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A single point mutation has pleiotropic effects on pp60v-src function

M J Welham1, J A Wyke

  • 1Imperial Cancer Research Fund Laboratories, St. Bartholomew's Hospital, London, United Kingdom.

Journal of Virology
|June 1, 1988
PubMed

Insights

A specific mutation in Rous sarcoma virus (RSV) pp60v-src protein causes temperature sensitivity in tyrosine kinase activity and cell periphery localization. This single alanine substitution at residue 507 is responsible for the temperature-sensitive phenotype.

Area of Science:

  • Molecular Biology
  • Virology
  • Cell Biology

Background:

  • Rous sarcoma virus (RSV) encodes pp60v-src, a non-receptor tyrosine kinase crucial for cell transformation.
  • Temperature-sensitive (ts) mutants of pp60v-src are valuable tools for studying its function.
  • The tsLA29 mutant exhibits temperature-dependent defects in both kinase activity and cellular localization.

Purpose of the Study:

  • To identify the specific mutation(s) responsible for the temperature-sensitive phenotype of pp60v-src in the tsLA29 mutant.
  • To investigate the functional consequences of identified mutations on pp60v-src activity and localization.
  • To elucidate the role of specific amino acid residues in pp60v-src function and cellular localization.

Main Methods:

  • Site-directed mutagenesis and construction of chimeric proteins involving wild-type and mutant pp60v-src.
  • Temperature shift experiments to assess tyrosine kinase activity and cellular localization.
  • Analysis of cell morphology in cells infected with viruses encoding specific pp60v-src mutants.

Main Results:

  • The tsLA29 pp60v-src molecule shows temperature sensitivity in both tyrosine kinase activity and peripheral localization.
  • A single amino acid substitution (alanine for proline at residue 507) in pp60v-src accounts for all temperature-sensitive characteristics.
  • A second-site mutation at residue 427 can restore the wild-type phenotype.
  • Cells expressing only the alanine substitution at position 507 display a fusiform morphology.

Conclusions:

  • The proline to alanine substitution at residue 507 is the primary determinant of temperature sensitivity in pp60v-src.
  • This mutation affects pp60v-src localization and kinase activity, impacting cellular morphology.
  • Subtle functional effects of the alanine substitution may be compensated by other mutations in the native tsLA29 context.

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