Differing Roles of Akt and Serum- and Glucocorticoid-regulated Kinase in Glucose Metabolism, DNA Synthesis, and Oncogenic Activity*
Hideyuki Sakoda
,
Yukiko Gotoh
,
Hideki Katagiri ¶,
Mineo Kurokawa ||,
Hiraku Ono
,
Yukiko Onishi
,
Motonobu Anai
,
Takehide Ogihara ||,
Midori Fujishiro ||,
Yasushi Fukushima ||,
Miho Abe ||,
Nobuhiro Shojima ||,
Masatoshi Kikuchi
,
Yoshitomo Oka ¶,
Hisamaru Hirai || and
Tomoichiro Asano || **
From the
Institute for Adult Diseases, Asahi Life
Foundation, 1-9-14 Nishishinjuku, Shinjuku-ku, Tokyo 116, Japan, the
||Department of Internal Medicine, Graduate School
of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113, Japan,
the
Department of Molecular Biology, Institute
of Molecular and Cellular Biosciences, University of Tokyo, 1-1-1 Yayoi,
Bunkyo-ku, Tokyo 113-0032, Japan, and the
¶Division of Molecular Metabolism and Diabetes,
Department of Internal Medicine, Tohoku University Graduate School of
Medicine, 2-1 Seiryou, Aoba-ku, Sendai 980-8575, Japan
Received for publication, February 3, 2003
, and in revised form, April 30, 2003.
 |
ABSTRACT
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Serum- and glucocorticoid-regulated kinase (SGK) is a serine kinase that
has a catalytic domain homologous to that of Akt, but lacks the pleckstrin
homology domain present in Akt. Akt reportedly plays a key role in various
cellular actions, including glucose transport, glycogen synthesis, DNA
synthesis, anti-apoptotic activity, and cell proliferation. In this study, we
attempted to reveal the different roles of SGK and Akt by overexpressing
active mutants of Akt and SGK. We found that adenovirus-mediated
overexpression of myristoylated (myr-) forms of Akt resulted in high glucose
transport activity in 3T3-L1 adipocytes, phosphorylated glycogen synthase
kinase-3 (GSK3) and enhanced glycogen synthase activity in hepatocytes, and
the promotion of DNA synthesis in interleukin-3-dependent 32D cells. In
addition, stable transfection of myr-Akt in NIH3T3 cells induced an oncogenic
transformation in soft agar assays. The active mutant of SGK (D-SGK,
substitution of Ser422 with Asp) and myr-SGK were shown to
phosphorylate GSK3 and to enhance glycogen synthase activity in hepatocytes in
a manner very similar to that observed for myr-Akt. However, despite the
comparable degree of GSK3 phosphorylation between myr-Akt and D-SGK
or myr-SGK, D-SGK and myr-SGK failed to enhance glucose transport
activity in 3T3-L1 cells, DNA synthesis in 32D cells, and oncogenic
transformation in NIH3T3 cells. Therefore, the different roles of SGK and Akt
cannot be attributed to ability or inability to translocate to the membrane
thorough the pleckstrin homology domain, but rather must be attributable to
differences in the relatively narrow substrate specificities of these kinases.
In addition, our observations strongly suggest that phosphorylation of GSK3 is
either not involved in or not sufficient for GLUT4 translocation, DNA
synthesis, or oncogenic transformation. Thus, the identification of substrates
selectively phosphorylated by Akt, but by not SGK, may provide clues to
clarifying the pathway leading from Akt activation to these cellular
activities.
 |
INTRODUCTION
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PI1 3-kinase has
been implicated in the regulation of numerous cellular processes
(13).
The lipid product of PI 3-kinase reportedly activates several AGC kinases,
including Akt, atypical protein kinases C, p70 S6 kinase, and SGK
(3,
45). Among these AGC kinases,
Akt was found to mediate various insulin- and growth factor-induced cellular
responses such as stimulation of GLUT4 translocation to the plasma membrane
(10,
11,
46,
47), inhibition of GSK3
(22), and promotion of cell
survival by inhibiting apoptosis
(48).
