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Vol. 17, Issue 9, 3940-3951, September 2006
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*Institut National de la Santé et de la Recherche Médicale U36 and ||Institut National de la Santé et de la Recherche Médicale U114, Collège de France, 75231 Paris, France
Submitted June 6, 2006;
Accepted June 22, 2006
Monitoring Editor: John York
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Other key mediators of growth are the extracellular signal-regulated kinases (ERKs) 1/2. ERK1/2 belong to the MAPK family and lie downstream of the cascade of Ras/Raf/MEK kinases. The nuclear translocation of ERK1/2 is a critical step to transduce cell growth (Brunet et al., 1999
). In their phosphorylated and activated forms, ERK1/2 transmit extracellular stimuli from the plasma membrane to the nucleus by phosphorylating and activating a variety of transcription factors. Among them, Elk-1 is a key element involved in the induction of immediate early genes such as cFos (Pearson et al., 2001; Peyssonnaux and Eychene, 2001).
The stimulation of both pathways by many common ligands raises the possibility that cross-talk between the PI3K/Akt and ERK1/2 pathways could play a major role in regulating cell proliferation under particular conditions. Functional interactions between these two pathways have been reported for the regulation of various cellular functions depending on cell types. Indeed, constitutively active Akt can negatively regulate the Ras/Raf/MEF/ERK1/2 cascade via phosphorylation and inactivation of the kinase Raf (Zimmermann and Moelling, 1999), leading to the phenotype modulation of differentiated myotubes (Rommel et al., 1999) or of vascular smooth muscle cells (Reusch et al., 2001). An additional level of interaction between Akt and the ERK1/2 pathway has been reported downstream of Ras, Raf, and MEK that involves the down-regulation of the transcription factor Elk-1 (Figueroa and Vojtek, 2003; Galetic et al., 2003). To date, the molecular mechanisms and the functional cellular consequences of this cross-talk remain poorly investigated.
A good candidate for mediating direct cross-talk between Akt and ERK1/2 is the phosphoprotein enriched in astrocytes of 15 kDa, PEA-15 (also called PED). PEA-15 is a small protein abundantly present in brain astrocytes (Araujo et al., 1993) but also widely expressed in other human tissues (Danziger et al., 1995; Estelles et al., 1996; Ramos et al., 1998). PEA-15 regulates multiple cellular functions (Renault et al., 2003), including Ras suppression of integrin activation, and protection against Fas- and tumor necrosis factor 
-induced apoptosis (Condorelli et al., 1999; Estelles et al., 1999; Kitsberg et al., 1999). Besides, we have already demonstrated that PEA-15 binds to ERK1/2 and prevents their nuclear translocation, which results in blockade of ERK-dependent transcription and cell proliferation in response to serum (Formstecher et al., 2001). More recently, PEA-15 has been identified as a novel Akt substrate (Trencia et al., 2003). These authors reported that phosphorylation by Akt of PEA-15 on Ser116 determines resistance to apoptosis induced by tumor necrosis factor-related apoptosis-inducing ligand or serum deprivation. However, the balanced cross-talk between Akt, PEA-15 and ERK1/2 as well as its consequences on ERK1/2 subcellular localization and cell responses (proliferation/survival) has not been defined.
We have previously reported that angiotensin II (AngII) stimulates cell proliferation through the angiotensin type 1A receptor (AT1A) by activation of the endogenous PI3K and ERK1/2 pathways in transfected Chinese hamster ovary (CHO) cells (CHO-AT1A) and in rat aortic smooth muscle cells (Dugourd et al., 2003). In this process, endogenous Akt and ERK1/2 were independently stimulated but were both necessary for AngII-induced cell proliferation. Therefore, changes in the balance of activation between Akt and ERK1/2 could generate new cross-talk, possibly through PEA-15, and have drastic functional consequences.
