Growth factors can activate ATF2 via a two-step mechanism: phosphorylation of Thr71 through the Ras-MEK-ERK pathway and of Thr69 through RalGDS-Src-p38
2002; Springer Nature; Volume: 21; Issue: 14 Linguagem: Inglês
10.1093/emboj/cdf361
ISSN1460-2075
Autores Tópico(s)Cell death mechanisms and regulation
ResumoArticle15 July 2002free access Growth factors can activate ATF2 via a two-step mechanism: phosphorylation of Thr71 through the Ras–MEK–ERK pathway and of Thr69 through RalGDS–Src–p38 D.Margriet Ouwens D.Margriet Ouwens Department of Molecular Cell Biology, Section of Signal Transduction, Leiden University Medical Centre Utrecht, Universiteitsweg 100, 3584 CG Utrecht, The Netherlands Search for more papers by this author Nancy D. de Ruiter Nancy D. de Ruiter Department of Physiological Chemistry and Centre for Biomedical Genetics, University Medical Centre Utrecht, Wassenaarseweg 72, 2333 AL Leiden, The Netherlands Search for more papers by this author Gerard C.M. van der Zon Gerard C.M. van der Zon Department of Molecular Cell Biology, Section of Signal Transduction, Leiden University Medical Centre Utrecht, Universiteitsweg 100, 3584 CG Utrecht, The Netherlands Search for more papers by this author Andrew P. Carter Andrew P. Carter Department of Molecular Cell Biology, Section of Signal Transduction, Leiden University Medical Centre Utrecht, Universiteitsweg 100, 3584 CG Utrecht, The Netherlands Present address: Medical Research Council Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 2QH UK Search for more papers by this author Jan Schouten Jan Schouten Centre for Biomedical Genetics, Section Gene Regulation, Leiden University Medical Centre, Wassenaarseweg 72, 2333 AL Leiden, The Netherlands Search for more papers by this author Corina van der Burgt Corina van der Burgt Centre for Biomedical Genetics, Section Gene Regulation, Leiden University Medical Centre, Wassenaarseweg 72, 2333 AL Leiden, The Netherlands Search for more papers by this author Klaas Kooistra Klaas Kooistra Centre for Biomedical Genetics, Section Gene Regulation, Leiden University Medical Centre, Wassenaarseweg 72, 2333 AL Leiden, The Netherlands Present address: Leadd BV, Wassenaarseweg 72, 2333 AL Leiden, The Netherlands Search for more papers by this author Johannes L. Bos Johannes L. Bos Department of Physiological Chemistry and Centre for Biomedical Genetics, University Medical Centre Utrecht, Wassenaarseweg 72, 2333 AL Leiden, The Netherlands Search for more papers by this author J.Antonie Maassen J.Antonie Maassen Department of Molecular Cell Biology, Section of Signal Transduction, Leiden University Medical Centre Utrecht, Universiteitsweg 100, 3584 CG Utrecht, The Netherlands Search for more papers by this author Hans van Dam Corresponding Author Hans van Dam Centre for Biomedical Genetics, Section Gene Regulation, Leiden University Medical Centre, Wassenaarseweg 72, 2333 AL Leiden, The Netherlands Search for more papers by this author D.Margriet Ouwens D.Margriet Ouwens Department of Molecular Cell Biology, Section of Signal Transduction, Leiden University Medical Centre Utrecht, Universiteitsweg 100, 3584 CG Utrecht, The Netherlands Search for more papers by this author Nancy D. de Ruiter Nancy D. de Ruiter Department of Physiological Chemistry and Centre for Biomedical Genetics, University Medical Centre Utrecht, Wassenaarseweg 72, 2333 AL Leiden, The Netherlands Search for more papers by this author Gerard C.M. van der Zon Gerard C.M. van der Zon Department of Molecular Cell Biology, Section of Signal Transduction, Leiden University Medical Centre Utrecht, Universiteitsweg 100, 3584 CG Utrecht, The Netherlands Search for more papers by this author Andrew P. Carter Andrew P. Carter Department of