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IEX-1: a new ERK substrate involved in both ERK survival activity and ERK activation

2002; Springer Nature; Volume: 21; Issue: 19 Linguagem: Inglês

10.1093/emboj/cdf488

1460-2075

Josefina Garcia, Yunbin Ye, Valérie Arranz, Claire Letourneux, Guillaume Pézeron, Françoise Porteu,

Peptidase Inhibition and Analysis

Article1 October 2002free access IEX-1: a new ERK substrate involved in both ERK survival activity and ERK activation Josefina Garcia Josefina Garcia Department of Hematology, Institut Cochin, INSERM U567, CNRS UMR 8104, Université René Descartes, 27 rue du Fg St Jacques, 75014 Paris, France Search for more papers by this author Yunbin Ye Yunbin Ye Department of Hematology, Institut Cochin, INSERM U567, CNRS UMR 8104, Université René Descartes, 27 rue du Fg St Jacques, 75014 Paris, France Search for more papers by this author Valérie Arranz Valérie Arranz Department of Hematology, Institut Cochin, INSERM U567, CNRS UMR 8104, Université René Descartes, 27 rue du Fg St Jacques, 75014 Paris, France Search for more papers by this author Claire Letourneux Claire Letourneux Department of Hematology, Institut Cochin, INSERM U567, CNRS UMR 8104, Université René Descartes, 27 rue du Fg St Jacques, 75014 Paris, France Search for more papers by this author Guillaume Pezeron Guillaume Pezeron Department of Hematology, Institut Cochin, INSERM U567, CNRS UMR 8104, Université René Descartes, 27 rue du Fg St Jacques, 75014 Paris, France Search for more papers by this author Françoise Porteu Corresponding Author Françoise Porteu Department of Hematology, Institut Cochin, INSERM U567, CNRS UMR 8104, Université René Descartes, 27 rue du Fg St Jacques, 75014 Paris, France Search for more papers by this author Josefina Garcia Josefina Garcia Department of Hematology, Institut Cochin, INSERM U567, CNRS UMR 8104, Université René Descartes, 27 rue du Fg St Jacques, 75014 Paris, France Search for more papers by this author Yunbin Ye Yunbin Ye Department of Hematology, Institut Cochin, INSERM U567, CNRS UMR 8104, Université René Descartes, 27 rue du Fg St Jacques, 75014 Paris, France Search for more papers by this author Valérie Arranz Valérie Arranz Department of Hematology, Institut Cochin, INSERM U567, CNRS UMR 8104, Université René Descartes, 27 rue du Fg St Jacques, 75014 Paris, France Search for more papers by this author Claire Letourneux Claire Letourneux Department of Hematology, Institut Cochin, INSERM U567, CNRS UMR 8104, Université René Descartes, 27 rue du Fg St Jacques, 75014 Paris, France Search for more papers by this author Guillaume Pezeron Guillaume Pezeron Department of Hematology, Institut Cochin, INSERM U567, CNRS UMR 8104, Université René Descartes, 27 rue du Fg St Jacques, 75014 Paris, France Search for more papers by this author Françoise Porteu Corresponding Author Françoise Porteu Department of Hematology, Institut Cochin, INSERM U567, CNRS UMR 8104, Université René Descartes, 27 rue du Fg St Jacques, 75014 Paris, France Search for more papers by this author Author Information Josefina Garcia1, Yunbin Ye1, Valérie Arranz1, Claire Letourneux1, Guillaume Pezeron1 and Françoise Porteu 1 1Department of Hematology, Institut Cochin, INSERM U567, CNRS UMR 8104, Université René Descartes, 27 rue du Fg St Jacques, 75014 Paris, France ‡J.Garcia and Y.Ye contributed equally to this work *Corresponding author. E-mail: [email protected] The EMBO Journal (2002)21:5151-5163https://doi.org/10.1093/emboj/cdf488 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info IEX-1 is an early response and NF-κB target gene implicated in the regulation of cellular viability. We show here that IEX-1 is a substrate for ERKs and that IEX-1 and ERK regulate each other's activities. IEX-1 was isolated by phosphorylation screening with active ERK2 and found subsequently phosphorylated in vivo upon ERK activation. IEX-1 interacts with phosphorylated ERKs but not with c-jun N-terminal kinase (JNK) or p38. Upon phosphorylation by ERKs, IEX-1 acquires the ability to inhibit cell death induced by various stimuli. In turn, IEX-1 potentiates ERK activation in response to various growth factors. By using various IEX-1 mutants in which the ERK phosphoacceptor and/or