Department of Biochemistry, Semmelweis University, Budapest H-1094, Hungary.
Molecular Channelopathies Research Group, Hungarian Centre of Excellence for Molecular Medicine - Semmelweis University, Budapest H-1094, Hungary.
Proc Natl Acad Sci U S A. 2024 Nov 12;121(46):e2407728121. doi: 10.1073/pnas.2407728121. Epub 2024 Nov 4.
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.
囊性纤维化跨膜电导调节因子（CFTR）是囊性纤维化（CF）患者突变的阴离子通道，它被蛋白激酶 A 的催化亚基（PKA-C）激活。PKA-C 通过结合非催化地激活 CFTR，并通过 CFTR 调节（R）域中多个丝氨酸的磷酸化催化地激活 CFTR。在这里，我们确定了 CFTR/PKA-C 相互作用的关键分子决定因素，这些决定因素对于这些过程至关重要。通过比较在存在 ATP 或不适合磷酸转移的 ATP 类似物的情况下 CFTR 电流的激活，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，它们分别与 CFTR 相互作用以引起非催化和催化 CFTR 刺激。虽然“底物位点”介导 CFTR 的磷酸化，但一个独特的疏水区（“对接位点”）负责非催化 CFTR 的激活，其通过稳定 R 域处于允许通道门控的“释放”构象来实现。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 端豆蔻酰基团的直接膜固定是 CFTR 激活的一个未被认识的基本要求：PKA-C 去豆蔻酰作用会消除非催化作用，并极大地减缓催化 CFTR 刺激。对于存在于约 90%CF 患者中的 F508del CFTR 突变体，与豆蔻酰 PKA-C 相比，去豆蔻酰 PKA-C 对其的最大激活作用降低了约 10 倍。最后，在含有常见 CF 病原体的细菌属中，我们鉴定了体外去豆蔻酰 PKA-C 的毒力因子，这增加了这样一种可能性，即在 CF 患者反复细菌感染期间，PKA-C 去豆蔻酰作用可能导致肺部疾病恶化。
