Skip to main content
PMC home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1994 Aug 30;91(18):8512–8516. doi: 10.1073/pnas.91.18.8512

Membrane-targeting potentiates guanine nucleotide exchange factor CDC25 and SOS1 activation of Ras transforming activity.

L A Quilliam 1, S Y Huff 1, K M Rabun 1, W Wei 1, W Park 1, D Broek 1, C J Der 1
PMCID: PMC44636  PMID: 8078913

Abstract

Growth factor-triggered activation of Ras proteins is believed to be mediated by guanine nucleotide exchange factors (CDC25/GRF and SOS1/2) that promote formation of the active Ras GTP-bound state. Although the mechanism(s) of guanine nucleotide exchange factor regulation is unclear, recent studies suggest that translocation of SOS1 to the plasma membrane, where Ras is located, might be responsible for Ras activation. To evaluate this model, we generated constructs that encode the catalytic domains of human CDC25 or mouse SOS1, either alone (designated cCDC25 and cSOS1, respectively) or terminating in the carboxyl-terminal CAAX membrane-targeting sequence from K-Ras4B (designated cCDC25-CAAX and cSOS1-CAAX, respectively; in CAAX, C is Cys, A is an aliphatic amino acid, and X is Ser or Met). We then compared the transforming potential of cCDC25 and cSOS1 with their membrane-targeted counterparts. We observed that addition of the Ras plasma membrane-targeting sequence to the catalytic domains of CDC25 and SOS1 greatly enhanced their focus-forming activity (10- to 50-fold) in NIH 3T3 transfection assays. Similarly, we observed that the membrane-targeted versions showed a 5- to 10-fold enhanced ability to induce transcriptional activation from the Ets/AP-1 Ras-responsive element. Furthermore, whereas cells that stably expressed cCDC25 or cSOS1 exhibited the same morphologies as untransformed NIH 3T3 cells, cells expressing cCDC25-CAAX or cSOS1-CAAX displayed transformed morphologies that were indistinguishable from the elongated and refractile morphology of oncogenic Ras-transformed cells. Thus, these results suggest that membrane translocation alone is sufficient to potentiate guanine nucleotide exchange factor activation of Ras.

