FIF [Fibroblast Growth Factor-2 (FGF-2)-Interacting-Factor], a Nuclear Putatively Antiapoptotic Factor, Interacts Specifically with FGF-2
Loïc Van den Berghe1,
Henrik Laurell1,
Isabelle Huez,
Catherine Zanibellato,
Hervé Prats and
Béatrix Bugler
INSERM U 397 Institut Louis Bugnard 31403 Toulouse Cedex 4,
France
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ABSTRACT
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Numerous evidence indicates that some of the
activities of fibroblast growth factor 2 (FGF-2) depend on an
intracrine mode of action. Recently, we showed that three high
molecular mass (HMM) nuclear forms of FGF-2 are part of a
320-kDa protein complex while the cytoplasmic AUG-initiated form is
included in a 130-kDa complex. Consequently, the characterization of
FGF endogenous targets has become crucial to allow the elucidation of
their endogenous activities. Through the screening of GAL4-based yeast
two-hybrid expression libraries, we have isolated a gene encoding a
nuclear protein of 55 kDa, FIF (FGF2-interacting-factor), which
interacts specifically with FGF-2 but not with FGF-1, FGF-3, or FGF-6.
In this system, FIF interacts equally well with the NH2-extended 24-kDa
FGF form as with the 18-kDa form, indicating that the FIF-binding motif
is located in the last 155 amino acids of FGF-2. Nevertheless,
coimmunoprecipitation experiments showed an exclusive association with
HMM FGF-2. The predicted protein contains a canonical leucine zipper
domain and three overlapping hydrophobic heptad repeats. The region
spanning these repeats is, together with a region located in the
N-terminal part of the FIF protein, implicated in the binding to FGF-2.
In contrast to the full-length FIF protein, several deletion constructs
were able to transactivate a lac-Z reporter gene. Furthermore, the
COOH-terminal part, but not the full-length FIF protein, has previously
been shown to exhibit antiapoptotic properties. Thus we discuss the
possibility that these activities could reflect a physiological
function of FIF through its interaction with FGF-2. constitute a family of at least
20 homologous proteins (1, 2) that act on a variety of cells by
stimulating mitogenesis or by inducing morphological changes and
differentiation. One of them, FGF-2 or basic FGF, is involved in
developmental processes, wound healing, and angiogenesis as well as in
tumorigenesis (for review, see Refs. 35). Five FGF-2 isoforms of 18,
22, 22.5, 24, and 34 kDa are synthesized through an alternative
translational initiation process (68). These isoforms differ only in
their NH2 extremities, which confer a nuclear
localization to the four high molecular mass CUG-initiated forms (HMM)
while the smaller AUG-initiated protein of 18 kDa is cytoplasmic or
localized in the extracellular compartment (9, 10).
These FGF-2s can exert their effects through different pathways.
Extracellular FGF-2 binds to high-affinity transmembrane tyrosine
kinase receptors (FGFR) and low-affinity receptors (heparan
sulfate-containing proteoglycans) (1113). Receptor activation
stimulates intracellular mitogen-activated protein kinase (MAPK)
and/or phospholipase C signaling pathways (1416). But FGF-2 can also
be internalized with both kinds of receptors into the cytoplasm
(1719) and translocate into the nucleus during the
G1 phase of the cell cycle (20) by a mechanism
distinct from that of nuclear endogenous FGF-2 (21). The nuclear HMM
forms are involved in cell proliferation (22) and in oncogenesis (23,
24) while the AUG-initiated 18-kDa protein stimulates the migration
(25), down-regulates its own receptor (22), and stimulates integrin
synthesis (26). The inhibition of the expression of all FGF-2 isoforms
leads to a loss of tumorigenicity in nude mice (27). A newly identified
34-kDa FGF-2 isoform has been recently characterized as a survival
factor (8) while intravenous injection of 24 kDa-producing cells led to
extensive lung metastases in nude mice (28). However, the details
regarding the mechanisms by which the endogenous FGF-2s exert their
intracellular effects remain to be elucidated.
We have previously shown that the three HMM (22, 22.5, and 24 kDa) and
the 18 kDa (LMM) intracellular FGF-2s are found as components of large
protein complexes of 320 and 130 kDa, respectively. Moreover, the
coimmunoprecipitation of distinct proteins by anti-chloramphenicol
acetyl transferase (CAT) antibodies in cells transfected with HMM-CAT
and LMM-CAT fusion constructs, could reflect different activities of
HMM and LMM FGF-2 (29). In an attempt to identify such associated
proteins, we used the yeast two-hybrid system to screen a human
lymphocyte cDNA library (30). We here report the characterization of a
nuclear protein, FIF (FGF-2 interacting factor) which is able to bind
HMM and LMM FGF-2 isoforms in the two-hybrid system and in in
vitro binding assays but not some other members of the FGF family.
Interestingly, coimmunoprecipitation experiments suggest that the
FGF-2/FIF complex is nuclear in vivo. The FIF protein
contains a leucine zipper, three hydrophobic heptad repeats, an acidic
region, and a nuclear localization signal (NLS). Moreover, some
truncated constructs revealed transactivating capacities.
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RESULTS
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Cloning and Analysis of a New Endogenous FGF-2 Binding Protein
To identify genes encoding proteins that associate with FGF-2, we
used the yeast two-hybrid system to screen a human lymphocyte cDNA
library (30). The yeast strain Y190 was first transformed with the bait
plasmid encoding the 155 amino acids (aa) FGF-2 in fusion with the DNA
binding domain of GAL4 in the pAS2 vector. The yeast cells were
subsequently transformed with the cDNA library in fusion with the
activating domain of GAL4 in the pACT vector. Twenty-eight clones grew
on selection medium and were positive for ß-galactosidase activity in
color filter assay. However, only two clones exhibited no activity when
the isolated pACT plasmids were retransformed with an empty pAS2
vector. These two clones contained the same insert comprising a
1,320-bp open reading frame and a 196-bp downstream noncoding sequence.
The protein was named FIF for FGF2-Interacting Factor, and the isolated
clones were denoted FIF
N1.2
Due to the lack of an initiation codon [Fig. 1
: the glutamic acid 72 is the first
amino acid of the isolated clone], a screening was performed on a
human hepatocyte cDNA library to isolate the full-length cDNA clone.
