Akt, also known as protein kinase B, is a
protein-serine/threonine kinase that is activated by growth factors in
a phosphoinositide (PI) 3-kinase-dependent manner.
Although Akt mediates a variety of biological activities, the
mechanisms by which its activity is regulated remain unclear. The
potential role of the
isozyme of protein kinase C (PKC) in the
activation of Akt induced by insulin has now been examined. Expression
of a kinase-deficient mutant of PKC
(
KD), but not that of
wild-type PKC
or of kinase-deficient mutants of PKC
or PKC
,
with the use of adenovirus-mediated gene transfer inhibited the
phosphorylation and activation of Akt induced by insulin in Chinese
hamster ovary cells or L6 myotubes. Whereas the
KD mutant did not
affect insulin stimulation of PI 3-kinase activity, the phosphorylation
and activation of Akt induced by a constitutively active mutant of PI
3-kinase were inhibited by
KD, suggesting that
KD affects insulin
signaling downstream of PI 3-kinase. PDK1
(3'-phosphoinositide-dependent kinase 1) is thought to
participate in Akt activation. Overexpression of PDK1 with the use of
an adenovirus vector induced the phosphorylation and activation of Akt;
KD inhibited, whereas wild-type PKC
had no effect on, these
actions of PDK1. These results suggest that
KD inhibits the
insulin-induced phosphorylation and activation of Akt by interfering
with the ability of PDK1 to phosphorylate Akt.
 |
INTRODUCTION |
Akt, also known as protein kinase B, is activated by growth
factors such as platelet-derived growth factor
(PDGF)1 and insulin (1-3),
by cytokines (4), by ligands of G protein-coupled receptors (5), and by
cellular stresses such as hyperosmolarity, heat shock, fluid shear
stress, and hydrogen peroxide-induced oxidative stress (6-8). The
activation of Akt by these stimuli contributes to a variety of their
biological effects, including promotion of cell survival and protection
from apoptosis (4, 9), induction of meiosis in oocytes (10), regulation
of vascular contractility (8, 11), activation of the transcription
factor NF-
B (12, 13), and various metabolic actions of insulin
(14-17).
Activation of Akt is mediated by phosphorylation of a threonine residue
in the kinase activation loop (Thr308 in Akt1) and a serine
residue in the COOH-terminal region (Ser473 in Akt1) (18).
Akt mutants in which these residues are replaced by neutral amino acids
are not activated in cells (15, 18). The phosphorylation and activation
of Akt induced by growth factors are blocked by pharmacological or
molecular biological inhibitors of phosphoinositide (PI) 3-kinase
(1-3, 15), indicating that PI 3-kinase is required for the
phosphorylation of Thr308 and Ser473 and
activation of Akt in response to growth factors.
PDK1 (3'-phosphoinositide-dependent kinase 1), originally
identified as a kinase that selectively phosphorylates
Thr308 of Akt in vitro, is thought to contribute
to Akt activation (19-21). However, the mechanism by which PDK1
phosphorylates Akt in intact cells remains unclear. Although the
phosphorylation of Akt by PDK1 is stimulated in the presence of
3'-phosphoinositides in vitro (19-21), the kinase activity
of PDK1 immunoprecipitated from cells is not affected by prior
treatment of the cells with either growth factors or pharmacological
inhibitors of PI 3-kinase (22), suggesting that PDK1 is constitutively
active in cells. Given that Akt translocates from the cytosol to the
membrane fraction of cells in response to growth factors (23), and that
membrane-targeted mutants of Akt are constitutively active in quiescent
cells (23, 24), it is thought that membrane-associated Akt is
phosphorylated by PDK1.
Mutational analysis has revealed that phosphorylation of both
Thr308 and Ser473 is required for Akt
activation (18). Although a kinase that phosphorylates
Ser473 of Akt has been tentatively designated PDK2, its
nature remains unclear. Balendran et al. (25) recently
showed that PDK1 phosphorylates Ser473 of Akt in
vitro only in the presence of a glutathione
S-transferase (GST) fusion protein that contains the
COOH-terminal portion of protein kinase C (PKC)-related kinase 2 (PRK2)
or of synthetic peptides corresponding to this region of PRK2. In
contrast, the phosphorylation of p70 S6 kinase by PDK1 was inhibited in
the presence of the same GST fusion protein or peptides (26). Moreover, when overexpressed in intact cells, this same region of PRK2 prevented ligand-induced activation of p70 S6 kinase (26). These observations suggest that the interactions of PDK1 with its substrates, at least
those with Akt and p70 S6 kinase, are regulated by another kinase.
PKC
is a member of the novel subfamily of PKC isozymes (27, 28).
When expressed in fibroblasts also expressing various mutant PDGF
receptors, PKC
contributed to the transactivation of the TPA
(12-O-tetradecanoylphorbol 13-acetate)-responsive element induced by PDGF in a PI 3-kinase-dependent manner (29),
suggesting that PKC
participates in signaling downstream of PI
3-kinase. We have therefore now investigated whether PKC
plays a
role in activation of Akt. We examined the effects of overexpression of wild-type or a kinase-deficient mutant of PKC
on Akt activation induced by insulin, heat shock, or hydrogen peroxide. Expression of the
kinase-deficient mutant of PKC
inhibited the phosphorylation and
activation of Akt in response to all three stimuli, probably by
affecting the ability of PDK1 to phosphorylate Akt.
