Temporal activation of p70 S6 kinase and Akt1 by insulin: PI
3-kinase-dependent and -independent mechanisms
Romel
Somwar1,2,
Satoru
Sumitani1,
Celia
Taha1,2,
Gary
Sweeney1, and
Amira
Klip1,2
1 Programme in Cell Biology,
The Hospital for Sick Children and
2 Department of Biochemistry,
University of Toronto, Toronto, Ontario, Canada M5S 1A8
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ABSTRACT |
Several studies have suggested that activation
of p70 ribosomal S6 kinase (p70 S6 kinase) by insulin may be mediated
by the phosphatidylinositol 3-kinase (PI 3-kinase)-Akt pathway.
However, by temporal analysis of the activation of each kinase in L6
muscle cells, we report that the activation of the two serine/threonine kinases (Akt and p70 S6 kinase) can be dissociated. Insulin stimulated p70 S6 kinase in intact cells in two phases. The first phase (5 min) of
stimulation was fully inhibited by wortmannin
(IC50 = 20 nM) and LY-294002 (full
inhibition at 5 µM). After this early inhibition, p70 S6 kinase was
gradually stimulated by insulin in the presence of 100 nM wortmannin.
After 30 min, the stimulation was 65% of the maximum attained in the
absence of wortmannin. The IC50 of
wortmannin for inhibition of this second phase was ~150 nM. In
contrast, activation of Akt1 by insulin was completely inhibited by 100 nM wortmannin at all time points investigated. Inhibition of
mitogen-activated protein kinase/extracellular signal-regulated protein
kinase kinase with PD-098059 (10 µM) or treatment with the protein
kinase C inhibitor bisindolylmaleimide (10 µM) had no effect on the
late phase of insulin stimulation of p70 S6 kinase. We have previously
shown that GLUT-1 protein synthesis in these cells is stimulated by
insulin via the mTOR-p70 S6 kinase pathway, based on its sensitivity to
rapamycin. We therefore investigated whether the signals leading to
GLUT-1 synthesis correlated with the early or late phase of stimulation
of p70 S6 kinase. GLUT-1 synthesis was not inhibited by wortmannin (100 nM). In summary, insulin activates p70 ribosomal S6 kinase in L6 muscle
cells by two mechanisms, one dependent on and one independent of the
activation of PI 3-kinase. In addition, activation of Akt1 is fully
inhibited by wortmannin, suggesting that Akt1 does not participate in
the late activation of p70 S6 kinase. Wortmannin-sensitive PI 3-kinases and Akt1 are not required for insulin stimulation of GLUT-1 protein biosynthesis.
glucose transporters; mTOR; wortmannin; rapamycin
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INTRODUCTION |
THE RIBOSOMAL p70 S6 kinase is a serine/threonine
kinase that is activated in response to many growth factors by
hierarchical phosphorylation of multiple serine and threonine residues
in discrete functional domains (34). In vivo, the kinase controls
protein synthesis by increasing the rate of translation of a family of mRNAs that encode essential components of the protein synthetic machinery (32). In addition, we have recently reported that the
macrolide immunosuppressant rapamycin, which prevents the activation of
p70 S6 kinase, blocks the insulin-induced upregulation of glucose
transporter 1 (GLUT-1) expression (39, 40). The molecular mechanisms
underlying the activation of p70 S6 kinase by insulin and other growth
factors are poorly understood, and some of the intermediate
participants that link growth factor receptors to the phosphorylation
and activation of this serine/threonine kinase remain elusive.
Phosphatidylinositol 3-kinase (PI 3-kinase) is a lipid kinase that
phosphorylates inositol lipids at the D-3 position (7), and several
lines of evidence support the view that it is one of the primary
signaling molecules linking growth factor receptors to the activation
of p70 S6 kinase (8, 10). For example, platelet-derived growth factor
(PDGF) failed to stimulate p70 S6 kinase in Chinese hamster ovary (CHO)
cells overexpressing a mutant PDGF receptor lacking the binding sites
for the p85 regulatory subunit of PI 3-kinase (Y740 and Y751) (10).
Moreover, two structurally different inhibitors of PI 3-kinase,
wortmannin and LY-294002, blocked the activation of p70 S6 kinase by
insulin in 3T3-L1 adipocytes (8) and by PDGF in CHO cells
overexpressing wild-type PDGF receptors (10). On the other hand,
mutation of Y740 in the PDGF receptor completely abolished activation
of PI 3-kinase by PDGF but did not prevent the activation of p70 S6
kinase (27). In addition, insulin can activate p70 S6 kinase in CHO
cells and Rat1 fibroblasts, both overexpressing a dominant
negative p85
subunit of PI 3-kinase, which lacks the binding site
for the p110 catalytic subunit (17). Surprisingly, wortmannin (50 nM)
completely inhibited insulin-induced activation of p70 S6 kinase in
both cell types expressing dominant negative p85
(17). These results suggest that a PI 3-kinase-independent but wortmannin-sensitive pathway
exists for the activation of p70 S6 kinase by insulin.
