Inhibition of cystic fibrosis transmembrane conductance regulator by novel interaction with the metabolic sensor AMP-activated protein kinase.
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 alpha1 (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 alpha1-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-alpha1-AMPK interaction and also identified alpha2-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.
Hallows
K R
KR
Renal-Electrolyte and Hypertension Division, Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, USA.
Raghuram
V
V
Kemp
B E
BE
Witters
L A
LA
Foskett
J K
JK
D055372:AMP-Activated Protein Kinases; D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D003001:Cloning, Molecular; D006224:Cricetinae; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics; D015723:Gene Library; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D008297:Male; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000378:metabolism*; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013737:Testis / Q000378:metabolism; D014162:Transfection
C505032:CFTR protein, human; D009097:Multienzyme Complexes; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D005982:Glutathione Transferase; C526181:PRKAA2 protein, human; D017346:Protein Serine-Threonine Kinases; D055372:AMP-Activated Protein Kinases; C526180:PRKAA1 protein, human