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Title

Activity-dependent phosphorylation of SNAP-25 in hippocampal organotypic cultures

Authors
Genoud, S.
Pralong, W.
Riederer, B. M.
Eder, L.
Catsicas, S.
Published in Journal of Neurochemistry. 1999, vol. 72, no. 4, p. 1699-1706
Abstract 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.
Keywords AnimalsBicuculline/pharmacologyBlotting, WesternCarcinogens/pharmacologyElectrophoresis, Gel, Two-DimensionalEnzyme Activation/drug effectsExcitatory Amino Acid Antagonists/pharmacologyExocytosis/physiologyGABA Antagonists/pharmacologyHippocampus/ chemistry/ enzymologyLong-Term Potentiation/physiologyMembrane Proteins/metabolismNerve Tissue Proteins/analysis/ metabolismNeuronal Plasticity/physiologyNeurotransmitter Agents/metabolismOrgan Culture TechniquesPhorbol 12,13-Dibutyrate/pharmacologyPhosphorus Radioisotopes/diagnostic usePhosphorylationProtein Kinase C/metabolismRatsRats, WistarSNARE ProteinsSynaptosomal-Associated Protein 25Vesicular Transport Proteins
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PMID: 10098880
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