Download AMP-activated protein kinase phosphorylates transcription factors of

J Appl Physiol 104: 429–438, 2008.
First published December 6, 2007; doi:10.1152/japplphysiol.00900.2007.
AMP-activated protein kinase phosphorylates transcription factors
of the CREB family
D. M. Thomson, S. T. Herway, N. Fillmore, H. Kim, J. D. Brown, J. R. Barrow, and W. W. Winder
Department of Physiology and Developmental Biology, Brigham Young University, Provo, Utah
Submitted 21 August 2007; accepted in final form 27 November 2007
20 years ago, acetyl-CoA carboxylase (ACC) and
3-hydroxy-3-methylglutaryl (HMG)-CoA reductase were identified as phosphorylation targets for the newly discovered
AMP-activated protein kinase (AMPK) (11). Since that time,
an increasing number of cellular proteins have been identified
as direct downstream targets (49). In liver, phosphorylation
of ACC and HMG-CoA reductase by AMPK results in decreased fatty acid and cholesterol synthesis, in keeping with
this kinase’s general role of enhancing ATP production and
reducing ATP utilization during times of energy stress. Specific transcription factors (e.g., p300, HNF4-␣, ChREBP,
TORC2) are phosphorylated by AMPK in liver, which results
in decreased expression of lipogenic and gluconeogenic en-
zyme proteins (11, 49). In skeletal muscle, activation of AMPK
in response to contraction is important in stimulation of glucose uptake and fatty acid oxidation . Phosphorylation/inhibition of ACC in muscle results in a decrease in malonyl-CoA,
relieving inhibition of carnitine acyl transferase and allowing
fatty acyl-CoA to enter the mitochondrial matrix where oxidation occurs (31, 37, 52). Although activation of AMPK using
5-aminoimidazole-4-carboxamide-1-␤-D-ribofuranoside (AICAR)
results in GLUT4 translocation and stimulation of glucose
uptake into muscle, the specific signaling pathway is unknown
(13, 28). In addition to acute effects on glucose uptake and
fatty acid oxidation, recurrent activation of AMPK results in
increases in expression of mitochondrial proteins, hexokinase,
and GLUT4, thus increasing capacity of the muscle to produce
ATP in response to contraction (16, 19, 35, 54). The increase
in GLUT4 is thought to be due in part to direct phosphorylation
of a transcription factor, GLUT4 enhancing factor (GEF), and
also to stimulation of nuclear localization of myocyte enhancing factor (MEF2) (17). Both of these transcription factors
have binding sites on the GLUT4 promoter. Recently, peroxisome proliferator-activated receptor-␥ coactivator-1␣ (PGC1␣) has been identified as one target of AMPK in the Thr177
and Ser538 sites (18). Phosphorylation at these sites appears to
be important for PGC-1␣ induction of the PGC-1␣ promoter
and induction of mitochondrial biogenesis (18). Other phosphorylation targets of AMPK responsible for the increase in
hexokinase and mitochondrial enzymes have not yet been
completely elucidated.
Examination of the promoter region of the hexokinase II
gene reveals a site for cAMP-response element (CRE) binding
protein (CREB) and activating transcription factor 1 (ATF1)
(34). Both homodimers and heterodimers of CREB and
ATF1 bind to this response element and enhance the rate of
transcription. The increase in muscle hexokinase II expression in response to AICAR infusion was reported to be due
to an increase in the rate of transcription (44). The increase
in hexokinase II mRNA in response to AICAR was reported
to be eliminated by knockout of the ␣2-subunit of AMPK
(20). In addition, PGC-1␣, the coactivator responsible in
part for regulating expression of many mitochondrial enzyme genes, has a CRE in the promoter region of its gene
(10, 14, 15, 23). A complex of LKB1, STRAD, and MO25
proteins serves as the major upstream kinase for AMPK in
skeletal muscle (26, 39, 48). Expression of PGC-1␣, cytochrome c, and citrate synthase each was diminished in red quadriceps muscle of muscle-specific LKB1 knockout (MLKB1-KO)
mice (48).
Address for reprint requests and other correspondence: W. W. Winder, Dept.
of Physiology and Developmental Biology, 545 WIDB, Brigham Young Univ.,
Provo, UT 84602 (e-mail: [email protected]).
The costs of publication of this article were defrayed in part by the payment
of page charges. The article must therefore be hereby marked “advertisement”
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
activating transcription factor 1; adenosine 5⬘-cyclic monophosphateresponsive element modulator; LKB1; transcription control
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Thomson DM, Herway ST, Fillmore N, Kim H, Brown JD,
Barrow JR, Winder WW. AMP-activated protein kinase phosphorylates transcription factors of the CREB family. J Appl
Physiol 104: 429–438, 2008. First published December 6, 2007;
doi:10.1152/japplphysiol.00900.2007.—AMP-activated protein kinase (AMPK) has been identified as a regulator of gene transcription, increasing mitochondrial proteins of oxidative metabolism as
well as hexokinase expression in skeletal muscle. In mice, musclespecific knockout of LKB1, a component of the upstream kinase of
AMPK, prevents contraction- and 5-aminoimidazole-4-carboxamide-1-␤-D-ribofuranoside (AICAR)-induced activation of AMPK
in skeletal muscle, and the increase in hexokinase II protein that is
normally observed with chronic AICAR activation of AMPK.
