From INSERM U504, Glycobiologie et Signalisation Cellulaire, 16, avenue Paul-Vaillant-Couturier, 94807 Villejuif Cedex, France
Received for publication, October 28, 2002, and in revised form, January 31, 2003
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ABSTRACT |
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Activation of ERK1/2 stimulates macroautophagy in
the human colon cancer cell line HT-29 by favoring the phosphorylation
of the G Macroautophagy is a lysosomal catabolic route characterized by the
sequestration of cytoplasmic material by a membrane of uncertain origin
to form an autophagosome that can receive input of material from the
endocytic pathway before its fusion with the lysosomal compartment
(1-4). There is now mounting evidence for the importance of
macroautophagy in physiological processes such as the formation of
pulmonary surfactant (5) and maturation of erythrocytes (6) and for its
implication in tumor progression (7), cardiomyopathy (8), and
programmed cell death (9). In recent years, our understanding of the
molecular controls of macroautophagy has greatly increased because of
the identification of a set of genes conserved from yeast to human that
are involved in the signaling and formation of autophagic vacuoles
(10-12). However, apg/aut genes are not the only actors
playing a role in macroautophagy that demonstrate the complexity of
this cell process. Phosphoinositide 3-kinase enzymes are involved both
in the signaling and formation of autophagic vacuoles (13-15). Similar to other cytoplasmic membrane dynamics, macroautophagy is controlled by
cytoplasmic GTPases (16, 17). When the cytoplasmic trimeric Gi3 protein is bound to GDP, the formation of autophagic
vacuoles is stimulated in human colon cancer HT-29 cells, whereas the
binding of GTP to the G In this work, we investigated whether or not amino acids are able to
control the ERK1/2 signaling pathway ending with the phosphorylation of
GAIP. The results reported here demonstrate that amino acids are able
to control the activity of the Raf-1 kinase, which acts upstream of the
MEK1/2 kinases in the ERK1/2 signaling pathway (25, 26). Our results
demonstrate a novel target for amino acid signaling in human intestinal
cells and underline the cross-talk existing between amino acid
signaling and trimeric G-proteins in the regulation of macroautophagy.
Reagents--
3-MA and all other chemicals were purchased from
Sigma. Cell culture reagents, the eukaryotic expression vectors
pcDNA3 and pcDNA3.1/c-Myc/HisC-term, and the
XPressTM system synthetic oligonucleotides were from
Invitrogen (Cergy Pontoise, France). Nitrocellulose membranes were from
Schleicher & Schüll (Dassel, Germany).
The radioisotopes L-[U-14C]valine (288.5 mCi/mmol), [ Cell Culture and Transfection of HT-29 Cells--
HT-29 cells
were cultured as previously described (18). Plasmid
pKR5-Myc-Ha-Ras(S17N) was a generous gift from Alan Hall (Medical
Research Council, Laboratory for Molecular Cell Biology, University College London, London, UK). Plasmids
pKR5-Myc-Ha-Ras(G12V,T35S), pKR5-Myc-Ha-Ras(G12V,E37G), and
pKR5-Myc-Ha-Ras(G12V,Y40C) were kindly provided by Luzheng Xue and
Aviva Tolkovsky (Department of Biochemistry, University of Cambridge,
Cambridge, UK). Plasmid pCMV6-HA-myrPKB was a generous gift
from Thomas F. Franke (Columbia University, New York, NY). Five µg of
cDNA constructions were transfected into exponentially growing
HT-29 cells using the Superfect kit according to the supplier's
recommendations. Cells were used 72 h after transfection.
Amino Acid Mixture--
The final concentration of amino acids
in the mixture was a multiple (4 × AA) of the preprandial
rat plasma concentration in the portal vein: 60 µM
asparagine, 100 µM isoleucine, 250 µM leucine, 300 µM lysine, 40 µM methionine,
50 µM phenylalanine, 100 µM proline,
180 µM threonine, 70 µM tryptophan, 180 µM valine, 400 µM alanine, 30 µM aspartate, 100 µM glutamate, 350 µM glutamine, 300 µM glycine, 60 µM cysteine, 60 µM histidine, 200 µM serine, 75 µM tyrosine, and 100 µM ornithine.
Immunoprecipitation of GAIP--
Metabolic labeling of HT-29
cells with 0.25 mCi/ml [32P]orthophosphoric acid was
carried out for 3 h in nutrient-free medium (HBSS) in the presence
or absence of amino acids. When required, ATA (100 µM),
EGF (100 ng/ml), or 4 × AA were added at the beginning of the
labeling period. Cells were then rinsed three times with phosphate-buffered saline and scraped into buffer A (20 mM
Tris (pH 7.5), 150 mM NaCl, 0.25 M sucrose, 5 mM EDTA, 5 mM EGTA, and 0.5% Triton X-100)
containing a mixture of protease and phosphatase inhibitors.
