Am J Physiol Heart Circ Physiol 292: H1460 –H1469, 2007.
First published November 10, 2006; doi:10.1152/ajpheart.01133.2006.
Expression of an active LKB1 complex in cardiac myocytes results in
decreased protein synthesis associated with phenylephrine-induced
hypertrophy
Anna A. Noga, Carrie-Lynn M. Soltys, Amy J. Barr,
Suzanne Kovacic, Gary D. Lopaschuk, and Jason R. B. Dyck
Cardiovascular Research Group, Departments of Pediatrics and Pharmacology,
Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Alberta, Canada
Submitted 16 October 2006; accepted in final form 7 November 2006
that was originally identified
as a tumor suppressor mutated in patients with Peutz-Jeghers
syndrome (17). Patients with mutations in LKB1 are at risk in
developing cancers in a number of different tissues (see Ref. 6
for review) and are also predisposed to developing both benign
and malignant tumors in their gastrointestinal tracts (17, 24).
Although the exact mechanism(s) by which LKB1 suppresses
tumor formation is not well understood, loss of LKB1 function
may contribute to cellular proliferation (2). Indeed, LKB1 has
been associated with proteins that are involved in controlling
cellular proliferation, including p53, Brg1, Lip1, and mammalian target of rapamycin (mTOR) (25, 30, 37, 38). Whereas the
exact substrate of LKB1 that may be involved in cell proliferation has not yet been identified, recent evidence has shown
that LKB1 can phosphorylate and activate AMP-activated
protein kinase (AMPK), raising the possibility that AMPK may
be involved in cellular growth control (18). While tumorigenesis is not common in the heart, the LKB1-AMPK pathway
may be involved in more heart-prevalent growth-control processes such as cardiac hypertrophy.
AMPK is a heterotrimeric protein [consisting of one catalytic subunit (␣) and two regulatory subunits (␤, ␥)] that is
centrally involved in controlling cellular energy homeostasis.
AMPK is activated by metabolic stresses that deplete cellular
ATP and increase AMP levels (see Ref. 14 for review). When
the AMPK holoenzyme binds AMP, the ␣-subunit is phosphorylated by upstream AMPK kinases (9) such as LKB1 (15, 18).
This phosphorylation occurs at Thr-172 of the ␣-subunit and
significantly increases AMPK activity (16, 40).
Once activated, AMPK switches on energy-producing pathways and switches off energy-consuming pathways in an
attempt to restore cellular ATP levels. We have previously
shown that AMPK activation can inhibit ATP-consuming processes such as protein synthesis and that this can inhibit cardiac
myocyte hypertrophy (7). Our previous study has also shown
that the inhibition of protein synthesis associated with hypertrophic growth occurs via the eukaryotic elongation factor-2
(eEF2) kinase-eEF2 signaling axis and/or the p70S6 kinase
pathway (7). Indeed, LKB1 and AMPK have emerged as major
regulators of protein synthesis in multiple cell types due to the
modification of several additional mediators of protein synthesis, including the tuberous sclerosis complex (TSC) 2 (also
called tuberin) (8, 21), mTOR (4, 20, 26), and p70S6 kinase (4,
11, 26, 28). Together, the AMPK-induced inhibition of the
TSC-mTOR-p70s6 kinase pathway leads to the inhibition of
mRNA translation by 40S ribosomal protein S6, which culminates in reduced protein synthesis (33).
Despite the evidence linking AMPK to the control of the
mTOR-p70s6 kinase pathway, there is still controversy as to
whether AMPK activation is involved in the mTOR-p70S6
kinase signaling pathway and subsequent inhibition of protein
synthesis in the cardiac myocyte. For example, it has been
suggested that the mTOR-p70S6 kinase pathway is not inhibited by AMPK in the heart and that an mTOR-independent
Address for reprint requests and other correspondence: J. R. B. Dyck, 474
Heritage Medical Research Centre, Univ. of Alberta, Edmonton, Alberta,
Canada (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.
adenosine 5⬘-monophosphate-activated protein kinase; hypertrophy;
protein synthesis; cardiac myocyte
LKB1 IS A SERINE-THREONINE KINASE
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0363-6135/07 $8.00 Copyright © 2007 the American Physiological Society
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Noga AA, Soltys C-L, Barr AJ, Kovacic S, Lopaschuk GD,
Dyck JR. Expression of an active LKB1 complex in cardiac
myocytes results in decreased protein synthesis associated with
phenylephrine-induced hypertrophy. Am J Physiol Heart Circ
Physiol 292: H1460 –H1469, 2007. First published November 10,
2006; doi:10.1152/ajpheart.01133.2006.—AMP-activated protein kinase (AMPK) is a major metabolic regulator in the cardiac myocyte.
Recently, LKB1 was identified as a kinase that regulates AMPK.
Using immunoblot analysis, we confirmed high expression of LKB1
in isolated rat cardiac myocytes but show that, under basal conditions,
LKB1 is primarily localized to the nucleus, where it is inactive. We
examined the role of LKB1 in cardiac myocytes, using adenoviruses
that express LKB1, and its binding partners Ste20-related adaptor
protein (STRAD␣) and MO25␣. Infection of neonatal rat cardiac
myocytes with all three adenoviruses substantially increased
LKB1/STRAD␣/MO25␣ expression, LKB1 activity, and AMPK␣
phosphorylation at its activating phosphorylation site (threonine-172).
