Activators of protein kinase C increase the phosphorylation of the synapsins at sites phosphorylated by cAMP-dependent and Ca2+/calmodulin-dependent protein kinases in the rat hippocampal slice

Authors

  • Michael D. Browning,

    Corresponding author
    1. Department of Pharmacology C-236, University of Colorado Health Sciences Center, Denver, Colorado 80262
    • Dept. of Pharmacology C-236, Univ. of Colorado, Health Sciences Center, 4200 E. 9th, Denver, CO 80262
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  • Ellen M. Dudek

    1. Department of Pharmacology C-236, University of Colorado Health Sciences Center, Denver, Colorado 80262
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Abstract

Previous studies have shown that activators of protein kinase C (C kinase) produce synaptic potentiation in the hippocampus. For example, the C kinase activator phorbol dibutyrate has been shown to increase transmitter release in the hippocampus. In addition, a role for C kinase in long-term potentiation has been proposed. A common assumption in such studies has been that substrates for C kinase were responsible for producing these forms of synaptic potentiation. However, we have recently shown that phorbol dibutyrate increased the phosphorylation of synapsin II (formerly protein III, Browning et al., 1987) in chromaffin cells (Haycock et al., 1988). Synapsin II is a synaptic vesicle-associated phosphoprotein that is a very poor substrate for C kinase but an excellent substrate for cAMP-dependent and Ca2+/calmodulin-dependent protein kinase. We felt, therefore, that activation of C kinase might lead to activation of a kinase cascade. Thus effects of C kinase activation might be produced via the phosphorylation of proteins that are not substrates for C kinase. In this report we test the hypothesis that activators of C kinase increase the phosphorylation of synapsin II and an homologous protein synapsin I. Our data indicate that PdBu produced dose-dependent increases in the phosphorylation of synapsin I and synapsin II. We also performed phospho-site analysis of synapsin I using limited proteolysis. These studies indicated that PdBu increased the phosphorylation of multiple sites on synapsin I. These sites have previously been shown to be phosphorylated by both cAMP-dependent protein kinase and the multifunctional Ca2+/calmodulin-dependent protein kinase II. The ability of C kinase activators to produce a net increase in the phosphorylation state of these sites on synapsin was confirmed by a “back phosphorylation assay” with exogenous cAMP- and Ca2+/calmodulin-dependent protein kinases. Previous studies have provided direct evidence that synapsin I regulates neurotransmitter release at the squid giant synapse and that this effect is controlled by phosphorylation of synapsin I by Ca2+/calmodulin-dependent protein kinase II. Accordingly we hypothesize that the phosphorylation of synapsin I and the related protein, synapsin II, may play an important role in mediating the increased transmitter release elicited by activators of C kinase. Moreover, we suggest that the role played by C kinase in long-term potentiation may involve, in part, the phosphorylation of the synapsins.

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