hABCF3, a TPD52L2 interacting partner, enhancesthe proliferation of human liver cancer cell lines in vitro

Juan Zhou • Ying Lin • Huili Shi • Keke Huo •

Yanhong Li

Received: 8 January 2013 / Accepted: 14 September 2013 / Published online: 20 September 2013

� Springer Science+Business Media Dordrecht 2013

Abstract In the present study, we characterized an evo-

lutionarily conserved non-transmembrane ATP-binding

cassette protein: hABCF3. Subcellular immunofluores-

cence staining demonstrated that hABCF3 localizes pref-

erentially in cytoplasm, unlike its paralog protein hABCF1,

which localizes in both cytoplasm and nucleus. Quantita-

tive realtime PCR analysis revealed that hABCF3 is

expressed in all tissues examined, with high expression

level in heart, liver, and pancreas. Interestingly, ectopic

hABCF3 promoted proliferation of human liver cancer cell

lines. Moreover, knock down of hABCF3 protein expres-

sion by siRNA inhibited cell proliferation. In addition, we

identified TPD52L2 (Tumor Protein D52-like 2) as a

hABCF3 interacting protein via yeast two-hybrid. This

interaction was further confirmed by in vivo co-immuno-

precipitation and co-localization assays. Furthermore, we

identified the interactional region of hABCF3 to be the first

200 amino acids uncharacterized region. Notably, the

truncated version of hABCF3, which lacks the TPD52L2

binding region, remarkably impaired hABCF3-mediated

cell proliferation. Taken together, these findings suggest

that hABCF3 positively regulates cell proliferation, at least

partially through the interaction with a tumor protein D52

protein family member: TPD52L2.

Keywords ABC transporters � ATP-binding

cassette (ABC) � Cell localization � Cell proliferation �hABCF3 � Yeast-two-hybrid (Y2H)

Introduction

The ATP-binding cassette (ABC) protein superfamily is

one of the largest and most highly conserved proteins

families, which contains transporters that bind ATP and use

the energy to drive the translocation of various molecules

across extracellular and intracellular membranes, including

metabolic products, lipid and drugs [1, 2]. There are seven

mammalian ABC gene subfamilies named ABCA to

ABCG [3, 4]. Typical ABC family proteins are comprised

of two transmembrane domains (TMD) and two nucleo-

tide-binding domains (NBD, or ABC), such as members in

the ABCA–ABCD and ABCG subfamilies. These proteins

are also called ABC transporters [5, 6]. The ABCE and

ABCF subfamilies are clearly derived from ABC trans-

porters, as they do not contain transmembrane domain and

are not known to be involved in any membrane transport

functions [7].

Recently, we systematically studied the protein expression

and interaction network in human liver samples [8]. We found

members of transmembrane ABC transporters were expressed

and interacting with other molecules to transfer metabolic

products, such as ABCA1, ABCB1, ABCB4, and ABCC2,

which are consistent with previous observations [9–12].

Besides the ABC transporters of the ABC superfamily, three

members of the ABCF (GCN20) subfamily, hABCF1, hAB-

CF2, and hABCF3, were expressed in human liver and par-

ticipated in the interaction network based on the proteomic

scale profiling [8]. hABCF1 (ABCF50) is the ortholog of the

yeast GCN20 in human; it contains two ABC domain in the C

Electronic supplementary material The online version of thisarticle (doi:10.1007/s11033-013-2679-z) contains supplementarymaterial, which is available to authorized users.

J. Zhou � Y. Lin � H. Shi � K. Huo (&) � Y. Li (&)

State Key Laboratory of Genetic Engineering, School of Life

Sciences, Institute of Genetics, Fudan University, 220 Handan Rd.,

Shanghai 200433, People’s Republic of China

e-mail: kkhuo@fudan.edu.cn

Y. Li

e-mail: lyhong@fudan.edu.cn

123

Mol Biol Rep (2013) 40:5759–5767

DOI 10.1007/s11033-013-2679-z

terminus, with an eIF2-interacting region in the N-terminus

[13]. hABCF1 and its yeast ortholog GCN20 play positive

roles in translation initiation [14]. hABCF2 and hABCF3 are

homologs of hABCF1. hABCF2 interacts with a-actinin-4 to

regulate cell volume and anion channels in human epithelial

cells [15]. Escherichia coli effector protein EspF can interact

with hABCF2 of the host to interfere with putative protective

function of hABCF2 [16]. The only one known binding

partner of hABCF3 is Oas1b, which confers resistance to

flavivirus-induced disease in mice [17]. The functions of

hABCF3 and its interacting partner(s) are still largely unclear.

