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FIF [Fibroblast Growth Factor-2 (FGF-2)-Interacting-Factor], a Nuclear Putatively Antiapoptotic Factor, Interacts Specifically with FGF-2

INSERM U 397 Institut Louis Bugnard 31403 Toulouse Cedex 4, France

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.

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