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. 1997 Oct;17(10):5739–5747. doi: 10.1128/mcb.17.10.5739

Enhanced signaling and morphological transformation by a membrane-localized derivative of the fibroblast growth factor receptor 3 kinase domain.

M K Webster 1, D J Donoghue 1
PMCID: PMC232422  PMID: 9315632

Abstract

Fibroblast growth factor (FGF) receptors (FGFRs) are membrane-spanning tyrosine kinase receptors that mediate regulatory signals for cell proliferation and differentiation in response to FGFs. We have previously determined that the Lys650-->Glu mutation in the activation loop of the kinase domain of FGFR3, which is responsible for the lethal skeletal dysplasia thanatophoric dyplasia type II (TDII), greatly enhances the ligand-independent kinase activity of the receptor. Here, we demonstrate that expression of this construct induces a c-fos promoter construct approximately 10-fold but does not lead to proliferation or morphological transformation of NIH 3T3 cells. In contrast, the isolated kinase domain of activated FGFR3, targeted to the plasma membrane by a myristylation signal, is able to stimulate c-fos expression by 40-fold, induce proliferation of quiescent cells, and morphologically transform fibroblasts. This result suggests that the extracellular and transmembrane domains of FGFRs exert a negative regulatory influence on the activity of the kinase domain. Targeting of the activated kinase domain to either the cytoplasm or the nucleus does not significantly affect biological signaling, suggesting that signals from FGFR3 resulting in mitogenesis originate exclusively from the plasma membrane. Furthermore, our novel observation that expression of a highly activated FGFR3 kinase domain is able to morphologically transform fibroblasts suggests that dysregulation of FGFR3 has the potential to play a role in human neoplasia.

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Selected References

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  1. Adnane J., Gaudray P., Dionne C. A., Crumley G., Jaye M., Schlessinger J., Jeanteur P., Birnbaum D., Theillet C. BEK and FLG, two receptors to members of the FGF family, are amplified in subsets of human breast cancers. Oncogene. 1991 Apr;6(4):659–663. [PubMed] [Google Scholar]
  2. Aronheim A., Engelberg D., Li N., al-Alawi N., Schlessinger J., Karin M. Membrane targeting of the nucleotide exchange factor Sos is sufficient for activating the Ras signaling pathway. Cell. 1994 Sep 23;78(6):949–961. doi: 10.1016/0092-8674(94)90271-2. [DOI] [PubMed] [Google Scholar]
  3. Bargmann C. I., Hung M. C., Weinberg R. A. Multiple independent activations of the neu oncogene by a point mutation altering the transmembrane domain of p185. Cell. 1986 Jun 6;45(5):649–657. doi: 10.1016/0092-8674(86)90779-8. [DOI] [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. Bongarzone I., Monzini N., Borrello M. G., Carcano C., Ferraresi G., Arighi E., Mondellini P., Della Porta G., Pierotti M. A. Molecular characterization of a thyroid tumor-specific transforming sequence formed by the fusion of ret tyrosine kinase and the regulatory subunit RI alpha of cyclic AMP-dependent protein kinase A. Mol Cell Biol. 1993 Jan;13(1):358–366. doi: 10.1128/mcb.13.1.358. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Buss J. E., Der C. J., Solski P. A. The six amino-terminal amino acids of p60src are sufficient to cause myristylation of p21v-ras. Mol Cell Biol. 1988 Sep;8(9):3960–3963. doi: 10.1128/mcb.8.9.3960. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Chen C., Okayama H. High-efficiency transformation of mammalian cells by plasmid DNA. Mol Cell Biol. 1987 Aug;7(8):2745–2752. doi: 10.1128/mcb.7.8.2745. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Chesi M., Nardini E., Brents L. A., Schröck E., Ried T., Kuehl W. M., Bergsagel P. L. Frequent translocation t(4;14)(p16.3;q32.3) in multiple myeloma is associated with increased expression and activating mutations of fibroblast growth factor receptor 3. Nat Genet. 1997 Jul;16(3):260–264. doi: 10.1038/ng0797-260. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Curtis B. M., Widmer M. B., deRoos P., Qwarnstrom E. E. IL-1 and its receptor are translocated to the nucleus. J Immunol. 1990 Feb 15;144(4):1295–1303. [PubMed] [Google Scholar]
  10. Delli Bovi P., Curatola A. M., Kern F. G., Greco A., Ittmann M., Basilico C. An oncogene isolated by transfection of Kaposi's sarcoma DNA encodes a growth factor that is a member of the FGF family. Cell. 1987 Aug 28;50(5):729–737. doi: 10.1016/0092-8674(87)90331-x. [DOI] [PubMed] [Google Scholar]
  11. Friesel R. E., Maciag T. Molecular mechanisms of angiogenesis: fibroblast growth factor signal transduction. FASEB J. 1995 Jul;9(10):919–925. doi: 10.1096/fasebj.9.10.7542215. [DOI] [PubMed] [Google Scholar]
  12. Goldfarb M., Deed R., MacAllan D., Walther W., Dickson C., Peters G. Cell transformation by Int-2--a member of the fibroblast growth factor family. Oncogene. 1991 Jan;6(1):65–71. [PubMed] [Google Scholar]
  13. Golub T. R., Barker G. F., Lovett M., Gilliland D. G. Fusion of PDGF receptor beta to a novel ets-like gene, tel, in chronic myelomonocytic leukemia with t(5;12) chromosomal translocation. Cell. 1994 Apr 22;77(2):307–316. doi: 10.1016/0092-8674(94)90322-0. [DOI] [PubMed] [Google Scholar]
  14. Hattori Y., Odagiri H., Nakatani H., Miyagawa K., Naito K., Sakamoto H., Katoh O., Yoshida T., Sugimura T., Terada M. K-sam, an amplified gene in stomach cancer, is a member of the heparin-binding growth factor receptor genes. Proc Natl Acad Sci U S A. 1990 Aug;87(15):5983–5987. doi: 10.1073/pnas.87.15.5983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hayman M. J., Enrietto P. J. Cell transformation by the epidermal growth factor receptor and v-erbB. Cancer Cells. 1991 Aug;3(8):302–307. [PubMed] [Google Scholar]
  16. Horlick R. A., Stack S. L., Cooke G. M. Cloning, expression and tissue distribution of the gene encoding rat fibroblast growth factor receptor subtype 4. Gene. 1992 Oct 21;120(2):291–295. doi: 10.1016/0378-1119(92)90108-2. [DOI] [PubMed] [Google Scholar]
  17. 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]
  18. Hu Q., Milfay D., Williams L. T. Binding of NCK to SOS and activation of ras-dependent gene expression. Mol Cell Biol. 1995 Mar;15(3):1169–1174. doi: 10.1128/mcb.15.3.1169. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. 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]
  20. Jans D. A. Nuclear signaling pathways for polypeptide ligands and their membrane receptors? FASEB J. 1994 Aug;8(11):841–847. doi: 10.1096/fasebj.8.11.8070633. [DOI] [PubMed] [Google Scholar]
  21. Jaye M., Schlessinger J., Dionne C. A. Fibroblast growth factor receptor tyrosine kinases: molecular analysis and signal transduction. Biochim Biophys Acta. 1992 Jun 10;1135(2):185–199. doi: 10.1016/0167-4889(92)90136-y. [DOI] [PubMed] [Google Scholar]
  22. Jiang L. W., Schindler M. Nucleocytoplasmic transport is enhanced concomitant with nuclear accumulation of epidermal growth factor (EGF) binding activity in both 3T3-1 and EGF receptor reconstituted NR-6 fibroblasts. J Cell Biol. 1990 Mar;110(3):559–568. doi: 10.1083/jcb.110.3.559. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Johnson D. E., Williams L. T. Structural and functional diversity in the FGF receptor multigene family. Adv Cancer Res. 1993;60:1–41. doi: 10.1016/s0065-230x(08)60821-0. [DOI] [PubMed] [Google Scholar]
