THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 282, NO. 16, pp. 11687–11695, April 20, 2007
© 2007 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

Protein Kinase A Suppresses Sterol Regulatory
Element-binding Protein-1C Expression via
Phosphorylation of Liver X Receptor in the Liver*
Received for publication, December 29, 2006 Published, JBC Papers in Press, February 12, 2007, DOI 10.1074/jbc. M611911200

Takashi Yamamoto‡, Hitoshi Shimano‡§1, Noriyuki Inoue‡, Yoshimi Nakagawa‡§, Takashi Matsuzaka‡§,
Akimitsu Takahashi‡§, Naoya Yahagi§, Hirohito Sone‡§, Hiroaki Suzuki‡, Hideo Toyoshima‡, and Nobuhiro Yamada‡From the ‡Department of Internal Medicine, Metabolism and Endocrinology, Graduate School of Comprehensive Human Sciencesand §Center for Tsukuba Advanced Research Alliance, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan

In mammals, carbohydrates are an essential energy resource.
When consumed in excess, carbohydrates are converted to lipids by lipogenic enzymes in preparation for times of energy
deficiency. These processes are known to be regulated at thetranscription level, and several different transcription factors
are known to contribute to this regulation. Sterol regulatory
element-binding protein (SREBP),2 a transcription factor
belonging to the basic helix-loop-helix-leucine zipper family,* This work was supported by grants-in-aid from the Ministry of Science, Education, Culture, and Technology of Japan. The costs of publication of thisarticle were defrayed in part by the payment of page charges. This article
must therefore be hereby marked “advertisement” in accordance with 18
U.S.C. Section 1734 solely to indicate this fact.
1
To whom correspondence and requests should be addressed. Fax: 81-29853-317; E-mail: shimano-tky@umin.ac.jp.
2
The abbreviations used are: SREBP, sterol regulatory element-binding protein; dbcAMP, dibutyryl-cAMP; NID, nuclear receptor interaction domain;
DBD, DNA binding domain; LBD, ligand binding domain; LXR, liver X receptor ␣; PKA, protein kinase A; CREB, cAMP-response element-binding protein; CBP, CREB-binding protein; HA, hemagglutinin; CMV, cytomegalovi-

APRIL 20, 2007 • VOLUME 282 • NUMBER 16

regulates triglyceride synthesis and cholesterol metabolism (1).
Three different SREBP isoforms have been identified.
SREBP-1c plays a role primarily in triglyceride synthesis (2),whereas SREBP-2 regulates cholesterol metabolism (3).
SREBP-1a is expressed in growing cells and activates an entire
array of genes involved in both triglyceride and cholesterol synthesis (4). In liver SREBP-1c controls transcription of genes
involved in fatty acid and triglyceride synthesis (2, 5). SREBP-1c
expression is highly regulated in response to the nutritional
condition. Expression of SREBP-1c is undetectable during fasting, whereas its expression is strongly induced in a fed state
followed by similar adapted nutritional changes in lipogenic
genes (6).
To understand the molecular mechanism of nutritional regulation of SREBP-1c expression, the SREBP-1c gene promoter
has been extensively studied. In the proximal region of themouse SREBP-1c promoter, Sp1, NF-Y, USF, SREBP, and LXRbinding sites have been identified (7–9). It has been observedthat insulin (10) and glucose (11, 12) are factors that induce
hepatic SREBP-1c in vivo and in vitro, although precise mechanisms are yet to be elucidated. Adenoviral overexpression in rat
primary hepatocytes suggested that the signal transducer, Akt,
could be a potential regulator of SREBP-1c transcription (13).
Liver X receptors (LXR) belong to a nuclear receptor superfamily. The LXR subfamily consists of two members, LXR␣ and
LXR␤, which are activated by oxysterols (14). The expression
pattern of LXR␣ is restricted mainly to liver, adipocytes, small
intestine, and macrophages, whereas LXR␤ is expressed ubiquitously. Although early reports revealed involvement of LXRs
in cholesterol homeostasis (15, 16), recent studies suggest that
LXR negatively regulates gluconeogenesis (17) and inflammatory responses (18, 19). While investigating the pharmacological effect of LXR in rodent models, it has been observed that
LXR ligands are protective against the development of atherosclerotic lesions (20) and ameliorates conditions of high blood
glucose and impaired glucose tolerance (21). LXRs directly bindthe cis element on the SREBP-1c promoter as heterodimers
with RXR, leading to transcriptional activation (8). Several
studies have established LXRs as dominant activators of
SREBP-1c expression. LXR␣/␤ double knock-out micerus; DTT, dithiothreitol; EMSA, electrophoretic mobility shift assay; ChIP,
chromatin immunoprecipitation; LXRE, LXR responsive element; RE,
responsive element.

