data provide strong additional support for the
requirement of the MAP kinase pathway in PC-12 cell differentiation.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.