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Mol Cell Biol, July 1998, p. 3708-3717, Vol. 18, No. 7
Second Department of Internal Medicine,
Received 12 December 1997/Returned for modification 28 January
1998/Accepted 20 March 1998
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Requirement for Activation of the Serine-Threonine
Kinase Akt (Protein Kinase B) in Insulin Stimulation of Protein
Synthesis but Not of Glucose Transport
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
ACKNOWLEDGMENTS
REFERENCES
SUMMARY
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A wide variety of biological activities including the major metabolic actions of insulin is regulated by phosphatidylinositol (PI) 3-kinase. However, the downstream effectors of the various signaling pathways that emanate from PI 3-kinase remain unclear. Akt (protein kinase B), a serine-threonine kinase with a pleckstrin homology domain, is thought to be one such downstream effector. A mutant Akt (Akt-AA) in which the phosphorylation sites (Thr308 and Ser473) targeted by growth factors are replaced by alanine has now been shown to lack protein kinase activity and, when overexpressed in CHO cells or 3T3-L1 adipocytes with the use of an adenovirus vector, to inhibit insulin-induced activation of endogenous Akt. Akt-AA thus acts in a dominant negative manner in intact cells. Insulin-stimulated protein synthesis, which is sensitive to wortmannin, a pharmacological inhibitor of PI 3-kinase, was abolished by overexpression of Akt-AA without an effect on amino acid transport into the cells, suggesting that Akt is required for insulin-stimulated protein synthesis. Insulin activation of p70 S6 kinase was inhibited by ~75% in CHO cells and ~30% in 3T3-L1 adipocytes, whereas insulin-induced activation of endogenous Akt was inhibited by 80 to 95%, by expression of Akt-AA. Thus, Akt activity appears to be required, at least in part, for insulin stimulation of p70 S6 kinase. However, insulin-stimulated glucose uptake in both CHO cells and 3T3-L1 adipocytes was not affected by overexpression of Akt-AA, suggesting that Akt is not required for this effect of insulin. These data indicate that Akt acts as a downstream effector in some, but not all, of the signaling pathways downstream of PI 3-kinase.
INTRODUCTION
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Akt is a pleckstrin homology (PH) domain-containing protein serine-threonine kinase whose kinase domain shares structural similarity with protein kinase C (PKC) isozymes and cyclic AMP-dependent protein kinase (PKA) (3). Thus, Akt has also been termed RAC-PK (protein kinase related to A and C kinases) (19) and PKB (protein kinase B) (7). Insulin and various other growth factors activate Akt, and this activation is inhibited by pharmacological blockers of phosphatidylinositol (PI) 3-kinase or by a dominant negative mutant of PI 3-kinase (4, 14, 25). Furthermore, Akt is activated by overexpression of a constitutively active mutant of PI 3-kinase in quiescent cells (11, 23). These observations indicate that Akt is a downstream effector of PI 3-kinase.
PI 3-kinase, which consists of an 85-kDa regulatory subunit and a 110-kDa catalytic subunit (5), is implicated in various metabolic effects of insulin (18, 59). A dominant negative mutant of PI 3-kinase as well as various pharmacological inhibitors, such as wortmannin and LY294002, have been used to block specific signaling pathways that include this enzyme (6, 16, 31, 39, 61). The metabolic actions of insulin that are sensitive to either a dominant negative mutant or pharmacological inhibitors of PI 3-kinase include stimulation of glucose uptake, antilipolysis, activation of fatty acid synthase and glycogen synthase, and stimulation of amino acid transport and protein synthesis (6, 16, 34, 37, 47, 48, 54, 55). Moreover, regulation of the amounts of specific protein participants in metabolic pathways by insulin is mediated by this lipid kinase (52). Although these observations indicate that PI 3-kinase is a major regulator of the metabolic effects of insulin, the roles of the various downstream effectors of PI 3-kinase in each of these actions remain unclear.