ERK docking sites were mutated, we show that the IEX-1 pro-survival effect is dependent on its phosphorylation state but not on its ability to potentiate ERK activation. Conversely, IEX-1-induced modulation of ERK activation requires ERK–IEX-1 association but is independent of IEX-1 phosphorylation. Thus, IEX-1 is a new type of ERK substrate that has a dual role in ERK signaling by acting both as an ERK downstream effector mediating survival and as a regulator of ERK activation. Introduction Mammalian cells respond to a variety of extracellular stimuli via activation of specific mitogen-activated protein kinases (MAPK) that orchestrate the delivery of the signal from receptors at the cell surface to the nucleus. Three main classes of MAPK are recognized: the classic MAPK, also known as extra-regulated kinases, ERK1 and ERK2, c-jun N-terminal kinase (JNK) and p38 MAPK, which are activated by dual phosphorylation at neighboring threonine and tyrosine residues in the activation loop and dephosphorylation of either residue results in MAPK inactivation. The general scheme of ERK activation involves a cascade of phosphorylation events initiated by stimulation of the Ras proto-oncogene following activation of growth factors receptors. The cascade starts with the activation of one or more Raf family kinases, which phosphorylate and activate the MAPK kinases (MEK1/2). MEK in turn catalyzes dual phosphorylation and activation of ERKs (for reviews see Schaeffer and Weber, 1999; Chang and Karin, 2001). Once activated, ERKs accumulate in the nucleus by a yet to be defined mechanism that seems to involve the dissociation of ERK from MEK (Fukuda et al., 1997) and/or a blocade of its export from the nucleus by neo-synthesized nuclear anchors (Lenormand et al., 1998). The ERK pathway has been implicated in diverse cellular processes including proliferation, differentiation and survival (for reviews see Marshall, 1995; Schaeffer and Weber, 1999; Ballif and Blenis, 2001). The variable responses elicited by this cascade in different cell types is presumably determined by the cell-specific combination of downstream substrates. More than 50 different ERK substrates have been identified so far. These include ubiquitous or lineage-restricted transcription factors, the kinases Rsk and Mnk and proteins involved in nucleotide biosynthesis, cytoskeleton organization, ribosomal transcription or membrane traffic (Sturgill et al., 1988; Marais et al., 1993; Treisman, 1996; Fukunaga and Hunter, 1997; Graves et al., 2000; Lewis et al., 2000; Stefanovsky et al., 2001). The net result of ERK activation varies also in a given cell according to the stimulus (Marshall, 1995). This suggests the existence of mechanisms allowing ERK to discriminate between the various substrates available. Key parameters for setting this choice include the control of the amplitude and duration of the signal and the spatial organization of the components of the ERK cascade and its substrates. The importance of these mechanisms is illustrated by data showing that sustained, but not transient, ERK activation is associated with its nuclear translocation and leads to growth factor-induced gene regulation, cell cycle entry or differentiation (Traverse et al., 1992; Marshall, 1995; Rouyez et al., 1997; Brunet et al., 1999). Interestingly, MAPKs themselves regulate the strength and duration of their activation by phosphorylating various substrates: MAPKs phosphorylate, and thereby inhibit, receptor tyrosine kinases (Countaway et al., 1992), the Ras-exhange factor SOS (Waters et al., 1995) and MEK (Brunet et al., 1994); MAPK also increases the activity and half-life of the MAPK phosphatases (Pulido et al., 1998; Brondello et al., 1999). ERKs interact with their substrates through conserved docking sites (Gavin and Nebreda, 1999; Jacobs et al., 1999; Smith et al., 1999). ERK substrates could therefore also play a role in the subcellular localization of ERKs. Such a regulation has already been reported in other MAPK pathways. For instance, in the fission yeast, the transcription factor Atf1 regulates the nuclear localization of the stress-activated kinase spc1 (Gaits et al., 1998). Likewise, phosphorylation of mammalian MAPKAP kinase 2 by p38 facilitates the