Effects of mutations of a phosphorylation site in an exposed loop in acidic fibroblast growth factor.

Acidic fibroblast growth factor (aFGF) contains a phosphorylation site recognized by protein kinase C. A non-mitogenic mutant growth factor is devoid of this phosphorylation site. We have changed amino acids in and close to the phosphorylation site and studied the consequences of this for binding of the growth factor to high affinity receptors as well as to heparin. We have also studied the ability of the mutants to stimulate DNA synthesis and cell proliferation as well as phosphorylation of mitogen-activated protein kinase and the ability of the growth factor mutants to be transported to the nucleus. The results indicate that while the mutations strongly affect the ability of the growth factor to bind to heparin, they do not affect much the binding to the specific FGF receptors, activation of mitogen-activated protein kinase or transport of the growth factor to the nucleus. The mutations affect to various extents the ability of the growth factor to stimulate DNA synthesis and to induce cell multiplication. We find that phosphorylation of aFGF is not required for mitogenic activity. The data suggest that altered interaction of the growth factor with a cellular component different from the receptor, possibly a component in the nucleus, is the reason for the different mitogenicity of the different growth factor mutants.     

Requirement of phosphatidylinositol 3-kinase activity for translocation of exogenous aFGF to the cytosol and nucleus

Acidic fibroblast growth factor (aFGF) is a potent mitogen for many cells. Exogenous aFGF is able to enter the cytosol and nucleus of sensitive cells. There are indications that both activation of the receptor tyrosine kinase and translocation of aFGF to the nucleus are of importance for mitogenesis. However, the mechanism of transport of aFGF from the cell surface to the nucleus is poorly understood. In this work we demonstrate that inhibition of phosphatidylinositol (PI) 3-kinase by chemical inhibitors and by expression of a dominant negative mutant of PI 3-kinase blocks translocation of aFGF to the cytosol and nucleus. Translocation to the cytosol and nucleus was monitored by cell fractionation, by farnesylation of aFGF modified to contain a farnesylation signal, and by phosphorylation by protein kinase C of aFGF added externally to cells. If aFGF is fused to diphtheria toxin A-fragment, it can be artificially translocated from the cell surface to the cytoplasm by the diphtheria toxin pathway. Upon further incubation, the fusion protein enters the nucleus due to a nuclear localization sequence in aFGF. We demonstrate here that upon inhibition of PI 3-kinase the fusion protein remains in the cytosol. We also provide evidence that the phosphorylation status of the fusion protein does not regulate its nucleocytoplasmic distribution.     

FGF-1 and FGF-2 require the cytosolic chaperone Hsp90 for translocation into the cytosol and the cell nucleus

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.     

Phosphorylation-regulated nucleocytoplasmic trafficking of internalized fibroblast growth factor-1

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)delta. 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 PKCdelta 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.     

Phosphorylation of fibroblast growth factor (FGF) receptor 1 at Ser777 by p38 mitogen-activated protein kinase regulates translocation of exogenous FGF1 to the cytosol and nucleus

Exogenous fibroblast growth factor 1 (FGF1) signals through activation of transmembrane FGF receptors (FGFRs) but may also regulate cellular processes after translocation to the cytosol and nucleus of target cells. Translocation of FGF1 occurs across the limiting membrane of intracellular vesicles and is a regulated process that depends on the C-terminal tail of the FGFR. Here, we report that translocation of FGF1 requires activity of the alpha isoform of p38 mitogen-activated protein kinase (MAPK). FGF1 translocation was inhibited after chemical inhibition of p38 MAPK or after small interfering RNA knockdown of p38alpha. Translocation was increased after stimulation of p38 MAPK with anisomycin, mannitol, or H2O2. The activity level of p38 MAPK was not found to affect endocytosis or intracellular sorting of FGF1/FGFR1. Instead, we found that p38 MAPK regulates FGF1 translocation by phosphorylation of FGFR1 at Ser777. The FGFR1 mutation S777A abolished FGF1 translocation, while phospho-mimetic mutations of Ser777 to Asp or Glu allowed translocation to take place and bypassed the requirement for active p38 MAPK. Ser777 in FGFR1 was directly phosphorylated by p38alpha in a cell-free system. These data demonstrate a crucial role for p38alpha MAPK in the regulated translocation of exogenous FGF1 into the cytosol/nucleus, and they reveal a specific role for p38alpha MAPK-mediated serine phosphorylation of FGFR1.     

