Mass Spectrometric Analysis of TRPM6 and TRPM7 Phosphorylation Reveals Regulatory Mechanisms of the Channel-Kinases.
Mass Spectrometric Analysis of TRPM6 and TRPM7 Phosphorylation Reveals Regulatory Mechanisms of the Channel-Kinases.
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DOI：
10.1038/srep42739
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发表时间：
2017-02-21
影响因子：
4.6
通讯作者：
Runnels LW
中科院分区：
文献类型：
--
作者：
Cai N;Bai Z;Nanda V;Runnels LW
TRPM7和TRPM6是最早发现的含有各自激活区的双功能通道，但对这些通道的调控机制知之甚少。以前的研究发现了TRPM7上的大量磷酸化位点，但对TRPM6的磷酸化或TRPM6转磷酸化的位点知之甚少。我们对同质和异质TRPM7和TRPM6通道的质谱分析确定了这两种蛋白质上的磷酸化位点，以及TRPM7上的几个重要部位，这些部位通常通过TRPM6的自磷酸化和转磷化来修饰。我们进行了一系列的氨基酸替换分析，确定TRPM7的S催化区的S1777和TRPM7的S交换区的S1565是潜在的调节位点。模拟磷化的S1777D取代破坏了催化活性，很可能是通过在活性部位引起静电扰动。S1565D的仿磷取代也使该激酶失活，但没有干扰该激酶的二聚化。分子模拟表明，S1565的磷酸化被预测在结构上影响TRPM7的S功能保守的N/D环，这被认为影响底物对活性部位口袋的访问。我们认为S1565在交换结构域内的磷酸化是控制TRPM7催化活性的调节开关。
TRPM7 and TRPM6 were the first identified bifunctional channels to contain their own kinase domains, but how these channel-kinases are regulated is poorly understood. Previous studies identified numerous phosphorylation sites on TRPM7, but very little is known about TRPM6 phosphorylation or sites on TRPM7 transphosphorylated by TRPM6. Our mass spectrometric analysis of homomeric and heteromeric TRPM7 and TRPM6 channels identified phosphorylation sites on both proteins, as well as several prominent sites on TRPM7 that are commonly modified through autophosphorylation and transphosphorylation by TRPM6. We conducted a series of amino acid substitution analyses and identified S1777, in TRPM7’s catalytic domain, and S1565, in TRPM7’s exchange domain that mediates kinase dimerization, as potential regulatory sites. The phosphomimetic S1777D substitution disrupted catalytic activity, most likely by causing an electrostatic perturbation at the active site. The S1565D phosphomimetic substitution also inactivated the kinase but did so without interfering with kinase dimerization. Molecular modeling indicates that phosphorylation of S1565 is predicted to structurally affect TRPM7’s functionally conserved N/D loop, which is thought to influence the access of substrate to the active site pocket. We propose that phosphorylation of S1565 within the exchange domain functions as a regulatory switch to control TRPM7 catalytic activity.