Na/K-ATPase tethers phospholipase C and IP3 receptor into a calcium-regulatory complex.
Na/K-ATPase tethers phospholipase C and IP3 receptor into a calcium-regulatory complex.
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DOI：
10.1091/mbc.e05-04-0295
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发表时间：
2005-09
影响因子：
3.3
通讯作者：
Zhaokan Yuan;T. Cai;Jiang Tian;A. Ivanov;D. Giovannucci;Zijian Xie
中科院分区：
文献类型：
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作者：
Zhaokan Yuan;T. Cai;Jiang Tian;A. Ivanov;D. Giovannucci;Zijian Xie
我们已经表明，小窝Na/K-ATP酶通过多个信号复合体传递哇巴因信号。为了获得这种复合物的组成信息，我们从大鼠肾脏外髓质的Na/K-ATP酶分离成两个不同的馏分洗涤剂处理和密度梯度离心。轻组分的分析表明，PLC-γ 1和IP 3受体（亚型2和3，IP 3R 2和IP 3R 3）与Na/K-ATP酶，小窝蛋白-1和Src共富集。GST pulldown分析显示Na/K-ATP酶α 1亚基的中心环与PLC-γ 1相互作用，而N-末端与IP 3R 2和IP 3R 3结合，表明Na/K-ATP酶信号转导可能将PLC-γ 1和IP 3受体连接在一起，形成Ca（2+）-调节复合物。以下调查结果支持了这一观点。首先，PLC-gamma 1和IP 3R 2与Na/K-ATP酶和哇巴因共免疫沉淀在LLC-PK 1细胞中以剂量和时间依赖性方式增加了这种相互作用。胆固醇的消耗消除了哇巴因对这种相互作用的影响。其次，哇巴因诱导PLC-γ 1在Tyr（783）处的磷酸化，并以Src依赖性方式激活PLC-γ 1，导致PIP 2水解增加。它还刺激Src依赖的IP 3R 2酪氨酸磷酸化。最后，哇巴因通过激活LLC-PK 1细胞的IP 3受体诱导细胞内Ca（2+）释放。这种效应需要哇巴因诱导的PLC-γ 1激活。抑制Src或降低胆固醇也可消除哇巴因对细胞内Ca（2+）的影响。
We have shown that the caveolar Na/K-ATPase transmits ouabain signals via multiple signalplexes. To obtain the information on the composition of such complexes, we separated the Na/K-ATPase from the outer medulla of rat kidney into two different fractions by detergent treatment and density gradient centrifugation. Analysis of the light fraction indicated that both PLC-gamma1 and IP3 receptors (isoforms 2 and 3, IP3R2 and IP3R3) were coenriched with the Na/K-ATPase, caveolin-1 and Src. GST pulldown assays revealed that the central loop of the Na/K-ATPase alpha1 subunit interacts with PLC-gamma1, whereas the N-terminus binds IP3R2 and IP3R3, suggesting that the signaling Na/K-ATPase may tether PLC-gamma1 and IP3 receptors together to form a Ca(2+)-regulatory complex. This notion is supported by the following findings. First, both PLC-gamma1 and IP3R2 coimmunoprecipitated with the Na/K-ATPase and ouabain increased this interaction in a dose- and time-dependent manner in LLC-PK1 cells. Depletion of cholesterol abolished the effects of ouabain on this interaction. Second, ouabain induced phosphorylation of PLC-gamma1 at Tyr(783) and activated PLC-gamma1 in a Src-dependent manner, resulting in increased hydrolysis of PIP2. It also stimulated Src-dependent tyrosine phosphorylation of the IP3R2. Finally, ouabain induced Ca(2+) release from the intracellular stores via the activation of IP3 receptors in LLC-PK1 cells. This effect required the ouabain-induced activation of PLC-gamma1. Inhibition of Src or depletion of cholesterol also abolished the effect of ouabain on intracellular Ca(2+).