The molecular mechanism underlying the activation of p70 S6 kinase is
complex, but one current model suggests that a PI 3-kinase-dependent kinase phosphorylates Thr389, one
of the most critical residues for p70 S6 kinase activation (12). Akt is
a serine/threonine kinase that has been demonstrated to be a downstream
target of PI 3-kinase in cells stimulated with insulin (23), PDGF,
epidermal growth factor (EGF), or basic fibroblast growth factor (4,
15), since activation of Akt by these growth factors is blocked by
wortmannin and LY-294002. Activation of Akt was also lost in cells
overexpressing a dominant negative p85
subunit of PI 3-kinase (4).
In addition, Akt could not be activated by PDGF in cells overexpressing
the PDGF-receptor mutants described above (4, 15). On the basis of the
characteristics of the activation of Akt and the observation that
overexpression of a gag fusion of Akt constitutively activates p70 S6
kinase (4), it has been speculated that Akt could be the protein kinase that mediates the PI 3-kinase-dependent phosphorylation of
Thr389 of p70 S6 kinase. On the
other hand, Akt cannot phosphorylate p70 S6 kinase in vitro (33), and a
kinase-deficient mutant of Akt believed to have dominant negative
action was unable to block p70 S6 kinase activation in vivo (22).
Given the controversy mentioned above, we have investigated the time
course of p70 S6 kinase activation vis-à-vis its sensitivity to
inhibition of PI 3-kinase. Surprisingly, two mechanisms of activation
were uncovered, one dependent on and one independent of PI 3-kinase,
based on sensitivity to wortmannin and LY-294002. We further
demonstrate that, in contrast, activation of Akt1 is fully dependent on
PI 3-kinase and can therefore be dissociated from the activation of p70
S6 kinase at longer times.
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MATERIALS AND METHODS |
Materials.
-MEM, FBS, and other tissue culture reagents were purchased from
GIBCO BRL (Burlington, ON, Canada). Human insulin (Humulin) was
obtained from Eli Lilly Canada (Toronto, ON, Canada). Protein A-Sepharose was from Pharmacia (Uppsala, Sweden). Polyclonal
anti-GLUT-1 glucose transporter antiserum was from East Acres
Laboratories (South Bridge, MA). The monoclonal antibody 6H to the
1-subunit of the
Na+-K+-ATPase
was a kind gift from Dr. M. Caplan (Dept. of Cellular and Molecular
Physiology, Yale University). Polyclonal anti-p70 S6 kinase, anti-Akt1,
and anti-IRS-1 antibodies, p70 S6 kinase peptide substrate, and protein
kinases A (PKA) and C (PKC) inhibitor peptides were purchased from
Santa Cruz Biotechnology (Santa Cruz, CA). Akt1 substrate peptide,
Crosstide, was purchased from Upstate Biotechnology (Lake
Placid, NY).
[
-32P]ATP (6,000 Ci/mmol) and enhanced chemiluminescence reagents were purchased from
Amersham (Oakville, ON, Canada). Purified L-
-phosphatidylinositol (PI)
was purchased from Avanti Polar Lipids (Alabaster, AL). Oxalate-treated
TLC silica gel H plates (250 µm) were from Analtech (Newark, DE).
Wortmannin was from Sigma (St. Louis, MO). PD-098059 was a kind gift
from Dr. Alan Saltiel (Parke-Davis, Ann Arbor, MI). LY-294002 and
okadaic acid were from Biomol (Plymouth Meeting, PA). Rapamycin and
bisindolylmaleimide (BIM) were from Calbiochem (La Jolla, CA).
SB-203580 was a generous gift from Dr. John Lee (SmithKline Beecham
Pharmaceuticals, King of Prussia, PA). All electrophoresis and
immunoblotting reagents were purchased from Bio-Rad (Mississauga, ON,
Canada). All other reagents were of the highest analytical grade.
Cell culture and incubations.
A clonal cell line of L6 rat skeletal muscle cells selected for high
fusion potential was grown in
-MEM containing 2% FBS; these cells
were allowed to fuse and differentiate as previously described (29).
Cultures were studied at >90% maximal fusion into fully
differentiated myotubes. For total membrane preparation, L6 myotubes
were incubated in the presence or absence of insulin (100 nM) for a
total of 18 h. Incubation media were replaced every 5 h. Where
indicated, the cells were incubated in the presence of 100 nM
wortmannin or 20 ng/ml rapamycin for the entire period that insulin was
present. For the kinetic analysis of the activation of p70 S6 kinase,
the cells were serum deprived in 0.1% FBS-
-MEM for 12 h and
stimulated with 100 nM insulin for the indicated time. For PI 3-kinase
assay, the serum-deprived cells were stimulated with insulin as
indicated in figure legends. All inhibitors were added 30 min before
insulin stimulation and were present throughout the incubations.