Molecular Cell Biology, Section of Signal Transduction, Leiden University Medical Centre Utrecht, Universiteitsweg 100, 3584 CG Utrecht, The Netherlands Present address: Medical Research Council Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 2QH UK Search for more papers by this author Jan Schouten Jan Schouten Centre for Biomedical Genetics, Section Gene Regulation, Leiden University Medical Centre, Wassenaarseweg 72, 2333 AL Leiden, The Netherlands Search for more papers by this author Corina van der Burgt Corina van der Burgt Centre for Biomedical Genetics, Section Gene Regulation, Leiden University Medical Centre, Wassenaarseweg 72, 2333 AL Leiden, The Netherlands Search for more papers by this author Klaas Kooistra Klaas Kooistra Centre for Biomedical Genetics, Section Gene Regulation, Leiden University Medical Centre, Wassenaarseweg 72, 2333 AL Leiden, The Netherlands Present address: Leadd BV, Wassenaarseweg 72, 2333 AL Leiden, The Netherlands Search for more papers by this author Johannes L. Bos Johannes L. Bos Department of Physiological Chemistry and Centre for Biomedical Genetics, University Medical Centre Utrecht, Wassenaarseweg 72, 2333 AL Leiden, The Netherlands Search for more papers by this author J.Antonie Maassen J.Antonie Maassen Department of Molecular Cell Biology, Section of Signal Transduction, Leiden University Medical Centre Utrecht, Universiteitsweg 100, 3584 CG Utrecht, The Netherlands Search for more papers by this author Hans van Dam Corresponding Author Hans van Dam Centre for Biomedical Genetics, Section Gene Regulation, Leiden University Medical Centre, Wassenaarseweg 72, 2333 AL Leiden, The Netherlands Search for more papers by this author Author Information D.Margriet Ouwens1, Nancy D. de Ruiter2, Gerard C.M. van der Zon1, Andrew P. Carter1,3, Jan Schouten4, Corina van der Burgt4, Klaas Kooistra4,5, Johannes L. Bos2, J.Antonie Maassen1 and Hans van Dam 4 1Department of Molecular Cell Biology, Section of Signal Transduction, Leiden University Medical Centre Utrecht, Universiteitsweg 100, 3584 CG Utrecht, The Netherlands 2Department of Physiological Chemistry and Centre for Biomedical Genetics, University Medical Centre Utrecht, Wassenaarseweg 72, 2333 AL Leiden, The Netherlands 3Present address: Medical Research Council Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 2QH UK 4Centre for Biomedical Genetics, Section Gene Regulation, Leiden University Medical Centre, Wassenaarseweg 72, 2333 AL Leiden, The Netherlands 5Present address: Leadd BV, Wassenaarseweg 72, 2333 AL Leiden, The Netherlands *Corresponding author. E-mail: [email protected] The EMBO Journal (2002)21:3782-3793https://doi.org/10.1093/emboj/cdf361 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Transcription factor ATF2 regulates gene expression in response to environmental changes. Upon exposure to cellular stresses, the mitogen-activated proteinkinase (MAPK) cascades including SAPK/JNK and p38 can enhance ATF2's transactivating function through phosphorylation of Thr69 and Thr71. How ever, the mechanism of ATF2 activation by growth factors that are poor activators of JNK and p38 is still elusive. Here, we show that in fibroblasts, insulin, epidermal growth factor (EGF) and serum activate ATF2 via a so far unknown two-step mechanism involving two distinct Ras effector pathways: the Raf–MEK–ERK pathway induces phosphorylation of ATF2 Thr71, whereas subsequent ATF2 Thr69 phosphorylation requires the Ral–RalGDS–Src–p38 pathway. Cooperation between ERK and p38 was found to be essential for ATF2 activation by these mitogens; the activity of p38 and JNK/SAPK in growth factor-stimulated fibroblasts is insufficient to phosphorylate ATF2 Thr71 or Thr69 + 71 significantly by