Molecular interaction of NADPH oxidase 1 with betaPix and Nox Organizer 1

It is well established that growth-factor-induced Reactive Oxygen Species (ROS) act as second messengers in cell signaling. We have previously reported that betaPix, a guanine nucleotide exchange factor for Rac, interacts with NADPH Oxidase 1 (NOX1) leading to EGF-induced ROS generation. Here, we report the identification of the domains of NOX1 and betaPix responsible for the interaction between the two proteins. GST pull-down assays show that the PH domain of betaPix binds to the FAD-binding region of NOX1. We also show that overexpression of the PH domain of betaPix results in inhibition of superoxide anion generation in response to EGF. Additionally, NADPH Oxidase Organizer 1 (NoxO1) is shown to interact with the NADPH-binding region of NOX1. These results suggest that the formation of the complex consisting of NOX1, betaPix, and NoxO1 is likely to be a critical step in EGF-induced ROS generation.