(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 [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.
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
[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).
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
massml
), 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.
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
[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).
Figure 2:
Effect
of rapamycin on the phosphorylation of ribosomal protein S6 in isolated
rat hepatocytes. Hepatocytes (5 mg of dry
massml
), 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.
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
massml
) 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), 10
M; 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.
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
[C]sucrose confirmed that rapamycin stimulates
the autophagic process (Fig. 5).
Figure 4:
Effect of rapamycin on autophagic
proteolysis. Hepatocytes (5-10 mg of dry
massml
) 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.
Figure 5:
Effect of rapamycin on
[C]sucrose sequestration.
[
C]Sucrose-loaded hepatocytes (15 mg of dry
mass
ml
) 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).
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 [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.
Figure 6:
Amino acids do not affect S6
dephosphorylation. Hepatocytes were incubated under iso-osmotic
conditions with [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.
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, initial
autophagic vacuole; AV
, degradative
autophagic vacuole.
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.