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J. Biol. Chem., Vol. 277, Issue 40, 37542-37550, October 4, 2002
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From the Department of Cellular and Molecular Physiology, Yale
University, New Haven, Connecticut 06510 and the Mount Desert
Island Biological Laboratory, Salsbury Cove, Maine 04672
Received for publication, June 24, 2002, and in revised form, July 16, 2002
The secretory Na-K-Cl cotransporter NKCC1 is
activated by secretagogues through a
phosphorylation-dependent mechanism. We found a
phosphorylation stoichiometry of 3.0 ± 0.4 phosphorylated residues/NKCC1 protein harvested from shark rectal gland tubules maximally stimulated with forskolin and calyculin A, showing that at
least three sites on the cotransporter are phosphorylated upon stimulation. Three phosphoacceptor sites were identified in the N-terminal domain of the protein (at Thr184,
Thr189, and Thr202) using high pressure
liquid chromatography and matrix-assisted laser desorption ionization
time-of-flight mass spectrometry to analyze tryptic fragments of the
radiolabeled cotransporter. None of these residues occurs in the
context of strong consensus sites for known Ser/Thr kinases. The
threonines and the surrounding amino acids are highly conserved between
NKCC1 and NKCC2, and similarities are also present in the Na-Cl
cotransporter NCC (or TSC). This strongly suggests that the
phosphoregulatory mechanism is conserved among isoforms. Through
expression of shark NKCC1 mutants in HEK-293 cells,
Thr189 was found to be necessary for activation of the
protein, whereas phosphorylation at Thr184 and
Thr202 was modulatory, but not required. In conjunction
with the recent finding (Darmen, R. B., Flemmer, A., and
Forbush, B. (2001) J. Biol. Chem. 276, 34359-34362) that
protein phosphatase-1 binds to residues 107-112 in the shark NKCC1
sequence, these results demonstrate that the N terminus of NKCC1
constitutes a phosphoregulatory domain of the transporter.
The secretory Na-K-Cl cotransporter NKCC1 is a major pathway for
net influx of Cl In order that the processes of regulatory volume increase, secretion,
and absorption may be tightly controlled, the Na-K-Cl cotransporter is
subject to strict regulation. The cotransporter is inactive in the
basolateral membranes of secretory cells until the application of
secretagogues or cell shrinkage causes a
phosphorylation-dependent activation of the cotransporter
(7-10). Studies on shark rectal gland secretory epithelia have shown
an increase in phosphorylation at serines and threonines in response to
forskolin or hypertonic stress, and the N terminus of NKCC1 has been
shown to be the locus of at least some of the phosphoregulatory sites
(7, 11). Although, in many cells,
PKA1 agonists effect an
increase in Na-K-Cl cotransporter activity, the cotransporter does not
appear to be directly phosphorylated by PKA (12, 13). Current evidence
indicates that the principal regulatory pathway involves PKA-mediated
stimulation of apical Cl The salt-secreting rectal gland of the shark has proven to be an
excellent physiological and biochemical model system for the study of
fluid secretion. In this study, we exploit the rectal gland epithelium
to examine phosphorylation sites on NKCC1 and demonstrate that the N
terminus functions as a regulatory phosphorylation domain. Of the three
phosphoacceptors identified, Thr189 is shown to be critical
for activation, whereas Thr184 and Thr202 are
necessary for optimal sensitivity and full activation of transport.
Materials and Reagents--
Dogfish sharks were obtained by net
off the coast of Mt. Desert Island, ME, and rectal glands were removed
after killing the animals. Calyculin A was purchased from LC
Laboratories/Alexis, collagenase A from Roche Molecular Biochemicals,
[32P]H3PO4 and 86RbCl
from PerkinElmer Life Sciences, rabbit anti-mouse antibodies from
Jackson ImmunoResearch Laboratories, Inc., and other reagents from Sigma.
Preparation of Shark Rectal Gland Tubules--
Suspensions of
rectal gland tubules from dogfish (Squalus acanthias) rectal
glands were produced using the collagenase digestion method previously
described (14). Briefly, rectal gland slices were incubated under
gentle agitation at 15 °C with 1 mg/ml collagenase A in shark
Ringer's solution (240 mM NaCl, 4 mM KCl, 2.5 mM CaCl2, 1.25 mM
MgSO4, 20 mM HEPES, 70 mM
trimethylamine N-oxide, 350 mM urea, 1.0 mM Na2HPO4, and 5.6 mM
D-glucose, adjusted to pH 7.8 at room temperature and
bubbled with O2). Tubule suspensions were isolated at three
or four 20-min intervals by low speed centrifugation and held on ice.
Phosphorylation of NKCC in Rectal Gland Tubules--
Suspensions
of rectal gland tubules (3% cytocrit) were gently agitated at
15 °C with 1.5 µCi/ml 32PO4 for 40 min. As
shown in Fig. 1 (upper panel), the enzymatic incorporation
of 32PO4 into cellular ATP and ADP was linear
through 60 min. The fraction of label in the
After labeling, tubules were rinsed with shark Ringer's solution and
stimulated with 50 µM forskolin in a 10-min incubation to
activate Cl Phosphopeptide Analysis--
Gel slices were washed and
equilibrated in 50% CH3CN and 200 mM
NH4HCO3, incubated with 45 mM
dithiothreitol at 37 °C for 20 min and then with 100 mM
methyl-4-nitrobenzene sulfonate for an additional 40 min, washed twice
with CH3CN/NH4HCO3, crushed through
nylon mesh, dried, and then rehydrated in a solution of 0.5 µg of
modified trypsin (Promega)/15 µl of gel in 15 µl of 10 mM NH4HCO3. After 10 min, an
additional 20 µl of 10 mM NH4HCO3 was added, and samples were incubated at 37 °C for 24 h.