In the present study, we report that hABCF3 localizes in

the cytoplasm, similar to its homologous protein hABCF2.

This is different from hABCF1, which is found in cyto-

plasm and nucleus. Interestingly, overexpressed hABCF3

enhances human liver cancer cell proliferation in vitro.

hABCF3 interacts with TPD52L2 via its N-terminal part.

The disruption of this interaction significantly decreases

cell growth. Our report here represents the first character-

ization of expression and localization of hABCF3, as well

as its cellular function and interaction partner.

Materials and methods

Plasmid construction

Human full-length ABCF3 gene was obtained by PCR from

human fetal liver cDNA library (Clontech), further cloned in-

frame into pEF-FLAG for expressing in mammalian cells,

pEGFP-C1 vector (Clontech) for cell localization assay, or

pDBLeu vector (Invitrogen) for yeast two-hybrid (Y2H)

assay. Various deletion version of hABCF3 were generated by

PCR, and then inserted into pDBLeu vector for Y2H analysis.

Immunofluorescence staining

Transfected Hep3B cells were washed with PBS and fixed

with 4 % paraformaldehyde (PFA, pH 7.4) in PBS for

10 min. After permeabilization with 0.5 % Triton X100 in

PBS for 10 min, cells were stained with an anti-FLAG mAb

(1: 500, Sigma), then Alexa Fluor 488-conjugated anti-

mouse IgG antibody (1:500, Molecular Probes), Slides were

embedded in an anti-fade aqueous mounting medium con-

taining 1 lg/ml DAPI (Sigma), then evaluated and photo-

graphed on an Olympus BX60 fluorescence microscope

equipped with an Olympus Olympus DP70 digital camera.

Quantitative realtime PCR analysis

Quantitative realtime PCR (qRT-PCR) was performed on

the ABI 7300 real-time PCR thermal cycler instrument

(Applied Biosystems) using SYBR Green realtime PCR

Master Mix (Toyobo, Japan). After an initial denaturation

at 95 �C for 1 min, amplification was performed with 40

cycles of 95 �C for 30 s, 60 �C for 30 s. For each sample,

reactions were set up in triplicate to ensure the reproduc-

ibility of the results. For expression profile of ABCF3,

human fetal and adult multiple-tissue cDNA (MTC) panels

(Clontech) were used as templates. The realtime PCR

primers are as following: hABCF3-FP: GGGGCATCA

GACACGCTCAC; hABCF3-RP: GTTGGGGCAGGGCA

TAGTCAT. b-actin was used as the internal control (FP:

CCTGGCACCCAGCACAAT and RP: GGGCCGGACTC

GTCATACT). Data were normalized as previously

described [18] prior to comparative analysis using 2-DDCt

method.

Cell proliferation analysis

Cell proliferation analysis was performed as described

previously [19]. Briefly, cells were plated in 96-well plates

at 2,000–4,000 cells per well and cultured in the growth

medium after transfection. At the indicated time points,

10 ll Cell Counting Kit-8 (CCK-8, Dojindo, Japan) solu-

tion was added to each well and incubated for 1 h, and the

cell numbers in triplicate wells were determined by reading

the OD at 450 nm. All experiments were repeated at least

three times with similar results. Error bars represent stan-

dard deviations from three independent experiments.

p values were compared between sample and control using

a Student’s t test.

RNA interference and RNA isolation

Synthetic siRNAs (GenePharma, Shanghai, China) were

delivered into Huh-7 cells by Lipofectamine 2000 reagent.

The nucleotide sequences of the siRNA were 50UUGAG

AACUUUGAUGUGUCdTdT30 (si-hABCF3-670), 50CCA

CAAAGCCAGGGAAAUAdTdT30 (si-hABCF3-362) and

50UUCUCCGAACGUGUCACGUdTdT30 (negative con-

trol). RNA was isolated from cultured cells using the Trizol

reagent (Invitrogen) according to manufacturer’s instruc-

tion. 5 lg RNA sample was treated with Dnase I (New

England Biolabs) and reverse transcribed by the M-MLV

reverse transcriptase (Promega) according to the manu-

facturer’s instructions.

