- Email: [email protected]
E1AF degradation by a ubiquitin–proteasome pathway
BBRC Biochemical and Biophysical Research Communications 327 (2005) 575–580 www.elsevier.com/locate/ybbrc
E1AF degradation by a ubiquitin–proteasome pathway Akiko Takahashia,1,2, Fumihiro Higashinoa,2, Mariko Aoyagia, Koichi Yoshidab, Miyuki Itohb, Masanobu Kobayashic, Yasunori Totsukaa, Takao Kohgoa, Masanobu Shindoha,* a
c
Department of Oral Pathobiological Science, Hokkaido University Graduate School of Dental Medicine, North 13 West 7, Kita-ku 060-8586, Sapporo, Japan b Department of Biology, Sapporo Medical University School of Medicine, South 1 West 17, Chuo-ku 060-8556, Sapporo, Japan Division of Cancer Pathobiology, Institute for Genetic Medicine, Hokkaido University, North 15 West 7, Kita-ku 060-0815, Sapporo, Japan Received 3 December 2004 Available online 18 December 2004
Abstract E1AF is a member of the ETS family of transcription factors. In mammary tumors, overexpression of E1AF is associated with tumorigenesis, but E1AF protein has hardly been detected and its degradation mechanism is not yet clear. Here we show that E1AF protein is stabilized by treatment with the 26S protease inhibitor MG132. We found that E1AF was modified by ubiquitin through the C-terminal region and ubiquitinated E1AF aggregated in nuclear dots, and that the inhibition of proteasome-activated transcription from E1AF target promoters. These results suggest that E1AF is degraded via the ubiquitin–proteasome pathway, which has some effect on E1AF function. Ó 2004 Elsevier Inc. All rights reserved. Keywords: E1AF; ETS; PEA3; Proteasome; Ubiquitin; Degradation
ETS proteins have a DNA-binding domain consisting of approximately 85 amino acids (ETS-domain) and function as transcription factors by binding to a GGA core motif in enhancers of various genes [1]. E1AF is a member of the ETS oncogene family that was cloned for its ability to bind to the enhancer elements of the adenovirus E1A gene [2]. Analysis of deduced amino acids from the cDNA has shown that E1AF is a human counterpart of mouse PEA3 (polyomavirus enhancer activator 3) [3]. The PEA3 subfamily also includes ER81 [4] and ERM [5], which bind to the PEA3 specif*
Corresponding author. Fax: +81 11 706 4237. E-mail address: [email protected] (M. Shindoh). 1 Present address: Division of Protein Information, Institute for Genome Research, The University of Tokushima, 3-18-15, Kuramotocho 770-8503, Tokushima-city, Tokushima, Japan. 2 These two authors contributed equally to this work. 0006-291X/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2004.12.045
ic-binding motif. E1AF has been shown to activate transcription derived from matrix metalloproteinase (MMP) genes [6] associated with cancer cell invasion [7,8]. Overexpression of E1AF/PEA3 is associated with breast cancer [9] and is overexpressed in all mouse mammary tumors arising in transgenic mice engineered to overexpress murine HER2/neu in their mammary glands [10]. Although the mRNA level of E1AF is upregulated in human breast tumor cells, the endogenous protein of E1AF has never been detected ([11,12], our unpublished data). Therefore, we assume that the expression of E1AF is modulated after transcription. The ubiquitin–proteasome pathway is associated with the degradation of various proteins. The covalent attachment of ubiquitin to lysine residues in a substrate protein requires the action of a cascade of enzymes. Commonly, the target protein is ubiquitinated by three different enzymes, E1 (ubiquitin-activating enzyme), E2
576
A. Takahashi et al. / Biochemical and Biophysical Research Communications 327 (2005) 575–580
(ubiquitin-conjugating enzyme), and E3 (ubiquitin ligase). Finally, 26S proteasome degrades multi-ubiquitinated protein. Recently, many transcription factors have been shown to be associated with the ubiquitin– proteasome pathway [13,14]. However, none of the ETS family has been reported to undergo post-transcriptional modification by ubiquitin. Here we show that E1AF is modified by ubiquitin and is degraded through the ubiquitin–proteasome pathway. The ubiquitination target was the carboxylterminal region of E1AF, including the ETS-domain. Ubiquitinated E1AF was localized in small dot-like structures in the nucleus. The inhibition of 26S proteasome resulted in the upregulation of E1AF target promoters. We suppose that the regulating system involving the ubiquitin–proteasome pathway exists in malignant tumor cells.
HA-ubiquitin by FuGENE6. After 24 h, the cells were fixed with 4% formaldehyde and permeabilized with 0.1% Triton X-100 (Wako). After blocking in 3% bovine serum albumin (BSA, Sigma), the cells were incubated with 1 lg/ml of primary antibodies, anti-FLAG (Sigma) and anti-HA (3F10, Roche), diluted in PBS/BSA. After washing in PBS/BSA, cells were stained with Rhodamine- or FITC-conjugated secondary antibodies, (1:200, Molecular Probes). Confocal images were obtained using a Zeiss LSM 510 confocal laser-scanning microscope. Reporter gene assay. CE and NE cells were cotransfected with reporter plasmids (pGL2-MMP1 or pBSB-3XEBS) and pRL-tk vector using FuGENE6. After 24 h, the transfected cells were treated with 5 lM MG132 for 6 h. The lysates were assayed using a Luminometer (Lumat LB 9507, Berthold) with a Dual-luciferase reporter assay system (Promega).
