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NOX5 is expressed at the plasma membrane and generates superoxide in response to protein kinase C activation
|Published in||Biochimie. 2007, vol. 89, no. 9, p. 1159-1167|
|Abstract||NOX5 is a ROS-generating NADPH oxidase which contains an N-terminal EF-hand region and can be activated by cytosolic Ca(2+) elevations. However the C-terminal region of NOX5 also contains putative phosphorylation sites. In this study we used HEK cells stably expressing NOX5 to analyze the size and subcellular localization of the NOX5 protein, its mechanisms of activation, and the characteristics of the ROS released. We demonstrate that NOX5 can be activated both by the protein kinase C activating phorbol esther PMA and by the Ca(2+) ionophore ionomycin. The PMA- but not the ionomycin-dependent activation can be inhibited by protein kinase C inhibitors. NOX5 activity is inhibited by submicromolar concentrations of diphenyl iodonium (DPI), but not by apocynin. Western blot analysis showed a lower ( approximately 70 kDa) than expected (82 kDa) molecular mass. Two arguments suggest that NOX5 is at least partially expressed on the plasma membrane: (i) the membrane-impermeant superoxide was readily detected by extracellular probes, and (ii) immunofluorescent labeling of NOX5 detected a fraction of the NOX5 protein at the plasma membrane. In summary, we demonstrate that NOX5 can be found intracellularly and at the cell surface. We also describe that it can be activated through protein kinase C, in addition to its Ca(2+) activation.|
|Keywords||Acetophenones/pharmacology — Amino Acid Sequence — Blotting, Western — Cell Line — Cell Membrane/ enzymology/metabolism — Dose-Response Relationship, Drug — Electrophoresis, Polyacrylamide Gel — Enzyme Activation/drug effects — Fluorescent Antibody Technique — Humans — Ionomycin/pharmacology — Membrane Proteins/antagonists & inhibitors/genetics/ metabolism — Microscopy, Confocal — Molecular Sequence Data — NADPH Oxidase/antagonists & inhibitors/genetics/ metabolism — Phosphorylation/drug effects — Protein Kinase C/antagonists & inhibitors/ metabolism — Protein Kinase Inhibitors/pharmacology — Reactive Oxygen Species/metabolism — Sequence Homology, Amino Acid — Superoxides/ metabolism — Transfection|