Sequential Activation of Phosphatidylinositol 3-Kinase, βPix, Rac1, and Nox1 in Growth Factor-Induced Production of H₂O₂

Abstract

The generation of reactive oxygen species (ROS) in cells stimulated with growth factors requires the activation of phosphatidylinositol 3-kinase (PI3K) and the Rac protein. We report here that the COOHterminal region of Nox1, a protein related to gp91 phox (Nox2) of phagocytic cells, is constitutively associated with ␤Pix, a guanine nucleotide exchange factor for Rac. Both growth factor-induced ROS production and Rac1 activation were completely blocked in cells depleted of ␤Pix by RNA interference. Rac1 was also shown to bind to the COOH-terminal region of Nox1 in a growth factor-dependent manner. Moreover, the depletion of Nox1 by RNA interference inhibited growth factor-induced ROS generation. These results suggest that ROS production in growth factor-stimulated cells is mediated by the sequential activation of PI3K, ␤Pix, and Rac1, which then binds to Nox1 to stimulate its NADPH oxidase activity.Reactive oxygen species (ROS), such as superoxide anions and hydrogen peroxide (H 2 O 2 ), are produced in mammalian cells in response to the activation of various cell surface receptors and contribute to intracellular signaling and to the regulation of various biological activities, including host defense and metabolic conversion (15,23,35). Receptor-mediated ROS production has been studied extensively in phagocytic cells. The enzyme NADPH oxidase of such cells is composed of at least five protein components, namely two transmembrane flavocytochrome b components (gp91 phox and p22 phox ) and three cytosolic components (p47 phox , p67 phox , and p40 phox ) (2). The exposure of resting phagocytic cells to an appropriate stimulus results in extensive phosphorylation of the cytosolic components of NADPH oxidase and their association with the transmembrane flavocytochrome b components (1,11,33,36). The assembled oxidase complex catalyzes the transfer of an electron to molecular oxygen to yield the superoxide anion, which is then spontaneously or enzymatically converted to H 2 O 2 . The small GTPase Rac is also required for the activation of NADPH oxidase in phagocytic leukocytes (12, 28). Hematopoietic cell-specific Rac2 and the ubiquitously expressed protein Rac1 are the major and minor Rac isoforms, respectively, in human neutrophils (19).Nonphagocytic cells also produce superoxide anions in response to a variety of extracellular stimuli, including plateletderived growth factor (PDGF) and epidermal growth factor (EGF) (3, 5, 35, 38) Several homologs (Nox1, Nox3, Nox4, Nox5, Duox1, and Duox2) of gp91 phox (Nox2) have been identified in various nonphagocytic cells (8,13,21,23,37). Nox proteins contain binding sites for FAD, NADPH, and heme, and their NH 2 -terminal portions contain a cluster of six hydrophobic segments that are predicted to form transmembrane ␣ helices (23). Some of the gp91 phox homologs likely associate with p22 phox to form functional cytochrome b in nonphagocytes, given that the latter protein is widely expressed (8) and that p22 phox antisense RNA was shown to inhibit angiotensin II-induced ROS gener...

Translationally controlled tumor protein induces human breast epithelial cell transformation through the activation of Src

“…It has been shown that growth factor-induced ROS generation is a result of sequential activation of EGFR, PI3K, bPix, Rac and NOX1 (Bae et al, 1997;Park et al, 2004). Here, we found remarkable increase in serum-independent, TCTPinduced ROS generation from NADPH oxidase, without cell death (Figures 4a and b).…”

“…As ROS generation by growth factor is related to sequential activation of EGFR and PI3K (Park et al, 2004), and Src has an important role in TCTP-induced EGFR transactivation (as demonstrated in this study), we examined whether TCTP-induced ROS generation depends on Src or PI3K, using Src-and PI3K-specific inhibitors, namely, PP2 and LY294002, respectively. Both PP2 and LY294002 completely blocked TCTPinduced ROS generation in MCF10A.…”

Gene network analysis of oxidative stress-mediated drug sensitivity in resistant ovarian carcinoma cells

“…This reduced mRNA expression and protein level in Cbl cos cells may be essential for inhibition of tumorigenicity, such as cell proliferation and spreading, before apoptosis and may induce apoptosis as observed in other tumor cells. 28 DNA-PKc, a critical DNA repair protein, travels to the cytoplasm and interacts with caspases 29 to induce apoptosis. Co-immunoprecipitation studies (Figures 3e and f) with the ARHGEF6 antibody suggest that DNA-PKc physically interacts with ARGEF6 in Cbl cos -treated cells.…”

“…ARHGEF6, a Gcoupled receptor activated by phosphatidylinositol 3-kinase, modulates the cellular ROS level through Rac1. It also modulates ROS production from nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity in a growth factor-dependent manner, 28 and downregulation of ARHGEF6 inhibits cell spreading and adhesion. 27 Reduction of expression in Cbl cos may act downstream of DNA repair signaling and help to signal cells to maintain excess ROS, to stop proliferation and further adhesion of cells.…”

Leukocyte transendothelial migration: A local affair

“…For instance a-Pix acts as a scaffold to integrate signals that arise from GPCRs with the activation of Cdc42 to drive chemotaxis. 90 In a related example b-Pix tethers NADPH oxidase-1 to Rac1 for activation, 91 a pathway that regulates production of reactive oxygen species that is important to kill pathogenic bacteria.…”