Results and discussion 26S proteasome-dependent degradation of E1AF
Materials and methods Cells and plasmids. The cell lines used in this study were COS1, NIH3T3, 293 T, HeLa, and H1299. The cells were grown at 37 °C with 5% CO2 in DulbeccoÕs modified EagleÕs medium (DMEM) containing 10% fetal bovine serum (Gibco) with penicillin/streptomycin (Sigma). pcDNA3-E1AF and pBSB-3XEBS are as described [15,16]. pCI-neo-HA-ubiquitin was a generous gift from Dr. Nakagawa. Complementary DNA encoding E1AF and its deletion mutants, dl13 (86– 485), dl10 (155–485), dl7 (251–485), dl4 (294–485), dl1 (336–485), and dlETS (1–336), tagged at its NH2 terminus with the FLAG epitope were generated by polymerase chain reaction using Pfu DNA polymerase (Promega) and they were subcloned into pcDNA3 (Invitrogen) to produce pcDNA3-FLAG-E1AF and its mutants. The MMP1 promoter fragment was subcloned and inserted into luciferase reporter vector pGL2-Basic (Promega) to produce pGL2-MMP1. To produce E1AF expressing COS1 (CE), NIH3T3 (NE), and their control (CC and NC) cells, either pcDNA3-E1AF or pcDNA3 was transfected into COS1 or NIH3T3 cells by the calcium phosphate precipitation method and stable transfectants were selected with 400 lg/ml G418 (Sigma). Analysis of protein stability. CE, NE, CC, and NC cells were treated with 25 lM MG132 (Calbiochem) or dimethyl sulfoxide (DMSO) for 1 h and the lysates were subjected to immunoblotting for E1AF or b-actin. COS1 cells were transfected with pcDNA3-FLAG-E1AF or pcDNA3-FLAG-E1AFdlETS using FuGENE6 transfection reagent (Roche). After 36 h, the transfected cells were treated with 25 lM MG132 or DMSO for 1 h and then with cycloheximide (Sigma) at a final concentration of 25 lg/ml for 0, 2, 4, and 6 h. The expression level of protein was assessed by immunoblotting analysis using a FLAG antibody (Sigma). The band intensities were counted with Kodak Digital Science 1D Image Analysis software, version 3.0 (Eastman Kodak). Immunoprecipitation assay. 293 T cells were transfected with pcDNA3-FLAG-E1AF, the expression constructs for E1AF mutants described above or pcDNA3 with pCI-neo-HA-ubiquitin or pCI-neo by the calcium phosphate precipitation method. After 36 h, the transfected cells were treated with 5 lM MG132 for 12 h. Cells were washed twice with phosphate-buffered saline (PBS) before lysis in buffer containing 250 mM NaCl/50 mM Hepes, pH 7.0/0.1% Nonidet P-40 with protease inhibitor cocktails (Sigma) at the manufacturerÕs recommended concentration. The lysates were subjected to immunoprecipitation as described [17]. Immunofluorescence staining. HeLa and H1299 cells grown on glass coverslips were transfected with pcDNA3-FLAG-E1AF and pCI-neo-
To examine the proteasome-dependent degradation of E1AF, we prepared COS1 (CE1 and CE2) and NIH3T3 (NE1 and NE2) cells expressing E1AF. Judging from immunoblotting analysis, endogenous E1AF, even overexpressed E1AF, was not detected (Fig. 1A; lanes 1, 3, 5, 7, 9, and 11). On the other hand, when cells were treated with MG132, a specific inhibitor of the 26S proteasome, E1AF appeared within 12 h after treatment in both cell lines (Fig. 1A; lanes 4, 6, 10, and 12). However, in control cells (CC or NC), it did not appear, even if the cells were treated with MG132 (Fig. 1A; lanes 2 and 8). To determine whether the proteasome inhibitor directly affected the degradation of E1AF, we analyzed the stability of E1AF in which protein synthesis had been blocked by cycloheximide (CHX). As the expression level of endogenous E1AF was too low to detect, we transiently overexpressed E1AF in COS1 cells. The cells were then subjected to MG132 addition followed by treatment with CHX. As shown in Fig. 1B (upper panel), MG132 treatment, but not solvent DMSO, induced the accumulation of E1AF protein (Fig. 1B; lower panel), suggesting that E1AF was a substrate of the 26S proteasome in vivo. The inhibition of 26S proteasome alone could not completely prevent the degradation of E1AF in 4 and 6 h samples, suggesting that another degradation mechanism might be involved in the post-transcriptional modification of E1AF. To investigate whether E1AF was ubiquitinated in vivo, 293 T cells were cotransfected with FLAG-tagged E1AF and HA-tagged ubiquitin expression plasmids, and then the cells were treated with MG132. Twelve hours after treatment, FLAG-E1AF was immunoprecipitated from whole cell extracts using an anti-FLAG antibody and the samples were subjected to immunoblotting using an anti-HA antibody to detect ubiquitin-conjugated E1AF. A ladder of higher molecular weight-ubiquitinated products was detected only in the
A. Takahashi et al. / Biochemical and Biophysical Research Communications 327 (2005) 575–580
577
right). These results indicate that E1AF protein is multi-ubiquitinated when both E1AF and ubiquitin are overexpressed in cells. The C-terminal region of E1AF is required for ubiquitination To determine the target region of the ubiquitination, we constructed various deletion mutants of E1AF (Fig. 2A). These deletion mutants were coexpressed in 293 T cells with ubiquitin and subjected to immunoprecipitation assay. The ubiquitinated E1AFs were detected when the expression constructs for dl13, dl10, dl7, dl4, and dl1 were transfected into the cells, although no ubiquitinated protein existed in dlETS-expressing cells (Fig. 2B; upper panel). Although the same amount of expression plasmids was transfected into 293 T cells, the expression level of each E1AF mutant varied and the quantity of the precipitated ubiquitinated protein seemed to accord with the expression level of each protein (Fig. 2B; lower panel). We could see higher molecular weight forms only when HA-tagged ubiquitin was coexpressed in the cells (data not shown). Moreover, we observed that the dlETS was long-lived compared to wild-type E1AF (Fig. 2C). These results indicate that the carboxyl terminal region, including the ETS-domain, is essential for the ubiquitination of E1AF. Since the ETS-domain is highly conserved among the ETS family of proteins, the stability of other ETS family members is possibly controlled through the ubiquitin–proteasome pathway. As far as we know, no other ETS family is regulated by the ubiquitin–proteasome pathway, but it would be interesting to examine this possibility. Ubiquitinated E1AF localized in nuclear dots
Fig. 1. Effects of the proteasome-specific inhibitor on the stability of E1AF protein. (A) Cos1 and NIH3T3 cells expressing E1AF (CE and NE) or harboring its empty vector (CC and NC) were treated with proteasome inhibitor MG132 or its solvent dimethyl sulfoxide (DMSO). Expressions of E1AF and b-actin were analyzed by immunoblotting. (B) Cos1 cells were transfected with the FLAGE1AF expression plasmid and then cells were treated with MG132 or the same amount of DMSO for 1 h followed by cycloheximide (CHX) treatment for the indicated periods. The expression levels of E1AF and b-actin were assessed by immunoblotting. Lower panel shows the relative amount of E1AF analyzed by band intensities from three independent experiments. (C) 293 T cells were cotransfected with the indicated combinations of FLAG-E1AF and HA-ubiquitin expression plasmids and whole cell extracts were subjected to anti-FLAG immunoprecipitation followed by anti-HA (left, upper panel) or antiFLAG (left, lower panel) immunoblotting. To check the expression level of HA ubiquitin, the same cell lysates were directly subjected to immunoblotting (right panel).