Since activation of AMPK can inhibit hypertrophic growth and since
LKB1 is upstream of AMPK, we hypothesized that expression of an
active LKB1 complex would also inhibit protein synthesis associated
with hypertrophic growth. Expression of the LKB1/STRAD␣/MO25␣
complex in neonatal rat cardiac myocytes inhibited the increase in
protein synthesis observed in cells treated with phenylephrine (measured via [3H]phenylalanine incorporation). This was associated
with a decreased phosphorylation of p70S6 kinase and its substrate
S6 ribosomal protein, key regulators of protein synthesis. In addition,
we show that the pathological cardiac hypertrophy in transgenic mice
with cardiac-specific expression of activated calcineurin is associated
with a significant decrease in LKB1 expression. Together, our data
show that increased LKB1 activity in the cardiac myocyte can decrease hypertrophy-induced protein synthesis and suggest that LKB1
activation may be a method for the prevention of pathological cardiac
hypertrophy.
LKB1 ACTIVATION INHIBITS PROTEIN SYNTHESIS
signaling pathway might be responsible for the reduction in
protein synthesis (19). Therefore, the role of AMPK and its
activating kinase LKB1 in controlling p70S6 kinase activity in
the cardiac myocyte is still in question. However, because of
the involvement of AMPK in regulating multiple pathways that
control cell growth, it is likely that the LKB1-AMPK signaling
axis is also a key regulator of protein synthesis associated with
cardiac hypertrophy. Investigating the role of the LKB1AMPK axis in protein synthesis associated with cardiac hypertrophy will allow a better understanding of the complex pathways involved in the hypertrophic response in the cardiac
myocyte.
MATERIALS AND METHODS
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using EcoRI sites, FLAG-tagged human STRAD␣ was released via
EcoRI/BamHI digestion, and Myc-tagged human MO25␣ was digested with BamHI. After confirmation that all three constructs were
successfully subcloned into pBluescript II KS⫺, they were all subcloned into pAdTrack CMV shuttle vector utilizing XhoI and XbaI
sites. Concomitant generation of recombinant adenoviral plasmids,
propagation in HEK-293 cells, and isolation of adenoviruses were
performed as previously described (13).
Isolation and culture of neonatal rat cardiac myocytes. Newborn
(1- to 3-day-old) rats were rapidly decapitated, and the hearts were
excised and cardiac myocytes were prepared as previously described
(7, 27). Cells were plated at a density of 1.5–2.0 ⫻ 106 cells per
35-mm plate. Cells used for immunocytochemistry were plated at a
density of 7.5 ⫻ 105 cells per 35-mm plate on fibronectin-coated
coverslips.
Adenoviral infection and cell treatments. After isolation, the cells
were plated and incubated overnight at 37°C in 5% CO2. The cells
were then rinsed 2⫻ with serum-free DMEM/F-12 media containing
50 ␮g/ml gentamicin, after which they were cultured in the same media
containing insulin-transferrin-sodium-selenite liquid media supplement
and 10 ␮M cytosine ␥-D-arabinofuranoside to prevent fibroblast
growth. After replacement of media, cells were immediately infected
with a combination of Ad.LKB1, Ad.STRAD␣, and Ad.MO25␣, each
at a multiplicity of infection (moi) of 7. Individual expression of
LKB1, STRAD␣, or MO25␣ involved infection of cells with each
adenovirus also at an moi of 7. In all experiments, the moi was kept
constant at 21 moi using equivalent amounts of Ad.GFP (moi of 14).
To determine whether endogenous rat LKB1 could be activated by
Ad.STRAD␣ and Ad.MO25␣, cells were infected with a combination
of Ad.GFP, Ad.STRAD␣, and Ad.MO25␣ each at an moi of 7.
Control cells were infected with Ad.GFP at an moi of 21. Twenty-four
hours postinfection the medium was replaced with fresh serum-free
media. For experiments involving phenylephrine, cells were treated at
this time with either 10 ␮M phenylephrine or vehicle. Cells were
harvested 48 h postinfection.
Cell lysis. To harvest the cells, the dishes were rinsed 2⫻ with
ice-cold 1⫻ PBS and scraped into 150 ␮l LKB1 lysis buffer [50 mM
Tris䡠HCl, pH 7.5, 1 mM EGTA, 1 mM EDTA, 1% (wt/vol) Triton-X
100, 1 mM sodium orthovanadate, 50 mM sodium fluoride, 5 mM
sodium pyrophosphate, 0.27 M sucrose, 0.1% (vol/vol) 2-mercaptoethanol, protease inhibitor cocktail, and phosphatase inhibitor
cocktail I]. The cells were lysed for 10 min on ice and then centrifuged at 800 g for 10 min at 4°C. The protein concentration of the
supernatant was determined using the Bradford assay (Bio-Rad), and
samples were subjected to SDS-PAGE and immunoblot analysis.
To study the intracellular localization of LKB1, cells were rinsed
with 1⫻ PBS as above and then treated with 300 ␮l, 1.497 mM
digitonin (Calbiochem) in 1⫻ PBS for 10 min at 4°C on a rocking
platform. The digitonin fraction (cytosol) was immediately removed,
and the cells were briefly rinsed with 1⫻ PBS. The intracellular
organelles were scraped into 200 ␮l LKB1 lysis buffer. Both fractions
were spun at 14,000 g for 5 min. The supernatants were saved, and
protein concentration was determined as described above. The proteins were utilized for SDS-PAGE and immunoblot analysis.
Immunoblot analysis. Cell homogenates were subjected to SDSPAGE in gels containing 10% acrylamide and were transferred to
nitrocellulose as previously described (7, 27). Membranes were
blocked for 1 h in 5% milk-TBS-0.1% Tween 20 and then immunoblotted overnight at 4°C in 5% BSA-TBS-0.1% Tween 20 containing
a 1:1,000 dilution of primary antibody, with the exception of mouse
anti-FLAG (1:3,000). The blots then underwent extensive washes in
TBS-0.1% Tween 20 and were incubated for 1 h in the appropriate
secondary antibody (1:2,000 dilution in blocking solution). After
further washes, the immunoblots were visualized using the Pharmacia
enhanced chemiluminescence detection system.
