CREB is activated by UVC through a p38/HOG-1-dependent protein kinase
1997; Springer Nature; Volume: 16; Issue: 5 Linguagem: Inglês
10.1093/emboj/16.5.1009
ISSN1460-2075
Autores Tópico(s)NF-κB Signaling Pathways
ResumoArticle1 March 1997free access CREB is activated by UVC through a p38/HOG-1-dependent protein kinase M. Iordanov M. Iordanov Forschungszentrum Karlsruhe, Institute für Genetik, Postfach 3640, D-76021 Karlsruhe, Germany Oregon Health Sciences University, Department of Cell and Developmental Biology, Portland, OR, 97201 USA Search for more papers by this author K. Bender K. Bender Forschungszentrum Karlsruhe, Institute für Genetik, Postfach 3640, D-76021 Karlsruhe, Germany Search for more papers by this author T. Ade T. Ade für Toxikologie, Postfach 3640, D-76021 Karlsruhe, Germany Search for more papers by this author W. Schmid W. Schmid Deutsches Krebsforschungszentrum, Abteilung für Molekularbiologie der Zelle I, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany Search for more papers by this author C. Sachsenmaier C. Sachsenmaier Forschungszentrum Karlsruhe, Institute für Genetik, Postfach 3640, D-76021 Karlsruhe, Germany Search for more papers by this author K. Engel K. Engel Max-Delbrück-Centrum für Molekulare Medizin, AG Stressproteine, Robert-Rössle-Strasse 10, D-13122 Berlin, Germany Search for more papers by this author M. Gaestel M. Gaestel Max-Delbrück-Centrum für Molekulare Medizin, AG Stressproteine, Robert-Rössle-Strasse 10, D-13122 Berlin, Germany Search for more papers by this author H.J. Rahmsdorf H.J. Rahmsdorf Forschungszentrum Karlsruhe, Institute für Genetik, Postfach 3640, D-76021 Karlsruhe, Germany Search for more papers by this author P. Herrlich Corresponding Author P. Herrlich Forschungszentrum Karlsruhe, Institute für Genetik, Postfach 3640, D-76021 Karlsruhe, Germany Universität Karlsruhe, Institut für Genetik, Postfach 3640, D-76021 Karlsruhe, Germany Search for more papers by this author M. Iordanov M. Iordanov Forschungszentrum Karlsruhe, Institute für Genetik, Postfach 3640, D-76021 Karlsruhe, Germany Oregon Health Sciences University, Department of Cell and Developmental Biology, Portland, OR, 97201 USA Search for more papers by this author K. Bender K. Bender Forschungszentrum Karlsruhe, Institute für Genetik, Postfach 3640, D-76021 Karlsruhe, Germany Search for more papers by this author T. Ade T. Ade für Toxikologie, Postfach 3640, D-76021 Karlsruhe, Germany Search for more papers by this author W. Schmid W. Schmid Deutsches Krebsforschungszentrum, Abteilung für Molekularbiologie der Zelle I, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany Search for more papers by this author C. Sachsenmaier C. Sachsenmaier Forschungszentrum Karlsruhe, Institute für Genetik, Postfach 3640, D-76021 Karlsruhe, Germany Search for more papers by this author K. Engel K. Engel Max-Delbrück-Centrum für Molekulare Medizin, AG Stressproteine, Robert-Rössle-Strasse 10, D-13122 Berlin, Germany Search for more papers by this author M. Gaestel M. Gaestel Max-Delbrück-Centrum für Molekulare Medizin, AG Stressproteine, Robert-Rössle-Strasse 10, D-13122 Berlin, Germany Search for more papers by this author H.J. Rahmsdorf H.J. Rahmsdorf Forschungszentrum Karlsruhe, Institute für Genetik, Postfach 3640, D-76021 Karlsruhe, Germany Search for more papers by this author P. Herrlich Corresponding Author P. Herrlich Forschungszentrum Karlsruhe, Institute für Genetik, Postfach 3640, D-76021 Karlsruhe, Germany Universität Karlsruhe, Institut für Genetik, Postfach 3640, D-76021 Karlsruhe, Germany Search for more papers by this author Author Information M. Iordanov1,2, K. Bender1, T. Ade3, W. Schmid4, C. Sachsenmaier1,5, K. Engel6, M. Gaestel6, H.J. Rahmsdorf1 and P. Herrlich 1,7 1Forschungszentrum Karlsruhe, Institute für Genetik, Postfach 3640, D-76021 Karlsruhe, Germany 2Oregon Health Sciences University, Department of Cell and Developmental Biology, Portland, OR, 97201 USA 3für