 -sensitive Protein Phosphorylation
in Apical Membranes from Ovine Airway Epithelium
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Abstract |
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
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We have previously shown that nucleotide species (adenosine triphosphate [ATP] or guanosine triphosphate [GTP]), [Cl
], and anion species determine the steady-state phosphorylation of apical membrane
proteins within human airway epithelium in vitro. We found that a Cl-regulated 37-kD protein (p37)
principally phosphorylated with GTP but not ATP as substrate. Here we show that apical membranes from
sheep tracheal epithelium also contain a Cl-regulated 37-kD phosphoprotein (p37s) and characterize one
of the kinases involved in the regulation of p37s. Analysis of phosphorylation of apical membrane proteins
with 
[32P]GTP in the presence of MgCl2 showed that two proteins circa 19 and 21 kD (p19s and p21s)
were transiently phosphorylated before p37s. Renaturation of apical membrane proteins within polyacrylamide gels showed that p19s and p21s autophosphorylated with either [32P]GTP or [32P]ATP as substrates, suggesting that the two proteins were kinases. Immunoblotting and immunoprecipitation with a
specific polyclonal antibody showed that p21s was a membrane-bound isoform of nucleoside diphosphate
kinase (NDPK, EC 2.7.4.6), a protein kinase which catalyzes transfer of terminal phosphate from ATP to
diphosphate nucleotides and is, among other functions, essential for cell secretion. Incubation of apical
membrane proteins in the presence of [32P]ATP and guanosine diphosphate (GDP) (but not GDP
S) resulted in enhancement of phosphorylation of p37s. Dephosphorylation of NDPK was stimulated by the addition of Mg2+, Mn2+, and Co2+ (but not Zn2+ or Ca2+). Our data show that ovine trachea is a good model
for further characterization of the chloride-dependent cascade in airway epithelium.
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Introduction |
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
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The intracellular chloride concentration ([Cl]i) is dependent on the balance between Cl influx and exit across the
plasma membrane. Cl crosses membranes via transporters and channels and in epithelia, [Cl]i itself regulates
both Na+ and Cl transport (1). However, the mechanisms
which sense [Cl]i and thereby regulate Na+ and Cl
fluxes are obscure. [Cl]i regulates such a diversity of cellular functions
including the conductance of Na+ channels
(1) and the CFTR Cl channel (2), cell volume homeostasis (3), and G protein activity (4)that there must be a
specific mechanism for each function and/or an unidentified common regulatory pathway. We have shown that
[Cl] and anion species are independent determinants of
the phosphorylation state of apical membrane proteins from
human airway epithelium in vitro (5). This work challenged the assumption that the apical membrane is an inert structure with respect to [Cl] because we found that
Cl regulated the steady-state level of phosphorylation of
a 37-kD membrane protein (p37h: the suffix denotes species; h = human) via a membrane-associated protein kinase(s) (non-PKA or -PKC) which utilized guanosine triphosphate (GTP) as a phosphate donor. In addition, we
observed that the nucleotide species altered the steady-state phosphorylation of apical membrane proteins, probably by activating distinct membrane-bound kinases. For
example, when GTP was replaced with adenosine triphosphate (ATP), a different anion-dependence of the profile
of phosphorylated membrane proteins was generated.
The present study had two aims: first, to show that an
apical fraction from sheep tracheal epithelium also manifested Cl-dependent phosphorylation equivalent to the
human airway (5); and second, to use the increased quantity of tissue available from the sheep to characterize the
nonsteady-state phosphorylation events prior to p37 phosphorylation. Our results show that sheep tracheal apical
membrane contains p37s (the suffix denotes species: s = sheep), a protein of equivalent molecular weight to the human Cl-dependent phosphoprotein (p37h) and that p37s
phosphorylation is enhanced by guanosine diphosphate
(GDP) and preceded by the transient phosphorylation of
nucleoside diphosphate kinase (NDPK). Our data confirm
that ovine tracheal epithelium provides a useful source of
material to characterize the molecular components of the
Cl-sensitive phosphorylation system.
