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 substra te from rat brain that is phosphorylated in nerve terminals and dephos phorylated upon stimulation of exocytosis and synaptic vesicle recycli ng. Phosphorylation activates an intrinsic GTPase activity and dephosp hin may play a role in endocytosis [Robinson P. J. et al. (1993) Natur e 365, 163-166]. In this study a specific polyclonal antiserum to deph osphin was used to investigate its distribution in rat brain by immuno blotting 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 m uscle. Immunoblotting of various regions of the brain revealed high le vels of dephosphin, particularly in the hippocampus, cerebellum and ce rebral cortex, but its absence from the anterior pituitary. Synaptosom es were prepared from these three regions and labelled with P-32(i) fo r 60 min, followed by incubation in control or 41 mM K+ depolarizing b uffer. Dephosphin was present in each region and was stoichiometricall y dephosphorylated by depolarization, indicating the presence and regu lation of dephosphin in intact cerebellar nerve terminals. The cerebel lum was selected for detailed study, using conventional light and conf ocal microscopy, owing to its ordered and well-characterized structure . Immunostaining was abundant within the cerebellar cortex and deep ce rebellar nuclei, but almost entirely absent from the medulla. In the c ortex many neuronal cells contained dephosphin-like immunoreactivity w hich was also evident in perikarya, axons, and nerve terminals. Dephos phin-like immunoreactivity was not detected in the radial Bergman glia l cells. The greatest concentrations were observed in synaptic termina ls, 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, wh ere it is regulated by phosphorylation and dephosphorylation. This dis tribution suggests a role for dephosphin in synaptic vesicle cycling i n nerve terminals.