|
Short Communication |
Identification
of Interaction between PAI-2 and IRF-3
ZHANG Yu-Qing, LI Ping, HOU Min,
WANG Xia, FAN Jing, TAN Li, ZHU Yun-Song*
(Department
of Molecular Genetics, Shanghai Medical School of Fudan University; Key
Laboratory of Molecular Medicine,
Ministry of Education, Shanghai 200032, China)
Abstract HeLa
cells transfected with plasminogen activator inhibitor-2 ( PAI-2 ) were protected
from TNF-α-induced apoptosis. The apoptosis protection by PAI-2 is dependent on
a 33 amino acids fragment between helix C and D of PAI-2, which may be due to
the interaction of PAI-2 with some intracellular proteins. In this study, the
yeast two-hybrid system was used to screen a HeLa cells cDNA library
constructed during apoptosis with the fragment between helix C and D of PAI-2
as bait. We retrieved a clone that encodes 98 amino acids of C-terminus of
interferon regulatory factor-3 (IRF-3). Co-immunoprecipitation experiments
confirmed the interaction between PAI-2 and IRF-3 in vivo. IRF-3 belongs to a
family of the IRF transcription factors and has been shown to participate in a
large number of biological processes. These data suggest that IRF-3 may be
involved in the apoptosis protection and antiviral function of PAI-2.
Key words
plasminogen
activator inhibitor type-2 ( PAI-2 ); interferon regulatory factor-3 (IRF-3 );
yeast two-hybrid system; co-immunoprecipitation
Plasminogen
activator inhibitor type-2(PAI-2), a member of the serine protease inhibitor
(serpin) super family, is a multifunctional protein which is involved in the
regulation of fibrinolysis, invasion and metastasis of cancer cells, and in
regulation of apoptosis. HeLa cells transfected with PAI-2 were protected from
TNF-α-induced apoptosis. It is known that the antiapoptotic activity of PAI-2
is dependent on a 33 amino acids fragment between helix C and D of PAI-2 and
this may be due to the interaction of PAI-2 with some unknown intracellular
proteins[1, 2]. Here a yeast two-hybrid system was applied to explore which
proteins could interact with PAI-2. We used the fragment between helix C and D
of PAI-2 as bait to screen a HeLa cells cDNA library constructed during
apoptosis and retrieved a clone that encodes 98 amino acids of C-terminus of
interferon regulatory factor-3 (IRF-3). Using RT-PCR, we got the full-length
cDNA of IRF-3. Co-immunoprecipitation experiments confirmed the interaction
between PAI-2 and IRF-3 in vivo.
IRF-3 belongs to
a family of the IRF transcription factors and has been shown to participate in
a large number of biological processes including the regulation of cell
proliferation, hematopoietic, development, antiviral defense, and response to
DNA damage. These data suggest that IRF-3 may also be involved in the apoptosis
protection and antiviral function of PAI-2.
1 Materials and Methods
1.1 Reagents
The yeast
two-hybrid system was purchased from Clontech, Inc.. Trizol reagent was
purchased from Invitrogen, Inc.. Antibodies to PAI-2 and the HA epitope were
purchased from Santa Cruz Biotech, Inc.. A HeLa cells cDNA library was
constructed during apoptosis by us and amplified according to the
manufacturer’s instructions. G418 was product of Gibco BRL Life Tech. Protein-G-agarose
was from Roche Inc.. Kit for purification of plasmid DNA was from Watson
Biotech (Shanghai). Primers were synthesized by Ji Kang Biotech (Shanghai). ECL
kit was from Amersham Pharmacia Biotech.
1.2 Plasmid constructs
The BD vector pAS2-1NE
used for the yeast two-hybrid system was a gift from Dr. TIAN Yu (Harvard
University). To obtain the interhelical region of C and D of PAI-2 as the bait
to screen a HeLa cells cDNA described above, the helix C and D of PAI-2 was
created by PCR. The primers were A1 (NheI): 5′-AAAGCTAGCATGGCCAAGGTGCTTCAG-3′
and A2 （EagI），
5′-AAACGGCCGGGATGAATGGATTTTA-TC-3′. The PCR condition was as follow: 80 s at 94
℃, 60 s at 58 ℃, 40 s at 72 ℃, 25 cycles. After digested by NheI and EagI, the
fragment was inserted in frame into pAS2-1NE excised by the same two enzymes.