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Materials and Methods |
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Preparation of Apical Membrane from Human and Ovine Airway
Human. The subjects studied were all healthy young adults undergoing surgery for reasons unrelated to nasal mucosal disease. Informed written consent and Ethical Committee approval were obtained. The method is detailed elsewhere (5). Briefly, immediately after anesthesia, respiratory epithelial cells were brushed from the inferior nasal turbinate epithelium and dislodged into a nutrient medium (Medium 199; ICN, Thame, UK). After washing in medium 199 (1 g), cells were disrupted by sonication and a postnuclear supernatant was centrifuged through discontinuous sucrose gradients at 150,000 × g for 1 h at 2°C in a swing-out rotor (Kontron TST55.5; Kontron Instruments, Watford, UK). Sheep. Tracheas from sheep freshly slaughtered at a local abattoir were transported in ice-cold isotonic saline, trimmed, cut open, and washed with saline solution as previously described (6). Briefly, following incubation at 37°C for 30 min in saline solution containing glucose (5 mM) and dithiothreitol (5 mM), the epithelium was scraped with a glass slide and placed in ice-cold homogenization buffer (250 mM sucrose, 10 mM triethanolamine, pH 7.6) containing the following inhibitors: 1 µg/ml deoxyribonuclease I, 5 µg/ml leupeptin, 13 µg/ml aprotonin, 5 µg/ml pepstatin A, 800 µg/ml benzamidine, and 174 µg/ml PMSF. Apical membrane proteins were prepared from homogenates of these epithelial scrapings using one of two methods: 1. Mg2+ precipitation: Apical membranes were prepared using a procedure previously described for purifying apical membrane vesicles from guinea-pig lung (7, based on the method of Kemp and colleagues [8]). The pellet resulting from the final spin (i.e., the membranes not precipitated by Mg2+) was resuspended in homogenization buffer. 2. Sucrose density centrifugation: In order to study the role of magnesium or chloride ions on membrane phosphorylation, sheep apical membranes were additionally prepared using a magnesium/chloride-free sucrose gradient technique as previously described (5). Briefly, epithelial scrapings were homogenized using a Polytron homogenizer (Silverson, Chesam, UK) for 5 min at 4°C. The homogenate was spun at 260 × g for 20 min. The pellet was discarded and the supernatant spun at 15,000 × g for 30 min. The new pellet was resuspended in homogenization buffer and loaded (3 ml) onto a discontinuous sucrose gradient (2 ml each of 20%, 30%, 40%, 50%, and 60% [wt/vol] sucrose) in precooled 15-ml polyallomer tubes (Kontron) and spun at 150,000 × g (Kontron TFT41.14 swing-out rotor) at 2°C for 60 min. The gradient was unloaded from the top, preserving the interfaces: 20-30%, 30-40%, 40-50%, and 50-60%. Each interface was then diluted with ice-cold homogenization buffer and spun at 100,000 × g for 20 min at 4°C. For each method, subcellular fractions were assayed using alkaline phosphatase, an apical membrane protein marker. Cytosolic contamination was measured using lactate dehydrogenase (LDH) activity as a marker (Sigma kit; Sigma, Poole, UK). Procedures used to determine protein concentration and purity of apical membrane preparations are described elsewhere (5, 7, 8). Aliquots of each membrane pellet were stored in liquid nitrogen until required.Phosphorylation and Dephosphorylation of Apical Membrane Proteins
Phosphorylation of apical membrane proteins by endogenous kinase. Aliquots (15 µg) of apically enriched membranes in 10 mM MOPS, pH 7.9, containing 5 mM DTT, 2% DMSO, and 0.05% Triton X-100 (total incubation volume, 25 µl) were incubated with 37 kBq[32P]GTP (final
concentration of GTP, 16 nM) at either 4 or 30°C as previously described (5). In all experiments, 1 µl of labeled nucleotide was spotted onto the side of the tube and the reaction started by a rapid spin to mix reagents. Unless
otherwise stated, phosphorylation was terminated by adding 5× Laemmli (9) sample buffer (5 µl) followed by rapid
mixing.
