Tài liệu Báo cáo khoa học: Casein kinase 2 specifically binds to and phosphorylates the carboxy termini of ENaC subunits ppt

Casein kinase 2 specifically binds to and phosphorylates the carboxy
termini of ENaC subunits
Haikun Shi
1
, Carol Asher
1
, Yuval Yung
2
, Luba Kligman
1
, Eitan Reuveny
1
, Rony Seger
2
and Haim Garty
1
1
Department of Biological Chemistry, and
2
Department of Biological Regulation, The Weizmann Institute of Science, Rehovot, Israel
A number of findings have suggested the involvement of
protein phosphorylation in the regulation of the epithelial
Na
+
channel (ENaC). A recent study has demonstrated that
the C tails of the b and c subunits of ENaC are subject to
phosphorylation by at least three protein kinases [Shi, H.,
Asher, C., Chigaev, A., Yung, Y., Reuveny, E., Seger, R. &
Garty, H. (2002) J. Biol. Chem. 277, 13539–13547]. One of
them was identified as ERK which phosphorylates bT613
and cT623 and affects the channel interaction with Nedd4.

The current study identifies a second protein kinase as casein
kinase 2 (CK2), or CK-2-like kinase. It phosphorylates
bS631, a well-conserved serine on the b subunit. Such
phosphorylation is observed both in vitro using glutathi-
one-S-transferase–ENaC fusion proteins and in vivo in
ENaC-expressing Xenopus oocytes. The c subunit is weakly
phosphorylated by this protein kinase on another residue
(cT599), and the C tail of a is not significantly phosphory-
lated by this kinase. Thus, CK2 may be involved in the
regulation of the epithelial Na
+
channel.
Keywords: casein kinase 2; ENaC; epithelial Na
+
channel;
phosphorylation.
Active Na
+
reabsorption in kidney collecting duct, distal
colon, lung, and exocrine glands is mediated by an apical
Na
+
specific channel, termed ENaC (epithelial Na
+
-
channel) [1–3]. The channel is a major target of the action of
several hormones such as the mineralocorticoid aldosterone,
the anti-diuretic peptide vasopressin, and insulin [1,4]. It is
composed of three homologous subunits (a, b,andc)which
transverse the membrane twice so that both the C and N

termini are intracellular [5–8]. Cell surface expression of
ENaC is determined by an interaction between the C tails of
b and c and the ubiquitin ligase Nedd4. The WW domains
of Nedd4 bind to the proline-rich PY motifs of b and
cENaC, leading to channel ubiquitination, internalization
and degradation [9,10]. Recently it was demonstrated that
the aldosterone-induced kinase sgk (serum and glucocorti-
coid dependent kinase) phosphorylates Nedd4-2 on two
serines and thereby reduces its interaction with the channel
[11,12]. In addition, aldosterone and insulin, as well as
intracellular signalling components such as protein kinase C
and protein kinase A, were found to increase the in vivo
phosphorylation of the C termini of both b and cENaC [13].
We have previously demonstrated phosphorylation of the
C termini of ENaC subunits, expressed as glutathione-S-
transferase (GST) fusion proteins by crude epithelial
cytosolic fractions [14]. Fractionating rat colon cytosol by
ion exchange chromatography revealed at least three
kinases phosphorylating b and cENaC [15]. One of these
was identified as ERK (extracellular regulated kinase) which
phosphorylates two conserved threonines in the immediate
vicinity of the PY motif bT613 and cT623. Phosphorylation
of these residues increases the channel’s affinity towards
WW sequences and down-regulates the channel activity [15].
A second peak corresponded to an as yet unidentified
protein kinase which phosphorylates bS623 and cT630
[14,15]. The third, major peak of protein kinase activity is
reported here. In the current paper we provide evidence that
this protein kinase is likely to be casein kinase 2 (CK2) and
demonstrate that the residues phosphorylated by it are

bS631 and cT599.
EXPERIMENTAL PROCEDURES
Materials
32
P orthophosphate (10 mCiÆmL
)1
), [c-
32
P] ATP
(10 mCiÆmL
)1
, 3000 Ci mmol
)1
)and[c-
32
P] GTP
(10 mCiÆmL
)1
, 5000 CiÆmmol
)1
)werefromAmersham
Pharmacia Biotech; glutathione–agarose beads, dephospho-
rylated casein, heparin, 2,3-diphosphoglycerate and rat liver
CK2 (a mixture of a
2
b
2
and aa¢b
2
)werefromSigma-

