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INTRODUCTION |
Mammalian AMP-activated protein kinase
(AMPK)1 plays a key role in
the regulation of energy homeostasis and is highly conserved among
animals, plants, and fungi (reviewed in Refs. 1-3). AMPK is a
heterotrimeric enzyme consisting of a catalytic subunit (
) and two
regulatory subunits (
and
), and it is activated by the cellular
stress causing ATP depletion, which in turn leads to elevation of the
AMP:ATP ratio (reviewed in Refs. 1-3). These stresses include heat
shock, ischemia/hypoxia in cardiac muscle, and exercise in skeletal
muscle. In addition to allosteric activation by AMP, AMPK is activated
by phosphorylation by an upstream kinase termed AMPK kinase (4). Once
activated, AMPK suppresses the key enzymes involved in ATP-consuming
anabolic pathways such as fatty acid and cholesterol synthesis (5, 6).
Besides, AMPK initiates a series of compensatory changes that increase
cellular ATP supply by activating the rate of fatty acid oxidation (7, 8) and glucose uptake in cardiac and skeletal muscle (9, 10). Thus,
AMPK has been speculated to play a role as a "fuel gauge" that
recognizes ATP depletion and maintains ATP level (reviewed in Refs.
1-3). Most of the currently identified substrates of AMPK are
metabolic enzymes. However, the recent implications of AMPK in
transcriptional control (11, 12), insulin secretion in pancreatic
cell (13), and regulation of endothelial NO synthase (14) and Raf-1
kinase (15) suggest that it might be involved in the regulation of many
cellular processes other than those that have been identified. Thus, it
appears that numerous novel targets remain to be discovered.
Recently, Sprenkle et al. (15) identified the principal
Raf-1 Ser621 kinase activity present in cytosolic
extracts of NIH-3T3 cells as AMPK by analyzing cytosolic fractions for
Ser621 peptide kinase activity. They also demonstrated that
AMPK phosphorylated Ser621 of Raf-1, which was expressed in
Escherichia coli or Sf9 insect cell (15).
The Ser/Thr Raf-1 kinase is a key intermediate in the transduction of
growth factor signals from the cell membrane to the nucleus (16, 17).
Activation of receptor tyrosine kinases stimulates the small
GTP-binding protein Ras, which interacts with cytoplasmic inactive Raf
and recruits it to the plasma membrane where further activation steps
occur (18, 19). Activated Raf in turn phosphorylates and activates
MAPK/Erk kinase 1 (MEK1), which phosphorylates and stimulates
mitogen-activated protein kinases/extracellular signal-regulated
kinases (MAPKs/Erks). This pathway (Ras
Raf
MEK
Erk) has
been known to play a significant role in the transmission of cellular
proliferation and developmental signals (20). Although the exact
regulatory mechanisms for Raf-1 activity are not fully understood
despite extensive investigations, they include phosphorylation of the
enzyme (17). The role of Raf-1 Ser621 phosphorylation has
been controversial. Ser621 was reported to be a
constitutive phosphorylation site, and the mutation of this site to
alanine resulted in a total loss of the kinase activity (21). On the
other hand, it was also claimed that phosphorylation of
Ser621 by cAMP-dependent protein kinase (PKA)
confers negative regulation (22).
In the present study, we have taken a pharmacological and molecular
approach to determine whether AMPK plays a role in Raf-1-involved signaling pathways. Our results suggest that AMPK can attenuate Ras
activation induced by IGF-1 or EGF and that AMPK can also further
stimulate Ras-independent Raf activation induced by EGF. Phosphorylation of Raf-1 Ser621, however, was not involved
in the AMPK-mediated regulation of Erk cascades. To our knowledge, this
is the first report demonstrating that AMPK is involved in the
regulation of growth factor-induced multiple signaling pathways.
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EXPERIMENTAL PROCEDURES |
Materials--
Dulbecco's modified Eagle's medium
(DMEM), DMEM/Ham's F-12 medium, and the other cell culture products
were purchased from Life Technologies, Inc. Forskolin and PD098059 were
obtained from Calbiochem. EGF and IGF-1 were from Calbiochem and Sigma,
respectively. 5-Aminoimidazole-4-carboxamide-1-
-D-ribofuranoside
(AICAR) and other chemicals were from Sigma. [
-32P]ATP
(6000 Ci/mmol) and [methyl-3H]thymidine (6.7 Ci/mmol)
were purchased from PerkinElmer Life Sciences.
Cell Culture and AICAR, PD098059, and Forskolin
Treatment--
NIH-3T3, 3T3-L1 preadipocytes, and COS-7 cells were
maintained in DMEM containing 10% calf serum. H9c2 cardiomyoblasts
were proliferated in DMEM/Ham's F-12 medium supplemented with 10%
calf serum. When confluent, cells were induced to differentiate for 6 days with medium containing 1% horse serum. About 80-90% confluent cells or H9c2 cardiomyotube were serum-starved for 16 h, and then the indicated pretreatment (1 mM AICAR, 50 µM
PD098059, or 50 µM forskolin) was performed for 1 h
in Krebs-Ringer buffer (25 mM HEPES, pH 7.4, 118 mM NaCl, 4.8 mM KCl, 1.3 mM
CaCl2, 1.2 mM KH2PO4,
1.3 mM MgSO4, 5 mM
NaHCO3, 0.07% bovine serum albumin, and 5.5 mM
glucose). Then cells were stimulated with 50 nM IGF-1 or
100 ng/ml EGF for the indicated time period.