The kinase activity of Akt fused with a viral Gag protein or tagged with a myristoylation signal sequence is higher than that of wild-type Akt (Akt-WT) (4, 26). Overexpression of these mutant Akt proteins induced activation of p70 S6 kinase (4, 26), which is also activated by insulin in a wortmannin-sensitive manner (42, 43). Expression of these active Akt mutants also promoted glucose uptake and translocation of GLUT4 glucose transporters in quiescent adipocytes (27, 53). These observations have suggested that Akt is a downstream effector of PI 3-kinase that mediates insulin-induced activation of p70 S6 kinase and glucose uptake. This proposal could be tested further by investigating the effects of specific inhibition of Akt activity on these actions of insulin. However, a mutant Akt that exerts dominant negative effects has not previously been described.
The mechanism by which Akt is activated in response to growth factor stimulation is not fully understood. PI 3,4-bisphosphate, one of the products of PI 3-kinase action, stimulates Akt activity in vitro (15, 24). Furthermore, Akt mutants with substitutions in or lacking the PH domain were not activated by this phospholipid (15, 24), suggesting that Akt is activated as a result of direct interaction of its PH domain with the lipid. On the other hand, other studies have suggested the importance of phosphorylation of Akt on serine and threonine residues in regulation of its activity. Akt is phosphorylated in vivo in response to various growth factors that stimulate Akt activity (4, 14, 26), and dephosphorylation of in vivo-activated Akt by a serine-threonine phosphatase abolished its enzymatic activity (26). Akt is phosphorylated on Thr308 and Ser473 in response to insulin in vivo, and Akt mutants in which either Thr308 or Ser473 was substituted were not activated (1). Moreover, a protein kinase which phosphorylates and activates Akt has been cloned and characterized (2, 44, 50, 51). These data have suggested that Akt is primarily activated as a result of its phosphorylation on serine and threonine residues by an upstream kinase.
We have now shown that when overexpressed in CHO cells or 3T3-L1 adipocytes, a mutant Akt in which growth factor-targeted serine and threonine phosphorylation sites are replaced with alanine exerted a dominant negative effect on endogenous Akt activity stimulated by insulin. With the use of this dominant negative Akt, we have investigated the roles of Akt in insulin-stimulated protein synthesis, p70 S6 kinase activation, and glucose transport in these cells.
MATERIALS AND METHODS
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Cells, plasmids, and antibodies.
CHO cells were routinely
maintained and 3T3-L1 preadipocytes were maintained and induced to
differentiate into adipocytes as described previously (48).
To establish CHO cells that stably express FLAG epitope-tagged Akt
(CHO-Akt cells), we transfected CHO cells with pSV40-hgh, which confers
resistance to hygromycin, and a PECE vector encoding FLAG
epitope-tagged rat Akt1 (RAC-PK
) (30). Transfected cells
were selected and cloned as described previously (22). PECE
vectors encoding FLAG epitope-tagged rat Akt2 (RAC-PK
)
(30) and RAC-PK
(29) were as described
previously. Monoclonal antibodies to the hemagglutinin (HA) epitope tag
(12CA5) or to the FLAG epitope tag were obtained from Boehringer
Mannheim and Kodak Scientific Imaging Systems, respectively. Polyclonal antibodies to Akt were generated against a glutathione
S-transferase fusion protein containing amino acids 428 to
480 of rat Akt1. Polyclonal antibodies to mitogen-activated protein
(MAP) kinase (C92) were as described previously (48).
Polyclonal antibodies to p70 S6 kinase were generated against a
synthetic peptide corresponding to amino acids 2 to 23 of the rat
enzyme (33).
Construction of adenovirus vectors.
Adenovirus vectors
encoding a dominant negative PI 3-kinase (AxCA
p85) or a dominant
negative SOS (AxCASOS) were as described previously (48).