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Sterol regulatory element-binding protein (SREBP)-1c is atranscription factor that controls synthesis of fatty acids andtriglycerides in the liver and is highly regulated by nutrition andhormones. In the current studies we show that protein kinase A(PKA), a mediator of glucagon/cAMP, a fasting signaling, suppresses SREBP-1c by modulating the activity of liver X receptor
␣ (LXR␣), a dominant activator of SREBP-1c expression. Activation of PKA repressed LXR-induced SREBP-1c expression
both in rat primary hepatocytes and mouse livers. Promoter
analyses revealed that the LXR␣-binding site in the SREBP-1c
promoter is responsible for PKA inhibitory effect on SREBP-1ctranscription. In vitro and in vivo PKA directly phosphorylated
LXR␣, and the two consensus PKA target sites (195, 196 serinesand 290, 291 serines) in its ligand binding/heterodimerization
domain were crucial for the inhibition of LXR signaling. PKA
phosphorylation of LXR␣ caused impaired DNA binding activity by preventing LXR␣/RXR dimerization and decreased its
transcription activity by inhibiting recruitment of coactivator
SCR-1 and enhancing recruitment of corepressor NcoR1. These
results indicate that LXR␣ is regulated not only by oxysterol
derivatives but also by PKA-mediated phosphorylation, whichsuggests that nutritional regulation of SREBP-1c and lipogenesis could be regulated at least partially through modulation of
LXR.

PKA Inhibits LXR Signaling

EXPERIMENTAL PROCEDURES
Materials—Anti-LXR␣, anti-RXR, and anti-SREBP-1 antibodies were purchased from Santa Cruz Biotechnology Inc.
(Santa Cruz, CA). Anti-CREB and anti-phospho-CREB (Ser133) antibodies were purchased from Cell Signaling Technology Inc. (Beverly, MA). Anti-His 5 antibody was purchasedfrom Qiagen Inc. Anti-HA and anti-Myc antibodies were purchased from Roche Applied Science. Anti-FLAG antibody waspurchased from Promega (Madison, WI). Dibutyryl-cAMP waspurchased from Promega. T0901317 as synthetic LXR ligand
was purchased from WAKO.
Animals—Male mice (C57BL/6J) were obtained from CLEA
Japan (Tokyo, Japan). For fasting and refeeding, mice were
fasted for 24 h and then fed a high sucrose/low fat diet for 12 h.
Isolation and Culture of Hepatocytes—Primary hepatocytes
were isolated form male Sprague-Dawley rats (200 –300 g)(CLEA Japan). Cell were resuspended in Dulbecco’s modified
Eagle’s medium containing penicillin and streptomycin supplemented with 10% fetal bovine serum before being seeded on
100-mm collagen-coated dishes at 2 ⫻ 106 cells/dish. Afterincubation for 4 h to allow attachment, medium was replaced,and experiments were performed.
Northern Blotting—Total RNA from mouse liver and rat
primary hepatocytes was isolated as described previously(29). The cDNA probes for mouse SREBP-1, phosphoenolpyruvate carboxykinase, acidic ribosomal phosphoprotein
PO (36B4), ABCA1, and LXR␣ were prepared as previously
described (8, 29, 30).
Plasmids—A series of mouse SREBP-1c promoter linked topGL2 basic were previously described (7). LXRE-enhancer
construct linked to pGL2 promoter vector was previously

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described (8). Human ABCA1 promoter (⫺919 to ⫹239)
linked to pGL3 basic were previously described (30). Expression vector for Gal4-DBD-LXR␣ ligand binding domain(LBD) fusion protein and Gal4 RE Luc vector were previously described (8). Expression vectors for mouse LXR␣(wild type, 195A 196A and 290A 291A) were constructed in pcDNA3.1(⫹). Expression vector for PKA c subunit was constructed in pFA. Expression vector for
V5-tagged PKA regulatory subunit (dominant negativeform) was constructed in pcDNA3.1(⫺) according to a previous report for the mutated form of the regulatory type 1
subunit of PKA (31). The expression vector for HA-tagged
human LXR␣ was constructed in pcDNA3. Expression vectors for FLAG-tagged wild type human RXR and FLAGtagged mutant RXR in which the serine 27 residue was substituted to alanine were constructed in pcDNA3.1(⫹).
Expression vectors for histidine-tagged recombinant mouse
LXR␣ (full-length, amino acids 1–163, 162–326, and 325–445) were constructed in pET28a(⫹). The expression vector
for glutathione S-transferase fusion protein was constructedin pGEX4T-2. For the mammalian two-hybrid system, themouse LXR␣-coding region was ligated into pACT. Mouse
SRC-1 nuclear receptor interaction domain (amino acids
568 –779) and mouse NcoR1 nuclear receptor interaction
domain (amino acids 1944 –2453) were ligated into pM. For
in vitro translation, SRC-1 nuclear receptor interaction
domain (NID; amino acids 576 –779) was ligated into
pcDNA3.1(⫹). The expression vector for FLAG-tagged CBP
was previously described. The expression vector for
HA-tagged mouse CREB was constructed in pcDNA3.
Transfections and Luciferase Assay—293 cells and 293T cells
were grown in Dulbecco’s modified Eagle’s medium containing
penicillin and streptomycin supplemented with 10% fetal
bovine serum at 37 °C in 24-well plate overnight before transfection. Cells were transfected with reporter vector, expression
vector using FuGENE 6. Total amounts of DNA were adjusted
to 0.5 ␮g/well using empty vector. After 24 h of incubation, theamounts of firefly luciferase activity in transfectants were measured. Firefly luciferase activity was normalized by the amountsof Renilla luciferase activity expressed from CMV or SV40 promoter linked-Renilla luciferase unless otherwise indicated.
In Vitro Kinase Assay—Histidine-tagged recombinant mouse
LXR␣ proteins were expressed in Escherichia coli (BL21, DE3)
and purified using standard techniques and purification kit
Mag Extractor (TOYOBO) according to the manufacturer’s
protocol. Briefly, bacterial culture containing kanamycin weregrown at 37 °C. After induction of recombinant proteins for
2–3 h, cells were resuspended in lysis buffer (6 M guanidinehydrochloride, 5 M NaCl (pH 8.0)) and lysed by sonication. The
centrifuged supernatant was mixed for 30 min at room temperature on a rotator with magnetic nickel beads. Protein-absorbed beads were washed with lysis buffer, and recombinant
LXR␣ proteins were eluted with elution buffer. After dialysis to
exclude guanidine, the concentration and size of histidinetagged proteins were estimated by SDS-PAGE followed by Coomassie Blue staining and immunoblotting using a known quantity of molecular weight standards. The purified proteins werestored at ⫺80 °C until experiments were performed.
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revealed dramatically reduced expression of SREBP-1c (9). We
have used an expression cloning strategy to show that LXRs are
the primary activators of the SREBP-1c promoter (8). Polyunsaturated fatty acids are the only known dietary lipid capable ofnegatively regulating hepatic SREBP-1c expression and lipogenesis (22). A portion of these effects is mediated at the transcriptional level through repression of LXR activity (23).
Considering that hepatic SREBP-1c expression is dominated by
LXRs and eliminated by fasting, it is probable that there is a mechanism by which LXR mediates the repressed SREBP-1c expression
during fasting. Consistently, it has been reported that glucagons
and its signal mediator, cAMP, suppresses SREBP-1c in rat primary hepatocytes (24). Protein kinase A (PKA) is a cAMP-dependent protein kinase that consists of both a catalytic and regulatory
subunit and regulates numerous cellular functions in eukaryotic
cells by phosphorylating target proteins. In regard to energy
metabolism, PKA is subordinated to glucagon and adrenalin and,
therefore, is classically recognized as a fasting signal to activate
gluconeogenesis and ␤-oxidation and to oppose triglycerides synthesis and glucose utilization. It is known that several nuclear
receptors (e.g. estrogen receptor, retinoic acid receptor, peroxisome proliferator-activated receptor, and hepatocyte nuclear factor-4␣) are phosphorylated by PKA leading to modification of
their trans activities via diverse mechanism (25–28). In the current
study we investigated effects of cAMP/PKA on the SREBP-1c
expression and LXR signaling system.