Antibodies and DNA Constructs--
The anti-dual
phospho-specific antibody that recognizes the active Erk1/2 was from
New England Biolabs, Inc. Erk1/2 antibody that recognizes the total
Erk1/2 regardless of their phosphorylation was from Transduction
Laboratories. Antibodies for Raf-1 (C-12), EGF receptor (EGFR, 1005),
IGF-1 receptor
subunit (IGF-1R, C-20), Grb2 (C-23), phosphotyrosine
(PY20), and c-Myc (9E10) were obtained from Santa Cruz Biotechnology
Inc. Phosphospecific Raf Ser621 antibody was kindly
provided by Dr. Andrey S. Shaw (Center for Immunology and Department of
Pathology, Washington University, St. Louis, MO). The AMPK
1-specific antibody and the AMPK pan-
antibody were kindly
provided by Dr. Ian Salt and Dr. D. Grahame Hardie (Biochemistry
Department, The University, Dundee, UK). Raf-1 mutant constructs
(Raf-wt, RafS621A, and RafR89L in pEFm vector fused with Myc epitope at
its N terminus) were generous gifts from Dr. Richard Marais and
Christopher J. Marshall (Cancer Research Campaign Center for Cell and
Molecular Biology, Institute of Cancer Research, UK).
GST-MEK(
) plasmid was provided by Dr. Kun-Liang Guan
(Department of Biochemistry, University of Michigan, town, MI).
A dominant negative Ras (RasS17N) was subcloned into pCMV-tag2B vector.
NIH-3T3 Cells Expressing an Antisense RNA for AMPK--
cDNA
corresponding to a portion of the coding region of AMPK
1 (amino
acids 1-392) was amplified by polymerase chain reaction. It was
subcloned into pcDNA 3 in such an orientation as to express an
antisense RNA. The plasmid was transfected into NIH-3T3 cells using
LipofectAMINE (Life Technologies, Inc.), and stable transfectants were
obtained in the presence of G418. Individual clones were further
isolated and examined for the effect of an antisense RNA expression.
AMPK Activity Assay--
Cells were lysed with digitonin buffer
(50 mM Tris-HCl, pH 7.3, 50 mM NaF, 30 mM glycerol phosphate, 250 mM sucrose, 1 mM sodium metavanadate, and 0.4 mg/ml digitonin) on ice for
2 min. AMPK was partially purified from cell lysates by adding
saturated ammonium sulfate to final 35% (v/v) concentration on ice for
15 min. AMPK activity was determined as previously described (23) with
these fractionated proteins in kinase assay buffer (62.5 mM
HEPES, pH 7.0, 62.5 mM NaCl, 62.5 mM NaF, 6.25 mM sodium pyrophosphate, 1.25 mM EDTA, 1.25 mM EGTA, and 1 mM dithiothreitol) containing 200 µM AMP, ATP mixture (200 µM ATP and 1.5 µCi of [
-32P]ATP), with or without 250 µM SAMS peptide (HMRSAMSGLHLVKRR) at 30 °C
for 10 min. The reaction was terminated by spotting the reaction
mixture on phosphocellulose paper (P81), and the paper was extensively
washed with 150 mM phosphoric acid. The radioactivity was
measured with a scintillation counter.
Raf-1 Kinase Assay--
Raf-1 kinase assay was performed
essentially as described by Ziogas et al. (24) with the
following modifications. After cell lysis, 500 µg of protein extracts
were subjected to immunoprecipitation with a Raf-1 antibody (C-12) or
c-Myc antibody (9E10) coupled to protein G-agarose. After extensive
washing of the immunoprecipitates, the kinase assay was performed at
30 °C for 30 min in a kinase assay buffer containing the Raf-1
immune complex, 10 µM ATP, 10 µCi of
[
-32P]ATP, and 1 µg of kinase defective
GST-MEK(
) as a specific substrate of Raf-1. After
reaction, samples were centrifuged, and the supernatants were separated
on 10% SDS/polyacrylamide gel electrophoresis. Proteins were blotted
on nitrocellulose membranes, and the radioactivity incorporated into
the substrates was measured by PhosphorImager. The resultant pellets
were also resolved on the 10% SDS/polyacrylamide gel electrophoresis
for immunoblotting with a Raf-1 antibody to compare the amount of Raf-1
present in the immune complex.
Activated Ras Affinity Precipitation Assay--
Raf Ras-binding
domain (RBD) fragment (Raf-1 amino acid residues 1-149) fused to GST
was purchased from Upstate Biotechnology. The fusion protein was
immobilized on glutathione-agarose. The activated Ras affinity
precipitation assay was performed as described according to the
manufacturer's protocol. Briefly, 500 µg of cell extracts were
incubated with 5 µg of GST-RBD complexes for 30 min at 4 °C. After
extensive washing of the agarose beads five times with
immunoprecipitation washing buffer (25 mM HEPES, pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 10 mM
MgCl2, 1 mM EDTA, and 2% glycerol), the active
Ras (Ras-GTP) bound to GST-RBD complexes was released by addition of
2× SDS/polyacrylamide gel electrophoresis loading buffer. The amount
of active Ras was determined by immunoblotting with an anti-pan Ras
monoclonal antibody.
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RESULTS |
AMPK Inhibits Erk Activation Induced by IGF-1 but Not by
EGF--
To investigate a possible involvement of AMPK in the
Ras/Raf/MEK/Erk signaling pathway, dose- and time-dependent
effects of AICAR on IGF-1-induced Erk1 and Erk2 activation were
first examined (Fig. 1, A and
B). AICAR becomes a potent activator of AMPK after its
intracellular phosphorylation to AMP-mimetic AICA-ribotide (ZMP) (5).
