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.
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.
© 2013.