title: Intracellular mobility and nuclear trafficking of the stress-activated kinase JNK1 are impeded by hyperosmotic stress.
abstract: The c-Jun N-terminal kinases (JNKs) are a group of stress-activated protein kinases that regulate gene expression changes through specific phosphorylation of nuclear transcription factor substrates. To address the mechanisms underlying JNK nuclear entry, we employed a semi-intact cell system to demonstrate for the first time that JNK1 nuclear entry is dependent on the importin α2/β1 heterodimer and independent of importins α3, α4, β2, β3, 7 and 13. However, quantitative image analysis of JNK1 localization following exposure of cells to either arsenite or hyperosmotic stress did not indicate its nuclear accumulation. Extending our analyses to define the dynamics of nuclear trafficking of JNK1, we combined live cell imaging analyses with fluorescence recovery after photobleaching (FRAP) protocols. Subnuclear and subcytoplasmic bleaching protocols revealed the slowed movement of JNK1 in both regions in response to hyperosmotic stress. Strikingly, while movement into the nucleus of green fluorescent protein (GFP) or transport of a GFP-T-antigen fusion protein as estimated by initial rates and time to reach half-maximal recovery (t1/2) measures remained unaltered, hyperosmotic stress slowed the nuclear entry of GFP-JNK1. In contrast, arsenite exposure which did not alter the initial rates of nuclear accumulation of GFP, GFP-T-antigen or GFP-JNK1, decreased the t1/2 for nuclear accumulation of both GFP and GFP-JNK1. Thus, our results challenge the paradigm of increased nuclear localization of JNK broadly in response to all forms of stress-activation and are consistent with enhanced interactions of stress-activated JNK1 with scaffold and substrate proteins throughout the nucleus and the cytosol under conditions of hyperosmotic stress.
authors.affiliation: Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Victoria 3010, Australia.
authors.forename: Mariya
authors.initials: M
authors.lastname: Misheva
authors.affiliation: Department of Biochemistry and Molecular Biology, Monash University, Victoria 3800, Australia; European Molecular Biology Laboratory (EMBL) Australia, Australian Regenerative Medicine Institute, Monash University, Clayton, Victoria 3800, Australia.
authors.forename: Gurpreet
authors.initials: G
authors.lastname: Kaur
authors.affiliation: Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Victoria 3010, Australia.
authors.forename: Kevin R W
authors.initials: KR
authors.lastname: Ngoei
authors.affiliation: Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Victoria 3010, Australia.
authors.forename: Yvonne Y
authors.initials: YY
authors.lastname: Yeap
authors.affiliation: Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Victoria 3010, Australia; Department of Biochemistry and Molecular Biology, Monash University, Victoria 3800, Australia.
authors.forename: Ivan H W
authors.initials: IH
authors.lastname: Ng
authors.affiliation: Department of Biochemistry and Molecular Biology, Monash University, Victoria 3800, Australia.
authors.forename: Kylie M
authors.initials: KM
authors.lastname: Wagstaff
authors.affiliation: Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Victoria 3010, Australia.
authors.forename: Dominic C H
authors.initials: DC
authors.lastname: Ng
authors.affiliation: Department of Biochemistry and Molecular Biology, Monash University, Victoria 3800, Australia.
authors.forename: David A
authors.initials: DA
authors.lastname: Jans
authors.affiliation: Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Victoria 3010, Australia. Electronic address: marieb@unimelb.edu.au.
authors.forename: Marie A
authors.initials: MA
authors.lastname: Bogoyevitch
mesh_terms: D000818:Animals; D000952:Antigens, Polyomavirus Transforming / Q000378:metabolism; D018053:Arsenites / Q000494:pharmacology; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D036681:Fluorescence Recovery After Photobleaching; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D042541:Intracellular Space / Q000187:drug effects / Q000378:metabolism*; D028884:Karyopherins / Q000378:metabolism; D007700:Kinetics; D051379:Mice; D048055:Mitogen-Activated Protein Kinase 8 / Q000378:metabolism*; D009997:Osmotic Pressure / Q000187:drug effects; D010766:Phosphorylation / Q000187:drug effects; D021381:Protein Transport / Q000187:drug effects; D051381:Rats; D013012:Sorbitol / Q000494:pharmacology*; D013312:Stress, Physiological / Q000187:drug effects; D013347:Subcellular Fractions / Q000201:enzymology
chemical_list: D000952:Antigens, Polyomavirus Transforming; D018053:Arsenites; D028884:Karyopherins; D049452:Green Fluorescent Proteins; D013012:Sorbitol; D048055:Mitogen-Activated Protein Kinase 8; C015001:arsenite
keywords: 5-iodoacetamidofluorescein; CLSM; ERK; FRAP; Fc; Fluorescence recovery after photobleaching; Fn; Fn/c; GFP; GST; HTC cells; IAF; Imp; JNK; Live-imaging; MAPK; MAPK kinase; MEFs; MKK; NLS; NTS; Nuclear import; SEM; Simian Virus SV40 large tumor antigen; Sorbitol; TAg; TR70; Texas Red-labeled dextran; c-Jun N-terminal kinase; confocal laser scanning microscopy; cytoplasmic fluorescence; extracellular signal-regulated kinase; fluorescence recovery after photobleaching; glutathione S-transferase; green fluorescent protein; importin; mitogen-activated protein kinase; murine embryonic fibroblasts; nuclear factor erythroid 2 (NF-E2); nuclear fluorescence; nuclear localization sequence; nuclear translocation signal; p45; rat hepatoma tissue culture cells; standard error of the mean; t(1/2); the nuclear to cytoplasmic ratio; time to reach half-maximal recovery