Toxikologie, Postfach 3640, D-76021 Karlsruhe, Germany 4Deutsches Krebsforschungszentrum, Abteilung für Molekularbiologie der Zelle I, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany 5Fred Hutchinson Cancer Research Center, A2-025, 1124, Columbia Street, Seattle, WA, 98104 USA 6Max-Delbrück-Centrum für Molekulare Medizin, AG Stressproteine, Robert-Rössle-Strasse 10, D-13122 Berlin, Germany 7Universität Karlsruhe, Institut für Genetik, Postfach 3640, D-76021 Karlsruhe, Germany The EMBO Journal (1997)16:1009-1022https://doi.org/10.1093/emboj/16.5.1009 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Changes in environmental conditions such as the addition of growth factors or irradiation of cells in culture first affect immediate response genes. We have shown previously that short wavelength UV irradiation (UVC) elicits massive activation of several growth factor receptor-dependent pathways. At the level of the immediate response gene c-fos, these pathways activate the transcription factor complex serum response factor (SRF)–p62TCF which mediates part of the UV-induced transcriptional response. These studies have, however, suggested that more that one pathway is required for full UV responsiveness of c-fos. Using appropriate promoter mutations and dominant-negative cAMP response element (CRE)-binding protein (CREB), we now find that UVC-induced transcriptional activation depends also on the CRE at position −60 of the c-fos promoter and on the functionality of a CREB. Upon UV irradiation, CREB and ATF-1 are phosphorylated at serines 133 and 63, respectively, preceded by and dependent on activation of p38/RK/HOG-1 and of a p38/RK/HOG-1-dependent p108 CREB kinase. Although p90RSK1 and MAPKAP kinase 2 are also activated by UV, p90RSK1 does not, at least not decisively, participate in this signalling pathway to CREB and ATF-1 as it is not p38/RK/HOG-1 dependent, and CREB is a poor substrate for MAPKAP kinase 2 in vitro. On the basis of resistance to the growth factor receptor inhibitor suramin and of several types of cross-refractoriness experiments, the UVC-induced CREB/ATF-1 phosphorylation represents an as yet unrecognized route of UVC-induced signal transduction, independent of suramin-inhibitable growth factor receptors and different from the Erk 1,2–p62TCF pathway. Introduction Many of the cellular reactions to short wavelength radiation (UVC) such as skin erythema and carcinogenesis, UVC-induced programmed cell death (apoptosis) and cellular survival seem to require UVC-induced gene expression (Dévary et al., 1992; Friedberg et al., 1995; Schreiber et al., 1995). Two series of experiments support this interpretation. The changes of gene expression after UVC irradiation depend on the activity of protein tyrosine kinases (Dévary et al., 1992; Sachsenmaier et al., 1994). Accordingly, tyrosine kinase inhibitors prevent gene induction and also increase the sensitivity of cells to UVC killing (Dévary et al., 1992). The second type of evidence is based on elimination of a critical transcription factor. Both mutagenic reactions and survival are impaired in the absence of the transcription factor subunit Fos. Antisense c-fos oligonucleotides suppress UVC-induced chromosomal aberrations (van den Berg et al., 1991) and embryonic fibroblasts from c-fos −/− mice show increased sensitivity to UVC (Schreiber et al., 1995; Haas and Kaina, 1995). In addition, UVC influences not only the programme of genes expressed in the irradiated cells, but also the fate of inner organismic cells that are not reached directly by UVC, by inducing the synthesis and secretion of growth factors (Schorpp et al., 1984; Rotem et al., 1987; Krämer et al., 1993). The fact that adverse agents such as UVC can trigger complex macromolecule synthesis requires the existence of primary sensors which react with the adverse agent or absorb the radiation energy and then translate into cellular language, i.e. activate molecules normally involved in