MAP kinase phosphorylation-dependent activation of Elk-1 leads to activation of the co-activator p300
2003; Springer Nature; Volume: 22; Issue: 2 Linguagem: Inglês
10.1093/emboj/cdg028
ISSN1460-2075
Autores Tópico(s)Ubiquitin and proteasome pathways
ResumoArticle15 January 2003free access MAP kinase phosphorylation-dependent activation of Elk-1 leads to activation of the co-activator p300 Qi-Jing Li Qi-Jing Li Department of Cell Biology and Neuroscience, University of California, Riverside, CA, 92521 USA Search for more papers by this author Shen-Hsi Yang Shen-Hsi Yang School of Biological Sciences, University of Manchester, 2.205 Stopford Building, Oxford Road, Manchester, M13 9PT UK Search for more papers by this author Yutaka Maeda Yutaka Maeda Department of Cell Biology and Neuroscience, University of California, Riverside, CA, 92521 USA Search for more papers by this author Frances M. Sladek Frances M. Sladek Department of Cell Biology and Neuroscience, University of California, Riverside, CA, 92521 USA Search for more papers by this author Andrew D. Sharrocks Andrew D. Sharrocks School of Biological Sciences, University of Manchester, 2.205 Stopford Building, Oxford Road, Manchester, M13 9PT UK Search for more papers by this author Manuela Martins-Green Corresponding Author Manuela Martins-Green Department of Cell Biology and Neuroscience, University of California, Riverside, CA, 92521 USA Search for more papers by this author Qi-Jing Li Qi-Jing Li Department of Cell Biology and Neuroscience, University of California, Riverside, CA, 92521 USA Search for more papers by this author Shen-Hsi Yang Shen-Hsi Yang School of Biological Sciences, University of Manchester, 2.205 Stopford Building, Oxford Road, Manchester, M13 9PT UK Search for more papers by this author Yutaka Maeda Yutaka Maeda Department of Cell Biology and Neuroscience, University of California, Riverside, CA, 92521 USA Search for more papers by this author Frances M. Sladek Frances M. Sladek Department of Cell Biology and Neuroscience, University of California, Riverside, CA, 92521 USA Search for more papers by this author Andrew D. Sharrocks Andrew D. Sharrocks School of Biological Sciences, University of Manchester, 2.205 Stopford Building, Oxford Road, Manchester, M13 9PT UK Search for more papers by this author Manuela Martins-Green Corresponding Author Manuela Martins-Green Department of Cell Biology and Neuroscience, University of California, Riverside, CA, 92521 USA Search for more papers by this author Author Information Qi-Jing Li1, Shen-Hsi Yang2, Yutaka Maeda1, Frances M. Sladek1, Andrew D. Sharrocks2 and Manuela Martins-Green 1 1Department of Cell Biology and Neuroscience, University of California, Riverside, CA, 92521 USA 2School of Biological Sciences, University of Manchester, 2.205 Stopford Building, Oxford Road, Manchester, M13 9PT UK *Corresponding author. E-mail: [email protected] The EMBO Journal (2003)22:281-291https://doi.org/10.1093/emboj/cdg028 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info CBP/p300 recruitment to enhancer-bound complexes is a key determinant in promoter activation by many transcription factors. We present a novel mechanism of activating such complexes and show that pre-assembled Elk-1–p300 complexes become activated following Elk-1 phosphorylation by changes in Elk-1–p300 interactions rather than recruitment. It is known that Elk-1 binds to promoter in the absence of stimuli. However, it is unclear how activation of Elk-1 by mitogen-acivated protein kinase (MAPK)-mediated phosphorylation leads to targeted gene transactivation. We show that Elk-1 can interact with p300 in vitro and in vivo in the absence of a stimulus through the Elk-1 C-terminus and the p300 N-terminus. Phosphorylation on Ser383 and Ser389 of Elk-1 by MAPK enhances this basal binding but, most importantly, Elk-1 exhibits new interactions with p300. These interaction changes render a strong histone acetyltransferase activity in the Elk-1-associated complex that could play a critical role in chromatin remodeling and gene activation. The pre-assembly mechanism may greatly accelerate transcription activation, which is important in regulation of expression of immediate-early response genes, in particular those involved in