 |
EXPERIMENTAL PROCEDURES |
Cells and Antibodies--
L6 myoblasts were maintained and
induced to differentiate into myotubes as described previously (30). We
amplified a full-length mouse serum- and glucocorticoid-regulated
protein kinase (SGK) 2 cDNA (31) by the polymerase chain reaction
(PCR) with cDNA synthesized from RNA extracted from mouse liver as
template. The amplified SGK2 cDNA was tagged with the HA epitope at
its NH2 terminus with the use of PCR. To establish CHO-IR
cells that express (in addition to insulin receptors) FLAG
epitope-tagged Akt1 or HA epitope-tagged SGK2 (CHO-IR/Akt cells and
CHO-IR/SGK2 cells, respectively), we transfected CHO-IR cells with
pSV40-hgh (which confers resistance to hygromycin) and a PECE vector
encoding FLAG-tagged rat Akt1 (RAC-PK
) (6) or a pCMV4 vector
encoding HA-tagged mouse SGK2. Transfected cells were selected and
cloned as described previously (32). CHO-IR cells that express
phosphodiesterase 3B (PDE3B), designated CHO-IR/PDE3B-WT cells, were
previously described (16). Polyclonal antibodies to Akt, to
mitogen-activated protein (MAP) kinase, and to PDE3B were generated as
described previously (15, 16, 33). Polyclonal antibodies to PKC
as well as monoclonal antibodies to PKC
and to PKC
were obtained from Santa Cruz Biotechnology and Transduction Laboratories,
respectively. Monoclonal antibodies to the HA epitope tag (12CA5) or to
the FLAG epitope tag were obtained from Roche Molecular Biochemicals. Polyclonal antibodies specific for phospho-Thr308 or
phospho-Ser473 forms of Akt or for the phosphorylated form
of MAP kinase were obtained from New England BioLabs.
Construction of and Infection with Adenovirus
Vectors--
Adenovirus vectors encoding a kinase-deficient mutant of
PKC
in which Lys273 is replaced by glutamate (AxCA
KD)
(32), or a constitutively active mutant of PI 3-kinase (AxCAMyr-p110)
(16, 34) were described previously. Complementary DNAs for wild-type
PKC
(
WT, Ref. 27) and a kinase-deficient mutant of PKC
in
which Lys437 is replaced by methionine (
KD, Ref. 35)
were modified with the use of PCR to encode the T7 or FLAG epitope tags
at the NH2 termini of the respective proteins. A
kinase-deficient mutant of PKC
in which Thr566 is
replaced by alanine (
T566A) was as described (36). A constitutively active mutant of PKC
in which Ala159 is replaced by
glutamate (
A159E) was constructed from the cDNA that encodes
FLAG epitope-tagged
WT with the use of a QuickChange site-directed
mutagenesis kit (Stratagene). Complementary DNAs encoding T7-tagged
WT, the FLAG-tagged
KD,
T566A, FLAG-tagged
A159E, Myc
epitope-tagged PDK1 (21), kindly provided by L. Stephens (The Babraham
Institute), or a kinase-deficient mutant of PKC
in which
Lys368 is replaced with arginine (37) were subcloned into
pAxCAwt (38), and adenoviral vectors containing these cDNAs were
generated with the use of an adenovirus expression kit (Takara, Tokyo,
Japan) as described previously (15, 16). The resultant adenovirus vectors were termed AxCA
WT, AxCA
KD, AxCA
T566A, AxCA
A159E, AxCAPDK1, and AxCA
KD, respectively. CHO-IR/Akt cells or
differentiated L6 myotubes were infected with adenovirus vectors at the
indicated multiplicity of infection (MOI), expressed in plaque-forming
units (PFU) per cell, as described previously (33). The cells were subjected to experiments 24-48 h after infection.
Kinase Assays--
L6 myotubes or CHO-IR/Akt cells were deprived
of serum for 16-20 h, incubated in the absence or presence of 100 nM insulin for the indicated time, and then immediately
frozen with liquid nitrogen. The assay for MAP kinase activity was
performed with immunoprecipitates prepared with antibodies to MAP
kinase, as described previously (33). For assay of PI 3-kinase
activity, cells were lysed and subjected to immunoprecipitation with
antibodies to phosphotyrosine (PY20; Transduction Laboratories); the
resulting immunoprecipitates were washed and PI 3-kinase activity in
the washed precipitates was assayed as described previously (33). For
assay of Akt activity, cells were lysed and subjected to
immunoprecipitation with polyclonal antibodies to Akt as described
(15). The immunoprecipitates were then mixed with 30 µl of kinase
reaction mixture containing 50 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 1 mM dithiothreitol, 1 mM of the specific peptide inhibitor of
cAMP-dependent protein kinase (PKI), 5 µM
nonradioactive ATP, 2 µCi of [
-32P]ATP (4000 Ci/mmol), and 5 µM Crosstide peptide (GRPRTSSFAEG) (39)
and then incubated for 30 min at 30 °C. Assay of SGK activity was
performed essentially as described (40) with the following modifications. Cells were lysed in a solution containing 50 mM Tris-HCl, pH 7.5, 120 mM NaCl, 1% Triton
X-100, 1 mM benzamidine, 25 mM NaF, 40 mM
-glycerophosphate, 1 mM
phenylmethylsulfonyl fluoride, and leupeptin (10 µg/ml). The lysate
was centrifuged, and the resulting supernatant was subjected to
immunoprecipitation with antibodies to HA. The immunoprecipitates were
washed once with the lysis buffer containing 500 mM NaCl
and with the lysis buffer and then twice with 50 mM
Tris-HCl, pH 7.5 containing 0.1% (v/v) of
-mercaptoethanol. The
immunoprecipitates were then mixed with 30 µl of kinase reaction
mixture containing 60 mM Tris-HCl, pH 7.5, 12 mM MgCl2, 0.12 mM EDTA, 0.12%
(v/v)
-mercaptoethanol, 3 µg/ml of PKI, 5 µM
nonradioactive ATP, 4 µCi of [
-32P]ATP (4000 Ci/mmol), and 36 µM Crosstide peptide, and then incubated for 60 min at 30 °C. The kinase recation mixture for Akt or SGK was
spotted onto a P81 phosphocellulose filter (Whatman), the filters were
washed three times with 0.5% (w/v) orthophosphoric acid, and the
radioactivity remaining on the filters was measured.