signalling and gene regulation. There has been only limited progress in unravelling the primary target(s) of UVC absorption relevant for signal transduction to the transcription factors. Comparison of dose–response curves for induced gene expression in DNA repair-proficient and −deficient cells has yielded support for DNA damage as an intermediate of induction (Miskin and Ben-Ishai, 1981; Schorpp et al., 1984; Stein et al., 1989). Thus DNA seems to be one of the relevant absorbing molecules. A second class of 'sensors' has been found recently in the plasma membrane by systematically following the signal pathways in the 'upstream' direction starting from transcription factors mediating the UV response (Mai et al., 1989; Dévary et al., 1992, 1993; Radler-Pohl et al., 1993; Sachsenmaier et al., 1994). On the basis of the inhibition of the UV response by either suramin (a drug that poisons plasma membrane receptors) or by pre-treatment of cells with growth factors (a procedure which induces, after transient activation, the inactivation and internalization of growth factor receptors), and from the finding that several growth factor receptors are phosphorylated at tyrosine residues within a fraction of a minute after UV irradiation, it has been concluded that growth factor receptors are part of the relevant pathway(s) used by UV to reach, in particular, the immediate response genes (Sachsenmaier et al., 1994; Knebel et al., 1996). According to these data, UVC-induced transcription of c-fos in HeLa cells is mediated to a large extent by epidermal growth factor receptor (EGFR), interleukin-1 receptor (IL-1R) and basic fibroblast growth factor receptor (bFGFR). The c-fos promoter elements required for UVC-induced transcription have been delimited by deletion mutation (Büscher et al., 1988). The serum response element (SRE) is required and its deletion reduces induction to ∼50%. Indeed, the UV-induced growth factor-dependent signal transduction pathways lead to activation of the ternary complex factor p62TCF (Elk-1; Sap-1; Sachsenmaier et al., 1994; Price et al., 1996) which, in conjunction with the serum response factor (SRF), binds to the SRE (reviewed by Treisman, 1994). Using new promoter mutants, we now find that the response of the c-fos promoter depends also on the presence of the cAMP response element (CRE)-binding protein (CREB) binding site at position −60 of the c-fos promoter. This not only indicates that promoter elements are complex cooperating assemblies but also shows the existence of still another UVC signalling pathway to c-fos and possibly to other CRE-regulated promoters. CREB and ATF-1 are indeed phosphorylated in response to UVC, at serines 133 and 63 respectively. A p38/RK/HOG-1-dependent p108 serine/threonine protein kinase is identified as a UVC-induced CREB kinase in HeLa cells. The UVC-induced CREB and ATF-1 phosphorylation, on the basis of suramin resistance and of various types of cross-refractoriness experiments, represents a novel UVC signalling pathway independent of activation of the growth factor receptors described so far. Results Several promoter elements contribute to the UVC response of the c-fos promoter: the proximal CRE as a new UV response element Dissection of the promoter has yielded evidence that the p62TCF/Elk-1–(SRF)2 complex formed over the SRE (at positions −320/−300), is important for the UVC response of c-fos (Büscher et al., 1988; Sachsenmaier et al., 1994; Price et al., 1996). When cloned in front of a heterologous UVC-non-responsive promoter, the SRE suffices to confer UVC inducibility, and inducibility of the c-fos promoter in 3T3 cells is inhibited by 50% upon deletion of the SRE. To screen for other UVC targets in the c-fos promoter, we examined deletion mutants of the proximal CRE (−60 CRE). In HeLa cells transiently transfected with c-fos promoter–CAT reporter constructs, deletion of the CRE reduced both the UVC and the cAMP inducibility to 50% both the UVC