Aldrich Fine Chemicals. Human recombinant CK2
(Escherichia coli) was from Calbiochem. An antibody
directed against the a and a¢ subunits of CK2 was kindly
provided by D. W. Litchfield, University of Western
Ontario, Canada.
Methods
Construction, expression and purification of GST fusion
proteins containing the cytoplasmic C-tail domains of
ENaC subunits, were carried out as described before [14].
Point mutations were introduced by using the Quik-
Change
TM
site-directed mutagenesis kit (Stratagene) and
verified by sequencing. Extraction of rat colon cytosol, its
fractionation by ion exchange chromatography and phos-
phorylation of GST–ENaC fusion proteins by either
Correspondence to H. Garty, Department of Biological Chemistry,
The Weizmann Institute of Science, Rehovot 76100 Israel.
Fax: +972 8 9344177, Tel.: +972 8 9342706,
E-mail:
Abbreviations: ENaC, epithelial Na
+
channel; CK2, casein kinase 2;
GST, glutathione S-transferase; DPG, 2,3-diphosphoglycerate;
HA, hemagglutinin A.
(Received 15 March 2002, revised 7 July 2002, accepted 30 July 2002)
Eur. J. Biochem. 269, 4551–4558 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03154.x
fractionated cytosol or purified enzyme are detailed in Shi
et al. [15]. Kinetic parameters were determined by phos-
phorylating GST–bENaC with human recombinant CK2 at

different substrate concentrations or for different time
periods. Stochiometry was determined by excising the
phosphorylated band from the gel and determining its
radioactivity and protein content. Phosphoamino-acid
analysis was performed as described previously [14].
Specific binding of kinases to GST fusion proteins was
determined by coprecipitation. A 200-lL aliquot of the
desired cytosolic fraction was diluted with 70 lL water and
30 lL10· binding buffer composed of 1.5
M
NaCl,
220 m
M
Hepes pH 7.7, 20 m
M
MgCl
2
, 0.75% Triton
X-100, 200 m
M
b-glycerolphosphate, 1 m
M
EDTA, 1 m
M
sodium orthovanadate and protease inhibitors (1 m
M
phenylmethanesulfonyl fluoride, 10 lgÆmL
)1
aprotinin,
10 lgÆmL

)1
leupeptin, 2 lgÆmL
)1
pepstatin A). The solution
was mixed with 5–20 lg GST-fusion protein immobilized
on glutathione-agarose beads, and mixtures were rotated at
4 °C for 2 h. Beads were sedimented by centrifugation and
washed six times using the following protocol: (a) two
washes in 0.5
M
LiCl, 100 m
M
Tris/HCl pH 8.0; (b) two
more washes in 1
M
NaCl plus HB1B buffer (20 m
M
Hepes
pH 7.7, 50 m
M
NaCl, 0.1 m
M
EDTA, 25 m
M
MgCl
2
,
0.05% Triton X-100); (c) one wash in buffer A [50 m
M
b-glycerophosphate pH 7.3, 1.5 m

M
EGTA, 1.0 m
M
EDTA, 1.0 m
M
dithiothreitol, and 0.1 m
M
de-aerated
sodium orthovanadate]; (d) A final wash in a kinase assay
buffer composed of 20 m
M
Hepes pH 7.7, 20 m
M
MgCl
2
,
20 m
M
2-glycerolphosphate, 2 m
M
dithiothreitol, 0.1 m
M
sodium orthovanadate. Beads were suspended in 30 lLof
the above kinase assay buffer, a second substrate was added,
and phosphorylation was initiated by the addition of 2 l
M
ATP plus 2 lCi [c-
32
P]ATP. The suspension was incubated
for 30 min at 30 °C and the reaction was stopped by

washing. Beads were suspended in 30 lL Laemmli sample
buffer, boiled for 5 min, resolved by 12% acrylamide SDS/
PAGE and visualized by autoradiography.
ÔIn-gelÕ assays were performed as described in [16]. In
brief, SDS/polyacrylmide gels were cast with 0.5 mgÆmL
)1
GST-b,GST-c, or casein in the gel polymerization solution.
Cytosolic fractions (50 lL) and purified CK2 (0.05 U) were
applied to different wells of the gel and resolved by
electrophoresis. The gel was then soaked in 20% 2-propra-
nol in 50m
M
Tris/HCl, pH 8.0, and then in 50 m
M
Tris/HCl
pH 8.0 plus 5 m
M
2-mercaptoethanol. Proteins were dena-
turated by soaking the gel in 6
M
urea and then renaturated
in 50 m
M
Tris/HCl, pH 8.0, 0.04% Tween-20 and 5 m
M
2-mercaptoethanol. For in-gel phosphorylation, the gel was
first preincubated for 30 min at 30 °Cin20m
M
Hepes
pH 8.0 plus 20 m

