Signaling disrupts mSin3A binding to the Mad1-like Sin3-interacting domain of TIEG2, an Sp1-like repressor
2002; Springer Nature; Volume: 21; Issue: 10 Linguagem: Inglês
10.1093/emboj/21.10.2451
ISSN1460-2075
Autores Tópico(s)Cell Adhesion Molecules Research
ResumoArticle15 May 2002free access Signaling disrupts mSin3A binding to the Mad1-like Sin3-interacting domain of TIEG2, an Sp1-like repressor Volker Ellenrieder Volker Ellenrieder Gastroenterology Research Unit, Mayo Clinic, Rochester, MN, 55905 USA Department of Internal Medicine, University of Ulm, Ulm, Germany Search for more papers by this author Jin-San Zhang Jin-San Zhang Gastroenterology Research Unit, Mayo Clinic, Rochester, MN, 55905 USA Search for more papers by this author Joanna Kaczynski Joanna Kaczynski Gastroenterology Research Unit, Mayo Clinic, Rochester, MN, 55905 USA Tumor Biology Program, Mayo Clinic, Rochester, MN, 55905 USA Search for more papers by this author Raul Urrutia Corresponding Author Raul Urrutia Gastroenterology Research Unit, Mayo Clinic, Rochester, MN, 55905 USA Tumor Biology Program, Mayo Clinic, Rochester, MN, 55905 USA Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN, 55905 USA Search for more papers by this author Volker Ellenrieder Volker Ellenrieder Gastroenterology Research Unit, Mayo Clinic, Rochester, MN, 55905 USA Department of Internal Medicine, University of Ulm, Ulm, Germany Search for more papers by this author Jin-San Zhang Jin-San Zhang Gastroenterology Research Unit, Mayo Clinic, Rochester, MN, 55905 USA Search for more papers by this author Joanna Kaczynski Joanna Kaczynski Gastroenterology Research Unit, Mayo Clinic, Rochester, MN, 55905 USA Tumor Biology Program, Mayo Clinic, Rochester, MN, 55905 USA Search for more papers by this author Raul Urrutia Corresponding Author Raul Urrutia Gastroenterology Research Unit, Mayo Clinic, Rochester, MN, 55905 USA Tumor Biology Program, Mayo Clinic, Rochester, MN, 55905 USA Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN, 55905 USA Search for more papers by this author Author Information Volker Ellenrieder1,2, Jin-San Zhang1, Joanna Kaczynski1,3 and Raul Urrutia 1,3,4 1Gastroenterology Research Unit, Mayo Clinic, Rochester, MN, 55905 USA 2Department of Internal Medicine, University of Ulm, Ulm, Germany 3Tumor Biology Program, Mayo Clinic, Rochester, MN, 55905 USA 4Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN, 55905 USA *Corresponding author. E-mail: [email protected] The EMBO Journal (2002)21:2451-2460https://doi.org/10.1093/emboj/21.10.2451 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info A Sin3-interacting domain (SID) originally described in Mad proteins is necessary for both transcriptional repression and growth suppression by these transcription factors. We recently reported that a structurally and functionally related Mad1-like SID is also present in five Sp1-like repressor proteins (TIEG1, TIEG2, BTEB1, BTEB3 and BTEB4), demonstrating that SID–mSin3A interactions have a wider functional impact on transcriptional repression. SID–mSin3A interaction is necessary for the anti-proliferative function of Mad, TIEG and BTEB proteins. It remains to be established, however, whether the SID–mSin3A interaction is constitutive or regulated. Here, we describe that the Mad1-like SID domain of the Sp1-like repressor TIEG2 is inhibited by the epidermal growth factor (EGF)–Ras–MEK1–ERK2 signaling pathway, via phosphorylation of four serine/threonine sites adjacent to the SID. This phenomenon disrupts the SID–mSin3A interaction and thereby inhibits TIEG2's repression activity. Thus, these results show for the first time that the repression of a SID-containing protein is regulated by signaling rather than functioning in a constitutive manner, extending our understanding of how the function of SID-containing repressors may be controlled. Introduction A large number of studies have firmly established a key role for transcriptional repression in most cellular functions that regulate embryogenesis, maintain homeostasis and participate in mechanisms of disease, including cancer. An emerging concept in this field is that short peptide motifs are a common mechanism mediating repressor–corepressor interactions. For example, Hairy and