Phosphorylation-dependent 14-3-3 protein interactions regulate CFTR biogenesis.
Phosphorylation-dependent 14-3-3 protein interactions regulate CFTR biogenesis.
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DOI：
10.1091/mbc.e11-08-0662
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发表时间：
2012-03
影响因子：
3.3
通讯作者：
Frizzell RA
中科院分区：
文献类型：
--
作者：
Liang X;Da Paula AC;Bozóky Z;Zhang H;Bertrand CA;Peters KW;Forman-Kay JD;Frizzell RA
cAMP/PKA 刺激引起翻译后 CFTR 表达增加以及特定 14-3-3 蛋白与 R 区内磷酸化位点的相互作用。这提高了新生 CFTR 生物合成的效率，并减少了其与 COPI 逆行修复机制的相互作用，使更多的 CFTR 可用于阴离子分泌。囊性纤维化跨膜电导调节器 (CFTR) 是一种 cAMP/蛋白激酶 A (PKA) 调节的氯离子通道，其磷酸化控制跨上皮细胞顶膜的阴离子分泌。我们基于 CFTR 内预测的 14-3-3 结合基序和毛喉素诱导的 CFTR 表达，检验了 cAMP/PKA 刺激在翻译后调节 CFTR 生物发生的假设。 14-3-3β、γ 和 ε 同工型在气道细胞中表达，并在免疫共沉淀测定中与 CFTR 相互作用。 Forskolin 刺激（15 分钟）增加了 14-3-3β 和 ε 与未成熟和成熟 CFTR（条带 B 和 C）的结合，14-3-3 过表达增加了 CFTR 条带 B 和 C 以及细胞表面条带 C。在脉冲追踪实验中，14-3-3β 增加了未成熟 CFTR 的合成，降低了其降解率，并增加了未成熟到成熟 CFTR 的转化。相反，14-3-3β敲除降低了CFTR B和C带（70%和55%），并引起细胞表面CFTR和毛喉素刺激的阴离子流出的平行减少。在体外，14-3-3β与CFTR调节区相互作用，通过核磁共振分析，这种相互作用发生在已知的PKA磷酸化位点。在免疫共沉淀试验中，毛喉素刺激 CFTR/14-3-3β 相互作用，同时减少 CFTR 与外壳蛋白复合物 1 (COP1) 的相互作用。因此，14-3-3 与磷酸化 CFTR 的结合通过减少 CFTR 逆向内质网的恢复来增强其生物发生。这种机制允许 cAMP/PKA 刺激，使更多的 CFTR 可用于阴离子分泌。
cAMP/PKA stimulation elicited posttranslational increases in CFTR expression and the interaction of specific 14-3-3 proteins with phosphorylated sites within the R region. This improved the efficiency of nascent CFTR biogenesis and reduced its interaction with the COPI retrograde retrieval mechanism, making more CFTR available for anion secretion. Cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP/protein kinase A (PKA)–regulated chloride channel whose phosphorylation controls anion secretion across epithelial cell apical membranes. We examined the hypothesis that cAMP/PKA stimulation regulates CFTR biogenesis posttranslationally, based on predicted 14-3-3 binding motifs within CFTR and forskolin-induced CFTR expression. The 14-3-3β, γ, and ε isoforms were expressed in airway cells and interacted with CFTR in coimmunoprecipitation assays. Forskolin stimulation (15 min) increased 14-3-3β and ε binding to immature and mature CFTR (bands B and C), and 14-3-3 overexpression increased CFTR bands B and C and cell surface band C. In pulse-chase experiments, 14-3-3β increased the synthesis of immature CFTR, reduced its degradation rate, and increased conversion of immature to mature CFTR. Conversely, 14-3-3β knockdown decreased CFTR B and C bands (70 and 55%) and elicited parallel reductions in cell surface CFTR and forskolin-stimulated anion efflux. In vitro, 14-3-3β interacted with the CFTR regulatory region, and by nuclear magnetic resonance analysis, this interaction occurred at known PKA phosphorylated sites. In coimmunoprecipitation assays, forskolin stimulated the CFTR/14-3-3β interaction while reducing CFTR's interaction with coat protein complex 1 (COP1). Thus 14-3-3 binding to phosphorylated CFTR augments its biogenesis by reducing retrograde retrieval of CFTR to the endoplasmic reticulum. This mechanism permits cAMP/PKA stimulation to make more CFTR available for anion secretion.