In Vivo Phosphorylation of PDE3B--
The in vivo
phosphorylation of PDE3B was assayed as described previously (16). In
brief, CHO-IR/PDE3B-WT cells, previously infected (or not) with
AxCA
KD, were labeled with [32P]orthophosphate,
incubated in the absence or presence of 100 nM insulin for
15 min, and lysed. Cell lysates were subjected to immunoprecipitation
with polyclonal antibodies to PDE3B, the resulting precipitates were
separated by SDS-polyacrylamide gel electrophoresis on a 7% gel, and
the incorporation of radioactivity into PDE3B was visualized and
quantitated with a Fuji BAS2000 image analyzer.
 |
RESULTS |
Effects of Wild-type and a Kinase-deficient Mutant of PKC
on
Insulin-induced Activation of Akt--
Infection of CHO-IR/Akt cells,
which stably express both human insulin receptors and rat Akt1, with an
adenovirus vector that encodes a kinase-deficient mutant of PKC
(AxCA
KD) resulted in a dose-dependent increase in the
amount of PKC
protein as assessed by immunoblot analysis; the amount
of PKC
protein in cells infected at an MOI of 10 PFU/cell was
~10-20 times that of endogenous PKC
(Fig.
1A). Exposure of noninfected
CHO-IR/Akt cells to insulin resulted in a >30-fold increase in the
amount of Akt activity measured in immunoprecipitates prepared with
antibodies to Akt (Fig. 1A). Expression of the
kinase-deficient mutant of PKC
(
KD) in the cells inhibited
insulin-induced activation of Akt in a dose-dependent
manner. Infection of CHO-IR/Akt cells with AxCA
KD also inhibited
insulin-induced phosphorylation of both Thr308 and
Ser473 of Akt, as assessed by immunoblot analysis with
antibodies specific for either phospho-Thr308 or
phospho-Ser473 forms of Akt (Fig. 1A).
Expression of a structurally distinct kinase deficient mutant of PKC
(
T566A) also exerted similar inhibitory effects on insulin-induced
activity (Fig. 1B) and phosphorylation (data not shown) of
Akt in CHO-IR/Akt cells.

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Fig. 1.
Effects of wild-type and kinase-deficient
mutants of PKC on insulin-induced
phosphorylation and activation of Akt. CHO-IR/Akt cells
(A and B) or L6 mytotubes (C and
D) that had been infected with either AxCA KD
(A and C), AxCA T566A (B) or
AxCA WT (D) at the indicated MOI (PFU/cell) were incubated
in the absence or presence of 100 nM insulin for 10 min and
then lysed. The cell lysates were either subjected to
immunoprecipitation with antibodies to Akt and thereby assayed for Akt
activity (upper panels), or subjected to immunoblot analysis
with antibodies to PKC , to the phospho-Thr308
(pT308), or the phospho-Ser473
(pS473) forms of Akt, or to total Akt (lower
panels). Quantitative data are means ± S.E. from three
experiments. Immunoblot data are representative of three independent
experiments.
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We next investigated whether
KD exerted a similar inhibitory effect
on Akt activation in physiological target cells of insulin. Insulin
induced an approximately 6-fold increase in Akt activity in L6 myotubes
(Fig. 1C). Expression of
KD inhibited insulin-induced phosphorylation and activation of Akt in a dose-dependent
manner (Fig. 1C). In contrast, overexpression of wild-type
PKC
in L6 myotubes did not affect insulin-induced phosphorylation
and activation of Akt (Fig. 1D). Expression of wild-type
PKC
also had no effect on the activation of Akt in response to
insulin in CHO-IR/Akt cells, and expression of either wild-type PKC
or a constitutively active mutant of PKC
(
A159E), of which kinase
activity was ~10-fold higher than that of wild-type PKC
(data not
shown), inconsistent with a previous report (41), did not induce Akt
activation in the absence of insulin in CHO/IR Akt cells or in L6
myotubes (data not shown). These results suggest that
KD inhibits
insulin-induced activation of Akt by preventing the phosphorylation of
Thr308 and Ser473, and that signal mediated
through PKC
alone is not sufficient to activate Akt in both
CHO-IR/Akt cells and L6 myotubes.
Effects of Kinase-deficient Mutants of Various PKC Isozymes on
Insulin-induced Activation of Akt--
We investigated whether
kinase-deficient mutants of other PKC isozymes also affected the
activation of Akt by insulin. Infection of L6 myotubes with adenovirus
vectors encoding
KD, wild-type PKC
, or kinase-deficient mutants
of PKC
(
KD) or PKC
(
KD) resulted in marked expression of
the encoded proteins (Fig.