Since previous reports show a cAMP response element in the
promoter region of the hexokinase II gene, we hypothesized that
the cAMP-response element (CRE) binding protein (CREB) family
of transcription factors could be targets of AMPK. Using radioisotopic kinase assays, we found that recombinant and rat liver and
muscle AMPK phosphorylated CREB1 at the same site as cAMPdependent protein kinase (PKA). AMPK was also found to phosphorylate activating transcription factor 1 (ATF1), CRE modulator
(CREM), and CREB-like 2 (CREBL2), but not ATF2. Treatment of
HEK-293 cells stably transfected with a CREB-driven luciferase
reporter with AICAR increased luciferase activity approximately
threefold over a 24-h time course. This increase was blocked with
compound C, an AMPK inhibitor. In addition, AICAR-induced
activation of AMPK in incubated rat epitrochlearis muscles resulted in an increase in both phospho-acetyl-CoA carboxylase and
phospho-CREB. We conclude that CREB and related proteins are
direct downstream targets for AMPK and are therefore likely
involved in mediating some effects of AMPK on expression of
genes having a CRE in their promoters.
430
AMPK PHOSPHORYLATES CREB, ATF1, AND CREM
We hypothesized first that induction of hexokinase II by
chronic AICAR injection would be prevented in mice lacking
LKB1/AMPK signaling in their muscles. A previous study
designed to develop AMPK assays using relatively small
synthetic peptides with sequence homology to CREB suggested that CREB could be phosphorylated by AMPK (29). In
addition, intracerebroventricular (icv) injection of AICAR induced a large increase in phospho-CREB in the arcuate nucleus, detected by immunohistochemistry (25). These observations along with scanning of peptide sequences for AMPK
recognition motifs led to the hypothesis that CREB and other
members of the CREB family of proteins are direct downstream targets for AMPK and that activation of AMPK would
result in an increase in phospho-CREB in skeletal muscle and
other tissues.
MATERIALS AND METHODS
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Animal care and generation of LKB1-KO mice. Experimental
procedures were approved by the Institutional Animal Care and Use
Committee of Brigham Young University. Mice were bred and housed
in a temperature-controlled (21–22°C) room with a 12:12-h light-dark
cycle, with free access to standard chow and water. Muscle-specific
knockout of LKB1 (LKB1-KO) was achieved by cross-breeding
LKB1 conditional mice (3) (provided by R. DePinho and N. Bardeesy,
Dana-Farber Cancer Institute, Boston, MA) in which the LKB1 allele
contains loxP sites in introns 2 and 6 with MCK-Cre transgenic mice
(4) (provided by C. R. Kahn, Joslin Diabetes Center, Boston, MA) in
which Cre recombinase is expressed constitutively in skeletal muscle
and heart under the creatine kinase promoter. In the resultant
LKB1-KO mice, the skeletal muscle-specific expression of Cre results
in Cre-mediated recombination between loxP sites and deletes exons
of the LKB1 gene between the two sites. Presence of the floxed LKB1
and the MCK-Cre genes was assessed by PCR ear-snip analysis as
described previously (48).
Treatment of wild-type and MLKB1-KO mice with AICAR. Wildtype and MLKB1-KO mice were given a subcutaneous injection (0.5
mg/g body wt) of AICAR 5 days in succession and were anesthetized
(pentobarbital sodium, 45 mg/kg ip) for tissue removal 1 h after the
final injection. The white region of the quadriceps was removed for
quantitation of hexokinase II, phospho-AMPK, and phospho-ACC by
Western blot.
Phosphorylation of CREB in incubated epitrochlearis muscles.
Wistar strain rats (95–130 g body wt) were anesthetized with pentobarbital sodium (45 mg/kg ip). After rats had been under anesthesia
for 45 min to 1 h, the epitrochlearis muscles were quickly removed
and incubated in cell culture medium (DMEM) for 60 min at 37°C in
the presence or absence of 2 mM AICAR. During the incubation
period, the flasks were gassed continuously with 95% oxygen-5%
carbon dioxide. Muscles were frozen in liquid nitrogen. Homogenates
were prepared in medium containing 50 mM Tris, 250 mM mannitol,
50 mM NaF, 5 mM sodium pyrophosphate, 1 mM EDTA, 1 mM
EGTA, 1% Triton X-100, 1 mM DTT, 1 mM benzamidine, 0.1 mM
phenylmethylsulfonyl fluoride (PMSF), and 5 ␮g/ml soybean trypsin
inhibitor, pH 7.4. Homogenates were freeze-thawed three times to
ensure rupture of the nuclei and then centrifuged at 1,200 g to remove
particulate matter before blotting. Western blots for phosphorylated
AMPK (pThr172, Cell Signaling no. 2531, Danvers, MA), phosphorylated ACC (Cell Signaling no. 3661), and phospho-CREB (pSer133)
(Upstate, Charlottesville, VA) were run on the supernatants using
standard blotting procedures as described previously (48).
In an additional experiment, LKB1, phospho-AMPK, and phospho-CREB were quantitated by Western blot in gastrocnemius,
tibialis anterior, and heart muscle from wild-type and MLKB1-KO
mice (n ⫽ 6 per genotype), using similar procedures as outlined
above, except that the mouse homogenates were clarified by
centrifugation at 5,000 g.