One clone, named FIF-504 (with a 504 aa open reading frame) was
isolated.3 This clone
contained 326 additional bp in the 5'-end compared with the FIF N1
clones. The downstream coding sequences were identical apart
from a divergence in the most 3'-region, comprising a 20-bp coding
sequence and a 189-bp noncoding sequence specific for FIF-504,
vs. 32 bp and 200 bp, respectively, which were specific for
FIF N1 (Fig. 1A
). These two 3'-extremities probably originated
from an alternative splicing since the two first divergent
nucleotides GT in the clone 510 could correspond to the canonical
splice site that would be used to generate the FIF-504. Both
3'-sequences are well represented in the EST database, indicating that
both alternatively spliced forms of FIF are expressed in various
tissues. In FIF-504, the first methionine, surrounded by a good Kozak
consensus sequence for initiation of translation, is preceded 11 bp
upstream by an in-frame stop codon, thus avoiding any upstream
initiation. Based on the fact that there is no divergence among EST
sequences corresponding to the 5'-region of FIF, we presume that the
missing 5'-sequence in N1 is identical to that of FIF-504. The
5'-extended N1 clone was therefore denoted FIF-510 (Fig. 1A
). Since no
difference in biochemical properties has yet been observed between both
FIF-504 and FIF-510, the two proteins will be considered as FIF. The
protein comprises two acidic domains (aa 164190 and aa 323335 in
Fig. 1B
), a leucine-zipper motif (aa 370391), and three overlapping
heptad repeats of hydrophobic amino acids (aa 259314). One EST clone
(W40304) corresponds to an alternatively spliced form of FIF, in which
a 77-bp deletion introduces a stop codon 14 bp downstream from
the splice junction (Fig. 1A
). We have recreated this COOH-truncated
FIF clone and named it FIF C1, which lacks the last 109 aa. During the
process of this investigation, two studies have reported the cloning of
a cDNA identical (API5-L1) (31) and highly similar
(AAC11long) (32) to that of FIF-504.
AAC11long and FIF-504 differ only by three amino
acids (residues 371373, Fig. 1A
); thus both proteins are likely to be
identical.

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Figure 1. Nucleotide and Deduced Amino Acid Sequences of the
FIF Protein
A, Comparison of the nucleotide sequences and the deduced amino acid
sequence of human FIF 510 and FIF 504. Divergent nucleotides and
residues with AAC-11 are boxed (from nt 1605 to nt 1817,
aa 314, from aa 370 to aa 373). , The start of the FIF N1 clone, isolated in the initial two-hybrid screening. ***
Indicates stop codons. The sequence in italics
corresponds to the FIF C1 construct and the alternatively spliced EST
clone W40304. The bracket indicates the putative splice
site which generates the two divergent 3'-extremities in the cDNA of
the FIF-504 and 510 sequences. B, Amino acid sequences of the human
FIF-510 and FIF-504. The circled glutamic acid residue
indicates the start of the N1 clone. The amino acids involved in the
canonical and the three overlapping heptad repeat motifs are
squared and linked. The LXXLL motif is
underlined and the nuclear localization signals (NLS)
are boxed. The small brackets delineate
the two acidic regions.
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FIF Interacts Specifically with FGF-2 Isoforms
To test the specificity of the FIF-FGF-2 interaction, we analyzed
the coexpression in yeast of three other representative members of the
FGF family, FGF-1, FGF-3, and FGF-6, as well as the larger 24-kDa FGF-2
(210 aa) with the FIF protein. As shown in Fig. 2A
, FIF (FIF 504 and 510 gave the same
results) was only able to interact specifically with the two FGF-2
tested, i.e. the 18- and 24-kDa isoforms. FIF and FGF-2
interacted mutually in both bait and prey configuration. The
pAS-myc was used as a negative control. We did not detect
any homotypic interaction between FIF molecules indicating its
inability to form dimers at least in its fusion state in yeast.
Conversely, both FGF-2 isoforms dimerize as already described (33). The
strength of the FIF/FGF-2 interaction is notable since several FIF
constructs, such as FIF C1 (FIF-510 lacking the last 109 aa),
interacted with FGF-2 with similar efficiency as the positive control
proposed by CLONTECH Laboratories, Inc. (Palo Alto, CA)
(SV-40 large-T antigen binding to p53) as measured by ß-galactosidase
activity in yeast extracts (Figs. 2B
and 7A
).

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Figure 2. Specificity of the FIF-FGF2 Interaction
A, In addition to the 18-kDa and the 24-kDa FGF-2 isoforms,
three members of the FGF family, FGF-1, FGF-3, and FGF-6 as well as the
c-myc protooncogene as a control, were tested for their
interaction with the FIF protein in the yeast two-hybrid system by the
color filter assay. Homodimerizations of FIF and FGF-2 were also
investigated. FGF-1, FGF-3, FGF-6, and c-myc were cloned in
the pAS2 vector, whereas FIF and FGF-2 were cloned in both pAS2 and
pACT2 vectors to test heterogeneous interaction and homodimerization.
B, Relative comparison between three different types of positive
interactions: FGF-2/FIF, FGF-2/FIF C1, SV40 large-T antigen/p53. DNA
BD, DNA binding domain; TAD, transactivating domain. (ß-Galactosidase
activity reflecting interaction was quantified in triplicates from at
least two independent yeast transformations and was normalized for
protein content.)
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Figure 7. FIF Protein Expression and Subcellular Localization
A, Western blot analysis of various cell lysates using immunopurified
polyclonal antibody against endogenous FIF and COS-transfected FIF. B,
In vitro-translated FIF-510 and 504 in rabbit
reticulocyte lysates. C, Subcellular fractionation of COS-7, NIH-3T3,
and SK-Hep-1 cells. Total, Total cell extract; Nuc., nuclear fraction;
Cyto., cytoplasmic fraction. D, Comparison of the p53 and the FIF NLS.
The identical residues are boxed and the conserved
glycines in +4 position after the last lysine are
underlined. Lysine and arginine residues are in
bold. E, Immunolocalization of COS-7 cells, transfected
with the plasmids encoding either the full-length FIF or the FIF C1
lacking the NLS. Magnification x100.
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FIF and FGF-2 Interact in Mammalian Cells
Since the first observations of the FIF/FGF-2 interaction were
made in yeast, we wanted to verify this association in mammalian cells.
