A new evaluation method for quantifying PI3K activity by HTRF assay

Biochem Biophys Res Commun. 2008 Dec 19;377(3):941-5. doi: 10.1016/j.bbrc.2008.10.083. Epub 2008 Oct 24.


PIK3CA, coding a catalytic subunit of PI3K p110alpha, is frequently mutated in cancer. In previous studies, p110alpha with hotspot mutations such as E545K and H1047R were shown to be gain-of-function mutations. However, quantitative evaluation of these mutants was not well established. Recently, a new method for measuring PI3K activity using homogeneous time-resolved fluorescence (HTRF) has been developed. Using this method, we constructed a quantitative evaluation system for PI3K activity. Serial dilutions of standard PIP3 were subjected to the PI3K-HTRF assay in order to establish a regression line for calibration. The recombinant FLAG-tagged p110alpha proteins were engineered together with a regulatory subunit p85alpha in human embryonic kidney 293T cells. Anti-FLAG-Ig immunoprecipitates were then subjected to the assay, which enabled us to quantitatively evaluate the activities of hotspot mutants of p110alpha. We believe this method will also be applicable to the evaluation of p110alpha having uncharacterized mutations found in cancer.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Line
  • Class I Phosphatidylinositol 3-Kinases
  • Fluorescence Resonance Energy Transfer / methods*
  • Humans
  • Mutation
  • Neoplasms / enzymology
  • Phosphatidylinositol 3-Kinases / analysis*
  • Phosphatidylinositol 3-Kinases / biosynthesis
  • Phosphatidylinositol 3-Kinases / genetics
  • Protein Engineering
  • Recombinant Proteins / analysis
  • Recombinant Proteins / biosynthesis


  • Recombinant Proteins
  • Phosphatidylinositol 3-Kinases
  • Class I Phosphatidylinositol 3-Kinases
  • PIK3CA protein, human