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Am J Physiol Heart Circ Physiol 292: H1460-H1469, 2007. First published November 10, 2006; doi:10.1152/ajpheart.01133.2006
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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


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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{alpha}) and MO25{alpha}. Infection of neonatal rat cardiac myocytes with all three adenoviruses substantially increased LKB1/STRAD{alpha}/MO25{alpha} expression, LKB1 activity, and AMPK{alpha} 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{alpha}/MO25{alpha} 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.

adenosine 5'-monophosphate-activated protein kinase; hypertrophy; protein synthesis; cardiac myocyte

LKB1 IS A SERINE-THREONINE KINASE 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 ({alpha}) and two regulatory subunits (beta, {gamma})] 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 {alpha}-subunit is phosphorylated by upstream AMPK kinases (9) such as LKB1 (15, 18). This phosphorylation occurs at Thr-172 of the {alpha}-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 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 LKB1-AMPK 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
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
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-FLAG-human ste20-related adaptor protein (STRAD{alpha}), and pCMV5-Myc-human-MO25{alpha} 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-{alpha}-AMPK (Thr-172), rabbit anti-AMPK, rabbit anti-phospho-p70S6 kinase (Thr-389), rabbit anti-phospho-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 anti-mouse 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'-6-diamidino-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 {gamma}-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 [{gamma}-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-FLAG-human STRAD{alpha}, and pCMV5-Myc-human-MO25{alpha}) were subcloned into pBluescript II KS–. FLAG-tagged mouse LKB1 was removed using EcoRI sites, FLAG-tagged human STRAD{alpha} was released via EcoRI/BamHI digestion, and Myc-tagged human MO25{alpha} 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 x 106 cells per 35-mm plate. Cells used for immunocytochemistry were plated at a density of 7.5 x 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 2x with serum-free DMEM/F-12 media containing 50 µg/ml gentamicin, after which they were cultured in the same media containing insulin-transferrin-sodium-selenite liquid media supplement and 10 µM cytosine {gamma}-D-arabinofuranoside to prevent fibroblast growth. After replacement of media, cells were immediately infected with a combination of Ad.LKB1, Ad.STRAD{alpha}, and Ad.MO25{alpha}, each at a multiplicity of infection (moi) of 7. Individual expression of LKB1, STRAD{alpha}, or MO25{alpha} 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{alpha} and Ad.MO25{alpha}, cells were infected with a combination of Ad.GFP, Ad.STRAD{alpha}, and Ad.MO25{alpha} 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 µM phenylephrine or vehicle. Cells were harvested 48 h postinfection.

Cell lysis. To harvest the cells, the dishes were rinsed 2x with ice-cold 1x PBS and scraped into 150 µl LKB1 lysis buffer [50 mM Tris·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 1x PBS as above and then treated with 300 µl, 1.497 mM digitonin (Calbiochem) in 1x 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 1x PBS. The intracellular organelles were scraped into 200 µl 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 SDS-PAGE 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 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 anti-rabbit IgG was utilized as the secondary antibody. To stain the nuclei, fixed cells were treated for 15 min with 300 nM DAPI in 1x 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-{alpha} 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 µg 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 µCi/ml) in the presence or absence of 10 µM phenylephrine. After an additional 24 h, cells were washed three times with ice-cold 1x 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 µl of LKB1 lysis buffer. Protein content was assayed and 20 µg of protein was used for SDS-PAGE and immunoblot analysis.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Expression of exogenous LKB1, STRAD{alpha}, and MO25{alpha} 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{alpha}/MO25{alpha} heterotrimeric complex forms an active AMPKK (15) that is capable of phosphorylating AMPK{alpha} at itsactivating phosphorylation site (Thr-172) (29). To investigate whether we could reconstitute an active LKB1/STRAD{alpha}/MO25{alpha} 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{alpha} (Ad.STRAD{alpha}), Myc-tagged human MO25{alpha} (Ad.MO25{alpha}), 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{alpha}) or anti-Myc (MO25{alpha}) 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).


