Down-regulation of NOX2 activity in phagocytes mediated by ATM-kinase dependent phosphorylation

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-ΔNIS 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.

CRK2 and C-terminal Phosphorylation of NADPH Oxidase RBOHD Regulate Reactive Oxygen Species Production in Arabidopsis

“…Phosphorylation of the C terminus is critical for the regulation of human NOXs. Phosphorylation of the NOX2 C terminus by protein kinase C enhances assembly of the multimeric NOX2 complex and its activity, whereas phosphorylation by ATM kinase inhibits NOX2 activity (Raad et al, 2009;Beaumel et al, 2017). NOX5 activity is regulated by Ca 21 binding to EF hands in the N terminus (Bánfi et al, 2004), but NOX5 is also activated by phosphorylation of the C terminus by protein kinase C a or calcium/calmodulin-dependent kinase II (Pandey et al, 2011;Chen et al, 2014).…”

NADPH oxidase activation in neutrophils: Role of the phosphorylation of its subunits

“…Interestingly, PKC phosphorylation increased the electron transfer activity of the gp91 phox flavoprotein and the binding of its cytosolic domain to the cytosolic proteins Rac2, p67 phox and p47 phox . Recently , Beaumel et al have shown that gp91 phox is also phosphorylated on Ser486 located in the insertion sequence called NIS in PMA‐stimulated neutrophil‐like PLB‐985 cells . Ser486 is located within a consensus sequence targeted by the ataxia telangiectasia ‐mutated kinase (ATM kinase).…”

“…22 Recently, Beaumel et al have shown that gp91 phox is also phosphorylated on Ser486 located in the insertion sequence called NIS in PMA-stimulated neutrophil-like PLB-985 cells. 23 Ser486 is located within a consensus sequence targeted by the ataxia telangiectasia-mutated kinase (ATM kinase). Interestingly, inhibiting the ATM kinase enhanced NADPH oxidase activity, as did mutating Ser486 to Ala486, whereas the Ser486Glu mutation inhibited it.…”

The Emerging Roles of Nicotinamide Adenine Dinucleotide Phosphate Oxidase 2 in Skeletal Muscle Redox Signaling and Metabolism

“…Phosphorylation by protein kinase C (PKC) isoform at undetermined sites in the Cterminal domain was reported to increase electron flow activity and binding to p47phox, p67phox, and Rac2 in human neutrophils (291). More recently, Ser486 in the so-called NOX-specific insertion sequence in the C-terminal NADPHbinding region was suggested to be an inhibitory phosphorylation site targeted by ataxia telangiectasia mutated kinase (ATM) (26). Since the NOX2 complex activates ATM (26,387), this might constitute a negative feedback loop to terminate the NOX2 activity.…”

“…More recently, Ser486 in the so-called NOX-specific insertion sequence in the C-terminal NADPHbinding region was suggested to be an inhibitory phosphorylation site targeted by ataxia telangiectasia mutated kinase (ATM) (26). Since the NOX2 complex activates ATM (26,387), this might constitute a negative feedback loop to terminate the NOX2 activity. Interestingly, both ATM and PKC are expressed in skeletal muscle.…”