NIH-3T3 cells were serum-starved for 16 h, pretreated with AICAR,
and then challenged with 50 nM IGF-1 for 10 min. In the
absence of AICAR, IGF-1 rapidly stimulated phosphorylation of Erk1 (p44
MAPK) and Erk2 (p42 MAPK) when examined by immunoblotting with an
antibody specific for the active biphosphorylated form of Erk1 and
Erk2. The pretreatment with AICAR inhibited IGF-1-dependent Erk activation in a dose- and time-dependent manner, and a
maximum effect was observed at 1 mM of AICAR for 1 h
of preincubation (Fig. 1, A and B). The total
amount of Erk proteins was essentially the same at each condition. The
kinetics of Erk phosphorylation correlated well with the enzyme
activity, which was directly measured by an immune complex using mylein
basic protein as a substrate (data not shown). As expected, AMPK was
activated by AICAR in a dose- and time-dependent manner,
and the maximum 3-fold activation was observed in 1 h at 1 mM AICAR (Fig. 1, C and D). These
results suggest that AMPK is probably involved in the negative
regulation of Erk cascades. The AMPK activation conditions found in
this study are consistent with other reports showing that the maximum effect of AICAR on AMPK activity or cellular processes regulated by
AMPK was observed in the range of 0.5-1 mM (5-12).
Therefore, this pretreatment condition (1 mM AICAR/1 h of
incubation) was used throughout the present study.

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Fig. 1.
Effects of AICAR on IGF-1-induced Erk
activation and AMPK activity. NIH-3T3 cells were incubated with
the indicated concentrations of AICAR for 1 h (A) or
with 1 mM AICAR for the indicated time period
(B) and then stimulated with 50 nM IGF-1 for 10 min. The phosphorylation level and the total protein amount of Erk1 and
Erk2 were determined by immunoblotting with an anti-dual
phospho-specific Erk antibody (P-ERK) and an antibody for
Erk1 and Erk2 regardless of their phosphorylation (ERK),
respectively. The effect of AICAR on AMPK activity was also examined
from the dose-dependently (C) and
time-dependently (D) treated cells. Partially
purified AMPK was subjected to the activity assay using SAMS peptide as
a substrate in the presence of 200 µM AMP. The data shown
represent the means ± S.E. for three separate experiments in
duplicate.
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Because of the lack of molecular approaches for manipulating AMPK
activity or a specific inhibitor, AICAR has been widely used to
demonstrate a role of AMPK in various cell lines and tissues (5-12).
However, AMPK-independent effects of AICAR were also reported in some
cases (25, 26). Thus, to substantially demonstrate the role of AMPK in
Erk cascades, we established NIH-3T3 cells stably expressing an
antisense RNA for the catalytic core region of AMPK catalytic
1
subunit, a predominantly expressed isoform in these cells, as described
under "Experimental Procedures." Two isoforms of the AMPK catalytic
subunit (
1 and
2) were identified, and
1 is ubiquitously
expressed, whereas
2 is highly expressed in skeletal muscle, heart,
and liver (27, 28). Both isoforms show an identical molecular mass (63 kDa) and have 90% amino acid sequence identity within the catalytic
core (28). For simplicity, we use anti-
1 to refer to NIH-3T3 cells
expressing an antisense RNA. The immunoblot analysis using either an
AMPK
1-specific antibody or an AMPK pan-
antibody
that recognizes both
1 and
2 revealed that the expression level
of AMPK
was substantially reduced in anti-
1 cells compared with
the vector-transfected control cells (Fig.
2A). The reduced expression
level of the
subunit resulted in a ~30-40% decrease in the
basal and the AICAR-stimulated AMPK activity (Fig. 2B). When
anti-
1 cells were pretreated with AICAR and then challenged with
IGF-1, the inhibitory effect of AICAR on Erk activation was
significantly diminished (Fig. 2C). These results indicate
that the effect of AICAR is indeed mediated by AMPK.

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Fig. 2.
Expression of an antisense RNA for the AMPK
catalytic subunit decreased the AMPK activity and diminished the
inhibitory effect of AICAR on IGF-1-induced Erk activation.
NIH-3T3 cells expressing an antisense RNA for AMPK 1
(Anti- 1) were established as described under
"Experimental Procedures." A, the expression level of
the AMPK subunit of control and anti- 1 cells was compared by
immunoblot analysis with AMPK 1-specific antibody (upper
panel) or with AMPK pan- antibody that recognizes both the 1
and 2 isoforms (lower panel). B, the basal and
AICAR-stimulated AMPK activities of the control and anti- 1 cell. The
data shown represent the means ± S.E. for two independent
experiments in duplicate. C, the control and anti- 1 cells
were pretreated with or without 1 mM AICAR and then
stimulated with 50 nM IGF-1 for 10 min. The phosphorylation
level (P-ERK) and the total protein amount of Erk
(ERK) were compared by immunoblot analysis with each
antibody.
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In contrast to IGF-1, AICAR did not block EGF (100 ng/ml)-induced Erk
activation, whereas PD098059, a specific inhibitor of MEK, almost
completely inhibited Erk activation by two growth factors (Fig.
3A). One explanation for the
selective effects of AICAR is that EGF could suppress the
AICAR-stimulated AMPK activity. To this end, the effects of IGF-1 and
EGF on AMPK activity were examined (Fig. 3B). However, the
results revealed that these two growth factors practically had no
effect on the basal and the AICAR-stimulated AMPK activities (Fig.
