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(Received for publication, September 19, 1994; and in revised form, November 7,
1994) From the
In rat hepatocytes, autophagy is known to be inhibited by amino
acids. Insulin and cell swelling promote inhibition by amino acids.
Each of the conditions leading to inhibition of autophagic proteolysis
was found to be associated with phosphorylation of a 31-kDa protein
that we identified as ribosomal protein S6. A combination of leucine,
tyrosine, and phenylalanine, which efficiently inhibits autophagic
proteolysis, was particularly effective in stimulating S6
phosphorylation. The relationship between the percentage inhibition of
proteolysis and the degree of S6 phosphorylation was linear. Thus,
inhibition of autophagy and phosphorylation of S6 are under the control
of the same signal transduction pathway. Stimulation of S6
phosphorylation by the presence of amino acids was due to activation of
S6 kinase and not to inhibition of S6 phosphatase. The inhibition by
amino acids of both autophagic proteolysis and autophagic sequestration
of electro-injected cytosolic [ We conclude that the fluxes through
the autophagic and protein synthetic pathways are regulated in an
opposite manner by the degree to which S6 is phosphorylated. Possible
mechanisms by which S6 phosphorylation can cause inhibition of
autophagy are discussed. During autophagy, part of the cytoplasm is surrounded by a
membrane to form an autophagosome in which, upon fusion with a
lysosome, degradation of macromolecular material
occurs(1, 2) . The origin of the autophagosomal
membrane is the ribosome-free part of the rough endoplasmic
reticulum(3, 4) . During starvation, autophagic
degradation of protein is accelerated in order to produce amino acids
for gluconeogenesis and other essential metabolic pathways. Regulation
of autophagy has mostly been studied in the liver of rats and mice (cf. (1) and (2) ). In these animals, during
the first 48 h of starvation, autophagic breakdown of protein in the
liver is higher than in other tissues(1) . Autophagic flux
is inhibited by amino acids(1, 2) . Insulin (1, 2) and cell swelling (5) promote
inhibition by low concentrations of amino acids, whereas glucagon (1) reduces it. However, little is known about the mechanism by
which these factors interact with the autophagic system except that
they primarily affect the first step in the pathway, i.e. the
formation of initial autophagosomes(1, 2) . In this
paper we show that amino acids added alone or in combination with
insulin, hypotonic cell swelling, or glucagon also affect
phosphorylation of ribosomal protein S6. In the literature it is
generally assumed that S6 phosphorylation is involved in the regulation
of protein synthesis(6, 7) . Evidence is now provided
suggesting that S6 phosphorylation is also directly involved in the
control of autophagic sequestration.
Incubation of rat hepatocytes with
[
Figure 1:
Effect of amino acids on the
phosphorylation of the 31-kDa protein in isolated rat hepatocytes and
its time course. In A, hepatocytes (10 mg of dry
mass
Cell
fractionation showed that the protein was exclusively localized in the
ribosomal fraction. Because the molecular mass of the protein was close
to that of ribosomal protein S6, we suspected that it was identical to
S6. Indeed, immune precipitation of the
[
Figure 2:
Effect
of rapamycin on the phosphorylation of ribosomal protein S6 in isolated
rat hepatocytes. Hepatocytes (5 mg of dry
mass
In perfused rat liver, cell swelling
mimicks the antiproteolytic effect of insulin(17) . Incubation
of isolated hepatocytes under hypo-osmotic conditions enhances the
sensitivity of autophagic proteolysis to inhibition by amino
acids(5) , which is similar to the effect of insulin (18, 19) (cf. Fig. 3, F and H). However, insulin and hypo-osmolarity have no effect on
their own, i.e. in the absence of amino acids. Exactly the same
phenomenon was observed with regard to the phosphorylation of S6. Both
hypo-osmolarity and insulin increased phosphorylation of S6 at low, but
not at high, concentrations of amino acids while having no effect in
the absence of amino acids (Fig. 3, B and D).
