title: Death receptors 4 and 5 activate Nox1 NADPH oxidase through riboflavin kinase to induce reactive oxygen species-mediated apoptotic cell death.
abstract: Stimulation of the proapoptotic tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) receptors, death receptors 4 (DR4) and 5 (DR5), conventionally induces caspase-dependent apoptosis in tumor cells. Here we report that stimulation of DR4 and/or DR5 by the agonistic protein KD548-Fc, an Fc-fused DR4/DR5 dual-specific Kringle domain variant, activates plasma membrane-associated Nox1 NADPH oxidase to generate superoxide anion and subsequently accumulates intracellular reactive oxygen species (ROS), leading to sustained c-Jun N-terminal kinase activation and eventual apoptotic cell death in human HeLa and Jurkat tumor cells. KD548-Fc treatment induces the formation of a DR4/DR5 signaling complex containing riboflavin kinase (RFK), Nox1, the Nox1 subunits (Rac1, Noxo1, and Noxa1), TNF receptor-associated death domain (TRADD), and TNF receptor-associated factor 2 (TRAF2). Depletion of RFK, but not the Nox1 subunits, TRADD and TRAF2, failed to recruit Nox1 and Rac1 to DR4 and DR5, demonstrating that RFK plays an essential role in linking DR4/DR5 with Nox1. Knockdown studies also reveal that RFK, TRADD, and TRAF2 play critical, intermediate, and negligible roles, respectively, in the KD548-Fc-mediated ROS accumulation and downstream signaling. Binding assays using recombinantly expressed proteins suggest that DR4/DR5 directly interact with cytosolic RFK through RFK-binding regions within the intracellular death domains, and TRADD stabilizes the DR4/DR5-RFK complex. Our results suggest that DR4 and DR5 have a capability to activate Nox1 by recruiting RFK, resulting in ROS-mediated apoptotic cell death in tumor cells.
authors.affiliation: Department of Molecular Science and Technology, Ajou University, Suwon 443-749, Korea.
authors.forename: Kyung-Jin
authors.initials: KJ
authors.lastname: Park
authors.forename: Chang-Han
authors.initials: CH
authors.lastname: Lee
authors.forename: Aeyung
authors.initials: A
authors.lastname: Kim
authors.forename: Ki Jun
authors.initials: KJ
authors.lastname: Jeong
authors.forename: Chul-Ho
authors.initials: CH
authors.lastname: Kim
authors.forename: Yong-Sung
authors.initials: YS
authors.lastname: Kim
mesh_terms: D017209:Apoptosis / Q000502:physiology*; D055785:Gene Knockdown Techniques; D006367:HeLa Cells; D006801:Humans; D048031:JNK Mitogen-Activated Protein Kinases / Q000235:genetics / Q000378:metabolism; D000074624:NADPH Oxidase 1; D019255:NADPH Oxidases / Q000235:genetics / Q000378:metabolism*; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics / Q000378:metabolism*; D053220:Receptors, TNF-Related Apoptosis-Inducing Ligand / Q000235:genetics / Q000378:metabolism*; D013481:Superoxides / Q000378:metabolism*; D053199:TNF Receptor-Associated Death Domain Protein / Q000235:genetics / Q000378:metabolism; D047992:TNF Receptor-Associated Factor 2 / Q000235:genetics / Q000378:metabolism; D020830:rac1 GTP-Binding Protein / Q000235:genetics / Q000378:metabolism
chemical_list: C107953:RAC1 protein, human; D053220:Receptors, TNF-Related Apoptosis-Inducing Ligand; D053199:TNF Receptor-Associated Death Domain Protein; D047992:TNF Receptor-Associated Factor 2; C105190:TNFRSF10A protein, human; D013481:Superoxides; D000074624:NADPH Oxidase 1; D019255:NADPH Oxidases; C402799:NOX1 protein, human; D017853:Phosphotransferases (Alcohol Group Acceptor); C022532:riboflavin kinase; D048031:JNK Mitogen-Activated Protein Kinases; D020830:rac1 GTP-Binding Protein