Isolation of total membranes.
Total membranes of L6 myotubes were prepared as previously described
(28). Briefly, cells were rinsed twice with cold homogenization buffer
[250 mM sucrose, 20 mM HEPES (pH 7.4), 5 mM
NaN3, and 2 mM EGTA]. Cell
monolayers were scraped into cold homogenization buffer containing 1 µM leupeptin, 1 µM pepstatin, 10 µM E-64, and 200 µM
phenylmethylsulfonyl fluoride (PMSF), homogenized with 20 strokes
(Dounce type A homogenizer), and then centrifuged at 700 g for 5 min at 4°C. The
supernatant from this low-speed spin was then centrifuged for 1 h at
190,000 g to obtain total membranes. Immunoblotting was carried out as previously described (28).
Preparation of cell extracts for p70 S6 kinase or PI 3-kinase
assays.
For p70 S6 kinase activity, L6 myotubes grown in 10-cm dishes were
washed twice with ice-cold PBS and lysed in 1 ml of
buffer A [50 mM HEPES (pH 7.5),
150 mM NaCl, 20 mM
-glycerophosphate, 10 mM EDTA, 10 mM sodium
pyrophosphate, 100 mM NaF, 1 mM
Na3VO4, and 1% (vol/vol) NP-40] containing a mixture of protease
inhibitors (1 µM leupeptin, 1 µM pepstatin A, 10 µM E-64, and 200 µM PMSF). After 15 min of slow agitation and centrifugation (15,000 g for 15 min), the supernatant was
subjected to immunoprecipitation. For PI 3-kinase activity, cell
extracts were prepared the same way except for using
buffer D [20 mM Tris (pH 7.5),
137 mM NaCl, 1 mM MgCl2, 1 mM
CaCl2, 100 mM
Na3VO4,
1% (vol/vol) NP-40, and 10% (vol/vol) glycerol] and the same
mixture of protease inhibitors.
Immunoprecipitation and assay of p70 S6 kinase activity.
p70 S6 kinase was immunoprecipitated by using 0.5 ml of cell extract
and 1 µg of a rabbit polyclonal p70 S6 kinase antibody. The p70 S6
kinase immunocomplex was washed three times with
buffer B [50 mM Tris acetate (pH
8), 50 mM NaF, 5 mM sodium pyrophosphate, 5 mM
-glycerophosphate, 1 mM
Na3VO4,
1 mM EDTA, 1 mM EGTA, 10 nM okadaic acid, and 0.1% (vol/vol)
-mercaptoethanol], including all the protease inhibitors used
above, and twice with buffer C
[20 mM MOPS (pH 7.2), 25 mM
-glycerophosphate, 5 mM EGTA, 2 mM
EDTA, 20 mM MgCl2, 2 mM
Na3VO4,
and 1 mM dithiothreitol (DTT)]. p70 S6 kinase activity was
assayed essentially as described (24) in a final volume of 50 µl of
buffer C containing 1 µM PKA and PKC
inhibitor peptides, 0.2 mM S6 peptide, and 0.25 mM
Mg-[
-32P]ATP at
30°C for 10 min. Aliquots (30 µl) were transferred onto Whatman p81 filter papers and washed three times for 15 min with 175 mM
phosphoric acid (35). 32P
incorporated into the S6 peptide was measured by liquid scintillation counting. One unit of protein kinase activity corresponds to 1 µM of
32P incorporated into the
substrate peptide under the assay conditions.
Immunoprecipitation and assay of PI 3-kinase activity.
PI 3-kinase activity was measured on IRS-1 immunoprecipitates as
described (41). Immunoprecipitation was performed as described above
but using anti-IRS-1-antibody instead of anti-p70 S6 kinase antibody.
The immunoprecipitates were washed three times with wash buffer 1 [PBS containing
1% (vol/vol) NP-40 and 100 µM
Na3VO4], three times with wash buffer 2 [100 mM Tris (pH 7.5) containing 500 mM LiCl and 100 µM
Na3VO4],
and twice with wash buffer 3 [10 mM Tris (pH 7.5) containing 100 mM NaCl, 1 mM EDTA, and 100 µM Na3VO4].
The pellets were resuspended in 50 µl of 10 mM Tris (pH 7.5), 100 mM
NaCl, 1 mM EDTA, 100 µM
Na3VO4,
10 µl of 100 mM MgCl2, and 10 µl of PI (2 mg/ml) in 10 mM Tris (pH 7.5) and 1 mM EGTA. The reaction
was initiated by the addition of 5 µl of 440 µM ATP containing 10 µCi of [
-32P]ATP.
