Aldosterone induces Ras methylation in A6
epithelia
N. F.
Al-Baldawi,
J. D.
Stockand,
O. K.
Al-Khalili,
G.
Yue, and
D. C.
Eaton
Center for Cell and Molecular Signaling, Department of Physiology,
Emory University School of Medicine, Atlanta, Georgia 30322
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ABSTRACT |
Aldosterone increases Na+ reabsorption by renal
epithelial cells: the acute actions (<4 h) appear to be promoted by
protein methylation. This paper describes the relationship between
protein methylation and aldosterone's action and describes
aldosterone-mediated targets for methylation in cultured renal cells
(A6). Aldosterone increases protein methylation from 7.90 ± 0.60 to 20.1 ± 0.80 methyl ester cpm/µg protein. Aldosterone
stimulates protein methylation by increasing methyltransferase activity
from 14.0 ± 0.64 in aldosterone-depleted cells to 31.8 ± 2.60 methyl ester cpm/µg protein per hour in aldosterone-treated cells. Three known methyltransferase inhibitors reduce the
aldosterone-induced increase in methyltransferase activity. One of
these inhibitors, the isoprenyl-cysteine methyltransferase-specific
inhibitor,
S-trans,trans-farnesylthiosalicylic acid, completely blocks aldosterone-induced protein methylation and also aldosterone-induced short-circuit current. Aldosterone induces
protein methylation in two molecular weight ranges: near 90 kDa and
around 20 kDa. The lower molecular weight range is the weight of small
G proteins, and aldosterone does increase both Ras protein 1.6-fold and
Ras methylation almost 12-fold. Also, Ras antisense oligonucleotides
reduce the activity of Na+ channels by about fivefold. We
conclude that 1) protein methylation is essential for
aldosterone-induced increases in Na+ transport;
2) one target for methylation is p21ras; and
3) inhibition of Ras expression or Ras methylation inhibits Na+ channel activity.
protein methylation; sodium transport; A6 cells; epithelial
transport
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INTRODUCTION |
ALDOSTERONE IS THE MOST
IMPORTANT systemic modulator of apical Na+
permeability in mammalian collecting tubule principal cells. The final
effect of this hormone is to increase the amount of Na+
uptake by Na+ channels in the apical membranes of
transporting cells; however, the mechanisms by which aldosterone
increases Na+ transport are not at all clear, and many of
the proposed mechanisms are controversial. The aldosterone-mediated
increase in Na+ transport occurs in two phases: an initial
phase that increases transport approximately fivefold in the first 4 to
6 h, and a late phase that requires 12-48 h and increases
transport another three- to fourfold. How the early and late phases are
related is unclear. In this report, we will concentrate on the initial phase.
Little is known about the cellular mechanisms of aldosterone's action
except that gene expression and protein synthesis are required and that
the final result is an increase in Na+ transport
(5, 13, 32). Originally, because
of the observation that protein synthesis was required for aldosterone
to increase Na+ transport, it was postulated that
aldosterone induced Na+ channel synthesis and insertion.
However, earlier studies in A6 cells suggest that aldosterone increases
Na+ entry at the apical membrane by changing the activity
of channels that are already present in the apical membrane and not by
increasing the number of channels (19). Data obtained with
biochemical methods support this observation, indicating that the
number of channels in the apical membrane does not increase in the
presence of aldosterone (at least in the first 2-4 h when the
increase in Na+ transport is most dramatic)
(13, 22). However, in rat cortical collecting
tubules and A6 cells, aldosterone appears to increase the number of
Na+ channels in the apical membrane based on single channel
and noise analysis (16, 33). Similar
conclusions were reached from studies in toad urinary bladder and A6
cells with the use of fluctuation analysis of amiloride-induced current
noise (3, 34). Additionally, Xenopus
laevis oocytes that were injected with RNA from tissue that had
been treated with aldosterone for 3-24 h expressed more channel
activity, suggesting that aldosterone increases the transcription of
the Na+ channel gene itself (30). Thus it
appears that depending on the experimental approach or system used to
obtain the data, the answer to the question of whether aldosterone
increases Na+ transport by increasing the transcription and
translation of the Na+ channel itself remains unresolved.
However, it is now possible to directly address this question because
of the identification of the genes that encode epithelial Na+ channel (ENaC) (9). Recently, Middleton et
al. (31) directly addressed in A6 cells the question of
whether aldosterone increases mRNA levels and/or protein levels of any
or all of the three ENaC subunits. Their data show that increases in
short-circuit current after 4 h exposure to aldosterone can occur
without corresponding increases in mRNA or protein levels for
-,
-, or
-ENAC. These results are consistent with the findings by
Marunaka et al. (30), who measured mRNA levels of
ENaCs in kidney cells of rats whose aldosterone levels had been changed
either by diet or exposure to corticosteroids. No differences for
-
or
-ENaC message were found between rats on a normal vs. a low-salt
diet, and only small increases in
-xENaC message were observed. Thus
in renal tissues (including A6 cells), the electrophysiological
evidence varies, but the biochemical evidence all supports the idea
that aldosterone (in the first 2-4 h) modulates the activity of
preexisting channels rather than promoting the synthesis or insertion
of new channels.
