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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

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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.