Runt transcription factors function by recruiting the corepressor Groucho via the WRPW or VWRPY motifs (Chen and Courey, 2000). NCoR and SMRT interact with nuclear hormone receptors via the I/LXXI/VI motif, and ligand binding regulates this interaction (Minucci and Pelicci, 1999; Nagy et al., 1999). Also, a consensus motif, PVDLS/T, is required for the interaction of several repressor proteins with mCtBP2 (Turner and Crossley, 2001). Since repression by proteins containing these domains supports different types of cellular functions, the mechanisms of repressor–corepressor interactions are critical for the regulation of homeostasis. Therefore, significant efforts from many laboratories are focused on defining the repertoire of short motifs participating in repression. The results of these experiments will no doubt extend our understanding of gene silencing mechanisms and their impact on cell biology. An important example of these repression mechanisms is the interaction between the corepressor mSin3A and a small motif called SID (Sin3-interacting domain) that contains the consensus φZZφφXφAAXXLE/D core sequence (Eilers et al., 1999; Brubaker et al., 2000). The SID was described originally in the Mad family of basic helix–loop–helix transcription factors and was also shown to be necessary for both their repressor and growth suppressive activities (Schreiber-Agus and DePinho, 1998). Interestingly, we have recently reported that five Sp1-like transcriptional repressors (TIEGs and BTEBs) also recruit the mSin3A–histone deacetylase complex through a conserved, α-helical motif with the core AA/VXXL sequence that is structurally and functionally similar to the Mad1 SID (Zhang et al., 2001). Other groups have also provided evidence for a wider participation of SID-like domains in transcriptional repression, such as the mammalian Pf1 (Yochum and Ayer, 2001) and the yeast Ume6 (Washburn and Esposito, 2001). Together, these studies have established that the SID represents a small structural motif that mediates interaction of various transcriptional repressors with the corepressor mSin3A. Furthermore, they reveal that SID–mSin3A interactions have been conserved throughout evolution, probably in part because they represent an efficient mechanism of repression selected to the organism's advantage. Currently, however, whether repression by SID-containing proteins is constitutive or regulated remains to be established and, if regulated, the mechanisms underlying such a phenomenon need to be defined. By analogy to nuclear receptors, we hypothesize that the repression activity of SID-containing proteins is likely to be regulated by differential association with the corepressors in various biological contexts. Potential mechanisms for this regulation include those that may act at the level of the SID domain itself, the corepressor mSin3A or modifications in the repressor that fall outside of the SID domain, affecting repressor–corepressor interactions. In contrast to nuclear receptors, the SID-containing proteins described thus far do not bind intracellular ligands. Therefore, other mechanisms, such as signaling-induced post-translational modification, at any of the levels mentioned above, are more likely to modulate their interactions with mSin3A. In this study, we have tested these hypotheses by studying the regulation of the SID-mediated transcriptional repression activity of the Sp1-like repressor TIEG2 by the epidermal growth factor (EGF)–Ras–MEK1–ERK2 signaling pathway. The results of these experiments show that the EGF–Ras–MEK1–ERK2 signaling pathway inhibits the repression activity of TIEG2 by mediating the phosphorylation of four sites immediately adjacent to the SID. This signaling-induced relief of the transcriptional repression of the TIEG2 SID results from a disruption of the binding between this domain and mSin3A. Therefore, TIEG2 is the first member of the TIEG/BTEB subgroup of Sp1-like transcriptional repressors shown to be regulated in this manner. Moreover, this is also the first evidence of a SID-containing protein that is regulated by signaling-induced phosphorylation of sites that exist outside of this motif. Thus, both concepts contribute to extend our understanding of the potential mechanisms