Dephosphin/dynamin is a neuronal phosphoprotein concentrated in nerve terminals: evidence from rat cerebellum

Dephosphin/dynamin is a 94,000/96,000 mol. wt protein kinase C substrate from rat brain that is phosphorylated in nerve terminals and dephosphorylated upon stimulation of exocytosis and synaptic vesicle recycling. Phosphorylation activates an intrinsic GTPase activity and dephosphin may play a role in endocytosis [Robinson P. J. et al. (1993) Nature 365, 163-166]. In this study a specific polyclonal antiserum to dephosphin was used to investigate its distribution in rat brain by immunoblotting and immunocytochemistry. Immunoblots of various organs of the rat showed that dephosphin was detectable only in the whole brain and not in the testes, lung, kidney, adrenals, heart, liver or skeletal muscle. Immunoblotting of various regions of the brain revealed high levels of dephosphin, particularly in the hippocampus, cerebellum and cerebral cortex, but its absence from the anterior pituitary. Synaptosomes were prepared from these three regions and labelled with 32Pi for 60 min, followed by incubation in control or 41 mM K+ depolarizing buffer. Dephosphin was present in each region and was stoichiometrically dephosphorylated by depolarization, indicating the presence and regulation of dephosphin in intact cerebellar nerve terminals. The cerebellum was selected for detailed study, using conventional light and confocal microscopy, owing to its ordered and well-characterized structure. Immunostaining was abundant within the cerebellar cortex and deep cerebellar nuclei, but almost entirely absent from the medulla. In the cortex many neuronal cells contained dephosphin-like immunoreactivity which was also evident in perikarya, axons, and nerve terminals. Dephosphin-like immunoreactivity was not detected in the radial Bergman glial cells. The greatest concentrations were observed in synaptic terminals, particularly in granular layer glomeruli and basket cell terminals surrounding Purkinje cell bodies and dendrites. Dephosphin therefore appears to be exclusive to neuronal tissue, but is distributed widely throughout the brain. It is located in many neuronal cell types of the cerebellum and may be particularly enriched in synaptic terminals, where it is regulated by phosphorylation and dephosphorylation. This distribution suggests a role for dephosphin in synaptic vesicle cycling in nerve terminals.