In addition, as shown by the v-Akt data, constitutively activated Akt has
transforming activity (9).
These cellular activities have been shown to be induced by overexpression of
constitutively activated Akt or its membrane-targeted mutant. For example,
glucose uptake is reportedly increased in both constitutively activated
Akt-overexpressing 3T3-L1 adipocytes and L6 myotubes
(10,
11), whereas Akt2-deficient
mice show impaired glucose tolerance due to decreased insulin-induced glucose
uptake in skeletal muscle and increased hepatic glucose production
(12).
On the other hand, SGK, the expression level of which is increased by
glucocorticoid and serum stimulation in cultured cells
(13,
48), is the one member of the
AGC kinase family with a highly conserved kinase domain compared with that of
Akt (54% identical amino acids)
(14,
15). Indeed, the substrate
specificity of the kinase domain of SGK has been reported to be similar to
that of Akt (16). Furthermore,
it was also reported that the PI 3-kinase pathway activates SGK through
PDK1/2, i.e. in the same manner as for Akt
(17,
49). However, the most
apparent difference in the structures of SGK and Akt is that SGK lacks the PH
domain that Akt has. Thus, SGK is considered to not be able to translocate to
the membrane like Akt in response to growth factor stimulation. In fact, it
was reported that insulin stimulation induces the translocation of Akt to the
membrane fraction, but that this does not occur with mutant Akt lacking the PH
domain or with SGK (18).
Although recent studies revealed a possible role of SGK in
aldosterone-induced apical translocation of the epithelial sodium channel in
distal nephrons
(1921),
it seems that our understanding of the roles of SGK remains limited. In this
study, we investigated whether SGK induces the cellular functions known to be
induced by activated Akt. Akt activation reportedly plays a key role in
glucose metabolism, including increased glycogen synthase and GLUT4
translocation to the plasma membrane, as well as in inhibition of apoptosis
and promotion of cell growth. The induction of these cellular functions by Akt
has been well demonstrated, and they are also induced by overexpressing
myr-Akt (10,
11,
22). Thus, we constructed the
active mutant of SGK and myr-SGK and investigated whether these SGK mutants
can exert the same actions as myr-Akt.
This is the first report clearly demonstrating the different roles of Akt
and SGK. In addition, interestingly, these differences are attributable not to
the presence or absence of the PH domain responsible for membrane targeting,
but very possibly to differences in the substrate specificities of the kinase
domains of SGK and Akt.
 |
EXPERIMENTAL PROCEDURES
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AntibodiesAnti-Myc tag (clone 9E10),
anti-phospho-Ser21 GSK3
, and anti-SGK antibodies were
purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). Anti-Akt and
anti-phospho-Ser473 Akt antibodies were purchased from Cell
Signaling Technology (Beverly, MA).
Cell CultureHepatocytes were isolated from fasted rats by
collagenase perfusion as described previously
(23) and plated in
collagen-coated 25-cm2 flasks at a density of 1.0 x
105 cells/cm2 in DMEM supplemented with 10% FCS, 0.5
µg/ml insulin, 1 µM dexamethasone, and 10 ng/ml epidermal
growth factor. After a 6-h attachment period, hepatocytes were transfected
using adenoviral gene transfer. 3T3-L1 fibroblasts were maintained in DMEM
supplemented with 10% donor calf serum (Invitrogen) under a 10% CO2
atmosphere at 37 °C. 2 days after the fibroblasts reached confluence,
differentiation was induced by incubating the cells for 48 h in DMEM
supplemented with 10% fetal bovine serum, 0.5 mmol/liter
3-isobutyl-1-methylxanthine, and 4 mg/ml dexamethasone. Thereafter, the cells
were maintained in DMEM supplemented with 10% fetal bovine serum for an
additional 410 days; once >90% of the cells expressed the adipocyte
phenotype, the cells were used for experimentation. 32D cells were maintained
in RPMI 1640 medium with 10% FCS and 0.25 ng/ml murine IL-3
(24). NIH3T3 cells were
maintained in DMEM with 10% FCS. Each cDNA was transfected into NIH3T3 cells
with an expression vector (pCAGGs), and the permanently expressing clones were
selected in DMEM containing 500 mg/liter Geneticin.
