Depolarization‐Induced Phosphorylation of the Protein Kinase C Substrate B‐50 (GAP‐43) in Rat Cortical Synaptosomes

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Abstract
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.

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