Inhibition of cystic fibrosis transmembrane conductance regulator by novel interaction with the metabolic sensor AMP-activated protein kinase
Inhibition of cystic fibrosis transmembrane conductance regulator by novel interaction with the metabolic sensor AMP-activated protein kinase
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DOI：
10.1172/jci9622
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发表时间：
2000-06-01
影响因子：
15.9
通讯作者：
Foskett, JK
中科院分区：
文献类型：
--
作者：
Hallows, KR;Raghuram, V;Foskett, JK
囊性纤维化跨膜传导调节因子（CFTR）是一种ATP门控Cl-通道，通过未知机制调节其他上皮转运蛋白。我们采用酵母双杂交筛选使用的COOH-末端70个残基的CFTR，以确定可能参与这种相互作用的蛋白质。AMP活化蛋白激酶（AMPK）的α 1（催化）亚基被鉴定为一种占主导地位的新型相互作用蛋白。这种相互作用由CFTR中的残基1420-1457和α 1-AMPK的COOH末端调节结构域介导。CFTR中38个氨基酸区域内的两个蛋白质运输基序的突变各自破坏了相互作用。体外和转染细胞中的GST融合蛋白下拉测定证实了CFTR-α 1-AMPK相互作用，并且还鉴定了α 2-AMPK作为与CFTR的相互作用物。AMPK在CFTR表达细胞系中共表达，并且在大鼠鼻上皮中与CFTR共享顶端分布。AMPK在体外磷酸化全长CFTR，并且AMPK与CFTR在非洲爪蟾卵母细胞中共表达抑制cAMP激活的CFTR全细胞Cl-电导约35- 50%。由于AMPK是细胞中的代谢传感器并且响应于细胞ATP的变化，因此AMPK对CFTR的调节在代谢应激条件下抑制CFTR中可能是重要的，从而将跨上皮转运与细胞代谢状态联系起来。
The cystic fibrosis transmembrane conductance regulator (CFTR) is an ATP-gated Cl- channel that regulates other epithelial transport proteins by uncharacterized mechanisms. We employed a yeast two-hybrid screen using the COOH-terminal 70 residues of CFTR to identify proteins that might be involved in such interactions. The alpha 1 (catalytic) subunit of AMP-activated protein kinase (AMPK) was identified as a dominant and novel interacting protein. The interaction is mediated by residues 1420-1457 in CFTR and by the COOH-terminal regulatory domain of alpha 1-AMPK. Mutations of two protein trafficking motifs within the 38-amino acid region in CFTR each disrupted the interaction. GST-fusion protein pull-down assays in vitro and in transfected cells confirmed the CFTR-alpha 1-AMPK interaction and also identified alpha 2-AMPK as an interactor with CFTR AMPK is coexpressed in CFTR-expressing cell lines and shares an apical distribution with CFTR in rat nasal epithelium. AMPK phosphorylated full-length CFTR in vitro, and AMPK coexpression with CFTR in Xenopus oocytes inhibited cAMP-activated CFTR whole-cell Cl- conductance by approximately 35-50%. Because AMPK is a metabolic sensor in cells and responds to changes in cellular ATP, regulation of CFTR by AMPK may be important in inhibiting CFTR under conditions of metabolic stress, thereby linking transepithelial transport to cell metabolic state.