stress responses. Introduction Elk-1 (Ets-like protein-1), a member of the Ets family of transcription activators, shares a similar DNA-binding domain with Ets-1/2 (Rao et al., 1989). It is an essential component in the serum response ternary complex and has been shown to regulate expression of immediate-early response genes such as Zif268, MKP-1 (Sgambato et al., 1998) and chemokine cIL-8 (cCXCL8/cCAF; Li et al., 1999) that are not directly related to cell division, making it also a primary regulator of gene transcription involved in responses to stress. Furthermore, in some cases, Elk-1 does not require serum response factor to bind to DNA (Li et al., 1999, 2000), and it can be bound in the absence of a stimulus (Sharrocks, 1995; Li et al., 1999, 2000). In the case of c-fos, the Elk-1-binding site also is constitutively occupied in vivo (Herrera et al., 1989). The functional domains of Elk-1 are well defined (Yang et al., 1998b). Activation of Elk-1 depends on phosphorylation of Ser383 and Ser389 in the transcriptional activation domain by mitogen-activated protein kinase (MAPK) (Janknecht et al., 1993; Gille et al., 1995), which causes a conformational change that enhances DNA binding affinity (Gille et al., 1995; Sharrocks, 1995; Yang et al., 1999; Li et al., 2000). Most 'activation' domains of transcription factors are adaptors for recruitment of transcription co-activators. p300 and CBP are well-known coactivators that function as downstream scaffold and integrators for signal transduction. They contain multiple modules for protein–protein interactions and can serve as adaptors for transcription machinery assembly and recruitment by bridging the transcription activator with general transcription factors such as TATA box-binding protein (TBP) or TFIIB. Importantly, they contain intrinsic or associated extrinsic histone acetyltransferase (HAT) activity that acetylates core histones and releases repression of transcription initiation (Vo and Goodman, 2001). Although p300 and CBP are highly homologous and share similar biochemical characteristics, they are not redundant in many of their functions, as shown by the different phenotypes exhibited by heterozygous knockout mice for either co-activator and by the fact that homozygous mutations in CBP or p300 are lethal (Yao et al., 1998; Kung et al., 2000). Furthermore, transcription factor pathways show differences in function between the two co-activators (e.g. Vo and Goodman, 2001). Therefore, critical questions remain concerning the identification of cofactors that cooperate with Elk-1 and how their cooperation leads to specific gene expression. For many genes, inducible interactions between transcription factors and p300/CBP are the primary mechanism that turns on gene transcription (Xu et al., 1999). However, this apparently is not the case for genes under the control of Elk-1. The evidence indicates a pre-assembled Elk-1–CBP co-activator complex associated with target enhancer elements (Janknecht and Nordheim, 1996; Nissen et al., 2001). Elk-1 controls primarily immediate-early response genes, all of which have low levels of basal transcription but can reach very high levels <6 h after stimulation. In the case of cIL-8, elevation of mRNA occurs <7 min after thrombin stimulation (Li et al., 2000). Considering that there are a multitude of gene activation processes driven by the limited concentrations of p300/CBP inside cells, the pre-assembly mechanism may greatly accelerate transcription activation. Several pieces of evidence suggest Elk-1-mediated gene activation mechanisms in these complexes. For example, phosphorylation of the Elk-1 activation domain leads to a conformational change and to enhancement of DNA binding in the N-terminus of Elk-1 (Yang et al., 1999; Li et al., 2000), but it is not yet known how this bound factor activates gene transcription. It also has been suggested that activated MAPK is able to phosphorylate the C-terminus of CBP, contributing to gene activation (Janknecht and Nordheim, 1996). However, this alone does not explain the prerequisite for Elk-1 phosphorylation in controlling gene activation. Given that pre-assembled complexes exist, it is critical to determine the switching mechanisms associated with the upstream signal