Skip to main content
PMC home page
Genetics logoLink to Genetics
. 1997 Jun;146(2):553–565. doi: 10.1093/genetics/146.2.553

Mechanism of Activation of the Caenorhabditis Elegans Ras Homologue Let-60 by a Novel, Temperature-Sensitive, Gain-of-Function Mutation

D M Eisenmann 1, S K Kim 1
PMCID: PMC1207997  PMID: 9178006

Abstract

The Caenorhabditis elegans let-60 gene encodes a Ras protein that mediates induction of the hermaphrodite vulva. To better understand how mutations constitutively activate Ras and cause unregulated cell division, we have characterized ga89, a temperature-sensitive, gain-of-function mutation in let-60 ras. At 25°, ga89 increases let-60 activity resulting in a multivulva phenotype. At 15°, ga89 decreases let-60 activity resulting in a vulvaless phenotype in let-60(ga89)/Df animals. The ga89 mutation causes a leucine (L) to phenylalanine (F) substitution at amino acid 19, a residue conserved in all Ras proteins. We introduced the L19F change into human H-Ras protein and found that the in vitro GTPase activity of H-Ras became temperature-dependent. Genetic experiments suggest that LET-60(L19F) interacts with GAP and GNEF, since mutations that decrease GAP and GNEF activity affect the multivulva phenotype of let-60(ga89) animals. These results suggest that the L19F mutation primarily affects the intrinsic rate of GTP hydrolysis by Ras, and that this effect may be sufficient to account for the activated-Ras phenotype caused by let-60(ga89). Our results suggest that a mutation in a human ras gene analogous to ga89 might contribute to oncogenic transformation.

Full Text

The Full Text of this article is available as a PDF (2.4 MB).