Immunocytochemistry. To visualize the intracellular localization of
LKB1, cardiac myocytes plated on glass coverslips were treated as
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Animal care. The University of Alberta Animal Policy and Welfare
Committee, which adheres to the principles for biomedical research
involving animals developed by the Council for International Organizations of Medical Sciences (CIOMS) and complies with the Canadian Council on Animal Care guidelines, approved all experimental
procedures involving animals.
Materials. The pCMV5-mouse-FLAG-LKB1, pCMV5-FLAGhuman ste20-related adaptor protein (STRAD␣), and pCMV5-Mychuman-MO25␣ cDNA constructs were kindly provided by Dr. Dario
Alessi (University of Dundee). The plasmids pAdTrack-CMV and
pAdEasy1 were a gift from Dr. Bert Vogelstein (The Johns Hopkins
University Medical Institutions). pBluescript II KS⫺, as well as the
BJ5183 with pAdEasy electroporation competent cells, were purchased from Stratagene. The T4 DNA ligase and all restriction enzymes
and buffers were obtained from Invitrogen except for Pac1 and Pme1,
which were from New England Biolabs.
The primary antibodies utilized in this study, including rabbit
anti-LKB1, rabbit anti-phospho-␣-AMPK (Thr-172), rabbit antiAMPK, rabbit anti-phospho-p70S6 kinase (Thr-389), rabbit antiphospho-p70S6 kinase (Thr-421/Ser-424), rabbit anti-phospho-S6
ribosomal protein (Ser-235/236), rabbit anti-S6 ribosomal protein,
rabbit anti-lamin A/C, and mouse anti-Myc tag were all purchased
from Cell Signaling Technology. The rabbit anti-phospho-acetyl CoA
carboxylase (ACC) (Ser-79) antibody was from Upstate Biotechnology and peroxidase-labeled streptavidin was from Kirkegaard and
Perry Labs. The LKB1(M18) and the anti-actin primary antibodies,
along with the secondary antibodies for goat anti-rabbit, goat antimouse and donkey anti-goat, were all from Santa Cruz Biotechnology.
Rabbit anti-lactate dehydrogenase (LDH) was obtained from Abcam
Limited. The primary antibody for anti-mouse FLAG M2 was from
Sigma. For the immunocytochemistry studies, both the Rhodamine
tetramethylrhodamine isothiocyanate (TRITC)-conjugated anti-rabbit
IgG and the Texas Red dye-conjugated F(ab⬘)2 donkey anti-mouse
IgG were from Jackson ImmunoResearch Laboratories. The 4⬘-6diamidino-2-phenylindole (DAPI) stain and the Prolong Antifade Kit
were from Molecular Probes. Immunoprecipitations were performed
using Anti-FLAG M2-Agarose from Sigma.
For cell culture work, gentamicin, horse serum, trypsin-EDTA,
and all tissue culture solutions were purchased from Invitrogen.
Dulbecco’s modified Eagle’s medium-nutrient mixture F-12 Ham
(DMEM/F-12), minimal essential medium Eagle’s, fetal bovine serum,
insulin-transferrin-sodium-selenite media supplement, cytosine ␥arabinofuranoside, fibronectin, protease, and phosphatase cocktails as
well as l-phenylephrine were obtained from Sigma. Worthington was
the source for DNAse, collagenase, and trypsin. [3H]phenylalanine
and [␥-32P]ATP were from Amersham Biosciences. All other chemicals and reagents were from standard commercial sources.
Construction of recombinant adenoviruses. Initially, the inserts of
all three constructs (pCMV5-mouse-FLAG-LKB1, pCMV5-FLAGhuman STRAD␣, and pCMV5-Myc-human-MO25␣) were subcloned
into pBluescript II KS⫺. FLAG-tagged mouse LKB1 was removed
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LKB1 ACTIVATION INHIBITS PROTEIN SYNTHESIS
RESULTS
Expression of exogenous LKB1, STRAD␣, and MO25␣ in
neonatal rat cardiac myocytes occurs mainly in the cytosol.
LKB1 is a serine-threonine kinase that complexes with two
other regulatory proteins called the STRAD (3) and MO25 (5).
STRAD is considered to be a pseudokinase (3), whereas MO25
appears to be a scaffolding protein (5). Together the LKB1/
STRAD␣/MO25␣ heterotrimeric complex forms an active
AMPKK (15) that is capable of phosphorylating AMPK␣ at its
activating phosphorylation site (Thr-172) (29). To investigate
whether we could reconstitute an active LKB1/STRAD␣/
MO25␣ complex in the cardiac myocyte, neonatal rat cardiac
myocytes were infected with the individual adenoviruses harboring the cDNAs for FLAG-tagged mouse LKB1 (Ad.LKB1),
FLAG-tagged human STRAD␣ (Ad.STRAD␣), Myc-tagged
human MO25␣ (Ad.MO25␣), or a combination of all three
adenoviruses. Immunoblot analysis of cell lysates from these
transduced neonatal rat cardiac myocytes demonstrated the
successful expression of all three proteins (Fig. 1). The expression of the tagged proteins was detected using anti-FLAG
(LKB1 and STRAD␣) or anti-Myc (MO25␣) antibodies,
whereas expression of endogenous LKB1 was detected by the
anti-LKB1 antibody. Since the FLAG tagged LKB1 protein
has a higher molecular weight than endogenous LKB1, both
proteins could be visualized using the anti-LKB1 antibody
(Fig. 1, top panel).