2A); at an MOI of 20 PFU/cell;
the amount of each recombinant protein was at least 10 times that of
the corresponding endogenous PKC isoform. Whereas expression of
KD
inhibited insulin-induced activation of Akt,
KD had no effect on
this action of insulin (Fig. 2A). The expression of
KD
resulted in a slight enhancement of the effect of insulin on Akt
activity, consistent with previous observations (42).

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Fig. 2.
Effects of kinase-deficient mutants of
various PKC isozymes on insulin-induced activation of Akt
(A) and effects of KD on Akt
activation induced by heat shock or hydrogen peroxide
(B). A, L6 mytotubes that had been
infected (or not) with either AxCA KD ( KD), AxCA WT ( WT),
AxCA KD ( KD), or AxCA KD ( KD) at an MOI of 20 PFU/cell were
incubated in the absence or presence of 100 nM insulin for
10 min and then lysed. Cell lysates were either subjected to
immunoprecipitation with antibodies to Akt and thereby assayed for Akt
activity (upper panel), or subjected to immunoblot analysis
with antibodies to PKC , to PKC , or to PKC (lower
panel). B, CHO-IR/Akt cells that had been infected (or
not) with AxCA KD at an MOI of 10 PFU/cell were either incubated for
10 min in the absence or presence of 8.8 mM hydrogen
peroxide or subjected to heat shock for 10 min at 45 °C. Cell
lysates were then prepared and either subjected to immunoprecipitation
with antibodies to Akt and thereby assayed for Akt activity
(upper panel), or subjected to immunoblot analysis with
antibodies to PKC or to phospho-Thr308 or to
phospho-Ser473 forms of Akt. Quantitative data are
means ± S.E. from three experiments. Immunoblot data are
representative of three independent experiments.
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Effects of a Kinase-deficient Mutant of PKC
on Akt Activation
Induced by Heat Shock or Hydrogen Peroxide--
Akt is also activated
by heat shock and by hydrogen peroxide (6, 7). These stimuli induced
~10- and 20-fold increases in Akt activity, respectively, in
CHO-IR/Akt cells (Fig. 2B). Expression of
KD inhibited
the activation of Akt in response to either heat shock or hydrogen
peroxide. The phosphorylation of Akt on Thr308 and
Ser473 induced by these stimuli was also inhibited by
expression of
KD (Fig. 2B).
Effect of a Kinase-deficient Mutant of PKC
on Insulin-induced
Phosphorylation of PDE3B--
PDE3B was recently identified as a
direct substrate of Akt (16). In CHO-IR cells expressing PDE3B, insulin
induced an approximately 2-fold increase in the extent of
phosphorylation of PDE3B (Fig. 3).
Infection of the cells with AxCA
KD at an MOI of 3 PFU/cell, a virus
dose that inhibited insulin-induced activation of Akt by ~50% in
CHO-IR/Akt cells (Fig. 1A), resulted in ~50% inhibition of insulin-induced phosphorylation of PDE3B. This observation suggests
that inhibition by
KD of Akt activation attenuates signaling downstream of Akt.

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Fig. 3.
Effect of KD on
insulin-induced phosphorylation of PDE3B. CHO-IR/PDE3B-WT cells
that had been infected (or not) with AxCA KD at an MOI of 3 PFU/cell
were labeled with [32P]orthophosphate, incubated in the
absence or presence of 100 nM insulin for 15 min, and
lysed. Cell lysates were subjected to immunoprecipitation with
antibodies to PDE3B, the resulting precipitates were subjected to
electrophoresis, and 32P incorporation into PDE3B was
visualized (lower panel) or quantitated (upper
panel) with an image analyzer. Quantitative data are means of two
experiments, and the image shown is representative of two independent
experiments.
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Effect of a Kinase-deficient Mutant of PKC
on Signaling
Downstream of PI 3-Kinase--
We further attempted to identify the
step of the insulin signaling pathway leading to activation of Akt that
is affected by
KD. Overexpression of
KD had no effect on the
insulin-induced increase in the activity of PI 3-kinase
immunoprecipitated from L6 myotubes with antibodies to phosphotyrosine
(Fig. 4A), suggesting that
KD affects insulin-induced activation of Akt at a step downstream of
PI 3-kinase. To verify this conclusion, we examined the effect of
KD
on the activation of Akt by a constitutively active mutant of PI
3-kinase, Myr-p110, which comprises the catalytic subunit of PI
3-kinase ligated to a myristoylation signal sequence at its
NH2 terminus (16, 34). Infection of L6 myotubes with an adenovirus vector encoding Myr-p110 (AxCAMyr-p110) induced both the
phosphorylation and activation of Akt (Fig. 4B).
Coexpression of
KD inhibited in a dose-dependent manner
the phosphorylation and activation of Akt induced by Myr-p110 (Fig.
4B); expression of
KD did not affect the amount Myr-p110
protein as assessed by immunoblot analysis (data not shown). These
observations are thus consistent with the notion that
KD inhibits
insulin-induced activation of Akt by affecting signaling downstream of
PI 3-kinase. Expression of
KD did not affect insulin-induced
phosphorylation and activation of MAP kinase (Fig. 4C),
indicating that early events of insulin signaling, such as activation
of the insulin receptor kinase and phosphorylation of its substrates,
are not prevented by
KD.

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Fig. 4.
Effects of KD on
insulin-induced activation of PI 3-kinase (A) and MAP
kinase (C) as well as on Akt activation by Myr-p110
(B). A and C, L6 mytotubes
that had been infected with AxCA KD at the indicated MOI (PFU/cell)
were incubated in the absence or presence of 100 nM insulin
for 10 min and then lysed. The lysates were subjected to
immunoprecipitation with antibodies to phoshotyrosine (A) or
to MAP kinase (C), and the resulting precipitates were
assayed for PI 3-kinase and MAP kinase activities, respectively.
