Cystic Fibrosis Transmembrane Conductance Regulator (CFTR), the anion channel mutated in cystic fibrosis (CF) patients, is activated by the catalytic subunit of protein kinase A (PKA-C). PKA-C activates CFTR both noncatalytically, through binding, and catalytically, through phosphorylation of multiple serines in CFTR’s regulatory (R) domain. Here, we identify key molecular determinants of the CFTR/PKA-C interaction essential for these processes. By comparing CFTR current activation in the presence of ATP or an ATP analog unsuitable for phosphotransfer, as well as pseudosubstrate peptides of various lengths, we identify two distinct specific regions of the PKA-C surface which interact with CFTR to cause noncatalytic and catalytic CFTR stimulation, respectively. Whereas the “substrate site” mediates CFTR phosphorylation, a distinct hydrophobic patch (the “docking site”) is responsible for noncatalytic CFTR activation, achieved by stabilizing the R domain in a “released” conformation permissive to channel gating. Furthermore, by comparing PKA-C variants with different posttranslational modification patterns, we find that direct membrane tethering of the kinase through its N-terminal myristoyl group is an unappreciated fundamental requirement for CFTR activation: PKA-C demyristoylation abolishes noncatalytic, and profoundly slows catalytic, CFTR stimulation. For the F508del CFTR mutant, present in ~90% of CF patients, maximal activation by demyristoylated PKA-C is reduced by ~10-fold compared to that by myristoylated PKA-C. Finally, in bacterial genera that contain common CF pathogens, we identify virulence factors that demyristoylate PKA-C in vitro, raising the possibility that during recurrent bacterial infections in CF patients, PKA-C demyristoylation may contribute to the exacerbation of lung disease.

中文翻译：

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蛋白激酶 A 的结构决定因素对 CFTR 通道激活至关重要


囊性纤维化跨膜电导调节因子 （CFTR） 是在囊性纤维化 （CF） 患者中突变的阴离子通道，由蛋白激酶 A （PKA-C） 的催化亚基激活。PKA-C 通过结合以非催化方式激活 CFTR，并通过 CFTR 调节 （R） 结构域中多个丝氨酸的磷酸化以催化方式激活 CFTR。在这里，我们确定了对这些过程至关重要的 CFTR/PKA-C 相互作用的关键分子决定因素。通过比较 ATP 或不适合磷酸转移的 ATP 类似物存在下的 CFTR 电流激活，以及各种长度的假底物肽，我们确定了 PKA-C 表面的两个不同的特定区域，它们与 CFTR 相互作用分别引起非催化和催化 CFTR 刺激。虽然“底物位点”介导 CFTR 磷酸化，但一个独特的疏水贴片（“停靠位点”）负责非催化性 CFTR 激活，这是通过将 R 结构域稳定在允许通道门控的“释放”构象中来实现的。此外，通过比较具有不同翻译后修饰模式的 PKA-C 变体，我们发现激酶通过其 N 末端肉豆蔻酰基的直接膜栓系是 CFTR 激活的一个未被重视的基本要求：PKA-C 脱肉豆酰化消除了非催化性，并大大减慢了催化性 CFTR 刺激。对于存在于 ~90% 的 CF 患者中的 F508del CFTR 突变体，与肉豆蔻酰化 PKA-C 相比，脱肉藻酰化 PKA-C 的最大激活减少了 ~10 倍。 最后，在含有常见 CF 病原体的细菌属中，我们鉴定了在体外脱糖酰化 PKA-C 的毒力因子，这增加了在 CF 患者反复细菌感染期间，PKA-C 脱肉豆酰化可能导致肺部疾病恶化的可能性。