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1.
IUBMB Life ; 68(3): 242-51, 2016 Mar.
Article in English | MEDLINE | ID: mdl-26840910

ABSTRACT

Besides its classical mode of action through activation of specific receptors at the cell surface, fibroblast growth factor 1 (FGF1) can also cross the cellular membrane and translocate into the cytosol and further to the nucleus. The mechanism of this translocation is described partially, but the role of FGF1 inside the cell remains unknown. The aim of our work was to identify novel binding partners of FGF1 to predict its intracellular functions. We combined three methods of identification of such partners based on different principles: yeast two-hybrid screen and mass spectrometry (MS) analysis of complexes obtained by Tandem Affinity Purification (TAP) or by co-precipitation from cell lysate using recombinant FGF1. Altogether, we identified twenty novel intracellular proteins interacting with FGF1. For selected proteins, their direct interaction with FGF1 was confirmed by pull-down assays and SPR measurements. Interestingly, half of the proteins found are involved in processes related to cell viability, such as apoptosis, cell proliferation, and cell cycle regulation. Thus, our study indicates that the role of intracellular FGF1 is to protect the cell against stress conditions by providing an additional signal for cell survival, independently of receptor-activated signaling cascades.


Subject(s)
Fibroblast Growth Factor 1/metabolism , Animals , Apoptosis , Chemical Precipitation , Chromatography, Affinity , HEK293 Cells , Humans , Mice , NIH 3T3 Cells , Protein Interaction Maps , Two-Hybrid System Techniques
2.
PLoS One ; 9(3): e90687, 2014.
Article in English | MEDLINE | ID: mdl-24595027

ABSTRACT

Extracellular fibroblast growth factor 1 (FGF1) acts through cell surface tyrosine kinase receptors, but FGF1 can also act directly in the cell nucleus, as a result of nuclear import of endogenously produced, non-secreted FGF1 or by transport of extracellular FGF1 via endosomes and cytosol into the nucleus. In the nucleus, FGF1 can be phosphorylated by protein kinase C δ (PKCδ), and this event induces nuclear export of FGF1. To identify intracellular targets of FGF1 we performed affinity pull-down assays and identified nucleolin, a nuclear multifunctional protein, as an interaction partner of FGF1. We confirmed a direct nucleolin-FGF1 interaction by surface plasmon resonance and identified residues of FGF1 involved in the binding to be located within the heparin binding site. To assess the biological role of the nucleolin-FGF1 interaction, we studied the intracellular trafficking of FGF1. In nucleolin depleted cells, exogenous FGF1 was endocytosed and translocated to the cytosol and nucleus, but FGF1 was not phosphorylated by PKCδ or exported from the nucleus. Using FGF1 mutants with reduced binding to nucleolin and a FGF1-phosphomimetic mutant, we showed that the nucleolin-FGF1 interaction is critical for the intranuclear phosphorylation of FGF1 by PKCδ and thereby the regulation of nuclear export of FGF1.


Subject(s)
Cell Nucleus/metabolism , Fibroblast Growth Factor 1/metabolism , Phosphoproteins/metabolism , RNA-Binding Proteins/metabolism , Active Transport, Cell Nucleus , Animals , Cell Line , Fibroblast Growth Factor 1/analysis , Fibroblast Growth Factor 2/metabolism , HEK293 Cells , Humans , Mice , NIH 3T3 Cells , Phosphorylation , Nucleolin
3.
Traffic ; 13(5): 650-64, 2012 May.
Article in English | MEDLINE | ID: mdl-22321063

ABSTRACT

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 Kpnß1 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 Kpnß1, 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 Kpnß1. 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.