Rat Akt1 was tagged with the HA epitope by PCR with a sense primer
(5'-ACT AAG CTT GCC ATG TAC CCA TAC GAT GTT CCG GAT TAC GCT AAC GAC GTA
GCC ATT GTG AAG G), an antisense primer (5'-GAT GAA TTC ACT GGG TGA ACC
TGA CCG G), and a full-length rat Akt1 cDNA as template. Both
Thr308 and Ser473 of HA-tagged rat Akt1 were
replaced by alanine with the use of a Quick Change site-directed
mutagenesis kit (Stratagene). Lys179 of HA-tagged rat Akt1
was replaced with asparate by the use of PCR. These mutants were termed
Akt-AA and AktK179D, respectively. DNA encoding the HA-tagged wild-type
and mutant (Akt-AA or AktK179D) Akt proteins was subcloned into pAxCAwt
(36), and adenovirus vectors containing these cDNAs were
generated by transfecting 293 cells with the corresponding pAxCAwt
plasmid together with a DNA-terminal protein complex (36),
as described previously (48). The resulting vectors were
termed AxCAAkt-WT, AxCAAkt-AA, and AxCAAkt-K179D, respectively. CHO
cells or 3T3-L1 adipocytes were infected with adenovirus vectors at the
indicated multiplicity of infection (MOI) as described previously
(48, 57). The cells were subjected to experiments 48 h
after infection.
Kinase assays. CHO cells or 3T3-L1 adipocytes were deprived of serum for 16 to 20 h, incubated in the absence or presence of insulin, and then immediately frozen with liquid nitrogen. The MAP kinase assay was performed with immunoprecipitates prepared with antibodies to MAP kinase as described previously (47, 48).
For p70 S6 kinase assays, the frozen cells were lysed in a solution containing 50 mM Tris-HCl (pH 8.0), 120 mM NaCl, 20 mM NaF, 1 mM benzamidine, 1 mM EDTA, 6 mM EGTA, 15 mM sodium pyrophosphate, 1% Nonidet P-40, 30 mM p-nitrophenyl phosphate, 0.5 mM dithiothreitol, and 0.1 mM phenylmethylsulfonyl fluoride. The lysate was centrifuged (at 15,000 × g for 20 min), and the resulting supernatant was subjected to immunoprecipitation with polyclonal antibodies to p70 S6 kinase. After being washed three times with HEPES-buffered saline (pH 7.5) containing 0.1% Triton X-100, the immunoprecipitates were incubated for 30 min at 25°C in a reaction mixture (30 µl) containing 50 mM morpholine propanesulfonic acid (pH 7.2), 5 mM MgCl2, 1 mM dithiothreitol, 10 mM p-nitrophenyl phosphate, 100 µM unlabeled ATP, 3.0 µCi of [-32P]ATP, and 100 µM S6 synthetic peptide
(KRRRLSSLRASTSKSESSQK) as the substrate. The reaction
mixture was then centrifuged (at 15,000 × g for 3 min), and the resulting supernatant was transferred to P81 (Whatman)
filter paper. After extensive washing of the filters with 0.5%
phosphoric acid, 32P incorporation into the peptide was
determined by liquid scintillation spectroscopy.
To assay the activities of FLAG- or HA-tagged Akt kinase, we lysed the
cells in a solution containing 50 mM HEPES-NaOH (pH 7.6), 150 mM NaCl,
1% Triton X-100, bacitracin (1 mg/ml), 1 mM phenylmethylsulfonyl
fluoride, 1 mM EDTA, 1 mM sodium orthovanadate, 10 mM NaF, and 30 mM
sodium pyrophosphate. The lysates were subjected to immunoprecipitation
with antibodies to HA or to FLAG. After being washed three times with
HEPES-buffered saline (pH 7.5) containing 0.1% Triton X-100, the
immunoprecipitates were incubated for 30 min at 30°C with 3.0 µCi
of [-32P]ATP in a reaction mixture (30 µl)
containing 20 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 25 µM
unlabeled ATP, 1 µM protein kinase inhibitor, and histone 2B (0.2 mg/ml) as the substrate. The reaction was terminated by the addition of
sodium dodecyl sulfate (SDS) sample buffer, and the samples were then
fractionated by SDS-polyacrylamide gel electrophoresis on a 15% gel.
The radioactivity incorporated into histone 2B was determined with a
Fuji BAS 2000 image analyzer.
