From the Third Department of Internal Medicine,
Faculty of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo
113, Japan, § The Institute for Diabetes Care and Research,
Asahi Life Foundation, 1-6-6 Marunouchi, Chiyoda-ku, Tokyo 100, Japan,
¶ Laboratory for Physiological Chemistry, Utrecht
University, Universiteitsweg 100, 3584 CG Utrecht, The Netherlands, and
Department of Pulmonary Disease, University Hospital Utrecht,
Heidelberglaan 100, 3584 CX Utrecht, The Netherlands
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ABSTRACT |
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Various biological responses stimulated by insulin have been thought to be regulated by phosphatidylinositol 3-kinase, including glucose transport, glycogen synthesis, and protein synthesis. However, the molecular link between phosphatidylinositol 3-kinase and these biological responses has been poorly understood. Recently, it has been shown that protein kinase B (PKB/c-Akt/Rac) lies immediately downstream from phosphatidylinositol 3-kinase. Here, we show that expression of a constitutively active form of PKB induced glucose uptake, glycogen synthesis, and protein synthesis in L6 myotubes downstream of phosphatidylinositol 3-kinase and independent of Ras and mitogen-activated protein kinase activation. Introduction of constitutively active PKB induced glucose uptake and protein synthesis but not glycogen synthesis in 3T3L-1 adipocytes, which lack expression of glycogen synthase kinase 3 different from L6 myotubes. Furthermore, we show that deactivation of glycogen synthase kinase 3 and activation of rapamycin-sensitive serine/threonine kinase by PKB in L6 myotubes might be involved in the enhancement of glycogen synthesis and protein synthesis, respectively. These results suggest that PKB acts as a key enzyme linking phosphatidylinositol 3-kinase activation to multiple biological functions of insulin through regulation of downstream kinases in skeletal muscle, a major target tissue of insulin.
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INTRODUCTION |
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Insulin promotes a wide variety of biological responses in vivo, including regulation of glucose metabolism and protein synthesis (1, 2). One of the most important metabolic responses by insulin is the stimulation of facilitated glucose transport in muscle and adipose tissues, primarily due to translocation of glucose transporter isoform 4 (GLUT4) from an intracellular pool to the plasma membrane (3, 4). Another major aspect of the glucose metabolism regulated by insulin is the activation of glycogen synthesis through the activation of glycogen synthase (GS)1 in skeletal muscle (5-8). Insulin also serves as the major regulator of protein synthesis by phosphorylation of eukaryotic initiation factor 4E (eIF-4E)-binding protein 1 (4E-BP1) and its dissociation from eIF-4E, thereby increasing the eIF-4E available for initiation of mRNA translation (9, 10). Many processes of these biological actions in response to insulin have been demonstrated to be regulated by phosphatidylinositol 3-kinase (PI3-K) (11-17). However, the molecular link between PI3-K activation and final biological functions is unknown. Recent reports revealed that protein kinase B (PKB) is regulated by PI3-K through the direct interaction of its pleckstrin homology domain and the phosphorylated products of PI3-K, leading to phosphorylation in its serine/threonine residues and activation by a putative upstream kinase (18-23). Moreover, PKB has been shown to phosphorylate and inhibit glycogen synthase kinase 3 (GSK3) in vitro (24), suggesting the possibility that it might be involved in glycogen synthesis regulated by PI3-K. Nevertheless, expression of a constitutively active form of PKB failed to stimulate glycogen synthesis in 3T3L-1 adipocytes, although it enhanced glucose transport activity (25). These findings prompted us to study the roles of PKB in multiple biological responses of insulin. Here, we show that expression of GagPKB (18), a constitutive active form of PKB, promotes glucose transport activity and protein synthesis rate in both 3T3L-1 adipocytes and L6 myotubes, which are demonstrated to be regulated by PI3-K, independent of mitogen-activated protein kinase. We also show that expression of GagPKB promotes glycogen synthesis in L6 myotubes but not in 3T3L-1 adipocytes that lack expression of GSK3. These data suggest that PKB might be a key player in insulin-stimulated multiple metabolic actions in skeletal muscles.