The aim of this study was to analyze the effects of modifying the balance between the PI3K/Akt and ERK1/2 pathways on the proliferative response to AngII by possible cross-talk through recruitment of endogenous PEA-15. We found that Akt1 overexpression in CHO-AT1A decreased cell proliferation induced by AngII. Investigating the molecular mechanisms lying between Akt and PEA-15 revealed that Akt bound to PEA-15 and that their binding did not require interaction of PEA-15 with ERK1/2. Furthermore, both ERK1/2 binding to PEA-15 and Akt activation elevated PEA-15 half-life and its protein level. Stabilization of endogenous PEA-15 by overexpressed Akt resulted in similar functional effects than overexpression of PEA-15 itself, namely, inhibition of Elk-1–dependent transcription and cFos induction. This mechanism due to the blockade of ERK nuclear accumulation led to the inhibition of AngII-induced proliferation. Importantly, the use of PEA-15 antisense counteracting endogenous PEA-15 stabilization upon Akt overactivation increased cFos induction. Together, these results demonstrate the important role of Akt/PEA-15 cross-talk in controlling ERK1/2 nuclear activity.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). PEA-15 antisense (5'-TGACGCCTCTGGAGCTGAGC-3') and mock (5'-GACATGGCCTTGCACGCGTG-3') oligonucleotides were a kind gift from Prof. Francesco Beguinot (University of Naples, Naples, Italy). The antibodies directed against the following molecules were used: polyclonal anti-ERK1/2, monoclonal anti-phospho-ERK1/2, and polyclonal anti-glycogen synthase kinase (GSK)3 antibodies from Upstate Biotechnology (Charlottesville, VA); polyclonal anti-phospho-GSK3/
(Ser21/9) antibody, polyclonal anti-Akt, and polyclonal and monoclonal anti-phospho-Ser473 Akt antibodies from Cell Signaling Technology (Beverly, MA); polyclonal anti-HA, anti-ERK1, and anti-cFos antibodies from Santa Cruz Biotechnology (Santa Cruz, CA); monoclonal anti-GFP and anti-HA antibodies from Roche Molecular Biochemicals (Mannheim, Germany); monoclonal M2 anti-FLAG antibody from Sigma-Aldrich (St. Louis, MO); and monoclonal anti-Smad2 antibody from BD Biosciences (Palo Alto, CA). The polyclonal antibody directed against PEA-15 was described previously (Formstecher et al., 2001). AngII and IGF-1 were obtained from Sigma-Aldrich. LY294002 and U0216 were obtained from Calbiochem (San Diego, CA) and Promega (Madison, WI), respectively.
Cell Culture and Establishment of Stable Cell Lines
CHO-AT1A cells, stably expressing the rat AT1A receptor (Teutsch et al., 1992) were maintained at 37°C in 5% CO2, under 750 µg/ml G418 selection (Invitrogen) in Ham’s F-12 medium (Invitrogen) supplemented with 7.5% fetal calf serum, 0.5 mM glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin (all from Roche Molecular Biochemicals). CHO-AT1A cells were stably or transiently transfected using the LipofectAMINE 2000 agent according to the manufacturer’s recommendations (Invitrogen). For establishment of stable clones, HA-Akt1, GFP, GFP-PEA-15, and GFP-D74A and FLAG-PEA-15–transfected CHO-AT1A cell lines were cultivated under 500 µg/ml hygromycin selection (Invitrogen) and cloned either by limiting dilution for HA-Akt1 or by fluorescence-activated cell sorting for the GFP-containing constructs. Clones were then screened by immunoblotting and by immunofluorescence to select cells with homogenous expression. Two independent clones were analyzed for each construct. NCI H295R cells were generously provided by Dr. Alessandro Capponi (Faculty of Medicine, University of Geneva, Geneva, Switzerland). Cells were grown at 37°C in 5% CO2 in DMEM/Ham’s F-12 containing 1% ITS (BD Biosciences), 2% UltroSer SF (Ciphergen, Fremont, CA), 100 U/ml penicillin, 100 µg/ml streptomycin, and 0.25 µg/ml fungizone. Transfection of PEA-15 antisense or mock DNAs in H295R cells was performed using the jetPEI reagent according to the manufacturer’s recommendations (Polyplus transfection, Illkirch, France).