Figure 3:
Phosphorylation of ribosomal protein S6
and inhibition of proteolysis. Hepatocytes (5 mg of dry
mass
In contrast to insulin and cell swelling, glucagon
decreases the sensitivity of autophagic proteolysis to inhibition by
amino acids ((20) ; Fig. 3F). Likewise, in the
presence of low, but not of high, concentrations of amino acids,
phosphorylation of S6 was decreased by glucagon (Fig. 3B). In the absence of added amino acids, little
effect of glucagon was noted, either on the phosphorylation of S6 (cf. (9) ) or on autophagic proteolysis. From our
previous results with cultured hepatocytes isolated from perinatal
rats, we concluded that among the 20 amino acids, a combination of 3
amino acids, i.e. leucine, phenylalanine, and tyrosine, is
particularly effective in inhibiting autophagic
proteolysis(21) . This specificity also applied to the
phosphorylation of S6 in hepatocytes isolated from adult rats (Fig. 3, A, C, E, and G); especially under hypo-osmotic conditions, the
phosphorylation of S6 in the presence of a complete mixture of amino
acids could (like the inhibition of proteolysis) largely be mimicked by
the combination of these 3 amino acids or by the combination of leucine
with either phenylalanine or tyrosine. Even leucine alone had some
effect (Fig. 3, C and G). Fig. 3I shows the relationship between the
phosphorylation of S6 and the percentage inhibition of autophagic
proteolysis under the conditions discussed. A striking linear
relationship between the two parameters was observed. Because
cycloheximide was included in our measurements of proteolytic rates (cf. (5) ), we tested whether or not deletion of this
compound from the incubations had any effect on S6 phosphorylation. At
25 µM, a concentration sufficient to inhibit protein
synthesis by more than 95% and with no effect on the rate of
proteolysis(5) , cycloheximide did not affect phosphorylation
of S6, neither in the absence nor in the presence of amino acids (Fig. 3, B and D). 3-Methyladenine, often
used as a specific inhibitor of autophagic sequestration(2) ,
strongly inhibited proteolysis but had no effect on the phosphorylation
of S6 (Fig. 3, A, C, E, G,
and I). Apparently, the mechanism by which this compound
inhibits autophagy is unrelated to that caused by amino acids. This
result also rules out the possibility that increased phosphorylation of
S6 by amino acids was the consequence of the antiproteolytic effect of
amino acids. Because the linear relationship between S6
phosphorylation and the inhibition of autophagic proteolysis
demonstrated that these two processes are controlled by the same
regulatory factors, the question arose whether S6 phosphorylation was
directly involved in the control of autophagic proteolysis. Rapamycin,
which inhibits S6 phosphorylation (cf. Fig. 2), could
provide an answer to this question. Fig. 4shows that rapamycin
stimulated proteolysis especially in the presence of a complete mixture
of amino acids. However, the inhibitory effect of amino acids was only
partially relieved by rapamycin. The stimulation of proteolysis by
rapamycin was prevented by 3-methyladenine (not shown), indicating that
autophagic proteolysis is involved in this stimulation. Direct
measurement of autophagic sequestration by sequestration of cytosolic
[
Figure 4:
Effect of rapamycin on autophagic
proteolysis. Hepatocytes (5-10 mg of dry
mass
Figure 5:
Effect of rapamycin on
[
[ In
order to gain insight into the underlying mechanism by which amino
acids increase the phosphorylation state of S6, i.e. either by
activation of S6 kinase, by inhibition of S6 phosphatase, or by a
combination of both, the following experiment was performed.
Hepatocytes were prelabeled with [
Figure 6:
Amino acids do not affect S6
dephosphorylation. Hepatocytes were incubated under iso-osmotic
conditions with [
In the literature, it
is generally assumed that S6 phosphorylation is involved in the
regulation of protein
synthesis(6, 7, 22, 23) .
Measurement of protein synthesis in our experimental system revealed
that under some conditions there may be a correlation between the rate
of protein synthesis and S6 phosphorylation but that this does not
apply to all conditions. As shown in Table 1, under iso-osmotic
conditions with increasing amino acid concentrations, there was indeed
a correlation between S6 phosphorylation and protein synthesis.
However, under hypo-osmotic conditions in the absence of amino acids,
when phosphorylation of S6 was unchanged, protein synthesis was higher
than under iso-osmotic conditions (Table 1; cf. (5) ). Also, under all conditions examined, addition of
rapamycin inhibited protein synthesis by 10-30%, even though S6
phosphorylation was completely prevented.
In a variety of cell systems, phosphorylation of S6 was shown
to be stimulated by serum, growth factors, and
insulin(24, 25, 26, 27, 28) .