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EXPERIMENTAL PROCEDURES |
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Cell Culture and Adenovirus-mediated Gene Transfer--
3T3L-1
cells and L6 cells were maintained in Dulbecco's modified Eagle's
medium (DMEM) and induced to differentiate into adipocytes and myotubes
as described in Refs. 26 and 27, respectively. The differentiated cells
were cultured in media containing the adenoviruses for 1 h at
37 °C, and DMEM supplemented with fetal calf serum was added and
cultured for 24 h. One day later, when cells were subjected to
assays, they were serum-starved for 20 h. The adenoviruses were
applied at a concentration of 3 × 108 plaque-forming
units/cm2 dish. Under these conditions, LacZ gene
expression was observed in more than 90% of 3T3L-1 cells and L6 cells
on postinfection from day 1 through day 3 measured by -galactosidase
assay. Expression of GagPKB was confirmed by Western blot analysis and
immune complex kinase assay as described below. Expression of dominant
negative Ras (DNRas) and that of mitogen-activated protein kinase
(MAPK) were confirmed by measurement of MAPK activity in response to insulin as described (28). Insulin-induced MAPK activity was completely
inhibited in cells expressing DNRas and increased 5-8-fold in cells
expressing MAPK compared with that in cells expressing LacZ.
Recombinant Adenoviruses--
cDNA of GagPKB was constructed
as described (18). cDNA of DNRas (K-Ras substituted Ser-17 to Asn
by polymerase chain reaction method) was kindly provided by Dr. Takai
(Osaka University). cDNA of MAPK was constructed as described (28).
cDNA of GSK3 (29) was kindly provided by Dr. He (NCI, National
Institutes of Health) and fused to hemagglutinin (HA) epitope sequence
substituted for its original stop codon (GSK3
-HA). cDNA
fragments including the whole coding region of GagPKB, DNRas, MAPK, and
GSK3
-HA were introduced to the expression cosmid cassette with blunt
end ligation into the SwaI site, respectively, as described
(30). The recombinant adenoviruses, Adex1CAGagPKB, Adex1CADNRas,
Adex1CAMAPK, and Adex1CAGSK3a-HA were constructed by homologous
recombination between the expression cosmid cassette and parental virus
genome as described (30). The control adenovirus Adex1CALacZ and the
cosmid cassette were kindly provided by Dr. Saito (University of
Tokyo).
Antibodies--
Rabbit polyclonal anti-PKB antibodies
(PKB-CT) were generated against a peptide corresponding to the
sequence of 465-480 human PKB (Upstate Biotechnology Inc.). Rabbit
polyclonal anti-p70 S6 kinase antibodies (C-18) were generated against
a peptide corresponding to the sequence of 485-502 of rat p70 S6
kinase (Santa Cruz Biotechnology). Anti-PP1G antibodies were generated
against a peptide corresponding to the sequence surrounding site 1 of
rabbit skeletal muscle PP1G (SPQPSRRGSESSEE) as described (31). Rabbit
polyclonal anti-GSK3 antibodies (
GSK3) were generated against a
peptide corresponding to the sequence of 462-475 of rat GSK3
, as
described (32). GSK3
monoclonal antibodies were generated by
immunizing against the N-terminal region (1-160) of rat GSK3
(Transduction Laboratory). Monoclonal anti-HA antibodies (12CA5) were
generated against a peptide (YPYDVPDYA) corresponding to the sequence
of influenza hemagglutinin (Boehringer Mannheim). Rabbit polyclonal
anti-4E-BP1 antibodies were generated against a glutathione
S-transferase 4E-BP1 fusion protein (33).
Immunoprecipitations were performed as described below. Immunoblots
were developed using the chemiluminescence Western blotting kit
(Boehringer Mannheim) and subjected to autoradiography.
In Vitro Kinase Assays--
Cells were starved for 20 h,
treated without or with insulin, and lysed with the lysis buffer
containing 20 mM Tris-HCl, pH 7.5, 25 mM
-glycerophosphate, 100 mM NaCl, 1 mM sodium
orthovanadate, 2 mM EGTA, 5 µg/ml leupeptin, 5 µg/ml
aprotinin, and 1 mM phenylmethylsulfonyl fluoride. Cell
lysates were subjected to immunoprecipitation with
PKB-CT, C-18, or
GSK3 followed by PKB kinase assay as described (Cross et
al. (24, 42)), S6 kinase assay as described (14), or GSK3 kinase
assay. Briefly, in PKB and S6 kinase assays, the immunoprecipitates
with
PKB-CT or C-18 were washed and resuspended in 50 mM
Tris-HCl, pH 7.5, 10 mM MgCl2, 1 mM
dithiotreitol to which 50 µM ATP, 3 µCi of
[
32P]ATP, and 1 µg of cross-tide in PKB assay or 1 µg of S6 peptide (32-mer peptide from the C-terminal sequence of
ribosomal S6 protein; Life Technologies Inc.) in S6 kinase assay had
been added. After 20 min at 30 °C, the reaction was stopped, and the
aliquots were spotted on squares of P-81 paper, washed, and counted by
Cherenkov. GSK3 kinase activity was determined by the method as
described (34) with modification. The immunoprecipitates with
GSK3
were resuspended in 25 mM
-glycerophosphate, 40 mM HEPES, pH 7.2, 10 mM MgCl2, and
2 mM protein kinase inhibitor adding 50 µM
ATP, 5 µCi of [
32P]ATP, and 1 µg of
phosphoglycogen synthase peptide (Upstate Biotechnology Inc.). After 20 min at 30 °C, the reaction was stopped, and the aliquots were
spotted on squares of P-81 paper, washed, and counted by Cherenkov.