  24. Johnston C. L., Cox H. C., Gomm J. J., Coombes R. C. Fibroblast growth factor receptors (FGFRs) localize in different cellular compartments. A splice variant of FGFR-3 localizes to the nucleus. J Biol Chem. 1995 Dec 22;270(51):30643–30650. doi: 10.1074/jbc.270.51.30643. [DOI] [PubMed] [Google Scholar]
  25. Kamps M. P., Buss J. E., Sefton B. M. Mutation of NH2-terminal glycine of p60src prevents both myristoylation and morphological transformation. Proc Natl Acad Sci U S A. 1985 Jul;82(14):4625–4628. doi: 10.1073/pnas.82.14.4625. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Kan M., Wang F., To B., Gabriel J. L., McKeehan W. L. Divalent cations and heparin/heparan sulfate cooperate to control assembly and activity of the fibroblast growth factor receptor complex. J Biol Chem. 1996 Oct 18;271(42):26143–26148. doi: 10.1074/jbc.271.42.26143. [DOI] [PubMed] [Google Scholar]
  27. Kanai M., Göke M., Tsunekawa S., Podolsky D. K. Signal transduction pathway of human fibroblast growth factor receptor 3. Identification of a novel 66-kDa phosphoprotein. J Biol Chem. 1997 Mar 7;272(10):6621–6628. doi: 10.1074/jbc.272.10.6621. [DOI] [PubMed] [Google Scholar]
  28. Kobrin M. S., Yamanaka Y., Friess H., Lopez M. E., Korc M. Aberrant expression of type I fibroblast growth factor receptor in human pancreatic adenocarcinomas. Cancer Res. 1993 Oct 15;53(20):4741–4744. [PubMed] [Google Scholar]
  29. Kouhara H., Kurebayashi S., Hashimoto K., Kasayama S., Koga M., Kishimoto T., Sato B. Ligand-independent activation of tyrosine kinase in fibroblast growth factor receptor 1 by fusion with beta-galactosidase. Oncogene. 1995 Jun 15;10(12):2315–2322. [PubMed] [Google Scholar]
  30. Leevers S. J., Paterson H. F., Marshall C. J. Requirement for Ras in Raf activation is overcome by targeting Raf to the plasma membrane. Nature. 1994 Jun 2;369(6479):411–414. doi: 10.1038/369411a0. [DOI] [PubMed] [Google Scholar]
  31. Lobie P. E., Wood T. J., Chen C. M., Waters M. J., Norstedt G. Nuclear translocation and anchorage of the growth hormone receptor. J Biol Chem. 1994 Dec 16;269(50):31735–31746. [PubMed] [Google Scholar]
  32. Lorenzi M. V., Horii Y., Yamanaka R., Sakaguchi K., Miki T. FRAG1, a gene that potently activates fibroblast growth factor receptor by C-terminal fusion through chromosomal rearrangement. Proc Natl Acad Sci U S A. 1996 Aug 20;93(17):8956–8961. doi: 10.1073/pnas.93.17.8956. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. MacArthur C. A., Lawshé A., Shankar D. B., Heikinheimo M., Shackleford G. M. FGF-8 isoforms differ in NIH3T3 cell transforming potential. Cell Growth Differ. 1995 Jul;6(7):817–825. [PubMed] [Google Scholar]
  34. Maher P. A. Identification and characterization of a novel, intracellular isoform of fibroblast growth factor receptor-1(FGFR-1). J Cell Physiol. 1996 Nov;169(2):380–390. doi: 10.1002/(SICI)1097-4652(199611)169:2<380::AID-JCP18>3.0.CO;2-D. [DOI] [PubMed] [Google Scholar]
  35. Maher P. A. Nuclear Translocation of fibroblast growth factor (FGF) receptors in response to FGF-2. J Cell Biol. 1996 Jul;134(2):529–536. doi: 10.1083/jcb.134.2.529. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Marics I., Adelaide J., Raybaud F., Mattei M. G., Coulier F., Planche J., de Lapeyriere O., Birnbaum D. Characterization of the HST-related FGF.6 gene, a new member of the fibroblast growth factor gene family. Oncogene. 1989 Mar;4(3):335–340. [PubMed] [Google Scholar]