Peptides were eluted with 0.05% trifluoroacetic acid and 5%
acetonitrile and separated on a Vydac C18 reverse-phase
column (1 mm × 25 cm, 5-µm particle size, 300 pore size)
with a gradient mobile phase (buffer A, 0.06% trifluoroacetic acid;
and buffer B, 0.052% trifluoroacetic acid and 80% acetonitrile). Mass
spectra of fractions containing significant 32P were
obtained by MALDI mass spectrometry on a Micromass TofSpec SE mass
spectrometer operating in the positive linear ion mode at an
accelerating voltage of 25 kV; the instrument was equipped with a
nitrogen laser (337 nm), a reflectron, delayed extraction, and a
post-acceleration detector. Samples were analyzed before and after
treatment with calf intestinal alkaline phosphatase (New England
Biolabs Inc.). Aliquots of fractions were dried, resuspended in 4 µl
of 200 mM NH4HCO3 containing 10 units of alkaline phosphatase, and incubated for 2 h at 37 °C.
The reactions were dried and redissolved in 50% CH3CN and
0.05% trifluoroacetic acid prior to analysis. Amino-terminal
sequencing was carried out by Edman degradation using an Applied
Biosystems sequencer equipped with an on-line HPLC system.
Phosphorylation Stoichiometry--
The stoichiometry of
32P incorporation into NKCC1 was determined as the ratio of
cpm incorporated per mol of cotransporter protein to cpm incorporated
per mol of 32P in the Mutational Analysis of Phosphorylation Sites and Analysis of NKCC
Function--
Point mutants of phosphorylation sites on sNKCC1 were
prepared and expressed in HEK-293 cells as previously described
(5). Na-K-Cl cotransporter activity was assessed by measuring
86Rb influx into a confluent monolayer of HEK-293
cells grown on poly-D-lysine-coated microtiter plates
(Biocoat, BD Biosciences). Following 10-60-min preincubation in the
specified media and a 2-min 86Rb influx period (in regular
flux medium (see below)), the flux was terminated with a rinse with
high K+ buffer (135 mM potassium gluconate, 5 mM sodium gluconate, 1 mM
CaCl2/MgCl2, 1 mM
Na2HPO4/Na2SO4, and 15 mM NaHEPES, pH 7.4) and a second rinse with isotonic
MgCl2 (110 mM) and allowed to dry. The flux
experiments reported here were performed in an automated 96-well plate
flux machine that performs each solution change as a wash procedure
without removing all of the fluid from the well (Fluxomatic, B. Forbush III). Cellular 86Rb uptake was determined by
PhosphorImage analysis of the 96-well plate. Because, as shown in Fig.
7A, virtually all of the ouabain-insensitive 86Rb influx in these cells was bumetanide-sensitive, we did
not carry out bumetanide controls on a routine basis.
Solutions used in these flux experiments were as follows. Basic medium
contained 135 mM NaCl, 5 mM RbCl, 1 mM CaCl2/MgCl2, 1 mM
Na2HPO4/Na2SO4, and 15 mM NaHEPES, pH 7.4. 86Rb influxes were carried
out in regular flux medium, consisting of basic medium with ~1
µCi/ml 86Rb and 10
The relative amount of NKCC (wild-type and mutant) was determined in
most experiments by dot blotting with the J3 antibody, a monoclonal
antibody specific for sNKCC1 (16). Cells from single wells of the
96-well flux plate were solubilized in 100 µl of SDS sample buffer
and serially diluted four times at a 1:5 ratio; 20-µl aliquots of
each dilution were blotted in a 96-well blotter; and NKCC was detected
with the J3 antibody, Pierce West Dura luminescent reagent, and a
cooled CCD camera. Additionally, total protein was determined in
individual wells using the Bio-Rad DC assay and a 96-well plate
reader after solubilizing cells in 50 µl of 1% SDS (3-12 wells for
each line on a plate). On a routine basis, the uniformity of cells in
the 96-well plate was checked by visual inspection of the cell
monolayer following the flux experiment.
Immunofluorescence Analysis--
Visualization of sNKCC1
in transfected HEK-293 cells was performed as previously described (6).
Briefly, cells were fixed in the culture dish with
periodate-lysine-paraformaldehyde fixative, scraped from the
dish, and pelleted at low speed; semi-thin cryosections were cut from
cryopreserved specimens. The J4 monoclonal antibody (anti-shark
NKCC1) was used to detect sNKCC1 with an Alexa 488 anti-mouse
secondary antibody (Molecular Probes, Inc.) in a Zeiss Axiophot
fluorescence microscope equipped with an Axiovision CCD camera.
