Involvement of protein kinase C-ε in activity-dependent potentiation of large dense-core vesicle exocytosis in chromaffin cells
Involvement of protein kinase C-ε in activity-dependent potentiation of large dense-core vesicle exocytosis in chromaffin cells
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DOI：
10.1523/jneurosci.2828-06.2006
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发表时间：
2006-08-30
影响因子：
5.3
通讯作者：
Kim, Kyong-Tai
中科院分区：
文献类型：
--
作者：
Park, Yong-Soo;Hur, Eun-Mi;Kim, Kyong-Tai
神经递质的释放以一种依赖活动的方式受到调节。我们先前表明，烟碱型乙酰胆碱受体（nAChR）的重复刺激可诱导嗜铬细胞中大致密核心囊泡（LDCV）胞吐作用的依赖活动的增强（ADP）。在此我们报道，蛋白激酶C（PKC）-ε在ADP中起关键作用。nAChR的刺激诱导PKC - ε的激活，并且通过表达PKC - ε的显性负性突变体（DN - PKC - ε）或针对PKC - ε的小干扰RNA（siRNA）对PKC - ε进行抑制，可通过降低融合囊泡的频率和量子大小而消除ADP，但不影响基础胞吐作用，这表明PKC - ε特异性地参与ADP。电子显微镜显示，对PKC - ε的抑制会破坏ADP所需的活动诱导的囊泡转运。我们还表明，富含十四烷基化丙氨酸的C激酶底物（MARCKS）（已知为PKC - ε的下游靶点）参与LDCV胞吐作用的ADP。磷酸化MARCKS的水平与ADP的时间进程相关，并且通过转染DN - PKC - ε而降低。由MARCKS磷酸化诱导的肌动蛋白丝解聚也因转染DN - PKC - ε而被显著阻断。此外，通过siRNA敲低MARCKS导致ADP受到抑制以及融合囊泡数量减少。总之，我们提供的证据表明，LDCV胞吐作用的ADP是由PKC - ε及其下游靶点MARCKS通过调节囊泡转运来调控的。
Neurotransmitter release is modulated in an activity-dependent manner. We showed previously that repetitive stimulation of nicotinic acetylcholine receptor (nAChR) induced activity-dependent potentiation (ADP) of large dense-core vesicle (LDCV) exocytosis in chromaffin cells. Here we report that protein kinase C (PKC)-epsilon is critically involved in ADP. Stimulation of nAChR induced activation of PKC-epsilon, and inhibition of PKC-epsilon by expression of the dominant-negative mutant of PKC-epsilon (DN-PKC-epsilon) or short interfering (siRNA) against PKC-epsilon abolished ADP via decreasing the frequency and quantal size of fused vesicles without affecting basal exocytosis, suggesting that PKC-epsilon is specifically involved in ADP. Electron microscopy revealed that inhibition of PKC-epsilon disrupts activity-induced vesicle translocation required for ADP. We also suggest the involvement of myristoylated alanine-rich C kinase substrate (MARCKS), which is known as a downstream target of PKC-epsilon, in ADP of LDCV exocytosis. The level of phospho-MARCKS correlated with the time course of ADP and was reduced by transfection with DN-PKC-epsilon. Actin filament disassembly induced by MARCKS phosphorylation was also significantly blocked by transfection of DN-PKC-epsilon. Furthermore, knockdown of MARCKS by siRNA resulted in inhibition of ADP and reduction of the number of fused vesicles. Together, we provide evidence that ADP of LDCV exocytosis is regulated by PKC-epsilon and its downstream target MARCKS via modulating vesicle translocation.