Long-term potentiation (LTP) is a well known experimental model for studying the activity-dependent enhancement of synaptic plasticity, and because of its long duration and its associative properties, it has been proposed as a system to investigate the molecular mechanisms of memory formation. At present, there are several lines of evidence that indicate that pre-and postsynaptic kinases and their specific substrates are involved in molecular mechanisms underlying LTP. Many studies focus on the involvement of protein kinase C (PKC). One way to investigate the role of PKC in long-term potentiation is to determine the degree of phosphorylation of its substrates after in situ phosphorylation in hippocampal slices. Two possible targets are the presynaptic membrane-associated protein B-50 (a.k.a. GAP 43, neuromodulin and F1), which has been implicated in different forms of synaptical plasticity in the brain such as neurite outgrowth, hippocampal LTP and neurotransmitter release, and the postsynaptic protein neurogranin (a.k.a. RC3, BICKS and p17) which function remains to be determined. This review will focus on the protein kinase C activity in pre-and postsynaptic compartment during the early phase of LTP and the possible involvement of its substrates B-50 and neurogranin.

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We studied the molecular events underlying K(+)-induced phosphorylation of the neuron-specific protein kinase C substrate B-50. Rat cortical synaptosomes were prelabelled with 32P-labelled orthophosphate. B-50 phosphorylation was measured by an immunoprecipitation assay. In this system, various phorbol esters, as well as a synthetic diacylglycerol derivative, enhance B-50 phosphorylation. K+ depolarization induces a transient enhancement of B-50 phosphorylation, which is totally dependent on extracellular Ca2+. Also, the application of the Ca2+ ionophore A23187 induces B-50 phosphorylation, but the magnitude and kinetics of A23187-induced B-50 phosphorylation differ from those induced by depolarization. The protein kinase inhibitors 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7), N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), and staurosporine antagonize K(+)- as well as PDB-induced B-50 phosphorylation, whereas trifluoperazine and calmidazolium are ineffective under both conditions. We suggest that elevation of the intracellular Ca2+ level after depolarization is a trigger for activation of protein kinase C, which subsequently phosphorylates its substrate B-50. This sequence of events could be of importance for the mechanism of depolarization-induced transmitter release.

To investigate a possible function of the nervous tissue-specific protein kinase C substrate B-50/GAP-43 in regulation of the dynamics of the submembranous cytoskeleton, we studied the interaction between purified B-50 and actin. Both the phosphorylated and dephosphorylated forms of B-50 cosedimented with filamentous actin (F-actin) in a Ca(2+)-independent manner. Neither B-50 nor phospho-B-50 had any effect on the kinetics of actin polymerization and on the critical concentration at steady state, as measured using pyrenylated actin. Light scattering of F-actin samples was not increased in the presence of B-50, suggesting that B-50 does not bundle actin filaments. The number of actin filaments, determined by [3H]cytochalasin B binding, was not affected by either phospho- or dephospho-B-50, indicating that B-50 has neither a severing nor a capping effect. These observations were confirmed by electron microscopic evaluation of negatively stained F-actin samples, which did not reveal any structural changes in the actin meshwork on addition of B-50. We conclude that B-50 is an actin-binding protein that does not directly affect actin dynamics.