that can regulate the function of these proteins. Results The repression activity of TIEG2 is regulated by EGF signaling We examined the regulation of the transcriptional repression activity of TIEG2 by proliferative and anti-proliferative signaling pathways. NIH 3T3 cells, responsive to a wide variety of growth factors, were co-transfected with FLAG-tagged TIEG2 and BTE reporter plasmids and treated with either the anti-proliferative transforming growth factor-β (TGF-β) or the mitogenic EGF. Treatment with TGF-β, even at high doses, has no effect on the transcriptional repression activity of TIEG2 (Figure 1A, left panel). In contrast, treatment with EGF resulted in a dose-dependent relief of TIEG2 repression activity and reached saturation at a concentration of 25 ng/ml (Figure 1A, right panel). Similar effects were also observed following treatment of NIH 3T3 cells with other mitogenic factors such as hepatocyte growth factor and fibroblast growth factor, however to a much lesser degree (data not shown). In parallel experiments, Chinese hamster ovary (CHO) cells, which are EGF receptor deficient, were used for the reporter assay, along with a constitutively active form of the EGF receptor, vErbB. Figure 1B (left panel) shows that vErbB co-expression strongly inhibits the repression activity of TIEG2, similarly to EGF in NIH 3T3 cells. In addition, CHO cells were co-transfected with other members of the EGF pathway, such as Ras, MEK1 and ERK2. Figure 1B shows that constitutively active mutants of Ras and MEK1 (caRas and caMEK1) relieve TIEG2 repression, similarly to vErbB (Figure 1B, left panel). In contrast, cotransfection of CHO cells with dominant-negative Ras or ERK2 (dnRas and dnERK2), or treatment with the MEK1 inhibitior PD089059, antagonized the inhibitory effect of vErbB on TIEG2 repression activity (Figure 1B, right panel). Together, these data suggest that the proliferative EGF–Ras–MEK1–ERK2 signaling cascade is both sufficient and required for EGF-induced inhibition of TIEG2 repression activity. Figure 1.EGF signaling regulates TIEG2 repression activity. (A) NIH 3T3 cells were transiently transfected with FLAG-tagged TIEG2 along with pBTE reporter plasmids. Cells were treated with increasing amounts of TGF-β (0.1, 1 and 10 ng/ml) or EGF (5, 25 and 25 ng/ml) for 18 and 24 h, respectively. Note that TGF-β does not affect the repression activity of TIEG2, whereas EGF strongly antagonizes TIEG2-mediated repression. (B) CHO cells were transiently transfected with FLAG-tagged TIEG2 along with constitutively activate forms of the EGF receptor (vErbB), Ras (caRas) and MEK1 (caMEK1) or vErbB along with dominant-negative constructs of Ras (dnRas) and ERK2 (dnERK2), as indicated. vErbB-transfected cells were also treated with PD089059 (50 μM) prior to reporter assay. Note that vErbB, caRas and caMEK1 similarly relieve TIEG2-mediated repression. In addition, the inhibition of TIEG2 repression activity in vErbB-transfected cells is reversed by co-expression of dnERK2 and dnRas, as well as treatment with PD089059. Control western blots using cell extracts from transfected NIH 3T3 and CHO cells show that expression of TIEG2 is not changed by the various co-transfections and treatments. Download figure Download PowerPoint Subsequently, we tested whether the antagonistic effect of EGF signaling changes the phosphorylation status of TIEG2 in vitro and in vivo. For the in vitro experiments, immunoprecipitated ERK2 was incubated with either GST alone or a GST fusion protein carrying full-length TIEG2 in the presence of [32P]ATP. Figure 2A illustrates that activated ERK2 (isolated from EGF-stimulated cells) phosphorylates TIEG2 in vitro (lane 4), but not GST alone (lane 2). However, ERK2 isolated from untreated cells does not phosphorylate TIEG2 (lane 3) or GST (lane 1). Control experiments in Figure 2B show that EGF treatment of NIH 3T3 cells causes ERK2 phosphorylation without changing its expression levels (lane 1 versus 2). Next, CHO cells were co-transfected with vErbB and FLAG-tagged TIEG2 followed by metabolic labeling with [32P]orthophosphate and immunoprecipitation with anti-FLAG antibodies. Figure 2C shows that co-transfection with vErbB results in increased phosphorylation of TIEG2 (lane 3 versus 4). Control experiments show that ERK2 is activated upon vErbB expression (lane 2 versus 4) and that cells transfected with vector alone do not exhibit TIEG2 phosphorylation (lanes 1 and 2). These results show that EGF signaling induces increased TIEG2 phosphorylation in vivo while in vitro assays demonstrate that TIEG2 is phosphorylatable by ERK2, suggesting that this kinase is a candidate to mediate the effects of this cascade. Because ERK2-mediated phosphorylation of transcription factors often changes their subcellular distribution (Cyert, 2001), we performed confocal laser scanning microscopy on CHO cells transfected with FLAG-tagged TIEG2 alone or in combination with caMEK1. Figure 2D demonstrates that FLAG-tagged TIEG2 is localized exclusively to the nucleus (red signal), which is consistent with previous reports (Cook et al., 1999), and that co-expression of caMEK1 does not alter its localization. Controls using an anti-phosphoERK2 antibody show that caMEK1 co-expression induces an increase in the signal of phosphorylated ERK2 in the cell nucleus (green). Overlaying the staining patterns demonstrates the nuclear localization of both proteins (yellow). Non-transfected cells or cells that were not incubated with the primary antibody did not exhibit immunostaining for FLAG-tagged TIEG2 (data not shown). Together, the results from the reporter, phosphorylation and localization experiments described above outline a mechanism of regulation of TIEG2 function whereby its repression activity is antagonized by EGF signaling-mediated phosphorylation via ERK2, which does not change the subcellular localization of this transcription factor. Figure 2.EGF signaling induces phosphorylation of TIEG2 in vitro and in vivo. (A) Total ERK2 from either EGF-treated (25 ng/ml for 15 min) or untreated NIH 3T3 cell extracts was immunopurified and incubated with GST–TIEG2 or GST alone. Coomassie Blue gel analysis shows comparable amounts of GST proteins used in these assays. EGF-activated immunoprecipitated ERK2 strongly phosphorylates GST–TIEG2 (lane 4) in vitro, whereas ERK2 from untreated cells does not (lane 3). GST alone (lanes 1 and 2) is not phosphorylated. (B) Immunoprecipitated ERK2 used in (A) was subjected to western blot analysis using anti-phosphoERK2 (pERK2) and anti-total ERK2 antibodies. Note that EGF treatment leads to ERK2 phosphorylation without changing its expression. (C) CHO cells were co-transfected with FLAG-tagged full-length TIEG2 or the parental vector along with vErbB, followed by metabolic labeling with [32P]orthophosphate and immunoprecipitation with anti-FLAG antibodies. TIEG2 expression and vErbB-induced ERK2 activation were monitored by immunoblotting using anti-TIEG2 and anti-phosphoERK2 antibodies. [32P]orthophosphate incorporation was detected by audioradiography. Note that co-transfection with vErbB leads to increased phosphorylation of TIEG2 in vivo (lane 4 versus 3). Controls show that ERK2 is activated when vErbB is co-expressed (lanes 2 and 4). TIEG2 expression levels are unchanged by vErbB co-expression (lanes 3 and 4). (D) CHO cells were transfected with FLAG-tagged TIEG2 alone or together with caMEK1 and subjected to immunofluorescence analysis. Note that TIEG2 (red signal) was detected exclusively in the nucleus of FLAG-TIEG2-transfected cells. Note that co-expression of caMEK1 did not alter the subcellular localization of TIEG2. As a control, caMEK1 expression causes increased staining for the phosphorylated form of endogenous ERK2 (green). The overlay of the staining patterns demonstrates nuclear localization of both proteins (yellow). Download figure Download PowerPoint Phosphorylation of TIEG2 occurs at ERK2 consensus sites adjacent to the Mad1-like SID In vitro kinase assays using deletion mutants that carry either the N-terminal transcriptional regulatory domain (NTD) or the C-terminal DNA-binding domain (CTD) of TIEG2 were carried out to begin delineating the regions that are phosphorylated by ERK2. Figure 3A shows that recombinant ERK2 phosphorylates TIEG2 NTD (lane 3 versus 4) and to a lesser degree CTD (lane 5 versus 6). GST alone is not phosphorylated (lane 1 versus 2). In addition, activated MAP kinases JNK and p38 fail to phosphorylate the