Activity-dependent phosphorylation of SNAP-25 in hippocampal organotypic cultures.
Synaptosomal-associated protein of 25 kDa (SNAP-25) is thought to play a key role in vesicle exocytosis and in the control of transmitter release. However, the precise mechanisms of action as well as the regulation of SNAP-25 remain unclear. Here we show by immunoprecipitation that activation of protein kinase C (PKC) by phorbol esters results in an increase in SNAP-25 phosphorylation. In addition, immunochemical analysis of two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels shows that SNAP-25 focuses as three or four distinct spots in the expected range of molecular weight and isoelectric point. Changing the phosphorylation level of the protein by incubating the slices in the presence of either a PKC agonist (phorbol 12,13-dibutyrate) or antagonist (chelerythrine) modified the distribution of SNAP-25 among these spots. Phorbol 12,13-dibutyrate increased the intensity of the spots with higher molecular weight and lower isoelectric point, whereas chelerythrine produced the opposite effect. This effect was specific for regulators of PKC, as agonists of other kinases did not produce similar changes. Induction of long-term potentiation, a property involved in learning mechanisms, and production of seizures with a GABA(A) receptor antagonist also increased the intensity of the spots with higher molecular weight and lower isoelectric point. This effect was prevented by the PKC inhibitor chelerythrine. We conclude that SNAP-25 can be phosphorylated in situ by PKC in an activity-dependent manner.
Genoud
S
S
Neuropharmacology, Centre Medical Universitaire, Geneva, Switzerland.
Pralong
W
W
Riederer
B M
BM
Eder
L
L
Catsicas
S
S
Muller
D
D
D015763:2-Amino-5-phosphonovalerate / Q000494:pharmacology; D000818:Animals; D001640:Bicuculline / Q000494:pharmacology; D015153:Blotting, Western; D002273:Carcinogens / Q000494:pharmacology; D015180:Electrophoresis, Gel, Two-Dimensional; D004789:Enzyme Activation / Q000187:drug effects; D018691:Excitatory Amino Acid Antagonists / Q000494:pharmacology; D005089:Exocytosis / Q000502:physiology; D018756:GABA Antagonists / Q000494:pharmacology; D006624:Hippocampus / Q000737:chemistry* / Q000201:enzymology*; D017774:Long-Term Potentiation / Q000502:physiology; D008565:Membrane Proteins / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000032:analysis / Q000378:metabolism*; D009473:Neuronal Plasticity / Q000502:physiology; D018377:Neurotransmitter Agents / Q000378:metabolism; D009924:Organ Culture Techniques; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D050600:SNARE Proteins; D050825:Synaptosomal-Associated Protein 25; D033921:Vesicular Transport Proteins
D002273:Carcinogens; D018691:Excitatory Amino Acid Antagonists; D018756:GABA Antagonists; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D018377:Neurotransmitter Agents; D010761:Phosphorus Radioisotopes; D050600:SNARE Proteins; C494210:Snap25 protein, rat; D050825:Synaptosomal-Associated Protein 25; D033921:Vesicular Transport Proteins; D015240:Phorbol 12,13-Dibutyrate; D015763:2-Amino-5-phosphonovalerate; D011493:Protein Kinase C; D001640:Bicuculline