From the Biosignal Research Center, Kobe University,
Kobe 657-8501, Japan and the § Diabetes Unit and Medical
Services, Massachusetts General Hospital and Harvard Medical School,
Boston, Massachusetts 02129
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ABSTRACT |
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Amino acid deprivation of Chinese hamster ovary
cells overexpressing human insulin receptors results in deactivation of
p70 S6 kinase (p70) and dephosphorylation of eukaryotic initiation factor 4E-binding protein 1 (4E-BP1), which become unresponsive to
insulin; readdition of amino acids restores these responses in a
rapamycin-sensitive manner, suggesting that amino acids and mammalian
target of rapamycin signal through common effectors. Contrarily,
withdrawal of medium amino acids from the hepatoma cell line H4IIE does
not abolish the ability of insulin to stimulate p70 and 4E-BP1. The
addition of 3-methyladenine (3MA) to H4IIE cells deprived of amino
acids inhibited the increment in protein degradation caused by amino
acid withdrawal nearly completely at 10 mM and also
strongly inhibited the ability of insulin to stimulate p70 and 4E-BP1
at 10 mM. Treatment of H4IIE cells with 3MA did not alter
the ability of insulin to activate tyrosine phosphorylation,
phosphoinositide 3-kinase, or mitogen-activated protein kinase. In
conclusion, the ability of H4IIE cells to maintain the insulin
responsiveness of the mammalian target of
rapamycin-dependent signaling pathways impinging on p70 and
4E-BP1 without exogenous amino acids reflects the generation of amino
acids endogenously through a 3MA-sensitive process, presumably
autophagy, a major mechanism of facultative protein degradation in liver.
Considerable progress has been achieved recently in the
understanding of the signal transduction pathways involved in
translational control. In addition to the many eukaryotic initiation
factors (eIFs)1 that are
regulated by phosphorylation, two translational regulatory proteins,
i.e. p70 S6 kinase (p70) and PHAS1/eIF-4E-binding protein 1 (4E-BP1), have been extensively studied as targets of growth factor-regulated signaling pathways (for review, see Refs. 1 and 2).
p70 is activated in response to insulin and mitogens through a
multisite phosphorylation and catalyzes the multiple phosphorylation of
40 S ribosomal protein S6 in vivo; this kinase plays a
critical regulatory role in the translation of a class of transcripts
that contain a 5' oligopyrimidine tract at their transcriptional start
site structure (3). 4E-BP1 exhibits rapid and multiple phosphorylation
in vivo in response to insulin and mitogens (2). The
initiation factor eIF-4E binds to the 5' cap structure of mRNA
(m7GpppN, where N is any nucleotide) and through its
binding to the scaffold protein eIF-4G recruits the mRNA to a
complex, known as the eIF-4F (4). 4E-BP1 in the dephosphorylated form
binds to eIF-4E (5) in a manner competitive with eIF-4G, thereby inhibiting translation of a subset of mRNAs (4). The insulin- and
mitogen-stimulated phosphorylation of 4E-BP1 inhibits 4E-BP1 binding to
eIF-4E, making eIF-4E available for incorporation into the eIF-4F
complex via eIF-4G (6) and restoring translation.
At least two distinct signaling pathways underlying activation of p70
and phosphorylation of 4E-BP1 have been identified. One input
controlling p70 and 4E-BP1 is provided by phosphoinositide 3-kinase
(PI3-k), which is recruited to the activated receptor tyrosine kinases
in response to insulin and mitogens. Thus, treatment of cells with
PI3-k inhibitors such as wortmannin inhibits activation of p70 and
phosphorylation of 4E-BP1 in response to insulin and mitogens (7, 8).
Most importantly, 3-phosphoinositide-dependent protein kinase-1,
phosphorylates p70 in vitro selectively at Thr-252, a
PI3-k-stimulated and wortmannin-sensitive phosphorylation site in
vivo (9), with a resultant increase in p70 activity (10, 11).
3-phosphoinositide-dependent protein kinase-1 was first identified as
an activator of protein kinase B (also called Akt and RacPK), another
downstream effector of PI3-k; protein kinase B activation in
vivo activates both p70 and 4E-BP1 indirectly, through an unknown
mechanism (12, 13). Another pathway contributing to activation of p70
and phosphorylation of 4E-BP1 is regulated by mammalian target of
rapamycin proteins (named FRAP, RAFT-1, RAPT-1, or mTOR; for review,
see Refs. 14 and 15). The macrolide immunosuppressant rapamycin is
known to cause a dephosphorylation and deactivation of p70 (for review,
see Refs. 14 and 15), and dephosphorylation of 4E-BP1 (16, 17).
Rapamycin, in complex with the cytosolic 12-kDa FK506-binding protein
(FKBP12), binds to mTOR and inhibits its protein kinase activity
in vitro (18), and recent studies have established that mTOR
is the rapamycin-sensitive upstream regulator of both p70 (18) and
4E-BP1 (19, 20). These two targets, however, are regulated by separate
mTOR-controlled pathways that bifurcate at or downstream of mTOR
(19, 21).
The phosphorylation of ribosomal S6, p70, and 4E-BP1 in vivo
is governed by the availability of amino acids (22-24). Rat
hepatocytes incubated in the absence of amino acids exhibit a decrease
in the phosphorylation of S6 that is rapidly reversed by addition of
amino acids but blocked by rapamycin (22). Withdrawal of amino acids
from the nutrient medium of CHO-IR or HEK293 cells results in a rapid
deactivation of endogenous or recombinant p70 and dephosphorylation of
4E-BP1, which become unresponsive to various agonists, including
insulin and recombinant PI3-k (24). Amino acid readdition restores p70
activity and the phosphorylation of 4E-BP1. The effect of amino acid
repletion on p70 and 4E-BP1 is blocked by rapamycin; moreover, the p70
mutant Cells regulate amino acid pools in part by the degradation of
endogenous proteins; this becomes the only source of amino acids when
exogenous amino acids are unavailable. A major mechanism for degrading
intracellular proteins in response to amino acid deprivation is
autophagy or macroautophagy (for review, see Refs. 25 and 26).
Autophagy is a major source of endogenous amino acids for
gluconeogenesis and other critical pathways early in starvation, and
extracellular amino acids have been shown to be the primary regulators
of autophagy in hepatocytes of perfused liver (27, 28) and isolated
hepatocytes (29). Autophagic proteolysis is enhanced in the absence of
amino acids, and it is suppressed by amino acids at concentrations
equivalent to the upper physiological limit (27, 30). The molecular
mechanisms underlying the control of autophagy by amino acids are
largely unknown. In yeast, autophagy is activated in response to
nutrient insufficiency and appears to be regulated by TOR, the yeast
homolog of mTOR (31).
In view of the prominent role of autophagy in hepatic proteolysis, and
the evidence for TOR regulation of autophagy in yeast, we reasoned that
cultured hepatocytes might provide an attractive system in which to
study the role of amino acids and mTOR signaling pathways in the
regulation of translation and autophagy. H4IIE cells, a cultured
hepatoma cell line, have been used to study the molecular mechanism of
p70 activation by various agonists, including insulin (32). In H4IIE
cells, in contrast to our previous finding in CHO-IR cells or HEK293
cells, withdrawal of amino acids from the nutrient medium did not
eradicate the ability of insulin to stimulate p70 activity and 4E-BP1
phosphorylation. The addition of 3-methyladenine (3MA), an inhibitor of
autophagy (33, 34), strongly and selectively inhibited the insulin
activation of p70 in amino acid-deprived H4IIE cells but inhibited p70
activity and 4E-BP1 phosphorylation only slightly in amino acid-replete cells. Thus, in cultured hepatoma cells deprived of exogenous amino
acids, autophagy serves to supply amino acids from endogenous proteins
in amounts sufficient to enable a full response of the p70 and 4E-BP1
to insulin.
Materials--
Dulbecco's modified Eagle's medium (DMEM),
minimal essential medium Antibodies--
The polyclonal antiserum against the C-terminal
104 amino acids of p70 used for immunoprecipitation of endogenous p70
and the polyclonal antibody against 4E-BP1 were described previously (9, 19). The anti-eIF-4E antibody and the anti-phosphotyrosine antibody
(PY20) were purchased from Transduction Laboratories. The
phospho-specific (Thr-202/Tyr-204) and the non-phospho-specific antibodies against p44/42 mitogen-activated protein kinase (MAPK) were
from New England Biolabs.
Cell Culture and Treatments--
Rat hepatoma H4IIE cells were
grown in minimal essential medium Immunoprecipitation and p70 S6 Kinase Assays--
p70 activity
was determined by immunocomplex assay using 40 S ribosomal subunits as
substrates. Immunoprecipitation of p70 and the kinase assay conditions
were as described (9, 19). Assay samples containing 32P-S6
were separated by SDS-PAGE on a 12% acrylamide gel, and radioactivity was quantified with a BAS-2000 Bioimaging analyzer (Fuji).
Immunoblot Analysis and 4E-BP1 Assay--
Aliquots of the
supernatants of cell extracts were heated for 7 min at 90 °C, cooled
on ice, and recentrifuged. The heat-soluble proteins were separated by
SDS-PAGE on 15% polyacrylamide gels, transferred onto a polyvinylidene
difluoride membrane, immunoblotted with the polyclonal antibody against
4E-BP1 (1:500 dilution), and visualized using the ABC kit according to
the manufacturer's protocol (Vectastain).