Immunoblotting
Before agonist stimulation, cells were maintained for 4 h of serum starvation. Cell lysates were prepared with 1% SDS containing 1 mM Na3VO4 and 10 mM -glycerophosphate or with ice-cold 1% Nonidet P-40 in 50 mM Tris-HCl, pH 7.4, containing 120 mM NaCl, 1 mM EDTA, 50 mM NaF, 0.1 mM Na3VO4, and 0.5 mM phenylmethylsulfonyl fluoride (PMSF). Lysates were then subjected to SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were probed overnight with primary antibody at 4°C. After incubation with peroxidase- or alkaline phosphatase-linked secondary antibodies, immunoreactive proteins were visualized by ECL reagent (GE Healthcare, Little Chalfont, Buckinghamshire, United Kingdom) or nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate (Promega) substrates, respectively. If necessary, quantitative analysis was performed using QuantityOne software (Bio-Rad, Hercules, CA).
Proliferation Assays
Cell growth was arrested by 48 h of serum starvation. For DNA synthesis quantification, [3H]thymidine incorporation experiments were performed as described previously (Dugourd et al., 2003). After G0 arrest, cells were stimulated with 100 nM AngII for 16 h and labeled with 1 µCi/ml [3H]thymidine for 2 h. After washing with trichloroacetic acid, radioactivity was measured by liquid scintillation counting. For cell number quantification, the Cell Titer 96 Aqueous NonRadioactive Cell Proliferation Assay (Promega) was used. After G0 arrest, cells were stimulated with 100 nM AngII for 24 h. After 1 h of incubation with a phenazine methosulfate/3,4-(5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfo-phenyl)-2H-tetrazolium salt (MTS) mix, the absorbance was measured at 490 nm.
Transcription Assay
Elk-1 transcription assay was performed as described previously (Inman et al., 2002). Eighty percent confluent cells were cotransfected with 150 ng of Nlex.ElkC, 750 ng of Lex-Op-luciferase reporter, and 100 ng of -galactosidase by using the LipofectAMINE 2000 agent. Nlex.ElkC encodes a fusion protein of the LexA DNA binding domain fused to the activation domain of Elk-1. Lex-Op-Luciferase has the luciferase reporter gene under the control of a synthetic promoter containing two repeats of the LexA operator. The -galactosidase plasmid was cotransfected to normalize the transfection efficiency. Cells were serum starved for 4 h and stimulated with 100 nM AngII for 6 h. Elk-1 transcription was measured by the expression of active luciferase using the Promega luciferase assay system according to the manufacturer’s instructions. -Galactosidase activity was assayed by adding the substrate chlorophenol--D-galactopyranoside to 20 µl of cell lysate and incubating at 37°C before measuring at 595 nm.
Immunofluorescence
CHO-AT1A cells stably expressing HA-Akt1, GFP-PEA-15 or GFP-D74A were seeded on 14-mm coverslips and cultivated for 40–48 h before analysis. In some experiments, 300,000 CHO-AT1A cells stably expressing HA-Akt1 were transiently transfected with GFP-PEA-15 and seeded 24 h later on 14-mm coverslips. After cell growth arrest induced by 16-h serum starvation, cells were stimulated by 100 nM AngII for 3 h in the presence or absence of the PI3K inhibitor LY294002 (10 µM). Cells were then fixed with ice-cold methanol for 3.5 min and washed with phosphate-buffered saline (PBS). Nonspecific binding was saturated with 10% normal goat serum (NGS) in PBS. Cells were then incubated for 2 h at room temperature with the polyclonal anti-ERK1 antibody in 1% NGS in PBS. After PBS washes, cells were incubated for 1 h at room temperature with a goat anti-rabbit secondary antibody coupled to Alexa Fluor-555 (Invitrogen). In some experiments, double immunostaining was performed by incubating the cells with the monoclonal anti HA-antibody in 1% NGS in PBS for 1 h at room temperature. After PBS washes, cells were incubated with a goat anti-mouse secondary antibody coupled to Alexa Fluo-647 (Invitrogen). Coverslips were washed with PBS and mounted with Mowiol (Sigma-Aldrich) for confocal microscopy.
Cells were examined with a Leica TCS SP2 confocal microscope (Leica Microsystems, Deerfield, IL). For double or triple detection of Alexa Fluor-555, Alexa Fluor-647, and GFP signal, fluorescence was analyzed in sequential scanning mode. Images (1024 x 1024 pixels) were obtained with an 63x oil immersion objective lens. Each confocal image corresponded to the medium level of the cells.