Figure 1
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  		Fig. 1. Expression of LKB1/STRAD{alpha}/MO25{alpha} in neonatal rat cardiac myocytes using recombinant adenoviruses. Neonatal rat cardiac myocytes were infected with Ad.GFP, the LKB1 complex Ad.LKB1/Ad.STRAD{alpha}/Ad.MO25{alpha}, 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{alpha}/MO25{alpha}. 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-FLAG-STRAD{alpha}. Bottom: representative immunoblot utilizing an anti-Myc antibody to recognize human-Myc-MO25{alpha}.

 
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{alpha}, and MO25{alpha} 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 non-cytosolic 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{alpha} and MO25{alpha} 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{alpha} and MO25{alpha}, 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).


Figure 2
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  		Fig. 2. Exogenous LKB1, STRAD{alpha}, and MO25{alpha} localizes to the cytosol. Neonatal rat cardiac myocytes were infected with Ad.GFP, the LKB1 complex Ad.LKB1/Ad.STRAD{alpha}/Ad.MO25{alpha}, 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. Forty-eight hours postinfection, cytosolic fractions and non-cytosolic 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 FLAG-LKB1 (~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{alpha} and MO25{alpha} 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{alpha}/Ad.STRAD{alpha} (LKB1 Complex) were fixed, permeabilized, and incubated with anti-LKB1 antibody followed by application of Rhodamine (TRITC)-conjugated secondary anti-rabbit IgG (LKB1-TRITC, B, ii and iv). Nuclei were stained with DAPI (Nuclei, B, i and iii). B: images are representative of three independent experiments.

 
To further demonstrate that coexpression of LKB1, STRAD{alpha}, and MO25{alpha} resulted in cytosolic LKB1, neonatal rat cardiac myocytes infected with Ad.GFP or Ad.LKB1/Ad.STRAD{alpha}/Ad.MO25{alpha} recombinant adenoviruses were used for immunocytochemistry experiments. Neonatal rat cardiac myocytes infected with Ad.GFP or Ad.LKB1/Ad.STRAD{alpha}/Ad.MO25{alpha} 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 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{alpha}/MO25{alpha} complex demonstrated an increase in cytosolic staining of LKB1 (Fig. 2B, iv). Neonatal rat cardiac myocytes infected with either Ad.STRAD{alpha} or Ad.MO25{alpha} 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).

Expression of the LKB1/STRAD{alpha}/MO25{alpha} complex results in increased LKB1 activity in neonatal rat cardiac myocytes. To investigate whether expression of the LKB1/STRAD{alpha}/MO25{alpha} complex in the cardiac myocyte displayed increased LKB1 activity, neonatal rat cardiac myocytes were infected with Ad.LKB1, Ad.STRAD{alpha}, or Ad.MO25{alpha} 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 FLAG-tagged 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{alpha}/MO25{alpha} complex exhibited a significant 10-fold increase in activity compared with Ad.GFP-infected cells, whereas immunoprecipitates from myocytes infected with Ad.LKB1, Ad.STRAD{alpha}, or Ad.MO25{alpha} alone did not have increased LKB1 activity compared with controls (Ad.GFP) (Fig. 3A).


Figure 3
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  		Fig. 3. Expression of the LKB1/STRAD{alpha}/MO25{alpha} complex results in increased LKB1 activity and phosphorylation of AMP-activated protein kinase (AMPK{alpha}) on Thr-172. Neonatal rat cardiac myocytes were infected with Ad.GFP, Ad.GFP/Ad.STRAD{alpha}/MO25{alpha}, or the LKB1 complex Ad.LKB1/Ad.STRAD{alpha}/Ad.MO25{alpha}. 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{alpha} at Thr-172 compared with cells infected with Ad.GFP alone or Ad.GFP/Ad.STRAD{alpha}/MO25{alpha}. 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.

