Skip to main content
PMC home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1995 Mar;15(3):1613–1619. doi: 10.1128/mcb.15.3.1613

Mechanism of activation of the ret proto-oncogene by multiple endocrine neoplasia 2A mutations.

N Asai 1, T Iwashita 1, M Matsuyama 1, M Takahashi 1
PMCID: PMC230385  PMID: 7532281

Abstract

Transforming activity of the c-ret proto-oncogene with multiple endocrine neoplasia (MEN) 2A mutations was investigated by transfection of NIH 3T3 cells. Mutant c-ret genes driven by the simian virus 40 or cytomegalovirus promoter induced transformation with high efficiencies. The 170-kDa Ret protein present on the cell surface of transformed cells was highly phosphorylated on tyrosine and formed disulfide-linked homodimers. This result indicated that MEN 2A mutations induced ligand-independent dimerization of the c-Ret protein on the cell surface, leading to activation of its intrinsic tyrosine kinase. In addition to the MEN 2A mutations, we further introduced a mutation (lysine for asparaginic acid at codon 300 [D300K]) in a putative Ca(2+)-binding site of the cadherin-like domain. When c-ret cDNA with both MEN 2A and D300K mutations was transfected into NIH 3T3 cells, transforming activity drastically decreased. Western blot (immunoblot) analysis revealed that very little of the 170-kDa Ret protein with the D300K mutation was expressed in transfectants while expression of the 150-kDa Ret protein retained in the endoplasmic reticulum was not affected. This result also demonstrated that transport of the Ret protein to the plasma membrane is required for its transforming activity.

Full Text

The Full Text of this article is available as a PDF (571.5 KB).