2-Deoxyglucose (2-DG) Uptake Assays-- 2-Deoxyglucose (2-DG) uptake assays were performed as described (26, 27) with modification. Cells were grown in 12-well plates and infected with adenoviruses as described above. Before initiating glucose uptake assays, cells were washed three times with phosphate-buffered saline and incubated in 1 ml of serum-free DMEM for 3 h at 37 °C. Next, cells were washed once with Krebs-Ringer phosphate-HEPES buffer (KRHB) containing 130 mM NaCl, 5 mM KCl, 1.3 mM CaCl2, 1.3 mM MgSO4, 10 mM Na2HPO4, and 25 mM HEPES, pH 7.4, and incubated in 1 ml of KRHB containing 0.1% bovine serum albumin without or with 100 nM insulin for 20 min at 37 °C. Glucose uptake was initiated by the addition of 2-deoxy-D-[2,6-3H]glucose to a final concentration of 0.5 µCi for 5 min at 37 °C and terminated by two washes with ice-cold KRHB. Cells were solubilized with 0.4 ml of 0.1% SDS and counted by scintillation counter. Nonspecific glucose uptake was measured in the presence of 20 µM cytochalasin B and 200 µM phloretin and was subtracted from total uptake in each assay to obtain specific uptake.
Glycogen Synthase Assays-- Glycogen synthase activity was measured as described previously (14) with slight modifications. Cells were infected with adenoviruses as described above and incubated in serum-free DMEM for 20 h. Then they were washed twice and incubated with KRHB without or with 100 nM insulin for 20 min. Cells were lysed with lysis buffer containing 25 mM Tris-HCl, pH 7.0, 30% glycerol, 10 mM EDTA, 100 mM KF, 1 mM phenylmethylsulfonyl fluoride. The lysates were centrifuged, and 30 µl of the supernatant was added to 60 µl of the assay mixture containing 50 mM Tris-HCl, pH 7.4, 25 mM NaF, 20 mM EDTA, 1 mg/ml glycogen, and 0.1 µCi of UDP-[14C]glucose and with 0.25 or 10 mM glucose 6-phosphate. After incubation at 30 °C for 30 min, aliquots were spotted on 3MM paper (Whatman), washed four times with ice-cold 70% ethanol, and counted radioactivity by scintillation counter.
Assays of Protein Synthesis Rate-- The protein synthesis rate was determined by measuring the incorporation of [3H]tyrosine into protein as described (35, 36). Briefly, cells were grown in 6-well dishes and infected with adenoviruses as described above. After starvation in serum-free DMEM for 20 h, cells were incubated in 1 ml of serum-free F-12 (Hams') medium without or with 100 nM insulin for 1 h. Then, the medium was replaced with medium containing the same additions plus L-[2,3,5,6-3H]tyrosine (5 µCi/ml). After 1 h, cells were rinsed twice with ice-cold phosphate-buffered saline and extracted with 1 ml of buffer containing 10 mM Tris-HCl, pH 7.5, 250 mM KCl, 10 mM MgCl2, 6 mM 2-mercaptothanol, 1 mM phenylmethylsulfonyl fluoride before centrifugation. For measurement of radioactivity in total soluble protein, aliquots of the supernatants (30 µg of protein) were spotted on 3MM paper strips, and the strips were kept in boiling 10% trichloroacetic acid for 5 min, washed, and the radioactivity was counted.