We determined the stoichiometry of NKCC1 phosphorylation in rectal
gland tubule cells by measuring the moles of 32P
incorporated per mol of Na-K-Cl cotransporter during a 10-min incubation period with forskolin and calyculin A. In this study, an
average of 3.0 ± 0.4 phosphates/mol of cotransporter was
determined in six experiments using the determined specific activity of
To identify individual phosphorylated residues in NKCC1, labeled
cotransporter protein was subjected to exhaustive trypsin digestion and
separated by HPLC. The labeled Na-K-Cl cotransporter elution profile
showed >15 fractions with levels of incorporation above background
(Fig. 2). As shown below, this profile
alone does not give a useful estimate of the number of phosphorylation sites on the cotransporter protein because incomplete trypsin digestion
can give rise to many partial cleavage products and because mono- and
diphosphorylated peptides will run with different mobility on the
column. Using MALDI-TOF spectroscopy, phosphorylated peptides were
identified by an appropriate mass shift in reflectron mode and/or by
alkaline phosphatase digestion to remove phosphates from the peptides.
The sequences of each peptide in the labeled fraction were ultimately
confirmed by Edman degradation. We were able to positively identify
four of the major phosphorylated peptides from the fractions with
measurable levels of 32P incorporation and to match these
with regions of the sNKCC1 protein (other fractions did not yield
useful MALDI data). Table I
presents a summary of the relevant analysis that led to the identification of the phosphorylation sites. Spectra from two of the
fractions are included in Figs. 3 and
4.
A Regulatory Locus of Phosphorylation in the N
Terminus of the Na-K-Cl Cotransporter, NKCC1*
and
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ABSTRACT
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in many cells (1, 2). Along with the
absorptive Na-K-Cl cotransporter NKCC2 and the thiazide-sensitive Na-Cl
transporter NCC (or TSC), NKCC1 belongs to the sodium-coupled branch of
the cation chloride cotransporter family; this family also includes the
K-Cl transporters (KCCs) (3) and two other major branches of putative
transporters, including human CIP (4) and SLC12-8 (GenBankTM/EBI accession number AAK94307), whose transport
functions are unknown. These proteins are all predicted to have 12 transmembrane-spanning domains that are responsible for ion transport
properties (5) and large cytoplasmic N and C termini that are
candidates for regulatory domains. NKCC1 is expressed in many cell
types and is involved in the regulation of both cell volume and
intracellular Cl concentration (2, 6). This isoform is
also a major component of the basolateral membrane of secretory cells,
mediating Cl influx, the first step in the
transepithelial movement of Cl. In contrast, the NKCC2
isoform is limited to the apical membranes of absorptive epithelia such
as the thick ascending limb of Henle's loop, where it is a major
determinant of NaCl reabsorption from the tubular fluid.
channels and subsequent
phosphorylation of NKCC1 in response to decreases in cell
Cl and volume by an as yet unknown kinase (12).
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-position
decreased somewhat over time (Fig. 1, lower panel), as an
increasing amount of radioactivity was incorporated into the
-position of ATP. For subsequent experimentation, a labeling period
of 40 min at 15 °C was chosen as an optimal point for cell
viability, overall cellular 32PO4 uptake, and
high fractional incorporation of label into the -position of ATP.
channels and thereby lower cell
[Cl]. Calyculin A (0.5 µM) was also added
to prevent NKCC dephosphorylation by protein phosphatase-1 (15). A
small portion of the tubules were treated with 5% trichloroacetic acid
or 1 M H3PO4 to precipitate protein, and the soluble extract was utilized for ATP analysis following neutralization. The remaining major portion of the tubule suspension was solubilized in an equal volume of ice-cold phosphatase inhibitory buffer (300 mM NaCl, 60 mM NaF, 10 mM EDTA, 30 mM Na2HPO4, 30 mM sodium pyrophosphate, 40 mM HEPES, 0.2 mM NaVO4, 0.5 µM calyculin A, and
4% Triton X-100, including protease inhibitors (7)) and frozen in
liquid N2. After thawing, Na-K-Cl cotransporter protein was
immunoprecipitated from Triton X-100-solubilized samples using the
J4 antibody, a monoclonal antibody specific for shark NKCC1
(16); NKCC-containing eluates were analyzed by SDS gel electrophoresis.
Following staining and destaining, gel slices containing purified
Na-K-Cl cotransporter were rinsed thoroughly with water and used for
either phosphoprotein determination or phosphopeptide analysis.
-position of ATP. Phosphoprotein
analysis of NKCC included determination of 32P in a gel
slice containing phosphorylated immunopurified NKCC by Cerenkov
radiation in a scintillation counter and determination of NKCC protein
by quantitative analysis of amino acids extracted from the gel slice
following a 16-h digestion at 115 °C with 6 N HCl. Total
ATP in tubule extracts was determined using the luciferin-luciferase reaction, with detection by a cooled CCD camera. The amount of [-32P]ATP was determined by TLC on
polyethyleneimine-cellulose plates in a 4.25 mM
KH2PO4 mobile phase utilizing
PhosphorImage analysis with 32P standards. The
fraction of 32P in the -position was determined by loss
of 32P from ATP (and appearance as Glc-P,
RF = 1.0) upon incubation with yeast hexokinase and
glucose and by TLC analysis as described above.
