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

Abstract

IEX-1 is an early response and NF-kappaB 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.

Fig. 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.

Fig. 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.

Fig. 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.

Fig. 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.

Fig. 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.

Fig. 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.

Fig. 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.

Fig. 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.

Fig. 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 treatment with STS for 7 h and measured in EGFP-transfected cells as described in Materials and methods. Results are mean ± SE of values from at least four independent transfections. (C) Total lysates from CHO cells transfected and treated as in (B) were analyzed by western blotting with anti-phosphoIEX-1 and anti-GFP antibodies.

Fig. 10. IEX-1 inhibits apoptosis independently of its ability to potentiate ERK activation. (A, B and E) CHO cells were transfected with the indicated plasmids encoding EGFP-tagged wild-type or mutant forms of IEX-1. After a 7 h treatment with STS in the presence or absence of 30 µM PD98059, cells were either lysed for western blotting analysis (A) or fixed for determination of cell death (B and E). Results of apoptosis are expressed as mean ± SE of four independent experiments. (C and D) CHO cells were transfected with HA-IEX-1 wild type or mutants, with (C) or without (D) HA-ERK1. HA immunoprecipitates were subjected to kinase assay and western blotting, as indicated.