Molecular Biology Reports

, Volume 23, Issue 2, pp 109–117

Positive and negative modulation of H-ras transforming potential by mutations of phenylalanine-28

  • Michael H. Ricketts
  • Glenda A. Durrheim
  • Honor M. North
  • Marthinus J. van der Merwe
  • Arthur D. Levinson
Research Article

DOI: 10.1007/BF00424436

Cite this article as:
Ricketts, M.H., Durrheim, G.A., North, H.M. et al. Mol Biol Rep (1996) 23: 109. doi:10.1007/BF00424436
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Abstract

Conserved amino-acids of H-ras from residues 25 to 34 were mutated in human H-ras cDNA with a pre-existing valine-12 activating mutation ([V12]p21), and built into SV40-driven expression vectors. The influence of the introduced mutations was initially screened by transfection of Rat-1 cells to score foci of transformed cells. Nonconservative mutations of amino-acids 25 (tryptophan for glutamine), 27 (asparagine for histidine) and 34 (alanine for proline) did not abrogate the transforming potential of [V12]p21. The conservative mutation of phenylalanine-28 to tryptophan ([V12W28]p21) was also still transforming. Significantly, in the absence of the valine-12 activating mutation, tryptophan-28-ras ([W28]p21) was weakly transforming while, in contrast, [V12D28]p21 was unable to transform Rat-1 cells and retarded cell growth. Analysis of the binding and dissociation of GTP and GDP to normal and mutated p21 expressed in Escherichia coli showed that [V12D28]p21 and [D28]p21 do not bind GTP. The dissociation rate of both GTP and GDP bound to [W28]p21 is increased, suggesting a mechanism for its transforming potential in Rat-1 cells. These studies illustrate the importance of phenylalanine-28 in guanine nucleotide binding by p21h-ras. The mutations described could be valuable tools in investigations of cellular signal transduction involving small GTP-binding proteins.

Key words

cellular transformation GTP-binding mutagenesis ras Rat-1 cells 

Copyright information

© Kluwer Academic Publishers 1996

Authors and Affiliations

  • Michael H. Ricketts
    • 1
    • 3
  • Glenda A. Durrheim
    • 2
    • 3
  • Honor M. North
    • 2
    • 3
  • Marthinus J. van der Merwe
    • 2
    • 3
  • Arthur D. Levinson
    • 2
    • 3
  1. 1.Department of PsychiatryUMDNJ, Robert Wood Johnson Medical SchoolPiscatawayUSA
  2. 2.US/MRC Centre for Molecular and Cellular Biology, Department of Medical BiochemistryUniversity of Stellenbosch Medical SchoolTygerbergSouth Africa
  3. 3.Genentech Inc.South San FranciscoUSA

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