title: IPO3-mediated Nonclassical Nuclear Import of NF-κB Essential Modulator (NEMO) Drives DNA Damage-dependent NF-κB Activation.
abstract: Activation of IκB kinase (IKK) and NF-κB by genotoxic stresses modulates apoptotic responses and production of inflammatory mediators, thereby contributing to therapy resistance and premature aging. We previously reported that genotoxic agents induce nuclear localization of NF-κB essential modulator (NEMO) via an undefined mechanism to arbitrate subsequent DNA damage-dependent IKK/NF-κB signaling. Here we show that a nonclassical nuclear import pathway via IPO3 (importin 3, transportin 2) mediates stress-induced NEMO nuclear translocation. We found putative nuclear localization signals in NEMO whose mutations disrupted stress-inducible nuclear translocation of NEMO and IKK/NF-κB activation in stably reconstituted NEMO-deficient cells. RNAi screening of both importin α and β family members, as well as co-immunoprecipitation analyses, revealed that a nonclassical importin β family member, IPO3, was the only importin that was able to associate with NEMO and whose reduced expression prevented genotoxic stress-induced NEMO nuclear translocation, IKK/NF-κB activation, and inflammatory cytokine transcription. Recombinant IPO3 interacted with recombinant NEMO but not the nuclear localization signal mutant version and induced nuclear import of NEMO in digitonin-permeabilized cells. We also provide evidence that NEMO is disengaged from IKK complex following genotoxic stress induction. Thus, the IPO3 nuclear import pathway is an early and crucial determinant of the IKK/NF-κB signaling arm of the mammalian DNA damage response.
authors.affiliation: Department of Oncology, University of Wisconsin, Madison, Wisconsin.
authors.forename: Byounghoon
authors.initials: B
authors.lastname: Hwang
authors.affiliation: Department of Oncology, University of Wisconsin, Madison, Wisconsin; Molecular and Cellular Pharmacology Program, University of Wisconsin, Madison, Wisconsin.
authors.forename: Kevin
authors.initials: K
authors.lastname: McCool
authors.affiliation: Department of Cell and Regenerative Biology, University of Wisconsin, Madison, Wisconsin.
authors.forename: Jun
authors.initials: J
authors.lastname: Wan
authors.affiliation: Department of Oncology, University of Wisconsin, Madison, Wisconsin; University of Wisconsin Carbone Cancer Center, University of Wisconsin, Madison, Wisconsin.
authors.forename: Shelly M
authors.initials: SM
authors.lastname: Wuerzberger-Davis
authors.affiliation: Department of Mechanical and Industrial Engineering and Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, Ontario M5S 3G8, Canada.
authors.forename: Edmond W K
authors.initials: EWK
authors.lastname: Young
authors.affiliation: Department of Oncology, University of Wisconsin, Madison, Wisconsin.
authors.forename: Eun Young
authors.initials: EY
authors.lastname: Choi
authors.affiliation: Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, Pennsylvania 19107.
authors.forename: Gino
authors.initials: G
authors.lastname: Cingolani
authors.affiliation: Department of Cell and Regenerative Biology, University of Wisconsin, Madison, Wisconsin; University of Wisconsin Carbone Cancer Center, University of Wisconsin, Madison, Wisconsin.
authors.forename: Beth A
authors.initials: BA
authors.lastname: Weaver
authors.affiliation: Department of Oncology, University of Wisconsin, Madison, Wisconsin; University of Wisconsin Carbone Cancer Center, University of Wisconsin, Madison, Wisconsin. Electronic address: smiyamot@wisc.edu.
authors.forename: Shigeki
authors.initials: S
authors.lastname: Miyamoto
mesh_terms: D021581:Active Transport, Cell Nucleus; D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D004249:DNA Damage; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D051550:I-kappa B Kinase / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D016328:NF-kappa B / Q000276:immunology*; D019913:Nuclear Localization Signals; D028961:beta Karyopherins / Q000276:immunology / Q000378:metabolism*
chemical_list: C491731:IKBKG protein, human; D016328:NF-kappa B; D019913:Nuclear Localization Signals; C503621:TNPO2 protein, human; D028961:beta Karyopherins; D051550:I-kappa B Kinase
keywords: DNA damage response; NEMO; NF-kappaB; cancer therapy; inflammation; nuclear translocation