32P-Labeled cell extract was lysed overnight. Anti-GAIP
polyclonal antibody (1:200) was bound to protein A-Sepharose.
32P-Labeled lysates were incubated with the anti-GAIP
antibody·protein A-Sepharose complex for 16 h at 4 °C.
Sepharose beads were washed three times with buffer A. Thereafter,
immunoprecipitates were analyzed by SDS-PAGE, transferred onto a
nitrocellulose membrane, and exposed to Eastman Kodak X-Omat film for
16 h at Immunoblotting--
HT-29 cells were scraped in lysis buffer (20 mM Tris (pH 7.5), 150 mM NaCl, 0.25 M sucrose, 5 mM EDTA, 5 mM EGTA,
0.5% Triton X-100, 10 µg/ml leupeptin, 10 µg/ml pepstatin, and 10 µg/ml aprotinin). The lysate was clarified by centrifugation at
500 × g for 15 min at 4 °C. One-hundred µg of
proteins were submitted to SDS-PAGE and transferred to nitrocellulose.
The membrane was incubated for 1 h in Tris-buffered saline (25 mM Tris (pH 7.5), 150 mM NaCl, and 0.05%
Tween 20) containing 5% nonfat dry milk. Primary antibodies were
incubated overnight at 4 °C in Tris-buffered saline supplemented with 2% bovine serum albumin. Antibodies were used at a 1:1000 dilution, except anti-phospho-ERK1/2 antibody, which was used at
a 1:4000 dilution. After three washes with Tris-buffered saline, membranes were incubated for 1 h at room temperature with the appropriate horseradish peroxidase-labeled secondary antibody (1:5000).
Bound antibodies were detected by enhanced chemiluminescence.
Raf-1 Activity--
The assay of Raf-1 activity was done
according to the supplier's instructions (Upstate
Biotechnology, Inc.). When required, parental or transfected HT-29
cells were incubated for the indicated time (see "Results") at
37 °C with 4 × AA, 100 µM ATA, 100 ng/ml EGF,
and 100 nM okadaic acid in different combinations. Lysates were then submitted to immunoprecipitation with 2 µg of anti-Raf-1 antibody. Protein G-agarose was pelleted and incubated with 0.4 µg of
inactivated MEK1 and 1 µg of inactivated ERK2. After a 30-min incubation at 30 °C, the supernatant was incubated with myelin basic
protein and [ Macroautophagic Parameters--
Measurement of the degradation
of [14C]valine-labeled long-lived proteins and lactate
dehydrogenase sequestration was monitored as reported previously
(18, 27).
Analysis of Protein Degradation--
HT-29 cells were incubated
for 18 h at 37 °C with 0.2 µCi/ml
L-[14C]valine. Unincorporated radioisotope
was removed by three rinses with phosphate-buffered saline (pH 7.4).
Cells were then incubated in nutrient-free medium (without amino acids
and in the absence of fetal calf serum) plus 0.1% bovine serum albumin
and 10 mM unlabeled valine. When required, 10 mM 3-MA, a potent inhibitor of the formation of autophagic
vacuoles (28), or 4 × AA were added throughout the chase period.
After the first hour of incubation, at which time short-lived proteins
were being degraded, the medium was replaced with the appropriate fresh
medium, and the incubation was continued for an additional 4-h period.
Cells and radiolabeled proteins from the 4-h chase medium were
precipitated in 10% (v/v) trichloroacetic acid at 4 °C. The
precipitated proteins were separated from soluble radioactivity by
centrifugation at 600 × g for 10 min and then
dissolved in 250 µl of Soluene 350. The rate of protein degradation
was calculated as acid-soluble radioactivity recovered from both cells
and the medium.
Analysis of Lactate Dehydrogenase Sequestration--
Briefly,
cells were gently washed three times with phosphate-buffered saline (pH
7.4) and then twice with homogenization buffer (50 mM
potassium phosphate, 1 mM EDTA, 1 mM
dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 300 mM sucrose, 100 µg/ml bovine serum albumin, and 0.01%
Tween 20 (pH 7.5)). Cells were homogenized in 1 ml of cold
homogenization buffer by 13 strokes in a glass/Teflon homogenizer on
ice. A post-nuclear supernatant was prepared by centrifugation at
300 × g for 10 min at 4 °C. Post-nuclear material was layered on 4 ml of buffered metrizamide/sucrose (10% sucrose, 8%
metrizamide, 1 mM EDTA, 100 µg/ml bovine serum albumin,
and 0.01% Tween 20 (pH 7.5)) and centrifuged at 7000 × g for 60 min. Finally, the pellet was washed once with
homogenization buffer and resuspended in buffer containing 2 mM Tris-HCl (pH 7.4), 50 mM mannitol, 1 mM phenylmethylsulfonyl fluoride, 0.5 µg/ml leupeptin, 0.1 µg/ml aprotinin, and 0.7 µg/ml pepstatin. The suspension was sonicated (VibraCellTM Model 72434 sonicator, power setting
3, microtip, 20 s at 20% charge) and centrifuged at 10,000 × g for 10 min. The lactate dehydrogenase activity was
assayed by measuring the oxidation of NADH with pyruvate as substrate
at 340 nm.