 
Since LKB1 is a known AMPKK, we also determined whether increased LKB1 activity in the cardiac myocytes resulted in an increase in AMPK{alpha} 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{alpha} (Thr-172) antibody. In accordance with the increase in LKB1 activity in the cells expressing the LKB1/STRAD{alpha}/MO25{alpha} complex, phosphorylation of AMPK{alpha} 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{alpha} phosphorylation in cells expressing LKB1/STRAD{alpha}/MO25{alpha}, phosphorylation of ACC at Ser-79 also increased (Fig. 3B). This was not observed in cells expressing STRAD{alpha}/MO25{alpha} alone (Fig. 3B). These data confirm that only cells infected with the combination of Ad.LKB1/Ad.STRAD{alpha}/Ad.MO25{alpha} expressed LKB1 as an active AMPKK.

Expression of the LKB1/STRAD{alpha}/MO25{alpha} 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{alpha}/MO25{alpha} complex, we infected neonatal rat cardiac myocytes with Ad.LKB1/Ad.STRAD{alpha}/Ad.MO25{alpha} recombinant adenoviruses. Immunoblot analysis of cell lysates from neonatal rat cardiac myocytes expressing the LKB1/STRAD{alpha}/MO25{alpha} 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{alpha}/MO25{alpha} (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{alpha}/MO25{alpha} complex compared with cells expressing GFP or GFP/STRAD{alpha}/MO25{alpha} (Fig. 4B).


Figure 4
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  		Fig. 4. Expression of the LKB1/STRAD{alpha}/MO25{alpha} complex negatively regulates p70S6 kinase. Neonatal rat cardiac myocytes were infected with Ad.GFP, Ad.GFP/Ad.STRAD{alpha}/Ad.MO25{alpha}, or the LKB1 complex Ad.LKB1/Ad.STRAD{alpha}/Ad.MO25{alpha}. 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.

 
Expression of the LKB1/STRAD{alpha}/MO25{alpha} 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{alpha}/MO25{alpha} complex would also inhibit protein synthesis associated with hypertrophic 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{alpha}/MO25{alpha} demonstrated significant 1.4- to 1.5-fold increases in protein synthesis compared with their vehicle-treated counterparts (Fig. 5). However, in the presence of the active LKB1/STRAD{alpha}/MO25{alpha} complex, phenylephrine-induced protein synthesis was significantly reduced compared with cells expressing both GFP and GFP/STRAD{alpha}/MO25{alpha} (Fig. 5). In addition, expression of the LKB1/STRAD{alpha}/MO25{alpha} 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{alpha}/MO25{alpha}. These data suggest that increased LKB1 activity can suppress both basal protein synthesis and enhanced protein synthesis induced by phenylephrine.


Figure 5
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  		Fig. 5. Expression of the LKB1/STRAD{alpha}/MO25{alpha} 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{alpha}/Ad.MO25{alpha}, or the LKB1 complex Ad.LKB1/Ad.STRAD{alpha}/Ad.MO25{alpha}. 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 µM PE or vehicle, as well as 1 µCi/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{alpha}/MO25{alpha} group. &P < 0.01, significantly different from the GFP +PE group. $P < 0.01, significantly different from the GFP/STRAD{alpha}/MO25{alpha} + PE group. n = 5.

 
To visualize the inhibition of phenylephrine-induced hypertrophic growth by the LKB1/STRAD{alpha}/MO25{alpha} complex, immunocytochemistry against {alpha}-actinin was performed on all groups of cells. Cells were infected with either Ad.GFP, Ad.GFP/Ad.STRAD{alpha}/Ad.MO25{alpha}, or Ad.LKB1/Ad.STRAD{alpha}/Ad.MO25{alpha} and treated with or without phenylephrine. The Ad.GFP and Ad.GFP/Ad.STRAD{alpha}/Ad.MO25{alpha}-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).


Figure 6
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  		Fig. 6. Expression of the LKB1/STRAD{alpha}/MO25{alpha} 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{alpha}/MO25{alpha} (B), or the LKB1 complex Ad.LKB1/Ad.STRAD{alpha}/Ad.MO25{alpha} (C), and {alpha}-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 µM PE or vehicle. After an additional 24 h, the cells were fixed and incubated with anti-{alpha}-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.

 
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 cardiac-specific 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).