themselves, while ERK cannot dual phosphorylate ATF2 Thr69 + 71 efficiently. These results reveal a so far unknown mechanism by which distinct MAPK pathways and Ras effector pathways cooperate to activate a transcription factor. Introduction ATF2 is a ubiquitously expressed member of the basic region-leucine zipper (bZIP) transcription factor family that can regulate gene expression in response to changes in the cellular environment. ATF2 plays an important role in placenta formation and skeletal and central nervous system development (Reimold et al., 1996; Maekawa et al., 1999) and is involved in oncogenic transformation and in adaptive responses of the cell to viral infections and (geno)toxic stresses (Liu and Green, 1990; Reimold et al., 1996; Ronai et al., 1998; Maekawa et al., 1999; Falvo et al., 2000; van Dam and Castellazzi, 2001). ATF2 binds its target promoter/enhancers as a homodimer or as a heterodimer with a restricted group of other bZip proteins, the most well known of which is the c-jun oncogene product. Heterodimerization of ATF2 appears to be crucial for at least some of its functions; for instance, the oncogenic activity of ATF2 in chicken cells critically depends on its ability to dimerize with cJun (Huguier et al., 1998). ATF2 is also assumed to play a role in cJun-dependent cell cycle progression, cell survival and apoptosis, in addition to the Fos family members (Johnson et al., 1993; Ham et al., 1995; Verheij et al., 1996; Bossy-Wetzel et al., 1997; Le Niculescu et al., 1999; Schreiber et al., 1999; Wisdom et al., 1999; Kolbus et al., 2000). cJun–ATF2 and ATF2–ATF2 complexes recognize sequence motifs (8 bp) different from the 7 bp motifs bound by cJun–Fos AP-1 complexes (Benbrook and Jones, 1990; Ivashkiv et al., 1990; Hai and Curran, 1991; Chatton et al., 1994), and on minimal promoters cJun–ATF2 heterodimers are more potent transcriptional activators than ATF2–ATF2 homodimers (Benbrook and Jones, 1990; Huguier et al., 1998; van Dam et al., 1998). cJun–ATF2 target genes implicated in growth control include c-jun itself, ATF3, cyclin D1 and cyclin A (Liang et al., 1996; Shimizu et al., 1998; Beier et al., 1999, 2000; Bakiri et al., 2000). A large number of stimuli, including cytokines, peptide growth factors, oncogenes, viruses and cellular stresses such as heat shock and DNA-damaging agents, induce cJun–ATF2 activity (Shaulian and Karin, 2001; van Dam and Castellazzi, 2001). One mechanism to establish this is by increasing the (limiting) levels of cJun, as ATF2 appears to be in excess in most cell types (van Dam and Castellazzi, 2001). Secondly, the transactivation capacities of the N-terminal domains of ATF2 and cJun can be enhanced through phosphorylation by the mitogen-activated protein kinase (MAPK) members p38 and JNK/SAPK (Davis, 2000; Chang and Karin, 2001; Kyriakis and Avruch, 2001). In the case of ATF2, this phosphorylation occurs at Thr69 and Thr71, which appears to enhance the intrinsic histone acetyltransferase activity of ATF2 and to regulate its degradation by the ubiquitin pathway (Davis, 2000; Fuchs et al., 2000; Kawasaki et al., 2000). In addition, ATF2's transactivating potential can be enhanced through direct or indirect binding to viral and cellular proteins, including adenovirus E1A (Liu and Green, 1990), the co-activator/acetyltransferase p300 (Kawasaki et al., 1998; Duyndam et al., 1999), the pX protein of hepatitis B virus, bZIP enhancer factor (bEF) and Tax, which stimulate DNA binding by increasing dimer formation and stability (Perini et al., 1999; Virbasius et al., 1999). Activation of c-jun and other cJun–ATF2 target genes can be established via Ras and/or the related Ral- and Rho-GTPases (Minden and Karin, 1997; Wolthuis and Bos, 1999; Bar-Sagi