M
MgCl
2
. ATP (100 lCi [c-
32
P]ATP plus
20 l
M
nonradioactive ATP) and 2 m
M
dithiothreitol were
added and the phosphorylation was allowed to proceed for
2hat30°C. The gel was washed in 5% (w/v) trichloro-
acetic acid plus 1% (w/v) sodium pyrophosphate, dried and
subjected to autoradiography.
Western blotting of CK2 was carried out as follows:
20 lL aliquots of different MonoQ fractions were resolved
by SDS/PAGE on a 10% acrylamide gel. Proteins were
blotted onto a nitrocellulose membrane, blocked with 5%
low fat milk, and probed with polyclonal antibodies against
the a and a¢ subunits of CK2 (dilution 1 : 1000). Blots were
overlaid with horse radish peroxidase conjugated goat
antirabbit antibody (1 : 10 000), and binding was detected
by enhanced chemiluminesence.
For functional expression in Xenopus oocytes, stage V–VI
oocytes were injected with cRNA mixtures containing
2.5 ng each ENaC subunit. Oocytes were incubated at
17 °C in a medium that contained 96 m
M
NaCl and 10 l

M
amiloride. Electrophysiological measurements were per-
formed 48–72 h after the injection by means of the two-
electrode voltage clamp technique. Channel activity was
determined as the amiloride-sensitive current amplitudes
monitored at )100 mV. To study phosphorylation of
bENaC, oocytes were injected with a cRNA mixture
containing a, c, and a hemagglutinin A (HA)-tagged
bENaC. The HA epitope was introduced in the ecto
domain at a position shown before not to affect channel
activity [17], and the construct was kindly provided by
B. Schwappach (Zentrum fu
¨
r Molekulare Biologie, Uni-
versita
¨
t Heidelberg, Germany). Injected oocytes were divi-
ded into two groups each containing  40 oocytes and
incubated with either 100 lCi [
35
S]methionine (2 days) or
3.3 mCi
32
Pi (4 h).
32
P- and
35
S-labelled oocytes were
washed and homogenized in buffer A containing protease
inhibitors and membranes were isolated by centrifugation

through a sucrose cushion. Membranes were solubilized in
1% Triton X-100 in buffer A and centrifuged for 5 min at
11 000 g to remove insoluble material. Aliquots of
 400 lL detergent soluble membrane protein extracts were
incubated for 12–16 h at 4 °Cwith2lg of a mouse mAb
anti-HA antibody (clone 12CA5, Roche Molecular Bio-
chemicals) and then for another 2 h with Protein A
Sepharose beads. The beads were sedimented, washed twice
in buffer A + 0.1% Triton-X-100, and a third time in buffer
A+0.5
M
LiCl. Immunopellets were suspended in SDS
sample buffer, resolved by SDS/PAGE (8% acrylamide gel)
and assayed for
35
Sand
32
P radioactivity by phosphor-
imaging.
RESULTS
Previous studies have demonstrated phosphorylation of the
C termini of ENaC subunits expressed as GST fusion
proteins by various cytosolic fractions extracted from rat
distal colon [14,15]. In particular, three peaks of kinase
activity which phosphorylate the C termini of b and cENaC
were identified by ion exchange chromatography. Two of
them have been studied before [14,15]. The third, a major
peak eluted at  0.25–0.27
M
NaCl, has not been charac-

terized yet. Fig. 1A depicts phosphorylation of GST-b and
-c by fractions 66–84 eluted from the monoQ column. In
both cases a peak was observed around fraction 76
( 0.26
M
NaCl, Fig. 1B). This kinase phosphorylated
GST-b better than GST-c, while GST and GST-a did not
incorporate a significant amount of
32
P (Fig. 1C). (Usually,
the GST–ENaC fusion proteins were partly degraded,
resulting in multiple bands by Coomassie blue staining, not
all of which are phosphorylated. This however, does not
pose a problem as phosphorylation could be quantified by
phosphorimaging of the band corresponding to the full-
length protein, and normalizing to the Coomassie blue
staining of the same band.)
It was next demonstrated that the kinase eluted in
fraction 76 tightly binds to both b and c C tails. In this
assay, GST, GST-b or GST-c immobilized on glutathione
4552 H. Shi et al. (Eur. J. Biochem. 269) Ó FEBS 2002
beads was first incubated with the above cytosolic fraction
in the absence of ATP. The beads containing the fusion
proteins (and other proteins associated with them) were
precipitated, washed stringently, and incubated with
[
32
P]ATP with no added kinases. Phosphorylation of the
fusion protein under these conditions is possible only if the
phosphorylating kinase is tightly bound to its substrate. As