Effect of AICAR on HEK-293 cells transfected with a CREB-driven
luciferase reporter. The HEK-293/CREB-luc cell line was obtained
from Panomics (Redwood City, CA). This line is stably transfected
with a luciferase reporter construct that has a CRE in its promoter.
Cells were cultured in DMEM supplemented with 10% FBS, 10,000
U penicillin and streptomycin/ml, and 100 ␮g hygromycin B/ml in a
humidified incubator at 37°C in 5% CO2. Cells were then transferred
to 96-well plates (⬃5 ⫻ 104 cells/well) and incubated overnight
before replacing medium with serum-free medium containing one of
the following inducing agents or vehicle: 10 ␮M forskolin or 1 mM
AICAR. Cells were then incubated for 1, 2, or 4 h before addition of
lysis buffer and determination of luciferase activity using the Promega
assay system kit (Madison, WI) according to instructions in the kit.
Bioluminescence was determined using a Biotec Synergy HT plate
reader (Winooski, VT). In a second experiment, HEK-293/CREB-luc
cells were incubated 24 h with and without 1 mM AICAR in the
presence and absence of 20 ␮M compound C (EMD Chemicals, San
Diego, CA), a potent AMPK inhibitor. Luciferase activity was measured as described above, and phospho-AMPK, phospho-ACC, phospho-CREB, and phospho-ATF1 (Cell Signaling no. 9191), total
AMPK (Cell Signaling no. 2532), total ACC (streptavidin-horseradish
peroxidase conjugate, GE Health Sciences, Piscataway, NJ), and total
CREB (Cell Signaling no. 9104) were quantitated by Western blotting, which was performed as follows. After the treatment period in
six-well plates, cells were lysed with lysis buffer (20 mM HEPES, pH
7.4, 50 mM NaF, 100 mM KCl, 1 mM EDTA, 50 mM ␤-glycerophosphate, 10 mM sodium pyrophosphate, 1% Triton X-100, 0.1 mM
PMSF, 1 mM benzamidine, 1 mM DTT, 1 mM NaVO4, 2.5 ␮g/ml
soybean trypsin inhibitor), then frozen at ⫺95°C (still in their wells).
After thawing at room temperature, lysates were pipetted up and
down, then transferred to tubes, and centrifuged at 1,500 g for 10 min.
An aliquot of supernatant containing 20 ␮g of protein was then
electrophoresed on 7.5% (phospho- and total AMPK␣ and ACC) or
10% (phospho-ATF1, phospho-CREB, and total CREB) gels and
Western blotted using standard procedures.
Recombinant transcription factors. The following recombinant
transcription factors were obtained from Panomics: ATF1 (accession
no. NM_005171), ATF2 (accession no. NM_005171), CREB1 (accession no. NM_004379), CRE modulator (CREM; variant 22, accession no. NM_183060), and CREB-like 2 (CREBL2, accession no.
NM_001310). All were purified by the vendor to 80 –90% purity
using His-Tag purification. Recombinant CREB coupled with maltose
binding protein was obtained from Active Motif (Carlsbad, CA).
Phosphorylation/activation of AMPK. Recombinant ␣2␤2␥2-AMPK
(rAMPK) was prepared as described previously (45, 46). rAMPK
(4 ␮g) was activated by incubation with 0.1 ␮g recombinant LKB1STRAD-MO25 (Upstate-Millipore) in 25 ␮l medium containing 40
mM HEPES, 0.2 mM AMP, 80 mM NaCl, 8% glycerol, 0.8 mM
EDTA, 0.8 mM DTT, 5 mM MgCl2, 0.2 mM ATP, pH 7.0, for 60 min
at 30°C.
Isolation of rat AMPK from liver and skeletal muscle. AMPK from
rat liver was purified as described by Hawley et al. (12) with
modifications for rat skeletal muscle as previously indicated (45).
Phosphorylation of CREB1 by AMPK and PKA. Recombinant
CREB1 (Panomics) (0.5 ␮g) was incubated 60 min in the presence of
the activated AMPK (0.3 ␮g) or 0.5 U PKA (Sigma-Aldrich, St.
Louis, MO; cat. no. 2645) in a final volume of 25 ␮l reaction mix
containing 40 mM HEPES, 0.2 mM AMP, 0.2 mM ATP, 80 mM
NaCl, 8% glycerol, 0.8 mM EDTA, 0.8 mM DTT, 5 mM MgCl2, and
[␥-32P]ATP (5 ␮Ci). The reaction was terminated by adding an equal
volume of 2⫻ sample buffer. Proteins in the reaction mix were
separated using SDS-PAGE on 10% gels. After washing, staining, and
drying the gels, phosphorylation of the target protein was determined
by autoradiography. This procedure was repeated for CREB-related
proteins, ATF1, ATF2, and CREM. In a control experiment, LKB1-
AMPK PHOSPHORYLATES CREB, ATF1, AND CREM
Table 1. Amino acid sequences of putative AMPK target
sites of CREB-related proteins
Name
Phosphorylation Site
Sequence
CREB
Ser98
Ser133
LKRLFSGTQI
LSRRPSYRKI
CREB1
Ser119
LSRRPSYRKI
ATF1
Ser63
Ser267
LARRPSYRKI
LKDLYSNKSV
ATF2
No consensus sites
CREM
Ser71
Ser192
CREBL2
No consensus sites
LSRRPSYRKI
VKCLESRVAV
AMPK, AMP-activated protein kinase; CREB, cAMP-response element
(CRE) binding protein; CREM, CRE modulator; CREBL2, CREB-like-2;
ATF1 and ATF2, activating transcription factor 1 and 2, respectively.
