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首页> 外文期刊>Neurochemistry International: The International Journal for the Rapid Publication of Critical Reviews, Preliminary and Original Research Communications in Neurochemistry >Involvement of the actin cytoskeleton in the regulation of serotonin transporter (SET) activity: possible mechanism underlying SET regulation by protein kinase C.
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Involvement of the actin cytoskeleton in the regulation of serotonin transporter (SET) activity: possible mechanism underlying SET regulation by protein kinase C.

机译：肌动蛋白细胞骨架参与血清素转运蛋白（SET）活性的调节：蛋白激酶C调节SET的潜在机制。

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Our previous report has revealed that PKC activation by 12-O-tetradecanoylphorbol 13-acetate (TPA) inhibited the uptake activity of serotonin transporter (SET), via an indirect mechanism unknown, but not likely via direct phosphorylation of SET by PKC (Sakai et al., 1997. J. Neurochem. 68, 2618-2624). To elucidate whether PKC can directly phosphorylate SET in vivo, FLAG-tagged SET (FLAG-SET) was expressed in COS-7 cells and the TPA-induced incorporation of (32)P into immunoprecipitated FLAG-SET was examined. PKC activation with TPA caused no phosphorylation of FLAG-SET expressed in COS-7 cells. On the other hand, morphological change associated with the disruption of filamentous actin (F-actin) was seen in TPA-treated COS-7 cells. Therefore, we studied the effects of cytochalasin D, an inhibitor of actin polymerization, on the uptake activity of the serotonin transporter (SET) to elucidate whether the actin cytoskeleton modulates the SET uptake activity. The treatment with cytochalasin D inhibited the uptake activity of both native and recombinant SET in a concentration-dependent manner. Eadie-Hofstee analysis revealed that cytochalasin D down-regulated the recombinant SET uptake activity by reducing the V(max), but not the K(m), mimicking the result observed in TPA-induced inhibition of SET activity (Sakai et al., 1997. J. Neurochem. 68, 2618-2624). The cytochalasin D-induced inhibition of SET activity was partially, but significantly, reversed by jasplakinolide, a cell permeable stabilizer of F-actin, whereas TPA-induced inhibition of SET activity was not reversed by jasplakinolide. To elucidate whether the subcellular localization of SET was changed in response to cytochalasin D or TPA, we expressed the SET fused with the green fluorescent protein (SET-GFP) in COS-7 cells and observed the subcellular distribution of SET-GFP under a confocal laser scanning fluorescent microscope. Neither cytochalasin D nor TPA markedly changed the SET-GFP cellular localization, although these drugs caused morphological change in the GFP-transfected COS-7 cells. In addition, SET activity was not altered by the treatment with either colchicine, an inhibitor of microtubule polymerization, or taxol, a stabilizer of microtubule polymerization. These results suggest that the SET uptake activity was regulated by the state of the actin cytoskeleton and that TPA exerts its inhibitory action on SET activity, in part, via disruption of F-actin and subsequent morphological change in cells.
机译：我们以前的报告表明，通过12-O-十四烷酰佛波醇13-乙酸酯（TPA）激活PKC可以抑制5-羟色胺转运蛋白（SET）的摄取活性，这是通过未知的间接机制实现的，但不太可能是通过PKC直接将SET磷酸化来实现的（Sakai等等，1997.J.Neurochem.68，2618-2624）。为了阐明PKC是否可以在体内直接磷酸化SET，在COS-7细胞中表达了FLAG标签的SET（FLAG-SET），并检测了TPA诱导的（32）P掺入免疫沉淀的FLAG-SET中。用TPA激活PKC不会导致COS-7细胞中表达的FLAG-SET磷酸化。另一方面，在TPA处理的COS-7细胞中发现了与丝状肌动蛋白（F-actin）破坏相关的形态变化。因此，我们研究了肌动蛋白聚合抑制剂细胞松弛素D对血清素转运蛋白（SET）摄取活性的影响，以阐明肌动蛋白细胞骨架是否调节SET摄取活性。用细胞松弛素D的处理以浓度依赖性方式抑制天然和重组SET的摄取活性。 Eadie-Hofstee分析显示，细胞松弛素D通过降低V（max）而不是K（m）来下调重组SET的摄取活性，这类似于在TPA诱导的SET活性抑制中观察到的结果（Sakai等， 1997.J.Neurochem.68，2618-2624）。细胞松弛素D诱导的SET活性的抑制被jasplakinolide（一种F-actin的细胞可渗透性稳定剂）部分逆转，但被jasplakinolide逆转了TPA诱导的SET活性抑制。为了阐明SET的亚细胞定位是否响应细胞松弛素D或TPA而改变，我们在COS-7细胞中表达了与绿色荧光蛋白（SET-GFP）融合的SET，并观察了共聚焦下SET-GFP的亚细胞分布激光扫描荧光显微镜。尽管细胞松弛素D和TPA均未显着改变SET-GFP细胞的定位，但是这些药物引起了GFP转染的COS-7细胞的形态变化。另外，通过用秋水仙碱（微管聚合的抑制剂）或紫杉酚（微管聚合的稳定剂）处理，SET活性没有改变。这些结果表明，SET的摄取活性受到肌动蛋白细胞骨架状态的调节，TPA对SET活性的抑制作用部分是通过破坏F-肌动蛋白和随后的细胞形态变化来实现的。

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