King JD , Lee J , Riemen CE , Neumann D , Xiong S , Foskett JK , Mehta A , Muimo R , Hallows KR .

R01 DK075048 NIDDK NIH HHS , 086370/Z/08/Z Wellcome Trust , P30 DK079307 NIDDK NIH HHS , T32 HL007563 NHLBI NIH HHS , P30 DK072506 NIDDK NIH HHS

	FIGURE 1. NDPK-A association with AMPK-α and CFTR in overlay assays. A, immunoblots of ovine tracheal epithelium membrane proteins (100 μg) performed in the presence or absence of an overlay solution of purified ovine tracheal epithelium NDPK-A (indicated by labels) and probed with either NDPK-A (lanes i and ii) or AMPK-α (lane iii) antibody. NDPK-A immunoreactivity was detected at ∼20 kDa and additionally at 63 kDa in overlaid blot. AMPK-α immunoreactivity was detected at 63 kDa. B, immunoblots (IB) of immunoprecipitated CFTR from membrane fractions of polarized 16HBE14o− cell lysates performed in the presence or absence of purified NDPK overlay and probed with NDPK-A antibody. NDPK-A immunoreactivity appears at 20 kDa (lanes i and ii) and additionally at 63 and 175 kDa in overlaid blot (lane ii), consistent with its binding to AMPK and CFTR present in the membrane, respectively. The results shown are representative of three replicate experiments.
	FIGURE 2. CFTR-1420-57 blocking peptide displaces binding of AMPK-α1 to CFTR in oocytes and HEK-293 cells. A, Xenopus oocytes expressing both CFTR and HA-tagged AMPK-α1 cRNA (injected 2 days prior) were injected with either CFTR-1420-57 peptide or vehicle (no peptide) 4 h prior to lysis. The lysates were used for IP (right) of CFTR using M24-1 antibody (+ Ab lanes) or no antibody as an IP control (No Ab lanes), followed by immunoblotting for CFTR (upper panel) or co-IP'd HA-AMPK-α1 (lower panel). The blots of 5% of whole cell lysates are also shown (left lanes). B, by densitometric analysis, the CFTR-1420-57 peptide reduced the amount of AMPK-α1 co-IP'd with CFTR (relative binding) by 75 ± 20%. #, p = 0.06, paired t test, n = 3 experiments. C, HEK-293 cells were co-transfected with plasmids to express CFTR and either NH2-terminal GST-tagged AMPK-α1 or GST alone 2 days prior to lysis. 100 μm CFTR-1420-57 peptide (or vehicle) was transduced into cells using the BioPORTER reagent 1 day before lysis as described under “Experimental Procedures.” After GST affinity purification of the cell lysates, the proteins were eluted in sample buffer and subjected to SDS-PAGE and immunoblotting for either CFTR (upper) or GST (lower). The amount of CFTR pulled down was substantially reduced following transduction of the blocking CFTR-1420-57 peptide (lane 6, top) relative to vehicle (lane 8, top). D, summary of relative binding (relative amount of CFTR pulled down by GST-AMPK-α1) in the presence of the CFTR-1420-57 peptide versus vehicle. By densitometric analysis, the blocking peptide reduced the relative binding of CFTR to AMPK-α1 by 41 ± 6%. *, p = 0.02, paired t test, n = 3 replicate experiments. Con, control; IB, immunoblot.
	FIGURE 3. CFTR-1420-57 blocking peptide transduced into Calu-3 cells enhances CFTR whole cell conductance in the presence of forskolin. A, representative current-time sweeps (gray) and current-voltage (I-V) plots (black) are shown from whole cell patch clamp recordings performed in the absence (panel i) or presence (panel ii) of CFTR-1420–57 blocking peptide. B, summary of mean (± S.E.) slope conductances following mock transduction or transduction with blocking peptide 1 day prior to measurements. *, p < 0.02, unpaired t test, n = 4–5 patches/condition.
	FIGURE 4. AMPK fails to inhibit CFTR in oocytes expressing catalytically inactive NDPK-A-H118F mutant. TEV recordings were performed 2 days after cRNA injection of CFTR and either wild-type NDPK-A (NDPK-A WT) or the catalytically inactive NDPK-A-H118F mutant into Xenopus oocytes. The oocytes were injected 2–4 h prior to recording with either potassium gluconate (Control) or the AMPK activator K-ZMP (ZMP). A and C, the mean (± S.E.) whole cell conductances are shown over time following addition of the cAMP agonists forskolin (1 μm) and IBMX (0.1 mm) at time 0 to activate CFTR in oocytes expressing either NDPK-A WT (A) or the NDPK-A-H118F mutant (C). B, ZMP injection inhibited the peak CFTR conductance in oocytes co-expressing CFTR and NDPK-A WT relative to control-injected oocytes. *, p < 0.001, ANOVA; data normalized to peak control-injected current. D, ZMP failed to inhibit CFTR conductance with co-expression of the catalytically inactive NDPK-A-H118F mutant (p > 0.10, ANOVA). Starting conductances were lower than peak conductances for all conditions. #, p < 0.001, ANOVA; n = 15–30 oocytes/condition from four to seven separate frogs for all conditions shown.
	FIGURE 5. Importance of both AMPK binding to CFTR and NDPK-A catalytic function for the inhibition of CFTR by AMPK in oocytes. TEV experiments were performed 2 days after microinjection of Xenopus oocytes with cRNAs to express CFTR and either NDPK-A WT or NDPK-A-H118F mutant. The oocytes were microinjected with either ZMP alone or ZMP + CFTR-1420-57 peptide 4 h prior to TEV recordings. Forskolin and IBMX were infused at time 0 to activate CFTR conductance. A, summary of mean (± S.E.) changes in whole cell conductance over time are shown in oocytes expressing NDPK-A WT. B, mean (± S.E.) conductances are shown relative to the peak conductance for the ZMP alone condition. The addition of the CFTR-1420-57 blocking peptide induced a significant increase in peak conductance relative to control. *, p = 0.002 (ANOVA). C, summary of mean (± S.E.) changes in whole cell conductance over time is shown in oocytes expressing NDPK-A-H118F. D, normalized data for CFTR and NDPKA-H118F condition show no significant change in either peak or starting conductances by the CFTR-1420-57 peptide. n = 23–25 oocytes from five separate frogs for all conditions shown).
	FIGURE 6. AMPK enhances NDPK-A autophosphorylation at His-118 in vitro. A, WT or indicated NDPK-A mutants were immunoprecipitated and subjected to in vitro phosphorylation by [γ-32P]ATP labeling in the presence (+) or absence (−) of purified active AMPK holoenzyme AMPK-α1-T172D, -β1, -γ1. Immunoblotting (lower panel) and phosphoscreen imaging (upper panel) were then performed on the same nitrocellulose membrane, as described under “Experimental Procedures.” B, summary of mean (± S.E.) NDPK-A phosphorylation signal normalized to expression levels and reported relative to WT NDPK-A phosphorylation in the presence of AMPK. *, p < 0.02, paired t test relative to same NDPK-A construct in presence of AMPK; #, p < 0.001 relative to WT NDPK-A in presence of AMPK, unpaired t-tests; data pooled from three to eleven experiments for each condition. These results suggest that AMPK does not directly phosphorylate NDPK-A at Ser-120 or Ser-122 (or any other site) but rather enhances an NDPK-A autophosphorylation event at His-118. C, in vitro phosphorylation assays were performed using active AMPK holoenzyme (AMPK-α1-T172D, -β1, -γ1), kinase-dead holoenzyme (AMPK-α1-D157A, -β1, -γ1), or no kinase, and mean (± S.E.) phosphorylation values relative to + active AMPK condition are shown. *, p < 0.001, unpaired t test relative to active AMPK; n = 8. D, acid (or control) washes (± addition of 10 μl of 1 n HCl to wash buffer to adjust the pH to 1.0 for 30 min) of the IP'd NDPK-A on protein A/G beads were performed following in vitro phosphorylation to detect whether phosphorylation had occurred at an acid-labile (i.e., His) residue in NDPK-A. *, p < 0.05; #, p < 0.05, relative to + AMPK condition of same NDPK-A species, unpaired t-tests, n = 3–4 experiments for each condition.
	FIGURE 7. NDPK-A S120 mutants disrupt the AMPK-dependent inhibition of CFTR in oocytes. TEV recordings were performed 2 days after cRNA injection of CFTR and NDPK-A WT or S120A and S120D mutants. 2–4 h prior to recording, oocytes were injected with either potassium gluconate (Con) or the AMPK activator K-ZMP (ZMP). Starting and peak conductances were measured before and after the addition of forskolin (1 μm) and IBMX (0.1 mm). A significant decrease in peak conductance was observed following ZMP treatment with co-expression of WT NDPK-A (*, p < 0.01 relative to Con, ANOVA), whereas this inhibition was lost with co-expression of NDPK-A-S120A and -S120D mutants (p > 0.10 between control and ZMP conditions, ANOVA). Starting conductances for all NDPK-A species were significantly lower than corresponding peak conductances. #, p < 0.001; n = 12 oocytes for each condition from three separate frogs.

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