The entry of exogenous fibroblast growth factor 2 (FGF-2) to the cytosolic/nuclear compartment was studied and compared with the translocation mechanism used by FGF-1. To differentiate between external and endogenous growth factor, we used FGF-2 modified to contain a farnesylation signal, a CaaX-box. Because farnesylation occurs only in the cytosol and nucleoplasm, farnesylation of exogenous FGF-2-CaaX was taken as evidence that the growth factor had translocated across cellular membranes. We found that FGF-2 translocation occurred in endothelial cells and fibroblasts, which express FGF receptors, and that the efficiency of translocation was increased in the presence of heparin. Concomitantly with translocation, the 18-kDa FGF-2 was N-terminally cleaved to yield a 16-kDa form. Translocation of FGF-2 required PI3-kinase activity but not transport through the Golgi apparatus. Inhibition of endosomal acidification did not prevent translocation, whereas dissipation of the vesicular membrane potential completely blocked it. The data indicate that translocation occurs from intracellular vesicles containing proton pumps and that an electrical potential across the vesicle membrane is required. Translocation of both FGF-1 and FGF-2 occurred during most of G(1) but decreased shortly before the G(1)-->S transition. A common mechanism for FGF-1 and FGF-2 translocation into cells is postulated.

† These authors contributed equally to this work.Fibroblast growth factor 1 (FGF1) taken up by cells into endocytic vesicles can be translocated across vesicular membranes into the cytosol and the nucleus where it has a growth regulatory activity. Previously, leucine-rich repeat containing 59 (LRRC59) was identified as an intracellular binding partner of FGF1, but its biological role remained unknown. Here, we show that LRRC59 is strictly required for nuclear import of exogenous FGF1. siRNA-mediated depletion of LRRC59 did not inhibit the translocation of FGF1 into cytosol, but blocked the nuclear import of FGF1. We also found that an nuclear localization sequence (NLS) in FGF1, Ran GTPase, karyopherin-α1 (Kpnα1), and Kpnβ1 were required for nuclear import of FGF1. Nuclear import of exogenous FGF2, which depends on CEP57/Translokin, was independent of LRRC59, but was dependent on Kpnα1 and Kpnβ1, while the nuclear import of FGF1 was independent of CEP57. LRRC59 is a membrane-anchored protein that localizes to the endoplasmic reticulum (ER) and the nuclear envelope (NE). We found that LRRC59 possesses NLS-like sequences in its cytosolic part that can mediate nuclear import of soluble LRRC59 variants, and that the localization of LRRC59 to the NE depends on Kpnβ1. We propose that LRRC59 facilitates transport of cytosolic FGF1 through nuclear pores by interaction with Kpns and movement of LRRC59 along the ER and NE membranes. Fibroblast growth factors (FGFs) control cellular functions through an evolutionarily conserved signaling module operative in invertebrates and vertebrates. The FGF family of proteins, including the prototype members FGF1 and FGF2, are potent regulators of cell proliferation, differentiation, migration and survival. Most FGFs exert a biological action through binding to and activation of a family of specific cell surface, high-affinity, tyrosine kinase FGF receptors (FGFR1-4). Activated FGFR initiates downstream signaling cascades such as Ras/MAPK, phosphoinositide 3-kinase (PI3K)/AKT and phospholipase Cγ/protein kinase C (PKC) pathways, as well as endocytosis leading to downregulation of the receptor signaling (1-3). Beyond this, the exogenous FGF1 and FGF2 are able to reach the cell cytosol and nucleus and thus have a dual mode of signal transduction (4-6). The nuclear-translocated exogenous FGF1 or FGF2 has been shown to regulate cell growth and rRNA synthesis (7-14).Accumulating evidence indicates that several exogenous growth factors and cytokines as well as their receptors can translocate to the nucleus (reviewed in 6,15-18), which often involves poorly understood, unconventional transport steps. Also, the secretion of FGF1 and FGF2 from cells involves unconventional transport across cellular membranes. FGF1 and FGF2 are synthesized in the cytosol without a leader sequence and are secreted by non-classical routes bypassing the endoplasmic reticulum (ER)-Golgi secretory pathway. FGF1 is released by a mechanism that involves stress-induced formation of multiprotein complexes comprising S10...

Fibroblast growth factor-1 (FGF-1) has both extraand intracellular functions. To identify intracellular binding partners for FGF-1, we isolated proteins from U2OS human osteosarcoma cells interacting speci®c-ally with FGF-1. One of the isolated proteins was identi®ed as protein kinase CK2 (CK2). We here provide evidence that FGF-1 binds to both the catalytic a-subunit and to the regulatory b-subunit of CK2. The interaction between FGF-1 and CK2a and b was characterized by surface plasmon resonance, giving K D values of 0.4 6 0.3 and 1.2 6 0.2 mM, respectively. By using a novel assay for intracellular protein interaction, FGF-1 and CK2a are shown to interact in vivo. In vitro, FGF-1 and FGF-2 are phosphorylated by CK2, and the presence of FGF-1 or FGF-2 was found to enhance the autophosphorylation of CK2b. A correlation between the mitogenic potential of FGF-1 mutants and their ability to bind to CK2a was observed. The possible involvement of CK2 in the FGF-induced stimulation of DNA synthesis is discussed.