4E-BP1 Binding Assay to eIF-4E--
Cell extracts were incubated
with m7GTP-Sepharose, and bound proteins were eluted in 20 mM Tris-HCl, pH 7.5, 100 mM KCl, 2 mM dithiothreitol, 2 mM EDTA, mixed with
SDS-sample buffer, and heated for 7 min at 90 °C. Samples were
separated by SDS-PAGE on 15% polyacrylamide gels, transferred onto a
polyvinylidene difluoride membrane, and immunoblotted with the
polyclonal antibody against 4E-BP1 (1:500 dilution) or the anti-eIF-4E
antibody (1:2000 dilution).
Autophagy Assay--
Autophagic activity was determined by
measurement of the degradation of long-lived proteins as described
previously (35) with a slight modification. H4IIE cells were incubated
for 6 h at 37 °C with serum-free minimal essential medium PI3-kinase Assay--
Aliquots of cell extracts lysed in
ice-cold buffer A containing 1 mM vanadate were subjected
to immunoprecipitation with the anti-phosphotyrosine antibody. PI3-k
activity in the immunoprecipitates was determined as described
previously (36).
The Ability of Insulin to Stimulate p70 Activity and 4E-BP1
Phosphorylation in H4IIE Cells Is Maintained Despite Amino Acid
Withdrawal--
As reported previously using CHO-IR and HEK293 cells
(24), we observed in H4IIE cells that brief (2-h) withdrawal of amino acids from serum-deprived culture medium diminished substantially the
activity of endogenous p70, and readdition of amino acids activated p70
activity (Fig. 1A, lanes
1 and 6). In the previous study in CHO-IR and HEK293
cells, withdrawal of amino acids from the culture medium inhibited
almost completely the ability of insulin to stimulate p70 activity
(24). By contrast, insulin activation of p70 in H4IIE cells was robust
despite the withdrawal of exogenous amino acids (Fig. 1A,
lane 2). The ability of insulin to stimulate p70 activity in
amino acid-deprived H4IIE cells was still maintained when the duration
of amino acid withdrawal was increased from 2 to 6 h (data not
shown). This pattern is similar to that exhibited when amino
acid-deprived CHO-IR cells are treated with very low levels of
exogenous amino acids. Readdition of amino acids to CHO-IR cells at
10-12% the level usually found in DMEM does not increase basal p70
activity but largely restores the responsiveness of p70 to insulin
(24).
Previous work showed that amino acid deprivation in CHO-IR and HEK293
cells abolished the insulin-stimulated phosphorylation of 4E-BP1 (24).
The effect of amino acid deprivation on 4E-BP1 and its association with
eIF-4E in H4IIE cells was therefore examined. 4E-BP1 binds to eIF-4E;
previous work has established that the insulin- and mitogen-stimulated
phosphorylation of 4E-BP1 inhibits its binding to eIF-4E (5), whereas
rapamycin-induced dephosphorylation of 4E-BP1 enhances its binding to
eIF-4E. Phosphorylation states of 4E-BP1 in H4IIE cell extracts were
monitored indirectly by shifts in the migration of 4E-BP1 polypeptides
on SDS-PAGE and by estimation of the amount of 4E-BP1 complexed with
eIF-4E, the latter isolated using m7GTP-Sepharose. H4IIE
cells deprived of amino acids for 2 h exhibited a predominance of
rapidly migrating bands (Fig.
2A, lane 1) and considerable binding of 4E-BP1 to eIF-4E (Fig. 2B,
lane 1). Readdition of amino acids diminished the abundance
of the more rapidly migrating bands, increased the abundance of the
slowest migrating, hyperphosphorylated bands (Fig. 2A,
compare lane 5 with lane 1), and decreased the amount of 4E-BP1 recovered in association with eIF-4E (Fig.
2B, compare lane 5 with lane 1).
Addition of insulin in the absence of amino acids also caused an
upshift in the mobility of 4E-BP1 (Fig. 2A, lane
2) and the decrease of 4E-BP1 complexed with eIF-4E (Fig.
2B, lane 2). Thus, in H4IIE cells, withdrawal of
exogenous amino acids promotes 4E-BP1 dephosphorylation but does not
abolish insulin stimulation of 4E-BP1 phosphorylation.