For this purpose, we developed a bigenic plasmid transfection system in
which both the FIF-504 and the FIF-510 sequences were cloned in fusion
with the GAL4 DNA-binding domain while FGF-2 was fused with the
VP16-activating domain in the same bigenic expression vector,
pTHM3-FIF (see Materials and Methods). Each of these
plasmids was cotransfected in SK-Hep-1 cells together with a reporter
plasmid encoding the luciferase protein. The ability of both fusion
proteins to associate in vivo was quantified by photon
emission reflecting the activation of the luciferase gene. As shown by
transfection with the plasmid encoding only the
FIF-GAL4-DNA-binding-domain fusion protein but lacking FGF-2, none of
the FIF proteins alone were able to transactivate the reporter gene
(Fig. 3
). However, coexpression of FGF
and FIF fusion proteins resulted in a high level of luciferase
activity. Similar results were obtained in HeLa cells (data not shown).
These results clearly demonstrate that the FIF and FGF-2 proteins
associate efficiently in mammalian cells.

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Figure 3. FIF-FGF-2 Interaction in a Mammalian Two-Hybrid
System
SK-Hep-1 cells were cotransfected with a reporter vector containing
three UAS sequences upstream of a cytomegalovirus (CMV) promoter
followed by the luciferase gene and a bigenic vector (pTHM3-FIF)
encoding two fusion proteins: a FIF-504 or 510-GAL4 DNA-binding-domain
protein and a FGF-VP16 transactivating-domain protein. Luciferase
activity reflecting FGF-2/FIF interaction was quantified in triplicates
from at least two independent transfections and was normalized for
protein content. Control experiments were performed with pTHM3 vector
lacking either the FIF or the FGF-2 encoding sequences (three first
histograms).
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FIF and FGF-2 Coimmunoprecipitate
To further study the FIF/FGF-2 interaction, coimmunoprecipitation
experiments were performed on human RPE cells, stably transfected with
a FGF-2 expression vector (pREP-hFGF2) that encodes 18- to 24-kDa FGF-2
isoforms. Rabbit antihuman FIF antibodies or monoclonal antihuman FGF-2
antibodies were used for immunoprecipitation, followed by Western
blotting with the reciprocal antibody. Under reducing SDS-PAGE, the
heavy chain of rabbit and mouse immunoglobulins comigrate with FIF,
rendering detection of this immunoprecipitated protein difficult. To
circumvent this problem, immunoprecipitates were dissolved in Laemmli
buffer lacking ß-mercaptoethanol and dithiothreitol (DTT) to maintain
the disulfide bonds between the light and heavy chains (Fig. 4
, A and B). Under these conditions,
anti-FGF-2 antibodies brought down a 55-kDa protein that was
detected by the anti-FIF antibodies (Fig. 4A
, lane 3). To verify that
the detected protein was not heavy chain immunoglobulins but FIF, the
blot was stripped and reincubated with rabbit antihuman FGF-2
antibodies (Fig. 4B
). Interestingly, in the reciprocal
coimmunoprecipitation experiment, anti-FIF antibodies brought down only
the nuclear HMM FGF-2 isoforms (22, 22.5, and 24 kDa) and not the
cytosolic 18-kDa FGF-2 form (Fig. 4C
, lane 2). This could suggest
either a nuclear localization of the FIF/FGF-2 complex and/or a higher
affinity for the HMM FGF-2 forms. Nevertheless, these results confirm
an in vivo association between FIF and FGF-2.

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Figure 4. Reciprocal Coimmunoprecipitation of FIF and FGF-2
Extracts from human stably transfected RPE-FGF cells were
immunoprecipitated with monoclonal anti-FGF or rabbit anti-FIF-2
antibodies as indicated (IP). Purified FIF (20 ng) (A and B, lane 1),
nonimmunoprecipitated cell extracts (A and B, lanes 2; C, lane 1), and
immunoprecipitated proteins were subjected to Western blotting with the
indicated antibodies (Blot). The samples of blot A (= B) were in
contrast to blot C, treated under nonreducing conditions. red.,
ß-mercaptoethanol and DTT.
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FIF and FGF-2 Interact in Vitro
To ascertain that the FIF/FGF-2 interaction is direct and
independent of a third partner, two in vitro-binding assays
were performed with purified proteins. First, we tested the ability of
purified His-tagged FIF-504 protein, immobilized on nickel agarose
column, to bind recombinant FGF-2. Both 18- and 24-kDa FGF-2 isoforms
were coeluted with the His-FIF protein by imidazole as visualized after
SDS-PAGE and Coomassie blue staining (Fig. 5A
, interaction test). FGF-2 alone did
not bind to the resin (Fig. 5A
, control). Furthermore, no binding of
any protein other than FGF-2 was observed when incubation was performed
with total Escherichia coli FGF-2 extract in place of
purified FGF-2 (data not shown). These results demonstrate the direct
and specific in vitro interaction between FIF and FGF-2.

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Figure 5. FIF-FGF-2 in Vitro Interaction
A, The FIF-504 protein was fused to a histidine tag, purified, and
immobilized on a nickel-agarose column (FIF). Purified 18- and 24-kDa
FGF-2 isoforms (FGF-18 and FGF-24) were added to the FIF column and
washed, and the retained proteins were eluted with imidazole. Ater
separation on SDS-PAGE the proteins were detected by Coomassie blue
staining (interaction test). The first three lanes correspond to the
three purified proteins loaded directly on the gel. (purified
proteins), and the two following lanes correspond to the binding
control of FGF to the column without FIF (control). B, In lanes 13,
purified biotinylated FGF-2, FIF C1, and full-length FIF were loaded
directly on the gel. Interaction test: biotinylated FGF-2 was fixed on
streptavidin-coated magnetic beads and incubated with purified FIF
protein in the absence or presence of DTT as indicated (lanes 45).
The same experiment was performed without DTT and with increasing
concentrations of the truncated protein FIF C1 (lanes 69). The eluted
proteins were analyzed by Western blotting using a mix of anti-FIF and
anti-FGF-2 antibodies. Lane 10 corresponds to the binding control of
FIF to the beads without FGF. Positions of FGF-2 monomers (biot FGF),
dimers, tetramers, FIF, FIF C1, and markers are indicated.
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We were able to verify this observation by performing the binding test
using biotinylated FGF-2 immobilized on streptavidin magnetic beads.
Biotinylation has previously been shown to have no effect on the
biological activity of FGF-2 (34) or on FGF-2 homodimerization (33).