Statistical Analysis--
Statistical analysis of differences
between groups was performed using Student's t test.
p < 0.005 was considered statistically significant.
Effect of the Stimulation of the ERK1/2 Pathway on the
Phosphorylation of GAIP--
We have previously shown that the
phosphorylation of Ser151 in the RGS domain of GAIP is
dependent upon the activity of ERK1/2 and is sensitive to amino acids
(22). To investigate the role of the Ras/Raf-1/MEK/ERK1/2 signaling
pathway in the phosphorylation of GAIP, cells were treated with either
EGF, a peptidic physiological activator (29), or ATA, a non-peptidic
activator of the MEK/ERK1/2 signaling pathway (30-32). According to
their effects on the tyrosine phosphorylation cascade, both EGF and ATA
increased the profile of tyrosine-phosphorylated proteins in HT-29
cells (Fig. 1A). However, ATA
and EGF had different effects on the phosphorylation of GAIP (Fig.
1B). ATA induced an increase in GAIP phosphorylation that was sensitive to the presence of amino acids. In contrast, EGF did not significantly modify the phosphorylation status of GAIP
compared with untreated cells. In agreement with our previous data
(22), the phosphorylation of GAIP was greatly reduced in EGF-treated
cells (Fig. 1B) and in control cells (data not shown).
ATA (but Not EGF) Stimulates Macroautophagy--
As we have
previously shown that ERK1/2-dependent phosphorylation of
GAIP controls macroautophagy in HT-29 cells (22), we next investigated
the effect of ATA and EGF on macroautophagy (Fig.
2). For this purpose, cells were
radiolabeled overnight with [14C]valine in complete
medium, and protein degradation was analyzed in nutrient-deprived
medium (HBSS) as described under "Experimental Procedures." ATA
induced a dose-dependent increase in
[14C]valine-labeled protein degradation (Fig.
2A). 3-MA, a well known inhibitor of the formation of
autophagic vacuoles (28), inhibited the increase in proteolysis induced
by ATA. The stimulatory effect of ATA on the macroautophagic pathway
was confirmed by assay of the sequestration of the cytosolic enzyme
lactate dehydrogenase into sedimentable material enriched in autophagic
vacuoles (Table I). In agreement with our
previous results (22), amino acids were as efficient as 3-MA in
inhibiting autophagy in HT-29 cells. Amino acids also reversed the
ATA-dependent stimulation of autophagy (Fig. 2 and Table
I). However, amino acids were less potent than 3-MA in competing with
ATA, suggesting that ATA can increase autophagy by mechanisms that are
sensitive and insensitive to amino acids (see below).
From the results described above and those previously reported on the
role of GAIP and the G EGF (but Not ATA) Elicits Cross-talk between Akt/PKB and
Raf-1 Pathways--
To understand the difference in macroautophagic
response after ATA or EGF challenge, we investigated their interference
with signal transduction (Fig. 3). After
treatment with EGF, we observed a rapid stimulation of the activation
of ERK1/2, followed by an attenuation phase. EGF also elicited a rapid
activation of Akt/PKB within 5 min of treatment as determined by its
phosphorylation of Thr308, a known regulatory site
essential for PKB activation (33). As PKB has been proposed to be a
kinase involved in the inactivating phosphorylation of Raf-1 at
Ser259 (34), we investigated the PKB-dependent
phosphorylation of Raf-1 in HT-29 cells after EGF treatment. As shown
in Fig. 3A, EGF treatment prolonged Raf-1
phosphorylation at Ser259. To verify that the
phosphorylation of Raf-1 at Ser259 was dependent upon the
activity of PKB, we repeated these experiments in the presence of the
PI3K inhibitor wortmannin (100 nM) (Fig. 3B). In
the presence of wortmannin, we observed an inhibition of PKB
phosphorylation and also an inhibition of Raf-1 phosphorylation at
Ser259. Furthermore, expression of a constitutively active
form of PKB (myrPKB) had an inhibitory effect on the
activity of Raf-1 (see Fig. 4).
ATA stimulated the activity of ERK1/2 with kinetics close to those
observed after EGF challenge (Fig. 3A). However, in contrast to EGF, (i) the ATA-dependent activation of ERK1/2 lasted
for a longer time (4 h versus 1 h), and (ii) ATA did
not activate PKB at early time points. Stimulation of PKB was only
detectable after 1 h. According to these results, we observed a
time-dependent dephosphorylation of Raf-1 at
Ser259 and a more sustained activation of ERK1/2. The
dephosphorylation of Raf-1 at Ser259 is in agreement with
recent data reporting that Raf-1 activation depends on the release of
inhibition through Ser259 dephosphorylation (35).