3B), supporting the general perspective that AMPK system is
stress-sensitive but not hormone-sensitive (1-3). Therefore, these
results suggest that the differential effects of AICAR are due to the
intrinsic differences between signal pathways leading to Erk activation
by IGF-1 and EGF.

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Fig. 3.
Selective effects of AICAR on Erk activation
induced by IGF-1 and EGF. A, NIH-3T3 cells were
pretreated with or without 1 mM AICAR or 50 µM PD098059 for 1 h and then stimulated with 50 nM IGF-1 or 100 ng/ml EGF for 10 min. The phosphorylation
level of active Erk (P-ERK) and the total amount of Erk
(ERK) in each sample were measured. B, NIH-3T3
cells were pretreated for 1 h with or without 1 mM
AICAR. After incubation with IGF-1 (50 nM) or EGF (100 ng/ml) for 10 min, AMPK activity was examined. The data shown represent
the means ± S.E. for three separate experiments in
duplicate.
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Next, to determine whether AMPK is involved in the regulation of Erk
pathways in other cell types, we examined the AICAR effects in the
COS-7, H9c2 cardiomyotube, and 3T3-L1 preadipocyte cell lines (Fig.
4). Although these cells showed slight
differences, IGF-1-induced Erk activation was inhibited, whereas
EGF-induced Erk activation was not affected by AICAR treatment in all
cell lines tested (Fig. 4). Therefore, the effect of AMPK on Erk
cascades is likely to be quite a widespread phenomena rather than
certain cell type-specific.

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Fig. 4.
Similar effects of AICAR on Erk activation
were observed in several cell lines. The effect of AICAR on Erk
activation was examined in H9c2 cardiomyotubes, 3T3-L1 preadipocytes,
and COS-7 cells. Each cell line was pretreated with or without 1 mM AICAR for 1 h and then stimulated with 50 nM IGF-1 or 100 ng/ml EGF for 10 min. Erk activities were
determined by immunoblotting with an anti-phosphospecific Erk antibody
(P-ERK).
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DNA Synthesis Induced by IGF-1 Was Significantly Inhibited by AICAR
Pretreatment--
Erk cascades are among the most intensively studied
signal transduction systems, and they have shown to participate in a
diverse array of cellular activities such as cell proliferation,
development, cell survival, and death (20). To investigate a
physiological significance of the AMPK-mediated Erk regulation, the
effect of AICAR on the growth factor-induced DNA synthesis was
examined. IGF-1 and EGF induced DNA synthesis ~2-fold after 9 h
of incubation and 4-fold after 19 h of incubation (Fig.
5A). In close correlation with
the Erk phosphorylation level, AICAR treatment led to ~70% inhibition of IGF-induced DNA synthesis after 9 h and 50%
inhibition after 19 h, whereas EGF-induced DNA synthesis was
relatively less affected, resulting in about 25% reduction (Fig.
5A). Furthermore, the inhibitory effect of AICAR on
IGF-1-dependent DNA synthesis was distinctively diminished
in the anti-
1 cell (Fig. 5B), in which IGF-1-induced Erk
activation is quite resistant to the AICAR-dependent down-regulation (Fig. 2C). Under these conditions, no
cytotoxic effects were observed (data not shown). A MEK inhibitor, 50 µM PD098059, almost completely blocked DNA synthesis
induced by both growth factors, indicating that Erk activation is
critical for cell proliferation. As shown in Fig. 3A, AICAR
exerted practically no effect on the basal Erk activity
(first and fourth lanes) or EGF-induced Erk
activation (third and sixth lanes). Nevertheless, this treatment resulted in statistically significant inhibition of the
basal as well as EGF-induced DNA synthesis (Fig. 5). Thus, besides Erk
cascades, some other mechanism(s) required for DNA synthesis seems to
be affected by AICAR. This possibility is considered later under
"Discussion."

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Fig. 5.
Effects of AICAR on DNA synthesis. About
60% confluent NIH-3T3 cells (A) or the vector-transfected
control cells and anti- 1 cells (B) were incubated in DMEM
supplemented with 0.5% calf serum for 16 h. After incubation with
or without 1 mM AICAR or 50 µM PD098059 for
1 h, these cells were further incubated for 9 or 19 h in the
presence of 50 nM IGF-1 or 100 ng/ml EGF. After the
addition of 3 µCi of [3H]thymidine for the last 3 h, cells were lysed by 0.1% SDS. The cellular DNA was precipitated
with 10% trichloroacetic acid, and the radioactivity was measured by
scintillation counter. Results are the means ± S.E. of at least
six determinations. *, p < 0.01.
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Activation of AMPK Did Not Induce Phosphorylation of
Ser621 of Raf-1--
The previous report demonstrated that
AMPK can phosphorylate Ser621 of Raf-1 in vitro
(15). The sequence around Raf-1 Ser621 exactly matches the
recognition motif of AMPK (29). Because it was also demonstrated that
phosphorylation of this residue by PKA leads to inhibition of Raf-1
kinase (22), we next examined the possibility that phosphorylation of
Raf-1 Ser621 is a mechanism responsible for AMPK-mediated
inhibition of Erk activation induced by IGF-1.
To this end, we first analyzed the effect of AICAR on the
phosphorylation level of Ser621 of Raf-1 (Fig.