After 10 min at 30°C, the reaction was terminated by the addition
of 20 µl of 8 M HCl and 180 µl of
CHCl3-methanol (1:1 vol/vol). The
samples were centrifuged for 5 min at maximal speed in a
microcentrifuge, and 50 µl of the lower organic phase were removed
and applied to a potassium oxalate (1%)-pretreated silica gel 60 TLC
plate that had been prebaked for at least 1 h. Lipids were separated by
TLC using
CHCl3-CH3OH-H2O-NH4OH
(60:47:11:3:2) as the running solvent. The detection and quantitation
of [32P]PI3P on the
TLC plates were done using a Molecular Dynamics Phosphorimager System
(Sunnyvale, CA).
Immunoprecipitation and assay of Akt1 protein kinase activity.
Immunoprecipitation of Akt1 and kinase assay were performed as
described (23) with modifications. Anti-Akt1 antibody was precoupled to
a mixture of protein A- and protein G-Sepharose beads by incubating 2 µg of antibody per condition with 20 µl of protein A-Sepharose (100 mg/ml) and 20 µl of protein G-Sepharose (100 mg/ml) for a minimum of
2 h. These anti-Akt1-bead complexes were washed twice with ice-cold PBS
and once with ice-cold lysis buffer. Akt1 was immunoprecipitated by
incubating 200 µg of total cellular protein with the anti-Akt1-bead
complex for 2-3 h under constant rotation (4°C). Akt1
immunocomplex was isolated and washed four times with 1 ml of wash
buffer [25 mM HEPES (pH 7.8), 10% glycerol (vol/vol), 1% Triton
X-100 (vol/vol), 0.1% BSA (vol/vol), 1 M NaCl, 1 mM DTT, 1 mM PMSF, 1 µM microcystin, and 100 nM okadaic acid] and twice with 1 ml
kinase buffer [50 mM Tris · HCl (pH 7.5), 10 mM
MgCl2, and 1 mM DTT]. This
was then incubated under constant agitation for 10 min at 30°C with
30 µl of reaction mixture (kinase buffer containing 5 µM ATP, 2 µCi of [
-32P]ATP,
and 100 µM Crosstide). After the reaction, 30 µl of the supernatant
were transferred onto Whatman p81 filter paper and treated as described
above for p70 S6 kinase assay.
Statistical analysis.
Autoradiograms of X-ray films exposed to produce bands within the
linear range for quantitation were scanned and quantitated by use of
the computer software NIH Image. Statistical analysis was performed
using the ANOVA test (Fisher, multiple comparisons). For analysis of PI
3-kinase, Akt1, and p70 S6 kinase activities, the Student's paired
t-test was applied.
 |
RESULTS |
Time course of p70 S6 kinase activation by insulin and effect of
inhibition of PI 3-kinase.
To understand the relationship between PI 3-kinase and the activation
of p70 S6 kinase, we examined the effect of wortmannin on
insulin-induced activation of the latter. Previous studies advocating
the positioning of PI 3-kinase upstream of p70 S6 kinase measured the
activity of p70 S6 kinase after only a 5-min stimulation of cells with
growth factors (8). To obtain a more detailed description of the
regulation of p70 S6 kinase by insulin, we used an in vitro kinase
assay to analyze the effect of wortmannin treatment on p70 S6 kinase
catalytic activity in L6 skeletal muscle cells. L6 myotubes were
pretreated for 30 min with 100 nM wortmannin before the addition of 100 nM insulin for 5-30 min. p70 S6 kinase was immunoprecipitated, and
the kinase activity associated with the immunocomplex was determined by
using an exogenous S6 peptide as substrate (a 9-amino acid peptide
derived from ribosomal S6 protein). Preliminary experiments confirmed
the linearity of the assay within the time frame used (data not shown).
As shown in Fig.
1A,
insulin increased p70 S6 kinase activity rapidly, reaching a peak in 10 min and remaining elevated for at least 30 min. Although wortmannin
(100 nM) completely abrogated insulin-stimulated p70 S6 kinase activity
at 5 min, insulin treatment gradually stimulated p70 S6 kinase activity
after this time in the continued presence of wortmannin. In two
independent experiments, insulin was not able to fully restore the
magnitude of p70 S6 kinase activity that was observed in the absence of
wortmannin for up to 60 min (data not shown).

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Fig. 1.
Effect of phosophatidylinositol (PI) 3-kinase inhibition on
insulin-stimulated p70 S6 kinase activation. L6 myotubes were
stimulated with 100 nM insulin for indicated time periods in absence
( ) or presence ( ) of 100 nM wortmannin (30-min pretreatment)
(A) or 5 or 30 min in
absence or presence of 100 nM wortmannin (30-min pretreatment) or 5 µM LY-294002 (30-min pretreatment)
(B). Cells were lysed, p70 S6 kinase
was immunoprecipitated, and an in vitro kinase assay was performed with
immunocomplex as described in MATERIALS AND
METHODS. Results are representative of 2 (A) or mean ± SE of 3 (B) independent experiments. In
B, insulin-stimulated p70 S6 kinase
activity at each time point in absence of inhibitors was considered as
100%. * P < 0.05, compared
with 5-min insulin-treated cells.