Whereas LKB1 resides predominantly in the nucleus, it has
been shown to translocate to the cytoplasm when associated
with STRAD and MO25 (5). To determine the subcellular
localization of the expressed LKB1, STRAD␣, and MO25␣
proteins, neonatal rat cardiac myocytes were infected as described in Fig. 1 and subsequently digitonin permeabilized to
extract the cytosolic contents of the myocytes. The noncytosolic components remaining on the tissue culture dish were
scraped and collected. Immunoblot analysis of the cytosolic
contents of the myocytes showed that the vast majority of
endogenous LKB1 was absent from the cytosolic fraction and
remained in the non-cytosolic fraction (Fig. 2A, compare lanes
1 and 6, top panels) regardless of expression of STRAD␣ and
MO25␣ proteins (Fig. 2A, compare lanes 3 and 4 with lanes 8
and 9, top panels). However, exogenous FLAG-tagged LKB1,
either on its own or when coexpressed with STRAD␣ and
MO25␣, was exclusively located in the cytosolic fraction (Fig.
2A, compare lanes 2 and 5 with lanes 7 and 10, second panels).
Immunoblot analysis of the cytosolic and non-cytosolic fractions using antibodies directed against LDH and lamin A/C
confirmed that the cytosolic fraction was not contaminated
with nuclear-associated proteins, and the non-cytosolic fraction
did not contain cytosolic proteins (Fig. 2A).
To further demonstrate that coexpression of LKB1,
STRAD␣, and MO25␣ resulted in cytosolic LKB1, neonatal
rat cardiac myocytes infected with Ad.GFP or Ad.LKB1/
Fig. 1. Expression of LKB1/STRAD␣/MO25␣ in neonatal rat cardiac myocytes using recombinant adenoviruses.
Neonatal rat cardiac myocytes were infected with Ad.GFP,
the LKB1 complex Ad.LKB1/Ad.STRAD␣/Ad.MO25␣,
or the individual proteins comprising the LKB1 holoenzyme complex. In all experiments, the multiplicity of
infection (moi) was kept constant using equivalent
amounts of adenovirus encoding for GFP. Forty-eight
hours postinfection, the cells were lysed, and immunoblot
analysis was performed to confirm expression of LKB1/
STRAD␣/MO25␣. Representative immunoblots from
three independent experiments are shown. Top: representative immunoblot utilizing an anti-LKB1 antibody to
recognize both endogenous and exogenous LKB1. Middle
two panels: representative immunoblots utilizing an
anti-FLAG antibody demonstrate there was successful
expression of mouse-FLAG-LKB1 and human-FLAGSTRAD␣. Bottom: representative immunoblot utilizing an
anti-Myc antibody to recognize human-Myc-MO25␣.
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described above and were fixed with 3.7% paraformaldehyde. The
incubation with rabbit anti-LKB1 (1:50) was performed as per the
manufacturer’s instructions, and Rhodamine (TRITC)-conjugated antirabbit IgG was utilized as the secondary antibody. To stain the nuclei,
fixed cells were treated for 15 min with 300 nM DAPI in 1⫻ PBS. To
visualize the hypertrophic effects of phenylephrine on the neonatal rat
cardiac myocytes, cells were also fixed with 3.7% paraformaldehyde and
incubated with mouse anti-␣ actinin (1:250) followed by Texas Red
dye-conjugated donkey anti-mouse IgG secondary antibody (1:250).
Immunoprecipitations and LKB1 assay. Exogenous FLAG-tagged
LKB1 was immunoprecipitated from 100 ␮g cellular protein using
anti-FLAG M2-Agarose. Immunoprecipitates were assayed for LKB1
activity using the LKBtide substrate (Alberta Peptide Institute), as
previously described (29).
[3H]Phenylalanine incorporation. Twenty-four hours postinfection,
neonatal rat cardiac myocytes were treated with [3H]phenylalanine
(1 ␮Ci/ml) in the presence or absence of 10 ␮M phenylephrine. After
an additional 24 h, cells were washed three times with ice-cold 1⫻
PBS, and proteins were precipitated over a 1-h period (4°C) in 10%
trichloroacetic acid. The samples were then briefly rinsed two times
with 95% ethanol and scraped in 1 M NaOH. The samples were
subsequently neutralized with 1 M HCl, and radioactivity was measured via scintillation counting as we have previously described (7).
Calcineurin transgenic mice. Transgenic mice with cardiac-specific
expression of activated calcineurin and their wild-type littermates
(a kind gift from Dr. Jeffery Molkentin, University of Cincinnati)
were euthanized with a 12-mg ip injection of pentobarbital sodium
at 8 –10 wk of age. Transgenic mice expressing calcineurin displayed marked cardiac hypertrophy in a similar fashion as previously described (31). Namely, these mice demonstrate a twofold
increase in heart-to-body weight ratios by 6 –10 wk of age
(J. Molkentin, personal communication). Heart tissue (15 mg) was
homogenized for 30 s on level 3 of a PowerGen-125 homogenizer
(Fisher Scientific) in 100 ␮l of LKB1 lysis buffer. Protein content
was assayed and 20 ␮g of protein was used for SDS-PAGE and
immunoblot analysis.