Figure 7
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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 cardiac-specific 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.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
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{alpha}, and MO25{alpha} 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{alpha}, and MO25{alpha} appeared higher when these proteins were expressed in combination rather than individually, suggesting that like the AMPK heterotrimer (12), the LKB1/MO25{alpha}/STRAD{alpha} heterotrimeric complex is stabilized and protected from degradation. In addition to detecting high levels of expressed LKB1/MO25{alpha}/STRAD{alpha} 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{alpha}, and MO25{alpha} 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{alpha}/MO25{alpha} 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{alpha} 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{alpha} and ACC were not altered when the neonatal rat cardiac myocytes were infected only with the LKB1-binding partners STRAD{alpha} and MO25{alpha} (GFP/STRAD{alpha}/MO25{alpha}) (Fig. 3B). Furthermore, changes observed 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{alpha}/MO25{alpha}, 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{alpha}, and MO25{alpha} 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 cytosol during these situations and/or whether this is necessary for LKB1 to fully activate AMPK, is currently unknown.

Although controversial, it has been shown that the p70S6 kinase pathway is not inhibited by AMPK in the heart (19). Therefore, the role of AMPK and its activating kinase LKB1 in controlling p70S6 kinase activity in the cardiac myocyte is still in question. In a previous report, we provided evidence that AMPK-induced inhibition of protein synthesis was indeed mediated via decreased p70S6 kinase activation (7). However, we did not investigate the effects of this decrease on other downstream targets of p70S6 kinase activity such as the 40S ribosomal protein S6. Ribosomal protein S6 has been proposed as a rapamycin-sensitive mechanism by which p70S6 kinase can activate the translation of 5'-TOP (tract of oligopyrimidine) mRNAs encoding for ribosomal proteins and elongation factors (22, 23). Thus inhibition of ribosomal protein S6 activity resulting from decreased p70S6 kinase activity may be one mechanism by which AMPK-induced inhibition of p70S6 kinase may contribute to the decreased rate of protein synthesis in the cardiac myocyte. Indeed, the data herein show that expression of the LKB1/STRAD{alpha}/MO25{alpha} complex results in a dramatic decrease in phosphorylation of p70S6 kinase at Thr-389 and Thr-421/Ser-424 compared with control cells (Fig. 4A). Moreover, as phosphorylation at these sites are closely related to p70S6 kinase activity (10, 32), we also observed a decrease in phosphorylation of ribosomal protein S6 at Ser-235/236, which indicates both a decrease in p70S6 kinase activity and an inhibition of ribosomal protein S6 activity. Together, these data indicate that activation of LKB1, presumably via increased AMPK activity, can decrease the rate of protein synthesis in the cardiac myocyte.

To investigate whether increased LKB1 activity is able to prevent phenylephrine-induced protein synthesis associated with cardiac myocyte hypertrophy, we treated cardiac myocytes with the {alpha}1-adrenergic receptor agonist phenylephrine. With this treatment, cardiac myocytes demonstrated the characteristic increase in cell size (Fig. 6), which was associated with a significant increase in protein synthesis (Fig. 5). However, protein synthesis was significantly decreased in the presence of the LKB1/STRAD{alpha}/MO25{alpha} complex (Fig. 5). This almost complete inhibition of phenylephrine-induced protein synthesis by active LKB1 was more dramatic than what we have previously reported using pharmacological activators of AMPK (7). This may be attributed to a more robust activation of AMPK using the LKB1/STRAD{alpha}/MO25{alpha} complex compared with pharmacological activators of AMPK. Despite this difference in AMPK activity between the two studies (the present study and Ref. 7), the decrease in phosphorylation of p70S6 kinase was virtually the same. This suggests that additional mechanisms controlling protein synthesis may be involved. Indeed, LKB1 also phosphorylates and activates at least 12 other members of the AMPK-related family of protein kinases (29), any of which could also be involved in regulating protein synthesis in our model.

Our gain-of-function data collected from transduced isolated myocytes support the concept that LKB1 activation can regulate protein synthesis associated with cardiac myocyte hypertrophy. In addition, the converse also appears to hold true. That is, hearts from transgenic mice with cardiac-specific expression of activated calcineurin develop profound hypertrophy, which is accompanied by a significant decrease in LKB1 expression (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{alpha} 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{alpha}/STRAD{alpha} 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 cardiac-specific 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.