** P < 0.05, compared with
30-min insulin-treated cells.
# P < 0.05, compared with
corresponding treatment at 5 min.
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We also tested the ability of 5 µM LY-294002, another inhibitor of PI
3-kinase, to inhibit the first (5 min) and second (30 min) phases of
p70 S6 kinase activation by insulin. In cells treated with insulin for
5 min in the presence of 5 µM LY-294002 or 100 nM wortmannin for
comparison, p70 S6 kinase activity was completely inhibited by both
agents (Fig. 1B), confirming the
involvement of PI 3-kinase. However, in cells treated with insulin for
30 min in the continued presence of 5 µM LY-294002 or 100 nM
wortmannin, 34 ± 13 or 65 ± 8%, respectively, of
insulin-stimulated p70 S6 kinase activity was still measurable.
To determine whether PI 3-kinase was inhibited by wortmannin under
these conditions, cells pretreated for 30 min with or without 100 nM
wortmannin were stimulated with insulin for 5 or 30 min. IRS-1-associated PI 3-kinase activity was then determined. Insulin (5 min) stimulated PI 3-kinase activity by 34 ± 6.2-fold above control, and in the presence of wortmannin, insulin-stimulated PI
3-kinase activity was 3.1 ± 1.4-fold above control
(P < 0.05, n = 5). Wortmannin also inhibited PI
3-kinase activity stimulated by 30 min of insulin treatment (insulin:
11.2-fold above control; wortmannin + insulin: 0.9-fold above control;
n = 1).
Dose-dependent effect of wortmannin on p70 S6 kinase
activation.
To confirm the existence of a PI 3-kinase-dependent pathway and a PI
3-kinase-independent pathway leading to the activation of p70 S6
kinase, we examined the dose-dependent inhibition of p70 S6 kinase
activity by wortmannin on the first (5 min) and second (30 min) phases
of activation by insulin. L6 myotubes were treated with 100 nM insulin
for 5 or 30 min in the presence of various concentrations of
wortmannin, and p70 S6 kinase activity was then assayed. In cells that
were treated with insulin for 5 min, wortmannin inhibited
insulin-stimulated p70 S6 kinase activity, with an
IC50 of ~20 nM (Table
1). In contrast, the
IC50 of wortmannin for the
inhibition of the second phase (30 min) of activation of p70 S6 kinase
stimulated was ~150 nM (Table 1). This concentration of wortmannin is
5- to 15-fold higher than that for inhibition of class IA PI 3-kinases
(11, 43). These results suggest that insulin stimulated the activation
of p70 S6 kinase, with two phases having distinct wortmannin
sensitivities.
Time course of Akt1 activation by insulin and inhibition by
wortmannin.
Overexpression of Akt was shown to activate p70 S6 kinase independent
of any stimulus (4). To determine whether Akt could mediate the second
phase (30 min) of insulin-stimulated p70 S6 kinase activation that has
a reduced sensitivity to wortmannin, we utilized an in vitro kinase
assay to investigate the time course of activation of Akt1 by insulin
along with the effect of wortmannin. L6 myotubes were treated for
5-30 min with 100 nM insulin in the absence or presence of 100 nM
wortmannin. Akt1 was immunoprecipitated, and the kinase activity
associated with the immunocomplex was determined by using an exogenous
substrate (Crosstide, a peptide derived from region of GSK-3
phosphorylated by Akt). Insulin rapidly stimulated Akt1 activity within
5 min (Fig. 2), and this level of
stimulation remained elevated throughout the entire period investigated. Maximal activation was achieved between 10 and 20 min.
Wortmannin pretreatment completely prevented the activation of Akt1 by
insulin at all time points investigated (Fig. 2), unlike the results
obtained for p70 S6 kinase (Fig. 1). Inhibition of insulin-stimulated
Akt1 activity by wortmannin is consistent with Akt1 being a downstream
target of PI 3-kinase. Because activation of Akt1 by insulin was
prevented by wortmannin, we conclude that Akt1 does not play a role in
the second phase (30 min) of activation of p70 S6 kinase.

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Fig. 2.
Effect of wortmannin on insulin-stimulated Akt1 activation. L6 myotubes
were stimulated with 100 nM insulin for indicated time periods in
absence ( ) or presence ( ) of 100 nM wortmannin (30-min
pretreatment). Akt1 was immunoprecipitated from total cell lysates and
an in vitro kinase assay performed as described in
MATERIALS AND METHODS. Results are
mean ± SE of 3-6 independent experiments.
* Significantly different from corresponding time point in
presence of wortmannin, P < 0.05.