export of p38 from the nucleus (Ben-Levy et al., 1998). Thus, discovery of ERK phosphorylation targets will help to get a better read-out of the regulation of both ERK multiple actions and spatio-temporal activation. To identify new ERK substrates involved in cell proliferation and survival, we used a solid phase phosphorylation assay (Fukunaga and Hunter, 1997) with active ERK2 to screen a phage expression library prepared from UT7, a cell line of hematopoietic origin, the growth of which is dependent on the presence of various cytokines such as interleukin-3, erythropoietin (EPO) and thrombopoietin (TPO) (Rouyez et al., 1997). One of the cDNAs isolated encoded IEX-1, a gene previously characterized as an early response and NF-κB-target gene regulated by X-irradiation, UV radiation and various growth factors, and reported to have contradictory functions in the control of cell survival (Charles et al., 1993; Kumar et al., 1998; Schafer et al., 1998; Wu et al., 1998; Arlt et al., 2001; Schilling et al., 2001). We show here that IEX-1 is a specific substrate of ERK1/2 and binds to the kinases in their active forms. The association of ERK and IEX-1 results in trans-regulation of their activities: IEX-1 acquires an anti-apoptotic function in response to various death triggers upon phosphorylation by ERKs, while in turn, IEX-1 potentiates ERK activation in response to growth factors. The cell survival effect of IEX-1 is dependent on its phosphorylation state but not its ability to enhance ERK signaling. Conversely, IEX-1-induced modulation of ERK activation is independent of IEX-1 phosphorylation but requires ERK binding. Thus, IEX-1 is a new type of ERK substrate that has a dual role in the ERK pathway by being both a signaling downstream effector mediating ERK survival activity and also a regulator of ERK activation. Results Identification of IEX-1 as a substrate of ERK MAPK in vitro and in vivo The cDNA encoding IEX-1 (Kondratyev et al., 1996) was isolated from the UT7 cell line during a phosphorylation screening with active ERK2 (see Materials and methods). To verify that IEX-1 was a substrate for ERKs, the IEX-1 coding region was expressed as a GST fusion protein, purified by glutathione–Sepharose chromatography and incubated with purified active ERK2 in an in vitro kinase assay. As shown in Figure 1A (upper panel), the GST–IEX1 protein was readily phosphorylated by ERK2 in vitro, whereas no radioactivity was incorporated into the GST protein. Figure 1.ERKs phosphorylate IEX-1 in vitro. (A) One microgram of purified GST or GST–IEX-1 fusion proteins wild-type (Wt) and mutants (T18A; T123AS126A; T/SA3; ΔBD) were reacted with purified recombinant active ERK2 in the presence of [32P]ATP. The products were analyzed by autoradiography or western blotting (WB) with the indicated antibodies. (B) Schematic representation of IEX-1 Wt and ERK-phosphorylation and/or binding sites mutants. Hatched area indicates the putative IEX-1 transmembrane domain. Download figure Download PowerPoint IEX-1 presents three putative ERK phosphorylation sites at positions T18, T123 and S126 (Kondratyev et al., 1996; Figure 1B). To determine whether ERK phosphorylates IEX-1 at one or several of these sites, we generated IEX-1 mutants in which either one, two or all three sites were converted to alanine. Whereas the GST–T123AS126A double mutant was phosphorylated to almost the same extent as the wild-type protein, the GST–T18A protein was only marginally phosphorylated by active ERK2 (Figure 1A), indicating that T18 of IEX-1 is the major ERK-catalyzed phosphorylation site in vitro. Mutation of the three ERK consensus phosphorylation sites (mutant T/SA3) completely abolished ERK phosphorylation (Figure 1A). To confirm these results, we raised rabbit antibodies to a phosphopeptide corresponding to T18. In an immunoblot assay, the anti-phosphoT18 peptide antibody recognized the wild-type but not the T18A IEX-1 fusion protein phosphorylated by ERK2 (Figure 1A, bottom panel), whereas the anti-IEX-1 antibody recognized both proteins. Thus, the anti-phosphoT18 antibody specifically recognizes IEX-1 phosphorylated in vitro by ERK. This antibody was therefore used to determine whether ERKs could phosphorylate IEX-1 at T18 in vivo. In