Subject(s)
Active Transport, Cell Nucleus , Endoplasmic Reticulum/metabolism , Fibroblast Growth Factor 1/metabolism , Membrane Proteins/physiology , alpha Karyopherins/metabolism , beta Karyopherins/metabolism , Biological Transport , Cell Nucleus/metabolism , Cytosol/metabolism , Humans , Nuclear Envelope/metabolism , Nuclear Localization Signals , Phosphorylation , Protein Kinase C-delta/metabolism , RNA, Small Interfering/metabolism , Subcellular Fractions/metabolism , ran GTP-Binding Protein/metabolism
4.
PLoS One ; 6(7): e21708, 2011.
Article in English | MEDLINE | ID: mdl-21779335

ABSTRACT

Endocytosis of tyrosine kinase receptors can influence both the duration and the specificity of the signal emitted. We have investigated the mechanisms of internalization of fibroblast growth factor receptor 3 (FGFR3) and compared it to that of FGFR1 which is internalized predominantly through clathrin-mediated endocytosis. Interestingly, we observed that FGFR3 was internalized at a slower rate than FGFR1 indicating that it may use a different endocytic mechanism than FGFR1. Indeed, after depletion of cells for clathrin, internalization of FGFR3 was only partly inhibited while endocytosis of FGFR1 was almost completely abolished. Similarly, expression of dominant negative mutants of dynamin resulted in partial inhibition of the endocytosis of FGFR3 whereas internalization of FGFR1 was blocked. Interfering with proposed regulators of clathrin-independent endocytosis such as Arf6, flotillin 1 and 2 and Cdc42 did not affect the endocytosis of FGFR1 or FGFR3. Furthermore, depletion of clathrin decreased the degradation of FGFR1 resulting in sustained signalling. In the case of FGFR3, both the degradation and the signalling were only slightly affected by clathrin depletion. The data indicate that clathrin-mediated endocytosis is required for efficient internalization and downregulation of FGFR1 while FGFR3, however, is internalized by both clathrin-dependent and clathrin-independent mechanisms.


Subject(s)
Clathrin/metabolism , Dynamins/metabolism , Endocytosis/physiology , Receptor, Fibroblast Growth Factor, Type 1/metabolism , Receptor, Fibroblast Growth Factor, Type 3/metabolism , Blotting, Western , Cell Line, Tumor , Cell Movement/genetics , Cell Movement/physiology , Clathrin/genetics , Dynamins/genetics , Endocytosis/genetics , Humans , Microscopy, Confocal , Receptor, Fibroblast Growth Factor, Type 1/genetics , Receptor, Fibroblast Growth Factor, Type 3/genetics , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction/genetics , Signal Transduction/physiology
5.
Exp Cell Res ; 317(7): 1005-15, 2011 Apr 15.
Article in English | MEDLINE | ID: mdl-21223966

ABSTRACT

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 24h 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.


Subject(s)
Active Transport, Cell Nucleus/physiology , Cell Nucleus/metabolism , Cytosol/metabolism , Fibroblast Growth Factor 1/metabolism , Receptor Protein-Tyrosine Kinases/metabolism , Animals , COS Cells , Chlorocebus aethiops , Fibroblast Growth Factor 1/genetics , Mice , NIH 3T3 Cells , Receptor Protein-Tyrosine Kinases/genetics , Receptor, Fibroblast Growth Factor, Type 1/genetics , Receptor, Fibroblast Growth Factor, Type 1/metabolism , Signal Transduction/physiology
6.
Mol Cancer Res ; 8(11): 1439-52, 2010 Nov.
Article in English | MEDLINE | ID: mdl-21047773

ABSTRACT

The fibroblast growth factor receptors (FGFR) play essential roles both during development and in the adult. Upon ligand binding, FGFRs induce intracellular signaling networks that tightly regulate key biological processes, such as cell proliferation, survival, migration, and differentiation. Deregulation of FGFR signaling can thus alter tissue homeostasis and has been associated with several developmental syndromes as well as with many types of cancer. In human cancer, FGFRs have been found to be deregulated by multiple mechanisms, including aberrant expression, mutations, chromosomal rearrangements, and amplifications. In this review, we will give an overview of the main FGFR alterations described in human cancer to date and discuss their contribution to cancer progression.