J Appl Physiol • VOL
previously phosphorylated as indicated above. After a 10-min incubation at 30°C, an aliquot of the reaction mix was spotted on a
1-cm-square P81 filter paper. The labeled ATP was washed out by six
washes with 1% phosphoric acid. The papers were immersed briefly
in acetone, dried, and added to scintillation vials. After addition of
Ecolite scintillation cocktail, radioactivity was determined in a liquid
scintillation counter. The Km for CREB-Ser133 was also determined
by measurement of activity at concentrations ranging from 4 ␮M to
100 ␮M CREB-Ser133. The Km was calculated using the enzyme
kinetics module of Sigma Plot (Aspire Software International,
Ashburn, VA).
RESULTS
The increase in white quadriceps hexokinase that occurs
with 5 days of chronic AICAR injections is prevented when
LKB1 is not expressed in muscle (Fig. 1B). Although phosphoAMPK did not appear to be increased 1 h following the final
injection with AICAR, phospho-ACC was markedly increased
in wild-type but not in MLKB1-KO mice (Fig. 1A). This
provides evidence for the ZMP-triggered allosteric activation
of phospho-AMPK. The MLKB1-KO mice, however, had
much less phospho-AMPK and phospho-ACC. ZMP derived
from phosphorylation of AICAR would not be expected to
activate the nonphosphorylated AMPK. rAMPK has no activity in the presence or absence of 0.2 mM AMP or 0.2 mM ZMP
unless first phosphorylated by LKB1-STRAD-MO25 (47).
CREB phosphorylation was lower in gastrocnemius, tibialis
anterior, and heart muscles (Fig. 2) from a separate group of
MLKB1-KO mice (in which AMPK phosphorylation is virtually eliminated), suggesting that AMPK may play a role in
maintaining basal levels of CREB phosphorylation. CREB
phosphorylation was not significantly altered in red and white
quadriceps muscles from MLKB1-KO mice (data not shown).
In incubated rat epitrochlearis muscles, phospho-AMPK is
significantly increased in response to 60 min incubation with
AICAR to activate AMPK (Fig. 3A). This is accompanied by
a concurrent increase in phosphorylation of the downstream
target, ACC (Fig. 3B). Under these conditions, phospho-CREB
is also significantly increased over muscles incubated with
medium only, without AICAR (Fig. 3C).
In the HEK-293 cells transfected with the luciferase reporter
gene, bioluminescense increased ⬃13-fold in response to 10
␮M forskolin over the course of a 4-h incubation (data not
shown). AICAR also stimulated transcription of the CREBdriven luciferase gene with a progressive increase in activity
over the course of the 4-h period (Fig. 4A). After 24 h an
approximate threefold increase occurred in luciferase activity
in response to AICAR compared with controls. Compound C
completely blocked this increase in luciferase expression triggered by incubation with AICAR (Fig. 4B). Phosphorylation of
AMPK (Fig. 4C), ACC (Fig. 4D), and CREB (Fig. 4E) were all
increased after 24 h of incubation with AICAR as well, and all
of these effects were likewise blocked by compound C.
Figure 5 shows that both AMPK and PKA phosphorylate
CREB1 with similar stoichiometry. Figure 6A indicates both
PKA and AMPK compete for phosphorylation at the same site
on CREB1. When CREB1 is first phosphorylated with PKA
with 0.2 mM ATP in the absence of radioactively labeled ATP,
little phosphorylation is observed with subsequent addition of
labeled ATP and AMPK. Likewise, when AMPK is added first
with 0.2 mM ATP and without label, Ser119 is already ester-
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STRAD-MO25 was added to the reaction mix with each of the
recombinant proteins but in the absence of rAMPK. No phosphorylation of any of the proteins was noted unless AMPK was present. In
some experiments, phosphorylation of CREB and other CREB-related
proteins was determined using Western blotting with an antibody
specific for the phospho-Ser133 site (Millipore, Billerica, MA). The
identical or very similar amino acid sequence in phospho-CREB1,
phospho-ATF1, and phospho-CREM is each detected with this antibody. CREB1 lacks a second AMPK site present in CREB.