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by freshly prepared elution buffer (1% SDS, 0.1 M NaHCO3).
Formaldehyde cross-linking was reversed by overnight incubation at 65 °C after adding 20 ␮l of 5 M NaCl. Samples were
purified with phenol:chloroform:isoamyl alcohol and precipitated with ethanol. DNA pellets were dissolved with TrisEDTA buffer and used as templates in PCR. Eight ␮l of 20 ␮l of
DNA solution was used for PCR amplification (1st step of 95 °C
for 3 min (one cycle), 2nd step of 94 °C for 1 min, 62.1 °C for
30 s, 72 °C for 1 min (25 cycles)). ChIP primers were 5⬘-GAACCAGCGGTGGGAACACAGAGC-3⬘ and 5⬘-GACGGCGGCAGCTCGGGTTTCTC.
Immunoblotting and Immunoprecipitation—COS7 cells
were seeded on 100-mm dishes and transfected with theexpression vectors. After incubation for 48 h, dbcAMP (100␮M), forskolin (10 ␮M), and isobutylmethylxanthine (100 ␮M)
were added into medium for 30 min, and then cells were harvested. Protein lysate was extracted in lysis buffer (25 mM
HEPES (pH 7.9), 50 mM KCl, 6% glycerol, 5 mM MgCl2, 0.5%
Triton X-100, 1 mM DTT, 50 mM NaF, 40 mM ␤-glycerophosphate, 25 mM sodium pyrophosphate, protease inhibitor mixture (Roche Applied Science)) on ice 20 min and centrifuge at
15,000 rpm at 4 °C for 10 min. Supernatants were subjected toimmunoprecipitation assay as previously described (29).
GST Pulldown Assay—GST and GST-LXR␣ proteins wereprepared using standard techniques according to the manufacturer’s protocol. Briefly, E. coli (BL21, DE3) transformed with
the GST or GST-LXR␣ expression vector were incubated
before induction at room temperature for 4 h with 0.4 mM isopropyl 1-thio-␤-D-galactopyranoside. The cells were harvested
by centrifugation and suspended in buffer A (25 mM HEPES(pH 7.9), 50 mM KCl, 6% glycerol, 5 mM EDTA, 5 mM MgCl2,
0.5% Triton X-100, protease inhibitor mixture, 1 mM DTT).
The cells were lysed by sonication, and cellular debris wasremoved by centrifugation at 15,000 rpm at 4 °C for 10 min.
Recombinant GST or GST-LXR␣ proteins were purified by glutathione-Sepharose beads according to the manufacturer’s protocol (Amersham Biosciences). GST fusion proteins were
eluted (50 mM Tris HCl (pH 8.0), 50 mM NaCl, 10 mM glutathione, protease inhibitor mixture, 1 mM DTT). Amounts andsizes of GST or GST-LXR␣ proteins were estimated by SDSPAGE followed by Coomassie Blue staining and immunoblotting using anti-LXR antibody. GST or GST-LXR␣ and the PKA
c subunit or equal amounts of bovine serum albumin weremixed in PKA buffer (50 mM Tris HCl (pH 7.5), 10 mM NaCl, 10mM MgCl2, 1 mM DTT, 50 mM NaF, 20 mM ␤-glycerophosphate) and incubated for 45 min at 30 °C. After adding glutathione beads and 35S-radiolabeled SRC-1 nuclear receptor interaction domain (amino acids 576 –779) synthesized in vitro tosamples and incubation for 2 h at 4 °C, beads were washed 3times with ice-cold phosphate-buffered saline containing 0.1%
Triton X-100. Proteins bound to beads were eluted with elution
buffer (50 mM Tris-HCl (pH 8.0), 50 mM glutathione), resolved
by SDS-PAGE, and visualized autoradiography.