Yeast two-hybrid screen

Two-hybrid screen was performed in the ProQuest two-

hybrid system (Invitrogen). pDBLeu-hABCF3 was used as

bait and was transformed into the yeast strain Mav203 to

generate stable BD-ABCF3 transformed yeast cells, then

these cells were transformed with the human liver library

(Invitrogen) to screen candidate interacting proteins. A

5760 Mol Biol Rep (2013) 40:5759–5767

123

total of 2 9 106 clones were screened on the selection agar

plates lacking histidine, leucine, and tryptophan (SC-HLT).

Positive clones were verified by b-galactosidase assay.

Plasmid DNA from His ?/Leu ?/LacZ ? colonies was

isolated and re-transformed into yeast along with either

pDBLeu-ABCF3 or other nonspecific bait plasmids to

verify the specific interaction. For the X-Gal filter assay,

colonies were lifted onto supported nitrocellulose filters

and then cracked open by freezing the filters in liquid

nitrogen three times. The thawed filters then were placed

on 3MM paper (Whatman) soaked in Z buffer (60 mM

Na2HPO4, 40 mM NaH2PO4, 10 mM KCl, 1 mM MgSO4,

pH 7.0) and 1.5 mg/ml X-Gal. The filters were incubated at

room temperature, and the appearance of blue color was

monitored over 3 h.

Cell culture and transfection

HEK293T (human embryonic kidney) cells were obtained

from American Type Culture Collection (ATCC) and cul-

tured in Dulbecco’s Modified Eagle Medium (DMEM)

supplemented with 10 % fetal bovine serum (FBS) in 5 %

CO2 at 37 �C. Plasmids were introduced into cells using

Lipofectamine reagent or Lipofectin 2000 (Invitrogen)

according to the manufacturer’s recommendation, and cells

were harvested at 48 h post-transfection.

MCF7, MDA-MB-231 and SK-BR-3 are different

human breast adenocarcinoma cell lines. SW480, SW620

and LoVo are human colon adenocarcinoma cell lines.

A549 and H1299 are human lung carcinoma cell lines.

SGC-7901 and BGC-823 are human gastric cancer cell

lines. Hep3B, HepG2, Huh-7, HCC-LM3 and HCC-LM6

are human hepatoma cell lines. All these cells cultured in

DMEM media with high glucose as HEK293T cells.

Co-immunoprecipitation and immunoblot assay

For immunoprecipitation, harvested cells were washed

with ice cold PBS and lysed in cell lysis buffer (150 mM

NaCl, 50 mM Tris–HCl pH 7.5, 1 % Nonidet P-40, 1 mM

DTT, 5 mM EDTA pH 8.0, protease inhibitors from

Sigma). Cell lysates were immunoprecipitated with 2 lg

monoclonal anti-HA (mouse IgG) covalently linked aga-

rose (Sigma) at 4 �C for 6 h. The bead-conjugated com-

plexes were washed with cell lysis buffer three times,

boiled in SDS sample buffer (0.125 M Tris–HCl at pH 6.8,

0.5 % SDS, 10 % glycerol) and subjected to SDS–PAGE

and immunoblot analysis.

After the SDS–PAGE separation, protein samples were

transferred to PVDF membranes (Millipore). After blocked

with 5 % non-fat milk in PBS, the membranes were incu-

bated with primary antibodies at 4 �C overnight, followed

by incubation of horseradish peroxidase-conjugated

secondary antibody. Immunoreactivity was visualized by

enhanced chemiluminescence (Millipore). Specific anti-

bodies we used included: mouse anti-Myc antibody (Santa

Cruz); mouse anti-FLAG (Sigma), rat anti-HA antibody

(Roche), and mouse anti-GAPDH antibody (Kangcheng,

Shanghai, China).

Laser-scanning confocal microscopy

Hep3B cells were treated essentially the same way as

described previously [20], except that the hABCF3-GFP

and TPD52L2-myc were co-transfected into the cells.

Mouse anti-Myc antibody (1:100, Santa Cruz) was

employed. The slides were analyzed using a laser-scanning

confocal microscope (LSM510 META, Zeiss) equipped

with a Zeiss AxioCam HR cooled CCD camera and a 409

oil immersion objective.