and Hall, 2000). Ras-dependent phosphorylation of cJun is established via the RalGDS pathway (de Ruiter et al., 2000). Whether ATF2 is a bona fide downstream target of Ras is as yet unclear. Other signaling enzymes involved in cJun–ATF2 activation are the Src(-related) tyrosine kinases, which are downstream targets of mitogen-induced Ral activity (de Ruiter et al., 2000; Goi et al., 2000) and also seem to play a role in JNK-dependent activation of cJun by UV and methyl methanesulfonate (MMS) (Devary et al., 1992; Liu et al., 1996). The observation that various growth factors activate cJun–ATF2-inducible genes in cells lacking functional cJun protein (M.Hamdi, D.M.Ouwens and H.van Dam, unpublished results) suggested that ATF2 is activated efficiently by mitogens. In this study, we have investigated this as yet unknown mechanism of ATF2 activation. We show that insulin, epidermal growth factor (EGF) and serum can increase the transactivation potential of ATF2 by enhancing the phosphorylation state of ATF2 Thr69 and Thr71 in a JNK-independent manner. The phosphorylation of ATF2 by growth factors was established via an as yet unknown two-step mechanism, requiring two distinct Ras effector pathways. The Raf–MEK–ERK pathway was found to induce only phosphorylation of ATF2 Thr71, whereas the Ral–RalGDS–Src-p38–pathway was found to be essential for the subsequent phosphorylation of ATF2 Thr69. Results Insulin activates ATF2 through phosphorylation of Thr69 and Thr71 Cellular stresses and certain cytokines induce ATF2-dependent transcription through JNK/SAPK- and p38-dependent phosphorylation of the N-terminal ATF2 transactivation domain. However, growth factors that activate JNK/SAPK and p38 only very weakly can still induce ATF2-dependent promoters efficiently. This observation prompted us to examine the mechanism of mitogen-induced cJun–ATF2 activation. As shown for A14 fibroblasts in Figure 1A, we found that insulin efficiently activated hybrid proteins containing the transactivation domain of ATF2 fused to the DNA-binding domain of the yeast transcription factor GAL4. Insulin activated the transactivation domain of ATF2 much more efficiently than the corresponding domain of cJun, while the alkylating agent MMS, a potent inducer of JNK and p38, activated ATF2 and cJun to the same extent. Insulin- and EGF-induced activation of GAL4-ATF2 was found to require Thr69 and Thr71, but not Ser90, a third MAPK site present in the ATF2 transactivation domain (Figure 1B; data not shown). We subsequently analyzed the phosphorylation state of endogenous ATF2 by western blot analysis, using anti-phospho-Thr71-ATF2, an antibody that recognizes Thr71-mono-phosphorylated ATF2, and anti-phospho-Thr69 + 71-ATF2, an antibody that recognizes Thr69 + 71-dual-phosphorylated ATF2 but not mono-phosphorylated ATF2 (see Materials and methods). In A14 cells and 3T3L1 adipocytes, insulin was found to induce the phosphorylation of Thr69 and Thr71 strongly within 5 min after addition (Figure 1C; data not shown). Also, other mitogens such as EGF and serum strongly enhanced endogenous ATF2 Thr69 + 71 phosphorylation, as depicted for A14 and primary human fibroblasts in Figure 1D. The levels of growth factor-induced ATF2 Thr69 + 71 phosphorylation were more or less comparable with those induced by cellular stresses, such as MMS, UVC and osmotic shock, although the mitogens had only weak stimulatory effects on the phosphorylation and activation of JNK/SAPK and p38 family members (Figure 1D, see below). Figure 1.Insulin activates ATF2 through phosphorylation of Thr69 and Thr71. (A) Insulin efficiently enhances the transactivating capacity of ATF2. A14 cells were transiently transfected with 2 μg of 5×GAL4-E4-luciferase