RESULTSPKA Represses Expression of SREBP-1 in Vivo—Expression of
SREBP-1c in the liver is highly regulated by nutrition; it is suppressed at fasting and induced in a refed state as previously
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Bovine purified PKA catalytic subunit was purchased from
Promega. PKA c subunit, histidine-tagged LXR␣, and[␥-32P]ATP were mixed in PKA buffer (50 mM Tris-HCl (pH7.5), 10 mM NaCl, 10 mM MgCl2, 1 mM DTT) and incubated for
45 min at 30 °C. Samples were analyzed by SDS-PAGE, andphosphorylation was visualized by autoradiography. Quantityof histidine-tagged protein was confirmed by immunoblotting
using anti-His 5 antibody. Histone H1 protein (Calbiochem)
was used as positive control in kinase assays.
In Vivo Kinase Assay—For in vivo kinase assay, COS7 cells
prepared in 100-mm collagen-coated dishes at 1 ⫻ 106 cells/
dish were transfected with control or expression vector for HAtagged human LXR␣ vectors. After transfection, the cells werestarved in phosphate-deficient medium for 12 h and then incubated for 2 h in the same medium containing 500 ␮Ci/ml[32P]orthophosphate. Cells were treated with PKA activators(forskolin 10 ␮M, dibutyryl-cAMP 1 mM, isobutylmethylxanthine 1 mM) for 30 min before harvesting.
At the end of the labeling period the cells were washed icecold phosphate-buffered saline, harvested, and lysed on ice.
Protein samples were extracted in lysis buffer mentioned elsewhere and centrifuged at 15,000 rpm 4 °C for 10 min. Supernatants were subjected to immunoprecipitation assay used withanti HA antibody (Roche Applied Science) as previously
described. The immunocomplex were eluted in sample buffer,resolved by SDS-PAGE (8% gel), visualized with autoradiography, and immunoblotted.
Electrophoretic Mobility Shift Assay—Nuclear extracts wereprepared from livers as previously described (32). RXR protein
was generated from expression vector using a coupled in vitro
reticulocyte transcription/translation system (Promega). Double-stranded oligonucleotides of LXR response element in
SREBP-1c promoter using in EMSA were prepared as previously described. In vitro synthetic protein lysate or nuclear
extracts (⬃1 ␮g) were incubated. DNA-protein complex wereresolved on a 4% polyacrylamide gel.
Chromatin Immunoprecipitation (ChIP) Assay—The ChIP
assay was conducted with a chromatin immunoprecipitation
kit (Upstate Biotechnology, Lake Placid, NY). After H2.35 cells
were treated with PKA activators (10 ␮M forskolin, 100 ␮M
dibutyryl-cAMP) or vehicle under T0901317 (10 ␮M) for 1 h at
37 °C, H2.35 cells were cross-linked for 10 min by adding formaldehyde directly to culture medium to a final concentration of
1%. Cross-linked cells were washed with ice-cold phosphatebuffered saline containing protease inhibitors, scraped, pelleted, resuspended in 200 ␮l of SDS lysis buffer (1% SDS, 10 mM
EDTA, 50 mM Tris-HCl (pH 8.0)) per 1 ⫻ 106 cells and incubated for 10 min on ice. The lysates were then sonicated for 30 seach by sonicator (Sonifer 250, Branson) with 20% duty cycleand 10% output power. The samples were on ice for 1 min
between the cycles. After sonication the samples were centrifuged, and supernatant was diluted 10-fold in ChIP dilution
buffer containing protease inhibitors. 2-ml aliquots wereprecleared with 75 ␮l of a 50% slurry of salmon sperm DNA/
protein A-Sepharose for 30 min at 4 °C and then incubatedovernight with 3 ␮g of anti-RXR antibody or control IgG. Antibody-protein-DNA complexes were immunoprecipitated with60 ␮l of protein A. After intensive washing, pellets were eluted

PKA Inhibits LXR Signaling

FIGURE 2. Effects of PKA on mRNA level of SREBP-1 induced by the synthetic LXR ligand, T0901317. Rat primary hepatocytes were incubated in
medium containing vehicle or T0901317 (1 ␮M) in the presence or absence of
dbcAMP (30 ␮M) for 24 h. Cells were harvested for extraction of total RNA
followed by northern blotting as described under “Experimental Procedures.”
PEPCK, phosphoenolpyruvate carboxykinase.