shown in Fig. 2 both b and c could be effectively
phosphorylated under these conditions (second and sixth
lanes). Moreover, it was found that the kinase precipitated
by one subunit could also phosphorylate the other. In this
case, the kinase was precipitated by one of the two fusion
proteins (1st) and the other subunit (2nd) was added only to
the final phosphorylation mixture. Both fusion proteins
could be phosphorylated irrespective of the order of
addition, indicating that the kinase bound to GST-b can
phosphorylate GST-c and vice versa. This protocol has also
been used to demonstrate that binding requires the ENaC
domain of the fusion protein and much less kinase activity is
precipitated by GST alone.
Next, b and c residues phosphorylated by this kinase were
identified. Phospho amino acid analysis demonstrated that
in b, all of the radioactivity is incorporated into serines,
while in c the phosphorylated residues are threonines
(Fig. 3A). No tyrosine phosphorylation could be detected
for any of the subunits. Identity of the phosphorylated
residues was further determined by assessing effects of
various point mutations on
32
P incorporation into GST-b
and -c. Accordingly, several serines and threonines on the C
termini of b and c weremutatedtoalanineandexamined
for phosphorylation by fraction 76. In b the major
phosphorylated residue was S631 and mutating it to alanine
blocked almost all
32
P incorporation (Fig. 3B). bS631 is

located past the PY motif and is well conserved among
known b sequences (Fig. 3D). Partial inhibition of b
Fig. 1. Phosphorylation of different fusion proteins by kinase enriched fractions. (A) Matched comparison of the phosphorylation of GST-b and
GST-c by monoQ fractions 64–84. (B) Quantification of the phosphorylation of b and c by fractions 66–86 (a different experiment from the one
shown in A). (C) GST fusion proteins containing the C termini of a, b,andcENaC were phosphorylated by fraction 76. Autoradiogram and
Coomassie blue staining are shown.
Fig. 2. Binding of kinases to GST-b and -c. GST, GST-b or GST-c
(1st) were first incubated with or without cytosolic fraction 76, and
then precipitated. The pellets were washed stringently as described in
Experimental procedures and phosphorylation was induced by the
addition of [c-
32
P]ATP in the presence or absence of a second substrate
(2nd). Autoradiogram and Coomassie blue staining are shown.
Ó FEBS 2002 CK2-mediated phosphorylation of ENaC (Eur. J. Biochem. 269) 4553
phosphorylation was seen also upon the mutation of a
neighbouring residue, S633. This residue may have an
indirect effect on the incorporation of
32
P into S631. The
analogous residue on the c subunit (cT644) did not
incorporate
32
P and this subunit appeared to be phospho-
rylated mainly on T599, a nonconserved threonine that
precedes the PY motif (Fig. 3C and D). Mutating other
conserved serine/threonines such as bS620 or cT623 and
cT630hadnoeffecton
32
P incorporation by fraction 76

(data not shown). bS631 and S633 have recently been shown
to be involved in the regulation of ENaC in mandibular
duct cells [18]. As above, the homologous c residue was not
involved in any such mechanism.
To determine whether bS631 is phosphorylated also
in vivo the a-, c-, and HA-tagged bENaC were expressed in
Xenopus oocytes and metabolically labelled with either
[
35
S]methionine or
32
Pi. The b subunit could be specifically
immunoprecipitated from oocytes by an anti-HA antibody
(Fig. 4A). It was endogenously phosphorylated, and the
amount of phosphorylation was considerably lower if S631
was mutated into alanine (Fig. 4B,C). Incorporation of
32
P
into bS631A was 0.55 ± 0.07 of the value obtained for wild
type b (mean ± SEM of three independent experiments).
Thus, S631 accounts for  50% of the endogenous phos-
phorylation of bENaC. Possible effects of this phosphory-
lation on channel activity were examined by recording the
amiloride-sensitive Na
+
current amplitudes in Xenopus
oocytes expressing wild-type and mutated ENaC. bS631
was mutated into either glutamic acid or alanine. It was
found that neither mutation evokes a significant effect on
the macroscopic amiloride blockable current measured in

this system (Fig. 5).
Both bS631 and cT599 are located in a cluster of acidic
residues and in particular have an acidic group at position
n + 3. This suggests that the phosphorylating protein kinase
might be CK2 [19]. The next set of experiments provided
further evidence that the cytosolic kinase phosphorylating
bS631 and cT599 is indeed likely to be CK2. First, Western
blot analysis of the active cytosolic fractions was performed
using polyclonal antibodies against the a and a¢ subunits of
CK2. The catalytic subunit of the enzyme could indeed be
detected in fractions 70–80 and the predominant isoform
was a (Fig. 6). (Protein samples from column fractions have
high concentration of NaCl resulting in somewhat diffused
bands.)
Next we showed that purified CK2 can effectively
phosphorylate GST-b and -c and that this phosphorylation
occurs primarily on bS631 and cT599 (Fig. 7A). Phospho-
rylation of bS631 was further investigated using human
recombinant CK2. The phosphorylation reaction was linear
for at least 40 min The fraction of protein phosphorylated
during a 3-h incubation corresponded to  10% of the total
protein, i.e. incorporation of
32
PintobS631 is not residual.
The K
m
for phosphorylation of bS631 was estimated to be
Fig. 3. Identification of the phosphorylated residues. (A) Amino acid analysis of phosphorylated GST-b and -c. The circles indicate positions of
marker phospho serine, threonine and tyrosine. (B and C) Phosphorylation of wild-type and mutated b and c subunits by fraction 76. Auto-
radiograms (top) and Coomassie blue stained gels (bottom) are shown. (D) Sequence alignments of the C termini of b and cENaC from different

