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Supplementary to
Involvement of Protein Kinase D in Phosphorylation andIncrease of DNA Binding of Activator Protein 2αααα to Down-
Regulate ATP-Binding Cassette Transporter A1Noriyuki Iwamoto, Sumiko Abe-Dohmae, Rui Lu, and Shinji Yokoyama
From Biochemistry, Nagoya City University Graduate School of Medical Sciences,
Kawasumi 1, Mizuho-cho, Mizuho-ku, Nagoya 467-8601, Japan
Materials and Methods
Reagents and Antibodies
Doxazosin mesylate, anti-β-actin antibody and rottlerin were purchased from Sigma.
Gö6976 and Gö6983 were obtained from Calbiochem. Mouse anti-AP2α antibody (SC-
25343), rabbit anti-PKD antibody (SC-639), nonimmune control mouse IgG (SC-2025),
and protein A/G agarose (SC-2003) were from Santa Cruz Biotechnology. Antibodies
against PKCα (2056), phospho-PKCα/βII substrate (Thr638/614) (9375), phospho-
(Ser) PKC substrate (2261), phospho-(Ser/Thr) PKA substrate (9261), phospho-
(Ser/Thr) PKD substrate (4381), phospho-MARCKS (Ser152/156) (2741), phospho-
PKD/PKCµ (Ser744/748)-PKD (2054) and PKD/PKCµ (2054) were from Cell
Signaling.
Cell Culture
THP-1 cells (Health Science Research Resources Bank, Han-nan, Japan) were cultured
in RPMI 1640 medium containing 10 % fetal bovine serum (FBS). The cells were
differentiated by 3.2 x 10-7 M PMA (Wako Pure Chemical) for 72 hours1. HEK293 cells
(Health Science Research Resources Bank) were cultured in DF medium (1:1 mixture of
Dulbecco's modified Eagle medium and Ham’s F12 medium) with 10 % FBS2. HepG2
cells (American Type Culture Collection) that lack AP2α protein expression were
cultured in minimum essential medium Eagle alpha modification with 10 % FBS. All
cultures were in a humidified atmosphere of 5 % CO2 and 95 % air at 37C°. NIH3T3-
L1 preadipocyte cells (American Type Culture Collection) were cultured in Dulbecco's
modified eagles medium with high glucose. The cells were allowed to grow for 2 days
to postconfluency and then differentiated to adipocytes in the same medium containing
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isobutylmethylxanthine (500 µM), dexamethasone (25 µM), and insulin (4 µg/ml) for
2 days and in the medium containing insulin for 3 additional days. The medium was
refreshed once every 3 days thereafter until the cells were fully differentiated.
Reverse Transcription Polymerase Chain Reaction (PCR)
Total RNA was isolated using ISOGEN (Nippon Gene) and reverse-transcribed by
SuperScriptIII (Invitrogen) with oligo-dT primers. Quantitative expression analysis was
performed in an ABI PRISM 7700 (Applied Biosystems) using SYBR Green technology.
The following primers were used in PCR: 5'-GCTTTCAATCATCCCCTGAA-3' and 5'-
CAGGTGTTTGCTTTGCTGA-3' for ABCA1; 5'-GACCTGCCGTCTAGAAAAACC-
3' and 5'-ACCACCTGGTGCTCAG-TGTAG-3' for glyceraldehydes-3-phosphate
dehydrogenase (GAPDH), forward and reverse, respectively. The results were
normalized for GAPDH.
Luciferase Reporter Gene Assay
To analyze promoter activity of ABCA1, HEK293 cells were subcultured in a 24-well
tray and incubated for 24 hours. The reporter luciferase plasmids (1 µg) and the phRL-
TK (Promega) encoding Renilla luciferase (for normalization; 50 ng) were transfected
by using Lipofectamine 2000. Expression plasmids of AP2α and its mutants or the
empty vector (mock), 250 ng per well, were co-transfected, and the cells were incubated
for 24 hours. Compounds were present for the last 16 hours, the cells were lysed, and the
luciferase activity was measured using the Dual-Luciferase Reporter system (Promega).
