Phosphorylation of protein kinase C sites in NBD1 and the R domain control CFTR channel activation by PKA
Phosphorylation of protein kinase C sites in NBD1 and the R domain control CFTR channel activation by PKA
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DOI：
10.1113/jphysiol.2002.035790
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发表时间：
2003-04-01
影响因子：
5.5
通讯作者：
Hanrahan, JW
中科院分区：
文献类型：
--
作者：
Chappe, V;Hinkson, DA;Hanrahan, JW
蛋白激酶C（PKC）可增强蛋白激酶A（PKA）对囊性纤维化跨膜传导调节因子（CFTR）通道的激活。然而，调节机制尚不清楚，PKC是否直接作用于CFTR或通过辅助蛋白的磷酸化作用仍然不确定。使用已用磷酸酶预处理的切除的补丁，我们发现，PKC暴露导致更大的PKA激活电流和PKA浓度依赖性的变化。为了检查这些作用是否由CFTR的直接PKC磷酸化介导，构建了突变体，其中CFTR上9个PKC共有序列处的丝氨酸或苏氨酸被丙氨酸取代（即“9 CA”突变体T582 A/T604 A/S641 A/T682 A/S686 A/S707 A/S790 A/T791 A/S809 A）。在切除的斑块中，9 CA通道对PKA的反应大大降低（即野生型的5- 10%），这并没有被PKC预处理增强，尽管根据碘化物流出测定，突变通道仍然有功能。氯代苯硫基-cAMP（cpt-cAMP）刺激碘流出在表达9 CA通道的细胞中延迟，并且当用PKC抑制剂白屈菜红碱处理表达野生型CFTR的细胞时观察到类似的延迟。这表明，在切除的补丁和缓慢的刺激碘流出完整的细胞中的PKA激活弱具体是由于PKC磷酸化的损失。最后，当磷酸酶预处理后添加到切除的斑块中时，PKC会引起野生型通道的轻微激活，但对突变体没有影响。我们的结论是，直接磷酸化的CFTR在一个或多个突变的9个位点在9 CA的部分激活所需的，PKC和其调制的CFTR对PKA的反应。
Activation of the cystic fibrosis transmembrane conductance regulator (CFTR) channel by protein kinase A (PKA) is enhanced by protein kinase C (PKC). However, the mechanism of modulation is not known and it remains uncertain whether PKC acts directly on CFTR or through phosphorylation of an ancillary protein. Using excised patches that had been pre-treated with phosphatases, we found that PKC exposure results in much larger PKA-activated currents and shifts the PKA concentration dependence. To examine if these effects are mediated by direct PKC phosphorylation of CFTR, a mutant was constructed in which serines or threonines at nine PKC consensus sequences on CFTR were replaced by alanines (i.e. the '9CA' mutant T582A/T604A/S641A/T682A/S686A/S707A/S79OA/T791A/S809A). In excised patches, 9CA channels had greatly reduced responses to PKA (i.e. 5-10 % that of wild-type), which were not enhanced by PKC pre-treatment, although the mutant channels were still functional according to iodide efflux assays. Stimulation of iodide efflux by chlorophenylthio-cAMP (cpt-cAMP) was delayed in cells expressing 9CA channels, and a similar delay was observed when cells expressing wild-type CFTR were treated with the PKC inhibitor chelerythrine. This suggests that weak activation by PKA in excised patches and slow stimulation of iodide efflux from intact cells are specifically due to the loss of PKC phosphorylation. Finally, PKC caused a slight activation of wildtype channels when added to excised patches after phosphatase pre-treatment but had no effect on the mutant. We conclude that direct phosphorylation of CFTR at one or more of the nine sites mutated in 9CA is required for both the partial activation by, PKC and for its modulation of CFTR responses to PKA.