There have been many suggestions about potential aldosterone-induced
posttranslational modifications, but in the context of our previous
results (4, 46, 47), one is
particularly interesting. Sariban-Sohraby et al. (38)
demonstrated that the amount of Na+ transport that could be
measured in apical membrane vesicles obtained from A6 cells, a
Na+-transporting, distal-nephron cell line, was markedly
enhanced by prior application of agents that methylate membrane
proteins. Because they also demonstrated that application of
aldosterone leads to the methylation of membrane protein and lipid,
their suggestion was that intracellular methyltransferases induced by aldosterone could be responsible for the methylation and, therefore, modulate the Na+ channel protein. As a posttranslational
modification, methylation is analogous to phosphorylation (for reviews,
see Refs. 2, 10, 36, and 48). Highly specific methyltransferases
promote the methylation of proteins at specific consensus sites, and
much less specific esterases promote demethylation of the proteins. The
difference between phosphorylation and methylation is that methylation
is in general more stable and, therefore, can be used to alter the
activity of proteins for a much longer period of time (2-4 h) than
is typical for phosphorylation. To understand the role of methylation
in altering cellular activity, it is useful to identify the protein
targets for methylation. Sariban-Sohraby et al. (40)
demonstrated that a 95-kDa protein was methylated in the presence of
aldosterone. Because this size is consistent with the size of a
glycosolated Na+ channel subunit, some investigators have
suggested that the target for methylation is one of the Na+
channel subunits (39). Indeed, in recent work, Rokaw et
al. (37) demonstrated in vitro methylation of the
-subunit of Na+ channels in A6 cells.
However, methylation of proteins that controls activity of proteins
usually occurs only at a very restrictive consensus sequence: the
so-called "CAAX" box (a cysteine residue followed by two aliphatic residues, followed by any residue at the COOH-terminal end of the
protein). None of the ENaC subunit sequences contain any cysteine residues that meet the criteria for methylation. However, there are
other possible cellular targets for aldosterone-induced methylation of
proteins that could regulate Na+ channels. In previous
work, using patch-clamp methods, Becchetti et al. (4) have
shown that addition of methyl donors to isolated, cell-free patches of
membrane to promote protein methylation increases the activity of
Na+ channels. Therefore, the target for methylation, if it
is not a channel subunit itself, must be a membrane-associated protein in close proximity to the channels. In addition, the same work by
Becchetti et al. and also work by Sariban-Sohraby et al.
(40) showed that methylation-induced activation of
Na+ channels is augmented by cytosolic GTP. Thus
methylation of excised, apical membranes induces Na+
channel activity similar to the activity produced by aldosterone, and
the methylation reaction is augmented by G protein stimulation. This is
a particularly interesting observation, because in one of the many
attempts to identify proteins differentially expressed by aldosterone,
Spindler and colleagues (42) found that mRNA for the
membrane-associated, small G protein K-Ras2a was increased in the
presence of aldosterone. K-Ras2a contains the consensus CAAX sequence
for methylation and is known to require both GTP and methylation for
activity and membrane association (36, 47, 48). The aim of this report is to confirm that aldosterone
activates the enzyme isoprenyl-cysteine methyltransferase and induces
an increase in cellular protein methylation, and to demonstrate that Ras protein is a target for aldosterone-induced methylation and that
Ras might play a role in methylation-induced stimulation of
Na+ transport. This work has been reported as a brief
communication (1).
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MATERIALS AND METHODS |
A6 cell culture preparation.
A6 cells from American Type Culture Collection (Rockville, MD) in the
68th passage (35) were prepared as previously described (25, 29). Experiments were carried out on
passages 70-80. Cells were maintained in plastic tissue
culture flasks (Corning, NY) at 26°C in a humidified incubator with
4% CO2 in air. The culture medium was a mixture of Coon's
medium F-12 (3 parts) and Leibovitz's medium L-15 (7 parts) modified
for amphibian cells with 104 mM NaCl-25 mM NaHCO3, pH 7.4, with a final osmolality of 240 mosmol/kgH2O.
Besides these components, 10% (vol/vol) fetal bovine serum (Irvine
Scientific), 1% streptomycin, and 0.6% penicillin (Hazleton
Biologics) were added. For the purpose of this study, A6 cells were
plated at confluent density on collagen-coated CM-permeable filters
(Millipore) in six-well plates. This sided preparation forms a
polarized monolayer with the apical surface oriented upward and net
Na+ transport moving from the apical to basolateral
surface. The bathing medium was supplemented with 1.5 µM aldosterone
and 10% fetal bovine serum during the growth phase before experiments.
O-COOH-methyltransferase activity.
Sets of confluent cells from the same passage were grown on permeable
supports in six-well plates as described above. After the cells reached
confluent density, the cells were incubated in aldosterone-free and
serum-free medium for 3 days. Then one-half of the cells were washed
with PBS (pH 7.4) and refed with serum-free medium containing 0.1 or
1.5 µM aldosterone. The remaining cells (aldosterone-free controls)
were washed with PBS followed by readdition of aldosterone-free and
serum-free medium. Cells were incubated for 4 h followed by
short-circuit current measurements with an epithelial volt-ohm meter
(World Precision Instrument, Sarasota, FL). Cells were then washed with
PBS and homogenized with 50 mM Tris · HCl, 1 mM NaEDTA (pH
8)-containing protease inhibitors, and centrifuged at 3,000 g at 4°C for 10 min. Supernatants were used as the enzyme
source. The enzyme activity assay was similar to that described
previously (14, 26). In brief, the incubation mixture (50 µl) contained 50 mM Tris · HCl buffer (pH 8),
720 nM [3H]adenosyl-methionine (15 Ci/mmol), 100 µM of
the methyltransferase substrate
N-acetyl-S-farnesyl-L-cysteine, a
blocking concentration of a methyltransferase inhibitor when
appropriate
[S-trans,trans-farnesylthiosalicylic acid (FTS), 3-deazaadenosine (DZA), or
S-adenosylhomocysteine (SAH)], and enzyme-containing
supernatant. After incubation for 1 h at 37°C, the reaction was
stopped by the addition of 50 µl of 20% TCA, and the reaction mix
vortexed for 10 s. The isoprenyl-cysteine methyl esters were
separated by the addition of 400 µl of heptane to the reaction mix. A
fraction (200 µl) of the organic top layer containing the methyl
esters was transferred to a small top-free Eppendorf tube and dried
under vacuum. Two hundred microliters of 1 M NaOH were added, and the
tube was placed upright in a vial containing a small volume of
scintillation fluid. The vials were sealed and incubated at 37°C
overnight. The strong base hydrolyzes the methyl esters, releasing
methanol vapor that partitions into the hydrophobic scintillant. The
methyl esters in the vials were counted by liquid scintillation
counting. The specific activity of the enzyme is defined as counts per
minute of methyl-3H group transferred per minute per
milligram of enzyme protein. Protein concentration was determined as
described by Bradford (7).
