Molecular interaction of NADPH oxidase 1 with βpix 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 βPix, 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 βPix responsible for the interaction between the two proteins. GST pull-down assays show that the PH domain of βPix binds to the FAD-binding region of Nox1. We also show that overexpression of the PH domain of βPix 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, βPix, and NoxO1 is likely to be a critical step in EGF-induced ROS generation.