Departments of 1 Biological Chemistry, 2 Immunology, and 3 Biological Regulation, The Weizmann Institute of Science, Rehovot 76100, Israel
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ABSTRACT |
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Recent findings have suggested the
involvement of protein phosphorylation in the regulation of the
epithelial Na+ channel (ENaC). This study reports the in
vitro phosphorylation of the COOH termini of ENaC subunits expressed as
glutathione S-transferase fusion proteins. Channel subunits
were specifically phosphorylated by kinase-enriched cytosolic fractions
derived from rat colon. The phosphorylation observed was not mediated by the serum- and glucocorticoid-regulated kinase sgk. For
the -subunit, phosphorylation occurred on a single, well-conserved threonine residue located in the immediate vicinity of the PY motif
(T630). The analogous residue on
(S620) was phosphorylated as well.
The possible role of
T630 and
S620 in channel function was
studied in Xenopus laevis oocytes. Mutating these residues to alanine had no effect on the basal channel-mediated current. They
do, however, inhibit the sgk-induced increase in channel activity but only in oocytes that were preincubated in low
Na+ and had a high basal Na+ current. Thus
mutating
T630 or
S620 may limit the maximal channel activity
achieved by a combination of sgk and low Na+.
epithelial sodium channel; serum- and glucocorticoid-dependent kinase; protein kinase; Nedd4; channel phosphorylation
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INTRODUCTION |
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ACTIVE NA+
reabsorption in kidney collecting duct, distal colon, lung, and
exocrine glands is mediated by an apical Na+-specific
channel, termed epithelial Na+ channel (ENaC) (3, 12,
15). The channel is composed of three homologous subunits,
denoted ,
, and
(9). They translate 85- to
95-kDa polypeptides that transverse the membrane twice so that both the
COOH and NH2 termini are intracellular (8, 24,
28). The crucial role of ENaC in maintaining salt and water
balance has been conclusively demonstrated by identifying genetic
diseases associated with mutations in ENaC subunits, as well as by the
phenotypic analysis of ENaC knockout mice (for review, see Ref.
16).
Regulation of ENaC in the kidney and intestine is done by a number of
hormones such as the mineralocorticoid aldosterone, the antidiuretic
peptide vasopressin, and insulin (12, 22, 32). Several
recent findings strongly suggest that protein phosphorylation is
involved in some of these mechanisms. First, it has been demonstrated that the serine/threonine kinase [serum- and glucocorticoid-dependent kinase (sgk)] is induced by aldosterone, and coexpressing
sgk with ENaC in Xenopus laevis oocytes results
in a several-fold increase in channel activity (10, 21,
26). Second, aldosterone and insulin were found to increase
phosphorylation of - and
-ENaC in transfected Madin-Darby canine
kidney (MDCK) cells (27). Finally, the response of A6
cells to both aldosterone and insulin, is inhibited by the
phosphoinositide 3-kinase blocker, LY-294002 (7, 23).
A major pathway controlling ENaC's cell surface expression (and
activity) involves its interaction with an underlying protein termed
Nedd4, which has an ubiquitin ligase activity. The WW domains of Nedd4
bind to the proline-rich PY motifs on the COOH termini of - and
-ENaC, leading to channel ubiquitination, internalization, and
degradation (30, 31). Mutating residues in the PY region of
and
impairs this interaction and increases cell surface expression of the channel as well as its open probability (1, 13,
17, 25, 29). This process is sensitive to changes in the
intracellular Na+ activity and is thought to play a role in
the modulation of transport by cell Na+ (11, 14,
18). Its involvement in other, hormone-induced channel-activating pathways has not been established.