To determine if AMPK phosphorylates the same site as PKA, a
competition assay was performed. For the first reactions, AMPK and
PKA were allowed (separately) to phosphorylate CREB1 for 60 min
in the presence of 0.2 mM ATP (unlabeled) and [␥-32P]ATP (labeled
ATP), as described above. In the next reactions, AMPK was allowed
to phosphorylate CREB1 for 30 min using only unlabeled ATP (0.2
mM). Then PKA was added to this reaction along with labeled ATP,
and the reaction was allowed to proceed for another 30 min. This
process was then reversed in another tube, with PKA added first in the
absence of label and AMPK added second along with the label. If
PKA and AMPK phosphorylate the same site on CREB1, then prior
phosphorylation (with unlabeled phosphate) of CREB1 by either PKA
or AMPK would prevent the subsequent incorporation of labeled
phosphate into CREB1 by the other kinase since the serine targets
would all be occupied by unlabeled phosphate. On the other hand, if
the two kinases did not phosphorylate the same site, or if one of the
kinases phosphorylated an additional site, significant incorporation of
labeled ATP would occur after preincubation with the other kinase
and unlabeled ATP.
To confirm that CREB1 could be phosphorylated by native AMPK
as well as by the rAMPK, CREB1 was added to the phosphorylation
mix indicated above along with the AMPK isolated from liver or
muscle. Autoradiography was used to determine incorporation of label
into the recombinant CREB1.
Phosphorylation of an artificial peptide having the sequence surrounding Ser133 of CREB. A peptide (CREB-Ser133) with the sequence ILSRRPSYRKILRR was prepared by BioPeptide (San Diego,
CA). This peptide has an amino acid sequence identical to the one
surrounding Ser133 of CREB, Ser119 of CREB1, and Ser71 of
CREM. With the exception of one amino acid (A replacing S),
CREB-Ser133 is identical to the sequence surrounding Ser63 of ATF1
(see Table 1). Phosphorylation of this peptide was compared with
peptides classically used for determination of AMPK activity (SAMS
from both BioPeptide and Zinsser Analytic and AMARA from Zinsser Analytic) at a concentration of 200 ␮M. Final concentrations in the
assay mix were 40 mM HEPES, 0.2 mM AMP, 80 mM NaCl, 8%
glycerol, 0.8 mM EDTA, 0.8 mM DTT, 5 mM MgCl2, 0.2 mM ATP,
pH 7.0. The reaction was initiated by addition of rAMPK (␣2␤2␥2)
431
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AMPK PHOSPHORYLATES CREB, ATF1, AND CREM
Figure 8 demonstrates that AMPK phosphorylates two preparations of CREB: recombinant CREB1 and recombinant
CREB fused with maltose binding protein. ATF1, CREM, and
CREBL were all found to be phosphorylated by AMPK. We
noted two distinct bands in the CREBL2. It is not clear if this
represents dimer formation. Recombinant ATF2 was not found
to be phosphorylated by AMPK (data not shown).
Figure 9 shows phosphorylation of synthetic peptides by
rAMPK. At concentrations of 200 ␮M, all peptides were
phosphorylated by rAMPK, although the CREB-Ser133 peptide was phosphorylated to a lesser extent than either preparation of SAMS or AMARA (Fig. 9A). The Km for CREBSer133 was determined to be 11.2 ⫾ 2.0 ␮M (Fig. 9B).
DISCUSSION
ified with cold phosphate so that subsequent incubation with
PKA ⫹ labeled ATP shows little incorporation of labeled
phosphate into the protein. Additional evidence that AMPK
and PKA phosphorylate the identical site of CREB1 is provided by the Western blot data using a specific antibody for
Ser133 phospho-CREB (Fig. 6B). After incubation with the
activated rAMPK or PKA, the phospho-serine-specific antibody detected similar amounts of phosphorylation by the two
kinases. Similar results were observed for ATF1 and CREM,
indicating that these two transcription factors are also phosphorylated at the same site by AMPK and PKA (data not
shown).
Figure 7 provides evidence that CREB1 can be phosphorylated by native AMPK isolated and partially purified from rat
liver and rat skeletal muscle. The phenomenon is not restricted
to the recombinant kinase.
J Appl Physiol • VOL
Fig. 2. Phosphorylation of cAMP-response element binding protein (CREB) at
Ser133 in heart, gastrocnemius (Gastroc), and tibialis anterior (TA) muscles
from WT and muscle-specific LKB1 knock-out mice (MLKB1-KO). Representative Western blot images are shown for each muscle. Values are means ⫾
SE for 6 mice. phospho-CREB, phosphorylated CREB. *Significantly different
from corresponding muscles from WT mice, P ⬍ 0.05.
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Fig. 1. Effect of 5 days of subcutaneous injections of 5-aminoimidazole-4carboxamide-1-␤-D-ribofuranoside (AICAR; 0.5 mg/g body wt) on phosphoacetyl-CoA carboxylase (phospho-ACC) (A) and hexokinase II (B) in white
quadriceps of wild-type (WT) and muscle-specific LKB1 knockout mice (KO).
Phospho-AMPK was barely detectable in the KO mice and was not increased
in response to the AICAR injection (data not shown). Values are means ⫾ SE
for 8 mice. Representative Western blot images are shown for each protein.
AIC, AICAR. *Significantly different from all other treatments, P ⬍ 0.05.
The failure of AICAR to induce an increase in hexokinase II
expression in the MLKB1-KO mice provided the first clue that
the CREB family of transcription factors may be targets for
AMPK. Since the hexokinase II gene has a CRE element in its
promoter, we hypothesized that AMPK could be stimulating
transcription of this gene by phosphorylating CREB or ATF1.