and the cAMP response of the full-length promoter (Figure 1, compare lanes 1–3 with lanes 4–6). This suggests that a factor with binding ability similar to CREBM1 was involved in the UVC response and that CREBM1 could not be activated by UVC (nor by cAMP) treatment of cells. The specificity of the inhibition by CREBM1 is demonstrated by the fact that it does not interfere with the UVC induction of a NF-κB-dependent promoter construct (−105/−79 HIV TATA CAT; Figure 1, compare lanes 14 and 17). Similar roles for the −60 CRE and for a CRE-binding factor in the UVC response of c-fos were observed in NIH3T3 mouse fibroblasts and in JEG-3 human chorionic carcinoma cells (not shown). The residual cAMP response of the CRE deletion mutant was reduced further by CREBM1 (Figure 1, compare lanes 9 and 12), probably because of the existence of two upstream CRE-like sequences located at positions −343/−336 and −295/−288 which presumably bind members of the CREB/ATF family (Berkowitz et al., 1989). Whether the upstream CREB/ATF-binding elements do participate in the UVC response cannot be decided unequivocally, since the contribution of the transcription factor complex binding to the SRE dominates (Figure 1, compare lanes 5, 8 and 11), but they probably do. The remaining cAMP response seen in lane 12 may be due to a non-homologous intragenic CRE which does not bind CREB (+18/+38, Härtig et al., 1991). UVC induces the transactivating property of CREB To show directly that the transactivating property of one of the CRE-binding factors, CREB (Sassone-Corsi et al., 1988), can be induced by UVC, we tested whether CREB fused to a different DNA-binding domain, that of the yeast transcription factor GAL4, could be activated by UVC to initiate transcription of a GAL4-dependent reporter. HeLa cells were transiently co-transfected with expression vectors encoding either the DNA-binding domain (DBD) of GAL4 or a chimeric protein consisting of the GAL4 DBD fused to the full-length CREB protein (Figure 2A) or to a CREB protein lacking the bZip region (Figure 2B), together with a GAL4 element-driven CAT reporter gene construct (Chrivia et al., 1993). Both cAMP and UVC promoted GAL4–CREB-dependent transcription but not that by the GAL4 DBD alone (Figure 2), indicating efficient activation of CREB by both UVC and cAMP. With constructs containing the complete CREB including the bZip region, UVC induction was less efficient than cAMP induction while the reverse was found for the truncated CREB (compare Figure 2A and B). The reason for this difference is unknown. Figure 2.(A) UVC induces the transactivation potential of CREB and of CBP. HeLa cells were transfected with 2 μg of GAL4–CAT reporter construct together with 3 μg of either GAL4 DBD (GAL 1–147), GAL4–CREB (full-length) or GAL4–CBP expression vectors as indicated. The cells were serum-starved for 24 h post-transfection and then stimulated with 30 J/m2 UVC or forskolin and IMX (20 μM and 1 mM, cAMP). After 24 h, CAT determinations were performed. (B) UV-induced activation of CREB depends on the presence of Ser133. HeLa cells were transfected with 2 μg of GAL4–CAT reporter construct together with 3 μg of either GAL4 DBD (GAL 1–147), GAL4–CREBΔbZip wild-type, GAL4–CREBΔbZip S133A or GAL4–CREBΔbZip S142A expression vectors as indicated. The cells were serum-starved for 24 h post-transfection and then stimulated with either 30 J/m2 UVC, forskolin and IMX (20 μM and 1 mM, cAMP) or EGF (20 ng/ml). After 24 h, CAT determinations were performed. Induction factors are indicated. The experiment presented is one of several independently performed determinations. Download figure Download PowerPoint Activation of CREB by cAMP depends on the phosphorylation of Ser133 by the cAMP-dependent protein kinase A (PKA; Gonzales and Montminy, 1989; Taylor et al., 1990). Accordingly, a GAL4–CREB gene construct in which Ser133 was mutated to alanine responded less well to cAMP treatment (Figure 2B, lane 11). UVC