After the incubation of biotinylated FGF-2 on streptavidin magnetic
beads with recombinant FIF-504, the resin was washed and the retained
proteins were eluted and separated by SDS-PAGE and subsequently
immunoblotted using a mix of specific antibodies against FGF-2 and FIF
(Fig. 5B
). FIF did not associate with streptavidin beads alone (Fig. 5B
, lane 10). However, FIF was found to interact with biotinylated
FGF-2 in a DTT-independent manner. The oligomeric nature of FGF-2 in
the nonreduced state disappeared with the addition of DTT,
indicating the involvement of disulfide bonds in the homotypic
interaction of FGF-2 (Fig. 5B
, lanes 4 and 5). FIF C1, used to
distinguish FIF from FGF-2 tetramers, was still able to associate FGF-2
in a dose-dependent manner and with the same efficiency as the
wild-type molecule (Fig. 5B
, lanes 69). The interaction is also
observed in the presence of 250 mM NaCl (data not shown).
Hence, these results confirm the data obtained with the immobilized
His-FIF protein (Fig. 5A
) and point out the fact that the FIF/FGF-2
interaction occurs directly. It also indicates that the last 109
COOH-terminal residues of the FIF protein, lacking in FIF C1, are not
required for FGF-2 binding as already shown (Fig. 2
).
Human FIF mRNA Expression Is Ubiquitous
To determine the size and tissue distribution of FIF
mRNA, a multiple tissue Northern blot with polyA mRNA extracted
from various human tissues (CLONTECH Laboratories, Inc.) was probed with a full-length FIF cDNA probe (Fig. 6
). One major RNA transcript with an
apparent size of 3.6 kb was detected in all the examined tissues and
reflects the ubiquitous expression of this messenger. The FIF mRNA
appeared relatively more abundant in heart, pancreas, and placenta,
whereas the signal obtained from liver mRNA was comparatively weaker.
Tewari and colleagues (32) reported, apart from the dominant transcript
of approximately 4 kb, the presence of some less abundant transcripts
in murine tissues. However, we were unable to detect these transcripts
in human tissues.

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Figure 6. Human FIF mRNA Distribution
Northern blot containing poly A mRNA extracted from cells of different
human tissues (CLONTECH Laboratories, Inc. MTNTM) and
probed with the complete P32-labeled FIF-504 cDNA.
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The FIF Expression Is Ubiquitously Expressed and Essentially
Nuclear
The expression pattern of endogenous FIF protein in different cell
types was studied by Western blotting using immunopurified anti-FIF
antibodies. A major protein with apparent molecular mass of 55 kDa was
detected in all tested cell lines from mouse, rat, monkey, bovine, and
human origin except for human skin fibroblast cells (Fig. 7A
). The 55-kDa protein is likely
corresponding to the characterized AUG-initiated full-length FIF since
it migrates as the FIF protein overexpressed in transfected COS-7 cells
(Fig. 7A
) as well as the in vitro-translated protein (Fig. 7B
). The similarity of the patterns of immunodetected FIF reflects the
high degree of conservation of this protein in various species [98%
identity between mouse and human protein (32)]. Nevertheless, a 53-kDa
protein, but not the 55 kDa isoform, was detected in normal human skin
fibroblast cells. This smaller form was also detected in several other
cell types such as MCF7, SK-Hep-1, HeLa human cells, and rat PC12 cells
(Fig. 7A
). A 40-kDa protein, immunologically detected by two different
sera raised against FIF, was observed in some cell lines of different
species (PC12 and SMC as well as in mouse NIH-3T3). The nature of the
40- and 53-kDa forms are presently unknown.
The subcellular localization of the FIF protein was studied both
by subcellular fractionation (endogenous FIF) and by immunofluorescence
labeling of FIF-transfected COS-7 cells. The Western blot presented in
Fig. 7C
shows that the endogenous 55-kDa protein could be detected both
in the nuclear and cytoplasmic compartments (COS-7, NIH-3T3) while the
40-kDa form protein in NIH-3T3 appears to be exclusively nuclear.
However, in SK-Hep-1 cells the 55-kDa protein was found exclusively in
the nuclear fraction. The analysis of the FIF amino acid sequence
revealed the presence of a potential bipartite NLS between the lysine
residues 454 and 475 (Fig. 1B
). This sequence is similar to the NLS of
the p53 tumor suppressor protein (Fig. 7D
). The C1 protein was found in
the cytoplasm in transfected COS-7 cells in contrast to the full-length
FIF that was essentially nuclear (Fig. 7E
). In some cells, however, the
protein was detected in the cytoplasm, confirming the results obtained
in subcellular fractionation of FIF-transfected COS-7 cells.
Overall, these results suggest that the full-length FIF protein is
essentially nuclear and that the sequence comprising residues 454475
does play a role of NLS in the targeting of the wild-type protein to
the nucleus. The nuclear localization of the 40-kDa protein (Fig. 7
, A
and C) is likely due to the presence of the NLS motif in the
COOH-terminal part, implying that this would correspond to a
NH2-truncated form of FIF.
The Interaction with FGF-2 Involves Two Distinct Regions in FIF
To define the FGF-binding region in the FIF sequence, progressive
deletion constructs were generated. As shown in Fig. 8A
, two
NH2-terminal deletion constructs, lacking the
first 71 residues (N1) and 95 residues (N2), retained the capacity to
bind FGF-2. In contrast, all the N3 constructs, lacking the first 107
residues, were unable to interact with FGF-2. These results suggest the
presence of a first FGF-2-binding region (FBR-1) spanning proline 96 to
glutamine 107 in the FIF protein. Since the COOH deletions C3 (1378),
C4 (1345), and C5 (1311) retained the ability to bind FGF-2, we can
conclude that the carboxy-terminal part of the protein beyond
phenylalanine 311 is not involved in the FGF-2 binding. Moreover, it
should be noted that in these deletions, as well as in several other
constructs, such as C1 (1426), N1 (72), and N1C2 (72402), the
binding capacity to FGF-2 appeared to be enhanced. This could be due to
differences in the structural conformation offering a better
accessibility to the FGF-2 binding sites through the lack of the
carboxy-terminal extremity.

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Figure 8. Characterization of FIF Regions Involved in FGF-2
Interaction and in FIF Transactivation Capacity
A, Schematic representation of truncated FIF proteins. The three heptad
repeats (HR12-3), the leucine zipper motif (LZ), the LXXLL motif, and
the NLS are shaded as indicated. The residues in the
boundaries of the deletion constructs are indicated in the full-length
FIF at the upper part of the figure. Deleted proteins
were fused to the transactivating or the DNA-binding domain of GAL4 and
were tested, respectively, for their capacity to interact with FGF-2
(left histogram) or to transactivate
(right histogram) through the LacZ reporter gene
activation. (ß-Galactosidase activity was measured and normalized for
protein content and is shown as the mean from at least three
independent transformations). B, Schematic representation of FIF
indicating the regions implicated in the interaction with FGF-2 (FBR)
or in the transcriptional activity of FIF.
