Role of Interaction and Nucleoside Diphosphate Kinase B in Regulation of the Cystic Fibrosis Transmembrane Conductance Regulator Function by cAMP-Dependent Protein Kinase A
Role of Interaction and Nucleoside Diphosphate Kinase B in Regulation of the Cystic Fibrosis Transmembrane Conductance Regulator Function by cAMP-Dependent Protein Kinase A
Lee A. Borthwick 0 1 2 3
Mathieu Kerbiriou 0 2 3
Christopher J. Taylor 0 2 3
Giorgio Cozza 0 2 3
Ioan Lascu 0 2 3
Edith H. Postel 0 2 3
Diane Cassidy 0 2 3
Pascal Trouvé 0 2 3
Anil Mehta 0 2 3
Louise Robson 0 2 3
Richmond Muimo 0 2 3
0 Current address: Inserm UMR 1078, Génétique, Génomique Fonctionnelle et Biotechnologies , 46 rue Félix Le Dantec, 29218, Brest Cedex2 , France
1 These authors are joint first authors on this work
2 1 Academic Unit of Respiratory Medicine, Department of Infection and Immunity, The University of Sheffield, The Medical School , Sheffield, S10 2RX , United Kingdom , 2 Academic Unit of Child Health, University of Sheffield , Stephenson Wing , Sheffield Children's Hospital , Sheffield, S10 2TH , United Kingdom , 3 Department of Biomedical Science, The University of Sheffield , Western Bank, Sheffield, S10 2TN , United Kingdom , 4 Department of Biomedical Sciences, University of Padova , Via Ugo Bassi 58/B 35131, Padova , Italy , 5 University of Bordeaux, France, and Institut de Biochimie et Genetique Cellulaires, Centre Nationale de la Recherche Scientifique UMR 5095 , 33077, Bordeaux, France, 6 Robert Wood Johnson Medical School, UMDNJ, New Brunswick , New Jersey, United States of America, 7 Medical Research Institute/CVS Diabetes Lung, Ninewells Hospital and Medical School, University of Dundee , Dundee, DD1 9SY , United Kingdom , 8 Inserm UMR 1078, Génétique , Génomique Fonctionnelle et Biotechnologies , 46 rue Félix Le Dantec, 29218, Brest Cedex2 , France
3 Editor: Tomohiko Ai, Indiana University , UNITED STATES
Cystic fibrosis results from mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-dependent protein kinase A (PKA) and ATP-regulated chloride channel. Here, we demonstrate that nucleoside diphosphate kinase B (NDPK-B, NM23-H2) forms a functional complex with CFTR. In airway epithelia forskolin/IBMX significantly
Data Availability Statement: All relevant data are
within the paper and its Supporting Information files.
Funding: This study was supported, in part, by
grants from the Wellcome Trust (086370/Z/08/Z)
(RM), the Cystic Fibrosis Trust and from the
association Gaëtan Salaün and the French CF
foundation “Vaincre la Mucoviscidose” (MK, PT).
Competing Interests: The authors have declared
that no competing interests exist.
increases NDPK-B co-localisation with CFTR whereas PKA inhibitors attenuate complex
formation. Furthermore, an NDPK-B derived peptide (but not its NDPK-A equivalent)
disrupts the NDPK-B/CFTR complex in vitro (19-mers comprising amino acids 36–54 from
NDPK-B or NDPK-A). Overlay (Far-Western) and Surface Plasmon Resonance (SPR) anal
ysis both demonstrate that NDPK-B binds CFTR within its first nucleotide binding domain
(NBD1, CFTR amino acids 351–727). Analysis of chloride currents reflective of CFTR or
outwardly rectifying chloride channels (ORCC, DIDS-sensitive) showed that the 19-mer
NDPK-B peptide (but not its NDPK-A equivalent) reduced both chloride conductances.
Additionally, the NDPK-B (but not NDPK-A) peptide also attenuated acetylcholine-induced
intestinal short circuit currents. In silico analysis of the NBD1/NDPK-B complex reveals an
extended interaction surface between the two proteins. This binding zone is also target of
the 19-mer NDPK-B peptide, thus confirming its capability to disrupt NDPK-B/CFTR
complex. We propose that NDPK-B forms part of the complex that controls chloride currents in
The importance of epithelial ion transport is highlighted by the disease cystic fibrosis (CF), a
monogenic disorder resulting from mutations in the cystic fibrosis transmembrane
conductance regulator (CFTR, ABCC7). CFTR is best characterized as a dual cAMP/PKA and
ATPregulated anion channel that is trafficked to the apical (luminal facing) membrane of polarized
epithelia such as gut, airway and reproductive tract [
]. CFTR is also expressed in
non-epithelial tissues such as lymphocytes and macrophages [
] and this might explain why clinical CF
disease manifests multiple cellular defects in addition to disrupted epithelial ion transport.
These ‘non-channel functions’ include defective autophagy , unchecked inflammation that
fails to resolve [
] and an excess of cancer [
]. The pleiotropic effects of CFTR mutation are
complex and no coherent model explains all aspects of the disease . However, recent
evidence show congenital abnormalities in various CF models suggesting defective airway
In 70–90% of CF patients, only one amino acid is deleted on one or both alleles to generate
a F508del-CFTR mutant that folds inefficiently. F508del-CFTR is an ER-associated mutant
that fails quality control and is not delivered to the plasma membrane [
], although there are
data that disagree with this notion [
]. Irrespective of this controversy, should some
fraction of F508del-CFTR reach the plasma membrane, its residence time is shortened and the
mutant (unlike wild type CFTR) additionally fails to recycle to the membrane . It is
established that CFTR does not act alone [
] and recent evidence demonstrates functional roles
for protein complexes bound to CFTR. For example, several transport-inhibitory proteins bind
to CFTR, including syntaxin 1A and AMPKα [
]. Correspondingly, reagents that disrupt
such complexes potentiate CFTR function [
]. In addition, the appellation “regulator” in the
naming of the CFTR channel describes the effect of CFTR mutation on the mis-control of
other ion channels such as the outwardly rectifying chloride channel (ORCC) [
]. Hence, the
signaling complexes and pathways that control CFTR are multiple and remain incompletely
Nucleoside diphosphate kinases (NDPK, nm23, nme) belong to an eight member protein
histidine kinase family divided into two groups (I and II). Only two closely homologous family
members (NDPK-A & B, from group I) have been extensively investigated. As reviewed
elsewhere, NDPK isoforms found in model systems that are similar to NDPK-A and -B control
] and tracheal development . These functions are in addition to the well
established catalytic function of group 1 members in the synthesis of non-adenine nucleoside
]. The pleiotropic effects of this protein family on cellular processes
including cell differentiation, growth and development, tumour metastasis and transcriptional
processing are well established [
]. NDPK-A and—B share 88% sequence similarity and
are thought to exist as heterohexamers in many cell types [
]. Increasing evidence suggests
that despite their highly homologous nature (their genes lie adjacent to one another), the
cellular actions of NDPK-A & -B isoforms differ substantially. For example, NDPK-B (nme2, or
nm23-H2), but not NDPK-A, binds and phosphorylates the G-protein β-subunit on a histidine
residue (H226) thereby enhancing the basal activity of the G-protein α-subunit [
2 / 25
Interestingly, regulation of the G protein-coupled receptor (thromboxane A2 receptor, TPβ)
and the calcium dependent potassium channel (KCa3.1) is also specific to NDPK-B [
this regard, the protein histidine kinase acivity of NDPK-B regulates a calcium-activated
potassium channel by direct transfer of its high energy phosphohistidine (on NDPK-B H118) to
another histidine on the channel protein . NDPK-B uses a similar ‘his-his’ energy transfer
mechanism to promote the basal rate of cAMP production found in cells in the absence of
Gprotein coupled receptor occupancy [
]. The combined data suggest that distinct regulatory
mechanisms control NDPK-A and -B function in vivo and that isoform NDPK-B is implicated
in signaling events close to the plasma membrane of many different cell types.
We recently demonstrated a functional interaction between CFTR, AMPKα1 and NDPK-A,
] which is independent of NDPK-B. These differences between NDPK-A and -B prompted
our interest because heterotrimeric G-proteins regulate CFTR channel activity [
] and there
exists sequence homology between G proteins and the region of the F508del-CFTR mutation
in the first nucleotide binding domain of CFTR (NBD1) [
]. Furthermore, we have previously
demonstrated that in epithelial membranes, NDPK histidine phosphorylation is itself regulated
by both chloride and cation concentration in vitro [
]. Previous work has also shown that
NDPK regulates the atrial muscarinic potassium channel but the mechanism is unknown [
Since cAMP not only plays a key role in CFTR-dependent chloride transport [
] but also
regulates NDPK in vitro [
], we investigated whether NDPK-B and CFTR might interact
functionally in epithelia in a pathway involving cAMP and/or PKA (see discussion). We report
that cAMP, acting through PKA regulates translocation of NDPK-B from the cytosol to the
apical membrane leading to the formation of a functionally relevant complex between
NDPK-B and CFTR. We demonstrate that the nucleotide binding domain 1 (NBD1, aa 351–
727) of CFTR constitutes an NDPK-B interaction site with CFTR. Thus our data suggest that
NDPK-B is important for the cAMP/PKA regulation of CFTR function.
Materials and Methods
Chemicals and reagents
All chemicals unless otherwise indicated were purchased from Sigma. PVDF membrane was
purchased from Millipore (Watford, UK), acrylamide and other electrophoretic reagents were
from BioRad (Hemel Hempsted, UK). N-[2-(p-bromocinnamylamino)
ethyl]-5-isoquinolinesulfonamide (H-89), and myristoylated protein kinase A inhibitor amide 14–22 were
purchased from Calbiochem (UK). Peptides (>95% purity) were obtained from Sigma Genosys
(Dorset, UK). Fetal calf serum was purchased from Invitrogen Life Technologies (Renfrew,
A wild type human bronchial epithelial cell line (16HBE14o-) [
]was grown in medium 199
containing fetal calf serum as described [
] until confluent. Membrane and cytosolic fractions
were prepared as previously described [
Ovine tracheal and human nasal epithelium (HNE)
Membrane and cytosolic fractions from ovine airway epithelia were prepared as described
]. HNE were obtained as described before from healthy young adults undergoing
surgery for reasons unrelated to nasal mucosal disease [
]. Local ethical committee (Tayside
Committee on Medical Research Ethics) approval and written informed consent were obtained
3 / 25
by AM (ref number 11/91). Nasal brushings were suspended in medium 199 until use or stored
in liquid nitrogen.
Expression and preparation of recombinant NBD1 domain
The CFTR fragment comprising the CFTR domains, nucleotide binding domain 1 (NBD1
domain—351 (TRQ>>) to 727 (GIEED)), R-domain -635 (NLQ>>) to 837 (FFDDM)) and
nucleotide binding domain 2 (NBD2, 1151 (IDV>>) to 1360 (LARSV)) were amplified by
PCR using human CFTR cDNA as template. The PCR product was then inserted into Xho1
restriction site of the bacterial pRSET-A plasmid (Invitrogen) carrying a 6xHis sequence
upstream the multiple cloning site or pGEX4T-1 plasmid. The vectors were transformed into
Escherichia coli strain BL21/DE3. The transformants were selected by ampicillin resistance
(100 μg/mL) and the correct positive clones were screened by restriction digestion and their
sequence checked by sequencing. Expression of recombinant proteins was induced by
isopropyl β-D-thiogalactopyranoside (IPTG) (100 μM) for 2 hours at 25°C, 180 rpm. The induced
cultures were centrifuged at 9,500 rpm for 20 minutes at 4°C and the bacterial pellet was then
resuspended with lysis buffer (50 mM Tris-HCl, 100 mM NaCl, 10% glycerol, 0.1% NP-40, 1
mM EDTA, 0.1% Triton X-100 and proteases inhibitors). After sonication, the lysates were
centrifuged 30 minutes at 9,500 rpm, 4°C and the supernatants added to a Ni-NTA (Qiagen,
30210)—Sepharose 4B (Sigma, CL4B200) beads mixture for the purification of the His-tagged
proteins or Glutathione Sepharose beads for the purification of the GST-tagged proteins. The
bound proteins were eluted with either Laemmli sample buffer (62.5 mM Tris-HCl, 25%
glycerol, 2% SDS, 0.01% bromophenol blue, pH 6.8) plus 5% β-mercaptoethanol, (710 mM) for
further far-western blot analysis or with HBS-EP buffer (10 mM HEPES, pH 7.4 containing 150
mM NaCl, 3 mM EDTA and 0.005% (v/v) Surfactant P20) for further Surface Plasmon
Resonance (SPR) analysis. The quantity and purity of the domains was confirmed by Coomassie
Blue staining of a 12% polyacrylamide gel.
Gut biopsy and short-circuit current measurements
With local ethical committee approval and written informed consent (ref number—04/Q2305/
83), a sheet of stripped intestine was obtained endoscopically from the distal ileum and the
potential difference (PD), SCC and tissue resistance measured using a modified Ussing
chamber technique as described previously [
]. Briefly, the sample was mounted in an Ussing
chamber with an aperture of 0.03 cm2 and incubated at 37°C in Krebs bicarbonate saline gassed
with 95% O2/5% CO2. The serosal fluid contained 10 mM glucose and the mucosal fluid 10
mM mannitol, each having a volume of 5 ml. The PD was measured using salt bridge electrodes
connected via calomel half cells to a differential input electrometer with output to a
two-channel chart recorder (Linseis L6512). Current was applied across the tissue via conductive plastic
electrodes and tissue resistance determined from the PD change induced by a 50 μA current
pulse, taking into account the fluid resistance. The SCC generated by the sheets was calculated
from PD and resistance measurements using Ohm's law. The tissue was allowed to stabilize for
10 min after mounting and then readings of electrical activity taken at 1-min intervals.
Acetylcholine (ACh, 1 mM) was added to the serosal solution after 5 min of basal readings and
readings continued for a further 5 min. Glucose (10 mM) was then added to the mucosal solution,
and readings taken for 10 min. Both mucosal and serosal solutions were then replaced with
fresh pre-warmed Krebs buffer. The procedure was repeated for the mucosal solution to
remove all traces of glucose. Peptide (NDPK-A or NDPK-B 36–54, 100 μM) was added to both
mucosal and serosal solutions and after 30 min stabilization, readings were taken for a further
4 / 25
10 min. Both mucosal and serosal solutions were then replaced with fresh pre-warmed Krebs
buffer. Glucose (10 mM) was added to the mucosal solution, and readings taken for 10 min.
Membrane proteins were re-suspended in immunoprecipitation buffer (10 mM Tris-HCl pH
7.4, 2 mM EDTA, 1 mM NaF, 1 mM DTT, 1% sodium deoxycholate, 1% NP-40, 0.3 μM
aprotonin, 0.2 μM PMSF). The mixture was pre-cleared with protein G-Sepharose beads (30 min at
4°C), centrifuged at 4°C at 350 g for 5 min and the supernatant incubated with primary
antibody for 60 min at 4°C. New beads were added and the mixture incubated overnight at 4°C.
The incubation mixture was centrifuged at 350 g for 5 min and the pelleted beads washed in 1
ml RIPA buffer (50 mM Tris-HCl pH 7.4, 1% NP-40, 0.5% sodium deoxycholate, 5 mM
EDTA, 1M NaCl). This wash step was repeated three times and then 50 μl of 5x Laemmli buffer
containing 100 mM DTT was added to the pellet, boiled for 2 min and finally, spun at 420 g for
5 min. An aliquot (20 μl) was then run on 12.5% polyacrylamide gels and blotted onto PVDF
Proteins (10–100 μg), separated by SDS-PAGE, were transferred to PVDF membrane
(Millipore). Pre-stained markers were used to confirm transfer. The blotted membrane was blocked
with 5% w/v non-fat dry milk and incubated at room temperature for 60 min. The blot was
washed with 1x TBS 0.1% Tween-20 (4 times for 15 min each) and then probed with
appropriate primary antibody. The blots were probed with appropriate Horseradish Peroxidase (HRP)
conjugated secondary antibody (1:2000) followed by supersignal™ West Pico chemiluminescent
detection (Pierce, UK).
Overlay or far Western assays
Proteins were separated by SDS-PAGE and blotted onto PVDF membranes. The blotted
membrane was blocked with 5% w/v non-fat dry milk, following which the cytosol protein (500 μg)
in 1x TBS containing 5% w/v non-fat dry milk was then overlaid onto the membrane and
incubated at room temperature for 60 min. The blot was washed with 1x TBS 0.1% Tween-20 (4
times for 15 min each) and then probed with antibody to NDPK-B.
Laser confocal microscopy
HNE were suspended in complete medium 199 and treated with either forskolin (FSK, 10 μm)/
3-isobutyl-1-methylxanthine (IBMX, 100 μM) for 30 min, H-89 (1 μM) or PKI (100 nM) for 5
min prior to the addition of FSK (10 μM)/IBMX (100 μM) for 30 min. Control cells were
incubated in complete medium 199 alone. The cells were fixed in 4% paraformaldehyde for 30 min
at room temperature and quenched with 100 mM glycine for 30 min. Cells were then
permeabilised using 1% Triton X-100 in 1x PBS (pH 7.2) for 30 min at room temperature, washed (x3)
and blocked with 1% BSA for 60 min at room temperature. Cells were incubated overnight at
4°C with primary antibody (1:100 anti-nm23-H2 mouse monoclonal) in PBS for 60 min,
washed (x3) and then incubated with the secondary antibodies [anti-mouse fluorescein
isothiocyanate (Sigma, UK) and anti-rabbit/goat rhodamine (Santa Cruz, CA)] at 1:100 dilution for a
further 60 min at room temperature. Cells were then washed 5 times with 1x PBS and
re-suspended in glycerol (70%). Slides were examined by laser confocal microscopy (LSM-510; Zeiss,
5 / 25
Biotinylation of surface membrane proteins
Surface biotinylation of glycosylated CFTR was performed as previously described [
some modifications. Briefly, cells grown to confluency were treated with FSK (10 μM)/IBMX
(100 μM) ± PKI (100 nM) for 30 min, washed with ice-cold 1x PBS and then biotinylated using
1mg/ml of EZ-Link sulfo-NHS-SS-biotin for 30 min at 4°C. Free biotin was removed by
washing twice with ice-cold 1x PBS containing 0.1% BSA and then with ice-cold 1x PBS. Cells were
then scraped in ice-cold homogenisation buffer (containing complete protease inhibitor
cocktails) and sonicated as previously [
]. The lysate was centrifuged at 300 g for 2 min and the
pellet discarded. Pre-washed avidin agarose beads suspended in PBS were added to the
supernatant and incubated for 30 min at room temperature with mild shaking. Avidin-bound
complexes were pelleted (350 g) for 2 min and washed five times. Biotinylated proteins were eluted
in Laemmli buffer, resolved by SDS-PAGE, and immunoblotted with appropriate antibody.
Whole cell recordings
Standard patch clamp experiments were used to examine whole cell currents in
16HBE14ocells grown on plastic coverslips (Hamill et al., 1981). Coverslips were placed in a Perspex bath
on the stage of an inverted microscope (Olympus IX70) and voltage protocols driven by an
IBM-compatible computer, equipped with a Digidata interface (Axon instruments, USA) and
the pClamp software, Clampex 8.0 (Axon Instruments, USA). A List EPC-7 amplifier was used
to make recordings.
The bath contained Na+ Ringer, which has the composition (in mM): 140 NaCl, 2 CaCl2, 1
MgCl2, 40 mannitol and 10 HEPES (titrated to pH 7.4 with NaOH). The pipette contained (in
mM): 135 CsCl (to inhibit K+ channels), 2 EGTA, 2 MgCl2, 2 Na2ATP and 10 HEPES (titrated
to pH 7.4 with CsOH). Whole cell currents were saved onto the hard disk of the computer
following low-pass filtering at 5 kHz. Cell potential was clamped at a holding potential of –40 mV
and then stepped to between +100 and –100 mV, in –20 mV steps. Cell area was calculated
from the capacity transients seen in response to a 20 mV potential step, with membrane
capacitance assumed to be 1 μF per cm2. Slope conductances for each cell were calculated over the
appropriate potential ranges; outward (Gout) +100 to +20 mV and inward (Gin) -20 to -100 mV
using Ohms law. To determine the magnitude of previously identified CFTR and
DIDS-sensitive Cl- conductances [
], whole cell current magnitude was initially measured and then
500 μM DIDS was added to the bath (OR Cl- channel magnitude) followed by 10 μM
CFTRinh172 (CFTR magnitude) [
Before obtaining the whole cell configuration, channels were activated by incubation of cells
with FSK (10 μM)/IBMX (100 μM) for 30 min. When the effect of the peptides was tested, cells
were incubated in the presence of the peptides (100 μM for each) for 30 min before an
additional 30 min in the presence of the peptides plus FSK (10 μM)/IBMX (100 μM). For these
experiments, a separate control dataset was obtained in the absence of peptide. These control
cells were incubated for 30 min in the control solution (no peptides) before the final 30 min
incubation in the presence of FSK (10 μM)/IBMX (100 μM).
Surface Plasmon Resonance (SPR) analysis
The human recombinant nucleoside diphosphate kinase B isoform (NDPK-B) was prepared
and provided by Professor Ioan Lascu. CM5 (carboxyl methyl dextran) sensor chips,
1-ethyl-3(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS),
ethanolamine hydrochloride and NaOH pH 8.5 were purchased from GE Healthcare. The
buffers used for the experiments are: immobilization buffer, 10mM sodium acetate buffer pH 4.0 to
6 / 25
5,5; HBS-EP buffer, 10 mM HEPES, pH 7.4 containing 150 mM NaCl, 3 mM EDTA and
0.005% (v/v) Surfactant P20.
The CM5 measurements were performed using the BIAcoreTM 3000 optical biosensor (GE
Healthcare) of the PurIProB platform (Inserm U1078, Brest, 29218, France). In accordance
with the pH-scouting wizard, the ligand (NDPK-B), diluted in 10 mM acetate buffers with
different pH (4.0, 4.5, 5.0 and 5.5), was injected on the sensor chip for 2 min at a flow rate of
20 μL/min to determine the appropriate immobilization pH conditions. For covalent
immobilization of the NDPK-B ligand on the CM5 sensor chip surface, the sensor surface was activated
with a freshly mixed solution of 0.4 M EDC and 0.1 M NHS (1:1) for 7 min at a flow rate of
5 μL/min. Purified NDPK-B (5561.5 RU final), was then injected over the activated chip
surfaces in 100 μL of 10 mM sodium acetate pH 5.5 (flow rate: 5 μL/min). The residual active
groups were inactivated with 1 M ethanolamine-HCl at pH 8.5 for 7 min (flow rate: 5 μL/min).
For binding measurements, ligand—protein interactions were monitored by injecting the
NBD1 domain dissolved at various concentrations in the HBS-EP running buffer at the flow
rate of 10 μL/min for 2 min. The binding of NBD1 domains was evaluated 20 s into the
dissociation phase. In order to continuously monitor the non-specific background binding of samples
to the carboxymethyl dextran substrate, a control flow cell was pre-activated and immediately
blocked with ethanolamine without exposure to the ligand. Interactions were estimated by
subtracting the response in the blank flow cell from the response in the cell with immobilized
NDPK-B. Bovine Serum Albumin (BSA, 500 ng) was injected as a negative control under
similar conditions. The interaction analyses were carried out in HBS-EP buffer at 25°C. The SPR
data analysis was done by applying the BIAevaluation v.4.1.1 software (BIAcore).
In silico analysis
Protein-protein docking analysis was performed using two FFT-based docking software PIPER
] and Zdock [
]. The in silico experiments were performed using NDPK-B crystal structure
(PDB code: 1NUE) as the probe and NBD1 crystal structure (PDB code: 1R0X) as the target
protein. 1000 complexes were obtained from both docking algorithms and clusterized using
the pairwise RMSD (Root Mean Square Deviation < 2Å) into 4 largest clusters. The final
complex was chosen according to the energy scoring function.
Molecular dynamics (MD) simulations of the final complex (parameterized with
AMBER99) were performed with NAMD 2.8 [
] in order to verify their stability over time; in
particular a 100ns of NPT (1atm, 300K) MD simulation were performed after an equilibration
phase of 1 ns (positional restraints were applied on carbon atoms to equilibrate the solvent
around the protein).
Antibodies used in this study
CFTR monoclonal antibody was from Labvision/Neomarkers (Runcorn, Cheshire, UK) and
CFTR polyclonal antibody (R&D Systems, Minneapolis, USA). The nm23-H1 antibody was
from Autogen Bioclear (Calne Wiltshire, UK) and has been described previously (Muimo
et al., 1998). The nm23-H2 monoclonal antibody was from Kamiya Biomedical Company
(Seattle, Washington, USA) and the nm23-H2 polyclonal antibodies have been described
previously (Ma et al., 2002a; Hippe et al., 2003). Anti-NDPK-A does not recognize NDPK-B, nor
vice versa, in western blot and immunostaining.
Osmolality of the experimental solutions was checked using a Roebling osmometer and
adjusted to 300 ± 1 mOsm.kg-1 H2O using mannitol or water as appropriate.
7 / 25
Results are presented as mean ± SEM. Effects of experimental interventions were assessed by
Student’s t-test and significance was assumed at the 5% level. Unless otherwise indicated all
immunoblots are representative of at least three independent experiments.
Distribution of NDPK-A and -B in airway epithelial cells
Among NDPK isoforms, NDPK-A and B share the highest sequence homology (88%), and
both have been detected in the cytoplasm and in the nucleus in various cell types [
28, 56, 57
However, the properties and function of membrane bound NDPK from mammalian cells is
less well characterized. We have previously shown that NDPK-A exists in apical membrane of
human and sheep airway epithelia [
] and that it forms a complex with AMPKα1 and CFTR
]. Western blot analysis shows that NDPK-B is also present in both membrane and cytosol
of 16HBE14o- cells and sheep tracheal epithelia. Interestingly, although NDPK-B is detectable
in the membrane fraction by immunoblot analysis, it is predominantly cytosolic in
16HBE14ocells (Fig 1a), sheep tracheal epithelia and rat thalamus (not shown). In contrast, NDPK-A
appears to be more uniformly distributed between the membrane and cytosol fractions of
16HBE14o- cells (Fig 1a), sheep tracheal epithelia and rat thalamus (not shown). Previous
studies have demonstrated that NDPK-A and -B form heterohexamers in human erythrocytes
]. However, since our data shows that the membrane fraction in airway epithelia contains
disproportionate amounts of NDPK-A and, NDPK-B the two isoforms may not always exist as
hetero-tetramers/hexamers or share the same subcellular distribution in airway epithelia.
Do NDPK A & NDPK B form complexes in airway epithelia?
The differential subcellular distribution of NDPK A and NDPK B in 16HBE14o- cells suggests
that the two isoforms are probably involved in different cellular processes. We have recently
provided evidence that NDPK A forms a functional complex with AMPKα [
]. Analysis of
AMPKα1 or AMPKα2 immunoprecipitates from membrane and cytosol of 16HBE14o- cells
or sheep tracheal epithelium (not shown) for the presence of NDPK-B (Fig 1b), shows that
while NDPK A co-immunoprecipitates with AMPKα1 (left panel), NDPK-B is undetectable.
Equally, AMPKα is undetectable on western blots containing immunoprecipitates of NDPK-B
(not shown). This suggests that NDPK B exists independently of the NDPK A/AMPKα1
complex. Furthermore, we investigated complex formation between NDPK A and NDPK B in both
membrane and cytosol of airway epithelia. Western blot analysis of NDPK-A and NDPK B
immunoprecipitates from airway epithelia shows that NDPKA is undetectable in NDPK B
immunoprecipitates and vice versa (Fig 1c). The failure to co-immunoprecipitate NDPKA and
NDPK B in airway epithelia indicates that the two proteins may not exist as heterohexamers
under normal cellular conditions in these cells. Furthermore, this data also suggests that the
two proteins are likely to localise to different subcellular compartments and engage in different
cellular events in airway epithelia.
NDPK-B interaction with CFTR is regulated by cAMP/PKA
Previous work shows that NDPK binds cAMP in vitro [
]. To analyse whether cAMP
might affect cellular distribution and function of NDPK-B in airway epithelial cells, we
immunolocalised NDPK-B in human nasal brushings treated with or without the well-established
stimulus that raises cyclic AMP (a combination of the adenylyl cyclase stimulator forskolin
(FSK) and a phosphodiesterase inhibitor (IBMX) to prevent cAMP degradation). Fig 1d shows
8 / 25
Fig 1. PKA regulates translocation of NDPK-B in airway epithelial cells. a) Distribution of NDPK A and NDPK B in 16HBE14o- cells. Western Blot of
membrane and cytosol (50 μg) from 16HBE14o- cells separated on a 15% SDS PAGE gel and transferred to PVDF membrane (Left panel, probed with
NDPK B rabbit polyclonal antibody (1/5000); Middle panel, probed with NDPK B monoclonal antibody (1/1000); Right panel, probed for NDPK A). b) NDPK B
is not detected in AMPKα immunoprecipitates. Left panel, western blot of AMPKα (pan) probed for NDPK A. Right panel, immunoprecipitates of AMPKα1 and
AMPKα2 probed for NDPK B. c) Absence of complex between NDPK A and B in airway epithelia. Left panel, immunoprecipitates of NDPK B and NDPK A
9 / 25
probed for NDPK A. Right panel, immunoprecipitates of NDPK A and NDPK B probed for NDPK B. Antibody staining detected with HRP antibody and a
chemiluminescent substrate. Results representative of at least four independent experiments. Modulation of PKA activity alters localisation and distribution of
NDPK-B in HNE. Immunocytochemical staining of HNE for NDPK-B in cells: d) untreated, e) treated with FSK/IBMX for 30 min, f) treated with PKI (5 min)
prior to FSK/IBMX for 30 min, g) No primary antibody control. Bar is 5 μM and arrows show position of apical membrane. The result is representative of three
confocal microscopy analysis of unstimulated human nasal epithelial cells stained with a
selective anti-NDPK-B antibody. Although NDPK-B staining was observed throughout the cell
including the nucleus, it was predominantly located within the cytoplasm within distinct
punctuate structures (as noted by others (18)). Following cell stimulation with FSK (10 μM)/IBMX
(100 μM), NDPK-B staining predominates at the apical membrane (Fig 1e) and is unexpectedly
also found associated with cilia suggestive of a secretory event (this was not explored further).
To determine whether PKA was involved, we inhibited PKA activity using PKI, a cell
permeable and selective peptide pseudo-substrate inhibitor of the catalytic activity of PKA (100 nM
myristoylated protein kinase A inhibitor amide 14–22 − (54)), added prior to FSK/IBMX
stimulation. PKA inhibition maintained the pre-stimulated pattern of NDPK-B staining within the
cytoplasm (Fig 1f). This suggests that PKA might regulate the distribution and or
translocation/secretion of NDPK-B in airway epithelia.
CFTR binding to cAMP has been proposed as a regulator of channel activation [
cAMP and PKA are cooperative regulators of CFTR (4, 5) and mature CFTR is apically
localised in airway epithelia [
], we analysed the interaction between CFTR and NDPK-B using
cell lysates. Fig 2a shows additional NDPK-B staining occurs at 175 kDa, when Western blots
of SDS-PAGE-separated membrane proteins from (CFTR-expressing) immortalised
16HBE14o- cells (100 μg) or ovine tracheal epithelial membranes (not shown) are overlaid
with a solution containing cytosol from the relevant cognate cells (Far-Western blot). Addition
of non-hydrolysable analogues of cAMP (8-bromo or di-octanoyl cAMP, 100 μM) to the
overlay solution strikingly increased NDPK-B staining at 175 kDa nearly 20-fold (Fig 2a and 2b).
This result strongly suggested cAMP might control the assembly of an NDPK-B/CFTR protein
complex at the apical membrane of airway epithelia. In order to determine whether the
interaction between NDPK-B was direct or involved a bridging protein, a Western blot of a CFTR
immunoprecipitate was first overlaid with purified NDPK-B and then probed for NDPK-B.
Enhanced NDPK-B staining is observed at 175 kDa in the presence of cAMP (100 μM) (Fig 2c
and 2d). To test the selectivity of the cAMP effect, we studied NDPK-A, and found that
although NDPK-A also binds at the expected molecular weight for CFTR in overlay assays, the
binding of this highly homologous family member is not enhanced by cAMP (not shown, see
also Fig 3a and 3b).
Western blot analysis of CFTR immunoprecipitates from membranes of 16HBE14o- cells
stimulated with FSK/IBMX shows that FSK significantly enhanced the amount of NDPK-B
coimmunoprecipitating with CFTR (Fig 3a, lane 2; quantitation Fig 3b). Pre-treatment of the
cells with PKA inhibitors, PKI (100 nM) or H-89 (1 μM, not shown) for 5 min prior to
stimulation with FSK/IBMX for 30 min, reduced the amount of NDPK-B interacting with CFTR (Fig
3a and 3b). Similarly, FSK-stimulation significantly increases the amount of CFTR
co-immunoprecipitating with NDPK-B (Fig 3c and 3d). On the other hand, analysis of NDPK-A
staining in CFTR immunoprecipitates shows no variation following cell stimulation with either
FSK/IBMX or PKI/FSK/IBMX suggesting that unlike NDPK-B, NDPK-A interaction with
CFTR is not regulated by changes in cellular cAMP/PKA activity (Fig 3a and 3b). Thus, our
data suggests cAMP and PKA selectively mediate promote the NDPK-B interaction with CFTR
and that despite its similar sequence, NDPK-A is not part of this process.
10 / 25
Fig 2. NDPK-B interaction with CFTR is regulated by cAMP. a) cAMP-dependent NDPK-B binding to a 175-kDa protein in overlay assays. Identical
immunoblots of (100 μg) probed for NDPK-B. Lanes: 1) control blot with no overlay, 2) blot overlaid with solution containing 16HBE14o- cytosol proteins (0.5
mg). 3, 4, 5) Blot overlaid with solution 2 above containing cAMP (100 μM), 8-Br-cAMP (100 μM) or N6,O2'- dioctanoyl-cAMP (Oco2cAMP) (100 μM),
respectively. Cyclic AMP enhanced NDPK-B staining at 175 kDa. Results are representative of four separate experiments. b) Quantification of the band
density of NDPK-B/CFTR complex at 175kDa (n = 4) * P<0.001 Student t-test. Dioctanoyl cAMP increased NDPK-B binding 20-fold. c) Recombinant
NDPK-B binds to a 175-kDa protein in cAMP-dependent manner in overlay assays. Identical western blots of membrane proteins from 16HBE14o- cells
(100 μg) probed for NDPK-B staining. Lanes 1) Control with no overlay. 2) Blot overlaid with solution containing 1 μg of recombinant NDPK-B. 3) Overlaid
with solution 2 containing cAMP (100 μM), 4) overlaid with solution 2 containing 8-Br-cAMP (100 μM). d) Quantification of the band density of NDPK-B at
175kDa (n = 4) * P<0.001 Student t-test. Br-cAMP increased NDPK-B binding 20-fold.
NDPK-B associates only with Cell surface CFTR
The cAMP/PKA-dependent translocation of NDPK-B to the apical membrane suggested that
NDPK-B might associate with cell surface CFTR. In order to confirm that the
cAMP/PKAinduced complex is associated with the cell surface CFTR and not CFTR within intracellular
organelles, 16HBE14o- cells were surface biotinylated with cell-impermeant EZ-Link
sulfoNHS-SS-biotin at 4°C for 30 min. Fig 4a (lane 2) shows increased levels of NDPK-B
co-precipitate with avidin-agarose in cells treated with FSK/IBMX. On the other hand, inhibition of
cellular PKA activity with PKI, prior to FSK/IBMX stimulation, reduces the amount of NDPK-B
precipitating with avidin-agarose (Fig 4a lane 3). This data is consistent with the notion that
NDPK-B translocation to the apical membrane leading to an association with cell surface
11 / 25
Fig 3. Forskolin (FSK) enhances NDPK-B interaction with CFTR in 16HBE14o- cells a) PKA regulates co-immunoprecipitation of CFTR with NDPK-B in
16HBE14o-. Immunoblot of CFTR immunoprecipitate from 16HBE14o- membranes showing that FSK increases the amount of NDPK-B, but not NDPK-A,
which co-immunoprecipitates with CFTR. Cells were untreated (lane 1), treated with FSK/IBMX for 30 min (lane 2) or with PKI for 5 min prior to FSK/IBMX
treatment (lanes 3) and probed for NDPK-A and NDPK-B. Equal loading of the CFTR immunoprecipitate, was confirmed by re-probing the same blot for
CFTR using a polyclonal antibody (R & D systems). b) Quantification of the band density of NDPK-B and NDPK-A shows a 3-fold increase in NDPK-B
coimmunoprecipitation with CFTR with FSK/IBMX (n = 4). * P<0.05 Student t-test. c) Immunoblot of NDPK-B immunoprecipitate from 16HBE14o- membrane
from cells: untreated (lane 1); treated with FSK/IBMX (lane 2) or PKI (5 min) prior to FSK/IBMX for a further 30 min (lane 3) and probed for CFTR. Equal
loading of immunoprecipitate was confirmed by re-probing the same blot for NDPK-B polyclonal antibody. PKI inhibited the impact of FSK/IBMX on NDPK-B
co-immunoprecipitation with CFTR. d) Quantitative analysis of the CFTR band density shows a 2-fold increase in CFTR co-immunoprecipitation with
NDPK-B with FSK/IBMX (n = 4) * P<0.05 Student t-test.
CFTR. The selectivity of this binding is demonstrated by our observing no change in the levels
of NDPK-A or CFTR co-precipitating with avidin-agarose following cell treatment with FSK/
IBMX (Fig 4a). To assess whether NDPK-B also associates with non-cell surface CFTR, CFTR
immunoprecipitates from the residual cell extracts (± FSK/IBMX-stimulation) already depleted
of cell surface/integral membrane proteins by avidin precipitation (post-avidin supernatant)
were probed for NDPK-B. Fig 4b and 4c show that, despite the presence of both proteins in the
post-avidin supernatant, NDPK-B did not co-immunoprecipitate with CFTR from this
fraction. Similarly, in the reverse experiment, CFTR staining is undetectable in NDPK-B
immunoprecipitates from supernatant depleted of biotin-labelled proteins. Thus, following cAMP/PKA
stimulation, there was increased association of NDPK-B, but not NDPK-A, with cell surface
12 / 25
Fig 4. NDPK-B binds cell surface CFTR in forskolin/IBMX stimulated cells. a) Immunoblots of avidin-agarose precipitates from lysates of
16HBE14ocells ± FSK or PKI/ FSK/IBMX, biotinylated for 30 min at 4°C and probed for CFTR, NDPK-A and NDPK-B. b) Non-cell surface CFTR does not associate with
NDPK-B. Immunoblots of CFTR immunoprecipitates from lysates of 16HBE14o- cells ± FSK or PKI/FSK/IBMX (post the avidin precipitation described in A,
above) probed for CFTR and NDPK-B. c) NDPK-B does not associate with non-cell surface CFTR. Immunoblots of NDPK-B immunoprecipitates from lysates
of 16HBE14o- cells ± FSK or PKI/FSK/IBMX (post the avidin-agarose precipitation described in A, above) probed for CFTR and NDPK-B. To confirm equal
loading of the immunoprecipitates, blots were stripped and re-probed with CFTR rabbit polyclonal antibody (R & D systems) (B) or NDPK polyclonal antibody
(C). The results are representative of two independent experiments.
CFTR. Complex formation is not observed with non-cell surface CFTR suggesting that
NDPKB tethers to cell surface CFTR.
NDPK-B interaction domain
NDPK-A and NDPK-B are reported to exist as heterohexamers and yet we find a difference in
their localization. In order to identify the NDPK-B domain responsible for interaction with
CFTR, we generated a 19-mer peptide corresponding to one of the regions of NDPK-B
showing the least sequence homology to NDPK-A comprising amino acids 36–54 (Fig 5a).
Immunoblot analysis shows that NDPK-B is released into the supernatant of the beads bearing the
CFTR immunoprecipitate and exposed to the peptide (Fig 5b lanes 3, 4, compare with lane 1).
These immunoprecipitates were from lysates of 16HBE14o- cells stimulated with FSK/IBMX
and incubated with the peptide NDPK-B 36–54 (V36AMKFLRAS44EEHLKQHYID54) for
30 min at 30°C. In control incubations, without peptide (Fig 5b lanes 1, 2) or with the
13 / 25
Fig 5. Analysis of NDPK-B interaction with CFTR. a) Position of the exposed side-chains of peptide 36–54, based on the published crystal structure of the
]. The CPK code was used: nitrogen is blue and oxygen is red. One subunit is shown in cyan to identify its border. Figure generated with
RASMOL. b) Immunoblots of CFTR immunoprecipitates from lysates of 16HBE14o- cells treated with FSK/IBMX, and probed for NDPK-B and CFTR show
peptide NDPK-B 36–54 (lanes 3, 4), but not peptide NDPK-A 36–54 (lanes 5, 6), released NDPK-B from CFTR immunoprecipitate. Control incubations with
buffer alone are shown in lanes 1, 2. c) Peptide NDPK-B 36–54 releases CFTR from complex with NDPK-B. Immunoblots of NDPK-B immunoprecipitates
from lysates of 16HBE14o- cells treated with FSK/IBMX, and probed for CFTR and NDPK-B show peptide NDPK-B 36–54 (lanes 3, 4), but not buffer alone
control (lanes 1, 2) or peptide NDPK-A 36–54 (lanes 5, 6), released CFTR from NDPK-B immunoprecipitate.
corresponding peptide from NDPK-A 36–54 (V36GLKFMQASEDLLKEHYVD54) (Fig 5b
lanes 5, 6), release of NDPK-B from the CFTR immunoprecipitate was never observed. The
selective release of NDPK-B induced by peptide NDPK 36–54 indicates that this peptide
disrupted the NDPK-B/CFTR complex and suggests this region of NDPK-B, may constitute a key
NDPK-B interaction domain for CFTR. Similar results were obtained in the reverse
experiment, the peptide NDPK 36–54 -induced CFTR release from beads containing the NDPK-B
immunoprecipitate (Fig 5c).
CFTR binding site for NDPK-B
In order to establish whether NDPK-B interacts directly with CFTR, we generated fusion
proteins of various CFTR domains: nucleotide binding domain 1 (NBD1, aa 351–727), R-domain
(635–837) and nucleotide binding domain 2 (NBD2, 1151–1360) and localised NDPK-B
binding to NBD1 by overlay on dot- blots (Fig 6a). Fig 6b shows the purity (by Coomassie blue
staining) of untagged NBD1 (various amounts) and of purified NDPK-B protein used for
overlay analysis. Fig 6c: Western blot and overlay analysis indicating direct interaction between
pure untagged NBD1 and NDPK-B (PVDF membrane containing untagged NBD1 was
overlaid with purified NDPK-B and then probed with anti-NDPK-B). The interaction was then
confirmed by Surface Plasmon Resonance (SPR) (Fig 6d–6g) assays. We injected several
quantities of His-tagged NBD1 (10, 20, 40 or 60 ng) over immobilized NDPK-B proteins and
showed the direct interaction between the NBD1 of CFTR and NDPK-B (Fig 6d–6g). The RU
values obtained twenty seconds into the dissociation phase were 2.2, 6.2, 13.5 and 20.9 for 10,
20, 40 and 60 ng of NBD1 respectively, and as such, increased linearly with the amount of
14 / 25
Fig 6. NDPK-B /NBD1 interaction analysis of by Surface Plasmon Resonance. a) Membrane (PVDF) was spotted with purified NDPK-B (lane 1),
GST-NBD1 (351–727), R domain (635–837) and GST-NBD2 (1151–1360) was overlaid with purified recombinant NDPK-B and probed for NDPK-B. Purified
NDPK-B bound to GST-NBD1. b) Coomassie blue staining of PVDF membrane showing the purity of NBD1 (28 kDa) (lanes 1–3; 900, 300 and 100 ng per
lane, respectively) and NDPK-B (lane 4, 16 kDa) and the amounts of the proteins loaded. c) Overlay analysis of the direct interaction between the NBD1
domain (351–727) of CFTR and NDPK-B. Overlay experiment showing western blot containing NBD1 (lanes 1–3; 900, 300 and 100 ng per lane,
respectively), NDPK-B (250 ng, lane 4) and BSA (250 ng, lane 5) overlaid with purified NDPK-B (50 μg) and then probed for NDPK-B staining using NDPK-B
specific antibodies. BSA was used as a control to exclude non-specific interactions with NDPK-B. d) SPR analysis showing the direct interaction between the
NBD1 of CFTR and NDPK-B. Example of sensorgrams obtained when several quantities of His-tagged NBD1 (10, 20, 40 or 60 ng) were injected over
immobilized NDPK-B proteins. In the inset, the RU = f(ng) curve shows that the RU values obtained twenty seconds into the dissociation phase linearly
increase with the amount of injected NBD1. e) SPR analysis showing the repeatability of the measurement of the direct interaction between the NBD1 of
CFTR and NDPK-B. Example of sensorgrams obtained from two separate measurements when 40 ng of His-tagged NBD1 were injected over immobilized
NDPK-B proteins. f) SPR analysis showing the specificity of the interaction between the NBD1 of CFTR and NDPK-B. Example of sensorgrams obtained
when 1 μg of untagged NBD1 was injected alone or with several quantities of NDPK-B peptide (10, 50 or 100 ng) over immobilized NDPK-B proteins. A
sensorgram obtained when 200 ng of NDPK-B peptide was injected alone over immobilized NDPK-B proteins is also shown. g) SPR analysis showing the
NDPK-B key residues for the NBD1-NDPK-B interaction. Example of sensorgrams obtained when 1 μg of untagged NBD1 was injected alone or with 100 ng
of different mutated NDPK-B peptides over immobilized NDPK-B proteins. Sensorgrams obtained when 1 μg of untagged NBD1 was injected with 100 ng of
NDPK-A or NDPK-B peptide over immobilized NDPK-B proteins are also shown.
injected NBD1 (Fig 6d). We evaluated the repeatability of the measurement of the direct
interaction between the NBD1 of CFTR and NDPK-B in two separate experiments whereby 40 ng
of His-tagged NBD1 injected over immobilized NDPK-B proteins provided a similar number
of RU in both cases (13.4 and 13.6) (Fig 6e). This showed the repeatability of the measurement.
To assess the specificity of the NBD1-NDPK-B interaction we used a 19 amino acid (36–54:
VAMKFLRASEEHLKQHYID) synthetic peptide of NDPK-B (Fig 5a) that disrupts the
NDPK-B/CFTR complex (Fig 5b and 5c) under similar conditions. We injected 1 μg of
untagged NBD1 alone or with several quantities of NDPK-B peptide (10, 50 or 100 ng) over
immobilized NDPK-B proteins and observed that the number of RU decreased with the
amount of injected NDPK-B peptide showing the specificity of the interaction between the
NBD1 of CFTR and NDPK-B (Fig 6f). As a control we also injected 200 ng of NDPK-B peptide
15 / 25
alone over immobilized NDPK-B proteins and observed no interaction confirming the
specificity of the interaction between the NBD1 of CFTR and NDPK-B (Fig 6f). First, our SPR data
show a specific interaction between the NBD1 of CFTR and NDPK-B, confirming the overlay
analysis (Fig 5a–5c). Secondly, these SPR results confirm the ability of NDPK-B peptide to
disrupt the NDPK-B/CFTR complex, showing that this region of NDPK-B (amino acid 36–63)
may constitute the NDPK-B interaction domain for CFTR.
To identify the NDPK-B key residues for the NBD1-NDPK-B interaction, we used different
mutated NDPK-B peptides as follows: NDPK-B peptide with I-V substitution, NDPK-B
peptide with H-L substitution and NDPK-B peptide with both I-V and H-L substitutions. By
looking at the sequence, we Figured residues I or H or both might be key to the interaction and that
the substitution may affect the ability of NDPK-B peptide to disrupt interaction and cause it to
behave like NDPK-A peptide which doesn’t disrupt the NDPK-B interaction with CFTR (Fig
5b and 5c). We injected 1 μg of untagged NBD1 alone or with 100 ng of different mutated
NDPK-B peptides over immobilized NDPK-B proteins and observed a similar signal in all
cases showing that theses amino acids substitutions affect the ability of NDPK-B peptide to
disrupt the NBD1-NDPK-B interaction (Fig 6g). We also injected 1 μg of untagged NBD1 with
100 ng of NDPK-A peptide over immobilized NDPK-B proteins and obtained an equivalent
response showing that these residues substitutions cause the mutated NDPK-B peptides to
behave like NDPK-A peptide (Fig 6g). These data suggest that I and H amino acids are key
residues for the NBD1-NDPK-B interaction. As a control we finally injected 1 μg of untagged
NBD1 with 100 ng of NDPK-B peptide over immobilized NDPK-B proteins and observed a
dissimilar signal, with a number of RU that became insignificant confirming the ability of
NDPK-B peptide to disrupt the NDPK-B interaction with CFTR (Fig 6g). These SPR results
first confirm the ability of the NDPK-B peptide to disrupt the CFTR/NDPK-B complex and
secondly identify that I and H amino acids as key residues for this NBD1-NDPK-B interaction
(NB. The transient signals are artefacts caused by the modification of the refractive index due
to buffer change just after the injection start/stop. These artefacts do not alter the analysis (RU
values are obtained twenty seconds into the dissociation phase)).
To clarify the binding motif of NBD1 and NDPK-B, protein protein docking experiments
were performed by using two FFT based software, PIPER [
] and Zdock [
] (see Methods
section). As shown in Fig 7a, the complex between NBD1 and NDPK-B involves a deep
interaction surface area (693 Å2). Electrostatic interactions are predominant, however a small
hydrophobic pattern has been revealed by in silico analysis. In detail, NDPK-B I53 is
submerged into a small hydrophobic pocket formed by V393, F446 and the methyl group of T390
of NBD1. This hydrophobic zone is surrounded by different electrostatic interactions, namely
between NBD1 K447 and NDPK-B D57, and between NBD1 E395 and NDPK-B H47,
furthermore interacting also with NDPK-B E46. On the other hand, no significant results was
obtained from the protein-protein docking analysis between NBD1 and NDPK-A, under the
same experimental condition used in the case of NDPK-B (see Methods section). Interestingly
NDPK-A presents a couple of substitutions in two key points of the interaction pattern
revealed by the in silico analysis of NBD1 and NDPK-B: the NDPK-B I53 and H47 are
substituted with V53 and L47 in the case of NDPK-A as shown in the table inset of Fig 7a.
Significant results were obtained also by studying the interaction between NBD1 and the
peptide 36–54 (VAMKFLRASEEHLKQHYID) derived from NDPK-B. The complex obtained
from the protein-protein docking analysis is partially superimposable with the complex
between NBD1 and the full NDPK-B protein (Fig 7b). The in silico results suggest that the
peptide 36–54 could directly and specifically compete with the interaction between NBD1 and
NDPK-B, by sharing the same binding zone, thus disrupting NDPK-B/CFTR complex. On the
other hand, the protein-protein docking analysis between NBD1 and the NDPK-B peptide
16 / 25
Fig 7. NDPK-B and NBD1 protein-protein docking analysis. a) i) Protein-protein docking complex between NBD1 (red) and NDPK-B (green); Analytic
Connolly’s surface has been highlighted. ii) Detailed interaction pattern between NBD1 and NDPK-B. Table inset: differences between NDPK-B and
NDPK-A at the interaction surface. b). i) Detailed interaction pattern between NBD1 (red) and the peptide 36–51 from NDPK-B (Yellow). ii) Superposition
between 36–51 peptide (yellow) and NDPK-B full protein (green). Table inset: statistical analysis of NBD1-NDPK peptides complexes.
17 / 25
carrying the single or the double I-V/H-L substitutions does not reveal statistically significant
complexes formation, thus confirming that this peptide, characterized by the NDPK-A
substitutions, is not able to interact as efficiently as the NDPK-B WT peptide. In particular, as shown
in the table inset of Fig 7b, while the NBD1-NDPK-B WT peptide complex present a high
statistical significance (653 complexes with an RMSD under 5Å), all the substituted peptides are
not able to produce statistically relevant complexes.
NDPK-B and CFTR function
Previous studies have demonstrated that NDPK regulates muscarinic K+ and calcium
-activated K+ (KCa3.1) channels [
]. To determine the functional significance of the cAMP/
PKA-dependent cell surface associated NDPK-B/CFTR complex, we analysed the impact of
peptide NDPK-A 36–54 or peptide NDPK-B 36–54 on whole cell currents in 16HBE14o- cells.
Fig 8a, shows that peptide NDPK-B 36–54, which disrupts the NDPK-B/CFTR complex (see
Fig 4), reduces the magnitude of both the DIDS-sensitive and CFTR-mediated chloride
conductances. The CFTR and OR mediated conductances were significantly smaller in the
presence of the peptide. The CFTR-sensitive Gout was 574 ± 164 μS/cm2 (n = 12) versus
187 ± 57.6 μS/cm2 (n = 13), in the absence and presence of NDPK-B 36–54, respectively. The
CFTRinh-sensitive Gin was 465 ± 132 μS/cm2 (n = 12) versus 165 ± 49.4 μS/cm2 (n = 13). The
DIDS-sensitive Gout was 341 ± 77.6 μS/cm2 (n = 13) versus 119 ± 37.9 μS/cm2 (n = 13), in
the absence and presence of NDPK-B 36–54, respectively. The DIDS-sensitive Gin was
172 ± 66.3 μS/cm2 (n = 13) versus 23.9 ± 13.8 μS/cm2 (n = 13). This suggests that the
NDPKFig 8. Effect of disruption of the NDPK-B/CFTR complex on the magnitude of FSK-dependent IDIDS and ICFTR in 16HBE14o- cells and the
AChinduced SCC in intestinal biopsy. a) Effect of peptide NDPK-B 36–54 on ICFTR and IDIDS. Cells were incubated with NDPK-B 36–54 (100 μM) for 30 min
prior to exposure to FSK/IBMX plus peptide NDPK-B 36–54 for 30 min. Control currents for each dataset were time and day matched. b) Lack of effect of
peptide NDPK-A 36–54 on the outward DIDS-sensitive and CFTRihn172- sensitive conductances. Cells were incubated for 30 min in the presence of the
peptide (100 μM), followed by incubation with the peptide plus FSK and IBMX for 30 min. Control currents for each dataset were time and day matched. c)
Effect of peptide NDPK-A or NDPK-B 36–54 on SCC in gut epithelia. SCC measurements obtained in response to ACh (Cl- flux) and glucose (Na+ flux)
stimulation of mounted gut epithelia biopsies. Measurements were taken in the presence or absence of the synthetic peptides NDPK-A or NDPK-B 36–54
(100 μM) (n = 3) **P<0.05 ANOVA.
18 / 25
B/CFTR complex is functionally significant and that NDPK-B regulates both the
cAMP/PKAdependent CFTR and the ORCC-mediated currents in epithelia. Since NDPK-A forms a
complex with AMPKα independently of NDPK-B in airway epithelia (25), we also analysed the
impact of peptide NDPK-A 36–54 on CFTR function. As expected, peptide NDPK-A 36–54,
which does not disrupt the NDPK-B/CFTR complex, was without effect on both the cAMP/
PKA-dependent ORCC and CFTR-mediated currents (Fig 8b). The CFTR and OR mediated
conductances were not significantly different in the presence of the peptide. (n = 11 or 12 as
stated). The CFTRinh-sensitive Gout was 510 ± 85.0 μS/cm2 (n = 11) versus 486 ± 149 μS/cm2
(n = 8), in the absence and presence of NDPK-B 36–54, respectively. The CFTRinh-sensitive
Gin was 447 ± 63.8 μS/cm2 (n = 11) versus 537 ± 219 μS/cm2 (n = 8). The DIDS-sensitive Gout
was 312 ± 58.5 μS/cm2 (n = 12) versus 322 ± 131 μS/cm2 (n = 7), in the absence and presence
of NDPK-B 36–54, respectively. The DIDS-sensitive Gin was 147 ± 56.6 μS/cm2 (n = 12) versus
67.4 ± 43.0 μS/cm2 (n = 7).
To confirm that NDPK-B effect on CFTR was not confined to our chosen airway cell line,
we analysed the impact of peptide NDPK-B 36–54 or NDPK-A 36–54 on chloride conductance
using short-circuit current measurements (SCC) in gut biopsies [
] (n = 3). ACh induced a
transient increase in SCC in the control measurements (ΔSCC, +32.7 ± 3.71 μA/cm2) (Fig 8c).
However, in the presence of peptide NDPK-B 36–54 (100 μM), ACh failed to induce an
increase in SCC (ΔSCC, -1.7 ± 1.64 μA/cm2) (Fig 8c). On the other hand, peptide NDPK-A
36–54 (100 μM) was without effect (ΔSCC, +27.6 ± 2.93 μA/cm2). The viability of the tissue
pre/post-treatment was confirmed using sodium linked glucose (10 mM) absorption (ΔSCC
+16.1 ± 1.6 μA/cm2 and +21 ± 1.9 μA/cm2, respectively). Thus, peptide NDPK-B 36–54
(100 μM) inhibited FSK-dependent CFTR and ORCC-mediated currents in 16HBE14o- cells
as well as ACh-induced SCC measured across a sheet of intestinal biopsy.
This study demonstrates that NDPK-B forms a cAMP/PKA-dependent complex with CFTR at
the apical membrane and is important for regulation of CFTR and ORCC channels by cAMP/
PKA. We propose that the cAMP/PKA-induced NDPK-B interaction with CFTR occurs
through a distinct domain within NDPK-B straddling serine 44. Second messenger cAMP
regulates many signalling events that control numerous processes in airway epithelia including
CFTR-dependent chloride transport [
]. In non-CF epithelia, phosphorylation of CFTR by
] is the major recognized intracellular signalling mechanism for activation of
CFTRdependent chloride flux. The inhibition of the ACh-dependent SCC by the peptide NDPK-B
36–54 in human gut biopsies provides evidence for a wider relevance of NDPK-B to epithelial
function and suggests that the NDPK-B/CFTR complex is likely to be relevant towards ion
transport in vivo across a range of epithelia given the universal expression of these isoforms of
NDPK. Association of NDPK-B with the CFTR nucleotide binding domain (NBDs) is
interesting because NDPKs play an important role in local provision of nucleotides to many cellular
processes and suggests NDPK-B could be important for NBD function. The two NBDs
dimerise to drive ATP hydrolysis and thereby regulate the opening and closing of the CFTR
Clchannel. Activation of CFTR function requires a combination of phosphorylation, ATP
binding and hydrolysis and one study suggests that cAMP binding may also occur [
]. A key
unexplained aspect of CFTR function is the discrepancy between the slow rates of ATP hydrolysis
observed with purified CFTR and the fast gating kinetics of channel opening [
speculate that, as for many aspects of NDPK function (G proteins, ion channel and dynamin
regulation), the fast turnover of NDPK (1000 per sec) could supply the missing link between the
biochemistry and the electrophysiology.
19 / 25
The precise mechanism by which PKA regulates the NDPK-B/CFTR complex is currently
unclear but likely involves protein phosphorylation, since catalytic inhibitors of PKA disrupt
both translocation and co-immunoprecipitation of NDPK-B with CFTR. CFTR is a
welldefined PKA substrate and possesses several physiologically relevant PKA target sites within its
regulatory domain [
]. Previous studies have demonstrated that apart from the
autophosphorylation of NDPK on histidine 118, phosphorylation also occurs on serine residues
(Ser44 and Ser-120/122, near to the catalytic H118) [
]. However, the significance of NDPK
Ser-44 phosphorylation remains uncertain. Ser-44 exists on the hexamer surface close to
negatively charged residues 45 and 46. Since the initial recognition of NDPK phosphorylation on
Ser-44, the number of NDPK isoforms known has increased and interestingly, sequence
analysis predicts a putative PKA target site at Ser-44 which is unique to NDPK-B and the testis
specific isoform , NM23-H5. Paradoxically, previous studies in vitro show that NDPK serine
phosphorylation is inhibited by cyclic AMP (with a Ki of 1 mM) [
]. At first glance the
physiological significance is unclear since the Ki was very high in the (mM) range but given
that NDPK is involved in energy charge regulation, it remains possible that a very high local
concentration of cyclic AMP in the vicinity of CFTR could remain relevant given that we find
(non-hydrolysable) cAMP can act as a bridge to enhance the binding of CFTR to NDPK in
vitro. Indeed it has been proposed that cAMP efflux pumps can co-localise with CFTR [
making such a scenario plausible should such efflux be inhibited.
Disruption of the NDPK-B/CFTR complex by peptide NDPK-B 36–54 demonstrates that
the region of diversity between NDPK-A & B corresponding to amino acids 36–69
encompassing the β-sheet of β2 and the α-helix αA, and which possesses an unusual leucine cluster and an
ideal structural surface location [
], is important for the cAMP/PKA-dependent interaction
with CFTR. In functional studies, the peptide NDPK-B 36-54-induced inhibition of CFTR
chloride conductance demonstrates that activation of CFTR-mediated currents by PKA is
dependent on NDPK-B forming a complex with CFTR. Given that NDPK-B interaction with
CFTR is cAMP/PKA-dependent, our application of peptide NDPK 36–54 to CFTR functional
analysis provides a novel tool to distinguish between effect of PKA-mediated CFTR
phosphorylation and the impact of NDPK-B/CFTR complex formation on CFTR function. In contrast,
peptide NDPK-A 36–54 neither disrupts the NDPK-B/CFTR complex nor inhibits cAMP/
PKA-dependent CFTR function providing both an ideal control and excluding non-specific
effects of a high peptide concentration. These data also suggest that NDPK-A may interact
with CFTR through a different mechanism.
Inhibition of CFTR function by peptide NDPK-B 36–54, despite PKA activation, also
highlights the fact that, in addition to CFTR phosphorylation, other cellular processes are required
for CFTR activation. However, this is in contradiction to O’Riordan et al, who found that PKA
alone regulates highly purified CFTR reconstituted into planer lipid bi-layers [
]. Our data
suggest that although PKA undoubtedly activates CFTR directly in planer lipid bilayers,
regulation of function in vivo is a more complex process. For example, increasing evidence shows
that a number of proteins, including syntaxin 1A/syntaxin 8/AMPKα, exist in complex with
CFTR under basal conditions and inhibit CFTR function. The impact of PKA on CFTR
function in lipid bilayers, in the presence of these naturally occurring inhibitory complexes is yet to
It has been repeatedly observed that CFTR interacts with and regulates a number of other
ion channels, including ORCC [
], ENaC [
] and ROMK [
]. Several possible mechanisms
have been proposed for these interactions, including changes in intracellular Cl-
concentrations, protein-protein interactions and a role of scaffolding proteins such as NHERF [
We measured whole cell cAMP-activated DIDS-sensitive (ORCC) and CFTR Cl- currents
(using DIDS and CFTRihn172). Incubation of the cells with peptide NDPK-B 36–54, decreased
20 / 25
both CFTR and ORCC current magnitude indicating that both ORCC and CFTR are regulated
by the interaction with NDPK-B. As ORCC are also regulated by CFTR, the interaction with
NDPK-B may control CFTR-mediated regulation of other ion channels. Alternatively,
NDPK-B may target ORCC independently of CFTR.
It is as yet unclear whether NDPK-B also binds to other CFTR domains (particularly
extracellular domains, since several reports show NDPK-B as well as NDPK-A are released
]). The interaction described herein between NDPK-B and NBD1 and its disruption
by NDPK-B 36–54 peptide strongly suggests that NDPK-B, following its
cAMP/PKA-dependent translocation to the plasma membrane, binds to CFTR via NBD1 domain. The
mechanism of cellular internalization of the NDPK-B 36–54 peptide is at present unclear. However,
recent reports show several types of peptides penetrate cells and traverse the plasma membrane
by various mechanisms including endocytosis and energy independent pathways [
]. In lower
organisms such as flies, worms, sponges and amoebae, it is now clear that NDPKs control the
balance between the different forms of cell nutrient uptake [
] (for example macro- versus
micro-pinocytosis in Amoebae) which has recently been linked to cell growth [
external peptide uptake might occur by interacting with the machinery governing nutrient uptake
In conclusion, our data identifies NDPK-B as a new modulator of the
cAMP/PKA-dependent ORCC and CFTR function in epithelia. Further understanding of NDPK-B regulation
should uncover novel pathways required for epithelial cell secretion and function. Given the
role of NDPK in accelerating nucleotide turnover (thus substantially increasing the activity of
proteins such as dynamin), NDPK could enhance the turnover of CFTR-dependent nucleotide
hydrolysis and further work will determine whether the discrepancy between fast gating and
slow ATP-ase activity of CFTR can be explained through NDPK-B’s augmentation and
interaction with CFTR. Recently, Amaral and Balch have set out a route map to understand the
pleiotropy in CF disease pathogenesis as a model of defective proteostasis [
]. Their review does not
focus on mechanism whereby a barely imperceptible 1 in 1480 amino acid deletion in CFTR
(the one) can lead to so many changes across networks. Our data on NDPK, AMPK and CFTR
could provide a new hypothesis to explain some of CF pleiotropy.
S1 File. Images of whole western blots from which cut-outs in Figs 3–5 were made.
We would like to thank Professor D Gruenert (San Francisco, USA) for the 16HBE14o- cells,
Ms Jean McGraw for her input regarding SCC measurements in gut epithelia, Professor Philip
Thomas and the Cystic Fibrosis Folding Consortium for the generous gift of NBD1 proteins,
Dr Mario Pagano for his input regarding NBD1 overlay experiments and Professor Claude
Férec for his valuable input in the project and for his on-going support for the first author.
Conceived and designed the experiments: RM AM CJT LR. Performed the experiments: LAB
MK GC DC IL EHP LR PT. Analyzed the data: LAB MK PT GC CJT IL DC AM RM.
Contributed reagents/materials/analysis tools: IL EHP. Wrote the paper: MK GC IL LR AM RM.
21 / 25
22 / 25
23 / 25
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