case of cotransfection of both expression constructs (Fig. 1C; left). HA ubiquitin was well expressed in cells transfected with its expression plasmid alone (Fig. 1C;
PEA3 subfamily members are transcription factors and their nuclear localization has been reported [5]. E1AF is dominantly located and diffuses in the nucleus (Fig. 3A; left panel), when an E1AF expression construct was introduced into HeLa cells, as in ERM and ER81. On the other hand, with the addition of MG132, E1AF accumulated in small dot-like structures in the nucleus (Fig. 3A; right panel). We then observed both ubiquitin and E1AF localization by transfection of the expression plasmids of these proteins into HeLa (Fig. 3B) and H1299 (Fig. 3C) cells. In these cells, E1AF was dominantly localized in the nucleus, which contained multiple condensed granules (Figs. 3B and C; left panels). On the other hand, HA-tagged ubiquitin was diffused throughout both the cytoplasm and the nucleus (Figs. 3B and C; middle panels), and we could see condensed dots of ubiquitin in the nucleus. Consistent with MERGE images (Figs. 3B and C; right panels), E1AF and ubiquitin were colocalized in nuclear dot-like structures, suggesting that ubiquitinated E1AF is local-
578
A. Takahashi et al. / Biochemical and Biophysical Research Communications 327 (2005) 575–580
Fig. 3. Subcellular localization of ubiquitinated E1AF. (A) E1AF expression vector was transfected with HeLa cells and cells were then treated with (right panel) or without (left panel) MG132. HeLa (B) and H1299 (C) cells were cotransfected with E1AF and ubiquitin expression plasmids and treated with MG132 for 12 h. Cells were stained with the primary antibodies followed by Rhodamine (red; E1AF)- or FITC (green; ubiquitin)-conjugated secondary antibodies. The right panels show overlapping images of the two proteins to detect colocalization (MERGE).
structures, including ubiquitinated E1AF, might be ND10. Inhibition of the proteasome activates E1AF target promoters
Fig. 2. The carboxyl-terminal region of E1AF was required for ubiquitination. (A) Schematic diagram of E1AF and its mutants, dl13 (86–485), dl10 (155–485), dl7 (251–485), dl4 (294–485), dl1 (336–485), and dlETS (1–336), is shown. (B) 293 T cells were transfected with FLAG-E1AF mutants and HA-ubiquitin expression plasmids, and immunoprecipitation was performed as described in Fig. 1C. Upper panel indicates the result of immunoblotting using an HA antibody. The expression of each mutant in the same cell lysate is shown in the lower panel. Cells transfected with both pcDNA3 and HA-ubiquitin expression constructs were used as a control (c). (C) The protein stability of both wild-type (WT) and dlETS E1AF was analyzed as described in Fig. 1B.
ized in the dot-like structure in the nucleus. Since cell treatment with a proteasome inhibitor such as MG132 caused an increase in the number of ND10 [18], dot-like
To examine the effect of proteasome-dependent degradation on transcriptional activation properties of E1AF, we performed a luciferase assay using a reporter containing three copies of ETS-binding sites (3XEBS; CAGGAAGT), responsive to the PEA3 family of the proteins [16,17] (Fig. 4A). In CE (upper panel) and NE (lower panel) cells, the 3XEBS reporter was never activated, however, when proteasome activity was inhibited by MG132, the transcriptional activity increased 4and 10-fold from the basal level in CC and NC cells, respectively. We then carried out a reporter assay, using a reporter including the promoter of the matrix metalloprotease 1 (MMP1) gene, since E1AF has been shown to stimulate transcription from the MMP1 gene [6]. As shown in Fig. 4B, as in the case of the 3XEBS reporter, inhibition of the 26S proteasome resulted in activation of the MMP1 reporter in both cell lines. These data suggest that inhibition of the 26S proteasome activates the transcription of the E1AF target gene.
A. Takahashi et al. / Biochemical and Biophysical Research Communications 327 (2005) 575–580
579
Fig. 4. Effects of the proteasome on transcriptional activity of E1AF. (A) Cos1 (CC and CE) cells (upper panel) and NIH3T3 (NC and NE) cells (lower panel) were transiently transfected with reporter plasmids (pBSB-3XEBS). After 24 h, the cells were treated with MG132 or DMSO for a further 12 h. Luciferase assay was performed and the activities in CC and NC cells were normalized to 1.0. All assays were repeated individually at least three times. (B) The same experiments were performed using pGL2-MMP1, COS1 (upper panel), and NIH3T3 (lower panel) cells.
We show here that E1AF is degraded by the ubiquitin–proteasome pathway and that ubiquitination has the potential to influence the activity of E1AF. E1AF/ PEA3 is known as a target of the MAPK-dependent phosphorylation pathway and is actually phosphorylated at serine residues by both activated ERK and SAPK [19]. Since phosphorylation resulted in the transcriptional activation of PEA3, the effect of protein stability of E1AF might be related to such a modification. Indeed, ER81 is acetylated by p300 and P/CAF through the MAPK signaling cascade and signal transduction changed its protein stability, whereas the mechanism of degradation remained unclear [20]. In many tumors, the expression of E1AF mRNA is elevated and is involved in their development, contributing to the development of a malignant character, whereas endogenous E1AF has hardly been detected. In stable transfectant CE and NE cells, overexpressed E1AF could not be detected without proteasome inhibition. We demonstrated the degradation mechanism of E1AF through the C-terminal region by the ubiquitin– proteasome pathway. However, the level of endogenous E1AF protein was still undetectable with the addition of MG132 (data not shown). We assume that a degradation mechanism besides the 26S proteasome also regu-
lates the protein stability of E1AF, and it is thought that a pathway regulating this proteasome activity exists in malignant tumor cells.
