Reactive oxygen species (ROS) formed by phagocytic leukocytes play a primary role in host defense, but also perpetuate both acute and chronic cellular inflammatory responses. Roles for ROS in non-phagocytic cells include intracellular signaling, proliferation, apoptosis, and innate immunity. It has been established that the activity of the human neutrophil ROS-generating NADPH oxidase (Nox2) is regulated by the small GTPase Rac2, and that Rac2 acts as a key """"""""molecular switch"""""""" for ROS formation in adherent neutrophils. Our previous work has suggested a two-step mechanism through which Rac2 regulates the process of electron transfer from NADPH to molecular oxygen in this multi-component system. A more detailed knowledge of the molecular basis for NADPH oxidase regulation by Rac GTPase is critical for understanding the physiological and pathological regulation of ROS formation in both immune- and non-immune cells.The molecular mechanisms regulating ROS formation by non-phagocyte Nox enzymes remain poorly defined. Our recent data indicates that Nox1, an abundant Nox in the vascular and gastrointestinal epithelium, is regulated by kinase-dependent pathways. We have shown that in various colon cancer cell lines there is a correlation between the levels of active Src present and Nox1 activity. Preliminary data suggest that Src may act through unique """"""""organizer"""""""" molecules that are structurally related to p40, p47, and Nox1, the known Nox regulatory proteins. We propose to investigate the molecular mechanisms used by Src kinases to regulate Nox1 activity and, more generally, to explore the biological role(s) of the new members of the Nox regulatory organizer family. To accomplish these goals, we will use a combination of biochemical, genetic, biophysical and cellular approaches. 1.0 We will investigate the role of Rac GTPase in regulation of Nox activity. Based upon our initial characterization of a Rac-binding site on Nox proteins, we will define the activity of Rac2 in regulating electron transfer reactions critical to phagocyte NADPH oxidase (phox) function. A detailed genetic analysis of the Rac-regulatory site on Nox protein(s) will be carried out. Fluorescence-based methods and other biophysical approaches will be used to evaluate Nox2 regulation by Rac GTPase. 2.0 We will dissect the molecular basis for Nox1 regulation by Src tyrosine kinase. We will use biochemical, cellular, and genetic means to investigate the role of Src-mediated NoxA1 phosphorylation in Nox1 regulation. The roles of novel p47-related """"""""organizers"""""""" in modulating Nox activities, localization, and responsiveness to stimuli will be determined using biochemical, cellular, genetic, and imaging approaches.

Public Health Relevance

The formation of reactive oxygen by enzymes known as NADPH oxidases plays important biological roles. We will investigate how the binding of Rac GTPase to NADPH oxidases (Nox) controls their activity. Nox1, abundant in the gut and vasculature, is also regulated by the Src kinase. We will study how a newly identified group of Src target proteins act to organize Nox assembly and localized activity. Understanding Nox regulation will have important implications for disease therapy.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Research Project (R01)
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Molecular and Integrative Signal Transduction Study Section (MIST)
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Charette, Marc F
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Scripps Research Institute
La Jolla
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Taulet, Nicolas; Delorme-Walker, Violaine D; DerMardirossian, Celine (2012) Reactive oxygen species regulate protrusion efficiency by controlling actin dynamics. PLoS One 7:e41342
Gianni, Davide; DerMardirossian, Céline; Bokoch, Gary M (2011) Direct interaction between Tks proteins and the N-terminal proline-rich region (PRR) of NoxA1 mediates Nox1-dependent ROS generation. Eur J Cell Biol 90:164-71
Gianni, Davide; Taulet, Nicolas; DerMardirossian, Celine et al. (2010) c-Src-mediated phosphorylation of NoxA1 and Tks4 induces the reactive oxygen species (ROS)-dependent formation of functional invadopodia in human colon cancer cells. Mol Biol Cell 21:4287-98
Gianni, Davide; Taulet, Nicolas; Zhang, Hui et al. (2010) A novel and specific NADPH oxidase-1 (Nox1) small-molecule inhibitor blocks the formation of functional invadopodia in human colon cancer cells. ACS Chem Biol 5:981-93
Kim, Jun-Sub; Bokoch, Gary M (2009) Anthrax edema toxin inhibits Nox1-mediated formation of reactive oxygen species by colon epithelial cells. J Innate Immun 1:145-52
Bokoch, Gary M; Diebold, Becky; Kim, Jun-Sub et al. (2009) Emerging evidence for the importance of phosphorylation in the regulation of NADPH oxidases. Antioxid Redox Signal 11:2429-41
Bielek, Heike; Anselmo, Anthony; Dermardirossian, Celine (2009) Morphological and proliferative abnormalities in renal mesangial cells lacking RhoGDI. Cell Signal 21:1974-83
Gianni, Davide; Diaz, Begoña; Taulet, Nicolas et al. (2009) Novel p47(phox)-related organizers regulate localized NADPH oxidase 1 (Nox1) activity. Sci Signal 2:ra54
Kim, Jun-Sub; Huang, Timothy Y; Bokoch, Gary M (2009) Reactive oxygen species regulate a slingshot-cofilin activation pathway. Mol Biol Cell 20:2650-60
Kao, Yu-Ya; Gianni, Davide; Bohl, Benjamin et al. (2008) Identification of a conserved Rac-binding site on NADPH oxidases supports a direct GTPase regulatory mechanism. J Biol Chem 283:12736-46

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