Results

hABCF3, a non-transmembrane cytoplasmic protein, is

expressed in various human tissues

Based on our recent liver proteomic profiling, ABC protein

subfamily members has expressed and participated in the

protein interaction network [8]. Human ABCF proteins are

highly evolutionarily conserved from yeast to human [21,

22]. Owing to the lack of the transmembrane domain, the

hABCF1, hABCF2, and hABCF3 cannot play a role as the

ABC transporter in liver for the lipid transportation

(Fig. 1a). Past studies have established hABCF1 as a factor

in protein translation initiation, which is similar to its yeast

ortholog GCN20 [14]. In the light of these past findings, we

are interested to know what are the functions and the

mechanisms in liver of its two paralogs, hABCF2 and

hABCF3. To investigate whether hABCF2 and hABCF3

are localized in cytoplasm as canonical non-transmem-

brane ABC proteins as the PSORT II (http://psort.nibb.ac.

jp/form2.html) and SOSUI (http://bp.nuap.nagoya-u.ac.jp/

sosui/) predictions, we transfected Hep3B cells with

hABCF2 and hABCF3 plasmids. As shown in Fig. 1b,

unlike the nuclear and cytoplasmic localization of hAB-

CF1, hABCF2 and hABCF3 exclusively localized in

cytoplasm for the lacking the nuclear localization sequence

in the N-terminal region (NTR). The possible nuclear

localization signal sequences of hABCF1 are: 73KKKR

and 82RRKK [22].

To examine if hABCF2 and hABCF3 are expressed

exclusively in liver tissue, we test the Clontech multi-tissue

cDNA samples by realtime PCR. As a result, hABCF3

mRNA showed high expression in heart and pancreas, in

addition to liver (Fig. 1c). Moderate expression was

Mol Biol Rep (2013) 40:5759–5767 5761

123

observed in brain, placenta, and kidney. Meanwhile, weak

signals were observed in lung and skeletal muscle. hAB-

CF2 is also expressed in multiple tissues as hABCF3 [21,

23]. These results suggest that the biological functions of

hABCF2 and hABCF3 may are different from their paralog

hABCF1 and most of the ABC family members, which

play important roles in substrate transport.

hABCF3 promotes cell proliferation in human cancer

cell lines

Since the non-transmembrane protein ABCE1 of the ABC

protein superfamily has shown the ability to proliferate the

small cell lung cancer cell line and promote the develop-

ment and progress of human lung adenocarcinoma [24, 25],

we wondered what cellular role hABCF2 and hABCF3

play. Thus, we measured the cellular proliferation of

Hep3B cells transfected with hABCF3. CCK-8 cell

counting kit was employed to determine cell proliferation.

As shown in Fig. 2a, the over-expressed hABCF3 pro-

motes the cellular proliferation of Hep3B cells, as com-

pared with vector-transfected control and hABCF2-

transfected cells (p \ 0.05). We found that hABCF3 plays

a similar function in HepG2 cells (Supplementary Fig. 1).

To further confirm the ability of hABCF3 to promote

cell proliferation, we employed the synthesized small

interference RNA (siRNA) to knockdown the endogenous

hABCF3 in cells where it highly expressed. As shown in

Fig. 2b, hABCF3 expressed in various human cancer cell

lines, especially in liver cancer cell line Huh-7. Therefore,

we transfected Huh-7 cells with si-hABCF3, and measured

the proliferation of these cells. As a result, siRNA against

hABCF3 can significantly inhibit the cell proliferation,

compared with the control (transfected with scrambled

siRNA) (Fig. 2c). Similar effect of hABCF3 on cell pro-

liferation was also achieved in human liver cancer cell line

Hep3B (data not shown). These observations indicate that

hABCF2 and hABCF3 have different biological function in

cells. By comparison of unobvious influence of hABCF2

on cell growth, hABCF3 enhances cell proliferation

significantly.