Construction of SGK Mutants and myr-Akt3A-SGK, a
dominant-negative form of SGK, was constructed by substituting lysine 127,
threonine 256, and serine 422 with alanines. D-SGK was constructed
by substituting serine 422 with aspartic acid. myr-SGK contains an
src myristoylation signal sequence at the N terminus. MAA-Akt, a
dominant-negative form of Akt, was constructed by substituting lysine 179,
threonine 308, and serine 473 with alanines. myr-Akt, which contains an
src myristoylation signal sequence, was described previously
(25). All the constructs were
designed to contain a Myc tag at the C terminus, and their structures are
shown in Fig. 1. Recombinant
adenoviruses were generated, purified, and concentrated using cesium chloride
ultracentrifugation as reported previously
(26,
27). Equal amounts of Akt and
SGK were overexpressed throughout all of the experiments.

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FIG. 1. Schematic presentation of recombinant adenoviruses of Akt and SGK.
3A-SGK was constructed by substituting lysine 127, threonine 256, and serine
422 with alanines. D-SGK was constructed by substituting serine 422
with aspartic acid. myr-SGK and myr-Akt contain an src myristoylation
signal sequence (myr) at the N terminus. MAA-Akt was constructed by
substituting lysine 179, threonine 308, and serine 473 with alanines. All
constructs were designed to contain a Myc tag at the C terminus.
a.a., amino acids.
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Gene Transfer into Each Cell TypeFor gene transfer into
hepatocytes, cells were incubated for 1 h at 37 °C in DMEM containing 1.0
x 108 plaque-forming units/ml recombinant adenovirus, after
which the virus-containing medium was replaced with normal growth medium.
Glycogen synthase assays were performed 36 h later. For gene transfer into
3T3-L1 adipocytes, cells were incubated for 6 h at 37 °C in DMEM
containing recombinant adenovirus. Experiments were performed 2 days later.
32D cells were incubated for 6 h at 37 °C in RPMI 1640 medium containing
recombinant adenovirus. 24 h after adenoviral gene transfer, the cells were
IL-3-starved for an additional 24 h, and then DNA synthesis assays were
performed. In 3T3-L1 adipocytes and 32D cells, all recombinant adenoviruses
were used at a concentration of 3.0 x 109 plaque-forming
units/ml. Under these conditions, none of the cells infected with LacZ
exhibited significant differences in glycogen synthesis or glucose transport
activity compared with non-infected cells, and 95
100% of the cells were
infected as judged by blue coloration after
5-bromo-4-chloro-3-indolyl-
-D-galactopyranoside (X-gal)
incubation 36
48 h after infection.
Immunoprecipitation and ImmunoblottingThe cells were
collected and boiled in Laemmli sample buffer containing 100 mmol/liter
dithiothreitol. SDS-PAGE and immunoblotting were performed as described
previously (27) using each
antibody as a probe.
Glycogen Synthase AssayAfter 3 h of serum starvation, cells
in 25-cm2 flasks were stimulated with or without 100 nM
insulin for 30 min. The cells were homogenized in homogenizing buffer
containing 50 mM Tris, 10 mM EDTA, and 100 mM
NaF. 50 µg of liver samples were then assayed in glycogen synthase buffer
containing 8.9 mM UDP-[6-3H]glucose (1 Ci/mmol) in the
absence or presence of 10 mM Glu-6-P for 20 min at 30 °C.
UDP-[6-3H]glucose incorporation into glycogen was measured by
liquid scintillation counting.