The present study investigates phosphorylation of ENaC subunits and its
possible involvement in channel function. The experiments exploited an
in vitro assay in which glutathione S-transferase (GST)
fusion proteins containing cytoplasmic domains of ENaC subunits are
phosphorylated by kinase-enriched cytosolic fractions. By using this
assay, we have demonstrated specific phosphorylation of the COOH
termini of the three ENaC subunits. Phosphorylation of the -subunit
rat ENaC (
-rENaC) by a specific cytosolic fraction occurs on T630, a
conserved residue located in the immediate vicinity of the PY motif.
The analogous residue on the
-subunit (S620) is phosphorylated too,
but in this case 32P incorporates into other residues as
well. Mutating either
S620 or
T630 into alanine inhibits the
sgk-induced increase in ENaC activity in oocytes
preincubated in low-Na+ buffer but not in oocytes
preincubated in normal Na+.
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MATERIALS AND METHODS |
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rDNA.
The COOH termini of the three rat ENaC subunits (-613-699,
-557-638, and
-564-650) were subcloned in downstream
GST in the bacterial expression vector pGEX3X. The desired fragments were amplified by PCR and ligated into the BamH
I/EcoR I site of the above vector in frame with GST. Point
mutations in pGEX3X were introduced by using a QuikChange site-directed
mutagenesis kit. Mutations in full-length ENaC subunits were introduced
by generating overlapping PCR products carrying the desired mutation and reamplifying them with the outside primers. All mutations were
verified by sequencing. The mouse sgk clone used has been described previously (26). cDNAs coding for the
-,
-,
-rENaC in pSPORT-1 were obtained from B. C. Rossier
(Institute of Pharmacology, University of Lausanne). The WWII domain of
rat Nedd4 cloned into pGEX-2TK was kindly provided by D. Rotin
(Hospital for Sick Children, Toronto, ON).
Purification of GST fusion proteins. pGEX plasmids were transformed into the protease-deficient Escherichia coli strain UT5600 (Genetic Stock Center, Yale University, strain 7092). Transformed bacteria were grown for 16-18 h at 37°C with shaking. Cultures were diluted with 400 ml LB-ampicillin and grown for an additional hour, and protein expression was stimulated by the addition of 0.1 mM isopropyl-D-thiogalactopyranoside. Cells were harvested 1 h later, and the fusion proteins were affinity purified on agarose-glutathione beads.
Extraction and fractionation of rat colon cytosol.
Rats (Wistar, 9-11 wk old) were used. Mineralocorticoid
stimulation of Na+ transport was induced by either in vivo
or in vitro incubation with 107-10
8 M
aldosterone for 2-2.5 h. The in vivo hormonal stimulation was done
by subcutaneous implantation of aldosterone-containing osmotic minipumps as described (6, 26). For in vitro incubation, the distal colon was cut longitudinally into two roughly equal portions
that were incubated on gelatine sponge rafts with and without
10
7 M aldosterone as described (5, 6).
The in vitro phosphorylation assay.
GST fusion proteins immobilized on agarose-glutathione beads were
washed in buffer A. Aliquots of a 10-µl bead slurry were mixed with 50 µl kinase-enriched cytosolic fraction and 47 µl buffer A. The reaction was initiated by the addition of 3 µl ATP mixture composed of 100 µl 1 M MgCl2, 50 µl
0.2 mM ATP, and 15 µl [32P]ATP (10 mCi/ml, 3,000 Ci/mmol). Suspensions were shaken for 30 min at 30°C and then
pelleted. Beads were washed several times in HB1B buffer (20 mM HEPES,
pH 7.7, 50 mM NaCl, 0.1 mM EDTA, 25 mM MgCl2, and 0.05%
Triton X-100) and then suspended in SDS gel sample buffer. Samples were
boiled for 5 min, resolved on a 12% SDS-PAGE gel, and exposed to X-ray
film. In some experiments, recombinant and activated human
sgk (Upstate Biotechnology) has been used instead of colonic
cytosol. The enzymatic activity of the purified kinase was determined
by phosphorylating the synthetic substrate Crosstide (19).