To analyze more precisely the effect of EGF and ATA on the class I PI3K
pathway, we investigated the activation of p70 S6 kinase, a downstream
target of the class I PI3K signaling pathway. ATA did not induce
phosphorylation of Thr389, a phosphorylation site essential
for the activation of p70 S6 kinase (36). In contrast, EGF treatment
induced phosphorylation of Thr389, suggesting that EGF (but
not ATA) is able to activate p70 S6 kinase.
From these results, we conclude that ATA stimulates macroautophagy
because it activates the ERK1/2 signaling pathway and only marginally
affects the PI3K signaling pathway. In contrast, the limited effect of
EGF is probably due to its capacity to stimulate two pathways known to
have opposite effects on macroautophagy in intestinal cells: the PI3K
pathway, which has an inhibitory effect, and the ERK1/2 signaling
pathway, which has a stimulatory effect. In addition, the
PKB-dependent inhibition of Raf-1 together with the
activation of p70 S6 kinase could be responsible for the low inhibitory
effect of EGF on macroautophagy observed in some experiments.
Effect of Amino Acids on Ras-dependent Signaling
Pathways--
The MAPK/ERK1/2 pathway starts with the membrane
recruitment of the Raf-1 kinase by GTP-bound Ras (25, 26). To
investigate whether amino acids are able to interfere with the function
of Ras, we analyzed the effect of amino acids on the kinase activity of
Raf-1 in cells transfected with mutants of Ras known to stimulate different signaling pathways (Fig. 4).
The first mutant we used was Ras(G12V,T35S), which specifically
activates the Raf-1/MEK/ERK1/2 pathway (37). In cells transfected with
the cDNA encoding Ras(G12V,T35S), we observed a stimulation of the
Raf-1 activity. In the presence of amino acids, the Raf-1 activity was
dramatically reduced in Ras(G12V,T35S)-expressing cells. This
inhibition was in the same range as that observed in HT-29 cells (see
Fig. 6).
In cells expressing either the dominant-negative Ras(S17N) mutant or
the PI3K pathway-stimulating Ras(G12V,Y40C) mutant (38), we observed a
reduction in the Raf-1 activity. The inhibitory effect of
Ras(G12V,Y40C) is probably due to the activation of PKB, which
phosphorylated Raf-1 at Ser259 (data not shown).
Accordingly, inhibition of the Raf-1 activity was also observed in
cells transfected with a constitutively active form of PKB
(myrPKB). As a control, the Ras(G12V,E37G) mutant, which
activates the Ral proteins (39) without affecting either the PI3K or
MAPK/ERK1/2 pathway, did not modify the activity of Raf-1.
The Ras(G12V,T35S) Mutant Stimulates Both GAIP Phosphorylation and
Autophagy in an Amino Acid-dependent Manner--
The
results reported above led us to investigate both GAIP phosphorylation
and autophagy in HT-29 cells expressing the Ras(G12V,T35S) mutant. As
expected, ERK1/2 was activated in Ras(G12V,T35S)-expressing cells,
whereas no activation was observed in HT-29 cells transfected with an
empty vector (Fig. 5A,
upper panel). GAIP phosphorylation was
significantly increased in Ras(G12V,T35S)-expressing cells compared
with control cells (Fig. 5A, lower panel). Both
the stimulation of GAIP phosphorylation and the activation of ERK1/2
were severely impaired in the presence of amino acids (Fig.
5A). According to these results, the rate of proteolysis was
increased in Ras(G12V,T35S)-expressing cells compared with HT-29 cells
transfected with an empty vector (Fig. 5B), and both amino
acids and 3-MA had the same potency in inhibiting
Ras(G12V,T35S)-dependent proteolysis. These results suggest
that amino acids interfere with GAIP-dependent
macroautophagy by targeting an element of the ERK1/2 signaling
pathway.
Raf-1 Is the Target of Amino Acids in the ERK1/2
Pathway--
The results reported above led us to consider the
possibility that amino acids interfere with the activity of Raf-1.
Amino acids were able to reverse the activation of Raf-1 in a
dose-dependent manner (Fig.
6). In the experiments reported above,
the total amount of Raf-1 detected by Western blotting was equivalent
in both the presence and absence of AA (data not shown). Accordingly, the activation of MEK, which is the kinase acting downstream of Raf-1
and is responsible for the activation of ERK1/2, was sensitive to the
presence of amino acids (Fig. 7). Amino
acids also partially reversed the ATA-dependent activation
of Raf-1 (Fig. 6). This incomplete inhibition is in agreement with the
partial inhibition of ATA-dependent macroautophagy
by amino acids (Fig. 2 and Table I). These results suggest that ATA
interferes with macroautophagy and the Raf-1-dependent
signaling pathway by amino acid-dependent and amino
acid-independent mechanisms.