transduction pathways. Thrombin activates cIL-8 via a G-protein-coupled receptor and transactivation of the epidermal growth factor receptor (EGFR) tyrosine kinase (Vaingankar and Martins-Green, 1998), followed by activation of the MAPK cascade and Elk-1 (Li et al., 2000). Using this system, here we elucidate the mechanisms of Elk-1 phosphorylation upon thrombin-stimulated transcription activation of the cIL-8 gene. We show that p300 is important in Elk-1 activation of transcription and that the interactions between Elk-1 and p300 change following Elk-1 phosphorylation, leading to a novel model for Elk-1-driven gene transcription. Results Thrombin and p300, but not CBP, enhance Elk-1-mediated transcription activation To examine whether CBP cooperates with the Elk-1 transcription factor to enhance transcription of stress response genes upon stimulation of cells by thrombin, we co-transfected primary fibroblasts with several plasmid constructs singly or in combinations. We used a luciferase reporter construct containing five Gal4 tandem elements (pFR-Luc) and two expression vectors, one containing a fusion protein with the Gal4 DNA-binding domain and Elk-1 activation domain (Gal4dbd–Elk-1307), and the other containing the co-activator CBP. pCH110 β-gal was used as internal control. To ensure that β-gal expression was not affected by co-activator transfection, we also normalized the luciferase activity to protein concentration. Exogenous expression of the Gal4dbd–Elk-1307 protein and CBP in the presence of thrombin treatment did not result in a significant increase in Elk-1-dependent gene transcription (Figure 1A, lanes 7 and 8). These observations led us to test whether thrombin-driven Elk-1 function is dependent on p300, a functional homolog of CBP. Overexpression of p300 in the presence of the Gal4dbd–Elk-1307 followed by thrombin treatment induced a synergistic effect of Elk-1-dependent transactivation (Figure 1A; compare lanes 2 and 5). Using an antibody that recognizes p300 and CBP and another that recognizes only p300, we verified that p300 and CBP are expressed at the same level in the cells (Figure 1A, insert), excluding the possibility that the lack of synergistic effect of CBP in the co-transfection experiments is due to lack of CBP expression. The same results were obtained in HEK293 cells (data not shown). Figure 1.Synergistic effects of thrombin and p300 on Elk-1 activation of transcription. A 1 μg aliquot of Gal4dbd–Elk-1307 plasmid, 1 μg of the Gal4-controlled luciferase reporter (pFRLuc) and 2 μg of the β-gal control plasmid pCH110 were co-transfected into fibroblasts. Luciferase activity was normalized to β-gal activity and to protein concentration. (A) Cells transfected with pFRLuc and treated with thrombin (9 U/ml) showed an ∼4-fold increase in transcription (lane 2). With p300 or CBP co-transfection alone (1 μg of plasmid), there was no difference from control (lanes 3 and 6). When co-transfected cells were treated with thrombin, a synergistic response was observed for p300 (lane 5) but was not significant for CBP (compare lanes 2 and 8). Gal4dbd served as negative control. Insert: immunoblots of extracts from lanes 5 and 8 to detect p300 and CBP levels using an antibody that recognizes both proteins (upper panel) and an antibody specific to the C-terminus of p300 (α-p300CT; lower panel). (B) p300–thrombin synergistic effects require phosphorylation of Ser383 and Ser389 on Elk-1. Odd lanes confirm the results in (A). Even lanes: cells transfected with Gal4dbd–Elk-1307 S-A (which carries S383A and S389A mutations) showing that loss of phosphorylation on these serine residues eliminates the thrombin response and p300 synergy. Insert: immunoblots of protein levels of exogenous Gal4 fusion proteins in lanes 7 and 8. (C) A schematic representation of a region of the cIL-8 promoter (p683; Li et al., 2000) depicts the Elk-1 DNA-binding elements. (D) To determine whether p300-mediated activation of the cIL-8 gene is direct and specific, Elk-1 and p300 expression vectors were transfected individually with the p683 reporter of cIL-8. Increasing amounts of p300 gave a linear response in luciferase activity upon thrombin stimulation, but increasing Elk-1 showed synergy. Download figure Download