induction of this hybrid transcription factor was reduced to the same extent (lane 10), indicating that Ser133 is also instrumental in UV-induced transactivation of CREB. It is not clear how the residual activity in the Ala133 mutant is generated. Perhaps some CBP (see below) can interact with the mutant protein. Residual activation could then be caused by CBP activation (see below). Interestingly, a second phosphorylation site, Ser142, usually found to be inhibitory and the target of Ca2+/calmodulin-dependent protein kinase II (Sun et al., 1994), does not seem to be addressed by UVC. Mutation of this serine to alanine did not significantly enhance forskolin- or UV-induced transactivation of GAL4–CREB (Figure 2B, compare lanes 7 and 15 and lanes 6 and 14). The activated CREB communicates with the basal transcription factor TFIIB through the bridging protein CBP (Kwok et al., 1994). A GAL4–CBP fusion protein should be able to activate transcription directly. When fused to the DNA-binding domain of GAL4, the C-terminal portion of CBP (amino acids 1678–2441) could indeed activate transcription, strongly suggesting the presence in this region of an autonomous spontaneously active transactivation domain. Interestingly, both cAMP and UVC enhanced transcription ∼2-fold, suggesting that the cAMP- and UVC-induced signalling pathways also caused an activating modification of CBP (Figure 2A). In conclusion, our experiments demonstrate that UVC activates a signalling pathway at the end of which CREB (and possibly other CRE-binding proteins) are located, and that CREB activation contributes to UVC-induced c-fos transcription. UVC is a potent inducer of CREB Ser133 and ATF-1 Ser63 phosphorylation At the c-fos promoter, the transcription factors are DNA bound at most or all times (Herrera et al., 1989). Activation must therefore involve post-translational modification of the factors in the DNA-bound state. The experiments with GAL4–CREB wild-type or with the Ser133 mutation (Figure 2) and with the dominant-negative CREBM1 (Figure 1) have already pointed to Ser133 as the site of modification after UVC as well as cAMP treatment. With cAMP as the inducer, Ser133 phosphorylation by the catalytic subunit of PKA (Gonzales and Montminy, 1989) has been shown to cause binding of CBP (Chrivia et al., 1993; Kwok et al., 1994). To examine directly the Ser133 phosphorylation state of CREB after UVC stimulation of cells, we used an antibody recognizing only the phosphorylated form of CREB: the amino acid sequence 123–136 (KRREILSRRPS133YRK, Herdegen et al., 1994) with Ser133 carrying the phosphate group, and investigated by immunofluorescence the presence of this epitope in NIH3T3 mouse fibroblasts. Since the phosphorylated epitope is conserved to 100% between CREB and its family member ATF-1, the antibody used also recognizes ATF-1 phosphorylated at Ser63 [this residue corresponds functionally to Ser133 of CREB (see sequence in Figure 3) and is phosphorylated by PKA; Lee and Masson, 1993, and references therein]. In untreated cells, the nuclei remained dark after staining with antibody (Figure 3A), consistent with the assumption that CREB and ATF-1 were in the non-phosphorylated state. Treatment of the cells with either forskolin or UVC (30 J/m2, a dose which leads to efficient c-fos transcription in these cells) resulted in strongly positive nuclear staining consistent with the efficient phosphorylation of nuclear CREB and/or ATF-1 at serines 133 and 63, respectively. A Western blot analysis with this antibody, using whole cell extracts, indeed revealed both cAMP- and UVC-induced phosphorylation of the same proteins (Figure 3B), with the characteristic molecular weights of CREB (43 kDa, Montminy and Bilezikjian, 1987) and ATF-1 (35 kDa, Hai et al., 1989). The β-splice form of CREB (Blendy et al., 1996) and the CRE modulator protein CREM (Foulkes et al., 1991) were not detected. A protein of 80 kDa and of unknown nature reacted strongly with the antibody irrespective of the source of the extract. In both untreated and treated cells, this crossreactivity is equally distributed in nucleus and cytoplasm and there is no induced translocation (not shown). Figure 3.Forskolin and UVC irradiation induce Ser133 phosphorylation of CREB and Ser63 phosphorylation of ATF-1 in NIH3T3 mouse fibroblasts. (A) NIH3T3 cells were serum-starved for 24 h (DMEM, 0.5% FCS) and then stimulated with forskolin (10 μM) or UVC (30 J/m2). 