Evidence using a green fluorescent protein-glucocorticoid receptor chimera that the Ran/TC4 GTPase mediates an essential function independent of nuclear protein import

The Ran/TC4 GTPase is required for the nuclear accumulation of artificial karyophiles in permeabilized cell assays. To investigate Ran function in a physiologically intact setting using mammalian cells, we examined the effects of several Ran mutants on cell growth and on the nuclear translocation of a glucocorticoid receptor-green fluorescent protein fusion (GR-GFP). Glucocorticoid receptor is cytosolic in the absence of ligand, but translocates to the nucleus on binding the agonist dexamethasone. After transfection into baby hamster kidney cells (BHK21), GR-GFP was detectable in living cells by direct fluorescence microscopy. Addition of dexamethasone caused a rapid translocation of the chimeric protein from the cytosol into the nucleus (t1/2 approximately 5 min). Cotransfection with epitope-tagged, wild- type Ran led to expression of HA1-Ran that was approximately 1.6-fold higher than the level of the endogenous protein, but it had no deleterious effect on nuclear import of the GR-GFP. However, expression of the Ran mutants G19V, T24N, or a COOH-terminal deletion (delta C) mutant dramatically reduced the accumulation of GR-GFP in the nuclei. An L43E mutant of Ran was without significant effect on nuclear GR-GFP import. Identical results were obtained following micro-injection of recombinant Ran mutants into cells expressing GR-GFP. Significantly, all of the Ran mutants, including L43E, strongly inhibited cell growth. These results demonstrate the use of GR-GFP in real-time imaging of nuclear transport. They also show that multiple types of Ran mutant exert dominant effects on this process, and that normal Ran function requires cycling between the GTP- and GDP-bound states of the protein. Most importantly, the results with the L43E Ran mutant provide strong evidence that Ran mediates a function essential to cell viability that is independent of nuclear protein import.     

Translocation of exogenous FGF1 into cytosol and nucleus is a periodic event independent of receptor kinase activity

Fibroblast growth factor 1 (FGF1) has the property to become translocated from the extracellular space into the cell cytosol and nucleus. Membrane translocation of FGF1 occurs subsequent to endocytic uptake and is strictly FGF-receptor (FGFR) dependent. Here we have investigated the timing of FGF1 translocation in relation to FGFR1 signalling. We found that the translocation of FGF1 is a periodic event that occurs with 24 h intervals. Serum-starved cells translocated the growth factor with peak occurrences ~ 6 h, ~ 30 h, and ~ 54 h after the addition of FGF1. The periodic FGF1 translocation was totally independent of the FGFR1 tyrosine kinase activity as it proceeded unchanged when the kinase activity was chemically inhibited or the kinase domain was deleted. Furthermore, FGF1 translocation was not restricted to a particular phase of the cell cycle or dependent on cell cycle progression. The results demonstrate that the FGF1/FGFR1 complex constitutes a signalling module that independently of the receptor tyrosine kinase can convey a signal that initiates a strictly timed and periodic release of endocytosed FGF1 into the cytosol/nucleus.     

Structural requirements of FGF-1 for receptor binding and translocation into cells

FGF-1 binds to and activates specific transmembrane receptors (FGFRs) and is subsequently internalized and translocated to the interior of the cell. To elucidate the role of the receptor in the translocation process, we studied the effects of the elimination of distinct sites of the ligand-receptor interaction. On the basis of the structure of the FGF-1-FGFR1 complex, we substituted four key amino acid residues of FGF-1 from the FGF-receptor binding site with alanines, constructing four point mutants and one double mutant. We determined by in vivo assays in NIH 3T3 cells the ability of the mutants to bind to specific FGF receptors, to stimulate DNA synthesis, and to activate downstream signaling pathways. We found that correct binding to the receptor is necessary for optimal stimulation of DNA synthesis. All four single mutants became phosphorylated to different extents, indicating that they were translocated to the cytosol/nucleus with varying efficiency. This indicates that despite a low affinity for FGFR, translocation to the cytosol/nucleus can still occur. However, simultaneous substitution in two of the positions led to a total loss of biological activity of the growth factor and prevented its internalization, implying that there is only one strongly receptor-dependent, productive way of translocating FGF-1. We also found that the process of translocation did not correlate with the thermal stability of the protein. Additionally, we observed a clear negative correlation between the stability of the FGF-1 mutants and the efficiency of their phosphorylation, which strongly suggests that protein kinases prefer the unfolded state of the protein substrate.     