The assay mixture contained 25 ng purified enzyme, 1 nmol Crosstide,
and 10 µCi [-32P]ATP in 50 µl of the following
buffer (in mM): 5 MOPS (pH = 7.2), 5
-glycerophosphate, 1 EGTA,
0.2 deaerated sodium orthovanadate, 0.2 dithiothreitol, 15 MgCl2, and 100 µM nonradioactive ATP. Each observation
reported below was confirmed by at least three independent measurements
using different cytosolic preparations.
Phosphoamino acid analysis.
Gel pieces containing 32P-labeled bands were cut out and
minced into small fragments in a solution composed of 50 mM ammonium bicarbonate, 0.1% SDS, and 5 mM -mercaptoethanol. Proteins were extracted by a 2-h incubation with shaking, and gel pieces were removed
by centrifugation. 32P-labeled proteins were precipitated
with trichloroacetic acid, pellets were dried, suspended in 6N HCl, and
hydrolyzed for 2 h at 110°C. The hydrolyzed amino acids were
mixed with phosphoamino acid markers and resolved on a TLC plate.
Markers were visualized by spraying the plate with ninhydrin and
comparing them with positions of the 32P-labeled spots.
Functional expression in X. laevis oocytes. Stage V-VI oocytes were injected with cRNA mixtures containing 2.5 ng of each ENaC subunit and sgk. Oocytes were maintained for 2-5 days at 17°C in either high-Na+ (96 mM NaCl + 10 µM amiloride) or low-Na+ (10 mM NaCl + 86 mM choline chloride) solutions. To monitor channel activity, oocytes were perfused in high-Na+ medium without amiloride (irrespective of the preincubation buffer), and channel densities were evaluated by recording the amiloride-sensitive (10 µM) current levels as described (26). For in vitro phosphorylation, groups of ~10 oocytes were homogenized in buffer A using a loose-fitting glass homogenizer. They were sedimented at 15,000 g, and the supernatant was collected and used in an in vitro phosphorylation assay.
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RESULTS |
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To study phosphorylation of the COOH termini of ENaC subunits by
cellular kinases, rat colon cytosolic extracts were fractionated on a
DE-52 column. Four fractions eluted with 0-0.5 M NaCl were selected for further investigation. They were reacted with the COOH
tails of ENaC subunits expressed as GST fusion proteins, in the
presence of [-32P]ATP, Mg2+, and
phosphatase inhibitors. Figure
1A depicts the incorporation of 32P into the
-COOH tail by the four column fractions.
The highest activity was obtained with the third fraction (0.2 M NaCl),
and it was therefore used for all subsequent experiments. A much
smaller signal was obtained with whole cytosol, presumably due to its high-phosphatase activity or the presence of other ATP-hydrolyzing enzymes. Phosphorylation of
- and
-tails was observed as well, whereas GST and the WWII domain of Nedd4 did not incorporate
significant amounts of 32P (Fig. 1, B and
C). Usually, the GST-ENaC fusion proteins were partly
degraded, resulting in multiple bands in Coomassie staining, not all of
which are phosphorylated (cf. Fig. 1B). In all experiments, phosphorylation was quantified by phosphoimaging of the band
corresponding to the full-length protein, and values were normalized to
the Coomassie staining of the same band. Thus results reported below are independent of protein degradation.
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An amino acid analysis of the phosphorylated species is depicted in
Fig. 2A. For -GST,
32P incorporated into threonines, whereas in the case of
, both serines and threonines were labeled. No phosphorylation of
tyrosines could be detected for any of the subunits. Next, we attempted to identify the phosphorylated residues in these subunits by mutating various conserved serines and threonines. For
, phosphorylation by
the above cytosolic fraction took place only on T630. Mutating this
residue into alanine completely abolished 32P incorporation
into the fusion protein (Fig. 2B). Mutating the comparable
residue on
(S620A) had a partial effect. In three independent
experiments using different cytosolic preparations, the mutation S620A
inhibited 56.4 ± 5.1% of the amount of 32P
incorporated into
. Thus this residue is likely to be the
phosphoserine detected in the amino acid analysis. However, another,
yet unidentified, residue is the major phosphorylation site.
