Dohrman Douglas P, Chen Hui-min, Gordon Adrienne S, Diamond Ivan
Ernest Gallo Clinic and Research Center, Department of Neurology, University of California-San Francisco, 5858 Horton Street, Emeryville, CA 94608, USA.
Alcohol Clin Exp Res. 2002 Mar;26(3):407-15.
Cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA) regulates cellular functions. The specificity of PKA-mediated phosphorylation is determined primarily by PKA localization to sub-cellular sites. Chronic exposure to ethanol causes sustained translocation of the PKA catalytic subunit (C) from the Golgi to the nucleus in NG108-15 cells. Here we find that this is preceded by a transient short-term ethanol-induced translocation of PKA C. Different molecular mechanisms appear to underlie early and late phases of ethanol-induced translocation of PKA subunits.
The time course and localization of PKA C and regulatory (RII) subunits was assessed by immunocytochemistry in NG108-15 cells in the presence of ethanol, adenosine receptor (A2) blockade, and inhibitors of PKA activity and RNA and protein synthesis.
Ethanol induces an early phase (<30 min) of C translocation to the cytoplasm and nucleus. This requires cAMP via adenosine A2 receptor activation. C then returns to the Golgi area after 60 min. A second phase of C translocation occurs during continuing exposure to ethanol (>12 hr). Re-accumulation of nuclear C no longer requires A2 or cAMP. RII also translocates to the nucleus during chronic treatment with ethanol. Both C and RII remain in the nucleus as long as ethanol is present. Unlike the early phase of ethanol induced translocation, the second phase of PKA subunit translocation requires protein and RNA synthesis.
We identify two distinct phases of ethanol-induced PKA translocation which appear to be regulated by different molecular mechanisms. The first requires A2 signaling and cAMP; the later phase requires RNA and protein synthesis. The two phases of ethanol-induced PKA translocation observed in cell lines may contribute to changes in PKA signaling, cAMP-dependent gene expression, and the initiation and maintenance of sustained drinking behavior in experimental animals.
环磷酸腺苷（cAMP）依赖性蛋白激酶A（PKA）调节细胞功能。PKA介导的磷酸化特异性主要由PKA在亚细胞位点的定位决定。长期暴露于乙醇会导致PKA催化亚基（C）在NG108 - 15细胞中从高尔基体持续转运至细胞核。在此，我们发现这之前存在乙醇诱导的PKA C短暂短期转运。乙醇诱导的PKA亚基转运的早期和晚期阶段似乎有不同的分子机制。
通过免疫细胞化学在存在乙醇、腺苷受体（A2）阻断剂以及PKA活性、RNA和蛋白质合成抑制剂的情况下，评估NG108 - 15细胞中PKA C和调节（RII）亚基的时间进程和定位。
乙醇诱导C在早期阶段（<30分钟）转运至细胞质和细胞核。这需要通过腺苷A2受体激活cAMP。60分钟后C返回高尔基体区域。在持续暴露于乙醇（>12小时）期间发生C转运的第二阶段。细胞核中C的重新积累不再需要A2或cAMP。在乙醇长期处理期间，RII也转运至细胞核。只要有乙醇存在，C和RII都留在细胞核中。与乙醇诱导转运的早期阶段不同，PKA亚基转运的第二阶段需要蛋白质和RNA合成。
我们确定了乙醇诱导的PKA转运的两个不同阶段，它们似乎由不同的分子机制调节。第一阶段需要A2信号传导和cAMP；后期阶段需要RNA和蛋白质合成。在细胞系中观察到的乙醇诱导的PKA转运的两个阶段可能有助于PKA信号传导、cAMP依赖性基因表达的变化以及实验动物持续饮酒行为的启动和维持。