Because serum contains amino acids and because effects of growth
factors and insulin have not been reported in the absence of amino
acids, it is likely that phosphorylation of S6 was mediated, either
directly or indirectly, by amino acids. Phosphorylation of S6 occurs
at several serine residues; 1 or 2 of these are phosphorylated by
protein kinase A (which are probably not involved in the control of
protein synthesis(26) ), whereas 3-4 other residues are
phosphorylated under the influence of insulin and growth-promoting
compounds(25, 26, 27, 28) . We did
not observe increased S6 phosphorylation by glucagon as described by
Wettenhall et al.(25) . Under our experimental
conditions, basal activity of protein kinase A was possibly sufficient
to cause phosphorylation in the absence of added amino acids. Our
result with cycloheximide in vitro (no effect on S6
phosphorylation) is at variance with that obtained with cycloheximide
in rat liver in vivo, which causes increased phosphorylation
of S6(29) . However, administration of cycloheximide in
vivo is likely to cause an increase in concentrations of
circulating amino acids and insulin(30) , and these may have
been responsible for the increased phosphorylation of S6 reported in (29) . Rapamycin completely blocked the effect of amino
acids on S6 phosphorylation. Because in the presence of rapamycin, the
rate of S6 dephosphorylation was identical in the absence or presence
of amino acids whether under iso-osmotic or under hypo-osmotic
conditions, we conclude that the degree of S6 phosphorylation was
determined by changes in the activity of p70 S6 kinase and not by
changes in the activity of S6 phosphatase. S6 phosphorylation was
not affected by either okadaic acid (20 nM) or by microcystin
(50-200 nM), inhibitors of type I and IIa phosphatases
(results not shown). Thus, S6 phosphatase is not a type I or IIa
protein phosphatase(31, 32) . It also indicates that
inhibition of autophagic proteolysis by these phosphatase inhibitors,
as found by Holen et al.(33) , can not be due to an
effect on S6 phosphorylation. According to these authors, type I and
IIa phosphatase inhibitors may affect the structure of the
cytoskeleton(33) . The close parallelism between S6
phosphorylation and inhibition of autophagic proteolysis observed in
our experiments indicates that these processes are controlled by the
same signal transduction pathway. The present data obtained with
rapamycin, as shown in Fig. 4and Fig. 5, strongly
suggest that S6 phosphorylation is directly involved in the control of
autophagic proteolysis. Also, the time required for S6 phosphorylation
and S6 dephosphorylation (20-30 min; Fig. 1B and Fig. 6) agrees well with the time required by amino acids to
inhibit autophagy maximally or for autophagy to regain its maximal flux
after inhibition by amino acids(1) . Although the effect of
rapamycin on S6 phosphorylation was complete (Fig. 2), the
compound partially relieved the inhibitory effect of amino acids on
autophagic proteolysis. This indicates that amino acids also inhibited
autophagic proteolysis by an S6-independent mechanism. The stimulatory
effect of rapamycin on the rate of autophagic sequestration (Fig. 5) was equal to the effect on total autophagic flux (Fig. 4), implying that most of the control of the flux (as
defined in (34) ) through the autophagic proteolytic pathway
resides in the sequestration step. This conclusion is also supported by
the finding that addition of methylamine did not affect the rate of
autophagic sequestration (Fig. 5) even though it completely
blocked lysosomal protein degradation (not shown), indicating that the
rate of formation of autophagosomes is not feedback controlled by the
accumulation of autophagosomes under these conditions. It is
generally believed that S6 phosphorylation is required for stimulation
of protein
synthesis(24, 25, 26, 27, 28) ,
even though the degree of S6 phosphorylation does not always parallel
the rate of protein synthesis (for a review, see (27) ). In our
experimental system, phosphorylation of S6 and protein synthesis were
stimulated by the addition of amino acids, indicating that both
processes are related. This relationship was also found by Morley and
Traugh in 3T3-L1 cells (35) . However, one should bear in mind
that amino acids are also substrates for protein synthesis. Because of
this, it is possible that the observed relationship between S6
phosphorylation and protein synthesis is coincidental. The regulatory
role of S6 phosphorylation in protein synthesis could be resolved by
the addition of rapamycin, which separates the effect of amino acids on
S6 phosphorylation from their function as substrates for protein
synthesis. These experiments revealed that S6 phosphorylation accounts
for 10-30% of the stimulation of protein synthesis by amino acids (Table 1). Interestingly, the opposite effects of rapamycin on
protein synthesis and degradation were of similar magnitude. This
strongly suggests that S6 phosphorylation is directly involved in the
co-regulation of both processes and can thus be considered as a
regulatory switch between these anabolic and catabolic processes. In
summary, the following sequence of events can be envisaged to
accomodate the data (Fig. 7). Amino acids inhibit autophagic
proteolysis and stimulate S6 phosphorylation. The mechanism by which
amino acids stimulate phosphorylation of S6 is unknown but could be the
consequence of binding of amino acids to a receptor in the plasma
membrane, followed by activation of a protein kinase cascade. The
existence of such a receptor molecule was postulated previously (36, 37, 38) . The effect of cell swelling on
autophagic proteolysis and S6 phosphorylation, making the processes
more sensitive to low concentrations of amino acids, may be mediated by
an increased affinity of the amino acid receptor for amino acids
following membrane stretch. Inhibition of autophagosome formation is
not only caused by S6 phosphorylation but also by some component in the
signal transduction pathway preceding S6 phosphorylation (Fig. 7).
Figure 7:
Schematic overview of the co-regulation of
autophagic proteolysis and protein synthesis by amino acids and cell
swelling. Amino acids (AA) stimulate a protein kinase cascade (X) via a plasma membrane receptor resulting in
phosphorylation of ribosomal protein S6 (S6P). Autophagic
sequestration is inhibited by both phosphorylated S6 and a component of
the signal transduction pathway preceding S6 phosphorylation.