With the aim of identifying new intracellular binding partners for acidic fibroblast growth factor (aFGF), proteins from U2OS human osteosarcoma cells were adsorbed to immobilized aFGF. One of the adsorbed proteins is a member of the leucine-rich repeat protein family termed ribosome-binding protein p34 (p34). This protein has previously been localized to endoplasmic reticulum membranes and is thought to span the membrane with the N terminus on the cytosolic side. Confocal microscopy of cells transfected with Myc-p34 confirmed the endoplasmic reticulum localization, and Northern blotting determined p34 mRNA to be present in a multitude of different tissues. Cross-linking experiments indicated that the protein is present in the cell as a dimer. In vitro translated p34 was found to interact with maltose-binding protein-aFGF through its cytosolic coiled-coil domain. The interaction between aFGF and p34 was further characterized by surface plasmon resonance, giving a K D of 1.4 ؎ 0.3 M. Even though p34 interacted with mitogenic aFGF, it bound poorly to the non-mitogenic aFGF(K132E) mutant, indicating a possible involvement of p34 in intracellular signaling by aFGF.

Similarly to many protein toxins, the growth factors fibroblast growth factor 1 (FGF-1) and FGF-2 translocate from endosomes into the cytosol. It was recently found that certain toxins are dependent on cytosolic Hsp90 for efficient translocation across the endosomal membrane. We therefore investigated the requirement for Hsp90 in FGF translocation. We found that low concentrations of the specific Hsp90 inhibitors, geldanamycin and radicicol, completely blocked the translocation of FGF-1 and FGF-2 to the cytosol and the nucleus. The drugs did not interfere with the initial binding of FGF-1 to the growth factor receptors at the cell-surface or with the subsequent internalization of the growth factors into endosomes. The activation of known signaling cascades downstream of the growth factor receptors was also not affected by the drugs. The data indicate that the drugs block translocation from endosomes to the cytosol implying that Hsp90 is required for translocation of FGF-1 and FGF-2 across the endosomal membrane. FGF-13 and FGF-2 bind to and activate FGFRs on the surface of target cells. Several downstream signaling cascades such as the Ras/MAPK, phospholipase C-␥/PKC, and PI 3-kinase/Akt pathways are then initiated (1). FGF signaling is important in several cellular processes such as proliferation, angiogenesis, migration, survival, and differentiation (2, 3).In addition, FGF-1 and FGF-2 have the peculiar ability to translocate through the endosomal membrane into the cytosol and then be transported into the nucleus (4). Several laboratories have presented data indicating that the nuclear targeting is involved in the proliferation of cells. Thus, elimination of the N-terminal nuclear localization signal of FGF-1 resulted in considerably reduced mitogenic activity even if binding and activation of the receptors was not much changed (5). When FGF-1 was introduced into the cytosol as a fusion protein with diphtheria toxin, it stimulated DNA synthesis in cells lacking FGFRs arguing for an intracellular role of the growth factor (6). Similarly, nuclear FGF-2 has been shown to activate rDNA transcription and to be associated with cell proliferation (7, 8). Nuclear FGF-2 has also been proposed to be involved in the survival of carcinoma cells important for lung metastasis (9).We recently found that after endocytosis by the specific FGFRs, FGF-1 and -2 translocate from endosomes into the cytosol (10, 11). The positive inside membrane potential was found to be crucial for translocation across the endosomal membrane. Furthermore, we have shown that FGF-1 is then transported further from the cytosol to the nucleus by the concerted action of two nuclear localization signals (12). In the nucleus FGF-1 is phosphorylated by PKC␦ and then rapidly exported to the cytosol by a leptomycin B-sensitive protein, probably exportin-1, and subsequently degraded (13). We have previously shown that the translocation of FGF-1 is prevented by PI 3-kinase inhibitors suggesting that it is regulated by signaling events (14).Another class of pro...

Current methods to detect protein-protein interactions are either laborious to implement or not adaptable for mammalian systems or in vitro methods. By adding a peroxisomal targeting signal (PTS) onto one protein, binding partners lacking a targeting signal were cotransported into the peroxisomes in a "piggy-back" fashion, as visualized by confocal and electron microscopy. A fragment of colicin E2 and its tightly interacting immunity protein, ImmE2, were both expressed in the cytosol. When either one contained a PTS tag, both proteins were co-localized in the peroxisomes. The cytokine-independent survival kinase (CISK) containing a PTS tag was not efficiently targeted to the peroxisomes unless the Phox homology (PX) domain, attaching the protein to endosomal membranes, was removed. However, PTS-tagged CISK with deleted PX domain was able to direct 3-phosphoinositide-dependent protein kinase-1 (PDK-1) into the peroxisomes. This demonstrates that the two proteins interact in vivo. Mutating Ser 486 , which is phosphorylated in activated CISK, to Ala prevented the interaction, indicating that CISK and PDK-1 interact in a phosphorylation-dependent manner. The method therefore allows assessment of protein-protein interactions that depend on post-translational modifications that are cell-specific or dependent on the physiological state of the cell.

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