Depolarization of cerebellar granule cells increases phosphorylation of rabphilin-3A

J Neurochem. 1998 Oct;71(4):1661-9. doi: 10.1046/j.1471-4159.1998.71041661.x.


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 32P-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 mM) in the presence of external Ca2+ (1 mM). Stimulation of protein kinase C with a phorbol ester (phorbol 12,13-dibutyrate) also enhanced the phosphorylation of rabphilin-3A (217 +/- 21%). Inhibitors of Ca2+/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 Ca2+-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 Ca2+-activated K+ depolarization.

MeSH terms

  • Adaptor Proteins, Signal Transducing
  • Animals
  • Calcium / metabolism
  • Cells, Cultured
  • Cerebellum / cytology
  • Cerebellum / enzymology
  • Cerebellum / metabolism*
  • Cerebellum / physiology*
  • Enzyme Activation / drug effects
  • GTP-Binding Proteins / biosynthesis
  • GTP-Binding Proteins / metabolism*
  • Intracellular Fluid / enzymology
  • Intracellular Fluid / metabolism
  • Nerve Tissue Proteins / biosynthesis
  • Nerve Tissue Proteins / metabolism*
  • Neurons / enzymology
  • Neurons / metabolism
  • Neurons / physiology
  • Phorbol 12,13-Dibutyrate / pharmacology
  • Phosphorylation / drug effects
  • Potassium / metabolism
  • Protein Kinase C / metabolism
  • Rats
  • Rats, Wistar
  • Vesicular Transport Proteins
  • rab GTP-Binding Proteins*


  • Adaptor Proteins, Signal Transducing
  • Nerve Tissue Proteins
  • Vesicular Transport Proteins
  • rabphilin-3A
  • Phorbol 12,13-Dibutyrate
  • Protein Kinase C
  • GTP-Binding Proteins
  • rab GTP-Binding Proteins
  • Potassium
  • Calcium