LKB1 ACTIVATION INHIBITS PROTEIN SYNTHESIS
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Ad.STRAD␣/Ad.MO25␣ recombinant adenoviruses were used
for immunocytochemistry experiments. Neonatal rat cardiac myocytes infected with Ad.GFP or Ad.LKB1/Ad.STRAD␣/
Ad.MO25␣ were fixed, permeabilized, and incubated with
rabbit anti-LKB1 antibody. Nuclei were also stained with
DAPI. After application of Rhodamine (TRITC)-conjugated
secondary anti-rabbit IgG antibody, cells were visualized for
TRITC-labeled LKB1 and DAPI-stained nuclei (Fig. 2B). Neonatal rat cardiac myocytes infected with Ad.GFP (control) demonstrated the majority of LKB1 to be located in the nucleus as
AJP-Heart Circ Physiol • VOL
seen with its colocalization with DAPI stain (Fig. 2B, i and ii).
This finding is consistent with endogenous LKB1 being primarily located in the nucleus. Cells expressing the LKB1/
STRAD␣/MO25␣ complex demonstrated an increase in cytosolic staining of LKB1 (Fig. 2B, iv). Neonatal rat cardiac
myocytes infected with either Ad.STRAD␣ or Ad.MO25␣
alone resembled Ad.GFP-infected cells (data not shown). Together, these results confirm that expression of exogenous
LKB1 is located within the cytosol where it has previously
been shown to be highly active (5).
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Fig. 2. Exogenous LKB1, STRAD␣, and MO25␣
localizes to the cytosol. Neonatal rat cardiac myocytes
were infected with Ad.GFP, the LKB1 complex
Ad.LKB1/Ad.STRAD␣/Ad.MO25␣, or the individual
proteins comprising the LKB1 holoenzyme complex. In
all experiments the moi was kept constant using equivalent amounts of adenovirus encoding for GFP. Fortyeight hours postinfection, cytosolic fractions and noncytosolic fractions of the neonatal rat cardiac myocytes
were collected. Immunoblot analysis of these fractions
using an anti-LKB1 antibody demonstrates that cells
infected with Ad.LKB1 express exogenous FLAGLKB1 (⬃52 kDa) protein primarily in the cytosolic
fraction, whereas endogenous LKB1 (⬃50 kDa) is predominantly in the noncytosolic fraction. Immunoblot
analysis with anti-FLAG or anti-Myc antibodies demonstrates that exogenous STRAD␣ and MO25␣ proteins
are almost exclusively found in the cytosolic fraction.
Immunoblot analysis using anti-lactate dehydrogenase
(LDH) and anti-lamin A/C antibodies confirmed that the
two cellular fractions were highly enriched for cytosolic
proteins and nuclear proteins, respectively. A: representative immunoblot of three independent experiments. To
visualize the intracellular localization of both endogenous and exogenous LKB1 in intact cells, neonatal rat
cardiac myocytes infected with Ad.GFP (Control) or
Ad.LKB1/Ad.MO25␣/Ad.STRAD␣ (LKB1 Complex)
were fixed, permeabilized, and incubated with antiLKB1 antibody followed by application of Rhodamine
(TRITC)-conjugated secondary anti-rabbit IgG (LKB1TRITC, B, ii and iv). Nuclei were stained with DAPI
(Nuclei, B, i and iii). B: images are representative of
three independent experiments.
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LKB1 ACTIVATION INHIBITS PROTEIN SYNTHESIS
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Fig. 3. Expression of the LKB1/STRAD␣/MO25␣ complex results in
increased LKB1 activity and phosphorylation of AMP-activated protein kinase
(AMPK␣) on Thr-172. Neonatal rat cardiac myocytes were infected with
Ad.GFP, Ad.GFP/Ad.STRAD␣/MO25␣, or the LKB1 complex Ad.LKB1/
Ad.STRAD␣/Ad.MO25␣. In all experiments, the moi was kept constant using
equivalent amounts of adenovirus encoding for GFP. Forty-eight hours postinfection, the cells were lysed, FLAG-tagged LKB1 was immunoprecipitated,
and the immunoprecipitates were assayed for LKB1 activity (A). Neonatal rat
cardiac myocytes infected with the LKB1 complex also exhibit a substantial
increase in phosphorylation of AMPK␣ at Thr-172 compared with cells
infected with Ad.GFP alone or Ad.GFP/Ad.STRAD␣/MO25␣. An increase in
the phosphorylation of ACC at Ser-79 is indicative of the level of AMPK
activation in these cells. Again, a substantial increase was observed only in the
cells infected with the LKB1 complex. Comparisons between all groups were
performed using a repeated measures analysis of variance followed by a
Bonferroni post test. *Significantly different from all other groups (P ⬍ 0.001).
For the LKB1 activity assay, all data are presented as means ⫾ SE.
B: representative immunoblots of three independent experiments are shown.
Expression of the LKB1/STRAD␣/MO25␣ complex inhibits
phenylephrine-induced protein synthesis in neonatal rat cardiac myocytes. Previous studies from our laboratory have
demonstrated that pharmacological activation of AMPK can
inhibit hypertrophic growth stimulated in neonatal rat cardiac
myocytes by phenylephrine treatment or increased Akt activity
(7). Since LKB1 is upstream of AMPK, we hypothesized that
overexpression of the LKB1/STRAD␣/MO25␣ complex
would also inhibit protein synthesis associated with hypertro-
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Expression of the LKB1/STRAD␣/MO25␣ complex results
in increased LKB1 activity in neonatal rat cardiac myocytes.