4 M ouabain.
Low chloride preincubation solutions utilized basic medium as a base
(but with potassium rather than rubidium), substituting gluconate for
chloride to achieve the appropriate Cl concentration (3, 30, 70, and 100 mM Cl). In the
Cl activation series (see Fig. 7), the solutions were
supplemented with 50 µM furosemide to prevent
transporter-specific alterations in ionic balance (15). Low
Cl hypotonic medium contained a 3 mM
Cl solution diluted 2-fold with water. 0Na-0K-130Cl
hypertonic medium contained N-methylglucamine to replace all
of the sodium and potassium/rubidium in basic medium and an additional
130 mM N-methylglucamine gluconate to make the
solution ~2× hypertonic. High K+ medium was basic medium
with an additional 10 mM K+.
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-label in cellular ATP (Fig. 1). This
value is close to that reported by Lytle (17) for cotransporter
phosphorylation in avian red cells; as discussed below, the
determination of stoichiometry represents a minimal estimate of the
number of phosphoacceptor sites. We have noted that there is little
incorporation of label into the cotransporter under resting conditions.
In numerous experiments (data not shown) and as previously reported
(7), <20% of 32P label is incorporated in the absence of
stimulatory agents; and we have been unable to isolate individual peaks
of radioactivity when control samples were digested with trypsin and
peptides were separated by HPLC. A stoichiometric relationship >1 for
phosphate incorporation into NKCC1 is consistent with a causal
relationship between cotransporter phosphorylation and increased
activity.
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Fig. 1.
Incorporation of
32PO4 into cellular pools in rectal gland
tubules. Rectal gland tubules were incubated with
[32P]H3PO4 for 10-40 min; the
incubation was terminated, and protein was precipitated with
trichloroacetic acid. [32P]ATP, [32P]ADP,
and 32Pi were determined by TLC of supernatant
samples (upper panel) using hexokinase to remove the
-phosphate from ATP (lower panel). Error bars
indicate the range in duplicate samples; similar results were obtained
in two other experiments.
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Fig. 2.
HPLC of tryptic peptides of immunopurified
Na-K-Cl cotransporter. Upper panel, peptide elution
profile of trypsin digest of NKCC1, separated on a C18 HPLC
column; lower panel, profile of 32P incorporated
in peptides. abs., absorbance.
Summary of sNKCC1 phosphorylation site identification
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Fig. 3.
MALDI-TOF spectrometry of peptides in
fraction 61. Samples were ionized and analyzed in linear positive
mode (A and B) or reflectron mode (C)
to observe post-source decay products. A shows the mass
spectrum of untreated fraction 61. The sample in B was
treated with alkaline phosphatase prior to MALDI-TOF analysis.
Two major peptides appeared in the linear mode spectrum of fraction 61: one with a mass of 1105 Da, and the other with a mass of 1489 Da (Fig. 3A). Based on mass alone, the 1105-Da peak best matches the non-phosphoacceptor peptide 1105MKEEEPWR1112 in the C-terminal domain of the sNKCC1 sequence. With the mass resolution available in this instrument, the 1489-Da peak had a possible match with two predicted phosphopeptides: diphosphorylated 184TFGHNTIDAVPR195 and monotyrosine-phosphorylated 826AFYAPVYAEDLR837. To determine whether these peptides were phosphorylated, fraction 61 was treated with alkaline phosphatase and again analyzed by MALDI (Fig. 3B). Whereas the 1105-Da peak was not changed, the 1489-Da peak was lost, and a new peak appeared at 1327 Da. A mass shift of 162 Da is consistent with the enzymatic removal of two HPO3 moieties from 184TFGHNTIDAVPR195. The spectrum of fraction 61 taken in reflectron mode (Fig. 3C) lacked the 1489-Da peptide, whereas a peak appeared at 1300 Da, a 189-Da shift consistent with the post-source decay of two HPO3 + H2O moieties from a phosphopeptide with a mass 1489 Da (18). Finally, microsequencing of fraction 61 by Edman degradation confirmed the presence of both the 1105MKEEEPWR1112 and 184TFGHNTIDAVPR195 peptides. Thus, it is clear that fraction 61 contains a diphosphopeptide containing Thr(P)184 and Thr(P)189.
In additional mass spectrometric analysis, the monophosphorylated form of the 184TFGHNTIDAVPR195 peptide was found to constitute the radioactivity in HPLC fraction 67 (data not shown). It was not determined which of the two phosphoacceptors was phosphorylated in this fraction (most likely the peak contains a mixture of the two potential monophosphorylated peptides). The Thr189 phosphoacceptor site has been previously identified by Lytle and Forbush (7) in a trypsin/CNBr digest fragment, presumably corresponding to fraction 67. In that study, it was not possible to identify Thr184 as a phosphoacceptor because of the high background in Edman sequencing of the initial residue.
Mass spectrometric analysis of HPLC fraction 55 resulted in the
identification of a second peptide with a novel phosphorylation site.