Glucose Transport Assay3T3-L1 adipocytes in 24-well culture
dishes were serum-starved for 3 h in DMEM containing 0.2% bovine serum
albumin. They were next incubated for 45 min in glucose-free Krebs-Ringer
phosphate buffer (137 mM NaCl, 4.7 mM KCl, 10
mM sodium phosphate (pH 7.4), 0.5 mM MgCl2,
and 1 mM CaCl2) and then incubated with or without 100
nM insulin for 15 min. Basal and stimulated uptakes of
2-deoxy-D-[3H]glucose were then measured as described
previously (28).
DNA Synthesis Assay32D cells were maintained in RPMI 1640
medium supplemented with 10% FCS and 0.25 ng/ml IL-3. After incubation in
IL-3-free RPMI 1640 medium for 24 h, the cells were incubated with BrdUrd
labeling solution for 6 h. BrdUrd incorporation was measured using the BrdUrd
labeling and detection kit III (Roche Applied Science)
(44).
Soft Agar AssaysFor the soft agar assay, cells of each
transfected derivative were trypsinized, suspended in DMEM containing 0.3%
agar and 20% FCS, and plated onto a bottom layer containing 0.6% agar. Cells
were plated at a density of 2 x 104 cells/3.5-cm dish, and
colonies >0.5 mm in diameter were counted after 14 days
(29). All procedures were
performed in three independent experiments, and similar results were
obtained.
Statistical AnalysisResults are expressed as means ±
S.E. Comparisons were made using unpaired Student's t test. Values of
p <0.05 were considered statistically significant.
 |
RESULTS
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Effect of SGK or Akt Overexpression on Glucose Transport
WT-Akt, MAA-Akt, myr-Akt, WT-SGK, 3A-SGK, and myr-SGK were overexpressed in
3T3-L1 adipocytes. Immunoblotting with anti-Myc tag antibody revealed the
expression levels of Akt and SGK and their mutants to be comparable
(Fig. 2A, first
panel). In addition, the expression levels of WT-Akt, MAA-Akt, and
myr-Akt were approximately five times those of endogenously expressed Akt
judging from the immunoblotting results using anti-Akt antibody
(Fig. 2A, second
panel), and basal phosphorylation was observed in myr-Akt (third
panel). On the other hand, endogenously expressed SGK was not detectable
in 3T3-L1 adipocytes, whereas overexpressed WT-SGK, 3A-SGK, and myr-SGK were
detected by immunoblotting (Fig.
2A, fourth panel). Increased phosphorylation of
GSK3 was observed in 3T3-L1 adipocytes overexpressing myr-Akt,
D-SGK, and myr-SGK, whereas overexpression of wild-type and
dominant-negative mutants of Akt and SGK did not increase GSK3 phosphorylation
(Fig. 2A, fifth
panel). Finally, interestingly, although
2-deoxy-D-[3H]glucose uptake into 3T3-L1 adipocytes was
increased 8-fold by overexpression of myr-Akt compared with that of LacZ,
D-SGK or myr-SGK overexpression had essentially no effect on
2-deoxy-D-[3H]glucose uptake
(Fig. 2B).

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FIG. 2. 2-Deoxy-D-[3H]glucose uptake by Akt- or
SGK-overexpressing 3T3-L1 adipocytes. A, after 3 h of serum
starvation, cells overexpressing LacZ, Akt, or SGK were washed twice with cold
phosphate-buffered saline. 3T3-L1 cells were then lysed and boiled in Laemmli
buffer. Western blotting was performed using anti-Myc (first panel),
anti-Akt (second panel), anti-phospho-Ser473 Akt
(pAkt; third panel), anti-SGK (fourth panel), and
anti-phospho-Ser21 GSK3 (pGSK3; fifth
panel) antibodies. B, after 3 h of serum starvation, cells
overexpressing LacZ, Akt, or SGK were incubated in glucose-free Krebs-Ringer
phosphate buffer for 45 min. The cells were then incubated with or without 100
nM insulin (Ins) for 15 min, and
2-deoxy-D-[3H]glucose (2DG) uptake was
measured. Bars depict means ± S.E. of three independent
experiments. IB, immunoblot.