Protein methylation.
Sets of confluent cells from the same passage were incubated in
aldosterone-free, serum-free, and methionine-free medium for 3 days as
described above. On the last day, the media were replaced with
equivalent methionine-free medium containing tracer amounts (0.05 mM)
of L-[methyl-3H]methionine (Amersham Life
Sciences, Arlington Heights, IL), and the cells were allowed to
incubate for 24 h to allow the methyl donor,
S-adenosyl-L-methionine, to reach labeling
equilibrium. Then one-half of the cells were washed with PBS (pH 7.4)
and refed with serum-free and methionine-free medium containing 0.1 or
1.5 µM aldosterone. The remaining cells (aldosterone-free controls) were washed with PBS followed by readdition of aldosterone-free, serum-free, and methionine-free bathing medium. Cells were incubated for 4 h followed by short-circuit current measurements. For
protein extraction, cells were washed with PBS and centrifuged at 4,000 g for 10 min at 4°C. The cell pellet was treated with 0.5 ml of RIPA buffer (PBS, Na deoxycholate, SDS) to lyse the cells. After incubation for 2 h at 4°C, the sample was spun at 10,000 g for 30 min at 4°C. The supernatant was retained for
further examination. To investigate the overall methylation of
proteins, the total protein in samples was determined as described by
Bradford (7). Aliquots of supernatants with equivalent
protein were placed in top-free Eppendorf tubes containing 1 M NaOH,
and the tubes were placed upright into scintillation vials containing a
small amount of scintillation cocktail. The vials were sealed and
incubated overnight at 37°C, and the base labile counts were
determined. To investigate the methylation of individual proteins,
aliquots of supernatants from lysate protein samples were either used
as such or the apical membranes were enriched as described by
Sariban-Sohraby et al. (41) and run on gels in paired
lanes. One lane was stained with Coomassie blue, whereas the other lane
was carefully cut from the gel and sliced into small segments that were
placed into Eppendorf tubes containing 1 M NaOH, and the tubes were
treated to extract base labile methyl esters as described above and as has been described by others (6, 37-40,
49). The counts remaining in the gel are a measure of
total protein in the gel slice, because [3H]methionine
not only leads to incorporation of counts into
S-adenosyl-L-methionine but is also incorporated
(as methionine) into protein. Thus the base labile counts are a measure
of methylation and the nonbase labile counts are a measure of protein.
Isolation of Ras.
After methylation as described above, supernatants from lysates were
immunoprecipitated with the specific anti-Ras antibody (1 µg/ml
H-Ras 259 antibody-agarose conjugate, SC-35 AC;
Santa Cruz Biotechnology) and allowed to incubate for 24 h at
4°C, followed by centrifugation at 3,500 g for 5 min at
4°C. The pellets were retained and washed three times with buffer.
The proteins were released from the agarose by addition of sample
loading buffer (glycerol, mercaptoethanol, SDS, Tris · HCl, pH
6.7, bromphenol blue) and boiled at 95°C for 5 min, followed by
centrifugation at 10,000 g for 30 min at 4°C. The proteins
in the supernatants were separated by SDS-gel electrophoresis (12%) at
pH 6.8 to separate the proteins. The proteins on one gel were detected
by staining with Coomassie blue, and the other lane was prepared as
described above to measure methylation of proteins in the
immunoprecipitate. The Coomassie-stained lane was destained and
transferred to nitrocellulose for Western blotting to determine whether
methylated protein(s) run at the same location as Ras. Briefly,
proteins on gels were transferred to nitrocellulose after standard
procedures. The nonspecific binding was blocked using 5% milk in Tween
20 and Tris-buffered saline and incubation for 1 h at 4°C,
followed by conjugation with the specific anti-Ras antibody for 2 h at 4°C followed by washing and exposure to secondary antibody (0.1 µg/ml v-H-Ras Ab-1 antibody, OP01; Oncogene Research
Products, Calbiochem, San Diego, CA). To detect the proteins, the
membrane was treated with enhanced chemiluminescence Western blotting
analysis following the protocol provided by Amersham (Amersham Life Sciences).
Methylation of Ras.
Sets of confluent cells from the same passage were incubated in
aldosterone-free and serum-free medium for 3 days. Then
one-half of the cells were washed with PBS (pH 7.4) and refed with
serum-free medium containing 0.1 µM aldosterone. The remaining cells
(aldosterone-free controls) were washed with PBS followed by readdition
of aldosterone-free and serum-free medium. Cells were incubated for
4 h followed by short-circuit current measurements with an
epithelial volt-ohm meter (World Precision Instrument). Cells were then
washed with PBS and homogenized with 50 mM Tris · HCl, 1 mM
NaEDTA (pH 8)-containing protease inhibitors and centrifuged at 3,000 g at 4°C for 10 min. Supernatants were used as the enzyme
source, and exogenous c-K-Ras (Calbiochem) was added as a target for
methylation. After incubation for 1 h at 37°C, Ras was separated
from the lysate by immunoprecipitation as described above. To
investigate the methylation of Ras, aliquots of immunoprecipitates were
run on gels in paired lanes. Ras protein in one lane was detected by
Western blotting, whereas methylation of proteins was detected in the
other lane by exposure of the dried gel on a phosphoimager.
