After neurons have received a depolarizing signal, the resulting influx of Ca2+ triggers the fusion of secretory vesicles with the plasma membrane and the release of neurotransmitters. Neurotrophic factors such as nerve growth factor (NGF) enhance this process, although the mechanism responsible is not known. Mori et al. expressed a fusion protein of neuropeptide Y and a fluorescent protein (NPY-Venus) in PC12 neuroendocrine cells and then monitored the release of NPY-Venus after applying a depolarizing signal to the cells. The authors confirmed that pretreatment of the cells with NGF enhanced the depolarization-induced secretion of NPY-Venus. Inhibition of c-Jun N-terminal kinase (JNK) blocked the enhancing effect of NGF on NPY-Venus secretion. Western blotting analyses showed that treatment of PC12 cells with NGF resulted in the phosphorylation of JNK and its substrate c-Jun. Yeast two-hybrid studies identified synaptotagmin 4 (Syt 4), a membrane-trafficking protein that regulates exocytosis, as a binding partner for JNK, and this interaction was confirmed in immunoprecipitation experiments. In vitro kinase assays showed that JNK phosphorylated Syt 4 at Ser135, and NGF treatment of PC12 cells resulted in JNK-mediated phosphorylation of Syt 4 at Ser135. Knockdown of Syt 4 in PC12 cells by short hairpin RNA blocked the enhancement of depolarization-induced secretion by NGF, compared with that in control cells, but had no effect on basal secretion. Finally, immunofluorescence studies showed that NGF stimulated the JNK-dependent localization of Syt 4 to mature secretory vesicles. Together, these data suggest a mechanism by which neurotrophins might enhance basal secretion of neurotransmitters.
Y. Mori, M. Higuchi, Y. Hirabayashi, M. Fukuda, Y. Gotoh, JNK phosphorylates synaptotagmin-4 and enhances Ca2+-evoked release. EMBO J. 27, 76-87 (2008). [PubMed]