Alternatively, cell lysates from (C) were subjected to
immunoblot analysis with antibodies to phospho-MAP kinase or to total
MAP kinase (MAPK, lower panel). B, L6
mytotubes were infected (or not) with AxCAMyr-p110 at an MOI of 10 PFU/cell and, after 12 h, with AxCA KD at the indicated MOI
(PFU/cell). After an additional 36 h, the cells were lysed and the
lysates were either subjected to immunoprecipitation with antibodies to
Akt and thereby assayed for Akt activity (upper panel) or
subjected to immunoblot analysis with antibodies to PKC , to the
phospho-Ser473 form of Akt, or to total Akt (lower
panel). Quantitative data are means ± S.E. from three
experiments. Immunoblot data are representative of three independent
experiments.
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Effect of a Kinase-deficient Mutant of PKC
on PDK1-induced
Activation of Akt--
PDK1 is a serine/threonine kinase that
phosphorylates and activates Akt in vitro (19-21). We thus
investigated the effect of
KD on PDK1-induced activation of Akt.
Infection of CHO-IR/Akt cells with an adenovirus vector encoding PDK1
(AxCAPDK1) induced the phosphorylation of Akt on both
Thr308 and Ser473 as well as the activation of
this enzyme (Fig. 5). Coexpression of
KD with PDK1 resulted in inhibition of the PDK1-induced
phosphorylation and activation of Akt, with no effect on the amount of
PDK1 protein. In contrast, expression of wild-type PKC
had no effect
on the phosphorylation and activation of Akt induced by PDK1. These
results suggest that
KD inhibited the ability of PDK1 to
phosphorylate and activate Akt.

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Fig. 5.
Effects of KD on
PDK1-induced phosphorylation and activation of Akt. CHO-IR/Akt
cells were infected (or not) with AxCAPDK1 at an MOI of 10 PFU/cell
and, after 12 h, with either AxCA KD or AxCA WT also at an MOI
of 10 PFU/cell. After an additional 36 h, the cells were lysed and
the lysates were either subjected to immunoprecipitation with
antibodies to Akt and thereby assayed for Akt activity (upper
panel), or subjected to immunoblot analysis with antibodies to
PKC , to the phospho-Thr308 or the
phospho-Ser473 forms of Akt, or to Myc (for PDK1)
(lower panel). Quantitative data are means ± S.E. from
three experiments. Immunoblot data are representative of three
independent experiments.
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Effect of a Kinase-deficient Mutant of PKC
on Insulin-induced
Activation of SGK--
PDK1 has recently been shown to contribute to
the phosphorylation and the activation of SGK (31, 40, 43). We finally investigated the effect of
KD on insulin-induced activation of SGK.
CHO-IR/SGK2 cells, which stably express both human insulin receptors
and HA-tagged mouse SGK2, were incubated in the absence or presence of
insulin for 10 min, lysed, and immunoprecipitated with antibodies to
HA, and then SGK kinase activity toward Crosstide was assayed in the
immunoprecipitates. Insulin induced ~6-fold increase in the activity
of SGK, and overexpression of
KD in the cells inhibited
insulin-induced activation of SGK in a dose-dependent manner (Fig. 6).

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Fig. 6.
Effect of KD on
insulin-induced activation of SGK. CHO-IR/SGK2 cells that had been
infected (or not) with AxCA KD at the indicated MOI (PFU/cell) were
incubated in the absence or presence of 100 nM insulin for
10 min and then lysed. The cell lysates were subjected to
immunoprecipitation with antibodies to HA and assayed for SGK activity.
Data are means ± S.E. from three experiments.
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|
 |
DISCUSSION |
We have shown that overexpression of kinase-deficient mutants of
PKC
(
KD or
T566A) with the use of adenovirus-mediated gene
transfer inhibited the phosphorylation and activation of Akt induced by
insulin. An adenovirus vector encoding wild-type PKC
had no effect
on the insulin-induced increase in Akt activity, and the virus encoding
KD had no effect on insulin-induced activation of either MAP kinase
or PI 3-kinase, suggesting that the inhibition of Akt activation by
KD is due neither to nonspecific effects of viral infection nor to
general inhibition of insulin signaling. The phosphorylation and
activation of Akt induced by heat shock or hydrogen peroxide were also
inhibited by
KD, suggesting that this mutant protein affects a
common component of the Akt activation pathways triggered by various
extracellular stimuli. PI 3-kinase acts as an upstream mediator of Akt
activation induced by several stimuli (1-3, 5, 7). However, our
observations that
KD both did not inhibit insulin-induced activation
of PI 3-kinase and prevented Akt activation by a constitutively active
mutant of PI 3-kinase indicate that
KD affects a signaling component that acts downstream of PI 3-kinase.
PDK1 was originally identified as a kinase that phosphorylates
Thr308 of Akt in vitro (20, 21), and an
unidentified kinase that phosphorylates Ser473 of Akt has
been termed PDK2. We have now shown that overexpression of PDK1 induced
the phosphorylation of Akt on both Thr308 and
Ser473 in intact cells. PDK1 has been shown to
phosphorylate Ser473 of Akt in vitro in the
presence of a GST fusion protein containing the COOH-terminal region of
PRK2 or of synthetic peptides that encompass this region of PRK2 (25).