reporter in the presence or absence of 2 μg of pRSV-GAL4-cJun-N, pRSV-GAL4-ATF2-N or an empty expression vector. At 20 h after transfection, the cells were incubated for 16 h with 10 nM insulin or 1 mM MMS. Transactivation by GAL4-cJun and GAL4-ATF2 in the absence of insulin and MMS was 2.4- and 46-fold, respectively. For comparison, only the fold activation (mean ± SD) by insulin and MMS is depicted, which represents the ratio between relative luciferase activity in the presence and absence of insulin or MMS. (B) Insulin-induced transactivation by ATF2 requires Thr69 and Thr71. A14 cells were transiently transfected with 0.5 μg of 5×GAL4-E4-luciferase reporter plasmid in the presence or absence of 2 μg of the indicated pC2-GAL4-ATF2 expression vectors, encoding either the wild-type (wt) ATF2 transactivation domain, or the corresponding domain in which Thr69 (T69A), Thr71 (T71A) or both (T69/71A) are replaced by alanine. At 6 h after transfection, the cells were stimulated for 16 h with 10 nM insulin. The relative activity is the enhancement of promoter activity by the various GAL4-ATF2 fusion proteins in the absence and presence of insulin, and is presented as the mean ± SD of two independent experiments performed in triplicate. Note the different scaling of the left and right y-axis. (C) Insulin-induced ATF2 Thr71 and Thr69 + 71 phosphorylation. Serum-starved A14 cells were stimulated with 10 nM insulin for the indicated times. Total cell extracts (30 μg of protein) were analyzed by SDS–PAGE and immunoblotting. Ponceau S staining confirmed that the filters contained equal amounts of protein extracts. The faster migrating bands seen by the phospho-specific ATF2 antibodies seem to represent shorter, alternatively spliced, ATF2 products (Georgopoulos et al., 1992). (D) Mitogen-induced ATF2 Thr69 + 71 phosphorylation does not correlate with JNK Thr183/Tyr185 and p38 Thr180/Tyr182 phosphorylation in A14 and primary human VH10 fibroblasts. Serum-starved cells were stimulated for the indicated times with 10 nM EGF, 10 nM insulin, 500 mM NaCl (O.S.), 10% FCS or 30 J/m2 UVC. Total cell extracts (30 μg of protein) were analyzed for the levels of the indicated proteins by SDS–PAGE and subsequent immunoblotting. Download figure Download PowerPoint Differential phosphorylation of ATF2 Thr71 and Thr69 by the Raf–MEK and RalGDS–Ral effector pathways of Ras As insulin and EGF, but not MMS and osmotic stress, rapidly activate the Ras proteins (Burgering et al., 1991; D.M.Ouwens and H.van Dam, data not shown), we examined whether insulin- and EGF-induced ATF2 Thr69 + 71 phosphorylation is established via a Ras-dependent signaling cascade. The dominant-negative Ras mutant RasN17 efficiently inhibited insulin and EGF-induced ATF2 Thr69 + 71 phosphorylation as well as insulin- and EGF-induced activation of GAL4-ATF2, while having no inhibitory effect on ATF2 activation by osmotic stress and MMS (Figure 2A and B; data not shown). In line with this, a constitutively active mutant of Ras, RasL61, induced phosphorylation of ATF2 Thr69 + 71 to the same extent as osmotic stress (Figure 2C) and activated GAL4-ATF2 in a Thr69- and Thr71-dependent manner (Figure 2D). This indicates that an increase in Ras-GTP levels is sufficient to trigger ATF2 activation. Figure 2.Ras-dependent growth factor-induced activation of ATF2. (A) Inhibition of insulin-induced ATF2 phosphorylation by dominant-negative Ras and Ral. A14 cells were transiently transfected with 0.5 μg of pMT2-HA-ATF2 in the presence or absence of 2 μg of pRSV-RasN17, pMT2-HA-RalN28 or an empty expression vector. Fugene reagent was used in order to obtain high levels of transfection efficiency (>40%). At 24 h after transfection, the cells were stimulated with