Metabolic Labeling and Immunoprecipitation
CHO-AT1A cells (150,000) stably expressing GFP-PEA-15 or GFP-D74A were seeded in 12-well plates. Forty hours later, cells were growth arrested by 4-h serum starvation and then labeled for 20 min with 100 µCi/ml [35S]methionine and cysteine (pulse) (ProMix; GE Healthcare). Cells were stimulated with 10 nM insulin-like growth factor (IGF)-1 for various times (0, 4, 15, 25, and 40 h) in the presence or absence of 10 µM LY294002 (chase) and extracted on ice with 1% Nonidet P-40 in 50 mM Tris-HCl, pH 7.4, containing 120 mM NaCl, 1 mM EDTA, 50 mM NaF, 0.1 mM Na3VO4, and 0.5 mM PMSF. Lysates were centrifuged for 10 min at 13,000 rpm at 4°C. The supernatants were then incubated for 2 h with anti-GFP antibody, and the immune complexes were precipitated using protein G coupled to Sepharose (GE Healthcare). Immunoprecipitated proteins were resolved by SDS-PAGE. Gels were dried and exposed to a Biomax film (Eastman Kodak, Rochester, NY). In some experiments, 600,000 CHO-AT1A cells were transiently transfected with FLAG-PEA-15 and split 24 h later into 12-well plates. Cells were then processed for metabolic labeling as described above with different times for the chase (0, 4, 8, 16, and 24 h). Immunoprecipitation of FLAG-PEA-15 was performed using a polyclonal anti-PEA-15 antibody. To measure the degradation rate of PEA-15, quantitative analysis of radiolabeled material was performed using an FX Molecular Imager and QuantityOne software (Bio-Rad). Protein half-life was calculated from each degradation–chase time curve by linear regression analysis.
In Vitro Binding Assays
Using the plasmid pcDNA3.1-HA-Akt1, [35S]methionine-radiolabeled proteins were synthesized in a coupled in vitro transcription/translation reaction (TnT T7; Promega). Radiolabeled proteins (2 µl of 50 µl of translation reaction) were incubated with 1 µg of GST-PEA-15, 1 µg of GST-D74A, or 1 µg of GST alone (negative control) adsorbed on glutathione-Sepharose beads as described previously (Formstecher et al., 2001). Proteins were resolved by SDS-PAGE. Gels were dried and exposed to a Biomax film (Eastman Kodak). Radiolabeled HA-Akt was detected using an FX Molecular Imager (Bio-Rad). Alternatively, cell lysates of CHO-AT1A cells stably expressing HA-Akt1 were prepared with 1% Nonidet P-40. Lysates (500 µg) were incubated with 50 µl of Sepharose beads bound to the GST constructs described above (
2 µg) for 2 h at 4°C. Proteins were resolved by SDS-PAGE, transferred to PVDF membranes, and processed by standard immunoblotting technique with polyclonal anti-HA antibody.
Statistical Analysis
Quantitative data were expressed as mean ± SEM and compared using a one-way analysis of variance followed by intergroup comparisons with a Student’s t test. p < 0.05 was considered statistically significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). The modification of the precise balance between these two endogenous pathways remains poorly investigated even though it could lead to dramatic changes in the cellular response. For this purpose, we stably transfected the CHO-AT1A cell line with human recombinant HA-tagged Akt1. Twenty-four clones expressing various levels of HA-Akt1 were isolated and analyzed. As shown in Figure 1A (right), one representative cell line of this clone expressed a 10-fold increase in Akt level compared with the endogenous level detected in wild-type CHO-AT1A cells. When an equivalent amount of total Akt was loaded, CHO-AT1A cells stably expressing Akt1 showed a similar level of Akt phosphorylation to wild-type CHO-AT1A cells upon AngII stimulation (Figure 1A, left), indicating an overall increase of Akt activation in response to AngII in cells overexpressing Akt. In both cell lines, phosphorylated Akt was inhibited by the PI3K inhibitor LY294002 (Figure 1A, left) but unaffected by the MEK inhibitor U0126 (our unpublished data), as already shown for endogenous Akt (Dugourd et al., 2003). These results indicate that Akt overexpression led to an increased Akt activation upon AngII stimulation that is PI3K dependent and MEK/ERK1/2 independent. In CHO-AT1A overexpressing Akt, the increased level of AngII-induced Akt phosphorylation was correlated with an enhanced ability of Akt to phosphorylate its cytosolic substrates, GSK3 and - (Figure 1A, right). Thus, Akt overexpression in CHO-AT1A cells correlated with an enhanced level of Akt activity upon AngII stimulation.