Amino Acids and Okadaic Acid Have a Similar Effect on the
Activation of Raf-1--
The effect of amino acids on the
phosphorylation of Raf-1 at Ser259 can be due either to the
stimulation of phosphorylation or to the impairment of
dephosphorylation. However, our previous results discredit a role for
PKB in the amino acid-sensitive inhibition of Raf-1 because amino acids
do not stimulate the activity of PKB in HT-29 cells (40). Okadaic acid,
which specifically inhibits PP2A when used at 100 nM (41),
has been shown to interfere with Raf-1 activation by maintaining the
hyperphosphorylation of Ser259 (42). Okadaic acid at 100 nM impaired the ATA-dependent activation of
Raf-1 (Fig. 8). In fact, okadaic acid and
AA inhibited Raf-1 activation to the same extent. No further inhibition
was observed when okadaic acid and AA were added in combination,
suggesting that they can act on the same target to interfere with the
activation of Raf-1.
The role of amino acids as intermediates in metabolic pathways is
well known; but recently, their role in signal transduction has
been described (23). Amino acids have been reported to inhibit macroautophagy by interfering with the stimulation of mTOR and the
phosphorylation of its downstream effector p70 S6 kinase (24). Here we
have shown that amino acids interfere with the ERK1/2 signaling-dependent control of macroautophagy in human
colon cancer cells. Activation of this pathway increases the
macroautophagic capacities of HT-29 cells via phosphorylation of the
GTPase-activating protein GAIP and the accumulation of the GDP-bound
form of the G By both a pharmacological (ATA) and a genetic (Ras(G12V,T35S))
approach, we have demonstrated that amino acids interfere with the
activation of Raf-1 and consequently with that of its downstream effectors MEK and ERK1/2. In addition, the use of the Ras(G12V,T35S) mutant led us to propose that Raf-1 is the target of amino acids in the
GAIP-dependent control of macroautophagy in human
intestinal cells.
Amino acids sustained the phosphorylation of Raf-1 at
Ser259. Dephosphorylation of this site by PP2A has been
recently shown to be a crucial step during the activation of Raf-1
(42). The effect of amino acids on the activation of Raf-1 is
reminiscent of that observed in the presence of okadaic acid, a PP2A
inhibitor. This result suggests that amino acids can impair the
PP2A-dependent dephosphorylation of Raf-1. Although this
has to be firmly demonstrated, the results reported in Fig. 8 suggest
that amino acids and okadaic acid can act on the same target to inhibit
Raf-1 activation. Our working model is that amino acids can modulate
either the interaction between Raf-1 and PP2A or the activity of PP2A.
In this context, it is interesting to note that amino acids have been
proposed to inhibit the dephosphorylation of p70 S6 kinase by
decreasing the activity of PP2A by an mTOR-dependent
restraining mechanism (47). This suggests that the control of PP2A
activity could be a pivotal amino acid target to control both
stimulatory and inhibitory signaling of macroautophagy. Amino
acid-dependent inactivation of PP2A would suppress
macroautophagy by down-regulating ERK1/2 signaling and by up-regulating
mTOR/p70 S6 kinase signaling. This hypothesis is in line with the
inhibitory effect of okadaic acid on the macroautophagic pathway in
liver cells (48) as well as in human intestinal cell lines (49).
However, inhibition of other PP2A-dependent events such as
the control of the intermediate filament network cannot be excluded in
the inhibition of the formation of autophagic vacuoles as suggested in
liver cells (50, 51).
Whether or not the ERK1/2 signaling control of macroautophagy can be
extended to other cell lines and tissues remains to be investigated. In
liver cells, the p38 MAPK pathway controls the regulation of
macroautophagy by cell volume, but ERK1/2 does not seem to be involved
in this process (52). However, the control of macroautophagy by ERK1/2
signaling is relevant to different biological situations in the
intestinal tract. Activation of the ERK1/2 signaling pathway has been
shown to regulate intestinal epithelial differentiation (53).
Loss of activation of ERK1/2 is an important event during the
differentiation process of HT-29 cells in goblet cells and enterocytes.
This down-regulation of ERK1/2 is in agreement with the previously
reported decrease in macroautophagy during the differentiation process
of HT-29 cells (49). In addition, blockade of the ERK1/2 pathway
suppresses the growth of colon tumors and the invasiveness of HT-29
cells (54). Recent evidence suggests that activation of the signaling pathway via Ki-Ras mutation can be a route for the development of
intestinal cancer in animal models (55).
It is tempting to speculate that the suppression of macroautophagy
correlates with the loss of proliferative capacities in these tumors.