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RESULTS AND DISCUSSION |
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Expression of GagPKB in 3T3L-1 Adipocytes and L6 Myotubes--
We
investigated insulin-sensitive cell lines, 3T3L-1 adipocytes and L6
myotubes expressing the constitutively active form of PKB (GagPKB)
using adenovirus vector (30). Western blot analysis revealed that the
amount of GagPKB expressed was comparable to that of endogenous PKB
(Fig. 1A). PKB activity,
assessed as the ability to phosphorylate Crosstide, a peptide
corresponding to the sequence surrounding the phosphorylation site
Ser-21 in GSK3 (24), was 2-3-fold higher at the basal state in
cells expressing GagPKB than in control cells expressing LacZ. In fact,
basal PKB activity in cells expressing GagPKB was already comparable to that detected in control cells after insulin stimulation (Fig. 1B). Increase in PKB activity was maintained significantly
higher after insulin stimulation in cells expressing GagPKB as compared with control cells. Concomitantly, p70 S6 kinase activity in cells expressing GagPKB was increased in both basal and insulin-stimulated states in these cell lines (Fig. 1C). In cells expressing
LacZ, pretreatment with wortmannin, which completely inhibited PI3-K, also completely inhibited the insulin-induced activation of endogenous PKB (Fig. 1B). In cells expressing GagPKB, however, PKB
activity was still about 2-fold higher than that in cells expressing
LacZ at the basal state without wortmannin, whereas an increase in the
kinase activity by insulin stimulation was not noted (Fig. 1B). Although not statistically significant, it was noted
that wortmannin treatment reduced PKB activity in both cells expressing LacZ and GagPKB in 3T3L-1 adipocytes and L6 myotubes, suggesting the
involvement of wortmannin-sensitive pathway in the PKB activation even
in the basal condition. Neither expression of DNRas or MAPK nor
treatment with PD98059 (MAP kinase kinase inhibitor) affected PKB or
p70 S6 kinase activity in either of the cell lines (data not shown),
although Ras has been reported to be involved in activation of PKB in
some cell lines (19).
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Activation of PKB Mimics Insulin-induced Glucose Transport Activity Regulated by PI3-K both in 3T3L-1 Adipocytes and L6 Myotubes-- Expression of GagPKB induced 2-DG uptake in the absence of insulin in 3T3L-1 adipocytes to almost the same levels as detected after insulin treatment of cells expressing LacZ (Fig. 2A). This is consistent with the recent results using another type of constitutively active PKB in 3T3L-1 adipocytes (25) and the findings that expression of GagPKB promotes GLUT4 appearance at the cell surface in isolated adipocytes (37). In L6 myotubes, expression of GagPKB also induced fundamentally similar effects (Fig. 2B). Pretreatment with wortmannin, which completely inhibited both PI3-K and insulin-sensitive endogenous PKB (Fig. 1B), also completely inhibited 2-DG uptake stimulated by insulin in 3T3L-1 adipocytes and L6 myotubes expressing LacZ (Fig. 2, A and B). In cells expressing GagPKB treated with wortmannin, basal 2-DG uptake was still higher than that in 3T3L-1 adipocytes and L6 myotubes expressing LacZ, although insulin stimulation was not noted (Fig. 2, A and B). This was consistent with the results that GagPKB was constitutively active even after wortmannin treatment to the same extent as PKB activity after insulin stimulation in cells expressing LacZ (Fig. 1B). In addition, expression of neither DNRas nor MAPK affected 2-DG uptake (Fig. 2, A and B), indicating that the Ras/MAPK pathway is neither sufficient nor required for glucose uptake by insulin in these cell lines. These findings suggest that PKB activated by insulin via PI3-K might stimulate glucose transport activity independent of Ras/MAPK activation in skeletal muscle and adipose tissue, the major tissues for glucose disposal by insulin.