The plasmid pcDNA3-HA which encodes a hemagglutinin (HA) epitope tag at the
N-terminus was a gift from Dr. CHEN She (Fudan University). Inserting the
full-length human IRF-3 cDNA in frame into pcDNA3-HA generated the mammalian
recombinant vector pcDNA3-HA-IRF-3. The deletion mutant of PAI-2, which encodes
PAI-2 protein without the interhelical region of C and D, has been constructed
before[1].
1.3 Cell culture and Transfection
HeLa cells were
maintained in RPMI 1640 containing 10% bovine calf serum, 2 mmol/L glutamine,
50 u/ml of penicillin, and 50 g/L streptomycin. Transfections were performed
using Lipofectamine (Invitrogen) as instructed by the manufacturer.
1.4 The yeast two-hybrid screen and
colony-lift filter assay
The interhelical
region of C and D of PAI-2 was used as the bait to screen a HeLa cells cDNA
library described above. The screen was done with Saccharomyces cerevisiae
strain AH109（MATa， Trp1－901, Leu2－3, Ura3－52, His3－200）, which expresses
reporter genes conferring selective auxotrophy and β-galactosidase activity.
The screen was carried out in Ade/His/Leu/Trp-deficient medium at 30 ℃. 10 d
after cotransformation, clones were screened and transferred onto a filter. It
was rapidly lysed by being dipped twice into liquid nitrogen and allowed to
thaw at room temperature. Carefully place the filter, colony side up, on
another filter presoaked with Z buffer (60 mmol/L Na2HPO4・7H2O, 40 mmol/L
NaH2PO4・H2O, 10 mmol/L KCl, 0.1 mmol/L MgSO4・7H2O, pH 7.0) containing 1 g/L X-gal and
0.27%β-mercaptoethanol. Filters were incubated at 37 ℃ until the blue colonies
appeared.
1.5 Isolation of the plasmids from
positive clones
Plasmids from positive
clones were isolated as described by Hoffman et al.[3]. In brief, a large fresh
positive colony was inoculated into 5 ml SD/-Trp and was incubated at 30 ℃
overnight with shaking at 250 r/min. After centrifugation, the pellets were
resuspended in 200 μl lysis buffer [2％ Triton X-100, 1% SDS, 100 mmol/L NaCl, 10 mmol/L Tris・HCl (pH 8.0),
1 mmol/L EDTA]. 0.2 μg glass beads (Sigma) and 200 μl phenol / chloroform (1∶1)
were added to the lysate and the mixture was vortexed thoroughly for 10 min.
They were dipped into liquid nitrogen for 10 min and allowed to thaw at room
temperature. After vortexed again for 10 min, the supernatant was collected by
centrifugation at 12 000 r/min for 10 min. 400 μl ice-cold ethanol was added
and the pellet was spinned down by centrifugation.
1.6 Analysis of homology of sequence of
positive clones
The plasmids
isolated from the positive clones were introduced into E.coli strain KC8 cells
by electroporation. The sequence of the plasmid DNA was analyzed by Ji Kang
Inc. and subjected to GenBank at the NCBI to analyze the homology using BLAST
program.
1.7 Amplification of the full-length
human IRF-3 cDNA by RT-PCR
The full-length
human IRF-3 cDNA was generated by RT-PCR. Briefly, total cellular RNA from HeLa
cells was prepared by Trizol (Invitrogene). RNA was controlled by agarose gel
electrophoresis and spectro photometrically quantified. dT15-primers and
AMV-Rtase were used for first strand synthesis. Primers for IRF-3 cDNA were B1（EcoRI）: 5′-AAAGAATTCATAGGAACCCCAAA-3′ and B2:（XhoI）5′-AAACTCGAGTCAGCTCTCCCCAG-3′。 1 μl total cDNA product was mixed with Taq DNA polymerase, 50
pmol/L of each appropriate primer, 200 μmol/L each dNTP in a buffer containing
10 mmol/L Tris・HCl (pH 8.3), 50 mmol/L KCl, 0.1 g/L BSA, 2 mmol/L MgCl2 in an
end volume of 100 μl RNA. The samples were amplified for 28 cycles and the PCR
condition was as follow: 40 s at 94 ℃, 40 s at 58 ℃, 80 s at 72 ℃.