It is thus possible that, in intact cells, PDK1 phosphorylates both
Thr308 and Ser473 of Akt, which would explain
why overexpression of PDK1 alone resulted in the activation of Akt. We
cannot, however, exclude the possibility that expression of PDK1
resulted in the activation of PDK2, and that PDK1 and PDK2 coordinately
phosphorylate and activate Akt.
We have shown that
KD inhibited PDK1-induced phosphorylation and
activation of Akt, suggesting that
KD affects the insulin signaling
pathway at a step downstream of PDK1 action. PDK1 has been shown, at
least in vitro, to phosphorylate not only Akt but various
other kinases including p70 S6 kinase (44), SGK (31, 40, 43),
p90RSK (45), cAMP-dependent protein kinase
(46), and PKC isozymes including PKC
and PKC
(47), all of which
belong to the AGC family of protein kinases. Thus, the observation that
KD inhibited insulin-induced activity of SGK is also consistent with
this hypothesis that
KD interfere with the insulin signaling at a
step downstream of PDK1. Given that PKC
is a member of this family
of kinases, it also might serve as a substrate for PDK1. However, it is
not likely that
KD inhibits insulin-induced activation of Akt simply by competing with Akt for PDK1, because expression of wild-type PKC
did not inhibit this effect of insulin.
The mechanism by which
KD inhibits the ability of PDK1 to activate
Akt remains unclear. It is possible that endogenous PKC
phosphorylates an unidentified substrate that is important for the
interaction between PDK1 and Akt, and that
KD exerts a
dominant-negative effect on endogenous PKC
. Our observation that
overexpression of wild-type PKC
or a constitutively active form of
PKC
alone did not increase Akt activity in the absence of insulin
may be explained if such a substrate is constitutively phosphorylated in cells, so that overexpression of the wild-type or a constitutively active enzyme would not alter the phosphorylation state of the substrate.
The COOH-terminal region of PRK2, which includes a consensus
sequence for a PDK2 phosphorylation site similar to that present in Akt
with the exception that the residue equivalent to Ser473 is
aspartic acid, modulates PDK1 activity (25, 26). Analysis of the
three-dimensional structure of PDK1 suggests the presence in the kinase
domain of a hydrophobic pocket that interacts with this region of PRK2
(48). Given that PKC
also contains a sequence similar to the
COOH-terminal region of PRK2, it is possible that
KD affects the
ability of PDK1 to phosphorylate Akt by interacting with the
hydrophobic pocket of PDK1. Whereas serine and threonine residues in
the COOH-terminal region of wild-type PKC
II are phosphorylated in
intact cells, those of a kinase-deficient mutant of the enzyme are not
(49). It is thus possible that the phosphorylation status of wild-type
PKC
and
KD differ and that the difference in the abilities of
these molecules to affect Akt activation might be because of such a
difference in phosphorylation status.
Published, JBC Papers in Press, January 22, 2001, DOI 10.1074/jbc.M011093200
The abbreviations used are:
PDGF, platelet-derived growth factor;
PI, phosphoinositide;
PDK, 3'-phosphoinositide-dependent kinase;
GST, glutathione
S-transferase;
PKC, protein kinase C;
PRK2, PKC-related
kinase 2;
SGK, serum- and glucocorticoid-regulated protein kinase;
PCR, polymerase chain reaction;
PDE3B, phosphodiesterase 3B;
MAP, mitogen-activated protein;
MOI, multiplicity of infection;
PFU, plaque-forming unit;
CHO, Chinese hamster ovary;
WT, wild-type.
1.
|
Burgering, B. M. T.,
and Coffer, P. J.
(1995)
Nature
376,
599-602[CrossRef][Medline]
[Order article via Infotrieve]
|
2.
|
Franke, T. F.,
Yang, S. I.,
Chan, T. O.,
Datta, K.,
Kazluskas, A.,
Morrison, D. K.,
Kaplan, D. R.,
and Tsichlis, P. N.
(1995)
Cell
81,
727-736[Medline]
[Order article via Infotrieve]
|
3.
|
Kohn, A. D.,
Kovacina, K. S.,
and Roth, R. A.
(1995)
EMBO J.
14,
4288-4295[Abstract]
|
4.
|
de Peso, L.,
Gonzalez-Galcia, M.,
Page, C.,
Herrera, R.,
and Nunez, G.
(1997)
Science
278,
687-689[Abstract/Free Full Text]
|
5.
|
Murga, C.,
Laguinge, L.,
Wetzker, R.,
Cuadrado, A. M.,
and Gutkind, J. S.
(1998)
J. Biol. Chem.
273,
19080-19085[Abstract/Free Full Text]
|
6.
|
Konishi, H.,
Matsuzaki, H.,
Tanaka, M.,
Ono, Y.,
Tokunaga, C.,
Kuroda, S.,
and Kikkawa, U.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
7639-7643[Abstract/Free Full Text]
|
7.
|
Morag, S.,
Cohen, P.,
and Alessi, D. R.
(1998)
Biochem. J.
336,
241-246[Medline]
[Order article via Infotrieve]
|
8.
|
Dimmeler, S.,
Fleming, I.,
Fisslthaler, B.,
Hermann, C.,
Busse, R.,
and Zeiher, A. M.
(1999)
Nature
399,
601-605[CrossRef][Medline]
[Order article via Infotrieve]
|
9.
|
Dudek, H.,
Datta, S. R.,
Franke, T. F.,
Birnbaum, M. J.,
Yao, R.,
Cooper, G. M.,
Segal, R. A.,
Kaplan, D. R.,
and Greenberg, M. E.