In linear mode (Fig. 4A), two
major peptides appeared in the fraction, one at 1751 Da and the other
at 923 Da. The 923-Da peak was insensitive to alkaline phosphatase
(Fig. 4B) and did not shift under conditions of limited
post-source decay. The 1751-Da peak was shifted by 
83 Da in the
spectra of alkaline phosphatase-treated samples and by 98 Da in the
reflectron spectrum (Fig. 4C). In both cases, the shifts
were consistent with loss of a single phosphate species from a serine
or threonine residue on the peptide. Edman degradation showed that
there were three peptides in the sample: one, an IgG contaminant;
another, VYTMGPPR, with a mass 923 Da; and the third corresponding to
sequence 196IDHYRHTVAQLGEK209, an incomplete
trypsin digest product with a non-phosphorylated apparent mass
of 1668 Da. Only a single threonine occurs on this peptide, and the gap
in the sequencing data is consistent with its being the
phosphoacceptor. The product of the complete digest, 201HTVAQLGEK209, was also identified as the
phosphorylated component in fraction 40 (data not shown.).
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To test the functional significance of these phosphorylation sites in
regulation of the cotransporter, point mutants of the phosphoacceptor
sites were stably expressed in HEK-293 cells. Each of these mutations
was successfully produced and delivered to the plasma membrane, as
illustrated by immunofluorescence in Fig.
5. The J4 antibody is specific for shark
NKCC1 and did not detect the native HEK-293 cell cotransporter, as
illustrated in Fig. 5 (upper right and lower right
panel pairs). It is shown that for some of the sNKCC1 constructs,
there was considerable transporter retained in intracellular
compartments; but in each case, NKCC1 appeared to be present in the
cell membrane and thus should be potentially active in a
86Rb influx assay.
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NKCC1 is activated under conditions in which the cell is depleted of
cell chloride, and we have routinely used low Cl
preincubation to stimulate transport in HEK-293 cells (5, 19, 20). As
illustrated in Fig. 6, replacing the
Thr189 phosphoacceptor with alanine resulted in a mutant
with little detectable cotransporter flux, presumably because the
mutation prevents the activation of the cotransporter by a low
Cl stimulus. In fact, as discussed further below, these
mutants actually have lower transport activity than the native HEK-293 cells (21) and the lines transfected with vector alone, i.e. the mutant cotransporters exert a dominant-negative effect.
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In some, but not all, proteins regulated by phosphorylation, the substitution of a phosphoacceptor serine or threonine with an acidic residue can mimic the effect of phosphorylation at that locus (22, 23). However, for Thr189 of the Na-K-Cl cotransporter, replacement with aspartate did not restore activity. The T189D mutant showed neither a constitutive activation nor the ability to be activated in response to the stimulus (the latter is shown in Fig. 6). Similarly, a threonine-to-glutamate triple mutation of the identified phosphoacceptor sites resulted in a non-activable cotransporter (Fig. 6).
We have also altered the Thr189 site to meet the consensus criterion for PKA phosphorylation by replacement of the sequence 186GHNT with 186RRNT ("PKA-1") and, in another mutant, by 186RKNT ("PKA-2"). As with the other Thr189 mutations, these replacements eliminated the transport function of the cotransporter (Fig. 6), and there was no evidence of an increase in activity by elevation of cAMP levels using 50 µM forskolin (data not shown; forskolin actually produced some inhibition of 86Rb influx in HEK-293 cells (see Fig. 8). We presume either that the structural context of Thr189 renders it inaccessible to PKA or that the altered context of the site renders it ineffective in bringing about the necessary cotransporter conformational change.
In contrast to the results with Thr189, mutagenesis of the
Thr184 and Thr202 phosphoacceptors resulted in
NKCCs that were capable of 86Rb transport under optimal
activation conditions (Fig. 6). To more carefully examine the
activation of these mutants by lowered intracellular
[Cl], we determined 86Rb influx after
preincubation in media of differing Cl concentrations.
Fig. 7A presents the results
from one such experiment in which the effect of bumetanide is also
shown; Fig. 7B summarizes the results from more than four
experiments with each mutant. As shown here, T184A mutants were less
sensitive to Cl changes compared with wild-type
sNKCC1, showing activity only after preincubation at the lowest
Cl concentrations (Fig. 7, A, lower left
panel; and B, upper panel). This behavior is
consistent with a mechanism in which increased phosphorylation at other
residues can partially compensate for the absence of phosphorylated
Thr184. As with Thr189, phosphorylation at
Thr184 could not be mimicked by replacement with fixed
negative charge; the characteristics of T184E were similar to those of
T184A, but with slightly greater chloride sensitivity (Fig.
7B, upper panel).
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The T202A mutant was also found to be less sensitive to
[Cl] changes compared with wild-type sNKCC1, although
the difference was less pronounced than for the mutation at
Thr184 (Fig. 7B, upper panel). Again,
a glutamate substitution failed to restore the phenotype of
wild-type sNKCC1, and T202E was actually less sensitive to
changes in [Cl] compared with T202A (Fig.
7B, upper panel). The T184E/T202E double mutant
was similar to the Thr202 single mutants in its activation
profile (Fig. 7, A, lower right panel; and
B, lower panel), whereas the flux of T184A/T202A
was very low and not greatly different from that in vector-transfected cells (Figs. 6 and 7B).
In the region immediately upstream of Thr184, there are many potential phosphoacceptor serines and threonines, some of which (e.g. Thr177-Thr179) are in a very similar context to Thr184/Thr189. Speculating that these residues might also be involved in activation of the transporter, we prepared a T177A/T179A mutant. In fact, as illustrated in Fig. 7B (lower panel), the behavior of this construct was very close to that of wild-type NKCC1, with a small tendency for the mutant to be less activated by a given stimulus.
