Functional characterization of Src-interacting Na/K-ATPase using RNA interference assay
Functional characterization of Src-interacting Na/K-ATPase using RNA interference assay
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DOI：
10.1074/jbc.m512240200
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发表时间：
2006-07-14
影响因子：
4.8
通讯作者：
Xie, Zi-Jian
中科院分区：
文献类型：
--
作者：
Liang, Man;Cai, Ting;Xie, Zi-Jian
我们已经表明，Na/K-ATP酶和Src形成一个信号受体复合物。在这里，我们确定了Na/K-ATP酶的量和性质的改变如何影响基础Src活性和哇巴因诱导的信号转导。通过用表达α 1特异性小干扰RNA的载体转染LLC-PK 1细胞产生几种α 1亚基敲低细胞系。虽然α 1敲低导致Na/K-ATP酶活性显著降低，但它增加了Src效应物的基础Src活性和粘着斑激酶的酪氨酸磷酸化。同时，它也消除了哇巴因诱导的Src和ERK 1/2的激活。当敲除的细胞被大鼠α 1拯救时，Na/K-ATP酶活性和基础Src活性都恢复。此外，哇巴因能够以高得多的浓度刺激获救细胞中的Src和ERK 1/2，这与猪和大鼠α 1之间哇巴因敏感性的既定差异一致。最后，荧光共振能量转移分析和免疫共沉淀分析表明，泵空大鼠α 1（D371 E）突变体也可以结合Src。该突变体的表达恢复了基础Src活性和粘着斑激酶酪氨酸磷酸化。总之，新的发现表明，LLC-PK 1细胞含有一个Src相互作用的Na/K-ATP酶池，不仅调节Src活性，而且作为哇巴因激活蛋白激酶的受体。
We have shown that the Na/K-ATPase and Src form a signaling receptor complex. Here we determined how alterations in the amount and properties of the Na/K-ATPase affect basal Src activity and ouabain-induced signal transduction. Several alpha 1 subunit knockdown cell lines were generated by transfecting LLC-PK1 cells with a vector expressing alpha 1-specific small interference RNA. Although the alpha 1 knockdown resulted in significant decreases in Na/K-ATPase activity, it increased the basal Src activity and tyrosine phosphorylation of focal adhesion kinase, a Src effector. Concomitantly it also abolished ouabain-induced activation of Src and ERK1/2. When the knockdown cells were rescued by a rat alpha 1, both Na/K-ATPase activity and the basal Src activity were restored. In addition, ouabain was able to stimulate Src and ERK1/2 in the rescued cells at a much higher concentration, consistent with the established differences in ouabain sensitivity between pig and rat alpha 1. Finally both fluorescence resonance energy transfer analysis and co-immunoprecipitation assay indicated that the pumping-null rat alpha 1 (D371E) mutant could also bind Src. Expression of this mutant restored the basal Src activity and focal adhesion kinase tyrosine phosphorylation. Taken together, the new findings suggest that LLC-PK1 cells contain a pool of Src-interacting Na/K-ATPase that not only regulates Src activity but also serves as a receptor for ouabain to activate protein kinases.