Subject(s)
Cell Transformation, Neoplastic/metabolism , Receptors, Fibroblast Growth Factor/metabolism , Adult , Animals , Humans , Neoplasms/enzymology , Neoplasms/metabolism , Neoplasms/pathology , Receptor Protein-Tyrosine Kinases/metabolism , Signal Transduction
7.
Biochimie ; 92(1): 71-80, 2010 Jan.
Article in English | MEDLINE | ID: mdl-19799962

ABSTRACT

Sialic acid-binding dwarf elder agglutinin (SEA) present only in rhizomes of the medicinal plant Sambucus ebulus L., was found to be a tetrameric glycoprotein consisting of two covalently-associated dimers of an enzymic A chain with rRNA N-glycosidase activity (EC 3.2.2.22) linked to a B chain with agglutinin properties. The lectin inhibited protein synthesis by a cell-free system and depurinated ribosomes. Cloning of the corresponding gene and molecular modeling of the deduced amino acid sequence demonstrated that SEA has a three-dimensional structure which resembles that reported for other two tetrameric type 2 RIPs from Sambucus (SNAI and SSA). The lectin agglutinated red blood cells and displayed sugar affinity for sialic acid residues apart from d-galactose, binding to the mucin-producing gut goblet cells. Since sialic acid is present in animal cells, especially in epithelial lining gut cells, but not in plants, SEA could play a role in the defense against insect attack. The nucleotide sequence reported in this paper has been submitted to the GenBank nucleotide database under accession number AM981401.


Subject(s)
Glycoside Hydrolases/metabolism , N-Acetylneuraminic Acid/metabolism , Nucleic Acids/metabolism , Plant Lectins/chemistry , Plant Lectins/metabolism , Sambucus , Amino Acid Sequence , Animals , COS Cells , Chemical Phenomena , Chlorocebus aethiops , Cloning, Molecular , Hemagglutination/drug effects , Models, Molecular , Molecular Sequence Data , Phylogeny , Plant Lectins/genetics , Plant Lectins/pharmacology , Protein Structure, Quaternary , Rhizome , Ribosome Inactivating Proteins, Type 2/chemistry , Ribosome Inactivating Proteins, Type 2/metabolism
8.
J Biol Chem ; 284(37): 25388-403, 2009 Sep 11.
Article in English | MEDLINE | ID: mdl-19574212

ABSTRACT

Human FGF1 (fibroblast growth factor 1) is a powerful signaling molecule with a short half-life in vivo and a denaturation temperature close to physiological. Binding to heparin increases the stability of FGF1 and is believed to be important in the formation of FGF1.fibroblast growth factor receptor (FGFR) active complex. In order to reveal the function of heparin in FGF1.FGFR complex formation and signaling, we constructed several FGF1 variants with reduced affinity for heparin and with diverse stability. We determined their biophysical properties and biological activities as well as their ability to translocate across cellular membranes. Our study showed that increased thermodynamic stability of FGF1 nicely compensates for decreased binding of heparin in FGFR activation, induction of DNA synthesis, and cell proliferation. By stepwise introduction of stabilizing mutations into the K118E (K132E) FGF1 variant that shows reduced affinity for heparin and is inactive in stimulation of DNA synthesis, we were able to restore the full mitogenic activity of this mutant. Our results indicate that the main role of heparin in FGF-induced signaling is to protect this naturally unstable protein against heat and/or proteolytic degradation and that heparin is not essential for a direct FGF1-FGFR interaction and receptor activation.


Subject(s)
Fibroblast Growth Factor 1/chemistry , Heparin/chemistry , Receptors, Fibroblast Growth Factor/chemistry , Animals , Binding Sites , Cell Proliferation , Humans , Mice , Mutagenesis, Site-Directed , NIH 3T3 Cells , Protein Binding , Protein Conformation , Protein Stability , Signal Transduction , Thermodynamics
9.
Biochemistry ; 48(30): 7209-18, 2009 Aug 04.
Article in English | MEDLINE | ID: mdl-19558187