To investigate whether alternative activation modalities differentially
affected the phosphorylation site mutants, we recorded time courses of
86Rb influx following preincubations in various media. As
illustrated in Fig. 8 (upper
panel), sNKCC1 in HEK-293 cells was most rapidly activated by the
low Cl hypotonic stimulus (black), but was
also activated by exposure to hypertonic media. Hypertonic sucrose
solutions (blue) produced various degrees of activation,
from ~30% (Fig. 8, upper panel) to ~80% in other
experiments (data not shown). 0Na-0K-130Cl hypertonic medium
(red) usually produced near-maximal activation, usually more
slowly than low Cl hypotonic medium. Interestingly, as
observed consistently in these experiments, forskolin slowed the
response to this intervention without abrogating the maximal level
(Fig. 8, compare + and 
); this provides at least a phenomenological
explanation for the forskolin inhibition of NKCC-mediated flux
sometimes seen at intermediate levels of stimulation.
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, +, O), low Cl
), low Cl
), hypertonic sucrose medium (blue;
). A 2-min 86Rb
influx was then carried out in all cells in regular flux medium. Each
panel presents the results from a single 96-well plate, with each point
showing the value of the flux in a single well. The two columns of
panels are from two separate experiments. Similar results were obtained
in at least four other experiments for each line.
In contrast to the behavior of wild-type sNKCC1, under hypertonic
conditions, the Thr184 and T202E mutants were activated
very poorly (T184A (Fig. 8, middle right panel) and
T184A/T202A and T184E/T202E (data not shown)) or not at all (T202E
(lower left panel) and T184E (data not shown)). The
difference is especially striking in the case of T202E; not only was
the transporter not activated by hypertonic media, but if previously
activated in low Cl hypotonic medium, it was seen to
rapidly deactivate upon transition to 0Na-0K-130Cl
hypertonic medium. It is clear that this cannot simply be explained by
a lower sensitivity of the mutant transporters to all activating
stimuli, for if that were the case, all curves would be down-shifted in
the same way. Rather, the results of Fig. 8 suggest that different
activation modalities may exert their effects preferentially through
different subsets of phosphorylated residues.
In the course of these experiments, we found that stable cell lines
containing Thr189 mutants exhibited maximal
86Rb influx that was generally lower than that of
vector-transfected cell lines; this phenomenon was particularly evident
at intermediate levels of cotransporter activation (Fig.
9). This dominant-negative effect of
inactive NKCCs has been noted in other mutagenesis studies in our
laboratory (5) and has also been seen with a truncation variant of
NKCC2 (24), in inactive mutants of KCC1 (25), and in an interaction
between human CIP and NKCC2 (4). At this point, it is unclear
whether the present effects are indicative of functional
protein-protein interaction at the plasma membrane, competition for an
unknown cellular component, or overexpression bottlenecks in the
synthesis and delivery of NKCC.
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The analysis of 32P-labeled shark rectal gland Na-K-Cl
cotransporter protein presented here led to a minimal estimate of a 3:1 phosphorylation stoichiometry and to the identification of three phosphoacceptor sites (at Thr184, Thr189, and
Thr202). These sites are proposed to form a regulatory
locus in a region of the N-terminal cytoplasmic region conserved
between NKCC isoforms. The functional role of each of the
phosphoacceptors was examined by testing point mutants of sNKCC1
expressed in HEK-293 cells. In this way, phosphorylation of
Thr189 was found to be absolutely necessary and possibly
sufficient for cotransporter activation, and Thr184 and
Thr202 were identified as important modulatory loci. The
latter sites appear to be required both for maximal activation and for
optimal sensitivity to Cl and volume changes.
A phosphorylation stoichiometry 
1 is consistent with the hypothesis
that phosphorylation of at least one residue is responsible for
activation of the protein. It should be noted that any determination of
phosphorylation stoichiometry is likely to be an underestimate of the
number of phosphoacceptor sites because some dephosphorylation may
occur during biochemical analysis and because there may be incomplete
phosphorylation during cellular stimulation. This is particularly true
in our dissociated cell system, where it is certain that some of the
isolated cells have been damaged during enzymatic and mechanical
treatment; this component would, of course, contribute NKCC protein
without a corresponding component of 32P incorporation.
Indeed, the isolation of the peptide containing Thr184 and
Thr189 as a monophosphorylated species in fraction 67 illustrates that not every acceptor site is fully phosphorylated in our
experimental samples. With this considered, we must defer to the
measurement of 5:1 stoichiometry for NKCC1 in duck erythrocyte
cotransporter maximally stimulated by hyperosmotic stress (17),
supporting an estimate of at least five phosphoacceptor sites in NKCC1.
It is also true that, although we have identified three threonine residues, previous work has demonstrated the presence of phosphoserine as well as well as phosphothreonine in activated NKCC1 from both shark
and avian sources (7, 17).