Effect of Wortmannin and Rapamycin on Insulin- and Amino
Acid-induced p70 Activation and 4E-BP1 Phosphorylation in H4IIE
Cells--
The ability of insulin to maintain p70 activity in amino
acid-deprived H4IIE cells enabled us to examine whether the upstream inputs required for the insulin-induced increase in p70 activity are
also necessary for the increase in p70 activity caused by amino acid
repletion. H4IIE cells were subjected to amino acid withdrawal for
2 h, and they were pretreated with wortmannin, rapamycin (each for
30 min), or PD98059 (for 1 h) before stimulation with insulin
(Fig. 1A, lanes 3-5) or during readdition of
amino acids (Fig. 1A, lanes 7-9). As expected,
insulin activation of p70 was strongly inhibited by wortmannin and
rapamycin but minimally altered by the mitogen-activated protein kinase
kinase-1 inhibitor PD98059. The increase in p70 activity induced by
amino acid readdition exhibited a sensitivity to inhibition by
rapamycin indistinguishable from that shown by p70 activated in
response to insulin (Fig. 1, A, compare lane 8 with lane 4, and C); however, the activation of
p70 by amino acids was considerably more resistant to inhibition by
wortmannin than was the activation induced by insulin (Fig. 1,
A, compare lane 7 with lane 3, and
B). Inhibition of the insulin-activated p70 by wortmannin
was complete and maximal at 30 nM (Fig. 1B), whereas much higher concentrations of wortmannin failed to inhibit amino acid-induced p70 activation to the extent achieved by much lower
concentrations of wortmannin acting on insulin-stimulated cells (Fig.
1B).
With regard to 4E-BP1, pretreatment of H4IIE cells with 100 ng/ml
rapamycin inhibited the ability of both insulin and amino acids to
cause the upshift of 4E-BP1 (Fig. 2A, lanes 4 and
7) and the decrease of 4E-BP1 binding to eIF-4E (Fig.
2B, lanes 4 and 7). Pretreatment with
100 nM wortmannin before insulin stimulation resulted in a
maximal downshift of 4E-BP1 mobility and increment in eIF-4E binding
(Fig. 2, A, lane 3, and B, lane
3). However, as with p70, the ability of amino acids to promote
the phosphorylation of 4E-BP1 was much less sensitive to inhibition by
wortmannin than was the insulin-stimulated 4E-BP1 phosphorylation (Fig.
2, A, lane 6, and B, lane
6).
Taken together, these data show that in H4IIE cells after amino acid
withdrawal, as in amino acid-replete cells, both rapamycin- and
wortmannin-sensitive signaling pathways are required for the insulin
stimulation of p70 activity and 4E-BP1 phosphorylation. Because mTOR is
the rapamycin-sensitive molecule in both circumstances, a conclusion
supported by the observation is that inhibition of mTOR in
vivo by rapamycin abolishes the response to amino acids and
insulin with a parallel sensitivity and to a comparable extent. By
contrast, the much lower potency of wortmannin in inhibiting the
response of p70 and 4E-BP1 to amino acids compared with insulin could
reflect the requirement for a lesser input from PI3-k in the response
to amino acids compared with insulin. Alternatively, the ability of
wortmannin to inhibit the response to amino acids may not reflect
wortmannin inhibition of PI3-k but the ability of the higher
concentrations of wortmannin to inhibit mTOR (8).
Amino Acid Release by Autophagy--
The sustained responsiveness
of p70 and 4E-BP1 to insulin in H4IIE cells after amino acid
withdrawal, mimicking the behavior of CHO-IR cells incubated in the
presence of the low concentrations of exogenous amino acids (24),
suggested that H4IIE cells might be better able than are CHO-IR cells
to generate amino acids from endogenous sources during the amino acid deprivation.
Autophagy is a ubiquitous mechanism for the degradation of endogenous
proteins and is especially active in liver and isolated hepatocytes
(28-30). Amino acid deprivation is known to enhance and probably
initiate this process. We assessed the effects of amino acid
sufficiency on the degradation of endogenous H4IIE proteins by labeling
cells with [14C]valine for 16 h. After washing in
phosphate-buffered saline to remove the bulk of unincorporated
[14C]valine, the cells were transferred to the medium
containing or lacking amino acids, and the degradation of long-lived
proteins was determined by the release of TCA-soluble
[14C]valine. Approximately 3-4% of total
[14C]valine was released per hour during 2 h of
incubation in the presence of exogenous amino acids;
[14C]valine release was ~50% higher when H4IIE cells
were incubated in medium lacking amino acids (Fig.
3, A and B, compare
lane 1 with lane 2). Moreover, 3MA, a widely used
inhibitor of autophagy (33, 34) inhibited [14C]valine
release from amino acid-deprived H4IIE cells to an extent similar to
that achieved by amino acid readdition (Fig. 3B, compare lane 3 with lane 2). Addition of 3MA in the
presence of exogenous amino acids did not decrease the rate of
[14C]valine release beyond the lower rate induced by the
readdition of amino acids per se (data not shown). These findings are
consistent with earlier reports and support the conclusion that
autophagy is the major mechanism for the facultative increase in
endogenous protein degradation in H4IIE cells in response to amino acid
withdrawal and can be inhibited by amino acid readdition or 3MA.