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To establish that the same kinase phosphorylates S620 and
T630,
we further purified the above kinase activity on a monoQ column. A
particular fraction eluted from the column by ~0.19 M NaCl
incorporated 32P into
S620 with minimal phosphorylation
of other residues, and the same fraction also phosphorylated
T630
(Fig. 2D).
T630 and
T620 are located in the immediate
vicinity of the PY motif and are well conserved in evolution (Fig.
2E). Another well-conserved threonine in this region (i.e.,
T613 or
T623) is not phosphorylated by the above cytosolic
fraction (Fig. 2, B and C). It should however, be
emphasized that identity of the major phosphorylated residue was
strongly affected by the cytosol fractionation procedure. A detailed
fractionation on a monoQ column did identify a kinase acting on
-T623, as well as on other residues (Shi H, Seger R, and Garty H,
unpublished observations).
Next, we examined whether pretreatment of rat colon with aldosterone
increases the ability of cytosolic kinases to phosphorylate ENaC's
COOH tails. Two experimental protocols were used. The first was an in
vitro incubation of tissue segments with
108-10
7 M aldosterone or diluent for
2.5 h. The second was implantation of aldosterone containing
osmotic minipumps and in vivo aldosterone perfusion. Both
protocols have been shown before to manifest the hormonal effects on
ENaC and sgk (6, 26). The results of these
experiments were quite variable. In some experiments, a clear
aldosterone-induced stimulation of
- and
-phosphorylation was
apparent, but in general this effect was not very reproducible.
It has been recently demonstrated that the serine/threonine kinase
sgk is strongly induced by aldosterone and it can activate ENaC on coexpression in X. laevis oocytes (10, 21,
26). Thus sgk may be either the
channel-phosphorylating kinase or an "upstream" kinase, whose
induction leads to the activation of the channel-phosphorylating
kinase. These possibilities were assessed in the experiments summarized
in Figs. 3 and 4. First, it was tested whether recombinant-activated
sgk can directly phosphorylate GST fusion proteins. No
significant incorporation of 32P into any of the channel
subunits or the WWII domain of Nedd4 could be detected in these
experiments (Fig. 3A). This is
in contrast to a strong phosphorylation of by epithelial cytosol,
measured in parallel (last lane in Fig. 3A). The recombinant
sgk used was active and effectively phosphorylated the
synthetic peptide Crosstide (Fig. 3B). Thus sgk
is not likely to be the COOH-tail-phosphorylating kinase monitored in
this study.
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The possibility that sgk is an upstream kinase mediating the
current phosphorylation was assessed by the following two protocols. In
the first, whole cytosol or the kinase-enriched cytosolic fraction was
preincubated with purified recombinant sgk and then assayed for phosphorylation of GST-. Such pretreatment was found to have no
effect on
-COOH-tail phosphorylation (data not shown). A second approach was to compare in vitro phosphorylation of the GST fusion proteins by fractionated cytosol extracted from X. laevis
oocytes, which do or do not overexpress sgk. In these
experiments oocytes were injected with ENaC ± sgk and
assayed for channel activity 3 days later (see below). After an
sgk-induced activation of ENaC was established, oocytes were
homogenized, and the cytosol was fractionated and assayed for
phosphorylation of GST-
by the above in vitro protocol. In
this case, a significant but very small sgk-induced increase
of GST-
phosphorylation was detected (Fig. 3C).
Finally, we tested whether mutating T630 or
S620 into alanine
affects channel activity in X. laevis oocytes. It has been previously reported that mutating these residues into either alanine or
glutamic acid has no effect on the current amplitude under normal
conditions (25). To further explore a possible role of these residues under specific conditions, wild-type and mutated ENaC
subunits were expressed with and without sgk. Oocytes were incubated in two different solutions for 48-72 h. In the first, they were maintained in a low-Na+ medium, a treatment that
has been shown before to maximalize ENaC's activity by impairing its
interaction with Nedd4 (11, 14, 18). In the second,
oocytes were maintained in the regular high-Na+ buffer.