Acknowledgments We thank Dr. Koji Nakagawa for providing the pCI-neo-HA-ubiquitin plasmid and Dr. Kohichi Nakajima for providing the pBSB-3XEBS plasmid. This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan.
References [1] V.I. Sementchenko, D.K. Watson, Ets target genes: past, present and future, Oncogene 19 (2000) 6533–6548. [2] F. Higashino, K. Yoshida, Y. Fujinaga, K. Kamio, K. Fujinaga, Isolation of a cDNA encoding the adenovirus E1A enhancer binding protein: a new human member of the ets oncogene family, Nucleic Acids Res. 21 (1993) 547–553. [3] J.H. Xin, A. Cowie, P. Lachance, J.A. Hassell, Molecular cloning and characterization of PEA3, a new member of the Ets oncogene family that is differentially expressed in mouse embryonic cells, Genes Dev. 6 (1992) 481–496.
580
A. Takahashi et al. / Biochemical and Biophysical Research Communications 327 (2005) 575–580
[4] T.A. Brown, S.L. McKnight, Specificities of protein–protein and protein–DNA interaction of GABP alpha and two newly defined ets-related proteins, Genes Dev. 6 (1992) 2502–2512. [5] D. Monte, J.L. Baert, P.A. Defossez, Y. de Launoit, D. Stehelin, Molecular cloning and characterization of human ERM, a new member of the Ets family closely related to mouse PEA3 and ER81 transcription factors, Oncogene 9 (1994) 1397– 1406. [6] F. Higashino, K. Yoshida, T. Noumi, M. Seiki, K. Fujinaga, Etsrelated protein E1A-F can activate three different matrix metalloproteinase gene promoters, Oncogene 10 (1995) 1461–1463. [7] M. Kaya, K. Yoshida, F. Higashino, T. Mitaka, S. Ishii, K. Fujinaga, A single ets-related transcription factor, E1AF, confers invasive phenotype on human cancer cells, Oncogene 12 (1996) 221–227. [8] M. Shindoh, F. Higashino, M. Kaya, M. Yasuda, K. Funaoka, M. Hanzawa, K. Hida, T. Kohgo, A. Amemiya, K. Yoshida, K. Fujinaga, Correlated expression of matrix metalloproteinases and ets family transcription factor E1A-F in invasive oral squamouscell-carcinoma-derived cell lines, Am. J. Pathol. 148 (1996) 693– 700. [9] C.C. Benz, R.C. OÕHagan, B. Richter, G.K. Scott, C.H. Chang, X. Xiong, K. Chew, B.M. Ljung, S. Edgerton, A. Thor, J.A. Hassell, HER2/Neu and the Ets transcription activator PEA3 are coordinately upregulated in human breast cancer, Oncogene 15 (1997) 1513–1525. [10] M.S. Trimble, J.H. Xin, C.T. Guy, W.J. Muller, J.A. Hassell, PEA3 is overexpressed in mouse metastatic mammary adenocarcinomas, Oncogene 8 (1993) 3037–3042. [11] G.K. Scott, J.C. Daniel, X. Xiong, R.A. Maki, D. Kabat, C.C. Benz, Binding of an ETS-related protein within the DNase I hypersensitive site of the HER2/neu promoter in human breast cancer cells, J. Biol. Chem. 269 (1994) 19848–19858. [12] J.L. Baert, D. Monte, E.A. Musgrove, O. Albagli, R.L. Sutherland, Y. de Launoit, Expression of the PEA3 group of ETS-
[13]
[14]
[15]
[16]
[17]
[18]
[19]
[20]
related transcription factors in human breast-cancer cells, Int. J. Cancer 70 (1997) 590–597. A. Marti, C. Wirbelauer, M. Scheffner, W. Krek, Interaction between ubiquitin-protein ligase SCFSKP2 and E2F-1 underlies the regulation of E2F-1 degradation, Nat. Cell Biol. 1 (1999) 14– 19. K. Nakagawa, H. Yokosawa, Degradation of transcription factor IRF-1 by the ubiquitin–proteasome pathway. The C-terminal region governs the protein stability, Eur. J. Biochem. 267 (2000) 1680–1686. A. Takahashi, F. Higashino, M. Aoyagi, K. Yoshida, M. Itoh, S. Kyo, T. Ohno, T. Taira, H. Ariga, K. Nakajima, M. Hatta, M. Kobayashi, H. Sano, T. Kohgo, M. Shindoh, EWS/ETS fusions activate telomerase in EwingÕs tumors, Cancer Res. 63 (2003) 8338–8344. K. Nakae, K. Nakajima, J. Inazawa, T. Kitaoka, T. Hirano, ERM, a PEA3 subfamily of Ets transcription factors, can cooperate with c-Jun, J. Biol. Chem. 270 (1995) 23795–23800. M. Aoyagi, F. Higashino, M. Yasuda, A. Takahashi, Y. Sawada, Y. Totsuka, T. Kohgo, H. Sano, M. Kobayashi, M. Shindoh, Nuclear export of adenovirus E4orf6 protein is necessary for its ability to antagonize apoptotic activity of BH3-only proteins, Oncogene 22 (2003) 6919–6927. R.D. Everett, P. Freemont, H. Saitoh, M. Dasso, A. Orr, M. Kathoria, J. Parkinson, The disruption of ND10 during herpes simplex virus infection correlates with the Vmw110- and proteasome-dependent loss of several PML isoforms, J. Virol. 72 (1998) 6581–6591. R.C. OÕHagan, R.G. Tozer, M. Symons, F. McCormick, J.A. Hassell, The activity of the Ets transcription factor PEA3 is regulated by two distinct MAPK cascades, Oncogene 13 (1996) 1323–1333. A. Goel, R. Janknecht, Acetylation-mediated transcriptional activation of the ETS protein ER81 by p300, P/CAF, and HER2/Neu, Mol. Cell Biol. 23 (2003) 6243–6254.