Effects of 3MA on the Ability of Insulin to Stimulate p70 Activity
and 4E-BP1 Phosphorylation in H4IIE Cells after Amino Acid
Withdrawal--
Reasoning that any effects of 3MA that are abrogated
by amino acid repletion can be attributed to the ability of 3MA to
inhibit autophagy, we next examined the effect of 3MA on insulin
activation of p70 and 4E-BP1 phosphorylation in both the presence and
absence of exogenous amino acids. H4IIE cells were serum-deprived for 24 h, followed by incubation for an additional 2 h in DMEM or DMEM lacking amino acids, in the absence or presence of 10 mM 3MA. 100 nM insulin was then added to some
incubations, and the cells were harvested 10 min thereafter. In H4IIE
cells incubated in the absence of exogenous amino acids, the ability of
insulin to activate p70 was inhibited ~90% by 3MA (Fig.
4A, compare lane 3 with lane 4). By contrast, the insulin activation of p70 in amino acid-replete cells was inhibited only modestly (~30%
inhibition) by the same concentration of 3MA (Fig. 4A,
compare lane 5 with lane 6). We also examined
dose-dependent effects of 3MA on insulin- and amino
acid-induced activation of p70 during incubation in the culture medium
lacking exogenous amino acids. The inhibition of insulin activation of
p70 by 3MA was observed in a dose-dependent manner (Fig.
4B). By contrast, 3MA barely inhibited activation of p70 by
amino acids (Fig. 4B), suggesting that the inhibitory effect
of 3MA on insulin activation of p70 in amino acid-deprived cells is not
attributable to the direct inhibition of p70 activity by 3MA in
situ.
With regard to 4E-BP1 phosphorylation, the ability of insulin to
upshift the mobility of 4E-BP1 (Fig.
5A, lane 3) and
decrease the amount of 4E-BP1 complexed with eIF-4E (Fig.
5B, lane 3) in amino acid-deprived cells was
completely inhibited by 10 mM 3MA (Fig. 5, A,
lane 4, and B, lane 4), whereas the
inhibitory effect of 3MA on 4E-BP1 phosphorylation in amino
acid-replete H4IIE cells was greatly attenuated; in amino acid-replete
cells, considerable 4E-BP1 persisted in the most slowly migrating band,
and the amount of 4E-BP1 complexed with eIF-4E greatly diminished
despite the presence of 10 mM 3MA (Fig. 5, A and
B, compare lanes 4 and 6). Thus, 3MA
inhibits insulin-induced p70 activation and 4E-BP1 phosphorylation to a
much greater extent in amino acid-deprived cells than in amino
acid-replete cells, consistent with the conclusion that in H4IIE cells
incubated without exogenous amino acids, amino acids derived from
autophagic proteolysis are critical to maintain the responsiveness of
p70 and 4E-BP1 to insulin.
3MA Does Not Inhibit Insulin Activation of Insulin Receptor Kinase,
PI3-k, or MAPK--
We examined effects of 3MA on several steps of
insulin signaling upstream of p70 and 4E-BP1. Treatment of H4IIE cells
with 3MA had no significant inhibitory effects on insulin-stimulated receptor tyrosine phosphorylation and tyrosine phosphorylation of
endogenous insulin receptor substrate proteins (Fig.
6A, lanes 3, 4,
7, and 8) or on the insulin-stimulated increase
in PI3-k immunoprecipitated by anti-phosphotyrosine antibodies (Fig.
6B, lanes 3, 4, 7, and 8),
either in the presence or absence of exogenous amino acids. The
insulin-stimulated phosphorylation at activating sites (TEY) on the
endogenous p44/42 MAPK, monitored by immunoblotting with the
phosphopeptide antibody against p44/42 MAPK, was not significantly
altered by 3MA (Fig. 6C, lanes 3, 4,
7, and 8). Thus, the inhibitory effects of 3MA on
insulin stimulation of p70 and 4E-BP1 in amino acid-deprived H4IIE
cells reflect the inhibition of a signal transduction response clearly
distinguished from those controlled directly by insulin. Nevertheless,
the provision of endogenous amino acids through autophagy is
indispensable to the ability of insulin to effectively increase the
phosphorylation of p70 and 4E-BP1 in amino acid-deprived cells. When
amino acids are supplied exogenously, however, autophagy is largely
dispensable for insulin signaling to p70 and 4E-BP1.
Conclusion--
Previous work in several cell lines had shown that
removal of amino acids from the nutrient medium blocked selectively the ability of insulin to regulate the phosphorylation of p70 and 4E-BP1
(24, 37). The present study was undertaken to explore the mechanism
that enabled H4IIE hepatoma cells deprived of amino acids to maintain
the responsiveness of p70 and 4E-BP1 to insulin. The resistance of
hepatoma cells to amino acid withdrawal was also observed by Patti
et al. (38), who reported that insulin was able to activate
p70 activity and 4E-BP1 phosphorylation in FAO hepatoma cells after the
complete omission of amino acids from the nutrient medium. The
similarity in the responses to insulin shown by H4IIE cells deprived
completely of exogenous amino acids and CHO-IR cells replete with low
concentrations of amino acids (<0.25 of concentrations usually present
in DMEM) led us to inquire as to whether H4IIE cells had a means to
generate amino acids from endogenous sources.