According to this hypothesis, macroautophagy is more active in
proliferating than in non-proliferating cultured human colon cancer
cells (27, 49). It is largely admitted that tumorigenesis and
proliferation have a down-regulating effect on macroautophagy (4, 7).
However, the situation may be more complex because macroautophagy can
depend upon cell-specific signaling pathways and upon the variable
expression of proteins with tumor suppressor and oncogenic properties
in tumors from different origins (reviewed in Ref. 56). Unraveling the
mechanism of amino acid-dependent Raf-1 activation will be
important to better understand the role of the ERK1/2 signaling pathway
in the control and function of intestinal macroautophagy during
differentiation and tumor progression.
-interacting protein (GAIP) in an amino
acid-dependent manner (Ogier-Denis, E., Pattingre, S., El
Benna, J., and Codogno, P. (2000) J. Biol. Chem. 275, 39090-39095). Here we show that ERK1/2 activation by
aurintricarboxylic acid (ATA) treatment induces the phosphorylation of
GAIP in an amino acid-dependent manner. Accordingly, ATA
challenge increased the rate of macroautophagy, whereas epidermal
growth factor did not significantly affect macroautophagy and GAIP
phosphorylation status. In fact, ATA activated the ERK1/2 signaling
pathway, whereas epidermal growth factor stimulated both the ERK1/2
pathway and the class I phosphoinositide 3-kinase pathway, known to
decrease the rate of macroautophagy. Amino acids interfered with the
ATA-induced macroautophagy by inhibiting the activation of the kinase
Raf-1. The role of the Ras/Raf-1/ERK1/2 signaling pathway in the GAIP-
and amino acid-dependent control of macroautophagy was
confirmed in HT-29 cells expressing the Ras(G12V,T35S) mutant.
Similar to the protein phosphatase 2A inhibitor okadaic acid, amino
acids sustained the phosphorylation of Ser259, which is
involved in the negative regulation of Raf-1. In conclusion, these
results add a novel target to the amino acid
signaling-dependent control of macroautophagy in intestinal cells.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
i3 protein inhibits the
formation of autophagic vacuoles (18, 19). More recently, we have shown
that GAIP,1 a protein
belonging to the RGS (regulator of G-protein
signaling) family (20), stimulates the macroautophagic
pathway by accelerating the GTPase activity of the G
i3
protein (21). Interestingly, the activity of GAIP for the
G
i3 protein has been shown to be dependent upon its
state of phosphorylation at a conserved serine residue
(Ser151) in the RGS domain of GAIP (22). The
phosphorylation of Ser151 has been shown in cultured HT-29
cells and in vitro to be dependent upon the activity of the
MAPKs ERK1 and ERK2. In addition, the phosphorylation of GAIP is
impaired by amino acids that are strong physiological inhibitors of
macroautophagy (22). Several different mechanisms can account for the
inhibitory effect of amino acids on macroautophagy in mammalian cells
(3, 23). Their inhibitory effect on the formation of autophagic
vacuoles has been suggested to depend on their interference with the
mTOR/p70 S6 kinase signaling pathway (24).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]ATP (3000 Ci/mmol), and
[32P]orthophosphoric acid (5 mCi/ml) were from
PerkinElmer Life Sciences (Les Ulis, France). The ECLTM
Western blotting detection kit, secondary antibodies, and protein A-Sepharose was purchased from Amersham Biosciences (Les Ulis). The
alkaline phosphatase detection kit was from Bio-Rad (Marne la Coquette,
France). Rabbit anti-GAIP polyclonal antibody was obtained as described
previously (19). Mouse
anti-phospho-Thr202/Tyr204 ERK1/2
monoclonal antibody was from New England Biolabs Inc. (Beverly,
MA). Anti-phosphotyrosine antibody was a gift from Anne Nègre-Salvayre (INSERM U466). The Raf-1 immunoprecipitation
kinase assay kit was from Upstate Biotechnology, Inc. (Lake Placid,
NY). Anti-ERK1/2, anti-phospho-Ser259 Raf-1, anti-MEK,
anti-phospho-Ser217/Ser221 MEK, anti-p70 S6
kinase, anti-phospho-Thr389 p70 S6 kinase, and
anti-phospho-Thr308 Akt/PKB antibodies were from Cell
Signaling (Montigny le Bretonneux, France). Anti-Akt/PKB antibody was
from Santa Cruz Biotechnology (Santa Cruz, CA). EGF was from BD
Biosciences (Bedford, UK), and ATA was from Calbiochem
(Fontenay-Sous-Bois, France). The Superfect kit was from QIAGEN Inc.
(Les Ulis, France).
80 °C. The same blot was used to perform immunoblot
experiments with anti-GAIP antibody and developed using the ECL kit.