Known target proteins of AMPK have amino acid sequences
surrounding the phosphorylated serine residue with the following characteristics: 1) an amino acid with a bulky hydrophobic
R-group (M, L, I, F, V) at position P (phospho-serine) minus 5;
2) an amino acid with a bulky hydrophobic R-group at P plus
4; 3) an amino acid with a basic R-group (R, K, H) at either P
minus 4 or P minus 3 (49). Table 1 shows the sequences in
the proteins we investigated, demonstrating that all except
CREBL2 have one or two putative sites that could be phosphorylated by AMPK. Note that CREB (Ser133), CREB1
(Ser119), and CREM (Ser71 in this variant and Ser120 in
full-length human CREM) all have identical sequences surrounding one AMPK recognition site. ATF1 (Ser63) is identical except for an alanine in place of serine in the P minus 4
position.
AMPK PHOSPHORYLATES CREB, ATF1, AND CREM
In these experiments, CREB1 was found to be phosphorylated at the same site by both PKA and AMPK evidenced by
competition for the same site in the labeled ATP assays and by
the Western blots using the antibody targeting Ser133 of
CREB. This antibody also detected phosphorylation of ATF1
and CREM by AMPK. The sequence recognition motif for
PKA is -XRRXSX- (X ⫽ any amino acid), which is present for
CREB, CREB1, ATF1, and CREM within the AMPK targeting
J Appl Physiol • VOL
site (8, 42). The synthetic peptide CREB-Ser133 was found to
be a good substrate for AMPK. The Km for this peptide was
relatively low (11.2 ⫾ 2 ␮M) and comparable to reported
values for SAMS peptide (26 ␮M) (41) and that reported for the
best model substrate, a peptide based on rat ACC1 (5 ␮M) (40).
CREB has previously been identified as a target for a
number of different kinases, including protein kinase C (PKC),
Akt, MSK-1, RSK2, p70S6K, MAPKAP-K2, and CaMKII
and -IV (8, 30, 42). These CREB-related transcription factors
have the capacity therefore to integrate a number of upstream
regulatory signals. In the present experiment, it is possible that
one or more kinases in addition to AMPK may have been
activated by AICAR. For example, atypical forms of PKC have
been reported to be activated with AICAR treatment, and
evidence is presented that this activation is downstream from
AMPK activation (6). Thus AMPK may not only phosphorylate CREB directly but also may activate other kinases capable
of phosphorylating CREB.
Phosphorylation of CREB, ATF1, and CREM has previously been demonstrated to be important for enhancement of
transcriptional activity. Although phosphorylation is not essential for binding to the CRE on promoter regions of genes,
recruitment of essential coactivators (CREB binding protein or
CBP, and p300) to the CREB-CRE complex is greatly enhanced by the phosphorylation (8, 30, 42). Each of these
factors may bind as homo- or heterodimers to the palindromic
CRE motif (TGACGTCA). Single-motif CREs also exist
(GTCA). CBP and p300 interact with TFIIB, TBP, and an
RNA helicase and also have histone acetyltransferase activity
(42, 43). The net effect of phosphorylation of CREB, ATF1,
and CREM is an increase in rate of transcription of the target
gene.
A large number of genes have been found to have CRE
elements in their promoters. Transcription factors of the CREB
family influence expression of a vast number of proteins
involved in many physiological processes. These include metabolic regulation, neurotransmitter and neurotransmitter receptor synthesis, memory, long-term potentiation, expression of
growth factors, immune regulation, structural protein expression, cell cycle regulation, DNA repair, and transport (30).
Those of particular importance to muscle include genes for
lactate dehydrogenase, cytochrome c, amino-levulinate synthase, carnitine palmitoyl-transferase, phosphoenolpyruvate carboxykinase, hexokinase II, pyruvate dehydrogenase, and PGC1␣, to name a few (30).
Evidence that phosphorylation of CREB by AMPK may be
an important cellular regulatory mechanism is supported by the
experiment with the HEK-293 cells transfected with the
CREB-driven luciferase reporter. The well-known activator of
AMPK, AICAR, stimulates a significant increase in both
CREB phosphorylation and transcription of the reporter gene
resulting in accumulation of the luciferase product. The AMPK
inhibitor, compound C, completely inhibits the 24-h response
to AICAR in the HEK-293 cells. The presence of LKB1
signaling is necessary for induction of an increase in hexokinase II expression in muscle by chronic injection with AICAR
for 5 days. These observations, coupled with a previous report
indicating reduced expression of PGC-1, cytochrome c, and
citrate synthase in skeletal muscles and heart of MLKB1-KO
mice compared with wild type suggest a physiological role for
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Fig. 3. Effect of 2 mM AICAR on phosphorylation of AMP-activated protein
kinase (AMPK) (A), ACC (B), and CREB (C) in incubated rat epitrochlearis
muscle. Muscles were removed from male Wistar rats and incubated for 1 h in
DMEM without (control) or with 2 mM AICAR under 95% oxygen-5% carbon
dioxide. After freezing the muscles, homogenates were prepared for Western
blots. Values are means ⫾ SE. Representative Western blot images are shown
for each protein. *Significantly different from control, P ⬍ 0.05; n ⫽ 8.