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Effect of SGK or Akt Overexpression on Phosphorylation of GSK3 and
Glycogen SynthesisTo confirm that phosphorylated GSK3 elevates
glycogen synthesis irrespective of whether Akt or SGK phosphorylates GSK3,
active mutants of Akt (myr-Akt) and SGK (D-SGK and myr-SGK) were
overexpressed in primary cultured rat hepatocytes. The expression levels of
myr-Akt, D-SGK, and myr-SGK were comparable, as shown by immunoblotting using
anti-Myc tag antibody (Fig.
3A). Similar to the observations in 3T3-L1 adipocytes,
overexpression of these active mutants of Akt and SGK induced an apparent
increase in phosphorylation of GSK3, as did insulin stimulation
(Fig. 3B). As shown in
Fig. 3C, glycogen
synthase activity was shown to be significantly increased (
1.5-fold)
either by 30 min of stimulation with insulin or by overexpression of myr-Akt,
D-SGK, or myr-SGK compared with the control. Thus, it is clear that
phosphorylation of GSK3 results in the elevation of glycogen synthesis
irrespective of phosphorylation by either Akt or SGK.

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FIG. 3. Phosphorylation of GSK3 and glycogen synthesis in primary cultured rat
hepatocytes. A and B, after 3 h of serum starvation,
cells were stimulated with or without 100 nM insulin for 30 min.
The cells were washed twice with cold phosphate-buffered saline and then lysed
and boiled. Western blotting was performed using anti-Myc (A) and
anti-phospho-Ser21 GSK3 (pGSK3; B)
antibodies. C, the cells were homogenized in homogenizing buffer. The
lysate was then assayed in glycogen synthase buffer in the absence and
presence of 10 mM Glu-6-P (G6P) for 20 min at 30 °C.
UDP-[6-3H]glucose incorporation into glycogen was measured by
liquid scintillation counting. Bars depict means ± S.E. of
three independent experiments. IB, immunoblot.
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Effect of SGK or Akt Overexpression on DNA SynthesisWT-Akt,
MAA-Akt, myr-Akt, WT-SGK, 3A-SGK, and myr-SGK were overexpressed in 32D cells
by adenoviral transfer so that their expression levels were similar
(Fig. 4A), and the
effects on DNA synthesis were examined by measuring BrdUrd incorporation.
Because 32D is an IL-3-dependent cell line, removal of IL-3 from the medium
reportedly induces apoptosis, which can be shown to be the result of
suppressed DNA synthesis. Overexpression of myr-Akt, D-SGK, and myr-SGK
induced GSK3 phosphorylation to a similar degree, whereas that of WT-Akt,
MAA-Akt, WT-SGK, and 3A-SGK did not (Fig.
4B). However, interestingly, overexpression of only
myr-Akt significantly enhanced the protection from apoptosis achieved by lack
of IL-3 (Fig. 4C). On
the other hand, overexpression of D-SGK or myr-SGK had no
inhibitory effect on apoptosis, despite phosphorylation of GSK3.

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FIG. 4. DNA synthesis in 32D cells. A and B, 32D cells
were maintained in RPMI 1640 medium supplemented with 10% FCS and 0.25 ng/ml
IL-3. 48 h after adenoviral transfer, SDS-PAGE and Western blotting were
performed using anti-Myc (A) and anti-phospho-Ser21
GSK3 (pGSK3; B) antibodies. C, after
incubation in IL-3-free RPMI 1640 medium for 24 h, the cells were incubated
with BrdUrd labeling solution for 6 h. BrdUrd incorporation was measured using
the BrdUrd labeling and detection kit III. Bars depict means ±
S.E. of three independent experiments. IB, immunoblot.
