JNK is a volume-sensitive kinase that phosphorylates the
Na-K-2Cl cotransporter in vitro
Janet D.
Klein1,
S. Todd
Lamitina1, and
W. Charles
O'Neill1,2
Renal Division, 1 Department of
Medicine, and 2 Department of
Physiology, Emory University School of Medicine, Atlanta, Georgia
30322
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ABSTRACT |
Cell shrinkage
phosphorylates and activates the Na-K-2Cl cotransporter (NKCC1),
indicating the presence of a volume-sensitive protein kinase. To
identify this kinase, extracts of normal and shrunken aortic
endothelial cells were screened for phosphorylation of NKCC1 fusion
proteins in an in-the-gel kinase assay. Hypertonic shrinkage activated
a 46-kDa kinase that phosphorylated an
NH2-terminal fusion protein, with
weaker phosphorylation of a COOH-terminal fusion protein. This
cytosolic kinase was activated by both hypertonic and isosmotic
shrinkage, indicating regulation by cell volume rather than osmolarity.
Subsequent studies identified this kinase as c-Jun
NH2-terminal kinase (JNK).
Immunoblotting revealed increased JNK activity in shrunken cells; there
was volume-sensitive phosphorylation of
NH2-terminal c-Jun fusion protein;
immunoprecipitation of JNK from shrunken cells but not normal cells
phosphorylated NKCC1 in gel kinase assays; and treatment of cells with
tumor necrosis factor, a known activator of JNK, mimicked the effect of
hypertonicity. We conclude that JNK is a volume-sensitive kinase in
endothelial cells that phosphorylates NKCC1 in vitro. This is the first
demonstration of a volume-sensitive protein kinase capable of
phosphorylating a volume-regulatory transporter.
cell volume; c-Jun
NH2-terminal kinase; stress-activated protein kinase; phosphorylation; endothelia
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INTRODUCTION |
ACUTE REGULATION OF CELL VOLUME is accomplished through
the activation of specific ion transporters, but the mechanism by which
cell volume regulates these transporters remains a mystery. NKCC1, the
ubiquitous "secretory" or "basolateral" isoform of the
Na-K-2Cl cotransporter, is phosphorylated and activated by cell
shrinkage. Protein phosphorylation has also been implicated in the
activation by shrinkage of the Na/H antiporter (NHE1), another
important volume-regulatory transporter, although phosphorylation of
the transporter itself is not increased (5, 6). The K-Cl cotransporter,
which is activated by cell swelling and regulates cell volume in the
opposite direction, appears to be inhibited by phosphorylation. These
data suggest that protein phosphorylation may play a central role in
the sensing and regulation of cell volume.
It is of interest that several kinases exhibit activation by
hyperosmolarity, most notably members of the mitogen-activated protein
kinase (MAPK) family, including p42,44 MAPK (2, 10), p38 MAPK (19), and
c-Jun NH2-terminal kinase (JNK),
also known as stress-activated protein kinase (4). However, it is not known whether activation of these serine/threonine kinases is due to
increased osmolarity or to decreased cell volume. Recently, two members
of the Src family of tyrosine kinases were shown to be activated
specifically by shrinkage in neutrophils (14). None of these
osmosensitive or volume-sensitive kinases have been implicated in the
activation of volume-regulatory transporters. We have previously
presented evidence that cell shrinkage activates myosin light chain
kinase (MLCK), another serine/threonine kinase (12, 21). Although MLCK
activation is required for activation of NKCC1 and NHE1 in shrunken
cells, this does not occur through transporter phosphorylation. A
specific volume-sensitive kinase that phosphorylates a
volume-regulatory transporter has yet to be identified.
We have previously shown that endothelial cells cultured from bovine
aortas exhibit abundant NKCC1 activity and rapid volume recovery after
shrinkage (18). Cell shrinkage increases the phosphorylation of NKCC1
(16), and kinetic data suggest activation of a protein kinase rather
than inhibition of a protein phosphatase (13) as the regulatory event.
