Depolarization of cerebellar granule cells increases phosphorylation of rabphilin-3A.
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.
Fykse
E M
EM
Norwegian Defence Research Establishment, Division for Environmental Toxicology, Kjeller.
D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D002531:Cerebellum / Q000166:cytology / Q000201:enzymology / Q000378:metabolism* / Q000502:physiology*; D004789:Enzyme Activation / Q000187:drug effects; D019204:GTP-Binding Proteins / Q000096:biosynthesis / Q000378:metabolism*; D007424:Intracellular Fluid / Q000201:enzymology / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000096:biosynthesis / Q000378:metabolism*; D009474:Neurons / Q000201:enzymology / Q000378:metabolism / Q000502:physiology; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects; D011188:Potassium / Q000378:metabolism; D011493:Protein Kinase C / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D033921:Vesicular Transport Proteins; D020691:rab GTP-Binding Proteins; D000097624:Rabphilin-3A
D048868:Adaptor Proteins, Signal Transducing; D002118:Calcium; D019204:GTP-Binding Proteins; D009419:Nerve Tissue Proteins; D015240:Phorbol 12,13-Dibutyrate; D011188:Potassium; D011493:Protein Kinase C; D033921:Vesicular Transport Proteins; D020691:rab GTP-Binding Proteins; D000097624:Rabphilin-3A