We suggest that the region just N-terminal to Thr184 is a
likely candidate for regulatory phosphorylation by the same kinase. This region is highly conserved between shark and human (Fig. 10), and it contains a number of
threonines and serines in a context very similar to that of
Thr184/Thr189. In an initial test of two of
these potential phosphoacceptors, we found that the T177A/T179A mutant
is almost indistinguishable from wild-type NKCC1 (Fig. 7B),
but there are 16 more candidate residues in this region, seven of which
are conserved between shark and human. In biochemical analysis, the
residues upstream of Thr184 would be found in an 8-kDa
tryptic fragment (residues 108-183); possibly this large fragment is
present in HPLC fractions 80-92, for which we were unable to obtain
useful mass spectrometric data.
|
Importantly, the identified phosphoacceptors Thr184, Thr189, and Thr202 all occur in a highly conserved region of the N terminus of NKCC (Fig. 10). The sequences flanking these phosphorylation sites are almost identical between shark and human NKCC1 (similarity indicated by color in Fig. 10), arguing for an essential regulatory role of this domain. The current finding of three phosphoacceptors in the N terminus of NKCC1 extends our earlier observation that Thr189 is phosphorylated (7), and it is fully consistent with the work of Kurihara et al. (11) that all of the regulatory phosphorylation of NKCC1 in rat parotid gland is found in the N-terminal cytoplasmic domain. The identified phosphoregulatory region is very well conserved between NKCC1 and NKCC2, with only two amino acid differences occurring in the region corresponding to sNKCC1 residues 181-194. The fact that the domain has been conserved in NKCC2 as well as in NKCC1 argues that both isoforms are activated by essentially the same mechanism, and recent data from our laboratory strongly support this hypothesis (26); alternative mechanisms for regulation of NKCC2 have also been suggested (24). Although there is less similarity than between the two NKCC isoforms, the Na-Cl cotransporter NCC retains potential phosphoacceptors corresponding to Thr184, Thr189, and Thr202 in a very similar context. Therefore, we speculate that NCC is also regulated largely by phosphoacceptors in its N terminus.
The data presented here add to a growing body of evidence that the N
terminus of NKCC is the major regulatory domain of the protein. The
phosphorylation locus identified in this study is 80 amino acids
downstream of a protein phosphatase-1-binding site identified by its
RVXFXD sequence. Recently, we have shown that the
NKCC1-protein phosphatase-1 interaction mediated by this site enhances
regulatory dephosphorylation of the cotransporter (15). This has the
functional consequence that the cotransporter is less active at any
given intracellular [Cl]; and thus, cell
[Cl] is maintained at a lower level.
The regulatory kinase that phosphorylates NKCC in response to cellular
stimuli remains elusive. None of the proposed phosphoacceptor threonines lies within strong consensus sequences for a known kinase,
and we have shown that NKCC1 phosphorylation is prevented by high
intracellular [Cl] even when PKA is maximally activated
with forskolin (12). Although it has been proposed that serum- and
glucocorticoid-inducible kinase (27) or c-Jun N-terminal kinase (28) is
the volume- and Cl-sensitive kinase, there is little
evidence that either of these directly phosphorylates NKCC1 in
vivo.
Previous evidence has strongly supported the idea that one final common
kinase can mediate the response to multiple stimuli. Lytle (17) has
shown that phosphopeptide maps of cotransporter from cells stimulated
with isoproterenol, hyperosmotic medium, calyculin A, and fluoride are
essentially identical, and we have obtained similar results for
forskolin and hypertonic stimulation of shark
NKCC1.2 On the other hand,
data presented here demonstrate that with alterations in
Thr184 or Thr202, the modality of cotransporter
regulation is appreciably changed: although these mutants were
activated by incubation in low Cl hypotonic medium, some
were not activated in hypertonic media, including 0Na-0K-130Cl
hypertonic medium, which yielded maximal activation of wild-type NKCC.
This behavior cannot be explained simply by a lower sensitivity of the
regulatory response. This finding is clearly inconsistent with the
simplest models involving a final common pathway, although the
possibility remains that the way in which a single kinase interacts
with different regions of NKCC is differentially affected by different stimuli.
In evaluating the results of mutations at Thr184 and Thr202, we should consider the possibility that, rather than having a direct mechanistic effect, the alteration at these sites changes the sensitivity profile by decreasing phosphorylation at Thr189. Although it is generally difficult to examine this experimentally, the utilization of the anti-phospho-NKCC antibody has allowed us to investigate the results of the T202E mutation. As described in the accompanying paper (29), the changes in sensitivity seen in the activation of 86Rb influx are mirrored by the Thr184/Thr189 phosphorylation of the T202E mutant. This demonstrates that Thr202 indeed alters Thr184/Thr189 phosphorylation, rather than exerting a direct effect.
The identification of specific phosphorylation sites on NKCC allows a
closer examination of the physiological significance of activation of
the protein. As described in the accompanying paper (29), we have
recently prepared an antibody (anti-phospho-NKCC1 antibody R5)
to a diphosphorylated peptide containing Thr184 and
Thr189. The antibody is highly specific for NKCC and is
able to discriminate between phosphorylated and non-phosphorylated
proteins with better than a 100:1 ratio. The R5 antibody will enable
ready determination of NKCC activation in physiological circumstances
and thus facilitate a further understanding of the regulatory
mechanisms involved in control of membrane transport.
| |
ACKNOWLEDGEMENTS |
|---|
We thank Katherine Stone and Kenneth Williams (W. M. Keck Facility, Yale University) for protein chemistry, including HPLC, amino acid analysis, and mass spectrometry, as well as for helpful discussions, and Sue Ann Mentone for expert preparation of histological samples. We are grateful to Grace Dillard for excellent technical assistance and to Ignacio Giménez and Brian Dowd for comments on the manuscript.
| |
FOOTNOTES |
|---|
* This work was supported by National Institutes of Health Grant DK47661.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Molecular
Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School,
330 Brookline Ave., Boston, MA 02215. Tel.: 617-667-2923; Fax:
617-667-8040; E-mail: rdarman@caregroup.harvard.edu.