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Analysis of second phase of p70 S6 kinase activation by insulin.
The observation that insulin can activate p70 S6 kinase by a PI
3-kinase-independent pathway prompted us to examine other signaling
pathway(s) that may mediate this activation of p70 S6 kinase. L6
myotubes were treated for 30 min with 100 nM insulin in the absence or
presence of inhibitors of two insulin-stimulated kinases that have been
implicated in the regulation of p70 S6 kinase: mitogen-activated
protein kinase (MAPK) (14) and PKC (38). The results are shown in Table
2. As expected, rapamycin completely
abrogated insulin-stimulated p70 S6 kinase activity (1 ± 0.1% of control, n = 3). In
concert with the results in Fig. 1A,
wortmannin only partially inhibited the stimulation of p70 S6 kinase by
insulin (allowing 65 ± 8% of maximal stimulation, n = 3). BIM (10 µM), an inhibitor of
all PKC isoforms at this concentration (26), had no effect on
insulin-stimulated p70 S6 kinase activity (Table 2; 106 ± 17% of
control, n = 3). This suggests that
insulin does not utilize the PKC pathway to stimulate p70 S6 kinase.
Similarly, the MAPK/extracellular signal-regulated protein kinase
kinase (MEK) inhibitor PD-098059 (10 µM) had no effect on p70 S6
kinase activity (91 ± 5% of control). p38 MAPK, a stress-activated
MAPK, has been shown to be activated by insulin in 3T3-L1 adipocytes
and fibroblasts (6), and its phosphorylation is stimulated by insulin
in L6 cells (40, 42). The specific inhibitor of p38 MAPK, SB-203580
(25), had no inhibitory effect on insulin-stimulated p70 S6 kinase
activity (133 ± 44% of control, n = 3), indicating that p38 MAPK is not necessary for the regulation of
p70 S6 kinase by insulin.
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Table 2.
Effect of inhibition of several kinases on stimulation of p70 S6
kinase activity by 30-min insulin stimulation
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Wortmannin does not inhibit insulin-stimulated biosynthesis of
GLUT-1.
We have previously demonstrated that insulin-induced synthesis of
GLUT-1 protein is dependent on the mTOR-p70 S6 kinase signaling pathway
in L6 myotubes (39, 40). We therefore investigated which of the two
phases of activation of p70 S6 kinase by insulin participates in this
end-point response. L6 myotubes were treated with or without 100 nM
insulin in the absence or presence of 100 nM wortmannin or 20 ng/ml
rapamycin for a total of 18 h. To ensure the stability and
bioavailability of wortmannin during the 18-h incubation period, we
replaced the incubation medium with medium containing freshly added
wortmannin every 5 h. Total membranes were then prepared as described
under MATERIALS AND METHODS. As shown
in Fig. 3A
and quantified in Fig. 3B, insulin
elicited a significant increase in GLUT-1 protein immunoreactivity
above control (1.8 ± 0.6-fold, n = 3, P < 0.05). Consistent with our previous report (39), this increase was completely abolished by 20 ng/ml rapamycin (data not shown). Here we show that, in the presence of
wortmannin, insulin still elicited a significant increase in GLUT-1
protein content above control (1.7 ± 0.2-fold, n = 3, P < 0.05). In the absence of
insulin, the levels of GLUT-1 were not altered by either wortmannin or
rapamycin treatment. After a 5-h pretreatment of L6 myotubes with 100 nM wortmannin, a 5-min insulin challenge was unable to stimulate PI
3-kinase activity associated with IRS-1 immunoprecipitates (insulin:
6.5-fold above control; insulin + wortmannin: 0.9-fold above control). This underscores that wortmannin was still capable of completely inhibiting PI 3-kinase activity 5 h after its addition to cells. Taken
together, these results suggest that wortmannin-sensitive PI 3-kinases
are not essential for insulin stimulation of GLUT-1 protein synthesis
in L6 skeletal muscle cells.

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Fig. 3.
Effect of wortmannin on insulin-stimulated GLUT-1 protein synthesis.
Total membranes were prepared as described in
MATERIALS AND METHODS from L6 myotubes
that were untreated (B) or treated for a total of 18 h with 100 nM
insulin (I) or treated with 100 nM wortmannin in absence (W) or
presence (WI) of 100 nM insulin. Membrane protein (50 µg) was
resolved by 10% SDS-PAGE, transferred onto PVDF membranes, and
immunoblotted with either
anti- 1-subunit of the
Na+-K+-ATPase (1:1,000,
A,
top) to ensure equality of loading
or anti-GLUT-1 (1:1,000, A,
bottom) antiserum. Immunoreactive
bands were detected using enhanced chemiluminesence method.
A: representative immunoblots;
B: means ± SE of 3 independent
experiments quantitated using software NIH Image.
* P < 0.05, compared
with basal cells. # P < 0.05, compared with wortmannin-treated control.
