Skip to main content
PMC home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1996 Oct;16(10):5518–5526. doi: 10.1128/mcb.16.10.5518

A splicing variant of the RON transcript induces constitutive tyrosine kinase activity and an invasive phenotype.

C Collesi 1, M M Santoro 1, G Gaudino 1, P M Comoglio 1
PMCID: PMC231551  PMID: 8816464

Abstract

The Ron tyrosine kinase receptor shares with the members of its subfamily (Met and Sea) a unique functional feature: the control of cell dissociation, motility, and invasion of extracellular matrices (scattering). The mature Ron protein is a heterodimer of disulfide-linked alpha and beta chains, originated by proteolytic cleavage of a single-chain precursor of 185 kDa. In a human gastric cancer cell line (KATO-III), we found abnormal accumulation of an uncleaved single-chain protein (delta-Ron) of 165 kDa; this molecule is encoded by a transcript differing from the full-length RON mRNA by an in-frame deletion of 49 amino acids in the beta-chain extracellular domain. The deleted transcript originates by an alternatively spliced cassette exon of 147 bp, flanked by two short introns. The delta-Ron tyrosine kinase is constitutively activated by disulfide-linked intracellular oligomerization because it contains an uneven number of cysteine residues. Oligomerization and constitutive tyrosine phosphorylation of the full-size Ron was obtained by site-directed mutagenesis of a single cysteine residue in the region encoded by the cassette exon, mimicking that occurring in the delta-Ron isoform. Inhibition of thiol-mediated intermolecular disulfide bonding prevented delta-Ron oligomerization. The intracellular activation of Ron is followed by acquisition of invasive properties in vitro. These data (i) provide a novel molecular mechanism for posttranscriptional activation of a tyrosine kinase receptor protein and (ii) suggest a role for the Ron receptor in progression toward malignancy.

