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. 1998 Nov 15;336(Pt 1):213–222. doi: 10.1042/bj3360213

Cloning of an intracellular protein that binds selectively to mitogenic acidic fibroblast growth factor.

E Kolpakova 1, A Wiedłocha 1, H Stenmark 1, O Klingenberg 1, P O Falnes 1, S Olsnes 1
PMCID: PMC1219860  PMID: 9806903

Abstract

In addition to its extracellular action, there is evidence that acidic fibroblast growth factor (aFGF) acts inside cells. To identify intracellular proteins interacting with aFGF, we screened a HeLa cell library in the yeast two-hybrid system using pLex-aFGF as a bait. A clone binding to aFGF, but not to the non-mitogenic mutant aFGF-K132E, was isolated and characterized. The insert contained an open reading frame corresponding to a novel protein of 42 kDa. The protein, termed aFGF intracellular binding protein (FIBP), is mainly hydrophilic and does not contain an N-terminal signal sequence. In vitro-translated FIBP bound specifically to a fusion protein of maltose-binding protein and aFGF. FIBP became post-translationally associated with microsomes added to the cell-free protein synthesizing system, and the membrane-associated protein bound aFGF with high efficiency. Immunoblots and fluorescence microscopy demonstrated that the protein is present in nuclei and, to a lesser extent, associated with mitochondria and other cytoplasmic membranes. The possibility is discussed that FIBP may be involved in the mitogenic action of aFGF.