Western-Blotting
Cell pellets were harvested with sonication in RIPA buffer (Tris-HCl 50mM, pH 7.5,
NaCl 150 mM, EDTA5 mM, imidazol1 mM, NaF1 mM, Na3VO4, 1 mM containing
1 % NP40 and 0.1 % sodium deoxycholate) supplemented with protease inhibitor
cocktail (Sigma). Insoluble debris was removed by centrifugation at 12,000 x g for
2 min at 4 °C. The supernatant was collected and protein concentration was measured by
a BCA method (Pierce). Equal amounts of protein were separated by
sodiumdodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE), and transferred
to a polyvinylidene difluoride membrane for immunoblotting.
RNA Interference
Specific small interfering (si)RNA for human and mouse PKD and respective scramble
control were obtained from Invitrogen and transfected into THP-1 cells or NIH3T3-L1
adipocytes using Amaxa transfection machine (Amaxa Inc) according to the protocol
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provided by the manufacture. Immediately after transfection, THP-1 cells were
differentiated, and specific mRNA expression and proteins were analyzed.
In Vitro Protein Phosphorylation Assay
For analysis of PKA- and PKD-mediated phosphorylation of AP2α in the cells and in
the organs, AP2α was immunoprecipitated with an anti-AP2α antibody and was further
analyzed by immunoblotting, with anti-phospho-PKA-substrate antibody that
specifically recognizes phospholyrated Ser or Thr in the RRXS/T sequence, or by using
an antibody against phospho-PKD-substrate that recognizes phosphorylated Ser or Thr
in LXRXXS/T sequence. For detection of specific phosphorylation site, expression
vectors of wild-type and mutated AP2α with glutathione S-transferase (GST) at their N-
terminus were prepared by introducing the EcoRI/XhoI fragment from pSG-neo-AP2α
into pGEX-5X-1 vector (Amersham Biosciences). GST-AP2α fusion proteins were
induced in E. coli BL-21 DE3 (Nippon Gene) and purified from the cell pellet by
solubilization with 50 mM Tris-HCl containing 10 mM glutathione by glutathione-
Sepharose chromatography. One microgram of purified GST-AP2 α fusion proteins was
suspended in 30 µl kinase buffer (50 mM HEPES, pH 8.0, 10 mM MgCl2, 2.5 mM
EGTA, 0.1mM dithiothreitol, 1mM NaF, 0.1 mM Na3VO4), 185 kBq of [γ-32P]ATP, and
10 µM ATP (cold), and incubated with 10 ng of PKD catalytic subunit (Calbiochem) at
37 °C for 30 min. The kinase reaction was stopped by adding 6-volume of SDS-PAGE
sample buffer. Protein was resolved by electrophoresis in a 4 - 20 % gradient SDS-
PAGE gel followed by autoradiography of the dried gel.
Gel Shift Assay
Approximately 2 µg of purified GST-AP2α fusion protein was incubated with a double-
strand DNA fragment of the human ABCA1 promoter –279 to –317 that was labeled at
the 3' end with digoxigenin-11-ddUTP by using a DIG Gel Shift Kit (Roche Applied
Science) with or without 10 ng of PKD catalytic subunit. Electrophoresis bands were
visualized with an anti-digoxigenin antibody conjugated with alkaline phosphatase.
ChIP assay
ChIP assay was performed as described previously3, 4. The human ABCA1 promoter
region containing the AP2-binding site was amplified with specific primers: 5'-
CGGGAACGTGGACTAGAGAG-3' and 5'-TGGAGGGTACAGCAGGTGTC-3' 5.
Cellular Lipid Release Assay
THP-1 macrophage was incubated with 10 µg/mL apoA-I in RPMI 1640 medium
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containing 0.1 % bovine serum albumin for 16 hours, and cholesterol in the medium
were enzymatically measured1, 6.
In Vivo Experiments
Gö6983 1mg/kg per day was given to 8-week-old male C57BL/6 mice for 3 days by
using a gastric tube. Blood was collected by cardiac puncture under anesthesia. The
animals were euthanized by cervical dislocation, and the livers and abdominal adipose
tissues were immediately removed for analysis of mRNA and protein expression.