PowerPoint To confirm that this synergistic effect is specific, we performed the same experiments using Gal4dbd–Elk-1307 with double mutation of Ser383 and Ser389 (Figure 1B), two serine residues critical for phosphorylation of Elk-1 by MAPK which leads to transcription activation (Janknecht et al., 1993; Gille et al., 1995). Mutation of these two serines completely eliminated the thrombin effects and thrombin–p300 synergy, even though the wild-type and mutant fusion proteins were expressed at the same level (Figure 1B, insert), showing that the synergistic effects in Figure 1A depend on Elk-1 Ser383 and Ser389 phosphorylation. To test for this synergy in a native eukaryotic promoter, we performed similar experiments using a luciferase reporter previously developed by us for cIL-8 (p683, Figure 1C). This reporter contains −683 to +32 bp of the cIL-8 promoter, responds to thrombin treatment, and the response is controlled by Elk-1 (Li et al., 2000). p683 was co-transfected with Elk-1- and/or p300-expressing vectors (Figure 1D). Exogenous expression of either Elk-1 or p300 alone affected the constitutive basal levels of transcription and enhanced cIL-8 gene transcription in response to thrombin. In the case of Elk-1, there was a clear dose-dependent synergistic effect, whereas, in the case of p300, the ratio of gene transcription in response to thrombin relative to the quiescent state was independent of concentration. Interactions between Elk-1 and p300 in vitro To determine whether Elk-1 and p300 physically interact, we used GST pull-down assays (Figure 2). Serial N-terminal deletions of Elk-1 cDNA were used to produce several C-terminal Elk-1 proteins that were fused to GST at their N-termini (Yang et al.,1998b). The inputs of GST–Elk-1 fusion proteins to be used in the pull-down assays were evaluated by western blot analysis and densitometry. Similarly, constructs containing various domains of p300 were expressed in vitro (Avantaggiati et al., 1997). All four constructs were used as templates for in vitro transcription and translation to obtain the various radiolabeled truncated proteins that were then run on gels for quantitation (Figure 2C). This procedure allowed us to use the same relative molar amounts for the various p300 truncated proteins, enabling detection of interactions between Elk-1 and p300 as well as changes in those interactions after exposure to activated ERK2 (Figure 3). Figure 2.GST pull-down assays to map Elk-1 and p300 'basal' interactions in vitro. GST–Elk-1 fusion proteins on beads and 35S-labeled p300 truncated proteins, prepared by in vitro transcription and translation, were used. (A) A schematic diagram of full-length Elk-1 and several GST–Elk-1 fusion proteins. (B) A schematic diagram of the p300 truncated proteins. (C) p300 proteins used in GST pull-down binding assays. One-tenth of p300 inputs in the binding assays were examined by SDS–PAGE and autoradiography. (D) Molecular interactions between Elk-1 and p300 truncated proteins. GST beads alone were used as negative control (panel I). All three Elk-1–GST fusion proteins tested contain the activation domain (C domain) and bind to the N-terminus (1–743) of p300 (panels II–IV). However, the Elk-1 protein that contains only the activation domain (panel IV) binds with a higher affinity to p3001–743 and also binds, albeit more weakly, to p3001514–1922. Download figure Download PowerPoint Figure 3.Phosphorylation of Elk-1 changes its interactions with p300. (A) For the GST fusion proteins, flag-tagged full-length Elk-1 (flag-Elk-1FL), GST–Elk-1310 and GST–Elk-1310 S-A fusion proteins were phosphorylated on beads by activated ERK2. Immunoblot analysis was performed with an antibody against phospho Ser383. The same membrane stripped and reprobed with an antibody against Elk-1 shows equal loading of proteins. (B) The unphosphorylated and phosphorylated Elk-1 proteins were subjected to GST or anti-Flag pull-down assays with radiolabeled p300 truncated proteins as described in Figure 2. Download figure Download PowerPoint The GST pull-down experiments show that the various Elk-1 truncated proteins bind to p3001–743 without requiring phosphorylation (Figure 2D, panels II and III). This was also true for the full-length Elk-1 protein (not