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Depolarization of Cerebellar Granule Cells Increases Phosphorylation of Rabphilin‐3A
Author(s) -
Fykse Else Marie
Publication year - 1998
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1046/j.1471-4159.1998.71041661.x
Subject(s) - phosphorylation , depolarization , microbiology and biotechnology , protein phosphorylation , autophosphorylation , protein kinase c , biology , protein kinase a , chemistry , biochemistry , biophysics
Studies performed over the past several years have provided evidence that phosphorylation of proteins is important in the regulation of neurotransmitter release. In this study, it is shown that rabphilin‐3A is present in cerebellar granule cells as a phosphoprotein, by using 32 P‐labeling of cerebellar granule cells, immunoprecipitation, phosphoamino acid analysis, and phosphopeptide mapping. The level of phosphorylation was increased (224 ± 13%) (mean ± SEM) on depolarization of the cells with K + (56 m M ) in the presence of external Ca 2+ (1 m M ). Stimulation of protein kinase C with a phorbol ester (phorbol 12,13‐dibutyrate) also enhanced the phosphorylation of rabphilin‐3A (217 ± 21%). Inhibitors of Ca 2+ /calmodulin‐stimulated protein kinases or protein kinase C reduced the depolarization‐enhanced phosphorylation of rabphilin‐3A, indicating that rabphilin‐3A is one of the targets for Ca 2+ ‐activated protein kinases in the nerve terminal. Costimulation of cells with phorbol 12,13‐dibutyrate and K + depolarization produced an increased level of phosphorylation of rabphilin‐3A compared with either stimulus alone (287 ± 61%). Phosphoamino acid analysis showed that serine was the main phosphorylated residue. A slight increase in the threonine phosphorylation could also be detected, whereas tyrosine phosphorylation could not be detected at all. These results suggest that rabphilin‐3A is phosphorylated in vivo and undergoes synaptic activity‐dependent phosphorylation during Ca 2+ ‐activated K + depolarization.