  37. Mohammadi M., Schlessinger J., Hubbard S. R. Structure of the FGF receptor tyrosine kinase domain reveals a novel autoinhibitory mechanism. Cell. 1996 Aug 23;86(4):577–587. doi: 10.1016/s0092-8674(00)80131-2. [DOI] [PubMed] [Google Scholar]
  38. Muenke M., Schell U. Fibroblast-growth-factor receptor mutations in human skeletal disorders. Trends Genet. 1995 Aug;11(8):308–313. doi: 10.1016/s0168-9525(00)89088-5. [DOI] [PubMed] [Google Scholar]
  39. Nakamoto T., Chang C. S., Li A. K., Chodak G. W. Basic fibroblast growth factor in human prostate cancer cells. Cancer Res. 1992 Feb 1;52(3):571–577. [PubMed] [Google Scholar]
  40. Park W. J., Bellus G. A., Jabs E. W. Mutations in fibroblast growth factor receptors: phenotypic consequences during eukaryotic development. Am J Hum Genet. 1995 Oct;57(4):748–754. [PMC free article] [PubMed] [Google Scholar]
  41. Peters K., Ornitz D., Werner S., Williams L. Unique expression pattern of the FGF receptor 3 gene during mouse organogenesis. Dev Biol. 1993 Feb;155(2):423–430. doi: 10.1006/dbio.1993.1040. [DOI] [PubMed] [Google Scholar]
  42. Podlecki D. A., Smith R. M., Kao M., Tsai P., Huecksteadt T., Brandenburg D., Lasher R. S., Jarett L., Olefsky J. M. Nuclear translocation of the insulin receptor. A possible mediator of insulin's long term effects. J Biol Chem. 1987 Mar 5;262(7):3362–3368. [PubMed] [Google Scholar]
  43. Prudovsky I. A., Savion N., LaVallee T. M., Maciag T. The nuclear trafficking of extracellular fibroblast growth factor (FGF)-1 correlates with the perinuclear association of the FGF receptor-1alpha isoforms but not the FGF receptor-1beta isoforms. J Biol Chem. 1996 Jun 14;271(24):14198–14205. doi: 10.1074/jbc.271.24.14198. [DOI] [PubMed] [Google Scholar]
  44. Prudovsky I., Savion N., Zhan X., Friesel R., Xu J., Hou J., McKeehan W. L., Maciag T. Intact and functional fibroblast growth factor (FGF) receptor-1 trafficks near the nucleus in response to FGF-1. J Biol Chem. 1994 Dec 16;269(50):31720–31724. [PubMed] [Google Scholar]
  45. Robbins J., Dilworth S. M., Laskey R. A., Dingwall C. Two interdependent basic domains in nucleoplasmin nuclear targeting sequence: identification of a class of bipartite nuclear targeting sequence. Cell. 1991 Feb 8;64(3):615–623. doi: 10.1016/0092-8674(91)90245-t. [DOI] [PubMed] [Google Scholar]
  46. Rodrigues G. A., Park M. Dimerization mediated through a leucine zipper activates the oncogenic potential of the met receptor tyrosine kinase. Mol Cell Biol. 1993 Nov;13(11):6711–6722. doi: 10.1128/mcb.13.11.6711. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Rousseau F., el Ghouzzi V., Delezoide A. L., Legeai-Mallet L., Le Merrer M., Munnich A., Bonaventure J. Missense FGFR3 mutations create cysteine residues in thanatophoric dwarfism type I (TD1). Hum Mol Genet. 1996 Apr;5(4):509–512. doi: 10.1093/hmg/5.4.509. [DOI] [PubMed] [Google Scholar]
  48. Sawyers C. L., Denny C. T. Chronic myelomonocytic leukemia: Tel-a-kinase what Ets all about. Cell. 1994 Apr 22;77(2):171–173. doi: 10.1016/0092-8674(94)90307-7. [DOI] [PubMed] [Google Scholar]
  49. Shu H. K., Pelley R. J., Kung H. J. Tissue-specific transformation by epidermal growth factor receptor: a single point mutation within the ATP-binding pocket of the erbB product increases its intrinsic kinase activity and activates its sarcomagenic potential. Proc Natl Acad Sci U S A. 1990 Dec;87(23):9103–9107. doi: 10.1073/pnas.87.23.9103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. 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]
