Tetanus toxin abolishes exocytosis of ROMK1 induced by inhibition of protein tyrosine kinase.
Tetanus toxin abolishes exocytosis of ROMK1 induced by inhibition of protein tyrosine kinase.
复制标题
破伤风毒素可消除因抑制蛋白酪氨酸激酶而诱导的 ROMK1 胞吐作用。
DOI：
10.1152/ajprenal.00309.2002
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发表时间：
2003
期刊：
影响因子：
--
通讯作者：
Wang,Wen-Hui
中科院分区：
文献类型：
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作者：
Sterling,Hyacinth;Lin,Dao-Hong;Wei,Yuan;Wang,Wen-Hui
在转染c-Src和绿色荧光蛋白(GFP)-ROMK1的HEK-293细胞中，我们使用共聚焦显微镜、片钳和生物素标记技术来检测可溶性-甲基丙烯酰亚胺敏感因子附着蛋白受体（SNARE）蛋白在介导PTK抑制ROMK1运输中的作用。用herbyycin A抑制c-Src可显著降低ROMK1酪氨酸磷酸化水平。膜片钳研究表明，添加herbyycin A可增加细胞贴附膜片中ROMK1的活性。共聚焦显微镜成像显示，herbimycin A降低了GFP-ROMK1的细胞内强度。生物素标记技术表明，c-Src的抑制使表面ROMK1增加了110%。相比之下，抑制c-Src并没有增加转染R1Y337A的HEK细胞中的K通道数量，R1Y337A是一种ROMK1突变体，酪氨酸残基337突变为丙氨酸。这表明酪氨酸残基337对于herbycin a诱导的表面ROMK1通道的增加至关重要。为了确定SNARE蛋白是否参与介导c-Src抑制诱导的ROMK1胞外分泌，我们检测了herbyycin A对破伤风毒素处理细胞中ROMK1运输的影响。细胞在含有破伤风毒素的培养基中孵育消除了herby霉素a诱导的表面ROMK1数量的增加。相比之下，c-Src的抑制仍然增加了煮沸破伤风毒素处理的细胞表面ROMK1的数量。我们得出结论，酪氨酸去磷酸化增强了ROMK1的胞吐，而SNARE蛋白是抑制PTK诱导的胞吐所必需的。
We used confocal microscopy, patch-clamp, and biotin-labeling techniques to examine the role of solubleN-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins in mediating the effect of inhibition of PTK on ROMK1 trafficking in HEK-293 cells transfected with c-Src and green fluorescent protein (GFP)-ROMK1. Inhibition of c-Src with herbimycin A significantly decreased the tyrosine phosphorylation level of ROMK1. Patch-clamp studies demonstrated that addition of herbimycin A increased the activity of ROMK1 in cell-attached patches. Confocal microscopic imaging showed that herbimycin A decreased the intracellular intensity of GFP-ROMK1. The biotin-labeling technique demonstrated that the inhibition of c-Src increased surface ROMK1 by 110%. In contrast, inhibition of c-Src did not increase the K channel number in HEK cells transfected with R1Y337A, a ROMK1 mutant in which tyrosine residue 337 was mutated to alanine. This suggests that tyrosine residue 337 is essential for the herbimycin A-induced increase in surface ROMK1 channels. To determine whether SNARE proteins are involved in mediating exocytosis of ROMK1 induced by the inhibition of c-Src, we examined the effect of herbimycin A on ROMK1 trafficking in cells treated with tetanus toxin. The incubation of cells in a medium containing tetanus toxin abolished the herbimycin A-induced increase in the number of surface ROMK1. In contrast, inhibition of c-Src still increased the numbers of surface ROMK1 in cells treated with boiled tetanus toxin. We conclude that tyrosine dephosphorylation enhances the exocytosis of ROMK1 and that SNARE proteins are required for exocytosis induced by inhibition of PTK.