ABSTRACT

After binding to its receptor on the surface of mammalian cells and subsequent endocytosis, FGF1 is translocated across the membrane into the cytosol. The growth factor is then further transported into the nucleus. In order to characterize more closely the translocation mechanism utilized by FGF1, we introduced additional amino acids into FGF1 to test the size dependence of the translocated substrate. We constructed mutants containing an increasing number of copies of the myc tag (1-13 copies) in a surface loop of the FGF1 molecule. All of the constructs bound to specific FGF receptors and to heparin and were taken up by endocytosis. However, only FGF1 mutants harboring up to three myc tags (53 amino acids) were translocated while mutants with five myc tags (77 amino acids) or more were not translocated through the membrane. We further showed that insertion of other, unrelated polypeptides into FGF1, i.e., 3xFLAG tag (22 amino acids) and streptavidin binding peptide (50 amino acids), was also translocated. Larger insertions into FGF1, like the CBP-SBP tag (82 amino acids) or ricin A-chain (272 amino acids), resulted in fusion proteins that failed to translocate. The presented data imply that it is possible to employ FGF1 to import various polypeptides into the cytosol and nucleus of cells. Furthermore, the strict size dependence of FGF1 fusion proteins in membrane translocation argues against simple leakage of FGF1 from ruptured endosomal membranes but rather points to a specific translocation apparatus involving a proteinaceous pore.


Subject(s)
Endosomes , Fibroblast Growth Factor 1/metabolism , Intracellular Membranes/metabolism , Recombinant Fusion Proteins/metabolism , Acetylcysteine/analogs & derivatives , Acetylcysteine/metabolism , Amino Acid Sequence , Animals , Cysteine Proteinase Inhibitors/metabolism , Endosomes/metabolism , Endosomes/ultrastructure , Fibroblast Growth Factor 1/chemistry , Fibroblast Growth Factor 1/genetics , Humans , Mice , Models, Molecular , Molecular Sequence Data , Mutagenesis, Site-Directed , NIH 3T3 Cells , Protein Structure, Tertiary , Protein Transport/physiology , Receptors, Fibroblast Growth Factor/genetics , Receptors, Fibroblast Growth Factor/metabolism , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Signal Transduction/physiology , Thermodynamics
10.
Mol Biol Cell ; 19(8): 3390-403, 2008 Aug.
Article in English | MEDLINE | ID: mdl-18480409

ABSTRACT

Endocytosis and targeting of growth factor receptors for lysosomal degradation have been associated with ubiquitination of the intracellular part of the receptors. To elucidate the role of receptor ubiquitination in internalization and sorting of fibroblast growth factor receptor (FGFR), we constructed several mutants of FGFR1 in which lysines, potential ubiquitination sites, were substituted for arginines. Substitution of all lysine residues in the intracellular part of FGFR1 resulted in inactivation of the tyrosine kinase domain of the receptor. However, several multilysine FGFR1 mutants, where up to 26 of 29 lysines in the intracellular part of the receptor were mutated, retained tyrosine kinase activity. The active multilysine mutants were poorly ubiquitinated, but internalized normally, indicating that ubiquitination of the receptor is not required for endocytosis. In contrast, degradation of the multilysine mutants was dramatically reduced as the mutants were inefficiently transported to lysosomes but rather sorted to recycling endosomes. The altered sorting resulted in sustained signaling. The duration of FGFR1 signaling seems to be tightly regulated by receptor ubiquitination and subsequent sorting to the lysosomes for degradation.


Subject(s)
Endocytosis , Endosomes/metabolism , Gene Expression Regulation , Lysosomes/metabolism , Mutation , Cell Line, Tumor , Cytoplasm/genetics , Down-Regulation , Humans , Lysine/chemistry , Models, Biological , Protein Transport , Receptor, Fibroblast Growth Factor, Type 1/genetics , Receptor, Fibroblast Growth Factor, Type 1/metabolism , Receptors, Fibroblast Growth Factor/metabolism , Ubiquitin/chemistry
11.
Mol Cell Biol ; 28(12): 4129-41, 2008 Jun.
Article in English | MEDLINE | ID: mdl-18411303

ABSTRACT

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.


