Casein kinase 2 specifically binds to and phosphorylates the carboxy termini of ENaC subunits Haikun Shi1, Carol Asher1, Yuval Yung2, Luba Kligman1, Eitan Reuveny1, Rony Seger2and Haim G
Trang 1Casein kinase 2 specifically binds to and phosphorylates the carboxy termini of ENaC subunits
Haikun Shi1, Carol Asher1, Yuval Yung2, Luba Kligman1, Eitan Reuveny1, Rony Seger2and Haim Garty1 1
Department of Biological Chemistry, and2Department 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, and c) 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
E X P E R I M E N T A L P R O C E D U R E S
Materials
32P orthophosphate (10 mCiÆmL)1), [c-32P] ATP (10 mCiÆmL)1, 3000 Ci mmol)1) and [c-32P] GTP (10 mCiÆmL)1, 5000 CiÆmmol)1) were from Amersham Pharmacia Biotech; glutathione–agarose beads, dephospho-rylated casein, heparin, 2,3-diphosphoglycerate and rat liver CK2 (a mixture of a2b2 and aa¢b2) were from Sigma-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-ChangeTM 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: h.garty@weizmann.ac.il
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)
Trang 2X-100, 200 mM b-glycerolphosphate, 1 mM EDTA, 1 mM
sodium orthovanadate and protease inhibitors (1 mM
phenylmethanesulfonyl fluoride, 10 lgÆmL)1 aprotinin,
10 lgÆmL)1leupeptin, 2 lgÆmL)1pepstatin A) The solution
was mixed with 5–20 lg GST-fusion protein immobilized
on glutathione-agarose beads, and mixtures were rotated at
4C for 2 h Beads were sedimented by centrifugation and
washed six times using the following protocol: (a) two
washes in 0.5M LiCl, 100 mM Tris/HCl pH 8.0; (b) two
more washes in 1MNaCl plus HB1B buffer (20 mMHepes
pH 7.7, 50 mM NaCl, 0.1 mM EDTA, 25 mM MgCl2,
0.05% Triton X-100); (c) one wash in buffer A [50 mM
b-glycerophosphate pH 7.3, 1.5 mM EGTA, 1.0 mM
EDTA, 1.0 mM dithiothreitol, and 0.1 mM de-aerated
sodium orthovanadate]; (d) A final wash in a kinase assay
buffer composed of 20 mMHepes pH 7.7, 20 mMMgCl2,
20 mM 2-glycerolphosphate, 2 mM dithiothreitol, 0.1 mM
sodium orthovanadate Beads were suspended in 30 lL of
the above kinase assay buffer, a second substrate was added,
and phosphorylation was initiated by the addition of 2 lM
ATP plus 2 lCi [c-32P]ATP The suspension was incubated
for 30 min at 30C 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
In-gel 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 50mMTris/HCl, pH 8.0, and then in 50 mMTris/HCl
pH 8.0 plus 5 mM2-mercaptoethanol Proteins were
dena-turated by soaking the gel in 6Murea and then renaturated
in 50 mM Tris/HCl, pH 8.0, 0.04% Tween-20 and 5 mM
2-mercaptoethanol For in-gel phosphorylation, the gel was
first preincubated for 30 min at 30C in 20 mM Hepes
pH 8.0 plus 20 mMMgCl2 ATP (100 lCi [c-32P]ATP plus
20 lMnonradioactive ATP) and 2 mMdithiothreitol were
added and the phosphorylation was allowed to proceed for
2 h at 30C 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
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 [35S]methionine (2 days) or 3.3 mCi 32Pi (4 h) 32P- and 35S-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 4C with 2 lg 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 + 0.5M LiCl Immunopellets were suspended in SDS sample buffer, resolved by SDS/PAGE (8% acrylamide gel) and assayed for 35S and 32P radioactivity by phosphor-imaging
R E S U L T S
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 0.25–0.27MNaCl, 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 ( 0.26M NaCl, Fig 1B) This kinase phosphorylated GST-b better than GST-c, while GST and GST-a did not incorporate a significant amount of32P (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
Trang 3beads 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
[32P]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 on32P incorporation into GST-b and -c Accordingly, several serines and threonines on the C termini of b and c were mutated to alanine and examined for phosphorylation by fraction 76 In b the major phosphorylated residue was S631 and mutating it to alanine blocked almost all 32P 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, and cENaC 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.
Trang 4phosphorylation was seen also upon the mutation of a
neighbouring residue, S633 This residue may have an
indirect effect on the incorporation of32P into S631 The
analogous residue on the c subunit (cT644) did not
incorporate32P 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
cT630 had no effect on32P 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 vivothe a-, c-, and HA-tagged bENaC were expressed in
Xenopus oocytes and metabolically labelled with either
[35S]methionine or32Pi 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 of32P
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 of32P into bS631 is not residual The K 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.
Trang 51.4 lM (Fig 7B) This value compares well to the Km
measured for b-casein (36.5 lM) 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 in-gel 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
D I S C U S S I O N
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 They were 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 from35S- and32P-labelled oocytes (C) Quantification of the incorporation of32P into wild-type and mutated b.32P/35S ratios 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 ).
Trang 6and the identification of cytosolic fractions that incorporate
32P 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 incorporates32P into bS620 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–
0.27MNaCl, 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) with a K 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 In-gel phosphorylation was performed as des-cribed in Experimental procedures In all cases the same 40–42 kDa doublet was identified.
Trang 7Phosphorylation 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 blocked32P 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 Km of 1.4 lM, 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 expressed in MDCK cells has been
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
A C K N O W L E D G E M E N T S
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.
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