either 10 nM insulin (15 min) or 500 mM NaCl (O.S.) (15 min). A 30 μg aliquot of total cell extract was analyzed by SDS–PAGE/immunoblotting. Ectopically expressed RasN17, HA-ATF2 and HA-RalN28 were detected using monoclonal Y13-259 Ras and HA antibodies, respectively. Comparison of the extracts of mock-transfected cells with those of HA-ATF2 transfected cells verified that only exogenous HA-ATF-2 is detected on the exposures shown [data not shown; compare with (C) and Figure 5F]. (B) Insulin-induced GAL4-ATF2-dependent transactivation is inhibited by RasN17 and RalN28. A14 cells were transiently transfected with 0.5 μg of 5×GAL4-E4-luciferase reporter together with 2 μg of expression vectors for RasN17 and RalN28, or an empty control vector, in the presence or absence of either 1 μg of pRSV-GAL4-ATF2-N or an empty expression vector. Cells were serum starved for 24 h, followed by stimulation with 10 nM insulin for 14 h. Depicted is the enhancement of promoter activity by GAL4-ATF2 in the absence and presence of insulin and/or the inhibitors (mean ± SD). Note the different scaling of the left and right y-axis. (C) Active RasL61 induces ATF2 Thr69 + 71 phosphorylation. A14 cells were kept untreated (−), or transfected with 0.5 μg of pMT2-HA-ATF2 in the presence or absence of 2 μg of RasL61 expression vector, or an empty vector (−). Fugene reagent was used in order to obtain high levels of transfection efficiency (>40%). At 24 h after transfection, cells were stimulated with NaCl (O.S.), as described for Figure 1D, when indicated, and total cell lysates were prepared and analyzed by SDS–PAGE and immunoblotting. (D) Active RasL61 enhances transactivation by ATF2 via ATF2 Thr69 and Thr71. A14 cells were transiently transfected with 2 μg of 5×GAL4-E4-luciferase reporter plasmid together with 2 μg of pRSV-GAL4-ATF2 expression vectors containing full-length (wt) ATF2, or the corresponding domain in which Thr69 (T69A), Thr71 (T71A) or both (T69/71A) are replaced by alanine. In addition to these GAL4 fusion constructs, 3 μg of pRSV-RasL61, or an empty expression vector, was co-transfected. At 40 h after transfection, cells were harvested and analyzed for luciferase activity. The fold activation depicted represents the ratio between luciferase activity (mean ± SD) in the presence and absence of RasL61. Download figure Download PowerPoint Since Ras can induce gene expression via three different effector pathways, Raf–MEK, phosphatidylinositol 3-kinase (PI3-K)–PKB and RalGDS–Ral (Bos, 1998), we next analyzed the involvement of these pathways in ATF2 activation. Inhibition of the PI3-K–PKB pathway by wortmannin and/or LY294002 had no effect on the induction of ATF2 Thr69 + 71 phosphorylation by mitogens (data not shown). In contrast, the dominant-negative Ral mutant RalN28 efficiently blocked insulin-induced activation of GAL4-ATF2 and the insulin-induced phospho-Thr69 + 71 signal (Figure 2A and B). However, in contrast to RasN17, RalN28 appears to inhibit insulin-induced ATF2 Thr71 mono-phosphorylation only partially (Figure 2A). Efficient inhibition of the phospho-Thr71 signal was only observed when the cells were, in addition to RalN28, also pre-treated with the MEK inhibitor U0126 (Figures 2A and 3A). Like dominant-negative Ras, the combination of RalN28 and U0126 had no effect on osmotic stress-induced ATF2 phosphorylation (Figure 3A). Figure 3.Differential phosphorylation of ATF2 Thr71 and Thr69 by the Raf–MEK and RalGDS–Ral effector pathways of Ras. (A) Inhibition of insulin-induced ATF2 phosphorylation by RalN28 and the MEK inhibitor U0126. A14 cells were transfected and stimulated with either insulin or NaCl (O.S.) as described for Figure 2A. Where indicated, the cells were pre-treated for 30 