15 min later the cells were fixed in 4% p-formaldehyde and processed for immunofluorescence analysis. The antibody used recognizes only CREB and ATF-1 phosphorylated at serines 133 and 63, respectively. The dark-stained nuclei are indicative of the non-phosphorylated state of CREB and ATF-1. The forskolin- and UVC-induced phosphorylations are manifested by positive nuclear staining. (B) Western blot using the same antibody. The cells were treated as in (A) (forskolin = Forsk), lysed in boiling Laemmli buffer to avoid dephosphorylation and degradation of the proteins and processed for Western blot analysis. The amino acid sequences of phosphorylated CREB and ATF-1 recognized by the antibody are indicated. Mock: treated as for UV irradiation, but without irradiation. Download figure Download PowerPoint We conclude that CREB and ATF-1 are indeed phosphorylated at the appropriate serines after UVC irradiation, which can explain the mechanism of UVC-induced activation of CRE-binding proteins and their transcriptional role in the UV response of c-fos. UV-induced CREB and ATF-1 phosphorylation is an immediate early event. It occurs with a lag period of <5 min after irradiation of 3T3 (not shown) or HeLa cells, with initial phosphorylations already seen at 1 min (Figure 4). The kinetics and magnitude of CREB/ATF-1 phosphorylation and of subsequent dephosphorylation after cAMP and UV resemble each other. Figure 4.Kinetics of UV- and cAMP-induced CREB and ATF-1 phosphorylation. HeLa cells were treated with forskolin and IMX (20 μM and 1 mM) or irradiated with UVC (30 J/m2) as indicated. At the time points indicated, cells were harvested and assayed for the phosphorylation of CREB and ATF-1 as described in Figure 3. Download figure Download PowerPoint UV-induced p108 CREB kinase Which protein kinase(s) could be responsible for UVC-induced CREB activation? A major criterion for its (their) identification could be that UVC enhanced its (their) activity. We therefore examined CREB-accepting protein kinases by 'in-gel' kinase assay (see Materials and methods). Nuclear extracts from untreated or from UVC-irradiated HeLa cells were resolved by SDS–PAGE with specific substrate added prior to acrylamide polymerization: the non-phosphorylated CREB peptide KRREILSRRPS133YRK (Figure 5, left two panels). In control gels, the peptide was omitted (Figure 5, right panel) or an irrelevant peptide was added to the gel (not shown). As one might expect from the resolution of total nuclear extracts, several protein kinase activities are detectable. Bands of ∼90 and 75 kDa autophosphorylate in the absence of CREB peptide (Figure 5, right panel). The important finding is that only two protein kinase activities are enhanced by UVC irradiation of the cells and that this enhancement depended entirely on the presence of the CREB peptide: a 90 kDa band (thin arrow) was increased after UVC treatment, a band of ∼108 kDa (thick arrow) is only visible in the lanes with extracts from UVC-irradiated cells (Figure 5, lanes 2 and 7, marked with an asterisk). We did not detect renaturable UVC-induced kinase activities at lower molecular weights (not shown). The UVC-induced enhancements of protein kinase activities were transient, raised at 15 min (lane 7) and gone at 4 h (lane 8). Both protein kinases were not activated in cells treated with forskolin plus iso-butylmethylxanthine (IMX) (Figure 5, lane 4, cAMP). Figure 5.UVC induces a 108 kDa CREB kinase in HeLa cells. Serum-starved