NKCC1 contains 12 putative membrane-spanning domains flanked by a
280-amino acid NH2 terminus and a
400-amino acid COOH terminus, both of which appear to be cytoplasmic
(23) and phosphorylated (15). Recent data point to regulatory
phosphorylation of a specific threonine residue in the
NH2 terminus of the shark NKCC1
(1). To identify the volume-sensitive kinase that phosphorylates NKCC1,
we developed an in vitro assay of NKCC1 phosphorylation within
polyacrylamide gels of electrophoretically separated endothelial proteins, using both cytoplasmic domains of NKCC1 as substrates.
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MATERIALS AND METHODS |
Cell cultures and treatments.
Endothelial cells were cultured from bovine aortas using DMEM
containing 10% fetal bovine serum as previously described (11). All
studies were performed on confluent cells in plastic culture flasks or
multiwell plates, with fresh medium applied 12-24 h before
experiments. For experiments, cells were incubated for 30 min in a
HEPES-buffered, balanced salt solution with or without 150 mM sucrose
or 2.8 nM human tumor necrosis factor-
(TNF-
).
Fusion proteins. Two overlapping cDNAs
encompassing the full-length cDNA for human NKCC1 (TEF1-1 and
TEF11a) were obtained from Dr. John Payne (Univ. of California, Davis,
CA). TEF11a was excised with Pst I and
cloned into the pQE-30 expression plasmid (Qiagen, Valencia, CA),
yielding a hexahistidine protein that encodes the first 277 amino acids
of NKCC1. A 1.4-kb portion of cotransporter cDNA encoding most of the
putative COOH-terminal cytoplasmic region including a unique
BamH I site was amplified from
TEF1-1 by PCR, with a Sal I
restriction site added to the 3' end. This was then cloned into
the pQE-30, yielding a protein that contains amino acids 758-1,157
(full-length NKCC1 is 1,212 amino acids). The cDNA for smooth muscle
myosin light chain 2 from chicken gizzard (kindly provided by Dr. Paul
Zavodny, Schering-Plough Pharmaceutical) was cloned into the expression
plasmid as a 759-bp fragment containing the entire coding region. The
pQE plasmid containing the sequence for dihydrofolate reductase was
purchased from the vendor. The structures of all constructs were
confirmed by sequencing. The resulting
NH2-terminal hexahistidine
proteins were produced in bacterial strain M15[pRep4],
extracted with guanidinium hydrochloride, and purified in a denatured
form on an Ni-NTA-agarose column according to the vendor's
instructions. A cDNA encoding a glutathione
S-transferase fusion protein
containing c-Jun1-135 was
kindly provided by Dr. S. R. Price and transformed into
Escherichia coli (DH5 strain). The
expressed protein was purified on a hexylglutathione column (Sigma
Chemical, St. Louis, MO) and eluted with glutathione.
Kinase assay. Medium was removed and
cells were extracted with 2% SDS and 1%
-mercaptoethanol. After
solubilization by scraping and shearing through a 27-gauge
needle, the samples were boiled for 1 min and then electrophoresed into
a 10% polyacrylamide gel containing 1 mg/ml fusion protein. Kinase
activity was then determined within the gel as described by Hutchcroft
et al. (9) with minor modifications. Briefly, gels were washed free of
SDS with 40 mM HEPES, pH 7.5, with exchanges every 30 min for ~4 h.
Gels were then incubated with phosphorylation buffer (10 mM
MgCl2, 0.1 µM cold ATP, and
[
-32P]ATP at 5 µCi/ml and 25 mM HEPES, pH 7.5) for 4 h with gentle agitation.
Unincorporated ATP was removed by repetitive 30-min washes with 40 mM
HEPES, pH 7.5, 1%
Na4P2O7
over 4-6 h. After fixation in 50% methanol and 10% acetic acid,
gels were reequilibrated in water, dried, and exposed to X-ray film.
Immunoprecipitations. Endothelial
cells were solubilized in RIPA buffer (10 mM Tris, pH 7.4, 2.5 mM EDTA,
50 mM NaF, 1 mM Na4P2O7 · 10 H2O, 1% Triton X-100, 10%
glycerol, 1% deoxycholate, 1 µg/ml aprotinin, 0.18 mg/ml
phenylmethylsulfonyl fluoride, 0.18 mg/ml orthovanadate) and
centrifuged at 4,000 g, and
the supernatant fractions were incubated with 10 µg of
anti-phosphotyrosine antibody (Transduction Laboratories,
Lexington, KY) or 20 µg of anti-JNK bound to agarose beads (Santa
Cruz Biotechnology, Santa Cruz, CA) overnight at 4°C. Beads were
washed three times with RIPA buffer, SDS-PAGE sample buffer was added,
and the samples were boiled for 1 min. Proteins were separated on 10%
gels containing NKCC1 NH2-terminal
fusion protein and assayed in the gel for kinase activity.
Western blots. Samples were prepared
as above, followed by separation on a 10% polyacrylamide gel and
electroblotting onto a polyvinylidene difluoride
membrane. The membrane was blocked with 5% nonfat dry milk in
Tris-buffered saline (TBS) and incubated overnight with anti-ERK1/2
(Transduction Laboratories) or anti-activated JNK (Promega, Madison,
WI). After the membrane was washed with TBS containing 0.5% Tween 20, it was incubated with horseradish peroxidase-linked donkey
anti-rabbit antibody for 2 h. Proteins were visualized by
enhanced chemiluminescence (Amersham, Arlington Heights, IL).
 |
RESULTS |
Extracts of untreated endothelial cells exhibited several
bands when polyacrylamide gels containing either NKCC1 fusion protein were exposed to
[32P]ATP (Fig.
1, A
and B, left
lanes). The catalytic subunit of protein kinase A
(PKA), which phosphorylates many proteins, was also electrophoresed
into the gel as a positive control, resulting in a single band at the
appropriate size (Fig. 1A,
right lane). This phosphorylation by
PKA is probably not physiologically relevant, since a relatively large
amount of PKA was used and since no region of NKCC1 matches the
consensus motif for PKA phosphorylation (15). One kinase in the cell
extracts, migrating slightly higher than the PKA catalytic subunit at
~46 kDa, was consistently activated in hypertonic medium and
phosphorylated both the
NH2-terminal fusion protein (Fig.
1A, middle
lane) and COOH-terminal fusion protein (Fig.
1B, right
lane). This band was always more prominent with the
NH2-terminal NKCC1 fusion protein,
suggesting that it is a better substrate. Figure
1A also shows a faint,
hypertonically activated kinase at ~90 kDa. This kinase was only
occasionally seen in other assays. When fusion protein was omitted from
the gel, bands were observed only when exposure time was substantially increased (Fig. 1C). None of these
bands were volume sensitive, and they most likely represent
autophosphorylating kinases. Kinase assays were performed with other,
unrelated proteins as substrates to determine the specificity of this
kinase and to confirm that phosphorylation was occurring on the fusion
proteins and not on a contaminating bacterial protein.
There was no hypertonically induced phosphorylation of fusion proteins
containing dihydrofolate reductase or myosin light chain (prepared
using the same expression and purification procedure) or of protamine
(Fig. 2). With myosin light chain as substrate, a faint
band is observed at ~40 kDa but was not reproducible. Although we
have previously shown that cell shrinkage activates MLCK, we would not
expect to see this in the gel assays, since MLCK requires calmodulin as
a cofactor. There was no volume-sensitive phosphorylation of histone II
AS or casein (not shown).

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Fig. 1.
Gel kinase assays of SDS extracts from control (Iso) and hypertonically
shrunken (Hyper) cells and of protein kinase A (PKA) catalytic subunit,
in gels containing NH2-terminal
NKCC1 fusion protein (A),
COOH-terminal NKCC1 fusion protein
(B), or no added protein
(C). Exposure time for
C was increased 3-fold to visualize
the bands. Each autoradiogram is representative of at least 4 additional experiments.
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Fig. 2.
Gel kinase assays of extracts from control (Iso) and hypertonically
shrunken (Hyper) cells using substrates unrelated to NKCC1.
A: hexahistidine fusion protein
containing dihydrofolate reductase (DHFR).
B: hexahistidine fusion protein
containing myosin light chain (MLC).
C: protamine at 1 mg/ml. PKA,
catalytic subunit of protein kinase A. Each autoradiogram is
representative of at least 1 additional experiment. Concentration of
each substrate was 1 mg/ml.