E1AF degradation by a ubiquitin–proteasome pathway Akiko Takahashia,1,2, Fumihiro Higashinoa,2, Mariko Aoyagia, Koichi Yoshidab, Miyuki Itohb, Masanobu Kobayashic, Yasunori Totsukaa, Takao Kohgoa, Masanobu Shindoha,* a
c
Department of Oral Pathobiological Science, Hokkaido University Graduate School of Dental Medicine, North 13 West 7, Kita-ku 060-8586, Sapporo, Japan b Department of Biology, Sapporo Medical University School of Medicine, South 1 West 17, Chuo-ku 060-8556, Sapporo, Japan Division of Cancer Pathobiology, Institute for Genetic Medicine, Hokkaido University, North 15 West 7, Kita-ku 060-0815, Sapporo, Japan Received 3 December 2004 Available online 18 December 2004
Abstract E1AF is a member of the ETS family of transcription factors. In mammary tumors, overexpression of E1AF is associated with tumorigenesis, but E1AF protein has hardly been detected and its degradation mechanism is not yet clear. Here we show that E1AF protein is stabilized by treatment with the 26S protease inhibitor MG132. We found that E1AF was modified by ubiquitin through the C-terminal region and ubiquitinated E1AF aggregated in nuclear dots, and that the inhibition of proteasome-activated transcription from E1AF target promoters. These results suggest that E1AF is degraded via the ubiquitin–proteasome pathway, which has some effect on E1AF function. Ó 2004 Elsevier Inc. All rights reserved. Keywords: E1AF; ETS; PEA3; Proteasome; Ubiquitin; Degradation
ETS proteins have a DNA-binding domain consisting of approximately 85 amino acids (ETS-domain) and function as transcription factors by binding to a GGA core motif in enhancers of various genes [1]. E1AF is a member of the ETS oncogene family that was cloned for its ability to bind to the enhancer elements of the adenovirus E1A gene [2]. Analysis of deduced amino acids from the cDNA has shown that E1AF is a human counterpart of mouse PEA3 (polyomavirus enhancer activator 3) [3]. The PEA3 subfamily also includes ER81 [4] and ERM [5], which bind to the PEA3 specif*
Corresponding author. Fax: +81 11 706 4237. E-mail address: [email protected] (M. Shindoh). 1 Present address: Division of Protein Information, Institute for Genome Research, The University of Tokushima, 3-18-15, Kuramotocho 770-8503, Tokushima-city, Tokushima, Japan. 2 These two authors contributed equally to this work. 0006-291X/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2004.12.045
ic-binding motif. E1AF has been shown to activate transcription derived from matrix metalloproteinase (MMP) genes [6] associated with cancer cell invasion [7,8]. Overexpression of E1AF/PEA3 is associated with breast cancer [9] and is overexpressed in all mouse mammary tumors arising in transgenic mice engineered to overexpress murine HER2/neu in their mammary glands [10]. Although the mRNA level of E1AF is upregulated in human breast tumor cells, the endogenous protein of E1AF has never been detected ([11,12], our unpublished data). Therefore, we assume that the expression of E1AF is modulated after transcription. The ubiquitin–proteasome pathway is associated with the degradation of various proteins. The covalent attachment of ubiquitin to lysine residues in a substrate protein requires the action of a cascade of enzymes. Commonly, the target protein is ubiquitinated by three different enzymes, E1 (ubiquitin-activating enzyme), E2
576
A. Takahashi et al. / Biochemical and Biophysical Research Communications 327 (2005) 575–580
(ubiquitin-conjugating enzyme), and E3 (ubiquitin ligase). Finally, 26S proteasome degrades multi-ubiquitinated protein. Recently, many transcription factors have been shown to be associated with the ubiquitin– proteasome pathway [13,14]. However, none of the ETS family has been reported to undergo post-transcriptional modification by ubiquitin. Here we show that E1AF is modified by ubiquitin and is degraded through the ubiquitin–proteasome pathway. The ubiquitination target was the carboxylterminal region of E1AF, including the ETS-domain. Ubiquitinated E1AF was localized in small dot-like structures in the nucleus. The inhibition of 26S proteasome resulted in the upregulation of E1AF target promoters. We suppose that the regulating system involving the ubiquitin–proteasome pathway exists in malignant tumor cells.
HA-ubiquitin by FuGENE6. After 24 h, the cells were fixed with 4% formaldehyde and permeabilized with 0.1% Triton X-100 (Wako). After blocking in 3% bovine serum albumin (BSA, Sigma), the cells were incubated with 1 lg/ml of primary antibodies, anti-FLAG (Sigma) and anti-HA (3F10, Roche), diluted in PBS/BSA. After washing in PBS/BSA, cells were stained with Rhodamine- or FITC-conjugated secondary antibodies, (1:200, Molecular Probes). Confocal images were obtained using a Zeiss LSM 510 confocal laser-scanning microscope. Reporter gene assay. CE and NE cells were cotransfected with reporter plasmids (pGL2-MMP1 or pBSB-3XEBS) and pRL-tk vector using FuGENE6. After 24 h, the transfected cells were treated with 5 lM MG132 for 6 h. The lysates were assayed using a Luminometer (Lumat LB 9507, Berthold) with a Dual-luciferase reporter assay system (Promega).