shown in Figure 2, but see Figure 3B, panel III). No binding of Elk-1 to p300964–1922 was observed. However, weak but repeatable binding of GST–Elk349 to p3001514–1922 was seen. Interestingly, the truncated GST–Elk-1 proteins do not bind to p300Δ242–1737, indicating that the sequence of amino acids between residues 243 and 743 is necessary for the interaction between these proteins and p300. In addition, our results show that the MAPK docking domain of Elk-1 (D-domain) does not participate in the interaction of the Elk-1 proteins with p3001–743 because the GST–Elk-1349 protein (that does not contain the D-domain) binds to this region of p300, and does so with a slightly higher efficiency than the D-domain-containing proteins (Figure 2D, panel IV). Moreover, when the D-domain is absent (Elk-1349), there is an additional weak affinity binding to the region of p300 that contains CH2 and CH3 alone (p3001514–1922; Figure 2D, panel IV), suggesting that the presence of the D-domain interferes with binding of Elk-1 to the HAT activity domain of p300 under quiescent conditions. These results taken together show that Elk-1 interacts with the p300 N-terminal region (amino acids 242–743) through the transcription activation domain and without requiring phosphorylation; hence, we refer to this interaction of Elk-1 with p300 as 'basal' binding. Elk-1 phosphorylation leads to changes in interactions with p300 Elk-1 constitutively binds to immediate-early response gene enhancer elements without requiring activation. As shown above, Elk-1 also can bind to p300 in vitro without requiring phosphorylation/activation. However, phosphorylation of Ser383 and Ser389 is a pre-requisite for Elk-1 function in activation of gene transcription in vivo (Janknecht et al., 1993); it causes a conformational change in Elk-1, which is accompanied by an increase in DNA-binding affinity of the Elk-1 N-terminus (Yang et al., 1999). Therefore, we tested whether phosphorylation of Elk-1 also affects its interactions with p300 and is important for activation of this co-activator. GST–Elk-1310 fusion protein contains both the MAPK docking motif and the activation domain of Elk-1, hence we used it and the Elk-1 full-length protein (Elk-1FL) in the presence of activated ERK2 to investigate the role of Elk-1 phosphorylation in co-activator binding. Figure 3A shows that recombinantly produced GST–Elk-1310 and flag-Elk-1FL become phosphorylated on Ser383 in the presence of activated ERK2. As expected, ERK2 does not phosphorylate the GST–Elk-1310 S-A protein and this latter protein cannot be detected with a specific antibody against phosphorylated Ser383. Figure 3B shows that phosphorylated and unphosphorylated forms of Elk-1 interact differently with p300. After phosphorylation by ERK2, GST–Elk-1310 not only has a higher affinity for the N-terminus of p300 (Figure 3B, panel I, compare lanes 1 and 5), but the most significant change occurs in the pairing of GST–Elk-1310 and the region of p300 that contains CH2 and CH3 (Figure 3B, panel I, compare lanes 4 and 8) which is associated with the HAT activity of this co-activator. This latter interaction is Elk-1 serine phosphorylation specific because such changes do not occur in GST–Elk-1310 S-A (Figure 3B, panel II, compare lanes 4 and 8). The small increase in binding of GST–Elk-1310 S-A to p3001–743 after exposure to activated ERK2 could be the result of phosphorylation of other serine or threonine residues present in the Elk-1 C-terminus. Interestingly, phosphorylated GST–Elk-1310 did not interact with p300964–1922 which also contains the CH2 and CH3 domains (Figure 3B, panel I, lane 7). This may be because the extra sequences present N-terminally of CH2, when not in the context of the whole molecule, interfere with the binding of GST–Elk-1310 to p3001514–1922. For example, p3001514–1922 may be an independently folding unit, but p300964–1922 fails to fold correctly. To further these studies, we performed similar experiments with flag-tagged Elk-1FL (Figure 3B, panel III). Much like Elk-1310, flag- Elk-1FL also interacts with p3001–743 and with p3001514–1922. However, in addition, it interacts with p300964–1922 which not only contains the CH2 and CH3 domains but also has the bromo domain, suggesting N-terminal