For assay of endogenous Akt activity in CHO cells or 3T3-L1 adipocytes
that had been infected with AxCAAkt-AA, the cells were lysed as
described for determination of the activity of epitope-tagged Akt. The
lysates were subjected to three sequential immunoprecipitations for 90 min at 4°C (in a final volume of 400 µl) with 20 µl of protein
G-Sepharose (Pharmacia) that had been coupled with 20 µg of
antibodies to HA. The final supernatant was then subjected to
immunoprecipitation with polyclonal antibodies to Akt, and Akt kinase
assays were performed with the resulting immunoprecipitates as
described above.
Glucose uptake and translocation of GLUT4. Glucose uptake was assayed as described previously (16, 48). In brief, CHO cells and 3T3-L1 adipocytes cultured in six-well plates were incubated for 16 h in Ham's F12 and Dulbecco's modified Eagle's media, respectively, containing 5.6 mM glucose and 0.5% fetal bovine serum. The cells were washed twice with DB buffer (140 mM NaCl, 2.7 mM KCl, 1 mM CaCl2, 1.5 mM KH2PO4, 8 mM Na2HPO4 [pH 7.4], 0.5 mM MgCl2) and incubated with 100 nM insulin for 15 min, after which 1 ml of DB buffer containing bovine serum albumin (BSA; 1 mg/ml) and 0.1 mM 2-deoxy-D-[1,2-3H]glucose (1 µCi) was added to each well. After 5 min, the cells were washed and then solubilized with 0.1% SDS. The radioactivity incorporated into the cells was measured by liquid scintillation spectroscopy.
Translocation of GLUT4 to the plasma membrane was measured by the plasma membrane lawn assay as previously described (48).Protein synthesis and amino acid transport. Insulin-stimulated protein synthesis was assayed essentially as described previously (34), with the following modifications. CHO cells or 3T3-L1 adipocytes were deprived of serum for 24 h and then incubated for 1 h either with a mixture of Ham's F12 and DB buffer (1:100 [vol/vol]) or with methionine- and cysteine-free Dulbecco's modified Eagle's medium (Sigma), respectively. After addition of Tran35S-label (16 µCi/ml; ICN) and 100 nM insulin, the cells were incubated for an additional 1 h and the medium was then aspirated. The cells were washed three times with phosphate-buffered saline containing 10 mM methionine and then lysed in a solution containing 30 mM Tris-HCl (pH 7.5), 140 mM NaCl, and 0.5% Nonidet P-40. Proteins were precipitated by the addition of ice-cold trichloroacetic acid (final concentration, 10% [wt/vol]) containing 10 mM methionine. The protein precipitates were washed three times with ice-cold phosphate-buffered saline containing 10% trichloroacetic acid and 10 mM methionine and then solubilized in 1 M NaOH at 37°C for 10 min. The protein-associated radioactivity was assayed by liquid scintillation spectroscopy.
Amino acid uptake was assayed as described previously (54), with the following modifications. CHO cells cultured in six-well plates were deprived of serum for 16 h, washed twice with DB buffer, and incubated for 20 min with 2 ml of DB buffer containing BSA (1 mg/ml) and 10 mM unlabeled-methylaminoisobutyrate (MeAIB). One milliliter
of DB buffer containing BSA (1 mg/ml), 10 mM unlabeled MeAIB, 10 µM
[14C]MeAIB (1 µCi/ml), and 100 nM insulin was then
added to each well; after 30 min, the cells were washed three times
with ice-cold DB buffer containing BSA (1 mg/ml) and solubilized with
0.1% SDS. The amount of radioactivity incorporated into the cells was
measured by liquid scintillation spectroscopy.
Apoptosis assay. Apoptosis was assayed by measuring characteristic DNA laddering. DNA laddering was analyzed essentially as described previously (20), with the following modifications. CHO cells cultured in 6-cm-diameter plates were infected with AxCAAkt-AA or AxCAAkt-WT at the indicated MOI (PFU/cell). After 32 h, the infected cells were deprived of serum for 16 h, washed once in phosphate-buffered saline, and lysed in 0.1 ml of a buffer containing 0.5% Triton X-100, 10 mM Tris-HCl (pH 7.5), and 10 mM EDTA. The lysates were incubated for 20 min at 4°C and then centrifuged at 15,000 × g for 20 min. The DNA-containing soluble fraction was extracted from the resultant supernatants with phenol-chloroform, ethanol precipitated, resuspended in a buffer containing 10 mM Tris-HCl (pH 8.0), 10 mM EDTA, and 20 µg of RNase A per ml, and then incubated at 37°C for 1 h. DNA was loaded onto a 1.5% agarose gel; after electrophoresis, the gel was stained with ethidium bromide and photographed.