Published, JBC Papers in Press, July 26, 2002, DOI 10.1074/jbc.M206293200
2 R. B. Darman and B. Forbush, unpublished data.
| |
ABBREVIATIONS |
|---|
The abbreviations used are: PKA, protein kinase A; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; HPLC, high pressure liquid chromatography; sNKCC1, shark NKCC1.
| |
REFERENCES |
|---|
|
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
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C. F. Simard, N. D. Daigle, M. J. Bergeron, G. M. Brunet, L. Caron, M. Noel, V. Montminy, and P. Isenring Characterization of a Novel Interaction between the Secretory Na+-K+-Cl- Cotransporter and the Chaperone hsp90 J. Biol. Chem., November 12, 2004; 279(46): 48449 - 48456. [Abstract] [Full Text] [PDF]
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B. Lenart, D. B. Kintner, G. E. Shull, and D. Sun Na-K-Cl Cotransporter-Mediated Intracellular Na+ Accumulation Affects Ca2+ Signaling in Astrocytes in an In Vitro Ischemic Model J. Neurosci., October 27, 2004; 24(43): 9585 - 9597. [Abstract] [Full Text] [PDF]
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H. Zhao, R. Hyde, and H. S Hundal Signalling mechanisms underlying the rapid and additive stimulation of NKCC activity by insulin and hypertonicity in rat L6 skeletal muscle cells J. Physiol., October 1, 2004; 560(1): 123 - 136. [Abstract] [Full Text] [PDF]
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C. F. Simard, G. M. Brunet, N. D. Daigle, V. Montminy, L. Caron, and P. Isenring Self-interacting Domains in the C Terminus of a Cation-Cl- Cotransporter Described for the First Time J. Biol. Chem., September 24, 2004; 279(39): 40769 - 40777. [Abstract] [Full Text] [PDF]
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 G. O. Andersen, T. Skomedal, M. Enger, A. Fidjeland, T. Brattelid, F. O. Levy, and J.-B. Osnes {alpha}1-AR-mediated activation of NKCC in rat cardiomyocytes involves ERK-dependent phosphorylation of the cotransporter Am J Physiol Heart Circ Physiol, April 1, 2004; 286(4): H1354 - H1360. [Abstract] [Full Text] [PDF]
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E. Gagnon, M. J. Bergeron, G. M. Brunet, N. D. Daigle, C. F. Simard, and P. Isenring Molecular Mechanisms of Cl- Transport by the Renal Na+-K+-Cl- Cotransporter: IDENTIFICATION OF AN INTRACELLULAR LOCUS THAT MAY FORM PART OF A HIGH AFFINITY Cl--BINDING SITE J. Biol. Chem., February 13, 2004; 279(7): 5648 - 5654. [Abstract] [Full Text] [PDF]
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K. Piechotta, N. Garbarini, R. England, and E. Delpire Characterization of the Interaction of the Stress Kinase SPAK with the Na+-K+-2Cl- Cotransporter in the Nervous System: EVIDENCE FOR A SCAFFOLDING ROLE OF THE KINASE J. Biol. Chem., December 26, 2003; 278(52): 52848 - 52856. [Abstract] [Full Text] [PDF]
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B. F. X. Dowd and B. Forbush PASK (Proline-Alanine-rich STE20-related Kinase), a Regulatory Kinase of the Na-K-Cl Cotransporter (NKCC1) J. Biol. Chem., July 18, 2003; 278(30): 27347 - 27353. [Abstract] [Full Text] [PDF]
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I. Gimenez and B. Forbush Short-term Stimulation of the Renal Na-K-Cl Cotransporter (NKCC2) by Vasopressin Involves Phosphorylation and Membrane Translocation of the Protein J. Biol. Chem., July 11, 2003; 278(29): 26946 - 26951. [Abstract] [Full Text] [PDF]
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J. P. Dehaye, A. Nagy, A. Premkumar, and R. J. Turner Identification of a Functionally Important Conformation-sensitive Region of the Secretory Na+-K+-2Cl- Cotransporter (NKCC1) J. Biol. Chem., March 28, 2003; 278(14): 11811 - 11817. [Abstract] [Full Text] [PDF]
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 S. L. Schomberg, J. Bauer, D. B. Kintner, G. Su, A. Flemmer, B. Forbush, and D. Sun Cross Talk Between the GABAA Receptor and the Na-K-Cl Cotransporter Is Mediated by Intracellular Cl- J Neurophysiol, January 1, 2003; 89(1): 159 - 167. [Abstract] [Full Text] [PDF]
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A. W. Flemmer, I. Gimenez, B. F. X. Dowd, R. B. Darman, and B. Forbush Activation of the Na-K-Cl Cotransporter NKCC1 Detected with a Phospho-specific Antibody J. Biol. Chem., September 27, 2002; 277(40): 37551 - 37558. [Abstract] [Full Text] [PDF]
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