-32P]ATP (5 µCi) for 10 min at
30 °C. Thereafter, aliquots were spotted on P-81 paper and counted.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (35K):
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Fig. 1.
ATA (but not EGF) stimulates GAIP
phosphorylation. A, HT-29 cells were cultured overnight
in serum-free medium. Thereafter, cells were incubated for 15 min with
HBSS or with HBSS supplemented with 100 µM ATA or 100 ng/ml EGF. After SDS-PAGE and immunoblotting, phosphoproteins were
revealed with anti-phosphotyrosine antibody (1:7000). B,
upper panel, HT-29 cells were radiolabeled with 0.25 mCi/ml
[32P] orthophosphoric acid for 3 h in HBSS. ATA
(100 µM) or EGF (100 ng/ml) was added at the beginning of
the labeling period in the presence or absence of 4 × AA. GAIP
was immunoprecipitated with anti-GAIP polyclonal antibody and submitted
to SDS-PAGE followed by autoradiography (32P).
Western blotting (WB) of the immunoprecipitate was performed
using anti-GAIP antibody. Lower panel, the ratio
32P/WB was determined after scanning. Results are
representative of three experiments.
View larger version (13K):
[in a new window]
Fig. 2.
ATA (but not EGF) stimulates macroautophagy
in HT-29 cells. The rate of [14C]valine-labeled
long-lived protein degradation was measured in HT-29 cells incubated in
nutrient-free medium (HBSS) in the presence or absence of 10 mM 3-MA or 4 × AA. When used, ATA or EGF was added at
the beginning of the chase period. The values reported are the
means ± S.D. of three independent experiments.
Effects of ATA and amino acids on autophagic sequestration in HT-29
cells
i3 protein in the control of autophagic vacuole formation in HT-29 cells (18, 21, 22), we conclude
that ATA has a stimulatory effect at an early step during
autophagy via activation of the
G
i3-dependent control of macroautophagy. In
contrast, EGF has no significant effect on the rate of proteolysis
along the macroautophagic pathway in the concentration range used (Fig.
2B). This result is in agreement with the lack of an
EGF-dependent effect on GAIP phosphorylation (see Fig.
1B).
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Fig. 3.
ATA and EGF stimulate the ERK1/2 pathway, but
only EGF activates the PI3K pathway. A, HT-29 cells
were incubated for 4 h in HBSS. When used, 100 µM
ATA or 100 ng/ml EGF was added for the indicated times. B,
HT-29 cells were incubated for 4 h in HBSS. When used, 100 ng/ml
EGF and 100 nM wortmannin (Wn) were added for
the indicated times. After SDS-PAGE and Western blotting, proteins were
revealed by immunodetection (anti-phospho (P)-ERK1/2
antibody, 1:4000; all other antibodies, 1:1000).
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Fig. 4.
Effect of amino acids and Ras
mutants on the activity of Raf-1. Proliferating HT-29 cells were
transfected with 5 µg of cDNA encoding myrPKB
or different Ras mutants: Ras(S17N), Ras(G12V,E37G),
Ras(G12V,T35S), and Ras(G12V,Y40C). Immunoprecipitations were
performed using anti-Raf-1 antibody in parental HT-29 cells (control
cells) and in the different cell populations 72 h after
transfection. Beads were incubated for 30 min at 30 °C with 0.4 µg
of inactivated MEK1 and 1 µg of inactivated ERK2. The resulting
supernatant was incubated for 10 min at 30 °C with myelin
basic protein and [ -32P]ATP and the level of
[32P] incorporation in myelin basic protein was
determined. The values reported are the means ± S.D. of three
independent experiments.
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Fig. 5.
Effect of the Ras(G12V,T35S) mutant on GAIP
phosphorylation and autophagy. A, upper
panels, HT-29 cells transfected with an empty vector or the
Ras(G12V,T35S) mutant were incubated for 4 h in HBSS in the
presence or absence of 4 × AA. After SDS-PAGE and Western
blotting, proteins were revealed by immunodetection (anti-phospho
(P)-ERK1/2 antibody, 1:4000; and anti-ERK1/2 antibody,
1:1000). Lower panels, HT-29 cells transfected with an empty
vector or the Ras(G12V,T35S) mutant were radiolabeled with 0.25 mCi/ml
[32P]orthophosphoric acid for 3 h in HBSS in the
presence or absence of 4 × AA. GAIP was immunoprecipitated with
anti-GAIP polyclonal antibody and submitted to SDS-PAGE followed by
autoradiography (32P). Western blotting
(WB) of the immunoprecipitate was performed using anti-GAIP
antibody. The ratio 32P/WB was determined after scanning.
B, the rate of [14C]valine-labeled long-lived
protein degradation was measured in HT-29 cells transfected with either
an empty vector or the Ras(G12V,T35S) mutant. Cells were incubated in
nutrient-free medium (HBSS) in the absence or presence of 10 mM 3-MA or 4 × AA. The values reported are the
means ± S.D. of three independent experiments.