Selected References

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

  1. Aroian R. V., Koga M., Mendel J. E., Ohshima Y., Sternberg P. W. The let-23 gene necessary for Caenorhabditis elegans vulval induction encodes a tyrosine kinase of the EGF receptor subfamily. Nature. 1990 Dec 20;348(6303):693–699. doi: 10.1038/348693a0. [DOI] [PubMed] [Google Scholar]
  2. Aroian R. V., Sternberg P. W. Multiple functions of let-23, a Caenorhabditis elegans receptor tyrosine kinase gene required for vulval induction. Genetics. 1991 Jun;128(2):251–267. doi: 10.1093/genetics/128.2.251. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Barbacid M. ras genes. Annu Rev Biochem. 1987;56:779–827. doi: 10.1146/annurev.bi.56.070187.004023. [DOI] [PubMed] [Google Scholar]
  4. Beitel G. J., Clark S. G., Horvitz H. R. Caenorhabditis elegans ras gene let-60 acts as a switch in the pathway of vulval induction. Nature. 1990 Dec 6;348(6301):503–509. doi: 10.1038/348503a0. [DOI] [PubMed] [Google Scholar]
  5. Boguski M. S., McCormick F. Proteins regulating Ras and its relatives. Nature. 1993 Dec 16;366(6456):643–654. doi: 10.1038/366643a0. [DOI] [PubMed] [Google Scholar]
  6. Bos J. L. ras oncogenes in human cancer: a review. Cancer Res. 1989 Sep 1;49(17):4682–4689. [PubMed] [Google Scholar]
  7. Brenner S. The genetics of Caenorhabditis elegans. Genetics. 1974 May;77(1):71–94. doi: 10.1093/genetics/77.1.71. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Duffy J. B., Perrimon N. Recent advances in understanding signal transduction pathways in worms and flies. Curr Opin Cell Biol. 1996 Apr;8(2):231–238. doi: 10.1016/s0955-0674(96)80070-6. [DOI] [PubMed] [Google Scholar]
  9. Eisenmann D. M., Kim S. K. Signal transduction and cell fate specification during Caenorhabditis elegans vulval development. Curr Opin Genet Dev. 1994 Aug;4(4):508–516. doi: 10.1016/0959-437x(94)90065-b. [DOI] [PubMed] [Google Scholar]
  10. Fasano O., Aldrich T., Tamanoi F., Taparowsky E., Furth M., Wigler M. Analysis of the transforming potential of the human H-ras gene by random mutagenesis. Proc Natl Acad Sci U S A. 1984 Jul;81(13):4008–4012. doi: 10.1073/pnas.81.13.4008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Feig L. A., Cooper G. M. Inhibition of NIH 3T3 cell proliferation by a mutant ras protein with preferential affinity for GDP. Mol Cell Biol. 1988 Aug;8(8):3235–3243. doi: 10.1128/mcb.8.8.3235. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Feig L. A., Pan B. T., Roberts T. M., Cooper G. M. Isolation of ras GTP-binding mutants using an in situ colony-binding assay. Proc Natl Acad Sci U S A. 1986 Jul;83(13):4607–4611. doi: 10.1073/pnas.83.13.4607. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Gideon P., John J., Frech M., Lautwein A., Clark R., Scheffler J. E., Wittinghofer A. Mutational and kinetic analyses of the GTPase-activating protein (GAP)-p21 interaction: the C-terminal domain of GAP is not sufficient for full activity. Mol Cell Biol. 1992 May;12(5):2050–2056. doi: 10.1128/mcb.12.5.2050. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Han M., Aroian R. V., Sternberg P. W. The let-60 locus controls the switch between vulval and nonvulval cell fates in Caenorhabditis elegans. Genetics. 1990 Dec;126(4):899–913. doi: 10.1093/genetics/126.4.899. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Han M., Sternberg P. W. let-60, a gene that specifies cell fates during C. elegans vulval induction, encodes a ras protein. Cell. 1990 Nov 30;63(5):921–931. doi: 10.1016/0092-8674(90)90495-z. [DOI] [PubMed] [Google Scholar]
  16. Hoskins R., Hajnal A. F., Harp S. A., Kim S. K. The C. elegans vulval induction gene lin-2 encodes a member of the MAGUK family of cell junction proteins. Development. 1996 Jan;122(1):97–111. doi: 10.1242/dev.122.1.97. [DOI] [PubMed] [Google Scholar]
  17. Katz W. S., Hill R. J., Clandinin T. R., Sternberg P. W. Different levels of the C. elegans growth factor LIN-3 promote distinct vulval precursor fates. Cell. 1995 Jul 28;82(2):297–307. doi: 10.1016/0092-8674(95)90317-8. [DOI] [PubMed] [Google Scholar]
  18. Kim S. K., Horvitz H. R. The Caenorhabditis elegans gene lin-10 is broadly expressed while required specifically for the determination of vulval cell fates. Genes Dev. 1990 Mar;4(3):357–371. doi: 10.1101/gad.4.3.357. [DOI] [PubMed] [Google Scholar]
