DEPOLARIZATION OF CEREBELLAR GRANULE CELLS INCREASES PHOSPHORYLATION OF RABPHILIN-3A

Studies performed over the past several years have provided evidence t hat phosphorylation of proteins is important in the regulation of neur otransmitter release. In this study, it is shown that rabphilin-3A is present in cerebellar granule cells as a phosphoprotein, by using P-32 -labeling of cerebellar granule cells, immunoprecipitation, phosphoami no acid analysis, and phosphopeptide mapping. The level of phosphoryla tion was increased (224 +/- 13%) (mean +/- SEM) on depolarization of t he cells with K+ (56 mM) in the presence of external Ca2+ (1 mM). stim ulation of protein kinase C with a phorbol ester(phorbol 12,13-dibutyr ate) also enhanced the phosphorylation of rabphilin-3A (217 +/- 21%), Inhibitors of Ca2+/calmodulin-stimulated protein kinases or protein ki nase C reduced the depolarization-enhanced phosphorylation of rabphili n-3A, indicating that rabphilin-3A is one of the targets for Ca2+-acti vated protein kinases in the nerve terminal. Costimulation of cells wi th phorbol 12,13-dibutyrate and K+ depolarization produced an increase d level of phosphorylation of rabphilin-3A compared with either stimul us alone (287 +/- 61%). Phosphoamino acid analysis showed that serine was the main phosphorylated residue. A slight increase in the threonin e phosphorylation could also be detected, whereas tyrosine phosphoryla tion could not be detected at all. These results suggest that rabphili n-3A is phosphorylated in vivo and undergoes synaptic activity-depende nt phosphorylation during Ca2+-activated K+ depolarization.