Simultaneously, protein synthesis is stimulated. Cell swelling
potentiates the effect of amino acids via a change in the affinity of
the receptor for amino acids. AV
Jefferies et al.(39) recently
showed in 3T3 cells that phosphorylation of S6 promotes binding of a
subclass of mRNAs (the ``polypyrimidine tract'' mRNA family)
to the ribosomes. We hypothesize that in rat hepatocytes S6
phosphorylation stimulates binding to the ribosomes of mRNAs, which
encode for proteins that have to be synthesized at the endoplasmic
reticulum. S6 phosphorylation would thus result in stimulation of
ribosome binding to the rough endoplasmic reticulum. This would reduce
the availability of ribosome-free regions of the rough endoplasmic
reticulum, which are the source of the autophagosomal
membrane(3, 4) , for initial autophagosome formation.
In this way, simultaneous inhibition of autophagic sequestration and
stimulation of endoplasmic reticulum-linked protein synthesis would
occur. An alternative possibility is that inhibition of autophagosome
formation does not result from the binding of ribosomes to the
endoplasmic reticulum but rather from the nascent polypeptide chains
plugged through the membrane of the endoplasmic reticulum.
Volume 270,
Number 5,
Issue of February 3, 1995 pp. 2320-2326
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
C]sucrose was
partially prevented by rapamycin, a compound known to inhibit
activation of p70 S6 kinase. In addition, rapamycin partially inhibited
the rate of protein synthesis.
Preparation of Hepatocytes
Hepatocytes were isolated from 18-24 h starved male
Wistar rats (200-250 g) as described by Groen et
al.(8) .Determination of Protein Phosphorylation
For measurement of protein phosphorylation, hepatocytes were
incubated at 37 °C for 50 min (9) (unless otherwise
indicated) in Krebs-Henseleit bicarbonate medium (2 ml; gas phase,
O
/CO = 95:5) plus 20 mM glucose, 0.2 mM [P]phosphate (100
µCi), and the additions indicated in the legends to the figures. At
the end of the incubations, cells were diluted 5-fold with ice-cold
Krebs-Henseleit bicarbonate medium and collected by centrifugation (2
min, 50
g). The cell pellets were extracted with 0.6
ml of sample buffer (10) and brought to 90 °C for 5 min; an
amount equivalent to about 100 µg of protein was analyzed by SDS
polyacrylamide (10%) gel electrophoresis. Gel slabs were dried and
subjected to autoradiography. Protein phosphorylation was quantified
with a PhosphorImager(TM) (Molecular Dynamics, Inc.).Measurement of Proteolysis
Proteolysis was measured as production of valine (11) after 90 min of incubation at 37 °C in Krebs-Henseleit
bicarbonate medium plus 20 mM glucose, 25 µM cycloheximide. and the additions or omissions indicated in the
legends to the figures. Cycloheximide was present in order to prevent
simultaneous protein synthesis. At this concentration, cycloheximide
did not affect flux through the autophagic pathway(5) .Measurement of Protein Synthesis
Protein synthesis was measured as incorporation of L-[
H]valine according to Meijer and
Hensgens (12) .Composition of the Complete Mixture of Amino Acids
The concentration of each amino acid in this mixture was
equal to either 1 (1 amino acids) or 4 (4 amino acids)
times its concentration in the portal vein of a starved rat. The
composition of the 1 mixture was as described in (13) ,
except that the concentration of leucine was 250 µM. When
proteolysis was measured, valine was omitted from the mixture because
its production was used to monitor proteolytic rates.Determination of the Intracellular Localization of the
Phosphorylated 31-kDa Protein
Cells were collected by centrifugation (2 min, 50 g) and resuspended in 4 ml of an ice-cold medium containing 50
mM Tris/HCl (pH 7.2), 50 mM NaF, 2.5 mM MgCl, 1 mM EGTA, 1 mM EDTA, 1 mM dithiothreitol, 1 mM sodium pyrophosphate, and 1 mM phenylmethylsulfonyl fluoride (medium A). After homogenization of
the cells with a Dounce homogenizer, 1 ml of 1.25 M sucrose in
medium A was added to bring the final concentration of sucrose to 250
mM. Cell ghosts were removed by centrifugation in a
microcentrifuge for 1 s. The resulting homogenate was centrifuged (5
min, 1000 g) to obtain nuclei and plasma membranes.