Antisense inhibition of Ras.
To inhibit expression of Ras, we used methods that we have previously
used to inhibit expression of another membrane-associated epithelial
protein, cystic fibrosis transmembrane conductance regulator
(24). Sense and antisense phosphorothioate
oligonucleotides corresponding to 21 nt beginning at nt 181 of X. laevis Ras (accession no. AF085280) were synthesized by the Emory
University Microchemical Facility. This sequence should inhibit
expression of both Xenopus H-Ras and K-Ras2a. A search of
GenBank revealed no similarity of this 21-mer with sequences other than
Ras. On the 5th to 10th day after plating, selected A6 cells were
loaded with Ras sense or antisense oligonucleotides. Cellular uptake
was enhanced with the use of a lipid transfection agent (Lipofectamine,
Life Technologies), according to the manufacturer's instructions.
Briefly, cells were incubated for >4 h with 10 µg/ml Lipofectin
containing 10 µM oligonucleotide in serum-free A6 medium added to the
basolateral and apical baths, followed by a 2-day incubation in
serum-free medium containing 10 µM oligonucleotide and 1 µM
aldosterone. For patch clamping, the disks were transferred to the
stage of an inverted microscope (Nikon), and experiments were performed
as described above. In our previous work, we demonstrated in
antisense-treated cells by Western blotting and immunocytochemical
localization that p21-Ras protein was reduced to levels 5- to 20-fold
below that found in sense-treated or untreated cells (47).
Statistics.
Tests for significance were performed using a t-test or
one-way ANOVA with Student-Newman-Keuls posttests. Results were
considered significant if P < 0.05.
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RESULTS |
Aldosterone and methylation.
To confirm the previously described effect of aldosterone on total
protein methylation (38), two batches of six-transwell plates with confluent A6 cells were treated as described in
Protein methylation. Figure
1 shows that in three separate
experiments, aldosterone significantly increases protein
methylation ~2.5-fold over aldosterone-free cells (from 7.90 ± 0.60 to 20.1 ± 0.80 methyl ester cpm/µg protein;
P < 0.001, n = 3). On the basis of
these data, we decided to measure the activity of the enzyme
responsible for this methylation. After having reached confluence for 2 days, the control and test samples consisting of six-transwell plates each were deprived of aldosterone. Then aldosterone was added to the
test samples 4 h before extraction. The enzyme activity was
determined as described in O-COOH-methyltransferase
activity. Figure 2 shows that in
three separate experiments, methylase activity was significantly
increased from 14.0 ± 0.64 in aldosterone-depleted cells to
31.8 ± 2.60 methyl ester cpm/µg protein per hour in
the aldosterone-treated cells (P = 0.003, n = 3). These observations suggest that methylation
might be involved in aldosterone-mediated epithelial Na+
transport. To examine the characteristics of this methylation, we used
three known inhibitors of methyltransferases: FTS, SAH, and DZA. SAH is
an end-product inhibitor; DZA causes an increase in SAH levels; and FTS
is a strong, competitive inhibitor of isoprenyl-cysteine binding. Of
the three, under our experimental conditions, we would expect, as we
observed, that FTS should be the most effective blocker, although all
three blockers reduced aldosterone-induced methylase activity (Fig.
3). Protein methylase activity
significantly increased from 16.1 ± 1.40 in aldosterone-depleted
cells to 43.5 ± 4.80 methyl ester cpm/µg protein per hour in
the aldosterone-treated cells (P < 0.05, n = 3). In cells treated with aldosterone and 100 µM
FTS for 4 h, methylase activity was dramatically reduced to
0.75 ± 0.21 methyl ester cpm/µg protein per hour (significantly less than all other treatments; P < 0.05;
n = 3). One hundred micromolar SAH and 300 µM DZA
treatment for 4 h reduced methylase activity to 20.0 ± 0.34 and 22.51 ± 1.50 methyl ester cpm/µg protein per hour,
respectively (both were significantly different from aldosterone-treated cells, but not different from untreated cells; P < 0.05, n = 3). To examine whether
inhibition of methyltransferase activity could alter Na+
transport, we examined the effect of the most effective
methyltransferase inhibitor, FTS, on short-circuit current, a direct
measure of Na+ flux in A6 cells (15). The
medium of two six-well plates was replaced 4 h before the
short-circuit current was measured in the samples. Three wells each
received, respectively, medium, medium containing 100 µM FTS, medium
containing 0.1 µM aldosterone, and medium containing 100 µM FTS,
medium, and 0.1 µM aldosterone. Figure
4A shows that, as expected in
three separate experiments, application of aldosterone increases
short-circuit current >13-fold (from 0.56 ± 0.06 to 7.33 ± 0.49 µA/cm2, significantly different from all other
treatments; P < 0.05). Application of FTS reduces
short-circuit current in the absence of aldosterone (to
1.07 ± 0.33), but completely blocks any aldosterone-induced increase in
short-circuit current (after FTS and aldosterone current is
0.94 ± 0.37 µA/cm2, significantly different from untreated
and aldosterone-treated cells; P < 0.05; but not
different from FTS treatment in the absence of aldosterone). The effect
of FTS is not to disrupt the integrity of the A6 monolayer (despite the
negative short-circuit current), because FTS does not alter the
resistance of monolayer (Fig. 4B), although aldosterone (in
the presence or absence of FTS) does produce a small increase in
resistance. The effect of FTS suggests a relationship between
isoprenyl-cysteine methylation and the aldosterone-induced activity of
amiloride-blockable Na+ channels.

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Fig. 1.
Aldosterone alters protein methylation in A6 cells. In 3 experiments with 2 groups of 6 transwells in each experiment containing
confluent A6 cells, 1 group treated with aldosterone-free medium
( aldosterone) and the other group in aldosterone-containing medium
(+aldosterone), the extent of methylation was significantly higher in
the aldosterone-treated cells (from 7.90 ± 0.60 to 20.1 ± 0.80 methyl ester cpm/µg protein, P < 0.001, n = 3).
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Fig. 2.
Aldosterone stimulates protein methylation by increasing
protein methylase activity in A6 cells. In 3 experiments consisting of
2 groups of 6 transwells containing confluent A6 cells, 1 treated with
aldosterone-free medium ( aldosterone) and the other in
aldosterone-containing medium (+aldosterone), protein methylase
activity increased significantly in the aldosterone-treated cells (from
14.0 ± 0.64 to 31.8 ± 2.60 methyl ester cpm/µg protein
per hour, P < 0.003, n = 3).
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Fig. 3.
Methyltransferase inhibitors reduce the
aldosterone-induced increase in methylase activity. To examine the
characteristics of this methylation, we used 3 known inhibitors of
methyltransferases:
S-trans,trans-farnesylthiosalicylic
acid (FTS), S-adenosylhomocysteine (SAH), and
3-deazaadenosine (DZA). Under our experimental conditions with 6 transwells for each condition, the direct blocker, FTS, was the most
effective blocker, although all 3 inhibitors reduced
aldosterone-induced methylase activity. Protein methylase activity
increased significantly from 16.1 ± 1.40 in aldosterone-depleted
cells to 43.5 ± 4.80 methyl ester cpm/µg protein per hour in
the aldosterone-treated cells (P < 0.05, n = 3). In cells treated with aldosterone and 100 µM
FTS, methylase activity was dramatically reduced to 0.75 ± 0.21 methyl ester cpm/µg protein per hour (significantly less than all
other treatments; P < 0.05; n = 3).
One hundred micromolar SAH and 300 µM DZA reduced methylase activity
to 20.0 ± 0.34 and 22.51 ± 1.50 methyl ester cpm/µg
protein per hour, respectively (both were significantly different from
aldosterone-treated cells, but not different from untreated cells;
P < 0.05, n = 3).
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Fig. 4.
The methyltransferase-specific inhibitor FTS reduces
short-circuit current but not resistance in aldosterone-free and
aldosterone-treated A6 cells. Four groups of cells (3 wells each) in 3 separate experiments were used: 1 group treated with aldosterone-free
medium (-aldosterone), a second treated with aldosterone-free media
containing 100 µM FTS (-aldosterone + FTS), a third treated with
0.1 µM aldosterone medium (+aldosterone), and a fourth treated with
100 µM FTS + 0.1 µM aldosterone (+aldosterone + FTS).
A: as expected, application of aldosterone increased
short-circuit current >13-fold (from 0.56 ± 0.06 to 7.33 ± 0.49 µA/cm2, significantly different from all other
treatments; P < 0.05). Application of FTS reduced
short-circuit current in the absence of aldosterone (to 1.07 ± 0.33), but completely blocked any aldosterone-induced increase in
short-circuit current (after FTS and aldosterone, current was
0.94 ± 0.37 µA/cm2, significantly different from
untreated and aldosterone-treated cells; P < 0.05, but
not different from FTS treatment in the absence of aldosterone). The
effect of FTS is not to disrupt the integrity of the A6 monolayer
(despite the negative short-circuit current), because FTS does not
alter the resistance of monolayer (B), although aldosterone
(in the presence or absence of FTS) does produce a small increase in
resistance.
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Targets for protein methylation.
On the basis of the above observations, we decided to investigate
possible target proteins for methylation in A6 cells. Two sets of two
six-well plates of cells from the same passage were treated to
label-methylated proteins (as described in Protein methylation), total cellular protein was extracted, and
the methylated proteins were resolved on a gel and quantitated by
slicing the gel into molecular weight regions. The base labile counts
are a measure of the extent of cysteine methyl esterification (Table 1). The ratio of the base labile counts
from the aldosterone-treated vs. untreated cells is a measure of the
aldosterone-induced methylation. Figure 5
and Table 1 show in three separate experiments that there are two
prominent peaks of methylation, one corresponding to a region near 90 kDa and another in a region around 20 kDa. The high-molecular-weight
region may correspond to the 95-kDa methylated protein previously
described by Sariban-Sohraby et al. (40). On the other
hand, we thought the low-molecular-weight band might actually be due to
methylation of a small molecular weight G protein like Ras because it
is known to require methylation for activity and membrane association
(36, 48), it requires cytosolic GTP for
activation (just like methylation-induced activation of Na+
channels), and Spindler et al. (42) and work by us
(47) has shown that at least one membrane-associated small
G protein, K-Ras2a, is induced by aldosterone. First, we examined A6
cells for the measurable presence of Ras protein. Figure
6 shows that an antibody that recognizes
the G protein binding domain of several small G proteins including
K-Ras2a and H-Ras can specifically detect p21ras in a
Western blot (A) and that the antibody is capable of
immunoprecipitating Ras (B). In each case, the band detected
by the antibody could be competed by the antigenic peptide (+ peptide
lanes). When the amount of total cellular Ras is examined as the
density of bands in Western blots or after immunoprecipitation, there
is an effect of aldosterone on the amount of Ras protein. Ras
protein increases 1.58 ± 0.22-fold (C; means ± SE, n = 11), which is marginally different from 1 (P = 0.026). However, because the antibody precipitates more than one isoform of Ras, larger changes in one type of Ras (like
K-Ras) might be obscured if that type were only a small fraction of
total Ras protein. Despite the fact that there was only a 58% increase
in total Ras protein, aldosterone might still induce Ras activation by
methylation. To test this possibility, we labeled cells to equilibrium
with [3H]methionine to tritiate the methyl esters in
methylated proteins (as described in Protein methylation),
after which total cellular protein was immunoprecipitated with the
Ras-specific antibody used in Fig. 6. The methylated proteins
in the immunoprecipitate were resolved on a gel and quantitated by
slicing the gel into molecular weight regions. The ratio of the base
labile counts from the aldosterone-treated vs. untreated cells is a
measure of the aldosterone-induced methylation. Figure
7A shows in three separate
experiments that there is now only one peak at the expected molecular
weight for Ras and that aldosterone produces an 11.9 ± 0.40-fold
increase in Ras methylation even though the protein content of the gel
slice is not changed dramatically.

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Fig. 5.
Aldosterone induces methylation of proteins in 2 molecular weight ranges. Two groups of cells were used, 1 treated with
aldosterone-free medium, and 1 treated with aldosterone-containing
medium. After protein methyl esters were labeled to equilibrium by
overnight incubation with [3H]methionine, proteins from
cell lysates were resolved on a gel and quantitated by slicing the gel
into molecular weight regions. For 5 separate experiments, the ratio of
the base labile counts from the aldosterone-treated vs. untreated cells
is shown on the vertical axis and is a measure of the
aldosterone-induced methylation. There are 2 prominent peaks of
methylation, 1 corresponding to a region near 90 kDa and another in a
region around 20 kDa.
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Fig. 6.
Aldosterone increases the amount of Ras protein.
A: Western blot analysis of A6 whole cell lysate; ( ) were
from aldosterone deprived cell lysate, (+) were from 4 h
aldosterone-treated cell lysates. Peptides were probed with antibody
(Ab) + 20-fold H-Ras control peptide (SC-35P). B:
immunoprecipitation (IP) of Ras from A6 cell lysate; ( ) and (+)
lysates were treated as above. Peptides were probed with antibody + 20-fold H-Ras control peptide (SC-35P). C: mean
fold change in density of p21Ras bands from aldosterone-free and
aldosterone-treated cell lysates calculated for 11 Western blots in
which aldosterone-free and aldosterone-treated cell lysates were run in
adjacent lanes and the band densities measured. Ras protein increases
1.58 ± 0.22-fold (means ± SE), which is marginally
different from 1 (P = 0.026).
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Fig. 7.
Aldosterone induces Ras methylation. A: in 3 separate experiments, 2 groups of cells were used, 1 treated with
aldosterone-free medium and 1 treated with aldosterone-containing
medium. After protein methyl esters were labeled to equilibrium by
overnight incubation with [3H]methionine, proteins were
immunoprecipitated from cell lysates with anti-Ras antibody. The
proteins in the immunoprecipitate were resolved on a gel, and the
extent of methylation was quantitated by slicing the gel into molecular
weight regions. The ratio of the base labile counts from the
aldosterone-treated vs. untreated cells is shown on the vertical axis
and is a measure of the aldosterone-induced methylation. After the
immunoprecipitation, there was only 1 peak at the expected molecular
weight for Ras, and aldosterone-induced methylation of the peak had
increased 11.93 ± 0.40-fold, even though the protein content of
the gel slice was unchanged (errors for most points except for the
20-25 kDa point are smaller than the symbol size). The
inset shows the autoradiogram from 1 experiment of cell
lysates from aldosterone-treated and aldosterone-free cells to which
exogenous 14C-SAM and K-Ras had been added and that were
subsequently immunoprecipitated with anti-Ras antibody. This result
demonstrates that K-Ras can act as a substrate for methylation and that
aldosterone increases the extent of methylation. B: the
amount of methylation in anti-Ras total immunoprecipitates from cells
grown in the presence and absence of aldosterone. These results
demonstrate that aldosterone increases methylation and that the
antibody is specific for Ras because coincubation with the Ras
antigenic peptide reduces methylation to baseline levels.
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Even though we would anticipate that the immunoprecipitation of Ras
demonstrates that Ras is specifically methylated in response to
aldosterone, we also wanted to show directly that Ras protein could be
methylated in aldosterone-treated A6 cells. In two batches of cells
confluent for 2 days, the control and test samples were deprived of
aldosterone. Then aldosterone was added to the test cells 4 h
before cell lysates were prepared from both batches of cells.
14C-S-adenosyl-L-methionine
(SAM) and commercially available K-Ras protein was added to
the lysates. After a 30-min incubation period, Ras protein was
immunoprecipitated as described above, and the immunoprecipitated
protein was separated on gels. To verify the efficacy of the
immunoprecipitation, some of the protein was transferred to
nitrocellulose and blotted with anti-Ras antibody with results similar
to those shown in Fig. 6. The remainder of the immunoprecipitated protein was also separated on a gel that was subsequently exposed to a
phosphoimager screen to resolve the 14C-methylated
proteins. The inset of Fig. 7A shows one such
autoradiogram and shows that lysates from aldosterone-treated cells
produced significantly more methylated Ras. Figure 7B and
Table 2 show a summary of methylase
activity when K-Ras is used as a substrate for methyltransferase in
cellular lysates. Aldosterone is capable of significantly increasing
Ras methylation, and the methylation is specific for Ras because it can
be inhibited by coincubation of antibody with K-Ras antigenic peptide
during the immunoprecipitation.
Aldosterone increases Ras methylation, and inhibiting cellular protein
methylation reduces Na+ transport. To determine if Ras
activation was directly related to increases in Na+ channel
activity, we inhibited Ras expression by treating the A6 cells with
antisense oligonucleotides directed against sequences near the
translation start site of Ras. Cells were incubated overnight with
either Ras antisense oligonucleotides or sense oligonucleotides (as
controls), and both groups of cells were treated with aldosterone for
4 h. Patches were examined on the apical surface of the cells, and
Na+ channel activity measured as NPo
(the product of the number of channels times the open probability of
the channels), and then open probability, Po,
was calculated. In cells treated with Ras antisense,
Po was significantly decreased (from 0.40 ± 0.03 in sense-treated cells to 0.08 ± 0.01; P < 0.0001, n = 11), showing that inhibition of Ras
expression decreased Na+ channel activity (Fig.
8). These observations support the idea that epithelial Na+ transport requires expression of a
small G protein that must be methylated to promote Na+
channel activity.

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Fig. 8.
Ras antisense oligonucleotides reduce the activity of
Na+ channels. To determine if Ras activation was
directly related to increases in Na+ channel activity, we
inhibited Ras expression by treating the A6 cells with antisense
oligonucleotide-directed sequences near the translation start site of
Ras. Cells were incubated overnight with either Ras antisense
oligonucleotides or sense oligonucleotides (as controls), and both
groups of cells treated with aldosterone for 4 h. Patches were
examined on the apical surface of the cells. Two representative single
channel records from a sense-treated cell (left) and an
antisense-treated cell (right) are shown (top).
Na+ channel activity measured as
NPo, and then Po was
calculated (bottom). In cells treated with Ras antisense,
Po was significantly decreased (from 0.40 ± 0.03 in sense-treated cells to 0.08 ± 0.01; P < 0.0001, n = 11), showing that inhibition of Ras
expression decreased Na+ channel activity.
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|
 |
DISCUSSION |
All of the available biochemical evidence suggests that the number
of Na+ channel proteins in the apical membrane of A6 cells
does not change in the initial period after aldosterone treatment
(13, 22, 23, 31,
34). Our laboratory, using single channel measurements,
has demonstrated that aldosterone increases Na+ transport
in A6 epithelia by increasing the open time and open probability of the
channels (20), possibly through a posttranslational modification of the channel (38, 49). One
possible aldosterone-induced modification is methylation of membrane
protein and lipid (38). In this scenario, intracellular
methyltransferases induced by aldosterone would be responsible for the
methylation and modulation of Na+ channel proteins. Indeed,
our laboratory, using patch-clamp methods, has shown that methylation
occurs in isolated, cell-free patches of membrane (4) and
that methylation-induced activation of Na+ channels is
augmented by cytosolic GTP, as others have also demonstrated (40). This indicates that the target for methylation must
be a membrane-associated protein in close proximity to the channel. If
alterations in methylation are responsible for aldosterone-induced increases in Na+ transport, then aldosterone must alter
methyltransferase activity. Our data (Fig. 1) show that aldosterone
does increase total protein methylation by activating methyltransferase
activity (Fig. 2). These two observations confirm the idea that
aldosterone alters protein methylation by, in some way, regulating
methyltransferase activity. One way of regulating active
methyltransferases is by controlling the cytosolic concentrations of
endogenous inhibitors. The most common inhibitor is the metabolic end
product of methylation, SAH. This is the product that is produced when
the endogenous methyl donor, SAM, sometimes referred to as SAMe or
AdoMet, transfers its methyl group to a target protein. SAH is a potent
inhibitor for all methyltransferases, although the half-maximal
inhibitory concentration does vary among different transferases
(10, 21). The inhibitory action of SAH or
DZA, which prevents the hydrolysis of SAH (52), is obvious
in Fig. 3. In other work, we have demonstrated that one
aldosterone-mediated regulatory mechanism for methyltransferases is by
regulating cellular levels of SAH (45). Figure 3 also shows that a potent prenylated protein methyltransferase inhibitor, FTS
(28), will inhibit methylase activity, and that the same inhibitor dramatically reduced aldosterone-stimulated Na+
transport (measured as short-circuit current; Fig. 4). These data
suggest that aldosterone modulates Na+ channel activity in
A6 epithelia by altering isoprenyl-cysteine methylation. This
conclusion is similar to that reached by others using somewhat
different approaches (6, 39,
47).
However, on the basis of the results described above and the
observation that at least one Ras gene, K-Ras2a, is an
aldosterone-inducible gene (42, 47), we
thought that one potential target that was known to be activated by
both methylation and GTP was a small G protein like Ras. Therefore, we
examined the effect of aldosterone treatment on methylation of Ras
protein. Figure 5 shows that in the molecular weight range expected for
Ras, there is a significant increase in methylation even though the
protein content of the gel slice is altered very little. Although there
is no significant change in the total protein within the gel slice that
includes Ras's molecular weight range, there still might be a change
in the amount of Ras protein if Ras makes up only a small fraction of
total protein. Figure 6 shows that, in fact, there is a significant change in the total cellular amount of Ras protein. One additional caveat is that the antibody used to detect Ras will detect most cellular forms of Ras. Thus if one type of Ras (like H-Ras) was present
at high concentration, then it might be difficult to demonstrate a
larger change in a rarer species (like K-Ras). The question remained of
whether the methylated protein in the 20-kDa gel slice is actually Ras
instead of some other low-molecular-weight protein. In Fig. 7, we show
that Ras is a substrate for methylation (Fig. 7B), that
aldosterone produces a large increase in the methylation of Ras
protein, and that methylated Ras is detectable in autoradiograms and is
increased after aldosterone treatment. This demonstrates that although
there might be some other low-molecular-weight protein that is also
methylated, Ras is strongly methylated in response to aldosterone.
Finally, we found it interesting that Ras is methylated in response to
aldosterone, whereas others have shown that methylation activates Ras
(36, 48); but the question remained of
whether Ras has anything to do with Na+ transport. Figure 8
shows that when Ras is inhibited by antisense oligonucleotides, there
is a significant reduction in channel activity.
All of these results demonstrate that methylation of Ras is necessary
for aldosterone-induced increases in Na+ transport, but
methylation alone may not be sufficient by itself to increase
Na+ transport. Rather, we feel that Ras methylation is
permissive. As we and others have pointed out in other work, neither of
the two main enzymes associated with methylation, methyltransferase, or
SAH hydrolase, are proteins in which cellular concentration is
increased by aldosterone; that is, aldosterone does not increase transcription and translation of either protein (1,
46, 47). Nonetheless, activity of both
proteins increases (possibly due to posttranslational phosphorylation
of methyltransferase). On the other hand, at least one Ras protein,
K-Ras2a, is apparently an aldosterone-induced gene (42,
47), and our results also suggest that some Ras proteins
are induced by aldosterone. However, to be active, K-Ras2a must be
methylated (36). Therefore, if the magnitude of the
aldosterone-induced increase in Na+ transport is to be
proportional to the extent of K-Ras2a transcription and translation,
then aldosterone must alter methyltransferase activity to ensure that
the rate of Ras methylation does not become the rate-limiting step in
the induction of ENaC. As mentioned above, this increase in
methyltransferase activity is due to activation of preexisting
methyltransferase, not to transcription and translation of new methyltransferase.
We have attempted in our work to limit our examination to Ras
methylation and its effect on channel activity. However, our results do
not preclude the possibility of alternative types of methylation or
methylation of alternative proteins; Ras may not be the only protein
that is methylated by aldosterone. After all, Fig. 5 shows that there
is also a significant increase in methylation in the 80- to 100-kDa
range. This range is consistent with previous reports of
aldosterone-induced methylation of a 95-kDa protein and methylation of
the
-subunit of ENaC (37, 39,
40). Methylation of other proteins besides Ras would be
consistent with a general aldosterone-induced increase in cellular
methyltransferase activity. Under such conditions, it would not be
surprising that
-ENaC is methylated, as reported by Rokaw et al.
(37). However, the real issue is what sort of methylation
is occurring and whether methylated ENaC by itself in the apical
membrane of A6 cells is more active than nonmethylated ENaC. In our
experiments, we tried to focus on isoprenyl-cysteine methyltransferase,
the methyltransferase responsible for cysteine methylation at the
consensus CAAX sequence and which is known to methylate Ras proteins.
There are no CAAX sequences in any of the ENaC subunits, and,
therefore, the subunits cannot be the target of isoprenyl-cysteine
methyltransferase. On the other hand, there are other known
methyltransferases, but few are known to directly activate proteins.
One that does modifies a COOH-terminal leucine (8,
50), but
-ENaC contains no such residue (
-ENaC
does). Alternatively, many proteins are methylated on basic and acidic
residues.
-ENaC contains many such residues (but
and
do,
also). In eukaryotes, methylation of acidic or basic residues tends to
mark proteins for degradation. Methylation of the
-subunit in this
context would be intriguing because some investigators have suggested
that turnover and degradation of
-ENaC regulates the surface
expression of functional channels (43, 44).
This does not, however, explain why
-ENaC appears to be activated in
lipid bilayers by methylation donors (like SAM) to increase the open
probability of Na+ channels (at least ones treated with
dithiothreitol) (18). The difficulty with these
experiments is that the channels are reconstituted from oocyte proteins
(that may contain other proteins that are targets for methylation, but
that must necessarily associate with
-ENaC), and methylation enzymes
are found in a total cell cytosolic lysate. Therefore, the oocyte
lysate may contain many methyltransferases other than
isoprenyl-cysteine methyltransferase that could methylate
-ENaC or
associated regulatory proteins.
An alternative possibility is that Ras protein forms a complex with one
of the ENaC subunits as part of the activation process, and the higher
molecular weight band represents methylation of Ras in such a complex.
The question of Ras-ENaC association underscores the question of the
mechanism by which Ras activation induces channel activity. There are
several reports of activation of ion channels by Ras protein
(11, 12, 17, 27,
30, 32, 51). In most of these
reports, some intracellular factor (such as the cytosolic tyrosine
kinase, Src, or the Ras GTPase activating protein, Ras GAP) is
necessary to support Ras activity. For these mediators, it seems
unlikely that there would be Ras activity in excised, inside-out
patches as we have previously reported (4). However, there
is at least one report of Ras activity in excised patches in which Ras
is still capable of activating a nonselective cation channel in
mesangial cells (27). In this case, channel activation and
Ras activity could be inhibited by application of an anti-Ras antibody
to the cytosolic surface of the excised patch. Thus it would appear
that all the necessary components for Ras activation of ion channels
can be associated with isolated membranes.
We, therefore, conclude that 1) protein methylation is
essential for aldosterone-induced increases in Na+
transport; 2) one of the targets for methylation is
p21ras; and 3) inhibition of Ras expression or
Ras methylation inhibits Na+ channel activity. A schematic
diagram of the components of aldosterone-signaling cascade described in
this paper is shown in Fig. 9.

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Fig. 9.
A schematic diagram of some of the possible
components of the aldosterone-signaling cascade depicting the
involvement of Ras and methylation. SAM,
S-adenosyl-L-methionine.
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|
 |
ACKNOWLEDGEMENTS |
The authors thank B. J. Duke for exceptional patience and good
humor in maintaining the A6 cell cultures.
 |
FOOTNOTES |
This work was supported by National Institute of Diabetes and Digestive
and Kidney Diseases Fellowship F32-DK-09729 (to J. D. Stockand)
and Grant R01-DK-37963 (to D. C. Eaton).
Address for reprint requests and other correspondence: D. C. Eaton, Center for Cell and Molecular Signaling, Dept. of Physiology, Emory Univ. School of Medicine, 1648 Pierce Drive, Atlanta, GA 30322.
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.
Received 29 July 1999; accepted in final form 21 February 2000.
 |
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