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 |
DISCUSSION |
Two pathways leading to p70 S6 kinase activation.
Here we show that activation of p70 S6 kinase by insulin in muscle
cells occurs in two phases, distinguishable by their different sensitivity to inhibitors of PI 3-kinase. The
IC50 of inhibition of the first
phase by wortmannin (20 nM) is consistent with that for inhibition of
class IA PI 3-kinases (11, 44). In vitro, the
IC50 of wortmannin for the
inhibition of p85/p110 PI 3-kinases is 2 nM (36, 43) and in intact
cells is 10-30 nM (43). On the other hand, the higher
IC50 value of wortmannin for
inhibition of the second phase (150 nM) suggests that class IA PI
3-kinases are not involved and that wortmannin targets another upstream activator, possibly mTOR. This notion is supported by two recent reports that demonstrate that wortmannin irreversibly inhibits the
serine-specific autokinase activity (3) and protein kinase activity (2)
of mTOR with IC50 of ~200 nM.
The higher sensitivity of the second phase of p70 S6 kinase activation
to LY-294002 than to wortmannin also supports this hypothesis, since
Brunn et al. (3) demonstrated that the
IC50 of LY-294002 for inhibition of both mTOR and PI 3-kinase is 5 µM. The yeast and mammalian TOR
proteins are now recognized as members of a growing family of
high-molecular-weight kinases whose catalytic domains resemble those of
PI 3-kinases (19) and are necessary for the activation of p70 S6 kinase
by all known stimuli, including insulin (9, 32, 34). Although mTOR
protein is upregulated as 3T3-L1 fibroblasts differentiate into the
more insulin-responsive adipocytes (46), it is unknown how insulin
regulates the kinase activity of this protein.
The protein kinase activity of mTOR is stimulated by serum in vivo, and
this precedes the activation of p70 S6 kinase (5). Full activation of
p70 S6 kinase by growth factors is achieved with phosphorylation of
three critical sites (Thr229,
Thr389, and
Ser404) (16, 31), and
phosphorylation of these sites is prevented by both wortmannin and
rapamycin (16, 31). The high sensitivity of
Thr389 phosphorylation to
wortmannin suggests that this site is phosphorylated by an as yet
unidentified PI 3-kinase-dependent kinase (12, 33). On the other hand,
the high sensitivity of Thr389
phosphorylation to rapamycin may be explained by the recent observation that mTOR phosphorylates this site in vitro. Taken together, these observations suggest that Thr389
could be phosphorylated by two different kinases that may be able to
functionally substitute for each other. We therefore propose a model in
which insulin stimulates p70 S6 kinase via two pathways. The first is
rapid, completely dependent on PI 3-kinase and mTOR. In the absence of
functional class IA PI 3-kinases (i.e., in presence of 100 nM
wortmannin), mTOR may unilaterally mediate the phosphorylation of
Thr389, leading to the second
phase activation of p70 S6 kinase, which is the PI 3-kinase-independent
pathway. Alternatively, the second phase of activation of p70 S6 kinase
could be mediated by PI 3-kinases that are less sensitive to
wortmannin, such as the class II PI 3-kinases (11, 44). It is unknown
at present whether any of these PI 3-kinases are activated by insulin.
Analysis of second phase of activation of p70 S6 kinase.
To further understand the stimulation of p70 S6 kinase by insulin, we
sought to define the signaling mechanism(s) of the PI 3-kinase-independent pathway. We utilized specific pharmacological inhibitors that act on kinases known to be stimulated by insulin and
implicated in the regulation of p70 S6 kinase. EGF stimulates p70 S6
kinase in Swiss 3T3 fibroblasts in a biphasic manner (38), with the
second phase requiring conventional isoforms of PKC (cPKC) (37, 38).
However, PDGF and insulin stimulate p70 S6 kinase in the same cells in
a cPKC-independent manner (38). Here, by utilizing BIM at a
concentration known to inhibit all isoforms of PKC (26), we demonstrate
that none of the known PKC isoforms is involved in the second, PI
3-kinase-independent phase of insulin-stimulated p70 S6 kinase
activation. MAPK was originally thought to be the proline-directed
kinase that phosphorylates the COOH-terminal autoinhibitory region of
p70 S6 kinase (30). However, this remains debatable, since other
studies have reported results to the contrary (27). Inhibition of the
MAPK pathway with the MEK inhibitor, PD-098059 (1), had no effect on
p70 S6 kinase activation by insulin (30-min stimulation). It has also
been suggested that the stress-activated MAPK, p38 MAPK, mediates the
activation of p70 S6 kinase by sodium arsenite in cardiomyocytes (45).
Because this kinase is also activated by insulin (6, 40, 42), we tested
its involvement in insulin-stimulated p70 S6 kinase activity. The
specific p38 MAPK inhibitor, SB-203580 (25), had no effect on
insulin-induced p70 S6 kinase activity (30-min stimulation).