min with 10 μM U0126. A 30 μg aliquot of total cell extract was analyzed by SDS–PAGE/immunoblotting. The positions of the exogenously expressed HA-ATF2 and the faster migrating endogenous (end.) ATF2 are indicated on the right. (B) Activation of the Raf–MEK pathway induces ATF2 Thr71 mono-phosphorylation. Serum-starved A14 cells were incubated for 30 min in the absence or presence of 10 μM U0126 prior to treatment with 10 nM insulin or 100 nM TPA. Total cell extracts (30 μg of protein) were prepared after 15 min, and analyzed by SDS–PAGE/immunoblotting. (C) Differential effects of SB203580 on ATF2 Thr71 phosphorylation induced by growth factors and stresses. Serum-starved JNK1+2−/− fibroblasts were incubated for 30 min in the absence or presence of 5 μM SB203580 prior to treatment with 10 nM EGF, 20% FCS, 30 J/m2 UVC or 1 mM MMS as indicated. Total cell extracts (30 μg of protein) were prepared after 15 min (except for MMS: 2 h), and analyzed by SDS–PAGE and subsequent immunoblotting. (D) Differential effects of SB203580 and U0126 on ATF2 Thr71 and Thr69 + 71 phosphorylation. Serum-starved JNK1+2−/− fibroblasts were treated for 30 min with 5 μM SB203580 and/or 10 μM U0126 prior to stimulation for 15 min with 10 nM insulin or 10 nM EGF as indicated. Total cell extracts (30 μg of protein) were analyzed by SDS–PAGE and immunoblotting. Download figure Download PowerPoint In the absence of RalN28, insulin-induced ATF2 Thr71 and Thr69 + 71 phosphorylation was only partially (∼55%) inhibited by U0126, despite the fact that MEK-dependent ERK phosphorylation was completely abrogated (Figure 3B). As this residual ATF2 phosphorylation seems to be due to the slight potentiating effect of U0126 on insulin-induced JNK and p38 activity (data not shown), we used the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) to examine further the role of the Raf–MEK pathway in ATF2 activation. TPA is a potent inducer of Raf and MEK in A14 cells, but does not activate RalGDS (Wolthuis et al., 1998a) and JNK/p38 (data not shown). Importantly, in contrast to insulin or EGF treatment, TPA treatment did not induce detectable ATF2 Thr69 + 71 phosphorylation, but did induce a strong phospho-Thr71 signal, which was completely prevented upon pre-treatment with U0126 (Figure 3B). These results indicate that activation of the Raf–MEK pathway is only sufficient for ATF2 Thr71 mono-phosphorylation and that ATF2 Thr69 + 71 dual phosphorylation requires additional activation of the RalGDS–Ral pathway. We next examined the effect of the p38 inhibitor SB203580 on insulin- and EGF-induced ATF2 Thr69 + 71 phosphorylation. Since SB203580 was found to enhance the activation of JNK by these mitogens strongly in fibroblasts (data not shown), we used JNK1+2−/− fibroblasts (K.Sabapathy, K.Hochedlinger, A.Bauer, L.Chang, M.Karin and E.Wagner, submitted). Interestingly, also in the presence of SB203580, differential regulation of ATF2 Thr71 mono-phosphorylation and ATF2 Thr69 + 71 dual phosphorylation by growth factors was observed: SB203580 completely blocked the insulin-, EGF- and serum-induced phospho-ATF2 Thr69 + 71 signal, but only very weakly suppressed insulin-induced ATF2 Thr71 phosphorylation and did not inhibit EGF-induced ATF2 Thr71 phosphorylation at all (Figure 3C and D). In contrast, MMS- and UV-induced ATF2 Thr69 + 71 and ATF2 Thr71 phosphorylation were both completely inhibited by SB203580 (Figure 3C). As previously found for A14 cells (Figure 3B), inhibition of MEK with U0126 also only partially (50–70%) prevented insulin-induced ATF2 Thr71 phosphorylation in JNK−/− cells (Figure 3D). Since the residual insulin-induced Thr71 phosphorylation could be inhibited by SB203580, both MEK and p38 appear to be required for