LAUSR

Abstract NADPH oxidases (NOX) have many biological roles, but their regulation to control production of potentially toxic ROS molecules remains unclear. A previously identified insertion sequence of 21 residues (called NIS) influences NOX activity, and its predicted flexibility makes it a good candidate for providing a dynamic switch controlling the NOX active site. We constructed NOX2 chimeras in which NIS had been deleted or exchanged with those from other NOXs (NIS1, 3 and 4). All contained functional heme and were expressed normally at the plasma membrane of differentiated PLB‐985 cells. However, NOX2‐&Dgr;NIS and NOX2‐NIS1 had neither NADPH‐oxidase nor reductase activity and exhibited abnormal translocation of p47phox and p67phox to the phagosomal membrane. This suggested a functional role of NIS. Interestingly after activation, NOX2‐NIS3 cells exhibited superoxide overproduction compared with wild‐type cells. Paradoxically, the Vmax of purified unstimulated NOX2‐NIS3 was only one‐third of that of WT‐NOX2. We therefore hypothesized that post‐translational events regulate NOX2 activity and differ between NOX2‐NIS3 and WT‐NOX2. We demonstrated that Ser486, a phosphorylation target of ataxia telangiectasia mutated kinase (ATM kinase) located in the NIS of NOX2 (NOX2‐NIS), was phosphorylated in purified cytochrome b558 after stimulation with phorbol 12‐myristate‐13‐acetate (PMA). Moreover, ATM kinase inhibition and a NOX2 Ser486Ala mutation enhanced NOX activity whereas a Ser486Glu mutation inhibited it. Thus, the absence of Ser486 in NIS3 could explain the superoxide overproduction in the NOX2‐NIS3 mutant. These results suggest that PMA‐stimulated NOX2‐NIS phosphorylation by ATM kinase causes a dynamic switch that deactivates NOX2 activity. We hypothesize that this downregulation is defective in NOX2‐NIS3 mutant because of the absence of Ser486. Graphical abstract No caption available. HighlightsNIS sequence is a flexible structure supporting a functional role in NOX enzymes.NIS sequence of NOX2 regulates its NADPH oxidase activity in phagocytes.ATM kinase is activated by ROS production in phagocytes after PMA stimulation.Ser486 phosphorylation in NIS‐NOX2 by ATM inhibits the NADPH oxidase activity.