The postnuclear supernatant was centrifuged (10 min at 10,000 g) to obtain the mitochondrial/lysosomal fraction. One part of
the postmitochondrial supernatant was centrifuged (150 min, 105,000
g) to obtain microsomes and cytosol. Another part of
the postmitochondrial supernatant was treated with a mixture of 1%
Triton X-100 and 1% deoxycholate, layered over 5 ml of 0.5 M sucrose in medium A, and centrifuged (150 min, 105,000 g) to obtain ribosomes. The various fractions were
characterized by measurement of specific marker enzymes and of
ribosomal RNA (not shown).Immune Precipitation of the Ribosomal 31-kDa Protein
Ribosomes were suspended in medium A, supplemented with 1%
SDS, and brought to 90 °C (5 min). 60 µg of ribosomal protein
(5 µl) was diluted 40-fold with a solution containing 50 mM Tris/HCl (pH 8.0), 120 mM NaCl, 20 mM NaF, 1%
Nonidet-P40, 1 mM EDTA, 1 mM EGTA, 1 mM sodium pyrophosphate, 1 mM dithiothreitol, and 1 mM phenylmethylsulfonyl fluoride (medium B). 5 µl of either
preimmune serum or antiserum was then added and allowed to interact
with the ribosomal protein for 2 h at 0 °C. 50 µl of 50%
protein A-Sepharose (prewashed in medium B) was then added, and the
mixture was incubated for 1 h at 4 °C. After this period, the
Sepharose particles were collected by centrifugation in a
microcentrifuge for 1 s, washed 6 times with 1-ml portions of medium B
followed by a washing step with 1 ml of a medium containing 20 mM Mops (
)(pH 7.5), 0.2% Triton X-100, and 10 mM MgCl. The final pellet was dissolved in sample buffer (10) and brought to 90 °C for 5 min. After centrifugation,
the supernatant was analyzed by SDS-polyacrylamide (10%) gel
electrophoresis. The gel slab was dried and subjected to
autoradiography.Measurement of [
Loading of hepatocytes with [C]Sucrose
SequestrationC]sucrose
and its autophagic sequestration was carried out with the
electropermeabilization procedure as described by Seglen and Gordon (14) .Statistical Determination
Statistical significance was determined using Student's t test (p 
0.05).Materials
Rapamycin
Rapamycin was a gift from Wyeth-Ayerst
Research, Princeton, NJ. Rapamycin was dissolved in MeSO.
Control incubations received equal amounts of MeSO (final
concentration, 0.5% v/v), which did not affect the processes tested.[14C]Sucrose
[C]Sucrose
(specific activity 630 µCi/µmol) was obtained from Amersham
Corp.[
[H]ValineH]valine
was obtained from Amersham Corp.
P]phosphate in the presence of a complete
mixture of amino acids greatly enhanced the phosphorylation of a
protein with a molecular mass of 31 kDa (Fig. 1A).
Phosphorylation of this protein in the presence of amino acids was
completed in 20-30 min (Fig. 1B).
ml), were incubated for 50 min with
[
P]phosphate, in the absence (laneA) or presence of a complete mixture of amino acids (1
amino acids, laneB; 4 amino acids, laneC). In B, hepatocytes were preincubated
for 50 min with [P]phosphate in the absence of
added amino acids. After this period, the mixture of amino acids (4
amino acids ( AA)) was added and, at the times
indicated, cells were collected and analyzed as described under
``Experimental Procedures.'' In the control (no amino acids
added), incubation was continued in the absence of added amino acids.
The intensity of the phosphorylated 31-kDa protein after the 50-min
preincubation period in the absence of amino acids was arbitrarily set
at 1.
P]phosphate-labeled protein with an antibody
directed against the C-terminal part
(Arg
-Lys
) of S6 confirmed this (data
not shown). An additional argument that the 31-kDa protein was
identical to S6 was provided by the observation that its
phosphorylation was completely prevented by low concentrations of
rapamycin (Fig. 2), which is an inhibitor of the signal
transduction pathway leading to activation of p70 S6
kinase(15, 16) . Inhibition by high concentrations of
rapamycin was complete (Fig. 2) and almost instantaneous (within
1-2 min, not shown).
ml), were incubated with
[
P]phosphate (100 µCi) in the presence of 4
the complete mixture of amino acids. Rapamycin was added at the
concentrations indicated at the top of the lanes.
After 90 min of incubation, cells were collected and analyzed by SDS
gel electrophoresis.
ml) were incubated and analyzed as
described under ``Experimental Procedures.'' Hypo-osmolarity
was obtained by decreasing the NaCl concentration of the incubation
medium from 120 mM (A, B, E, and F) to 70 mM (C, D, G, and H). The concentrations were as follows: leucine (L),
1000 µM; phenylalanine (F), 200 µM;
tyrosine (Y), 300 µM. These concentrations are
identical to the concentrations of these amino acids in a 4
complete mixture of amino acids. Other concentrations were as follows:
insulin (INS), 10M; glucagon (GLCN), 10
M; 3-methyladenine (3MA), 10 mM. All indicates a complete
4
mixture of amino acids. In B, D, F and H: hatchedbars, no amino acids
added; whitebars, a complete 1 amino acid
mixture added; blackbars, a complete 4 amino
acid mixture added. In I, 
indicates iso-osmotic
conditions, and indicates hypo-osmotic conditions. The degree
of S6 phosphorylation under all conditions was related to that observed
in the absence of added amino acids under iso-osmotic conditions.