Subject(s)
Gene Expression Regulation, Enzymologic , Receptor, Fibroblast Growth Factor, Type 1/physiology , Serine/chemistry , p38 Mitogen-Activated Protein Kinases/metabolism , Animals , COS Cells , Cell Nucleus/metabolism , Chlorocebus aethiops , Cricetinae , Humans , MAP Kinase Signaling System , Mice , Models, Biological , NIH 3T3 Cells , Phosphorylation , Protein Transport , Receptor, Fibroblast Growth Factor, Type 1/metabolism
12.
J Biol Chem ; 282(36): 26245-56, 2007 Sep 07.
Article in English | MEDLINE | ID: mdl-17616529

ABSTRACT

Receptor-bound and endocytosed fibroblast growth factor-1 (FGF-1) is able to cross the vesicle membrane and translocate to cytosol and nucleus. This suggests an intracellular role of FGF-1, which also signals by activating transmembrane FGF receptors. Phosphorylation of internalized FGF-1 by nuclear protein kinase C delta induces rapid export from the nuclei by a leptomycin B-sensitive pathway. In the present work, we have searched for and identified a Leu-rich nuclear export sequence (NES) at the C terminus of FGF-1 required for its nuclear export and able to confer nuclear export activity to a reporter protein in an in vivo system. Mutants where hydrophobic amino acids within the NES were exchanged for alanine exhibited reduced or abolished nuclear export. As demonstrated in co-immunoprecipitation experiments, a complex containing FGF-1, exportin-1, and its co-factor Ran-GTP, was formed in vitro. Formation of this complex in vivo was demonstrated by a peroxisomal targeting assay. Formation of the FGF-1-exportin-1-Ran-GTP complex in vitro as well as nuclear export of FGF-1 in vivo was dependent on phosphorylation of FGF-1, and it was abolished by leptomycin B. The FGF-1 NES was found to be situated along a beta-strand, which has not been reported before, since NESs usually are alpha-helical.


Subject(s)
Cell Nucleus/metabolism , Fibroblast Growth Factor 1/metabolism , Signal Transduction/physiology , Active Transport, Cell Nucleus/drug effects , Active Transport, Cell Nucleus/physiology , Amino Acid Substitution , Animals , Antibiotics, Antineoplastic/pharmacology , Cell Nucleus/genetics , Cell-Free System/metabolism , Endocytosis/drug effects , Endocytosis/physiology , Fatty Acids, Unsaturated/pharmacology , Fibroblast Growth Factor 1/genetics , Karyopherins/genetics , Karyopherins/metabolism , Mice , Multiprotein Complexes , Mutation, Missense , NIH 3T3 Cells , Nuclear Export Signals , Phosphorylation , Protein Kinase C-delta/genetics , Protein Kinase C-delta/metabolism , Protein Structure, Secondary , Receptors, Cytoplasmic and Nuclear/genetics , Receptors, Cytoplasmic and Nuclear/metabolism , Signal Transduction/drug effects , ran GTP-Binding Protein/genetics , ran GTP-Binding Protein/metabolism , Exportin 1 Protein
13.
Biochem Biophys Res Commun ; 357(1): 144-9, 2007 May 25.
Article in English | MEDLINE | ID: mdl-17407762

ABSTRACT

Shiga toxin has a protease-sensitive site in the disulfide loop region of the A-chain. Cleavage of this site by furin is essential for rapid intoxication of cells by Shiga toxin. We have here investigated whether in addition to the Arg-X-X-Arg sequence, there are other structural requirements in the disulfide loop region for furin cleavage. A toxin mutant (Shiga-2D toxin) still containing the consensus motif for cleavage by furin, but lacking ten amino acids in the disulfide loop, was generated. Trypsin was able to cleave Shiga-2D toxin in vitro, demonstrating that the protease-sensitive region is intact. However, Shiga-2D toxin was not efficiently cleaved by furin either in vitro or in vivo. Furthermore, unless it was precleaved with trypsin, Shiga-2D toxin was much less toxic than wild type Shiga toxin in LoVo cells expressing functional furin. In contrast, LoVo/neo cells lacking functional furin were unable to activate both wild type Shiga toxin and Shiga-2D toxin. In conclusion, an extended loop structure is required for furin-induced cleavage of Shiga toxin.


