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.

Activation of ERK1/2 stimulates macroautophagy in the human colon cancer cell line HT-29 by favoring the phosphorylation of the Galpha-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 Ser(259), 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.

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)(2)(3)(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)(14)(15). Similar to other cytoplasmic membrane dynamics, macroautophagy is controlled by cytoplasmic GTPases (16,17). When the cytoplasmic trimeric G i3 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␣ 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 Gprotein 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 (Ser 151 ) in the RGS domain of GAIP (22). The phosphorylation of Ser 151 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).
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.

EXPERIMENTAL PROCEDURES
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 XPress TM system synthetic oligonucleotides were from Invitrogen (Cergy Pontoise, * 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. This 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.
Amino Immunoprecipitation of GAIP-Metabolic labeling of HT-29 cells with 0.25 mCi/ml [ 32 P]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 M), EGF (100 ng/ml), or 4 ϫ 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. 32 P-Labeled cell extract was lysed overnight. Anti-GAIP polyclonal antibody (1:200) was bound to protein A-Sepharose. 32 P-Labeled lysates were incubated with the anti-GAIP antibody⅐protein 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 Ϫ80°C. The same blot was used to perform immunoblot experiments with anti-GAIP antibody and developed using the ECL kit.
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 ϫ AA, 100 M ATA, 100 ng/ml EGF, and 100 nM okadaic acid in different combinations. Lysates were then submitted to immunoprecipitation with 2 g of anti-Raf-1 antibody. Protein G-agarose was pelleted and incubated with 0.4 g of inactivated MEK1 and 1 g of inactivated ERK2. After a 30-min incubation at 30°C, the supernatant was incubated with myelin basic protein and [␥-32 P]ATP (5 Ci) for 10 min at 30°C. Thereafter, aliquots were spotted on P-81 paper and counted.
Macroautophagic Parameters-Measurement of the degradation of [ 14 C]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 Ci/ml L-[ 14 C]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 ϫ 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 ϫ g for 10 min and then dissolved in 250 l of Soluene 350. The rate of protein degradation was calculated as acid-soluble radioactivity recovered from both cells and the medium.
Statistical Analysis-Statistical analysis of differences between groups was performed using Student's t test. p Ͻ 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 Ser 151 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 phosphoryla-tion 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 [ 14 C]valine in complete medium, and protein degradation was analyzed in nutrient-deprived medium (HBSS) as described under "Experimental Procedures." ATA induced a dosedependent increase in [ 14 C]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␣ 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).
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 Thr 308 , 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 Ser 259 (34), we investigated the PKBdependent phosphorylation of Raf-1 in HT-29 cells after EGF treatment. As shown in Fig. 3A, EGF treatment prolonged Raf-1 phosphorylation at Ser 259 . To verify that the phosphorylation of Raf-1 at Ser 259 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 Ser 259 . Furthermore, expression of a constitutively active form of PKB ( myr PKB) 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 Ser 259 and a more sustained activation of ERK1/2. The dephosphorylation of Raf-1 at Ser 259 is in agreement with recent data reporting that Raf-1 activation depends on the release of inhibition through Ser 259 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 Thr 389 , a phosphorylation site essential for the activation of p70 S6 kinase (36). In contrast, EGF treatment induced phosphorylation of Thr 389 , 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 PKBdependent 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 Ser 259 (data not shown). Accordingly, inhibition of the Raf-1 activity was also observed in cells transfected with a constitutively active form of PKB ( myr PKB). 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 Ser 259 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 Ser 259 (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.

DISCUSSION
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␣ i3 protein (22). In human intestinal cells, amino acids can suppress macroautophagy through their interaction with two signaling path- ways: their stimulatory effect on the mTOR signaling pathway 2 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)(44)(45). Interestingly, in renal cells, the suppression of proteolysis by EGF is dependent upon the activation of the class I PI3K pathway (46).
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 Ser 259 . 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 downregulation 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.