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.

Abstract

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*

Substances

  • 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