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PEA-15 Directly Binds Akt Independently of Its Binding to ERK1/2
We previously reported that PEA-15 blocks serum-induced proliferation through its binding with ERK1/2 and the prevention of ERK1/2 nuclear localization (Formstecher et al., 2001). More recently, PEA-15 was identified as an Akt substrate (Trencia et al., 2003). To elucidate the molecular mechanisms leading to the inhibition of cell proliferation induced by Akt overexpression, we investigated possible cross-talk between the PI3K/Akt and the ERK1/2 signaling pathways. We first tested the interaction between Akt and PEA-15 by using pull-down assay. Lysates from CHO-AT1A cells overexpressing HA-Akt1 were incubated with GST-PEA-15 fusion protein. Bound proteins were blotted with anti-HA antibody. Recombinant HA-Akt1 specifically bound GST-PEA-15 but not GST alone (Figure 2A). Furthermore, 35S in vitro-labeled HA-Akt1 bound to immobilized GST-PEA-15 fusion protein but not to GST alone, demonstrating a direct interaction between Akt and PEA-15 (Figure 2B). To examine whether this interaction required ERK1/2 binding to PEA-15, the same experiments were performed with the D74A mutant of PEA-15. We have previously shown that this mutation of an aspartate in the death effector domain of PEA-15 abrogates its binding to ERK1/2 (Formstecher et al., 2001), although this mutant possesses the same structure as wild-type PEA-15 protein (Hill et al., 2002). Similar to wild-type PEA-15, the D74A mutant was able to interact with HA-Akt1 using either cell lysates or 35S in vitro-labeled HA-Akt1 (Figure 2, A and B). These results demonstrate the direct binding of PEA-15 to Akt. Furthermore, we show for the first time that this interaction did not require ERK1/2 binding to PEA-15.
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We then analyzed whether this increase of PEA-15 half-life had a detectable consequence on its expression level. In CHO-AT1A cell line stably expressing FLAG-PEA-15, PEA-15 protein levels were significantly higher under serum or IGF-1 exposure than under serum starvation (Figure 4A). These increases were associated with Akt phosphorylation as shown on Figure 4A (left). In addition, IGF-1– or serum-induced increase of PEA-15 level was reversed by LY294002 pretreatment. These results indicate that the active form of Akt was associated with the stabilization of PEA-15 and subsequently with an increase of its intracellular protein content. We then investigated the cellular relevance of the overactivation of endogenous Akt on endogenous PEA-15 protein level. For this purpose, we used a human adrenocortical carcinoma cell line H295R known to activate several signaling pathways through the AngII AT1 receptor that leads notably to the secretion of aldosterone (Bird et al., 1993). In those cells, endogenous phosphorylation of Akt was already detectable under serum deprivation and was not further increased after serum or AngII stimulation (Figure 4B, top), as reported previously (Zheng and Bollag, 2003). We further showed that, although constitutive, Akt phosphorylation was still PI3K-dependent, because it was blocked by LY294002. Interestingly, blockade of AngII- or serum-induced Akt phosphorylation by 24-h LY294002 treatment was associated with a concomitant significant reduction of the cellular level of PEA-15 (Figure 4B, bottom left and right). Inhibiting Akt activity decreased PEA-15 protein levels only by regulating the protein stability, because no modification of its mRNA expression was observed after LY294002 treatment (our unpublished data). Moreover, modulation of PEA-15 protein level by overactive Akt is specific for PEA-15. Indeed, Smad2 level is unaffected by similar treatments, although it is another protein with a short half-life subject to proteasomal degradation (Figure 4, A and B, left). Together, our results indicate that, in CHO or H295R cells, the variation of the level of phosphorylated Akt was associated with the regulation of the cellular protein level of recombinant tagged PEA-15 as well as endogenous PEA-15.