1.8 Co-immunoprecipitation of PAI-2 and
IRF-3
HeLa cells were
grown as a monolayer in 10-cm-diameter dishes and transfected with both
pcDNA3-PAI-2 and pcDNA3-HA-IRF-3 (4 μg of each) or both pcDNA3- PAI-2CD and
pcDNA3-HA-IRF-3 (4 μg of each) using LipofectAMINE. 24 h after transfection,
100 IU/ml TNF-α and 10 mg/L cycloheximide were added to induce apoptosis. 8 h later,
cells were scraped from the dish, washed with ice-cold phosphate buffered
saline (PBS), and lysed with ice-cold lysis buffer [137 mmol/L NaCl, 20 mmol/L
Tris・HCl (pH 8.0), 0.1 mmol/L CaCl2, 1 mmol/L MgCl2, 1% NP40, 10% glycerol, 1 mmol/L PMSF, 1mg/L aprotinin]
for 15 min at 4 ℃ on a rotating platform. Samples were immunoprecipitated with
1 μg of polyclonal anti-PAI-2 antibody (Santa Cruz) for 2 h at 4 ℃ on a
rotating platform. 40 μl protein G beads (Roche) was added to the lysate and
the mixture was incubated through night at 4 ℃ on a rotating platform. The
beads were washed with ice-cold lysis butter for three times and resuspended in
100 μl loading
buffer and boiled. After centrifugation, samples were separated by 10% SDS-PAGE
and transferred to nitrocellulose membrane (Amersham Pharmacia). The membranes were blocked with 5%
skimmed milk and sequentially incubated with monoclonal anti-HA antibody (Santa
Cruz) and horseradish peroxidase-conjugated secondary antibodies (Santa Cruz).
Results were analyzed by ECL (Amersham Pharmacia) by exposing the member to
X-ray film (Kodak).
2 Results
2.1 Library screening
Yeast two-hybrid
screen was used to identify proteins that interact with PAI-2 via the
interhelical region of C and D of PAI-2. Before screening the library, the
recombinant vector pAS2-1NE-PAI-2CD was transformed with blank vector pACT2
into AH109, and no clones appeared. It indicated that PAI-2CD itself had no
transcriptional activity. 10 d after cotransformation, 40 positive clones were
screened and 36 clones showed the β-galactosidase activities verified by the
colony-lift filter assay.
2.2 Identification of the positive
clones and analysis of homology
36 plasmids DNA
isolated from candidate clones were cotransformed with blank vector pAS2-1NE
into AH109. Those, which showed transcriptional activities, were excluded for
further analysis. Thus, 24 clones were considered to be the candidates of PAI-2
partners. The cDNAs of these clones were amplified by PCR and their sequences
were subjected to GenBank at the NCBI to analyze the homology using BLAST
program. Our result suggested that one clone showed 100% homology to the
C-terminus of interferon regulatory factor 3 (IRF-3), which encodes 98 amino
acids of IRF-3.
2.3 Amplification of human full-length IRF-3
cDNA and linkage with pcDNA3-HA
The full-length
human IRF-3 cDNA was generated by RT-PCR. The agarose gel electrophoresis
showed the fragment was about 1.3 kb, according with our expectation (Fig.1).