RESULTS
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Dominant negative effects of a mutant Akt with alanine
substitutions at Thr308 and Ser473.
A rat
Akt1 (RAC-PK) (28) mutant (termed Akt-AA) in which
Thr308 and Ser473 were replaced by alanine and
rat Akt-WT were tagged with the HA epitope at their NH2
termini and expressed in CHO cells with the use of adenovirus vectors
(AxCAAkt-WT or AxCAAkt-AA) containing the corresponding cDNA. The
infected cells were then incubated in the absence or presence of
insulin, after which recombinant Akt was immunoprecipitated with
antibodies to HA and assayed for Akt kinase activity with histone 2B as
the substrate. Consistent with previous observations (1),
insulin increased the activity of Akt1-WT about 20-fold but had no
effect on the activity of Akt-AA (Fig. 1A
to C).
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p85 (48), an adenovirus encoding a dominant
negative mutant of PI 3-kinase, inhibited insulin-induced activation of Akt (Fig. 2A). In contrast, infection of CHO-Akt cells with an adenovirus encoding a dominant negative mutant of SOS (AxCASOS) (48, 57), even at a virus concentration sufficient for
almost complete inhibition of insulin-induced activation of MAP kinase activity in CHO cells (data not shown), had no effect on Akt activity (Fig. 2B). Infection of the cells with AxCAAkt-AA resulted in a
dose-dependent inhibition of Akt activity precipitated with antibodies
to FLAG (Fig. 3B and C); the extent of
inhibition paralleled the expression of Akt-AA protein (Fig. 3A), with
~75% inhibition apparent at an MOI of 20 PFU/cell. We also
constructed an adenovirus vector that encodes a mutant Akt in which
Lys179 in the kinase domain was replaced by asparate
(Akt-K179D). This mutant Akt did not exhibit kinase activity (data not
shown). Infection of the cells with AxCAAkt-K179D had little effect on
Akt activity precipitated with antibodies to FLAG (Fig. 3B and C),
whereas the extent of expression of Akt-K179D protein assessed by
immunoblot analysis with antibodies to HA was similar to that of Akt-AA
protein in the cells infected with AxCAAkt-AA (Fig. 3A).
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), Akt2 (RAC-PK), and RAC-PK,
which were transiently expressed in COS cells (Fig. 4). Because these antibodies recognize
both endogenous and recombinant Akt proteins, Akt-AA was immunodepleted
from cell lysates with antibodies to HA before endogenous Akt was
immunoprecipitated with the polyclonal antibodies and assayed for
kinase activity toward histone 2B. Infection of CHO cells with
AxCAAkt-AA resulted in the expression of Akt-AA, the electrophoretic
mobility of which was slightly less than that of endogenous Akt because
of the presence of the epitope tag, in an MOI-dependent manner (Fig.
5A). At an MOI of 20 PFU/cell, the
abundance of Akt-AA was ~30 to 50 times that of endogenous Akt (data
not shown). After three sequential immunoprecipitations with antibodies
to HA, the amounts of Akt protein remaining in the supernatant were
similar in infected and noninfected cells (Fig. 5A), indicating that
Akt-AA was removed by this procedure. The insulin-induced activation of
endogenous Akt was found to be inhibited by AxCAAkt-AA in an
MOI-dependent manner, with 95% inhibition apparent at an MOI of 20 (Fig. 5B). In contrast, insulin-induced activation of MAP kinase was
not affected by the expression of Akt-AA (Fig. 5C). Furthermore,
infection of the cells with a control virus containing the
lacZ gene (AxCALacZ) at an MOI of 20 had no effect on
insulin-stimulated Akt activity (data not shown), suggesting that the
inhibition of insulin-induced Akt activation by AxCAAkt-AA was not due
to a nonspecific effect of viral infection.