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Activation of PKB Mimics Insulin-induced Glycogen Synthesis Regulated by PI3-K in L6 Myotubes but Not in 3T3L-1 Adipocytes-- Next, we investigated GS activity regulating glycogen synthesis, a major glucose utilization pathway by insulin. In 3T3L-1 adipocytes, expression of GagPKB did not affect either basal or insulin-stimulated GS activity (Fig. 3A), whereas inhibition of PI3-K by wortmannin resulted in complete inhibition of insulin-dependent GS activity in cells expressing either LacZ or GagPKB (Fig. 3A). Moreover, expression of neither DNRas nor MAPK affected GS activity (Fig. 3A). These results suggest that a PI3-K-dependent but PKB-independent pathway plays a key role in activation of GS activity in 3T3L-1 adipocytes. In contrast, expression of GagPKB in L6 myotubes enhanced basal GS activity to essentially the same levels as the insulin-stimulated activity in cells expressing LacZ. Increase in insulin-stimulated activity in cells expressing GagPKB compared with cells expressing LacZ did not reach statistical significance (Fig. 3B). After treatment with wortmannin in L6 myotubes, GS activity in cells expressing GagPKB still remained significantly higher than that in cells expressing LacZ (Fig. 3B), suggesting that wortmannin inhibited both PI3-K and the insulin-induced activation of endogenous PKB but that constitutively active GagPKB could mimic PI3-K-dependent GS activation in L6 myotubes. In L6 myotubes as well as 3T3L-1 adipocytes, expression of neither DNRas nor MAPK affected GS activity (Fig. 3B). These findings indicate that constitutively active PKB is sufficient to stimulate GS activity in L6 myotubes and that the Ras/MAPK pathway is not involved in GS activation.
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Activation of PKB Inhibits GSK3 Activity in L6 Myotubes--
What
is it that causes the difference between 3T3 L-1 adipocytes and L6
myotubes? GS activation by insulin is thought to be mediated by
promotion of dephosphorylation and activation of GS due to either
activation of protein phosphatase 1 by phosphorylation of its G subunit
(PP1G) (6, 38, 39) or inactivation of GSK3 by phosphorylation (8, 24,
40) or both. We addressed the possibility that the difference in the
relative abundance of PP1G and GSK3 between 3T3L-1 adipocytes and L6
myotubes might lead to alterations of the predominant signaling
pathways regulating GS activity by insulin. Thus, we investigated the
expression levels of PP1G and GSK3 by Western blot and compared the two
cell lines. Fig. 3C shows that PP1G was expressed in both
3T3L-1 adipocytes and L6 myotubes and seemed to be more abundant in the
former. In contrast, expression of GSK3 (both and
) was observed
only in L6 myotubes but not in differentiated 3T3L-1 adipocytes as described previously (41). These findings raise the possibility that
PKB might stimulate GS activity by inactivation of GSK3 in L6 myotubes,
and this mechanism might not work in 3T3L-1 adipocytes. In fact,
expression of GagPKB significantly inhibited GSK3 activity at the basal
state to essentially the same levels as insulin-stimulated GSK3
activity in cells expressing LacZ in L6 myotubes (Fig. 3D), whereas the activity could not be detected in 3T3L-1 adipocytes, even
at the basal state (data not shown). Moreover, expression of GSK3
resulted in a decrease in basal GS activity (about 30%) in 3T3L-1
adipocytes compared with cells expressing LacZ, consistent with the
results in 293 cells (40), whereas such suppression of GS activity was
not observed in cells coexpressing GSK3 and GagPKB (Fig.
3E). From these data, we conclude that PKB stimulates GS
activity by regulating GSK3 activity in L6 myotubes. Our data also
suggested that regulation of GS activity by PP1G via PI3-K, independent
of PKB, might be dominant in 3T3L-1 adipocytes. This may explain the
lack of stimulation of glycogen synthesis by a constitutively active
PKB in 3T3L-1 adipocytes (this study and Ref. 25). Taken together,
these findings suggested the possibility that PKB might act as a key
regulator in glycogen synthesis in skeletal muscle. Recent studies have
demonstrated that GSK3 is detected, and the kinase activity is
negatively regulated by insulin in isolated fat cells (42, 43),
different from the results with 3T3L-1 adipocytes shown in this study.
It suggests the possibility that PKB/GSK3 signaling pathway in adipose
tissue might be also involved in the regulation of glycogen synthesis
in vivo.