Subject(s)
Furin/chemistry , Furin/metabolism , Shiga Toxin/chemistry , Shiga Toxin/metabolism , Amino Acid Sequence , Furin/genetics , Molecular Sequence Data , Protein Binding , Shiga Toxin/genetics , Structure-Activity Relationship
14.
J Cell Physiol ; 212(1): 148-56, 2007 Jul.
Article in English | MEDLINE | ID: mdl-17311277

ABSTRACT

STAT transcription factors signal from the plasma membrane to the nucleus in response to growth factors and cytokines, but little is known about activation of STAT1 from intracellular sites. Here we show that transient transfection of COS cells with fibroblast growth factor receptors (FGFRs) led to ligand-independent phosphorylation of the receptors, including intracellular immature forms. FGF-independent activation of STAT1 was demonstrated at the Golgi apparatus where it was colocalized with FGFRs. Both FGFR1 and FGFR2 induced strong phosphorylation of STAT1 causing redistribution of the Golgi apparatus, while FGFR3 and FGFR4 induced less phosphorylation of STAT1 and little or no redistribution of the Golgi apparatus. Upon expression of a cytosolic mutant of FGFR4 lacking the transmembrane as well as the extracellular region (CytR4), STAT1 was phosphorylated and transferred to the nucleus. The results indicate that immature forms of FGFRs form incomplete signaling complexes on Golgi membranes trapping phospho-STAT1 on this organelle.


Subject(s)
Golgi Apparatus/metabolism , Receptors, Fibroblast Growth Factor/metabolism , STAT1 Transcription Factor/metabolism , Active Transport, Cell Nucleus/physiology , Animals , Breast Neoplasms/metabolism , COS Cells , Cell Line, Tumor , Cell Nucleus/metabolism , Chlorocebus aethiops , Gene Expression Regulation , Humans , Phosphorylation , Receptors, Fibroblast Growth Factor/genetics , STAT1 Transcription Factor/genetics , Transfection
15.
Biochemistry ; 45(51): 15338-48, 2006 Dec 26.
Article in English | MEDLINE | ID: mdl-17176056

ABSTRACT

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.


Subject(s)
Fibroblast Growth Factor 1/chemistry , Fibroblast Growth Factor 1/metabolism , Receptor, Fibroblast Growth Factor, Type 1/metabolism , Amino Acid Substitution/genetics , Animals , Fibroblast Growth Factor 1/genetics , Humans , Mice , Mutagenesis, Site-Directed , NIH 3T3 Cells , Phosphorylation , Protein Binding/genetics , Protein Folding , Protein Transport/genetics , Receptor, Fibroblast Growth Factor, Type 1/chemistry
16.
J Cell Sci ; 119(Pt 20): 4332-41, 2006 Oct 15.
Article in English | MEDLINE | ID: mdl-17003104

ABSTRACT

Members of the fibroblast growth factor family bind to one or more of the four closely related membrane-spanning FGF receptors. In addition to signaling through the receptors, exogenous FGF-1 and FGF-2 are endocytosed and translocated to the cytosol and nucleus where they stimulate RNA and DNA synthesis. Here we have studied the ability of the four FGF receptors to facilitate translocation of exogenous FGF-1 to the cytosol and nucleus. FGFR1 and FGFR4 were able to mediate translocation, whereas FGFR2 and FGFR3 completely lacked this ability. By analyzing mutant FGFRs we found that the tyrosine kinase domain could be deleted from FGFR1 without abolishing translocation, whereas the C-terminal tail of the FGFRs, constituted by approximately 50 amino acids downstream of the kinase domain, plays a crucial role in FGF-1 translocation. Three amino acids residues within the C-terminal tail were found to be of particular importance for translocation. For FGFR2, the two amino acid substitutions Q774M and P800H were sufficient to enable the receptor to support FGF-1 translocation. The results demonstrate a striking diversity in function of the four FGFRs determined by their C-terminal domain.