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p85 and incubated in the absence or presence of
insulin. The activity of endogenous Akt was then assayed after immunodepletion of Akt-AA. As with CHO cells, three sequential immunoprecipitations with antibodies to HA removed Akt-AA (Fig. 6B). Infection of the adipocytes with
AxCAp85 inhibited insulin-induced activation of endogenous Akt in an
MOI-dependent manner, with ~65% inhibition apparent at an MOI of 30 (Fig. 6A). Infection of the cells with AxCAAkt-AA also inhibited
insulin-induced activation of endogenous Akt, with ~80% inhibition
apparent at an MOI of 200 (Fig. 6C). DNA laddering was not evident with
3T3-L1 adipocytes infected with AxCAAkt-AA at an MOI of 200 (data not
shown). Infection of 3T3-L1 adipocytes with AxCAAkt-AA did not affect
either the abundance of GLUT4 protein or morphological characteristics
of the adipocytes (data not shown), suggesting that Akt-AA did not cause a change in phenotype of the adipocytes during the time course of
the experiments. Thus, Akt-AA exerts a dominant negative effect in both
CHO cells and 3T3-L1 adipocytes.
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Effects of Akt-AA on insulin-stimulated protein synthesis in CHO cells and 3T3-L1 adipocytes. We next investigated the effect of Akt-AA on insulin-stimulated bulk protein synthesis, which has previously been shown to be regulated by PI 3-kinase (10, 34). Insulin stimulated an ~1.8-fold increase in protein synthesis in CHO cells within 1 h (Fig. 7A). Cells that had been exposed to wortmannin before treatment with insulin showed a level of protein synthesis that was less than the basal value. Infection of cells with AxCAAkt-AA inhibited insulin-stimulated protein synthesis in an MOI-dependent manner, without affecting the basal level; insulin-stimulated protein synthesis was completely abolished at an MOI of 20 PFU/cell.
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SOS at an MOI
sufficient for almost complete inhibition of insulin-induced activation
of MAP kinase activity in the adipocytes had no effect on
insulin-induced protein synthesis (data not shown).
Effects of Akt-AA on insulin-stimulated p70 S6 kinase activity. We next investigated the effects of Akt-AA on insulin-induced activation of p70 S6 kinase, the activity of which has previously been shown to be increased as a result of overexpression of a membrane-targeted mutant Akt or a Gag-Akt fusion protein (4, 26). Insulin induced an approximately sixfold increase in p70 S6 kinase activity in CHO cells (Fig. 8A). Infection of the cells with AxCAAkt-AA inhibited insulin-stimulated activation of p70 S6 kinase in an MOI-dependent manner, with ~75% inhibition apparent at an MOI of 20 PFU/cell.
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Effects of Akt-AA on insulin-stimulated glucose uptake and GLUT4
translocation.
Finally, we examined the effects of Akt-AA on
insulin-stimulated glucose uptake. Insulin induced an approximately
twofold increase in glucose uptake in CHO cells, an effect that was
inhibited by infection of the cells with AxCAp85 (Fig.
9A), consistent with our previous data
(16). However, infection of CHO cells with AxCAAkt-AA had no
effect on insulin-stimulated glucose uptake (Fig. 9B), even at a virus
concentration (MOI of 20 PFU/cell) that inhibited insulin-induced
activation of endogenous Akt by ~95% (Fig. 5B). Wortmannin abolished
insulin stimulation of glucose uptake in cells that had been infected
with AxCAAkt-AA (data not shown).
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p85
inhibited insulin-stimulated glucose uptake in an MOI-dependent manner
(Fig. 9C). In contrast, AxCAAkt-AA had no effect on glucose uptake
(Fig. 9D) even at an MOI of 200. Furthermore, a dose-response curve of
insulin-stimulated glucose uptake in the cells infected with AxCAAkt-AA
at an MOI of 200 was similar to that of noninfected cells (Fig. 9E). We
examined the effect of Akt-AA on insulin-induced translocation of GLUT4
by the plasma membrane lawn assay. Whereas plasma membrane lawns
prepared from quiescent adipocytes showed little GLUT4
immunoreactivity, insulin induced a marked increase in the amount of
the glucose transporter in the plasma membrane (Fig.