To investigate whether expression of the LKB1/STRAD␣/
MO25␣ complex in the cardiac myocyte displayed increased
LKB1 activity, neonatal rat cardiac myocytes were infected
with Ad.LKB1, Ad.STRAD␣, or Ad.MO25␣ individually or in
combination. Forty-eight hours postinfection, homogenates
from these neonatal rat cardiac myocyte were immunoprecipitated using the anti-FLAG antibody to isolate the FLAGtagged LKB1 protein. Immunoprecipitated proteins were
subsequently assayed for their ability to phosphorylate the
LKBtide substrate, a 22-mer peptide representing the ideal
phosphorylation motif for LKB1 (29). Cardiac myocytes expressing the LKB1/STRAD␣/MO25␣ complex exhibited a significant 10-fold increase in activity compared with Ad.GFPinfected cells, whereas immunoprecipitates from myocytes
infected with Ad.LKB1, Ad.STRAD␣, or Ad.MO25␣ alone
did not have increased LKB1 activity compared with controls
(Ad.GFP) (Fig. 3A).
Since LKB1 is a known AMPKK, we also determined
whether increased LKB1 activity in the cardiac myocytes
resulted in an increase in AMPK␣ phosphorylation at its
primary activation site Thr-172. Homogenates from neonatal
rat cardiac myocytes infected as above were subjected to
immunoblot analysis using the anti-AMPK␣ (Thr-172) antibody. In accordance with the increase in LKB1 activity in the
cells expressing the LKB1/STRAD␣/MO25␣ complex, phosphorylation of AMPK␣ at Thr-172 was also dramatically
increased (Fig. 3B). Immunoblot analysis of phosphorylated
ACC at Ser-79 (the known phosphorylation site of AMPK) was
also performed to confirm AMPK activation. Concomitant
with increased AMPK␣ phosphorylation in cells expressing
LKB1/STRAD␣/MO25␣, phosphorylation of ACC at Ser-79
also increased (Fig. 3B). This was not observed in cells
expressing STRAD␣/MO25␣ alone (Fig. 3B). These data confirm that only cells infected with the combination of Ad.LKB1/
Ad.STRAD␣/Ad.MO25␣ expressed LKB1 as an active
AMPKK.
Expression of the LKB1/STRAD␣/MO25␣ complex alters
phosphorylation of p70S6 kinase and S6 ribosomal protein in
neonatal rat cardiac myocytes. We have previously shown that
pharmacological activation of AMPK results in the inhibition
of p70S6 kinase phosphorylation (7). To investigate whether
this is also the case in cardiac myocytes expressing the LKB1/
STRAD␣/MO25␣ complex, we infected neonatal rat cardiac
myocytes with Ad.LKB1/Ad.STRAD␣/Ad.MO25␣ recombinant adenoviruses. Immunoblot analysis of cell lysates from
neonatal rat cardiac myocytes expressing the LKB1/STRAD␣/
MO25␣ complex showed a significant decrease in phosphorylation of p70S6 kinase at Thr-389 and Thr-421/Ser-424 compared with cells expressing GFP or GFP/STRAD␣/MO25␣
(Fig. 4A). Because phosphorylation of these sites is closely
related to a decrease in p70S6 kinase activity (10, 32), we also
examined whether there was a decrease in the phosphorylation
of its substrate S6 ribosomal protein. Indeed, immunoblot
analysis revealed a decrease in phosphorylation of S6 ribosomal protein at Ser-235/236 in neonatal rat cardiac myocytes
expressing the LKB1/STRAD␣/MO25␣ complex compared
with cells expressing GFP or GFP/STRAD␣/MO25␣ (Fig. 4B).
LKB1 ACTIVATION INHIBITS PROTEIN SYNTHESIS
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Fig. 4. Expression of the LKB1/STRAD␣/MO25␣ complex negatively regulates p70S6 kinase. Neonatal rat cardiac myocytes were infected with Ad.GFP,
Ad.GFP/Ad.STRAD␣/Ad.MO25␣, or the LKB1 complex Ad.LKB1/Ad.STRAD␣/Ad.MO25␣. In all experiments, the moi was kept constant using equivalent
amounts of adenovirus encoding for GFP. Forty-eight hours postinfection, the cells were harvested, and cellular extracts were subjected to immunoblot analysis
using anti-phospho-p70S6 kinase (Thr-389), anti-phospho-p70S6 kinase (Thr-421/Ser-424), and anti-actin antibodies. A: representative immunoblots of three
independent experiments. Immunoblots were also blotted with anti-phospho-S6 ribosomal protein (Ser-235/236) and anti-S6 ribosomal protein antibody.
B: immunoblots of three independent experiments. For all immunoblots, densitometry, and subsequent statistical analysis were performed, and the graphical
representations of the data are shown. All data are presented as means ⫾ SE. Statistical comparisons were performed using ANOVA for both the p70s6 kinase
and the ribosomal S6 protein immunoblots. *Differences were judged to be significant when P ⬍ 0.05.
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LKB1 ACTIVATION INHIBITS PROTEIN SYNTHESIS
Fig. 5. Expression of the LKB1/STRAD␣/MO25␣ complex inhibits phenylephrine (PE)-induced hypertrophic growth in neonatal rat cardiac myocytes.
Neonatal rat cardiac myocytes were infected with Ad.GFP, Ad.GFP/
Ad.STRAD␣/Ad.MO25␣, or the LKB1 complex Ad.LKB1/Ad.STRAD␣/
Ad.MO25␣. In all experiments the moi was kept constant using equivalent
amounts of adenovirus encoding for GFP. Twenty-four hours postinfection, the
cells were treated with 10 ␮M PE or vehicle, as well as 1 ␮Ci/ml [3H]phenylalanine. After an additional 24 h, total cellular proteins were precipitated,
and radioactivity was measured to determine levels of protein synthesis as a
marker of hypertrophic growth. Data are presented as means ⫾ SE of the [3H]
incorporated into the cellular proteins. Differences between individual groups
were performed using a repeated measures analysis of variance followed by a
Student-Newman-Keuls multiple comparisons post test. Differences were
judged to be significant when P ⬍ 0.05. *P ⬍ 0.05, significantly different from
GFP group. #P ⬍ 0.01, significantly different from the GFP/STRAD␣/MO25␣
group. &P ⬍ 0.01, significantly different from the GFP ⫹PE group. $P ⬍ 0.01,
significantly different from the GFP/STRAD␣/MO25␣ ⫹ PE group. n ⫽ 5.
