The integral membrane protein p22phox is an indispensable component of the superoxide-generating phagocyte NADPH oxidase, whose catalytic core is the membrane-associated gp91 phox (also known as Nox2). p22 phox associates with gp91 phox and, through its proline-rich C terminus, provides a binding site for the tandem Src homology 3 domains of the activating subunit p47 phox . Whereas p22phox is expressed ubiquitously, its participation in regulating the activity of other Nox enzymes is less clear. This study investigates the requirement of p22 phox for Nox enzyme activity and explores the role of its proline-rich region (PRR) for regulating activity. Coexpression of specific Nox catalytic subunits (Nox1, Nox2, Nox3, Nox4, or Nox5) along with their corresponding regulatory subunits (NOXO1/NOXA1 for Nox1; p47 phox / p67 phox /Rac for Nox2; NOXO1 for Nox3; no subunits for Nox4 or Nox5) resulted in marked production of reactive oxygen. Small interfering RNAs decreased endogenous p22 phox expression and inhibited reactive oxygen generation from Nox1, Nox2, Nox3, and Nox4 but not Nox5. Truncated forms of p22 phox that disrupted the PRR-inhibited reactive oxygen generation from Nox1, Nox2, and Nox3 but not from Nox4 and Nox5. Similarly, p22 phox (P156Q), a mutation that disrupts Src homology 3 binding by the PRR, potently inhibited reactive oxygen production from Nox1 and Nox2 but not from Nox4 and Nox5. Expression of p22 phox (P156Q) inhibited NOXO1-stimulated Nox3 activity, but co-expression of NOXA1 overcame the inhibitory effect. The P157Q and P160Q mutations of p22 phox showed selective inhibition of Nox2/p47 phox /p67 phox , and selectivity was specific for the organizing subunit (p47 phox or NOXO1) rather than the Nox catalytic subunit. These studies stress the importance of p22 phox for the function of Nox1, Nox2, Nox3, and Nox4, and emphasize the key role of the PRR for regulating Nox proteins whose activity is dependent upon p47 phox or NOXO1.

gp91 phox (Nox2), the catalytic subunit of the superoxide-generating respiratory burst oxidase, is regulated by subunits p47 phox and p67 phox . Nox1, a homolog of gp91 phox , is regulated by NOXO1 and NOXA1, homologs of p47 phox and p67 phox , respectively. For both Nox1 and gp91 phox , an organizer protein (NOXO1 or p47 phox ) cooperates with an activator protein (NOXA1 or p67 phox ) to regulate the catalytic subunit. Herein, we investigate the subunit regulation of Nox3 compared with that of other Nox enzymes. Nox3, like gp91 phox , was activated by p47 phox plus p67 phox . Whereas gp91 phox activity required the protein kinase C activator phorbol myristate acetate (PMA), Nox3 activity was already high without PMA, but was further stimulated ϳ30% by PMA. gp91 phox was also activated by NOXO1/NOXA1 and required PMA for high activity. gp91 phox regulation required an intact activation domain in the activator protein, as neither p67 phox (V204A) nor NOXA1(V205A) were effective. In contrast, p67 phox (V204A) was effective (along with p47 phox ) in activating Nox3. Unexpectedly, Nox3 was strongly activated by NOXO1 in the absence of NOXA1 or p67 phox . Nox3 activity was regulated by PMA only when p47 phox but not NOXO1 was present, consistent with the phosphorylation-regulated autoinhibitory region in p47 phox but not in NOXO1. Deletion of the autoinhibitory region from p47 phox rendered this subunit highly active in the absence of PMA toward both gp91 phox and Nox3, and high activity required an activator subunit. The unique regulation of Nox3 supports a model in which multiple interactions with regulatory subunits stabilize an active conformation of the catalytic subunit.The Nox family of NAD(P)H oxidases has recently been described (1-10). These enzymes are structural homologs of gp91 phox (a.k.a. Nox2), the catalytic subunit of the phagocyte NADPH oxidase, and are distributed in a variety of non-phagocytic tissues such as colon, kidney, vascular smooth muscle, and brain. Reactive oxygen species generated by these novel enzymes are proposed to function in signal transduction related to cell growth and cancer (1,(11)(12)(13)(14)(15)(16)(17), angiogenesis (18), and in innate immunity (15), e.g. in barrier cells such as epithelium. In addition, reactive oxygen species from these enzymes are proposed to be causally linked to pathological states including atherosclerosis (19), cancer (11,15,20,21), and diabetes (22).The human Nox enzymes are encoded by at least 7 genes. NOX1, NOX3, and NOX4 encode proteins that are similar in size and domain structure to gp91 phox . These consist of a Cterminal flavoprotein domain containing both an FAD-binding site and an NADPH-binding site, and an N-terminal membrane-associated hydrophobic domain consisting of 6 transmembrane ␣ helices that provide binding sites for 2 hemes (3). Nox5 consists of these same domains along with an N-terminal calcium-binding domain (9); this enzyme is dormant when expressed in cells, but is activated by calcium. Duox1 and Duox2 build upon the Nox5 str...

The catalytic subunit gp91phox (Nox2) of the NADPH oxidase of mammalian phagocytes is activated by microbes and immune mediators to produce large amounts of reactive oxygen species (ROS) which participate in microbial killing. Homologs of gp91phox, the Nox and Duox enzymes, were recently described in a range of organisms, including plants, vertebrates, and invertebrates such as Drosophila melanogaster. While their enzymology and cell biology are being extensively studied in many laboratories, little is known about in vivo functions of Noxes. Here, we establish and use an inducible system for RNAi to discover functions of dNox, an ortholog of human Nox5 in Drosophila. We report here that depletion of dNox in musculature causes retention of mature eggs within ovaries, leading to female sterility. In dNox-depleted ovaries and ovaries treated with a Nox inhibitor, muscular contractions induced by the neuropeptide proctolin are markedly inhibited. This functional defect results from a requirement for dNox-for the proctolin-induced calcium flux in Drosophila ovaries. Thus, these studies demonstrate a novel biological role for Nox-generated ROS in mediating agonist-induced calcium flux and smooth muscle contraction.

Increased cellular reactive oxygen species (ROS) can act as mitogenic signals in addition to damaging DNA and oxidizing lipids and proteins, implicating ROS in cancer development and progression. To analyze the effects of Nox1 expression and its relation to cellular ROS and signal transduction involved in cellular proliferation, Nox1RNAi constructs were transfected into DU145 prostate cancer cells overexpressing Nox1, causing decreased Nox1 message and protein levels in the Nox1RNAi cell lines. Increased ROS and tumor growth in the Nox1-overexpressing DU145 cells were reversed in the presence of the Nox1RNAi. Analysis and comparison of the message levels in the overexpression and RNAi cells demonstrated that Nox1 overexpression leads to changes in message levels of a variety of proteins including c-fos-induced growth factor, interleukin-8, and Cav-1. Finally, we found that Nox1 protein overexpression is an early event in the development of prostate cancer using a National Cancer Institute prostate cancer tissue microarray (CPCTR). Tumor (86%) was significantly more likely to have Nox1 staining than benign prostate tissue (62%) (P = 0.0001). These studies indicate that Nox1 overexpression may function as a reversible signal for cellular proliferation with relevance for a common human tumor.