Full Text

The Full Text of this article is available as a PDF (544.5 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Alberini C. M., Bet P., Milstein C., Sitia R. Secretion of immunoglobulin M assembly intermediates in the presence of reducing agents. Nature. 1990 Oct 4;347(6292):485–487. doi: 10.1038/347485a0. [DOI] [PubMed] [Google Scholar]
  2. Albini A., Iwamoto Y., Kleinman H. K., Martin G. R., Aaronson S. A., Kozlowski J. M., McEwan R. N. A rapid in vitro assay for quantitating the invasive potential of tumor cells. Cancer Res. 1987 Jun 15;47(12):3239–3245. [PubMed] [Google Scholar]
  3. Andreadis A., Gallego M. E., Nadal-Ginard B. Generation of protein isoform diversity by alternative splicing: mechanistic and biological implications. Annu Rev Cell Biol. 1987;3:207–242. doi: 10.1146/annurev.cb.03.110187.001231. [DOI] [PubMed] [Google Scholar]
  4. 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]
  5. Breitbart R. E., Andreadis A., Nadal-Ginard B. Alternative splicing: a ubiquitous mechanism for the generation of multiple protein isoforms from single genes. Annu Rev Biochem. 1987;56:467–495. doi: 10.1146/annurev.bi.56.070187.002343. [DOI] [PubMed] [Google Scholar]
  6. Chomczynski P., Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987 Apr;162(1):156–159. doi: 10.1006/abio.1987.9999. [DOI] [PubMed] [Google Scholar]
  7. Cooper C. S., Park M., Blair D. G., Tainsky M. A., Huebner K., Croce C. M., Vande Woude G. F. Molecular cloning of a new transforming gene from a chemically transformed human cell line. Nature. 1984 Sep 6;311(5981):29–33. doi: 10.1038/311029a0. [DOI] [PubMed] [Google Scholar]
  8. Coulier F., Kumar R., Ernst M., Klein R., Martin-Zanca D., Barbacid M. Human trk oncogenes activated by point mutation, in-frame deletion, and duplication of the tyrosine kinase domain. Mol Cell Biol. 1990 Aug;10(8):4202–4210. doi: 10.1128/mcb.10.8.4202. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Don R. H., Cox P. T., Wainwright B. J., Baker K., Mattick J. S. 'Touchdown' PCR to circumvent spurious priming during gene amplification. Nucleic Acids Res. 1991 Jul 25;19(14):4008–4008. doi: 10.1093/nar/19.14.4008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Downward J., Yarden Y., Mayes E., Scrace G., Totty N., Stockwell P., Ullrich A., Schlessinger J., Waterfield M. D. Close similarity of epidermal growth factor receptor and v-erb-B oncogene protein sequences. Nature. 1984 Feb 9;307(5951):521–527. doi: 10.1038/307521a0. [DOI] [PubMed] [Google Scholar]
  11. Gaudino G., Follenzi A., Naldini L., Collesi C., Santoro M., Gallo K. A., Godowski P. J., Comoglio P. M. RON is a heterodimeric tyrosine kinase receptor activated by the HGF homologue MSP. EMBO J. 1994 Aug 1;13(15):3524–3532. doi: 10.1002/j.1460-2075.1994.tb06659.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Giordano S., Di Renzo M. F., Narsimhan R. P., Cooper C. S., Rosa C., Comoglio P. M. Biosynthesis of the protein encoded by the c-met proto-oncogene. Oncogene. 1989 Nov;4(11):1383–1388. [PubMed] [Google Scholar]
  13. Giordano S., Ponzetto C., Di Renzo M. F., Cooper C. S., Comoglio P. M. Tyrosine kinase receptor indistinguishable from the c-met protein. Nature. 1989 May 11;339(6220):155–156. doi: 10.1038/339155a0. [DOI] [PubMed] [Google Scholar]
  14. Huff J. L., Jelinek M. A., Borgman C. A., Lansing T. J., Parsons J. T. The protooncogene c-sea encodes a transmembrane protein-tyrosine kinase related to the Met/hepatocyte growth factor/scatter factor receptor. Proc Natl Acad Sci U S A. 1993 Jul 1;90(13):6140–6144. doi: 10.1073/pnas.90.13.6140. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Humphrey M. B., Bryan J., Cooper T. A., Berget S. M. A 32-nucleotide exon-splicing enhancer regulates usage of competing 5' splice sites in a differential internal exon. Mol Cell Biol. 1995 Aug;15(8):3979–3988. doi: 10.1128/mcb.15.8.3979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Hurtley S. M., Helenius A. Protein oligomerization in the endoplasmic reticulum. Annu Rev Cell Biol. 1989;5:277–307. doi: 10.1146/annurev.cb.05.110189.001425. [DOI] [PubMed] [Google Scholar]
  17. Itoh H., Hattori Y., Sakamoto H., Ishii H., Kishi T., Sasaki H., Yoshida T., Koono M., Sugimura T., Terada M. Preferential alternative splicing in cancer generates a K-sam messenger RNA with higher transforming activity. Cancer Res. 1994 Jun 15;54(12):3237–3241. [PubMed] [Google Scholar]
  18. Klausner R. D., Sitia R. Protein degradation in the endoplasmic reticulum. Cell. 1990 Aug 24;62(4):611–614. doi: 10.1016/0092-8674(90)90104-m. [DOI] [PubMed] [Google Scholar]
  19. Kobayashi H., Ohi H., Sugimura M., Shinohara H., Fujii T., Terao T. Inhibition of in vitro ovarian cancer cell invasion by modulation of urokinase-type plasminogen activator and cathepsin B. Cancer Res. 1992 Jul 1;52(13):3610–3614. [PubMed] [Google Scholar]
  20. Lee C. C., Yamada K. M. Alternatively spliced juxtamembrane domain of a tyrosine kinase receptor is a multifunctional regulatory site. Deletion alters cellular tyrosine phosphorylation pattern and facilitates binding of phosphatidylinositol-3-OH kinase to the hepatocyte growth factor receptor. J Biol Chem. 1995 Jan 13;270(2):507–510. doi: 10.1074/jbc.270.2.507. [DOI] [PubMed] [Google Scholar]
  21. Longati P., Bardelli A., Ponzetto C., Naldini L., Comoglio P. M. Tyrosines1234-1235 are critical for activation of the tyrosine kinase encoded by the MET proto-oncogene (HGF receptor). Oncogene. 1994 Jan;9(1):49–57. [PubMed] [Google Scholar]