involvement of full-length Elk-1 in binding to this region of p300. Taken together, these results show a novel role for Ser383 and Ser389 phosphorylation in Elk-1 function. Not only does this change enhance the N-terminal DNA-binding activity of Elk-1, but it also alters the interactions and enhances the affinity of the C-terminus of Elk-1 for the p300 co-activator, which potentially can have important effects in downstream gene activation. Interactions between Elk-1 and p300 in vivo Using confocal microscopy, we determined that Elk-1 is transiently activated by thrombin in primary fibroblasts; the peak of activation occurs ∼15 min after stimulation, at which time Elk-1 and p300 co-localize in the nucleus. The nuclear Ser383 and Ser389 phosphorylation signal was attenuated after 30 min (data not shown). To address further the phosphorylation effects on Elk-1–p300 interaction in vivo, we performed competition experiments using the Gal4 fusion protein and analyzed them by co-immunoprecipitation. The fibroblasts were co-transfected with pCMV-Elk-1FL, Gal4dbd, Gal4dbd–Elk-1260 or Gal4dbd–Elk-1260 S-A. The three latter proteins were transfected in excess (twice the amount of full-length Elk-1) to facilitate detection of competition. Nuclear extracts were prepared after 15 min in the presence or absence of thrombin treatment, and subjected to two types of immunoprecipitation. One used an antibody against the Elk-1 DNA-binding domain (anti-Elk-1DBD) that does not interfere with the Elk-1–p300 binding region (which occurs through the C-terminus of Elk-1) to pull-down endogenous and overexpressed Elk-1FL. The other used an antibody to the Gal4 DNA-binding domain (anti-Gal4dbd) to pull-down Gal4–Elk-1 fusion proteins. The precipitates from both antibodies were examined for (i) p300 or CBP by immunoblotting with antibodies that specifically recognize these two co-activators (anti-p300 and anti-CBP); (ii) Elk-1 activation with an antibody that recognizes phosphorylated Elk-1 (α-P-Elk-1); and (iii) expression levels with an antibody that recognizes the Elk-1 protein (α-Elk-1). Using this approach, we compared the interaction between p300/CBP and Elk-1 in vivo (Figure 4): p300 was co-immunoprecipitated with both the endogenous Elk-1 (Figure 4A, panel I, lane 1) and overexpressed Elk-1 (panel I, lane 2), and this interaction was enhanced significantly by thrombin treatment (panel I, compare lanes 1 and 2 with lanes 6 and 7). Furthermore, Gal4dbd–Elk-1260 competed with Elk-1FL for p300 binding under both conditions (panel I, lanes 4 and 9), whereas the mutated form Gal4dbd–Elk-1260 S-A could not compete out phosphorylated Elk-1 binding to p300 (panel I, lanes 5 and 10), strongly suggesting that Elk-1 phosphorylation enhances Elk-1–p300 interactions in vivo. Indeed, the Ser383 and Ser389 phosphorylated form of Elk-1 (Figure 4A, panel II, lane 9) had a higher affinity for p300 than the S-A mutant, which cannot be phosphorylated (panel II, lane 10), even though the amount of S-A mutant is in excess. This competition is specific for Elk-1 because the control fusion protein Gal4dbd did not compete out p300 binding to full-length Elk-1 (Figure 4A, panel I, lanes 3 and 8). These results are summarized in Figure 4B. All of these affinity changes correspond to the in vitro GST pull-down data (Figure 3). These results indicate that in vivo, thrombin can control the affinity of Elk-1–p300 interactions by phosphorylation of Elk-1 on Ser383 and Ser389. Similar results were obtained when the recombinant GST–Elk-1310 was used as competitor in the co-immunoprecipitation assays (data not shown). Figure 4.In vivo competition assays to examine Elk-1–p300 interaction. (A) Cells overexpressing p300/CBP (2 μg of plasmid) and full-length Elk-1 (0.2 μg of plasmid) in the presence or absence of Gal4–Elk-1260 or Gal4–Elk-1260 S-A (0.4 μg of plasmid each) were treated with thrombin (9 U/ml) for 15 min and nuclear extracts prepared. Differential co-immunoprecipitation experiments were performed with antibody against the DNA-binding domain of Elk-1 (α-Elk-1DBD) or with antibody against the Gal4 DNA-binding domain (α-Gal4dbd). Panels I and II: p300 binding to Elk-1FL and Gal4dbd–Elk-1260. Panels III and IV: CBP binding to Elk-1FL and