HeLa cells were treated with either UVC (60 J/m2), EGF (20 ng/ml) or forskolin plus IMX (10 μM and 0.5 mM, cAMP) for 15 min or, where indicated, for 4 h. Nuclear extracts were prepared and 'in gel kinase assays' were performed as described in Materials and methods. In lanes 11–16, no CREBtide has been added to the gel. Mock = mock-treated cells. The p90 kDa CREB kinase is indicated by a thin arrow, the thick arrow points to the p108 kDa CREB kinase. Where detectable, the p108 CREB kinase is also indicated in the gel by an asterisk. The peptide used for the in-gel kinase assay ('CREBtide') is shown below the figure. Download figure Download PowerPoint The protein kinases known to phosphorylate CREB, the activity of the catalytic subunit of PKA and the Ca2+/calmodulin-dependent kinases, were not enhanced in UVC-treated cells (not shown, below the molecular weight range of the gel in Figure 5). Experiments addressing the upstream pathway to be described below also speak against an involvement of either PKA or Ca2+/calmodulin-dependent protein kinases. CREB phosphorylation upon growth factor stimulation Interestingly, the 90 and 108 kDa protein kinases were also transiently activated upon EGF treatment of cells (Figure 5, lanes 3 and 9). This finding prompted an examination of the CREB and ATF-1 phosphorylation states in cells treated with one of several growth stimuli. CREB Ser133 and ATF-1 Ser63 were indeed phosphorylated in response to phorbol ester, bFGF, IL-1α and EGF, as well as UVC (Figure 6) and tumour necrosis factor (TNF)-α (not shown). EGF also causes Ser133-dependent activation of GAL4–CREB (Figure 2B, lanes 8, 12 and 16), although the enhancement was only slight. Thus CREB and ATF-1 phosphorylation is not at all exclusive for elevated cAMP and it appears that several signalling pathways address CREB and ATF-1, and probably involve the same protein kinases. These data match those of other laboratories demonstrating CREB Ser133 phosphorylation in PC12 cells in response to nerve growth factor (NGF) and in melanocytes in response to several growth factors (Ginty et al., 1994; Böhm et al., 1995). A protein kinase induced after NGF migrated with 105 kDa (Ginty et al., 1994) and could be identical with p108 CREB kinase. Figure 6.EGF, IL-1α, bFGF and the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA) induce phosphorylation of CREB and ATF-1 in HeLa cells. Serum-starved HeLa cells were stimulated with either forskolin and IMX (10 μM and 0.5 mM, cA), UVC (30 J/m2), EGF (20 ng/ml), IL-1α (2 ng/ml), bFGF (10 ng/ml) or TPA (200 ng/ml). After 15 min, cell lysates were prepared for Western blot analyses. M = mock-treated cells. Download figure Download PowerPoint Dissection of upstream signalling Could one of these putative growth factor-triggered pathways be relevant for the induction by UVC? We have shown previously that several UVC-initiated signal transduction cascades inducing c-fos and c-jun gene transcription depend on the presence on the cell surface of activatable growth factor receptors (Sachsenmaier et al., 1994). UVC strongly stimulates ligand-independent tyrosine phosphorylation of several receptor tyrosine protein kinases (Sachsenmaier et al., 1994; Coffer et al., 1995; Knebel et al., 1996). To try and identify putative signalling components shared by the UVC response of CREB, specific down-modulation experiments were performed (Sachsenmaier et al., 1994) and inhibitors were applied. Specific down-modulation experiments are based on the following principle: a stimulus, e.g. EGF, activates components of a signal transduction pathway in a transient manner. In the case of EGF, the EGFR undergoes autophosphorylation followed by receptor internalization. Restimulation by the same stimulus, e.g. EGF, is not possible for some period of time. Such down-modulation has been found for many stimuli. Pre-treatment of cells with one stimulus m
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