reported (6). One potential mechanism of this suppression at
fasting could be mediated through the cAMP/PKA system thatmediates the glucagon signal. C57BL/6J mice in a refed state
were administrated with dibutyryl-cAMP and theophylline foractivation of PKA to mimic fasting signal. The SREBP-1c
mRNA that was highly induced by refeeding was robustly
repressed by cAMP/theophylline administration (Fig. 1A).
Reciprocally, phosphoenolpyruvate carboxykinase, a gene that
controls gluconeogenesis and a well known target of the glucagon/cAMP/PKA signal, was suppressed by refeeding but
restored by these PKA activators. The reduction of SREBP-1
mRNA level by PKA activation was associated with a robust
decrease in active SREBP-1c protein in hepatic nuclear extracts(Fig. 1B). SREBP-1c expression is dominantly controlled by
LXR/RXR that binds directly to the two LXREs in the SREBP-1c
promoter (8, 9). As shown in Fig. 1B, the expression of LXR/
RXR was not affected by the PKA activation. Therefore,
SREBP-1c suppression by PKA was not due to reduction in theamounts of LXR/RXR.
PKA Activation Represses LXR-mediated SREBP-1 Induction—
The effect of PKA on LXR activation of SREBP-1c was estimated. In rat primary hepatocytes, the SREBP-1c mRNAlevel was essentially undetectable but was highly induced by
T0901317, an LXR agonist. This induction was substantiallysuppressed by the addition of dibutyryl-cAMP whereas
expression of LXR and RXR was not increased at the mRNAlevel (Fig. 2). These data suggest that PKA repression of
SREBP-1c was mediated not through changes in LXR expression but through modulating the LXR activity. Meanwhile,
LXR agonist-induced expression of ABCA1 (ATP binding
cassette transporter A1), an LXR target gene, was only
slightly affected by PKA activation.

LXRE Response Element Is Responsible for PKA Inhibition of
SREBP-1c Promoter—To further investigate the PKA effect on
SREBP-1 transcription, in vitro reporter assays for SREBP-1c
promoter were performed. Luciferase reporters linked to different sizes of SREBP-1c promoter region around the LXRbinding sites (LXREs) and expression vectors of LXR␣ and the
PKA catalytic subunit for PKA activation were transfected into
HepG2 cells (Fig. 3A). PKA expression did not affect the basal
level of SREPB-1c promoter activity in HepG2 cells. Meanwhile, PKA co-expression strongly suppressed LXR␣-mediatedactivation of SREBP-1c promoter. This was only observed in
LXRE-containing promoters (⫺2.6 kilobase pairs and ⫺550bp). The shorter construct lacking LXREs (90 bp) did not show
LXR activation or PKA inhibition. Thus, the inhibitory effect of
PKA on SREBP-1c promoter was conceivably mediatedthrough LXREs. This was supported by the LXRE enhancer
construct assays, demonstrating that endogenous PKA activation by forskolin, an adenylate cyclase activator, dose-dependently inhibited LXRE enhancer activity induced by the LXR
ligand, and this inhibitory effect was abolished by co-expressionof the PKA regulatory subunit dominant negative form (Fig.
3B). The mechanism against the PKA suppression of LXR activity was further explored the using Gal4 DNA binding domain(DBD) protein fused to LXR␣ LBD in HepG2 (Fig. 3C). LXRLBD was highly activated in this system by the LXR synthetic
ligand as previously reported. This activation was completely
abolished by co-transfection of PKA, suggesting that PKA activation inhibits ligand-mediated activation of LXR. PKA-mediated inhibition of LXR transcriptional activity was supported byluciferase assays using the promoter of ABCA1, another LXR
target gene. Mouse ABCA1 promoter activity was also dose-dependently inhibited by PKA expression in both basal and LXRinduced conditions (Fig. 3D).

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FIGURE 1. Effects of PKA activation on expression of mouse hepatic
SREBP-1. C57BL6/J mice fasted for 24 h (Fasted) followed by refeeding with ahigh sucrose high fat diet for 12 h (Refed) were intravenously treated withvehicle or a combination of PKA activators, dbcAMP (35 mg/kg) and theophylline (30 mg/kg) (PKA). Two hours post-injection mice were sacrificed, andtotal liver RNA and nuclear extracts were prepared. Total RNA (10 ␮g) andnuclear extracts (40 ␮g of protein) were subjected to Northern (panel A)
and immunoblotting (panel B), respectively.

