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A High Resolution Method to Monitor Phosphorylation-dependent Activation of IRF3
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A new evaluation method for quantifying PI3K activity by HTRF assay.

Hironobu Sugita1, Shingo Dan, Dexin Kong

  • 1Division of Molecular Pharmacology, Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, 3-10-6 Ariake, Koto-ku, Tokyo 135-8550, Japan.

Biochemical and Biophysical Research Communications
|October 28, 2008
PubMed
Summary

This study quantifies PI3K p110alpha activity in cancer using a novel HTRF assay. The developed system accurately measures hotspot PIK3CA mutations, aiding cancer research.

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Oncology

Background:

  • The PIK3CA gene encodes a key subunit of phosphoinositide 3-kinase (PI3K) p110alpha, frequently altered in various cancers.
  • Hotspot mutations like E545K and H1047R in p110alpha are known to confer gain-of-function activity, but quantitative assessment has been limited.

Purpose of the Study:

  • To establish a robust quantitative evaluation system for PI3K p110alpha activity, particularly for cancer-associated mutants.
  • To validate a new homogeneous time-resolved fluorescence (HTRF) assay for precise PI3K activity measurement.

Main Methods:

  • Development of a PI3K activity assay utilizing homogeneous time-resolved fluorescence (HTRF).
  • Calibration of the assay using serial dilutions of standard phosphatidylinositol (3,4,5)-trisphosphate (PIP3) to establish a regression line.
  • Expression and purification of recombinant FLAG-tagged p110alpha and p85alpha subunits in HEK293T cells.
  • Quantitative evaluation of p110alpha hotspot mutant activities via anti-FLAG immunoprecipitation followed by the PI3K-HTRF assay.

Main Results:

  • A quantitative PI3K activity evaluation system was successfully constructed using the PI3K-HTRF assay.
  • The assay enabled precise measurement of the enzymatic activities of well-characterized p110alpha hotspot mutants (E545K, H1047R).

Conclusions:

  • The developed PI3K-HTRF assay provides a reliable method for quantitatively assessing PI3K p110alpha activity.
  • This system is expected to be valuable for evaluating uncharacterized PIK3CA mutations found in cancer, advancing our understanding of oncogenesis.