Plasma lipoprotein profile was analyzed by high performance liquid chromatography7 at
Skylight Biotech (Akita, Japan). Doxazosin, 25 mg/kg, was also given to the mice in the
same procedure and the liver was collected after sacrifice. PKD-mediated
phosphorylation of AAP2a was examined as described above for THP-1 cells. The
experimental protocol was pre-approved by the institutional committee for animal
welfare.
Other Methods
Intensity of photo-image was digitalized by scanning using an EPSON GT-X700 and
analyzed with Adobe Photoshop Software. Statistical analysis of the data was
performed by 1-way ANOVA followed by Scheffé's test. Values represent average ±
SD.
Results of Searching Empirical Selective PKD inhibitors
PKD phosphorylation by PMA was strongly inhibited by Gö6983 while the inhibition
was only moderate by Gö6976, rottlerin and doxazosin in HEK293 cells and THP-1
macrophages (Supplementary Figures IIAB) even when PKCα was strongly down-
regualted by PMA (data not shown). Finally, doxazosin reduced the PMA-induced PKD
phosphorylation (Supplementary Figures IIC). On the other hand, PKCα
phosphorylation was interfered by neither of the PKC inhibitors (400 nM) although
phosphorylation MARCKS, a PKCα substrate peptide, was inhibited by Gö6983
(Supplementary Figures IID). The effects of these inhibitors were analyzed for the
ABCA1-related parameters in THP-1 in macrophage (Supplementary Figure III).
Expression of ABCA1 gene was increased by the most prominent inhibitor of PKD
phosphorylation, Gö6983, and that of apoE gene, which is positively regulated by AP2,
was increased with the same treatment (Supplementary Figures IIIAB). The results were
parallel to the ABCA1 protein level and HDL biogenesis measured as cellular lipid
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release by apoA-I (Supplementary Figures IIICD). The results here were consistent with
those of the experiments of PKD knock-down by siRNA (Figure 4).
References
1. Arakawa R, Abe-Dohmae S, Asai M, Ito J, Yokoyama S. Involvement of
caveolin-1 in cholesterol-enrichment of HDL during its assembly by apolipoprotein and
THP-1 cells. J. Lipid Res. 2000; 41: 1952-1962.
2. Abe-Dohmae S, Ikeda Y, Matsuo M, Hayashi M, Okuhira K, Ueda K,
Yokoyama S. Human ABCA7 supports apolipoprotein-mediated release of cellular
cholesterol and phospholipid to generate high density lipoprotein. J. Biol. Chem. 2004;
279: 604-611.
3. Shang Y, Hu X, DiRenzo J, Lazar MA, Brown M. Cofactor dynamics and
sufficiency in estrogen receptor-regulated transcription. Cell 2000; 103: 843-52.
4. Iwamoto N, Abe-Dohmae S, Sato R, Yokoyama S. ABCA7 expression is
regulated by cellular cholesterol through the SREBP2 pathway and associated with
phagocytosis. J Lipid Res 2006; 47: 1915-27.
5. Iwamoto N, Abe-Dohmae S, Ayaori M, Tanaka N, Kusuhara M, Ohsuzu F,
Yokoyama S. ATP-binding cassette transporter A1 gene transcription is downregulated
by activator protein 2alpha. Doxazosin inhibits activator protein 2alpha and increases
high-density lipoprotein biogenesis independent of alpha1-adrenoceptor blockade. Circ.
Res. 2007; 101: 156-165.
6. Abe-Dohmae S, Suzuki S, Wada Y, Aburatani H, Vance DE, Yokoyama S.
Characterization of apolipoprotein-mediated HDL generation induced by cAMP in a
murine macrophage cell line. Biochemistry 2000; 39: 11092-9.
7. Okazaki M, Usui S, Ishigami M, Sakai N, Nakamura T, Matsuzawa Y,
Yamashita S. Identification of unique lipoprotein subclasses for visceral obesity by
component analysis of cholesterol profile in high-performance liquid chromatography.