Identification of the interacting partner of hABCF3:

TPD52L2

To further investigate the molecular mechanism of hAB-

CF3 function on cell proliferation, we carried out the yeast

two-hybrid assay (Y2H) to identify the interacting partners

of hABCF3 in human liver cDNA Y2H library. Sequence

analysis showed that among twenty positive preys, four

encode the C-terminal portion of TPD52L2. To confirm the

interaction in yeast, the full length TPD52L2 was cloned

A

ABC

eIF2

NTR ABC 807

hABCF1

DAPI Merge

Hep3B

ABCNTR ABC 752

ABC ABC 709

ABC ABC 634

B

C

0

25

place

nta

rela

tive

hAB

CF3

mR

NA

leve

l

5

10

15

20

kidne

y

muscle

panc

reaslun

gbr

ainhe

art

liver

hABCF1

hAB

CF1

hAB

CF2

hAB

CF3

hABCF2

hABCF3

scGCN20

hABCF3

DAPI Merge

Hep3B

hABCF2

DAPI Merge

Hep3B

655hABCG2 ABC TM

Fig. 1 ABCF3 is a ubiquitously

expressed cytoplasmic protein.

a Schematic map of the ABCF

subfamily members. hABCF1:

human ABCF1, scGCN20: S.

cerevisiae GCN20. ABC: ATP-

binding cassette domain. NTR:

N-terminal region (eIF2 binding

region). b Subcellular

localization of ABCF subfamily

members in human liver cancer

cell line Hep3B. Bar = 5 lm.

c Quantitative realtime PCR

analysis of expression pattern of

hABCF3 in multiple human

tissues using SYBR Green

method. b-actin was used as an

internal control

5762 Mol Biol Rep (2013) 40:5759–5767

123

into pPC86 and co-transformed with pDBLeu-hABCF3

into the yeast strain MaV203. As shown in Fig. 3a, the

reporter LacZ gene is activated in the hABCF3 and

TPD52L2 co-transformed yeast cells, compared to cells

with only hABCF3or TPD52L2 alone. Y2H controls serve

as references to indicate the strength of protein interaction

from negative (a) to strongly positive (e) (see Fig. 3a lower

panel).

To verify the interaction in mammalian cells, immuno-

fluorescence microscopy was utilized to visualize the

subcellular localization of the two proteins in Hep3B cells.

We found that most of TPD52L2 is localized in cell

cytoplasm region surrounding the cell membrane, as

indicated by the Myc–tag protein. Meanwhile, hABCF3

exists evenly in cell cytoplasm, as shown by GFP-labeled

green fluorescence (Fig. 3b). Importantly, significant co-

localization of TPD52L2 and hABCF3 can be easily

viewed in the region near cell membrane, suggesting that

the interaction of TPD52L2 and hABCF3 is physiologi-

cally relevant.

To further confirm the interaction of TPD52L2 and

hABCF3 in mammalian cells, HA-tagged TPD52L2 and

FLAG-tagged hABCF3 were co-transfected into HEK293T

cells to examine that if TPD52L2 and hABCF3 can be co-

immunoprecipitated with specific antibodies. As shown in

Fig. 3c, FLAG-tagged hABCF3 was co-precipitated with

A

B

C

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1Day

Day

cell

viab

ility

(A

450

nm)

2 3 4 5

Vec

hABCF2-FLAG

hABCF3-FLAG

0

10

20

30

40

50

60

70

80

90

MCF7

MDA-MB-2

31

SK-BR-3

SW48

0

SW62

0Lo

Vo A

549

H1299

SGC-790

1

BGC-823

Hep3B

HepG2

Huh-7

HCC-LM3

HCC-LM6

HEK293T

Vec hABCF2-

FLAG

hABCF3-

FLAG

rela

tive

hAB

CF3

mR

NA

leve

l

rela

tive

hAB

CF3

mR

NA

leve

l

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

1 2 3 4 5 6

Ctrl-siRNAsi-hABCF3-670si-hABCF3-362 Ctrl-siRNA

IB: FLAG

IB: GAPDH

si-hABCF3-670si-hABCF3-362

0123456789

10

cell

viab

ility

(A

450

nm)

Hep3B *

*

*

*

*

** *

**

**

*

**

Fig. 2 hABCF3 promotes cell

proliferation in human cancer

cell lines. a Growth curves of

ABCF3-transfected Hep3B

cells. Cellular proliferation was

measured by using the Cell

Counting Kit (CCK-8). Empty

vector was also transfected in

cells and used as control. The

right panel shows the Western

blot of the transfected cells.

