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

Authors

Sylvain Beaumel, Univ. Grenoble Alpes, CNRS, TIMC-IMAG, F-38000 Grenoble, France; CDiReC, Pôle Biologie, CHU de Grenoble, Grenoble F-38043, France.
Antoine Picciocchi, Univ. Grenoble Alpes, CNRS, CEA, Institut de Biologie Structurale, F-38044 Grenoble, France.
Franck Debeurme, Univ. Grenoble Alpes, CNRS, TIMC-IMAG, F-38000 Grenoble, France.
Corinne Vivès, Univ. Grenoble Alpes, CNRS, CEA, Institut de Biologie Structurale, F-38044 Grenoble, France.
Anne-Marie Hesse, 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.
Myriam Ferro, Univ. Grenoble Alpes, INSERM, CEA, Laboratoire de Biologie à Grande Echelle, Grenoble F-38054, France.
Didier Grunwald, Univ. Grenoble Alpes, CEA, INSERM, Laboratoire de Biologie du Cancer et de l'infection, Grenoble F-38000, France.
Heather Stieglitz, Department of Molecular and Cellular Biology, Kennesaw State University, Kennesaw, GA, USA.
Pahk Thepchatri, Department of Molecular and Cellular Biology, Kennesaw State University, Kennesaw, GA, USA.
Susan M. Smith, Department of Molecular and Cellular Biology, Kennesaw State University, Kennesaw, GA, USA.
Franck Fieschi, Univ. Grenoble Alpes, CNRS, CEA, Institut de Biologie Structurale, F-38044 Grenoble, France.
Marie José Stasia, 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.

Department

Molecular and Cellular Biology

Document Type

Article

Publication Date

12-1-2017

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 p47 and p67 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 V 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 b 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.

Journal Title

Free radical biology & medicine

Volume

113

First Page

1

Last Page

15

Digital Object Identifier (DOI)

10.1016/j.freeradbiomed.2017.09.007

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