|
|||||
|
|
A new evaluation method for quantifying PI3K activity by HTRF assay |
|
| Authors: | Sugita Hironobu Dan Shingo Kong Dexin Tomida Akihiro Yamori Takao |
| Institution: | a Division of Molecular Pharmacology, Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, 3-10-6 Ariake, Koto-ku, Tokyo 135-8550, Japan b Biomedical Science PhD Program, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8510, Japan c Division of Genome Research, Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, 3-10-6 Ariake, Koto-ku, Tokyo 135-8550, Japan |
| Abstract: | PIK3CA, coding a catalytic subunit of PI3K p110α, is frequently mutated in cancer. In previous studies, p110α 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 p110α proteins were engineered together with a regulatory subunit p85α 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 p110α. We believe this method will also be applicable to the evaluation of p110α having uncharacterized mutations found in cancer. |
| Keywords: | PI3K PIK3CA HTRF assay Gain-of-function mutation |
| 本文献已被 ScienceDirect PubMed 等数据库收录！ | |