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Expression of LKB1 is downregulated in a mouse model of
pathological cardiac hypertrophy. To investigate whether
pathological cardiac hypertrophy is associated with alterations
in LKB1 expression, we analyzed transgenic mice with cardiacspecific expression of activated calcineurin and their wild-type
littermates. Extracts from hearts were subjected to immunoblot
analysis using anti-LKB1(M18) antibody (Fig. 7). Densitometric analysis of the immunoblots demonstrate that hypertrophic
hearts from calcineurin transgenic mice had a significant reduction in the amount of LKB1 expression compared with
wild-type littermates (Fig. 7).
DISCUSSION
To investigate the potential role of LKB1 in the regulation
of protein synthesis associated with phenylephrine-induced
cardiac hypertrophy, adenoviruses harboring the cDNAs for
LKB1, STRAD␣, and MO25␣ were constructed. Transduction
of neonatal rat cardiac myocytes with these adenoviruses
resulted in high levels of these proteins being expressed (Fig.
1). Interestingly, the expression of LKB1, STRAD␣, and
MO25␣ appeared higher when these proteins were expressed
in combination rather than individually, suggesting that like the
AMPK heterotrimer (12), the LKB1/MO25␣/STRAD␣ heterotrimeric complex is stabilized and protected from degradation.
In addition to detecting high levels of expressed LKB1/
MO25␣/STRAD␣ proteins, we also observed that endogenous
LKB1 is expressed in the cardiac myocyte. Previous studies
have observed LKB1 transcripts in human hearts (34) and
protein expression in mouse (35) and rat heart homogenates
(1). Our work clearly shows that the contractile cells of the
heart, i.e., cardiac myocytes, also contain LKB1. Despite
possessing LKB1, our data show that the majority of endogenous LKB1 in the cardiac myocyte is localized primarily to the
nucleus (Fig. 2, A and B). Previous studies have suggested that
nuclear localized LKB1 is inactive, at least as an AMPKK, and
needs to translocate to the cytoplasm and associate with
STRAD and MO25 before becoming a fully active AMPKK
(18). Although the conditions that induce LKB1 translocation
to the cytosol in the cardiac myocyte have not been extensively
studied, it is possible that translocation of LKB1 may be an
additional mechanism by which AMPK activity can be regulated in the heart. Presently, however, it is unknown what role
nuclear LKB1 plays in the heart.
Whereas endogenous LKB1 resides primarily in the nucleus,
expression of exogenous LKB1, STRAD␣, and MO25␣ proteins is almost exclusively cytosolic (Fig. 2A). This cytosolic
localization suggests an increased potential for elevated LKB1
kinase activity. Indeed, LKB1 activity assays performed using
LKB1 immunoprecipitates from neonatal rat cardiac myocytes
expressing the LKB1/STRAD␣/MO25␣ complex demonstrate
a significant 10-fold increase in LKB1 activity using an in vitro
peptide substrate assay (Fig. 3A). In accordance with this
increase in LKB1 activity, phosphorylation of AMPK␣ at
Thr-172 was also dramatically increased as well as phosphorylation of ACC at Ser-79 (Fig. 3B), confirming increased
cellular AMPKK and AMPK activity, respectively. As expected, phosphorylation of AMPK␣ and ACC were not altered
when the neonatal rat cardiac myocytes were infected only
with the LKB1-binding partners STRAD␣ and MO25␣ (GFP/
STRAD␣/MO25␣) (Fig. 3B). Furthermore, changes observed
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phic growth. As a quantitative measurement of protein synthesis, incorporation of [3H]phenylalanine into protein was utilized in cells treated with or without phenylephrine. As expected, phenylephrine-treated cells expressing GFP or GFP/
STRAD␣/MO25␣ demonstrated significant 1.4- to 1.5-fold
increases in protein synthesis compared with their vehicletreated counterparts (Fig. 5). However, in the presence of the
active LKB1/STRAD␣/MO25␣ complex, phenylephrine-induced protein synthesis was significantly reduced compared
with cells expressing both GFP and GFP/STRAD␣/MO25␣
(Fig. 5). In addition, expression of the LKB1/STRAD␣/
MO25␣ complex resulted in a small, yet significant, reduction
in protein synthesis compared with the cells expressing GFP
alone but not the cells expressing GFP/STRAD␣/MO25␣.
These data suggest that increased LKB1 activity can suppress
both basal protein synthesis and enhanced protein synthesis
induced by phenylephrine.
To visualize the inhibition of phenylephrine-induced hypertrophic growth by the LKB1/STRAD␣/MO25␣ complex, immunocytochemistry against ␣-actinin was performed on all
groups of cells. Cells were infected with either Ad.GFP,
Ad.GFP/Ad.STRAD␣/Ad.MO25␣, or Ad.LKB1/Ad.STRAD␣/
Ad.MO25␣ and treated with or without phenylephrine. The
Ad.GFP and Ad.GFP/Ad.STRAD␣/Ad.MO25␣-infected cells
treated with phenylephrine clearly demonstrate an increase in
cell size (Fig. 6, A and B). However, expression of the active
LKB1 complex prevented this phenylephrine-induced hypertrophic growth (Fig. 6C).
