The superoxide-producing NAD(P)H oxidase Nox4 was initially identified as an enzyme that is highly expressed in the kidney and is possibly involved in oxygen sensing and cellular senescence. Although the oxidase is also abundant in vascular endothelial cells, its role remains to be elucidated. Here we show that Nox4 preferentially localizes to the nucleus of human umbilical vein endothelial cells (HUVECs), by immunocytochemistry and immunoelectron microscopy using three kinds of affinity-purified antibodies raised against distinct immunogens from human Nox4. Silencing of Nox4 by RNA interference (RNAi) abrogates nuclear signals given with the antibodies, confirming the nuclear localization of Nox4. The nuclear fraction of HUVECs exhibits an NAD(P)Hdependent superoxide-producing activity in a manner dependent on Nox4, which activity can be enhanced upon cell stimulation with phorbol 12-myristate 13-acetate. This stimulant also facilitates gene expression as estimated in the present transfection assay of HUVECs using a reporter regulated by the Maf-recognition element MARE, a DNA sequence that constitutes a part of oxidative stress response. Both basal and stimulated transcriptional activities are impaired by RNAi-mediated Nox4 silencing. Thus Nox4 appears to produce superoxide in the nucleus of HUVECs, thereby regulating gene expression via a mechanism for oxidative stress response.

Protein-phosphoinositide interaction participates in targeting proteins to membranes where they function correctly and is often modulated by phosphorylation of lipids. Here we show that protein phosphorylation of p47 phox , a cytoplasmic activator of the microbicidal phagocyte oxidase (phox), elicits interaction of p47 phox with phosphoinositides. Although the isolated phox homology (PX) domain of p47 phox can interact directly with phosphoinositides, the lipid-binding activity of this protein is normally suppressed by intramolecular interaction of the PX domain with the C-terminal Src homology 3 (SH3) domain, and hence the wild-type full-length p47 phox is incapable of binding to the lipids. The W263R substitution in this SH3 domain, abrogating the interaction with the PX domain, leads to a binding of p47 phox to phosphoinositides. The findings indicate that disruption of the intramolecular interaction renders the PX domain accessible to the lipids. This conformational change is likely induced by phosphorylation of p47 phox , because protein kinase C treatment of the wild-type p47 phox but not of a mutant protein with the S303͞ 304͞328A substitution culminates in an interaction with phosphoinositides. Furthermore, although the wild-type p47 phox translocates upon cell stimulation to membranes to activate the oxidase, neither the kinase-insensitive p47 phox nor lipid-bindingdefective proteins, one lacking the PX domain and the other carrying the R90K substitution in this domain, migrates. Thus the protein phosphorylation-driven conformational change of p47 phox enables its PX domain to bind to phosphoinositides, the interaction of which plays a crucial role in recruitment of p47 phox from the cytoplasm to membranes and subsequent activation of the phagocyte oxidase. O ne of the most dominant themes in current cell biology is acute and sophisticated targeting of proteins to new cellular locations, e.g., to membranes, the nucleus, and so forth. Recruitment of proteins to cell membranes is often triggered by phosphorylation of the lipid phosphatidylinositol (PtdIns), which can create targeting sites for proteins (1, 2). The phosphorylation or hydrolysis of inositol-containing lipids in cell membranes is currently known to orchestrate numerous complex cellular events (3, 4). A variety of protein modules such as pleckstrin homology and FYVE domains recognize specific phosphoinositides (phosphorylated forms of PtdIns) to recruit proteins to appropriate cell membranes (1, 2).The phagocyte oxidase (phox) homology (PX) domain (5), also known as the phox and Bem1p 2 (PB2) domain (6, 7), occurs in the phox proteins p47 phox and p40 phox in mammals, the polarity establishment protein Bem1p in budding yeast, and a variety of eukaryotic proteins involved in membrane trafficking. We have determined the NMR structure of the PX domain of p47 phox and demonstrated that it interacts with the C-terminal Src homology 3 (SH3) domain of this protein (8). The p47 phox PX domain consists of an antiparallel ␤-sheet formed by three strand...

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Component Affected g p9 1-phox gp91-phox gp91-phox p22-ph0~ p22-phox p47-ph0~ p67-ph0~ Cytochrome 458 Heme Spectrum