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, 105 (12), 1711-21

Inhibition of Cystic Fibrosis Transmembrane Conductance Regulator by Novel Interaction With the Metabolic Sensor AMP-activated Protein Kinase

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Inhibition of Cystic Fibrosis Transmembrane Conductance Regulator by Novel Interaction With the Metabolic Sensor AMP-activated Protein Kinase

K R Hallows et al. J Clin Invest.

Abstract

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.

Figures

Figure 1
Figure 1
Yeast two-hybrid interaction maps of α1-AMPK and CFTR COOH-terminal tail. (a) Determination of regions of α1-AMPK important for interaction with CFTR-1411–1480. Functional regions of α1-AMPK are shown schematically at the top, as estimated by sequence homology to yeast Snf1p (38). Constructs containing residues upstream of amino acid 161 were made by high-fidelity PCR amplification using appropriate primers and full-length rat α1-AMPK cDNA (31) as template. Other constructs were either original prey clones identified in the two-hybrid screen (α1-161-550 and 294-550) or were generated by PCR using α1-294-550 prey plasmid as a template. (b) Determination of region and specific residues within CFTR-1411–1480 important for interaction with α1-294-550. Expression of the CFTR-1420–1443 fragment could not be detected by Western analysis. Because lack of interaction observed may have resulted from no expression, it is uncertain whether residues 1444–1457 are required for strong interaction.
Figure 2
Figure 2
Confirmation of AMPK-CFTR interaction by GST pull-down assays. (a) CFTR COOH-terminal tail (GST-CFTR-1411–1480) pulls down α1-AMPK in vitro. Purified liver AMPK holoenzyme (15 ng) was loaded in first lane as reference. (b) GST-α1-AMPK fusion protein expressed in CHO-BQ2 cells pulls down full-length CFTR in vivo. Ten micrograms of the total soluble cellular protein was loaded in first and third (lysate) lanes as reference for CFTR. Second and fourth (beads) lanes show eluate from GSH-Sepharose beads after affinity purification and washing. (c) Comparison of binding strengths of CFTR with NH2-terminal and COOH-terminal GST-α1-AMPK fusion proteins in CHO-BQ2 cells. Lower panel is the same membrane probed with anti-GST antibodies to allow comparisons of different CFTR bands in panel above. (d) GST-α2-AMPK fusion protein expressed in CHO-BQ2 cells pulls down CFTR in vivo. All results shown are representative of at least three replicate experiments.
Figure 3
Figure 3
Western blot of lysates from various cell lines. Total protein (10–20 μg per lane) was loaded on a 10% gel and immunoblotted with anti-α1-AMPK antibody (1:1,000), followed by anti-rabbit IgG-HRP (1:10,000).
Figure 4
Figure 4
Immunohistochemistry of tissue sections from rat nasal mucosa. Contiguous sections were stained using (a) anti-CFTR or (b) anti-α1-AMPK antibodies, followed by biotinylated secondary antibodies and rhodamine-avidin. Both sections are also DAPI-nuclear stained (blue). Bar, 100 μm. Additional contiguous sections stained without addition of primary antibody revealed little nonspecific staining (not shown).
Figure 5
Figure 5
In vitro phosphorylation of CFTR by AMPK. CFTR was immunoprecipitated from CHO-BQ2 cell lysates (lanes 2–5) before phosphorylation, SDS-PAGE, and autoradiography. In lane 2, no exogenous kinase was added to the phosphorylation buffer. In lanes 3 and 4, affinity-purified AMPK holoenzyme was added to immunoprecipitated CFTR in the absence or presence of 0.2 mM AMP, respectively. Affinity-purified α1-1-312 was added to immunoprecipitate in lane 5. As a negative control, AMPK holoenzyme was added to an immunoprecipitate from CHO cells that do not express CFTR (lane 1). Results shown are representative of three replicate experiments.
Figure 6
Figure 6
Cyclic AMP-activated CFTR whole-cell conductances in Xenopus oocytes in the presence or absence of AMPK subunits. Oocytes were injected with 20 ng CFTR cRNA ± 5–15 ng total of AMPK subunits. Two-electrode voltage clamp measurements were performed 2–4 days after injections. (a) Representative traces of CFTR Cl conductance activation in presence or absence of coexpressed AMPK holoenzyme. Dashed vertical line indicates when oocytes were perfused with solution containing 10 μM forskolin plus 1 mM IBMX to stimulate CFTR Cl conductance. μS, microsiemens. (b) Relative stimulated CFTR conductances in oocytes expressing CFTR plus different AMPK subunits. Bars represent the mean change in conductance (± SEM) of voltage-clamped oocytes expressing CFTR with indicated AMPK subunits relative to that of oocytes expressing CFTR alone from the same batch (horizontal dashed line at 100%). Number of replicates for each condition and P values compared with the CFTR alone condition are shown.
Figure 7
Figure 7
ClustalW alignment of peptide sequences from various ABC transporters to the α-AMPK-interacting region of the CFTR COOH-terminal tail. All sequences shown are human, except CDR2 and YCF1, which are yeast. A consensus sequence for AMPK phosphorylation [Hyd-(Basic, X)-X-X-S/T-X-X-X-Hyd] (30, 37) is preserved in COOH-termini of MDR, ABC1, TAP1, and CDR2. GenBank accession numbers for sequences shown are: CFTR (P13569); MDR1 (P08183), MDR3 (P21439); MRP1 (P33527), MRP2 (Q92887), MRP4 (NP 005836); SUR1 (Q09428), SUR2A (AAC16057), SUR2B (AAC16058); ABC1 (NP 005493); TAP1 (A41538); CDR2 (P78595); and YCF1 (NP 010419). MDR, multidrug resistance protein; MRP, multidrug resistance-associated protein; MDP, multidrug resistance protein; SUR, sulfonylurea receptor; ABC1, ATP-binding cassette transporter 1; TAP1, transporter for antigen processing 1; CDR2, multidrug resistance protein; YCF1, metal resistance protein.

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