GST–NTD (data not shown). Next, we determined whether the N-terminus of TIEG2 is phosphorylated in vivo. For this purpose, CHO cells were transfected with TIEG2 NTD cloned as a GAL4 DNA-binding domain (DBD) fusion construct (GAL4 NTD) or GAL4 DBD alone, with or without caMEK1, followed by [32P]orthophosphate labeling and anti-GAL4 immunoprecipitations. Figure 3B shows that co-transfection with caMEK1 resulted in a significant increase of TIEG2 NTD phosphorylation (lane 3 versus 4). Similar results were obtained when GAL4 NTD was co-transfected with vErbB (data not shown). Expression of caMEK1 does not phosphorylate GAL4 DBD alone used as control (lane 1 versus 2). Subsequently, we focused on characterizing the effects of EGF–Ras–MEK1–ERK2 signaling on the N-terminal repression domain using GAL4-based reporter assays. GAL4 NTD or DBD alone was co-transfected into CHO cells along with a GAL4 luciferase reporter plasmid and vErbB, caRas or caMEK1. Figure 3C shows that GAL4 NTD represses transcription ∼6.6-fold (0.79 ± 0.1 GAL4 DBD versus 0.12 ± 0.04 GAL4 NTD). Co-transfection with vErbB, caRas or caMEK1 reduced GAL4 NTD-mediated repression activity to ∼1.3-fold (0.59 ± 0.1, 0.51 ± 0.09 and 0.51 ± 0.1, respectively). On the other hand, the MEK1 inhibitor PD089059 reverses vErbB inhibition of TIEG2 repression activity (0.20 ± 0.04). In contrast to the potent effect of the EGF–Ras–MEK1–ERK2 pathway on TIEG2 NTD repression activity, anisomycin, an activator of JNK and p38, only slightly affects the repression activity (0.29 ± 0.05). Control gel-shift assays using the GAL4 site show that vErbB and caMEK1 alter neither the expression nor the DNA-binding activity of these GAL4 constructs (data not shown). In addition, we performed gel-shift assays to determine whether the phosphorylation of the C-terminus affects TIEG2 DNA binding. Figure 3D shows that the rERK2-phosphorylated TIEG2 C-terminus retains its ability to bind a previously characterized TIEG2-binding site (lane 4 versus 5) (Cook et al., 1998). Taken together, these data suggest that TIEG2 repression activity is inhibited by the EGF–Ras–MEK1–ERK2 pathway through regulation of the N-terminal domain. Figure 3.EGF signaling inhibits TIEG2 N-terminal repression activity. (A) Equal amounts of GST fusion proteins carrying the N-terminal (GST–NTD) or the C-terminal (GST–CTD) domains of TIEG2 or GST alone were incubated with recombinant ERK2 (rERK2) (20 ng/μl) and [γ-32P]ATP as described in Materials and methods. Phosphorylation of the GST proteins was visualized by autoradiography. Note that rERK2 strongly phosphorylates GST–NTD (lane 3 versus 4) and to a lesser degree GST–CTD (lane 5 versus 6). GST alone is not phosphorylated (lane 1 versus 2). (B) CHO cells were transfected with TIEG2 NTD expressed as a GAL4 DBD fusion protein (GAL4 NTD) or GAL4 DBD alone (GAL4), with or without caMEK1. At 24 h post-transfection, cells were metabolically labeled with [32P]orthophosphate and subjected to anti-GAL4 immunoprecipitation. Phosphorylation of TIEG2 NTD was detected by autoradiography. Note that caMEK1 expression leads to increased phosphorylation of TIEG2 NTD (lane 3 versus 4). GAL4 DBD alone is not phosphorylated (lane 1 versus 2). To control for TIEG2 NTD expression, transfected CHO cells were labeled with [35S]methionine and analyzed by anti-GAL4 immunoprecipitation as described above. Note that TIEG2 NTD is expressed at comparable levels. (C) GAL4-based reporter assays were performed in CHO cells transfected with GAL4 DBD alone or GAL4 NTD along with a GAL4 luciferase reporter construct. Note that TIEG2 NTD repression (0.12 ± 0.04 versus 0.79 ± 0.1 GAL4 DBD alone) is strongly antagonized by co-expression of caMEK1 (0.51 ± 0.1), caRas (0.51 ± 0.09) or vErbB (0.59 ± 0.1). The MEK1 inhibitor PD089059 reverses the inhibition of TIEG2 NTD by vErbB (0.20 ± 0.04). Also note that anisomycin, a JNK and p38 activator, slightly reduces the NTD-mediated repression (0.29 ± 0.05). (D) GST–CTD and GST alone were subjected to rERK2 phosphorylation and used in a gel-shift assay with a probe containing a GC-rich binding site for TIEG2. The specific complex that forms between the GST–CTD and probe is indicated on the right (asterisk). Note that the DNA-binding activity