Antagonistic Cross-talk between Rac and Cdc42 GTPases Regulates Generation of Reactive Oxygen Species

Abstract

Cross-talk between Rho GTPase family members (Rho, Rac, and Cdc42) plays important roles in modulating and coordinating downstream cellular responses resulting from Rho GTPase signaling. The NADPH oxidase of phagocytes and nonphagocytic cells is a Rac GTPaseregulated system that generates reactive oxygen species (ROS) for the purposes of innate immunity and intracellular signaling. We recently demonstrated that NADPH oxidase activation involves sequential interactions between Rac and the flavocytochrome b 558 and p67 phox oxidase components to regulate electron transfer from NADPH to molecular oxygen. Here we identify an antagonistic interaction between Rac and the closely related GTPase Cdc42 at the level of flavocytochrome b 558 that regulates the formation of ROS. Cdc42 is unable to stimulate ROS formation by NADPH oxidase, but Cdc42, like Rac1 and Rac2, was able to specifically bind to flavocytochrome b 558 in vitro. Cdc42 acted as a competitive inhibitor of Rac1-and Rac2-mediated ROS formation in a recombinant cell-free oxidase system. Inhibition was dependent on the Cdc42 insert domain but not the Switch I region. Transient expression of Cdc42Q61L inhibited ROS formation induced by constitutively active Rac1 in an NADPH oxidase-expressing Cos7 cell line. Inhibition of Cdc42 activity by transduction of the Cdc42-binding domain of Wiscott-Aldrich syndrome protein into human neutrophils resulted in an enhanced fMetLeuPhe-induced oxidative response, consistent with inhibitory cross-talk between Rac and Cdc42 in activated neutrophils. We propose here a novel antagonism between Rac and Cdc42 GTPases at the level of the Nox proteins that modulates the generation of ROS used for host defense, cell signaling, and transformation.The process by which cells produce reactive oxygen species (ROS) 1 has gained much interest because of the diverse functions attributed to this class of molecules. In nonphagocytic cells, oxidants affect a variety of cellular processes, including transcription factor activation, proliferation, transformation, and apoptosis. In neutrophils and other phagocytes, oxidants play an important role in cellular innate immune responses. A critical component of the bactericidal activity of phagocytes is the NADPH oxidase, also referred to as "phox" (phagocytic oxidase) (1-3), which generates superoxide anion and, subsequently, a number of other ROS. The phagocyte NADPH oxidase is a multiprotein system whose activity is regulated by the RhoGTPase Rac2 in human cells (4 -6). Electrons are transferred from NADPH to molecular oxygen through the action of an integral membrane flavocytochrome b 558 (cyt b 558 ), composed of subunits gp91 phox and p22 phox . In addition to Rac2, the activity of the NADPH oxidase is regulated by the cytosolic components p47 phox , p67 phox , and p40 phox , which exist as a heterotrimeric complex in the cytosol of unstimulated neutrophils (7). In a separate cytosolic complex are Rac2 (or Rac1 in certain species) and GDP dissociation inhibitor (8). When neutrophils are activate...

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