GAPDH was used as internal

control. The antibody against

FLAG or GAPDH was used in a

dilution of 1:3000 and 1:1000,

respectively. Error bars

represent standard deviations

from three independent

experiments. p values were

compared between hABCF3

and empty vector control using

a Student’s t test. * p \ 0.05;

** p \ 0.01. b The expression

pattern of ABCF3 in human

cancer cell lines. Quantitative

realtime RT–PCR analysis of

hABCF3 on 17 different human

cancer cell lines. c Growth

curves of si-hABCF3

transfected Huh-7 cells. Cellular

proliferation was measured by

using CCK-8. The right panel

shows the knockdown of

hABCF3 using siRNA. The

scrambled siRNA was used as

control. These values were

obtained from triplicate

experiments and are indicated as

means and standard deviation.

Error bars represent standard

deviations from three

independent experiments.

p values were compared

between si-hABCF3 and control

siRNA using a Student’s t test.

* p \ 0.05; ** p \ 0.01

Mol Biol Rep (2013) 40:5759–5767 5763

123

HA-tagged TPD52L2, while the control vector did not.

These results suggest that that TPD52L2 and hABCF3

interact in vivo.

Identification of the domain of hABCF3 involved

in the TPD52L2 and hABCF3 interaction

After having shown that TPD52L2 can bind hABCF3 in

Y2H and in human cells, we next asked which region or

domain of the hABCF3 is involved in their interaction.

Since hABCF3 contains two separate ATP-binding cassette

domain ABC, we constructed five truncation mutants of

hABCF3, labeled hABCF3-1 to hABCF3-5 (Fig. 4a).

Notably, all of the truncated versions of hABCF3 with

N-terminal region (amino acid 1–200) retain the binding

activity with TPD52L2, suggesting that the N-terminal of

hABCF3 is responsible for this interaction between

TP52L2 and hABCF3 in the yeast two-hybrid assay

(Fig. 4b).

Inasmuch as the full-length hABCF3 enhance cell pro-

liferation significantly and the N-terminal 200 amino acids

(aa) is involved the interaction with TPD52L2, we tried to

examine whether hABCF3 without the N-terminal 200 aa

(e.g. hABCF3-1) still promote cell growth to the same extent

A

B C

hABCF3-Y2H

Y2H (controls)

hABCF3TPD52L2

hABCF3AD

a b c d e

BDTPD52L2

DAPI

TPD52L2-Myc

hABCF3-GFP

Merge

Vec TPD52L2-HA

hABCF3-FLAG

IB: FLAG

TPD52L2-HA

IB: HA

IP: H

A

IN IP IN IP

hABCF3-FLAG

Fig. 3 The interaction between ABCF3 and TPD52L2. a Yeast two-

hybrid LacZ reporter gene assay for interaction between hABCF3 and

TPD52L2. Yeast strain MaV203 was co-transformed with pDBLeu-

hABCF3/pPC86-TPD52L2, pDBLeu- hABCF3/pPC86 (AD) or pPC86-

TPD52L2L/pDBLeu (BD). Blue color indicates positive interactions.

White color indicates no direct interaction. The lower panel is MaV203

yeast two-hybrid controls with different degrees of protein–protein

interaction. Controls (a) to (e) represent the Y2H result from negative to

strongly positive. b colocalization analysis of hABCF3 and TPD52L2 in

Hep3B cells. Hep3B cells were co-transfected with TPD52L2-Myc and

hABCF3-GFP plasmids. Immunofluorescence staining was performed

with an anti-c-Myc antibody. Colocalized positions of hABCF3 and

TPD52L2 were marked with arrow. These images were visualized by a

confocal laser-scanning microscope. Bar = 5 lm. c co-IP confirmation

of interaction between hABCF3 and TPD52L2 in HEK293T cells.

hABCF3-FLAG and TPD52L2-HA plasmids were co-transfected into

HEK293T cells; Empty FLAG-tagged vector and TPD52L2-HA were

co-transfected as control. Lysate was immunoprecipitated with anti-HA

agarose (mouse monoclonal antibody-linked agarose), and then analyzed

by immunoblotting with mouse anti-FLAG antibody (1:3000) or rat anti-

HA antibody (1: 3000). IN: Input, 1 % entire lysate. IP: immunoprecip-

itation. (Color figure online)

5764 Mol Biol Rep (2013) 40:5759–5767

123

of full-length hABCF3. Interestingly, as shown in Fig. 4c,

hABCF3-1 slightly enhanced cell growth, compared to the

vector control. In contrast, full-length hABCF3 promoted

cell proliferation significantly, suggesting that deleting the

N-terminal 200 amino acids substantially impaired the

capability of hABCF3 to enhance cell proliferation.