After sequencing, the RT-PCR product was confirmed to encode IRF-3 protein. The
plasmid pcDNA3-HA, which encodes a hemagglutinin (HA) epitope tag at the
N-terminus, was inserted with full-length human IRF-3 cDNA to generate the
mammalian recombinant vector pcDNA3-HA-IRF-3. The restriction enzymes analysis
was identical to our expectation (Fig.2).
|
Fig. 1 RT-PCR amplification of IRF-3 cDNA M, |
Fig. 2 Restriction enzymes identification of
|
2.4 PAI-2 can bind IRF-3 via its
interhelical region of C and D in vivo
In order to
further investigate the interaction of PAI-2 and IRF-3, we tested whether they
interact with each other in mammalian cells. The IRF-3 protein was tagged at
its N-terminus with a HA epitope. Both pcDNA3-PAI-2 and pcDNA3-HA-IRF-3 were
transiently co-transfected in HeLa cells. 24 h after transfection, 100 IU/ml
TNF-α and 10 mg/L cycloheximide were added to induce apoptosis. Whole lysates
of transfected HeLa cells were co-immunoprecipitated with anti-PAI-2 antibody.
The immunoprecipitates were resolved by SDS-PAGE and immunoblotted with anti-HA
antibody. As shown in Fig.3, PAI-2 could be co-immunoprecipitated with IRF-3
regardless of apoptosis or not, while it could not be detected in the control
co-immunoprecipitation. In addition, we have constructed the deletion mutant of
PAI-2, which encodes PAI-2 protein without the interhelical region of C and D.
The data suggested that
Fig.
3 Western blotting of co-immunoprecipitation between PAI-2 and IRF-3
1, HeLa; 2, PAI-2CD+IRF-3; 3, PAI-2+IRF-3;
4, HeLa; 5, PAI-2CD+IRF-3; 6, PAI-2+IRF-3. this mutant can’t interact with
IRF-3 (Fig. 3). Thus we proved that PAI-2 could interact with IRF-3 via its
interhelical region of C and D in mammalian cells as well as in yeast.
3
Discussion
IRF-3 belongs to
a family of the IRF transcription factors, members of which play diverse roles
in the immune response to pathogens, immunomodulation, hematopoietic
development and mediate of cellular resistance against viral infection. In
unstimulated cells, IRF-3 is present in an inactive cytoplasmic form without
binding some other proteins. Once stimulated, such as viral infection,
treatment with dsRNA, IRF-3 will be phosphorylated rapidly and be driven from
cytoplasm to nuclear, where it associates with the transcriptional coactivator
CBP/p300, and stimulates the DNA binding and transcriptionally activates
virus-inducible genes[4]. Recently, Heylbroeck et al.[5] discovered that
overexpression of IRF-3 can commit cells to apoptosis, if infected with Sendai
virus, and the results of Weaver et al.[6] also demonstrated that apoptosis is
promoted by the dsRNA-activated IRF-3 during viral infection independent of the
action of interferon or p53. These data indicated that IRF-3 may participate in
the regulation of apoptosis, other than respond to viral infection by mediating
the expression of IFN.
TNF-α is known
to initiate apoptosis[7] and some evidences suggested that there might exist
some crosstalk in signal transductions induced by IFN and TNF-α. NF-κB can
repress apoptosis by transcriptional activation of some apoptosis repressive
proteins, and through STAT1 signal pathway, IFN-α can regulate the activity of
NF-κB induced by TNF-α[8]. Since IRF-3 is the most important transcriptional
factor for activation of IFN-α, we speculate that IRF-3 may be involved in
regulation of apoptosis via this signal pathway.
It is known that
TNF-α can up-regulate the expression of PAI-2[9], and in this study, it is the
first time to confirm that PAI-2 can interact with IRF-3 via its interhelical
region of C and D, which is necessary to protect cells from apoptosis induced
by TNF-α. Accumulated evidences indicate that multiple serine and threonine
residues locate in C-terminus of IRF-3, such as Ser385, Ｓer386, Ｓer396, Ｓer398 and Thr404, which can be
phosphorylated by some unknown kinase, and the post-translational modification
may play a critical role in activation of IRF-3[10]. Since PAI-2 can interact
with the C-terminus of IRF-3, it would possibly mask these posttranslational
residues, and then disturb the phosphorylation of IRF-3.