433
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AMPK PHOSPHORYLATES CREB, ATF1, AND CREM
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Fig. 4. Effect of 1 mM AICAR and 20 ␮M compound C (CC) on HEK-293 cells stably transfected with a CREB-driven luciferase reporter gene. A: luciferase
activity after cells were incubated in serum-free DMEM (control) or serum-free DMEM ⫹ AICAR for the times indicated. B: luciferase activity after incubation
of cells for 24 h with the indicated treatments. Control, serum free media ⫹ vehicle (DMSO); AIC, 1 mM AICAR; CC, 20 ␮M compound C. C: AMPK
phosphorylation after incubation of cells for 24 h with the indicated treatments. D: ACC phosphorylation after incubation of cells for 24 h with the indicated
treatments. E: CREB phosphorylation after incubation of cells for 24 h with the indicated treatments. For all experiments, values are means ⫾ SE for 6 incubations
per treatment group. Representative images are shown for the Western blotting data. *Significantly different from control at same time point, P ⬍ 0.05.
the LKB1/AMPK signaling pathway in controlling CREB
phosphorylation and gene expression in skeletal muscle.
Additional evidence for a physiological role of AMPK
phosphorylation of CREB comes from studies on the arcuate
nucleus in the hypothalamus (25). C75, a fatty acid synthetase
blocker, reduced AMPK phosphorylation in the hypothalamus
and reduced food intake. Infusion of AICAR reversed this
effect on food intake. Blocking the action of AMPK using
compound C also caused a marked reduction in food intake.
J Appl Physiol • VOL
Infusion of AICAR (icv) into the hypothalamus caused a
marked increase in phospho-CREB. The immunoreactivity of
phospho-CREB fluctuated similarly to phospho-AMPK in response to C75 and fasting. It was hypothesized that AMPK
phosphorylates and activates CREB, which then increases
neuropeptide Y (NPY) expression. The increase in NPY then
triggers the increase in food intake. More recent studies in our
laboratory show a concurrent increase in AMPK activation
along with an increase in phospho-CREB in response to ex-
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AMPK PHOSPHORYLATES CREB, ATF1, AND CREM
435
Little data is available regarding effect of muscle contraction
on phosphorylation of CREB. One study on human subjects
exercising one leg for 1 h demonstrated an increase in phospho-CREB in the nonexercising leg 1 h postexercise, but not in
the contracting muscle, although there appeared to be a trend
toward an increase at the end of exercise in the contracting
muscle (51). Subjects were working at ⬃70% of maximal O2
uptake. A more recent one-leg training study reports phosphoCREB to be elevated 15 h postexercise in muscle biopsies from
both nontrained and 3-wk trained muscle (36).
We have previously reported increases in mitochondrial
enzyme and hexokinase II expression in muscle in response to
perimental hyperthyroidism in the rat (unpublished data from
our laboratory). These hyperthyroid rats also show increases in
protein expression of several genes with CREs in their promoters. AMPK is clearly activated in muscle in response to
contraction, as the free concentration of AMP increases (50,
52, 53). In other tissues, any energy challenge, such as hypoxia
or substrate deficiency, can activate AMPK (5, 11, 24, 49). The
system is also subject to regulation by hormones, including
IL-6, leptin, and adiponectin (21, 32, 33, 56). Since several
CREB proteins are clearly downstream targets for AMPK as
shown from our in vitro experiments, AMPK must be added to
the list of kinases that can regulate the CREB family of
transcription factors.
Because of the large number of kinases that can phosphorylate CREB, it may prove difficult to consistently demonstrate
changes in CREB phosphorylation state in a tissue by removal
or activation of a specific kinase, such as AMPK. When the
influence of one of these kinases is removed, others may
compensate under many circumstances. For instance, we observed reduced CREB phosphorylation in the gastrocnemius,
tibialis anterior, and heart muscles from MLKB1-KO mice, but
not in the quadriceps muscles. Likewise, it was only after
several unsuccessful trials that we were able to define conditions where an increase in phospho-CREB could be seen in
incubated muscle in response to AMPK activation with
AICAR. First we found it necessary to use serum-free and
albumin-free medium with no hormones added. Before collection of the epitrochlearis, rats were anesthetized for 45 min to
1 h to allow stress of the injection of anesthetic to subside. Rats
in the 95–130 g range of body weight were utilized so that the
epitrochlearis muscles were small and could be oxygenated
adequately by continuous gassing of the incubation flask with
95% oxygen-5% CO2. Without these stringent conditions, we
found the response to AMPK activation with AICAR to be
somewhat variable with respect to phospho-CREB content,
although trends were noted with less stringent conditions.
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Fig. 5. Phosphorylation of CREB1 by AMPK and cAMP-dependent protein
kinase (PKA) (n ⫽ 5). CREB was incubated with recombinant AMPK or PKA
in the presence of 0.2 mM ATP, radiolabeled ATP, and 0.2 mM AMP in
phosphorylation buffer for 60 min. After addition of sample buffer, the
reaction mix was subjected to autoradiography. Gels were dried and subjected
to autoradiography. A representative autoradiogram is shown above. Values
are means ⫾ SE. AMPK, AMPK added alone; PKA, PKA added alone;
CREB1/AMPK, CREB1 added along with AMPK; CREB1/PKA, CREB1
added along with PKA.