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(Fig. 4A). Functional domains of the
LXR␣ protein were also tested using
this in vitro kinase assay (Fig. 4B).
Every domain of LXR␣ was significantly phosphorylated. In the aminoacid sequence of PKA, there are twopotential PKA target sites (serines
195 and 196; threonine 290 and serine 291) in the LBD/heterodimerization domain. Introduction of
mutation in which these serine residues were replaced by alanine
caused resistance to PKA inhibitionof SREBP-1c promoter activation(Fig. 4C). To assess whether LXR␣ isphosphorylated by PKA in vivo,
COS7 cells were transfected withexpression vector for HA-tagged
LXR␣ and subsequently labeledwith [32P]orthophosphate. After
treatment with PKA activators, thephosphorylation state of LXR␣ was
examined by autoradiography. As
evident from autoradiography andimmunoblots (Fig. 4D), PKA stimulation induced the phosphorylation
state of LXR␣. These results indicate that PKA directly phosphorylates LXR protein, which is crucial
for PKA inhibition of LXR activity.
DNA Binding Activity of LXR/RXR Heterodimer Was Decreased by
PKA Phosphorylation of LXR␣—The effect of LXR␣ phosphorylation
by PKA on DNA binding was tested
by EMSA assays (Fig. 5A). Recombinant LXR␣ protein requires RXR
FIGURE 3. Effects of PKA on SREBP-1c promoter activity induced by LXR␣. A, HepG2 cells were transfected for its binding to LXRE. The signal
with SREBP-1c promoter Luc vector (300 ng/well), CMV Renilla luciferase vector as an internal reference (2.5 of LXR/RXR bound to LXRE was
ng/well), expression vectors for LXR␣ (100 ng/well), and PKA catalytic subunit (100 ng/well). After transfection, partially decreased by PKA treatthe cells were further incubated with the medium containing vehicle or T0901317 (T, 1 ␮M) for 20 h. kbp,
kilobase pairs. RLU, relative light units. B, HepG2 cells were transfected with LXRE enhancer Luc vector (200 ment of LXR. This effect required
ng/well), CMV Renilla luciferase vector (2.5 ng/well), and expression vectors for LXR␣ (100 ng/well) and PKA the incubation of LXR with PKA at
regulatory subunit dominant negative form (DN PKA) (300 ng/well). After transfection cells were incubated in
30 °C, indicating the PKA inhibimedium containing vehicle or T0901317 (1 ␮M) and forskolin (0, 0.1, 0.3, 1, 3 ␮M) for 20 h. Assays were performed as described under “Experimental Procedures.” C, HepG2 cells were transfected with expression vec- tion was mediated through its
tors for Gal4-DBD-LXR␣ ligand binding domain (150 ng/well), PKA Gal4 RE LUC vector (150 ng/well), PKA (3, 10, kinase activity. To confirm this30, 100, 200 ng/well), and CMV Renilla luciferase vector (2.5 ng/well). After each transfection cells were incubated in medium containing vehicle or T0901317 (1 ␮M)) for 20 h and subjected luciferase and Renilla reporter effect in vivo, nuclear extracts ofassays as in described under “Experimental Procedures.” D, 293 cells were transfected with human ABCA1 livers from dibutyryl-cAMP/theopromoter Luc vector (300 ng/well), expression vector for LXR␣ (100 ng/well), PKA (0, 3, 10, 30, 100 ng/well), and phylline-administrated mice were
CMV Renilla luciferase vector for internal reference (2.5 ng/well). After each transfection cells were incubated in
prepared and subjected to EMSA
medium containing vehicle or T0901317 (1 ␮M) for 20 h.
assay (Fig. 5B). PKA activation in
LXR␣ Is Phosphorylated by PKA in Vitro—These data led us these nuclear extracts was confirmed by phosphorylation ofto speculate that PKA modulates LXR activity, most likely CREB, a well known PKA target in mouse livers, as shown by
through direct phosphorylation of LXR␣. To test this hypoth- immunoblot analysis. EMSAs of these nuclear extracts demonesis, in vitro kinase assays of LXR␣ were performed. Purified strated that the signal of LXRE binding mainly from LXR/RXR,
recombinant protein of histidine-tagged LXR␣ was incubated as confirmed by supershift with LXR and RXR antibodies, waswith the PKA catalytic subunit. LXR␣ protein was phosphoryl- dose-dependently diminished by PKA administration. Next, toated by PKA as well as PKA autophosphorylation and histone determine whether PKA activation reduced DNA binding
H1 phosphorylation as positive controls of PKA target protein activity of LXR/RXR heterodimer, ChIP assay analysis on

PKA Inhibits LXR Signaling

extracts from H2.35 cells, a mouse hepatocyte-derived cell line,
was conducted. Anti-RXR antibody for immunoprecipitationof LXR/RXR heterodimer, and primers to detect LXRE on
SREBP-1c promoter for PCR were used. As shown in Fig. 5C,
the interaction of LXR/RXR heterodimer with LXRE wasdecreased upon PKA activation in vivo. These data suggest thatthe modification of LXR activity by PKA is attributed to reduction of DNA binding activity of LXR/RXR heterodimer via PKA
phosphorylation of LXR␣.

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FIGURE 5. Phosphorylation of LXR␣ by PKA decreases binding of LXR␣/RXR to LXRE. A, decreased binding of LXR␣/RXR to LXRE by PKA treatment
with LXR␣ in electrophoretic mobility shift assays. Increasing amounts ofrecombinant LXR␣ (rLXR␣, left panel) were incubated with bovine serum albumin (used as nonspecific protein control) or PKA catalytic subunit and thenincubated with radiolabeled LXRE probe and in vitro synthesized RXR for
EMSA as described under “Experimental Procedures.” In the right panel,
recombinant LXR␣ was preincubated with bovine serum albumin or PKA at
30 or 0 °C (on ice) (right panel) and then incubated with radiolabeled LXRE
probe and in vitro synthesized RXR for EMSA as described under “Experimental Procedures.” IVT, in vitro translated. B, in vivo PKA activation decreased the
DNA binding activity of LXR/RXR complex in EMSA (left panel). The various
doses of PKA activators (vehicle, 35 mg/kg dbcAMP ⫹ 30 mg/kg theophylline,
105 mg/kg dbcAMP ⫹ 30 mg/kg theophylline) were administrated to refed
C57BL6/J mice. Nuclear protein extracts prepared from livers were incubatedwith the 32P-labeled LXRE probe. The signal from LXR/RXR was confirmed bysupershift assays after the addition of LXR and RXR antibodies (ab) to the
incubations. Nuclear protein (30 ␮g/lane) was resolved by SDS-PAGE (10%gel) and subjected to immunoblotting with anti phospho (P)-CREB, antiCREB, and anti-LXR␣ antibodies (right panel). C, PKA activation decreases DNA
binding of LXR/RXR complex to LXRE on SREBP-1c promoter in vivo. H2.35
cells were treated with PKA activators (10 ␮M forskolin, 100 ␮M dibutyrylcAMP) or vehicle for 60 min. Because LXR antibody suitable for immunoprecipitation is not currently available (43), ChIP analysis was performed with
anti-RXR antibody or control IgG. Primers specific to the LXRE region of mouse
SREBP-1c promoter were used for PCR analysis. As a positive control (3%INPUT), the assay was performed with the same primers on the genomic DNA
on which the ChIP assays were performed.