6). The Myc-tagged Raf-wild type
(mycRaf-wt) plasmid was transiently transfected in NIH-3T3 cells, and
in 48 h post-transfection, cells were treated for 1 h with 1 mM AICAR or 50 µM forskolin, which enhances
cAMP accumulation and in turn activates PKA. The expressed mycRaf-wt
protein was immunoprecipitated with a c-Myc epitope-specific monoclonal
antibody 9E10 and then immunoblotted with a phosphospecific Raf
Ser621 antibody. The basal phosphorylation level of
Ser621 of mycRaf-wt was hardly detectable under our
experimental conditions, and AICAR did not exert any effect on this
residue, whereas forskolin markedly increased the phosphorylation level
of Ser621 (Fig. 6, upper panel). Under the
identical condition, forskolin did not exert any effect on mycRafS621A,
in which Ser621 is replaced with alanine. There was
essentially no difference in the amount of the immunoprecipitated
mycRaf-wt and mycRafS621A (Fig. 6, lower panel). This result
supports the previous finding that Ser621 of Raf-1 can be
phosphorylated by PKA (22). However, in contrast to the report
describing that AMPK in vitro phosphorylated
Ser621 of the overexpressed Raf-1 from bacteria and insect
cell (15), our results argue that this may not be the case in intact
cells.

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Fig. 6.
Ser621 of Raf-1 was not
phosphorylated by AICAR treatment in NIH-3T3 cells. NIH-3T3 cells
were transiently transfected with Myc-tagged Raf-wt or RafS621A
plasmid. 48 h post-transfection, cells were treated with 1 mM AICAR or 50 µM forskolin for 1 h. The
expressed Raf-1 kinases were immunoprecipitated with a c-Myc antibody
(IP: c-myc), and the phosphorylation level of
Ser621 was examined by immunoblotting with a
phosphospecific Raf Ser621 antibody (IB: Phospho-Raf
S621). The membrane was stripped off and reprobed with a Raf-1
antibody (IB: Raf-1).
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The Mechanism by Which Raf-1 Is Regulated by AMPK Differs
from the PKA-mediated One--
Besides Ser621, Raf-1
contains multiple phosphorylation sites critical for the kinase
activity (17). Although phosphorylation of Ser621 was not
induced by AICAR, we next examined whether or not Raf-1 kinase activity
is directly affected by AICAR treatment (Fig. 7). NIH-3T3 cells were pretreated with
AICAR and then challenged with IGF-1 (50 nM) or EGF (100 ng/ml) for 5 min. Endogenous Raf-1 was immunoprecipitated with a Raf-1
antibody, and in vitro kinase assays were performed using
the recombinant kinase-defective GST-MEK(
) as a
Raf-1-specific substrate. IGF-1 and EGF stimulated Raf-1 activity
~3-fold in 5 min, and AICAR pretreatment almost completely blocked
Raf-1 activation by IGF-1, but it did not interfere with EGF-induced
Raf-1 activation (Fig. 7A). This result is consistent with
the Erk phosphorylation profile presented in Fig. 3A,
indicating that Raf-1 is a main activator of MEK and Erk in NIH-3T3
cells. Next, Myc-tagged Raf-wild type (mycRaf-wt) and mycRafS621A
plasmids were transiently transfected in NIH-3T3 cells, and the effects of AICAR and forskolin on each Raf kinase form were compared. Consistent with the results of the endogenous Raf-1 activity (Fig. 7A), AICAR pretreatment blocked IGF-induced Raf-wt activity
but not EGF-induced Raf-wt activity (Fig. 7B, upper
panels). In contrast, forskolin inhibited Raf-wt activation
induced by EGF and IGF-1 (Fig. 7B, upper panels,
sixth and seventh lanes). RafS621A was activated
about 1.7-fold by IGF-1 or EGF (Fig. 7B, lower
panels), but the degree of activation was relatively mild compared
with 3-fold activation of endogenous Raf or Raf-wt. Notably, RafS621A activation by IGF-1, but not by EGF, was blocked by AICAR (Fig. 7B, lower panels, fourth and
fifth lanes), whereas activation of this mutant form by IGF
or EGF was resistant to the PKA-mediated down-regulation (Fig.
7B, lower panels, sixth and
seventh lanes). The effects of forskolin on Raf-wt and
RafS621A activation together with the results shown in Fig. 6 support
the previous finding that Raf Ser621 serves as a negative
regulation site by PKA phosphorylation (22). Therefore, these results
indicate that the mechanism by which Raf-1 is regulated by AMPK should
differ from the PKA-mediated one.

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Fig. 7.
AICAR treatment blocked the Raf-1 activation
induced by IGF-1 but not by EGF. A, NIH-3T3 cells
pretreated with or without 1 mM AICAR for 1 h were
stimulated with 50 nM IGF-1 or 100 ng/ml EGF for 5 min.
Then the endogenous Raf-1 kinase was immunoprecipitated with a Raf-1
antibody, and in vitro kinase activity was measured using
kinase inactive recombinant GST-MEK( ) as a substrate.
B, NIH-3T3 cells transfected with Myc-tagged Raf-wt
(upper panels) or RafS621A plasmid (lower panels)
were pretreated with or without 1 mM AICAR or 50 µM forskolin for 1 h and then stimulated with IGF-1
(50 nM) or EGF (100 ng/ml) for 5 min. Each expressed Raf
form was immunoprecipitated with a c-Myc antibody, and the Raf kinase
activity assay was performed. The amounts of immunoprecipitated
endogenous Raf-1 (A), mycRaf-wt, and mycRafS621A
(B) were detected by immunoblotting with a Raf-1 antibody as
indicated by an arrow. The kinase activity was normalized
for the amount of Raf-1 protein present in each immune complex and
expressed as a fold induction of the basal activity. Experiments were
repeated 2-4 times with similar results, and a representative result
is shown.