Autophosphorylation of the kinase "in-gel."
Unlabeled
apical membranes, prepared by the sucrose gradient
method, were solubilized in 50 mM MOPS, pH 7.9, containing 5 mM DTT, 2% DMSO, 0.05% Triton X-100, and
5× Laemmli sample buffer. Using a method derived from
Hutchcroft and associates (10) the sample mixture was
heated at 70°C for 5 min and proteins separated by electrophoresis (12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis [SDS-PAGE], 0.25 mg protein/lane).
Following electrophoresis, all incubations were performed
at room temperature on a shaker. Gels were washed 6 times in 250 ml of 50 mM MOPS, pH 7.9, containing 20%
isopropanol, over a period of 6 h and then incubated with
50 ml of 25 mM MOPS, pH 7.9, 10 mM MnCl2, and 0.05% Triton X-100 containing 9.25 MBq [32P]ATP or [32P]-
GTP (specific activity 6,000 Ci/mmol) for 3 h. Gels were
rinsed briefly in water and incubated for a further 4 h in two
changes of 400 ml of 50 mM MOPS, pH 7.9, and 10 g activated charcoal in dialysis tubing to remove residual [32P]-
ATP or GTP. The gel was then dried and labeled proteins
were detected by electronic autoradiography.
Dephosphorylation of endogenous proteins.
A three-stage protocol was devised to characterize endogenous
phosphatase activity in isolation from kinase activity and
vice versa. The first two incubation stages were at 4°C in
order to minimize endogenous protein phosphatase activity
(11). First, proteins were phosphorylated with [32P]GTP
for 1 min using endogenous kinase(s) and then the kinase activity was terminated with 5' guanylylimidodiphosphate
(Gpp[NH]p, 0.25 mM). This nonhydrolyzable analogue of
GTP had been found to inhibit phosphorylation in sheep
tracheal membrane preparations (see RESULTS). Phosphorylated membrane proteins were then incubated at 30°C
for up to 60 min under various conditions to study dephosphorylation in isolation.
Quantitation of phosphorylation.
Proteins were separated by SDS-PAGE on a Protean II slab cell apparatus
(BioRad, Hemel Hempstead, UK). The gels were dried at
80°C and, for qualitative analysis, autoradiographed against pre-flashed Hyperfilm MP (Amersham, Slough, UK) at
70°C. Incorporation of 32PO42 into individual proteins
was measured by electronic autoradiography (Canberra-Packard Instant Imager, Pangbourne, UK) as previously described (5). In order to compensate for variation in intensity of phosphorylation between experiments, data were
expressed as percentage maximal phosphorylation of the
protein of interest for each individual experiment ("% of
maximum"). Background phosphorylation, defined as an
area of the lane within the gel adjacent to the protein of interest but containing no phosphorylated proteins, was subtracted from each band.
Detection of Nucleoside Diphosphate Kinase
Immunoprecipitation. Apical membrane protein (200- 500 µg) was phosphorylated with 37 kBq[32P]GTP (16 nM) in 10 mM MOPS, pH 7.9, for 2 min at 4°C in a volume of 100 µl. The reaction was terminated with 50 mM EDTA
followed by the addition of 9 volumes of immunoprecipitation buffer (10 mM Tris-HCl, pH 7.4, 25 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 precleared with preimmune serum (1 µg) and protein G-Sepharose beads (1 h at 4°C) and centrifuged at 4°C at 200 × g
for 5 min, and the supernatant was incubated with the appropriate antiserum (1 µg; see next section) for 1 h 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 5 times
(10 min) in 1 ml RIPA buffer (50 mM Tris-HCl, pH 7.4, 1%
NP-40, 0.5% sodium deoxycholate, 5 mM EDTA, 150 mM
NaCl). The washed beads were resuspended in Laemmli
sample buffer (50 µl) containing 100 mM DTT and left at
room temperature for 30 min. Following a second spin at
200 × g for 5 min, an aliquot (20 µl) of the supernatant
was subjected to SDS-PAGE using 12.5% gels.