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). We then analyzed the functional consequences of PEA-15 overexpression on the activation of the nuclear targets of ERK1/2, such as Elk-1. In response to AngII, PEA-15 overexpression decreased Elk-1–dependent transcription and suppressed, downstream of Elk-1, cFos induction (Figure 5, C and D). The induction of cFos by AngII was abolished by U0126 but unaffected by LY294002 in both wild-type and PEA-15–overexpressing cells (Supplemental Figure 1). The ability of PEA-15 to down-regulate Elk-1–dependent transcription relied on its capacity to bind to ERK1/2, because the D74A mutant, unable to bind ERK, did not modify Elk-1–dependent transcription and cFos expression in response to AngII (Figure 5, C and D).
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). To test whether PEA-15 overexpression decreased ERK1/2 nuclear activity by inhibiting their nuclear ERK1/2 accumulation, we assessed the subcellular localization of ERK1/2 in CHO-AT1A cells expressing GFP-PEA-15, GFP-D74A, or GFP alone under AngII stimulation. Immunofluorescence revealed that in the presence of GFP alone, ERK1/2 localization was predominantly cytosolic in serum-starved cells and mostly nuclear in response to AngII for 3 h (Figure 6, top). In contrast, AngII failed to stimulate nuclear accumulation of ERK1/2 in cells overexpressing GFP-PEA-15 (Figure 6, middle). The cytosolic localization of GFP-PEA-15 was unaffected by the presence or absence of AngII. The cytosolic retention of ERK1/2 required its binding to PEA-15, because the D74A mutant failed to block ERK1/2 nuclear localization in response to AngII (Figure 6, bottom). As for wild-type PEA-15, the subcellular localization of GFP-D74A remained cytosolic in the presence or absence of AngII. ERK1/2 nuclear localization was unaffected by LY294002 pretreatment in the three cell lines (Supplemental Figure 2). Thus, overexpression of PEA-15–regulated ERK1/2 nuclear localization induced by AngII through the AT1 receptor. Together, our results demonstrate for the first time that increased expression of PEA-15 was able to down-regulate AngII-induced Elk1-dependent transcription, cFos induction, and cell proliferation through impaired ERK accumulation in the nucleus without any modification of their phosphorylation state.
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). We thus tested whether the cytosolic retention of ERK1/2 induced by overexpressed Akt would lead to a sustained activation of ERK1/2 in the cytosol, as a result of a decreased nuclear ERK1/2 inactivation. After 5 min of AngII exposure, CHO-AT1A cells overexpressing or not HA-Akt1 or FLAG-PEA-15 were rinsed and maintained in serum-free medium for various times to detect ERK1/2 dephosphorylation rate in the cytosol. Overexpression of PEA-15 in CHO-AT1A cells prolonged AngII-induced ERK1/2 phosphorylation in the cytosol (Figure 9, middle), in a similar way to those observed under serum stimulation (Ramos et al., 2000; Formstecher et al., 2001). Interestingly, overexpression of Akt led to the same effect than PEA-15 overexpression, i.e., a decrease in ERK1/2 dephosphorylation kinetics in the cytosol (Figure 9, right). Together, these results indicate that Akt overactivation through PEA-15 down-regulated ERK1/2 nuclear localization, leading to the suppression of AngII-induced proliferative responses.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). The present study shows that overexpression of Akt sequestered ERK1/2 in the cytosol of CHO-AT1A cells via PEA-15 with a consequent abolition of the mitogenic response to AngII. This study is the first report to delineate the molecular mechanism responsible for the down-regulation of ERK1/2 transcriptional activity induced by Akt without inhibiting ERK1/2 activation.