Inasmuch as autophagy offered an attractive candidate mechanism for the
provision of endogenous amino acids, we examined the effect of 3MA, a
relatively potent, widely used inhibitor of autophagy (33, 34), on the
ability of insulin to regulate p70 and 4E-BP1 in the presence and
absence of exogenous amino acids in H4IIE cells. Omission of amino
acids from the medium increased the rate of endogenous proteolysis
([14C]valine release) presumably by autophagy, resulting
in the release of amino acids into the nutrient medium (Fig.
3A). This increment in proteolysis was inhibited by 3-MA
(Fig. 3B). The inhibition of autophagy in amino
acid-deprived H4IIE cells by 3MA was accompanied by a dephosphorylation
of 4E-BP1 (Fig. 5, A and B), a strong inhibition of the ability of insulin to promote 4E-BP1 phosphorylation (Fig. 5,
A and B) and the activation of p70 (Fig. 4,
A and B). The inhibitory effects of 3MA on basal
and insulin-stimulated 4E-BP1 phosphorylation and p70 activation were
greatly attenuated by reintroduction of amino acids into the medium
(Figs. 4, A and B, and 5, A, and
B). Moreover, 3MA did not inhibit signal transduction
pathways activated by insulin (Fig. 6). These data support the
conclusion that the inhibition of autophagy by 3MA resulted in the
reduction of amino acid supply and a shutoff of amino acid-activated,
rapamycin-sensitive signal that is necessary to maintain the
responsiveness of these translational regulators to insulin. Further
studies are necessary to clarify the signaling components that regulate
translational control and autophagy via amino acid and mTOR
signaling pathways.
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results & Discussion
References
2-46/
CT104, which is resistant to inhibition by
rapamycin, is also resistant to inhibition by amino acids withdrawal,
suggesting that the amino acids and mTOR signal to p70 through a common
effector mechanism (24). Based on these findings, we proposed that
amino acid insufficiency and loss of mTOR activity both act as override
switches, which inhibit p70 activation and 4E-BP1 phosphorylation
irrespective of the receptor tyrosine kinase and PI3-k signals.
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results & Discussion
References
, and fetal calf serum were purchased from
Life Technologies, Inc. Protein G-Sepharose 4FF and
m7GTP-Sepharose were from Amersham Pharmacia Biotech.
Radioisotopes were obtained from Amersham Pharmacia Biotech. Human
insulin was supplied from Boehringer Mannheim. 3-Methyladenine and
wortmannin were purchased from Sigma. PD98059 and rapamycin were
obtained from New England Biolabs and Calbiochem, respectively.
with 10% fetal calf serum on
6-cm culture dishes. Cells were first incubated in minimal essential
medium
without fetal calf serum for 24 h, washed once with
DMEM lacking amino acids, and incubated in the same medium for the
times indicated for up to 2 h. Readdition of amino acids restored
the concentration of amino acids to the level found in complete DMEM.
Treatments of cells were terminated by removal of the medium followed
by freezing down with liquid nitrogen; then cells were stored at
80 °C until lysis. Cells were extracted into ice-cold buffer A (50 mM Tris-HCl, pH 8.0, 1% Nonidet P-40, 120 mM
NaCl, 20 mM NaF, 1 mM EDTA, 6 mM
EGTA, 20 mM
-glycerophosphate, 0.5 mM
dithiothreitol, 50 µM p-amidinophenylmethanesulfonyl fluoride hydrochloride, 1 µg/ml aprotinin, 1 µg/ml leupeptin) and the extracts were
centrifuged at 10,000 × g for 20 min at 4 °C before analysis.
and labeled in the same medium containing 0.2 µCi/ml
[14C]valine for 18 h at 37 °C. After three rinses
with phosphate-buffered saline, pH 7.4, to remove unincorporated
radioisotope, the cells were incubated in DMEM lacking amino acids,
complete DMEM, or DMEM lacking amino acids with 10 mM 3MA,
containing 0.1% of bovine serum albumin and 10 mM
unlabeled valine for 1 h, at which time short-lived proteins were
being degraded. Then incubation to measure the degradation of
long-lived proteins was initiated by replacing the medium with the same
fresh medium and continued for up to 2 h. At the indicated times,
the cells and media were collected separately. The cells were then
frozen and scraped into 0.5 ml of phosphate-buffered saline.