Results and discussion 26S proteasome-dependent degradation of E1AF
Materials and methods Cells and plasmids. The cell lines used in this study were COS1, NIH3T3, 293 T, HeLa, and H1299. The cells were grown at 37 °C with 5% CO2 in DulbeccoÕs modified EagleÕs medium (DMEM) containing 10% fetal bovine serum (Gibco) with penicillin/streptomycin (Sigma). pcDNA3-E1AF and pBSB-3XEBS are as described [15,16]. pCI-neo-HA-ubiquitin was a generous gift from Dr. Nakagawa. Complementary DNA encoding E1AF and its deletion mutants, dl13 (86– 485), dl10 (155–485), dl7 (251–485), dl4 (294–485), dl1 (336–485), and dlETS (1–336), tagged at its NH2 terminus with the FLAG epitope were generated by polymerase chain reaction using Pfu DNA polymerase (Promega) and they were subcloned into pcDNA3 (Invitrogen) to produce pcDNA3-FLAG-E1AF and its mutants. The MMP1 promoter fragment was subcloned and inserted into luciferase reporter vector pGL2-Basic (Promega) to produce pGL2-MMP1. To produce E1AF expressing COS1 (CE), NIH3T3 (NE), and their control (CC and NC) cells, either pcDNA3-E1AF or pcDNA3 was transfected into COS1 or NIH3T3 cells by the calcium phosphate precipitation method and stable transfectants were selected with 400 lg/ml G418 (Sigma). Analysis of protein stability. CE, NE, CC, and NC cells were treated with 25 lM MG132 (Calbiochem) or dimethyl sulfoxide (DMSO) for 1 h and the lysates were subjected to immunoblotting for E1AF or b-actin. COS1 cells were transfected with pcDNA3-FLAG-E1AF or pcDNA3-FLAG-E1AFdlETS using FuGENE6 transfection reagent (Roche). After 36 h, the transfected cells were treated with 25 lM MG132 or DMSO for 1 h and then with cycloheximide (Sigma) at a final concentration of 25 lg/ml for 0, 2, 4, and 6 h. The expression level of protein was assessed by immunoblotting analysis using a FLAG antibody (Sigma). The band intensities were counted with Kodak Digital Science 1D Image Analysis software, version 3.0 (Eastman Kodak). Immunoprecipitation assay. 293 T cells were transfected with pcDNA3-FLAG-E1AF, the expression constructs for E1AF mutants described above or pcDNA3 with pCI-neo-HA-ubiquitin or pCI-neo by the calcium phosphate precipitation method. After 36 h, the transfected cells were treated with 5 lM MG132 for 12 h. Cells were washed twice with phosphate-buffered saline (PBS) before lysis in buffer containing 250 mM NaCl/50 mM Hepes, pH 7.0/0.1% Nonidet P-40 with protease inhibitor cocktails (Sigma) at the manufacturerÕs recommended concentration. The lysates were subjected to immunoprecipitation as described [17]. Immunofluorescence staining. HeLa and H1299 cells grown on glass coverslips were transfected with pcDNA3-FLAG-E1AF and pCI-neo-
To examine the proteasome-dependent degradation of E1AF, we prepared COS1 (CE1 and CE2) and NIH3T3 (NE1 and NE2) cells expressing E1AF. Judging from immunoblotting analysis, endogenous E1AF, even overexpressed E1AF, was not detected (Fig. 1A; lanes 1, 3, 5, 7, 9, and 11). On the other hand, when cells were treated with MG132, a specific inhibitor of the 26S proteasome, E1AF appeared within 12 h after treatment in both cell lines (Fig. 1A; lanes 4, 6, 10, and 12). However, in control cells (CC or NC), it did not appear, even if the cells were treated with MG132 (Fig. 1A; lanes 2 and 8). To determine whether the proteasome inhibitor directly affected the degradation of E1AF, we analyzed the stability of E1AF in which protein synthesis had been blocked by cycloheximide (CHX). As the expression level of endogenous E1AF was too low to detect, we transiently overexpressed E1AF in COS1 cells. The cells were then subjected to MG132 addition followed by treatment with CHX. As shown in Fig. 1B (upper panel), MG132 treatment, but not solvent DMSO, induced the accumulation of E1AF protein (Fig. 1B; lower panel), suggesting that E1AF was a substrate of the 26S proteasome in vivo. The inhibition of 26S proteasome alone could not completely prevent the degradation of E1AF in 4 and 6 h samples, suggesting that another degradation mechanism might be involved in the post-transcriptional modification of E1AF. To investigate whether E1AF was ubiquitinated in vivo, 293 T cells were cotransfected with FLAG-tagged E1AF and HA-tagged ubiquitin expression plasmids, and then the cells were treated with MG132. Twelve hours after treatment, FLAG-E1AF was immunoprecipitated from whole cell extracts using an anti-FLAG antibody and the samples were subjected to immunoblotting using an anti-HA antibody to detect ubiquitin-conjugated E1AF. A ladder of higher molecular weight-ubiquitinated products was detected only in the
A. Takahashi et al. / Biochemical and Biophysical Research Communications 327 (2005) 575–580
577
right). These results indicate that E1AF protein is multi-ubiquitinated when both E1AF and ubiquitin are overexpressed in cells. The C-terminal region of E1AF is required for ubiquitination To determine the target region of the ubiquitination, we constructed various deletion mutants of E1AF (Fig. 2A). These deletion mutants were coexpressed in 293 T cells with ubiquitin and subjected to immunoprecipitation assay. The ubiquitinated E1AFs were detected when the expression constructs for dl13, dl10, dl7, dl4, and dl1 were transfected into the cells, although no ubiquitinated protein existed in dlETS-expressing cells (Fig. 2B; upper panel). Although the same amount of expression plasmids was transfected into 293 T cells, the expression level of each E1AF mutant varied and the quantity of the precipitated ubiquitinated protein seemed to accord with the expression level of each protein (Fig. 2B; lower panel). We could see higher molecular weight forms only when HA-tagged ubiquitin was coexpressed in the cells (data not shown). Moreover, we observed that the dlETS was long-lived compared to wild-type E1AF (Fig. 2C). These results indicate that the carboxyl terminal region, including the ETS-domain, is essential for the ubiquitination of E1AF. Since the ETS-domain is highly conserved among the ETS family of proteins, the stability of other ETS family members is possibly controlled through the ubiquitin–proteasome pathway. As far as we know, no other ETS family is regulated by the ubiquitin–proteasome pathway, but it would be interesting to examine this possibility. Ubiquitinated E1AF localized in nuclear dots
Fig. 1. Effects of the proteasome-specific inhibitor on the stability of E1AF protein. (A) Cos1 and NIH3T3 cells expressing E1AF (CE and NE) or harboring its empty vector (CC and NC) were treated with proteasome inhibitor MG132 or its solvent dimethyl sulfoxide (DMSO). Expressions of E1AF and b-actin were analyzed by immunoblotting. (B) Cos1 cells were transfected with the FLAGE1AF expression plasmid and then cells were treated with MG132 or the same amount of DMSO for 1 h followed by cycloheximide (CHX) treatment for the indicated periods. The expression levels of E1AF and b-actin were assessed by immunoblotting. Lower panel shows the relative amount of E1AF analyzed by band intensities from three independent experiments. (C) 293 T cells were cotransfected with the indicated combinations of FLAG-E1AF and HA-ubiquitin expression plasmids and whole cell extracts were subjected to anti-FLAG immunoprecipitation followed by anti-HA (left, upper panel) or antiFLAG (left, lower panel) immunoblotting. To check the expression level of HA ubiquitin, the same cell lysates were directly subjected to immunoblotting (right panel).