Acidic fibroblast growth factor autocrine system as a mediator of calcium-regulated parathyroid cell growth.

Both parathyroid hormone secretion and cell growth are negatively regulated by extracellular calcium in parathyroid cells. The mechanism of growth regulation by calcium has been unknown. Previously, we reported that clonal parathyroid cells (PT-r cells) bear two high affinity receptors for acidic fibroblast growth factor (aFGF) and that at least a subpopulation of the receptors with a higher molecular mass carries heparan sulfate (HS) glycosaminoglycan chains which give the receptor higher affinity (Sakaguchi, K., Yanagishita, M., Takeuchi, Y., and Aurbach, G. D. (1991) J. Biol. Chem. 266, 7270-7278). Here, I have found that the parathyroid cells expressed aFGF and that aFGF receptors with lower affinity apparently translocated in response to changing extracellular calcium concentrations. Expression of both aFGF mRNA and peptide was suppressed by calcium. Cells had more ligand-accessible receptors on the cell surface at lower calcium concentrations. This apparent translocation was temperature-dependent but independent of de novo protein synthesis. Heparin or HS glycosaminoglycans are a prerequisite for the FGF receptor encoded by flg gene to bind basic FGF (Yayon, A., Klagsbrun, M., Esko, J. D., Leder, P., and Ornitz, D. M. (1991) Cell 64, 841-848). In PT-r cells, major cellular HS proteoglycans redistribute between intracellular and extracellular compartments with more HS proteoglycans expressed on the cell surface at lower calcium concentrations (Takeuchi, Y., Sakaguchi, K., Yanagishita, M., Aurbach, G. D., and Hascall, V. C. (1990) J. Biol. Chem. 265, 13661-13668). However, this redistribution of HS proteoglycans cannot explain the difference in bindability of radiolabeled aFGF to its receptors in different calcium concentrations, since addition of heparin did not change the binding of radiolabeled aFGF to the receptors either at high or low calcium conditions. In concordance with the apparent translocation of aFGF receptors, thymidine incorporation was stimulated by decreasing extracellular calcium concentrations with further stimulation by added aFGF. Anti-aFGF antibody inhibited thymidine incorporation by more than 32% in the cells exposed to 0.05 mM Ca2+ shortly before adding [3H]thymidine, whereas the incorporation was not significantly affected by the antibody at 0.7 mM Ca2+. Cell growth was also stimulated by low calcium. Anti-aFGF antibody inhibited cell growth significantly only at low calcium concentrations. From these observations, an aFGF autocrine system including the apparent translocation of aFGF receptors may explain, if not entirely, the mechanism by which calcium regulates parathyroid cell growth.     

Nuclear import of exogenous FGF1 requires the ER-protein LRRC59 and the importins Kpnα1 and Kpnβ1

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.     

Protein folding does not prevent the nonclassical export of FGF1 and S100A13

Newly synthesized proteins are usually exported through the endoplasmic reticulum (ER) and Golgi due to the presence in their primary sequence of a hydrophobic signal peptide that is recognized by the ER translocation system. However, some secreted proteins lack a signal peptide and are exported independently of ER-Golgi. Fibroblast growth factor (FGF)1 is included in this group of polypeptides, as well as S100A13 that is a small calcium-binding protein critical for FGF1 export. Classically secreted proteins are transported into ER in their unfolded states. To determine the role of protein tertiary structure in FGF1 export through the cell membrane, we produced the chimeras of FGF1 and S100A13 with dihydrofolate reductase (DHFR). The specific DHFR inhibitor, aminopterin, prevents its unfolding. We found that aminopterin did not inhibit the release of FGF1:DHFR and S100A13:DHFR. Thus, FGF1 and S100A13 can be exported in folded conformation.     