Phosphorylation was measured after 50 min of incubation (A-D). Proteolysis was measured after 90 min of
incubation (E-H). The basal rate of proteolysis under
iso-osmotic conditions, in the absence of added amino acids (0%
inhibition), was equal to 16.6 ± 0.9 µmol valine produced
per g of dry mass of cells/90 min. In the absence of cycloheximide (-CH), production of valine does not indicate the true
proteolytic rate because of simultaneous protein synthesis; for this
reason, valine production data were not plotted under these conditions (F and H). Data are the means (±S.E.) from
experiments carried out with 3-5 different hepatocyte
preparations.
C]sucrose confirmed that rapamycin stimulates
the autophagic process (Fig. 5).
ml) were incubated in the absence or
presence of the complete mixture of amino acids and analyzed as
described under ``Experimental Procedures.'' Hypo-osmolarity
was obtained by decreasing the NaCl concentration of the incubation
medium from 120 mM to 70 mM. Whitebars, rapamycin absent; blackbars,
rapamycin (100 nM) added. Proteolysis was measured after 90
min of incubation. The basal rate of proteolysis under iso-osmotic
conditions, in the absence of added amino acids (0% inhibition), was
equal to 16.6 ± 0.9 µmol valine produced per g of dry mass
of cells/90 min. Data are the means (±S.E.) from experiments
carried out with 3-5 different hepatocyte preparations. *,
significantly different from the same condition in the absence of
rapamycin.
C]sucrose sequestration.
[C]Sucrose-loaded hepatocytes (15 mg of dry
massml) were incubated for 90 min in the
absence or presence of 100 nM rapamycin and the additions
indicated. CTRL, control; RAPA, rapamycin; 4
AA, 4 times the complete mixture of amino acids; 3MA, 3-methyladenine; MA, methylamine. *,
significantly different from the same condition in the absence of
rapamycin.
C]Sucrose sequestration was measured as
described by Seglen and Gordon(14) .
[C]Sucrose, normally impermeant and inert to
hepatocytes, was injected into the cytosol by electropermeabilization.
Preloaded cells were then incubated, and
[C]sucrose was taken up by the autophagic
system. About 50% of the radioactivity was taken up by the mitochondria
(not shown) and was resistant to inhibition by amino acids, in
agreement with the data of Seglen and Gordon(14) . The amount
of [C]sucrose sequestered in the lysosomal
fraction in a certain period of time, is a measure of the rate of
autophagic sequestration. As shown in Fig. 5, amino acids
strongly inhibited autophagic sequestration. The effect of amino acids
was comparable with the effect of 3-methyladenine (cf. (14) ). Rapamycin partially reversed inhibition of autophagic
sequestration by amino acids. Addition of methylamine, a lysosomotropic
agent, did not affect the rate of autophagic sequestration (Fig. 5) but strongly inhibited proteolysis (not shown).P]phosphate in
the presence of high concentrations of amino acids. These preloaded
cells were then further incubated with rapamycin (to block S6 kinase)
in the absence or presence of amino acids, and the phosphorylation
state of S6 was followed in time. In the presence of rapamycin, S6
became rapidly dephosphorylated to its basal level in 20-30 min (Fig. 6). The rate of dephosphorylation was not significantly
affected by the presence of amino acids or by hypo-osmolarity. Thus,
the stimulation of S6 phosphorylation by amino acids and
hypo-osmolarity must have been due to stimulation of p70 S6 kinase
rather than to inhibition of S6 phosphatase.
P]phosphate (100
µCi
ml) for 50 min in the presence of 4
times the complete mixture of amino acids. After this period, cells
were washed (time zero in the graph) and re-incubated under iso-osmotic (A) or hypo-osmotic (B) conditions in the absence
(
, ) or presence of 4 times the complete mixture of amino
acids (
, ), and both in the absence (opensymbols) or presence (filledsymbols)
of rapamycin (100 nM). Phosphorylation of S6 was measured at
the time points indicated.
, initial
autophagic vacuole; AV
, degradative
autophagic vacuole.