the first set of experiments, phosphorylation was assessed in UT7 cells stably expressing an HA epitope-tagged version of IEX-1 in which ERK activation was induced by TPO stimulation. As shown in Figure 2A, the anti-phosphoT18 antibody recognized HA-IEX-1 species only in TPO-stimulated cells. The signal detected disappeared when TPO was added in the presence of the MEK inhibitor PD98059, indicating that the MEK–ERK pathway is responsible for the phosphorylation of IEX-1 at T18 in vivo upon TPO stimulation. Figure 2.Expression, phosphorylation and subcellular localization of IEX-1 in UT7 cells. (A and B) UT7 cells stably expressing HA-tagged IEX-1 (A) or control (B) were deprived of cytokine and stimulated with TPO for various times in the absence or presence of 20 μM PD98059, as indicated. ERK activation and the presence of phosphorylated and non-phosphorylated forms of IEX-1 were monitored by immunoblotting of total cell lysates. (C and D) UT7 cells expressing an empty vector (C), or HA-tagged Wt-IEX-1 or T/SA3-IEX-1 (D) were stimulated for 3 h with TPO, lysed and subjected to differential centrifugation. Proteins (100 μg) from each cellular fraction were loaded: heavy membrane (HM), light membrane (LM), cytoplasm (C) and nucleus (N). Antibodies specific for cytochrome c (cyt c, mitochondrial marker), caspase-3 (cytosolic marker) and PARP (nuclear marker) were used to characterize the fractions. Download figure Download PowerPoint IEX-1 has been described as an early gene regulated by some growth factors (Charles et al., 1993). In agreement with these reports, TPO induced a rapid expression of IEX-1 in UT7 cells (Figure 2B). Western blot with anti-phosphoT18 antibodies showed that the appearance of phosphorylated IEX-1 species correlated with that of IEX-1 protein and with ERK activation. Thus, like overexpressed IEX-1, endogenous IEX-1 protein is phosphorylated at T18 upon ERK activation. Subcellular fractionation studies of UT7 cells showed that the endogenous IEX-1 protein induced by TPO stimulation was mainly located in the heavy membrane fraction that contains mitochondrial membranes, with only low levels of the protein detected in the light membrane fraction and the nucleus (Figure 2C). Since induction of IEX-1 expression by TPO required ERK activation (data not shown), the effect of IEX-1 phosphorylation on its localization was studied using stable clones of UT7 cells expressing either wild type or the non-phosphorylable T/A3-IEX-1 mutant. Like the endogenous protein, most of the HA-IEX-1 species, either wild type or T/SA3, migrated with the heavy membrane fraction whether the cells were stimulated (Figure 2D) or not (data not shown) with TPO to induce ERK activation. Thus, ERK activation does not affect IEX-1 subcellular localization. IEX-1 binds specifically to the active forms of ERK1/2 but not to p38 and JNK MAPK The capacity of MAPKs to phosphorylate their substrates is controlled by their docking to specific amino acid sequences on the substrates (Gavin and Nebreda, 1999; Jacobs et al., 1999; Smith et al., 1999). Therefore, we examined whether IEX-1 could interact with ERKs by reacting Sepharose-bound GST–IEX-1 fusion proteins with lysates prepared from UT7 cells that had been stimulated with TPO to activate ERKs. GST–IEX-1 precipitated ERK1 and ERK2, but only from lysates of activated cells (Figure 3A), indicating that IEX-1 associates with the active but not with the resting forms of ERK1 and ERK2. This binding to ERK was specific since the GST–IEX-1 Sepharose was unable to precipitate p38 or JNK MAPKs in their resting or anisomycin-induced phosphorylated forms (Figure 3B). Figure 3.IEX-1 binds specifically to the active forms of ERK1/2. (A and B) Sepharose-bound GST or GST–IEX-1 wild type and mutants were incubated with lysates from UT7 cells either untreated (0) or stimulated with 10 nM TPO or 100 ng/ml anisomycin (Aniso) for 30 min, as indicated. MAPKs were detected in GST precipitates or in samples of total lysates (TL) by immunoblotting with antibodies directed against the active forms of ERK, JNK or p38. (C and D) Cos7 cells were transfected with 2 μg of pcDNA-HA-IEX-1 (Wt or ΔBD mutant) or empty vector (V), starved overnight and stimulated (+) or not (−) with 100 