It is
interesting that intracellular, but not extracellular, PAI-2 can protect cells
from the rapid cytopathic effects of alphavirus infection, and this protective
function did not appear to be related to effect on anti-apoptosis[11].
Meanwhile, Shafren et al.[12] demonstrated that cytoplasmic expression
of PAI-2 affords a high level of protection from lytic infection by multiple
human picornaviruses. These data suggested that PAI-2 may also response to the
viral infection. Now our study revealing the interaction between PAI-2 and
IRF-3 may provide an important clue to study the antiviral function of PAI-2.
But the exact molecular mechanism of the interaction still remains to be
investigated.
Acknowledgements We thank Dr.
TIAN Yu and Dr. CHEN She for their kind gifts of pAS2-1-NE and pcDNA3-HA. We
also thank Dr. ZHU Xiao-Yu for her helpful discussions.
References
1 Tian
Y， Shen JQ， Li P， Zhu YS. The protection function of the PAI-2 from TNF-α is
denpendent upon its protein binding domain. Acta Biochim Biophys Sin, 2000, 32:
175-178
2 Dickinson JL， Norris BJ， Jensen PH， Antalis TM. The CD interhelical
domain of the serpin plasminogen activator inhibitor-type2 is required for
prtection from TNF-α induced apoptosis. Cell Death Differ， 1998， 5: 163－171
3 Hoffman
CS, Winston F. A ten-minute DNA preparation from yeast efficiently releases
autonomous plasmids for transformation of Escherichia coli. Gene, 1987, 57: 267－272
4 Kumar
KP, McBride KM, Weaver BK, Dingwall C, Reich NC. Regulated nuclear-cytoplasmic localization
of interferon regulatory factor 3, a subunit of double-stranded RNA-activated
factor 1. Mol Cell Biol, 2000, 20: 4159－4168
5 Heylbroeck
C, Balachandran S, Servant MJ, DeLuca C, Barber GN, Lin R, Hiscott J. The IRF-3
transcription factor mediates Sendai virus-induced apoptosis. J Virol, 2000,
74: 3781－3792
6 Weaver
BK, Ando O, Kumar KP, Reich NC. Apoptosis is promoted by the dsRNA-activated
factor (DRAF1) during viral infection independent of the action of interferon
or p53. FASEB J, 2001, 15: 501－515
7 Schulze-Osthoff
K, Ferrari D, Los M, Wesselborg S, Peter ME. Apoptosis signaling by death
receptors. Eur J Biochem, 1998, 254: 439－459
8 Suk
K, Kim YH, Chang I, Kim JY, Choi YH, Lee KY, Lee MS. IFN alpha sensitizes
ME-180 human cervical cancer cells to TNFalpha-induced apoptosis by inhibiting
cytoprotective NF-kappaB activation. FEBS Lett, 2001, 495: 66－70
9 Medcalf
RL, Kruithof EK, Schleuning WD. Plasminogen activator inhibitor 1 and 2 are
tumor necrosis factor / cachectin-responsive genes. J Exp Med, 1988, 168: 751－759
10 Lin
R, Mamane Y, Hiscott J. Structural and functional analysis of interferon
regulatory factor 3: Localization of the transactivation and aut oinhibitory
domains. Mol Cell Biol, 1999, 19: 2465－2474
11 Antalis
TM, la Linn M, Donnan K, Mateo L, Gardner J, Dickinson JL, Buttigieg K et al.
The serine proteinase inhibitor (serpin) plasminogen activation inhibitor type
2 protects against viral cytopathic effects by constitutive interferon α/β
priming. J Exp Med, 1998, 187: 1799－1811
12 Shafren
DR, Gardner J, Mann VH, Antalis TM, Suhrbier A. Picornavirus receptor
down-regulation by plasminogen activator inhibitor type 2. J Virol, 1999, 73:
7193－7198
_______________________________________
Received: March 10, 2003Accepted: April 11,
2003
The work was supported by a grant from the
National Natural Sciences Foundation of China ( No. 30070412 )
*Corresponding author: Tel, 86-21-54237278;
e-mail, [email protected]