(1997)
Science
275,
661-667[Abstract/Free Full Text]
|
10.
|
Andersen, C. B.,
Roth, R. A.,
and Conti, M.
(1998)
J. Biol. Chem.
273,
18705-18708[Abstract/Free Full Text]
|
11.
|
Fulton, D.,
Gratton, J. P.,
McCabe, T. J.,
Fontana, J.,
Fujio, Y.,
Walsh, K.,
Franke, T. F.,
Papapetropoulos, A.,
and Sessa, W. C.
(1999)
Nature
399,
597-601[CrossRef][Medline]
[Order article via Infotrieve]
|
12.
|
Ozes, O. N.,
Mayo, L. D.,
Gustin, J. A.,
Pfeffer, S. R.,
Pfeffer, L. M.,
and Donner, D. B.
(1999)
Nature
401,
82-85[CrossRef][Medline]
[Order article via Infotrieve]
|
13.
|
Romashkova, J. A.,
and Makarov, S. S.
(1999)
Nature
401,
86-90[CrossRef][Medline]
[Order article via Infotrieve]
|
14.
|
Kohn, A. D.,
Summers, S. A.,
Birnbaum, M. J.,
and Roth, R. A.
(1996)
J. Biol. Chem.
271,
31372-31378[Abstract/Free Full Text]
|
15.
|
Kitamura, T.,
Ogawa, W.,
Sakaue, H.,
Hino, Y.,
Kuroda, S.,
Takata, M.,
Matsumoto, M.,
Maeda, T.,
Konishi, H.,
Kikkawa, U.,
and Kasuga, M.
(1998)
Mol. Cell. Biol.
18,
3708-3717[Abstract/Free Full Text]
|
16.
|
Kitamura, T.,
Kitamura, Y.,
Kuroda, S.,
Hino, Y.,
Ando, M.,
Kotani, K.,
Konishi, H.,
Matsuzaki, H.,
Kikkawa, U.,
Ogawa, W.,
and Kasuga, M.
(1999)
Mol. Cell. Biol.
19,
6286-6296[Abstract/Free Full Text]
|
17.
|
Takata, M.,
Ogawa, W.,
Kitamura, T.,
Hino, Y.,
Kuroda, S.,
Kotani, K.,
Klip, A.,
Gingras, A.-C.,
Sonenberg, N.,
and Kasuga, M.
(1999)
J. Biol. Chem.
274,
20611-20618[Abstract/Free Full Text]
|
18.
|
Alessi, D. R.,
Andjelkovic, M.,
Caudwell, B.,
Cron, P.,
Morrice, N.,
Cohen, P.,
and Hemmings, B. A.
(1996)
EMBO J.
15,
6541-6551[Abstract]
|
19.
|
Alessi, D. R.,
Deak, M.,
Casamayor, A.,
Caudwell, F. B.,
Morrice, N.,
Norman, D. G.,
Gaffney, P.,
Reese, C. B.,
MacDougall, C. N.,
Harbison, D.,
Ashworth, A.,
and Bownes, M.
(1997)
Curr. Biol.
7,
776-789[Medline]
[Order article via Infotrieve]
|
20.
|
Alessi, D. R.,
James, S. R.,
Downes, C. P.,
Holmes, A. B.,
Gaffney, P. R.,
Reese, C. B.,
and Cohen, P.
(1997)
Curr. Biol.
7,
261-269[Medline]
[Order article via Infotrieve]
|
21.
|
Stephens, L.,
Anderson, K.,
Stokoe, D.,
Erdjument-Bromage, H.,
Painter, G. F.,
Holmes, A. B.,
Gaffney, P. R.,
Reese, C. B.,
McCormick, F.,
Tempst, P.,
Coadwell, J.,
and Hawkins, P. T.
(1998)
Science
279,
710-714[Abstract/Free Full Text]
|
22.
|
Dong, L. Q.,
Zhang, R.-B.,
Langlais, P.,
He, H.,
Matthew, C.,
Zhu, L.,
and Liu, F.
(1999)
J. Biol. Chem.
274,
8117-8122[Abstract/Free Full Text]
|
23.
|
Andjelkovic, M.,
Alessi, D. R.,
Meier, R.,
Fernandez, A.,
Lamb, N. J.,
Frech, M.,
Cron, P.,
Cohen, P.,
Lucocq, J. M.,
and Hemmings, B. A.
(1997)
J. Biol. Chem.
272,
31515-31524[Abstract/Free Full Text]
|
24.
|
Kohn, A. D.,
Takeuchi, F.,
and Roth, R. A.
(1996)
J. Biol. Chem.
271,
21920-21926[Abstract/Free Full Text]
|
25.
|
Balendran, A.,
Casamayor, A.,
Deak, M.,
Paterson, A.,
Gaffney, P.,
Currie, R.,
Downes, C. P.,
and Alessi, D. R.
(1999)
Curr. Biol.
9,
393-404[CrossRef][Medline]
[Order article via Infotrieve]
|
26.
|
Balendran, A.,
Currie, R.,
Armstrong, C. G.,
Avruch, J.,
and Alessi, D. R.
(1999)
J. Biol. Chem.
274,
37400-37406[Abstract/Free Full Text]
|
27.
|
Ono, Y.,
Fujii, T.,
Ogita, K.,
Kikkawa, U.,
Igarashi, K.,
and Nishizuka, Y.