These oocytes expressed ~3-fold lower Na+ currents and
also had lower reversal potentials (6.2 ± 0.7 vs. 30 ± 3.3 mV).1 Hence, the decreased current is likely
to reflect downregulation of ENaC by an increased intracellular
Na+ (18). In these low-current oocytes
the two-point mutations were without effect on the macroscopic current
or its activation by sgk (Fig.
4A). A profound effect of
these mutations on the response to sgk was seen, however, in
the oocytes that were maintained in a low-Na+ buffer (Fig.
4B). In the wild-type channel, the stimulatory actions of
low Na+ and sgk were additive, and each of the
two stimuli substantially increased the current amplitude. However, in
oocytes expressing mutated subunits, sgk could not increase
the macroscopic current beyond the level already achieved by
preincubation in a low-Na+ solution. A similar phenomenon
has been noticed by first recording channel activity in oocytes
incubated in high Na+ and then transferring the same
oocytes to a low-Na+ medium for 24-48 h (not shown).
Table 1 averages data from a large number
of oocytes obtained from three to four different batches of oocytes.
The results clearly demonstrate the lack of response to sgk
by the mutated ENaC in oocytes incubated in low-Na+ but not
in high-Na+ solutions. We have also found that truncating
the whole COOH tail of
or
, but not
, blunts the response to
sgk, and this effect is independent of the incubation
conditions (Table 1).
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Possible interpretations of the above findings are discussed in the
next section. One possibility, however, is that different cytosolic
Na+ activities in the two groups of oocytes affect the
phosphorylation efficiencies of - and
-ENaC. To evaluate such an
option, we have tested for effects of Na+ on the in vitro
incorporation of 32P into GST-
. In these experiments,
the fusion protein was phosphorylated in modified solutions that
contained either 50 or <1 mM Na+. The Na+
concentration in the reaction mixture was found to have no effect on
the phosphorylation efficiency (data not shown). Thus the different response with high and low Na+ is not likely to reflect a
direct effect of Na+ on the phosphorylation reaction.
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DISCUSSION |
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The present study examined phosphorylation of the COOH termini of
ENaC subunits using an in vitro assay. It was motivated by reports
indicating a protein kinase-dependent regulation of the channel
(7, 10, 20, 21, 27). Indeed, kinase-enriched cytosolic
fractions could substantially phosphorylate all three subunits, whereas
no incorporation of 32P into GST or the WWII domain of
Nedd4 has been seen. Specificity of the response was further
established by demonstrating that mutating a single, well-conserved S/T
in the immediate vicinity of the PY motif fully blocked incorporation
of 32P into the - and
-fusion proteins. We suggest
that these residues are specifically phosphorylated by a cytosolic
kinase and such phosphorylation plays a role in channel function. One
cannot, however, rule out the possibility that the above mutations
indirectly affect incorporation of 32P at another site. It
should also be stressed that the relative intensities of different
phosphorylation events largely depend on the cytosol fractionation
procedure and the presence of different phosphatases in the tested
fraction. Thus no 32P incorporation could be seen when the
fusion proteins were phosphorylated by whole cytosol. On the other
hand, a more detailed fractionation by fast-performance liquid
chromatography identified additional phosphorylated residues, not
detected using the crude DE-52 fraction (Shi H, Reuveny E, Seger R, and
Garty H, unpublished observations).