ng/ml EGF for 10 min. Lysates were immunoprecipitated (IP) with anti-HA or anti-ERK1 antibodies and analyzed by western blotting. Expressions of ERK and HA-IEX-1 are shown in total lysates (TL). (E) UT7 cells (50 × 107) were treated with TPO for 3 h to induce endogenous IEX-1 protein expression. The presence of IEX-1 and phosphorylated ERK was monitored in anti-IEX-1, anti-phosphoERK or control (C) immunoprecipitates (IP), as indicated. As a control, phosphoERK and IEX-1 expressions are shown in total lysates (TL) from 1 × 106 cells. Download figure Download PowerPoint To examine whether the interaction between IEX-1 and phosphorylated ERKs could occur within the cells, an HA-tagged IEX-1 construct was transiently transfected into Cos7 cells and the presence of ERK in anti-HA immunoprecipitates was analyzed by immunoblotting. As shown in Figure 3C, anti-HA antibodies were found to precipitate endogenous ERK1/2 only from the cells stimulated with EGF prior to lysis. Conversely, HA-IEX-1 was detected in anti-ERK1 immunoprecipitates from EGF-treated cells but not from resting cells. In TPO-stimulated UT7 cells, both endogenous IEX-1 and phosphorylated ERKs were found in the heavy membrane fraction (Figure 2C and D) and, more importantly, the two endogenous proteins could be co-immunoprecipitated from these lysates (Figure 3E). Thus, IEX-1 binds specifically to the active form of ERK1 and ERK2 MAPK in vivo. Two different MAPK docking site consensus sequences have been identified so far in MAPK substrates: the D-domain and the FXF (or DEF) motif (Jacobs et al., 1999). Analysis of IEX-1 amino acid sequence revealed the presence of the FTF sequence just downstream of the major phosphorylation site T18. To determine whether this sequence could be responsible for the capacity of IEX-1 to bind ERK, we generated a IEX-1 mutant in which the three residues FTF were converted to alanine (Δ binding domain or ΔBD mutant; Figure 1B). In constrast to the wild-type (Wt)-IEX-1, the ΔBD-IEX-1 mutant could not precipitate ERK from stimulated cell lysates in either pull-down (Figure 3A) or co-precipitation assays (Figure 3D). This demonstrates that the FXF site is necessary for the docking of active ERK on IEX-1. Disruption of this site led to a complete loss of phosphorylation of the GST–ΔBD-IEX-1 protein by active ERK2 in vitro (Figure 1A). Thus, phosphorylation of IEX-1 is dependent on its capacity to bind ERK. In contrast, IEX-1 binding to ERK occurs independently of its phosphorylation, as shown by the capacity of Sepharose-bound GST–T/SA3-IEX-1 to pull down ERK as efficiently as Wt-IEX-1 (Figure 3A). IEX-1 increases ERK activation While performing ERK/IEX-1 co-precipitation experiments, we noticed that ERK phosphorylation was regularly increased in cells transfected with IEX-1, suggesting that IEX-1 may modulate ERK activity. To analyze this possibility further, CHO cells were transiently transfected with vectors encoding His-tagged IEX-1 and HA-ERK1, and ERK activation was examined in anti-HA immunoprecipitates. Figure 4A shows that HA-ERK1 phosphorylation and kinase activity were significantly increased in cells transfected with Wt-IEX-1 as compared with control cells. In seven independent experiments, IEX-1 increased ERK activity by 20 ± 14-fold (mean ± SE). Transient transfection of IEX-1 into UT7 cells also stimulates endogenous ERK activity, as detected by an increase in Elk1-dependent transcription (data not shown). Likewise, endogenous ERK activity was also increased upon TPO stimulation in two stable clones of UT7 cells expressing different levels of HA-IEX-1 as compared with control cells (Neo) expressing the empty vector (Figure 4B). Figure 4.IEX-1 potentiates ERK activity. (A) CHO cells were co-transfected with 1 μg of pcDNA-HA-ERK1 and 1 μg of either empty vector (V) or plasmid encoding His-Wt-IEX, as indicated. Twenty-four hours later, ERK activity was measured in anti-HA immunoprecipitates (IP) by western blotting or kinase assay using MBP as a substrate. (B) Stable clones of UT7 cells expressing different levels of HA-IEX-1 or control cells (Neo) were stimulated for 30 min with TPO and ERK activation was measured by western blotting with anti-phosphoERK antibodies. Download figure Download