of TIEG2 CTD is unaffected by rERK2 treatment (lane 4 versus 5). GST alone does not bind the GC probe (lanes 2 and 3); lane 1 shows the mobility of the GC-rich probe in the absence of binding proteins. Download figure Download PowerPoint Sequence analysis shown in Figure 4A reveals the presence of four putative MAPK phosphorylation sites (T56, S94, S107 and S149) in the region immediately adjacent to the N-terminal SID domain of TIEG2 that conform to the ERK2 consensus target sequence of P-X-S/T-P (Jacobs et al., 1999). We created alanine substitution mutations of the consensus sites in the context of the GAL4 NTD construct. For the remainder of the paper, we will refer to these mutations as M1, M2, M3 and M4, corresponding to residues T56, S94, S107 and S149 mutated to alanine, respectively, and M5, corresponding to the mutant carrying alanine mutations of all four serine/threonine sites (Figure 4B). We then performed a GAL4 reporter assay in the absence or presence of caMEK1. Figure 4B shows that the repression activity of both the wild-type and mutant GAL4 NTD constructs is comparable in the absence of caMEK1 (lanes 2–7). Co-transfection with caMEK1 antagonizes the repression activity of wild-type NTD. The repression activity of the NTD mutants is also inhibited by caMEK1, although to a lesser degree than wild-type NTD. The M5 mutation leads to the most significant reduction of caMEK1-induced inhibition of the TIEG2 NTD repression (lane 7). Expression of caMEK1 does not modify the activity of the GAL4 DBD control (lane 1). To determine the effect of the alanine mutations on NTD phosphorylation, we performed in vivo phosphorylation studies combined with anti-GAL4 immunoprecipitation. Figure 4C shows that co-transfection with caMEK1 resulted in increased phosphorylation of wild-type NTD (lane 1 versus 2). The phosphorylation of TIEG2 NTD was slightly reduced by single mutations of the ERK2 sites (lanes 3, 4, 5 and 6). In contrast, the caMEK1-induced phosphorylation was abolished in the M5 mutant (lane 7). From these studies, we conclude that the four ERK2–MAPK consensus sites adjacent to the TIEG2 SID are targets of the EGF–Ras–MEK1–ERK2 pathway in vivo and that all four sites are needed for the antagonistic effect of this cascade on TIEG2 repression activity. Figure 4.ERK2 phosphorylation sites are adjacent to the TIEG2 SID. (A) A schematic representation of TIEG2 protein structure shows the three zinc finger motifs that bind DNA and repression domains SID, R2 and R3 within the NTD. Sequence analysis of the linker region found between SID and R2 reveals four putative MAP kinase phosphorylation sites (PLT56P, PQS94P, PVS107P and PSS149P). An additional site is present C-terminal to the zinc finger domains. (B) Alanine substitution mutants of the ERK2 sites were generated. GAL4 constructs encoding wild-type NTD (NTD WT), NTD with single point mutations (NTD M1–M4) or NTD with combined point mutations (NTD M5) were co-transfected into CHO cells along with the GAL4 reporter and caMEK1 as indicated. Note that co-transfection with caMEK1 antagonizes the repression activity of the wild-type NTD (lane 2) and to a lesser degree that of the GAL4 NTD mutants M1–M4 (lanes 3–6). The M5 mutant leads to the greatest reduction of MEK1-induced inhibition of the transcriptional repression activity. (C) As indicated, CHO cells were transfected with GAL4 NTD wild-type or mutant constructs along with caMEK1. Cells were then metabolically labeled with [32P]orthophosphate and anti-GAL4 immunoprecipitations were performed followed by audioradiography. Note that wild-type NTD phosphorylation is increased when caMEK1 is co-expressed (lanes 1 versus 2). CaMEK1-mediated phosphorylation of NTD M1–M4 (lanes 3, 4, 5 and 6, respectively) is reduced. NTD M5 abolishes caMEK1-induced phosphorylation (lane 7). Expression of the constructs was monitored by anti-TIEG2 western blots of cell lysates and shows that all constructs are expressed at comparable levels. Download figure Download PowerPoint EGF-induced phosphorylation of TIEG2 disrupts its binding to mSin3A Since the TIEG2 NTD is phosphorylated by ERK2 at sites adjacent to the SID, we hypothesized that the EGF–Ras–MEK1–ERK2 pathway may alter the activity of the SID