LKB1 ACTIVATION INHIBITS PROTEIN SYNTHESIS
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in cell signaling (Fig. 4), protein synthesis (Fig. 5), and cellular
growth (Fig. 6) in the cells infected with the LKB1 complex
were not observed in those cells expressing GFP/STRAD␣/
MO25␣, suggesting the activation of nuclear localized endogenous LKB1 is more complex than the availability of these two
binding partners. Therefore, our data show that infecting neonatal
rat cardiac myocytes with recombinant adenoviruses harboring
the cDNAs for LKB1, STRAD␣, and MO25␣ is an effective
approach to increasing LKB1 activity. This is especially important given that no reagents are currently available that can
be used to increase LKB1 activity in the cardiac myocyte.
Related to this, we also show that expression of the LKB1
complex is a powerful, nonpharmacological tool that can be
used for activating endogenous AMPK in the cardiac myocyte.
Taken together, these localization data show that endogenous LKB1 resides primarily in the nucleus within the neonatal
rat cardiac myocyte, suggesting that it may not be involved in
regulating cardiac AMPK during normal conditions. However,
since LKB1 has recently been shown to be necessary for the
ischemia-induced activation of cardiac AMPK (36), it is likely
that LKB1 may play a more central role in mediating AMPK
activity in stressed and/or pathological situations. In addition to
ischemia, this may also include pathological cardiac hypertrophy. Whether LKB1 translocates from the nucleus to the
Fig. 6. Expression of the LKB1/STRAD␣/MO25␣ complex inhibits increases
in cell size in PE-treated neonatal rat cardiac myocytes. To qualitatively
visualize PE-induced hypertrophic growth, neonatal rat cardiac myocytes were
infected with Ad.GFP (A), Ad.GFP/Ad.STRAD␣/MO25␣ (B), or the LKB1
complex Ad.LKB1/Ad.STRAD␣/Ad.MO25␣ (C), and ␣-actinin was visulaized. In all experiments, the moi was kept constant using equivalent amounts
of adenovirus encoding for GFP. Twenty-four hours postinfection, the cells
were treated with 10 ␮M PE or vehicle. After an additional 24 h, the cells were
fixed and incubated with anti-␣-actinin antibody followed by Texas-Red
conjugated secondary anti-mouse IgG. Corresponding images of GFP fluorescence are also included. Representative images of 3 independent experiments
are shown.
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Fig. 7. Endogenous LKB1 expression is decreased in a model of pathological
cardiac hypertrophy. Heart extracts from 8 transgenic mice with cardiacspecific expression of activated calcineurin (and 5 control littermates) were
subjected to immunoblot analysis. A representative immunoblot performed
using anti-LKB1 antibody shows a decrease in LKB1 expression in hearts from
transgenic mice with cardiac-specific expression of activated calcineurin.
Representative samples from the immunoblot along with densitometric analysis are shown. All data are presented as means ⫾ SE. Comparisons between
the two groups was performed using a Student’s two-tailed t-test. *Differences
were judged to be significant when P ⬍ 0.05.
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LKB1 ACTIVATION INHIBITS PROTEIN SYNTHESIS
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(Fig. 7). Whereas we did not measure LKB1 or AMPK activity
in these calcineurin hearts, a recent report in skeletal muscle of
insulin-resistant Zucker rats has shown that a decrease in
LKB1 expression corresponds to a significant decrease in
phosphorylation of AMPK␣ at Thr-172 (39), suggesting the
same may be true in our model. While tumorigenesis is not
common in the heart, pathological cardiac hypertrophy, like
cancer, also involves abnormal cellular growth. Therefore, the
decrease in LKB1 expression identified in hearts from transgenic mice with cardiac-specific expression of activated calcineurin is consistent with a loss of cellular growth control.
When combined with our LKB1 gain-of-function results, these
data further support the notion that LKB1 activation is inversely correlated with hypertrophic growth.
Interestingly, LKB1 knockout mice with skeletal and cardiac
muscle LKB1 ablation have been reported and appear to have
no overt phenotype (35). A subsequent study has indicated that
the hearts from these mice were actually smaller in size than
hearts from wild-type mice (36). This observation is not
consistent with the data presented herein, although it is consistent with the hypothesis that LKB1 may not be an integral
component of the hypertrophic growth process. Whereas it is
possible that LKB1 is not a necessary component of hypertrophic signaling, our data suggest that heart-specific activation of
LKB1 may be an effective approach to preventing pathological
hypertrophy. The novel model that we have developed in this
study will allow us to fully investigate this hypothesis.
In conclusion, we show that expression of the LKB1/
MO25␣/STRAD␣ complex in the cytosol results in a significant increase in LKB1 activity, which is sufficient to inhibit
protein synthesis associated with phenylephrine-induced cardiac hypertrophy. Our data also show that a major effect of
LKB1 activation is to inhibit protein synthesis via the p70S6
kinase/ribosomal S6 pathway. We also show that the pathological cardiac hypertrophy in transgenic mice with cardiacspecific expression of activated calcineurin is associated with a
significant decrease in LKB1 expression, suggesting that
LKB1 may have a major regulatory role in the development of
pathological cardiac hypertrophy. Finally, we have developed
a novel model by which the complete role of LKB1 in the
regulation of the signaling events controlling cardiac hypertrophy can be fully explored.
ACKNOWLEDGMENTS
A. A. Noga is an Alberta Heritage Foundation for Medical Research
(AHFMR) Fellow. J. R. B. Dyck is a Senior Scholar of the AHFMR and a
Canada Research Chair in Molecular Biology of Heart Disease and Metabolism. This research was supported by the Canadian Institutes of Health
Research Grant MOP53088.
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