10A and B). Infection of the cells with
AxCAp85 inhibited the insulin-induced increase in GLUT4
immunoreactivity in the membrane (Fig. 10C), whereas AxCAAkt-AA had no
effect on this action of insulin (Fig. 10D).
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DISCUSSION
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We have investigated the roles of Akt in cells by specifically inhibiting the activity of the endogenous enzyme. Such inhibition was achieved by expression of a mutant Akt (Akt-AA) in which the sites of ligand-induced phosphorylation are mutated to alanine and that acts in a dominant negative manner. Akt-AA was introduced into both CHO cells and 3T3-L1 adipocytes with the use of an adenovirus expression system, which has previously been used to express a variety of genes with high efficiency (36). The maximal abundance of Akt protein in CHO cells or 3T3-L1 adipocytes infected with AxCAAkt-AA was 30 to 50 times that of endogenous Akt.
We tested several mutants of Akt for dominant negative effects, including a protein in which Lys179 in the kinase domain was replaced by asparate (Akt-K179D). Although Akt-K179D did not exhibit kinase activity, it was less effective than Akt-AA in inhibiting the activity of endogenous Akt when overexpressed by the adenovirus system. Activity-deficient mutants of protein kinases in which phosphorylation sites targeted by extracellular stimuli have been replaced by neutral amino acids have previously been shown to act in a dominant negative manner. For example, mutants of MAP kinase in which either Thr192 or Tyr194 is replaced by alanine (40) act in a dominant negative manner. Furthermore, a MEK protein in which extracellular stimulus-dependent phosphorylation sites were changed to alanine also behaved as a dominant negative mutant (8). Such a mutant MEK showed increased binding to Raf, a kinase upstream of MEK, compared with wild-type MEK (60). These data, together with our observation that Akt-K179D is phosphorylated in vivo in response to insulin (unpublished data) and by a putative upstream kinase in vitro (51), suggest that Akt-AA may interact with its upstream kinase with higher affinity than does either wild-type Akt or Akt-K179D and that this higher-affinity interaction underlies its dominant negative effects.
Overexpression of a membrane-targeted mutant Akt or a Gag-Akt fusion protein, the kinase activity of both of which is greater than that of Akt-WT, was previously shown to increase glucose uptake or translocation of GLUT4 in quiescent adipocytes (27, 53). However, we have now shown that inhibition of endogenous Akt activity by Akt-AA had no effect on glucose transport. The simplest explanation for these observations would be that Akt is not necessary for insulin-stimulated glucose uptake, although activated Akt is sufficient to increase glucose uptake under certain conditions. A constitutively active mutant of Ras also increases glucose uptake and translocation of GLUT4 (32, 45), whereas Ras activation and its downstream signaling are not required for insulin stimulation of glucose uptake (12, 17, 45, 48).
It is possible that the inhibition of endogenous Akt activity by
overexpression of Akt-AA is not sufficient to affect insulin-stimulated glucose uptake. However, the same extent of inhibition of
insulin-stimulated Akt activity achieved by a dominant negative mutant
of PI 3-kinase (p85) was sufficient to inhibit insulin-induced
glucose transport. The polyclonal antibodies used to examine endogenous
Akt activity are capable of precipitating all three known isoforms of
Akt (Akt1 [RAC-PK], Akt2 [RAC-PK], and RAC-PK). Thus, our
data indicate that the activities of all three Akt isoforms are
inhibited by Akt-AA. However, we cannot exclude the possibility that an
unidentified isoform of Akt that is resistant to Akt-AA is present in
the cells studied and responsible for insulin-stimulated glucose
uptake.