  19. Lackner M. R., Kornfeld K., Miller L. M., Horvitz H. R., Kim S. K. A MAP kinase homolog, mpk-1, is involved in ras-mediated induction of vulval cell fates in Caenorhabditis elegans. Genes Dev. 1994 Jan;8(2):160–173. doi: 10.1101/gad.8.2.160. [DOI] [PubMed] [Google Scholar]
  20. Lewis J. A., Fleming J. T. Basic culture methods. Methods Cell Biol. 1995;48:3–29. [PubMed] [Google Scholar]
  21. Lowy D. R., Willumsen B. M. Function and regulation of ras. Annu Rev Biochem. 1993;62:851–891. doi: 10.1146/annurev.bi.62.070193.004223. [DOI] [PubMed] [Google Scholar]
  22. Powers S., O'Neill K., Wigler M. Dominant yeast and mammalian RAS mutants that interfere with the CDC25-dependent activation of wild-type RAS in Saccharomyces cerevisiae. Mol Cell Biol. 1989 Feb;9(2):390–395. doi: 10.1128/mcb.9.2.390. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Pronk G. J., Bos J. L. The role of p21ras in receptor tyrosine kinase signalling. Biochim Biophys Acta. 1994 Dec 30;1198(2-3):131–147. doi: 10.1016/0304-419x(94)90010-8. [DOI] [PubMed] [Google Scholar]
  24. Quilliam L. A., Zhong S., Rabun K. M., Carpenter J. W., South T. L., Der C. J., Campbell-Burk S. Biological and structural characterization of a Ras transforming mutation at the phenylalanine-156 residue, which is conserved in all members of the Ras superfamily. Proc Natl Acad Sci U S A. 1995 Feb 28;92(5):1272–1276. doi: 10.1073/pnas.92.5.1272. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Run J. Q., Steven R., Hung M. S., van Weeghel R., Culotti J. G., Way J. C. Suppressors of the unc-73 gene of Caenorhabditis elegans. Genetics. 1996 May;143(1):225–236. doi: 10.1093/genetics/143.1.225. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Sigal I. S., Gibbs J. B., D'Alonzo J. S., Temeles G. L., Wolanski B. S., Socher S. H., Scolnick E. M. Mutant ras-encoded proteins with altered nucleotide binding exert dominant biological effects. Proc Natl Acad Sci U S A. 1986 Feb;83(4):952–956. doi: 10.1073/pnas.83.4.952. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Simske J. S., Kim S. K. Sequential signalling during Caenorhabditis elegans vulval induction. Nature. 1995 May 11;375(6527):142–146. doi: 10.1038/375142a0. [DOI] [PubMed] [Google Scholar]
  28. Stacey D. W., Feig L. A., Gibbs J. B. Dominant inhibitory Ras mutants selectively inhibit the activity of either cellular or oncogenic Ras. Mol Cell Biol. 1991 Aug;11(8):4053–4064. doi: 10.1128/mcb.11.8.4053. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Sternberg P. W., Horvitz H. R. Pattern formation during vulval development in C. elegans. Cell. 1986 Mar 14;44(5):761–772. doi: 10.1016/0092-8674(86)90842-1. [DOI] [PubMed] [Google Scholar]
  30. Sternberg P. W. Intercellular signaling and signal transduction in C. elegans. Annu Rev Genet. 1993;27:497–521. doi: 10.1146/annurev.ge.27.120193.002433. [DOI] [PubMed] [Google Scholar]
  31. Sternberg P. W. Lateral inhibition during vulval induction in Caenorhabditis elegans. Nature. 1988 Oct 6;335(6190):551–554. doi: 10.1038/335551a0. [DOI] [PubMed] [Google Scholar]
  32. Sundaram M., Han M. The C. elegans ksr-1 gene encodes a novel Raf-related kinase involved in Ras-mediated signal transduction. Cell. 1995 Dec 15;83(6):889–901. doi: 10.1016/0092-8674(95)90205-8. [DOI] [PubMed] [Google Scholar]
  33. Valencia A., Chardin P., Wittinghofer A., Sander C. The ras protein family: evolutionary tree and role of conserved amino acids. Biochemistry. 1991 May 14;30(19):4637–4648. doi: 10.1021/bi00233a001. [DOI] [PubMed] [Google Scholar]
  34. Wu XG, Dev G, Jain JK. Mixed-spin incompressible states in the fractional quantum Hall effect. Phys Rev Lett. 1993 Jul 5;71(1):153–156. doi: 10.1103/PhysRevLett.71.153. [DOI] [PubMed] [Google Scholar]
  35. Wu Y., Han M. Suppression of activated Let-60 ras protein defines a role of Caenorhabditis elegans Sur-1 MAP kinase in vulval differentiation. Genes Dev. 1994 Jan;8(2):147–159. doi: 10.1101/gad.8.2.147. [DOI] [PubMed] [Google Scholar]
  36. de Vos A. M., Tong L., Milburn M. V., Matias P. M., Jancarik J., Noguchi S., Nishimura S., Miura K., Ohtsuka E., Kim S. H. Three-dimensional structure of an oncogene protein: catalytic domain of human c-H-ras p21. Science. 1988 Feb 19;239(4842):888–893. doi: 10.1126/science.2448879. [DOI] [PubMed] [Google Scholar]

Articles from Genetics are provided here courtesy of Oxford University Press

RESOURCES