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.
Kawahara
Tsukasa
T
Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, GA 30322, USA. tkawaha@emory.edu
Lambeth
J David
JD
D000595:Amino Acid Sequence; D000818:Animals; D019921:Biotinylation; D002462:Cell Membrane / Q000378:metabolism; D006801:Humans; D007313:Insecta; D008055:Lipids / Q000737:chemistry; D008565:Membrane Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D000074664:NADPH Oxidase 5; D019255:NADPH Oxidases / Q000378:metabolism*; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000737:chemistry*; D010716:Phosphatidylinositols / Q000737:chemistry; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D017382:Reactive Oxygen Species / Q000737:chemistry
D008055:Lipids; D008565:Membrane Proteins; D019269:Phosphatidylinositol 4,5-Diphosphate; D010716:Phosphatidylinositols; D017382:Reactive Oxygen Species; D000074664:NADPH Oxidase 5; D019255:NADPH Oxidases; C431571:NOX5 protein, human