Radiolabeled proteins in the cells and media were precipitated in 10%
trichloroacetic acid (TCA) at 4 °C. The precipitated proteins were
separated from the soluble radioactivity by centrifugation at 600 × g for 10 min and then dissolved in 1 ml of Soluene 350 (Packard). The radioactivities in the fractions soluble and insoluble
in TCA recovered from the cells and media were measured by
scintillation counting. The rate of protein degradation attributable to
autophagic activity was calculated as a percentage of the
radioactivities in the fractions soluble in TCA recovered from the
cells and media to total radioactivity recovered from the cells and media.
RESULTS AND DISCUSSION
Top
Abstract
Introduction
Procedures
Results & Discussion
References
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Fig. 1.
Effects of wortmannin, rapamycin,
and PD98059 on the ability of insulin and amino acids to stimulate p70
activity after amino acid withdrawal. A, after serum
starvation for 24 h, H4IIE cells were incubated with DMEM lacking
amino acids for 2 h. Five plates (lanes 1-5) were
incubated for another 10 min with vehicle (lane 1) or 100 nM insulin (lanes 2-5). Before insulin
stimulation, the cells were pretreated with vehicle (lane
2), 100 nM wortmannin (lane 3), or 100 ng/ml rapamycin (lane 4) for 30 min or with 40 µM PD98059 (lane 5) for 1 h. After
incubation with DMEM lacking amino acids for 2 h, another four
plates (lanes 6-9) were incubated in complete DMEM
containing vehicle (lane 6), 100 nM wortmannin
(lane 7), or 100 ng/ml rapamycin (lane 8) for 30 min or 40 µM PD98059 (lane 9) for 1 h.
p70 activity was determined as described under "Experimental
Procedures." Numbers at the top of each
lane represent 32P incorporated into S6
expressed as a percentage of that catalyzed by p70 immunoprecipitated
from cell extracts treated with 100 nM insulin after amino
acid withdrawal (lane 2). B and C,
after serum starvation for 24 h, H4IIE cells were incubated with
DMEM lacking amino acids for 2 h. The cells were then incubated
for another 10 min with 100 nM insulin (open
circles). Before insulin stimulation, cells were pretreated with
the indicated concentrations of wortmannin (B) or rapamycin
(C) for 30 min. After amino acid deprivation, cells were
incubated in complete DMEM (closed circles) containing the
indicated concentrations of wortmannin (B) or rapamycin
(C) for 30 min. p70 activity was determined as described
under "Experimental Procedures." p70 activity stimulated with
either 100 nM insulin or complete DMEM in the absence of
inhibitors was regarded as maximal stimulation. Data are expressed as a
percentage of maximal stimulation of p70 activity by either insulin or
amino acids. Data are the means ± S.D. of triplicates.
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Fig. 2.
Effects of wortmannin and rapamycin on the
ability of insulin and amino acids to stimulate 4E-BP1 phosphorylation
and the dissociation of 4E-BP1 from eIF-4E after amino acid
withdrawal. After serum starvation for 24 h, H4IIE cells were
incubated with DMEM lacking amino acids for 2 h. Four plates
(lanes 1-4) were incubated for another 10 min with vehicle
(lane 1) or 100 nM insulin (lanes
2-4). Before insulin stimulation, the cells were pretreated with
vehicle (lane 2), 100 nM wortmannin (lane
3), or 100 ng/ml rapamycin (lane 4) for 30 min. After
amino acid deprivation, another three plates (lanes 5-7)
were incubated in complete DMEM containing vehicle (lane 5),
100 nM wortmannin (lane 6), or 100 ng/ml
rapamycin (lane 7) for 30 min. The phosphorylation of 4E-BP1
(A) and the binding of 4E-BP1 to eIF-4E (B) were
analyzed as described under "Experimental Procedures." In
B, positions of eIF-4E and 4E-BP1 are shown by
arrows.
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Fig. 3.
Amino acid release by autophagy. H4IIE
cells were labeled with [14C]valine, and incubation to
measure the degradation of long-lived proteins was initiated as
described under "Experimental Procedures." A, the cells
were incubated in DMEM lacking amino acids (open circles) or
complete DMEM (closed circles) for up to 2 h. At the
indicated times, the cells and media were collected. The
radioactivities in the fractions soluble and insoluble in TCA recovered
from the cells and media were measured, and the rate of protein
degradation was calculated as described under "Experimental
Procedures." Data are the means ± S.D. of triplicates.
B, the cells were incubated with DMEM lacking amino acids
(lane 1), complete DMEM (lane 2), or DMEM lacking
amino acids with 10 mM 3MA (lane 3) for 2 h. The cells and media were collected at 0 h and 2 h. The
rate of protein degradation was measured as described under
"Experimental Procedures." Data are expressed as subtraction of
values at 0 h from those at 2 h. Data are the means ± S.D. of triplicates.
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Fig. 4.
Effects of 3MA on the ability of insulin to
stimulate the activation of p70 following amino acids withdrawal.