species.
4554 H. Shi et al. (Eur. J. Biochem. 269) Ó FEBS 2002
1.4 l
M
(Fig. 7B). This value compares well to the K
m
measured for b-casein (36.5 l
M
) or specific substrate
peptides [20].
A unique feature of CK2 is its ability to use both ATP
and GTP as phosphate donors [21]. Other criteria for CK2-
mediated phosphorylation are inhibition by heparin and
2,3-diphosphoglycerate as well as the ability to phosphory-
late casein. Experiments summarized in Fig. 8 indicate that
the ENaC phosphorylating kinase eluted in fraction 76 does
indeed have CK2 characteristics. The two fusion proteins
could be phosphorylated by GTP, and the GTP-dependent
phosphorylation took place at bS631 and cT599 (Fig. 8A).
Both heparin and DPG inhibited phosphorylation by
fraction 76 of either GST-b or GST-c.
Finally, we used an Ôin-gelÕ assay to determine the
electrophoretic mobility of the kinase eluted in fraction 76,
and compare it to that of CK2. In this assay, GST, GST-b,
GST-c and casein were copolymerized with polyacrylamide
to form substrate-containing SDS gels. Fraction 76 and
purified CK2 were resolved electrophoretically, gels were
re-naturated, and phosphorylation reactions were carried
out within the gels. The radioactive bands detected in such
an assay correspond to the electrophoretic mobility of the

kinase acting on the in-gel substrate. The kinase eluted in
fraction 76 appeared as a 40–42 kDa doublet which
phosphorylated b or cENaC but not GST (Fig. 9A). The
same bands were visualized using b, c or casein, indicating
that the same protein kinase phosphorylates all three
substrates (Fig. 9A and B). These bands were also visualized
using purified CK2 instead of fraction 76 (Fig. 9C). Taken
together, the data summarized in Figs 6–9 strongly suggest
that CK2 is the cytosolic kinase phosphorylating bS631 and
cT599 and that this phosphorylation is of high affinity.
DISCUSSION
The current study identifies CK2 as one of the kinases that
specifically phosphorylates bENaC. It was motivated by
accumulating data suggesting the involvement of phosphate
transfer reactions in the cellular regulation of this channel,
Fig. 4. Phosphorylation of bENaC in Xenopus oocytes. Oocytes were injected with cRNA mixtures coding for a-, c- and HA-tagged b.Theywere
metabolically labelled with either [
35
S]methionine or
32
Pi. bENaC was immunoprecipitated with an anti-HA antibody and resolved by SDS/PAGE.
(A) Autoradiogram of
35
S-labelled proteins immunoprecipitated from ENaC injected (b) and noninjected (–) oocytes. (B) HA-tagged b and b
S631A
immunoprecipitated from
35
S- and
32
P-labelled oocytes. (C) Quantification of the incorporation of

32
Pintowild-typeandmutatedb.
32
P/
35
Sratios
were calculated for three independent experiments each averaging  40 oocytes. Means ± SEM are shown.
Fig. 5. Functional expression of ENaC in Xenopus oocytes. Oocytes
were injected with cRNA mixtures corresponding to a, c, and either b
or b
S631A
or b
S631E
. Amiloride blockable current amplitudes at
)100 mV were measured 3 days later, as described in Experimental
procedures. Data were normalized to the average current in oocytes
injected with the wild-type constructs (11.38 ± 0.94 lA). Means ±
SEM of 16–20 oocytes from two different frogs are shown.
Fig. 6. Detection of CK2 in ENaC phosphorylating cytosolic fractions.
Western blot hybridization of cytosolic fractions with anti-a and -a¢
CK2 antibody was performed as described in Experimental proce-
dures. The last lane contains 20 lg of rat brain CK2 (aa’b
2
).
Ó FEBS 2002 CK2-mediated phosphorylation of ENaC (Eur. J. Biochem. 269) 4555
and the identification of cytosolic fractions that incorporate
32
P into the C termini of b and cENaC [13,15,22–24].
Detailed fractionation of rat colon cytosol by ion
exchange chromatography has identified several kinase