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The LXXLL motif (where L is leucine and X is any amino acid, Fig. 1B
)
has been shown to mediate binding between transcriptional coactivators
and nuclear receptors (35). This motif is present in FBR-1
(L102L106). The replacement of the conserved leucine residues 102 and
105 by alanine (mutations generally done to disable this functional
binding motif) did not diminish the FGF binding, suggesting that this
short sequence is not involved in the FIF/FGF-2 interaction (data not
shown).
With respect to the deletions of the C terminus, a large difference in
FGF-2-binding capacity was observed between the C5 (1311) and the C6
(1273) constructs, indicating that the sequence including threonine
274 to phenylalanine 311 represents a second important region for the
FIF/FGF-2 interaction (Fig. 8B
, FBR-2). Furthermore, the data suggest
that the two FBRs are necessary for FGF-2 binding since C6, lacking the
FBR-2, vs. N3C3, N4C3, N5C3, N3C5, and N6, only lacking the
FBR-1, did not exhibit any FGF-2 binding activity.
Taken together, these results indicate the presence of two mutually
dependent regions in the FIF protein (spanning residues 96107 and
274311, respectively) which are both required for the interaction
with FGF-2.
Truncation-Dependent Transactivation Capacity in the FIF
Protein
To determine whether the FIF protein could act as a
transactivator, each deleted-FIF cDNA was fused to a GAL4-DNA binding
sequence in the pAS2 plasmid. Yeast cells were transformed with each
construct, and the ß-galactosidase activity was measured from liquid
cultures.
As shown in Fig. 8
, the results obtained with the N6C4 (231345), C5
(1311), and C6 (1273) fusion proteins indicate that a potential
transactivation region is comprised between residues 231 and 273.
Notably, all constructs with a COOH terminus beyond glutamine 378
failed to transactivate the reporter gene. Moreover, in constructs with
only a COOH-terminal truncation, the region between phenylalanine 311
and leucine 346 seems to play an important role in the regulation of
the transactivating activity of FIF [compare C5 (1311) with C4
(1345)]. A region rich in acidic amino acids between the glutamate
residues 323 and 335 (pI = 2.8) (present in the C4 but not in the
C5 construct) could play a role in the inhibition of the
transactivation. However, this repressing activity appears to be
dependent on the nature of the N-terminal part of the protein (compare
C4 and N3C4 with N6C4). Conceivably, some intramolecular interactions
between the region from residues 108 to 231, and the acidic region
between residues 311 and 345, could contribute to this inhibition
(compare N4C3 or N3C4 with N6C4). This is further supported by the fact
that all constructs comprising both regions, except N3C3, did not show
any transactivating activity.
As mentioned previously, the N3C3 construct differed from the other
similar truncated forms like N2C3 and N4C3 as it displayed
transactivation capacities. This could imply a potentially inhibitory
role of FBR-1 (aa 96107) on transactivation. Likewise, one could
assume that the sequence comprising residues 108149, without any
further NH2 extension, could adopt a specific conformation and/or allow
the association of a specific binding factor leading to
transactivation.
In conclusion, a region delimited from residues 231273 can confer a
transactivating potential of the FIF protein that is probably masked in
the full-length protein.
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DISCUSSION
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Numerous studies have suggested the presence of signaling pathways
for intracellular FGF-2 involving either the internalized or the
endogenously produced factor. We recently showed that the FGF-2 exists
as a component in endogenous molecular complexes (29). We therefore
used two types of two-hybrid systems to screen and characterize
proteins able to bind to FGF-2. In this paper we describe a protein,
FIF, that binds FGF-2 in a potentially regulated manner.
The interaction, initially observed in yeast, was reproduced in
mammalian cells (Fig. 3
) and appears specific, since other FGF members
tested, such as FGF-1, FGF-3, and FGF-6, fail to interact with FIF
protein despite the high degree of sequence homology between these
different FGF members (Fig. 2
). FGF-2 and FIF also coimmunoprecipitated
reciprocally (Fig. 4
). The direct association between FIF and FGF-2 was
demonstrated by two reciprocal in vitro-binding assays (Fig. 5
). Therefore, it appears that FIF constitutes a specific intracellular
partner of FGF-2.
We show that FIF is widely expressed in multiple human tissues (Fig. 6
)
as well as in several other species such as monkey, rat, mouse, and
hamster (Fig. 7A
). All the tested cells, namely fibroblasts, neural
(PC12), epithelial (RPE), smooth muscle (SMC), carcinoma (HeLa), and
hepatoma cells (SK Hep-1) express FIF. We can also note a parallel
ubiquitous expression of the FGF-2 in these cells (data not shown).
Two FIF cDNAs were cloned which differ in their 3'-extremities,
generating FIF-504 and FIF-510 (504 and 510 residues, respectively)
with an apparent molecular mass of 55 kDa. This corresponds well with
the theoretical weight of 56,734 and 57,725 Daltons, respectively.
These proteins, however, did not exhibit any detectable difference with
respect to their affinity for FGF-2. Two other proteins, immunorelated
to the full-length 55 kDa, with apparent molecular masses of 40 and 53
kDa were detected in some cell types. The protein
AAC-11long, cloned by Tewari et al.
(32), is identical to FIF apart from three residues that could come
from an inversion between mouse and human sequences as confirmed in a
recent publication concerning the localization of the FIF gene on the X
chromosome (31). Furthermore, no human EST clones containing the
sequence reported by Tewari et al. have been reported.
AAC-11 was described as two isoforms with apparent molecular masses of
55 and 25 kDa. However, a 25-kDa form was never recognized by our
antibodies.
FIF does not present any homology with two recently cloned FGF-binding
proteins such as the FGF-1 intracellular binding protein (FIBP) (36),
or the ribosomal L6/TAX-responsive element-binding 107 protein, which
is able to associate with different FGF-2 isoforms (37).
FIF appears mainly located in the nucleoplasm (SK-Hep-1; Fig. 7
, E and
C) even if fractionation assays in COS-7 and NIH-3T3 cells, and
in situ detection assays in COS-7, revealed its presence in
the cytoplasm. The nuclear localization is due to the presence of a
bipartite NLS in the C terminus, which is homologous to the one
described for the p53 protein (38). The functionality of this NLS was
demonstrated by in situ immunocytochemistry experiments
(Fig. 7E
). The 40-kDa protein, which could either correspond to a
maturation product of the 55-kDa protein or a protein originating from
a downstream alternative initiation codon, is entirely nuclear and is
hence likely to contain the C-terminal portion of the full-length
FIF.
