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

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)P-2], 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)P-2 levels in intact cells altered cell surface localization of Nox5 in parallel with extracellular ROS generation. Mutations in PBR-N prevented PtdIns(4,5)P-2- 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)P-2, but not to PtdIns, whereas mutations in the PBR-N peptide abrogated the binding to PtdIns(4,5)P-2. Arginine-197 in PBR-N was a key residue to regulate subcellular localization of Nox5 and its interaction with PtdIns(4,5)P-2. In contrast, mutation in PBR-C did not affect localization. Thus, extracellular ROS production by Nox5 is modulated by PtdIns(4,5)P-2 by localizing Nox5 to the plasma membrane.