We have shown that Akt-AA inhibited insulin-stimulated p70 S6 kinase activity, suggesting that Akt is required for activation of p70 S6 kinase. It is not clear why insulin-induced activation of p70 S6 kinase in CHO cells was inhibited by only ~75% whereas insulin activation of endogenous Akt was almost completely abolished in these cells. However, previous evidence has suggested that growth factor stimulation of p70 S6 kinase is mediated by redundant signaling pathways (43, 58). Thus, interruption of only one pathway (the Akt pathway) may be insufficient for complete inhibition of p70 S6 kinase activation. The relatively small inhibitory effect of Akt-AA on insulin activation of p70 S6 kinase in 3T3-L1 adipocytes may reflect a minor contribution of Akt to this action of insulin in these cells.
Wortmannin inhibits insulin-stimulated bulk protein synthesis (10, 34). Furthermore, we have recently shown that a dominant negative mutant of PI 3-kinase inhibited insulin-stimulated protein synthesis in CHO cells (52a). These data suggest that PI 3-kinase is important for insulin-stimulated bulk protein synthesis. However, it has not been known at which of the multiple steps of protein synthesis (21) PI 3-kinase exerts its effect. Tsakiridis et al. (54) showed that wortmannin inhibits insulin-stimulated amino acid transport in L6 myoblast cells. We have now shown that Akt-AA inhibited protein synthesis but not amino acid transport in CHO cells. These data demonstrate that the Akt pathway affects protein synthesis at a step distinct from amino acid transport. However, we cannot exclude the possibility that upstream kinases of Akt have physiological substrates other than Akt and that the dominant negative effects of Akt-AA are, at least in part, due to the inhibition of phosphorylation of these substrates but not of Akt.
p70 S6 kinase is thought to contributes to the regulation of protein
synthesis by phosphorylating ribosomal protein S6 (43). Phosphorylation of S6 in intact cells correlates with increased protein
synthesis (21). However, the inhibitory effect of Akt-AA on
bulk protein synthesis is probably not due to the inhibition of p70 S6
kinase because rapamycin, which prevents insulin activation of p70 S6
kinase (41, 43), has only a small effect on
insulin-stimulated protein synthesis (10, 34). We recently
showed that insulin-induced activation of guanine nucleotide exchange
activity toward translation initiation factor eIF-2 in total lysates of
CHO-IR cells was completely inhibited by overexpression of a dominant
negative PI 3-kinase (57). The eIF-2B factor is thought to
mediate this guanine nucleotide exchange activity and subsequently to
regulate recruitment of the initiator Met-tRNA to the 40S ribosomal
subunit (46), an essential step for initiation of
translation. Glycogen synthase kinase-3 (GSK-3), a putative
downstream effector of Akt (9), has been suggested to
participate in the regulation of eIF-2B (56). Although the
effects of Akt-AA on regulation of GSK-3 and eIF-2B remain to be
elucidated, Akt-AA may affect bulk protein synthesis by inhibiting the
GSK-3-eIF-2B pathway.
In summary, we have identified a dominant negative mutant of Akt and,
with the use of an adenovirus encoding this protein, shown that Akt
mediates some, but not all, signaling pathways downstream of PI
3-kinase. Because PI 3-kinase contributes not only to the metabolic
actions of insulin but to a variety of biological effects, it will be
important to determine which other signaling pathways downstream of PI
3-kinase are mediated through Akt. An atypical PKC isozyme, PKC
, is
a putative downstream effector of PI 3-kinase (38) and has
recently been shown to be required for insulin-stimulated bulk protein
synthesis (35). It is not clear which steps of protein
synthesis are regulated by PKC, and so it remains to be determined
whether Akt and PKC participate in the same signaling pathway or
whether each controls protein synthesis by regulating different steps.
ACKNOWLEDGMENTS
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We thank I. Saito for pAxCAwt, AxCALacZ, and technical advice on preparation of adenovirus vectors.
This work was supported by grants (to M.K.) from the Ministry of Education, Science, Sports, and Culture of Japan and from the Uehara Memorial Foundation.
FOOTNOTES
* Corresponding author. Second Department of Internal Medicine, Kobe University School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650, Japan. Phone: (81) 78-341-7451. Fax: (81) 78-382-2080. E-mail: ogawa{at}med.kobe-u.ac.jp.
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