Cross-talk between the PI3K/Akt and Ras/Raf/MEK/ERK1/2 occurs at different levels and exerts cooperative or antagonistic effects depending on external stimuli and cellular background. PI3K has been shown to stimulate integrin-mediated Raf activation in synergy with Ras (Chaudhary et al., 2000). Cooperative effects between these two signaling cascades have also been demonstrated in the regulation of the platelet-derived growth factor-induced proliferation (Choudhury et al., 1997) or in cell cycle progression and transformation (Sheng et al., 2001a, b). Alternatively, Akt has been shown to phosphorylate Raf-1, leading to the down-regulation of the ERK pathway in phorbol 12-myristate 13-acetate–stimulated MCF-7 cells (Zimmermann and Moelling, 1999) or in differentiated myotubes (Rommel et al., 1999). B-Raf activity is also inhibited by epidermal growth factor-induced Akt stimulation (Guan et al., 2000). Our study reveals an additional mechanism, because we show that overexpressed Akt down-regulated ERK/Elk-1–dependent transcriptional activity, an effect that was undetectable under endogenous Akt activation. In our system, inhibition of Ras, Raf, or MEK by overexpressed Akt cannot account for this negative regulation, because ERK1/2 phosphorylation was not affected in CHO-AT1A overexpressing Akt. In contrast, this result suggests that Akt acted downstream of ERK1/2 activation in the cytosol. In agreement with our data, a recent study reported that constitutively active Akt does not modify ERK1/2 phosphorylation (Galetic et al., 2003). In our study, the inhibition of ERK1/2 nuclear activity by overexpressed Akt resulted in a decrease in Elk-1–dependent transcription with a subsequent abolition of cFos expression. Increased Akt activation has already been shown to down-regulate the Elk-1 transcription factor by decreasing either its expression (Figueroa and Vojtek, 2003) or its activation (Galetic et al., 2003). Therefore, the critical step for the negative regulation of ERK1/2 by Akt lies between the cytosolic activation of ERK1/2 and its activity on nuclear substrates, such as Elk-1. In this context, our study describes a new cellular process of down-regulation of ERK/Elk-1–dependent transcription that implies the cytosolic retention of active ERK1/2 by active Akt (Figure 10). Indeed, only the phosphorylated form of Akt was able to retain ERK1/2 into the cytosol, because pretreatment with the PI3K inhibitor LY294002 that blocks Akt phosphorylation restored ERK1/2 nuclear translocation induced by AngII.
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recently reported that PEA-15 is a new Akt substrate and that its phosphorylation and stabilization by Akt participate in Akt-mediated survival signaling. Moreover, we have previously demonstrated that PEA-15 binds ERK1/2 and abolishes their nuclear translocation in response to serum (Formstecher et al., 2001). In the present study, we show that Akt binds to PEA-15 in vitro, in agreement with Trencia et al. (2003). This interaction did not require ERK1/2 binding to PEA-15, because the D74A mutant of PEA-15, which cannot bind to ERK1/2, was still able to bind Akt. Furthermore, the interaction between recombinant GST-PEA-15 and in vitro-radiolabeled Akt allowed us to conclude that a direct interaction occurs between Akt and PEA-15. Therefore, PEA-15 is a pivotal protein functionally linking Akt and ERK1/2. Although the molecular interactions between various motifs of ERK1/2 and PEA-15 have been investigated for their direct binding (Hill et al., 2002; Chou et al., 2003), the mechanisms of molecular interactions between Akt and PEA-15 remain to be determined. An important issue will be to assess whether the phosphorylation state of Akt affects its binding to PEA-15 and/or the binding of PEA-15 to ERK1/2. A better understanding of this interaction will clarify the preferential regulation of ERK1/2 or Akt binding to PEA-15, which could be modulated by kinase expression levels and lead to differential cellular responses. Regarding the functional consequence of this interaction, we show that Akt binds to PEA-15 and increases PEA-15 half-life. We further demonstrate that only the active form of Akt was responsible for PEA-15 stabilization, because IGF-1–induced increase in PEA-15 half-life was abrogated by LY294002 pretreatment. Interestingly, the half-life of the D74A mutant was also affected by the activation state of Akt. Together, these results demonstrate for the first time that endogenous Akt phosphorylation as well as ERK1/2 binding to PEA-15 exerted additive effects on the stability of PEA-15. Stabilization of PEA-15 by overexpressed Akt amplified the functional consequences of PEA-15 binding to ERK1/2. Overexpression of Akt resulted in the same regulation pattern of the ERK1/2 signaling as increased PEA-15 expression, i.e., exclusion of ERK1/2 from the nucleus and the consequent inhibition of Elk-1–dependent transcription and of cFos induction. When stabilization of PEA-15 by overactive Akt is blocked by specific PEA-15 antisense, ERK1/2-dependent transcription is rescued, demonstrating the important role of Akt/PEA-15 cross-talk in controlling ERK1/2 nuclear activity. Moreover, the lack of effect of LY294002 on ERK1/2 nuclear translocation and activity when PEA-15 is overexpressed rules out the possible involvement of parallel regulators of ERK1/2 signaling, other than PEA-15, downstream of Akt activation.