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NKCC1 is activated by isosmotic shrinkage as well as hypertonic
shrinkage, indicating regulation by cell volume rather than osmolarity
(18). To determine whether the NKCC1 kinase is similarly regulated,
cells were shrunk isosmotically by incubation in isosmotic Na-free,
K-free solution containing
N-methyl-D-glucamine
for 15 min as previously described (18). This reduces endothelial cell volume by ~30% and produces an activation of NKCC1 similar to that
seen with the addition of 150 mM sucrose (18). As shown in Fig.
3, activation of the NKCC1 kinase is similar with the two types of shrinkage, indicating that it is cell volume and not
osmolarity that activates the kinase. To determine the intracellular location of the volume-sensitive kinase, endothelial monolayers were
scraped into suspension and sonicated, and fractions were separated by
low-speed centrifugation followed by ultracentrifugation of the
supernatant (100,000 g for 60 min).
The resulting supernatant (cytosol) was concentrated by centrifugation
through a 10,000-mol-wt cutoff membrane and subjected to SDS-PAGE along
with the ultracentrifugation pellet (membranes). The resulting kinase
assay (Fig. 4) demonstrates that the volume-sensitive
NKCC1 kinase is present exclusively in cytosol.

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Fig. 3.
Comparison of isosmotic and hypertonic shrinkage. In-the-gel kinase
assay with NH2-terminal NKCC1
fusion protein as substrate, using extracts from isosmotically shrunken
cells (15-min incubation in isosmotic Na-free, K-free medium), control
cells (no shrinkage), and hypertonically shrunken cells (150 mM sucrose
for 15 min). PKA, catalytic subunit of protein kinase A. Arrow
indicates location of volume-sensitive kinase. Identical results were
obtained in 2 additional experiments.
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Fig. 4.
Cellular localization of NKCC1 kinase. In-the-gel kinase assay of
cytosol or membranes from normal (Iso) or hypertonically shrunken
(Hyper) cells using NH2-terminal
NKCC1 fusion protein as substrate. Approximately 15% of membrane
fraction and 2% of cytosol were loaded on the gel. PKA, catalytic
subunit of protein kinase A. Identical results were obtained in 1 additional experiment. Arrow indicates location of volume-sensitive
kinase.
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The apparent molecular weight of the volume-sensitive kinase suggested
several candidate kinases that were investigated in subsequent studies.
To determine whether volume-sensitive phosphorylation was mediated by
PKA, duplicate samples were electrophoresed into a gel containing NKCC1
NH2-terminal fusion protein, which
was then divided so that the gel kinase assay could be performed in the
absence (Fig.
5A)
and presence (Fig. 5B) of H-89, an
inhibitor of PKA. The intensity of several bands was reduced and the
PKA band was completely eliminated by H-89. However, the 46-kDa,
volume-sensitive band persisted despite complete inhibition of PKA,
indicating that PKA was not the volume-sensitive kinase. In the
presence of H-89, another volume-sensitive kinase is apparent at ~55
kDa. Despite the fact that the p42,44 MAPKs (ERK1 and
ERK2) are reported to be activated by hypertonicity (2, 10), there was
no evidence of this in endothelial cells. Neither ERK1 nor ERK2 was
precipitated by anti-phosphotyrosine antibodies in shrunken cells,
despite the fact that both kinases were present in cell extracts (Fig. 6). Furthermore, pretreatment of cells with PD-98059,
which blocks activation of p42,44 MAPK or with tyrphostin A23, an
inhibitor of tyrosine kinases, did not prevent activation of the
volume-sensitive kinase and did not block the stimulation of
bumetanide-sensitive K+ influx by
cell shrinkage (data not shown).

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Fig. 5.
Effect of PKA inhibition in vitro on phosphorylation of
NH2-terminal NKCC1 fusion protein.
Duplicate samples of extracts from normal (Iso) and hypertonically
shrunken (Hyper) cells and of PKA catalytic subunit were
electrophoresed into a gel containing
NH2-terminal NKCC1 fusion protein.
The gel was then divided and each half was incubated with
[32P]ATP in absence
(A) or presence
(B) of 10 µM H-89. PKA, catalytic
subunit of protein kinase A. Arrows indicate location of
volume-sensitive kinase. Results are representative of 2 additional
experiments.
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Fig. 6.
