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Phosphorylation of middle T by pp60c-src: a switch for binding of phosphatidylinositol 3-kinase and optimal

D A Talmage1, R Freund, A T Young

  • 1Department of Pathology, Harvard Medical School, Boston, Massachusetts 02115.

Cell
|October 6, 1989
PubMed

Insights

Replacing tyrosine 315 in polyoma virus middle T protein with phenylalanine reduces tumor formation. This modification prevents phosphatidylinositol 3-kinase binding, crucial for viral tumorigenesis.

Area of Science:

  • Virology
  • Molecular Biology
  • Oncology

Background:

  • Polyoma virus middle T (mT) protein is a key oncogene.
  • Phosphorylation of mT at tyrosine 315 by pp60c-src is essential for tumorigenesis.

Purpose of the Study:

  • To investigate the role of tyrosine 315 phosphorylation in mT protein function and viral tumorigenesis.
  • To determine the impact of this modification on pp60c-src association and phosphatidylinositol 3-kinase binding.

Main Methods:

  • Site-directed mutagenesis to substitute phenylalanine for tyrosine 315 in mT protein.
  • Co-immunoprecipitation assays to analyze protein-protein interactions (mT, pp60c-src, phosphatidylinositol 3-kinase).
  • Use of specific anti-peptide antiserum to detect phosphorylated mT.

Main Results:

  • The Phe315 mT mutant associated with and activated pp60c-src but failed to bind phosphatidylinositol 3-kinase.
  • Specific antiserum confirmed that mT lacking tyrosine 315 phosphorylation did not form ternary complexes with pp60c-src and phosphatidylinositol 3-kinase.
  • Ternary complexes were recovered when mT was phosphorylated on tyrosine 315.

Conclusions:

  • Phosphorylation of mT at tyrosine 315 by pp60c-src is critical for recruiting phosphatidylinositol 3-kinase.
  • This recruitment is essential for the full tumorigenic potential of polyoma virus.

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