Elsevier

Biochimie

Volume 89, Issue 9, September 2007, Pages 1159-1167
Biochimie

NOX5 is expressed at the plasma membrane and generates superoxide in response to protein kinase C activation

https://doi.org/10.1016/j.biochi.2007.05.004Get rights and content

Abstract

NOX5 is a ROS-generating NADPH oxidase which contains an N-terminal EF-hand region and can be activated by cytosolic Ca2+ 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 Ca2+ 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 (∼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 Ca2+ activation.

Introduction

Reactive oxygen species (ROS) have been implicated in a number of biological processes, such as differentiation, apoptosis, proliferation, changed transcription patterns, channel function among others (reviewed in [1], [2]). The NADPH oxidase (NOX) family of enzymes is a major source of ROS production in cells, with the sole apparent purpose of generating ROS. The NOX family contains seven members (NOX1-5 and DUOX1 and 2) which show distinct tissue distribution and mechanisms of activation. The best studied NOX member is the phagocyte NADPH oxidase, NOX2, which is predominantly expressed in neutrophils and macrophages. Activation of NOX2 is responsible for the massive ROS production observed during the respiratory burst which is involved in the killing of ingested microorganisms. NOX2 itself is a membrane protein with six transmembrane domains, but its expression requires stabilization by another membrane protein, p22phox. Activation of NOX2 requires translocation of cytoplasmic subunits (p47phox, p67phox, p40phox) and activators (Rac1 or Rac2) to the membrane-bound NOX2/p22phox. NOX2 activation mechanisms include the phosphorylation of its cytoplasmic subunit p47phox [3]. The mechanisms of activation of NOX1 and NOX3 are not fully understood, but also depend on cytosolic regulatory subunits and may require phosphorylation at least under some circumstances [4], [5], [6], [7], [8].

In contrast to NOX1, 2 and 3, NOX4 and NOX5 do not appear to require cytosolic subunits for activation. NOX4 is associated with p22phox and seems to be constitutively active in most cell types, while NOX5, with 4 N-terminal EF-hand domains, can be activated by elevations of the cytosolic free Ca2+ concentration, [Ca2+]c [9].

NOX5 is expressed in various fetal tissues, uterus, testis, spleen, lymph nodes and endothelial cells [10], [11]. Interestingly, rodents appear to have selectively lost NOX5, while other mammals have one NOX5 gene, and plants express a multitude of NOX5-like genes [12]. In humans, there are at least five splice variants of NOX5: NOX5α is expressed in lymphoid tissues; the NOX5β form is found in testis: NOX5γ for which evidence for a presence in a cellular type has not been confirmed to date; NOX5δ, which is found in human microvascular endothelial cells (HMEC-1) [13]; and NOX5ɛ, also called NOX5S, which is a shorter form without EF-hands expressed mainly in embryonic tissues [10]. An increased expression of NOX5 has been observed in certain pathologies, such as hairy cell leukemia [14], melanoma cells [15], prostate cancer cells [16] and Barret's mucosa [17].

Herein, we used HEK293 cells stably expressing human NOX5β as a tool to study NOX5 activation and localization. We demonstrate that NOX5 can be activated by Ca2+ and by a pathway involving PKC and inhibited by DPI, but not by apocynin, the most widely used NOX inhibitors. We observed a perinuclear localization of NOX5 as well as cell surface expression, and a migration of NOX5 on SDS gels slightly lower than predicted by computer models.

Section snippets

Chemicals, enzymes and buffers

Dulbecco's modified Eagle's medium (DMEM), Hank's buffered salt solution (HBSS), fetal calf serum, neomycin (G418, geneticin) and Amplex Red were purchased from Invitrogen. pGEM-T vector was purchased from Promega. Streptavidin–horseradish peroxidase conjugate was purchased from Amersham. Protein kinase inhibitors were purchased as a kit from Calbiochem. Penicillin, streptomycin, phorbol myristate acetate (PMA), ionomycin, apocynin, luminol, cytochrome C and diphenylene iodonium chloride (DPI)

Prediction of phosphorylation on NOX5

The NetPhos 2.0 Server was used for prediction of serine-threonine phosphorylation of NOX5 [18]. Eleven serine residues were considered to have a probability higher than 95% of being phosphorylated (Fig. 1a). S111, S114 are located in the specific intracellular NOX5 N terminal region between EF hands domains III and IV. S346 lies in the intracellular loop between transmembrane domains IV and V. S486, S490, S492, S498, S501, S502 and S505 are located in a serine rich region in NOX5 intracellular

Discussion

After the initial description of several EF-hand containing NOX5 isoforms [11], it appeared that from a biochemical point of view, NOX5 was the most straightforward of the NOX enzymes. With no need for subunits and phosphorylation steps, expression of the NOX5 protein and [Ca2+]c elevations appeared sufficient for activity [9]. The present study demonstrates that NOX5 is more complex than this, and can be activated by phosphorylation by protein kinase C, likely through phosphorylation of serine

Acknowledgements

The authors would like thank Marie-Claude Jacquot for excellent technical assistance.

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