Coactivator or Corepressor Recruitments of LXR␣ Are Reciprocally Modified by LXR␣ Phosphorylation by PKA—It wasreported that in LXR␣ or ␤-mediated transcription of SREBP1c; SRC-1 works as a coactivator, and NcoR1 works as a corepressor (33). To test the possibility of involvement of thesecofactors in PKA inhibition of LXR activity, mammalian twoVOLUME 282 • NUMBER 16 • APRIL 20, 2007

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FIGURE 4. Direct phosphorylation of recombinant LXR␣ (rLXR␣) protein
at the serine or threonine residues in the ligand binding domain is crucial for inhibition of LXR signaling by dbcAMP. A, in vitro kinase assay offull-length LXR␣. Histone H1 or recombinant histidine-tagged LXR␣ wereincubated with PKA and [32P]ATP and resolved by SDS-PAGE (8% gel). Phosphorylated protein was visualized by autoradiography. Phosphorylation ofhistone H1 and PKA itself was shown as a positive control of PKA target protein. B, in vitro kinase assay of several fragments of LXR␣. Amino acids 1–163,
162–326, and 325– 445, corresponding amino acids sequences of LXR␣ protein, respectively, were incubated with PKA. Samples were resolved by SDSPAGE (12% gel) and electrophoretically transferred to PVDF membranes.
Phosphorylated proteins were visualized by autoradiography. LXR␣ proteins
were estimated by immunoblotting with anti-histidine antibody. WB, Western blot. C, substitution of serine or threonine amino acid residues for alanine
in LXR␣ abolished the PKA-dependent repression of a luciferase reporter
derived by LXRE. Expression vectors for wild type LXR␣ (wt LXR␣) or an LXR␣mutant with S195A and S196A (195A196A) or with T290 and S290A (290A
291A) (100 ng/well) and CMV Renilla luciferase vector (2.5 ng/well) were
cotransfected with LXRE enhancer reporter plasmid (100 ng/well) into 293
cells. After transfection dbcAMP was added into the medium followed byincubation for 20 h. Luciferase activity was measured. Relative activity compared with each control was plotted. wt, wild type. D, PKA activation increases
phosphorylation of LXR␣ in vivo. COS7 cells transfected with HA-tagged LXR␣were metabolically labeled with [32P]orthophosphate and incubated for 30
min with PKA activators (10 ␮M, forskolin, 1 mM dibutyryl-cAMP, 1 mM isobutylmethylxanthine) before harvesting. Protein samples were immunoprecipitated (IP) with anti-HA antibody (ab). Labeled proteins resolved by SDS-PAGE
were autoradiographed and immunoblotted. [35S]Met is methionine-labeled
LXR␣, used for the positive control.

PKA Inhibits LXR Signaling

hybrid assays were preformed. The system detects the bindingof VP16-AD-LXR␣ fusion protein and Gal4-DBD-SRC-1-NID(a functional region that interacts with nuclear receptors (34))
in 293 cells. The addition of the LXR ligand markedly activatedthe signal, as evidenced by recruitment of SRC-1 to LXR. PKA
co-expression markedly and dose-dependently inhibited therecruitment of SRC-1 (Fig. 6A). Inversely, PKA co-expression
increased the recruitment of NcoR1-NID to AD-LXR␣ (Fig.
6B). Inhibition of the binding between LXR␣ and SRC-1 NID by
PKA was directly confirmed in pulldown assays. The labeled
SRC-1-NID was pulled down by GST-LXR␣, and the signal was
reduced by PKA phosphorylation (Fig. 6C). These data suggestthat PKA activation modifies LXR and enhances dissociation ofcoactivator and recruitment of corepressor, leading to repression of LXR transcriptional activity.
LXR␣/RXR Interaction Is Impaired by PKA—Finally, theeffect of PKA on heterodimerization of LXR/RXR was estimated by co-immunoprecipitation in vivo. Myc-tagged LXR␣and RXR were co-expressed in COS7 with or without PKA activation (Fig. 7A). RXR was co-immunoprecipitated with thetagged LXR␣. This binding between LXR␣ and RXR was nearly
eliminated by PKA activation. In contrast, PKA increased the
APRIL 20, 2007 • VOLUME 282 • NUMBER 16

co-immunoprecipitation of FLAG-tagged CBP and HA-tagged
CREB, well characterized targets of PKA (Fig. 7B). These data
demonstrated that the interaction of LXR␣ and RXR is
impaired by PKA activation, explaining at least partly PKA inhibition of LXR transactivation.
Contribution of RXR to PKA Inhibition of LXR/RXR Effect on
LXRE in SREBP-1c Promoter—In a previous report, RXR isshown as a substrate of PKA in vitro, and serine 27 is shown asessential for cAMP-mediated down-regulation of RXR transcriptional activation in COS cells by assessed in luciferase
assay (35). Therefore, the effect of PKA on RXR activity to regulate activity of SREBP-1c promoter was tested. HepG2 cells
were transfected with wild type RXR or mutant RXR (S27A). As
a result (Fig. 8), both wild type and mutant RXRs activate the
LXRE-luciferase reporter in response to LG268, a synthetic
RXR agonist, with a only slight (⬃25%) decrease in both basal
and ligand-stimulated response by the RXR mutation. PKA
activation dose-dependently inhibited this RXR-mediated
LXRE reporter. The RXR mutation modestly prevented thisnegative effect of PKA on RXR function especially at low dose
dbcAMP(10, 30 ␮M) but less at high dose dbcAMP(100 ␮M).
These results indicate that RXR phosphorylation is at least inpart involved in suppression of SREBP-1c promoter activity bycAMP/PKA signals.

