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

Abstract

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. However, the mechanism of ATF2 activation by growth factors that are poor activators of JNK and p38 is still elusive. Here, we show that in ®broblasts, 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 factorstimulated ®broblasts is insuf®cient to phosphorylate ATF2 Thr71 or Thr69 + 71 signi®cantly by themselves, while ERK cannot dual phosphorylate ATF2 Thr69 + 71 ef®ciently. These results reveal a so far unknown mechanism by which distinct MAPK pathways and Ras effector pathways cooperate to activate a transcription factor. Keywords: ATF2/ERK/mitogens/p38/Ras 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 . 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;. cJun±ATF2 target genes implicated in growth control include c-jun itself, ATF3, cyclin D1 and cyclin A ...

Aberrant Polycystin-1 Expression Results in Modification of Activator Protein-1 Activity, whereas Wnt Signaling Remains Unaffected

“…1A, upper two panels) in short TOP and FOP; the ␤-catenin Ser 33 (␤-cat S33) construct containing full-length ␤-catenin with a mutation at Ser 33 and its corresponding empty vector control, pcDNA3Zeo⌬MCS, in short pcDNA3Zeo (14); the 5ϫjun2 TATA pGL3, in short 5ϫjun, 5ϫcollTRE TATA pGL3, in short 5ϫcoll, TATA pGL3 (15,16) (Fig. 4A, upper panel), Ϫ1600/ϩ740 wt c-jun TATA pGL3, and Ϫ1600/ϩ740 m1 ϩ 2 c-jun TATA pGL3, luciferase reporter constructs (17); the Myc-tagged cdc42 V12 construct, encoding constitutively active Cdc42, and its corresponding empty vector control, pMT2 (18); and the HA-tagged ATF2 construct, HA-ATF2 (19). The p-AP-1-Luc or 7ϫAP-1 reporter construct (Stratagene, Cedar Creek, TX) was a kind gift from M. Karperien (Leiden University Medical Center, Department of Endocrinology, Leiden, The Netherlands).…”

“…4A, upper panel). The 5ϫcoll and 7ϫAP-1 reporters are activated by Jun-Fos heterodimers and can be strongly induced by TPA (19,27). The 5ϫjun reporter construct mainly monitors Jun-ATF activity and is hardly enhanced by TPA.…”

“…6B, right panel). This increased expression of ATF2 may reflect enhanced activity of more upstream extracellular signal-regulated kinases (ERK) (19). Furthermore, increased activity of the Jun-Fos-dependent AP-1 reporters, 5ϫcoll (Fig.…”

ATF-2 Regulates Fat Metabolism inDrosophila

“…Activation of ATF-2 by p38/JNK is thought to play a role in apoptosis (Gupta et al, 1995). ATF-2 is also activated by insulin, epidermal growth factor, and serum via a two-step mechanism involving two distinct Ras effector pathways (Ouwens et al, 2002). A group of ATF-2 target genes have been identified that are involved in multiple biological phenomena.…”

Activation of the ERK signal transduction pathway by Epstein–Barr virus immediate-early protein Rta

“…Furthermore, Rta activates the BZLF1 promoter through an ATF2 site in the ZII region (Adamson et al, 2000). Therefore, activation of the ERK signal transduction pathway by Rta may ultimately influence the transcription of BZLF1 through the activation of ATF2, a downstream target of the ERK signal transduction pathway (Morton et al, 2004;Ouwens et al, 2002). The first piece of evidence in support of this hypothesis is the fact that the binding capacity of GST-BRAP2-glutathione-Sepharose beads to KSR1 declined substantially when the beads were pre-incubated with cell lysates containing Rta (Fig.…”