Full text

PDF
8513

Images in this article

8513Image
on p.8513
8514Image
on p.8514
8514Image
on p.8514
8515Image
on p.8515

Selected References

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

  1. Barlat I., Schweighoffer F., Chevallier-Multon M. C., Duchesne M., Fath I., Landais D., Jacquet M., Tocque B. The Saccharomyces cerevisiae gene product SDC25 C-domain functions as an oncoprotein in NIH3T3 cells. Oncogene. 1993 Jan;8(1):215–218. [PubMed] [Google Scholar]
  2. Basu T. N., Gutmann D. H., Fletcher J. A., Glover T. W., Collins F. S., Downward J. Aberrant regulation of ras proteins in malignant tumour cells from type 1 neurofibromatosis patients. Nature. 1992 Apr 23;356(6371):713–715. doi: 10.1038/356713a0. [DOI] [PubMed] [Google Scholar]
  3. 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]
  4. Bruder J. T., Heidecker G., Rapp U. R. Serum-, TPA-, and Ras-induced expression from Ap-1/Ets-driven promoters requires Raf-1 kinase. Genes Dev. 1992 Apr;6(4):545–556. doi: 10.1101/gad.6.4.545. [DOI] [PubMed] [Google Scholar]
  5. Buday L., Downward J. Epidermal growth factor regulates p21ras through the formation of a complex of receptor, Grb2 adapter protein, and Sos nucleotide exchange factor. Cell. 1993 May 7;73(3):611–620. doi: 10.1016/0092-8674(93)90146-h. [DOI] [PubMed] [Google Scholar]
  6. Burgering B. M., Pronk G. J., van Weeren P. C., Chardin P., Bos J. L. cAMP antagonizes p21ras-directed activation of extracellular signal-regulated kinase 2 and phosphorylation of mSos nucleotide exchange factor. EMBO J. 1993 Nov;12(11):4211–4220. doi: 10.1002/j.1460-2075.1993.tb06105.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Buss J. E., Solski P. A., Schaeffer J. P., MacDonald M. J., Der C. J. Activation of the cellular proto-oncogene product p21Ras by addition of a myristylation signal. Science. 1989 Mar 24;243(4898):1600–1603. doi: 10.1126/science.2648572. [DOI] [PubMed] [Google Scholar]
  8. Cen H., Papageorge A. G., Vass W. C., Zhang K. E., Lowy D. R. Regulated and constitutive activity by CDC25Mm (GRF), a Ras-specific exchange factor. Mol Cell Biol. 1993 Dec;13(12):7718–7724. doi: 10.1128/mcb.13.12.7718. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Chardin P., Camonis J. H., Gale N. W., van Aelst L., Schlessinger J., Wigler M. H., Bar-Sagi D. Human Sos1: a guanine nucleotide exchange factor for Ras that binds to GRB2. Science. 1993 May 28;260(5112):1338–1343. doi: 10.1126/science.8493579. [DOI] [PubMed] [Google Scholar]
  10. Chevallier-Multon M. C., Schweighoffer F., Barlat I., Baudouy N., Fath I., Duchesne M., Tocqué B. Saccharomyces cerevisiae CDC25 (1028-1589) is a guanine nucleotide releasing factor for mammalian ras proteins and is oncogenic in NIH3T3 cells. J Biol Chem. 1993 May 25;268(15):11113–11118. [PubMed] [Google Scholar]
  11. Chow L. M., Fournel M., Davidson D., Veillette A. Negative regulation of T-cell receptor signalling by tyrosine protein kinase p50csk. Nature. 1993 Sep 9;365(6442):156–160. doi: 10.1038/365156a0. [DOI] [PubMed] [Google Scholar]
  12. Clark G. J., Quilliam L. A., Hisaka M. M., Der C. J. Differential antagonism of Ras biological activity by catalytic and Src homology domains of Ras GTPase activation protein. Proc Natl Acad Sci U S A. 1993 Jun 1;90(11):4887–4891. doi: 10.1073/pnas.90.11.4887. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Cox A. D., Der C. J. Protein prenylation: more than just glue? Curr Opin Cell Biol. 1992 Dec;4(6):1008–1016. doi: 10.1016/0955-0674(92)90133-w. [DOI] [PubMed] [Google Scholar]
  14. DeClue J. E., Papageorge A. G., Fletcher J. A., Diehl S. R., Ratner N., Vass W. C., Lowy D. R. Abnormal regulation of mammalian p21ras contributes to malignant tumor growth in von Recklinghausen (type 1) neurofibromatosis. Cell. 1992 Apr 17;69(2):265–273. doi: 10.1016/0092-8674(92)90407-4. [DOI] [PubMed] [Google Scholar]
