| Literature DB >> 18952065 |
Hironobu Sugita1, Shingo Dan, Dexin Kong, Akihiro Tomida, Takao Yamori.
Abstract
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.Entities:
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Year: 2008 PMID： 18952065 DOI： 10.1016/j.bbrc.2008.10.083
Source DB: PubMed Journal: Biochem Biophys Res Commun ISSN： 0006-291X Impact factor: 3.575