Western blots.
Membrane proteins (250-500 µg), separated by SDS-PAGE, were transferred to PVDF membrane (Millipore, Watford, UK) by semi-dry electrophoretic
transfer (Pharmacia) using 0.8 mA/cm2 for 1 h with 20%
methanol added to standard SDS-PAGE running buffer.
Prestained markers were used to confirm transfer. The primary antibody (1: 2,000) was an affinity-purified rabbit
polyclonal directed against the nm23-H1 peptide epitope of
NDPK (Santa Cruz Biotechnology, Heidelberg, Germany). Horseradish peroxidase-conjugated antirabbit secondary antibody (Sigma) and ECL detection (Amersham) were used to locate NDPK.
Chemical Reagents
All chemicals were of analytical grade and purchased from Sigma, BDH (Poole, UK), or Aldrich (Poole, UK) except for the following: the 32P nucleotides from NEN Du Pont (Stevenage, UK); acrylamide and other electrophoretic materials from BioRad; and antibodies to nm23-H1 (NDPK) from Santa Cruz Biotechnology. Okadaic acid was a gift from P. Cohen (Biochemistry, Dundee, Scotland, UK).
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Results |
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Apical Membrane Purity
Sucrose gradient and Mg2+ precipitation techniques generated apically enriched membrane fractions with approximately 20- and 15-fold enrichments, respectively, in alkaline phosphatase activity relative to homogenate. Assays for marker enzymes (5, 7, 8), showed that the fractions enriched in alkaline phosphatase activity did not contain LDH, a marker for cytosol (specific activity for LDH in apical membrane fractions was 1.6 ± 0.9 µmol/min/mg compared with 235 ± 15 and 54 ± 2 µmol/min/mg in the supernatant and homogenate, respectively; ± SEM, n = 4). We have previously shown that the sucrose gradient technique (5) generates an apical membrane fraction not enriched with markers for endoplasmic reticulum (NADPH cytochrome C reductase) or mitochondrial membranes (succinate dehydrogenase).
Sheep Airway Epithelium Phosphorylation as a Model for Human Airway
Sheep apical membranes prepared using Cl-free sucrose
gradient technique were incubated with [32P]GTP at 37°C
for 5 min. Figure 1A shows that this preparation contained
a 37-kD protein (p37s) which was sensitive to [Cl] with
[32P]GTP as phosphate donor. No phosphoproteins were
observed when membranes were incubated with 
[32P]GTP
(data not shown), thus excluding high-affinity GTP binding as an explanation for the phosphorylated bands. Incubation with increasing concentrations of the chloride salt of
N-methyl-D-glucamine (NMDG) as previously described (5)
showed that phosphorylation of p37s was maximal at 5 mM
NMDGCl (Figure 1A, left panel). In contrast, our earlier
data (human nasal airway epithelium) showed a peak of
phosphorylation for p37h at 40 mM NMDGCl (Figure 1B, left panel). Interestingly, for both ovine and human membranes MgCl2 (3 mM) induced a shift in the peak of phosphorylation of p37s and p37h to higher [Cl] (Figures 1A
and 1B, right panels). In the human studies (Figure 1B),
two additional proteins of low molecular weight (circa 19 and 21 kD) were not phosphorylated at low Cl but became visible as NMDGCl concentration increased above
50 mM. In sheep membranes, similar enhancement of
phosphorylation of a 19 and 21 kD doublet (p19s and p21s,
respectively) was observed at higher concentrations of
NMDGCl. The presence in sheep trachea epithelium of a
37-kD protein (p37s) whose phosphorylation state was sensitive to [Cl] suggested that sheep tracheas could serve as
a useful model to study the [Cl]-sensitive phosphorylation
system previously observed in human nasal epithelia (5).
