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


    ABSTRACT
 TOP
 ABSTRACT
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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. 3–5). Five FGF-2 isoforms of 18, 22, 22.5, 24, and 34 kDa are synthesized through an alternative translational initiation process (6–8). 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) (11–13). Receptor activation stimulates intracellular mitogen-activated protein kinase (MAPK) and/or phospholipase C signaling pathways (14–16). But FGF-2 can also be internalized with both kinds of receptors into the cytoplasm (17–19) 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.


    RESULTS
 TOP
 ABSTRACT
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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. 1Go: 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. 1AGo). 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. 1AGo). 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 164–190 and aa 323–335 in Fig. 1BGo), a leucine-zipper motif (aa 370–391), and three overlapping heptad repeats of hydrophobic amino acids (aa 259–314). 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. 1AGo). 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 371–373, Fig. 1AGo); 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). {dagger}, 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.

 
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. 2AGo, 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. 2BGo and 7AGo).



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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.

 
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. 3Go). 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).

 
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. 4Go, 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. 4AGo, 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. 4BGo). 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. 4CGo, 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.

 
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. 5AGo, interaction test). FGF-2 alone did not bind to the resin (Fig. 5AGo, 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 1–3, 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 4–5). The same experiment was performed without DTT and with increasing concentrations of the truncated protein FIF C1 (lanes 6–9). 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.

 
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. 5BGo). FIF did not associate with streptavidin beads alone (Fig. 5BGo, 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. 5BGo, 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. 5BGo, lanes 6–9). 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. 5AGo) 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. 2Go).

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. 6Go). 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.

 
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. 7AGo). 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. 7AGo) as well as the in vitro-translated protein (Fig. 7BGo). 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. 7AGo). 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. 7CGo 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. 1BGo). This sequence is similar to the NLS of the p53 tumor suppressor protein (Fig. 7DGo). 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. 7EGo). 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 454–475 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. 7Go, 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. 8AGo, 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 (1–378), C4 (1–345), and C5 (1–311) 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 (1–426), N1 (72), and N1C2 (72–402), 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 (HR1–2-3), 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.

 
The LXXLL motif (where L is leucine and X is any amino acid, Fig. 1BGo) has been shown to mediate binding between transcriptional coactivators and nuclear receptors (35). This motif is present in FBR-1 (L102–L106). 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 (1–311) and the C6 (1–273) constructs, indicating that the sequence including threonine 274 to phenylalanine 311 represents a second important region for the FIF/FGF-2 interaction (Fig. 8BGo, 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 96–107 and 274–311, 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. 8Go, the results obtained with the N6C4 (231–345), C5 (1–311), and C6 (1–273) 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 (1–311) with C4 (1–345)]. 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 96–107) on transactivation. Likewise, one could assume that the sequence comprising residues 108–149, 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 231–273 can confer a transactivating potential of the FIF protein that is probably masked in the full-length protein.


    DISCUSSION
 TOP
 ABSTRACT
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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. 3Go) 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. 2Go). FGF-2 and FIF also coimmunoprecipitated reciprocally (Fig. 4Go). The direct association between FIF and FGF-2 was demonstrated by two reciprocal in vitro-binding assays (Fig. 5Go). 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. 6Go) as well as in several other species such as monkey, rat, mouse, and hamster (Fig. 7AGo). 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. 7Go, 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. 7EGo). 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. 4CGo). 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. 8Go). It is inferred from these data that aa 96–107 are required for FGF binding (FBR-1). A second centrally located region in the full-length FIF (aa 274–311) 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. 1Go and 8Go). 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 231–273 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 AR4–2J cells rendered these cells serum independent (61). The 24-kDa isoform was found to render HeLa cells resistant toward {gamma}-radiation (62), and elevated levels of this isoform were also in correlation in enhanced mouse intestinal epithelial cells survival after {gamma}-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
 TOP
 ABSTRACT
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 {lambda} 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 50–500 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. 7AGo). 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
pSG5–504 and pSG5–510 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 1Go. The PCR primers used to generate some constructs are shown in Table 2Go. 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 1Go. The details regarding the construction of the pREP-hFGF2 plasmid, which expresses the CUG1–3- 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.


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Table 1. Plasmid Constructs

 

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Table 2. PCR Primers

 


    ACKNOWLEDGMENTS
 
We thank M. Oulad-Abdelghani for excellent technical assistance. We are grateful to Dr. Chambon’s 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 l’Association de la Recherche contre le Cancer and La Fondation de la Recherche Medicale.

1 These authors contributed equally to this work. Back

2 A schematic representation of the different truncated FIF constructs is shown in Fig. 8AGo. Back

3 The sequence data have been submitted to the GenBank database under accession numbers AF229253 and AF229254. Back

Received for publication February 7, 2000. Revision received July 31, 2000. Accepted for publication August 8, 2000.


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