PowerPoint To determine whether IEX-1 acts as a constitutive activator of the ERK pathway or can only enhance the signal induced by growth factors, CHO cells expressing the EPO receptor (CHO-ER) were co-transfected with IEX-1 and ERK1 expressing vectors and analyzed after overnight starvation in a serum-free medium and EPO stimulation. No phosphorylation of ERK could be detected in non-stimulated starved cells expressing IEX-1 or the empty vector (Figure 5A). However, IEX-1 expression resulted in a great increase in EPO-induced ERK phosphorylation. Similar results were obtained in Cos7 cells stimulated with EGF (data not shown). The effect of IEX-1 was more pronounced in response to low doses of growth factors, i.e. when ERK activation is not at its maximum (Figure 5A). Kinetic studies showed that IEX-1 expression resulted in a prolonged EPO-induced ERK activation in CHO-ER cells, although the effect of IEX-1 was stronger at short time points of stimulation (Figure 5B). Figure 5.Effect of IEX-1 on the kinetics and dose–response of growth factor-mediated ERK activation. CHO-ER cells were transfected with HA-ERK along with either empty pcDNA (V) or pcDNA-HA-IEX-1. Twenty-four hours post-transfection, cells were either harvested directly (non-starved) or starved of serum overnight prior to stimulation with 1–10 U/ml EPO for 10 min (A), or 4 U/ml EPO for various times (B). The activity of HA-tagged ERK was analyzed in anti-HA-immuno precipitates. Download figure Download PowerPoint The above results, together with the fact that IEX-1 is a growth factor–early gene product (Figure 2B; Charles et al., 1993) suggest that the ERK signaling might be prolonged in cells where endogenous IEX-1 is expressed after growth factor treatment. To test this hypothesis, the effect of siRNA duplexes targeting IEX-1 were examined on ERK phosphorylation at times of TPO stimulation when endogenous IEX-1 protein is not expressed (15 min) or is expressed (3, 10 and 14 h). Owing to the low transfection efficiency of UT7 cells, the effect of IEX-1 siRNA on ERK activation was assessed in cells co-transfected with HA-ERK1 and the siRNA duplex. A duplex of GFP was used as a control. As shown in Figure 6, phosphorylation of HA-ERK at late but not at early time points of TPO stimulation was greatly affected by transfection of the IEX-1 siRNA. The ability of the IEX-1 duplex to efficiently silence IEX-1 expression is indicated by the complete loss of HA-IEX-1 expression in cells co-transfected with HA-IEX-1 and IEX-1 siRNA. These results show that the induction of IEX-1 during TPO stimulation plays an important role in the the capacity of this cytokine to sustain ERK activation in UT7 cells. Figure 6.IEX-1 expression is involved in the long-lasting ERK activation induced by TPO in UT7 cells. UT7 cells were electroporated with plasmids encoding HA-ERK1 (upper panel) or HA-IEX-1 (bottom panel) together with either IEX-1 or GFP siRNA duplexes, as indicated. The activity of HA-ERK was assessed in anti-HA-immunoprecipitates following various times of TPO stimulation. Download figure Download PowerPoint In contrast to its ability to potentiate ERK activation, IEX-1 did not activate a co-transfected HA-JNK1, nor did it increase its activation by anisomycin (Figure 7A). Since this specific increase in ERK versus JNK activation recalls the ability of IEX-1 to bind ERK specifically, we examined whether ERK-stimulating activity of IEX-1 requires association with ERK. As shown in Figure 7B, the ΔBD-IEX-1 mutant was unable to enhance ERK phosphorylation. In contrast, the T/SA3-IEX-1 mutant (which still binds to ERK) was as efficient as the wild-type protein in stimulating ERK activation. Thus, IEX-1-induced stimulation of ERK activity correlates with its capacity to dock ERK kinases at the DEF motif. This stimulatory effect was specific for IEX-1 rather than a general function of ERK substrates docking ERK through a DEF motif, as shown by the inability of Elk1 (Jacobs et al., 1999) to affect ERK activation when expressed in CHO cells (Figure 7C). Figure 7.The ability of IEX-1 to stimulate ERK activity is specific and requires its DEF motif. (A) CHO cells were transfected with HA-IEX-1 together with HA-JNK1 or empty vector (V) and treated or not