Ciraolo Elisa, Gulluni Federico, Hirsch Emilio
Molecular Biotechnology Center, Department of Molecular Biotechnology and Health Sciences, University of Torino, Torino, Italy.
Molecular Biotechnology Center, Department of Molecular Biotechnology and Health Sciences, University of Torino, Torino, Italy.
Methods Enzymol. 2014;543:115-40. doi: 10.1016/B978-0-12-801329-8.00006-4.
Phosphoinositide-3-kinase (PI3K) signaling has been implicated in a panoply of cellular responses including survival, proliferation, protein synthesis, migration, and vesicular trafficking. In addition, alterations in the enzymatic activity of PI3Ks have been involved in the pathogenesis of multiple diseases, ranging from cancer to chronic inflammation. The emerging interest in PI3K as a pharmacological target has prompted the development of several molecules with inhibitory activity. In this context, the quantification of the second messenger generated by PI3Ks, phosphoinositide-3-phosphate, offers an opportunity to directly test variations in the lipid kinase activity of PI3K in physiological as well as pathological conditions. Here, we will describe common methods to measure the lipid kinase activity of PI3K in vitro and new techniques to follow the production of phosphoinositide-3-phosphate in vivo. These methods are relevant to study the alterations of the PI3K systems at the interface between signaling and oncometabolism.
磷酸肌醇-3-激酶（PI3K）信号传导与一系列细胞反应有关，包括细胞存活、增殖、蛋白质合成、迁移和囊泡运输。此外，PI3K酶活性的改变涉及多种疾病的发病机制，从癌症到慢性炎症。对PI3K作为药物靶点的兴趣日益浓厚，促使人们开发了几种具有抑制活性的分子。在此背景下，对PI3K产生的第二信使磷酸肌醇-3-磷酸的定量，为直接检测生理和病理条件下PI3K脂质激酶活性的变化提供了机会。在这里，我们将描述体外测量PI3K脂质激酶活性的常用方法以及体内追踪磷酸肌醇-3-磷酸产生的新技术。这些方法对于研究信号传导与肿瘤代谢之间界面处PI3K系统的改变具有重要意义。