Interestingly, in the coimmunoprecipitation experiments, the anti-FIF
antibodies only brought down the nuclear HMM FGF-2 isoforms (Fig. 4C
).
This indicate that the FIF/FGF-2 complex is nuclear in vivo
and/or that FIF exhibits a higher affinity for the HMM FGF-2 isoforms.
Nevertheless, we did not observe any significant differences in the
intensity of the interaction between FIF and the 18- or 24-kDa FGF-2
forms in two-hybrid experiments (data not shown). It is possible that
posttranslational events participate in the stabilization of the
FIF/FGF-2 complex in the nucleus. Several putative predicted
phosphorylation sites [protein kinase A (PKA), protein kinase C (PKC),
MAPK, casein kinase II (CK2), and ABL tyrosine kinase sites] are
located in or close to the NLS in FIF. Thus, phosphorylation might be
involved in the regulation of the compartmentalization of FIF within
the cell, as already shown in other nuclear proteins (39, 40, 41, 42).
Preliminary pull-down assays using antiphosphotyrosine antibodies on
total COS-7 cell extract immunoprecipitated the 55-kDa protein,
assuming a potential role of phosphates on tyrosine residues (data not
shown). Moreover, FGF-2 has been found to bind to the ß-subunit of
the CK2 (43). FIF contains six putative CK2 phosphorylation sites that
could be involved in the regulation of the localization and/or activity
of FIF.
To map the regions in the FIF protein involved in the interaction with
FGF-2, a set of truncated fusion constructs were cotransformed with the
FGF-2 into the yeast (Fig. 8
). It is inferred from these data that aa
96107 are required for FGF binding (FBR-1). A second centrally
located region in the full-length FIF (aa 274311) was also shown to
interact with FGF (FBR-2). Both regions are required for the
interaction. Thus, through these two binding domains, FGF-2 could
induce a structural refolding of FIF, allowing either a restrained or
activated state. We can notice that the FBR-II comprises at least one
heptad repeat but not the canonical leucine zipper (Figs. 1
and 8
). It
is possible that this hydrophobic structural feature may constitute a
spatial motif that is recognized by FGF-2. Nevertheless, the leucine
zipper motif in the L6/TAX-REB107 protein is not essential for the
binding to FGF-2 (37).
We can indeed observe that the transactivating activity of the FIF
occurs essentially when a COOH-terminal part of the tested constructs
is truncated. Intramolecular interactions between the COOH-terminal
region beyond residue 311 and a region
NH2-terminal to residue 231 could explain the
absence of transactivating activity of the full-length protein. The
region comprising residues 231273 is present in all constructs with
transactivating capacity, thus delineating a putative transactivating
domain. It is possible that posttranslational modifications and/or
alterations in the protein conformation could unmask and activate the
transactivating domain. Such conformational changes could potentially
be induced after phosphorylation of consensus sites. Following the
example of the heat shock factors (HSF) model, inactive monomeric HSF1
is bound to hsp70 or hsp90 in nonstressed cells (44, 45). Following
stress, however, the HSFs are released and subsequently acquire a
trimeric active conformation with transactivating properties.
Similarly, one could speculate that the FIF-FGF pair proceeds in a
similar manner toward the FIF activation in response to specific
signals. Preliminary results revealing homodimerization capacity for
some truncated forms of FIF strengthen this eventuality (data not
shown).
Numerous studies have shown that FGF-2 is able to protect cells from
apoptosis (46, 47, 48, 49). However, FGF-2 has also been reported to promote
apoptosis in some cell types, which suggests that the role of FGF-2 in
programmed cell death could be cell type specific or dependent on the
physiological status of the cell (50, 51, 52). The role of endogenously
produced FGF-2 on apoptosis has been addressed by antisense approaches
(46, 49) and by overexpression in vitro (47) and in
vivo (52). It is not clear to which extent the HMM FGF-2 isoforms
interfere in the regulation of apoptosis. It has been demonstrated,
however, that cell surface receptor-mediated activation of MAPKs
and a subsequent up-regulation of Bcl-2 and Bcl-xL levels are crucial
for the antiapoptotic effect of FGF-2, which at least in RPE cells is
mediated by a FGF2-induced production of FGF-1 (49, 53, 54).
Several studies suggest the necessity of a complementation between
FGFR-mediated signaling and the direct interaction between
intracellular proteins and internalized FGF-2 (20, 55) as well as
internalized FGF-1 (36, 56, 57) for normal proliferative FGF-mediated
activity. In addition the HMM FGF-2 forms have been attributed specific
functions, which often are correlated to malignant or
pathophysiological states (recently reviewed in Ref. 58). The
expression of HMM FGF-2 is enhanced in transformed and stressed cells
(24) and is negatively correlated to the cell density in normal cells
(59). Selective overexpression of HMM FGF-2 in NIH-3T3 fibroblasts
allowed growth and survival in low serum (8, 22, 60). Similarly,
expression of HMMW FGF-2 in pancreatic acinar AR42J cells rendered
these cells serum independent (61). The 24-kDa isoform was found to
render HeLa cells resistant toward
-radiation (62), and elevated
levels of this isoform were also in correlation in enhanced mouse
intestinal epithelial cells survival after
-irradiation (63).
Moreover, selective expression of 24-kDa FGF-2 in NBT-II cells was
shown to induce lung metastases after subcutaneous injection in nude
mice, in contrast to 18-kDa FGF2-producing cells (28).
Very little is known about the molecular mechanisms underlying HMM
FGF-2-specific signaling. FIF is, to our knowledge, the first nuclear
protein to interact directly with HMM FGF-2 isoforms and constitutes a
potential mediator of these signals.
The antiapoptotic property of the shorter AAC-11 form of FIF (32) could
be linked to the putative HSF-like behavior of FIF. The biophysical
status of FIF (phosphorylation and/or maturation with a parallel change
in the conformation) could be an important switch regulating
FGF-2-binding and transcriptional activity. Moreover, expression of the
HMM FGF-2 isoforms after serum stimulation of quiescent cells (59) and
heat shock-stressed cells (24) are enhanced. These FGF-2 nuclear
isoforms could then act concomitantly with FIF to stimulate or repress
specific genes in response to stress.
 |
MATERIALS AND METHODS
|
---|
Yeast Two-Hybrid System
The two-hybrid expression-cloning system was carried out as
described by Durfee et al. (64). The 18-kDa FGF-2 was used
as bait, and the corresponding cDNA was inserted in the
NcoI/SalI sites in the pAS2 vector. A human
lymphocyte cDNA library (generously provided by S. Elledge) cloned into
the pACT vector was screened in Saccharomyces cerevisiae
yeast strain Y190, containing the two reporter genes HIS3 and LacZ.
