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

M H Ricketts1, G A Durrheim, H M North

  • 1Department of Psychiatry, UMDNJ, Robert Wood Johnson Medical School, Piscataway 08854, USA.

Molecular Biology Reports
|January 1, 1996
PubMed

Insights

Mutations in conserved H-ras amino acids reveal phenylalanine-28

Area of Science:

  • Molecular Biology
  • Cellular Biology
  • Biochemistry

Background:

  • Ras proteins are key regulators of cellular signaling pathways.
  • Activating mutations in H-ras, such as Valine-12, are common in cancer.
  • Understanding the structure-function relationship of H-ras is crucial for cancer research.

Purpose of the Study:

  • To investigate the role of conserved amino acids (25-34) in H-ras function.
  • To determine the impact of specific mutations on the transforming potential of H-ras.
  • To elucidate the mechanism by which H-ras mutations affect guanine nucleotide binding.

Main Methods:

  • Site-directed mutagenesis of human H-ras cDNA.
  • Transfection of Rat-1 cells to assess cellular transformation.
  • Expression and purification of mutated H-ras p21 proteins in E. coli.
  • Analysis of GTP and GDP binding and dissociation kinetics.

Main Results:

  • Non-conservative mutations at positions 25, 27, and 34 did not abolish [V12]p21 transforming activity.
  • Conservative mutation of phenylalanine-28 to tryptophan ([V12W28]p21) retained transforming potential.
  • Tryptophan-28-ras ([W28]p21) showed weak transforming activity, while [V12D28]p21 and [D28]p21 failed to transform cells and bind GTP.
  • Increased GTP/GDP dissociation rates were observed for [W28]p21, suggesting a mechanism for its transforming potential.

Conclusions:

  • Phenylalanine-28 is critical for guanine nucleotide binding in p21H-ras.
  • Specific mutations can alter H-ras transforming potential by affecting GTP binding.
  • These mutated H-ras proteins serve as valuable tools for studying cellular signal transduction.

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