rather than other repression domains. To test this hypothesis, we constructed various deletion mutants of the N-terminus and performed GAL4-based assays. Figure 5A shows a physical diagram of these deletion mutants (D1–D3). D1 lacks the SID but contains the four phosphorylation sites and the previously characterized repression domains R2 and R3. D2 contains only the SID. D3 contains the SID and adjacent ERK2 sites. Figure 5A shows that SID-containing constructs (D2 and D3; lanes 4 and 5, respectively) display a repression activity that is similar to that of the entire TIEG2 NTD (lane 2), whereas the SID minus D1 mutant (lane 3) displays a less potent repression activity. This result is identical to previously described data from our laboratory (Cook et al., 1999). Interestingly, caMEK1 antagonizes the repression activity of D3 (lane 5), whereas the activity of D1 was only slightly relieved (lane 3). In addition, D2 repression activity is unaffected by caMEK1. These data suggest that the repression activity associated with the SID is the main target of EGF–Ras–MEK1–ERK2 signaling. Thus, we investigated the role of these ERK2 sites in the MEK1-mediated inhibition of the SID repression activity by mutating them in the D3 construct (M1–M5 mutants). Figure 5B shows that, in the absence of caMEK1, all mutant GAL4 D3 constructs retain the repression activity as compared with wild-type D3 (lane 2 versus 3–7). The caMEK1-mediated inhibition of D3 repression is partially blocked by single point mutations of the phosphorylation sites (lanes 3–6), and mutation of all four phosphorylation sites nearly abolishes caMEK1-mediated inhibition of GAL4 D3 repression activity (lane 7), a result similar to what was observed in Figure 4B for the entire N-terminus. Together, these results indicate that although the ERK2 phosphorylation sites are necessary for the modulation of the SID repression activity, there is not a predominant site that single handedly is responsible for this phenomenon. Figure 5.EGF signaling targets SID repression activity. (A) Deletions of TIEG2 NTD expressing D1, D2 and D3 were cloned as GAL4 DBD fusion constructs and co-transfected into CHO cells in the absence or presence of caMEK1, along with the GAL4 luciferase reporter plasmid. Note that co-expression of caMEK1 strongly antagonizes the repression activity of NTD (lane 2) and D3 (lane 5), but not D2 (lane 4). Also note that D1 repression activity is slightly antagonized by caMEK1 co-transfection (lane 5). (B) Mutations M1–M5 were generated in the context of the GAL4 D3 construct, and GAL4-based reporter assays were performed. Note that caMEK1 co-expression inhibits the repression activity of wild-type D3 (lane 2) and that mutations M1–M4 partially block caMEK1 inhibition of SID repression activity (lanes 3–6). CaMEK1-mediated inhibition is almost abolished in D3 M5 (lane 7). Download figure Download PowerPoint We have shown previously that the repression activity of TIEG2 SID requires interaction with mSin3A (Zhang et al., 2001). It is likely that the EGF–Ras–MEK1–ERK2 pathway regulates TIEG2 function by modulating the SID–mSin3A interaction. To test this hypothesis, we analyzed the effect of this pathway on the interaction of mSin3A with both full-length TIEG2 and the SID by immunoprecipitation assays combined with western blot analyses. Baseline experiments shown in Figure 6A illustrate the strong binding of endogenous mSin3A to TIEG2 (lane 1). In contrast, co-expression of either vErbB or caMEK1 markedly reduces the interaction of TIEG2 with mSin3A (lanes 2 and 3). Moreover, the vErbB-mediated disruption of the TIEG2–mSin3A interaction is reversed by treatment with the MEK1 inhibitor PD089059 (lane 4). We next studied whether the ERK2 sites adjacent to the SID are required for the interaction with mSin3A using the same deletion constructs as described in Figure 5A. Figure 6B illustrates that, in the absence of caMEK1, mSin3A co-immunoprecipitates with NTD, D2 and D3 (SID-containing constructs; lanes 2, 6 and 8, respectively) but not with D1 (SID minus construct; lane 4) or GAL4 DBD alone (lane 1). Co-expression of caMEK1, however, significantly reduces mSin3A binding by both the NTD
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