)
We thank H. R. Bandi for preparation of the anti-S6
antibody, H. Jefferies for instruction as to its use, G. Thomas for
interest in the work, W. H. Lamers for a gift of a ribosomal RNA probe,
and R. Benne and L. Boon for help and advice.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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M. Djavaheri-Mergny, M. Amelotti, J. Mathieu, F. Besancon, C. Bauvy, S. Souquere, G. Pierron, and P. Codogno NF-{kappa}B Activation Represses Tumor Necrosis Factor-{alpha}-induced Autophagy J. Biol. Chem., October 13, 2006; 281(41): 30373 - 30382. [Abstract] [Full Text] [PDF]
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T. Kabuta, Y. Suzuki, and K. Wada Degradation of Amyotrophic Lateral Sclerosis-linked Mutant Cu,Zn-Superoxide Dismutase Proteins by Macroautophagy and the Proteasome J. Biol. Chem., October 13, 2006; 281(41): 30524 - 30533. [Abstract] [Full Text] [PDF]
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M. Shibata, T. Lu, T. Furuya, A. Degterev, N. Mizushima, T. Yoshimori, M. MacDonald, B. Yankner, and J. Yuan Regulation of Intracellular Accumulation of Mutant Huntingtin by Beclin 1 J. Biol. Chem., May 19, 2006; 281(20): 14474 - 14485. [Abstract] [Full Text] [PDF]
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A. Panner, C. D. James, M. S. Berger, and R. O. Pieper mTOR Controls FLIPS Translation and TRAIL Sensitivity in Glioblastoma Multiforme Cells Mol. Cell. Biol., October 15, 2005; 25(20): 8809 - 8823. [Abstract] [Full Text] [PDF]
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E. M. Smith, S. G. Finn, A. R. Tee, G. J. Browne, and C. G. Proud The Tuberous Sclerosis Protein TSC2 Is Not Required for the Regulation of the Mammalian Target of Rapamycin by Amino Acids and Certain Cellular Stresses J. Biol. Chem., May 13, 2005; 280(19): 18717 - 18727. [Abstract] [Full Text] [PDF]
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J.-K. Chen, J. Chen, E. G. Neilson, and R. C. Harris Role of Mammalian Target of Rapamycin Signaling in Compensatory Renal Hypertrophy J. Am. Soc. Nephrol., May 1, 2005; 16(5): 1384 - 1391. [Abstract] [Full Text] [PDF]
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M. T.N. Moller, H. R. Samari, and P. O. Seglen Toxin-Induced Tail Phosphorylation of Hepatocellular S6 Kinase: Evidence for a Dual Involvement of the AMP-Activated Protein Kinase in S6 Kinase Regulation Toxicol. Sci., December 1, 2004; 82(2): 628 - 637. [Abstract] [Full Text] [PDF]
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T. Kanazawa, I. Taneike, R. Akaishi, F. Yoshizawa, N. Furuya, S. Fujimura, and M. Kadowaki Amino Acids and Insulin Control Autophagic Proteolysis through Different Signaling Pathways in Relation to mTOR in Isolated Rat Hepatocytes J. Biol. Chem., February 27, 2004; 279(9): 8452 - 8459. [Abstract] [Full Text] [PDF]
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H.-J. Lee, F. Khoshaghideh, S. Patel, and S.-J. Lee Clearance of {alpha}-Synuclein Oligomeric Intermediates via the Lysosomal Degradation Pathway J. Neurosci., February 25, 2004; 24(8): 1888 - 1896. [Abstract] [Full Text] [PDF]
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C. H. Lang, R. A. Frost, N. Deshpande, V. Kumar, T. C. Vary, L. S. Jefferson, and S. R. Kimball Alcohol impairs leucine-mediated phosphorylation of 4E-BP1, S6K1, eIF4G, and mTOR in skeletal muscle Am J Physiol Endocrinol Metab, December 1, 2003; 285(6): E1205 - E1215. [Abstract] [Full Text] [PDF]
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C. J. Lynch, B. Halle, H. Fujii, T. C. Vary, R. Wallin, Z. Damuni, and S. M. Hutson Potential role of leucine metabolism in the leucine-signaling pathway involving mTOR Am J Physiol Endocrinol Metab, October 1, 2003; 285(4): E854 - E863. [Abstract] [Full Text] [PDF]
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S. Pattingre, C. Bauvy, and P. Codogno Amino Acids Interfere with the ERK1/2-dependent Control of Macroautophagy by Controlling the Activation of Raf-1 in Human Colon Cancer HT-29 Cells J. Biol. Chem., May 2, 2003; 278(19): 16667 - 16674. [Abstract] [Full Text] [PDF]
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B. Ravikumar, A. Stewart, H. Kita, K. Kato, R. Duden, and D. C. Rubinsztein Raised intracellular glucose concentrations reduce aggregation and cell death caused by mutant huntingtin exon 1 by decreasing mTOR phosphorylation and inducing autophagy Hum. Mol. Genet., May 1, 2003; 12(9): 985 - 994. [Abstract] [Full Text] [PDF]
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J. L. Crespo and M. N. Hall Elucidating TOR Signaling and Rapamycin Action: Lessons from Saccharomyces cerevisiae Microbiol. Mol. Biol. Rev., December 1, 2002; 66(4): 579 - 591. [Abstract] [Full Text] [PDF]
