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Sensitivity of wild type and mutant ras alleles to Ras specific exchange factors: Identification of factor specific requirements

Oncogene volume 20, pages 2091–2100 (2001)Cite this article

Abstract

We have investigated the productive interaction between the four mammalian Ras proteins (H-, N-, KA- and KB-Ras) and their activators, the mammalian exchange factors mSos1, GRF1 and GRP, by using a modified Saccharomyces cerevisiae whose growth is dependent on activation of a mammalian Ras protein by its activator. All four mammalian Ras proteins were activated with similar efficiencies by the individual exchange factors. The H-Ras mutant V103E, which is competent for membrane localization, nucleotide binding, intrinsic and stimulated GTPase activity as well as intrinsic exchange, was defective for activation by all factors tested, suggesting that the integrity of this residue is necessary for catalyzed exchange. However, when other H-Ras mutants were studied, some distinct sensitivities to the exchange factors were observed. GRP-mediated, but not mSos1-mediated, exchange was blocked in additional mutants, suggesting different structural requirements for GRP. Analysis of Ras-mediated gene activation in murine fibroblasts confirmed these results.

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Acknowledgements

We thank Marianne Knudsen and Rikke Ingvorsen for technical assistance and Jeffrey DeClue for constructing the KB gene. The introduction to S. cerevisiae manipulations by Steen Holmberg is gratefully acknowledged, and we thank James Stone, Adrienne Cox, Alan Hall, Channing Der, and Douglas Lowy for providing materials; Douglas Lowy and Malene Hansen for reading the manuscript, and Kaare Teilum for help with Figure 6. This work was supported by NIH Grant CA41086 to JRB, and grants 97 100 13 from the Danish Cancer Society and 9600821 from the Danish Medical Research council to BM Willumsen.

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Authors and Affiliations

  1. Department of Molecular Cell Biology, University of Copenhagen, Oester Farimagsgade 2A, DK 1353 Copenhagen, Denmark

    Klaus Hvid Nielsen, Lars Gredsted & Berthe Marie Willumsen

  2. Molecular Biology, Princeton University, 303 Lewis Thomas Lab, 08544, Princeton, USA

    James R Broach

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Nielsen, K., Gredsted, L., Broach, J. et al. Sensitivity of wild type and mutant ras alleles to Ras specific exchange factors: Identification of factor specific requirements. Oncogene 20, 2091–2100 (2001). https://doi.org/10.1038/sj.onc.1204306

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