We prepared apical membranes using the magnesium precipitation technique and showed the presence of the same
proteins in this preparation (Figure 2, inset). The ease of
use of this technique relative to the sucrose method allowed us to prepare sufficient membrane to characterize
the phosphoproteins.
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Time Course of Phosphorylation of Apical Membrane Proteins from Sheep Airway
Prior to the appearance of p37s (circa 5 min), p19s and p21s were rapidly phosphorylated (by 10 s) and then dephosphorylated after 2 min (Figure 2). The label incorporated into p21s was always an order of magnitude less than that in p19s at equivalent time points and there appeared to be a temporal association between the dephosphorylation of p19s/p21s and the phosphorylation of p37s. The graph in Figure 2 shows that phosphate incorporated into p37s was always less than that lost from p19s (100% on the ordinate refers to the phosphate incorporated into p19s at [quasi] time zero for each experiment). This result suggests that either the phosphate on p19s/p21s was transferred to p37s or that p19s/p21s were dephosphorylated by a phosphatase, the latter event resulting in phosphorylation of p37s. However, it is equally possible that both processes were occurring simultaneously.
In order to understand the role of dephosphorylation in
the putative relationship between p19s/p21s and p37s, we
first developed a method to control the endogenous kinase
activity. Our earlier study had shown that this GTP kinase
could not be inhibited by classic protein kinase inhibitors
(5). This problem was circumvented by using nonhydrolyzable analogues of GTP; analogues containing substituted
NH or CH2 between the - phosphates. Dose-response studies showed that maximal inhibition of phosphorylation of p19s/p21s by Gpp[NH]p and --methyleneguanosine triphosphate (GppCp) occurred at 0.25 mM (Figure 3D). In addition, Gpp[NH]p (0.25 mM) abolished phosphorylation of p37s whereas an identical concentration of GppCp only attenuated its phosphorylation (Figures 3B and 3C). This result suggests that the kinase(s) involved were able to make the subtle distinction between
the presence of carbon or nitrogen on the - phosphates
of GTP. It was interesting to note that inhibition of p19s/
p21s phosphorylation by GppCp also advanced the onset
of phosphorylation of p37s (Figures 3A and 3C).
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We suspected a role for Mg2+ in the phosphorylation
state of p19s/p21s for the following reasons: in the presence of MgCl2, phosphorylation of p19s/p21s was rapid
and transient (Figure 2); in the presence of NMDGCl (no
Mg2+ added) p19s/p21s was phosphorylated only at high
[Cl] (Figure 1A, left panel) but addition of MgCl2 (3 mM)
reduced the amount of phosphate incorporated into the
doublet (Figure 1, right panels). We decided to investigate
the notion that a magnesium-dependent phosphatase was
involved in the process.
The Role of Magnesium
Study of the phosphorylation state of p19s and p21s by
pre-incubating membranes prepared by the Mg2+ precipitation technique with 100 mM EGTA, 1 mM orthovanadate, 10 µM okadaic acid, 50 µM calyculin A, or 100 µM
microcystin LR (separately or in combination) showed
that dephosphorylation of p19s/p21s could not be inhibited (data not shown). This result indicated that dephosphorylation of p19s/p20s was not due to the activity of protein phosphatases PP1, PP2A or PP2B. Figure 4 shows that another class of phosphatase was likely to be present
because a reduction of the reaction temperature to 4°C resulted in attenuation of dephosphorylation of p19s/p21s
low temperature disproportionately reduces the reaction
rate of phosphatases compared to kinases (11). When EDTA
(10 mM) was included in the third step of our dephosphorylation protocol, the rate of dephosphorylation of p19s and p21s was greatly reduced (data not shown).
