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

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.