Translocation of FGF-1 and FGF-2 across vesicular membranes occurs during G1-phase by a common mechanism

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.     

Analysis of endogenous and exogenous nuclear translocation of fibroblast growth factor-1 in NIH 3T3 cells.

Nuclear localization of fibroblast growth factors (FGF) have been reported by many laboratories. We demonstrate here that FGF-1, the precursor for acidic FGF contains a putative nuclear translocation sequence (NTS) NYKKPKL, which is able to direct the expression of the bacterial beta galactosidase (beta gal) gene to the nucleus of transfected NIH 3T3 cells. However, this NTS is unable to target either FGF-1 itself or a FGF-1-beta gal fusion protein into the nucleus, suggesting that FGF-1 may contain an additional sequence which prevents endogenously expressed FGF-1 from being translocated into the nucleus. Indeed, when FGF-1 was fused to the NTS derived from the yeast histone 2B gene, the chimeric construct also failed to be transported into the nucleus either by itself or as a beta gal fusion protein. Interestingly, when 125I-FGF-1 was used to stimulate quiescent NIH 3T3 cells, a significant amount of internalized 125I-FGF-1 (approximately 10%) was found within the nucleus and the nuclear localization of FGF-1 through the exogenous pathway could be significantly reduced by suramin, an inhibitor of the interaction of FGF-1 with its receptor. These data suggest that while FGF-1 contains a NTS, nuclear translocation requires an exogenous and not an endogenous pathway.     

Expression of acidic fibroblast growth factor cDNA confers growth advantage and tumorigenesis to Swiss 3T3 cells.

Acidic fibroblast growth factor (aFGF), a polypeptide with a mol. wt of approximately 16,000, is a potent mitogen for a variety of cells and shares 55% amino acid sequence identity with basic FGF. The recent isolation of three new oncogenes which share 35-45% amino acid sequence similarity with the FGFs suggests that the coding sequences for the FGFs themselves may be oncogenic under certain circumstances. To test this hypothesis, we cotransfected 3T3 NR6 cells with factors expressing the aFGF coding sequence and the bacterial neomycin gene. The aFGF produced by cotransfected cells was found only in the cellular homogenate and not in medium conditioned by the cells. Cells expressing aFGF grew to 10 times the density of control cells at saturation and were multilayered and disorganized, similar to transformed cells. The cotransfected cells do not grow in soft agar, but show enhanced soft agar growth relative to controls in the presence of added aFGF and heparin. The aFGF-producing cells formed small, non-progressive tumors when injected subcutaneously into nude mice. Our data suggest that expression of aFGF in NR6 cells results in enhanced growth, and that several traits characteristic of the transformed phenotype are partially expressed.     

Two nuclear localization signals required for transport from the cytosol to the nucleus of externally added FGF-1 translocated into cells

Externally added FGF-1 is transported into the nucleus of cells. It was earlier shown that FGF-1 contains an N-terminal nuclear localization signal (NLS) implicated in the stimulation of DNA synthesis. We here provide evidence that FGF-1 contains a second putative NLS (NLS2), which is located near the C-terminus. It is a bipartite NLS consisting of two clusters of lysines separated by a spacer of 10 amino acids. A fusion protein of GFP and the bipartite NLS was more efficiently transported into the nucleus than GFP alone, indicating that it can act as an NLS in the living cell. FGF-1 mutated in the N-terminal NLS (NLS1) or in the first cluster of the bipartite NLS2 bound to heparin and FGF receptors and activated downstream signaling similarly to the wild-type growth factor. Mutations in the second cluster of NLS2 resulted in impaired interaction with heparin and reduced stability. When radiolabeled FGF-1 with mutated NLS1 or the first lysine cluster of NLS2 was added to NIH/3T3 cells, it was translocated into the cytosol, but not transported efficiently to the nucleus. Phosphorylation of FGF-1 occurs normally in the nucleus, and while wild-type FGF-1 was phosphorylated after addition to cells, the NLS mutants were not. It therefore appears that both NLS1 and NLS2 are important for efficient transport of FGF-1 to the nucleus. Stimulation of DNA synthesis by FGF-1 with mutations in both NLSs was reduced considerably indicating that efficient transport to the nucleus may be involved in the stimulation of DNA synthesis.     