Several approaches have been used to elucidate cellular roles of the
phosphorylations observed. First, we explored whether pretreating the
tissue with aldosterone increases the incorporation of 32P
into GST- or -
. This hormone is a major regulator of ENaC activity and has been reported to increase COOH-tail phosphorylation in
transfected MDCK cells (27). The results were quite
variable and essentially negative. This, however, does not exclude
involvement of aldosterone in channel phosphorylation. Cell
homogenization and cytosol fractionation should cause a large dilution
of small factors and loss of compartmentalization, which enable new
protein/protein interactions. These may reverse or blunt an in vivo
activation of kinases by aldosterone.
Next, we evaluated the possible involvement of sgk in the
phosphorylation of T630 or
S620. The enzyme did not directly
phosphorylate the COOH termini of any of the three ENaC subunits (Fig.
3), nor did it facilitate phosphorylation by the kinase-enriched
cytosolic fraction. Some activation could be observed using cytosol
extracted from sgk-overexpressing oocytes, but this effect
was quite small (Fig. 3C). Again, these results do not
exclude an in vivo mediating role of sgk that is blunted
during cell homogenization and fractionation.
Finally, we examined effects of the phosphorylated residues on channel
activity in X. laevis oocytes. Mutating the phosphorylated residues into alanine had no effect on the basal current or its activation by Na+ withdrawal. They did, however, impair the
response to sgk measured in oocytes that were preincubated
in low-Na+-containing solution that had high channel
activity. A similar inhibition was recorded in oocytes that had a
high basal activity due to the truncation of or
; however, this
effect was independent of the Na+ activity in the
incubation medium (Table 1). This observation suggests a physiological
role of
T630 and
S620, but its interpretation is not trivial. The
simplest interpretation is that these mutations tend to limit the
maximal channel activity in the oocyte membrane. Both sgk
and low-external Na+ increase the amiloride-sensitive
Na+ currents by elevating cell surface expression of ENaC
without increasing the total amount of channel protein (4,
18). Thus one may speculate that mutating
T630 and/or
S620
decreases the size of an sgk-dependent intracellular pool
from which channels are recruited to the plasma surface. Such an effect
may have no consequence on the cell response to sgk in
oocytes expressing low levels of ENaC on the cell surface (i.e., have a
large intracellular pool), but it may be inhibitory if the
intracellular pool is further depleted by the preincubation in a
low-Na+ buffer. Alternatively, the response to
sgk may be mediated by a Na+-dependent
process(es) that also requires phosphorylation of the above residues.
According to these interpretations, phosphorylation of
S620 and
T630 is not directly related to the sgk response. The
first possibility also predicts that dissociating ENaC from Nedd4 by
truncating
and/or
should blunt the response to sgk, independently of external Na+. This indeed was found to be
the case (Table 1). This result, however, differs from the one reported
in a previous study (4). The reason for the difference
between the two sets of data is not clear. It may reflect different
expression levels, or the fact that the channels studied in
(4) lacked all three COOH-tails, whereas we have mutated
one subunit at a time. Another unexpected observation is that
incubation in high Na+ lowered the Na+ currents
expressed, in spite of the presence of amiloride during the incubation
period. This is likely to reflect Na+ loading through
amiloride-insensitive pathways as was indeed indicated by the lower
reversal potential. Other possibilities, such as a toxic effect of
amiloride cannot be ruled out.
In summary, the present study identifies phosphorylated residues in the
COOH termini of - and
-ENaC and provides an initial indication of
their physiological role.
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ACKNOWLEDGEMENTS |
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This study was supported by research grants from the Israel Science Foundation and The Dolfi and Lola Ebner Center for Biomedical Research (to H. Garty and E. Reuveny).
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FOOTNOTES |
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In both cases currents were recorded in the same high-Na+ buffer.
Address for reprint requests and other correspondence: H. Garty, Dept. of Biological Chemistry, The Weizmann Institute of Science, Rehovot 76100 Israel, (E-mail: h.garty{at}weizmann.ac.il).
1 In both cases, currents were recorded in the same high-Na+ buffer.
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.
Received 2 October 2000; accepted in final form 11 February 2001.
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