enriched fractions acting on b and c [15]. Two of them have
been studied in recent publications. The first is an as yet
unidentified kinase which incorporates
32
PintobS620 and
cT630 [14]. The second is ERK which phosphorylates
bT613 and cT623 and facilitates interactions between the
channel and Nedd4 [15]. However, the strongest phospho-
rylation activity was evoked by a kinase eluted with  0.25–
0.27
M
NaCl, which had not been characterized so far. The
current study demonstrates that this protein kinase is likely
to be CK2 and that it phosphorylates bS631 and cT599.
This identification is based on the ability of GTP to act as a
phosphate donor (Fig. 8A), the inhibitory effects of heparin
and DPG (Fig. 8B), the fact that purified CK2 can
phosphorylate bS631 and cT599 (Fig. 7A), the presence of
the a subunit of CK2 in the active cytosolic fractions
(Fig. 6), and the finding that the electrophoretic mobility of
the phosphorylating kinase corresponds to that of the a
subunit of CK2 (Fig. 9). However, other experiments
aiming to detect the b subunit of CK2 in the active cytosolic
fraction were inconclusive (data not shown). Thus the
possibility remains that the protein kinase characterized is
another, CK2-like enzyme.
CK2 is a ubiquitously expressed serine/threonine kinase,
composed of two catalytic (a and/or a¢) and two regulatory
(b) subunits [25–28]. It has a large number of substrates
which include components of signalling pathways, cytoskel-

etal elements, transcription factors and others [25]. Relat-
ively little is known about its function, and evidence has
been provided that the enzyme plays a role in cell survival,
division and proliferation, as well as synaptic development
and transmission [25,26]. It is generally believed that the
enzyme is constitutively active [26–28]. However, a number
of studies have reported activation of CK2 by extracellular
signals such as serum, steroid hormones and growth factors
[25].
Fig. 7. Phosphorylation of ENaC by CK2. (A) Wild-type and mutated
fusion proteins were phosphorylated as described in Experimental
procedures using 0.01 U of purified rat liver CK2. (B) Different
amounts of GST-b were phosphorylated for 40 min by human
recombinant CK2. The amount of phosphate incorporated was
determined by phosphorimaging (V) and the protein content of the
radioactive band (S) was estimated. A Lineweaver–Burk presentation
of the data could be fitted to straight line (R >0.98)withaK
m
of
1.4 l
M
.
Fig. 8. GTP-mediated phosphorylation of ENaC. (A) Phosphorylation
of wild-type and mutated GST constructs was carried out using equal
amounts [c-
32
P]ATP or [c-
32
P]GTP. (B) Phosphorylation of GST-b
and -c was performed with and without 10 l

M
heparin or 10 m
M
DPG. Autoradiograms and Coomassie blue stained gels are shown.
Fig. 9. ‘In-gel’ assay identifying the kinase in fraction 76. Various
substrates (GST, GST-b,GST-c and casein) were copolymerized with
the gel, and fraction 76 (A, B) or purified CK2 (C) were resolved
electrophoretically. ÔIn-gelÕ phosphorylation was performed as des-
cribed in Experimental procedures. In all cases the same 40–42 kDa
doublet was identified.
4556 H. Shi et al. (Eur. J. Biochem. 269) Ó FEBS 2002
Phosphorylation of bENaC by CK2 occurs on residue
S631. This residue and the acidic amino acids surrounding
it are well conserved in evolution (Fig. 3D). Because
mutating S631 fully blocked
32
P incorporation into GST-b
we assume that this is the only residue phosphorylated.
However S633 too is a conserved serine with a consensus
CK2 site, mutation of which inhibits phosphorylation.
Phosphorylation of such neighbouring serines may also be
interrelated [29]. Taken together with the fact that the
phosphorylation does take place also in whole cells, is
characterized by a K
m
of 1.4 l
M
, and that the kinase
tightly binds to its substrate, it is likely that the above
phosphorylation plays a role in the regulation of Na

+
transport. A role of the region that includes bS631 in
determining the Na
+
conductance of ENaC was inferred
from a number of studies. First it was shown that
truncating the last eight amino acids of b elevates channel
activity by  50% and a similar activation can be seen by
mutating the four acidic residues in this region [30]. A
peptide corresponding to the last 10 amino acids of b
inhibited the channel in planer bilayer [31] and in
mandibular duct cells [18]. In both cases, the analogous
c peptide had no or a much smaller effect. The b motif
involved in this interaction has been studied by Dinudom
et al. [18] and found to involve S631, D632 and S633. The
analogous c residues did not participate in such interac-
tion, and in this case another nonconserved serine
mediated channel inhibition. It was suggested that the
above serines participate in protein–protein interactions
which may involve their phosphorylation.
To further assess the above possibility we have deter-
mined the activity of ENaC in Xenopus oocytes expressing
wild-type and mutated b subunits. Substituting bS631 by
either alanine or glutamic acid had no effect on the mac-
roscopic Na
+
current (Fig. 5). This is in spite of the fact
that bS631 was endogenously phosphorylated in the
oocytes. It is however, possible that CK2 plays a role in
one of several ENaC regulatory processes that can be