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The effect of divalent ions on the dephosphorylation of p19s/p21s was studied using sheep apical membranes prepared by the sucrose gradient technique together with our dephosphorylation protocol (see MATERIALS AND METHODS). As predicted, a decreased rate of dephosphorylation of p19s/p21s (Figure 5A) was observed and the addition of Mg2+ restored the rapid dephosphorylation previously observed with membranes prepared using Mg2+ precipitation technique. Furthermore, Mg2+-induced dephosphorylation affected p21s more than p19s, an observation consistent with that found in membranes prepared by Mg2+ precipitation (Figure 2, inset). The threshold for the effect of Mg2+ on the dephosphorylation of p19s (at 10 min) was around 200 µM (Figure 5B) and dephosphorylation was abolished by the addition of EDTA (10 mM) (data not shown). Figure 6 shows that Mn2+ and Co2+ but not Zn2+ or Ca2+ (20 mM) increased the rate of dephosphorylation of p19s/p21s.
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Identification of p21s as Nucleoside Diphosphate Kinase
The rapid phosphorylation of p19s/p21s, occurring when
membranes were phosphorylated on ice (Figure 4), and
the absence of any other phosphoproteins suggested that
these two proteins were themselves kinases which underwent autophosphorylation. In order to test this hypothesis,
unlabeled membrane proteins were separated by SDS-PAGE, renatured "in gel" by removing the SDS, and then exposed to [32P]GTP or [32P]ATP. In-gel phosphorylation of p19s and p21s was observed (Figure 7), indicating
that the two proteins autophosphorylated with GTP and
ATP and were likely to be kinases. A literature search for kinases capable of utilizing both ATP and GTP and having
a molecular weight in the region of 20 kD indicated NDPK
as the most likely candidate for two principal reasons:
NDPK, when phosphorylated, exists as two isoforms of 19 and 21 kD (12) and, unlike other kinases such as PKA and
PKC, NDPK initiates nucleotide binding predominantly
via the phosphate moiety and can therefore utilize both
ATP and GTP (12). The presence of a membrane-bound
isoform of this protein in sheep apical membrane preparations devoid of cytosolic contamination was confirmed by
probing Western blots with an affinity-purified antibody
raised to a peptide epitope of nm23-H1 (Figure 8). The
molecular identity of p21s as NDPK was strengthened by
immunoprecipitation of a 21-kD phosphoprotein from sheep
apical membranes previously phosphorylated with [32P]-
GTP (Figure 8). The nm23-H1 antibody (but not an unrelated antibody raised against G protein -subunits) selectively immunoprecipitated a single 21-kD phosphoprotein.
Furthermore, Figure 8 also shows that no phosphorylated
protein was immunoprecipitated when the nm23-H1 antibody was pre-absorbed with the peptide epitope against which it was raised. These data strongly suggested that the
NDPK was the early phosphorylated species in apical
membranes and that its autophosphorylation was occurring followed by rapid dephosphorylation.
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Effect of GDP on the Phosphorylation of p37s
We have previously shown that phosphorylation of p37h
from human nasal epithelia was enhanced in the presence
of GTP as phosphate donor (5). Since NDPK catalyzes transfer of phosphate predominantly from ATP to diphosphate
nucleotides (13), we postulated that incubation of apical
membrane preparation in the presence of [32P]ATP and
GDP would result in increased phosphorylation of p37s by
promoting production of [32P]GTP. Apical membrane proteins, prepared by the sucrose gradient technique, were incubated at 37°C with [32P]ATP ± diphosphate nucleotide
(500 nM). Figure 9 shows a time course of phosphorylation
in the absence or presence of GDP or its hydrolysis-resistant analogue GDPS as control. Maximal phosphorylation of p37s occurred in the presence of GDP and was approximately 20-fold greater than buffer control (data not shown).
Interestingly, incubation with GDPS did not significantly
increase phosphorylation of p37s above buffer control.
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Discussion |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Ovine Model
This study describes the suitability of sheep tracheal epithelium for further study of the Cl-dependent phosphorylation cascade previously identified in the apical membrane of human airway epithelium. We demonstrate the presence of an ovine 37-kD protein, p37s, whose steady-state phosphorylation is dependent on Cl concentration.