Fig. 6. A: competition of AMPK and PKA in phosphorylating CREB1. In
lanes 1 and 3, AMPK or PKA was added to the reaction mix along with
[32P]ATP and 0.2 mM nonlabeled ATP. In lane 2, the CREB1 was incubated
first with PKA and 0.2 mM ATP in the absence of label followed by addition
of AMPK and [32P]ATP label. In lane 4 the CREB1 was incubated first with
AMPK and 0.2 mM ATP in the absence of [32P]ATP followed by addition of
PKA with the [32P]ATP (n ⫽ 4). Reaction mixes were then subjected to
electrophoresis. A representative autoradiogram of the respective reaction
mixes is shown above. B: CREB1 was first phosphorylated by AMPK or PKA
in a reaction mix containing 0.2 mM ATP and 0.2 mM AMP. The reaction mix
was subjected to electrophoresis. A representative Western blot showing
AMPK and PKA phosphorylation of CREB1 with an antibody specific for
phospho-Ser133 of CREB (phospho-Ser119 in CREB1); n ⫽ 5.
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AMPK PHOSPHORYLATES CREB, ATF1, AND CREM
Fig. 7. Autoradiogram demonstrating phosphorylation of CREB1 by AMPK
isolated from skeletal muscle (SM AMPK) or isolated from rat liver (Liv
AMPK) or by recombinant AMPK (rAMPK). Representative of 5 determinations for liver.
Fig. 8. Autoradiogram showing phosphorylation of CREB1, CREB-maltose
binding protein fusion protein (CREB-MBP), activating transcription factor 1
(ATF1), CRE modulator (CREM), and CREB-like 2 (CREBL2). Representative of 5 autoradiograms.
J Appl Physiol • VOL
Fig. 9. A: AMPK phosphorylation of SAMS (two vendors, Z and B), AMARA,
and CREB-Ser133 (CREB-S133) (sequence ⫽ ILSRRPSYRKILRR) at a peptide
concentration of 200 ␮M (n ⫽ 4). The reaction mix contained 0.2 mM ATP,
radiolabeled ATP, and 0.2 mM AMP in the phosphorylation buffer. B: determination of the Km for CREB-S133. The rate of phosphorylation was determined for
concentrations of the peptide (n ⫽ 3 for each concentration) ranging from 0 to 100
␮M. Data were fitted to the Michaelis-Menten equation using the enzyme kinetics
module of SigmaPlot. The Km was determined to be 11.2 ⫾ 2 ␮M.
phosphorylation/activation of the CREB proteins by AMPK is
balanced by the parallel negative signals mediated by SIK or
under what conditions the positive signal predominates. In
skeletal muscle, however, SIK immunoreactivity has been
reported to be undetectable (38), but TORC has been reported
to be a major stimulator of PGC-1␣ gene expression and mitochondrial biogenesis in mouse primary muscle cultures (55).
In summary, induction of an increase in hexokinase II with
chronic AICAR injection is prevented in the MLKB1-KO
mice. HEK-293 cells stably transfected with a CREB-driven
luciferase reporter show an increase in CREB phosphorylation
and luciferase expression on treatment with AICAR. These
increases are blocked with an AMPK inhibitor. When AMPK
is activated in incubated epitrochlearis with AICAR, an increase in phospho-CREB was observed after 1 h of incubation.
The recombinant transcription factors rCREB, rATF1, and
rCREM are all phosphorylated by recombinant AMPK.
CREB1 can be phosphorylated by both PKA and AMPK at the
same phosphorylation site. CREB1 is also phosphorylated by
native AMPK isolated from liver and skeletal muscle. A
synthetic peptide with sequence identical to the site surround-
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chemical activation of AMPK with AICAR (16, 54). PGC-1␣
is now well-established as being important in control of expression of mitochondrial oxidative enzymes (2, 9, 10, 23).
Genes for PGC-1␣, hexokinase II, and other muscle proteins
also have CREB-regulated elements in their promoters. A CRE
has been demonstrated to be essential for nerve stimulationinduced increase in PGC-1␣ promoter activity in mouse tibialis
anterior (1, 57). AMPK phosphorylation of CREB may therefore be responsible in part for increasing these proteins in
response to AICAR and possibly other physiological means of
AMPK activation.
Recent studies have identified additional levels of regulation
of CREB by the LKB1 signaling pathway (22). LKB1 is the
upstream kinase of salt-inducible kinase (SIK), which represses CREB by phosphorylating the transducer of regulated
CREB activity (TORC). TORC upregulates CREB activity by
a mechanism independent of Ser133 phosphorylation. When
phosphorylated by SIK, TORC is exported from the nucleus,
and its stimulatory effect on CREB is lost. In liver, AMPK can
also phosphorylate TORC directly, which induces retention in
the cytoplasm (7, 27). Currently, it is not clear how direct
AMPK PHOSPHORYLATES CREB, ATF1, AND CREM
ing Ser133 in CREB is phosphorylated with a Km lower than
that reported for SAMS peptide. We conclude that the LKB1/
AMPK signaling system exhibits the capacity to regulate the
CREB family of transcription factors by phosphorylation.
21.
22.
GRANTS
These studies were supported by National Institute of Arthritis and Musculoskeletal and Skin Diseases Grants AR-41438 and AR-051928.
23.
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