  51. Su W. C., Kitagawa M., Xue N., Xie B., Garofalo S., Cho J., Deng C., Horton W. A., Fu X. Y. Activation of Stat1 by mutant fibroblast growth-factor receptor in thanatophoric dysplasia type II dwarfism. Nature. 1997 Mar 20;386(6622):288–292. doi: 10.1038/386288a0. [DOI] [PubMed] [Google Scholar]
  52. Takahashi J. A., Mori H., Fukumoto M., Igarashi K., Jaye M., Oda Y., Kikuchi H., Hatanaka M. Gene expression of fibroblast growth factors in human gliomas and meningiomas: demonstration of cellular source of basic fibroblast growth factor mRNA and peptide in tumor tissues. Proc Natl Acad Sci U S A. 1990 Aug;87(15):5710–5714. doi: 10.1073/pnas.87.15.5710. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Tavormina P. L., Shiang R., Thompson L. M., Zhu Y. Z., Wilkin D. J., Lachman R. S., Wilcox W. R., Rimoin D. L., Cohn D. H., Wasmuth J. J. Thanatophoric dysplasia (types I and II) caused by distinct mutations in fibroblast growth factor receptor 3. Nat Genet. 1995 Mar;9(3):321–328. doi: 10.1038/ng0395-321. [DOI] [PubMed] [Google Scholar]
  54. Theillet C., Le Roy X., De Lapeyrière O., Grosgeorges J., Adnane J., Raynaud S. D., Simony-Lafontaine J., Goldfarb M., Escot C., Birnbaum D. Amplification of FGF-related genes in human tumors: possible involvement of HST in breast carcinomas. Oncogene. 1989 Jul;4(7):915–922. [PubMed] [Google Scholar]
  55. Webster M. K., D'Avis P. Y., Robertson S. C., Donoghue D. J. Profound ligand-independent kinase activation of fibroblast growth factor receptor 3 by the activation loop mutation responsible for a lethal skeletal dysplasia, thanatophoric dysplasia type II. Mol Cell Biol. 1996 Aug;16(8):4081–4087. doi: 10.1128/mcb.16.8.4081. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Webster M. K., Donoghue D. J. Constitutive activation of fibroblast growth factor receptor 3 by the transmembrane domain point mutation found in achondroplasia. EMBO J. 1996 Feb 1;15(3):520–527. [PMC free article] [PubMed] [Google Scholar]
  57. Webster M. K., Donoghue D. J. FGFR activation in skeletal disorders: too much of a good thing. Trends Genet. 1997 May;13(5):178–182. doi: 10.1016/s0168-9525(97)01131-1. [DOI] [PubMed] [Google Scholar]
  58. 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]
  59. Yamaguchi F., Saya H., Bruner J. M., Morrison R. S. Differential expression of two fibroblast growth factor-receptor genes is associated with malignant progression in human astrocytomas. Proc Natl Acad Sci U S A. 1994 Jan 18;91(2):484–488. doi: 10.1073/pnas.91.2.484. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Yamanaka Y., Friess H., Buchler M., Beger H. G., Uchida E., Onda M., Kobrin M. S., Korc M. Overexpression of acidic and basic fibroblast growth factors in human pancreatic cancer correlates with advanced tumor stage. Cancer Res. 1993 Nov 1;53(21):5289–5296. [PubMed] [Google Scholar]
  61. Yan G., Fukabori Y., McBride G., Nikolaropolous S., McKeehan W. L. Exon switching and activation of stromal and embryonic fibroblast growth factor (FGF)-FGF receptor genes in prostate epithelial cells accompany stromal independence and malignancy. Mol Cell Biol. 1993 Aug;13(8):4513–4522. doi: 10.1128/mcb.13.8.4513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  62. Zhan X., Bates B., Hu X. G., Goldfarb M. The human FGF-5 oncogene encodes a novel protein related to fibroblast growth factors. Mol Cell Biol. 1988 Aug;8(8):3487–3495. doi: 10.1128/mcb.8.8.3487. [DOI] [PMC free article] [PubMed] [Google Scholar]
  63. Zhan X., Hu X., Friesel R., Maciag T. Long term growth factor exposure and differential tyrosine phosphorylation are required for DNA synthesis in BALB/c 3T3 cells. J Biol Chem. 1993 May 5;268(13):9611–9620. [PubMed] [Google Scholar]

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