Arterioscler. Thromb. Vasc. Biol. 2005; 25: 578-584.
8. Williams T, Tjian R. Characterization of a dimerization motif in AP-2 and its
function in heterologous DNA-binding proteins. Science 1991; 251: 1067-1071.
9. Eckert D, Buhl S, Weber S, Jager R, Schorle H. The AP-2 family of
transcription factors. Genome Biol. 2005; 6: 246.1-246.8.
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10. Garcia MA, Campillos M, Marina A, Valdivieso F, Vazquez J. Transcription
factor AP-2 activity is modulated by protein kinase A-mediated phosphorylation. FEBS
Lett. 1999; 444: 27-31.
11. Ikeda K, Maegawa H, Ugi S, Tao Y, Nishio Y, Tsukada S, Maeda S,
Kashiwagi A. Transcription factor activating enhancer-binding protein-2beta. A negative
regulator of adiponectin gene expression. J. Biol. Chem. 2006; 281: 31245-31253.
Legends for Supplementary Figures
Supplementary Figure I. Potential Ser-phosphorylation sites of AP2α by PKA and
PKD. A: A schematic diagram of the human structure of transcriptional factor AP2α.
The proline and glutamine rich domain (P/Q-rich domain) composed of 89 amino acid
residues is responsible for tarnsactivation. The helix-span-helix motif of 131 amino
acids mediates dimerization of AP2α, which is necessary for its DNA binding in the
presence of the basic domain of 20 amino acids 8, 9. Ser239 is known phosphorylated by
PKA and independent of the DNA binding 10. Ser258 and Ser326 position are both in the
same motif of LXRXXS that represents a putative phosphorylation site by PKD. B:
Ser239 is included in a PKA-phosphorylation motif of RRXS. Ser258 and Ser326 are
both in a putative PKD-phosphorylation motif of LXRXXS. Ser 258 is highly conserved
with respect to this motif among species but Ser326 is not completely conserved.
Supplementary Figure II. Effects of protein kinase inhibitors on PKD phosphorylation.
HEK293 cells (A, C, D) and THP-1 macrophages (B) were treated with Gö6976 (400
nM), Gö6983 (400 nM), rottlerin (Ro) (1 µM), doxazosin (Dx) (25 µM) and DMSO (C)
for 1 h. The cells were then stimulated with PMA 320 nM for 15 min or indicated times.
Phopshorylated PKD, total PKD (A, B, C) and phosphorylated PKCα, MARCKS, and
total PKCα (D) were analyzed by Western blotting using respective specific antibody.
Supplementary Figure III. Increase of ABCA1 mRNA, protein and activity by protein
kinase inhibitiors. A and B: THP-1 macrophages were incubated with protein kinase
inhibitors for 16 h and relative mRNA levels of ABCA1 (A) and apoE (B) were
measured. C: ABCA1 protein was analyzed by Western blotting for 50 µg protein of
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THP-1 macrophages. D: Effects of protein kinase inhibitors on HDL biogenesis in THP-
1 macrophages. The cells were treated with the protein kinase inhibitors for 16 h in the
presence of 10 µg apoA-I and release of cholesterol and phospholipid into the medium
was measured. Data represent average ± SD for triplicate assays. * p < 0.05, ** p < 0.01
and *** p < 0.001 against control.
Supplementary Figure IV. A model diagram for regulation of ABCA1 genes by the
PKD/AP2 signaling system. PKD phosphorylates AP2α Ser258 and increases its
binding activity to the ABCA1 promoter to down-regulate ABCA1 expression. Gö6983
and Doxazosin apparently reverse this process and consequently increase ABCA1
expression. The PKD/AP2 pathway may involve regulation of the adiponectin gene in a
similar manner to regulation of ABCA1 but via AP2β 11. Inhibition of PKD is apparently
related to suppression of SERBP1 expression and reduction of TG in plasma. At the
current stage of investigation, it is unclear yet whether doxazosin decreases PKD-
mediated AP2α phosphorylation or increases AP2α dephosphorylation.