Yeast cells were transformed by the lithium acetate method (65) and
were grown on plates with appropriate selection medium
(-Trp/-Leu/-His/50 mM 3-aminotriazole). After 4
days, a ß-galactosidase assay was performed by a color filter assay
as previously described (33). Liquid ß-galactosidase assays
normalized for protein content were also performed to quantify the
intensity of the interaction between different FIF constructs and FGF-2
using a chemiluminescence assay kit as previously described (33),
following the instructions of the supplier (Tropix Inc., Bedford, MA).
The quantification was performed in an automatic microplate luminometer
(EG & G Berthold, Bad Wildbad, Germany).
Isolation of the Complete FIF cDNA
The FIF cDNA isolated in yeast was used as a probe to screen a
human hepatocyte-cDNA library inserted in the EcoRI site of
the
ZAP II vector following the protocol provided by the supplier
(Stratagene, La Jolla, CA). Automatic nucleotide
sequencing was performed using the AmpliTaq FS polymerase and a ABI
373A sequencer (Perkin Elmer Corp., Foster City, CA).
Two-Hybrid System in Mammalian Cells
For interaction studies in mammalian cells, we constructed a
bigenic plasmid that allowed expression of the bait (FGF-2 fused to
VP16 transactivating domain) and the prey (GAL4 DNA-binding domain
fused to FIF 504 or 510) (see Plasmid Constructs
below). In the reporter plasmid, the firefly luciferase gene was
under the control of a minimal thymidine kinase promoter downstream
from the upstream activating sequences (UAS). Forty eight hours after
cotransfection in SK-Hep-1 cells, the luciferase activity was
determined by chemiluminescence assay as described by the manufacturer
(Promega Corp., Madison, WI).
Coimmunoprecipitations
Human stable transfected RPE-FGF cells (2 x
106) were rinsed in cold PBS, scraped,
pelleted, and incubated for 30 min at 4 C in 200 µl lysis buffer
(RIPA buffer: 50 mM Tris-HCl, pH 7.5, 150 mM
NaCl, 10 mM MgCl2, 1% Triton X-100,
0.1% SDS, 0.5% deoxycholic acid, 1 mM DTT) plus protease
and phosphatase inhibitors (10 mM
Na3VO4, 1 µg/ml
leupeptin, 1 µg/ml aprotinin, 0.1 mg/ml PMSF, 50 mM NaF,
50 mM ß-glycerophosphate, 100 µg/ml
N-tosyl-L-phenylalanine
chloromethyl ketone, 50 µg/ml
N-tosyl-L-lysine chloromethyl ketone,
100 µg/ml soybean trypsin inhibitor). The DTT was omitted in
experiments under nonreducing conditions. Cell debris was removed by
centrifugation at 10,000 x g for 10 min at 4 C.
Extracts were precleared with 50 µl of a mix of protein A and protein
G-Sepharose beads (Amersham Pharmacia Biotech, Uppsala,
Sweden) for 1 h at 4 C. Nonimmune serum (1:100) was added and the
mix remained 1 h at 4 C on a rotating wheel. After centrifugation
for 15 sec at 10,000 x g, the supernatant was
incubated for 2 h at 4 C with the primary antibody (rabbit
polyclonal antihuman FIF antibodies (1:200) or monoclonal antihuman
FGF-2 antibodies (Upstate Biotechnology, Inc., Lake
Placid, NY) (1:200) and 50 µl of protein A and protein G-Sepharose
beads. The beads were washed four times with RIPA buffer and twice with
PBS. The immobilized proteins were released by boiling in Laemmli
sample buffer and then analyzed by SDS-PAGE. Under conditions in which
the protein of interest comigrated with either the heavy or light chain
of immunoglobulins, proteins were heated at 75 C in Laemmli sample
buffer devoid of ß-mercaptoethanol and DTT. The rabbit polyclonal
antihuman FGF-2 antibodies used were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).
Protein Production and Purification
The 18- and 24-kDa FGF-2 were produced and purified on a
heparin-Sepharose column by a FPLC (Amersham Pharmacia Biotech) as described previously (21). The His-FIF 504-, His-FIF
510-, and His-FIF C1-tagged proteins were produced using the inducible
pET expression system (pET-15b, Novagen, Madison, WI). in E.
coli BL21(DE3)pLysS and purified on a nickel-agarose column
(Ni-NTA, QIAGEN, Chatsworth, CA). For equilibration and
elution, 20 mM and 200 mM
imidazole, respectively, were used. An aliquot of each fraction was
analyzed in a SDS 10% polyacrylamide gel. The protein purity of the
fractions used was always higher than 95% as estimated by Coomassie
blue staining. Protein concentrations were determined by BCA assay
(Pierce Chemical Co., Rockford, IL).
In Vitro Interaction
The His-tagged recombinant FIF 504 and 510 purified on a nickel
agarose column (0.5 ml of FIF-NTA agarose = 100 µg of bound FIF)
were incubated with 20 µg of purified 18- or 24-kDa FGF-2 isoforms in
TKM50 (50 mM Tris-HCl, pH 7.4, 50 mM KCl, 5
mM MgCl2). After washing with 20
volumes of TKM100 (TKM50 but 100 mM KCl), the bound factors
were eluted with 0.5 ml of TKI (TKM100 + 1 M imidazole).
The eluted proteins were separated on SDS 12% acrylamide gel and
visualized by Coomassie blue staining. For FIF interaction with
biotinylated FGF-2, 200 ng of biotinylated FGF-2 were immobilized on 5
µl of streptavidin-coated-magnetic beads (Dynal, Oslo,
Norway) (33) and then incubated at 4 C in a Tween buffer (15
mM Tris-HCl, pH 7.5, 150 mM NaCl, 5
mM MgCl2, 0.1% Tween-20) with
50500 ng of each purified full-length His-FIF protein or the
truncated form, His-FIF C1. After three washes with Tween buffer, the
bound proteins were eluted in SDS-sample buffer and analyzed by Western
blotting.
Northern Blot Analysis
A multiple tissue Northern blot, with polyadenylated mRNA
extracted from human cells of various tissues (MTN, CLONTECH Laboratories, Inc. Palo Alto, CA), was probed with the FIF 504
cDNA labeled with 32P-dATP using a
nick-translation kit (Promega Corp.).