DISCUSSION
Our current studies demonstrate that PKA directly phosphorylates LXR␣ protein and inhibits its signaling, resulting in supJOURNAL OF BIOLOGICAL CHEMISTRY

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FIGURE 6. PKA modulates recruitment of coactivator and corepressor for
LXR␣. A, inhibition of recruitment of SRC-1 to LXR␣ by PKA in the mammalian
two-hybrid system. 293 cells were transfected with Gal4 RE Luc vector, CMV
Renilla luciferase vector (2.5 ng/well), and expression vectors for VP-16-LXR␣
(amino acids 1– 445). Gal4-DBD-SRC-1, DBD of Gal4 fused to NID of SRC-1 andPKA. Four hours after transfection the cells were further incubated with medium
containing vehicle or T0901317 (1 ␮M) for 20 h. AD, activation domain. B, activation of recruitment of NcoR-1 to LXR␣ by PKA. 293 cells were transfected with
Gal4 RE Luc vector, SV40 Renilla luciferase vector (25 ng/well), and expression
vectors for VP-16 LXR␣ ((amino acid 1– 445), Gal4-DBD-NcoR1, and PKA. 4 h after
transfection the cells were further incubated with medium containing vehicle orT0901317 (1 ␮M) for 20 h. C, PKA treatment inhibited the interaction of LXR␣ with
SRC-1 in vitro. Recombinant GST-LXR␣ fusion protein was treated with PKA or
bovine serum albumin (used as nonspecific control protein) at 30 °C and thenincubated with 35S-radiolabeled SRC-1-NID synthesized in vitro and glutathioneSepharose beads. Pulled-down proteins were resolved by SDS-PAGE (12% gel) as
described under “Experimental Procedures.”

FIGURE 7. PKA prevents interaction between LXR␣ and RXR in vivo. A, PKA
inhibition of co-immunoprecipitation of LXR␣ and RXR. B, PKA activation of
co-immunoprecipitation of CREB and CBP. Cos7 cells were transfected withexpression vector for Myc-tagged LXR␣ and RXR (A) or for FLAG-tagged CBPand HA-tagged CREB (B). After incubation for 48 h, cells were treated withmedium containing PKA activators (100 ␮M dbcAMP, 10 ␮M forskolin, 100 ␮Misobutylmethylxanthine) for 30 min and harvested. Immunoprecipitation (IP)and immunoblotting (WB) with the indicated antibodies (ab) were performed
as described under “Experimental Procedures.” Proteins were extracted bylysis buffer and resolved by SDS-PAGE (8% gel for LXR and RXR, 4% gel for CBP,and 10% gel for CREB). There is different exposure time between lysate andimmunoprecipitation samples for detection of FLAG-tagged CBP because
intensity of protein amount of FLAG-tagged CBP in lysate was too strong.

PKA Inhibits LXR Signalingpression of SREBP-1c transcription both in vitro and in vivo.
Phosphorylation of LXR␣, presumably through its conformational change, causes two consequences; suppression of RXR
dimerization leading to decreased binding to LXRE and suppression of the ligand activation followed by decreased recruitment of coactivator SRC-1 and increased recruitment ofcorepressor NcoR1. Both events lead to repression of LXR
transactivation for SREBP-1c. PKA inhibition was more prominent for LBD activation of LXR than for its binding to LXRE.
Post-translational modification of transcription factors byphosphorylation provides a rapid cellular response to environmental changes. We now show that the ligand-induced transactivation of LXR can be regulated in vivo, ex vivo, and in vitro
by PKA-dependent phosphorylation. The catalytic subunit of
PKA has been identified in the nucleus and phosphorylates
numerous transcription factors, modulating their transcriptional activities positively such as hepatocyte nuclear factor-4␣
and negatively such as L-type PK (28). Now, LXR can be added
to the list of PKA-modulating factors. Although PKA-dependent phosphorylation sites (RX0 –2(S/T)) lie within all domains of
LXR, the 195–196 serines and 290 –291 threonine and serine
residues might be crucial for ligand-induced conformationalchange since these sites in human LXR␣ completely match themost preferable consensus sequence (R(R/K)X(S/T)). It is also
possible that phosphorylation states of these critical sites control other potential phosphorylation sites. LXR/RXR also transactivate other genes such as the ABCA1 (36) and the ABCG
family (37, 38). The LXR␣-induced activity of the ABCA1 promoter containing LXRE was also repressed by PKA similarly to11694 JOURNAL OF BIOLOGICAL CHEMISTRY

Acknowledgments—We are grateful to Alyssa H. Hasty for criticalreading of this manuscript and Drs. Tomoaki Morioka and Hidenori
Koyama for help in generation of expression plasmid of mutated(dominant negative) form of the regulatory type 1 subunit of cyclic
AMP-dependent protein kinase.

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FIGURE 8. PKA modifies RXR activity induced by RXR ligand on LXRE in
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APRIL 20, 2007 • VOLUME 282 • NUMBER 16

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