for 10 min with 100 ng/ml anisomycin. Activated JNK was detected in anti-HA immunoprecipitates by immunoblotting with anti-phospho-JNK antibodies and by in vitro kinase assay using GST–c-jun as substrate. (B and C) CHO cells were transfected with HA-ERK, along with either empty vector (V), plasmids encoding the indicated HA-IEX-1 species or HA-Elk1. ERK activity was determined in anti-HA immunoprecipitates by in vitro kinase assay. Download figure Download PowerPoint Taken together these data show that IEX-1 is a direct phosphorylation target of ERK that has the ability to potentiate ERK activation in response to various growth factors (serum, EPO, TPO, EGF). The binding of active ERK to the DEF binding site of IEX-1 is necessary for both events to occur. IEX-1 is an inhibitor of apoptosis induced by various stimuli Previous studies have described conflicting data on the role of IEX-1 in cellular viability establishing IEX-1 either as an inducer or as an inhibitor of apoptosis (Wu et al., 1998; Arlt et al., 2001; Schilling et al., 2001). The above data showing that IEX-1 is a target of ERKs that potentiates growth factor-induced ERK activation prompted us to re-explore the possible involvement of IEX-1 in the control of apoptosis. First, we analyzed apoptosis in UT7 cells stably transfected with HA-tagged Wt-IEX-1 or with an empty vector. Death was induced by removing the cytokine (EPO) from the culture medium while maintaining the presence of serum, and was monitored by annexin V–FITC staining (Figure 8A) and by the appearance of active caspase-3 fragments (Figure 8B). Several clones expressing IEX-1 were uniformly more resistant to apoptosis induced by cytokine deprivation than control (Neo) cells, with the extent of protection correlating with the levels of HA-IEX-1 protein. Figure 8.IEX-1 inhibits cell death induced by various stimuli. (A and B) Clones of UT7 cells stably transfected with empty (Neo) or HA-IEX-1 (Wt) encoding plasmids were cultured with EPO. Apoptosis was induced by removing EPO from the culture medium and measured by annexin V–FITC staining (A) or immunoblotting with anti-caspase-3 antibodies (B). Anti-HA immunoblots show expression of the transgene in the different clones (lower panels). (C) CHO or (D) HeLa cells were transiently transfected with pEGFP or pEGFP-IEX-1. Twenty-four hours post-transfection, the cells were treated with STS (C) or TNF + CHX (D), as indicated. Apoptosis of the EGFP-positive transfected cells was determined as described in Materials and methods. (D) Mean ± SE of three independent transfections. Download figure Download PowerPoint The impact of IEX-1 overexpression on cell survival was also explored in cells transiently transfected with EGFP-IEX-1 proteins. As shown in Figure 8C, CHO cells transfected with EGFP-IEX-1 were considerably more resistant to apoptosis induced by staurosporin (STS) than cells expressing EGFP. Likewise, HeLa cells expressing EGFP-IEX-1 were partially protected against death induced by tumor necrosis factor (TNF) in the presence of cycloheximide (CHX) (Figure 8D). Thus, IEX-1 is able to decrease cell death in response to many stimuli and in different cell types. IEX-1 phosphorylation at T18 is required for its survival effect To determine whether IEX-1 phosphorylation by ERK is required for its anti-apoptotic effect, we examined the ability of IEX-1 phosphorylation mutants to decrease cell death in the various apoptotic assays described above. In contrast to the limited cell death observed in cells expressing Wt-IEX-1, apoptosis induced upon cytokine deprivation was similar in UT7 clones expressing HA- T/SA3-IEX-1 and HA-ΔBD-IEX-1, and in the Neo controls (Figure 9A). This indicates that phosphorylation of IEX-1 at ERK consensus sites is required for its pro-survival activity. Figure 9.IEX-1 phosphorylation is required for its survival effect. (A) UT7 clones stably expressing HA-tagged Wt-IEX-1, T/SA3-IEX-1, ΔBD-IEX-1 or an empty vector (Neo) were deprived of cytokine and stained with annexin V–FITC at the indicated times. Similar results were obtained in three independent experiments. (B) CHO cells were transiently transfected with EGFP or EGFP-IEX-1 wild-type or mutant fusion proteins. Apoptosis was induced by treatm