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myr-Akt Overexpression Alone Can Transform NIH3T3
CellsFinally, we analyzed the transforming ability of Akt and SGK
by evaluating the capacity for anchorage-independent growth. NIH3T3 cells
stably transfected with either Akt or SGK constructs were seeded in DMEM
containing 0.3% agar and 20% FCS, and colony formation was estimated as a
representative of anchorage-independent growth ability. Three independent
experiments were performed using three different clones of each derivative,
and a representative one is shown in Fig. 5
(A and B). Stable expression of Akt and SGK
constructs was confirmed by immunoblotting with anti-Myc tag, anti-Akt, and
anti-SGK antibodies (Fig.
5B). Similar to observations in cells transiently
overexpressing Akt or SGK due to adenoviral transfer, stable overexpression of
myr-Akt, D-SGK, and myr-SGK significantly increased the phosphorylation level
of GSK3, whereas that of other constructs did not. These cells were subjected
to soft agar assay to evaluate their transforming activity. As shown in
Fig. 5B (middle
panel), myr-Akt-expressing cells formed many macroscopic colonies within
14 days after being seeded. In contrast, although D-SGK- and
myr-SGK-expressing cells showed phosphorylation of GSK3, they apparently could
not make colonies (Fig.
5B, upper and lower panels). The
colonies (counted from the three independent clones for each of the Akt and
SGK constructs) are shown as bar graphs in
Fig. 5C.

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FIG. 5. Soft agar assays demonstrating transformation of NIH3T3 fibroblasts by
Akt and SGK. A, NIH3T3 cells stably transfected with either Akt
or SGK constructs were lysed and boiled in Laemmli buffer. Western blotting
was performed using anti-Myc (first panel), anti-Akt (second
panel), anti-phospho-Ser473 Akt (pAkt; third
panel), anti-SGK (fourth panel), and
anti-phospho-Ser21 GSK3 (pGSK3; fifth
panel) antibodies. B and C, for the soft agar assay,
cells of each transfected derivative were trypsinized, suspended in DMEM
containing 0.3% agar and 20% FCS, and plated onto a bottom layer containing
0.6% agar. Cells were plated at a density of 2 x 104
cells/3.5-cm dish. myr-Akt-expressing cells (but not other transfectants) made
macroscopic colonies (B). Colonies >0.5 mm in diameter were
counted after 14 days (C). Three independent experiments were
performed using three different clones of each derivative, and similar results
were obtained. IB, immunoblot.
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DISCUSSION
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Numerous serine/threonine kinases exist in the cell, and they play specific
roles by inducing individual cellular functions. As to the molecular
mechanisms underlying the various roles of each kinase protein, the substrate
specificity of the kinase domain and subcellular distribution are considered
to be major contributors. SGK and Akt are considered to be very similar kinase
proteins, both of which belong to the AGC family. They are activated via
phosphorylation by PDK1 and PDK2, located downstream from PI 3-kinase.
However, the presence and absence of a PH domain in Akt and SGK, respectively,
should result in different subcellular distributions of these two kinases. In
the case of Akt, Akt translocates to the membrane fraction via binding of the
PH domain with PI-3,4,5-P3 produced by PI 3-kinase, and
phosphorylation of Thr306 and Ser473 of Akt by PDK1 and
PDK2 takes place, thereby activating Akt kinase
(3032).
myr-Akt is located at the plasma membrane without stimulation and is
constitutively activated (10,
33). Then, Akt phosphorylates
several substrates that transmit signals
(6).
In this study, to investigate the different roles of Akt and SGK, active as
well as membrane-targeted active mutants of these kinases were overexpressed.
Previous studies have established the functions induced by and the roles of
activated Akt, and all or most of these cellular actions, for which Akt is
reportedly responsible, are indeed induced by overexpression of myr-Akt
(3337).