Gal4dbd–Elk-1260. Panels V and VI: immunoblots on the same membrane as panels I and II verify the phosphorylation status on Ser383 in each sample. The presence of more activated Elk-1 in lanes 4 and 9 of panel VI than that of panel V corresponds to higher levels of p300 binding to Gal4dbd–Elk-1260 in panel II, indicating that the competition shown in panels I/II and III/IV is specific. Panels VII and VIII: the same blots as V and VI reprobed with antibody against the C-terminus of Elk-1. (B and C) Summary of competition experiments. The p300/CBP band density in panels I and III was normalized to the Elk-1FL band density in VII. (D) Cells overexpressing Elk-1FL and p300FL were co-transfected with expression vectors for various p300 truncated proteins; the nuclear extracts were immunoprecipitated with an antibody to Elk-1 DBD and immunoblotted with an antibody to the N-terminus of p300. Without thrombin treatment, full-length p300 remained associated with Elk-1 in the presence of various p300 truncated proteins, except for p3001–743, and this competitor was detected by the antibody shown as the band below the full-length p300. Experiments were repeated with samples after thrombin treatment for 15 min. Competition was detected not only with p3001–743, but also with p3001514–1922. p3001514–1922 is not visible on the blot because the α-p300 antibody only recognizes the N-terminus of p300. (E) A 4 μg aliquot of p683 reporter, 1 μg of pCH110 and the same molar amount of expression vectors for the p300 truncated proteins were co-transfected into cells, and samples collected 6 h after thrombin treatment and analyzed for cIL-8 promoter activation. Overexpressed p300 truncated proteins bind to endogenous Elk-1 protein if they have affinity and titrate out the endogenous p300, impairing reporter gene activation. This effect was observed with p3001–743 in the absence and presence of thrombin, but detected significantly with p3001514–1922 only after thrombin treatment. **P < 0.01. Download figure Download PowerPoint Similar experiments to examine Elk-1–CBP interaction patterns showed that thrombin treatment enhances CBP interaction with Elk-1. However, this increase is much less pronounced and is not as dependent on Ser383 and Ser389 phosphorylation (Figure 4A; compare lanes 9 and 10 in panels I and II, and lanes 9 and 10 in panels III and IV). This is consistent with the lack of sygnergism observed when thrombin is applied to cells overexpressing CBP with Gal4–Elk1 fusion protein (Figure 1A). To confirm the changes in binding interactions between Elk-1 and p300 with and without thrombin treatment and to examine details of binding in vivo, we performed competition experiments with co-transfection of expression vectors for full-length p300 and various deletion fragments of p300. Nuclear extracts were collected and precipitated with an antibody specific to the Elk-1 DNA-binding domain followed by immunoblot analysis with antibody specific to the p300 N-terminus (Figure 4D). In the absence of thrombin, only p3001–743 competed with full-length p300 for Elk-1 binding, whereas in the presence of thrombin, p3001514–1922 showed affinity for Elk-1, confirming the in vitro GST pull-down assay (Figure 3). As a functional consequence, p3001–743 competes with the endogenous Elk-1–p300 interaction and results in a decrease of luciferase production in the cIL-8 p683 reporter assay in the presence or absence of thrombin, whereas p3001514–1922 only competes for binding to Elk-1 in the presence of thrombin (Figure 4E). Taken together, the in vitro and in vivo results strongly suggest that Elk-1 phosphorylation increases its affinity for p300 and that this increase involves a new interaction domain on p300. Changes in interaction of Elk-1 and p300 are accompanied by changes in p300-associated HAT activity p300 is a co-activator with intrinsic and associated acetyltransferase activity that normally functions as an important component of histone-modifying complexes. As shown in Figure 4, Elk-1 and p300 interact with each other and that interaction is enhanced after thrombin treatment. To determine whether this interaction leads to HAT function, standard HAT assays were performed with the same imm
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