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Selected References

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  1. Alterio J., Halley C., Brou C., Soussi T., Courtois Y., Laurent M. Characterization of a bovine acidic FGF cDNA clone and its expression in brain and retina. FEBS Lett. 1988 Dec 19;242(1):41–46. doi: 10.1016/0014-5793(88)80981-5. [DOI] [PubMed] [Google Scholar]
  2. Arfin S. M., Bradshaw R. A. Cotranslational processing and protein turnover in eukaryotic cells. Biochemistry. 1988 Oct 18;27(21):7979–7984. doi: 10.1021/bi00421a001. [DOI] [PubMed] [Google Scholar]
  3. Barondes S. H., Cooper D. N., Gitt M. A., Leffler H. Galectins. Structure and function of a large family of animal lectins. J Biol Chem. 1994 Aug 19;269(33):20807–20810. [PubMed] [Google Scholar]
  4. Basilico C., Moscatelli D. The FGF family of growth factors and oncogenes. Adv Cancer Res. 1992;59:115–165. doi: 10.1016/s0065-230x(08)60305-x. [DOI] [PubMed] [Google Scholar]
  5. Blobel G., Dobberstein B. Transfer of proteins across membranes. II. Reconstitution of functional rough microsomes from heterologous components. J Cell Biol. 1975 Dec;67(3):852–862. doi: 10.1083/jcb.67.3.852. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Boissel J. P., Kasper T. J., Bunn H. F. Cotranslational amino-terminal processing of cytosolic proteins. Cell-free expression of site-directed mutants of human hemoglobin. J Biol Chem. 1988 Jun 15;263(17):8443–8449. [PubMed] [Google Scholar]
  7. Bonnet H., Filhol O., Truchet I., Brethenou P., Cochet C., Amalric F., Bouche G. Fibroblast growth factor-2 binds to the regulatory beta subunit of CK2 and directly stimulates CK2 activity toward nucleolin. J Biol Chem. 1996 Oct 4;271(40):24781–24787. doi: 10.1074/jbc.271.40.24781. [DOI] [PubMed] [Google Scholar]
  8. Burgess W. H., Friesel R., Winkles J. A. Structure-function studies of FGF-1: dissociation and partial reconstitution of certain of its biological activities. Mol Reprod Dev. 1994 Sep;39(1):56–61. doi: 10.1002/mrd.1080390110. [DOI] [PubMed] [Google Scholar]
  9. Burgess W. H., Maciag T. The heparin-binding (fibroblast) growth factor family of proteins. Annu Rev Biochem. 1989;58:575–606. doi: 10.1146/annurev.bi.58.070189.003043. [DOI] [PubMed] [Google Scholar]
  10. Burgess W. H., Shaheen A. M., Ravera M., Jaye M., Donohue P. J., Winkles J. A. Possible dissociation of the heparin-binding and mitogenic activities of heparin-binding (acidic fibroblast) growth factor-1 from its receptor-binding activities by site-directed mutagenesis of a single lysine residue. J Cell Biol. 1990 Nov;111(5 Pt 1):2129–2138. doi: 10.1083/jcb.111.5.2129. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Burke C. J., Volkin D. B., Mach H., Middaugh C. R. Effect of polyanions on the unfolding of acidic fibroblast growth factor. Biochemistry. 1993 Jun 29;32(25):6419–6426. doi: 10.1021/bi00076a015. [DOI] [PubMed] [Google Scholar]
  12. Burrus L. W., Zuber M. E., Lueddecke B. A., Olwin B. B. Identification of a cysteine-rich receptor for fibroblast growth factors. Mol Cell Biol. 1992 Dec;12(12):5600–5609. doi: 10.1128/mcb.12.12.5600. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Chellaiah A. T., McEwen D. G., Werner S., Xu J., Ornitz D. M. Fibroblast growth factor receptor (FGFR) 3. Alternative splicing in immunoglobulin-like domain III creates a receptor highly specific for acidic FGF/FGF-1. J Biol Chem. 1994 Apr 15;269(15):11620–11627. [PubMed] [Google Scholar]
  14. Chien C. T., Bartel P. L., Sternglanz R., Fields S. The two-hybrid system: a method to identify and clone genes for proteins that interact with a protein of interest. Proc Natl Acad Sci U S A. 1991 Nov 1;88(21):9578–9582. doi: 10.1073/pnas.88.21.9578. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Christmas P., Callaway J., Fallon J., Jones J., Haigler H. T. Selective secretion of annexin 1, a protein without a signal sequence, by the human prostate gland. J Biol Chem. 1991 Feb 5;266(4):2499–2507. [PubMed] [Google Scholar]
  16. Crumley G., Bellot F., Kaplow J. M., Schlessinger J., Jaye M., Dionne C. A. High-affinity binding and activation of a truncated FGF receptor by both aFGF and bFGF. Oncogene. 1991 Dec;6(12):2255–2262. [PubMed] [Google Scholar]
  17. DiGabriele A. D., Lax I., Chen D. I., Svahn C. M., Jaye M., Schlessinger J., Hendrickson W. A. Structure of a heparin-linked biologically active dimer of fibroblast growth factor. Nature. 1998 Jun 25;393(6687):812–817. doi: 10.1038/31741. [DOI] [PubMed] [Google Scholar]
  18. Durfee T., Becherer K., Chen P. L., Yeh S. H., Yang Y., Kilburn A. E., Lee W. H., Elledge S. J. The retinoblastoma protein associates with the protein phosphatase type 1 catalytic subunit. Genes Dev. 1993 Apr;7(4):555–569. doi: 10.1101/gad.7.4.555. [DOI] [PubMed] [Google Scholar]