(1988)
J. Biol. Chem.
263,
6927-6932[Abstract/Free Full Text]
|
28.
|
Mellor, H.,
and Parker, P. J.
(1998)
Biochem. J.
332,
281-292[Medline]
[Order article via Infotrieve]
|
29.
|
Moriya, S.,
Kazlauskas, A.,
Akimoto, K.,
Hirai, S.,
Mizuno, K.,
Takenawa, T.,
Fukui, Y.,
Watanabe, Y.,
Ozaki, S.,
and Ohno, S.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
151-155[Abstract/Free Full Text]
|
30.
|
Mitsumoto, Y.,
Liu, Z.,
and Klip, A.
(1993)
Endocrine J.
1,
307-315
|
31.
|
Kobayashi, T.,
Deak, M.,
Morrice, N.,
and Cohen, P.
(1999)
Biochem. J.
344,
189-197[CrossRef][Medline]
[Order article via Infotrieve]
|
32.
|
Kotani, K.,
Ogawa, W.,
Matsumoto, M.,
Kitamura, T.,
Sakaue, H.,
Hino, Y.,
Miyake, K.,
Sano, W.,
Akimoto, K.,
Ohno, S.,
and Kasuga, M.
(1998)
Mol. Cell. Biol.
18,
6971-6982[Abstract/Free Full Text]
|
33.
|
Sakaue, H.,
Ogawa, W.,
Takata, M.,
Kuroda, S.,
Kotani, K.,
Matsumoto, M.,
Sakaue, M.,
Nishio, S.,
Ueno, H.,
and Kasuga, M.
(1997)
Mol. Endocrinol.
11,
1552-1562[Abstract/Free Full Text]
|
34.
|
Kotani, K.,
Ogawa, W.,
Hino, Y.,
Kitamura, T.,
Ueno, H.,
Sano, W.,
Sutherland, C.,
Granner, D. K.,
and Kasuga, M.
(1999)
J. Biol. Chem.
274,
21305-21312[Abstract/Free Full Text]
|
35.
|
Kuroda, S.,
Tokunaga, C.,
Kiyohara, Y.,
Higuchi, O.,
Konishi, H.,
Mizuno, K.,
and Kikkawa, U.
(1996)
J. Biol. Chem.
271,
31029-31032[Abstract/Free Full Text]
|
36.
|
Takahashi, M.,
Mukai, H.,
Oishi, K.,
Isagawa, T.,
and Ono, Y.
(2000)
J. Biol. Chem.
275,
34592-34596[Abstract/Free Full Text]
|
37.
|
Ohno, S.,
Konno, Y.,
Akita, Y.,
Yano, Y.,
and Suzuki, K.
(1990)
J. Biol. Chem.
265,
6296-6300[Abstract/Free Full Text]
|
38.
|
Miyake, S.,
Makimura, M.,
Kanegae, Y.,
Harada, S.,
Sato, Y.,
Takamori, K.,
Tokuda, C.,
and Saito, I.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
1320-1324[Abstract/Free Full Text]
|
39.
|
Cross, D. A. E.,
Alessi, D. R.,
Cohen, P.,
Andjelkovich, M.,
and Hemmings, B. A.
(1995)
Nature
378,
785-789[CrossRef][Medline]
[Order article via Infotrieve]
|
40.
|
Kobayashi, T.,
and Cohen, P.
(1999)
Biochem. J.
339,
319-328[CrossRef][Medline]
[Order article via Infotrieve]
|
41.
|
Genot, E. M.,
Parker, P. J.,
and Cantrell, D. A.
(1995)
J. Biol. Chem.
270,
9833-9839[Abstract/Free Full Text]
|
42.
|
Doornbos, R. P.,
Theelen, M.,
van der Hoeven, P. C.,
van Blitterswijk, W. J.,
Verkleij, A. J.,
and van Bergen en Henegouwen, P. M.
(1999)
J. Biol. Chem.
274,
8589-8596[Abstract/Free Full Text]
|
43.
|
Park, J.,
Leong, M. L.,
Buse, P.,
Maiyar, A. C.,
Firestone, G. L.,
and Hemmings, B. A.
(1999)
EMBO J.
18,
3024-3033[Abstract/Free Full Text]
|
44.
|
Pullen, N.,
Dennis, P. B.,
Andjelkovic, M.,
Dufner, A.,
Kozma, S. C.,
Hemmings, B.,
and Thomas, G.
(1998)
Science
279,
707-710[Abstract/Free Full Text]
|
45.
|
Jensen, C. J.,
Buch, M. B.,
Krag, T. O.,
Hemmings, B. A.,
Gammeltoft, S.,
and Frodin, M.
(1999)
J. Biol. Chem.
274,
27168-27176[Abstract/Free Full Text]
|
46.
|
Cheng, X.,
Ma, Y.,
Moore, M.,
Hemmings, B. A.,
and Taylor, S. S.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
9848-9854
|
47.
|
Le Good, J. A.,
Ziegler, W. H.,
Parekh, D. B.,
Alessi, D. R.,
Cohen, P.,
and Parker, P. J.
(1998)
Science
281,
2042-2045[Abstract/Free Full Text]
|
48.
|
Biondi, R. M.,
Cheung, P. C.,
Casamayor, A.,
Deak, M.,
Currie, R. A.,
and Alessi, D. R.
(2000)
EMBO J.
19,
979-988[Abstract/Free Full Text]
|
49.
|
Behn-Krappa, A.,
and Newton, A. C.
(1999)
Curr. Biol.
9,
728-737[CrossRef][Medline]
[Order article via Infotrieve]
|