case of cotransfection of both expression constructs (Fig. 1C; left). HA ubiquitin was well expressed in cells transfected with its expression plasmid alone (Fig. 1C;
PEA3 subfamily members are transcription factors and their nuclear localization has been reported [5]. E1AF is dominantly located and diffuses in the nucleus (Fig. 3A; left panel), when an E1AF expression construct was introduced into HeLa cells, as in ERM and ER81. On the other hand, with the addition of MG132, E1AF accumulated in small dot-like structures in the nucleus (Fig. 3A; right panel). We then observed both ubiquitin and E1AF localization by transfection of the expression plasmids of these proteins into HeLa (Fig. 3B) and H1299 (Fig. 3C) cells. In these cells, E1AF was dominantly localized in the nucleus, which contained multiple condensed granules (Figs. 3B and C; left panels). On the other hand, HA-tagged ubiquitin was diffused throughout both the cytoplasm and the nucleus (Figs. 3B and C; middle panels), and we could see condensed dots of ubiquitin in the nucleus. Consistent with MERGE images (Figs. 3B and C; right panels), E1AF and ubiquitin were colocalized in nuclear dot-like structures, suggesting that ubiquitinated E1AF is local-
578
A. Takahashi et al. / Biochemical and Biophysical Research Communications 327 (2005) 575–580
Fig. 3. Subcellular localization of ubiquitinated E1AF. (A) E1AF expression vector was transfected with HeLa cells and cells were then treated with (right panel) or without (left panel) MG132. HeLa (B) and H1299 (C) cells were cotransfected with E1AF and ubiquitin expression plasmids and treated with MG132 for 12 h. Cells were stained with the primary antibodies followed by Rhodamine (red; E1AF)- or FITC (green; ubiquitin)-conjugated secondary antibodies. The right panels show overlapping images of the two proteins to detect colocalization (MERGE).
structures, including ubiquitinated E1AF, might be ND10. Inhibition of the proteasome activates E1AF target promoters
Fig. 2. The carboxyl-terminal region of E1AF was required for ubiquitination. (A) Schematic diagram of E1AF and its mutants, dl13 (86–485), dl10 (155–485), dl7 (251–485), dl4 (294–485), dl1 (336–485), and dlETS (1–336), is shown. (B) 293 T cells were transfected with FLAG-E1AF mutants and HA-ubiquitin expression plasmids, and immunoprecipitation was performed as described in Fig. 1C. Upper panel indicates the result of immunoblotting using an HA antibody. The expression of each mutant in the same cell lysate is shown in the lower panel. Cells transfected with both pcDNA3 and HA-ubiquitin expression constructs were used as a control (c). (C) The protein stability of both wild-type (WT) and dlETS E1AF was analyzed as described in Fig. 1B.
ized in the dot-like structure in the nucleus. Since cell treatment with a proteasome inhibitor such as MG132 caused an increase in the number of ND10 [18], dot-like
To examine the effect of proteasome-dependent degradation on transcriptional activation properties of E1AF, we performed a luciferase assay using a reporter containing three copies of ETS-binding sites (3XEBS; CAGGAAGT), responsive to the PEA3 family of the proteins [16,17] (Fig. 4A). In CE (upper panel) and NE (lower panel) cells, the 3XEBS reporter was never activated, however, when proteasome activity was inhibited by MG132, the transcriptional activity increased 4and 10-fold from the basal level in CC and NC cells, respectively. We then carried out a reporter assay, using a reporter including the promoter of the matrix metalloprotease 1 (MMP1) gene, since E1AF has been shown to stimulate transcription from the MMP1 gene [6]. As shown in Fig. 4B, as in the case of the 3XEBS reporter, inhibition of the 26S proteasome resulted in activation of the MMP1 reporter in both cell lines. These data suggest that inhibition of the 26S proteasome activates the transcription of the E1AF target gene.
A. Takahashi et al. / Biochemical and Biophysical Research Communications 327 (2005) 575–580
579
Fig. 4. Effects of the proteasome on transcriptional activity of E1AF. (A) Cos1 (CC and CE) cells (upper panel) and NIH3T3 (NC and NE) cells (lower panel) were transiently transfected with reporter plasmids (pBSB-3XEBS). After 24 h, the cells were treated with MG132 or DMSO for a further 12 h. Luciferase assay was performed and the activities in CC and NC cells were normalized to 1.0. All assays were repeated individually at least three times. (B) The same experiments were performed using pGL2-MMP1, COS1 (upper panel), and NIH3T3 (lower panel) cells.
We show here that E1AF is degraded by the ubiquitin–proteasome pathway and that ubiquitination has the potential to influence the activity of E1AF. E1AF/ PEA3 is known as a target of the MAPK-dependent phosphorylation pathway and is actually phosphorylated at serine residues by both activated ERK and SAPK [19]. Since phosphorylation resulted in the transcriptional activation of PEA3, the effect of protein stability of E1AF might be related to such a modification. Indeed, ER81 is acetylated by p300 and P/CAF through the MAPK signaling cascade and signal transduction changed its protein stability, whereas the mechanism of degradation remained unclear [20]. In many tumors, the expression of E1AF mRNA is elevated and is involved in their development, contributing to the development of a malignant character, whereas endogenous E1AF has hardly been detected. In stable transfectant CE and NE cells, overexpressed E1AF could not be detected without proteasome inhibition. We demonstrated the degradation mechanism of E1AF through the C-terminal region by the ubiquitin– proteasome pathway. However, the level of endogenous E1AF protein was still undetectable with the addition of MG132 (data not shown). We assume that a degradation mechanism besides the 26S proteasome also regu-
lates the protein stability of E1AF, and it is thought that a pathway regulating this proteasome activity exists in malignant tumor cells.