  22. Martin-Zanca D., Oskam R., Mitra G., Copeland T., Barbacid M. Molecular and biochemical characterization of the human trk proto-oncogene. Mol Cell Biol. 1989 Jan;9(1):24–33. doi: 10.1128/mcb.9.1.24. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Medico E., Mongiovi A. M., Huff J., Jelinek M. A., Follenzi A., Gaudino G., Parsons J. T., Comoglio P. M. The tyrosine kinase receptors Ron and Sea control "scattering" and morphogenesis of liver progenitor cells in vitro. Mol Biol Cell. 1996 Apr;7(4):495–504. doi: 10.1091/mbc.7.4.495. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Mondino A., Giordano S., Comoglio P. M. Defective posttranslational processing activates the tyrosine kinase encoded by the MET proto-oncogene (hepatocyte growth factor receptor). Mol Cell Biol. 1991 Dec;11(12):6084–6092. doi: 10.1128/mcb.11.12.6084. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Neilson K. M., Friesel R. E. Constitutive activation of fibroblast growth factor receptor-2 by a point mutation associated with Crouzon syndrome. J Biol Chem. 1995 Nov 3;270(44):26037–26040. doi: 10.1074/jbc.270.44.26037. [DOI] [PubMed] [Google Scholar]
  26. Nelson K. K., Green M. R. Splice site selection and ribonucleoprotein complex assembly during in vitro pre-mRNA splicing. Genes Dev. 1988 Mar;2(3):319–329. doi: 10.1101/gad.2.3.319. [DOI] [PubMed] [Google Scholar]
  27. Oda T., Kanai Y., Oyama T., Yoshiura K., Shimoyama Y., Birchmeier W., Sugimura T., Hirohashi S. E-cadherin gene mutations in human gastric carcinoma cell lines. Proc Natl Acad Sci U S A. 1994 Mar 1;91(5):1858–1862. doi: 10.1073/pnas.91.5.1858. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Park M., Dean M., Kaul K., Braun M. J., Gonda M. A., Vande Woude G. Sequence of MET protooncogene cDNA has features characteristic of the tyrosine kinase family of growth-factor receptors. Proc Natl Acad Sci U S A. 1987 Sep;84(18):6379–6383. doi: 10.1073/pnas.84.18.6379. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Pelham H. R. Control of protein exit from the endoplasmic reticulum. Annu Rev Cell Biol. 1989;5:1–23. doi: 10.1146/annurev.cb.05.110189.000245. [DOI] [PubMed] [Google Scholar]
  30. Qiu F. H., Ray P., Brown K., Barker P. E., Jhanwar S., Ruddle F. H., Besmer P. Primary structure of c-kit: relationship with the CSF-1/PDGF receptor kinase family--oncogenic activation of v-kit involves deletion of extracellular domain and C terminus. EMBO J. 1988 Apr;7(4):1003–1011. doi: 10.1002/j.1460-2075.1988.tb02907.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Reddy P., Sparvoli A., Fagioli C., Fassina G., Sitia R. Formation of reversible disulfide bonds with the protein matrix of the endoplasmic reticulum correlates with the retention of unassembled Ig light chains. EMBO J. 1996 May 1;15(9):2077–2085. [PMC free article] [PubMed] [Google Scholar]
  32. Rodrigues G. A., Naujokas M. A., Park M. Alternative splicing generates isoforms of the met receptor tyrosine kinase which undergo differential processing. Mol Cell Biol. 1991 Jun;11(6):2962–2970. doi: 10.1128/mcb.11.6.2962. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Ronsin C., Muscatelli F., Mattei M. G., Breathnach R. A novel putative receptor protein tyrosine kinase of the met family. Oncogene. 1993 May;8(5):1195–1202. [PubMed] [Google Scholar]
  34. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Santoro M., Carlomagno F., Romano A., Bottaro D. P., Dathan N. A., Grieco M., Fusco A., Vecchio G., Matoskova B., Kraus M. H. Activation of RET as a dominant transforming gene by germline mutations of MEN2A and MEN2B. Science. 1995 Jan 20;267(5196):381–383. doi: 10.1126/science.7824936. [DOI] [PubMed] [Google Scholar]
  36. Siegel P. M., Dankort D. L., Hardy W. R., Muller W. J. Novel activating mutations in the neu proto-oncogene involved in induction of mammary tumors. Mol Cell Biol. 1994 Nov;14(11):7068–7077. doi: 10.1128/mcb.14.11.7068. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Sorokin A., Lemmon M. A., Ullrich A., Schlessinger J. Stabilization of an active dimeric form of the epidermal growth factor receptor by introduction of an inter-receptor disulfide bond. J Biol Chem. 1994 Apr 1;269(13):9752–9759. [PubMed] [Google Scholar]
  38. Takahashi M., Cooper G. M. ret transforming gene encodes a fusion protein homologous to tyrosine kinases. Mol Cell Biol. 1987 Apr;7(4):1378–1385. doi: 10.1128/mcb.7.4.1378. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Tanaka K., Watakabe A., Shimura Y. Polypurine sequences within a downstream exon function as a splicing enhancer. Mol Cell Biol. 1994 Feb;14(2):1347–1354. doi: 10.1128/mcb.14.2.1347. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Terranova V. P., Hujanen E. S., Loeb D. M., Martin G. R., Thornburg L., Glushko V. Use of a reconstituted basement membrane to measure cell invasiveness and select for highly invasive tumor cells. Proc Natl Acad Sci U S A. 1986 Jan;83(2):465–469. doi: 10.1073/pnas.83.2.465. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Terranova V. P., Hujanen E. S., Martin G. R. Basement membrane and the invasive activity of metastatic tumor cells. J Natl Cancer Inst. 1986 Aug;77(2):311–316. [PubMed] [Google Scholar]
  42. Watowich S. S., Yoshimura A., Longmore G. D., Hilton D. J., Yoshimura Y., Lodish H. F. Homodimerization and constitutive activation of the erythropoietin receptor. Proc Natl Acad Sci U S A. 1992 Mar 15;89(6):2140–2144. doi: 10.1073/pnas.89.6.2140. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Woolford J., McAuliffe A., Rohrschneider L. R. Activation of the feline c-fms proto-oncogene: multiple alterations are required to generate a fully transformed phenotype. Cell. 1988 Dec 23;55(6):965–977. doi: 10.1016/0092-8674(88)90242-5. [DOI] [PubMed] [Google Scholar]
  44. Xu R., Teng J., Cooper T. A. The cardiac troponin T alternative exon contains a novel purine-rich positive splicing element. Mol Cell Biol. 1993 Jun;13(6):3660–3674. doi: 10.1128/mcb.13.6.3660. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Molecular and Cellular Biology are provided here courtesy of Taylor & Francis

RESOURCES