title: S-nitrosylation regulates nuclear translocation of chloride intracellular channel protein CLIC4.
abstract: Nuclear translocation of chloride intracellular channel protein CLIC4 is essential for its role in Ca(2+)-induced differentiation, stress-induced apoptosis, and modulating TGF-beta signaling in mouse epidermal keratinocytes. However, post-translational modifications on CLIC4 that govern nuclear translocation and thus these activities remain to be elucidated. The structure of CLIC4 is dependent on the redox environment, in vitro, and translocation may depend on reactive oxygen and nitrogen species in the cell. Here we show that NO directly induces nuclear translocation of CLIC4 that is independent of the NO-cGMP pathway. Indeed, CLIC4 is directly modified by NO through S-nitrosylation of a cysteine residue, as measured by the biotin switch assay. NO enhances association of CLIC4 with the nuclear import proteins importin alpha and Ran. This is likely a result of the conformational change induced by S-nitrosylated CLIC4 that leads to unfolding of the protein, as exhibited by CD spectra analysis and trypsinolysis of the modified protein. Cysteine mutants of CLIC4 exhibit altered nitrosylation, nuclear residence, and stability, compared with the wild type protein likely as a consequence of altered tertiary structure. Moreover, tumor necrosis factor alpha-induced nuclear translocation of CLIC4 is dependent on nitric-oxide synthase activity. Inhibition of nitric-oxide synthase activity inhibits tumor necrosis factor alpha-induced nitrosylation and association with importin alpha and Ran and ablates CLIC4 nuclear translocation. These results suggest that S-nitrosylation governs CLIC4 structure, its association with protein partners, and thus its intracellular distribution.
authors.affiliation: Laboratory of Cancer Biology and Genetics, Center for Cancer Research, NCI, National Institutes of Health, Bethesda, MD 20892, USA.
authors.forename: Mariam
authors.initials: M
authors.lastname: Malik
authors.forename: Anjali
authors.initials: A
authors.lastname: Shukla
authors.forename: Palak
authors.initials: P
authors.lastname: Amin
authors.forename: Wendy
authors.initials: W
authors.lastname: Niedelman
authors.forename: Jessica
authors.initials: J
authors.lastname: Lee
authors.forename: Kasey
authors.initials: K
authors.lastname: Jividen
authors.forename: Juanita M
authors.initials: JM
authors.lastname: Phang
authors.forename: Jinhui
authors.initials: J
authors.lastname: Ding
authors.forename: Kwang S
authors.initials: KS
authors.lastname: Suh
authors.forename: Paul M G
authors.initials: PM
authors.lastname: Curmi
authors.forename: Stuart H
authors.initials: SH
authors.lastname: Yuspa
mesh_terms: D021581:Active Transport, Cell Nucleus; D000818:Animals; D002454:Cell Differentiation; D018118:Chloride Channels / Q000737:chemistry* / Q000378:metabolism; D015603:Keratinocytes / Q000166:cytology; D051379:Mice; D024101:Mitochondrial Proteins / Q000737:chemistry* / Q000378:metabolism; D009154:Mutation; D041681:NIH 3T3 Cells; D019001:Nitric Oxide Synthase / Q000378:metabolism; D009584:Nitrogen / Q000737:chemistry*; D010084:Oxidation-Reduction; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism; D028901:alpha Karyopherins / Q000378:metabolism; D020931:ran GTP-Binding Protein / Q000378:metabolism
chemical_list: C479423:CLIC protein, mouse; D018118:Chloride Channels; D024101:Mitochondrial Proteins; D014409:Tumor Necrosis Factor-alpha; D028901:alpha Karyopherins; D019001:Nitric Oxide Synthase; D020931:ran GTP-Binding Protein; D009584:Nitrogen