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Under our experimental conditions, the basal activity of RafS621A was
partially active showing about 30% of the Raf-wt activity in contrast
to the previous report demonstrating that the RafS621A mutant is
completely inactive as a kinase (21). Although we cannot completely
rule out the possibility that the observed kinase activity of RafS621A
is due to nonspecific protein kinases contaminated in an immune
complex, this possibility seems unlikely because c-Myc antibody
immunoprecipitates of vector-transfected cell lysates did not show any
activity exceeding the background level (data not shown). Rather the
discrepancy may be generated by experimental conditions because the
other mutant form, RafS621D, was reported to retain a low inducible
kinase activity when expressed in Sf9 insect cells
(22).
Ras-independent Raf-1 Activity Induced by EGF Is Further Stimulated
by AICAR Treatment--
Ras is the best characterized upstream
activator of Raf-1, but recent studies demonstrate that Raf-1 is also
activated via Ras-independent mechanisms (18, 19, 24, 30-32). Because
it is also known that EGF-induced Erk activation can occur via a Ras-independent as well as a Ras-dependent pathway (22,
33), we investigated the nature of the differential effects of AICAR on
IGF-1- and EGF-induced Raf activation using mycRafR89L, which is
defective for Ras binding (Fig.
8A). EGF indeed activated
RafR89L about 2-fold (Fig. 8A, first and
third lanes), whereas IGF showed very little effect (Fig.
8A, first and second lanes),
indicating that EGF signal can be transduced in a Ras-independent
pathway. In agreement with the earlier report (22), forskolin still
blocked EGF-induced RafR89L activation (Fig. 8A,
third and seventh lanes), indicating that Raf-1
is a direct target of PKA-mediated down-regulation. Unexpectedly, AICAR
further stimulated the EGF-induced RafR89L activity by 50% (Fig.
8A, third and fifth lanes), suggesting
that Ras-independent Raf-1 activation by EGF is positively regulated by
AICAR treatment. Additional evidence supporting this suggestion was
obtained by a cotransfection experiment with Raf-wt and a dominant
negative RasS17N (Fig. 8B). RasS17N expression, which blocks
Ras-mediated signal transmission, resulted in a drastic reduction of
IGF-1-dependent Raf-wt activation (Fig. 8B,
second and sixth lanes), but it exerted a
relatively less effect on EGF-dependent Raf-wt activation
(Fig. 8B, third and seventh lanes).
AICAR treatment clearly stimulated the EGF-dependent Raf-wt
activity that was partially blocked by RasS17N (Fig. 8B,
seventh and ninth lanes). Taken together, these
data demonstrate that IGF-1 transmits its signal to Raf-1 predominantly
via Ras, whereas the EGF signal can be transduced in a Ras-independent
as well as in a Ras-dependent manner. Furthermore, our data
suggest that Ras-independent Raf-1 activation by EGF is up-regulated by
AMPK. At this point, this result seems very contradictory because the
endogenous Raf-1 or mycRaf-wt activation by EGF was not affected by
AICAR (Fig. 7). Thus, we suspected that a compensatory mechanism such
as attenuation by AMPK of Ras-dependent Raf-1 activation by
EGF would exist.

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Fig. 8.
Ras-independent Raf-1 activation by EGF was
further induced by AICAR. A, NIH-3T3 cells transfected
with mycRafR89L plasmid were identically treated as described in Fig.
7B legend, and mycRafR89L kinase activity was compared at
each condition. B, NIH-3T3 cells were cotransfected with
mycRaf-wt and RasS17N at 1:3 ratio of DNA concentration. These cells
were pretreated with or without 1 mM AICAR for 1 h and
then stimulated with 50 nM IGF-1 or 100 ng/ml EGF for 5 min, and Raf-wt activity was determined. The arrow in the
lower panel indicates the amount of mycRafR89L
(A) or Raf-wt (B) present in each immune complex.
The experiments were repeated twice with similar results, and a
representative result is shown.
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AMPK Inhibits Ras Activation without Attenuating the Receptor
Tyrosine Kinase Activity--
To investigate the effect of AICAR on
Ras activity, we used the commercially available Ras activity assay kit
(upstate biotechnology), which is based on the fact that
only activated Ras (Ras-GTP) can bind to the RBD of Raf-1 (34, 35).
Active GTP-bound Ras was pulled down from cell lysates with the
GST-Raf-RBD coupled to glutathione agarose, and the fraction of
activated Ras was determined by immunoblotting with a Ras antibody
(Fig. 9). EGF and IGF-1 rapidly activated
Ras within 3 min; pretreatment of AICAR almost completely blocked this
activation, and this effect of AICAR on Ras was distinctively
diminished from the anti-
1 cells, indicating again that its effect
is mediated by AMPK (Fig. 9). This result together with the data in
Fig. 8 illustrates that two distinct signaling pathways originated from
an identical ligand (EGF) are differentially affected by AICAR.
Therefore, the receptor itself is not likely to be directly affected by
AICAR. Thus, to test this possibility, we examined the effect of AICAR
on phosphorylation of EGFR and IGF-1 receptor
subunit (IGF-1R)
(Fig. 10); EGF and IGF-1 start
transmitting signals by binding to their receptors, and this
interaction causes autophosphorylation of receptors on tyrosine
residues. These receptors were immunoprecipitated with each receptor
antibody and immunoblotted with an antibody specific for
phosphotyrosine. The results revealed that growth factor-enhanced phosphorylation of EGFR and IGF-1R on tyrosine residues was hardly affected by AICAR, and these results were reconfirmed by immunoblotting with the receptor antibody following immunoprecipitation with the
phosphotyrosine-specific antibody (Fig. 10). Additionally, we examined
the effect of AICAR on the interaction between the receptor and growth
factor receptor-bound protein 2 (Grb2) (Fig. 10). Grb2 forms a complex
with the guanine nucleotide-exchanging factor Sos in the cytoplasm.
Once the EGFR or IGF-1R is activated by autophosphorylation, Grb2-Sos
complex is recruited to the receptor and then catalyzes the formation
of the active GTP-bound form of Ras (36). Our result revealed that the
receptor-Grb2 interaction stimulated by each growth factor was not
affected by AICAR, either (Fig. 10). Consequently, our data suggest
that AMPK can down-regulate the growth factor-induced Erk pathway at
the Ras level. In addition, AMPK is likely to up-regulate a
Ras-independent pathway leading to Raf-1.

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Fig. 9.
Effects of AICAR on Ras activation induced by
IGF-1 and EGF. The control NIH-3T3 cells and anti- 1 cells were
incubated with or without 1 mM AICAR for 1 h and then
stimulated with 50 nM IGF-1 or 100 ng/ml EGF for 3 min,
respectively. The fraction of active Ras was determined by affinity
precipitation assay as described under "Experimental Procedures,"
and the Ras activity at each condition was expressed as a fold
induction of the basal activity. Experiments were repeated 2-4 times
with similar results, and a representative result is shown.
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Fig. 10.
IGF-1 and EGF receptor kinase activity was
not affected by AICAR treatment. NIH-3T3 cells pretreated or
untreated with AICAR were stimulated with 50 nM IGF-1 or
100 ng/ml EGF for 3 min, respectively. IGF-1 receptor subunit and
EGF receptor were immunoprecipitated with an antibody for each receptor
(IP: IGF-1R and IP: EGFR) followed by
immunoblotting with an antibody specific for phosphotyrosine (IB:
P-Y). The amount of each receptor in the immune complex was
simultaneously determined by immunoblotting with each receptor antibody
(IB: IGF-1R, EGFR). The experiment by an opposite way showed
a similar result: immunoprecipitation with a phosphotyrosine antibody
(IP: P-Y) followed by immunoblotting with each receptor
antibody. Under the identical experimental condition, the association
of Grb2 with each receptor was also examined by immunoprecipitation
with Grb2 antibody (IP: Grb2) followed by immunoblotting
with each receptor antibody.
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DISCUSSION |
In the present study, we have investigated the effects of AMPK
activation on the Erk pathway induced by two different growth factors,
IGF-1 and EGF. Pretreatment of AICAR, an activator of AMPK, drastically
inhibited the Ras activation induced by IGF-1 or EGF (Fig. 9). This
inhibitory effect of AICAR was distinctively diminished in NIH-3T3
cells in which AMPK activity was suppressed by expression of an
antisense RNA for the AMPK catalytic subunit (Figs. 2 and 9).
Therefore, these observations illustrate that the observed effects of
AICAR in the present work are indeed mediated by AMPK. Because IGF-1 or
EGF receptor activity was not affected by AICAR (Fig. 10), our results
suggest that AMPK can negatively regulate Erk pathway at Ras level. In
contrast to the previous report (15), phosphorylation of Raf-1
Ser621 was not involved in the AMPK-mediated regulation of
the Erk pathway (Fig. 6)
We have identified another signaling pathway that seems to be
positively affected by AMPK. In case of IGF-1-induced Erk cascades, Ras-dependent pathway is predominant as shown in Fig. 8,
and down-regulation of Ras activity was directly reflected to the
downstream effector Raf-1 and Erk activity (Figs. 1 and 7). Although
Ras activation induced by EGF was also inhibited by AICAR,
EGF-dependent Raf-1 and Erk activation was not attenuated
under this condition (Figs. 3A and 7). Our results revealed
that EGF transmits its signal to Raf-1 kinase through Ras-independent
as well as Ras-dependent pathways (Fig. 8), and we also
demonstrated that the Ras-independent pathway leading to Raf-1 was
further stimulated by AICAR (Fig. 8). As a consequence of these two
compensatory effects, the endogenous Raf-1 and the subsequent Erk
activation induced by EGF appear to be not affected by AICAR treatment
(Figs. 3A and 7).
The activity of AMPK with the cell is tightly regulated by the AMP:ATP
ratio. Because of adenylate kinase, which maintains its reaction (2ADP
ATP + AMP) close to equilibrium, the AMP:ATP ratio varies as the
square of the ADP:ATP ratio, and this makes the AMP:ATP ratio a very
sensitive indicator of cellular energy charge (reviewed in Ref. 1). In
cells with sufficient energy supply, the AMP:ATP ratio is very low, and
AMPK is inactive. However, any cellular stress, which causes ATP
depletion, can lead to a dramatic increase of the AMP:ATP ratio caused
by adenylate kinase reaction and the subsequent activation of AMPK in
even fully energized cells. Ras is a key regulator of cell growth in
all eukaryotic cells. Thus, down-regulation of Ras by AMPK could
represent the mechanism for inhibiting cell proliferation when
proliferating cells are exposed to stresses causing ATP depletion. The
fact that the yeast homologue of AMPK is involved in regulating the gene expression and the cell cycle supports this possibility (2). To
reveal the physiological significance of the AMPK-mediated down-regulation of Ras/Erk pathway, the effect of AMPK activation on
DNA synthesis was only examined in the present study (Fig. 5). However,
the role of AMPK may not be limited to the control of cell
proliferation because Ras has been implicated in a variety of cellular
activities including differentiation, development, and cell death (37).
