Membrane interactions of diphtheria toxin analyzed using in vitro synthesized mutants.

Journal of Biological Chemistry

Falnes

Rapak³

et al. 1995

Acidic fibroblast growth factor (aFGF) added externally to cells has been proposed to enter the nucleus and stimulate DNA synthesis, but it has remained controversial whether or not exogenous aFGF has the capability of crossing cellular membranes. To test this, a novel principle to study translocation of proteins to the cytosol was developed by fusing a C-terminal farnesylation signal, a CAAX tag (C ‫؍‬ Cys, A ‫؍‬ an aliphatic amino acid, and X ‫؍‬ any amino acid), onto aFGF. Farnesylation is only known to occur in the cytosol and possibly in the nucleus. When incubated with NIH3T3 cells overnight, about one-third of the cell-associated, CAAX-tagged growth factor was farnesylated, indicating that efficient translocation had taken place. Binding to specific FGF receptors was required for translocation to occur. Part of the farnesylated growth factor was found in the nuclear fraction. The data indicate that CAAX-tagged aFGF added externally to cells is able to cross cellular membranes and enter the cytosol and the nucleus.

Section: Methodsmentioning

confidence: 99%

Translocation to Cytosol of Exogenous, CAAX-tagged Acidic Fibroblast Growth Factor

Journal of Biological Chemistry

Falnes

Rapak³

et al. 1995

“…For in vitro transcription and translation, plasmid DNA was linearized downstream of the encoding gene and transcribed with T3 RNA polymerase as described previously (McGill et al, 1989). The mRNA was precipitated with ethanol and dissolved in H 2 O containing 10 mM dithiothreitol (DTT) and 0.1 U/l RNasin.…”

Section: In Vitro Transcription and Translationmentioning

confidence: 99%

Phosphorylation-regulated Nucleocytoplasmic Trafficking of Internalized Fibroblast Growth Factor-1

Nilsen

Wesche

et al. 2005

MBoC

Fibroblast growth factor-1 (FGF-1), which stimulates cell growth, differentiation, and migration, is capable of crossing cellular membranes to reach the cytosol and the nucleus in cells containing specific FGF receptors. The cell entry process can be monitored by phosphorylation of the translocated FGF-1. We present evidence that phosphorylation of FGF-1 occurs in the nucleus by protein kinase C (PKC)␦. The phosphorylated FGF-1 is subsequently exported to the cytosol. A mutant growth factor where serine at the phosphorylation site is exchanged with glutamic acid, to mimic phosphorylated FGF-1, is constitutively transported to the cytosol, whereas a mutant containing alanine at this site remains in the nucleus. The export can be blocked by leptomycin B, indicating active and receptor-mediated nuclear export of FGF-1. Thapsigargin, but not leptomycin B, prevents the appearance of active PKC␦ in the nucleus, and FGF-1 is in this case phosphorylated in the cytosol. Leptomycin B increases the amount of phosphorylated FGF-1 in the cells by preventing dephosphorylation of the growth factor, which seems to occur more rapidly in the cytoplasm than in the nucleus. The nucleocytoplasmic trafficking of the phosphorylated growth factor is likely to play a role in the activity of internalized FGF-1.

“…In Vitro Transcription and Translation-Plasmid DNA was linearized downstream of the encoding gene and transcribed with T3 RNA polymerase as described (21 SDS-PAGE-Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate was carried out in 13.5% gels as described by Laemmli (23). After electrophoresis, the gel was fixed for 30 min in 27% methanol, 4% acetic acid and then incubated for 30 min in 1 M sodium salicylate, 2% glycerol, pH 5.8.…”

Section: Materials Media and Buffers-[mentioning

confidence: 99%

FGF-1 and FGF-2 Require the Cytosolic Chaperone Hsp90 for Translocation into the Cytosol and the Cell Nucleus

Wesche

Małecki

Journal of Biological Chemistry

et al. 2006

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...