Acknowledgments We thank Dr. Koji Nakagawa for providing the pCI-neo-HA-ubiquitin plasmid and Dr. Kohichi Nakajima for providing the pBSB-3XEBS plasmid. This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan.
References [1] V.I. Sementchenko, D.K. Watson, Ets target genes: past, present and future, Oncogene 19 (2000) 6533–6548. [2] F. Higashino, K. Yoshida, Y. Fujinaga, K. Kamio, K. Fujinaga, Isolation of a cDNA encoding the adenovirus E1A enhancer binding protein: a new human member of the ets oncogene family, Nucleic Acids Res. 21 (1993) 547–553. [3] J.H. Xin, A. Cowie, P. Lachance, J.A. Hassell, Molecular cloning and characterization of PEA3, a new member of the Ets oncogene family that is differentially expressed in mouse embryonic cells, Genes Dev. 6 (1992) 481–496.
580
A. Takahashi et al. / Biochemical and Biophysical Research Communications 327 (2005) 575–580
[4] T.A. Brown, S.L. McKnight, Specificities of protein–protein and protein–DNA interaction of GABP alpha and two newly defined ets-related proteins, Genes Dev. 6 (1992) 2502–2512. [5] D. Monte, J.L. Baert, P.A. Defossez, Y. de Launoit, D. Stehelin, Molecular cloning and characterization of human ERM, a new member of the Ets family closely related to mouse PEA3 and ER81 transcription factors, Oncogene 9 (1994) 1397– 1406. [6] F. Higashino, K. Yoshida, T. Noumi, M. Seiki, K. Fujinaga, Etsrelated protein E1A-F can activate three different matrix metalloproteinase gene promoters, Oncogene 10 (1995) 1461–1463. [7] M. Kaya, K. Yoshida, F. Higashino, T. Mitaka, S. Ishii, K. Fujinaga, A single ets-related transcription factor, E1AF, confers invasive phenotype on human cancer cells, Oncogene 12 (1996) 221–227. [8] M. Shindoh, F. Higashino, M. Kaya, M. Yasuda, K. Funaoka, M. Hanzawa, K. Hida, T. Kohgo, A. Amemiya, K. Yoshida, K. Fujinaga, Correlated expression of matrix metalloproteinases and ets family transcription factor E1A-F in invasive oral squamouscell-carcinoma-derived cell lines, Am. J. Pathol. 148 (1996) 693– 700. [9] C.C. Benz, R.C. OÕHagan, B. Richter, G.K. Scott, C.H. Chang, X. Xiong, K. Chew, B.M. Ljung, S. Edgerton, A. Thor, J.A. Hassell, HER2/Neu and the Ets transcription activator PEA3 are coordinately upregulated in human breast cancer, Oncogene 15 (1997) 1513–1525. [10] M.S. Trimble, J.H. Xin, C.T. Guy, W.J. Muller, J.A. Hassell, PEA3 is overexpressed in mouse metastatic mammary adenocarcinomas, Oncogene 8 (1993) 3037–3042. [11] G.K. Scott, J.C. Daniel, X. Xiong, R.A. Maki, D. Kabat, C.C. Benz, Binding of an ETS-related protein within the DNase I hypersensitive site of the HER2/neu promoter in human breast cancer cells, J. Biol. Chem. 269 (1994) 19848–19858. [12] J.L. Baert, D. Monte, E.A. Musgrove, O. Albagli, R.L. Sutherland, Y. de Launoit, Expression of the PEA3 group of ETS-
[13]
[14]
[15]
[16]
[17]
[18]
[19]
[20]
related transcription factors in human breast-cancer cells, Int. J. Cancer 70 (1997) 590–597. A. Marti, C. Wirbelauer, M. Scheffner, W. Krek, Interaction between ubiquitin-protein ligase SCFSKP2 and E2F-1 underlies the regulation of E2F-1 degradation, Nat. Cell Biol. 1 (1999) 14– 19. K. Nakagawa, H. Yokosawa, Degradation of transcription factor IRF-1 by the ubiquitin–proteasome pathway. The C-terminal region governs the protein stability, Eur. J. Biochem. 267 (2000) 1680–1686. A. Takahashi, F. Higashino, M. Aoyagi, K. Yoshida, M. Itoh, S. Kyo, T. Ohno, T. Taira, H. Ariga, K. Nakajima, M. Hatta, M. Kobayashi, H. Sano, T. Kohgo, M. Shindoh, EWS/ETS fusions activate telomerase in EwingÕs tumors, Cancer Res. 63 (2003) 8338–8344. K. Nakae, K. Nakajima, J. Inazawa, T. Kitaoka, T. Hirano, ERM, a PEA3 subfamily of Ets transcription factors, can cooperate with c-Jun, J. Biol. Chem. 270 (1995) 23795–23800. M. Aoyagi, F. Higashino, M. Yasuda, A. Takahashi, Y. Sawada, Y. Totsuka, T. Kohgo, H. Sano, M. Kobayashi, M. Shindoh, Nuclear export of adenovirus E4orf6 protein is necessary for its ability to antagonize apoptotic activity of BH3-only proteins, Oncogene 22 (2003) 6919–6927. R.D. Everett, P. Freemont, H. Saitoh, M. Dasso, A. Orr, M. Kathoria, J. Parkinson, The disruption of ND10 during herpes simplex virus infection correlates with the Vmw110- and proteasome-dependent loss of several PML isoforms, J. Virol. 72 (1998) 6581–6591. R.C. OÕHagan, R.G. Tozer, M. Symons, F. McCormick, J.A. Hassell, The activity of the Ets transcription factor PEA3 is regulated by two distinct MAPK cascades, Oncogene 13 (1996) 1323–1333. A. Goel, R. Janknecht, Acetylation-mediated transcriptional activation of the ETS protein ER81 by p300, P/CAF, and HER2/Neu, Mol. Cell Biol. 23 (2003) 6243–6254.