Effect of cell volume on tyrosine phosphorylation of p42,44
mitogen-activated protein kinase. Cells were exposed to isotonic (290 mosM) or hypertonic (440 mosM) media for 20 min before extraction for
immunoprecipitation and/or immunoblotting with anti-phosphotyrosine
antibody or anti-ERK1/2 antibody as described in
MATERIALS AND METHODS.
A: cell extracts probed with
anti-ERK1/2 antibody. B:
anti-phosphotyrosine immunoprecipitates of same extracts probed with
anti-ERK1/2 antibody. C: same
anti-phosphotyrosine immunoprecipitates probed with
anti-phosphotyrosine antibody (7.5% acrylamide vs. 10% acrylamide in
the previous blots).
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Another member of the MAPK family that is activated by hyperosmolarity
in other cells is JNK, also known as stress-activated protein kinase
(4). Immunoblotting of cell extracts with an antibody specific for the
activated (phosphorylated) form of JNK revealed that this kinase is
activated by hyperosmolarity in endothelial cells as well (Fig.
7). This blot demonstrates activation of both the 46-kDa
(JNK1) and 55-kDa (JNK2) forms of JNK. The identity of the band at
~90 kDa is unknown. Activation of JNK was confirmed by showing
volume-sensitive phosphorylation of c-Jun
NH2-terminal fusion protein at 46 kDa, both by cell extracts and by JNK immunoprecipitates (Fig.
8). Treatment of cells with TNF, which
activates JNK in endothelial cells (17), mimicked the effect of
shrinkage on c-Jun phosphorylation. The additional, weaker band at 55 kDa in the immunoprecipitates is consistent with the larger form of JNK (7) as observed with Western blotting (Fig. 7). Identical results were
obtained with NKCC1 fusion protein as substrate (Fig.
9), demonstrating that JNK is activated in
shrunken cells, that activation of JNK by TNF mimics the effect of cell
shrinkage, and that JNK can phosphorylate NKCC1 in vitro.

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Fig. 7.
Immunoblot of endothelial cell extracts separated by SDS-PAGE, using
antibody against activated c-Jun
NH2-terminal kinase (JNK). Hyper,
cells treated with hypertonic medium for 30 min; Iso, isotonic control;
TNF, cells treated with 2.8 nM tumor necrosis factor- for 30 min.
Arrows indicate 46- and 55-kDa forms of JNK. Similar results were
obtained in 1 additional experiment.
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Fig. 8.
Volume-sensitive phosphorylation of c-Jun by JNK. In-the-gel kinase
assays of extracts (A) and JNK1
immunoprecipitates (B) from
hypertonically shrunken (Hyper), normal (Iso), and TNF-treated (TNF)
cells, using c-Jun1-135
glutathione S-transferase fusion protein as substrate.
Treatment with hypertonic medium or TNF (2.8 nM) was 30 min. Arrows
indicate location of 46-kDa form of JNK. Identical results were
obtained in 1 additional experiment.
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Fig. 9.
Volume-sensitive phosphorylation of NKCC1 by JNK. In-the-gel kinase
assays of extracts (A) and JNK1
immunoprecipitates (B) from
hypertonically shrunken (Hyper), normal (Iso), and TNF-treated (TNF)
cells, using NH2-terminal NKCC1
fusion protein as substrate. Treatment with hypertonic medium or TNF
(2.8 nM) was 30 min. Identical results were obtained in 1 additional
experiment.
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DISCUSSION |
Based on phosphorylation of the volume-regulatory Na-K-2Cl
cotransporter in vitro, we have detected a 46-kDa, cytosolic protein kinase that is regulated by cell volume. This is the first
demonstration of a volume-sensitive kinase that phosphorylates a
relevant target protein. Several findings indicate that this kinase is
the stress-activated protein kinase JNK. The apparent molecular weight
corresponds to that of the smaller form of JNK, and the larger
volume-sensitive kinase occasionally observed corresponds to the larger
form of JNK (7). The NH2 terminus
of c-Jun is a substrate for the volume-sensitive kinase, and TNF, which
activates JNK in aortic endothelial cells (17), also activates a 46-kDa
kinase that phosphorylates NKCC1. Last, JNK was activated in shrunken
cells, and immunoprecipitates of JNK demonstrated volume-sensitive
phosphorylation of NKCC1 at 46 kDa. A volume-sensitive kinase was
occasionally observed at ~90 kDa, the identity of which is unknown.