Subject(s)
Fibroblast Growth Factor 1/metabolism , Receptors, Fibroblast Growth Factor/metabolism , Amino Acid Sequence , Animals , Blotting, Western , COS Cells , Cattle , Chlorocebus aethiops , Electrophoresis, Polyacrylamide Gel , HeLa Cells , Humans , Kinetics , Mice , Molecular Sequence Data , Mutation/genetics , Phosphorylation , Protein Transport/physiology , Rats , Receptor, Fibroblast Growth Factor, Type 1/genetics , Receptor, Fibroblast Growth Factor, Type 1/metabolism , Receptor, Fibroblast Growth Factor, Type 1/physiology , Receptor, Fibroblast Growth Factor, Type 2/genetics , Receptor, Fibroblast Growth Factor, Type 2/metabolism , Receptor, Fibroblast Growth Factor, Type 2/physiology , Receptor, Fibroblast Growth Factor, Type 3/genetics , Receptor, Fibroblast Growth Factor, Type 3/metabolism , Receptor, Fibroblast Growth Factor, Type 3/physiology , Receptor, Fibroblast Growth Factor, Type 4/genetics , Receptor, Fibroblast Growth Factor, Type 4/metabolism , Receptor, Fibroblast Growth Factor, Type 4/physiology , Receptors, Fibroblast Growth Factor/genetics , Receptors, Fibroblast Growth Factor/physiology , Sequence Homology, Amino Acid
17.
Growth Factors ; 24(2): 111-9, 2006 Jun.
Article in English | MEDLINE | ID: mdl-16801131

ABSTRACT

The activating mutation FGFR3-R248C in the D2-D3 linker region of fibroblast growth factor receptor 3 leads as germline mutation to the neonatal lethal syndrome thanatophoric dysplasia type I (TD1). As somatic mutation it has been found in cancer. We introduced into the murine FGFR3 the mutation R242C that is orthologoues to the human mutation R248C. A strong reduction in binding of the 16 and 18 kDa forms of FGF1 to the mutant receptor was found, highlighting the importance of D2-D3 linker region of FGFR3 in determination of binding affinity to ligands. Another mutant, G374R, introduced into the murine FGFR3, is orthologoues to the human mutant FGFR3-G380R, and leads to achondroplasia (ACH). The binding of the 16 kDa and 18 kDa forms of FGF1 to this mutant receptor was the same as for wild-type FGFR3 in a cell-free system, but it was reduced in living cells. The data indicate a minor changes in conformation of FGFR3-G374R receptors at the cell surface that lead to reduced binding to FGF1.


Subject(s)
Fibroblast Growth Factor 1/metabolism , Receptor, Fibroblast Growth Factor, Type 3/metabolism , Achondroplasia/genetics , Achondroplasia/metabolism , Animals , Cells, Cultured , Gene Expression , Mice , Mutation , Receptor, Fibroblast Growth Factor, Type 3/genetics , Thanatophoric Dysplasia/genetics , Thanatophoric Dysplasia/metabolism
18.
J Biol Chem ; 281(16): 11405-12, 2006 Apr 21.
Article in English | MEDLINE | ID: mdl-16495214

ABSTRACT

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.


Subject(s)
Cell Nucleus/metabolism , Cytosol/metabolism , Fibroblast Growth Factor 1/physiology , Fibroblast Growth Factor 2/physiology , HSP90 Heat-Shock Proteins/metabolism , Animals , Benzoquinones , Cell Line , Cell Proliferation , Dose-Response Relationship, Drug , Electrophoresis, Polyacrylamide Gel , Endosomes/metabolism , Fibroblast Growth Factor 1/metabolism , Fibroblast Growth Factor 2/metabolism , Heparin/pharmacology , Humans , Lactams, Macrocyclic , Lactones/pharmacology , Lasers , MAP Kinase Signaling System , Macrolides/pharmacology , Mice , Microscopy, Confocal , Models, Biological , Monensin/pharmacology , NIH 3T3 Cells , Phosphorylation , Plasmids/metabolism , Protein Binding , Protein Biosynthesis , Protein Transport , Quinones/pharmacology , Signal Transduction , Subcellular Fractions , Time Factors , Transcription, Genetic
19.
Tidsskr Nor Laegeforen ; 125(18): 2477-9, 2005 Sep 22.
Article in Norwegian | MEDLINE | ID: mdl-16186864

