Phosphorylation of B‐50 (GAP43) Is Correlated with Neurotransmitter Release in Rat Hippocampal Slices

Abstract

Recent studies have demonstrated that phorbol diesters enhance the release of various neurotransmitters. It is generally accepted that activation of protein kinase C (PKC) is the mechanism by which phorbol diesters act on neurotransmitter release. The action of PKC in neurotransmitter release is very likely mediated by phosphorylation of substrate proteins localized in the presynaptic nerve terminal. An important presynaptic substrate of PKC is B-50. To investigate whether B-50 mediates the actions of PKC in neurotransmitter release, we have studied B-50 phosphorylation in intact rat hippocampal slices under conditions that stimulate or inhibit PKC and neurotransmitter release. The slices were labelled with [32P]orthophosphate. After treatment, the slices were homogenized, B-50 was immunoprecipitated from the slice homogenate, and the incorporation of 32P into B-50 was determined. Chemical depolarization (30 mM K+) and the presence of phorbol diesters, conditions that stimulate neurotransmitter release, separately and in combination, also enhance B-50 phosphorylation. Polymyxin B, an inhibitor of PKC and neurotransmitter release, decreases concentration dependently the depolarization-induced stimulation of B-50 phosphorylation. The effects of depolarization are not detectable at low extracellular Ca2+ concentrations. It is concluded that in rat hippocampal slices B-50 may mediate the action of PKC in neurotransmitter release.

Long-term potentiation activates the GAP-43 promoter: Selective participation of hippocampal mossy cells

“…The initial enhancement of LTP, lasting hours, could be maintained in part by increased phosphorylation of existing GAP-43 in mossy cell axon terminals, possibly regulating increased neurotransmitter release in the inner molecular layer along with the presumed increased release in the perforant path (30). Thus, a cooperativity of the perforant path and mossy cell axon inputs may be important for enhanced responses in the first hours after tetanus.…”

Phosphorylation of the N-Ethylmaleimide-sensitive Factor Is Associated with Depolarization-dependent Neurotransmitter Release from Synaptosomes

“…Several studies indicate that PKC translocation from cytosol to membrane is associated with both PKC activation and enhanced L-glutamate release (32,35,36). Moreover, phosphorylation of PKC substrates that are selectively localized in nerve terminals, such as B-50/GAP-43 and myristoylated alanine-rich PKC substrate, has been related to neurotransmitter release (37)(38)(39). Among candidate protein targets for PKC that have received scrutiny are those involved in the docking/fusion process.…”

Long-term potentiation and synaptic protein phosphorylation

“…Intracellular injection of PKC (19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31), a peptide fragment representing the pseudosubstrate region of the regulatory domain of PKC, prevented the maintenance of LTP in the injected cell whereas the neighbouring cells showed normal LTP [48]. These results indicate that postsynaptic PKC is involved in the maintenance of LTP.…”

“…At a molecular level, B-50 regulates these processes by modulating signal transduction pathways in growth cones and mature nerve terminals. Indeed, the degree of PKC-mediated B-50 phosphorylation has been correlated with plastic synaptic alterations in hippocampal neurons [ 17], enhanced neurotransmitter release from hippocampal slices and synaptosomes [26,28], inhibition of the PIP kinase activity [61] and more recently with growth cone mobility [31,64] and long-term potentiation in hippocampal slices [32,53]. Thus, it seems that PKCmediated B-50 phosphorylation is of major physiological importance.…”