Selected References

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

  1. Avantaggiato V., Dathan N. A., Grieco M., Fabien N., Lazzaro D., Fusco A., Simeone A., Santoro M. Developmental expression of the RET protooncogene. Cell Growth Differ. 1994 Mar;5(3):305–311. [PubMed] [Google Scholar]
  2. Carlson K. M., Dou S., Chi D., Scavarda N., Toshima K., Jackson C. E., Wells S. A., Jr, Goodfellow P. J., Donis-Keller H. Single missense mutation in the tyrosine kinase catalytic domain of the RET protooncogene is associated with multiple endocrine neoplasia type 2B. Proc Natl Acad Sci U S A. 1994 Feb 15;91(4):1579–1583. doi: 10.1073/pnas.91.4.1579. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. 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]
  4. Donis-Keller H., Dou S., Chi D., Carlson K. M., Toshima K., Lairmore T. C., Howe J. R., Moley J. F., Goodfellow P., Wells S. A., Jr Mutations in the RET proto-oncogene are associated with MEN 2A and FMTC. Hum Mol Genet. 1993 Jul;2(7):851–856. doi: 10.1093/hmg/2.7.851. [DOI] [PubMed] [Google Scholar]
  5. Edery P., Lyonnet S., Mulligan L. M., Pelet A., Dow E., Abel L., Holder S., Nihoul-Fékété C., Ponder B. A., Munnich A. Mutations of the RET proto-oncogene in Hirschsprung's disease. Nature. 1994 Jan 27;367(6461):378–380. doi: 10.1038/367378a0. [DOI] [PubMed] [Google Scholar]
  6. Eng C., Smith D. P., Mulligan L. M., Nagai M. A., Healey C. S., Ponder M. A., Gardner E., Scheumann G. F., Jackson C. E., Tunnacliffe A. Point mutation within the tyrosine kinase domain of the RET proto-oncogene in multiple endocrine neoplasia type 2B and related sporadic tumours. Hum Mol Genet. 1994 Feb;3(2):237–241. doi: 10.1093/hmg/3.2.237. [DOI] [PubMed] [Google Scholar]
  7. Hofstra R. M., Landsvater R. M., Ceccherini I., Stulp R. P., Stelwagen T., Luo Y., Pasini B., Höppener J. W., van Amstel H. K., Romeo G. A mutation in the RET proto-oncogene associated with multiple endocrine neoplasia type 2B and sporadic medullary thyroid carcinoma. Nature. 1994 Jan 27;367(6461):375–376. doi: 10.1038/367375a0. [DOI] [PubMed] [Google Scholar]
  8. Ikeda I., Ishizaka Y., Tahira T., Suzuki T., Onda M., Sugimura T., Nagao M. Specific expression of the ret proto-oncogene in human neuroblastoma cell lines. Oncogene. 1990 Sep;5(9):1291–1296. [PubMed] [Google Scholar]
  9. Iwamoto T., Taniguchi M., Asai N., Ohkusu K., Nakashima I., Takahashi M. cDNA cloning of mouse ret proto-oncogene and its sequence similarity to the cadherin superfamily. Oncogene. 1993 Apr;8(4):1087–1091. [PubMed] [Google Scholar]
  10. Kuma K., Iwabe N., Miyata T. Motifs of cadherin- and fibronectin type III-related sequences and evolution of the receptor-type-protein tyrosine kinases: sequence similarity between proto-oncogene ret and cadherin family. Mol Biol Evol. 1993 May;10(3):539–551. doi: 10.1093/oxfordjournals.molbev.a040033. [DOI] [PubMed] [Google Scholar]
  11. Maruyama S., Iwashita T., Imai T., Funahashi H., Ceccherini I., Luo Y., Romeo G., Matsuo S., Matsuyama M., Takahashi M. Germ line mutations of the ret proto-oncogene in Japanese patients with multiple endocrine neoplasia type 2A and type 2B. Jpn J Cancer Res. 1994 Sep;85(9):879–882. doi: 10.1111/j.1349-7006.1994.tb02962.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Mulligan L. M., Eng C., Healey C. S., Clayton D., Kwok J. B., Gardner E., Ponder M. A., Frilling A., Jackson C. E., Lehnert H. Specific mutations of the RET proto-oncogene are related to disease phenotype in MEN 2A and FMTC. Nat Genet. 1994 Jan;6(1):70–74. doi: 10.1038/ng0194-70. [DOI] [PubMed] [Google Scholar]
  13. Mulligan L. M., Kwok J. B., Healey C. S., Elsdon M. J., Eng C., Gardner E., Love D. R., Mole S. E., Moore J. K., Papi L. Germ-line mutations of the RET proto-oncogene in multiple endocrine neoplasia type 2A. Nature. 1993 Jun 3;363(6428):458–460. doi: 10.1038/363458a0. [DOI] [PubMed] [Google Scholar]
  14. Ozawa M., Engel J., Kemler R. Single amino acid substitutions in one Ca2+ binding site of uvomorulin abolish the adhesive function. Cell. 1990 Nov 30;63(5):1033–1038. doi: 10.1016/0092-8674(90)90506-a. [DOI] [PubMed] [Google Scholar]
  15. Pachnis V., Mankoo B., Costantini F. Expression of the c-ret proto-oncogene during mouse embryogenesis. Development. 1993 Dec;119(4):1005–1017. doi: 10.1242/dev.119.4.1005. [DOI] [PubMed] [Google Scholar]
  16. Romeo G., Ronchetto P., Luo Y., Barone V., Seri M., Ceccherini I., Pasini B., Bocciardi R., Lerone M., Käriäinen H. Point mutations affecting the tyrosine kinase domain of the RET proto-oncogene in Hirschsprung's disease. Nature. 1994 Jan 27;367(6461):377–378. doi: 10.1038/367377a0. [DOI] [PubMed] [Google Scholar]
  17. Roussel M. F., Downing J. R., Rettenmier C. W., Sherr C. J. A point mutation in the extracellular domain of the human CSF-1 receptor (c-fms proto-oncogene product) activates its transforming potential. Cell. 1988 Dec 23;55(6):979–988. doi: 10.1016/0092-8674(88)90243-7. [DOI] [PubMed] [Google Scholar]
  18. Santoro M., Rosati R., Grieco M., Berlingieri M. T., D'Amato G. L., de Franciscis V., Fusco A. The ret proto-oncogene is consistently expressed in human pheochromocytomas and thyroid medullary carcinomas. Oncogene. 1990 Oct;5(10):1595–1598. [PubMed] [Google Scholar]
  19. Schneider R. The human protooncogene ret: a communicative cadherin? Trends Biochem Sci. 1992 Nov;17(11):468–469. doi: 10.1016/0968-0004(92)90490-z. [DOI] [PubMed] [Google Scholar]
  20. Schuchardt A., D'Agati V., Larsson-Blomberg L., Costantini F., Pachnis V. Defects in the kidney and enteric nervous system of mice lacking the tyrosine kinase receptor Ret. Nature. 1994 Jan 27;367(6461):380–383. doi: 10.1038/367380a0. [DOI] [PubMed] [Google Scholar]
  21. Takahashi M., Asai N., Iwashita T., Isomura T., Miyazaki K., Matsuyama M. Characterization of the ret proto-oncogene products expressed in mouse L cells. Oncogene. 1993 Nov;8(11):2925–2929. [PubMed] [Google Scholar]
  22. Takahashi M., Buma Y., Hiai H. Isolation of ret proto-oncogene cDNA with an amino-terminal signal sequence. Oncogene. 1989 Jun;4(6):805–806. [PubMed] [Google Scholar]
  23. Takahashi M., Buma Y., Iwamoto T., Inaguma Y., Ikeda H., Hiai H. Cloning and expression of the ret proto-oncogene encoding a tyrosine kinase with two potential transmembrane domains. Oncogene. 1988 Nov;3(5):571–578. [PubMed] [Google Scholar]
  24. Takahashi M., Buma Y., Taniguchi M. Identification of the ret proto-oncogene products in neuroblastoma and leukemia cells. Oncogene. 1991 Feb;6(2):297–301. [PubMed] [Google Scholar]
  25. Tsuzuki T., Takahashi M., Asai N., Iwashita T., Matsuyama M., Asai J. Spatial and temporal expression of the ret proto-oncogene product in embryonic, infant and adult rat tissues. Oncogene. 1995 Jan 5;10(1):191–198. [PubMed] [Google Scholar]
  26. 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]
  27. 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]
  28. Yoshimura A., Longmore G., Lodish H. F. Point mutation in the exoplasmic domain of the erythropoietin receptor resulting in hormone-independent activation and tumorigenicity. Nature. 1990 Dec 13;348(6302):647–649. doi: 10.1038/348647a0. [DOI] [PubMed] [Google Scholar]

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

RESOURCES