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Fig. 6.
Amino acids inhibit Raf-1 activation.
HT-29 cells were incubated for 4 h at 37 °C in nutrient-free
medium (HBSS) in the presence or absence of 1 or 4 × AA. When
required, ATA was added at the indicated concentration together with
amino acids. Thereafter, cell lysates were prepared, and
immunoprecipitation was performed using anti-Raf-1 antibody. Beads were
incubated for 30 min at 30 °C with 0.4 µg of inactivated MEK1 and
1 µg of inactivated ERK2. The resulting supernatant was incubated for
10 min at 30 °C with myelin basic protein and
[ -32P]ATP and the level of [32P]
incorporation in myelin basic protein was determined. The values
reported are the means ± S.D. of three independent experiments.
*, p < 0.005 compared with the control (HBSS); #,
p < 0.005 compared with 1 × AA.
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Fig. 7.
Amino acids inhibit MEK phosphorylation.
HT-29 cells were incubated for 4 h in HBSS in the presence or
absence of amino acids. After SDS-PAGE and Western blotting, proteins
were revealed by immunodetection with anti-phospho (P)-MEK
antibody, and after scraping, with anti-MEK antibody. Both antibodies
were used at a 1:1000 dilution.
View larger version (15K):
[in a new window]
Fig. 8.
Amino acids and okadaic acid have a similar
effect on Raf-1 activation. HT-29 cells were incubated for 4 h at 37 °C in nutrient-free medium (HBSS) in the presence or absence
of 4 × AA. When required, 100 µM ATA and 100 nM okadaic acid (OA) were added for 30 and 45 min, respectively. Thereafter, cell lysates were prepared, and
immunoprecipitation was performed using anti-Raf-1 antibody. Beads were
incubated for 30 min at 30 °C with 0.4 µg of inactivated MEK1 and
1 µg of inactivated ERK2. The resulting supernatant was incubated for
10 min at 30 °C with myelin basic protein and
[ -32P]ATP and the level of [32P]
incorporation in myelin basic protein was determined. The values
reported are the means ± S.D. of three independent experiments.
*, p < 0.005 compared with ATA-treated cells; #,
p < 0.05 compared with ATA- and amino acid-treated
cells.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
i3 protein (22). In human intestinal cells,
amino acids can suppress macroautophagy through their interaction with
two signaling pathways: their stimulatory effect on the mTOR signaling
pathway2 and their inhibitory
effect on the ERK1/2 signaling pathway (this work and Ref. 22).
However, activation of the ERK1/2 pathway by EGF did not greatly affect
the macroautophagic pathway in HT-29 cells. A possible explanation for
this observation is that EGF activates both positive signaling (ERK1/2)
and negative signaling (class I PI3K) controls of macroautophagy (see
Fig. 3 and Ref. 13). Cell-specific cross-talk between the MAPK and
class I PI3K pathways could explain the variability of the effect of
EGF on intracellular proteolysis and autophagy observed in different cell types (43-45). Interestingly, in renal cells, the suppression of
proteolysis by EGF is dependent upon the activation of the class I PI3K
pathway (46).
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ACKNOWLEDGEMENTS |
---|
We thank Alan Hall, Luzheng Xue, Aviva Tolkovsky, and Thomas F. Franke for the gift of plasmids encoding Ha-Ras mutants and myrPKB and Anne Nègre-Salvayre for providing anti-phosphotyrosine antibody. We are grateful to Eric Ogier-Denis, who contributed to the preliminary experiments of this work. Thanks are also due to S. E. H. Moore for critical reading of the manuscript.
![]() |
FOOTNOTES |
---|
* This work was supported in part by INSERM and by Grant P 4622 from the Association pour la Recherche sur le Cancer (to P. C.).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.
Recipient of an Association pour la Recherche sur le Cancer fellowship.
§ To whom correspondence should be addressed. Tel.: 33-1-4559-5041; Fax: 33-1-4677-0233; E-mail: codogno@vjf.inserm.fr.
Published, JBC Papers in Press, February 27, 2003, DOI 10.1074/jbc.M210998200
2 S. Pattingre, C. Bauvy, and P. Codogno, unpublished data.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
GAIP, G-interacting protein;
MAPK, mitogen-activated protein kinase;
ERK, extracellular signal-regulated kinase;
MEK, mitogen-activated protein
kinase/extracellular signal-regulated kinase kinase;
3-MA, 3-methyladenine;
PKB, protein kinase B;
EGF, epidermal growth
factor;
ATA, aurintricarboxylic acid;
AA, amino acids;
HBSS, Hanks' balanced salt solution;
PI3K, phosphoinositide 3-kinase;
myrPKB, myristoylated protein kinase B;
PP2A, protein
phosphatase 2A.
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