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Free Radic Biol Med. 2017 Dec;113:1-15. doi: 10.1016/j.freeradbiomed.2017.09.007. Epub 2017 Sep 13.

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

Author information

1
Univ. Grenoble Alpes, CNRS, TIMC-IMAG, F-38000 Grenoble, France; CDiReC, Pôle Biologie, CHU de Grenoble, Grenoble F-38043, France.
2
Univ. Grenoble Alpes, CNRS, CEA, Institut de Biologie Structurale, F-38044 Grenoble, France.
3
Univ. Grenoble Alpes, CNRS, TIMC-IMAG, F-38000 Grenoble, France.
4
Univ. Grenoble Alpes, INSERM, CEA, Laboratoire de Biologie à Grande Echelle, Grenoble F-38054, France; Univ. Grenoble Alpes, CEA, INSERM, Laboratoire de Biologie du Cancer et de l'infection, Grenoble F-38000, France.
5
Univ. Grenoble Alpes, INSERM, CEA, Laboratoire de Biologie à Grande Echelle, Grenoble F-38054, France.
6
Univ. Grenoble Alpes, CEA, INSERM, Laboratoire de Biologie du Cancer et de l'infection, Grenoble F-38000, France.
7
Department of Molecular and Cellular Biology, Kennesaw State University, Kennesaw, GA, USA.
8
Univ. Grenoble Alpes, CNRS, TIMC-IMAG, F-38000 Grenoble, France; CDiReC, Pôle Biologie, CHU de Grenoble, Grenoble F-38043, France. Electronic address: mjstasia@chu-grenoble.fr.

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.

KEYWORDS:

Ataxia telangiectasia mutated (ATM); NADPH oxidase; NOX; NOX-specific Insertion Sequence (NIS); Neutrophil; Phosphorylation

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