Activation of PKB Mimics Insulin-induced Protein Synthesis both in
3T3L-1 Adipocytes and L6 Myotubes--
The stimulation of protein
synthesis is an important and early response by insulin and is observed
in a wide variety of cell types (44). The initiation phase of
translation is rate-limiting, and the regulation by insulin is exerted
at this step. The most important event is phosphorylation of 4E-BP1
(PHAS-I) in response to insulin and its dissociation from eIF-4E/4E-BP1
complex, leading to mRNA translation. MAPK was reported to
phosphorylate 4E-BP1 in vitro and to abolish binding of
4E-BP1 to eIF-4E (10). However, recent studies revealed that insulin
promotes the phosphorylation of multiple sites in 4E-BP1, one of which
is likely to involve PI3-K-p70 S6 kinase, since it occurs in a
rapamycin-sensitive and MAPK-independent manner (33, 45-48). 4E-BP1
detected by Western blot appears as three bands, designated ,
,
and
, representing different extents of phosphorylation in 3T3L-1
adipocytes (Fig. 4A), as
described (36). Indeed, wortmannin inhibited the increase in the amount
of
band in response to insulin, but expression of DNRas did not
alter phosphorylation levels of 4E-BP1 (Fig. 4A), suggesting
that such phosphorylation by insulin is PI3-K-dependent and
MAPK-independent. We next investigated the effect of GagPKB, a putative
regulatory molecule of downstream effectors of PI3-K such as p70 S6
kinase, on phosphorylation of 4E-BP1 and protein synthesis in response
to insulin in both cell lines. Expression of GagPKB resulted in much
higher phosphorylation of 4E-BP1 in basal and insulin-stimulated
states. In cells expressing GagPKB, the basal levels of phosphorylation
of 4E-BP1 were much higher than in cells expressing LacZ, even after
treatment with wortmannin (Fig. 4B), suggesting that GagPKB
can stimulate 4E-BP1 phosphorylation downstream of PI3-K. In L6
myotubes, essentially the same results were observed (data not shown).
These data suggested that activation of PKB might promote protein
synthesis by phosphorylating 4E-BP1. Expression of GagPKB indeed
increased the protein synthesis rate in both cell lines (Fig.
4C). Recently, a hypothetical model for the pathways
promoting phosphorylation of 4E-BP1 in response to insulin has been
proposed (46, 48, 49). According to this model, insulin stimulates
phosphorylation on multiple sites of 4E-BP1 that might be mediated by
the putative proline-directed serine/threonine kinase. Rapamycin
potently inhibits this serine/threonine kinase, thereby inhibiting
phosphorylation of both 4E-BP1 and p70 S6 kinase. (Very recently, Brunn
et al. (50)) has demonstrated that TOR (target of rapamycin)
may, in fact, directly phosphorylate PHAS-I.) Since expression of
GagPKB stimulated 4E-BP1 phosphorylation and p70 S6 kinase activity,
which were both inhibited by rapamycin in cells expressing LacZ (data
not shown), PKB may activate the common kinase downstream of PI3-K,
phosphorylating both 4E-BP1 and p70 S6 kinase in a TOR-sensitive
fashion; phosphorylation of 4E-BP1 and phosphorylation and activation
of p70 S6 kinase could cause a rise in cap-dependent
mRNA translation and phosphorylation of ribosomal S6 protein,
respectively, leading to increased protein synthesis.
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ACKNOWLEDGEMENTS |
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We thank P. Cohen for anti-PP1G antibody, N. Sonenberg for anti-4E-BP1 antibody, Y. Takai for the construct of
DNRas, X. He for the construct of GSK3, I. Saito for adenovirus
vector, and S. Kakinuma for technical assistance.
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FOOTNOTES |
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* This work was supported by Ministry of Education, Science, Sports, and Culture and Ministry of Health and Welfare of Japan grants (to T. K.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
** To whom correspondence should be addressed: Tel.: +81-3-3815-5411 (ext. 3111); Fax.: +81-3-5689-7209.
1 The abbreviations used are: GS, glycogen synthase; PI3-K, phosphatidylinositol 3-kinase; PKB, protein kinase B, Rac, related to A and protein kinase C; GSK3, glycogen synthase kinase 3; GLUT4, glucose transporter isoform 4; eIF-4E, eukaryotic initiation factor 4E; 4E-BP1, eIF-4E-binding protein 1; DMEM, Dulbecco's modified Eagle's medium; GagPKB, protein kinase B fused to Gag protein; MAPK, mitogen-activated protein kinase; DNRas, dominant negative Ras; PP1G, regulatory subunit of protein phosphatase 1; PDGF, platelet-derived growth factor; p85, the regulatory 85-kDa subunit of phosphatidylinositol 3-kinase; DG, deoxyglucose; HA, hemagglutinin; KRHB, Krebs-Ringer phosphate-HEPES buffer.
2 K. Ueki, R. Yamamoto-Honda, Y. Kaburagi, T. Yamauchi, K. Tobe, B. M. Th. Burgering, P. J. Coffer, I. Komuro, Y. Akanuma, Y. Yazaki, and T. Kadowaki, unpublished data.
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REFERENCES |
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