Depolarization-induced phosphorylation of the protein kinase C substrate B-50 (GAP-43) in rat cortical synaptosomes.
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.
Dekker
L V
LV
Division of Molecular Neurobiology, Rudolf Magnus Institute, University of Utrecht, The Netherlands.
De Graan
P N
PN
De Wit
M
M
Hens
J J
JJ
Gispen
W H
WH
D000818:Animals; D000001:Calcimycin / Q000494:pharmacology; D002540:Cerebral Cortex / Q000378:metabolism* / Q000648:ultrastructure; D004563:Electrochemistry; D019922:GAP-43 Protein; D008562:Membrane Glycoproteins / Q000378:metabolism*; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D011188:Potassium / Q000502:physiology; D011233:Precipitin Tests; D047428:Protein Kinase Inhibitors; D051381:Rats; D013574:Synaptosomes / Q000378:metabolism*
D019922:GAP-43 Protein; D008562:Membrane Glycoproteins; D009419:Nerve Tissue Proteins; D010750:Phosphoproteins; D047428:Protein Kinase Inhibitors; D000001:Calcimycin; D011188:Potassium