  15. Egan S. E., Giddings B. W., Brooks M. W., Buday L., Sizeland A. M., Weinberg R. A. Association of Sos Ras exchange protein with Grb2 is implicated in tyrosine kinase signal transduction and transformation. Nature. 1993 May 6;363(6424):45–51. doi: 10.1038/363045a0. [DOI] [PubMed] [Google Scholar]
  16. 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]
  17. Gale N. W., Kaplan S., Lowenstein E. J., Schlessinger J., Bar-Sagi D. Grb2 mediates the EGF-dependent activation of guanine nucleotide exchange on Ras. Nature. 1993 May 6;363(6424):88–92. doi: 10.1038/363088a0. [DOI] [PubMed] [Google Scholar]
  18. Huang D. C., Marshall C. J., Hancock J. F. Plasma membrane-targeted ras GTPase-activating protein is a potent suppressor of p21ras function. Mol Cell Biol. 1993 Apr;13(4):2420–2431. doi: 10.1128/mcb.13.4.2420. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Li N., Batzer A., Daly R., Yajnik V., Skolnik E., Chardin P., Bar-Sagi D., Margolis B., Schlessinger J. Guanine-nucleotide-releasing factor hSos1 binds to Grb2 and links receptor tyrosine kinases to Ras signalling. Nature. 1993 May 6;363(6424):85–88. doi: 10.1038/363085a0. [DOI] [PubMed] [Google Scholar]
  20. Liu B. X., Wei W., Broek D. The catalytic domain of the mouse sos1 gene product activates Ras proteins in vivo and in vitro. Oncogene. 1993 Nov;8(11):3081–3084. [PubMed] [Google Scholar]
  21. Lowenstein E. J., Daly R. J., Batzer A. G., Li W., Margolis B., Lammers R., Ullrich A., Skolnik E. Y., Bar-Sagi D., Schlessinger J. The SH2 and SH3 domain-containing protein GRB2 links receptor tyrosine kinases to ras signaling. Cell. 1992 Aug 7;70(3):431–442. doi: 10.1016/0092-8674(92)90167-b. [DOI] [PubMed] [Google Scholar]
  22. McCormick F. Signal transduction. How receptors turn Ras on. Nature. 1993 May 6;363(6424):15–16. doi: 10.1038/363015a0. [DOI] [PubMed] [Google Scholar]
  23. Olivier J. P., Raabe T., Henkemeyer M., Dickson B., Mbamalu G., Margolis B., Schlessinger J., Hafen E., Pawson T. A Drosophila SH2-SH3 adaptor protein implicated in coupling the sevenless tyrosine kinase to an activator of Ras guanine nucleotide exchange, Sos. Cell. 1993 Apr 9;73(1):179–191. doi: 10.1016/0092-8674(93)90170-u. [DOI] [PubMed] [Google Scholar]
  24. Pendergast A. M., Quilliam L. A., Cripe L. D., Bassing C. H., Dai Z., Li N., Batzer A., Rabun K. M., Der C. J., Schlessinger J. BCR-ABL-induced oncogenesis is mediated by direct interaction with the SH2 domain of the GRB-2 adaptor protein. Cell. 1993 Oct 8;75(1):175–185. [PubMed] [Google Scholar]
  25. 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]
  26. Quilliam L. A., Kato K., Rabun K. M., Hisaka M. M., Huff S. Y., Campbell-Burk S., Der C. J. Identification of residues critical for Ras(17N) growth-inhibitory phenotype and for Ras interaction with guanine nucleotide exchange factors. Mol Cell Biol. 1994 Feb;14(2):1113–1121. doi: 10.1128/mcb.14.2.1113. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Rozakis-Adcock M., Fernley R., Wade J., Pawson T., Bowtell D. The SH2 and SH3 domains of mammalian Grb2 couple the EGF receptor to the Ras activator mSos1. Nature. 1993 May 6;363(6424):83–85. doi: 10.1038/363083a0. [DOI] [PubMed] [Google Scholar]
  28. Satoh T., Nakafuku M., Kaziro Y. Function of Ras as a molecular switch in signal transduction. J Biol Chem. 1992 Dec 5;267(34):24149–24152. [PubMed] [Google Scholar]
  29. Simon M. A., Dodson G. S., Rubin G. M. An SH3-SH2-SH3 protein is required for p21Ras1 activation and binds to sevenless and Sos proteins in vitro. Cell. 1993 Apr 9;73(1):169–177. doi: 10.1016/0092-8674(93)90169-q. [DOI] [PubMed] [Google Scholar]
  30. Wei W., Mosteller R. D., Sanyal P., Gonzales E., McKinney D., Dasgupta C., Li P., Liu B. X., Broek D. Identification of a mammalian gene structurally and functionally related to the CDC25 gene of Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1992 Aug 1;89(15):7100–7104. doi: 10.1073/pnas.89.15.7100. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES