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INTRODUCTION |
Serum- and glucocorticoid-induced protein kinase
(Sgk)1 is a
serine/threonine-specific protein kinase that regulates sodium absorption by the amiloride sensitive sodium channel in kidney principal cells (1, 2). Transepithelial sodium transport is an
essential physiological function regulated by multiple hormone systems,
including aldosterone, vasopressin, and insulin. Recent evidence has
suggested that Sgk is an important molecular target that integrates the
multiple endocrine inputs regulating sodium transport. Expression of
the Sgk gene is regulated at the transcriptional level by serum,
glucocorticoids, and aldosterone, all of which up-regulate gene
transcription (2, 3). Furthermore, insulin as well as insulin-like
growth factors stimulate Sgk activity by a mechanism requiring the
participation of phosphatidylinositol 3-kinase. Activation of
phosphatidylinositol 3-kinase leads to activation of two downstream
protein kinase activities: phosphoinositide-dependent kinases (PDK)-1 and -2. Phosphorylation of Thr256 and
Ser422 by PDK-1 and -2, respectively, leads to activation
of Sgk (4, 5).
Thr369 of Sgk is contained within a consensus sequence for
phosphorylation by cyclic AMP-dependent protein kinase
(PKA) (6, 7). Because vasopressin leads to activation of adenylate
cyclase (8), we hypothesized that phosphorylation of Sgk by protein kinase A (PKA) might mediate the action of vasopressin to stimulate sodium transport. In the present study, we obtained evidence that cyclic AMP activates recombinant Sgk expressed in COS7 cells. Moreover,
cyclic AMP-induced activation of Sgk is inhibited by H89, a PKA
inhibitor, and also by mutation of the putative PKA phosphorylation
site (Thr369
Ala). In addition, we obtained evidence
that phosphorylation by PDK-1 and/or PDK-2 is required for the ability
of cyclic AMP to activate Sgk. These data provide additional evidence
elucidating the mechanisms whereby Sgk mediates endocrine regulation of
sodium transport.
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EXPERIMENTAL PROCEDURES |
Expression of Recombinant Sgk--
We searched
GenBankTM and identified several expressed sequence
tags corresponding to human Sgk. Clones 645185 and 42669 were obtained from Research Genetics (Huntsville, AL) and were found to
contain the complete coding sequence of Sgk1. Clone 645185, which
contained the complete coding sequence of the enzyme, was used in our
experiments. We used polymerase chain reaction (KlenTaq, CLONTECH, Palo Alto, CA) to introduce both a strong
Kozak consensus sequence at the translation start site and a
Myc-epitope tag at the N terminus. The cDNA, flanked by an
EcoRI site upstream and a NotI site downstream,
was ligated into the TA Cloning pCRTM 2.1 plasmid
(Invitrogen, Carlsbad, CA) and subcloned into the multiple cloning
sites of pcDNA 3.1+ (Invitrogen), pCI-neo (Promega, Madison, WI),
and pEGFP-C1 (CLONTECH). In addition, using a
polymerase chain reaction-based method (Quick change
Site-directed mutagenesis Kit; Stratagene, La Jolla, CA) we introduced
the following three mutations into the coding sequence of Sgk:
Thr369
Ala, Ser422
Ala, and
Thr256
Ala. All the mutations were confirmed by
automated DNA sequencing using reagents supplied in the DNA Sequencing
Kit (PerkinElmer Life Sciences-Applied Biosystems, Foster City, CA).
COS7 cells were plated at a density of 1.5 × 105
cells/ml in six-well plates and cultured overnight in DMEM-low glucose
(Life Technologies, Inc.) containing 10% fetal bovine serum (Life
Technologies, Inc.). The following day, cells were transfected with
expression plasmids using LipofectAMINE (Life Technologies, Inc.)
following the manufacturer's instructions. We used pCI-neo Myc-Sgk
(600 ng/well) to express Sgk in the studies of in vitro
kinase activity, and in most of the 32P labeling studies.
pEGFP-C1 Myc-Sgk (600 ng/well) was used for the 32P
labeling study of the phosphorylation of the Sgk-GFP fusion protein
(Fig. 6B). Three hours after transfecting the cells, the transfection medium was diluted 1:1 with DMEM medium containing 20%
fetal bovine serum. The next day the medium was replaced with DMEM
containing 10% fetal bovine serum plus antibiotics (penicillin, 100 units/ml and streptomycin, 100 µg/ml). Twelve hours later, the medium
was replaced with serum-free medium (DMEM containing insulin-free
bovine serum albumin (0.1%) plus antibiotics).
In Vitro Kinase Assay--
Cells were incubated at 37 °C in
serum-free medium in the presence or absence of human recombinant
insulin (1 µM; Sigma) or 8-(4-chlorophenylthio)-cAMP (0.2 mM; 8CPT-cAMP;
Sigma). Insulin was added 20 min prior to the time of cell lysis; and
8CPT-cAMP (9) was added 60 min prior to lysing the cells. In some
experiments, we added various inhibitors 100 min prior to lysing the
cells: wortmannin (100 nM) (5), H89 (10 µM)
(10), or okadaic acid (600 nM) (11). H89 was dissolved in
DMEM while wortmannin and okadaic acid were dissolved in
Me2SO. In each case, an equal volume of solvent was
added to cell cultures lacking the inhibitor. Wortmanin and H89 were
obtained from Calbiochem, while okadaic acid was supplied by Sigma.
The cell monolayers were then solubilized in solubilization buffer (300 µl/well) for 20 min at 4 °C: Tris, 50 mM, pH 7.8; NaCl, 300 mM; Triton X-100, 0.5%; protease inhibitors
CompleteTM (Roche Molecular Biochemicals) and phosphatase
inhibitors (NaF, 100 mM; sodium pyrophosphate, 5 mM; sodium orthovanadate, 2 mM; EDTA, 5 mM). Extracts from two replicate wells were pooled, and an
aliquot (100 µl) was analyzed by immunoblotting with rabbit anti-Myc
antibody (Santa Cruz Biotechnology) to assess the expression of
Myc-Sgk. A second aliquot (450 µl) was immunoprecipitated with anti-Myc antibody (30 µl) for the immune complex kinase assay (12).
Antibody was prebound to protein G-UltraLink (15 µl; Pierce) at
4 °C for 120 min. The immobilized antibody was sedimented, washed,
and incubated with cell extract overnight at 4 °C on a rotating
wheel. The immune complexes were sedimented and washed three times with
solubilization buffer and twice with a kinase buffer (Tris, 20 mM, pH 7.4; MgCl2, 10 mM). Pellets
were finally resuspended in kinase buffer containing ATP (5 µM), protein kinase A inhibitor (1 µM;
Sigma), dithiothreitol (1 mM),
[
-32P]ATP (0.02 mCi/sample), and
Arg-Pro-Arg-Thr-Ser-Thr-Phe peptide substrate (1 mM) (5).
The reaction was allowed to occur for 30 min at room temperature, with
gentle agitation, prior to stopping with 10 µl of stopping solution
(ATP, 1 mM; bovine serum albumin, 1%; HCl, 0.6% w/v). The
reaction mix was centrifuged in an Eppendorf microcentrifuge for 10 min
at 14,000 rpm. Supernatants (20 µl) were applied to a 2.1-cm diameter
p81 Whatman paper. After drying at room temperature, the filter was
washed four times with phosphoric acid (25 mM), once with
acetone, and then counted in a scintillation counter. Background
incorporation of radioactivity was estimated by assaying extracts
prepared from untransfected cells incubated in the presence or absence
of insulin or 8CPT-cAMP. To determine the kinase activity of Sgk, we
subtracted the average level of background 32P
incorporation from 32P incorporation catalyzed by extracts
of transfected cells.
32P Labeling of Intact Cells--
After ~16 h of
serum starvation (see above), the medium was replaced with 2 ml/well of
phosphate-free RPMI 1640 (Biofluids, Rockville, MD) containing
insulin-free bovine serum albumin (0.1%). Two hours later, the medium
was exchanged for 1 ml/well of the same medium supplemented with
[32P]orthophosphoric acid (0.25 mCi/ml) (PerkinElmer Life
Sciences) (13). After the cells were incubated in the presence
or absence of insulin (see above), the medium was aspirated, and the
cells were frozen on methanol-dry ice. Monolayers were then solubilized with 300 µl/well of lysis buffer (see above) for 20 min at 4 °C. For each experimental point, the extracts from two wells were pooled.
The Triton extracts were clarified by centrifugation in an Eppendorf
microcentrifuge (14,000 rpm, 20 min, 4 °C) and
immunoprecipitated with rabbit anti-Myc antibody (see above). However,
instead of washing with kinase buffer, the pellets were resuspended in
Laemmli sample buffer containing dithiothreitol (1 mM), boiled for 5 min, and analyzed by a SDS-polyacrylamide
gel electrophoresis using a 10% gel. The 32P-labeled
proteins were transferred to nitrocellulose (Protran, Schleicher & Schuell) and detected both by autoradiography and immunodetection with
mouse anti-Myc antibodies (Santa Cruz Biotechnology).
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RESULTS |
Regulation of Sgk by cAMP--
The amino acid sequence of Sgk
contains a potential protein kinase A phosphorylation site:
Lys-Lys-Ile-Thr-Pro (amino acid residues 366-370). Therefore, we
investigated the effects of cyclic AMP upon Myc-Sgk activity. After
transiently expressing Myc-Sgk in COS7 cells, the transfected cells
were incubated in the presence or absence of cyclic AMP, insulin, or
both. Subsequently, the cells were lysed in detergent, Myc-Sgk was
immunoprecipitated with anti-Myc antibody, and Sgk activity
was assayed in immune complexes. Incubation of cells in the presence of
8CPT-cAMP (0.2 mM) for 60 min led to a 2-fold activation of
Myc-Sgk (p = 0.0007; Fig.
1A, lane 3). As
expected, incubation with insulin for 20 min increased Myc-Sgk activity
by 3-fold (p = 0.0002; Fig. 1A, lane
2).

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Fig. 1.
Activation of Sgk by insulin and
8-CPT-cAMP. Myc-Sgk (600 ng of expression plasmid DNA) was
expressed in COS7 cells. Where indicated, the cells were stimulated
with insulin (1 µM for 20 min) and/or 8CPT-cAMP (0.2 mM for 60 min). Myc-Sgk was immunoprecipitated by rabbit
anti-Myc antibodies, and the immune complexes were assayed in an
in vitro kinase assay (A). Sgk activity was
calculated by subtracting the background incorporation of
32P catalyzed by immunoprecipitates of extracts from
nontransfected cells. To average data from multiple experiments, the
data were expressed as a fraction of the mean Sgk activity measured in
cells incubated in the presence of insulin. Data are means ± S.E.
of duplicate measurements obtained in eight independent experiments.
Duplicate aliquots of the anti-Myc immunoprecipitates were resuspended
in Laemmli sample buffer and analyzed by SDS-polyacrylamide gel
electrophoresis, followed by immunoblotting with monoclonal anti-Myc
antibodies. The blot was exposed to film for 30 s (B,
upper gel) or 5 min (B, lower gel). In the
immunoblots, several different bands with different mobilities were
detected. As described under "Results," B1 appeared not to
be phosphorylated, B2 was phosphorylated at relatively low
stoichiometry, and B3 was phosphorylated at high stoichiometry.
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Since neither insulin nor cAMP affected the quantity of Myc-Sgk
contained in the immunoprecipitates (Fig. 1B, lanes
2 and 3), we conclude that cAMP and insulin increased
the specific activity of the enzyme.
H89 (10 µM), an inhibitor of protein kinase A, blocked
the effect of 8CPT-cAMP (Fig.
2A, column 7), but
did not interfere with the ability of insulin to stimulate the kinase
activity of Myc-Sgk (Fig. 2A, column 6). As shown
previously (4, 5), wortmannin (100 nM) inhibited the effect
of insulin to activate Sgk (Fig. 2B, column 6).
However, unexpectedly, wortmannin also inhibited the ability of
8CPT-cAMP to activate Myc-Sgk (Fig. 2B, column 7)
and inhibited the combined effect of the insulin plus 8CPT-cAMP (Fig.
2B, column 8).

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Fig. 2.
Effects of H89 and wortmannin upon regulation
of Sgk activity. Cells were transfected and incubated as
described in the legend to Fig. 1A with the exception that
either H89 (10 µM; A) or wortmannin (100 nM; B) was added to the incubation medium 70 min
before lysing the cells where indicated. Anti-Myc immunoprecipitates
were used in an in vitro kinase assay, as indicated under
the "Experimental Procedures." Sgk activity was calculated by
subtracting the background incorporation of 32P catalyzed
by immunoprecipitates of extracts from nontransfected cells. Data are
expressed as a fraction of the mean insulin-stimulated Sgk
activity observed in the absence of wortmannin and H89. Data are
means ± S.E. obtained from four individual points in two
replicate experiments (A) and six individual points in three
replicate experiments (B).
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Mutational Analysis of Phosphorylation Sites--
-We carried out
mutational studies to investigate the role of the various
phosphorylation sites. Thr369
Ala mutant Myc-Sgk, which
lacks the predicted phosphorylation site for protein kinase A, is
unable to undergo activation in response to 8CPT-cAMP
(p = 0.39; Fig. 3,
column 7). This observation suggests that phosphorylation of
Thr369 ,presumably catalyzed by protein kinase A, mediates
activation of Sgk in response to cAMP. In contrast, Thr369
Ala mutant Myc-Sgk underwent normal activation in response to
insulin (p = 0.0002; Fig. 3, column 6).

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Fig. 3.
The protein kinase A phosphorylation site in
Sgk is required for activation by 8-CPT-cAMP. COS7 cells were
transfected with expression plasmids for either Myc-Sgk or the
Thr369 Ala mutant Myc-Sgk in experiments similar to
those described in the legend to Fig. 1. Sgk activity was
calculated by subtracting the background incorporation of
32P catalyzed by immunoprecipitates of extracts from
nontransfected cells. Data are expressed as a fraction of the mean
insulin-stimulated wild type Myc-Sgk. Data are means ± S.E. obtained from six individual points in three replicate
experiments.
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Thr256 and Ser422 of Sgk are the
phosphorylation sites for PDK1 and PDK2, respectively. As expected,
Ser422
Ala mutant Myc-Sgk was not activated by insulin
(Fig. 4, column 6). Moreover,
despite the fact that the protein kinase A phosphorylation site is
intact, the Ser422
Ala mutant does not undergo
activation in response to 8CPT-cAMP (Fig. 4, column 7).
Similar results were obtained with Thr256
Ala mutant
Myc-Sgk (data not shown). These results are consistent with the
observations reported above, demonstrating that wortmannin inhibits
activation of Myc-Sgk in response to either 8-CPT-cAMP or insulin (Fig.
2). Thus, phosphorylation by PKA is not sufficient to activate Myc-Sgk;
phosphorylation of Ser422 and Thr256,
presumably by PDK2 and PDK1, is also required.

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Fig. 4.
The PDK2 phosphorylation site in Sgk is
required for activation by insulin or 8-CPT-cAMP. COS7 cells were
transfected with expression plasmids for either Myc-Sgk or
Ser422 Ala-mutant Myc-Sgk in experiments similar to
those described in the legend to Fig. 1. Sgk activity was
calculated by subtracting the background incorporation of
32P catalyzed by immunoprecipitates of extracts from
nontransfected cells. Data are expressed as a fraction of the mean
insulin-stimulated wild type Myc-Sgk. Data are means ± S.E. obtained from four individual points in two replicate
experiments.
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Multiple Species of Sgk--
Using immunoblotting to analyze
solubilized extracts from transfected cells expressing Myc-Sgk, we
detected two major bands corresponding to recombinant Myc-Sgk: a dark
Mr 51,000 band and a light
Mr 49,000 band, designated B2 and B1,
respectively (Fig. 1B, upper panel, lane
1). Insulin did not alter the relative intensities of B1 and B2
compared with untreated samples (Fig. 1B, upper
panel, lane 2). In contrast, 8CPT-cAMP increased the
intensity of B1 when added alone (Fig. 1B, upper
panel, lane 3) or in combination with insulin (Fig.
1B, upper panel, lane 4). H89 and
wortmannin do not inhibit the ability of 8CPT-cAMP to increase the
intensity of the faster migrating band, B1 (Fig.
5A, lanes 4 and
8). Likewise, this effect was also observed with the two
mutant forms of Myc-Sgk: Thr369
Ala and
Ser422
Ala (Fig. 5B, lanes 2-4).
Thus, the shift in electrophoretic mobility does not appear to require
phosphorylation by PKA or PDK-2 and does not seem to be related to the
activation of the enzymatic activity of Sgk.

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Fig. 5.
Multiple species of Sgk with distinct
electrophoretic mobilities. A, COS7 cells expressing
recombinant wild type Myc-Sgk were stimulated with insulin (1 µM for 20 min) and/or 8CPT-cAMP (0.2 mM for
60 min). Where indicated, either wortmannin (100 nM) or H89
(10 µM) was added 70 min before lysing the cells.
B, COS7 cells expressing wild type Myc-Sgk (upper
gel), Thr369 Ala mutant Myc-Sgk (middle
gel), or Ser422 Ala-mutant Myc-Sgk (lower
gel). Where indicated cells were stimulated with insulin (1 µM for 20 min) and/or 8CPT-cAMP (0.2 mM for
60 min) and/or okadaic acid (600 nM for 70 min) before
lysing the cells. A and B, solubilized extracts were diluted
with Laemmli sample buffer and analyzed with SDS-polyacrylamide gel
electrophoresis followed by immunoblotting with rabbit anti-Myc
antibodies.
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When films were overexposed, additional weak bands of slower mobility,
designated B3, were detected in extracts of cells that had been
stimulated with either insulin or 8CPT-cAMP (Fig. 1B, lower panel, lanes 2-4). Intact cell
phosphorylation studies (see below) suggested that the cluster of low
mobility bands (B3) represents hyperphosphorylated forms of Sgk.
Intact Cell Phosphorylation--
Cells were labeled with
[32P]orthophosphate and stimulated by insulin and/or
8CPT-cAMP. Under these conditions, the fastest migrating species (B1)
detected by c-Myc antibody (Fig.
6A, left panel,
lanes 1-5) was not phosphorylated (Fig. 6A,
right panel, lanes 1-5). On the other hand,
insulin, 8CPT-cAMP, and okadaic acid induced the appearance of a highly
phosphorylated slower mobility form of Sgk (B3) (Fig. 6A,
right panel, lanes 1-5). The intensity of the
slow migrating band (B3) correlates well with the extent of activation
of Myc-Sgk, being maximal in cells incubated with both insulin and
8CPT-cAMP, but somewhat less intense in cells incubated with either
agent alone. Although band B2 is much more intense than band B3 in the
anti-Myc immunoblots, band B3 is easily detected in the 32P
labeling studies. This suggests that the B3 species is highly phosphorylated, presumably with multiple sites being phosphorylated. Nevertheless the B2 species also contains 32P, even in the
absence of insulin and 8CPT-cAMP.

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Fig. 6.
Incorporation of
[32P]orthophosphate into Sgk in intact cells.
A, COS7 cells were transfected with wild type Myc-Sgk.
Thereafter, transfected cells were labeled with
[32P]orthophosphate and stimulated with insulin (1 µM for 20 min) and/or 8CPT-cAMP 0.2 mM for 60 min. In some wells, okadaic acid (600 nM) was added 70 min
prior to lysing the cells. Rabbit anti-Myc immunoprecipitates were
separated by SDS-polyacrylamide gel electrophoresis. Proteins were
blotted on nitrocellulose and detected by mouse anti-Myc antibodies
followed by ECL (left panels) or by autoradiography
(right panels). B, COS7 cells were transfected
with LipofectAMINE in the absence (lane 1) or presence of
expression plasmids (600 ng) for Myc-Sgk (lanes 2 and
3) or Myc-GFP-Sgk (lanes 4 and 5).
Transfected cells were preincubated in the presence of
[32P]orthophosphate and subsequently, where indicated, in
the presence or absence of insulin (1 µM for 20 min).
Anti-Myc immunoprecipitates were separated by SDS-polyacrylamide gel
electrophoresis, blotted on nitrocellulose, and detected by
autoradiography.
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There are at least two possible interpretations of the significance of
the B3 band, the intensity of which is increased in response to
insulin, 8CPT-cAMP, and okadaic acid. First, because none of the Sgk
bands were present in nontransfected cells (Fig. 6A,
left and right panels, lane C), it
seemed likely that the B2 and B3 bands corresponded to different forms
(e.g. different phosphorylation states) of Myc-Sgk.
Alternatively, it was possible that one band might have represented a
tightly bound substrate for Sgk. To distinguish between these two
possibilities, we transfected cells with a Sgk-GFP fusion protein (Fig.
6B). By fusing Sgk to the GFP moiety, we shifted both bands
to higher molecular mass (Fig. 6B, lanes 4 and
5). This demonstrates that both bands are forms of the
Myc-Sgk molecule, rather than substrates for phosphorylation by
Sgk.
Effects of Okadaic Acid--
Incubation of cells in the presence
of okadaic acid, an inhibitor of protein phosphatases 1 and 2a, led to
a 10-fold increase in the activity of Myc-Sgk (Fig.
7, column 1 versus
column 5). Indeed, the effect of okadaic acid was greater than the
effects of either insulin or 8CPT-cAMP. Nevertheless, both agents were able to further increase Sgk activity even in the presence of okadaic
acid (600 nM): p = 0.033 (insulin) and
p = 0.039 (8CPT-cAMP). When the phosphorylation sites
for PDK-2 (Ser422Ala-mutant; Fig. 8,
columns 9-16) and PDK-2 (Thr256
Ala mutant;
data not shown) were abolished, this blocked the ability of okadaic
acid to activate Myc-Sgk. In contrast, abolishing the protein kinase A
phosphorylation site (Thr369
Ala mutant; Fig.
9, lanes 13-16) did not
impair the ability of okadaic acid to activate Myc-Sgk.

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Fig. 7.
Effect of okadaic acid upon recombinant Sgk
activity. Cells were transfected and incubated as described in the
legend to Figs. 1A and 2 with the exception that okadaic
acid (600 nM) was added instead of wortmannin or H89.
Anti-Myc immunoprecipitates were used in an in vitro kinase
assay, as indicated under "Experimental Procedures." Sgk
activity was calculated by subtracting the background incorporation of
32P catalyzed by immunoprecipitates of extracts from
nontransfected cells. Data are expressed as a fraction of the mean
insulin-stimulated Sgk activity observed in the absence okadaic
acid. Data are means ± S.E. obtained from five individual points
in two replicate experiments.
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Fig. 8.
The PDK2 phosphorylation site in Sgk is
required for activation by okadaic acid. COS7 cells were
transfected with expression plasmids for either Myc-Sgk or the
Ser422 Ala mutant Myc-Sgk in experiments similar to
those described in the legend to Fig. 1. Where indicated, okadaic acid
(600 nM) was added. Sgk activity was calculated by
subtracting the background incorporation of 32P catalyzed
by immunoprecipitates of extracts from nontransfected cells. Data are
expressed as a fraction of the mean insulin-stimulated wild type
Myc-Sgk. Data are means ± S.E. obtained from two
individual points in one experiment.
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Fig. 9.
The PKA phosphorylation site is not required
for activation of Sgk by okadaic acid. COS7 cells were transfected
with expression plasmids for either Myc-Sgk or the
Thr369 Ala-mutant Myc-Sgk in experiments similar to
those described in the legend to Fig. 1. Where indicated, okadaic acid
(600 nM) was added. Sgk activity was calculated by
subtracting the background incorporation of 32P catalyzed
by immunoprecipitates of extracts from nontransfected cells. Data are
expressed as a percentage fraction of the mean insulin-stimulated wild
type Myc-Sgk. Data are means ± S.E. obtained from seven
individual points in three replicate experiments.
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Okadaic acid (600 nM for 70 min) resulted in the appearance
of the hyperphosphorylated B3 band (Fig. 6A, right panels,
lane 5), consistent with Sgk kinase activation.
Interestingly, like 8CPT-cAMP, okadaic acid increased the relative
intensity of B1 (i.e. the B1:B2 ratio; Fig. 6A, left
panels, lanes 1, 2, and 5). The observation
was confirmed in separate experiments, where okadaic acid was added
alone or in combination with insulin and 8CPT-cAMP (Fig. 5B,
lanes 2-9).
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DISCUSSION |
Regulation of Sgk--
The amiloride-sensitive epithelial sodium
channel (ENaC) in principal cells of the kidney plays a central role in
determining sodium balance. Because this channel is responsible for
reabsorbing the majority of the sodium in the glomerular filtrate, even
small changes in activity of the channel can have large effects upon the quantity of sodium excreted in the urine. In keeping with its
important physiological role, the activity of ENaC is highly regulated.
It has recently been demonstrated that Sgk activates ENaC. Moreover,
Sgk is itself highly regulated. For example, transcription of the Sgk
gene has been reported to be up-regulated by both mineralocorticoids and glucocorticoids (2, 3) and also by follicle-stimulating hormone (14, 15). In addition, Sgk can be phosphorylated on Thr256 and Ser422 by PDK-1 and -2, respectively; these phosphorylations mediate the ability of insulin,
insulin-like growth factor-1, and serum to activate Sgk (4, 5). It has
been proposed that these two phosphorylations may occur in a sequential
fashion (5). First, insulin stimulates PDK-2 to phosphorylate
Ser422, and this renders Sgk a better substrate for PDK-1,
which phosphorylates Thr256. It is the phosphorylation of
Thr256 by PDK-1 that is believed to be responsible for
activation of Sgk (5).
In this publication, we demonstrate that cAMP can activate Sgk and that
this effect is mediated by PKA, which directly phosphorylates Thr369 in Sgk. This hypothesis is supported by two
principal lines of evidence: (a) activation of Sgk by
8CPT-cAMP is inhibited by H89, an inhibitor of PKA activity; and
(b) substitution of Ala for Thr369 abolishes the
PKA phosphorylation site in Sgk and also abolishes the ability of
8CPT-cAMP to activate Sgk. Taken together, these observations
demonstrate that phosphorylation by PKA is required for activation of
Sgk in response to 8CPT-cAMP. However, two observations suggest that
phosphorylation of Thr369 is not sufficient to activate
Sgk: (a) wortmannin markedly inhibits the ability of
8CPT-cAMP to activate Sgk; and (b) mutation of the PDK-1 and
PDK-2 phosphorylation sites (Thr256 and Ser422,
respectively) prevents activation of Sgk in response to 8CPT-cAMP. Although the requirement for multisite phosphorylation has not yet been
fully explained, we would like to propose one possible mechanism. When
PKA phosphorylates Thr369, this might make Sgk a better
substrate for phosphorylation of Thr256 and
Ser422 by PDK-1 and PDK-2, respectively. According to this
hypothesis, phosphorylation of Thr369 renders Sgk such a
good substrate that it can be phosphorylated by the basal activities of
PDK-1 and PDK-2 without the need for insulin to activate those two
enzymes. The resulting phosphorylation of Thr256 and
Ser422 is sufficient to activate Sgk (4-6).
In contrast, although phosphorylation of Thr256 and
Ser422 is required for insulin-stimulated activation of
Sgk, PKA-mediated phosphorylation of Thr369 is not required
for activation in response to insulin. Furthermore, Ser422
is required for activation of Sgk in response to okadaic acid, whereas
Thr369 is not required. These observations suggest that
phosphorylation by PDK-1 and/or PDK-2 are the principal protein kinases
responsible for activating Sgk. Okadaic acid, an inhibitor of protein
phosphatases 1 and 2a, also leads to activation of Myc-Sgk. Taken
together, these observations suggest that either protein phosphatase 1 or 2a is responsible for dephosphorylating Thr256 and
Ser422, thereby inactivating Sgk.
After this work was completed, Gonzalez-Robayna et
al. (15) reported that protein kinase A contributed to mediating
the action of follicle-stimulating hormone to increase Sgk
activity in ovarian granulosa cells. They particularly emphasized the
role of cAMP in inducing transcription of the Sgk gene. Moreover, they concluded that cAMP triggered a pathway that activated
phosphatidylinositol 3-kinase. Nevertheless, these mechanisms are not
sufficient to account for our observations. Our mutational studies
provide strong direct evidence that activation in response to cAMP
requires direct phosphorylation of Sgk by protein kinase A inasmuch as
mutation of the protein kinase A phosphorylation site abolishes the
response. In contrast, the same mutation does not impair the ability of the enzyme to be activated in response to insulin, a response that is
mediated by PDK-1 and -2 but that does not require of Thr369 in Sgk by protein kinase A.
Multiple Species of Sgk--
When Myc-Sgk was analyzed by
SDS-polyacrylamide gel electrophoresis followed by immunoblotting with
anti-Myc antibody, we detected two closely spaced bands with
Mr of 49,000 and 51,000 (designated as B1 and
B2, respectively). In addition, when the blots were overexposed,
we detected a third lower mobility band (designated B3) in extracts of
cells that were stimulated with insulin and/or 8CPT-cAMP.
Interestingly, 8CPT-cAMP increased the intensity of B1 (i.e.
the highest mobility band). The alteration in electrophoretic mobility
of B1 is likely due to a covalent modification of the protein. While
the nature of this covalent modification is unknown, 32P
labeling experiments carried out in intact cells suggest that B1 is not
phosphorylated. Rather, it is possible that 8CPT-cAMP triggers a
proteolytic cleavage that accounts for the decrease in apparent
molecular mass. Several lines of evidence suggest that this
8CPT-cAMP-induced gel shift is not related to Sgk activation. For
example, the mobility shift is observed with both Ser422
Ala- and Thr369
Ala-Myc-Sgk although these mutant
forms of the enzyme are resistant to activation by 8CPT-cAMP.
Furthermore, even at low concentrations (0.05-0.10 mM),
which did not activate Myc-Sgk, 8CPT-cAMP increased the intensity of
the B1 band (data not shown).
In contrast, B2 and B3 are both phosphorylated forms of Sgk. However,
by comparing the immunoblots with the autoradiographs of
32P-labeled Myc-Sgk, we conclude that the stoichiometry of
phosphorylation is higher for B3 than for B2. It is likely that the
hyperphosphorylation accounts for the decreased electrophoretic
mobility of B3. Furthermore, the intensity of B3 is increased in
response to either insulin and/or 8CPT-cAMP, which correlates with
activation of the enzyme.
In the immunoblotting experiments (Fig. 6A), band B3 is
considerably weaker than band B2, suggesting that only a small
percentage of Myc-Sgk molecules become hyperphosphorylated. If B3
represents the activated species, this observation suggests that a
relatively small minority of Myc-Sgk molecules are responsible for the
majority of Sgk activity. It is unclear why such a small percentage of Sgk molecules become hyperphosphorylated in response to insulin and/or cAMP.
Significance of cAMP-induced Activation of Sgk--
cAMP functions
as the second messenger for multiple signaling pathways. Thus, the
existence of a PKA phosphorylation site in Sgk provides opportunities
for regulation in many tissues. Three distinct isoenzymes of Sgk have
been reported. While Sgk1 and Sgk3 are widely expressed in many
tissues, Sgk2 has a more limited pattern of expression, i.e.
liver, kidney, pancreas, and brain (6). Interestingly, while the
protein kinase A phosphorylation site is conserved in both Sgk1 and
Sgk2, it does not appear to be present in the sequence of Sgk3. In
contrast, the phosphorylation sites for phosphoinositide kinases are
conserved in all three isoforms.
In the kidney, activation of Sgk by PKA may mediate the ability of
vasopressin to stimulate sodium transport (16). Interestingly, it has
recently been reported that wortmannin inhibits vasopressin-induced sodium transport (17), a result in agreement with our findings. Furthermore, there is evidence that cAMP exerts regulatory influences upon Sgk in extra-renal tissues. For example, it has been reported that
follicle-stimulating hormone activates Sgk in the ovary (14, 15). In addition to the role in normal physiology, it is possible that
Sgk may play important roles in the pathophysiology of various disease states.