title: Establishment of a human cell line stably overexpressing mouse Nip45 and characterization of Nip45 subcellular localization.
abstract: The nuclear factor of activated T cells, cytoplasmic, calcineurin dependent 2 interacting protein, Nfatc2ip (Nip45), has been implicated as a crucial coordinator of the immune response and of cellular differentiation in humans and mice, and contains SUMO-like domains in its C-terminal region. However, the significance of its N-terminal region and its correlation to the SUMO modification pathway remain largely uncharacterized. In this study, a human cultured cell line was established, in which FLAG-tagged mouse Nip45 (FLAG-mNip45) was stably overexpressed. Under standard, non-stressful conditions, we detected FLAG-mNip45 diffusely distributed in the nucleus. Intriguingly, proteasome inhibition by MG132 caused FLAG-mNip45, together with SUMOylated proteins, to localize in nuclear domains associated with promyelocytic leukemia protein. Finally, using an in vitro binding assay, we showed interaction of the N-terminal region of mNip45 with both free SUMO-3 and SUMO-3 chains, indicating that Nip45 may, in part, exert its function via interaction with SUMO/SUMOylated proteins. Taken together, our study provides novel information on a poorly characterized mammalian protein and suggests that our newly established cell line will be useful for elucidating the physiological role of Nip45.
authors.affiliation: Department of Biological Sciences, Graduate School of Science and Technology, Kumamoto University, Kumamoto, Japan.
authors.forename: Kohtaro
authors.initials: K
authors.lastname: Hashiguchi
authors.forename: Masumi
authors.initials: M
authors.lastname: Ozaki
authors.forename: Isao
authors.initials: I
authors.lastname: Kuraoka
authors.forename: Hisato
authors.initials: H
authors.lastname: Saitoh
mesh_terms: D000818:Animals; D002467:Cell Nucleus / Q000378:metabolism*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D057809:HEK293 Cells; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D007976:Leupeptins / Q000494:pharmacology; D051379:Mice; D009687:Nuclear Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D061988:Proteasome Inhibitors / Q000494:pharmacology; D025841:Small Ubiquitin-Related Modifier Proteins / Q000378:metabolism; D058207:Sumoylation; D014452:Ubiquitins / Q000378:metabolism
chemical_list: D015853:Cysteine Proteinase Inhibitors; D047908:Intracellular Signaling Peptides and Proteins; D007976:Leupeptins; C102566:Nfatc2ip protein, mouse; D009687:Nuclear Proteins; D061988:Proteasome Inhibitors; C117909:SUMO2 protein, human; C492189:SUMO3 protein, human; D025841:Small Ubiquitin-Related Modifier Proteins; D014452:Ubiquitins; D046988:Proteasome Endopeptidase Complex; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde