Attenuation of Phosphorylation-dependent Activation of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) by Disease-causing Mutations at the Transmission Interface.
Attenuation of Phosphorylation-dependent Activation of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) by Disease-causing Mutations at the Transmission Interface.
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DOI：
10.1074/jbc.m116.762633
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发表时间：
2017-02-03
期刊：
影响因子：
--
通讯作者：
Bear CE
中科院分区：
文献类型：
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作者：
Chin S;Yang D;Miles AJ;Eckford PDW;Molinski S;Wallace BA;Bear CE
囊性纤维化跨膜传导调节因子（CFTR）是一种多结构域的膜蛋白，作为磷酸化调节的阴离子通道发挥作用。其两个胞质核苷酸结合结构域和由从通道孔结构域延伸的胞内环赋予的偶联螺旋之间的界面被称为传输界面，并且被认为对于CFTR的调节通道活性至关重要。CFTR的调节结构域被蛋白激酶A（PKA）磷酸化是其通道活性所必需的。然而，目前尚不清楚磷酸化是否改变了传输界面。在这里，我们研究了纯化的全长CFTR蛋白使用光谱技术，以确定PKA介导的磷酸化的后果。同步辐射圆二色光谱证实，纯化的全长野生型CFTR折叠和结构响应磷酸化。CFTR的内源性色氨酸荧光研究表明，磷酸化减少碘化物介导的淬灭，与磷酸化在传输界面处掩埋色氨酸的作用一致。重要的是，磷酸化依赖性通道激活的速率受到在预测与核苷酸结合结构域1在界面处相互作用的两个偶联螺旋中的任一个中引入致病突变的影响。总之，这些结果表明磷酸化修饰了CFTR的催化结构域和孔结构域之间的界面，并且这种修饰促进了CFTR通道活化。
Cystic fibrosis transmembrane conductance regulator (CFTR) is a multidomain membrane protein that functions as a phosphorylation-regulated anion channel. The interface between its two cytosolic nucleotide binding domains and coupling helices conferred by intracellular loops extending from the channel pore domains has been referred to as a transmission interface and is thought to be critical for the regulated channel activity of CFTR. Phosphorylation of the regulatory domain of CFTR by protein kinase A (PKA) is required for its channel activity. However, it was unclear if phosphorylation modifies the transmission interface. Here, we studied purified full-length CFTR protein using spectroscopic techniques to determine the consequences of PKA-mediated phosphorylation. Synchrotron radiation circular dichroism spectroscopy confirmed that purified full-length wild-type CFTR is folded and structurally responsive to phosphorylation. Intrinsic tryptophan fluorescence studies of CFTR showed that phosphorylation reduced iodide-mediated quenching, consistent with an effect of phosphorylation in burying tryptophans at the transmission interface. Importantly, the rate of phosphorylation-dependent channel activation was compromised by the introduction of disease-causing mutations in either of the two coupling helices predicted to interact with nucleotide binding domain 1 at the interface. Together, these results suggest that phosphorylation modifies the interface between the catalytic and pore domains of CFTR and that this modification facilitates CFTR channel activation.