ABSTRACT

Lately there have been several incidents with the plant protein ricin, for instance in the capitol building of the American Senate in February 2004. By focusing on this toxin, the media have spread a fear that ricin will be used in terrorist acts. In this article, we want to give a review of the qualities and potentials of the use of ricin. Ricin is a highly toxic plant protein that is lethal in small doses; it kills cells by inactivating ribosomes but has potential for medical use.


Subject(s)
Antineoplastic Agents , Biological Warfare , Bioterrorism , Chemical Warfare Agents , Ricin , Animals , Antineoplastic Agents/chemistry , Antineoplastic Agents/therapeutic use , Chemical Warfare Agents/chemistry , Chemical Warfare Agents/poisoning , Chemical Warfare Agents/toxicity , Humans , Ribosomes/drug effects , Ricin/chemistry , Ricin/poisoning , Ricin/therapeutic use , Ricin/toxicity
20.
J Mol Biol ; 352(4): 860-75, 2005 Sep 30.
Article in English | MEDLINE | ID: mdl-16126225

ABSTRACT

Fibroblast growth factor 1 (FGF-1) shows strong angiogenic, osteogenic and tissue-injury repair properties that might be relevant to medical applications. Since FGF-1 is partially unfolded at physiological temperature we decided to increase significantly its conformational stability and test how such an improvement will affect its biological function. Using an homology approach and rational strategy we designed two new single FGF-1 mutations: Q40P and S47I that appeared to be the most strongly stabilizing substitutions among those reported so far, increasing the denaturation temperature by 7.8 deg. C and 9.0 deg. C, respectively. As our goal was to produce highly stable variants of the growth factor, we combined these two mutations with five previously described stabilizing substitutions. The multiple mutants showed denaturation temperatures up to 27 deg. C higher than the wild-type and exhibited full additivity of the mutational effects. All those mutants were biologically competent in several cell culture assays, maintaining typical FGF-1 activities, such as binding to specific cell surface receptors and activation of downstream signaling pathways. Thus, we demonstrate that the low denaturation temperature of wild-type FGF-1 is not related to its fundamental cellular functions, and that FGF-1 action is not affected by its stability. A more detailed analysis of the biological behavior of stable FGF-1 mutants revealed that, compared with the wild-type, their mitogenic properties, as probed by the DNA synthesis assay, were significantly increased in the absence of heparin, and that their half-lives were extensively prolonged. We found that the biological action of the mutants was dictated by their susceptibility to proteases, which strongly correlated with the stability. Mutants which were much more resistant to proteolytic degradation always displayed a significant improvement in the half-life and mitogenesis. Our results show that engineered stable growth factor variants exhibit enhanced and prolonged activity, which can be advantageous in terms of the potential therapeutic applications of FGF-1.


Subject(s)
Fibroblast Growth Factor 1 , Protein Conformation , Animals , Enzyme Activation , Fibroblast Growth Factor 1/chemistry , Fibroblast Growth Factor 1/genetics , Fibroblast Growth Factor 1/metabolism , Hot Temperature , Humans , MAP Kinase Signaling System/physiology , Mice , Models, Molecular , Mutation , NIH 3T3 Cells , Phospholipase C gamma , Protein Denaturation , Protein Serine-Threonine Kinases/metabolism , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins c-akt , Receptors, Fibroblast Growth Factor/metabolism , Type C Phospholipases/metabolism , p38 Mitogen-Activated Protein Kinases/metabolism
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