Phosphorylation of gp91phox/NOX2 in Human Neutrophils
The phagocyte NADPH oxidase NOX2 was the first NOX family member to be discovered. It is responsible for the production of reactive oxygen species that are required for bacterial killing and host defense. Activated NOX2 is an enzymatic complex composed of two membrane proteins, p22phox and gp91phox (renamed NOX2), which form the cytochrome b558, and four cytosolic proteins, p47phox, p67phox, p40phox, and the small GTPase Rac2. Except for Rac2, all proteins from the complex become phosphorylated during neutrophil activation, suggesting the importance of this process in NOX2 regulation. The phosphorylation of the cytosolic components, and in particular p47phox, has been extensively studied; however, the phosphorylation of the membrane proteins was less studied, in part due to the lack of good antibodies and accurate membrane solubilization techniques. In this chapter, we describe a method we have used to study NOX2 phosphorylation, which is based on the labeling of the intracellular ATP pool with 32P prior to applying a stimulus inducing protein phosphorylation. We also describe the solubilization of membrane-bound gp91phox/NOX2 and analysis by immunoprecipitation, polyacrylamide gel electrophoresis, electrophoretic transfer, phosphoamino acid analysis, and autoradiography. This protocol can also be used to study the possible phosphorylation of other NOX family members.
Key wordsNOX2 gp91phox NADPH oxidase Neutrophils Protein phosphorylation Respiratory burst
This work was supported by grants from the Institut National de la Santé et de la Recherche Médicale (INSERM), the Centre National de la Recherche Scientifique (CNRS), Université Paris Diderot, Labex Inflamex, and Association Vaincre la Mucoviscidose (VLM).
The authors also thank Dr. Martine Torres for her critical review and editing of the manuscript.
Houssam Raad, Riad Arabi Derkawi, contributed equally to this work.
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