   FOOTNOTES
 

Address for reprint requests and other correspondence: J. R. B. Dyck, 474 Heritage Medical Research Centre, Univ. of Alberta, Edmonton, Alberta, Canada (e-mail: Jason.Dyck{at}UAlberta.ca)

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.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Altarejos JY, Taniguchi M, Clanachan AS, Lopaschuk GD. Myocardial ischemia differentially regulates LKB1 and an alternate 5' -AMP-activated protein kinase kinase. J Biol Chem 280: 183–190, 2005.[Abstract/Free Full Text]
  2. Anderson KV, Ingham PW. The transformation of the model organism: a decade of developmental genetics. Nat Genet 33, Suppl: 285–293, 2003.
  3. Baas AF, Boudeau J, Sapkota GP, Smit L, Medema R, Morrice NA, Alessi DR, Clevers HC. Activation of the tumour suppressor kinase LKB1 by the STE20-like pseudokinase STRAD. EMBO J 22: 3062–3072, 2003.[CrossRef][Web of Science][Medline]
  4. Bolster DR, Crozier SJ, Kimball SR, Jefferson LS. AMP-activated protein kinase suppresses protein synthesis in rat skeletal muscle through down-regulated mammalian target of rapamycin (mTOR) signaling. J Biol Chem 277: 23977–23980, 2002.[Abstract/Free Full Text]
  5. Boudeau J, Baas AF, Deak M, Morrice NA, Kieloch A, Schutkowski M, Prescott AR, Clevers HC, Alessi DR. MO25alpha/beta interact with STRAD{alpha}/beta enhancing their ability to bind, activate and localize LKB1 in the cytoplasm. EMBO J 22: 5102–5114, 2003.[CrossRef][Web of Science][Medline]
  6. Boudeau J, Sapkota G, Alessi DR. LKB1, a protein kinase regulating cell proliferation and polarity. FEBS Lett 546: 159–165, 2003.[CrossRef][Web of Science][Medline]
  7. Chan AY, Soltys CL, Young ME, Proud CG, Dyck JR. Activation of AMP-activated protein kinase inhibits protein synthesis associated with hypertrophy in the cardiac myocyte. J Biol Chem 279: 32771–32779, 2004.[Abstract/Free Full Text]
  8. Cheng SW, Fryer LG, Carling D, Shepherd PR. Thr2446 is a novel mammalian target of rapamycin (mTOR) phosphorylation site regulated by nutrient status. J Biol Chem 279: 15719–15722, 2004.[Abstract/Free Full Text]
  9. Cheung PC, Salt IP, Davies SP, Hardie DG, Carling D. Characterization of AMP-activated protein kinase gamma-subunit isoforms and their role in AMP binding. Biochem J 346: 659–669, 2000.
  10. Dennis PB, Pullen N, Kozma SC, Thomas G. The principal rapamycin-sensitive p70(s6k) phosphorylation sites, T-229 and T-389, are differentially regulated by rapamycin-insensitive kinase kinases. Mol Cell Biol 16: 6242–6251, 1996.[Abstract]
  11. Dubbelhuis PF, Meijer AJ. Hepatic amino acid-dependent signaling is under the control of AMP-dependent protein kinase. FEBS Lett 521: 39–42, 2002.[CrossRef][Web of Science][Medline]
  12. Dyck JRB, Gao G, Widmer J, Stapleton D, Fernandez CS, Kemp BE, Witters LA. Regulation of 5' AMP-activated protein kinase activity by the noncatalytic beta and gamma subunits. J Biol Chem 271: 17798–17803, 1996.[Abstract/Free Full Text]
  13. Graham FL, Prevec L. Adenovirus-based expression vectors and recombinant vaccines. Biotechnology 20: 363–390, 1992.[Medline]
  14. Hardie DG, Carling D. The AMP-activated protein kinase–fuel gauge of the mammalian cell? Eur J Biochem 246: 259–273, 1997.[Web of Science][Medline]