Role of Akt1 in activation of p70 S6 kinase.
Akt has recently been identified as a direct downstream substrate of PI
3-kinase in many growth factor signaling pathways (18). However, Akt
could be activated by basic fibroblast growth factor without prior
activation of PI 3-kinase (21), whereas overexpression of Akt in cells
suggested that it had a role in the activation of p70 S6 kinase (4). To
determine whether the kinetics of activation of Akt matched those of
the activation of p70 S6 kinase, we first looked at the effect of
wortmannin on the activation of Akt1 by insulin. Activation of Akt1 was
fully inhibited by 100 nM wortmannin under conditions in which only 35% of insulin-stimulated p70 S6 kinase was inhibited. Thus Akt1 activation by insulin could not support the latent activation of p70 S6
kinase in the face of complete inhibition of PI 3-kinase. Together with
the results presented here, this suggests that if Akt1 is involved in
the regulation of p70 S6 kinase, it can mediate only the first phase of
p70 S6 kinase activation.
Akt2 and Akt3 are two additional isoforms of Akt that have been
suggested to be downstream of PI 3-kinase (13). No detailed time course
showing activation of these two isoforms has been reported. Hence, we
do not know whether the kinetics of Akt2 and Akt3 activation match
those for activation of p70 S6 kinase. More detailed studies of Akt2
and Akt3 are required before we can assign a role for either one in the
regulation of p70 S6 kinase. However, we can speculate that these two
isoforms may participate in the first phase of activation of p70 S6
kinase because their activation is PI 3-kinase dependent.
GLUT-1 protein synthesis occurs via second phase of p70 S6 kinase
activation.
Finally, we demonstrate that wortmannin was unable to block
insulin-stimulated GLUT-1 protein synthesis in L6 muscle cells, whereas
rapamycin did. It is unlikely that degradation of wortmannin during the
incubation caused reactivation of PI 3-kinase, since repetitive
addition of wortmannin every 5 h was still effective in preventing the
rapid stimulation of PI 3-kinase by insulin. A similar protocol for
prolonged treatment with wortmannin was shown to inhibit
differentiation of L6E9 cells (20). Wortmannin covalently modifies
Lys802 in the lipid kinase domain
of the p110 catalytic subunit, resulting in the irreversible inhibition
of PI 3-kinase activity (47). Thus PI 3-kinase is not required for
insulin to upregulate GLUT-1 protein, and it is likely that the second
phase of activation of p70 S6 kinase is sufficient to upregulate GLUT-1
protein content.
In summary, the activation of p70 S6 kinase by insulin in L6 skeletal
muscle cells is composed of two separable components: an early phase
dependent on class IA PI 3-kinase and possibly Akt1, and a second phase
independent of class IA PI 3-kinases (Fig.
4). The latter phase does not involve PKC,
MEK, or p38 MAPK. In addition, insulin upregulates GLUT-1 protein
despite complete inhibition of PI 3-kinase and Akt1 activation with
wortmannin. Because the second phase of activation of p70 S6 kinase
remains sensitive to higher levels of wortmannin, it is possible that PI 3-kinase-related mTOR or another PI 3-kinase enzyme is involved in
the maintenance of insulin-stimulated p70 S6 kinase activity and GLUT-1
protein biosynthesis.

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|
Fig. 4.
Schematic model of regulation of p70 S6 kinase and GLUT-1 protein
synthesis by insulin. Insulin stimulates p70 S6 kinase via 2 pathways,
one sensitive to inhibitors of PI 3-kinase (early phase, indicated by
solid arrows) and the other less sensitive to these inhibitors (late
phase, indicated by dashed arrows) at concentrations at which they
inhibit class IA PI 3-kinase. Both phases of activation are inhibited
by rapamycin. Late phase is probably sufficient to mediate
insulin-induced synthesis of GLUT-1, since this process is inhibited by
rapamycin but not wortmannin.
|
|
 |
ACKNOWLEDGEMENTS |
We thank Dr. Philip J. Bilan for critical reading of this paper.
 |
FOOTNOTES |
We are grateful to Dr. A. R. Saltiel for the supply of PD-098059,
Dr. J. C. Lee for the supply of SB-203580, Dr. M. Caplan for
the anti-Na+-K+-ATPase
1-antibody, and Dr. R. Roth for
advice on the Akt activity assay.
This work was supported by a grant from the Canadian Diabetes
Association to A. Klip. R. Somwar and S. Sumitani contributed equally
to the present work.
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. §1734 solely to indicate this fact.
Address for reprint requests: A. Klip, Programme in Cell Biology, The
Hospital for Sick Children, 555 University Ave., Toronto, ON, Canada
M5G 1X8.
Received 21 April 1998; accepted in final form 23 June 1998.
 |
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