Discussion

In the present study, we characterized a non-transmem-

brane ABC protein, hABCF3. The localization of hABCF3

and its paralog hABCF2 is similar, compared to the dif-

ferent localization of hABCF1. The protein domain com-

position of hABCF3 and hABCF2 is very similar: two

consecutive ABC domains in the C-terminus with an un-

charactized N-terminal region (Fig. 1a). In addition, they

share very high sequence homology (46 % positive, 33 %

identical) (Fig. 4d). Although hABCF2 and hABCF3 have

similar sequences and localization pattern, their biological

functions seem to be different. hABCF2 interacts with two

partners, EspF and ACTN4, to play protective roles in cells

[15, 16]. Previous researchers have reported that hABCF3

interacts with Oas1b to confer resistance to flavivirus-

A

C

B

D

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1 2 3 4 5 6

hABCF3

hABCF3-1

Vec

ABC ABC 709hABCF3

Y2H

hABCF3-1 ABC ABC 709201

1

ABC ABC 683hABCF3-5 1

ABC 709hABCF3-4 1

ABC ABC 709hABCF3-3

hABCF3hABCF2

1

1hABCF3-2

hABCF3

hABCF3-1

hABCF3-5

hABCF3-4

hABCF3-3

hABCF3-2

ABC 709201 401

401 517

517 683

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KDDAGIRAVCQRMYNTLRLAEPQSQGNSQVLLDAPIQLSKITENYDCGTKLPGLLPSDLAKKKAAKKKEAAKARQRPR-KGHE--------------ENGDVVTEPQVAE

KREQSSTVNAKKLEKAEARLKAKQEKRSEKDTLKTSNPLVLEEASASQAGSRKESKNE------ANGRETTEVDLLTK----------------ELEDFEMKKAAARAVT

RLESSGKNKSYDVRIENFDVSFGDRVLLAGADVNLAWGRRYGLVGRNGLGKTTLLGVLASHPN-STDVHIINLSLTFHGQELLSDTKLELNSGRRYGLIGLNGIGKSMLL

KMLATRSLRVPAHISLLHVEQEVAGDDTPALQSVLESDSVREDLLRRERELTAQISAIGKREVPIPEHIDIYHLTREMPPSDKTPLHCVMEVDTERAMLEK-------EA

AAGRAEGSEAAELAEIYAKLEEIEADKAPARASVILAGLGFTPKMQQQPTREFSGERLAHEDAECEKLMELYERLEELDADKAEMRASRILHGLGFTPAMQRKKLKDFSG

GWRMRLALARALFARPDLLLLDEPTNMLDVRAILWLENYLQTWPSTILVVSHDRNGWRMRVALARALFIRPFMLLLDEPTNHLDLDACVWLEEELKTFKRILVLVSHSQD

FLNAIATDIIHLHSQRLDGYRGDFETFIKSKQERLLNQQREYEAQQQYRQHIQVFFLNGVCTNIIHMHNKKLKYYTGNYDQYVKTRLELEENQMKRFHWEQDQIAHMKNY

IDRFRYN-ANRASQVQSKLKMLEKLPELKPVDKES--EVVMKFPDGFEKFSPPILIARFGHGSAKLARQAQSKEKTLQKMMASGLTERVVSDKTLSFYFPPCGKIPPPVI

QLDEVDFYYDPK-HVIFSRLSVSADLESRICVVGENGAGKSTMLKLLLGDLAPVRMVQNVSFKYTKDGPCIYNNLEFGIDLDTRVALVGPNGAGKSTLLKLLTGELLPTD

GIRHAHRNLKIGYFSQHHVEQLDLNVSAVELLARKFPGRPE-EEYRHQLGRYGISGMIRKHSHVKIGRYHQHLQEQLDLDLSPLEYMMKCYPEIKEKEEMRKIIGRYGLT

GELAMRPLASLSGGQKSRVAFAQMTMPCPNFYILDEPTNHLDMETIEALGRALNNGKQQVSPIRNLSDGQKCRVCLAWLAWQNPHMLFLDEPTNHLDIETIDALADAINE

FRGGVILVSHDERFIRLVCRELWVCEGGGVTRVEGGFDQYRALLQEQFRRE----FEGGMMLVSHDFRLIQQVAQEIWVCEKQTITKWPGDILAYKEHLKSKLVDEEPQL

---------G---FL---TKRTHNVCTLTLASLPRP

Bind to TPD52L2

_+

+

+++

hABCF3-

FLAG

hABCF3-

1-FLA

G

Vec

IB: FLAG

IB: GAPDH

cell

viab

ility

(A

450

nm)