  15. Hawley SA, Boudeau J, Reid JL, Mustard KJ, Udd L, Makela TP, Alessi DR, Hardie DG. Complexes between the LKB1 tumor suppressor, STRAD{alpha}/beta and MO25{alpha}/beta are upstream kinases in the AMP-activated protein kinase cascade (Abstract). J Biol 2: 28, 2003.[CrossRef][Medline]
  16. Hawley SA, Davison M, Woods A, Davies SP, Beri RK, Carling D, Hardie DG. Characterization of the AMP-activated protein kinase kinase from rat liver and identification of threonine 172 as the major site at which it phosphorylates AMP-activated protein kinase. J Biol Chem 271: 27879–27887, 1996.[Abstract/Free Full Text]
  17. Hemminki A, Markie D, Tomlinson I, Avizienyte E, Roth S, Loukola A, Bignell G, Warren W, Aminoff M, Hoglund P, Jarvinen H, Kristo P, Pelin K, Ridanpaa M, Salovaara R, Toro T, Bodmer W, Olschwang S, Olsen AS, Stratton MR, de la Chapelle A, Aaltonen LA. A serine/threonine kinase gene defective in Peutz-Jeghers syndrome. Nature 391: 184–187, 1998.[CrossRef][Medline]
  18. Hong SP, Leiper FC, Woods A, Carling D, Carlson M. Activation of yeast Snf1 and mammalian AMP-activated protein kinase by upstream kinases. Proc Natl Acad Sci USA 100: 8839–8843, 2003.[Abstract/Free Full Text]
  19. Horman S, Beauloye C, Vertommen D, Vanoverschelde JL, Hue L, Rider MH. Myocardial ischemia and increased heart work modulate the phosphorylation state of eukaryotic elongation factor-2. J Biol Chem 278: 41970–41976, 2003.[Abstract/Free Full Text]
  20. Horman S, Browne G, Krause U, Patel J, Vertommen D, Bertrand L, Lavoinne A, Hue L, Proud C, Rider M. Activation of AMP-activated protein kinase leads to the phosphorylation of elongation factor 2 and an inhibition of protein synthesis. Curr Biol 12: 1419–1423, 2002.[CrossRef][Web of Science][Medline]
  21. Inoki K, Zhu T, Guan KL. TSC2 mediates cellular energy response to control cell growth and survival. Cell 115: 577–590, 2003.[CrossRef][Web of Science][Medline]
  22. Jefferies HB, Fumagalli S, Dennis PB, Reinhard C, Pearson RB, Thomas G. Rapamycin suppresses 5' TOP mRNA translation through inhibition of p70s6k. EMBO J 16: 3693–3704, 1997.[CrossRef][Web of Science][Medline]
  23. Jefferies HB, Reinhard C, Kozma SC, Thomas G. Rapamycin selectively represses translation of the "polypyrimidine tract" mRNA family. Proc Natl Acad Sci USA 91: 4441–4445, 1994.[Abstract/Free Full Text]
  24. Jenne DE, Reimann H, Nezu J, Friedel W, Loff S, Jeschke R, Muller O, Back W, Zimmer M. Peutz-Jeghers syndrome is caused by mutations in a novel serine threonine kinase. Nat Genet 18: 38–43, 1998.[CrossRef][Web of Science][Medline]
  25. Karuman P, Gozani O, Odze RD, Zhou XC, Zhu H, Shaw R, Brien TP, Bozzuto CD, Ooi D, Cantley LC, Yuan J. The Peutz-Jegher gene product LKB1 is a mediator of p53-dependent cell death. Mol Cell 7: 1307–1319, 2001.[CrossRef][Web of Science][Medline]
  26. Kimura N, Tokunaga C, Dalal S, Richardson C, Yoshino K, Hara K, Kemp BE, Witters LA, Mimura O, Yonezawa K. A possible linkage between AMP-activated protein kinase (AMPK) and mammalian target of rapamycin (mTOR) signalling pathway. Genes Cells 8: 65–79, 2003.[Abstract]