Because antibodies against activated JNK also recognized a protein of
this size in extracts from shrunken cells, this kinase may be related
to JNK and possibly could represent a dimer. Several other kinases
phosphorylated NKCC1 in vitro but were not influenced by cell volume.
Both JNK1 and JNK2 are known to be activated by hypertonicity (4, 8)
and appear important for osmotic tolerance. Specifically, JNK1 rescues
a mutant yeast with defective growth in hyperosmolar medium (4), and
inhibition of JNK2 blocks osmotic tolerance of cells cultured from
inner medullary collecting ducts (22). However, previous studies have
not determined whether activation by JNK is a result of hypertonicity
or cell shrinkage. Our results provide the first demonstration that JNK
is activated by cell shrinkage in the absence of hyperosmolarity or
increased intracellular ionic strength. This explains why JNK is not
activated by the permeant solute urea (24). Hyperosmolar activation of
other members of the MAPK family has also been observed (10, 20), but
whether this is a response to cell shrinkage and has a functional role
is unknown. No activation of ERK1 or ERK 2 was detected in hypertonically shrunken endothelial cells. Another family of kinases, the Src tyrosine kinases, have been implicated in neutrophils, where
p59fgr and
p56/59hck are phosphorylated and
activated both by hypertonic and isosmotic shrinkage (14). Tyrosine
kinase inhibition prevented phosphorylation and activation of these
kinases and blocked volume-sensitive activation of NHE1, but a specific
role for these kinases has not been demonstrated. No hypertonic
activation of ERK1 or ERK2 was observed in these cells as well.
The results of this study demonstrate that JNK is capable of
phosphorylating NKCC1 in vitro, but this does not necessarily indicate
that JNK phosphorylates NKCC1 in vivo. However, several findings are
consistent with in vivo phosphorylation. The kinase was activated by
both hypertonic and isosmotic shrinkage and did not phosphorylate a
variety of other proteins, indicating some degree of specificity for
NKCC1. No other volume-sensitive kinases capable of phosphorylating
NKCC1 were consistently detected, although we cannot rule out other
kinases that do not survive the extraction procedure. Both the
NH2-terminal and COOH-terminal
putative cytoplasmic domains are phosphorylated, which is consistent
with recent phosphopeptide analysis of endogenously phosphorylated
NKCC1 from duck red blood cells (15). Although it is not possible to
quantitate phosphorylation in our assay, volume-sensitive
phosphorylation was more robust with the
NH2-terminal fusion protein as
substrate, suggesting that it is preferentially phosphorylated. This
would be consistent with the recent demonstration that activation of
shark NKCC1 requires phosphorylation on the
NH2 terminus (1).
The mechanism by which cell shrinkage activates JNK is unknown. The
fact that activation survives denaturing conditions indicates covalent,
rather than allosteric, modification, which is consistent with the
activation of JNK by phosphorylation (3). This indicates the existence
of a kinase cascade whereby JNK, which may not be inherently volume
sensitive, is phosphorylated and activated by an upstream,
volume-sensitive protein kinase. Candidates include several kinases
that are known to phosphorylate and activate JNK (3). This study thus
provides evidence for a volume-sensitive protein kinase cascade that
results in activation of JNK and subsequent phosphorylation of the
Na-K-2Cl cotransporter. Although further studies are needed to
demonstrate that JNK phosphorylates NKCC1 in vivo, this is the first
demonstration of a volume-sensitive protein kinase that can
phosphorylate a volume-regulating target. This kinase cascade may play
an important role in the regulation of cell volume and provide clues
about initial volume-sensing mechanisms. Because JNK is activated by
other stresses, the results suggest an important link between cellular
stress responses and the regulation of cell volume.
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ACKNOWLEDGEMENTS |
This work was supported by National Heart, Lung, and Blood
Institute Grant HL-47449.
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FOOTNOTES |
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: W. C. O'Neill,
Emory Univ. School of Medicine, Renal Division, WMB 338, 1639 Pierce
Dr., Atlanta, GA 30322 (E-mail: woneill{at}emory.edu).
Received 8 February 1999; accepted in final form 18 May 1999.
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