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C. J. Lynch, S. M. Hutson, B. J. Patson, A. Vaval, and T. C. Vary Tissue-specific effects of chronic dietary leucine and norleucine supplementation on protein synthesis in rats Am J Physiol Endocrinol Metab, October 1, 2002; 283(4): E824 - E835. [Abstract] [Full Text] [PDF]
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S. Patel, P. A. Lochhead, G. Rena, S. Fumagalli, M. Pende, S. C. Kozma, G. Thomas, and C. Sutherland Insulin Regulation of Insulin-like Growth Factor-binding Protein-1 Gene Expression Is Dependent on the Mammalian Target of Rapamycin, but Independent of Ribosomal S6 Kinase Activity J. Biol. Chem., March 15, 2002; 277(12): 9889 - 9895. [Abstract] [Full Text] [PDF]
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H. Tang, E. Hornstein, M. Stolovich, G. Levy, M. Livingstone, D. Templeton, J. Avruch, and O. Meyuhas Amino Acid-Induced Translation of TOP mRNAs Is Fully Dependent on Phosphatidylinositol 3-Kinase-Mediated Signaling, Is Partially Inhibited by Rapamycin, and Is Independent of S6K1 and rpS6 Phosphorylation Mol. Cell. Biol., December 15, 2001; 21(24): 8671 - 8683. [Abstract] [Full Text] [PDF]
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B. R. Dorn, W. A. Dunn Jr., and A. Progulske-Fox Porphyromonas gingivalis Traffics to Autophagosomes in Human Coronary Artery Endothelial Cells Infect. Immun., September 1, 2001; 69(9): 5698 - 5708. [Abstract] [Full Text] [PDF]
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A. Donati, G. Cavallini, C. Paradiso, S. Vittorini, M. Pollera, Z. Gori, and E. Bergamini Age-Related Changes in the Regulation of Autophagic Proteolysis in Rat Isolated Hepatocytes J. Gerontol. A Biol. Sci. Med. Sci., July 1, 2001; 56(7): B288 - 293. [Abstract] [Full Text] [PDF]
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B. Raught, A.-C. Gingras, and N. Sonenberg The target of rapamycin (TOR) proteins PNAS, June 19, 2001; 98(13): 7037 - 7044. [Abstract] [Full Text] [PDF]
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C. J. Lynch Role of Leucine in the Regulation of mTOR by Amino Acids: Revelations from Structure-Activity Studies J. Nutr., March 1, 2001; 131(3): 861S - 865. [Abstract] [Full Text]
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A. Petiot, E. Ogier-Denis, E. F. C. Blommaart, A. J. Meijer, and P. Codogno Distinct Classes of Phosphatidylinositol 3'-Kinases Are Involved in Signaling Pathways That Control Macroautophagy in HT-29 Cells J. Biol. Chem., January 14, 2000; 275(2): 992 - 998. [Abstract] [Full Text] [PDF]
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O. J. Shah, D. A. Antonetti, S. R. Kimball, and L. S. Jefferson Leucine, Glutamine, and Tyrosine Reciprocally Modulate the Translation Initiation Factors eIF4F and eIF2B in Perfused Rat Liver J. Biol. Chem., December 17, 1999; 274(51): 36168 - 36175. [Abstract] [Full Text] [PDF]
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U. Krause, M. H. Rider, and L. Hue Protein Kinase Signaling Pathway Triggered by Cell Swelling and Involved in the Activation of Glycogen Synthase and Acetyl-CoA Carboxylase in Isolated Rat Hepatocytes J. Biol. Chem., July 12, 1996; 271(28): 16668 - 16673. [Abstract] [Full Text] [PDF]
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S. Arico, A. Petiot, C. Bauvy, P. F. Dubbelhuis, A. J. Meijer, P. Codogno, and E. Ogier-Denis The Tumor Suppressor PTEN Positively Regulates Macroautophagy by Inhibiting the Phosphatidylinositol 3-Kinase/Protein Kinase B Pathway J. Biol. Chem., September 14, 2001; 276(38): 35243 - 35246. [Abstract] [Full Text] [PDF]
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S. Mordier, C. Deval, D. Bechet, A. Tassa, and M. Ferrara Leucine Limitation Induces Autophagy and Activation of Lysosome-dependent Proteolysis in C2C12 Myotubes through a Mammalian Target of Rapamycin-independent Signaling Pathway J. Biol. Chem., September 15, 2000; 275(38): 29900 - 29906. [Abstract] [Full Text] [PDF]
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E. Ogier-Denis, S. Pattingre, J. El Benna, and P. Codogno Erk1/2-dependent Phosphorylation of Galpha -interacting Protein Stimulates Its GTPase Accelerating Activity and Autophagy in Human Colon Cancer Cells J. Biol. Chem., December 8, 2000; 275(50): 39090 - 39095. [Abstract] [Full Text] [PDF]
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J. Rohde, J. Heitman, and M. E. Cardenas The TOR Kinases Link Nutrient Sensing to Cell Growth J. Biol. Chem., March 23, 2001; 276(13): 9583 - 9586. [Abstract] [Full Text] [PDF]
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