The phosphorylation state of two identified neural-specific protein kinase C substrates (the presynaptic protein B-50 and the postsynaptic protein neurogranin) was monitored after the induction of long term potentiation in the CA1 field of rat hippocampus slices by quantitative immunoprecipitation following 32Pi labeling in the recording chamber. B-50 phosphorylation was increased from 10 to 60 min, but no longer at 90 min after long term potentiation had been induced, neurogranin phosphorylation only at 60 min. Increased phosphorylation was not found when long term potentiation was blocked with the N-methyl-D-aspartate receptor antagonist D-2-amino-5-phosphonovalerate, when only low frequency stimulation was applied or tetanic stimulation failed to induce long term potentiation. Our data show that both B-50 and neurogranin phosphorylation are increased following the induction of long term potentiation, thus providing strong evidence for pre- and postsynaptic protein kinase C activation during narrow, partially overlapping, time windows after the induction of long term potentiation. The phosphorylation state of two identified neural-specific protein kinase C substrates (the presynaptic protein B-50 and the postsynaptic protein neurogranin) was monitored after the induction of long term potentiation in the CA1 field of rat hippocampus slices by quantitative immunoprecipitation following 32Pi labeling in the recording chamber. B-50 phosphorylation was increased from 10 to 60 min, but no longer at 90 min after long term potentiation had been induced, neurogranin phosphorylation only at 60 min. Increased phosphorylation was not found when long term potentiation was blocked with the N-methyl-D-aspartate receptor antagonist D-2-amino-5-phosphonovalerate, when only low frequency stimulation was applied or tetanic stimulation failed to induce long term potentiation. Our data show that both B-50 and neurogranin phosphorylation are increased following the induction of long term potentiation, thus providing strong evidence for pre- and postsynaptic protein kinase C activation during narrow, partially overlapping, time windows after the induction of long term potentiation.
Abstract: The nervous tissue‐specific protein B‐50 (GAP‐43), which has been implicated in the regulation of neurotransmitter release, is a member of a family of atypical calmodulin‐binding proteins. To investigate to what extent calmodulin and the interaction between B‐50 and calmodulin are involved in the mechanism of Ca 2+ ‐induced noradrenaline release, we introduced polyclonal anti‐calmodulin antibodies, calmodulin, and the calmodulin antagonists trifluoperazine, W‐7, calmidazolium, and polymyxin B into streptolysin‐O‐permeated synaptosomes prepared from rat cerebral cortex. Anti‐calmodulin antibodies, which inhibited Ca 2+ /calmodulin‐dependent protein kinase II autophosphorylation and calcineurin phosphatase activity, decreased Ca 2+ ‐induced noradrenaline release from permeated synaptosomes. Exogenous calmodulin failed to modulate release, indicating that if calmodulin is required for vesicle fusion it is still present in sufficient amounts in permeated synaptosomes. Although trifluoperazine, W‐7, and calmidazolium inhibited Ca 2+ ‐induced release, they also strongly increased basal release. Polymyxin B potently inhibited Ca 2+ ‐induced noradrenaline release without affecting basal release. It is interesting that polymyxin B was also the only antagonist affecting the interaction between B‐50 and calmodulin, thus lending further support to the hypothesis that B‐50 serves as a local Ca 2+ ‐sensitive calmodulin store underneath the plasma membrane in the mechanism of neurotransmitter release. We conclude that calmodulin plays an important role in vesicular noradrenaline release, probably by activating Ca 2+ /calmodulin‐dependent enzymes involved in the regulation of one or more steps in the release mechanism.
The neuron-specific phosphoprotein B-50 (Mr 48 kDa, isoelectric point, IEP, 4.5), which is identical to GAP43, is a member of a family of growth-associated proteins. Protein B-50 is a major phosphoprotein in nerve growth cones isolated from fetal rat brain. In a growth cone particulate fraction (GCp), endogenous B-50 phosphorylation is Ca2+-dependent and is unaffected by cAMP. Addition of purified protein kinase C (PKC) to GCP enhances B-50 phosphorylation. In heat-inactivated GCp, B-50 is one of the major substrates of purified PKC. Endogenous B-50 phosphorylation in GCP is stimulated in a dose-dependent manner by 4 beta-phorbol diesters, known to activate PKC, but not by the inactive 4 alpha-phorbol derivatives. In synaptic plasma membranes (SPM) isolated from adult rat brain, the degree of B-50 phosphorylation has been implicated in the modulation of receptor-mediated polyphosphoinositide (PPI) hydrolysis. In addition to B-50 and its kinase, PKC, the GCp fraction was also shown to contain all other components of such a modulatory system: the phosphatidylinositol 4-phosphate (PIP)-kinase, as shown on Western blots with affinity-purified IgGs against PIP-kinase, and the polyphosphoinosides, PIP and phosphatidylinositol 4,5-bisphosphate (PIP2), since the addition of gamma-32P-ATP to the GCp fraction not only results in B-50 phosphorylation but also in the labeling of phosphatidic acid (PA), PIP, and PIP2. ACTH1-24, which inhibits B-50 phosphorylation in the GCp fraction in a dose-dependent manner (IC50 = 5 x 10(-6) M), stimulates PIP2 labeling dose-dependently in the same preparation.(ABSTRACT TRUNCATED AT 250 WORDS)
A combined assay to measure neurite outgrowth and B-50/GAP-43 levels in PC12 cells is reported. During NGF-induced neuritogenesis, B-50/GAP-43 expression was monitored by enzyme-linked immunosorbent assay (ELISA). Neurite outgrowth was quantified at the same time by the use of video image analysis. Sensitivity and reliability of the methods are shown with a dose-response and time curve of beta-NGF-induced neuritogenesis. A linear increase in total length of neurites was induced by concentrations of beta-NGF greater than or equal to 5 ng/ml and was accompanied by a linear increase in the amount of B-50/GAP-43. The combined methods presented here can conveniently and reliably establish subtle changes in neurite outgrowth and intracellular protein contents.