We also identify an apical membrane-bound isoform of
NDPK and show that, in the presence of MgCl2, phosphorylation and dephosphorylation of this protein occur prior
to those of the Cl-dependent protein, p37s. Our data also
reveal that in the presence of labeled nucleotides, the
phosphorylation state of the apical membrane proteins is
dependent on the method used in their preparation; membrane proteins prepared using Mg2+ precipitation or sucrose gradient ultracentrifugation techniques showed very
distinct patterns of phosphate incorporation. The divalent
ion precipitation technique has been used for many years to isolate apical membrane from a variety of epithelia
(cited in refs. 6-8) but our data show that these ions alter
the phosphorylation state of apical membrane proteins.
NDPK
In eukaryotes, NDPK exists as a tetramer or hexamer (14- 16) and in denaturing gels as two different isoforms with molecular weights of 19 and 21 kD, similar to those found in our study. We have used commercially available antibodies to an epitope of nm23-H1 to identify p21s as NDPK. NDPK is known to generate cellular GTP from ATP and GDP by transphosphorylation (17). However, the membrane-delimited pool of GTP is different from its cytosolic equivalent because the former is channeled to G proteins in a hormone-sensitive manner (18). Kimura and Shimada (18) also showed that NDPK coimmunoprecipitates with at least one G protein. Furthermore, the location of NDPK within the secretory pathway from the Golgi to the apical membrane (19) provides a compelling function for its role as a GTP generator, GTP being an essential cofactor for secretion. NDPK has also been shown to differentially bind to some isoforms of calmodulin and may therefore affect calcium-regulated secretion (20). NDPK also regulates K+ channels which maintain a K+ leak pathway to balance ionic charge during cellular accumulation of chloride (21). Recently the role of NDPK as a kinase in its own right has been elucidated. It has been shown to phosphorylate ATP-citrate Lyase, a key regulator of energy metabolism (22).
Although in mammals membrane-bound isoforms of
NDPK have previously been described in rat liver (23) and
inside-out patches of atrial cells (21), our data localize
NDPK to a 15-fold enriched apical membrane fraction
from airway epithelium. Apical membranes play a key role
in vectorial ion transport (24), volume regulation (25), and
fluid secretion (26), processes which are known to require
protein kinases, Cl, and GTP. Could NDPK integrate
these activities in airway epithelium? At present we can
only speculate that, consistent with its known functions,
NDPK regulates the apical secretory path of airway epithelia by channeling GTP to the membrane. In addition,
several important biologic functions such as cell growth
(12), development/differentiation (27, 28), and tumor metastasis suppression (17, 29) have been attributed to NDPK
but the underlying biochemical mechanisms remain to be
defined. Understanding dual biochemical activities ascribed to this protein, i.e., GTP synthetase activity and a protein kinase activity, form the focus of our current work.
Phosphorylated NDPK
Autophosphorylation of NDPK from human and Myxococcus has been reported to occur on histidine and serine residues (12, 29). Phosphorylation on histidine forms a highly energized intermediate of NDPK responsible for phosphate transfer between nucleotides. On the other hand, the function and fate of the low-energy phosphate on serine is presently unclear. It is probable that the dual ability of NDPK to act as a kinase and GTP-synthetase are regulated by differential phosphorylation (and/or dephosphorylation). Differential targeting of NDPK isoforms by nucleotides was observed using "in-gel" assays where p19s preferentially autophosphorylated with GTP and the 21-kD isoform was preferentially autophosphorylated by ATP. Furthermore, dephosphorylation of NDPK that had been observed in reactions carried out prior to SDS-PAGE was not observed in the "in-gel" assays despite long periods of incubation in the presence of divalent metal ion (10 mM). This could have been due to the absence of acceptor nucleotide or separation of NDPK from a protein phosphatase.