measured in mammalian cells but not in the oocyte
expression system. In this respect it is interesting to note
that CK2 has been reported to be activated by both
insulin and dexamethasone [25]. The two hormones have
well-established effects on ENaC which may be mediated
by protein phosphorylation and also cannot be mimicked
in Xenopus oocytes [1,24,32]. An in vitro aldosterone- and
insulin-dependent phosphorylation of several residues in
the C tail of b expressedinMDCKcellshasbeen
reported previously [13]. These residues have not been
fully identified but one of them is a serine located between
amino acids 619 and 638. The only serines in this range
are S620, S631 and S633.
Also interesting is the fact that several studies have
documented a GTP-dependent regulation of ENaC [1]. The
underlying mechanism has not yet been determined and in
particular no apical, G-protein coupled receptor is known to
play a role in the regulation of Na
+
transport. The current
observations raise the possibility that GTP acts on the
channel directly by promoting a CK2-mediated phospho-
rylation, rather than by activating a G-protein.
In summary, this study demonstrates tight binding of
aCK2 to the C tail of ENaC and phosphorylation of a
conserved serine in the b subunit. The cellular role of this
event awaits further studies.
ACKNOWLEDGEMENTS
We thank D. W. Litchfield of the University of Western Ontario,
Canada for the anti-aCK2 antibody. This study was supported by

research grants from the Israel Science Foundation and the US-Israel
Binational Science foundation to H. G and E. R.
REFERENCES
1. Garty, H. & Palmer, L.G. (1997) Epithelial Na
+
channels: func-
tion, structure, and regulation. Physiol. Rev. 77, 359–396.
2. Horisberger, J.D. (1998) Amiloride-sensitive Na channels, Curr.
Opin. Cell Biol. 10, 443–449.
3. Alvarez de la Rosa, D., Canessa, C.M., Fyfe, G.K. & Zhang, P.
(2000) Structure and regulation of amiloride-sensitive sodium
channels. Annu. Rev. Physiol. 62, 573–594.
4. Palmer, L.G. & Garty, H. (2000) Epithelial Na
+
channels. In The
Kidney, Physiology and Pathophysiology (Seldin, D.W. &
Giebisch, G., eds), pp. 251–276, Lippincott Williams & Wilkins,
Philadelphia.
5. Canessa, C.M., Schild, L., Buell, G., Thorens, B., Gautschi, I.,
Horisberger, J D. & Rossier, B.C. (1994) Amiloride-sensitive
epithelial Na
+
channel is made of three homologous subunits.
Nature 367, 463–467.
6. Renard, S., Lingueglia, E., Voilley, N., Lazdunski, M. & Barbry,
P. (1994) Biochemical analysis of the membrane topology of the
amiloride-sensitive Na+ channel. J. Biol. Chem. 269, 12981–
12986.
7. Snyder, P.M., Mcdonald, F.J., Stokes, J.B. & Welsh, M.J. (1994)
Membrane topology of the amiloride-sensitive epithelial sodium

channel. J. Biol. Chem. 269, 24379–24383.
8. Canessa, C.M., Me
´
rillat, A M. & Rossier, B.C. (1994) Membrane
topology of the epithelial sodium channel. Am. J. Physiol. Cell
Physiol. 267, C1682–C1690.
9. Staub, O., Gautschi, I., Ishikawa, T., Breitschopf, K.,
Ciechanover, A., Schild, L. & Rotin, D. (1997) Regulation of
stability and function of the epithelial Na
+
channel (ENaC) by
ubiquitination. EMBO J. 16, 6325–6336.
10. Staub, O., Dho, S., Henry, P.C., Correa, J., Ishikawa, T.,
Mcglade, J. & Rotin, D. (1996) WW domains of Nedd4 bind to
the proline-rich PY motifs in the epithelial Na
+
channel deleted in
Liddle’s syndrome. EMBO J. 15, 2371–2380.
11. Debonneville, C., Flores, S.Y., Kamynina, E., Plant, P.J., Tauxe,
C.,Thomas,M.A.,Munster,C.,Chraibi,A.,Pratt,J.H.,
Horisberger, J.D., Pearce, D., Loffing, J. & Staub, O. (2001)
Phosphorylation of Nedd4-2 by Sgk1 regulates epithelial Na+
channel cell surface expression. EMBO J. 20, 7052–7059.
12. Snyder,P.M.,Olson,D.R.&Thomas,B.C.(2002)Serumand
glucocorticoid-regulated kinase modulates Nedd4-2-mediated
inhibition of the epithelial Na
+
Channel. J. Biol. Chem. 277, 5–8.
13. Shimkets, R.A., Lifton, R. & Canessa, C.M. (1998) In vivo
phosphorylation of the epithelial sodium channel. Proc. Natl