ATF2 Thr71 phosphorylation by insulin (Figure 3D). However, in the case of EGF, the residual Thr71 phosphorylation in the presence of U1026 seems to be due mainly to the potentiating effect of U0126 on the activation of p38 (Figure 3D; data not shown): U0126 and SB203580 completely blocked ATF2 Thr71 phosphorylation by EGF when added together, but SB203580 only inhibited Thr69 + 71 phosphorylation, and not Thr71 phosphorylation, when added alone (Figure 3D). These data strongly suggest that the induction of ATF2 Thr71 mono-phosphorylation by EGF occurs mainly via a MEK-dependent process, and that p38 only plays a role when the MEK pathway is inhibited. In summary, the results presented above indicate that growth factors can activate ATF2 via an as yet unknown two-step mechanism: Thr71 mono-phosphorylation executed predominantly by the Ras–Raf–MEK pathway and (subsequent) Thr69 + 71 dual phosphorylation via RalN28- and SB203580-inhibitable factors. Insulin- and EGF-induced ATF2 Thr71 mono-phosphorylation is mediated by ERK To identify the Ras–MEK-induced ATF2 Thr71 kinase, we performed anion-exchange chromatography of A14 cell extracts on MonoQ columns. As depicted in Figure 4A, the bulk of the insulin-induced ATF2 kinase activity (fractions 12 and 13) contained high levels of active (mobility-shifted and phosphorylated) ERK1 and ERK2, no p38 and only low amounts of (non-mobility-shifted) JNK (compare fractions 9 and 11 with fractions 12 and 13 in Figure 4A). The induction of the kinase activity in fraction 12 + 13 by insulin was inhibited specifically by pre-treatment with the MEK inhibitors U0126 or PD98059 (data not shown; compare Figure 4B). In contrast to the insulin-induced ATF2 kinase, the osmotic stress-induced ATF2 kinase activity co-purified with JNK/SAPK [fractions 9 and 11–13; predominantly containing (mobility-shifted active) JNK] and p38 (fraction 17; Figure 4A). Also, in EGF-treated JNK−/− cells, nearly all of the ATF2 kinase activity co-purified with phosphorylated ERK1/2 (90%) rather than with p38 (5%). Co-purification of ERK1/2 and this ATF2 kinase activity was still observed after subsequent monoQ chromatography of fraction 12 at pH 7.8 instead of pH 7.5, which led to a further separation of ERK1 and ERK2, and after refractionation on a MonoS column (data not shown). We subsequently analyzed immunopurified ERK for mitogen-inducible MEK-dependent ATF2 kinase activity. Insulin, but not osmotic stress, enhanced ERK-associated N-terminal ATF2 kinase activity ∼4.8-fold, which was inhibited by pre-treatment with the MEK inhibitors PD98059 and U0126 (Figure 4B; data not shown). Thus, in A14 and JNK−/− cells, the major mitogen-inducible ATF2 kinase appears to be ERK1/2 rather than JNK/SAPK and p38 (family members). Figure 4.Insulin- and EGF-induced ATF2 Thr71 mono-phosphorylation is mediated by ERK. (A) The main insulin-induced ATF2 N-terminal kinase activity co-purifies with ERK1/2 after MonoQ anion-exchange chromatography. Total cell lysates from A14 cells treated for 15 min with either 10 nM insulin or 500 mM NaCl (O.S.) were separated on a MonoQ column using a linear gradient of NaCl (dotted line). Fractions were analyzed for in vitro ATF2 kinase activity (filled circles) as described in Materials and methods, and for the presence of JNK, ERK1/2 and p38 by SDS–PAGE and immunoblotting. (B) Insulin induces ERK-associated ATF2 N-terminal kinase activity. Serum-starved A14 cells were either untreated or treated for 15 min with 10 nM insulin, with or without 15 min pre-treatment with 20 μM PD98059, as indicated. Total cell lysates were immunoprecipitated with antibodies that recognize both ERK1 and ERK2, and subsequently assa
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