Dekker L V, De Graan P N, De Wit M, Hens J J, Gispen W H
Division of Molecular Neurobiology, Rudolf Magnus Institute, University of Utrecht, The Netherlands.
J Neurochem. 1990 May;54(5):1645-52. doi: 10.1111/j.1471-4159.1990.tb01217.x.
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.
我们研究了钾离子诱导神经元特异性蛋白激酶C底物B-50磷酸化的分子机制。用32P标记的正磷酸盐对大鼠皮质突触体进行预标记。通过免疫沉淀测定法检测B-50的磷酸化。在该系统中，各种佛波酯以及一种合成二酰基甘油衍生物均可增强B-50的磷酸化。钾离子去极化可诱导B-50磷酸化的短暂增强，这完全依赖于细胞外钙离子。此外，应用钙离子载体A23187也可诱导B-50磷酸化，但A23187诱导的B-50磷酸化的幅度和动力学与去极化诱导的不同。蛋白激酶抑制剂1-(5-异喹啉磺酰基)-2-甲基哌嗪（H-7）、N-(6-氨基己基)-5-氯-1-萘磺酰胺（W-7）和星形孢菌素可拮抗钾离子以及佛波酯诱导的B-50磷酸化，而三氟拉嗪和氯米帕明在这两种情况下均无效。我们认为去极化后细胞内钙离子水平的升高是蛋白激酶C激活的触发因素，随后蛋白激酶C使其底物B-50磷酸化。这一系列事件可能对去极化诱导的递质释放机制具有重要意义。