  19. Ensoli B., Buonaguro L., Barillari G., Fiorelli V., Gendelman R., Morgan R. A., Wingfield P., Gallo R. C. Release, uptake, and effects of extracellular human immunodeficiency virus type 1 Tat protein on cell growth and viral transactivation. J Virol. 1993 Jan;67(1):277–287. doi: 10.1128/jvi.67.1.277-287.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Evan G. I., Lewis G. K., Ramsay G., Bishop J. M. Isolation of monoclonal antibodies specific for human c-myc proto-oncogene product. Mol Cell Biol. 1985 Dec;5(12):3610–3616. doi: 10.1128/mcb.5.12.3610. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Guarente L. Yeast promoters and lacZ fusions designed to study expression of cloned genes in yeast. Methods Enzymol. 1983;101:181–191. doi: 10.1016/0076-6879(83)01013-7. [DOI] [PubMed] [Google Scholar]
  22. Hou J. Z., Kan M. K., McKeehan K., McBride G., Adams P., McKeehan W. L. Fibroblast growth factor receptors from liver vary in three structural domains. Science. 1991 Feb 8;251(4994):665–668. doi: 10.1126/science.1846977. [DOI] [PubMed] [Google Scholar]
  23. Imamura T., Engleka K., Zhan X., Tokita Y., Forough R., Roeder D., Jackson A., Maier J. A., Hla T., Maciag T. Recovery of mitogenic activity of a growth factor mutant with a nuclear translocation sequence. Science. 1990 Sep 28;249(4976):1567–1570. doi: 10.1126/science.1699274. [DOI] [PubMed] [Google Scholar]
  24. Imamura T., Oka S., Tanahashi T., Okita Y. Cell cycle-dependent nuclear localization of exogenously added fibroblast growth factor-1 in BALB/c 3T3 and human vascular endothelial cells. Exp Cell Res. 1994 Dec;215(2):363–372. doi: 10.1006/excr.1994.1353. [DOI] [PubMed] [Google Scholar]
  25. Jackson A., Tarantini F., Gamble S., Friedman S., Maciag T. The release of fibroblast growth factor-1 from NIH 3T3 cells in response to temperature involves the function of cysteine residues. J Biol Chem. 1995 Jan 6;270(1):33–36. doi: 10.1074/jbc.270.1.33. [DOI] [PubMed] [Google Scholar]
  26. Johnson D. E., Lee P. L., Lu J., Williams L. T. Diverse forms of a receptor for acidic and basic fibroblast growth factors. Mol Cell Biol. 1990 Sep;10(9):4728–4736. doi: 10.1128/mcb.10.9.4728. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Johnson D. E., Lu J., Chen H., Werner S., Williams L. T. The human fibroblast growth factor receptor genes: a common structural arrangement underlies the mechanisms for generating receptor forms that differ in their third immunoglobulin domain. Mol Cell Biol. 1991 Sep;11(9):4627–4634. doi: 10.1128/mcb.11.9.4627. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Klingenberg O., Widlocha A., Rapak A., Muñoz R., Falnes P., Olsnes S. Inability of the acidic fibroblast growth factor mutant K132E to stimulate DNA synthesis after translocation into cells. J Biol Chem. 1998 May 1;273(18):11164–11172. doi: 10.1074/jbc.273.18.11164. [DOI] [PubMed] [Google Scholar]
  29. Kozak M. An analysis of 5'-noncoding sequences from 699 vertebrate messenger RNAs. Nucleic Acids Res. 1987 Oct 26;15(20):8125–8148. doi: 10.1093/nar/15.20.8125. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Lin L. F., Mismer D., Lile J. D., Armes L. G., Butler E. T., 3rd, Vannice J. L., Collins F. Purification, cloning, and expression of ciliary neurotrophic factor (CNTF). Science. 1989 Nov 24;246(4933):1023–1025. doi: 10.1126/science.2587985. [DOI] [PubMed] [Google Scholar]
  31. Loukianov E. V., Olsnes S. New cloning opportunities with the use of noncomplementary SfiI adaptors. Biotechniques. 1993 Dec;15(6):980–982. [PubMed] [Google Scholar]
  32. Mach H., Volkin D. B., Burke C. J., Middaugh C. R., Linhardt R. J., Fromm J. R., Loganathan D., Mattsson L. Nature of the interaction of heparin with acidic fibroblast growth factor. Biochemistry. 1993 May 25;32(20):5480–5489. doi: 10.1021/bi00071a026. [DOI] [PubMed] [Google Scholar]
  33. Maciag T., Friesel R. E. Molecular mechanisms of fibroblast growth factor-1 traffick, signaling and release. Thromb Haemost. 1995 Jul;74(1):411–414. [PubMed] [Google Scholar]
  34. Mascarelli F., Fuhrmann G., Courtois Y. aFGF binding to low and high affinity receptors induces both aFGF and aFGF receptors dimerization. Growth Factors. 1993;8(3):211–233. doi: 10.3109/08977199309011024. [DOI] [PubMed] [Google Scholar]
  35. Mason I. J. The ins and outs of fibroblast growth factors. Cell. 1994 Aug 26;78(4):547–552. doi: 10.1016/0092-8674(94)90520-7. [DOI] [PubMed] [Google Scholar]
  36. McGill S., Stenmark H., Sandvig K., Olsnes S. Membrane interactions of diphtheria toxin analyzed using in vitro synthesized mutants. EMBO J. 1989 Oct;8(10):2843–2848. doi: 10.1002/j.1460-2075.1989.tb08431.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Mergia A., Tischer E., Graves D., Tumolo A., Miller J., Gospodarowicz D., Abraham J. A., Shipley G. D., Fiddes J. C. Structural analysis of the gene for human acidic fibroblast growth factor. Biochem Biophys Res Commun. 1989 Nov 15;164(3):1121–1129. doi: 10.1016/0006-291x(89)91785-3. [DOI] [PubMed] [Google Scholar]