Furthermore, our data suggest that AMPK is likely to positively
regulate the Ras-independent pathway, which also plays an important
role in cells. For example, in mitotic cells, the Ras-independent
mechanism results in a cytoplasmic active Raf-1 (24), and interferon
activation of Raf-1 is mediated by Janus tyrosine kinase-1
independently of Ras (30). In case of EGF signaling, protein kinase C
or calcium influx was implicated in Ras-independent pathway for Erk
regulation (33); members of protein kinase C family have been also
widely implicated in the regulation of cell growth and differentiation
(38). Therefore, the possibility that AMPK is involved in the
regulation of various cellular processes in addition to cell
proliferation deserves further investigation.
Besides the known stress conditions, AMPK would be differentially
regulated in a multitude of cellular processes such as proliferation, differentiation, and cell death because changes in the intracellular level of ATP and AMP occur in these processes as well. For example, an
altered level of phosphometabolites including AMP and ATP has been
constantly observed among differentiated cells, proliferating cells,
and tumor cells (39). In addition, intracellular ATP levels are known
to be a determinant of cell death fate by apoptosis or necrosis (40).
Thus, although further studies are required, the current
implications of AMPK in the regulation of Ras activity and
Ras-independent signaling pathways suggest that AMPK may play an
important role in coupling the varying energy status of the cell to the
regulation of diverse cellular processes.
As an attempt to reveal the underlying mechanisms for the AMPK-mediated
regulation of signaling molecule(s), it would be of interest to
determine whether AMPK is localized in plasmalemmal caveolae, small
membrane invaginations present in most cells of higher eukaryotes.
These membrane specializations are known to function as signal
transduction organizing centers and compartmentalize a subset of signal
transducing molecules including G-protein
subunit, Ras, Src family
tyrosine kinase, eNOS, EGFR, and protein kinase C isoforms (41). A
portion of AMPK is membrane-bound probably because of the N-terminally
myristoylated
1 subunit of AMPK (42), which serves as a scaffold
protein for the formation of the trimeric complex with the
and
subunits. In addition, it was demonstrated that AMPK
coimmunoprecipitated with eNOS (14), which is rich in caveolae. Thus, a
signal module that couples the localized recognition of intracellular
energy charge with the regulation of signaling molecules might exist in caveolae.
Although the antisense RNA experiments support the possibility that the
AICAR effects are mediated by AMPK (Figs. 2, 5, and 9), it should be
carefully examined whether or not the AMPK-independent effects of AICAR
still interfere with and complicate the interpretation of our data.
AICAR could affect de novo purine synthesis because ZMP is a
natural intermediate in the pathway (26). In fact, AICAR was reported
to inhibit the cell growth of Chinese hamster fibroblasts, and this was
associated with a reduction of phosphoribosyl pyrophosphate and an
increase in purine nucleotides (43). Thus, these subsidiary effects
might be responsible for the partial inhibition by AICAR of the basal
and EGF-dependent DNA synthesis in an Erk-independent
manner (Fig. 5). However, the observation that IGF-1-induced DNA
synthesis was more drastically inhibited under the identical condition
cannot be explained solely by the derangement of purine synthesis
caused by AICAR, and moreover, IGF-1-dependent DNA
synthesis of anti-
1 cells was hardly inhibited by AICAR (Fig. 5).
Therefore, the AMPK-mediated down-regulation of Ras/Erk pathway must be
more critical for the AICAR-dependent inhibition of
IGF-1-induced DNA synthesis as demonstrated in our study. Furthermore,
similar effects of AICAR on EGF- and IGF-1-dependent Erk
activation were observed from several different cell types (Fig. 4),
which have the remarkable different rates of purine metabolism. This
further supports the possibility that the AICAR effects observed in the
present work are mediated by AMPK rather than caused by the secondary
effects of AICAR on the purine biosynthesis pathway.
More relevant to our study, a recent report shows that a combinatorial
action of 5'-AMP and small heat shock proteins mediate the inhibitory
effect on Src kinase in terminally differentiated adult cardiomyocytes
(44). Raf-1 activation can be mediated via tyrosine phosphorylation by
Src (45) or family tyrosine kinase such as Lck (46). In addition, the
oncogenic form of Src and Ras can synergize in phosphorylation and
activation of Raf-1 (45, 47). Because ZMP acts as 5'-AMP analogue,
down-regulation by AICAR of IGF-1-dependent Raf activation
could be interpreted as a result of ZMP-mediated inhibition of Src
without involvement of AMPK. However, this assumption is not in line
with our observation that EGF-dependent Raf-1 activation
was not blocked by AICAR (Fig. 7). Src family tyrosine kinase can also
interact with growth factor receptors including EGFR and IGF-1R (48).
These interactions are known to be bi-directional; Src can bind,
phosphorylate, and activate the receptor and vice versa (48). Under our
experimental conditions, however, EGF and IGF-1 receptor activities
were not affected by AICAR (Fig. 10). Therefore, inhibition of Src by
AMP-mimetic ZMP is not likely to be engaged in our system. Moreover,
the inhibitory effect of 5'-AMP on Src was predominantly observed in
heart tissue (44).
For optimal AMPK activity, the formation of a trimeric subunit complex
is necessary (49), and this has in part hindered the current progress
in transfection studies aimed at revealing the downstream effects of
AMPK. However, the molecular approaches that overcome this technical
difficulty are currently under development (50, 51). Thus, we expect
that these approaches will markedly enhance our understanding of the
physiological roles of AMPK within a few years.