biostudies-literatureKuihon SVNPAmerican Heart Association IncNational Institutes of Health107130https://www.ebi.ac.uk/biostudies/studies/S-EPMC10979099biostudies-literatureUnknown300(4)The actin cytoskeleton and reactive oxygen species (ROS) both play crucial roles in various cellular processes. Previous research indicated a direct interaction between two key components of these systems: the WAVE1 subunit of the WAVE regulatory complex (WRC), which promotes actin polymerization and the p47^phox subunit of the NADPH oxidase 2 complex (NOX2), which produces ROS. Here, using carefully characterized recombinant proteins, we find that activated p47^phox uses its dual Src homology 3 domains to bind to multiple regions within the WAVE1 and Abi2 subunits of the WRC, without altering WRC's activity in promoting Arp2/3-mediated actin polymerization. Notably, contrary to previous findings, p47^phox uses the same binding pocket to interact with both the WRC and the p22^phox subunit of NOX2, albeit in a mutually exclusive manner. This observation suggests that when activated, p47^phox may separately participate in two distinct processes: assembling into NOX2 to promote ROS production and engaging with WRC to regulate the actin cytoskeleton.The Journal of biological chemistryThe NADPH oxidase 2 subunit p47^phox binds to the WAVE regulatory complex and p22^phox in a mutually exclusive manner.PMC1097909919IPLOI34660134R35 GM128786Kuihon SVNPBayless KJAbbey CAChen BSevart BJfalseThe NADPH oxidase 2 subunit p47^phox binds to the WAVE regulatory complex and p22^phox in a mutually exclusive manner.The actin cytoskeleton and reactive oxygen species (ROS) both play crucial roles in various cellular processes. Previous research indicated a direct interaction between two key components of these systems: the WAVE1 subunit of the WAVE regulatory complex (WRC), which promotes actin polymerization and the p47^phox subunit of the NADPH oxidase 2 complex (NOX2), which produces ROS. Here, using carefully characterized recombinant proteins, we find that activated p47^phox uses its dual Src homology 3 domains to bind to multiple regions within the WAVE1 and Abi2 subunits of the WRC, without altering WRC's activity in promoting Arp2/3-mediated actin polymerization. Notably, contrary to previous findings, p47^phox uses the same binding pocket to interact with both the WRC and the p22^phox subunit of NOX2, albeit in a mutually exclusive manner. This observation suggests that when activated, p47^phox may separately participate in two distinct processes: assembling into NOX2 to promote ROS production and engaging with WRC to regulate the actin cytoskeleton.2024-01-01T00:00:00Z2024 Mar2025-04-26T11:27:42.358Z2025-04-06T13:39:20.45ZS-EPMC109790993843263010.1016/j.jbc.2024.107130