PIK3CA, coding a Catalytic Subunit of PI3K p110 alpha, is frequently Mutated in cancer. In previous studies, p110 alpha 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 p110 alpha. proteins were engineered together with a regulatory subunit p85 alpha in human embryonic kidney 293T cells. Anti-FLAG-Ig immunoprecipitates were then subjected to the assay, which enabled LIS to quantitatively evaluate the activities of hotspot mutants of p110 alpha. We believe this method will also be applicable to the evaluation of p110 alpha having uncharacterized mutations found in cancer. (C) 2008 Elsevier Inc. All rights reserved.