A, after serum starvation for 24 h, H4IIE cells were
incubated with DMEM lacking amino acids (lanes 1-4) or
complete DMEM (lanes 5 and 6) in the absence
(lanes 1, 3, and 5) or presence
(lanes 2, 4, and 6) of 10 mM 3MA for 2 h and stimulated with vehicle
(lanes 1 and 2) or 100 nM insulin
(lanes 3-6) for another 10 min. Then p70 activity was
determined as described under "Experimental Procedures."
B, after serum starvation for 24 h, cells were
incubated with DMEM lacking amino acids in the presence of the
indicated concentration of 3MA for 2 h followed by incubation for
another 10 min with 100 nM insulin (open
circles) or vehicle (open square), or for another 30 min with complete DMEM (closed circles). Then p70 activity
was determined as described under "Experimental Procedures." In
both A and B, p70 activity was expressed in
arbitrary units (photostimulated luminescence (PSL) units),
and data are the means ± S.D. of triplicates.
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Fig. 5.
Effects of 3MA on 4E-BP1 phosphorylation and
binding of 4E-BP1 to eIF-4E. After serum starvation for 24 h,
H4IIE cells were incubated with DMEM lacking amino acids (lanes
1-4) or complete DMEM (lanes 5 and 6) in
the absence (lanes 1, 3, and 5) or
presence (lanes 2, 4, and 6) of 10 mM 3MA for 2 h and then stimulated with vehicle
(lanes 1 and 2) or 100 nM insulin
(lanes 3-6) for another 10 min. The phosphorylation of
4E-BP1 (A) and the binding of 4E-BP1 to eIF-4E
(B) were analyzed as described under "Experimental
Procedures." In B, positions of eIF-4E and 4E-BP1 are
shown by arrows.
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Fig. 6.
Effects of 3MA on insulin
signaling. After serum starvation for 24 h, H4IIE cells were
incubated with DMEM lacking amino acids (lanes 1-4) or
complete DMEM (lanes 5-8) for 2 h, and 10 mM 3MA was added for the last 30 min to lanes 2,
4, 6, and 8. Cells were then
stimulated with vehicle (lanes 1, 2,
5, and 6) or 100 nM insulin
(lanes 3, 4, 7, and 8) for
another 10 min. Supernatants of cell extracts were prepared as
described under "Experimental Procedures." A and
B, tyrosine-phosphorylated proteins were immunoprecipitated
with the anti-phosphotyrosine antibody (PY20). In A,
tyrosine phosphorylation of -subunits of insulin receptors
(IR) and insulin receptor substrates (IRS) were
determined by immunoblotting with PY20 as the first antibody. Positions
of IRS and IR are shown by arrows. In
B, PI3-k activity in the immunoprecipitates was determined
as described under "Experimental Procedures." Positions of
phosphoinositide 3-phosphate (PIP) and thin layer
chromatography origin (Origin) are shown by
arrows. C, the supernatants were separated by
SDS-PAGE and immunoblotted with the non-phospho-specific antibody
against p44/42 MAPK (upper panel) or the phospho-specific
(Thr-202/Tyr-204) antibody against p44/42 MAPK (lower panel)
as the first antibody. Positions of p44 and p42 MAPK are shown by
arrows.
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ACKNOWLEDGEMENTS |
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We are grateful to Dr. Y. Nishizuka for encouragement and Drs. M. Kadowaki and S. Hidayat for valuable advice. The skillful secretarial assistance of M. Kusu is cordially acknowledged.
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FOOTNOTES |
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* This work was supported by Grant-in-Aid for Creative Basic Research and Scientific Research C from the Ministry of Education, Science, Sports and Culture of Japan, the Sankyo Foundation of Life Science, Juvenile Diabetes Foundation International, the Kato Memorial Bioscience Foundation, and Japan Foundation for Applied Enzymology.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: Biosignal Research Center, Kobe University, 1-1 Rokkodai-cho, Nada-ku, Kobe 657-8501, Japan. Tel.: 81-78-803-1253; Fax: 81-78-803-1259; E-mail: yonezawa{at}kobe-u.ac.jp.
The abbreviations used are: eIF, eukaryotic initiation factor; 4E-BP1, eIF-4E-binding protein 1; m7GpppN, 7-methylguanosine 5'-triphosphate-containing mRNA "cap"; CHO-IR, Chinese hamster ovary cells overexpressing human insulin receptors; 3MA, 3-methyladenine; DMEM, Dulbecco's modified Eagle's medium; PAGE, polyacrylamide gel electrophoresis; p70, p70 S6 kinase; PI3-k, phosphoinositide 3-kinase; mTOR, mammalian target of rapamycin; MAPK, mitogen-activated protein kinase; TCA, trichloroacetic acid.
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REFERENCES |
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