Alternative splicing in fibroblast growth factor receptor 2 is associated with induced epithelial-mesenchymal transition in rat bladder carcinoma cells.

We described previously that acidic fibroblast growth factor (aFGF), but not basic fibroblast growth factor (bFGF), can induce the rat carcinoma cell line NBT-II to undergo a rapid and reversible transition from epithelial to mesenchymal phenotype (EMT). We now find that NBT-II EMT is stimulated by keratinocyte growth factor (KGF) in cells grown at low density. Accordingly, a high-affinity receptor showing 98% homology to mouse FGF receptor 2b/KGF receptor was cloned and sequenced from NBT-II cells. Northern analysis indicated that mRNA for FGF receptor 2b/KGF receptor was drastically down-regulated within 1 wk in aFGF-induced mesenchymal NBT-II cells. This decrease coincided with an up-regulation of FGF receptor 2c/Bek, a KGF-insensitive, alternatively spliced form of FGF receptor 2b/KGF receptor. Functional studies confirmed that KGF could not maintain EMT induction on mesenchymal NBT-II cells. FGF receptor 1 and FGF receptor 2c/Bek could also support EMT induction when transfected into NBT-II cells in response to aFGF or bFGF. Such transfected cells could bind bFGF as well as aFGF. Therefore, EMT can be induced through different FGF receptors, but EMT may also regulate FGF receptor expression itself.     

Relevance of partially structured states in the non-classical secretion of acidic fibroblast growth factor

Acidic fibroblast growth factor (aFGF) is a signal peptide-less protein that is secreted into the extracellular compartment as part of a multiprotein release complex, consisting of aFGF, S100A13 (a calcium binding protein), and a 40 kDa (p40) form of synaptotagmin (Syt1), a protein that participates in the docking of a variety of secretory vesicles. p40 Syt1, and specifically its C2A domain, is believed to play a major role in the non-classical secretion of the aFGF release complex mediated by the interaction of aFGF and p40 Syt1with the phospholipids of the cell membrane inner leaflet. In the present study, we investigate the structural characteristics of aFGF and the C2A domain of p40 Syt1 under acidic conditions, using a variety of biophysical techniques including multidimensional NMR spectroscopy. Urea-induced equilibrium unfolding (at pH 3.4) of both aFGF and the C2A domain are non-cooperative and proceed with the accumulation of stable intermediate states. 1-Anilino-8-napthalene sulfonate (ANS) binding and size-exclusion chromatography results suggest that both aFGF and the C2A domain exist as partially structured states under acidic conditions (pH 3.4). Limited trypsin digestion analysis and 1H-15N chemical shift perturbation data reveal that the flexibility of certain portions of the protein backbone is increased in the partially structured state(s) of aFGF. The residues that are perturbed in the partially structured state(s) in aFGF are mostly located at the N- and C-terminal ends of the protein. In marked contrast, most of the interactions stabilizing the native secondary structure are preserved in the partially structured state of the C2A domain. Isothermal titration calorimetry data indicate that the binding affinity between aFGF and the C2A domain is significantly enhanced at pH 3.4. In addition, both aFGF and the C2A domain exhibit much higher lipid binding affinity in their partially structured states. The translocation of the multiprotein FGF release complex across the membrane appears to be facilitated by the formation of partially structured states of aFGF and the C2A domain of p40 Syt1.     

Protein translocation into mitochondria

The biogenesis of mitochondria requires the translocation of most mitochondrial proteins across two biological membranes. Mitochondrial preproteins are synthesized in the cytosol carrying targeting information, that is recognized by specific receptor proteins. The precursor polypeptides are transported across both mitochondrial membranes via three large integral membrane protein complexes forming specialized preprotein translocases. A soluble protein complex in the matrix provides the ATP-dependent translocation force, responsible for the movement and unfolding of the bulk polypeptide chain. After the removal of the targeting sequence, imported proteins fold into their native conformation with the help of chaperone proteins in the mitochondrial matrix.