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Vol. 19, Issue 10, 4020-4031, October 2008

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Phosphatidylinositol (4,5)-bisphosphate Modulates Nox5 Localization via an N-Terminal Polybasic Region

Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, GA 30322

Submitted December 10, 2007; Revised June 19, 2008; Accepted June 26, 2008
Monitoring Editor: John York

Nox5, an EF-hand–containing reactive oxygen species (ROS)-generating NADPH oxidase, contains two conserved polybasic regions: one N-terminal (PBR-N), located between the fourth EF-hand and the first transmembrane region, and one C-terminal (PBR-C), between the first and second NADPH-binding subregions. Here, we show that phosphatidylinositol (4,5)-bisphosphate [PtdIns(4,5)P₂], a major phosphoinositide in plasma membrane, binds to human Nox5 causing Nox5 to localize from internal membranes to the plasma membrane. Enzymatic modulation of PtdIns(4,5)P₂ levels in intact cells altered cell surface localization of Nox5 in parallel with extracellular ROS generation. Mutations in PBR-N prevented PtdIns(4,5)P₂-dependent localization of Nox5 to the plasma membrane and decreased extracellular ROS production. A synthetic peptide corresponding to PBR-N bound to PtdIns(4,5)P₂, but not to PtdIns, whereas mutations in the PBR-N peptide abrogated the binding to PtdIns(4,5)P₂. Arginine-197 in PBR-N was a key residue to regulate subcellular localization of Nox5 and its interaction with PtdIns(4,5)P₂. In contrast, mutation in PBR-C did not affect localization. Thus, extracellular ROS production by Nox5 is modulated by PtdIns(4,5)P₂ by localizing Nox5 to the plasma membrane.

Address correspondence to: Tsukasa Kawahara (tkawaha{at}emory.edu)

Abbreviations used: Nox, NADPH oxidase; Duox, dual oxidase; ROS, reactive oxygen species; SOD, superoxide dismutase; PBR-N, N-terminal polybasic region; PBR-C, C-terminal polybasic region; WT, wild type; PLC, phospholipase C; PtdIns(4,5)P₂, Phosphatidylinositol (4,5)-bisphosphate; PtdIns-3K, Phosphatidylinositol 3-kinase; PtdIns(4,5)P₂-5Ptase, Phosphatidylinositol (4,5)-bisphosphate 5-phoshatase; PtdIns(4)P-5K, Phosphatidylinositol 4-phosphate 5-kinase.