Stress-induced activation of c-Jun N-terminal kinase in sensory ganglion neurons: Accumulation in nuclear domains enriched in splicing factors and distribution in perichromatin fibrils

In response to cellular stress, the activation of the JNK cascade mediates phosphorylation of c-Jun that promotes its transactivation, which in turn activates the transcription of specific genes, In an experimental model of neuronal stress in vivo, by means of immunofluorescence and kinase assays we have found a reversible activation of JNK induced by the administration of the anti-cancer drug Adriamycin. In control neurons, a considerable basal level of the active, phosphorylated JNK was detected in neuronal nuclei, with a speckled distribution in addition to a diffuse nucleoplasmic signal. Adriamycin-induced neuronal stress was associated with a notable increase of this nuclear immunostaining, indicating activation of the JNK pathway which was confirmed by the increase of JNK enzymatic activity, while no changes in the total JNK were detected by Western blots, The JNK neuronal response to stress was also accompanied by an increase in the nuclear immunoreactivity for c-Jun and also by the de novo appearance of a strong nuclear phospho-c-Jun signal, These effects tend to revert to the control situation after 24 h of Adriamycin treatment, The nuclear compartmentalization of phospho-JNK and its substrate c-Jun was analyzed by confocal laser microscopy. Phospho-JNK strongly colocalizes with snRNPs in nuclear speckles, while the former was not concentrated in the coiled bodies, Upon stress induction, both c-Jun and phospho-c-Jun show a nucleoplasmic distribution in euchromatin domains, with the nucleoli free of immunolabeling. Furthermore, the nuclear speckles enriched in phospho-JNK exhibit a very low or undetectable signal with both c-Jun antibodies. Immunogold electron microscopy confirms the accumulation of phospho-JNK in interchromatin granule clusters (nuclear speckles), while in the nucleoplasm this kinase is mainly localized in perichromatin fibrils. Both c-Jun and phospho-c-Jun mere also detected in perichromatin fibrils, Double labeling experiments show the colocalization of phospho-JNK and phospho-c-Jun in certain perichromatin fibrils. These results indicate that the neuronal response to the Adriamycin-induced stress is mediated by the activation of the JNK pathway. The accumulation of phospho-JNK in nuclear speckles raises the possibility that this kinase may be involved in the phosphorylation of an unknown splicing factor. Moreover, the colocalization of phospho-JNK and c-Jun in perichromatin fibrils, which are associated with sites of active transcription, suggests that these nuclear structures may be putative sites for the phosphorylation of JNK substrates. (C) 2000 Academic Press.