Cell Types and Culture Conditions
Human retinal pigmentary epithelial cells (RPE) and human skin
fibroblasts were isolated and grown (
4 passages) as previously
described (24). Human RPE-FGF cells correspond to RPE cells stably
transfected with pREP-hFGF2 plasmid (see Plasmid Constructs)
and grow as RPE cells. Pheochromocytoma rat cells (PC12, kindly
provided by M. Weber) were grown in RPMI-1640 medium with 10% horse
serum and 5% FCS. The rat smooth muscle cells (SMC, kindly provided by
C. Vagner) were cultivated in DMEM with 10% calf serum. Cell lines
provided from ATCC, SK-Hep-1 (HTB 52), HeLa (CCL2), and
NIH-3T3 (HB-11601), were grown in DMEM with 10% FCS, whereas COS-7
cells (CRL 1654) and MCF-7 (HTB 22) were grown in DMEM with 5% FCS.
All cells were cultivated in 5% CO2 at 37 C.
Production and Immunopurification of FIF Antibodies
The sera of two rabbits were collected after three injections of
100 µg His-FIF 510 recombinant protein. To immunopurify antibodies, 5
mg of FIF 510 were linked to 500 µl of Affi gel-10 (Bio-Rad Laboratories, Inc.) in 0.1 M MOPS buffer, pH 7.5.
The unsaturated sites were blocked by ethanolamine before loading onto
a column. Ten milliliters of anti-His-FIF serum were added to the
column in a cyclic way and then washed with PBS/0.2% Triton, PBS/0.2%
Tween-20, and PBS. The immunoglobulins were eluted with 0.2
M glycine, pH 2.8/0.1% BSA and immediately neutralized
with 2 M Tris-HCl, pH 8. Protein concentrations were
determined by BCA assay (Pierce Chemical Co.).
DNA Transfection and Western Immunoblot Analysis
COS-7 monkey cells were transfected by the
diethylami-noethyl-dextran method as described previously (8). For
better separation of proteins in the size range of 40 to 60 kDa, we
used 10% SDS PAGE with Tris pH 8.3 in place of pH 8.8 (Fig. 7A
).
SK-Hep-1 and HeLa cells were transfected by the use of Lipofectin
(Life Technologies, Inc.) as described by the
manufacturer. Cell extracts were treated for immunoblotting as
previously described (29). FGF-2 and FIF proteins were immunodetected
using rabbit polyclonal anti-human-FGF-2 antibodies (Santa Cruz Biotechnology, Inc., 1:1000 dilution) and rabbit polyclonal
antihuman-FIF antibodies (1:1000, see above), respectively. The blotted
proteins were detected by ECL (Amersham Pharmacia Biotech)
using either donkey peroxidase-linked antirabbit IgG or sheep
peroxidase-linked antimouse IgG (Amersham Pharmacia Biotech).
In Situ Immunocytochemistry
COS-7 cells grown on coverslips were transfected with pSG5 504
and pSG5 C1 (2 µg/ml) and prepared for immunofluorescence microscopy
as previously described (8). They were viewed under a fluorescence
microscope (Leica Corp., Deerfield, IL) and photographed
using 400 ASA HP5 film (Ilford).
In Vitro Translation
pSG5504 and pSG5510 plasmids, encoding FIF 504 and 510,
respectively, were digested by BglII and translated in the
presence of 35S methionine (Amersham Pharmacia Biotech) as previously described (8). Translation
products were analyzed by SDS-PAGE (10%) followed by autoradiography
of dried gels.
Plasmid Constructs
The details of each construct have been grouped in Table 1
. The PCR primers used to generate some
constructs are shown in Table 2
. Full
construction details are available upon request. pACT, pACT2, and pAS2
plasmids derive from MATCHMAKER (CLONTECH Laboratories, Inc.) two-hybrid vectors. The cloning of FGF-2 into pAS2 and
pACT is described elsewhere (33). The pET-15b vector (Novagen) was used
for protein production in E. coli. pET-FGF 155 and pET FGF
210 encoding the 18-kDa and 24-kDa isoforms of FGF-2, respectively,
were constructed as described previously (33). For production of the
different His-tagged FIF proteins, pET 15b constructs are described in
Table 1
. The details regarding the construction of the pREP-hFGF2
plasmid, which expresses the CUG13- and AUG-initiated FGF-2 isoforms,
is described elsewhere (66). pSG5 FIF 504 and pSG5 FIF 510 were
constructed to analyze FIF protein in mammalian cells and for in
vitro translation experiments. pTHM3-FIF 510 and pTHM3-FIF 504
were designed to test the two-hybrid system in mammalian cells. The
corresponding reporter gene plasmid was derived from the SV40-pFlash
(SynapSys), which was modified through three successive insertions of a
double-stranded oligonucleotide containing a UAS recognition sequence
(sense oligo-sense: 5'-GCGGAGTACTGTCCTCCGGAGCTCG-3' with
SacI in bold; antisense oligo-antisense:
5'-AGCTGCGGAGTACTGTCCTCCGGAGC-TCGGATC-3'
(with a "sticky" degenerated SacI site, a wild-type
SacI, and a "sticky" BamHI site in
bold, respectively). After hybridization of the
complementary oligonucleotides, the double-stranded UAS fragment was
ligated into the SacI/BamHI sites of the
SV40-pFlash vector. The resulting construct was digested with
SacI/BamHI, and a second UAS fragment was
inserted. The procedure was repeated until three repeats had been
inserted. cDNA clones encoding mouse FGF-3 and human FGF-6 were kindly
provided by C. Dickson and F. Coulier, respectively.
 |
ACKNOWLEDGMENTS
|
---|
We thank M. Oulad-Abdelghani for excellent technical assistance.
We are grateful to Dr. Chambons laboratory for the production of one
of the FIF antisera and for the gift of the human hepatocyte cDNA
library.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Béatrix Bugler, U 397 INSERM, Institut Louis Bugnard, CHU Rangueil, Bât L3, 31 403 Toulouse Cedex 4, France. E-mail: bugler{at}rangueil.inserm.fr
This research was supported by CNRS and INSERM grants, and
lAssociation de la Recherche contre le Cancer and La Fondation de la
Recherche Medicale.
1 These authors contributed equally to this work. 
2 A schematic representation of the different
truncated FIF constructs is shown in Fig. 8A
. 
3 The sequence data have been submitted to
the GenBank database under accession numbers AF229253 and
AF229254. 
Received for publication February 7, 2000.
Revision received July 31, 2000.
Accepted for publication August 8, 2000.
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