AB

CA

BC

**

**

*

*

*

*

*

Fig. 4 Identification of domain of hABCF3 involved in the TPD52L2

and hABCF3 interaction. a Schematic map of the truncations of

hABCF3. All deletion regions are marked in the map. hABCF3-BD

and five hABCF3 deletion mutants were co-transformed with

TPD52L2-AD into yeast strain MaV203 separately. b b-galactosidase

reporter gene assay was utilized to test the interactional region. Blue

color indicates positive interactions. White color indicates no

detectable interaction. c Growth curves of hABCF3-1 or full-length

hABCF3 transfected Hep3B cells. Cellular proliferation was mea-

sured by using CCK-8. The right panel shows the Western blot of the

transfected cells. GAPDH was used as an internal control. These

values were obtained from triplicate experiments and are indicated as

means and standard deviation. The antibody against FLAG or

GAPDH was used in a dilution of 1:3000 and 1:1000, respectively.

Error bars represent standard deviations from three independent

experiments. p values were compared between wild-type or mutant

hABCF3 and empty vector control using a Student’s t test.

* p \ 0.05; ** p \ 0.01. d Alignment of hABCF3 and hABCF2.

Red Box: ABC domain. (Color figure online)

Mol Biol Rep (2013) 40:5759–5767 5765

123

induced disease in mice [17]. All these researches dem-

onstrated that hABCF2 and hABCF3 involve in cell pro-

tection. Here, we shown that hABCF3 could enhance

cancer cell proliferation when it exogenously expressed.

The observations in this work indicated that hABCF3 is a

new non-transmembrane ABC protein that plays a role in

promoting cell growth, like the recently identified protein

hABCE1 [24–26].

TPD52L2 (tumor protein D52–like 2) is a member of

TPD52 family. It is comprised of four members, of which

three (TPD52 or D52, TPD52L1 or D53, and TPD52L2 or

D54) are indicated to be widely expressed [27]. TPD52

family proteins are emerging as playing a role in the

development of solid tumors [28–31]. TPD52L2 and its

family members form homomeric and heteromeric inter-

actions to play a role in cell proliferation [32, 33]. The

present study demonstrated that hABCF3 interacts with

TPD52L2 via the N-terminal 200 aa of hABCF3. Disrup-

tion of the interaction between hABCF3 and TPD52L2

significantly impaired the ability of hABCF3 to enhance

cell proliferation. The major difference of primary

sequences between hABCF3 and hABCF2 is also localized

in the first 200 aa of hABCF3, which is consistent with our

observation that hABCF3, not its near paralog hABCF2,

promotes cell growth. We also observed that the truncated

hABCF3, without the first 200 aa, still has limited ability to

slightly enhance cell growth, compared with the vector

control. This result suggests that besides the N-terminal

TPD52L2 binding region, there are other region(s) of

hABCF3 also contribute to the ability of cell growth pro-

motion. To fully elucidate the molecular mechanisms of

the role of hABCF3 on cell proliferation, it is necessary to

further identify other interacting partner(s) and their spe-

cific binding region.

In summary, our study has identified that hABCF3

enhances cell proliferation, and provided one possible

molecular mechanism of how TP52L2 mediates the pro-

motion of cell proliferation. We proposed that the first 200

amino acids of hABCF3 are involved in the interaction

between hABCF3 and TPD52L22, at least partially by

which binding interface hABCF3 enhances human cell

proliferation.

Acknowledgments This work is supported by grants from the

Chinese Natural Science Foundation (30800174) and Science Foun-

dation for Young Scientists of Fudan University to Y. Li, and the

National Key Basic Research Program of China (2010CB912603,

2013CB531603), National Special Key Project of China

(2008ZX10003-006, 2009ZX09301-011) to K. Huo. We are grateful

to Dr. David Shultis and Shirley Lee for proofreading the manuscript

and critical discussions. We are also grateful to Dr. Bingbing Wan for

providing technical assistance.

Conflict of interest The authors have declared that no conflict of

interest exists.

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