Acad.Sci.USA95, 3301–3305.
14. Chigaev, A., Lu, G., Shi, H K., Asher, C., Xu, R., Latter, H.,
Seger, R., Garty, H. & Reuveny, E. (2001) In vitro phosphoryla-
tion of the carboxy termini of the epithelial Na
+
channel (ENaC)
and its effects on the channel activity in Xenopus oocytes. Am. J.
Physiol. Renal Fluid Electrolyte Physiol. 280, F1030–F1036.
15. Shi, H., Asher, C., Chigaev, A., Yung, Y., Reuveny, E., Seger, R.
& Garty, H. (2002) Interactions of beta and gamma ENaC with
Nedd4 can be facilitated by an ERK-mediated phosphorylation.
J. Biol. Chem. 277, 13539–13547.
16. Rubinfeld, H. & Seger, R. (1998) Detection of MAP kinase sig-
naling. In Current Protocols in Cell Biology. John Wiley & Sons,
Inc., New York.
17. Firsov, D., Schild, L., Gautschi, I., Merillat, A.M., Schneeberger,
E. & Rossier, B.C. (1996) Cell surface expression of the epithelial
Ó FEBS 2002 CK2-mediated phosphorylation of ENaC (Eur. J. Biochem. 269) 4557
Na channel and a mutant causing Liddle syndrome: a quantitative
approach. Proc. Natl Acad. Sci. USA 93, 15370–15375.
18. Dinudom, A., Harvey, K.F., Komwatana, P., Jolliffe, C.N.,
Young, J.A., Kumar, S. & Cook, D.I. (2001) The roles of the
carboxyl termini of a, b and c subunits of ENaC in regulating
epithelial Na
+
channels and in mediating their inhibition by
cytosolic Na
+
. J. Biol. Chem. 276, 13744–14749.
19. Meggio, F., Marin, O. & Pinna, L.A. (1994) Substrate specificity

of protein kinase CK2. Cell. Mol. Biol. Res. 40, 401–409.
20. Marin, O., Meggio, F. & Pinna, L.A. (1994) Design and synthesis
of two new peptide substrates for the specific and sensitive
monitoring of casein kinases-1 and -2. Biochem. Biophys. Res.
Commun. 198, 898–905.
21. Hathaway, G.M. & Traugh, J.A. (1982) Casein kinases – multi-
potential protein kinases. Curr. Top. Cell Regul. 21, 101–127.
22. Shigaev, A., Asher, C., Latter, H., Garty, H. & Reuveny, E. (2000)
Regulation of sgk by aldosterone and its effects on the epithelial
Na
+
channel. Am. J. Physiol Renal Fluid Electrolyte Physiol. 278,
F613–F619.
23. Blazer-Yost, B.L., Punescu, T.G., Helman, S.I., Lee, K.D. &
Vlahos, C.J. (1999) Phosphoinositide 3-kinase is required for
aldosterone-regulated sodium reabsorption. Am. J. Physiol Cell
Physiol. 277, C531–C536.
24. Record, R.D., Froelich, L.L., Vlahos, C.J. & Blazer-Yost, B.L.
(1998) Phosphatidylinositol 3-kinase activation is required for
insulin- stimulated sodium transport in A6 cells. Am.J.Physiol.
274, E611–E617.
25. Blanquet, P.R. (2000) Casein kinase 2 as a potentially important
enzyme in the nervous system. Prog. Neurobiol. 60, 211–246.
26. Pinna, L.A. & Meggio, F. (1997) Protein kinase CK2 (Ôcasein
kinase-2Õ) and its implication in cell devision and proliferation.
Prog.CellCycleRes.3, 77–97.
27. Guerra,B.,Boldyreff,B.,Sarno,S.,Cesaro,L.,Issinger,O.G.&
Pinna, L.A. (1999) CK2: a protein kinase in need of control.
Pharmacol. Ther. 82, 303–313.
28. Pinna, L.A. (1997) Protein kinase CK2. Int. J. Biochem. Cell. Biol.

29, 551–554.
29. Perich, J.W., Meggio, F., Reynolds, E.C., Marin, O. & Pinna,
L.A. (1992) Role of phosphorylated aminoacyl residues in gen-
erating atypical consensus sequences which are recognized by
casein kinase-2 but not by casein kinase-1. Biochemistry. 31, 5893–
5897.
30. Snyder, P.M., Price, M.P., Mcdonald, F.J., Adams, C.M., Volk,
K.A.,Zeiher,B.G.,Stokes,J.B.&Welsh,M.J.(1995)Mechanism
by which Liddle’s syndrome mutations increase activity of a
human epithelial Na
+
channel. Cell. 83, 969–978.
31. Ismailov, I.I., Berdiev, B.K., Fuller, C.M., Bradford, A.L., Lifton,
R.P., Warnock, D.G., Bubien, J.K. & Benos, D.J. (1996) Peptide
block of constitutively activated Na
+
channels in Liddle’s disease.
Am.JPhysiolCellPhysiol.270, C214–C223.
32. Garty, H. (2000) Regulation of the epithelial Na
+
channel by
aldosterone: open questions and emerging answers. Kidney Int. 57,
1270–1276.
4558 H. Shi et al. (Eur. J. Biochem. 269) Ó FEBS 2002