  27. Kovacic S, Soltys CL, Barr AJ, Shiojima I, Walsh K, Dyck JR. Akt activity negatively regulates phosphorylation of AMP-activated protein kinase in the heart. J Biol Chem 278: 39422–39427, 2003.[Abstract/Free Full Text]
  28. Krause U, Bertrand L, Hue L. Control of p70 ribosomal protein S6 kinase and acetyl-CoA carboxylase by AMP-activated protein kinase and protein phosphatases in isolated hepatocytes. Eur J Biochem 269: 3751–3759, 2002.[Web of Science][Medline]
  29. Lizcano JM, Goransson O, Toth R, Deak M, Morrice NA, Boudeau J, Hawley SA, Udd L, Makela TP, Hardie DG, Alessi DR. LKB1 is a master kinase that activates 13 kinases of the AMPK subfamily, including MARK/PAR-1. EMBO J 23: 833–843, 2004.[CrossRef][Web of Science][Medline]
  30. Marignani PA, Kanai F, Carpenter CL. LKB1 associates with Brg1 and is necessary for Brg1-induced growth arrest. J Biol Chem 276: 32415–32418, 2001.[Abstract/Free Full Text]
  31. Molkentin JD, Lu JR, Antos CL, Markham B, Richardson J, Robbins J, Grant SR, Olson EN. A calcineurin-dependent transcriptional pathway for cardiac hypertrophy. Cell 93: 215–228, 1998.[CrossRef][Web of Science][Medline]
  32. Pearson RB, Dennis PB, Han JW, Williamson NA, Kozma SC, Wettenhall RE, Thomas G. The principal target of rapamycin-induced p70s6k inactivation is a novel phosphorylation site within a conserved hydrophobic domain. EMBO J 14: 5279–5287, 1995.[Web of Science][Medline]
  33. Proud CG. Regulation of mammalian translation factors by nutrients. Eur J Biochem 269: 5338–5349, 2002.[Web of Science][Medline]
  34. Rowan A, Churchman M, Jefferey R, Hanby A, Poulsom R, Tomlinson I. In situ analysis of LKB1/STK11 mRNA expression in human normal tissues and tumours. J Pathol 192: 203–206, 2000.[CrossRef][Web of Science][Medline]
  35. Sakamoto K, McCarthy A, Smith D, Green KA, Grahame Hardie D, Ashworth A, Alessi DR. Deficiency of LKB1 in skeletal muscle prevents AMPK activation and glucose uptake during contraction. EMBO J 24: 1810–1820, 2005.[CrossRef][Web of Science][Medline]
  36. Sakamoto K, Zarrinpashneh E, Budas GR, Pouleur AC, Dutta A, Prescott AR, Vanoverschelde JL, Ashworth A, Jovanovic A, Alessi DR, Bertrand L. Deficiency of LKB1 in heart prevents ischemia-mediated activation of AMPK{alpha}2 but not AMPK{alpha}1. Am J Physiol Endocrinol Metab 290: E780–E788, 2006.[Abstract/Free Full Text]
  37. Shaw RJ, Bardeesy N, Manning BD, Lopez L, Kosmatka M, DePinho RA, Cantley LC. The LKB1 tumor suppressor negatively regulates mTOR signaling. Cancer Cell 6: 91–99, 2004.[CrossRef][Web of Science][Medline]
  38. Smith DP, Rayter SI, Niederlander C, Spicer J, Jones CM, Ashworth A. LIP1, a cytoplasmic protein functionally linked to the Peutz-Jeghers syndrome kinase LKB1. Hum Mol Genet 10: 2869–2877, 2001.[Abstract/Free Full Text]
  39. Sriwijitkamol A, Ivy JL, Christ-Roberts C, DeFronzo RA, Mandarino LJ, Musi N. LKB1-AMPK signaling in muscle from obese insulin-resistant Zucker rats and effects of training. Am J Physiol Endocrinol Metab 290: E925–E932, 2006.[Abstract/Free Full Text]
  40. Stein SC, Woods A, Jones NA, Davison MD, Carling D. The regulation of AMP-activated protein kinase by phosphorylation. Biochem J 345: 437–443, 2000.



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