Thus, although this study was carried out employing an overexpression system,
the cellular actions described herein are considered to be physiological
functions induced by Akt. For example, it is well established that Akt plays a
major role in glucose metabolism, including increased glycogen synthase and
GLUT4 translocation to the plasma membrane, functions that can be induced by
overexpressing myr-Akt (10,
11). Indeed, translocation of
Akt to the plasma membrane is suggested to be important for glucose transport.
In addition, the oncogenic activity of Akt is also well known since v-Akt has
been identified as an oncogenic protein, and this promotion of cell growth
activity is similarly observed upon overexpression of myr-Akt
(38,
39).
We obtained two important conclusions from this comparative study of SGK
and Akt mutants. The first is that SGK can mediate GSK3 phosphorylation and
the resultant glycogen synthesis, but not other cellular functions induced by
Akt such as increased glucose transport, inhibition of apoptosis, and
oncogenic transformation. Thus, it is possible that increased expression of
SGK inhibits the phosphorylation of Akt and the resultant cellular functions
such as increased glucose transport, inhibition of apoptosis, and cell
proliferation because these two kinases are similarly phosphorylated by PDK1
and PDK2. Conversely, an increase in SGK may contribute to increased glycogen
synthesis by increasing the phosphorylation of GSK3. In addition, a previous
report showed that overexpression of the myristoylated and PH domain-deleted
form of Akt induces an increase in glucose transport activity in 3T3-L1
adipocytes (10). Therefore,
these different roles of the two kinases are apparently not attributable to
membrane-targeting ability or an unidentified mechanism of signal transduction
from the PH domain, caused by the presence or absence of the PH domain,
because myristoylated and PH domain-deleted Akt, but not myr-SGK, enhanced
glucose transport, inhibition of apoptosis, and cell proliferation.
GSK3 is well established as a regulator of glycogen metabolism
(22,
4042).
Many proteins in this family are related to protein synthesis. Wnt signaling
and transcription factors have been reported to be substrates of GSK3, and
Somervaille et al.
(43) described GSK3 and Bax as
being involved in the suppression of apoptosis induced by growth factor
withdrawal. However, our results suggest that GSK3 is very likely to be
independent of Akt-induced cellular actions such as increased glucose
transport, inhibition of apoptosis, and cell proliferation.
As discussed above, the different cellular functions induced by Akt and SGK
cannot be attributed to the difference in membrane targeting through the PH
domain. Although it has been reported that substrate specificities for
synthetic peptides differ minimally between Akt and SGK based on an in
vitro experiment using glutathione S-transferase fusion proteins
and synthetic peptides (16),
we speculate that considerable differences in terms of in vivo
substrate specificity exist between these kinases, which account for the
apparent differences in the roles of Akt and SGK. In other words, there may be
some unidentified proteins phosphorylated by Akt, but not by SGK, that play
key roles in glucose transport and anti-apoptotic effects, whereas GSK3 plays
only minor roles in these functions.
In conclusion, we have demonstrated for the first time an important
difference between Akt and SGK and that this difference is not due to membrane
localization, but rather possibly to the difference in their kinase
activities. The identification of substrates selectively phosphorylated by
Akt, but not by SGK, may provide clues to clarifying the pathway leading from
Akt activation to the aforementioned cellular activities.
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FOOTNOTES
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* The costs of publication of this article were defrayed in part by the
payment of page charges. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section 1734
solely to indicate this fact. 
**
To whom correspondence should be addressed. Tel.: 81-3-3815-5411 (ext. 33133);
Fax: 81-3-5803-1874; E-mail:
asano-tky{at}umin.ac.jp.
1 The abbreviations used are: PI, phosphatidylinositol; SGK, serum- and
glucocorticoid-regulated kinase; GLUT4, glucose transporter-4; GSK3, glycogen
synthase kinase-3; PDK, phosphoinositide-dependent kinase; PH, pleckstrin
homology; myr-, myristoylated; DMEM, Dulbecco's modified Eagle's medium; FCS,
fetal calf serum; IL-3, interleukin-3; BrdUrd, bromodeoxyuridine; WT,
wild-type. 
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