While the loss of phosphate from histidine can be explained by its transfer to diphosphate nucleotides, loss of label from serine may be due to phosphatase activity. We have shown that rapid dephosphorylation of apical membrane-bound NDPK occurred in the presence of divalent cations (Mg2+, Mn2+, and Co2+) and could not be inhibited by inhibitors of PP1, PP2A, and PP2B. This result suggests that NDPK may be tightly regulated by a Mg2+-dependent phosphatase also present on the apical membrane. The molecular identity of our phosphatase(s) is currently unknown but recently a Mg2+-dependent, 10-kD phosphatase isolated from bacteria has been shown to be highly active against phosphorylated NDPK from bacteria and other sources (30). Protein phosphatase 2C (PP2C), a Mg2+-dependent phosphatase present in all cells, could also target NDPK. Unfortunately, there is currently no specific inhibitor for PP2C. PP2C may regulate osmotic swelling (31), a process known to be controlled by the concentration of chloride in epithelial cells (3). PP2C also regulates PKA-dependent epithelial secretory processes in rat parotid (32). We are currently isolating the phosphatase(s) responsible for dephosphorylating NDPK in apical membrane.
Evidence for an Interaction Between p37s and NDPK
The phosphorylation of NDPK was both rapid and transient relative to p37s. Although we do not understand the
relationship between NDPK and p37s, we suspect a link exists between the dephosphorylation of NDPK (p19s/p21s)
and the subsequent phosphorylation of p37s for the following reasons: When NDPK phosphorylation is abolished by GppCp, p37s phosphorylation has an earlier onset; when
NDPK dephosphorylation is prevented by a reduction of
temperature, p37s is not phosphorylated; both the time
course of dephosphorylation and effect of chloride on NDPK
phosphorylation are reciprocally related to the phosphorylation of p37s; and phosphorylation of p37s from ATP is
enhanced by GDP but not GDPS, a hydrolysis-resistant analogue. The most likely explanation for the failure of
GDPS to enhance p37s phosphorylation is the inability
of this thio analogue to form GTP via transphosphorylation. However, we cannot exclude a structural explanation
based on the inability of GDPS to bind to the GDP site
on NDPK. Our working model of the relationship between
NDPK and p37s is that NDPK synthesizes GTP from GDP
and ATP and channels this GTP either to a second kinase
targeting p37s or directly to p37s for autophosphorylation.
The putative second kinase would be inhibited by Gpp[NH]p
but not GppCp. We cannot discriminate between these two
mechanisms until we have purified p37s to homogeneity.
In summary, we have found that sheep tracheal epithelium provides a good model for future studies of the Cl-sensitive phosphorylation cascade. We have also demonstrated the presence of apical membrane-bound NDPK in
sheep tracheal epithelium and provided evidence for an
association between NDPK and p37s. Studies are currently underway to isolate p37s and to further characterize
its interaction with NDPK.
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Footnotes |
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Address correspondence to: Richmond Muimo, Ph.D., Centre for Research into Human Development, Ninewells Hospital Medical School, Dundee DD1 9SY, Scotland, UK. E-mail: r.muimo{at}dundee.ac.uk
(Received in original form November 21, 1996 and in revised form June 30, 1997).
Acknowledgments: Author R.M. was supported by the Wellcome Trust, S.J.B. was supported by the European Social Fund, and L.J.M. is a Wellcome Prize Student. The authors thank the Anonymous Trust, Tenovus (Scotland) and the European Community (network grant: BMH4-CT96-0602) for their generous support to purchase essential reagents and equipment. They are grateful to M. Hohenegger for useful discussions on NDPK and to P. J. Kemp and D. Meek for criticisms of the manuscript. This paper is dedicated to the memory of the late professor L. B. Strang.
Abbreviations
ATP, adenosine triphosphate;
GDP, guanosine diphosphate;
GppCp, --methyleneguanosine triphosphate;
Gpp[NH]p, 5' guanylylimidodiphosphate;
GTP, guanosine triphosphate;
LDH, lactate dehydrogenase;
NDPK, nucleoside diphosphate kinase;
NMDG, N-methyl-D-glucamine;
C), protein kinase (A;
C), PK(A;
PP, protein phosphatase;
SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
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