  38. Michalak M., Milner R. E., Burns K., Opas M. Calreticulin. Biochem J. 1992 Aug 1;285(Pt 3):681–692. doi: 10.1042/bj2850681. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Mohammadi M., Honegger A. M., Rotin D., Fischer R., Bellot F., Li W., Dionne C. A., Jaye M., Rubinstein M., Schlessinger J. A tyrosine-phosphorylated carboxy-terminal peptide of the fibroblast growth factor receptor (Flg) is a binding site for the SH2 domain of phospholipase C-gamma 1. Mol Cell Biol. 1991 Oct;11(10):5068–5078. doi: 10.1128/mcb.11.10.5068. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Muesch A., Hartmann E., Rohde K., Rubartelli A., Sitia R., Rapoport T. A. A novel pathway for secretory proteins? Trends Biochem Sci. 1990 Mar;15(3):86–88. doi: 10.1016/0968-0004(90)90186-f. [DOI] [PubMed] [Google Scholar]
  41. Pineda-Lucena A., Jiménez M. A., Lozano R. M., Nieto J. L., Santoro J., Rico M., Giménez-Gallego G. Three-dimensional structure of acidic fibroblast growth factor in solution: effects of binding to a heparin functional analog. J Mol Biol. 1996 Nov 22;264(1):162–178. doi: 10.1006/jmbi.1996.0631. [DOI] [PubMed] [Google Scholar]
  42. Prats A. C., Vagner S., Prats H., Amalric F. cis-acting elements involved in the alternative translation initiation process of human basic fibroblast growth factor mRNA. Mol Cell Biol. 1992 Oct;12(10):4796–4805. doi: 10.1128/mcb.12.10.4796. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Rapak A., Falnes P. O., Olsnes S. Retrograde transport of mutant ricin to the endoplasmic reticulum with subsequent translocation to cytosol. Proc Natl Acad Sci U S A. 1997 Apr 15;94(8):3783–3788. doi: 10.1073/pnas.94.8.3783. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Schiestl R. H., Gietz R. D. High efficiency transformation of intact yeast cells using single stranded nucleic acids as a carrier. Curr Genet. 1989 Dec;16(5-6):339–346. doi: 10.1007/BF00340712. [DOI] [PubMed] [Google Scholar]
  45. Spivak-Kroizman T., Lemmon M. A., Dikic I., Ladbury J. E., Pinchasi D., Huang J., Jaye M., Crumley G., Schlessinger J., Lax I. Heparin-induced oligomerization of FGF molecules is responsible for FGF receptor dimerization, activation, and cell proliferation. Cell. 1994 Dec 16;79(6):1015–1024. doi: 10.1016/0092-8674(94)90032-9. [DOI] [PubMed] [Google Scholar]
  46. Stenmark H., Vitale G., Ullrich O., Zerial M. Rabaptin-5 is a direct effector of the small GTPase Rab5 in endocytic membrane fusion. Cell. 1995 Nov 3;83(3):423–432. doi: 10.1016/0092-8674(95)90120-5. [DOI] [PubMed] [Google Scholar]
  47. Stöckli K. A., Lottspeich F., Sendtner M., Masiakowski P., Carroll P., Götz R., Lindholm D., Thoenen H. Molecular cloning, expression and regional distribution of rat ciliary neurotrophic factor. Nature. 1989 Dec 21;342(6252):920–923. doi: 10.1038/342920a0. [DOI] [PubMed] [Google Scholar]
  48. Vojtek A. B., Hollenberg S. M., Cooper J. A. Mammalian Ras interacts directly with the serine/threonine kinase Raf. Cell. 1993 Jul 16;74(1):205–214. doi: 10.1016/0092-8674(93)90307-c. [DOI] [PubMed] [Google Scholar]
  49. Walter P., Gilmore R., Blobel G. Protein translocation across the endoplasmic reticulum. Cell. 1984 Aug;38(1):5–8. doi: 10.1016/0092-8674(84)90520-8. [DOI] [PubMed] [Google Scholar]
  50. Wiedlocha A., Madshus I. H., Mach H., Middaugh C. R., Olsnes S. Tight folding of acidic fibroblast growth factor prevents its translocation to the cytosol with diphtheria toxin as vector. EMBO J. 1992 Dec;11(13):4835–4842. doi: 10.1002/j.1460-2075.1992.tb05589.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Wiedłocha A., Falnes P. O., Madshus I. H., Sandvig K., Olsnes S. Dual mode of signal transduction by externally added acidic fibroblast growth factor. Cell. 1994 Mar 25;76(6):1039–1051. doi: 10.1016/0092-8674(94)90381-6. [DOI] [PubMed] [Google Scholar]
  52. Wiedłocha A., Falnes P. O., Rapak A., Klingenberg O., Muñoz R., Olsnes S. Translocation of cytosol of exogenous, CAAX-tagged acidic fibroblast growth factor. J Biol Chem. 1995 Dec 22;270(51):30680–30685. doi: 10.1074/jbc.270.51.30680. [DOI] [PubMed] [Google Scholar]
  53. Wiedłocha A., Falnes P. O., Rapak A., Muñoz R., Klingenberg O., Olsnes S. Stimulation of proliferation of a human osteosarcoma cell line by exogenous acidic fibroblast growth factor requires both activation of receptor tyrosine kinase and growth factor internalization. Mol Cell Biol. 1996 Jan;16(1):270–280. doi: 10.1128/mcb.16.1.270. [DOI] [PMC free article] [PubMed] [Google Scholar]

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