Finally, this study is the first to report the functional consequence of the cross-talk between Akt, PEA-15, and ERK1/2. Negative regulation of ERK1/2 by overactivated Akt was associated with the inhibition of cell proliferation induced by AngII. Hence, changes in the expression of Akt can modulate the mitogenic response by decreasing ERK1/2 nuclear activity through their cytosolic retention. This result is important considering that Akt is overexpressed and/or overactivated under many pathophysiological circumstances, ranging from tumor growth to vascular diseases (Hixon et al., 2000; Testa and Bellacosa, 2001). Whereas Akt overexpression is mainly associated with an oncogenic phenotype, this pathological context is usually associated with mutation or modification of the expression of other components of the Akt pathway, such as PI3K and/or phosphatase and tensin homologue deleted on chromosome 10 (Luo et al., 2003). Besides, the dual involvement of Akt in mediating proliferative and/or antiapoptotic responses has to be considered inasmuch as both responses are intricate (Hu et al., 2004). Zhu et al. (2004) reported that Gab2 overexpression, which enhanced the phosphorylation of Akt but not of ERK1/2, reduced cell proliferation induced by granulocyte colony-stimulated factor (Zhu et al., 2004). Our results are in agreement with this recent study. Moreover, the molecular mechanism that we propose could be involved in the lack of proliferative effect observed upon Akt up-regulation in vascular smooth muscle cells (Hixon et al., 2000). This dual effect of Akt has to be correlated to contradictory effects of PEA-15. Indeed, PEA-15 is a strong inhibitor of death receptor-dependent apoptosis (Condorelli et al., 1999; Estelles et al., 1999; Kitsberg et al., 1999) but is associated with decreased cell proliferation (Formstecher et al., 2001; Gaumont-Leclerc et al., 2004). Furthermore, a recent study showed that sustained level of PEA-15 participated in Akt-dependent chemoresistance in human breast cancer cells (Stassi et al., 2005). Hence, according to our model of Akt/PEA-15 interaction, we hypothesized that, in some physiopathological context, overexpression of Akt could slow proliferation and render the cells quiescent and resistant to certain forms of apoptosis.
Akt overexpression generated a cross-talk between the PI3K and the MAPK pathways that relies on the pivotal role of PEA-15, because this protein could directly interact with Akt and with ERK1/2 (Figure 10). Variations in the expression of one of the three proteins can result in a modified balance of these complexes and shift the cell toward a proliferative or nonproliferative phenotype. Thus, in the presence of basal endogenous PEA-15 or Akt protein levels, the cross-talk between the ERK1/2 and Akt pathways does not occur. This is due to a high amount of ERK1/2 whose nuclear translocation cannot be clearly affected by PEA-15 binding and relocalization. However, higher amount of PEA-15, either by overexpression of the protein or by increase of its half-life regulated by overactivated Akt, can lead to a blockade of the ERK1/2 nuclear translocation and a subsequent blockade of cFos induction and cell proliferation. This new cross-talk between two main kinases mediated by a small noncatalytic protein leads to a better understanding of the cellular mechanisms necessary to switch the cell from a proliferative phenotype to a quiescent phenotype.
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ACKNOWLEDGMENTS |
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Footnotes |
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The online version of this article contains supplemental material at MBC Online (http://www.molbiolcell.org). 
This article was published online ahead of print in MBC in Press (http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E06-06-0501) on July 5, 2006.
These authors contributed equally to this work.
Present addresses: 
Unité Mixte de Recherche, Centre National de la Recherche Scientifique 7054, Centre de Recherches Chirurgicales, Hôpital Henri-Mondor, Créteil, France;
Département de la Recherche Clinique et de la Valorisation, Hôpital Cimiez CHU de Nice, Nice, France.
Address correspondence to: Catherine Monnot (catherine.monnot{at}college-de-france.fr)
Abbreviations used: AngII, angiotensin II; CHO, Chinese hamster ovary cells; ERK, extracellular signal-regulated kinases; GSK, glycogen synthase kinase; PEA-15, phosphoprotein enriched in astrocytes of 15 kDa; PI3K, phosphatidylinositol 3-kinase.
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REFERENCES |
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